Data-driven integration of hippocampal CA1 synaptic physiology in silico.

The anatomy and physiology of monosynaptic connections in rodent hippocampal CA1 have been extensively studied in recent decades. Yet, the resulting knowledge remains disparate and difficult to reconcile. Here, we present a data-driven approach to integrate the current state-of-the-art knowledge on the synaptic anatomy and physiology of rodent hippocampal CA1, including axo-dendritic innervation patterns, number of synapses per connection, quantal conductances, neurotransmitter release probability, and short-term plasticity into a single coherent resource. First, we undertook an extensive literature review of paired recordings of hippocampal neurons and compiled experimental data on their synaptic anatomy and physiology. The data collected in this manner is sparse and inhomogeneous due to the diversity of experimental techniques used by different groups, which necessitates the need for an integrative framework to unify these data. To this end, we extended a previously developed workflow for the neocortex to constrain a unifying in silico reconstruction of the synaptic physiology of CA1 connections. Our work identifies gaps in the existing knowledge and provides a complementary resource toward a more complete quantification of synaptic anatomy and physiology in the rodent hippocampal CA1 region.

The physiological variability of channel density in hippocampal CA1 pyramidal cells and interneurons explored using a unified data-driven modeling workflow.

Every neuron is part of a network, exerting its function by transforming multiple spatiotemporal synaptic input patterns into a single spiking output. This function is specified by the particular shape and passive electrical properties of the neuronal membrane, and the composition and spatial distribution of ion channels across its processes. For a variety of physiological or pathological reasons, the intrinsic input/output function may change during a neuron's lifetime. This process results in high variability in the peak specific conductance of ion channels in individual neurons. The mechanisms responsible for this variability are not well understood, although there are clear indications from experiments and modeling that degeneracy and correlation among multiple channels may be involved. Here, we studied this issue in biophysical models of hippocampal CA1 pyramidal neurons and interneurons. Using a unified data-driven simulation workflow and starting from a set of experimental recordings and morphological reconstructions obtained from rats, we built and analyzed several ensembles of morphologically and biophysically accurate single cell models with intrinsic electrophysiological properties consistent with experimental findings. The results suggest that the set of conductances expressed in any given hippocampal neuron may be considered as belonging to two groups: one subset is responsible for the major characteristics of the firing behavior in each population and the other is responsible for a robust degeneracy. Analysis of the model neurons suggests several experimentally testable predictions related to the combination and relative proportion of the different conductances that should be expressed on the membrane of different types of neurons for them to fulfill their role in the hippocampus circuitry.

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains.

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/ß subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, ß2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/ß2/γ2-expressing HEK293 cells, the α1, ß2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.

GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses.

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABAA receptors (GABAA Rs) themselves--the essential functional postsynaptic components of GABAergic synapses--can be sufficient to initiate formation of synaptic contacts, a novel co-culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e., α1/ß2/γ2-GABAA Rs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse-like contacts in these co-cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle-luminal domain-specific antibody; and they supported spontaneous and action potential-driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N-methyl-d-aspartate receptor-expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin-2 was co-expressed with GABAA Rs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABAA Rs can promote the adhesion of inhibitory axons and the development of functional synapses.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons.

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

SP-SR interneurones: a novel class of neurones of the CA2 region of the hippocampus.

The CA2 region of the hippocampus has distinctive properties and inputs and may be linked with the pathology of specific psychiatric and neurological disorders. It is, therefore, important to understand CA2 circuitry and its involvement in the circuitry of the hippocampus. Properties of CA2 basket cells have been reported. However, other classes of CA2 interneurones with cell bodies located in stratum pyramidale remained to be described. In this study, the unusual axonal arbors of a novel subclass of dendrite-preferring CA2 interneurones whose somata are located in the pyramidal cell layer was revealed following intracellular recordings and biocytin labeling. One to four apical dendrites emerged from the soma, branched in stratum radiatum (SR) forming a tuft, but rarely penetrated stratum lacunosum-moleculare (SLM). One or two basal dendrites branched close to the soma, the branches extended through stratum oriens (SO) and often reached the alveus. Unlike CA2 bistratified cells, the axons of these cells arborized almost exclusively in SR with few, if any, branches extending to stratum pyramidale (SP), SO, or SLM. These interneurones again, unlike bistratified cells, were immunonegative for parvalbumin and cholecystokinin. Electrophysiologically, they were similar to some CA2 basket and bistratified cells in that they presented a "sag" in response to hyperpolarizing current injections and displayed spike frequency adaptation. They targeted the apical dendrites of neighboring CA2 pyramidal cells and received inputs from them.

