Rodent Hippocampal Neurons Research Articles

On the Occasion of an Important Anniversary.

Previous articleNext article FreeEditorialOn the Occasion of an Important AnniversaryJames L. OldsJames L. Olds Search for more articles by this author PDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinked InRedditEmailQR Code SectionsMoreThe arrival of the current issue of The Biological Bulletin marks the 125th anniversary of our publisher, The Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts. It also marks a new chapter for MBL as it commences an affiliation with the University of Chicago. The virtual symposium within this issue, “Cell Biology of Gametes and Embryos: Insights from Marine Invertebrates” edited by Dominic Poccia of Amherst College, represents, in every sense, a commemoration of that anniversary. Any cursory history of cell biology would reveal the MBL’s central role in some of this field’s seminal discoveries over the last century and a quarter.Recently, as I had the pleasure of sailing through Alaska’s Glacier Bay National Park, with its abundant diversity of species, I thought a bit about the founding, in 1888, of the MBL: in proximity to the Gulf Stream, at the juncture of Vineyard Sound and Buzzards Bay, MBL enjoys a similar blessing of species diversity. The initial trajectory of the MBL took specific advantage of this species diversity, but the institution has since progressed in some very fruitful ways that go far beyond this initial advantage. At today’s MBL, you are as likely to find someone patch-clamping rodent hippocampal neurons as you are to wander past tanks of squid or sea urchins. MBL is of course unique with its long history; but it also singular, to my opinion, among America’s marine labs because of the breadth of its science, which now spans the complete realm of biological discovery.With breadth, also comes depth. In Ecology, Microbiology, Cell Biology, and Neuroscience, just to name a few, investigators at the laboratory continue to make high-impact discoveries, a number of which have led to a Nobel Prize. Over the years, several of those key discoveries have been published first within these pages. And beyond its contribution to research, the education programs at MBL, in particular, the summer courses, have been called a “national treasure.” Generations of biological scientists have had their careers forever altered by an MBL educational experience—and that includes myself three decades ago.So especially here, at this time, we celebrate the 125th anniversary of MBL. As it enters a new chapter we rejoice in what promises to be an exciting future, retaining its central role as a catalyst for biological discovery, while also opening up new opportunities with one of the world’s premier research universities.This journal, The Biological Bulletin, is also over a century old. At the ripe age of 116, we continue to reinvent ourselves, with novel forays into the open access space, position papers, and virtual symposia like the one in this issue, while at the same time maintaining the availability of our entire catalog since 1897 on-line at http://www.biolbull.org. Like our publisher, we have branched out significantly from the early focus on marine invertebrate models, although that biological thread remains clearly visible in every issue. Like our publisher, with breadth comes depth, as evidenced by the current virtual symposium in this issue.As a neuroscientist, I’ve found my own interest in gamete and embryo biology. Eighteen years ago, at the MBL, we used the sea urchin oocyte model system to visualize the activation of protein kinase C in close to real time after fertilization (Olds et al., Developmental Biology 1995). PKC is an enzyme family that also plays a key role in the neurobiology of learning and memory—a topic central to my own scientific interests. For us, the sea urchin oocyte represented a robust model system in which activation was under tight spatial and temporal control. For others, the cell biology of gametes has its own intrinsic interest: these cells are at the nexus of reproduction across eukaryotes. In any case, I hope that our readers will enjoy this issue as they celebrate the institution that founded and publishes this wonderful journal.Enjoy! Previous articleNext article DetailsFiguresReferencesCited by The Biological Bulletin Volume 224, Number 3June 2013 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv224n3p119 Views: 283Total views on this site © 2013 Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

Genome-Wide Association Study of Orthostatic Hypotension and Supine-Standing Blood Pressure Changes in Two Korean Populations

