Double Bouquet Cells Research Articles

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

Differential effects of group III metabotropic glutamate receptors on spontaneous inhibitory synaptic currents in spine-innervating double bouquet and parvalbumin-expressing dendrite-targeting GABAergic interneurons in human neocortex.

Diverse neocortical GABAergic neurons specialize in synaptic targeting and their effects are modulated by presynaptic metabotropic glutamate receptors (mGluRs) suppressing neurotransmitter release in rodents, but their effects in human neocortex are unknown. We tested whether activation of group III mGluRs by L-AP4 changes GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in 2 distinct dendritic spine-innervating GABAergic interneurons recorded in vitro in human neocortex. Calbindin-positive double bouquet cells (DBCs) had columnar "horsetail" axons descending through layers II-V innervating dendritic spines (48%) and shafts, but not somata of pyramidal and nonpyramidal neurons. Parvalbumin-expressing dendrite-targeting cell (PV-DTC) axons extended in all directions innervating dendritic spines (22%), shafts (65%), and somata (13%). As measured, 20% of GABAergic neuropil synapses innervate spines, hence DBCs, but not PV-DTCs, preferentially select spine targets. Group III mGluR activation paradoxically increased the frequency of sIPSCs in DBCs (to median 137% of baseline) but suppressed it in PV-DTCs (median 92%), leaving the amplitude unchanged. The facilitation of sIPSCs in DBCs may result from their unique GABAergic input being disinhibited via network effect. We conclude that dendritic spines receive specialized, diverse GABAergic inputs, and group III mGluRs differentially regulate GABAergic synaptic transmission to distinct GABAergic cell types in human cortex.

Preferential inputs from cholecystokinin-positive neurons to the somatic compartment of parvalbumin-expressing neurons in the mouse primary somatosensory cortex

Parvalbumin-positive (PV+) neurons in the cerebral cortex, mostly corresponding to fast-spiking basket cells, have been implicated in higher-order brain functions and psychiatric disorders. We previously demonstrated that the somatic compartment of PV+ neurons received inhibitory inputs mainly from vasoactive intestinal polypeptide (VIP)+ neurons, whereas inhibitory inputs to the dendritic compartment were derived mostly from PV+ and somatostatin (SOM)+ neurons. However, a substantial number of the axosomatic inputs have remained unidentified. Here we show preferential innervation of the somatic compartment of PV+ neurons by cholecystokinin (CCK)+ neurons in the mouse primary somatosensory cortex. CCK+ neurons, a minor population of GABAergic neurons (3.2%), displayed no colocalization with PV or SOM immunoreactivity but partial overlap with VIP immunoreactivity (27.7%). Confocal laser scanning microscopy observation of CCK+ synaptic inputs to PV+ neurons revealed that CCK+ neurons preferred the somatic compartment to the dendritic compartment of PV+ neurons and provided approximately 33% of the axosomatic inhibitory inputs to PV+ neurons. Additionally, 20.9% and 12.1% of the axosomatic inputs were derived from CCK+/VIP+ and CCK+/VIP-negative (−) neurons, presumably double bouquet and large basket cells, respectively. Furthermore, the densities of the axosomatic inputs from CCK+ and/or VIP+ neurons to PV+ neurons were not significantly different among the cortical layers. The present findings suggest that, by preferentially innervating the cell bodies of PV+ neurons, both CCK+/VIP− basket and CCK+/VIP+ double bouquet cells might efficiently interfere with action potential generation of PV+ neurons, and that the two types of CCK+ neurons might have a large impact on cortical activity through PV+ neuron inhibition.

Small-Scale Module of the Rat Granular Retrosplenial Cortex: An Example of the Minicolumn-Like Structure of the Cerebral Cortex