Local circuitry involving parvalbumin-positive basket cells in the CA2 region of the hippocampus.

There is a growing recognition that the CA2 region of the hippocampus has its own distinctive properties, inputs, and pathologies. The dendritic and axonal patterns of some interneurons in this region are also strikingly different from those described previously in CA1 and CA3. The local circuitry in this region, however, had yet to be studied in detail. Accordingly, using dual intracellular recordings and biocytin-filling, excitatory and inhibitory connections involving CA2 parvalbumin-positive basket cells were characterized for the first time. CA2 basket cells targeted neighboring pyramidal cells and received excitatory inputs from them. CA2 basket cells that resembled those in CA1 with a fast spiking behavior and dendritic tree confined to the region of origin received depressing excitatory postsynaptic potentials (EPSPs). In contrast, unlike CA1 basket cells but like CA1 Oriens-Lacunosum Moleculare (OLM) cells, the majority of CA2 basket cells had horizontally oriented dendrites in Stratum Oriens (SO), which extended into all three CA subfields, had an adapting firing pattern, presented a "sag" in their voltage responses to hyperpolarizing current injection, and received facilitating EPSPs. The expression of I(h) did not influence the EPSP time courses and paired pulse ratios (PPR). Estimates of the probability of release (p) for the depressing and facilitating EPSPs were correlated with the PPR. Connections with low probabilities of release had higher PPR. Quantal amplitude (q) for the facilitating connections was larger than q at depressing inputs onto fast spiking basket cells.

Circuits and Synapses: Hypothesis, Observation, Controversy and Serendipity - An Opinion Piece.

More than a century of dedicated research has resulted in what we now know, and what we think we know, about synapses and neural circuits. This piece asks to what extent some of the major advances - both theoretical and practical - have resulted from carefully considered theory, or experimental design: endeavors that aim to address a question, or to refute an existing hypothesis. It also, however, addresses the important part that serendipity and chance have played. There are cases where hypothesis driven research has resulted in important progress. There are also examples where a hypothesis, a model, or even an experimental approach - particularly one that seems to provide welcome simplification - has become so popular that it becomes dogma and stifles advance in other directions. The nervous system rejoices in complexity, which should neither be ignored, nor run from. The emergence of testable "rules" that can simplify our understanding of neuronal circuits has required the collection of large amounts of data that were difficult to obtain. And although those collecting these data have been criticized for not advancing hypotheses while they were "collecting butterflies," the beauty of the butterflies always enticed us toward further exploration.

Mechanisms underlying synapse-specific clustering of GABA(A) receptors.

A principle that arises from a body of previous work is that each presynaptic terminal recognises its postsynaptic partner and that each postsynaptic site recognises the origin of the synaptic bouton innervating it. In response, the presynaptic terminal sequesters the proteins whose interactions result in the dynamic transmitter release pattern and chemical modulation appropriate for that connection. In parallel, the postsynaptic site sequesters, inserts or captures the receptors and postsynaptic density proteins appropriate for that type of synapse. The focus of this review is the selective clustering of GABA(A) receptors (GABA(A)R) at synapses made by each class of inhibitory interneurone. This provides a system in which the mechanisms underlying transynaptic recognition can be explored. There are many synaptic proteins, often with several isoforms created by post-translational modifications. Complex cascades of interactions between these proteins, on either side of the synaptic cleft, are essential for normal function, normal transmitter release and postsynaptic responsiveness. Interactions between presynaptic and postsynaptic proteins that have binding domains in the synaptic cleft are proposed here to result in a local cleft structure that captures and stabilises only the appropriate subtype of GABA(A)Rs, allowing others to drift away from that synapse, either to be captured by another synapse, or internalised.