Orthostatic hypotension (OH) is defined by a 20-mm Hg difference of systolic blood pressure (dtSBP) and/or a 10-mm Hg difference of diastolic blood pressure (dtDBP) between supine and standing, and OH is associated with a failure of the cardiovascular reflex to maintain blood pressure on standing from a supine position. To understand the underlying genetic factors for OH traits (OH, dtSBP, and dtDBP), genome-wide association studies (GWASs) using 333,651 single nucleotide polymorphisms (SNPs) were conducted separately for two population-based cohorts, Ansung (n = 3,173) and Ansan (n = 3,255). We identified 8 SNPs (5 SNPs for dtSBP and 3 SNPs for dtDBP) that were repeatedly associated in both the Ansung and Ansan cohorts and had p-values of <1 × 10-5 in the meta-analysis. Unfortunately, the SNPs of the OH case control GWAS did not pass our p-value criteria. Four of 8 SNPs were located in the intergenic region of chromosome 2, and the nearest gene (CTNNA2) was located at 1 Mb of distance. CTNNA2 is a linker between cadherin adhesion receptors and the actin cytoskeleton and is essential for stabilizing dendritic spines in rodent hippocampal neurons. Although there is no report about the function in blood pressure regulation, hippocampal neurons interact primarily with the autonomic nervous system and might be related to OH. The remaining SNPs, rs7098785 of dtSBP trait and rs6892553, rs16887217, and rs4959677 of dtDBP trait were located in the PIK3AP1 intron, ACTBL2-3' flanking, STAR intron, and intergenic region, respectively, but there was no clear functional link to blood pressure regulation.

Serum IgG Antibodies Against the NR1 Subunit of the NMDA Receptor Not Detected in Schizophrenia

To the Editor: A consensus exists that schizophrenia is not a single disease, but the final pathway of a variety of still unknown neurobiological derangements. Recently, several treatable autoimmune brain disorders have been identified presenting with psychotic symptoms that, in some cases, resemble those found in schizophrenia (1). Antibodies target synaptic proteins, interrupting synaptic transmission in brain networks supporting cognition and emotion. Particularly relevant are auto-antibodies against the NR1 subunit of the N-acetyl methyl D-aspartate receptor (NMDAR), which result in diminished NMDAR activity, now considered a hallmark of schizophrenia (2). In patients harboring these antibodies, initial psychiatric symptoms are usually followed by dyskinetic movements or seizures and decreased respiratory drive, with a reduced level of consciousness, often requiring intensive care (1). However, it could be postulated that a limited form of the disease may result in a milder syndrome, akin to schizophrenia. We tested this hypothesis using sera from patients with their first psychotic episode referred to the regional psychiatric center of the province of Alava, Spain, and from healthy controls. All were enrolled after written informed consent according to protocols approved by the local IRB. Blood was drawn and sera were frozen for subsequent study, blinded to patient-control status. After a one-year follow up, sera of patients who then met DSM-IV-TR criteria for schizophrenia-spectrum disorders were tested for antibodies to NR1 and other cell surface antigens using three criteria (immunohistochemistry on rat brain slices and dissociated rodent hippocampal neurons, and a cell-based assay in which HEK cells recombinantly express NMDAR), as previously reported (1). Patients (n=80) and healthy controls (n=40) did not differ statistically in age (29.4±9.9 and 30.7±9.4 years), or sex (28 and 38 percent women). Anti-NR1 IgG antibodies were not detected in either group. Both had four cases with sera reactive to other, still unidentified, neuronal surface antigens. Our findings and a study of seven patients with schizophrenia (3) fail to support the hypothesis that NMDAR IgG antibodies are present in the sera of patients with schizophrenia. Although another study (4) did report NMDAR antibodies in the sera of some schizophrenia patients, it was performed without a control group and test specificity was lower (only one of the above criteria was applied); differences in the clinical diagnosis could also explain the discrepant findings. It should be noted, however, that our study does not rule out that some patients could have antibodies only in cerebrospinal fluid, but not in serum (1). Additionally, antibodies could be present in patients with acute psychosis not meeting DSM-IV diagnostic criteria at one-year.

Myelination of rodent hippocampal neurons in culture

Axons of various hippocampal neurons are myelinated mainly postnatally, which is important for the proper function of neural circuits. Demyelination in the hippocampus has been observed in patients with multiple sclerosis, Alzheimer's disease or temporal lobe epilepsy. However, very little is known about the mechanisms and exact functions of the interaction between the myelin-making oligodendrocytes and the axons within the hippocampus. This is mainly attributable to the lack of a system suitable for molecular studies. We recently established a new myelin coculture from embryonic day (E) 18 rat embryos consisting of hippocampal neurons and oligodendrocytes, with which we identified a novel intra-axonal signaling pathway regulating the juxtaparanodal clustering of Kv1.2 channels. Here we describe the detailed protocol for this new coculture. It takes about 5 weeks to set up and use the system. This coculture is particularly useful for studying myelin-mediated regulation of ion channel trafficking and for understanding how neuronal excitability and synaptic transmission are regulated by myelination.

