"Dormant basket cell" hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat

The "dormant basket cell" hypothesis suggests that postinjury hippocampal network hyperexcitability results from the loss of vulnerable neurons that normally excite insult-resistant inhibitory basket cells. We have reexamined the experimental basis of this hypothesis in light of reports that excitatory hilar mossy cells are not consistently vulnerable and inhibitory basket cells are not consistently seizure resistant. Prolonged afferent stimulation that reliably evoked granule cell discharges always produced extensive hilar neuron degeneration and immediate granule cell disinhibition. Conversely, kainic acid-induced status epilepticus in chronically implanted animals produced similarly extensive hilar cell loss and immediate granule cell disinhibition, but only when granule cells discharged continuously during status epilepticus. In both preparations, electron microscopy revealed degeneration of presynaptic terminals forming asymmetrical synapses in the mossy cell target zone, including some terminating on gamma-aminobutyric acid-immunoreactive elements, but no evidence of axosomatic or axoaxonic degeneration in the adjacent granule cell layer. Although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than basket cell death, because substance P receptor-positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived. These results confirm the inherent vulnerability of dendritically projecting hilar mossy cells and interneurons and the relative resistance of dentate inhibitory basket and chandelier cells that target granule cell somata. The variability of hippocampal cell loss after status epilepticus suggests that altered hippocampal structure and function cannot be assumed to cause the spontaneous seizures that develop in these animals and highlights the importance of confirming hippocampal pathology and pathophysiology in vivo in each case.     

Different output properties of perisomatic region‐targeting interneurons in the basal amygdala

The perisomatic region of principal neurons in cortical regions is innervated by three types of GABAergic interneuron, including parvalbumin‐containing basket cells (PVBCs) and axo‐axonic cells (AACs), as well as cholecystokinin and type 1 cannabinoid receptor‐expressing basket cells (CCK/CB1BCs). These perisomatic inhibitory cell types can also be found in the basal nucleus of the amygdala, however, their output properties are largely unknown. Here, we performed whole‐cell recordings in morphologically identified interneurons in slices prepared from transgenic mice, in which the GABAergic cells could be selectively targeted. Investigating the passive and active membrane properties of interneurons located within the basal amygdala revealed that the three interneuron types have distinct single‐cell properties. For instance, the input resistance, spike rate, accommodation in discharge rate, or after‐hyperpolarization width at the half maximal amplitude separated the three interneuron types. Furthermore, we performed paired recordings from interneurons and principal neurons to uncover the basic features of unitary inhibitory postsynaptic currents (uIPSCs). Although we found no difference in the magnitude of responses measured in the principal neurons, the uIPSCs originating from the distinct interneuron types differed in rise time, failure rate, latency, and short‐term dynamics. Moreover, the asynchronous transmitter release induced by a train of action potentials was typical for the output synapses of CCK/CB1BCs. Our results suggest that, despite the similar uIPSC magnitudes originating from the three perisomatic inhibitory cell types, their distinct release properties together with the marked differences in their spiking characteristics may contribute to accomplish specific functions in amygdala network operation.     

Neural activity and neurotransmission regulate the maturation of the innervation field of cortical GABAergic interneurons in an age-dependent manner

Neural activity guides the patterning of neuron synaptic territory in the developing nervous system. Evidence supporting this hypothesis comes from numerous studies on projection neurons in neuromuscular and visual systems. It is unknown whether the innervation field of GABAergic interneurons, which forms local dense innervations, follows similar rules. Cortical basket cells innervate hundreds of pyramidal cell somata and proximal dendrites. Thanks to this connectivity pattern, they can tightly control neural excitability and synchronization. Here we show that reducing excitation, and thus neurotransmitter release, in mouse cortical single basket cells in slice cultures decreases the number of innervated cells without changing the pattern of perisomatic innervation, both at the peak and after the proliferation phase of perisomatic synapse formation. Conversely, suppressing neurotransmitter release in single basket cells can have completely opposite effects depending on the developmental stage. Our results reveal a remarkably specific and age-dependent role of neural activity and neurotransmission levels in the establishment of the synaptic territory of cortical GABAergic cells.     