Synaptic alpha 5 subunit-containing GABAA receptors mediate IPSPs elicited by dendrite-preferring cells in rat neocortex.

Previous studies indicated that one class of dendrite-preferring hippocampal interneurones inhibits pyramidal cells via alpha 5 gamma-aminobutyric acid (GABA(A)) receptors whereas parvalbumin- and CCK-containing basket cells act via alpha1 and alpha2/3 GABA(A) receptors, respectively. This study asked whether there is selective insertion of different alpha subunit-containing GABA(A) receptors at neocortical inhibitory synapses innervated by specific classes of interneurones. The benzodiazepine site pharmacology of inhibitory postsynaptic potentials (IPSPs) elicited in neocortical pyramidal cells by 3 classes of interneurones was explored with dual whole-cell recordings in neocortical slices from juvenile rats (P18-23). Fast IPSPs activated by multipolar interneurones with narrow spikes and nonadapting firing patterns were powerfully enhanced by the alpha1-preferring agonist zolpidem, suggesting mediation via larger proportion of alpha1 GABA(A) receptors than those activated by multipolar, adapting interneurones, which were less strongly enhanced by zolpidem, but equally insensitive to the alpha 5-selective inverse agonist IA alpha 5 (MSD, Essex, UK) suggesting mediation predominantly via alpha2/3 GABA(A) receptors. In contrast, the IPSPs elicited by bitufted, dendrite-preferring interneurones were reduced by IA alpha 5 and by zinc and insensitive to zolpidem despite enhancement by the broad-spectrum agonist, diazepam. Thus insertion of GABA(A) receptors at synapses on neocortical pyramids is input-specific, with proximal inhibition employing alpha1 and alpha2/3 GABA(A) receptors and dendrite-preferring bitufted interneurones activating alpha 5 GABA(A) receptors.

Characterization of neurons in the CA2 subfield of the adult rat hippocampus.

The hippocampal cornu ammonis 2 (CA2) region is unique in being the only CA region receiving inputs from the hypothalamic supramammillary nucleus, of importance in modulating hippocampal theta rhythm, and is seizure resistant in temporal lobe epilepsy. CA2 has, however, been little studied, possibly because of its small size and difficulty encountered in defining its borders. To investigate the properties of CA2 interneurons, intracellular recordings with biocytin filling were made in adult hippocampal slices. Two types of basket cells were identified. A minority resembled those in CA1, with fast spiking behavior, vertically oriented dendrites, and axons confined to the region of origin. In contrast, the majority of parvalbumin-immunopositive CA2 basket and bistratified cells had long, horizontally oriented, sparsely spiny dendrites extending into all CA subfields in stratum oriens, adapting firing patterns and a pronounced "sag" in voltage responses to hyperpolarizing current, indicative of I(h). Broad CA2 basket cells innervated all three CA subfields and could thus provide CA1 and CA2 with feedforward and CA3 with feedback inhibition. In contrast, CA2 bistratified cell axons displayed striking subfield preference, innervating stratum oriens and stratum radiatum of CA2 and CA1 but stopping abruptly at the CA2/CA3 border, implying feedforward inhibition of CA2 and CA1. These unique features suggest that CA2 is more than a transitional region between CA1 and CA3. The pronounced slow sag current of many CA2 interneurons may contribute to coordination of pyramidal cell firing during theta, whereas the fast spiking behavior of a smaller population of interneurons supports more localized gamma.

Cornu Ammonis Regions-Antecedents of Cortical Layers?