Crucial polarity regulators in axon specification

Cell polarization is critical for the correct functioning of many cell types, creating functional and morphological asymmetry in response to intrinsic and extrinsic cues. Neurons are a classical example of polarized cells, as they usually extend one long axon and short branched dendrites. The formation of such distinct cellular compartments (also known as neuronal polarization) ensures the proper development and physiology of the nervous system and is controlled by a complex set of signalling pathways able to integrate multiple polarity cues. Because polarization is at the basis of neuronal development, investigating the mechanisms responsible for this process is fundamental not only to understand how the nervous system develops, but also to devise therapeutic strategies for neuroregeneration. The last two decades have seen remarkable progress in understanding the molecular mechanisms responsible for mammalian neuronal polarization, primarily using cultures of rodent hippocampal neurons. More recent efforts have started to explore the role of such mechanisms in vivo. It has become clear that neuronal polarization relies on signalling networks and feedback mechanisms co-ordinating the actin and microtubule cytoskeleton and membrane traffic. The present chapter will highlight the role of key molecules involved in neuronal polarization, such as regulators of the actin/microtubule cytoskeleton and membrane traffic, polarity complexes and small GTPases.

Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission

Optogenetic silencing strategies using light-driven ion fluxes permit rapid and effective inhibition of neural activity. Using rodent hippocampal neurons we show that silencing activity with a chloride pump can increase the probability of synaptically-evoked spiking in the period following light-activation, whereas this is not the case for a proton pump. This effect can be accounted for by changes to the GABAA receptor reversal potential and demonstrates an important difference between silencing strategies.

Brain‐derived neurotrophic factor produced by human umbilical tissue‐derived cells is required for its effect on hippocampal dendritic differentiation

The potential for nonembryonic cells to promote differentiation of neuronal cells has therapeutic implications for regeneration of neurons damaged by stroke or injury and avoids many ethical and safety concerns. The authors have assessed the capacity of human umbilical tissue-derived cells (hUTC) and human mesenchymal stromal cells (hMSC) to enhance differentiation of rodent hippocampal neurons. Co-culture of hippocampal cells with hUTC or hMSC in transwell inserts for 3 days resulted in increase of several dendritic parameters including the number and length of primary dendrites. The effect of hUTC or hMSC on dendritic maturation was only apparent on neurons grown for 2 weeks in vitro prior to co-culture. Changes in dendritic morphology in the presence of hUTC were also accompanied by increased expression of the presynaptic marker synaptotagmin and the postsynaptic marker postsynaptic density protein 95 kDa (PSD95) suggesting that there may also be an increase in the number of synapses formed in the presence of hUTC. The effect of hUTC and hMSC on hippocampal cells in co-culture was comparable to those induced by treatment with recombinant human brain-derived neurotrophic factor (BDNF) implying that a similar factor may be released from hUTC or hMSC. Analysis of hUTC-conditioned medium by ELISA demonstrated that BDNF was indeed secreted. An antibody that blocks the actions of BDNF partially inhibited the actions of hUTC on dendritic morphology suggesting that BDNF is at least one of the factors secreted from the cells to promote dendritic maturation. These results indicate that hUTC secrete biologically active BDNF, which can affect dendritic morphology.

Aβ neurotoxicity depends on interactions between copper ions, prion protein, and N -methyl- d -aspartate receptors