Structures associated with the small-scale module called “minicolumn” can be observed frequently in the cerebral cortex. However, the description of functional characteristics remains obscure. A significant confounding factor is the marked variability both in the definition of a minicolumn and in the diagnostic markers for identifying a minicolumn (see for review, Jones, 2000; DeFelipe et al., 2002; Rockland and Ichinohe, 2004). Within a minicolumn, cell columns are easily visualized by conventional Nissl staining. Dendritic bundles were first discovered with Golgi methods, but are more easily seen with microtubule-associated protein 2 immunohistochemistry. Myelinated axon bundles can be seen by Tau immunohistochemistry or myelin staining. Axon bundles of double bouquet cell can be seen by calbindin immunohistochemistry. The spatial interrelationship among these morphological elements is more complex than expected and is neither clear nor unanimously agreed upon. In this review, I would like to focus first on the minicolumnar structure found in layers 1 and 2 of the rat granular retrosplenial cortex. This modular structure was first discovered as a combination of prominent apical dendritic bundles from layer 2 pyramidal neurons and spatially matched thalamocortical patchy inputs (Wyss et al., 1990). Further examination showed more intricate components of this modular structure, which will be reviewed in this paper. Second, the postnatal development of this structure and potential molecular players for its formation will be reviewed. Thirdly, I will discuss how this modular organization is transformed in mutant rodents with a disorganized layer structure in the cerebral cortex (i.e., reeler mouse and shaking rat Kawasaki). Lastly, the potential significance of this type of module will be discussed.

Enhanced Ras activity preserves dendritic size and extension as well as synaptic contacts of neurons after functional deprivation in synRas mice

The monomeric GTP-binding protein p21Ras has been repeatedly implicated in neuronal stability and plastic changes of the adult nervous system. Recently, we have shown that expression of constitutively active Ras protein in transgenic synRas mice results in a significant increase in the dendritic size and complexity of differentiated pyramidal neurons as well as in increased synaptic connectivity. In the present study, we examined the organization of the vibrissae-barrel cortex in synRas mice and the effects of enhanced Ras activity on deprivation-induced dendritic reorganization after vibrissectomy. The results demonstrate a significant increase in vibrissae-barrel sizes and proportional spacing between barrels in synRas mice, suggesting that the neuronal target specificity of thalamocortical terminals is preserved. Accordingly, the arrangement of double bouquet cells at the borders of barrel columns ensuring functional distinctness is unchanged. Partial vibrissectomy is followed by significant dendritic regression of corresponding pyramidal neurons in the barrel cortex of wild-type mice, which, however, could not be observed in synRas mice. The results provide the first evidence for a role of Ras in preserving neuronal structure after functional deprivation in vivo.

Adrenomedullin Expression is Up‐regulated by Acute Hypobaric Hypoxia in the Cerebral Cortex of the Adult Rat

Hypobaric hypoxia can produce neuropsychological disorders such as insomnia, dizziness, memory deficiencies, headache and nausea. Here we report the changes in adrenomedullin (AM) expression observed in rats exposed to hypobaric hypoxia and different times of reoxygenation. AM immunoreactivity was transiently elevated in the cerebral cortex after 7 h of exposure to a simulated altitude of 8325 m (27 000 ft). This higher expression was seen in all pyramidal cells and in a subset of small interneurons. AM-positive nonpyramidal neurons contained also calbindin and calretinin, but no parvalbumin immunoreactivity, thus identifying them as bipolar and double bouquet cells. Small blood vessels and related astroglia also became immunoreactive following the hypobaric insult. AM up-regulation decreased progressively with the time of reoxygenation, reaching almost control levels after 5 days. Real-time PCR quantification of AM mRNA and Western blotting confirmed the up-regulation of AM expression following hypobaria. In addition, hypobaria modulates alternative splicing of the AM gene resulting in a higher production of AM. Our data show that AM expression regulation constitutes a cortical response to hypobaria, suggesting that AM modulation may provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaria.

Reduced density of calbindin-immunoreactive interneurons in the planum temporale in schizophrenia

Reduced density of calbindin-containing interneurons in the prefrontal cortex in schizophrenia has been reported (Beasley et al 2002; Biol Psych 52:708–715). Calbindin is a calcium-binding protein (CBP) present in a subpopulation of GABAergic neurons restricted mainly to layer II of the cortex. A paraffin-embedded, 10-μm-thick section from the planum temporale (PT) of each hemisphere was prepared from 12 patients with schizophrenia and 12 controls. Calbindin-containing cells were stained using an antibody (D-28K). Counting frames were superimposed to sample within layer II of the PT. A bilateral reduction (20%) in calbindin cell density was found in patients (controlling for fixation time). Furthermore, mean calbindin cell cross-sectional area was increased in female patients and reduced in male patients. Reduced CBP expression (reducing the excitability of interneurons) or reduced number of CBP-containing cells may cause disinhibition of pyramidal cells. The majority of calbindin-containing cells in the mature brain are double-bouquet cells with vertically oriented dendrites and axon bundles. By exercising inhibitory modulation of pyramidal cells in a columnar arrangement, they make possible cohesive vertical inhibition of minicolumns. Loss of columnar inhibition may result in reduced minicolumnar segregation and altered cell size may reflect altered minicolumn size.