Interneurons in the Hippocampal Dentate Gyrus: an In Vivo intracellular Study

Interneurons in the dentate area were characterized physiologically and filled with biocytin in urethane-anaesthetized rats. On the basis of axonal targets the following groups could be distinguished. (i) Large multipolar interneurons with spiny dendrites in the deep hilar region densely innervated the outer molecular layer and contacted both granule cells and parvalbumin-positive neurons (hilar interneuron with perforant pathway-associated axon terminals; HIPP cells). (ii) A pyramidal-shaped neuron with a cell body located in the subgranular layer innervated mostly the inner molecular layer and the granule cell layer (hilar interneuron with commissural-associational pathway-associated axon terminals; HICAP cell). It contacted both granule cells and interneurons. Axon collaterals of HIPP and HICAP neurons covered virtually the entire Septo-temporal extent of the dorsal dentate gyrus. (iii) Calbindin-immunoreactive neurons with horizontal dendrites in stratum oriens of the CA3c region gave rise to a rich axon arbor in strata oriens, pyramidale and radiatum and innervated almost the entire extent of the dorsal hippocampus, with some collaterals entering the subicular area (putative trilaminar cell). (iv) Hilar basket cells innervated mostly the granule cell layer and to some extent the inner molecular layer and the CA3c pyramidal layer. HIPP and trilaminar interneurons could be antidromically activated by stimulation of the fimbria. Only the HICAP cells could be monosynaptically discharged by the perforant path input. All interneurons examined showed phase-locked activity to the extracellularly recorded theta/gamma oscillations or to irregular dentate electroencephalogram spikes. These observations indicate that the interconnected interneuronal system plays a critical role in coordinating population activity of the dentate gyrus and Ammon's horn.     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons

The generation of emotional responses by the basolateral amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons, the pyramidal cells. The activity of these neurons is tightly controlled by gamma-aminobutyric acid (GABA)-ergic interneurons, especially a parvalbumin-positive (PV(+)) subpopulation that constitutes almost half of all interneurons in the basolateral amygdala. In the present semiquantitative investigation, we studied the incidence of synaptic inputs of PV(+) axon terminals onto pyramidal neurons in the rat basolateral nucleus (BLa). Pyramidal cells were identified by using calcium/calmodulin-dependent protein kinase II (CaMK) immunoreactivity as a marker. To appreciate the relative abundance of PV(+) inputs compared with excitatory inputs and other non-PV(+) inhibitory inputs, we also analyzed the proportions of asymmetrical (presumed excitatory) synapses and symmetrical (presumed inhibitory) synapses formed by unlabeled axon terminals targeting pyramidal neurons. The results indicate that the perisomatic region of pyramidal cells is innervated almost entirely by symmetrical synapses, whereas the density of asymmetrical synapses increases as one proceeds from thicker proximal dendritic shafts to thinner distal dendritic shafts. The great majority of synapses with dendritic spines are asymmetrical. PV(+) axon terminals form mainly symmetrical synapses. These PV(+) synapses constitute slightly more than half of the symmetrical synapses formed with each postsynaptic compartment of BLa pyramidal cells. These data indicate that the synaptology of basolateral amygdalar pyramidal cells is remarkably similar to that of cortical pyramidal cells and that PV(+) interneurons provide a robust inhibition of both the perisomatic and the distal dendritic domains of these principal neurons.     

Loss of interneurons innervating pyramidal cell dendrites and axon initial segments in the CA1 region of the hippocampus following pilocarpine-induced seizures

In the pilocarpine model of chronic limbic seizures, vulnerability of GABAergic interneurons to excitotoxic damage has been reported in the hippocampal CA1 region. However, little is known about the specific types of interneurons that degenerate in this region. In order to characterize these interneurons, we performed quantitative analyses of the different populations of GABAergic neurons labeled for their peptide or calcium-binding protein content. Our data demonstrate that the decrease in the number of GAD mRNA-containing neurons in the stratum oriens of CA1 in pilocarpine-treated rats involved two subpopulations of GABAergic interneurons: interneurons labeled for somatostatin only (O-LM and bistratified cells) and interneurons labeled for parvalbumin only (basket and axo-axonic cells). Stratum oriens interneurons labeled for somatostatin/calbindin or somatostatin/parvalbumin were preserved. The decrease in number of somatostatin- and parvalbumin-containing neurons was observed as early as 72 hours after the sustained seizures induced by pilocarpine injection. Many degenerating cell bodies in the stratum oriens and degenerating axon terminals in the stratum lacunosum-moleculare were observed at 1 and 2 weeks after injection. In addition, the synaptic coverage of the axon initial segment of CA1 pyramidal cells was significantly decreased in pilocarpine-treated animals. These results indicate that the loss of somatostatin-containing neurons corresponds preferentially to the degeneration of interneurons with an axon projecting to stratum lacunosum-moleculare (O-LM cells) and suggest that the death of these neurons is mainly responsible for the deficit of dendritic inhibition reported in this region. We demonstrate that the loss of parvalbumin-containing neurons corresponds to the death of axo-axonic cells, suggesting that perisomatic inhibition and mechanisms controlling action potential generation are also impaired in this model.     

Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity

Two neuronal calcium-binding proteins, calbindin-D28k (CaBP) and parvalbumin (PV), were localized in the normal rat hippocampus by using immunocytochemical methods to determine 1) their location and 2) whether a correlation exists between the presence of these two calcium-binding proteins and the selective vulnerability of different hippocampal neuronal populations to experimental seizure activity. CaBP-like immunoreactivity (CaBP-LI) is present in all dentate granule cells and some, but not all, CA1 and CA2 pyramidal cells. Some CA1 pyramidal cells lack CaBP-LI, and those that do are lightly stained compared to the dentate granule cells. CA3 pyramidal cells appear to contain neither CaBP- nor PV-LI, and no granule or pyramidal cells exhibit PV-LI. CaBP-LI is present in distinct populations of dentate and hippocampal interneurons but absent from others. In area dentata, CaBP-LI is present in a small number of interneurons of the molecular and granule cell layers and in a small population of presumed basket cells in or below the granule cell layer. Conversely, more presumed dentate basket cells exhibit PV-LI than CaBP-LI. In the hilus of area dentata, few cells are CaBP- or PV-immunoreactive. The hilar somatostatin/neuropeptide Y (NPY)-immunoreactive cells and hilar mossy cells, two distinct and large populations, lack CaBP- and PV-LI. In the CA3 region, CaBP-LI is present in a relatively small number of interneurons in each stratum. PV-immunoreactive interneurons in area CA3 are more numerous. In area CA1, CaBP-LI is present in many interneurons in strata radiatum and lacunosum-moleculare. Some, but relatively fewer, CaBP-positive interneurons are present in strata pyramidale and oriens. Conversely, PV-immunoreactive interneurons are numerous in strata pyramidale and oriens but rare in strata radiatum and lacunosum-moleculare. Staining with the particulate chromagen benzidine hydrochloride revealed a previously undescribed dense band of CaBP-LI in the inner dentate molecular layer, a lamina enriched with kainate-displaceable glutamate-binding sites and innervated by the apparently excitatory ipsilateral associational/commissural (IAC) pathway that originates in the CaBP-negative hilar mossy cells. Bilateral electrical stimulation of the perforant path was performed in order to destroy the hilar mossy cells and to determine if this band of CaBP-LI is normally present within the mossy cell terminals. Perforant path stimulation that destroyed hilar mossy cells throughout the dorsal portions of both hippocampi did not abolish the dense CaBP-like immunoreactivity in the inner molecular layer.     

Anatomically heterogeneous populations of CB1 cannabinoid receptor‐expressing interneurons in the CA3 region of the hippocampus show homogeneous input–output characteristics