N-methyl-d-aspartate receptors (NMDARs) mediate critical CNS functions, whereas excessive activity contributes to neuronal damage. At physiological glycine concentrations, NMDAR currents recorded from cultured rodent hippocampal neurons exhibited strong desensitization in the continued presence of NMDA, thus protecting neurons from calcium overload. Reducing copper availability by specific chelators (bathocuproine disulfonate, cuprizone) induced nondesensitizing NMDAR currents even at physiologically low glycine concentrations. This effect was mimicked by, and was not additive with, genetic ablation of cellular prion protein (PrP(C)), a key copper-binding protein in the CNS. Acute ablation of PrP(C) by enzymatically cleaving its cell-surface GPI anchor yielded similar effects. Biochemical studies and electrophysiological measurements revealed that PrP(C) interacts with the NMDAR complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor. Synthetic human Aβ(1-42) (10 nM-5 μM) produced an identical effect that could be mitigated by addition of excess copper ions or NMDAR blockers. Taken together, Aβ(1-42), copper chelators, or PrP(C) inactivation all enhance the activity of glycine at the NMDAR, giving rise to pathologically large nondesensitizing steady-state NMDAR currents and neurotoxicity. We propose a physiological role for PrP(C), one that limits excessive NMDAR activity that might otherwise promote neuronal damage. In addition, we provide a unifying molecular mechanism whereby toxic species of Aβ(1-42) might mediate neuronal and synaptic injury, at least in part, by disrupting the normal copper-mediated, PrP(C)-dependent inhibition of excessive activity of this highly calcium-permeable glutamate receptor.

AKAP79/150 Anchored PKA and Calcineurin are Critical Components in Calcium-Dependent Inactivation of Neuronal L-Type Calcium Channels

In neurons, L-type voltage-gated Ca2+ channels (L-channels) provide Ca2+ signals that help control gene expression. Consequently, regulation of L-channel activity via processes such as Ca2+-dependent inactivation (CDI) is thought to be important in neuronal function. CDI of CaV1.2 L-channels relies upon proteins associated with the channel's C-terminus, such as the Ca2+-sensor calmodulin. The C-terminus of the CaV1.2 α1 subunit also interacts with the A-kinase anchoring protein AKAP79/150 (79 = human, 150 = rodent), which binds both protein kinase A (PKA) and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN). To study the roles of AKAP79/150-anchored CaN and PKA in CDI, we recorded pharmacologically-isolated L-type Ca2+ currents from rodent hippocampal neurons maintained in culture for up to 6 days. Rates of Ca2+-dependent inactivation (1/τ) were estimated by curve-fitting the fast component of the decay of Ca2+ current (10 mM) that occurred during 500-msec step depolarizations (0.067 Hz) from the holding potential (−60 mV) to 0 mV. In control neurons L-current underwent CDI at a rate of ∼40 sec−1. CDI was slowed to < 10 sec−1 by disruption of CaN anchoring or activity via (1) RNAi knock-down of AKAP150 coupled with over-expression of a mutant human AKAP lacking the CaN binding motif (AKAP79ΔPIX); (2) block of CaN-AKAP150 interaction by an anchoring competitor (VIVIT peptide); (3) the CaN inhibitor cyclosporine A; or (4) expression of AKAP150ΔPIX in transgenic mice. CDI was slowed to less than 20 sec−1 by perturbation of the PKA-AKAP interaction via (1) AKAP150 knock-down coupled with over-expression of anchoring protein lacking the PKA binding site (AKAP79ΔPKA); (2) block of PKA-AKAP150 interaction with an anchoring competitor (Ht31 peptide); or (3) expression of AKAP150ΔPKA in transgenic mice. The PKA inhibitor PKI slowed CDI to < 7 sec−1.

Ubiquitination Acutely Regulates Presynaptic Neurotransmitter Release in Mammalian Neurons

The ubiquitin proteasome system (UPS) plays a crucial role in modulating synaptic physiology both presynaptically and postsynaptically, but the regulatory mechanisms remain obscure. To determine acute effects of proteasome inhibition on neurotransmission, we performed whole-cell voltage-clamp recordings from cultured rodent hippocampal neurons. We find that proteasome inhibitors induce a strikingly fast, severalfold increase in the frequency of both miniature (mini) and spontaneous synaptic currents at excitatory and inhibitory synapses. The lack of change in mini amplitude and kinetics indicates a presynaptic site of action. This effect does not depend on increased levels of presynaptic proteins, previously suggested as proteasomal targets. Furthermore, blockade of the UPS using E1-activating enzyme inhibitors also increases mini frequency, demonstrating that accumulation of ubiquitinated proteins is not required. Overall, these data suggest that the UPS not only orchestrates protein turnover, but also dynamically regulates the activity state of presynaptic proteins, thus crucially shaping synaptic transmission.

