Publications

Early life stress is an important vulnerability factor for the development of anxiety disorders, depression and late-onset cognitive decline. Recently, we demonstrated that juvenile stress (JS) lastingly enhanced long-term potentiation via reduction of steady-state glutamine synthetase mRNA expression and the associated dysregulation of the astrocytic glutamate-glutamine cycle in the rat ventral CA1. We now investigated the regulation of steady-state mRNA expression of neuronal gene products that determine GABAergic and glutamatergic neurotransmission in layers of the ventral and dorsal CA1 after JS. We further studied their interaction with stress in young adult age (AS) to address their putative role in psychopathology development. Strikingly, mRNA levels of the glutamic acid decarboxylase (GAD) isoforms GAD65 and of the GABA-A receptor α2 (Gabra2) were increased after single JS or AS, but not after combined JS/AS stress experience. In fact, JS/AS resulted in layer-specific reduction of Gabra2 and also of Gabra1 mRNA levels in the ventral CA1. Furthermore, GAD65 and Gabra2 mRNAs were correlated with glutamatergic AMPA and NMDA receptor subunit mRNAs after single JS and AS, but not after combined JS/AS. Together, these data indicate a loss of allostatic regulation of steady-state mRNA levels of key GABAergic components that may result in a dysregulation of excitation/ inhibition balance in the ventral CA1 upon dual stress exposure. Finally, individual differences in local glucocorticoid receptor mRNA expression may contribute to this regulation.

The interplay between excitation and inhibition is crucial for neuronal circuitry in the brain. Inhibitory cell fractions in the neocortex and hippocampus are typically maintained at 15 to 30%, which is assumed to be important for stable dynamics. We have studied systematically the role of precisely controlled excitatory/inhibitory (E/I) cellular ratios on network activity using mice hippocampal cultures. Surprisingly, networks with varying E/I ratios maintain stable bursting dynamics. Interburst intervals remain constant for most ratios, except in the extremes of 0 to 10% and 90 to 100% inhibitory cells. Single-cell recordings and modeling suggest that networks adapt to chronic alterations of E/I compositions by balancing E/I connectivity. Gradual blockade of inhibition substantiates the agreement between the model and experiment and defines its limits. Combining measurements of population and single-cell activity with theoretical modeling, we provide a clearer picture of how E/I balance is preserved and where it fails in living neuronal networks.

Alcohol dependence is one of the top priority public health problems on a global scale. The costs of medical treatments of patients with alcohol dependence, a decrease in labor productivity, an increased risk of developing somatic and mental disorders, and early mortality are all consequences of acute and chronic alcohol abuse. The brain is one of the main targets of alcohol intoxication. Extensive neurobiological studies have revealed a number of synaptic and extra-synaptic mechanisms, affected by alcohol. A primary target of it is GABAergic transmission. Nevertheless, the exciting and disinhibiting actions of alcohol at the system and cellular levels have not been satisfactorily elucidated. It remains unclear whether effects of ethanol are highly complex, manifested only at the level of entire brain or concerns also individual cells, their subcellular structures, organelles, ion channels and receptors. With this approach, small, cultured neural networks that are isolated from the rest of the brain are of particular interest. A serious problem of modern pharmaceuticals is the lack of drugs that have a therapeutic effect on alcohol toxicity of the brain and nervous system, despite the abundance of so-called \u201ctraditional medicines\u201d. Substances obtained from some herbs containing a mixture of biologically active substances that exhibit a wide range of properties are of particular interest. Among them - flavonoids, which are polyphenols of plant origin and often reveal a sign of sedative, neuroprotective, antidepressant properties, and may improve cognitive function. The aims of our study is to reveal the mechanisms of various concentrations of ethanol, as well as its chronic effects on the functional properties of neurons in small neural networks such as the primary neuronal culture of the rat hippocampus. We have also performed a complex neuropharmacology screening and the study of flavonoids, extracted from Scrophulariaceae plant family, which is known in the traditional medicine for its antialcohol properties.

The enhanced ability to visualize small neuronal compartments in live tissue, such as individual dendritic spine, is accompanied in recent years by a need for a precise, high temporal and spatial resolution ability to activate or suppress electrophysiological as well as biochemical properties within such compartments. Parallel rapid progress in molecular, cellular, and physics methodologies enabled the recruitment of novel technologies to the analysis of a wide spectrum of issues, from long lasting imaging of subcellular compartments in vitro as well as in vivo, to simultaneous recording of activities of networks of hundreds of neurons in behaving animals. In the present review, we will focus on a fast UV flash (4 ns) photolysis of caged molecules in a small sphere (

Disruption in calcium homeostasis is linked to several pathologies and is suggested to play a pivotal role in the cascade of events leading to Alzheimer's disease (AD). Synaptopodin (SP) residing in dendritic spines has been associated with ryanodine receptor (RyR), such that spines lacking SP release less calcium from stores. In this work, we mated SPKO with 3xTg mice (3xTg/ SPKO) to test the effect of SP deficiency in the AD mouse. We found that 6-month-old male 3xTg/ SPKO mice restored normal spatial learning in the Barns maze, LTP in hippocampal slices, and expression levels of RyR in the hippocampus that were altered in the 3xTg mice. In addition, there was a marked reduction in 3xTg-associated phosphorylated tau, amyloid beta plaques, and activated microglia in 3xTg/ SPKO male and female mice. These experiments indicate that a reduction in the expression of SP ameliorates AD-associated phenotype in 3xTg mice.

Palmitoyl-protein thioesterase 1 (PPT1) is a depalmitoylation enzyme that is mutated in cases of neuronal ceroid lipofuscinosis (NCL). The hallmarks of the disease include progressive neurodegeneration and blindness, as well as seizures. In the current study, we identified 62 high-confident PPT1-binding proteins. These proteins included a self-interaction of PPT1, two V-type ATPases, calcium voltage-gated channels, cytoskeletal proteins and others. Pathway analysis suggested their involvement in seizures and neuronal morphology. We then proceeded to demonstrate that hippocampal neurons from Ppt1-/- mice exhibit structural deficits, and further investigated electrophysiology parameters in the hippocampi of mutant mice, both in brain slices and dissociated postnatal primary cultures. Our studies reveal new mechanistic features involved in the pathophysiology of this devastating neurodegenerative disease.

Extracellular calcium ions support synaptic activity but also reduce excitability of central neurons. In the present study. the effect of calcium on excitability was explored in cultured hippocampal neurons. CaCl2 injected by pressure in the vicinity of a neuron that is bathed only in MgCl2 as the main divalent cation caused a depolarizing shift in action potential threshold and a reduction in excitability. This effect was not seen if the intracellular milieu consisted of Cs+ instead of K-gluconate as the main cation or when it contained ruthenium red, which blocks release of calcium from stores. The suppression of excitability by calcium was mimicked by caffeine, and calcium store antagonists cyclopiazonic acid or thapsigargin blocked this action. Neurons taken from synaptopodin-knockout mice show significantly reduced efficacy of calcium modulation of action potential threshold. Likewise. in Orail knockdown cells, calcium is less effective in modulating excitability of neurons. Activation of small-conductance K (SK) channels increased action potential threshold akin to that produced by calcium ions, whereas blockade of SK channels but not big K channels reduced the threshold for action potential discharge. These results indicate that calcium released from stores may suppress excitability of central neurons.NEW & NOTEWORTHY Extracellular calcium reduces excitability of cultured hippocampal neurons. This effect is mediated by calcium-gated potassium currents, possibly small-conductance K channels. Release of calcium from internal stores mimics the effect of extracellular calcium. It is proposed that calcium stores modulate excitability of central neurons.

Stress has a profound effect on ability to express neuronal plasticity, learning, and memory. Likewise, epileptic seizures lead to massive changes in brain connectivity, and in ability to undergo long term changes in reactivity to afferent stimulation. In this study, we analyzed possible long lasting interactions between a stressful experience and reactivity to pilocarpine, on the ability to produce long term potentiation (LTP) in a mouse hippocampus. Pilocarpine lowers paired pulse potentiation as well as LTP in CA1 region of the mouse hippocampal slice. When stress experience precedes exposure to pilocarpine, it protects the brain from the lasting effect of pilocarpine. When stress follows pilocarpine, it exacerbates the effect of the drug, to produce a long lasting reduction in LTP. These changes are accompanied by a parallel change in blood corticosterone level. A single exposure to selective mineralo- or gluco-corticosterone (MR and GR, respectively) agonists and antagonists can mimic the stress effects, indicating that GR's underlie the lasting detrimental effects of stress whereas MRs are instrumental in counteracting the effects of stress. These studies open a new avenue of understanding of the interactive effects of stress and epileptic seizures on brain plasticity.

ALBRECHT, A., MULLER, L, ARDI, Z, CALISKAN, G., GRUBER, D., IVENS, S., SEGAL, M., BEHR, J., HEINEMANN, U., STORK, O., and RICHTER-LEVIN, G. Neurobiological consequences of juvenile stress: A GABAergic perspective on risk and resilience. NEUROSCI BIOBEHAV REV XXX-XXX, 2016. - Childhood adversity is among the most potent risk factors for developing mood and anxiety disorders later in life. Therefore, understanding how stress during childhood shapes and rewires the brain may optimize preventive and therapeutic strategies for these disorders.To this end, animal models of stress exposure in rodents during their post-weaning and pre-pubertal life phase have been developed. Such 'juvenile stress' has a long-lasting impact on mood and anxiety like behavior and on stress coping in adulthood, accompanied by alterations of the GABAergic system within core regions for the stress processing such as the amygdala, prefrontal cortex and hippocampus. While many regionally diverse molecular and electrophysiological changes are observed, not all of them correlate with juvenile stress-induced behavioral disturbances. It rather seems that certain juvenile stress-induced alterations reflect the system's attempts to maintain homeostasis and thus promote stress resilience. Analysis tools such as individual behavioral profiling may allow the association of behavioral and neurobiological alterations more clearly and the dissection of alterations related to the pathology from those related to resilience. (C) 2017 Elsevier Ltd. All rights reserved.

The present chapter attempts to outline the rules that govern dendritic spine formation, plasticity, and elimination in relation to memory functions in the brain. The striking heterogeneity of spine morphologies on different types of neurons, as well as among adjacent spines in the same dendritic branch complicates this analysis. Major issues concerning the relationship between the morphology of dendritic spines and memory mechanisms and the functional, neuronal network relevance of such parameters remain unsettled; for example, what are the immediate and long-lasting changes in electrical properties of dendritic spines and their parent neurons following exposure to plasticity-producing stimulation? Are there different structural correlates for different classes of memories (e.g., episodic, semantic, motor)? What happens to the structural change when a memory is removed or moved from one brain area to another? Can we improve memory by changing the structure? A growing body of research indicates that indeed, dendritic spines are the best morphological correlates of different types of memories, in different brain regions. In this chapter, we will outline the main molecular properties of dendritic spines, as well as the changes observed in the hippocampus, amygdala, and cortical structures in relations to different forms of learning and memory.

In previous studies we and others have found that activation of ryanodine receptors (RyRs) facilitate expression of long-term potentiation (LTP) of reactivity to afferent stimulation in hippocampal slices, with a more pronounced action in the ventral hippocampus. We have also been able to link the involvement of synaptopodin (SP), an actin-binding protein, with neuronal plasticity via its interaction with RyRs. To test this link more directly, we have now compared the ability of ryanodine to convert short-term to LTP in hippocampal slices taken from normal and SP-knockout (SPKO) mice. Indeed, SPKO hippocampus expresses lower concentrations of RyRs and in slices of these mice ryanodine is unable to facilitate conversion of short-term to LTP. These observations link functionally SP with calcium stores.

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is a neurodevelopmental disorder caused by silencing of the FMR1 gene, which in FXS becomes inactivated during human embryonic development. We have shown recently that this process is recapitulated by in vitro neural differentiation of FX human embryonic stem cells (FX-hESCs), derived from FXS blastocysts. In the present study, we analyzed morphological and functional properties of neurons generated from FX-hESCs. Human FX neurons can fire single action potentials (APs) to depolarizing current commands, but are unable to discharge trains of APs. Their APs are of a reduced amplitudes and longer durations than controls. These are reflected in reduced inward Na+ and outward K+ currents. In addition, human FX neurons contain fewer synaptic vesicles and lack spontaneous synaptic activity. Notably, synaptic activity in these neurons can be restored by coculturing them with normal rat hippocampal neurons, demonstrating a critical role for synaptic mechanisms in FXS pathology. This is the first extensive functional analysis of human FX neurons derived in vitro from hESCs that provides a convenient tool for studying molecular mechanisms underlying the impaired neuronal functions in FXS.

