Large Basket Cells Research Articles

Noradrenergic Excitation and Inhibition of GABAergic Cell Types in Rat Frontal Cortex

Noradrenaline (NA) from the locus coeruleus and GABA from intracortical nonpyramidal cells exert strong influences on cortical activity. To assess possible interaction between the two, the effects of noradrenergic agonists on spontaneous GABAergic IPSCs as well as on the activity of identified GABAergic cell types were investigated by in vitro whole-cell recordings from the frontal cortex of 18- to 22-d-old rats. NA (3-50 microM) and an alpha-adrenergic agonist, 6-fluoronorepinephrine (FNE; 30-50 microM), induced an increase of IPSC frequency in pyramidal cells, but a beta-adrenergic agonist did not. This increase was reduced by tetrodotoxin, bicuculline, and alpha-adrenergic antagonists, suggesting that GABAergic cells are excited via alpha-adrenoceptors. Fast-spiking or late-spiking cells were depolarized by application of NA or FNE, but none demonstrated spike firings. The former morphologically included common multipolar cells with extended axonal arborizations as well as chandelier cells, and the latter neurogliaform cells. Most somatostatin-immunoreactive regular or burst-spiking cells, including Martinotti cells and wide arbor cells, were depolarized and accompanied by spike firing. In a few cases this was preceded by hyperpolarization. Cholecystokinin-immunoreactive regular or burst-spiking nonpyramidal cells, including large basket cells, were affected heterogeneously: depolarization, hyperpolarization followed by depolarization, or hyperpolarization resulted. The findings suggest that, similar to the effects of acetylcholine, the excitability of cortical GABAergic cell types is differentially regulated by NA and that NA actions are similar to cholinergic ones in some GABAergic cell types but not in others.

Evidence for a contribution of lateral inhibition to orientation tuning and direction selectivity in cat visual cortex: reversible inactivation of functionally characterized sites combined with neuroanatomical tracing techniques.

We have previously reported that cells in cat areas 17 and 18 can show increases in response to non-optimal orientations or directions, commensurate with a loss of inhibition, during inactivation of laterally remote, visuotopically corresponding sites by iontophoresis of gamma-aminobutyric acid (GABA). We now present anatomical evidence for inhibitory projections from inactivation sites to recording sites where 'disinhibitory' effects were elicited. We made microinjections of [3H]-nipecotic acid, which selectively exploits the GABA re-uptake mechanism, < 100 microm from recording sites where cells had shown either an increase in response to non-optimal orientations during inactivation of a cross-orientation site (n = 2) or an increase in response to the non-preferred direction during inactivation of an iso-orientation site with opposite direction preference (n = 5). Retrogradely labelled GABAergic neurons were detected autoradiographically and their distribution was reconstructed from series of horizontal sections. In every case, radiolabelled cells were found in the vicinity of the inactivation site (three to six within 150 microm). The injection and inactivation sites were located in layers II/III-IV and their horizontal separation ranged from 400 to 560 microm. In another experiment, iontophoresis of biocytin at an inactivation site in layer III labelled two large basket cells with terminals in close proximity to cross-orientation recording sites in layers II/III where disinhibitory effects on orientation tuning had been elicited. We argue that the inactivation of inhibitory projections from inactivation to recording sites made a major contribution to the observed effects by reducing the strength of inhibition during non-optimal stimulation in recurrently connected excitatory neurons presynaptic to a recorded cell. The results provide further evidence that cortical orientation tuning and direction selectivity are sharpened, respectively, by cross-orientation inhibition and iso-orientation inhibition between cells with opposite direction preferences.