P03-349 The potential effects of antidepressants in attenuating synaptic degeneration in depression and Alzheimer's disease

Depression is a highly prevalent psychiatric disorder. Furthermore, it is one of the most common neuropsychiatric presentations in Alzheimer's disease (AD). Common underlying neuropathological processes appear to exist between these conditions. Synaptic degeneration has been implicated in AD pathogenesis, while its role in depression is not well understood.ObjectivesThis study aims to investigate synaptic degeneration as a possible underlying mechanism for depression and whether antidepressants could alleviate the stated pathology.MethodsPrimary rodent hippocampal neurons treated with corticosterone were used as an in vitro model of depression. Toxicity of corticosterone was determined through the lactate dehydrogenase and caspase-3 activity assays. Immunocytochemical analysis of synaptic proteins was employed to investigate effects of corticosterone on synapses.ResultsNeurotoxicity was observed in hippocampal neurons after treatment with corticosterone (10μM) for 24 hours. Aggregations of synaptotagmin and synaptophysin were observed 24 hours after treatment with corticosterone (10μM). Similar effects were observed after sub-lethal treatments with corticosterone (0.5μM and 1μM) for 48 and 72 hours. Pre-treatment for 1 hour with imipramine and escitalopram (20μM and 40μM for both agents) were able to alleviate these toxic effects.ConclusionsThese results suggest the involvement of synaptic degeneration in corticosterone-induced toxicity and that commonly used antidepressants are able to alleviate synaptic derangements. Taken together, synaptic degeneration could be a common pathway for neuronal demise occurring in AD and depression, which can be attenuated by antidepressant administration. Future research to elucidate the precise mechanism for the synaptic protective effect of antidepressants is warranted.

Establishment of Axon-Dendrite Polarity in Developing Neurons

Neurons are among the most highly polarized cell types in the body, and the polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. Significant progress has been made in the identification of the cellular and molecular mechanisms underlying the establishment of neuronal polarity using primarily in vitro approaches such as dissociated culture of rodent hippocampal and cortical neurons. This model has led to the predominant view suggesting that neuronal polarization is specified largely by stochastic, asymmetric activation of intracellular signaling pathways. Recent evidence shows that extracellular cues can play an instructive role during neuronal polarization in vitro and in vivo. In this review, we synthesize the recent data supporting an integrative model whereby extracellular cues orchestrate the intracellular signaling underlying the initial break of neuronal symmetry leading to axon-dendrite polarization.

Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons

Signals received at distal synapses of neurons must be conveyed to the nucleus to initiate the changes in transcription that underlie long-lasting synaptic plasticity. The presence of importin nuclear transporters and of select transcription factors at synapses raises the possibility that importins directly transport transcription factors from synapse to nucleus to modulate gene expression. Here, we show that cyclic AMP response element binding protein 2 (CREB2)/activating transcription factor 4 (ATF4), a transcriptional repressor that modulates long-term synaptic plasticity and memory, localizes to distal dendrites of rodent hippocampal neurons and neurites of Aplysia sensory neurons (SNs) and binds to specific importin alpha isoforms. Binding of CREB2 to importin alpha is required for its transport from distal dendrites to the soma and for its translocation into the nucleus. CREB2 accumulates in the nucleus during long-term depression (LTD) but not long-term potentiation of rodent hippocampal synapses, and during LTD but not long-term facilitation (LTF) of Aplysia sensory-motor synapses. Time-lapse microscopy of CREB2 tagged with a photoconvertible fluorescent protein further reveals retrograde transport of CREB2 from distal neurites to the nucleus of Aplysia SN during phenylalanine-methionine-arginine-phenylalanine-amide (FMRFamide)-induced LTD. Together, our findings indicate that CREB2 is a novel cargo of importin alpha that translocates from distal synaptic sites to the nucleus after stimuli that induce LTD of neuronal synapses.