The ζ-inhibitory peptide (ZIP) is considered a candidate inhibitor of the atypical protein kinase Mζ(PKMζ). ZIP has been shown to reverse established LTP and disrupt several forms of long-term memory. However, recent studies have challenged the specificity of ZIP, as it was reported to exert its effect also in PKMζknock-out mice. These results raise the question of what are the targets of ZIP that may underlie its effect on LTP and memory. Here we report that ZIP as well as its inactive analog, scrambled ZIP, induced a dose-dependent increase in spontaneous activity of neurons in dissociated cultures of rat hippocampus. This was followed by a sustained elevation of intracellular calcium concentration ([Ca²⁺]i) which could not be blocked by conventional channel blockers. Furthermore, ZIP caused an increase in frequency of mEPSCs followed by an increase in membrane noise in patch-clamped neurons both in culture and in acute brain slices. Finally, at 510 μM, ZIP-induced excitotoxic death of the cultured neurons. Together, our results suggest that the potential contribution of cellular toxicity should be taken into account in interpretation of ZIPs effects on neuronal and behavioral plasticity.

Neuronal networks can generate complex patterns of activity that depend on membrane properties of individual neurons as well as on functional synapses. To decipher the impact of synaptic properties and connectivity on neuronal network behavior, we investigate the responses of neuronal ensembles from small (5-30 cells in a restricted sphere) and large (acute hippocampal slice) networks to single electrical stimulation: in both cases, a single stimulus generated a synchronous long-lasting bursting activity. While an initial spike triggered a reverberating network activity that lasted 2-5 seconds for small networks, we found here that it lasted only up to 300 milliseconds in slices. To explain this phenomena present at different scales, we generalize the depression-facilitation model and extracted the network time constants. The model predicts that the reverberation time has a bell shaped relation with the synaptic density, revealing that the bursting time cannot exceed a maximum value. Furthermore, before reaching its maximum, the reverberation time increases sub-linearly with the synaptic density of the network. We conclude that synaptic dynamics and connectivity shape the mean burst duration, a property present at various scales of the networks. Thus bursting reverberation is a property of sufficiently connected neural networks, and can be generated by collective depression and facilitation of underlying functional synapses.

The effects of chronic exposure to moderate concentrations of ethanol were studied in cultured hippocampal neurons. Network activity, assessed by imaging of [Ca²⁺]i variations, was markedly suppressed following 5 days of exposure to 0.25-1% ethanol. The reduced activity was sustained following extensive washout of ethanol, but the activity recovered by blockade of inhibition with bicuculline. This reduction of network activity was associated with a reduction in rates of mEPSCs, but not in a change in inhibitory synaptic activity. Chronic exposure to ethanol caused a significant reduction in the density of mature dendritic spines, without an effect on dendritic length or arborization. These results indicate that chronic exposure to ethanol causes a reduction in excitatory network drive in hippocampal neurons adding another dimension to the chronic effects of alcohol abuse.

Background: Down Syndrome (DS) is the most common chromosomal abnormality in humans, with many phenotypic characteristics-mental retardation is probably the most prominent one. Embryonic hippocampal neuronal cultures from two mouse models (TC1 and Ts65Dn) for DS were compared to control littermate cultures to detect possible cellular phenotypes of DS. Network behaviour was assessed using calcium imaging, and whole cell patch clamp technique was used to measure differences in single cell properties. Results: Calcium imaging, measured with fluo4, revealed smaller amplitude, shorter duration network bursts, with no change in basal calcium level, which was measured using Fura-2. Baclofen, a GABA-B agonist, suppressed network bursts with lower concentration in DS networks than in control networks. In patch clamped neurons, the threshold for excitation, after-hyperpolarization (AHP) amplitude and input resistance were reduced, while spike height was increased in DS cells compared to controls. Following blockage of sodium currents we found that potassium currents (slow and fast) are reduced in DS cells. Network activity was different with electrophysiological tools as well: bursts were shorter, the total amount of time the network spent bursting was shorter, and network bursts were less synchronized. Most of the differences appeared in both mouse models. Conclusions: DS cells were different both in the basic cell properties and in the assembly into a network. The changes in spike properties, along with reduced potassium currents all point to changes in potassium channels. We speculate that this may be due to possible overexpression of two potassium channel regulators, KCNE1 and KCNE2, whose genes reside on chromosome 21. The altered network activity along with baclofen experiments may be caused by increased inhibition. Two types of inwardly rectifying potassium channels genes reside on chromosome 21: KCNJ6, KCNJ15, and may be the cause of this increased network inhibition.

Despite decades of research, the role of calcium stores in dendritic spines structure, function and plasticity is still debated. The reasons for this may have to do with the multitude of overlapping calcium handling machineries in the neuron, including stores, voltage and ligand gated channels, pumps and transporters. Also, different cells in the brain are endowed with calcium stores that are activated by different receptor types, and their differential compartmentalization in dendrites, spines and presynaptic terminals complicates their analysis. In the present review we address several key issues, including the role of calcium stores in synaptic plasticity, their role during development, in stress and in neurodegenerative diseases. Apparently, there is increasing evidence for a crucial role of calcium stores, especially of the ryanodine species, in synaptic plasticity and neuronal survival.

Background: A possible contributing factor to the development of cognitive deficits in Alzheimer's disease (AD) patients involves the exposure to early life stress. Objective: We explored the impact of stress on synaptic plasticity (long-term potentiation, LTP) of 6-month-old triple-transgenic mice (3×Tg-AD). Methods: 3×Tg-AD and control (NonTg) mice were exposed to three stressors at the age of 2 and 4 months. Excitatory postsynaptic potentials were recorded in the stratum radiatum of the CA1 region of hippocampal slices, in a two-pathway paradigm. Results: Slices taken from 3×Tg-AD mice exhibited significant deficits in LTP compared with NonTg slices. Early stress led to a further decrease in LTP in these mice, while it did not affect NonTg mice. LTP in 3×Tg-AD and stressed 3×Tg-AD mice was rescued by pre-exposure to 0.2 μM ryanodine. In an attempt to find a molecular correlate for the effects of stress in the 3×Tg-AD mice, we found that stressed mice have an altered ratio of Aβ42/40 both in the cortex and hippocampus. Conclusions: Stress experiences in young adults may accelerate the cognitive loss in AD mice, adding another dimension to the plethora of factors that lead to AD.

Long-term effects of stress during pregnancy on brain and behavior have been analyzed extensively in recent years. These effects include changes in emotional behavior, a reduction in learning capacity, and ability to generate long-term potentiation (LTP) in the offspring. In earlier studies, we and others have described a difference in ability to express LTP in dorsal and ventral sectors of the hippocampus (DH and VH, respectively) and its modification by prior stress. We now found that norepinephrine (NE) facilitated conversion of short-term potentiation to LTP in the normal DH but not in VH. Prenatal stress (PS) switched the locus of the facilitating action of NE from the DH to the VH. The effects of NE are likely to be mediated by activation of calcium stores. PS also facilitated (S)-3,5-dihydroxyphenylglycine hydrate (DHPG)-induced LTD in the VH, assumed to be mediated by release of calcium from stores. These observations have important implications for the role of the hippocampus in cognitive and emotional memories.

Thrombin is a serine protease playing an essential role in the blood coagulation cascade. Recent work, however, has identified a novel role for thrombin-mediated signaling pathways in the central nervous system. Binding of thrombin to protease-activated receptors (PARs) in the brain appears to have multiple actions affecting both health and disease. Specifically, thrombin has been shown to lead to the onset of seizures via PAR-1 activation. In this perspective article, we review the putative mechanisms by which thrombin causes seizures and epilepsy. We propose a potential role of PAR-1 antagonists and novel thrombin inhibitors as new, possible antiepileptic drugs.

The role of stress hormones in the initiation of epileptic seizures has been studied extensively in the past decade, with conflicting observations, from suppression to exacerbation of spontaneous seizures. We have now studied the effects of an acute stress on reactivity of juvenile rats to kainic acid (KA), which produces epileptic seizures. With a short (30. s) stress-KA delay, stress exacerbated epilepsy via activation of mineralocorticosterone receptors (MR). With a long (60. min) stress-KA delay, seizures were suppressed through activation of a glucocorticosterone receptor (GR). In a parallel study with CA1 pyramidal neurons in acute hippocampal slices, activation of MRs reduced the frequency of mIPSCs, whereas activation of GRs produced a slow onset, 2.5 fold increase in amplitudes of mIPSCs. GR effects were not mediated by protein synthesis, but did require activation of some protein kinases. These experiments suggest that stress can either facilitate or suppress seizures, in a time and receptor dependent manner.

Synchronized network activity is an essential attribute of the brain. Yet the cellular mechanisms that determine the duration of network bursts are not fully understood. In the present study, synchronized network bursts were evoked by triggering an action potential in a single neuron in otherwise silent microcultures consisting of 4-30 hippocampal neurons. The evoked burst duration, ~ 2 s, depended on the recovery time after a previous burst. While interburst intervals of 35 s enabled full-length bursts, they were shortened by half at 5-s intervals. This reduction in burst duration could not be attributed to postsynaptic parameters such as glutamate receptor desensitization, accumulating afterhyperpolarization, inhibitory tone, or sodium channel inactivation. Reducing extracellular Ca ²⁺ concentration ([Ca ²⁺] _o) relieved the effect of short intervals on burst duration, while depletion of synaptic vesicles with α-latrotoxin gradually eliminated network bursts. Finally, a transient exposure to high [K ⁺] _o slowed down the recovery time following a burst discharge. We conclude that the limiting factor regulating burst duration is most likely the depletion of presynaptic resources.

Recent studies indicate that astrocytes play an integral role in neural and synaptic functioning. To examine the implications of these findings for neurobehavioral plasticity we investigated the involvement of astrocytes in memory and long-term potentiation (LTP), using a mouse model of impaired learning and synaptic plasticity caused by genetic deletion of the interleukin-1 receptor type I (IL-1RI). Neural precursor cells (NPCs), derived from either wild type (WT) or IL-1 receptor knockout (IL-1rKO) neonatal mice, were labeled with bromodeoxyuridine (BrdU) and transplanted into the hippocampus of either IL-1rKO or WT adult host mice. Transplanted NPCs survived and differentiated into astrocytes (expressing GFAP and S100β), but not to neurons or oligodendrocytes. The NPCs-derived astrocytes from WT but not IL-1rKO mice displayed co-localization of GFAP with the IL-1RI. Four to twelve weeks post-transplantation, memory functioning was examined in the fear-conditioning and the water maze paradigms and LTP of perforant path-dentate gyrus synapses was assessed in anesthetized mice. As expected, IL-1rKO mice transplanted with IL-1rKO cells or sham operated displayed severe memory disturbances in both paradigms as well as a marked impairment in LTP. In contrast, IL-1rKO mice transplanted with WT NPCs displayed a complete rescue of the impaired memory functioning as well as partial restoration of LTP. These findings indicate that astrocytes play a critical role in memory functioning and LTP, and specifically implicate astrocytic IL-1 signaling in these processes. The results suggest novel conceptualization and therapeutic targets for neuropsychiatric disorders characterized by impaired astrocytic functioning concomitantly with disturbed memory and synaptic plasticity.

Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.

Ivenshitz M, Segal M. Neuronal density determines network connectivity and spontaneous activity in cultured hippocampus. J Neurophysiol 104: 1052-1060, 2010. First published June 16, 2010; doi: 10.1152/jn.00914.2009. The effects of neuronal density on morphological and functional attributes of the evolving networks were studied in cultured dissociated hippocampal neurons. Plating at different densities affected connectivity among the neurons, such that sparse networks exhibited stronger synaptic connections between pairs of recorded neurons. This was associated with different patterns of spontaneous network activity with enhanced burst size but reduced burst frequency in the sparse cultures. Neuronal density also affected the morphology of the dendrites and spines of these neurons, such that sparse neurons had a simpler dendritic tree and fewer dendritic spines. Additionally, analysis of neurons transfected with PSD95 revealed that in sparse cultures the synapses are formed on the dendritic shaft, whereas in dense cultures the synapses are formed primarily on spine heads. These experiments provide important clues on the role of neuronal density in population activity and should yield new insights into the rules governing neuronal network connectivity.

The spine apparatus (SA) is an essential component of mature dendritic spines of cortical and hippocannpal neurons, yet its functions are still enigmatic. Synaptopodin (SP), an actin-binding protein, colocalizes with the SA. Hippocampal neurons in SP-knockout mice lack SA, and they express lower LTP. SP probably plays a role in synaptic plasticity, but only recently it is being linked mechanistically to synaptic functions. These authors and others have studied endogenous and transfected SP in dendritic spines of cultured hippocampal neurons. They found that spines containing SP generate twice as large responses to flash photolysis of caged glutamate than SP-negative ones. An N-methyl-D-aspartate receptor mediated chemical LTP caused accumulation of GFP-GIuR I in spine heads of control but not of shRNA transfected, SP-deficient neurons. SP is linked to calcium stores, because their pharmacological blockade eliminated SP-related enhancement of glutamate responses. Furthermore, release of calcium from stores produces an SP-dependent delivery of GIuR I into spines. Thus, SP plays a crucial role in the calcium store-associated ability of neurons to undergo long-term plasticity.

The neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote excitatory and inhibitory synapse development. However, a quantitative analysis of their influence on connectivity has proven in general difficult to achieve. In this work we use a novel experimental approach based on percolation concepts that provides a quantification of the average number of connections per neuron. In combination with electrophysiological measurements, we characterize the changes in network connectivity induced by BDNF and NT-3 in rat hippocampal cultures. We show that, on the one hand, BDNF and NT-3 accelerate the maturation of connectivity in the network by about 17 h. On the other hand, BDNF and NT-3 increase the number of excitatory input connections by a factor of about two, but without modifying the number of inhibitory input connections. This scenario of a dominant effect on the excitation is supported by the analysis of spontaneous population bursts in cultures treated with either BDNF or NT-3, which show burst amplitudes that are insensitive to the blockade of inhibition. A leaky integrate-and-fire model reproduces the experimental results well.