Studying neocortex and hippocampus in parallel, we are struck by the similarities. All three to four layered allocortices and the six layered mammalian neocortex arise in the pallium. All receive and integrate multiple cortical and subcortical inputs, provide multiple outputs and include an array of neuronal classes. During development, each cell positions itself to sample appropriate local and distant inputs and to innervate appropriate targets. Simpler cortices had already solved the need to transform multiple coincident inputs into serviceable outputs before neocortex appeared in mammals. Why then do phylogenetically more recent cortices need multiple pyramidal cell layers? A simple answer is that more neurones can compute more complex functions. The dentate gyrus and hippocampal CA regions-which might be seen as hippocampal antecedents of neocortical layers-lie side by side, albeit around a tight bend. Were the millions of cells of rat neocortex arranged in like fashion, the surface area of the CA pyramidal cell layers would be some 40 times larger. Even if evolution had managed to fold this immense sheet into the space available, the distances between neurones that needed to be synaptically connected would be huge and to maintain the speed of information transfer, massive, myelinated fiber tracts would be needed. How much more practical to stack the "cells that fire and wire together" into narrow columns, while retaining the mechanisms underlying the extraordinary precision with which circuits form. This demonstrably efficient arrangement presents us with challenges, however, not the least being to categorize the baffling array of neuronal subtypes in each of five "pyramidal layers." If we imagine the puzzle posed by this bewildering jumble of apical dendrites, basal dendrites and axons, from many different pyramidal and interneuronal classes, that is encountered by a late-arriving interneurone insinuating itself into a functional circuit, we can perhaps begin to understand why definitive classification, covering every aspect of each neurone's structure and function, is such a challenge. Here, we summarize and compare the development of these two cortices, the properties of their neurones, the circuits they form and the ordered, unidirectional flow of information from one hippocampal region, or one neocortical layer, to another.

GABAA receptor activity shapes the formation of inhibitory synapses between developing medium spiny neurons.

Basal ganglia play an essential role in motor coordination and cognitive functions. The GABAergic medium spiny neurons (MSNs) account for ~95% of all the neurons in this brain region. Central to the normal functioning of MSNs is integration of synaptic activity arriving from the glutamatergic corticostriatal and thalamostriatal afferents, with synaptic inhibition mediated by local interneurons and MSN axon collaterals. In this study we have investigated how the specific types of GABAergic synapses between the MSNs develop over time, and how the activity of GABAA receptors (GABAARs) influences this development. Isolated embryonic (E17) MSNs form a homogenous population in vitro and display spontaneous synaptic activity and functional properties similar to their in vivo counterparts. In dual whole-cell recordings of synaptically connected pairs of MSNs, action potential (AP)-activated synaptic events were detected between 7 and 14 days in vitro (DIV), which coincided with the shift in GABAAR operation from depolarization to hyperpolarization, as detected indirectly by intracellular calcium imaging. In parallel, the predominant subtypes of inhibitory synapses, which innervate dendrites of MSNs and contain GABAAR α1 or α2 subunits, underwent distinct changes in the size of postsynaptic clusters, with α1 becoming smaller and α2 larger over time, while both the percentage and the size of mixed α1/α2-postsynaptic clusters were increased. When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged. In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters. Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord.

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.

Physiological and morphological diversity of immunocytochemically defined parvalbumin- and cholecystokinin-positive interneurones in CA1 of the adult rat hippocampus.

To investigate the electrophysiological properties, synaptic connections, and anatomy of individual parvalbumin-immunoreactive (PV-IR) and cholecystokinin-immunoreactive (CCK-IR) interneurones in CA1, dual intracellular recordings using biocytin-filled microelectrodes in slices of adult rat hippocampus were combined with fluorescence labelling of PV- and CCK-containing cells. Of 36 PV-IR cells, 29 were basket cells, with most of their axonal arbours in the stratum pyramidale (SP). Six were bistratified cells with axons ramifying throughout stratum oriens (SO) and stratum radiatum (SR). One was a putative axo-axonic cell with an axonal arbour confined to half of the SP and a narrow adjacent region of the SO. Of 27 CCK-IR neurones, 13 were basket cells, with most of their axonal arbours in the SP, and included basket cells with somata in the SP (6), SO (3), and SR (2) and at the border between the stratum lacunosum-moleculare (SLM) and the SR (2). In addition, several dendrite-targeting cell classes expressed CCK-IR: 4 of 9 bistratified cells with axons ramifying in the SO and SR; all five Schaffer-associated cells whose axons ramified extensively in the SR; both cells classified as quadrilaminar because their axons ramified in the SO, SP, SR, and SLM; one SO-SO cell whose dendritic and axonal arbours were contained within the SO; and one perforant path-associated cell with axonal and dendritic arbours within the distal SR and SLM. The majority (31 of 36) of PV-IR neurones recorded were fast-spiking, and most fast-spiking cells tested (25 of 29 basket, 1 axo-axonic, and 5 of 6 bistratified cells) were PV-IR. However, 1 of 6 regular-spiking basket, 1 of 4 regular-spiking bistratified, and 3 of 5 burst-firing basket cells were also PV-IR. In contrast, the majority (17 of 27) of the CCK-IR neurones recorded were regular-spiking, 3 were burst-firing, and 7 were fast-spiking. These data confirm that the majority of PV-IR and CCK-IR axon terminals innervate proximal portions of CA1 pyramidal cells. Most PV-IR cells are fast-spiking, whereas most CCK-IR cells are regular-spiking. In both neurochemical classes basket cells predominate, but both groups included subpopulations of dendrite-targeting cells. Despite these similarities, the two populations exhibited very different somatic distributions, and each contained cellular morphologies not represented in the other.