Potential Anatomical Basis for Attentional Modulation of Hippocampal Neurons

Lesions of the hippocampus and related structures produce profound anterograde amnesia. The amnesia is specific to what has been called "explicit," "declarative," and "episodic" memory. These memories are frequently believed to be central to the human condition, requiring such advanced cognitive functions as attention and even consciousness. However, the hippocampus and associated structures are evolutionarily conserved, which argues that the memories of lower mammals should be qualitatively similar in nature. Just as attention and arousal are critical components of appropriate memory formation in humans, an emerging body of evidence suggests that these processes bear on the firing patterns of hippocampal neurons in rodents. Here the evidence favoring this hypothesis is discussed and then the potential anatomical basis for such modulation is considered.

Protection against β-amyloid induced abnormal synaptic function and cell death by Ginkgolide J

A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents Aβ1–42 induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by Aβ1–42. This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimer's disease.

Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unapposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.

Identification of Process-Localized mRNAs from Cultured Rodent Hippocampal Neurons

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapse-specific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 microm pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.

Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin

Genetically encoded fluorescent probes have become indispensable tools in the biological sciences. Studies of synaptic vesicle recycling have been facilitated by a group of GFP-derived probes called pHluorins. These probes exploit changes in pH that accompany exocytosis and recapture of synaptic vesicles. Here we describe how these synaptic tracers can be used in rodent hippocampal neurons to monitor the synaptic vesicle cycle in real time and to obtain mechanistic insights about it. Synapses can be observed in living samples using a wide-field fluorescence microscope and a cooled charge-coupled device camera. A simple specimen chamber allows electrical stimulation of synapses to evoke exocytosis in a precisely controlled manner. We present protocols to measure various parameters of the synaptic vesicle cycle. This technique can be easily adapted to study different classes of synapses from wild-type and mutant mice. Once cultured neurons expressing synaptopHluorin are available, the whole procedure should take about 2 h.

Hippocampal place cells, context, and episodic memory

Although most observers agree that the hippocampus has a critical role in learning and memory, there remains considerable debate about the precise functional contribution of the hippocampus to these processes. Two of the most influential accounts hold that the primary function of the hippocampus is to generate cognitive maps and to mediate episodic memory processes. The well-documented spatial firing patterns (place fields) of hippocampal neurons in rodents, along with the spatial learning impairments observed with hippocampal damage support the cognitive mapping hypothesis. The amnesia for personally experienced events seen in humans with hippocampal damage and the data of animal models, which show severe memory deficits associated with hippocampal lesions, support the episodic memory account. Although an extensive literature supports each of these hypotheses, a specific contribution of place cells to episodic memory has not been clearly demonstrated. Recent data from our laboratory, together with previous findings, indicate that hippocampal place fields and neuronal responses to task-relevant stimuli are highly sensitive to the context, even when the contexts are defined by abstract task demands rather than the spatial geometry of the environment. On the basis of these findings, it is proposed that place fields reflect a more general context processing function of the hippocampus. Hippocampal context representations could serve to differentiate contexts and prime the relevant memories and behaviors. Since episodic memories, by definition, include information about the time and place where the episode occurred, contextual information is a necessary prerequisite for any episodic memory. Thus, place fields contribute importantly to episodic memory as part of the needed context representations. Additionally, recent findings indicate that hippocampal neurons differentiate contexts at progressively finer levels of detail, suggesting a hierarchical coding scheme which, if combined with temporal information, could provide a means of differentiating memory episodes.

Regulation of Hippocampal Sharp Waves by Ca2+-Dependent Slow After Hyperpolarization

In rodent hippocampal pyramidal neurons, repetitive discharges are followed by a slow afterhyperpolarization (sAHP) as a result of activation of a Ca2+-dependent K+ current. The sAHP is sensitive to activation of several G-protein coupled neurotransmitter receptors and downstream signal cascades. Modulations of the sAHP have been shown to be closely associated with synaptic plasticity, learning, and aging processes. However, it is presently unclear whether the sAHP generation is involved in hippocampal network activities. We explored this issue using an in vitro (thick-slice) model of mouse hippocampal sharp waves. Our data show that the sAHP occurs in CA3 pyramidal neurons following each sharp wave event and sAHP suppression is associated with a large increase in occurrence frequency of spontaneous sharp waves. Considering that sharp waves are important for hippocampal-cortical communication and memory processes, we postulate that the sAHP serves as an intrinsic regulatory mechanism of sharp waves and plays a significant role in hippocampus-dependent cognitive functions.