The ventral hippocampus (VH) was recently shown to express lower-magnitude long-term potentiation (LTP) than the dorsal hippocampus (DH). An exposure to acute stress reversed this difference, and VH slices from stressed rats expressed larger LTP than controls, whereas LTP in the DH was suppressed by stress. We have now used long-term depression (LTD)-generating trains of stimulation to examine whether this differential LTP reflects a genuine difference in synaptic modifiability between the two sectors of the hippocampus. Surprisingly, slices of DH and VH express similar magnitudes of LTD. However, while prior stress enhanced LTD in the DH, it actually converted LTD to slow-onset, robust LTP in the VH. These two effects of stress on LTD were blocked by glucocorticosterone receptor (GR) and mineralocorticosterone receptor (MR) antagonists, respectively. Acute exposure of slices to a GR agonist dexamethasone facilitated LTD in slices of both DH and VH, while activation of MRs by aldosterone converted LTD to LTP in both regions. Thus, differential activation of the two species of corticosterone receptors determines the ability of the two sectors of the hippocampus to undergo plastic changes in response to LTD-inducing stimulation.

Environmental enrichment (EE) was found to facilitate memory functioning and neural plasticity in normal and neurologically impaired animals. However, the ability of this manipulation to rescue memory and its biological substrate in animals with specific genetically based deficits in these functions has not been extensively studied. In the present study, we investigated the effects of EE in two mouse models of impaired memory functioning and plasticity. Previous research demonstrated that mice with a deletion of the receptor for the cytokine interleukin-1 (IL-1rKO), and mice with CNS-specific transgenic over-expression of the IL-1 receptor antagonist (IL-1raTG) display impaired hippocampal memory and long-term potentiation (LTP). We report here a corrective effect of EE on spatial and contextual memory in IL-1 rKO and IL-1 raTG mice and reveal two mechanisms for this beneficial effect: Concomitantly with their disturbed memory functioning, LTP in IL-1rKO mice that were raised in a regular environment is impaired, and their dendritic spine size is reduced. Both of these impairments were corrected by environmental enrichment. No deficiencies in neurogenesis or hippocampal BDNF and vascular endothelial growth factor secretion were found in IL-1rKO mice that were raised in a regular environment, and both of these variables were increased to a similar degree in enriched IL-1rKO and wild-type mice. These findings suggest that exposure to an enriched environment may be beneficial for individuals with impaired learning and memory related to genetic impairments of IL-1 signaling (and possibly other genetic causes), by reversing impairments in dentate gyrus LTP and spine size and by promoting neurogenesis and trophic factors secretion.

The neurofibrillary-tangles (NTFs), characteristic of tauopathies including Alzheimer's-disease (AD), are the pathological features which correlate best with dementia. The objective of our study was to generate an authentic transgenic (tg) animal model for NFT pathology in tauopathy/AD. Previous NFT-tg mice were driven by non-related/non-homologous promoters. Our strategy was to use the natural tau promoter for expressing the human-tau (htau) gene with two mutations K257T/P301S (double mutant, DM) associated with severe phenotypes of frontotemporal-dementia in humans. Cellular, biochemical, behavioral and electrophysiological studies were subsequently conducted. The tg mice showed a tolerated physiological level of the DM-htau protein, mostly in cortex and hippocampus. The mice demonstrated tauopathy-like characteristics, which increased with age, that included NFT-related pathology, astrogliosis, argyrophilic plaque-like (amyloid-free) structures in brain, with memory deficits and signs of anxiety. Moreover, the tg mice showed a robust synaptic plasticity deficit selectively expressed in a severe impairment in their ability to maintain hippocampal long-term-potentiation (LTP) in response to stimulation of the perforant path, providing evidence that "tau-pathology only" is sufficient to cause this memory and learning-associated deficit. This is a unique mutant-htau-tg model which presents a wide spectrum of features characteristic of tauopathy/AD, which does not show unrelated motor deficits described in other models of tauopathy. In addition, expressing the DM-htau in a neuronal cell model resulted in tau-aggregation, as well as impaired microtubule arrangement. Both animal and cell models, which were regulated under the natural tau promoter (of rat origin), provide authentic and reliable models for tauopathy, and offer valuable tools for understanding the molecular events underlying tauopathies including AD.

The effects of thrombin, a blood coagulation serine protease, were studied in rat hippocampal slices, in an attempt to comprehend its devastating effects when released into the brain after stroke and head trauma. Thrombin acting through its receptor, protease-activated receptor 1 (PAR1), produced a long-lasting enhancement of the reactivity of CA1 neurons to afferent stimulation, an effect that saturated the ability of the tissue to undergo tetanus-induced long-term potentiation. This effect was mediated by activation of a PAR1 receptor, because it was shared by a PAR1 agonist, and was blocked by its selective antagonist. An independent effect of thrombin involved the lowering of the threshold for generating epileptic seizures in CA3 region of the hippocampus. Thus, the experiments in a slice mimicked epileptic and cognitive dysfunction induced by thrombin in the brain, and suggest that these effects are mediated by activation of the PAR1 receptor.

Glutamate receptor trafficking into dendritic spines is a pivotal step in synaptic plasticity, yet the relevance of plasticity-producing rise of [Ca²⁺]i and of spine morphology to subsequent delivery of glutamate receptors into dendritic spine heads are still not well understood. Following chemical induction of LTP, an increase in eGFP-GluR1 fluorescence in short but not long dendritic spines of cultured hippocampal neurons was found. Repeated flash photolysis of caged calcium, which produced a transient rise of [Ca²⁺]i inside spine heads caused a selective, actin and protein synthesis dependent increase of eGFP-GluR1 in these spines. Strikingly, GluR1 increase was correlated with the ability of a calcium transient generated in the spine head to diffuse into the parent dendrite, and inversely correlated with the length of the spine: short spines were more likely to raise GluR1 than long ones. These observations link, for the first time, calcium transients in dendritic spines with spine morphology and its ability to undergo synaptic plasticity.

The ability to express long-term potentiation (LTP) of reactivity to afferent stimulation along the septotemporal axis was explored in transverse rat hippocampal slices. The ventral pole of the hippocampus (VH) was found to be much impaired in ability to express LTP compared with the rest of the hippocampus. An exposure to acute stress before the rat was killed reversed this trend, and slices from VH now expressed a large LTP, whereas in the rest of the hippocampus, it was much suppressed. The enhanced LTP in VH was mediated by activation of a mineralocorticoid receptor (MR), whereas the suppressed LTP was mediated by activation of a glucocorticoid receptor, and indeed selective agonists of the respective steroid receptors mimicked the effects of stress, whereas selective antagonists blocked them. The MR-enhanced LTP in VH was not mediated by activation of the NMDA receptor but by enhancement of voltage-gated calcium channels. Because the VH has an unique efferent system to the hypothalamus, these results indicate that stress may activate this system while suppressing the ability of the rest of the hippocampus to express plastic properties under stressful conditions.

The recent finding that hippocampal slices from aged mice overexpressing the gene for superoxide dismutase (SOD) exhibit long-term potentiation (LTP) of reactivity to afferent stimulation that is significantly larger than that produced in aged wild-type (wt) mice has encouraged the exploration of the effects of reactive oxygen species (ROS) on learning in aged mice. In addition, young-adult and aged wt and SOD transgenic mice were used in an attempt to correlate adult neurogenesis with spatial learning. Aged wt and SOD mice exhibited a 90% reduction in doublecortin-labeled new dentate gyrus neurons as compared to young mice, with no significant difference between genotypes. In addition, aged SOD mice exhibited better performance than wt controls in both reference and working memory tasks in a water maze. These findings provide a behavioral measure to demonstrate that excessive production of H ₂O₂ is beneficial in aged mice.

Elevated levels of corticosteroid hormones, presumably occupying both mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs), have been reported to impair synaptic plasticity in the hippocampus as well as the acquisition of hippocampus-dependent memories. In contrast, recent evidence suggests that activation of MRs enhance cognitive functions. To clarify the roles of different steroid receptors in hippocampal plasticity, young adult rats were injected with the GR antagonist RU38486 (mifepristone) or the MR antagonist Spironolactone before the exposure to an acute swim stress. Hippocampal responses to perforant path stimulation were then recorded in anesthetized rats. Stress combined with RU38486 produced a striking facilitation of LTP. Spironolactone enabled only short-term potentiation that reversed to long-term depression (LTD) in the stressed animals. Finally, the blockade of both MRs and GRs led to impairment of long-term potentiation. These findings indicate that MRs and GRs assume opposite roles in regulation of synaptic plasticity after acute exposure to stressors.

A fundamental issue in understanding activity-dependent long-term plasticity of neuronal networks is the interplay between excitatory and inhibitory synaptic drives in the network. Using dual whole-cell recordings in cultured hippocampal neurons, we examined synaptic changes occurring as a result of a transient activation of NMDA receptors in the network. This enhanced transient activation led to a long-lasting increase in synchrony of spontaneous activity of neurons in the network. Simultaneous long-term potentiation of excitatory synaptic strength and a pronounced long-term depression of inhibitory synaptic currents (LTDi) were produced, which were independent of changes in postsynaptic potential and Ca²⁺ concentrations. Surprisingly, miniature inhibitory synaptic currents were not changed by the conditioning, whereas both frequency and amplitudes of miniature EPSCs were enhanced. LTDi was mediated by activation of a presynaptic GABA_B receptor, because it was blocked by saclofen and CGP55845 [(2S)-3-{[(15)-1-(3,4-dichlorophenyl)ethyl] amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid]. The cAMP antagonist Rp-adenosine 3,5-cyclic monophosphothioate abolished all measured effects of NMDA-dependent conditioning, whereas a nitric oxide synthase inhibitor was ineffective. Finally, network-induced plasticity was not occluded by a previous spike-timing-induced plasticity, indicating that the two types of plasticity may not share the same mechanism. These results demonstrate that network plasticity involves opposite affects on inhibitory and excitatory neurotransmission.

Dissociated neurons were cultured on lines of various lengths covered with adhesive material to obtain an experimental model system of linear signal transmission. The neuronal connectivity in the linear culture is characterized, and it is demonstrated that local spiking activity is relayed by synaptic transmission along the line of neurons to develop into a large-scale population burst. Formally, this can be treated as a one-dimensional information channel. Directional propagation of both spontaneous and stimulated bursts along the line, imaged with the calcium indicator Fluo-4, revealed the existence of two different propagation velocities. Initially, a small number of neighboring neurons fire, leading to a slow, small and presumably asynchronous wave of activity. The signal then spontaneously develops to encompass much larger and further populations, and is characterized by fast propagation of high-amplitude activity, which is presumed to be synchronous. These results are well described by an existing theoretical framework for propagation based on an integrate-and-fire model.

A recent flurry of time-lapse imaging studies of live neurons have tried to address the century-old question: what morphological changes in dendritic spines can be related to long-term memory? Changes that have been proposed to relate to memory include the formation of new spines, the enlargement of spine heads and the pruning of spines. These observations also relate to a more general question of how stable dendritic spines are. The objective of this review is to critically assess the new data and to propose much needed criteria that relate spines to memory, thereby allowing progress in understanding the morphological basis of memory.

Reactive oxygen species (ROS) have been considered for some time only in the context of oxidative stress-induced cell damage. In this review, we discuss the growing body of evidence that implicates ROS in general, and hydrogen peroxide (H₂O₂) in particular, in regulatory events underlying synaptic plasticity. H₂O₂ is regarded in this context as a specific diffusible signaling molecule. The action of H ₂O₂ is assumed to be carried out via the release of calcium ions from internal stores, modulating the activity of specific calcium-dependent protein phosphatases. These phosphatases eventually affect neuronal plasticity. We discuss the role of H₂O₂ in these systems, stressing the importance of cellular regulation of H₂O ₂ levels that are altered in aging individuals, in the ability to express plasticity. These studies highlight the function of H₂O ₂ in processes of learning and memory and their change in elderly individuals, irrespective of neurodegeneration found in Alzheimer's patients.

Down's syndrome (DS), the phenotypic manifestation of trisomy 21, involves overexpression of chromosome 21-encoded genes. The gene for amyloid precursor protein (APP), known to be involved in AD pathology, resides on chromosome 21 along with the gene for Cu/Zn superoxide dismutase (SOD1), a key enzyme in the metabolism of oxygen free radicals. We investigated the consequences of a combined increase in APP and SOD1, in a double-transgenic (tg)-APP-SOD1 mouse. These mice expressed severe impairment in learning, working and long-term memory. Expression of long-term potentiation in hippocampal slices was impaired in both tg-SOD and tg-APP-SOD mice, but not in tg-APP mice, indicating that increased APP by itself did not affect in vitro synaptic plasticity. In tg-APP-SOD mice, membrane-bound high molecular weight APP species accumulated while APP cleavage products did not increase and levels of secreted APP were unchanged. Severe morphological damage, including lipofuscin accumulation and mitochondria abnormalities, were found in aged tg-APP-SOD but not in the other mice. Thus, a combined elevation of the two chromosome 21 genes in tg-APP-SOD mice induced age-dependent alterations in morphological and behavioural functions.