A subpopulation of GABAergic cells in cortical structures expresses CB₁ cannabinoid receptors (CB₁) on their axon terminals. To understand the function of these interneurons in information processing, it is necessary to uncover how they are embedded into neuronal circuits. Therefore, the proportion of GABAergic terminals expressing CB₁ and the morphological and electrophysiological properties of CB₁‐immunoreactive interneurons should be revealed. We investigated the ratio and the origin of CB₁‐expressing inhibitory boutons in the CA3 region of the hippocampus. Using immunocytochemical techniques, we estimated that ～40% of GABAergic axon terminals in different layers of CA3 also expressed CB₁. To identify the inhibitory cell types expressing CB₁ in this region, we recorded and intracellularly labeled interneurons in hippocampal slices. CB₁‐expressing interneurons showed distinct axonal arborization, and were classified as basket cells, mossy‐fiber‐associated cells, dendritic‐layer‐innervating cells or perforant‐path‐associated cells. In each morphological category, a substantial variability in axonal projection was observed. In contrast to the diverse morphology, the active and passive membrane properties were found to be rather similar. Using paired recordings, we found that pyramidal cells displayed large and fast unitary postsynaptic currents in response to activating basket and mossy‐fiber‐associated cells, while they showed slower and smaller synaptic events in pairs originating from interneurons that innervate the dendritic layer, which may be due to dendritic filtering. In addition, CB₁ activation significantly reduced the amplitude of the postsynaptic currents in each cell pair tested. Our data suggest that CB₁‐expressing interneurons with different axonal projections have comparable physiological characteristics, contributing to a similar proportion of GABAergic inputs along the somato‐dendritic axis of CA3 pyramidal cells. © 2014 Wiley Periodicals, Inc.     

Selective destruction of mossy fibers and granule cells with preservation of the GABAergic network in the inferior region of the rat hippocampus after colchicine treatment

Lesions induced by colchicine injection into the rat hippocampus were investigated by means of electron microscopy and GABA immunocytochemistry. Granule cells were nearly completely destroyed 3 days after colchicine injection; since the necrosis of their axonal endings was delayed, an anterograde degeneration of the mossy fibers had probably taken place. The selectivity of the lesions was not limited to granule cells, for some pyramidal neurons in CA1 pyramidal layer were damaged. It was, however, striking to observe that throughout the hippocampal structure GABAergic neurons were spared from the effects of colchicine. For instance, GABAergic neurons were found in the vicinity of the completely destroyed granule cell layer. GABAergic neurons and terminals were also present in the CA3 region where the GABA-containing terminals formed a dense network of synapses with somata and dendrites of pyramidal cells. It was interesting to note that, consistent with previous studies, the GABAergic neurons in CA3 are innervated by mossy fibers. We conclude that after colchicine treatment the destruction of the granule cells was not associated with a lesion of the GABAergic network. This selective lesion provides a useful model with which to study the properties of CA3 neurons deprived of their major excitatory input but with an intact inhibitory network.     

Commissurally projecting inhibitory interneurons of the rat hippocampal dentate gyrus: a colocalization study of neuronal markers and the retrograde tracer Fluoro-gold.

Improved methods for detecting neuronal markers and the retrograde tracer Fluoro-Gold (FG) were used to identify commissurally projecting neurons of the rat hippocampus. In addition to the dentate hilar mossy cells and CA3 pyramidal cells shown previously to transport retrograde tracers after injection into the dorsal hippocampus, FG-positive interneurons of the dentate granule cell layer and hilus were detected in numbers greater than previously reported. FG labeling of interneurons was variable among animals, but was as high as 96% of hilar somatostatin-positive interneurons, 84% of parvalbumin-positive cells of the granule cell layer and hilus combined, and 33% of hilar calretinin-positive cells. By comparison, interneurons of the dentate molecular layer and all hippocampal subregions were conspicuously FG-negative. Whereas hilar mossy cells and CA3 pyramidal cells were FG-labeled throughout the longitudinal axis, FG-positive interneurons exhibited a relatively homotopic distribution. "Control" injections of FG into the neocortex, septum, and ventral hippocampus demonstrated that the homotopic labeling of dentate interneurons was injection site-specific, and that the CA1-CA3 interneurons unlabeled by contralateral hippocampal FG injection were nonetheless able to transport FG from the septum. These data suggest a hippocampal organizing principle according to which virtually all commissurally projecting hippocampal neurons share the property of being monosynaptic targets of dentate granule cells. Because granule cells innervate their exclusively ipsilateral target cells in a highly lamellar pattern, these results suggest that focal granule cell excitation may result in commissural inhibition of the corresponding "twin" granule cell lamella, thereby lateralizing and amplifying the influence of the initiating discharge.     