Double bouquet cell in the human cerebral cortex and a comparison with other mammals

Double bouquet cells (DBCs) are neocortical gamma-aminobutyric acid (GABA)ergic interneurons characterized by the vertical bundling of its axon, which are generally termed "bundles" or "horse-tails." Using immunocytochemistry for the calcium binding protein calbindin, we have analyzed the morphology, density, and distribution of DBC horse-tails in different cortical areas of the human cortex (Brodmann's areas 10, 4, 3b, 22, 18, and 17). Although DBC horse-tails were very numerous and regularly distributed in all cortical areas, variations were observed both in terms of morphology and density. We distinguished two major classes of DBC horse-tails: the thicker complex type (type I) that had more axon collaterals; and the simple type (type II). The density of DBC horse-tails was significantly higher in areas 17, 18, 22, and 4 than in areas 3b and 10. Moreover, the proportion of type I and type II DBC horse-tails varied in the cortical areas studied. We also examined the distribution of DBC horse-tails in frontal, parietal, and occipital areas of different mammalian species. We found DBCs to be present in carnivores but not in rodents, lagomorphs, or artiodactyls. In carnivores, relatively few DBC horse-tails can be identified and they were generally found in the occipital cortex. Therefore, there is significant variability in the morphology and distribution of DBC horse-tails in different species and cortical areas. We conclude that, although these interneurons may be an important element in the organization of cortical microcolumns in primates, this is not the case in other mammalian species.

Localization of Calcium-binding Proteins in Physiologically and Morphologically Characterized Interneurons of Monkey Dorsolateral Prefrontal Cortex

In the primate neocortex, little is known about the possible associations between functional subclasses of GABA neurons, their morphological properties and calcium-binding protein (CaBP) content. We used whole-cell current clamp recordings, combined with intracellular labeling and fluorescence immunohistochemistry, to determine these relationships for interneurons in layers 2-3 of monkey prefrontal cortex (PFC). Eighty-one interneurons were included in the analysis. Thirty-eight of these cells showed immunoreactivity for one of the three CaBPs tested. Co-localization of more than one CaBP was not observed in any of the interneurons examined. Interneurons with different CaBPs formed distinct populations with specific physiological membrane properties and morphological features. Parvalbumin (PV)-positive cells had the physiological properties characteristic of fast-spiking interneurons (FS) and the morphology of basket or chandelier neurons. Most calretinin (CR)-containing cells had the physiological properties ascribed to non-fast-spiking cells (non-FS) and a vertically oriented axonal morphology, similar to that of double bouquet cells. Calbindin (CB)-positive interneurons also had non-FS properties and included cells with double bouquet morphology or with a characteristic dense web of axonal collaterals in layer 1. Classification of the interneurons based on cluster analysis of multiple electrophysiological properties suggested the existence of at least two distinct groups of interneurons. The first group contained mainly PV-positive FS cells and the second group consisted predominantly of CR- and CB-positive non-FS interneurons. These findings may help to illuminate the functional roles of different groups of interneurons in primate PFC circuitry.

Chandelier cells control excessive cortical excitation: characteristics of whisker-evoked synaptic responses of layer 2/3 nonpyramidal and pyramidal neurons.