Hydrogen peroxide (H₂O₂), a reactive oxygen species, is assumed to have a detrimental effect on neuronal plasticity. Indeed, H ₂O₂ suppresses long-term potentiation (LTP) in hippocampal slices of normal rats and wild-type (wt) mice. Transgenic mice overexpressing superoxide dismutase (SOD) 1 (tg-SOD), which maintain high ambient H₂O₂, have also been shown to be impaired in their ability to express hippocampal LTP. Paradoxically, H₂O ₂, at a concentration (50 μM) that blocks LTP in wt mice, actually enhanced LTP in slices of 2-month-old tg-SOD mice. H₂O ₂-dependent LTP in tg-SOD was blocked by the protein phosphatase calcineurin inhibitor FK506, but not by rapamycin, an FK-binding protein 12 (FKBP12) inhibitor or by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a serine-kinase inhibitor. Interestingly, wt and tg-SOD mice expressed similar levels of the antioxidant enzyme catalase and similar activity of glutathione peroxidase. An opposite situation was found in 2-year-old mice. Aged wt mice were impaired in LTP in a manner that could be reversed by the addition of H₂O₂. Surprisingly, aged tg-SOD mice exhibited larger LTP than that found in wt mice, but this was now reduced by 50 μM H ₂0₂. Both young tg-SOD and aged control mice displayed altered protein phosphatase activity, compared with that of young controls; moreover, FK506 inhibited LTP in old tg-SOD as well as in old wt mice treated with H₂O₂. These data promoted a dual role for H ₂O₂ in the regulation of LTP, and proposed that it is mediated by the protein phosphatase calcineurin.

The role of afferent innervation in the formation of dendritic spines was studied in cultured rat striatum. The striatum is a unique structure in that it contains highly spiny GABAergic projection neurons, with no known local excitation. Grown alone in culture, striatal neurons did not express spontaneous network activity and were virtually devoid of dendritic spines. Adding GFP-expressing mouse cortical neurons to the striatal culture caused the appearance of spontaneous and evoked excitatory synaptic currents in the striatal neurons and a 10-fold increase in the density of spines on their dendrites. This effect was blocked by a continuous presence of TTX in the growth medium, while removal of the drug caused a rapid appearance of spines. Exposure to glutamate, or the presence of cortex-conditioned medium did not mimic the effect of cortical neurons on formation of spines in the striatal neurons. Also, the cortical innervation did not cause a selective enhancement of survival of specific subtypes of spiny striatal neurons. These experiments demonstrate that excitatory afferents are necessary for the formation of dendritic spines in striatal neurons.

Dendritic morphology of 2-week-old cultured neurons, taken from postnatal day 1 fragile X mental retardation gene1 knock out (FMR1-/-) mice hippocampus, were compared with cells taken from wild type mice. Under control conditions the FMR1-/- neurons displayed significantly lower spine densities compared to wild type neurons. Pharmacological stimulation of electrical activity, induced by bicuculline, caused a reduction in dendritic spine density in both the FMR1-/- and the wild type cells. In both groups, bicuculline induced a significant shrinkage of spines that were occupied by one or more synaptophysin-immunoreactive presynaptic terminals. The concentration of FMR1 in the wild type cultures was not affected by bicuculline treatment. These experiments indicate that FMR1 is not likely to be an essential factor in activity-modulated morphological plasticity of dendritic spines in cultured hippocampal neurons.

The cytokine interleukin-1 (IL-1) is produced by peripheral immune cells as well as glia and neurons within the brain; it plays a major role in immune to brain communication and in modulation of neural, neuroendocrine, and behavioral systems during illness. Although previous studies demonstrated that excess levels of IL-1 impaired memory processes and neural plasticity, it has been suggested that physiological levels of IL-1 are involved in hippocampal-dependent memory and long-term potentiation (LTP). To examine this hypothesis, we studied IL-1 receptor type I knockout (IL-1rKO) mice in several paradigms of memory function and hippocampal plasticity. In the spatial version of the water maze test, IL-1rKO mice displayed significantly longer latency to reach a hidden platform, compared with wild-type controls. Furthermore, IL-1rKO exhibited diminished contextual fear conditioning. In contrast, IL-1rKO mice were similar to control animals in hippocampal-independent memory tasks; i.e., their performance in the visually guided task of the water maze and the auditory-cued fear conditioning was normal. Electrophysiologically, anesthetized IL-1rKO mice exhibited enhanced paired-pulse inhibition in response to perforant path stimulation and no LTP in the dentate gyrus. In vitro, decreased paired-pulse responses, as well as a complete absence of LTP, were observed in the CA1 region of hippocampal slices taken from IL-1rKO mice compared with WT controls. These results suggest that IL-1 contributes to the regulation of memory processes as well as short- and long-term plasticity within the hippocampus. These findings have important implications to several conditions in humans, which are associated with long-term defects in IL-1 signaling, such as mutations in the IL-1 receptor accessory protein-like gene, which are involved in a frequent form of X-linked mental retardation.

We demonstrate third-harmonic microscopy of thin biological objects using a Ti:sapphire laser source. A very simple modification to the collection and detection systems of the microscope allows for the detection of the third-harmonic signal near 270 nm. We show that, using a Ti:sapphire laser, we can combine third-harmonic imaging with two-photon excitation fluorescence simultaneously. Compared to other possible laser sources for third-harmonic microscopy, the Ti:sapphire laser provides better optical resolution, better power availability, and is less absorbed in water. We find that the Ti:sapphire laser is the most suitable laser source for third-harmonic microscopy for thin biological specimens.

Regulation of dendritic spine motility was studied in dissociated cultures of the rat and mouse hippocampus, using green fluorescent protein-labeled neurons or neurons loaded with the calcium-sensitive dye Oregon Green-1. Cells were time-lapsephotographed on a confocal laser-scanning microscope at high resolution to detect movements as well as spontaneous fluctuations of intracellular calcium concentrations in their dendritic spines. Active presynaptic terminals attached to the spines were labeled with FM4-64, which marks a subset of synaptophysin-labeled terminals. Dendritic spines were highly motile in young, 4- to 7-d-old cells. At this age, neurons had little spontaneous calcium fluctuation or FM4-64 labeling. Within 2-3 weeks in culture, dendritic spines were much less motile, they were associated with active presynaptic terminals, and they expressed high rates of spontaneous calcium fluctuations. Irrespective of age, and even on the same dendrite, there was an inverse relationship between spine motility and presence of FM4-64-labeled terminals in contact with the imaged spines. Spine motility was blocked by latrunculin, which prevents actin polymerization, and was disinhibited by blockade of action potential discharges with tetrodotoxin. It is proposed that an active presynaptic terminal restricts motility of dendritic spines.

We have recently demonstrated that embryonic E16 hippocampal neurons grown in cultures are unable to form fast synaptic connections unless treated with BDNF or NT-3, This experimental system offers an opportunity to define the roles of neurotrophins in processes leading to formation of functional synaptic connections. We have used ultrastructural and electrophysiological methods to explore the cellular locations underlying neurotrophin action on synaptic maturation. The rate of spontaneous miniature excitatory postsynaptic currents (mEPSCs) evoked by hyperosmotic stimulation was 7-16-fold higher in neurotrophin-treated cells than in controls. In addition, the potent neurotransmitter-releasing drug alpha -latrotoxin was virtually ineffective in the control cells while it stimulated synaptic events in neurotrophin-treated cells. Likewise, the membrane-bound dye FM1-43 was taken up by terminals in neurotrophin-treated cultures five-fold more than in controls. Both the total number and the number of docked synaptic vesicles were increased by neurotrophin treatment. Activation of synaptic responses by neurotrophins occurred even when postsynaptic glutamate receptors and action potential discharges were pharmacologically blocked. These results are consistent with a presynaptic locus of action of neurotrophins to increase synaptic vesicle density which is critical for rapid synaptic transmission. They also suggest that neurotrophins can activate synapses in the absence of pre- and postsynaptic neuronal activity.

Transcranial magnetic stimulation (TMS) is being proposed as a method of choice for the treatment of clinical depression, yet its action in the brain is still not well understood. In previous studies we found that TMS has a long-term effect on reactivity of the hippocampus to perforant path stimulation. Since the efficacy of antidepressants is highly age-dependent, we studied possible age-related effects of TMS on hippocampal evoked responses. Young adult (3 months), aging (10 months) and aged (24-26 months) awake rats were subjected to daily TMS for one week, followed by measurements of several parameters of reactivity to perforant path stimulation in the anesthetized rat. TMS did not affect responses of the hippocampus to single perforant path stimulation, but reduced drastically paired-pulse and frequency dependent depression in the young and aging but not the old rats. Likewise, TMS increased LTP expression in the young but not the old rats, and reduced the efficacy of serotonin modulation of reactivity of the hippocampus, in the young but not the old rats. Thus, long term effects of chronic TMS on local GABAergic inhibition are highly age dependent.

Despite widespread interest in dendritic spines, little is known about the mechanisms responsible for spine formation, retraction, or stabilization. We have now found that a brief exposure of cultured hippocampal neurons to a conditioning medium that favors activation of the NMDA receptor produces long-term modification of their spontaneous network activity. The conditioning protocol enhances correlated activity of neurons in the culture, in a process requiring an increase in [Ca2+](i) and is associated with both formation of novel dendritic spines and pruning of others. The novel spines are likely to be touched by a presynaptic terminal, labeled with FM4-64 dye, whereas the absence of such terminals increases the likelihood of spine pruning. These results indicate that long-term functional changes are correlated with morphological modifications of dendritic spines of neurons in a network.

The recent advent of novel high-resolution imaging methods has created a flurry of exciting observations that address a century-old question: what are biological signals that regulate formation and elimination of dendritic spines? Contrary to the traditional belief that the spine is a stable storage site of long-term neuronal memory, the emerging picture is of a dynamic structure that can undergo fast morphological variations. Recent conflicting reports on the regulation of spine morphology lead to the proposal of a unifying hypothesis for a common mechanism involving changes in postsynaptic intracellular Ca²⁺ concentration, [Ca²⁺](i): a moderate rise in [Ca²⁺](i) causes elongation of dendritic spines, while a very large increase in [Ca²⁺](i) causes fast shrinkage and eventual collapse of spines. This hypothesis provides a parsimonious explanation for conflicting reports on activity-dependent changes in dendritic spine morphology, and might link these changes to functional plasticity in central neurons.

Dissociated hippocampal neurons, grown in culture for 2 to 3 weeks, tended to fire bursts of synaptic currents at fairly regular intervals, representing network activity. A brief exposure of cultured neurons to GABA caused a total suppression of the spontaneous network activity. Following a washout of GABA, the activity was no longer clustered in bursts and instead, the cells fired at a high rate tonic manner. The effect of removing GABA could be seen as long as 1 to 2 days after GABA withdrawal and is expressed as an increase in the number of active cells in a network, as well as in their firing rates. Such striking effects of GABA removal may underlie part of the GABA withdrawal syndrome seen elsewhere.

A recent series of exciting observations, using novel high-resolution time-lapse imaging of living cells, has provoked a major shift in our understanding of the dendritic spine, from a stable storage site of long-term memory to a dynamic structure that undergoes rapid morphological variations. Through these recent observations, the molecular mechanisms underlying spine plasticity are beginning to emerge. A common mechanism involving changes in intracellular Ca²⁺ concentration may control both the formation/elongation and the pruning/retraction of spines. Spine motility may be instrumental in the formation of synapses, may contribute to the anchoring/removing of glutamate receptors at spine heads, and may control the efficacy of existing synapses.

The ability to monitor ongoing changes in the shape of dendritic spines has important implications for the understanding of the functional correlates of the great variety of shapes and sizes of dendritic spines in central neurons. We have monitored and three-dimensionally reconstructed dendritic spines in cultured hippocampal neurons over several hours of observation in a confocal laser scanning microscope. In the absence of extrinsic stimulation, the dimensions of dendritic spines of 3-week-old cultured neurons did not change to any significant degree over 3-4 hr in the culture dish, unlike the case with younger cultures. Releasing calcium from stores with pulse application of caffeine causes a transient rise of [Ca²⁺](i) in dendrites and spines, monitored with the calcium dye Oregon-green. Application of caffeine to a dendrite imaged with calcein caused a fast and significant increase in the size of existing dendritic spines and could lead to formation of new ones. This effect is mediated by calcium released from the ryanodine- sensitive stores, as application of caffeine in the presence of ryanodine blocked this effect on the morphology of dendritic spines. Thus, release of calcium from stores is sufficient to produce significant changes in the shape of dendritic spines of cultured hippocampal neurons.

