Pyramidal Basket Research Articles

Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.

Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1−/− GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic generalized epilepsy syndromes.

Ih modulates theta rhythm and synchrony in computer model of CA3

Although the contributions of different hippocampal cell classes to oscillatory activity has been studied, the intrinsic membrane mechanisms contributing to the oscillations and neuronal synchronization are not well understood. The hyperpolarization-activated cation (HCN) channel is a voltage-gated ion channel potentially implicated in epilepsy. Ih, the current produced by HCN channels, plays an important role in regulating neuronal excitability, particularly in hippocampal and neocortical pyramidal neurons. Changes in HCN conductance have been associated with epilepsy, manifesting as discharges with aberrant synchrony [1]. In this study we investigated the role of Ih in pacing network oscillations and synchronizing activity. We studied the effects of modulating both the Ih time-constant and the Ih conductance level in a biophysical computer model of the CA3 region of the hippocampus using the NEURON simulator. Some of the manipulations could be realized in-vitro with pharmacological manipulation via the Ih blocker, ZD7288. Our network consisted of 800 five-compartment pyramidal cells, 200 one-compartment basket cell interneurons, and 200 one-compartment oriens lacunosum-moleculare (O-LM) interneurons. All cells contained leak current, transient sodium current and delayed rectifier current. Additionally, pyramidal cells contained potassium type A current and pyramidal and OLM cells had Ih current. Cell classes were interconnected probabilistically with AMPA/NMDA, and two classes of GABAA synapses. The O-LM cells formed synapses on the distal dendrites of pyramidal cells, while the basket cells synapsed proximally on pyramidal and other basket cells. Pyramidal cells synapsed on both types of interneurons with AMPA/NMDA synapses. All synapses were bombarded with external Poisson inputs to generate network activity. We used Kendall’s tau correlation to measure the synchrony between pairs of pyramidal cells and performed FFT analysis on local field potentials generated by the pyramidal cells to measure rhythmic activity. At baseline, OLM cells fired preferentially at the theta frequency, causing periodic inhibition/disinhibition of pyramidal cells [2]. Although lowering the Ih conductance of pyramidal cell distal dendrites did not change average firing rates of pyramidal cells, the delay to pyramidal cell synchronization increased. Delayed synchronization was associated with a delay in the emergence of pyramidal interneuron network gamma (PING; in PING, pyramidal cells drive basket cells via AMPA/NMDA receptors and basket cells in turn inhibit the pyramidal cells through GABAergic synapses). This mechanism depends on stabilization via pyramidal cell synchronization. Analysis of the simulated local field potential spectral power showed that Ih conductance level correlated with the peak theta rhythm, from ~6.5 - 8.5 Hz. Our model demonstrates that changes in conductance of HCN channels can modulate hippocampal network rhythms and synchrony. These effects could be tested in-vivo and in-vitro, under neuromodulatory or pharmacological control. Our model also predicts that hippocampal networks may become more prone towards epilepsy with alterations in the level of HCN channel expression.

CPP alters theta/gamma oscillations in rat hippocampus: simulation and experiment