Chandelier cells form inhibitory axo-axonic synapses on pyramidal neurons with their characteristic candlestick-like axonal terminals. The functional role of chandelier cells is still unclear, although the preferential loss of this cell type at epileptic loci suggests a role in epilepsy. Here we report an examination of whisker- and spontaneous activity-evoked responses in chandelier cells and other fast-spiking nonpyramidal neurons and regular-spiking pyramidal neurons in layer 2/3 of the barrel cortex. Fast-spiking nonpyramidal neurons, including chandelier cells, basket cells, neurogliaform cells, double bouquet cells, net basket cells, bitufted cells, and regular-spiking pyramidal neurons all respond to stimulation of multiple whiskers on the contralateral face. Whisker stimulation, however, evokes small, delayed EPSPs preceded by an earlier IPSP and no action potentials in chandelier cells, different from other nonpyramidal and pyramidal neurons. In addition, chandelier cells display a larger receptive field with lower acuity than other fast-spiking nonpyramidal neurons and pyramidal neurons. Notably, simultaneous dual whole-cell in vivo recordings show that chandelier cells, which rarely fire action potentials spontaneously, fire more robustly than other types of cortical neurons when the overall cortical excitation increases. Thus, chandelier cells may not process fast ascending sensory information but instead may be reserved to prevent excessive excitatory activity in neuronal networks.

Cholecystokinin-immunopositive basket and Schaffer collateral-associated interneurones target different domains of pyramidal cells in the CA1 area of the rat hippocampus

Two types of GABAergic interneurone are known to express cholecystokinin-related peptides in the isocortex: basket cells, which preferentially innervate the somata and proximal dendrites of pyramidal cells; and double bouquet cells, which innervate distal dendrites and dendritic spines. In the hippocampus, cholecystokinin immunoreactivity has only been reported in basket cells. However, at least eight distinct GABAergic interneurone types terminate in the dendritic domain of CA1 pyramidal cells, some of them with as yet undetermined neurochemical characteristics. In order to establish whether more than one population of cholecystokinin-expressing interneurone exist in the hippocampus, we have performed whole-cell current clamp recordings from interneurones located in the stratum radiatum of the hippocampal CA1 region of developing rats. Recorded neurones were filled with biocytin to reveal their axonal targets, and were tested for the presence of pro-cholecystokinin immunoreactivity. The results show that two populations of cholecystokinin-immunoreactive interneurones exist in the CA1 area ( n=15 positive cells). Cholecystokinin-positive basket cells (53%) preferentially innervate stratum pyramidale and adjacent strata oriens and radiatum. A second population of cholecystokinin-positive cells, previously described as Schaffer collateral-associated interneurones [Vida et al. (1998) J. Physiol. 506, 755–773], have axons that ramify almost exclusively in strata radiatum and oriens, overlapping with the Schaffer collateral/commissural pathway originating from CA3 pyramidal cells. Two of seven of the Schaffer collateral-associated cells were also immunopositive for calbindin. Soma position and orientation in stratum radiatum, the number and orientation of dendrites, and the passive and active membrane properties of the two cell populations are only slightly different. In addition, in stratum radiatum and its border with lacunosum of perfusion-fixed hippocampi, 31.6±3.8% (adult) or 26.8±2.9% (postnatal day 17–20) of cholecystokinin-positive cells were also immunoreactive for calbindin. Therefore, at least two populations of pro-cholecystokinin-immunopositive interneurones, basket and Schaffer collateral-associated cells, exist in the CA1 area of the hippocampus, and are probably homologous to cholecystokinin-immunopositive basket and double bouquet cells in the isocortex. It is not known if the GABAergic terminals of double bouquet cells are co-aligned with specific glutamatergic inputs. However, in the hippocampal CA1 area, it is clear that the terminals of Schaffer collateral-associated cells are co-stratified with the glutamatergic input from the CA3 area, with as yet unknown functional consequences. The division of the postsynaptic neuronal surface by two classes of GABAergic cell expressing cholecystokinin in both the hippocampus and isocortex provides further evidence for the uniform synaptic organisation of the cerebral cortex.

Distribution and patterns of connectivity of interneurons containing calbindin, calretinin, and parvalbumin in visual areas of the occipital and temporal lobes of the macaque monkey