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca²⁺](i) response to glutamate, and a large increase in [Ca²⁺](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co- incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B₁, completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca²⁺](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C₈- glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre- incubation with ryanodine, suggesting that [Ca²⁺](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Vasoactive intestinal peptide (VIP) expression is restricted to interneurons in the hippocampus of normal adult rats. However, 3-6 hours after a 60-minute walk in an activity wheel, VIP was transiently expressed in most pyramidal and granular neurons of the hippocampus. Locomotion was also associated with a dramatic increase in VIP immunoreactivity in the motor cortex, primarily in bipolar cells. Reverse transcriptase-polymerase chain reaction analysis indicated that VIP mRNA increases transiently by more than twofold, before the increases in peptide immunoreactivity in both the hippocampus and motor cortex. By comparison, another marker of inhibitory interneurons, glutamate decarboxylase, did not change its expression pattern after locomotion. The calcium binding protein, calbindin-D28K, normally expressed in interneurons, was now found also in glial cells of the hippocampus and motor cortex. Another marker of enhanced electrical activity, the immediate early gene, c-Fos, was expressed in pyramidal and granular neurons at 3 hours but not at 6 hours after locomotion. These results suggest that mapping of peptide expression in the brain of a docile, inactive rat may not reflect the real distribution and functions of a peptide in an active animal.

The role of dendritic spine morphology in the regulation of the spatiotemporal distribution of free intracellular calcium concentration ([Ca²⁺])(i)) was examined in a unique axial-symmetrical model that focuses on spine-dendrite interactions, and the simulations of the model were compared with the behavior of real dendritic spines in cultured hippocampal neurons. A set of nonlinear differential equations describes the behavior of a spherical dendritic spine head, linked to a dendrite via a cyclindrical spine neck. Mechanisms for handling of calcium (including internal stores, buffers, and efflux pathways) are placed in both the dendrites and spines. In response to a calcium surge, the magnitude and time course of the response in both the spine and the parent dendrite vary as a function of the length of the spine neck such that a short neck increases the magnitude of the response in the dendrite and speeds up the recovery in the spine head. The generality of the model, originally constructed for a case of release of calcium from stores, was tested in simulations of fast calcium influx through membrane channels and verified the impact of spine neck on calcium dynamics. Spatiotemporal distributions of [Ca²⁺](i) measured in individual dendritic spines of cultured hippocampal neurons injected with Calcium Green-1, were monitored with a confocal laser scanning microscope. Line scans of spines and dendrites at a

Cell cultures were used to analyze the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the development of synaptic transmission. Neurons obtained from embryonic day 18 (E18) rat hippocampus and cultured for 2 weeks exhibited extensive spontaneous synaptic activity. By comparison, neurons obtained from E16 hippocampus expressed very low levels of spontaneous or evoked synaptic activity. Neurotrophin treatment produced a sevenfold increase in the number of functional synaptic connections in the E16 cultures. BDNF induced formation of both excitatory and inhibitory synapses, whereas NT-3 induced formation of only excitatory synapses. These effects were independent of serum or the age of the gila bed used for the culture. They were not accompanied by significant changes in synaptic-vesicle-associated proteins or glutamate receptors. Treatment of the cultures with the neurotrophins for 3 d was sufficient to establish the maximal level of functional synapses. During this period, neurotrophins did not affect the viability or the morphology of the excitatory neurons, although they did produce an increase in the number and length of dendrites of the GABAergic neurons. Remarkably, only BDNF caused an increase in the number of axonal branches and in the total length of the axons of the GABAergic neurons. These results support a unique and differential role for neurotrophins in the formation of excitatory and inhibitory synapses in the developing hippocampus.

Local modulation of hippocampal-evoked responses to perforant path stimulation was studied by leaking drugs from the recording pipette placed in the dentate gyrus of anesthetized young (3 months old), aging (17 months old) and old (28 months old) rats. In old rats, the excitatory postsynaptic potential (EPSP) slope was much reduced compared to young: and aging rats. The population spike (PS) size was similar in all age groups. Bicuculline caused a marked increase in PS size relative to population EPSP, and reversed the response to the second pulse in a paired-pulse paradigm from inhibition to facilitation. The effect of bicuculline was only slightly reduced in old rats. The 5-HT1a agonist 8-OH-DPAT potentiated PSs in the dentate gyrus, while not affecting paired-pulse inhibition. The effect of 8-OH-DPAT was slightly reduced in old rats. Carbachol, a cholinergic agonist, reversed paired-pulse inhibition into facilitation in the young brain, but not in aging and old rats. These results demonstrate that age affects differentially the action of biogenic amines on hippocampal reactivity to efferent stimulation. (C) 1998 Elsevier Science Inc.

Physiological and morphological properties of cultured hippocampal neurons were measured to investigate whether NMDA receptors play a role in survival and differentiation. Neurons dissociated from mouse embryos with different NMDAR1 genotypes were grown in culture. Electrophysiological analysis verified the absence of NMDA receptor-mediated currents in neurons taken from homozygous mutant (NR1-/-) embryos. The number of surviving hippocampal neurons was 2.5-fold higher in cultures from the NR1-/- embryos compared with wild type (NR1+/+) and heterozygous (NR1+/-) controls. Despite the lack of NMDA receptor function, NR1-/- neurons formed synapsin I-positive presynaptic boutons associated with MAP2ab-positive dendrites in culture. Confocal microscopic analysis of Dil labelled neurons confirmed the presence of dendritic spines on NR1-/- neurons with 80% of the density found in NR1+/+ neurons. These results suggest that the NMDA receptor has little effect on general features of neuronal differentiation. In contrast, there is clear effect on neuronal survival. This finding establishes neuron number in standard culture conditions as a measure of NMDA receptor activity.

Copper/zinc superoxide dismutase (CuZn-SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene encoding CuZn-SOD resides on human chromosome 21 and is overexpressed in Down syndrome (DS) patients. Overexpression of CuZn-SOD in transgenic (Tg) mice and cultured cells creates chronic oxidative stress leading to enhanced susceptibility to degeneration and apoptotic cell death. We have now found that three lines of Tg-CuZn-SOD mice, one of which also overexpresses S100β, a glial calcium binding protein, are deficient in spatial memory. Furthermore, hippocampal slices taken from these mice have an apparently normal synaptic physiology, but are impaired in the ability to express long-term potentiation (LTP). This effect on hippocampal LTP was abrogated by treatment of slices with the H₂O₂ scavenger catalase or the antioxidant N-t-butyl-phenylnitrone (BPN). It is proposed that elevated CuZnSOD causes an increase in tetanic stimulation-evoked formation of H₂O₂ which leads to diminished LTP and cognitive deficits in these mice.

Activation of cyclic AMP dependent kinase is believed to mediate slow onset, long-term potentiation (LTP) in central neurons. Cyclic-AMP activates a cascade of molecular events leading to phosphorylation of the nuclear cAMP responsive element binding protein (pCREB). Whereas a variety of stimuli lead to activation of CREB, the molecular processes downstream of CREB, which may be relevant to neuronal plasticity, are yet largely unknown. We have recently found that following exposure to estradiol, pCREB mediates the large increase in dendritic spine density in cultured rat hippocampal neurons. We now extend these observations to include other stimuli, such as bicuculline, that cause the formation of new dendritic spines. Such stimuli share with estradiol the same mechanism of action in that both require activity-dependent CREB phosphorylation. Our observations suggest that CREB phosphorylation is a necessary, but perhaps not sufficient, step in the process leading to the generation of new dendritic spines and perhaps to functional plasticity as well.

While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.

Exposure of rat hippocampal slices to low concentrations of the muscarinic agonist carbachol (CCh) has been shown to produce a slow onset long-term potentiation (LTP) of reactivity to afferent stimulation in CA1 neurons. Although this potentiation shares a number of properties with tetanic LTP, muscarinic LTP (LTP(m)) is independent of activation of the NMDA receptor. We now demonstrate that low levels of hydrogen peroxide (H₂O₂) cause hippocampal slices to lose the ability to express LTP(m). This powerful effect of H₂O₂ is selective in that it does not affect the reactivity of hippocampal neurons to higher concentrations of CCh. In fact, H₂O₂ also blocks induction of a slow onset, non-NMDA-dependent tetanic LTP (NN-LTP). The functional relevance of this action of H₂O₂ is exemplified by the fact that the hippocampus of aged rats, which produces higher levels of endogenous H₂O₂ than that of young rats, lacks LTP(m) and expresses a markedly reduced NN-LTP. In aged rats, the lack of LTP(m) contrasts with an apparently normal muscarinic suppression of the EPSP slope induced by higher concentrations of CCh. When hippocampal slices from aged animals are treated with catalase, an enzyme that breaks down H₂O₂, LTP(m) is restored, and NN-LTP is enhanced. Thus, our study proposes a unique and novel age-dependent peroxide regulation of LTP(m) in the brain and provides a link between the cholinergic system, aging, and memory functions.

To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.

In previous studies we have demonstrated that raphe grafts, implanted into serotonin-depleted rat hippocampus can restore behavioral and physiological functions impaired by serotonin depletion. Since aging is associated with a reduction in serotonergic functions, we explored the possibility that grafting embryonic raphe tissue will ameliorate age-associated reduction of serotonergic functions in the hippocampus. Aged rats were implanted with E14 embryonic neural tissue, containing the raphe, or part of the parietal cerebral cortex. Three months later, the rats were anesthetized, and the responses of the dentate gyrus to perforant-path stimulation were measured. Serotonin-containing neurons were found in the raphe-grafted hippocampi. No differences were found between the two groups in the volume of the graft in the host brain. Raphe-grafted rats were not different from the cortex-grafted rats in reactivity to perforant path stimulation or in the response to a second of a pair of stimuli to the perforant path. They did, however, express a pronounced commissural inhibition, unlike the cortex-grafted rats. These results are similar to those found previously with a pharmacological enhancement of serotonergic neurotransmission. It is suggested that a graft of serotonergic neurons can ameliorate age-associated reduction in serotonergic functions in the hippocampus.

Rat hippocampal neurons, grown in dissociated culture for about 18 days, were exposed for 6 h to three days to stimuli which cause either an increase (GABA(A) antagonists, bicuculline or picrotoxin), or decrease (tetrodotoxin) in spontaneous neuronal activity. Individual neurons were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate and visualized with a confocal laser scanning microscope. GABA antagonists caused a marked, up to 60%, increase in spine density on secondary dendrites or cultured hippocampal neurons. This was associated with a small decrease in spine length. The rise in spine density was partially prevented by treatment with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetate, or by blockade of protein synthesis with cycloheximide. Tetrodotoxin caused a marked elongation of dendritic spines (but did not cause a decrease in spine density comparable to the increase caused by picrotoxin). This effect was seen primarily but not exclusively in spines with no distinct head. Both treatments were most effective within 24 h of exposure. There were no other systematic effects of the drugs on the morphology of the dendritic spines. These results indicate that dendritic spines in cultured neurons depend on ongoing synaptic activity to maintain their shape, and that neurons respond to an increase in synaptic demand by an increase in spine density. Thus, dendritic spines are likely to have a role in short-term synaptic interaction rather than to constitute a long-term memory storage device.

Numerous reports have shown that drugs which elevate 5-HT @hydroxytryptamine) function-remarkably such as dl- or d-fenfluramine (dexfenfluramine, DF) may reduce food and palatable fluid intake [I]. It has been reported that DF potently suppresses sucrose sham feeding in gastricfistulated rat [2]. It has recently been reported that, glucose induces strong conditioned preference based on its or ostimulant properties (taste) as well as its postingestive actions (calories) (3). The objective of this study was to analyse the effects of DF on flavour-preference learning and conditioning with glucose solutions.

Ever since their first description in neurons, dendritic spines could be visualized only in fixed tissue, using high-power light and electron microscopy. Recent studies have been able to measure the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in dendritic spines of live neurons, and the results suggest that the spine is an independent cellular Ca²⁺ compartment. Other recent observations have indicated that the density of spines on dendrites changes in a dynamic fashion depending on ongoing neuronal activity. Together, these findings have led to the proposal that the dendritic spine is not only a storage device for long-term memory but perhaps a means for isolating the cell from the harmful consequences of synaptically evoked surges in [Ca²⁺]_i. In other words, the dendritic spine is a neuroprotectant. This hypothesis has specific testable implications, including relating cell activity to spine density.

Individual cultured rat hippocampal neurons grown on glass coverslips were loaded with lucifer yellow (LY) and segments of their dendrites, containing dendritic spines, were exposed to the glutamate agonist N-methyl-D-aspartate (NMDA). Repeated pulse application of NMDA caused a reduction of spine length by 20%, within 1-2 h of exposure to the drug. No such changes were seen in spines exposed to NMDA in presence of the antagonist 2-aminophosphonovalerate or in spines on sister dendrites which were not exposed to NMDA. It is suggested that dendritic spines shrink following exposure to excessive synaptic stimulation.