Several different NMDA receptor (NMDAR) antagonists are used to produce pharmacological models for schizophrenia. These antagonists have different molecular mechanisms of action: non-competitive (e.g. ketamine, which binds at a site different from glutamate) and competitive (e.g. CPP, which displaces glutamate). Because competitive antagonism results in more glutamate availability, this may result in glutamate spill-over either onto other receptors of the same synapse or to neighboring synapses. In this study, we modeled the effects of competitive blockade by simultaneously blocking NMDAR and increasing activity of AMPAR (spillover). We looked at alterations of network oscillations and synchronization using our biophysical model of the CA1 region of hippocampus using the NEURON simulator. We then compared the results with data obtained from the CA1 region of hippocampus in rats injected with CPP. Our network consisted of 800 five-compartment pyramidal cells (PYR), 200 one-compartment basket cell interneurons (BAS), and 200 one-compartment oriens lacunosum-moleculare interneurons (OLM) [1]. All cells contained leak current, transient sodium current and delayed rectifier current. Additionally, pyramidal cells contained potassium type A current and pyramidal and OLM cells had Ih current. Cell classes were interconnected probabilistically with AMPA/NMDA synapses, and two classes of GABAa synapses. The OLM cells formed synapses on the distal dendrites of pyramidal cells, while the basket cells synapsed proximally on pyramidal and other basket cells. Pyramidal cells synapsed on both types of interneurons with AMPA/NMDA synapses. All synapses were bombarded with external Poisson inputs to generate network activity. Competitive inhibition was modeled by setting the conductance across NMDAR to zero, and increasing current conductance across AMPAR on the same set of synapses. We modeled this on synaptic sites on all cell populations. Also, given the possible variation of the affinity of competitive inhibitors to NMDAR on different cells, we studied this effect when it took place at the receptors of the 3 different cell populations separately. Experimental recordings were made from tetrode arrays implanted in the CA1 region of Long Evans rats chasing sugar pellets in a box. CPP at a dose of 5 mg/kg was injected intraperitoneally in the rats. Recordings were for 16 min sessions, separated by 30 min breaks during which the rats were returned to their cages. Recordings from CA1 of rat hippocampus under the effect of CPP, showed that there is a reduction in theta and an increase in gamma. Modeling competitive inhibition to glutamate at all synapse locations (on OLM, BAS, and PYR) resulted in a reduction in the theta power and an increase in gamma power, differing from the noncompetitive effect with ketamine, which reduced both theta and gamma when given across all synapses. However, this result was also seen when the effect of competitive inhibition was restricted to only OLM cell NMDARs. Therefore, our model demonstrates both of these interpretations of CPP action. We will examine further correlates between simulation and experiment to reduce this uncertainty.

Early signs of tinnitus in a simulation of the mammalian primary auditory cortex

The majority of tinnitus cases are related to cochlear dysfunction, leading to altered peripheral input to the central auditory system [1]. These alterations are believed to increase the basic level of neural activity during off-conditions of sound and to diminish the increase in neural activity when sound is presented [2]. As a compensatory means the affected region of primary auditory cortex tries to maximize the difference between basic level activity and sound-induced activity by adapting inhibitory and excitatory influences towards less GABAergic inhibition. This adaptation in turn triggers unmasking of dormant synapses and creation of new connections through axonal sprouting and finally results in a reorganization of tonotopic receptive fields and the manifestation of tinnitus [3]. To further investigate the processes involved in tinnitus manifestation we used neuroConstruct [4] to implement a simulation of the primary auditory cortex. It consists of two groups of different types of neurons, excitatory regular spiking pyramidal cells and inhibitory fast spiking basket cells with a group size of ? and ?, respectively. Its organization is based on experimental data from animal studies. Neurons were modeled as conductance-based multi-compartment neurons having numerous ionic conductances to achieve the desired firing properties. Model neurons posses glutamate- (AMPA and NMDA) and GABA-sensitive synaptic receptors. Afferent input from the auditory pathway was modeled as trains of spikes. Their frequency depended on the strength of the input whereas their target site depended on the frequency of the input. Additionally, synaptic background noise was introduced to the system as Gaussian noise. Excitatory cells made connections to other

Branched-chain amino acids and neurotransmitter metabolism: expression of cytosolic branched-chain aminotransferase (BCATc) in the cerebellum and hippocampus.