Distribution of interneurons in the CA2 region of the rat hippocampus.

The CA2 region of the mammalian hippocampus is a unique region with its own distinctive properties, inputs and pathologies. Disruption of inhibitory circuits in this region appears to be linked with the pathology of specific psychiatric disorders, promoting interest in its local circuitry, its role in hippocampal function and its dysfunction in disease. In previous studies, CA2 interneurons, including a novel subclass of CA2 dendrite-preferring interneurons that has not been identified in other CA regions, have been shown to display physiological, synaptic and morphological properties unique to this sub-field and may therefore play a crucial role in the hippocampal circuitry. The distributions of immuno-labeled interneurons in dorsal CA2 were studied and compared with those of interneurons in CA1 and CA3. Like those in CA1 and CA3, the somata of CA2 parvalbumin-immunoperoxidase-labeled interneurons were located primarily in Stratum Pyramidale (SP) and Stratum Oriens (SO), with very few cells in Stratum Radiatum (SR) and none in Stratum Lacunosum Moleculare (SLM). There was, however, a greater proportion of GAD-positive cells were immunopositive for PV in SP in CA2 than in CA1 or CA3. CA2 SP also contained a larger density of somatostatin-, calbindin-, and VIP-immunopositive somata than CA1 and/or CA3. Like those in CA1 and CA3, CCK-immunopositive somata in CA2 were mostly located in SR. Reelin- and NPY- immunolabeled cell bodies were located in all layers of the three CA regions. However, a higher density of Reelin-positive somata was found in SP and SR of CA2 than in CA1 or CA3.

Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Biocytin-labelling and its impact on late 20th century studies of cortical circuitry.

In recognition of the impact that a powerful new anatomical tool, such as the Golgi method, can have, this essay highlights the enormous influence that biocytin-filling has had on modern neuroscience. This method has allowed neurones that have been recorded intracellularly, 'whole-cell' or juxta-cellularly, to be identified anatomically, forming a vital link between functional and structural studies. It has been applied throughout the nervous system and has become a fundamental component of our technical armoury. A comprehensive survey of the applications to which the biocytin-filling approach has been put, would fill a large volume. This essay therefore focuses on one area, neocortical microcircuitry and the ways in which combining physiology and anatomy have revealed rules that help us explain its previously indecipherable variability and complexity.

Development of cortical GABAergic innervation.

The mature neocortex contains many different classes of GABAergic inhibitory interneurons, distributed, with some degree of selectivity, through six layers, and through many different regions. Some of the events in the early lives of these neurones that may determine their ultimate destination, their maturation and their selective innervation of targets appropriate for each subtype, are discussed. Both time and place of birth influence the class of interneuron that an early post-mitotic interneuronal precursor will become, driven by the selective expression of different combinations of transcription factors in different regions of their birth places in the ganglionic eminence and ventricular zone. The long distance migration of these precursors along tangential routes in marginal, subventricular, and intermediate zones and their final radial movement, into the developing cortex, is regulated by chemical cues, both attractant and repellent. Once they arrive at their final destination, they must integrate into the developing circuitry. As they mature within the cortex, their axons grow and branch in highly specific patterns that may be partially determined by the genetic blueprint for each interneuronal class and partly by the environment in which they find themselves. Finally, as each interneuron class begins to form synapses with only certain postsynaptic targets, cell-cell recognition, most probably via protein-protein interactions across the synaptic cleft, facilitate the formation of appropriate synapses.