We monitored developmental alterations in the morphology of dendritic spines in primary cultures of hippocampal neurons using confocal laser scanning microscopy (CLSM) and the fluorescent marker Dil. Dissociated rat hippocampal neurons were plated on polylysine-coated glass cover slips end grown in culture for 1-4 weeks. Fixed cultures were stained with Dil and visualized with the CLSM. Spine density, spine length, and diameters of spine heads and necks were measured. Some cultures were immunostained for synaptophysin and others prepared for EM analysis. In the 1-3 week cultures, 92-95% of the neurons contained spiny dendrites. Two subpopulations of spine morphologies were distinguished. At 1 week in culture, 'headless' spines constituted 50% of the spine population and were equal in length to the spines with heads. At 2, 3, and 4 weeks in culture headless spines constituted a progressively smaller fraction of the population and were, on average, shorter than spines with heads. Spines with heads had narrower necks than headless spines. At 3 weeks in culture, spines were associated with synaptophysin-immunoreactive labeling, resembling synaptic terminals. At 4 weeks in culture, only 70% of the Dil-filled cells had spiny dendrites, and the density of spines decreased. Ultrastructurally, the majority of dendritic spine-like structures at I week resembled long filopodia without synaptic contacts. The majority of axospinous synapses were on short 'stubby' spines. At 3 weeks in culture, the spines were characteristic of those seen in vivo. They contained no microtubules or polyribosomes, were filled with a characteristic, filamentous material, and formed asymmetric synapses. These studies provide the basis for further analysis of the rules governing the formation, development, and plasticity of dendritic spines under controlled, in vitro conditions.

Reactivity of the hippocampal system to stimulation of its main afferent, the perforant path, was studied in the intact, anesthetized rat. Parental administration of fenfluramine caused a marked elevation of population spike response to perforant path stimulation. An injection of atropine before, but not after fenfluramine, blocked the potentiating effect of fenfluramine. The atropine blockade was dose-dependent and not mimicked by the peripheral muscarinic receptor antagonist methyl atropine. This effect of fenfluramine was also prevented by an injection of the 5-HT receptor antagonist spiperone. The effect of fenfluramine was mimicked by the anticholinesterase physostigmine, which was not affected by spiperone pretreatment. It is proposed that release of 5-HT (5-hydroxytryptamine) by fenfluramine potentiates reactivity to afferent stimulation by interacting with cholinergic terminals in the hippocampus.

1. Variations in intracellular free Ca2+ concentration ([Ca2+]i) induced by alteration of the extracellular concentrations of Ca2+ ([Ca2+]o) and K+ ([K+]o) were imaged in single fluo3loaded C6 glioma cells. In addition, the effect of membrane potential on [Ca2+]i was investigated in fura2loaded, voltageclamped cells. 2. Step alterations of [Ca2+]o from 0 to 10 nM were followed by proportional variations in [Ca2+]i, with a maximum 7fold increase and an apparent halfmaximum at [Ca2+]o of 1.5 mM. 3. The time to halfmaximum change (t1/2) of [Ca2+]oassociated [Ca2+]i variations ranged between 10 and 50 s, and was inversely related to the amplitude of [Ca2+]o steps. 4. Transient, serotonininduced [Ca2+]i elevations, used as a measure of Ca2+ availability in inositol 1,4,5trisphosphatesensitive stores, were diminished within 10 min in 0 mM [Ca2+]o, but were unaffected by [Ca2+]o changes in the 15 mM range. 5. Restoration of normal [Ca2+]i following its elevation by serotonin was delayed by removal of external Na+ or Cl and was enhanced by warming the medium to 37 degrees C. These conditions did not affect [Ca2+]oassociated [Ca2+]i variations. 6. [Ca2+]oassociated [Ca2+]i variations were depressed by La3+ and Ba2+, while blockers of voltageactivated Ca2+ channels were ineffective. 7. Elevated [K+]o depressed the basal level of [Ca2+]i, and in high concentrations (70140 mM) also diminished the response to serotonin. 8. Depolarizing the membrane potential of voltageclamped cells reversibly reduced [Ca2+]i. These membranepotential associated [Ca2+]i variations were blocked by La3+, Ba2+ and TEA, all of which also depolarized membrane resting potential. 9. Apamin (at 110 microM), a blocker of [Ca2+]iactivated K+ channel, totally and reversibly prevented [Ca2+]oassociated [Ca2+]i variations. 10. These studies indicate that C6 cells are responsive to variations in [Ca2+]o, and that a K+ channel is a possible path through which Ca2+ penetrates into the cell.

1. We studied long-term cholinergic effects on synaptic transmission in submerged hippocampal slices using intra- and extracellular recording techniques. 2. Bath application of submicromolar concentrations of carbachol (CCh) produced a gradually developing, long-lasting increase in the CA1 excitatory postsynaptic potential and population spike. This potentiation was blocked by atropine and, hence, named muscarinic long-term potentiation (LTP(m)). Application of DL-2-amino-5-phosphonovaleric acid had no effect on LTP(m), indicating that this phenomenon is N-methyl-D-aspartate receptor independent. 3. These effects of CCh were not likely to be due to the blockade of one of several K⁺ conductances by the drug; the time and concentration dependence of LTP(m) were different from those associated with cholinergic blockade of K⁺ conductances. 4. Removal of extracellular calcium (Ca(o)/²⁺) from the bath blocked synaptic transmission. CCh added in calcium-free medium induced LTP(m), which was revealed upon removal of the drug by washing with normal calcium-containing medium. Neither cutting CA1- CA3 connections nor cessation of synaptic stimulation interfered with LTP(m) induction. 5. Application of thapsigargin or H-7 together with CCh blocked LTP(m), suggesting the involvement of intracellular calcium (Ca(i)/²⁺) stores and protein kinases, respectively, in the LTP(m) mechanism. 6. Subthreshold cholinergic stimulation coupled with subthreshold tetanic stimulation caused LTP. CCh had no effect when administered after the LTP mechanism had been saturated by repeated suprathreshold tetani. Tetanic stimulation failed to cause LTP when applied after LTP(m) had been induced by CCh. These experiments indicate that tetanus-induced potentiation and LTP(m) share a common mechanism and provide a direct link between ACh and mechanisms of synaptic plasticity.

Changes in cytosolic Na⁺ ([Na⁺](i)) caused by a toxic glutamate (GLU) or NMDA treatment of cultured hippocampal neurons were monitored by using SBFI fluorescent probe and imaging microscopy. Both GLU and NMDA (50 or 100 μM in Mg²⁺-free solution, 15 min) induced a marked increase in [Na⁺](i) (from 6-8 to 30-45 mM) which persisted after the termination of a treatment. The competitive NMDA antagonist, APV (100 μM) when applied in the post-NMDA period failed to decrease the elevated [Na⁺](i). The results obtained strongly suggest that the main reason for an impairment of Na⁺/Ca²⁺ exchange in the post-glutamate period is a reduction of the transmembrane Na⁺ gradient caused apparently by inhibition of Na⁺-K⁺ pump.

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AP102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the ml AChR subtype. Notably, the selectivity of the new ml agonists is reflected also by activation of select second messenger systems via distinct Gproteins. These compounds reflect a new pharmacological concept, tentatively defined as ligandselective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex on the ability of rats to perform a spatial memory watermaze task. Serotonin depletion or partial cholinergic lesion of the hippocampus (by injection of colchicine into the septum) did not affect the ability of rats to perform the task, but the combined treatment did. Double-lesioned rats, with raphe grafts in the hippocampus, but not in the entorhinal cortex, performed similar to control or single-lesioned rats. The results suggest that the functioning of the serotonergic innervation of the hippocampus, and not of its afferents, is crucial for the ability of rats to perform spatial memory tasks, especially when the septohippocampal cholinergic connection is disrupted.

GABA evoked a reversible rise of free intracellular calcium concentration ([Ca]i) in cultured rat hippocampal neurons, detected with Fluo3 fluorescence in a confocal laser scanning microscope. The GABAevoked change of [Ca]i was mimicked by muscimol and not by baclofen, but was only minimally affected by picrotoxin or bicuculline, indicating that this effect of GABA is not likely to be mediated by activation of GABA_B receptor or by a conventional chloridelinked GABA_A receptor. GABAevoked rise of [Ca]i expressed a marked desensitization; only 1020 minutes after a previous exposure to GABA was the response to a subsequent application fully expressed. This desensitization was not seen in electrophysiological responses to GABA or in [Ca]i changes evoked by NMDA in the same neurons. The GABA response appeared to be developmentally regulated and was seen in 17dayold more than in 2128dayold cells. It is suggested that GABA evokes a unique change of [Ca]i in young hippocampal neurons.

The effects of acetylcholine (ACh) on changes in [Ca]_i produced by the glutamate agonist N-methyl-d-aspartate (NMDA) were measured in cultured rat hippocampal neurons loaded with the fluorescent calcium indicator Fluo-3 in a confocal laser scanning microscope. NMDA produced a dose-dependent reversible rise in [Ca]_i. ACh had a smaller and less consistent effect on [Ca]_i but could cause a marked enhancement of the reactivity of neurons to NMDA. This effect was reversed by the presence of the muscarinic antagonist atropine. AMPA, another glutamate agonist which causes a rise in [Ca]_i by activating voltage gated calcium influx was less affected by ACh. Caffeine, which releases calcium from intracellular stores also enhanced reactivity of these neurons to NMDA. It is suggested that ACh can enhance reactivity to NMDA by releasing calcium from internal stores.

Changes in intracellular calcium concentration ([Ca²⁺]_i) in response to topical application of the glutamate agonists N-methyl-d-aspartate (NMDA), or amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were measured in cultured rat hippocampal neurons loaded with Fluo-3 and visualized in a confocal laser scanning microscope. These neurons were subsequently stained for the presence of the enzyme marker for γ-amino butyric acid (GABA), glutamate decarboxylase (GAD). GAD-positive, putative interneurons were less responsive to NMDA and AMPA than GAD-negative neurons. The time course of the rise and decay of [Ca²⁺]_i was similar in the two groups of neurons. Also, there was no clear difference in the shape and size of these two neuron groups indicating that the difference between them is not due to diffusion distances. These data indicate that interneurons are probably more able to handle a calcium load than other neurons, a difference that may underly their resistance to treatments which cause degeneration of other neurons in the hippocampus.

Cells in layers II-III or VI were activated by microdrop application of acetylcholine (ACh), while monitoring the intracellular response of layer V pyramidal cells. This enabled the tracing of functional connections between the cells of layers II-III or VI with those of layer V. ACh activation of layer II-III or VI cells resulted in a small depolarization of these cells, accompanied by a burst of excitatory postsynaptic potentials (EPSPs) from layer V pyramidal cells. These effects of ACh were blocked by tetrodotoxin (TTX), suggesting the involvement of actions potentials in their production. The input resistance of layer V pyramidal cells during and after the EPSP burst was not significantly different from control values, further suggesting an indirect effect of ACh on layer V pyramidal cells. Isolation of the supragranular layer, by horizontal cutting, did not prevent the EPSP burst evoked by ACh application to the lower layer VI, suggesting a direct input from layer VI to layer V pyramidal cells. ACh applied near pyramidal cells in layers II-III, V or VI caused transient hyperpolarization associated with a decrease in input resistance followed by a large depolarization, an increase in input resistance, and action potential discharges. The ACh- mediated hyperpolarization and the train of action potentials of layer II-III pyramidal cells were blocked by TTX. Thus the ACh-activated cells in layers II-III and VI make an excitatory synaptic contact with layer V pyramidal cells, producing the EPSP burst observed in layer V.

We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex, on the responsiveness of dentate granule cells to stimulation of the perforant path. Raphe grafts in the hippocampus reversed the hyperexcitability of granule cells, resulting from depletion of the serotonergic innervation. Such grafts also restored the responsiveness of the granule cells to application of a serotonin releasing drug, fenfluramine (FFA). In contrast, hyperexcitability was not reversed when the graft was placed in the entorhinal cortex. Furthermore, although some increase in population spike size was observed in these rats after application of FFA, this increase had a response profile which was different from that of control and of lesioned rats that were grafted in the hippocampus. These results suggest that the serotonergic innervation, within the hippocampus and not in the entorhinal cortex, modulates granule cells excitability.

We examined the effect of elevating intracellular calcium ([Ca²⁺]_i) on responses to iontophoretically applied N-methyl-d-aspartate (NMDA), and quisqualate in CA1 neurons of the hippocampal slice. Topical application of calcimycin (A23187), a calcium ionophore, potentiated responses to NMDA but not to quisqualate. This potentiation was prevented by loading cells with the calcium chelator, BAPTA, suggesting that the action of calcimycin on NMDA receptors was mediated by an elevation of [Ca²⁺]_i in the recorded cell. The potentiation was also recorded in voltage-clamped and in cesium-loaded cells, suggesting that it was not mediated by non-specific changes in voltage or input resistance of the cell that may have resulted from the rise in [Ca²⁺]_i. We propose that intracellular calcium plays a crucial role in regulating the activity of the NMDA subtype of l-glutamate receptor.

Acting at postsynaptic alpha-1 and beta-1-receptors, norepinephrine (NE) exerts a complex action in rat hippocampus. It is currently believed that beta-1-receptor activation enhances excitability of recorded neurons, whereas alpha-1 activation suppresses reactivity to afferent stimulation. These reported effects of alpha-agonists are not consistent with alpha-1 effects found elsewhere in the brain. We have conducted experiments in the anesthetized rat and found that an amphetamine-induced increase in the dentate gyrus population spike can be blocked by a beta-antagonist but also by an alpha-1-antagonist. We have conducted experiments in the brain slide preparation and found that an alpha-agonist, phenylephrine (PHE), selectively enhances responses to N-methyl-D-aspartate (NMDA) but not to quisqualate. We propose that the product of activation of both alpha- and beta-receptor types will enhance reactivity of hippocampal cells to afferent stimulation.