In the brain, catabolism of the branched-chain amino acids (BCAAs) provides nitrogen for the synthesis of glutamate and glutamine. Glutamate is formed through transfer of an amino group from BCAA to alpha-ketoglutarate in reaction catalyzed by branched-chain aminotransferases (BCAT). There are two isozymes of BCAT: cytosolic BCATc, which is found in the nervous system, ovary, and placenta, and mitochondrial BCATm, which is found in all organs except rat liver. In cell culture systems, BCATc is found only in neurons and developing oligodendrocytes, whereas BCATm is the isoform in astroglia. In this study, we used immunohistochemistry to examine the distribution of BCATc in the rat brain, focusing on the well-known neural architecture of the cerebellum and hippocampus. We show that BCATc is expressed only in neurons in the adult rat brain. In glutamatergic neurons such as granule cells of the cerebellar cortex and of the dentate gyrus, BCATc is localized to axons and nerve terminals. In contrast, in GABAergic neurons such as cerebellar Purkinje cells and hippocampal pyramidal basket cells, BCATc is concentrated in cell bodies. A common function for BCATc in these neurotransmitter systems may be to modulate amounts of glutamate available either for release as neurotransmitter or for use as precursor for synthesis of GABA. Particularly striking in our findings is the strong expression of BCATc in the mossy fiber pathway of the hippocampal formation. This result is discussed in light of the effectiveness of the anticonvulsant drug gabapentin, which is a specific inhibitor of BCATc.

Modulation of taurine release by metabotropic receptors in the developing hippocampus.

Taurine abounds in the hippocampus14, particularly in the immature hippocampus26, and taurine-like immunoreactivity has been located in hippocampal interneurons, pyramidal neurons and dentate granule cells15. Taurine inhibits the firing of hippocampal pyramidal neurons, increases membrane chloride conductance and causes hyperpolarization20. In the hippocampus, the taurine-synthesizing enzyme cysteine sulphinate decarboxylase has also been identified in pyramidal basket interneurons20. Taurine has been held to have a special role in immature brain tissue12,21. Besides being an osmoregulator and neuromodulator it seems to be essential for the development and survival of neural cells29. The excitatory glutamatergic innervation in the hippocampus is modulated by inhibitory GABA-releasing interneurons8. These mechanisms could also regulate taurine release. Ionotropic glutamate receptors modify taurine release in the hippocampus16,25, as do GABAA receptors in the cerebral cortex and cerebellum24. The hippocampal innervation also includes a large family of metabotropic glutamate receptors (mGluRs) coupled to second messenger systems via GTP-binding proteins. At least eight subtypes of them have been cloned, divided into three major groups based on pharmacology, second-messenger coupling and sequence homology6,22. Metabotropic GABAB receptors, which initiate slow inhibition via Gprotein-coupled action on Ca2+ and K+ channels18, also exist in the hippocampus7. We have now characterized the regulation of taurine release

A role for sodium and chloride in kainic acid-induced beading of inhibitory interneuron dendrites

Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors. In experiments reported here, transient (1 h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5 mM kainic acid, more than half of the immunopositive apical dendrites in area CA 1 had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300 mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl- d-aspartate receptor antagonists, the l-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading. Thus, we suggest that the movement of Na + and Cl −, rather than Ca 2+, into cells underlies the focal swellings of interneuron dendrites in hippocampus.

Post-mortem brain weight in schizophrenia and affective disorder

Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors.In experiments reported here, transient (1 h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5 mM kainic acid, more than half of the immunopositive apical dendrites in area CA1 had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300 mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl-d-aspartate receptor antagonists, the l-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading.Thus, we suggest that the movement of Na+ and Cl−, rather than Ca2+, into cells underlies the focal swellings of interneuron dendrites in hippocampus.

Morphological evidence for altered synaptic organization and structure in the hippocampal formation of seizure-sensitive gerbils.