Immunocytochemical techniques were used to examine the distribution of double-bouquet cells and chandelier cells that were immunoreactive (-ir) for the calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV) in the primary visual area (V1), the second visual area (V2), and cytoarchitectonic area TE in the macaque monkey. Furthermore, the connections between CB-, CR-, and PV-ir neurons in these visual areas were investigated at the light microscope level by using a dual-immunocytochemical staining procedure. The most significant findings were three-fold. First, the number and distribution of CB-ir and CR-ir double-bouquet cells and PV-ir chandelier cells differed considerably between different visual areas. In particular, the different distribution of double-bouquet cells was illustrated dramatically at the V1/V2 border, where CB-ir double-bouquet axons were very few or lacking in V1 but were very numerous in V2. Furthermore, PV-ir chandelier cell terminals were relatively sparse in V1, more frequent in V2, and most frequent in area TE. Second, the percentage of CB-, CR-, and PV-ir neurons receiving multiple contacts on their somata and proximal dendrites from other calcium-binding protein neurons varied between 22% and 85%. The highest percentage of contacts found between immunolabelled cells and multiterminals were for the combinations CR/CB (76-85%; percent of cells immunoreactive for CB that were innervated by multiterminals immunoreactive for CR), followed by the combination PV/CR (42-48%), and then by the other combinations that had similar percentages (22-32% for CR/PV; 26-37% for CB/CR; 29-42% for CR/PV). Third, differences in the relative proportions of CB, CR, and PV terminals in contact with CB-, CR-, and PV-ir neurons were consistent between the different cortical areas studied. Thus, certain characteristics of intraareal circuits differ, whereas others remain similar, in different areas of the occipitotemporal visual pathway. The differences may represent regional specializations related to the different processing of visual stimuli, whereas the similarities may be attributed to general functional requisites for interneuronal circuitry.

Differentially Interconnected Networks of GABAergic Interneurons in the Visual Cortex of the Cat

Networks of GABAergic neurons have been implicated in neuronal population synchronization. To define the extent of cellular interconnections, we determined the effect, number, and subcellular distribution of synapses between putative GABAergic neurons in layers II-IV of the cat visual cortex using paired intracellular recordings in vitro followed by correlated light and electron microscopy. All neurons having interneuronal electrophysiological properties were classified by their postsynaptic target profile and were identified as basket (BC; n = 6), dendrite-targeting (DTC; n = 1), and double bouquet (DBC; n = 2) cells. In four out of five anatomically fully recovered and reconstructed cell pairs, synaptic connections were found to be reciprocal. Generally BCs established synaptic junctions closer (21 +/- 20 micron) to postsynaptic somata than did DBCs (43 +/- 19 micron; p < 0.01). The unitary number of synapses (n values, 10, 7, and 20) in each of three BC-to-BC pairs was higher than that in three BC-to-DBC (n values, 1, 2, and 2) and three DBC-to-BC (n values, 1, 4, and 4) connections (p < 0.05). A BC innervated a DTC through two synaptic junctions. Unitary postsynaptic effects mediated by five BCs could be recorded in two BCs, two DBCs, and a DTC. The BCs elicited short-duration fast IPSPs, similar to those mediated by GABAA receptors. At a membrane potential of -55.0 +/- 6.4 mV, unitary IPSPs (n = 5) had a mean amplitude of 919 +/- 863 microV. Postsynaptic response failures were absent when an IPSP was mediated by several release sites. Thus, distinct GABAergic interneurons form reciprocally interconnected networks. The strength of innervation and the proximal placement of synapses suggest a prominent role for BCs in governing the activity of intracortical GABAergic networks in layers II-IV.

Double bouquet cell axons in the human temporal neocortex: relationship to bundles of myelinated axons and colocalization of calretinin and calbindin D-28k immunoreactivities

We have examined the distribution of double bouquet cell axons, immunocytochemically stained for the calcium-binding proteins calretinin and calbindin D-28k in the human temporal neocortex, in relation to bundles of myelinated axons (originating from pyramidal cells) and the colocalization of these calcium-binding proteins. The large number and regularity of distribution of double bouquet cell axons was clearly visualized in tangential Sections from cortical layers III–V. In these sections, we estimated that the mean number±standard deviation of double bouquet cell axons per 10 000 μm 2 was 11.65±0.44 with a mean diameter of 12.10±0.63 μm and a mean center-to-center spacing of 29.8±0.91 μm. These values are very similar to those previously reported in the monkey neocortex. The distribution of double bouquet cell axons was closely related to bundles of myelinated axons; there was overlapping with basically a one-to-one correspondence. Finally, double-label immunofluorescence experiments revealed that the vast majority of double bouquet cell axons immunoreactive for calbindin were also stained for calretinin. Since relatively few cell somata were double-labeled in the human temporal cortex, we concluded that double bouquet cells may represent a significant subpopulation of neurons that colocalize these calcium-binding proteins.