Expression of nerve growth factor and neurotrophin-3 mRNAs in hippocampal interneurons: Morphological characterization, levels of expression, and colocalization of nerve growth factor and neurotrophin-3

We have investigated the distribution and morphology of hippocampal interneurons that express the neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the rat. For this study, we combined in situ hybridization for the detection of NGF and NT-3 mRNAs and immunocytochemistry against the calcium-binding proteins parvalbumin (PARV), calretinin (CALR), and calbindin (CALB). Whereas the majority of PARV+ interneurons expressed NGF mRNA, only subsets of CALR- and CALB-immunoreactive interneurons (23% and 24%, respectively) displayed NGF hybridization. Most CALB/NGF+ cells were located in the stratum oriens/alveus of the CA3-CA1 regions, suggesting that they may include the population of CALB+, hippocamposeptal, nonpyramidal neurons. Most of the nonspiny CALR/NGF+ neurons were located within or in the vicinity of the pyramidal layer and had faint CALR immunostaining and stellate, thin dendrites. Regarding the spiny CALR-immunoreactive cells, we found that most of these neurons in the hilus were NGF+, whereas only 59% of displayed NGF hybridization in the stratum lucidum of the CA3 region. A small subset of PARV- and CALR-immunoreactive cells expressed NT-3 mRNA (16% and 13%, respectively). NT-3 message was not found in the large basket cells of the dentate gyrus, whereas the distribution and morphology of CALR/NT-3+ cells were similar to those of nonspiny CALR/NGF+ cells. In fact, double in situ hybridization analysis confirmed that most NT-3+ neurons also expressed NGF mRNA, indicating coexpression of both neurotrophins in subpopulations of PARV+ and CALR+ neurons. Moreover, the level of NGF mRNA expression was higher in PARV+ neurons than in CALR- and CALB-immunoreactive interneurons, whereas NT-3 message was expressed similarly in PARV+ and CALR+ neurons. The present findings show a differential expression of NGF and NT-3 mRNAs in subsets of hippocampal interneurons and suggest that the expression of these transcripts depends on factors intrinsic to particular cell types.

1507 Two types of large basket cells in rat frontal cortex

Acute ischemic stroke in the distribution of the anterior inferior cerebellar artery (AICA) can cause the vestibular dysfunction in the roll plane of the vestibuloocular reflex with abnormal ocular torsion (OT). There has been no systemic study that carefully investigates the nature of OT that occurs with AICA infarction.To investigate the frequency, the characteristic patterns of OT associated with AICA territory infarction, and the crucial site for determining the direction of OT in AICA territory infarction.We studied 12 consecutive cases of infarction in the territory of the AICA diagnosed by brain MRI. Fundus photography, prism cover test, and subjective visual vertical tilting test were performed to evaluate the function of the otolith system. Pure tone audiogram was also performed to evaluate the function of the auditory system.Nine (75%) of 12 patients exhibited pathological ocular torsion (OT). Two types of pathological OT were found: ipsiversive OT accompanying skew deviation (n = 6), and contraversive OT only (n = 3). Six patients with ipsiversive OT with skew deviation showed an audiovestibular loss with canal paresis and hearing loss ipsilaterally whereas three patients with contraversive OT without skew deviation had a normal audiovestibular response. In all cases with pathological OT, the direction of the subjective visual vertical tilt corresponded to the direction of the OT.Our findings emphasize that the peripheral vestibular structure with inner ear probably plays a crucial role in determining the direction of OT associated with AICA territory infarction.

Two types of large basket cells in rat frontal cortex

Acute ischemic stroke in the distribution of the anterior inferior cerebellar artery (AICA) can cause the vestibular dysfunction in the roll plane of the vestibuloocular reflex with abnormal ocular torsion (OT). There has been no systemic study that carefully investigates the nature of OT that occurs with AICA infarction.To investigate the frequency, the characteristic patterns of OT associated with AICA territory infarction, and the crucial site for determining the direction of OT in AICA territory infarction.We studied 12 consecutive cases of infarction in the territory of the AICA diagnosed by brain MRI. Fundus photography, prism cover test, and subjective visual vertical tilting test were performed to evaluate the function of the otolith system. Pure tone audiogram was also performed to evaluate the function of the auditory system.Nine (75%) of 12 patients exhibited pathological ocular torsion (OT). Two types of pathological OT were found: ipsiversive OT accompanying skew deviation (n = 6), and contraversive OT only (n = 3). Six patients with ipsiversive OT with skew deviation showed an audiovestibular loss with canal paresis and hearing loss ipsilaterally whereas three patients with contraversive OT without skew deviation had a normal audiovestibular response. In all cases with pathological OT, the direction of the subjective visual vertical tilt corresponded to the direction of the OT.Our findings emphasize that the peripheral vestibular structure with inner ear probably plays a crucial role in determining the direction of OT associated with AICA territory infarction.