Seizure-sensitive (SS) and seizure-resistant (SR) Mongolian gerbils were used for three experiments. In the first experiment, GABAergic neurons and terminals in the dentate gyrus were localized with GAD immunocytochemistry. GAD-positive puncta adjacent to cell bodies of GABAergic pyramidal basket cells were counted in light microscopic preparations. The pyramidal basket cells of SS gerbils displayed a significant threefold increase in the number of GAD-positive puncta associated with their cell bodies as compared to those from SR gerbils. These data indicate that the number of GABAergic synapses with pyramidal basket cell bodies in the dentate gyrus was greater in SS gerbils. An electron microscopic (EM) analysis of GAD immunocytochemical preparations showed GAD-positive axon terminals forming symmetric synapses with GAD-positive basket cell bodies. However, numerous terminals forming symmetric axosomatic synapses with basket cells were not immunopositive, and other synapses formed by terminals were not classified because reaction product in the cell bodies obscured postsynaptic densities. Therefore, routine EM preparations were analyzed for symmetric and asymmetric axosomatic synapses on pyramidal basket cells and granule cells of SS and SR gerbils. The data obtained from these preparations showed that the pyramidal basket cells of SS gerbils had a selective increase in the number of symmetric synapses per 10 microns of soma as compared to those of the SR gerbils. In contrast, the granule cells did not show any significant difference in the number of either symmetric or asymmetric axosomatic synapses between SS and SR gerbils. These results indicate that pyramidal basket cell bodies of SS gerbils have more inhibitory synapses than do those of SR gerbils. The third experiment used SS gerbils with lesions of the perforant pathway that stopped seizure activity (Ribak, C. E., and S. U. Khan (1987) The effects of knife cuts of hippocampal pathways on epileptic activity in the seizure-sensitive gerbil. Brain Res. 418:251-260). The percentage of axon terminal area occupied by synaptic vesicles and their packing density was determined in CA3 mossy fiber boutons and compared for lesioned and nonlesioned SS gerbils. The mossy fibers of nonlesioned SS gerbils showed a depletion of synaptic vesicles consistent with the previous results of Peterson et al. (Peterson, G. M., C. E. Ribak, and W. H. Oertel (1985) A regional increase in the number of hippocampal GABAergic neurons and terminals in the seizure-sensitive gerbil. Brain Res. 340:384-389).(ABSTRACT TRUNCATED AT 400 WORDS)

Parvalbumin‐ and calbindin D28K‐immunoreactive neurons in the hippocampal formation of the macaque monkey

Calcium-binding proteins calbindin D28k (CaBP) and parvalbumin (PV) were localized in neurons of the monkey hippocampal formation. CaBP immunoreactivity is present in all granule cells and in a large proportion of CA1 and CA2 pyramidal neurons, as well as in a distinct population of local circuit neurons. In the dentate gyrus, CaBP-immunoreactive nongranule cells are present in the molecular layer and in the hilar region, but they do not include the pyramidal basket cells at the hilar border. In the Ammon's horn, CaBP-positive, nonpyramidal neurons are more frequent in the CA3 area than in any other parts of the hippocampal formation. They are concentrated in the strata oriens and pyramidale of areas CA1-3, whereas only a few small neurons were found in the strata lucidum and radiatum of CA3 and in the stratum moleculare of the CA1 area. PV is exclusively present in local circuit neurons both in the dentate gyrus and in Ammon's horn. In the dentate gyrus the presumed basket cells at the hilar border exhibit PV immunoreactivity. In the hilar region and molecular layer only a relatively small number of cells are immunoreactive for PV. Most of these PV-positive cell bodies are located in the inner half of the molecular layer, with occasional horizontal cells at the hippocampal fissure. In Ammon's horn, strata oriens and pyramidale of areas CA1-3 contain a large number of PV-positive cells. There are no PV-immunoreactive cells in the strata lucidum, radiatum, or lacunosum moleculare. The CaBP- and PV-containing neurons form different subpopulations of cells in the monkey hippocampal formation. With the exception of a basket cell type in the monkey dentate gyrus, the CaBP- and PV-positive cell types were found to be remarkably similar in rodents and primates.

The mouse gene retinal degeneration ( rd) may reduce the number of neurons present in the adult hippocampal dentate gyrus

C57BL/6J and C57BL/6J- rd le Gus-s h mice, congenics at the rd locus, were compared with respect to number of granule cells and presumed pyramidal basket cells in the hippocampal dentate gyrus. Number of both types of neurons were less in rd/rd mice than in ±/± mice. The rd gene may be responsible.