Specific and localized lesions of the 5-HT fibers in the hippocampus induce homotypic collateral sprouting and enhance serotonergic fiber outgrowth from adult neurons and transplanted fetal tissue. In this study, hippocampal extracts were prepared and applied to primary cultures of fetal serotonergic neurons. The effects of plating density and serum additives were examined. The growth of the serotonergic neurons in the rostral brainstem dissociated cultures were estimated by measuring the specific uptake of [³H]5-HT. The results indicate the presence of a trypsin-sensitive factor which is active when prepared fresh at dilutions up to 1 10,000. The factor is higher in hippocampus than cerebellum. Young male tissue contained more activity than either female or aged hippocampus. Although both positive and negative effects are described, higher dilutions of factor ( 1 1,000) were generally stimulatory in high density cultures while lower dilutions ( 1 10) were inhibitory in low density cultures. Specific removal of 5-HT hippocampal afferents with fornix-fimbria microinjections of 5,7-dihydroxytryptamine resulted in an initial loss of activity (2 days and 2 weeks) followed by an enhanced activity (2 months) compared to normal hippocampal extract. Several possibilities are discussed as to the identity of the serotonergic growth factor from hippocampal supernatant.

The gating and tuning actions of noradrenaline (NA) at post-synaptic sites have been highly suggestive of an important role for the locus coeruleus (LC) in attention, learning and memory. By recording the activity of single units in the LC in behaving rats in a strictly controlled conditioning paradigm, direct evidence was provided that this nucleus is engaged during specific aspects of learning. The neuronal response to a discrete sensory stimulus was monitored as a function of the changing significance of the stimulus i.e., when it was novel, during habituation, associative learning, reversal and extinction. Both appetitive and aversive paradigms were used. We consistently observed differential conditioned responding with food reinforcement, while when footshock reinforcement was used, there was an increase in response to both CS+ and CS. In both paradigms, the LC response disappeared when the conditioning was expressed at a behavioral level, to reappear vigorously as soon as the stimulus reinforcement contingencies were changed, i.e., during reversal or extinction. These results suggest that the LC does not mediate specific sensory or associative information necessary for ongoing performance but shows remarkable plasticity of sensory responding as a function of changing cognitive significance of the stimulus.

THE dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes^1-6, at least some of which are regulated by intracellular calcium concentrations^7-11. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts^12,13, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca²⁺]_d) are frequently not parallelled in the spine ([Ca²⁺]_s). This isolation is not caused by a physical diffusion barrier. This report provides, to our knowledge, the first direct demonstration of autonomous spine function.

Serotonin modulating effects on hippocampal electrical activity were studied using serotonin releasing drugs (e.g. d-fenfluramine, FFA, and p-chloroamphetamine, PCA). FFA and PCA enhanced the reactivity of the dentate gyrus to stimulation of the perforant path (PP) in the anesthetized rat. The population spike (PS) but not the population EPSP (EPSP) was enhanced by FFA indicating that the drug effect is not exerted at the PP synapse, but at some postsynaptic site between the synapse and the spike generation mechanism. A depth profile of the response to PP stimulation indicated that the largest effect of FFA was present just below the granular cell layer. There were no systematic effects of FFA on the EPSP at any depth tested. The effect of FFA was much reduced in rats depleted of serotonin by p-chlorophenylalanine (PCPA) and restored when serotonin stores were repleted by the serotonin precursor 5-hydroxytryptophane (5-HTP). d-FFA was at least twice as effective as 1-FFA in enhancing responses in the dentate gyrus (DG). In noradrenaline (NA) depleted rats the increase in PS size was as in control rats. The effects of FFA were blocked by the 5-HT1a antagonist spiperone but not by the 5-HT2 antagonist mianserin. These results suggest that the effect of FFA is primarily due to release of serotonin from its terminals. At the gross electrographic level, FFA suppressed spontaneous sharp wave activity and reduced the magnitude of hippocampal EEG. Spontaneous extracellular single unit activity, recorded in the DG, was also inhibited by FFA concomitantly with the increase in the PS size. FFA did not affect paired-pulse depression. Transection of the commissural connection to the hippocampus (stimulation of which elicits feed forward inibition) markedly attenuated the effects of FFA. It is suggested that serotonin exerts a dual effect on DG granule cells; it suppresses spontaneous activity while enhancing excitability to afferent stimulation. Possibly, the effects of serotonin are exerted by modulation of commissural feed-forward inhibition.

The ability of midbrain raphe (MR) cells grafted into serotonin-depleted brain to restore hippocampal responses to synaptically released serotonin was studied using a serotonin-releasing drug, d-fenfluramine (FFA). In normal rats, FFA enhances reactivity of the dentate gyrus (DG) to perforant-path stimulation, while suppressing spontaneous activity. These effects of the drug were dependent on the presence of normal serotonin neutransmission in the hippocampus. Depletion of brain serotonin markedly attenuated DG reactivity to FFA. MR grafts (4-5 months after transplantation) restored the reactivity of the hippocampus to FFA. Immature MR grafts (3 weeks after transplantation) or septal control grafts could not reproduce the effects of mature grafts. These experiments demonstrate the possible functional incorporation of neural transplants in a host brain and the possible involvement of these grafts in regulation of normal host electrical activity. The combined utilization of a serotonergic graft and a releasing drug (i.e. FFA), in the serotonin-deprived brain, allows the study of serotonin functions in restricted parts of the brain.

We examined the electrophysiological properties of cholinergic and non-cholinergic neurons in the medial septum-diagonal band complex (MSDB) of the rat in the in vitro slice preparation. Cells were identified electrophysiologically, filled with Lucifer yellow, fixed and processed immunohistochemistry with fluorescent labeled anti-choline acetyltransferase (ChAT) antibody. Cholinergic and non-cholinergic neurons differed in action potential parameters, spike afterpotentials and in current-voltage relationships. In addition, cholinergic neurons expressed a potent transient outward rectification in response to a depolarizing current pulse.

Topical application of 4-aminopyridine (4-AP) onto hippocampal slices produced spontaneous repetitive large hyperpolarizing potentials in CA1 neurons. This effect of 4-AP was blocked by a new GABA_B receptor antagonist, 2-hydroxy-saclofen. 2-Hydroxy-saclofen also blocked slow IPSPs evoked by stimulation of stratum radiatum. It is suggested that 4-AP-evoked slow hyperpolarizing potentials are in fact slow IPSPs evoked by activation of a selective subset of interneurons which do not produce fast IPSPs.

A monoclonal antibody to the nerve growth factor receptor (NGFR) (IgG 192) was used to visualize differences in immunohistochemical labeling of young (10 months) and old (35 months) rats. Three parameters were analyzed; cell counts, immunoreactive cross-sectional surface area (SA) and optical density (OD) of labeled cells. Large reductions in all three parameters were recorded in the medial septum (MS). Both OD and immunoreactive SA were reduced in the VDB, while only OD was reduced in the HDB. This observation confirms that NGFR labeling is reduced in the aged rat septum and adds that the loss of labeling is differential, with greater deficits in the MS-VDB complex than in the HDB.

Behavioral effects of septal lesion and fornix-fimbria transection were compared in absence and presence of a septal transplant in the hippocampus. The transplant grew in the hippocampus and projected acetylcholinesterase (AChE)-containing fibers throughout the extent of the denervated hippocampus. There were no differences in graft size or AChE reinnervation pattern after septal lesion or fornix transection. An increase in the density of M1 binding sites seen in hippocampal CA3 region after a cholinergic lesion, was restored back to normal after reinnervation of the hippocampus by the graft. Fornix-transected rats were more impaired in water maze acquisition than septal-lesioned rats which were impaired compared to controls. Septal-grafted rats were not different from lesioned rats in the behavioral tasks. However, an injection of physostigmine improved their performance relative to lesioned non-grafted rats. These experiments indicate that grafts can ameliorate behavioral deficits when the efficacy of acetylcholine of graft origin is enhanced.

The ability of embryonic raphe cells grafted into the hippocampus to restore spatial learning ability was tested in rats with combined serotonergic/cholinergic deficits. Embryonic raphe cells (E14) were transplanted into the hippocampus of serotonin-depleted rats. Two to 3 months after transplantation, control, lesioned and grafted rats were tested in a spatial memory task (a water maze) with and without the addition of atropine. All 3 groups could negotiate the water maze equally well, in non-drug conditions. The injection of atropine cause a severe distruption of performance only in the serotonin depleted rats. The presence of an active serotonergic graft was examined in the intact hippocampus using the serotonin releasing drug fenfluramine (FFA). A pronounced depression of hippocampal EEG was observed in control and grafted but not in lesioned rats 15 min after the injection of FFA. These results suggest the involvement of serotonin in cognitive functions in the rat. Furthermore, it is suggested that an interaction between serotonergic and cholinergic neurotransmission occurs in the hippocampus.

A possible interaction between serotonergic and cholinergic neurotransmission was examined in relation to the performance of a spatial memory task. Blockade of cholinergic transmission with a high dose of atropine was sufficient to impair performance of a water maze task. A partial reduction of cholinergic transmission using a low dose of atropine had no effect on this performance. Reducing serotonin synthesis, using a specific inhibitor of tryptophane hydroxylase p-chlorophenylalanine (PCPA) also had no effect on performance of such a task. However, a combined treatment with a low dose of atropine and PCPA severely impaired the performance of rats in the water maze. The rats were impaired in both acquisition of the initial spatial task and in reacquisition of a new spatial position (= working memory). These findings suggest an interaction between cholinergic and serotonergic transmission in acquisition and retention of spatial information. Furthermore they propose that deficits in cognitive abilities, observed in aging or Alzheimer's disease, may result from the combined reduction in cholinergic and serotonergic transmission.

Cholinergic M1 and M2 muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated QNB in the presence of pirenzepine or carbachol. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most thalamic and hypothalamic nuclei. M1 and M2 receptors showed small but significant decreases in cortical regions and in the striatum. The largest effects were seen in M2 receptors of the ventral forebrain cholinergic nuclei where binding was reduced by up to 40%. No similar reductions were seen in the M1 receptor population in these regions. The results suggest that both muscarinic receptor subtypes show an anatomically selective pattern of decrease with age, with the M2 receptor subtype in the basal forebrain nuclei being specially vulnerable to the effects of aging.

Electrical stimulation of the perforant path produces a characteristic population EPSP and population spike in the dentate gyrus of the anesthetized rat. Parenteral administration of a serotonin releasing drug d-fenfluramine (FFA) caused a marked (30-100%) and highly significant increase in dentate gyrus population spike response to perforant path stimulation without affecting the slope of the population excitatory postsynaptic potential (EPSP). This indicates that FFA modifies granular cell excitability to afferent stimulation. The facilitatory effect of FFA was not present in rats depleted of serotonin following treatment with the synthesis inhibitor p-chlorophenylalanine (PCPA) but was restored after restoration of serotonin synthesis with the precursor 5-hydroxytryptophan indicating that presence of serotonin in terminals is required for the action of FFA.

The acquisition and retention of a water maze task was examined in 12 intact, young Wistar rats. Acetylcholinesterase activity in 43 discrete brain regions was then measured in the same rats by quantitative histochemistry. Individual learning and retention indices were found to be significantly correlated with acetylcholinesterase (AChE) levels in specific regions, e.g. cholinergic nuclei; the ventral pallidum and nucleus basalis; and in the dentate gyrus of the hippocampus. High levels of AChE in these regions predicted poor performance in the water maze. Thus cholinergic activity in selected regions of the rat brain might be involved in the performance of spatial memory tasks.

An injection of trimethyltin (TMT) into adult male Wistar rats produced a transient hyperirritability and aggressive behavior. When these effects of the drug subsided, the rats were still markedly impaired in performance of a spatial memory task examined in a watermaze. Activity of neurons in the hippocampus of these TMT-treated rats was studied in a slice preparation. A large (8 mg/kg) or double (7.5 and 3.5 mg/kg, two weeks apart) i.p. injection of TMT caused a loss of slow inhibitory postsynaptic potentials recorded in CA1 neurons in response to stimulation of stratum radiatum. A loss of accomodating properties of the neurons in response to a prolonged depolarizing current pulse was also seen in some of these cells. Other resting properties of the neurons were within the normal range. Topical application of acetylcholine (ACh) produces in normal hippocampal neurons a depolarizing response and a blockade of after-hyperpolarization. Only the latter response to ACh was seen in cells from TMT treated rats. No effect of the drug on reactivity to serotonin was detected. These effects of TMT were not seen when the drug was applied acutely while recording from cells in the slice indicating that it involves a degeneration process. These results suggest that the cognitive deficits in TMT treated rats might be associated with deficits in cholinergic functions.