GABAergic cell subtypes and their synaptic connections in rat frontal cortex.

Physiological, morphological and immunohistochemical characteristics of non-pyramidal cells in frontal cortex of young rats were studied in vitro by whole-cell recording and biocytin injection. Several groups of GABAergic non-pyramidal cells were identified: (i) parvalbumin fast-spiking (FS) cells with low input resistances and spikes of short duration, including extended plexus (basket) cells and chandelier cells. These cells showed abrupt episodes of non-adapting repetitive discharges; (ii) late-spiking (LS) cells exhibiting slowly developing ramp depolarizations, including neurogliaform cells; (iii) the remaining groups contained both burst-spiking (BS) or regular-spiking (RS) non-pyramidal (NP) cells. BSNP cells exhibited bursting activity (two or more spikes on slow depolarizing humps) from hyperpolarized potentials. Both these physiological types corresponded to a range of morphologies: (i) somatostatin-containing Martinotti cells with ascending axonal arbors to layer I (some were also positive for calbindin D28k); (ii) VIP-containing double bouquet cells with descending axonal arbors as well as arcade cells (these included small cells immunoreactive for CCK or calretinin). Each subtype of cells made GABAergic synapses onto relatively specific portions of cortical cells, but similar domains were innervated by multiple classes of GABA cells.

Massive Autaptic Self-Innervation of GABAergic Neurons in Cat Visual Cortex

Autapses are transmitter release sites made by the axon of a neuron on its own dendrites. We determined the numbers and precise subcellular position of autapses on different spiny and smooth dendritic cell types using intracellular biocytin filling in slices of adult neocortex. Potential self-innervation was light microscopically assessed on 10 pyramidal cells, 7 spiny stellate cells, and 41 smooth dendritic neurons from cortical layers II-V. Putative autapses occurred on each smooth dendritic neuron and on seven pyramids, but not on spiny stellate cells. However, electron microscopic examination of all light microscopically predicted sites on pyramids (n = 28) showed only one case of self-innervation with two autapses on dendritic spines. Interneurons were classified by postsynaptic target distribution () and all putative autapses of seven basket, three dendrite-targeting, and three double bouquet cells were scrutinized. All basket and dendrite-targeting cells established self-innervation, the number of autapses being 12 +/- 7 and 22 +/- 12 (mean +/- SD), respectively; only one of the double bouquet cells formed autapses (n = 3). Basket cell autapses (n = 74) were closer to the soma (12.2 +/- 22.3 microm) than autapses established by dendrite-targeting cells (51.8 +/- 49.9 microm; n = 66). The degree of self-innervation is cell type-specific. Unlike on spiny cells, autapses are abundant on GABAergic basket and dendrite-targeting interneurons, with subcellular location similar to that of synapses formed by the parent cell on other neurons. The extensive self-innervation may modulate integrative properties and/or the firing rhythm of the neuron in a manner temporally correlated with its own activity.

Fast IPSPs elicited via multiple synaptic release sites by different types of GABAergic neurone in the cat visual cortex.