Network of GABAergic large basket cells in cat visual cortex (area 18): Implication for lateral disinhibition

Anatomical and immunohistochemical data indicate that, in addition to pyramidal neurons, nonpyramidal cells are exposed to perisomatic inhibition mediated by γ-aminobutyric acid (GABA)-containing terminals. However, no direct information is available as yet for the origin of GABAergic inputs to morphologically identified GABAergic neurons. In the present paper, we studied the topographical and synaptic relationship between identified GABAergic large basket cells and their immunohistochemically characterized target neurons revealed by parvalbumin-(PV) and GABA immunostaining in the same material. Extracellularly applied biocytin labelled a total of 36 and 9 large basket cells in layers III and V, respectively. Of these, the axonal arborizations of two basket cells, BC1 and BC2, were reconstructed. The axon of BC1 occupied an area of about 2.3 × 2.2 mm² in layer III, providing a total of 2,755 terminals. The axon of BC2 showed an overall extent of 3.8 × 1.7 mm² in layer V elongated in the anteroposterior direction, and gave off 1,599 terminals. Immunostaining for PV was carried out to reveal putative nonpyramidal targets for BC1 and BC2. It was found that in addition to immunonegative cells, they established an average of 4–6 perisomatic contacts onto each of 58 (BC1) and 33 (BC2) PV-immunopositive neurons. For electron microscopic verification, 23 terminals apposing the somata of 12 PV-immunopositive neurons were selected. Each terminal was found to establish symmetrical (type II) contacts with its targeted cell. Furthermore, the distribution of soma area of the targeted PV-immunopositive cells and of identified large basket cells showed remarkable similarity, implying that the two populations were actually the same. In addition, the average horizontal distance between neighbouring PV-immunopositive target cells was found to be about 100 μm both in layers III and V. The results suggest that in area 18 the same large basket cell provides direct inhibition to certain pyramidal cells and facilitation to other pyramidal neurons, by inhibiting their presynaptic large basket cells at regular intervals. 1993 Wiley-Liss, Inc.     

Synaptic connections of cholecystokinin-immunoreactive neurons and terminals in the rat fascia dentata: a combined light and electron microscopic study

We report here on the fine structure and synaptic connections of neurons and axon terminals in the rat fascia dentata displaying immunoreactivity to antibodies against cholecystokinin octapeptide (CCK). In the fascia dentata and hilar region, CCK-immunoreactivity was confined to nonpyramidal neurons that were similar in appearance to basket cells known to use gamma-aminobutyric acid (GABA) as neurotransmitter. These neurons exhibited dense accumulations of endoplasmic reticulum and infolded nuclei, and established asymmetric and symmetric synaptic contacts with presynaptic terminals. Among those terminals that formed asymmetric synaptic contacts, giant mossy fiber boutons arising from granule cell axons were identified. Cholecystokinin-immunoreactive terminals established symmetric synaptic contacts on the cell bodies and dendrites of granule cells. Similar contacts were formed on nonimmunoreactive hilar neurons. Some of these hilar cells were identified as commissural neurons by retrograde filling with horseradish peroxidase (HRP) following injection of the tracer into the contralateral fascia dentata. Synaptic contacts were rarely observed between immunolabeled pre- and postsynaptic elements. The results are discussed with regard to inhibitory processes in the fascia dentata since other studies have shown that CCK is coexistent with GABA in hippocampal nonpyramidal neurons.     

Precision and Variability in Postsynaptic Target Selection of Inhibitory Cells in the Hippocampal CA3 Region

Non-pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea-pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo-axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritlc region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritlc segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum-moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo-axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo-axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramldale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons–often in conjunction with particular excitatory afferents (Han et at., Eur. J. Neurosci., 5, 395–410, 1993)–underlies functional differences in inhibitory synaptic actions.     

Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule

Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development.     

Distribution of GABAergic Elements Postsynaptic to Ventroposteromedial Thalamic Projections in Layer IV of Rat Barrel Cortex