Functional and Structural Topography of Horizontal Inhibitory Connections in Cat Visual Cortex

The functional organization of long-horizontal inhibitory connections was studied in cat visual cortical area 17, using a combination of electrophysiological recording and anatomical tracing in the same tissue. Orientation maps were obtained by recording multiunit activity from layer III at regular intervals (100-300 microns) in a region of approximately 1.3 mm2 of cortex at a depth corresponding to the location of the basket cell axons reconstructed later. Before the physiological mapping, the neuronal tracer biocytin had been iontophoretically injected at one functionally characterized site. On the basis of light microscopic features a total of five biocytin-labelled large basket axons, BC1-BC5, were reconstructed from series of horizontal sections of two cats. The parent somata and dendritic fields of three axons (BC1, BC4 and BC5) could also be reconstructed. The axonal field of basket cell BC1 had an overall lateral spread of 1.8 mm. The axons of basket cells BC4 and BC5 spanned a distance of 3.05 and 2.85 mm, respectively. The distribution pattern of histologically reconstructed recording sites and of five labelled basket cell axons were directly compared in the same sections. The results show that a single large basket cell provides input to regions representing the whole range of orientations, i.e. iso-orientation (+/- 30 degrees), oblique orientation (+/- [30-60] degrees) and cross-orientation (+/- [60-90] degrees) to that at the basket cell's soma. Furthermore, the differential effect mediated by the same large basket cell at sites of different orientation preference was numerically estimated for two basket cells (BC4 and BC5) whose preferred orientations could be determined on the basis of recording sites adjacent to their parent somata. We counted the number of axonal terminals of these basket cells at iso-, oblique- and cross-orientation sites and found no significant difference in the average density of terminals at sites of either orientation preference. The functional topography of large basket cell axons indicates that the same basket cell can mediate iso-, oblique- and cross-orientation inhibition at different sites. Hence, we assume that large basket cells serve a complex physiological role depending on the location of target cells in the orientation map.

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 gamma-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 x 2.2 mm2 in layer III, providing a total of 2,755 terminals. The axon of BC2 showed an overall extent of 3.8 x 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 microns 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.

Electron microscopic immunocytochemical study of the distribution of parvalbumin‐containing neurons and axon terminals in the primate dentate Gyrus and Ammon's horn

Five green monkeys were examined with light and electron microscopic preparations to explore the regional differences in the distribution of parvalbumin (PV)-positive neurons and axon terminals in the primate hippocampus. PV-positive neurons were mainly found in the hilus of the dentate gyrus and the strata oriens and pyramidale of Ammon's horn. In electron microscopic preparations, the PV-positive cells displayed nuclear infoldings, intranuclear rods, a large rim of perikaryal cytoplasm with numerous organelles and both asymmetric and symmetric axosomatic synapses. One prominent PV-positive cell type in CA1 was a large multipolar neuron that resembled the large basket cells of the neocortex. Although most PV-positive dendrites were aspiny and postsynaptic to numerous axon terminals, some PV-positive dendrites in the molecular layer of the dentate gyrus displayed filipodia-like appendages with no synapses or spines that were postsynaptic to multiple axon terminals. The PV-positive dendrites in the hilus and stratum oriens were apposed at specialized junctions that resembled gap junctions. PV-positive axons were concentrated in the principal cell layers, and formed axosomatic, axodendritic, and axon initial segment synapses. In cases where these axons were observed to appose the surface of granule cells for a long length, only one axosomatic symmetric synapse per cell was found. In the hilus, PV-positive axon terminals formed synapses onto thorny excrescences of spiny cells. Both semithin sections and electron microscopic preparations indicated that more PV-positive axon terminals formed symmetric axosomatic synapses with pyramidal cells in CA2 than in CA1 and CA3. Also, CA2 displayed a unique plexus of PV-positive axon terminals in stratum lacunosum moleculare. These results indicate that the PV-positive hippocampal cells form a subset of GABAergic local circuit neurons, including the basket and chandelier cells. The ubiquitous finding of PV-positive dendrites linked by gap junctions throughout the dentate gyrus and Ammon's horn adds further data to indicate that this subset of GABAergic neurons is linked electrotonically. The synaptic organization of PV-positive neurons in the hippocampus suggests their participation in both feedback and feedforward inhibition. The PV-positive neurons in the hippocampus are only a proportion of the basket and chandelier cells, whereas virtually all of these cells in neocortex are PV-positive.