1. The effects of synapses established by smooth dendritic neurones on pyramidal and spiny stellate cells were studied in areas 17 and 18 of the cat visual cortex in vitro. Paired intracellular recordings with biocytin-filled electrodes and subsequent light and electron microscopic analysis were used to determine the sites of synaptic interaction. 2. All smooth dendritic cells established type II synapses previously shown to be made by terminals containing GABA, therefore the studied cells are probably GABAergic. Three classes of presynaptic cell could be defined, based on their efferent synaptic target preference determined from random samples of unlabelled postsynaptic cells. (a) Basket cells (n = 6) innervated mainly somata (49.9 +/- 13.8%) and dendritic shafts (45.2 +/- 10.7%) and, to a lesser extent, dendritic spines (4.9 +/- 4.6%). (b) Dendrite-targeting cells (n = 5) established synapses predominantly on dendritic shafts (84.3 +/- 9.4%) and less frequently on dendritic spines (11.2 +/- 6.7%) or somata (4.5 +/- 4.7%). (c) Double bouquet cells (n = 4) preferred dendritic spines (69.2 +/- 4.2%) to dendritic shafts (30.8 +/- 4.2%) as postsynaptic targets and avoided somata. 3. Interneurones formed 5240 +/- 1600 (range, 2830-9690) synaptic junctions in the slices. Based on the density of synapses made by single interneurones and the volume density of GABAergic synapses, it was calculated that an average interneurone provides 0.66 +/- 0.20% of the GABAergic synapses in its axonal field. 4. The location of synaptic junctions on individual, identified postsynaptic cells reflected the overall postsynaptic target distribution of the same GABAergic neurone. The number of synaptic junctions between pairs of neurones could not be predicted from light microscopic examination. The number of electron microscopically verified synaptic sites was generally smaller for the dendritic domain and larger for the somatic domain than expected from light microscopy. All presynaptic cells established multiple synaptic junctions on their postsynaptic target cells. A basket cell innervated a pyramidal cell via fifteen release sites; the numbers of synapses formed by three dendrite-targeting cells on pyramidal cells were seventeen and eight respectively, and three on a spiny stellate cell; the interaction between a double bouquet cell and a postsynaptic pyramidal cell was mediated by ten synaptic junctions. 5. All three types of interneurone (n = 6; 2 for each type of cell) elicited short-latency IPSPs with fast rise time (10-90%; 2.59 +/- 1.02 ms) and short duration (at half-amplitude, 15.82 +/- 5.24 ms), similar to those mediated by GABAA receptors. 6. Average amplitudes of unitary IPSPs (n = 6) were 845 +/- 796 microV (range, 134-2265 microV). Variability of IPSP amplitude was moderate, the average ratio of IPSP and baseline noise variance was 1.54 +/- 0.96. High frequency activation of single presynaptic dendrite-targeting cells led to an initial summation followed by use-dependent depression of the averaged postsynaptic response. Double bouquet cell-evoked IPSPs, recorded in the soma, had a smaller amplitude than those evoked by the other two cell types. In all connections, transmission failures were rare or absent, particularly when mediated by a high number of release sites. 7. The results demonstrate that different types of neocortical GABAergic neurones innervate distinct domains on the surface of their postsynaptic target cells. Nevertheless, all three types of cell studied here elicit fast IPSPs and provide GABAergic input through multiple synaptic release sites with few, if any, failures of transmission.

Effect, number and location of synapses made by single pyramidal cells onto aspiny interneurones of cat visual cortex.

1. Dual intracellular recordings were made from synaptically coupled pyramidal cell-to-interneurone pairs (n = 5) of the cat visual cortex in vitro. Pre- and postsynaptic neurones were labelled with biocytin, followed by correlated light and electron microscopic analysis to determine all sites of synaptic interaction. 2. Pyramidal neurones in layers II-III elicited monosynaptic EPSPs in three distinct classes of smooth dendritic local-circuit neurones, namely basket cells (n = 3), a dendrite-targeting cell (n = 1) and a double bouquet cell (n = 1). Unitary EPSPs in basket cells were mediated by one, two, and two synaptic junctions, whereas the pyramid-to-dendrite-targeting cell and pyramid-to-double bouquet cell interaction were mediated by five and seven synaptic junctions, respectively. Recurrent synaptic junctions were found on all somato-dendritic compartments, with a tendency to be clustered close to the soma on the double bouquet and dendrite-targeting cells. The latter interneurones were reciprocally connected with pyramidal cells. 3. Unitary EPSPs had an average peak amplitude of 1005 +/- 518 microV, fast rise times (10-90%; 0.67 +/- 0.25 ms) and were of short duration (at half-amplitude, 4.7 +/- 1.0 ms). Their decay was monoexponential (tau = 7.8 +/- 4.3 ms) at hyperpolarized membrane potentials and appeared to be shaped by passive membrane properties (tau = 9.2 +/- 8.5 ms). All parameters of concomitantly recorded spontaneous EPSPs were remarkably similar (mean amplitude, 981 +/- 433 microV; mean rise time, 0.68 +/- 0.18 ms; mean duration, 4.7 +/- 1.7 ms). 4. In all three pyramidal-to-basket cell pairs, closely timed (10-50 ms) pairs of presynaptic action potentials resulted in statistically significant paired-pulse depression, the mean of the averaged second EPSPs being 80 +/- 11% of the averaged conditioning event. The overall degree of paired-pulse modulation was relatively little affected by either the amplitude of the preceding event or the inter-event interval. 5. The probability density function of the peak amplitudes of the unitary EPSPs could be adequately fitted with a quantal model. Without quantal variance, however, the minimum number of components in the model, excluding the failures, exceeded the number of electron microscopically determined synaptic junctions for all five connections. In contrast, incorporating quantal variance gave a minimum number of components which was compatible with the number of synaptic junctions, and which fitted the data equally well as models incorporating additional components but no quantal variance. For this model with quantal variance with the minimum number of components the estimate of the quantal coefficient of variation ranged between 0.33 and 0.46, and the corresponding quantal sizes ranged between 260 and 657 microV. The peak EPSP amplitudes in two of the four connections with more than one synaptic junction could be adequately described by a uniform binomial model for transmitter release. 6. In conclusion, at least three distinct interneurone classes receive local excitatory pyramidal cell input which they relay to different compartments on their postsynaptic target neurones. The reliability of transmission is high, but the fast time course of the EPSPs constrains their temporal summation. Due to the relatively small amplitude of unitary EPSPs several convergent inputs will therefore be required to elicit suprathreshold responses.