Immunohistochemical distribution of calcium‐activated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus

Intracellular accumulation of Ca2+ after brain ischemia is regarded as one of the principal causes of neuronal death, but details of the intracellular events occurring after Ca2+ accumulation have not yet been described. We propose that a calcium-activated neutral proteinase which can degrade neuronal cytoskeletal proteins might link Ca2+ accumulation and irreversible injury of the neuronal intracellular structure. First, therefore, we examined the distribution of calcium-activated neutral proteinase in normal brains. Immunohistochemical distribution of calcium-activated neutral proteinases (CANP) with high and low sensitivity to Ca2+ (muCANP and mCANP) and of endogenous CANP inhibitor was investigated in the dorsal hippocampus of the rabbit. muCANP-immunoreactivity was detected in almost all of the pyramidal cells and granule cells and in some other neurons. A full-length staining from perikarya to dendrites was shown in muCANP-positive neurons. mCANP-immunoreactivity was found mainly in four kinds of hippocampal interneurons: 1) basket cells in the stratum oriens of Ammon's horn, 2) pyramidal basket cells at the boundary of pyramidal cell layer and stratum oriens, 3) polymorphic cells in the hilar region of dentate gyrus, and 4) pyramidal or fusiform basket cells at the inner boundary of the granule cell layer and the hilar region. The distribution of these four kinds of neurons was similar to that of parvalbumin-containing GABAergic neurons. CANP inhibitor immunoreactivity was confined to pyramidal cells in the CA3-CA3c region and some hilar neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus.

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.

Colocalization of taurine- and cysteine sulfinic acid decarboxylase-like immunoreactivity in the hippocampus of the rat.

It is proposed that taurine is an inhibitory neurotransmitter/neuromodulator in the CNS. The present study localized taurine-containing neurons within the rat hippocampus with the use of a monoclonal antibody against conjugated taurine (Tau2) in conjunction with an antiserum against cysteine sulfinic acid decarboxylase (CSADC), a synthesizing enzyme for taurine. Taurine-like immunoreactivity Tau-LI) and CSADC-LI were colocalized in neurons of the dentate gyrus, CA1(/CA2), CA3, and CA4. Of all the cells examined, pyramidal basket cells within the granule cell layer of the dentate gyrus were most intensely stained with both Tau2 and CSADC. Granule cells were also double-labeled with Tau-LI and CSADC-LI. Cell nuclei and dendrites in the CA1 region stained more intensely with Tau2 than somata. CSADC-LI was colocalized with Tau-LI within these neurons. Light staining with both Tau2 and the CSADC antiserum was inconsistently present in CA3 and CA4 neurons and was found to be highly dependent on the type of fixation and delay to fixation. Tau-LI was more consistently present in increased numbers of neurons in CA3 when glutaraldehyde was added to the paraformaldehyde fixative solution. Hippocampi which were immersion-fixed in paraformaldehyde following a 0-, 6-, or 24-hour postmortem delay exhibited a lack of Tau2 staining in the CA3 region in the majority of animals studied, similar to some paraformaldehyde perfusion-fixed rats. These studies suggest that taurine was present in the majority of neurons within the major cell layers of the rat hippocampus, but Tau-LI was more easily lost from neurons in the CA3 region following delay to fixation. The localization of Tau-LI in excitatory neurons such as granule cells and pyramidal cells is not consistent with its proposed inhibitory transmitter role. However, the prominent Tau2 staining in dendrites of the CA1 region provides anatomical support for the hypothesis that taurine may be released from dendrites in the CA1 region and may function as a neuromodulator of calcium flux in these pyramidal neurons.