The synaptology of parvalbumin-immunoreactive neurons in the primate prefrontal cortex.

Electron microscopy and immunocytochemistry with a monoclonal antibody against parvalbumin (PV) were combined to analyze the distribution and morphology of PV-immunoreactive (PV-IR) neurons and the synaptology of PV-IR processes in the principal sulcus of the macaque prefrontal cortex. Parvalbumin-IR neurons are present in layers II-VI of the macaque principal sulcus (Walker's area 46) and are concentrated in a band centered around layer IV. PV-IR cells are exclusively non-pyramidal in shape and are morphologically heterogeneous with soma sizes ranging from less than 10 microns to greater than 20 microns. Well-labeled neurons that could be classified on the basis of soma size and dendritic configuration resembled large basket and chandelier cells. A novel finding is that supragranular PV-IR neurons exhibit dendritic patterns with predominantly vertical orientations, whereas infragranular cells exhibit mostly horizontal or oblique dendritic orientations. PV-IR cells within layer IV exhibit a mixture of dendritic arrangements. Vertical rows of PV-IR puncta, 15-30 microns in length, resembling the "cartridges" of chandelier cell axons were most dense in layers II, superficial III, and the granular layer IV but were not observed in the infragranular layers. Cartridges were often present beneath unlabeled, presumed pyramidal cells. PV-IR puncta also formed pericellular nests around pyramidal cell somata and proximal dendrites, suggestive of basket cell innervation. PV-IR axons were occasionally observed in the white matter underlying the principal sulcus. Electron microscopic analysis revealed that PV-IR somata and dendrites are densely innervated by nonimmunoreactive terminals forming asymmetric (Gray type I) synapses as well as by fewer terminals forming symmetric (Gray type II) synapses. The majority of terminals forming symmetric synapses with PV-IR post-synaptic structures were not immunolabeled; however, some of these boutons did contain PV-immunoreactivity. PV-IR boutons exclusively form symmetric synapses and heavily innervate layer II/III pyramidal cells. PV-IR axon cartridges formed numerous axo-axonic synapses with the axon initial segments of pyramidal cells 15-20 microns beneath the axon hillock and also terminated on large axonal spines of the initial segment. Furthermore, we failed to observe a mixture of PV-immunoreactive and non-immunoreactive boutons composing a single axon cartridge. Pyramidal cell somata and proximal dendrites were also heavily innervated by PV-IR boutons forming symmetric synapses, again, consistent with basket cell innervation. In addition, PV-IR axon terminals frequently formed symmetric synapses with dendritic shafts and spines of unidentified neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Soybean lectin binding neurons in the visual cortex of the rat contain parvalbumin and are covered by glial nets.