The organization of double bouquet cells in monkey striate cortex.

In previous publications we proposed a model of cortical organization in which the pyramidal cells of the cerebral cortex are organized into modules. The modules are centred around the clusters of apical dendrites that originate from the layer 5 pyramidal cells. In monkey striate cortex such modules have an average diameter of 23 microm and the outputs originating from the modules are contained in the vertical bundles of myelinated axons that traverse the deeper layers of the cortex. The present study is concerned with how the double bouquet cells in layer 2/3 of striate cortex relate to these pyramidal cell modules. The double bouquet cells are visualized with an antibody to calbindin, and it has been shown that their vertically oriented axons, or horse tails, are arranged in a regular array, such that there is one horse tail per pyramidal cell module. Within layer 2/3 the double bouquet cell axons run alongside the apical dendritic clusters, while in layer 4C they are closely associated with the myelinated axon bundles. However, the apical dendrites are not the principal targets of the double bouquet cell axons. Most of the neuronal elements post-synaptic to them are the shafts of small dendrites (60%) and dendritic spines, with which they form symmetric synapses. This regular arrangement of the axons of the double-bouquet cells and their relationship to the components of the pyramidal cells modules supports the concept that there are basic, repeating neuronal circuits in the cortex.

Calbindin-D28k-immunoreactive cells and fibres in the human amygdaloid complex

The distribution of calbindin-D28k-immunoreactive cells and fibres in five human amygdalae was analysed from sections that had been stained immunohistochemically with a monoclonal antibody raised against calbindin-D28k. The highest density of calbindin-D28k-positive neurons were found in the anterior cortical, medial, posterior cortical and accessory basal nuclei, in the parvicellular division of the basal nucleus and in the amygdalohippocampal area. The lowest densities of immunopositive neurons were found in the paralaminar nucleus, in the periamygdaloid cortex (PAC1 and PACo) and in some of the intercalated nuclei. The deep nuclei (lateral, basal and accessory basal nuclei) contained a high density of calbindin-D28k-immunoreactive fibres and terminals. The cortical nuclei and the central nucleus were characterized by intense neuropil labelling. Morphologically, a large majority of the calbindin-D28k-immunoreactive neurons were aspiny or sparsely spiny and resembled inhibitory local circuit neurons. A small population of lightly-stained, pyramidal-shaped neurons was also observed. In most of the amygdaloid nuclei, calbindin-D28k-immunoreactive fibres travelled close to each other and formed bundles, which suggests that some of the immunostained neurons were double-bouquet cells. In the paralaminar nucleus, the calbindin-D28k-immunoreactive axons formed tortuous plexus (100–200mm in diameter) that surrounded several unstained somata.This study provides baseline information on the morphology and distribution of calcium-binding protein-containing inhibitory cells and fibres immunoreactive for calbindin-D28k in the human amygdaloid complex. This information can be used in future studies on the pathogenesis of diseases known to damage the amygdala, such as Alzheimer's disease and temporal lobe epilepsy.