This chapter discusses the efficacy of using neural grafting to restore physiological and behavioral functions following damage to the cholinergic septohippocampal system. It presents a study in which embryonic septal regions were injected into the hippocampi of fimbriafornix (FF) transected rats. The grafts developed an extensive innervation of the host hippocampus and, when stimulated, produced a slow depolarizing response that was blocked by atropine and facilitated by physostigmine. There is evidence that the graft restored hippocampal theta rhythm, which had been eliminated by FF transactions or septal lesions. However, unlike unlesioned controls, theta in grafted animals is not correlated with movement. Performance in a water maze, which was markedly impaired by fornix transection, showed no improvement 6 months after transplantation, but was restored in septal lesioned, septal grafted rats. The results indicate that grafting may provide a useful tool for studying the action of neurotransmitter systems and their involvement in higher brain functions.

Embryonic midbrain raphe was grafted into serotonin-deficient adult rat hippocampus. Serotonin-containing neurons in the graft survive for at least 6 months after grafting. Grafted neurons develop physiological properties, not present on the day of grafting, identical to those of normal adult serotonin-containing neurons. These include (a) high input resistance and slow membrane time constant, (b) lack of inward rectification in response to hyperpolarizing current pulses and (c) a potent, 4-aminopyridine-sensitive transient outward rectification. The grafted neurons innervate the host tissue with axons that have a slow conduction velocity and refractoriness. It is suggested that grafted CNS neurons may possess normal physiological properties.

Noradrenaline(NA)-containing fibers originate in the nucleus locus coeruleus (LC) and innervate wide cortical and subcortical structures. Extensive evidence indicates that NA of LC origin can suppress apparent spontaneous activity of many neurons in the brain, yet increase their reactivity to certain stimuli. The type of stimuli as well as the nature of the enhancement are region specific. The observed effects of activation of LC are a product of an effect on remote afferents, on local interneurons, and on the projection neuron that is being monitored. The cellular mechanisms underlying these actions have been studied with intracellular recording techniques in the slice preparation. NA hyperpolarizes hippocampal neurons probably by activating an electrogenic Na-K pump. It enhances reactivity to afferent stimulation probably by acting presynaptically and also by suppressing a postsynaptic hyperpolarizing Ca-dependent K current. This results in enhancement of the signal-to-noise ratio of the monitored system and in a more efficient response to significant environmental simuli.

Extracellular single unit recordings were made in the median raphe nucleus from rats anaesthetized with urethane. Spontaneous firing as well as orthodromic and antidromic responses to stimulation of the fornix and the medial septum were studied. One hundred and twelve units (out of a total of 355) with a regular spontaneous firing rate of 0.2-3 spikes/s were classified as serotonin-containing neurons. Fifty nine of them were antidromically invaded from either the fornix or the medial septum (conduction velocity, 0.8 m/s) and 7 additional neurones from both the fornix and the medial septum. Antidromic action potentials were followed by a period of decreased probability of firing, that was already present below threshold for antidromic invasion, were proportional to the stimulation intensity and had a latency similar to orthodromic inhibition. No preferential topographical distribution within the median raphe nucleus was observed for the serotonin neurones, even those invaded antidromically. Twenty six neurones with a clear-cut anatomical location around the borders of the median raphe nucleus showed a spontaneous rhythmic activity (4-20 spikes/s) characterized by the presence of extremely prolonged silent periods (up to 5 min). Only one of these neurones was invaded antidromically from the medial septum and none from the fornix. Of the remaining non-serotonin neurones, 28 showed a very low firing rate consisting of single action potentials every 10-60 s while 189 had a spontaneous activity of 6-30 spikes/s. Regardless of their firing rate they were all antidromically invaded from the fornix and/or the medial septum and had a conduction velocity of 5 m/s. These experiments demonstrate the electrophysiological heterogeneity of the neuronal population of the median raphe nucleus, the presence of strong projections of both putative serotonin and non-serotonin neurones to the medial septum and, via the fornix, to the hippocampus, and the existence of axonal branching in both types of neurones.

1. Voltage-sensitive membrane-bound dyes and a matrix of 100 photodetectors were used to detect the spread of evoked electrical activity at the CA1 region of rat hippocampus slices. A display processor was designed in order to visualize the spread of electrical activity in slow motion.2. The stimulation of the Schaffer collateral-commissural path in the stratum radiatum evoked short latency (2-4 msec) fast optical signals, followed by longer latency (4-15 msec) slow signals which decayed within 20-50 msec. Multiple fast signals were frequently detected at the stratum pyramidale; they propagated toward the stratum oriens with an approximate conduction velocity of 0.1 m/sec.3. The fast signals were unaltered in a low Ca2+ high Mg2+ medium but were blocked by tetrodotoxin. These signals probably represent action potentials in the Schaffer collateral axons. Their conduction velocity was about 0.2 m/sec and their refractory period about 3-4 msec.4. The slow signals were absent in a low Ca2+ medium and probably represent excitatory post-synaptic potentials (e.p.s.p.s) generated in the apical dendrites of the pyramidal cells. They were generated in the stratum radiatum, where the presynaptic signals were seen, and spread into somata and basal dendrites (the stratum pyramidale and oriens, respectively).5. The timing of the signals with fast rise-time, which were detected at the statum pyramidale, approximately coincided with the timing of the extracellularly recorded field potentials. These multiple discharges probably represent action potentials of the pyramidal cells. They spread back into the apical dendrites but with significant attenuation of the amplitudes of the high frequency components of the pyramidal action potentials.6. Hyperpolarizing potentials could be detected when strong stimuli were applied to the stratum radiatum or alveus. The net hyperpolarizations were detected only in the stratum pyramidale and the border region between the stratum pyramidale and radiatum. Frequently the inhibition was masked by the large e.p.s.p.s. However, its existence could be demonstrated by treatment of the slice with picrotoxin or a low Cl- medium. Under these conditions a long-lasting depolarization of the apical dedrites was evoked by the stimulation. This was associated with an increase of the multiple discharges in the stratum pyramidale and oriens.7. These studies illustrate the usefulness of voltage-sensitive dyes in the analysis of passive and active electrical properties, pharmacological properties and synaptic connexions in mammalian brain slices, at the level both of small neuronal elements (dendrites, axons) and of synchronously active neuronal populations.

Properties of the norepinephrine- (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3-4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 sec but maximal stimulation was reached only after 5-10 min of incubation. Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12-15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation. It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.

Serotonin (5HT) is one of the main monoamine neurotransmitters in the brain. Its biochemistry and pharmacology (Bloom et al., 1972; Costa et al., 1974; Weight and Salmoiraghi, 1968; Bennett and Snyder, 1976; Nelson et al., 1978), brain distribution (Azmitia, 1978; Azmitia and Segal, 1978; Conrad et al., 1974; Bobillier et al., 1976; Moore and Halaris, 1975; Segal and Landis, 1974) and functions (Akil and Liebeskind, 1975; Jacobs et al., 1974; Jouvet, 1969; Lorens and Yunger, 1974) have been the subject of numerous investigations in the past decade. Compared with the wealth of information gathered in these disciplines, relatively little was known until recently about the postsynaptic action of 5HT and the mechanisms by which it exerts its overt behavioral effects.

Impulses in rat locus coeruleus neurons exhibit pronounced conduction latency decreases, followed by even larger latency increases (of over 20 msec in some cases) during a single train of antidromic activation. The magnitude of latency fluctuation varies as a function of basal antidromic latency, frequency of stimulation, and number of stimuli in a train. These and additional data indicate that this variability in latency is a consequence of altered impulse conduction velocity along the axons, perhaps reflecting reduced ion concentration gradients resulting from impulse propagation. These latency changes may allow thin unmyelinated axons to influence target cells most effectively with short bursts of activity, and suggest that myelination and large axon diameter provide for high fidelity as well as for high velocity of impulse flow in nervous tissue.

1. Intracellular activity was recorded from neurones in the CA1 pyramidal layer of slices of rat hippocampus maintained in vitro. 2. Application of 5HT in a droplet or via ionophoresis produced a 35 mV hyperpolarization associated with a 30% decrease in input resistance. 3. The response to 5HT was minimal with a drop concentration of 1 microM and maximal with 100 microM. The responses appeared to be blocked by methysergide applied in the superfusion medium. 4. The responses to 5HT were minimal when the drug was applied in the apical dendritic region and maximal when it was applied near the soma. 5. 5HT produced no substantial changes in e.p.s.p.s evoked by stimulation of the Schaffer collateralcommissural system or in i.p.s.p.s which were occasionally encountered following stimuli to the stratum radiatum. 6. The responses to 5HT are true postsynaptic responses and are not indirect effects since they are present in a Ca2+deficient Mg2+enriched medium which blocks synaptic transmission. 7. The responses to 5HT were not dependent on extracellular Cl concentration. 8. These experiments indicate that 5HT produces its effects in the rat hippocampus by activating K+ channels.

Transplants of the embryonic locus coeruleus (LC) region were implanted into the circuity of the hippocampal formation in adult rats in which the normal adrenergic afferents to the hippocampus had been removed. The growth of new adrenergic axons from the implant in the denervated hippocampus was followed for 1-14 months after surgery by means of fluorescence histochemistry, and the function of the implant-hippocampal connections was tested electrophysiologically after 2-3 months survival. In the successful cases the entire hippocampal formation was reinnervated from the LC implant within 3-6 months after operation, and the newly formed innervation still persisted unchanged by 14 months. The reinnervation was equally effective irrespective of the route by which the axons entered the hippocampus, i.e. along the lesioned fornix-fimbria or along a retrosplenial route. The pattern formed by the ingrowing LC axons mimicked to a large extent that of the normal LC afferents. Little growth was seen into denervated terminal fields of the commissural, septal or entorhinal afferents, pointing to a preference of the ingrowing LC fibers for the areas normally innervated by adrenergic afferents. In the electrophysiological experiments, stimulation of the LC implants caused (in 20 out of 29 cells monitored) an inhibition of the spontaneous activity of neurons in the host hippocampus. This inhibition had a relatively long latency and a long duration, similar to that observed after stimulation of the innate LC in the intact rat. As in the normal rat, the inhibitory responses were blocked by systemic or local application of the beta-adrenergic receptor blockers propranolol or sotalol. It is concluded that the adult rat brain is capable of carrying out all steps involved in correct functional reinnervation of a denervated region. Moreover, the implant-hippocampal preparation should be a highly suitable model system for functional studies of a central noradrenergic connection.

Pharmacologic studies of analgesia produced by stimulation of the nucleus locus coeruleus (LC) were conducted using the rat hot-plate test. A correlation between self-stimulation and analgesia produced by stimulation of LC was found. Analgesia produced by LC stimulation was attenuated by naloxone, a morphine antagonist, cyproheptidine, a serotonin antagonist, and WB-4101, an alpha-adrenergic antagonist. The analgesia was absent in 6-OHDA-treated rats. Catecholamine synthesis inhibition by a combination of reserpine and AMT or more specific inhibition of noradrenaline synthesis by DDC elevated latency to paw lick and yet did not affect stimulation-produced analgesia. It is suggested that morphinergic, serotonergic, and alpha-adrenergic mechanisms mediate LC stimulation produced analgesia.

The relationship between intracranial self-stimulation (ICSS) and stimulation-produced analgesia (SPA) was investigated in the rat employing an operant bar-press response and a modification of the hot-plate test. ICSS and SPA were elicited through bipolar electrodes chronically implanted in two catecholamine nuclei; the nucleus locus coeruleus (LC) and the substantia nigra (SN). These sites have previously been shown to yield both phenomena. SPA was shown to be of a magnitude similar to that of morphine. In addition, SPA deriving from both LC and SN was significantly reversed by the specific opiate antagonist, naloxone. The intensity of the stimulating current sufficient to induce SPA was found to be higher than that required for ICSS. The pharmacological susceptibilities of the two phenomena were tested by administering a number of drugs: haloperidol, propanolol, pimozide and AMPT attenuated ICSS in both LC and SN, while leaving SPA unaffected. In contrast, methysergide and PCPA blocked SPA and simultaneously facilitated ICSS. The present results indicate a dissociation of ICSS and SPA from LC and SN at both physiological and neurochemical levels; ICSS rates appeared to be a function of catecholaminergic tone, while SPA depended upon the integrity of serotonin transmission.

The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after ³Hproline microinjections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudateputamen and the parietotemporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body. Further studies were done to test whether the fiber tracts travelling in the MFB contained 5HT. Unilateral (left) injections of 5,7dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of ³Hproline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5hydroxytryptophan administration. The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5HT. The results agree with the neuroanatomical distribution of the 5HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.

Evoked hippocampal responses to stimulation of the contralateral hippocampus were recorded in the awake rat. The effects of priming stimulation applied to the nucleus locus coeruleus and the raphe nuclei on averaged evoked hippocampal responses to the interhemispheric stimulation were measured. It was found that priming stimulation of these monoamine-containing nuclei caused the formation of a late component in the interhemispheric potential without affecting the magnitude of the initial response component. Drugs which selectively interfere with noradrenergic or serotonergic transmission, antagonized the locus coeruleus or raphe primed late response component, respectively. Parenteral administration of d-amphetamine or l-amphetamine caused the appearance of a late response component which was similar to that seen after brainstem priming stimulation. It is suggested that the monoamines modify responses to afferent stimulation of particular pathways in the brain.