The spatial synaptic pattern formed by boutons, originating in the ventroposteromedial thalamic nucleus, with GABAergic neurons in the rat barrel cortex was mapped. The aim was to shed light on the structural basis by which inhibitory circuits may be activated at the first stage of cortical information processing. The thalamic afferent projection was labelled by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), whereas the GABAergic targets in layer IV of the rat barrel cortex were visualized by postembedding GABA immunogold-labelling or by pre-embedding parvalbumin immunocytochemistry. In the first set of experiments, we mapped barrels, contained in single ultrathin sections, by means of a computer-controlled electron microscope stage in their entire layer IV representation. From a total of 1199 asymmetric PHA-L-labelled synapses, only 98 were on GABAergic elements, mainly on dendritic shafts. This corresponded to 8.2% of all synapses counted. These synapses on GABAergic targets were essentially homogeneously distributed without a reliable relationship to barrel subdivisions, i.e. hollow versus wall; or layer IVa versus layer IVb. In the second part of the study, we demonstrated that parvalbumin-containing neurons represent the major GABAergic cell type targetted by thalamic afferents in layer IV of the barrel cortex, since all parvalbumin-positive cells investigated received multiple synaptic contacts (up to eight synapses per neuron) from the ventroposteromedial thalamic nucleus. These results imply that interneurons responsible for perisomatic inhibition (basket and chandelier cells known to contain parvalbumin) are likely to be strongly excited by thalamic afferents, despite the relatively low proportion of thalamic synapses on GABAergic elements compared to spines of principal cells, and participate in the early stages of cortical sensory information processing.     

Postnatal development of parvalbumin immunoreactivity in axon terminals of basket and chandelier neurons in monkey neocortex.

1. Two classes of GABAergic inhibitory interneurons, chandelier and basket cells, are known regulators of pyramidal neurons. Parvalbumin (PV) a calcium binding protein, has been shown to be a marker for axon terminals of subpopulations of these interneurons. 2. Immunohistochemical methods were used in this study to examine changes in the distribution of PV-immunoreactive (IR) chandelier and basket axon terminals during postnatal development of monkey neocortex. 3. Our results indicate a differential effect of postnatal development on PV-IR axon terminals of chandelier and basket neurons that is region-specific. 4. The differential regional, laminar and developmental pattern of PV-IR axon terminals of chandelier and basket cells may provide insight into the functional role of these classes of inhibitory neurons in primate neocortex.     

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)     

Recurrent mossy fibers preferentially innervate parvalbumin-immunoreactive interneurons in the granule cell layer of the rat dentate gyrus

Detection of vesicular zinc and immunohistochemistry against markers for different interneuron subsets were combined to study the postsynaptic target selection of zinc-containing recurrent mossy fiber collaterals in the dentate gyrus. Mossy fiber collaterals in the granule cell layer selectively innervated parvalbumin-containing cells, with numerous contacts per cell, whereas the granule cells were avoided. Under the electron microscope, those boutons made asymmetrical contacts on dendrites and somata. These findings suggest that, in addition to the hilar perforant path-associated (HIPP) interneurons, the basket and chandelier cells also receive a powerful feed-back drive from the granule cells, and thereby are able to control population synchrony in the dentate gyrus. On the other hand, the amount of monosynaptic excitatory feed-back among granule cells is shown to be negligible.     

Complete Axon Arborization of a Single CA3 Pyramidal Cell in the Rat Hippocampus, and its Relationship With Postsynaptic Parvalbumin-containing Interneurons

The complete axon arborization of a single CA3 pyramidal cell has been reconstructed from 32 (60 /μm thick) sections from the rat hippocampus following in vivo intracellular injection of neurobiotin. The same sections were double-immunostained for parvalbumin-a calcium-binding protein selectively present in two types of GABAergic interneurons, the basket and chandelier cells-in order to map boutons of the pyramidal cell in contact with dendrites and somata of these specific subsets of interneurons visualized in a Golgi-like manner. The axon of the pyramidal cell formed 15 295 boutons, 63.8% of which were in stratum oriens, 15.4% in stratum pyramidale and 20.8% in stratum radiatum. Only 2.1% of the axon terminals contacted parvalbumin-positive neurons. Most of these were single contacts (84.7%), but double or triple contacts (15.3%) were also found. The majority of the boutons terminated on dendrites (84.1%) of parvalbumin-positive cells, less frequently on cell bodies (15.9%). In order to estimate the proportion of contacts representing synapses, 16 light microscopically identified contacts between boutons of the filled pyramidal cell axon and the parvalbumin-positive targets were examined by correlated electron microscopy. Thirteen of them were found to be asymmetrical synapses, and in the remaining three cases synapses between the labelled profiles could not be confirmed. We conclude that the physiologically effective excitatory connections between single pyramidal cells and postsynaptic inhibitory neurons are mediated by a small number of contacts, mostly by a single synapse. This results in a high degree of convergence and divergence in hippocampal networks.