Electron microscopic localization of acetylcholinesterase in the dentate gyrus of young and adult rats

Acetylcholinesterase (AChE) histochemical staining occurred in neurons of the dentate gyrus at the day of birth and steadily increased in intensity and distribution during the first 3 postnatal weeks until the adult pattern was reached. Granule cells failed to display AChE staining; however, the somata of most non-principal cells in these regions showed AChE activity. It is interesting that most hilar neurons in the dentate gyrus were AChE-positive, but molecular layer local circuit neurons and pyramidal basket cells associated with the granule cell layer did not display AChE staining. AChE reaction product was localized to the nuclear envelope and cisternae of the granular endoplasmic reticulum in the labeled neuronal somata. In addition, the neuropil in the dentate gyrus displayed AChE staining associated with membranes. The possible cholinoceptive role of the AChE somata in the hilus is discussed.

Taurine in hippocampus: Localization and postsynaptic action

Both immunocytochemical and electrophysiological methods have been employed to determine whether the localization of the taurine synthetic enzyme, cysteine sulfinic acid decar☐ylase, (CSAD) and the postsynaptic action of taurine in the CA1 region of rat hippocampus are consistent with the hypothesis that taurine may be used as a neurotransmitter by some hippocampal neurons. At the light microscopic level, CSAD-immunoreactivity (CSAD-IR) was found in the pyramidal basket cells, and around pyramidal cells in stratum pyramidale and stratum radiatum. At the electron microscopic level, CSAD-IR was seen most often in the soma and the dendrites and was rather infrequent in the axon or the nerve terminals. Electrophysiological observations on the in vitro hippocampal slice demonstrated that pyramidal neurons respond to artifically applied taurine with inhibition that depended in large part upon an increased chloride conductance. Although electrophysiological observations are consistent with a neurotransmitter role for taurine, results from immunocytochemical studies suggest a minor role for taurine as a neurotransmitter. In fact, immunocytochemical observations suggested that taurine may be used as a neurotransmitter only by a small number of pyramidal basket interneurons, the vast majority of CSAD-positive neurons may use taurine for other functions.

Various types of non-pyramidal hippocampal neurons project to the septum and contralateral hippocampus

The morphology was studied of hippocampal neurons which had their somata in the hilus of the area dentata, and in stratum radiatum or stratum oriens of Ammon's horn, and which sent projections to the septum and contralateral hippocampus, respectively. The fluorescent marker Fast Blue was injected into the septum or contralateral hippocampus. Somata were then identified by their fluorescent label in slices of perfused brains. After intracellular injection of these somata with Lucifer Yellow, it was found that contralaterally projecting neurons were pyramidal cells, inverted fusiform and multipolar cells in CA3c, and stellate, fusiform and multipolar cells in the hilus. After septal injections, we identified two groups of aspiny stellate cells in the hilus; pyramidal basket cells, polygonal basket cells, horizontal basket cells in stratum oriens; and stellate cells in stratum radiatum of CA1 and CA3, as well as pyramid-like aspiny cells in stratum radiatum of CA1. These cells also had short locally arborizing axons, thus probably contributing to local circuits. Such cells may constitute a third class of hippocampal neurons combining the properties of principal cells and interneurons. These results support the opinion that the simple concept of separating hippocampal cells into projection neurons and local-circuit neurons needs reconsideration.

Neuropeptide Y innervation of the hippocampal region in the rat and monkey brain.

Using antibodies to neuropeptide Y (NPY) in combination with immunohistochemical techniques we have studied the distribution of cell bodies and nerve terminals containing NPY immunoreactivity (-i) in the hippocampal region of rats and monkeys (cynomolgus). In colchicine-pretreated rats a large number of NPY-positive cells are present in all areas of the hippocampal region. The NPY-i cells range in size from small (diameter across soma: 10-15 micron) to large (approximately 20 micron). Most of the NPY-i cells are situated in the hilus, in the subgranular zone of the area dentata, and in the stratum oriens of Ammon's horn. A majority of these are polymorphic cells but cells of different morphology are present in these layers as well. These include small spheroid cells and dentate pyramidal basket cells that are distinct from the polymorphic cells in the subgranular zone. The subicular complex (e.g., the subiculum, pre-, and parasubiculum) and the entorhinal area contain fewer NPY-i cells than the rest of the hippocampal region. In the dorsal parts of the pre- and parasubiculum numerous small cells are scattered throughout all layers, while in the entorhinal area the NPY-stained cells are situated primarily in the deep layers (V and VI). In the ventral part of the lateral entorhinal area large multipolar and bitufted cells are found in layers II-VI. In the untreated monkey brain NPY-positive cells are found in the hilus of the area dentata and in the deep (IV through VI) layers of both the medial and lateral entorhinal area. Fewer NPY-stained cells are present in the subicular complex and in the entorhinal area. In the monkey as well as in the rat, NPY-stained cells are present in the angular bundle and in the alveus. A dense network of NPY-i fibers innervates the entire hippocampal region in both the rat and the monkey. The hippocampal NPY-i preterminal processes are present primarily in stratum moleculare of Ammon's horn and in the outer one-third of this layer in the area dentata. The NPY-positive innervation of the dentate molecular layer is far more prominent in the monkey than in the rat brain. Numerous NPY-stained fibers are scattered in other areas as well. In all retrohippocampal structures, and in particular the entorhinal area, the NPY-i fibers form a massive network that innervates all layers to about the same extent, with the exception of the molecular layer, which is more densely innervated than the other layers.

Mossy fibres form synapses with identified pyramidal basket cells in the CA3 region of the guinea-pig hippocampus: a combined Golgi-electron microscope study

Mossy fibres, i.e. the axons of dentate granule cells, terminate with characteristic giant boutons on large spines or excrescences of the pyramidal cells in regio inferior of the hippocampus. In addition to pyramidal cells there are several types of non-pyramidal neuron which extend their dendrites into the termination zone of mossy fibres. By using the combined Golgi-electron microscope technique mossy fibre terminals were found, which established asymmetric synaptic contacts with both spines of pyramidal cells and smooth dendrites of identified (Golgi-stained) pyramidal basket cells in the CA3 region of the guinea-pig hippocampus. The observed synaptic connection with pyramidal basket cells suggests an involvement of the mossy fibre system in feed-forward inhibition of the hippocampal pyramidal neurons.

Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus.

Identified interneurons in the CA1 region of guinea pig hippocampus were examined using light and electron microscopic (EM) techniques. The HRP was intracellularly injected into cells, recorded in the in vitro slice preparation, which met physiological criteria for interneurons. These neurons were characterized at the light and electron microscopic level, and used as standards for identifying interneurons which had not been HRP-labeled. Pyramidal basket cell somata were found in stratum oriens and stratum pyramidale; they were easily identifiable by their convoluted nucleus, dense endoplasmic reticulum, and numerous organelles. Aspinous dendrites reached into stratum oriens, where they received profuse synaptic input (primarily asymmetric synapses). Many of the asymmetric synapses degenerated following commissural lesions, suggesting that much of the input to interneurons was from extrinsic afferents. Dendrites were characterized by their spindled appearance, especially at distal sites. They showed postsynaptic degenerative changes following commissural lesions. Interneuron axons were extremely fine, with regular enlargements or "beads" which made apparent synaptic contacts primarily on pyramidal cell somata. The axon of a single, HRP-injected interneuron made many apparent contacts on large numbers of pyramidal cells; axons arborized over distances of several hundred micra within stratum pyramidale. This study provides direct evidence that neurons, with an identified inhibitory interneuron function in hippocampus, can mediate feed-forward as well as feed-back (recurrent) inhibition. Interneuron output showed extreme divergence, with influence over large distances. The high density of intracellular organelles in these cells suggested high metabolic activity and demand, perhaps making these interneurons exceptionally vulnerable to trauma-induced damage.