We carried out qualitative and quantitative studies on the distribution of soybean agglutinin-labelled cells in the visual cortex of the rat. Lectin-positive nerve cells mostly showed the morphological characteristics of small and large multipolar basket cells. Only a few cells appeared to be bipolar with a vertical or horizontal orientation. By light microscopy, soybean agglutinin binding sites were seen as discontinuous, punctate perineuronal staining (or pericellular nets) on the surface of about 9% of cortical neurons. Lectin-positive cells were predominantly localized in layers IV and V (16.9 and 12.4% of all neurons), where the intensity of staining was also the strongest. Combining lectin- and immunohistochemistry on cryo semithin sections, lectin-positive cells were shown to contain parvalbumin. Nerve cells in the visual cortex containing the related calcium binding-proteins, calbindin or calretinin were never soybean agglutinin-positive. Some glial cells and their processes were also soybean agglutinin-positive. The structure of the soybean agglutinin-positive pericellular nets was similar to that of glial nets visualized with the Golgi-method. Electron microscopy revealed that lectin binding sites were localized on the membranes and cytoplasm of glial processes ensheathing the axon terminals that impinged upon neurons and their proximal dendrites. Synaptic clefts and axon terminals were never reactive, thus explaining the discontinuous punctate labelling of the neuronal surface. Lectin binding sites were also found on the trans-face of the Golgi-complex of some lectin positive neurons suggesting that N-acetylgalactosamine-containing glycoconjugates, which are selectively detected by the lectin, are synthesized, at least partly, within the labelled neurons. We therefore consider possible the existence of specific interactions between parvalbumin positive basket cells and the glial network surrounding them, by which the neuron may determine the conditions of its own microenvironment.

Distribution of phosphate‐activated glutaminase in the human cerebral cortex

Phosphate-activated glutaminase (PAG), which catalyses conversion of glutamine to glutamate, is a potential marker for glutamatergic, and possibly GABA, neurons in the central nervous system. A polyclonal antibody, raised in rabbits against rat brain PAG, was applied to postmortem human brain tissue to reveal the distribution of PAG in the cerebral cortex. PAG immunoreactivity was observed in pyramidal and non-pyramidal neurons but not in glial cells. In the neocortex, large to medium-sized pyramidal neurons in layers III and V were stained most intensely, while the majority of smaller pyramidal cells were labeled either lightly or moderately. Such modified pyramids as the giant Betz cells, the large pyramidal cells of Meynert, and the solitary cells of Ramón y Cajal were also stained intensely. Fusiform cells in layer VI showed moderate to intense labeling. A number of cortical non-pyramidal neurons of various sizes stained moderately to intensely. These included large basket cells which were identified by their characteristic morphology and size in primary cortical areas. Pyramidal cells in the hippocampal formation as well as basket cells of the stratum oriens stained moderately to intensely. Since pyramidal cells are believed to be glutamatergic and large basket cells GABAergic, these results suggest that PAG plays a role in generating not only transmitter glutamate, but also GABA precursor glutamate.

Modulatory influences of a moving visual noise background on bar-evoked responses of cells in area 18 of the feline visual cortex

The modulatory influence of a synchronously moving visual noise background on responsiveness to an optimally-oriented moving bar stimulus was investigated in visual cortical area 18 of the lightly-anaesthetized cat. The bar and noise background were swept along the axis orthogonal to bar orientation, with the same phase, velocity and amplitude of motion. Cells which were insensitive to motion of visual noise per se or weakly responsive to individual 'grains' in the noise sample showed suppression of bar-evoked responses by simultaneous motion of the noise background. Percent suppression declined with increase in bar length, over a range which could exceed the maximum estimate of receptive field length. The decline in percent suppression was non-linear, becoming progressively flatter in slope as bar length was increased until an asymptotic value was reached; observations on end-stopped cells and on end-free cells with restricted length summation verified that percent suppression was related specifically to the length of the comparison bar and not to the strength of response it evoked. Percent suppression and the extent over which it declined with increase in bar length were comparable for preferred and opposite directions of bar motion even in cells with radically different length-response functions in the two directions, including end-stopped cells with direction-selective end-zones. In contrast to end-inhibition, which was maximal at or near the preferred velocity for a bar of optimal length, percent suppression by motion of the noise background was essentially velocity-invariant; in velocity tuned and velocity high-pass cells, background motion reduced the slope(s) of the velocity-response function, implying that the suppressive action of moving noise backgrounds is divisive rather than subtractive. It is argued that the suppression derives predominantly from an axo-somatic noise-sensitive inhibitory input from superficial- and deep-layer, large basket cells in orientation 'columns' at some distance from those of their target cells.

Directional tuning of cells in area 18 of the feline visual cortex for visual noise, bar and spot stimuli: a comparison with area 17

Directional tuning for visual noise, bar and single spot stimuli was compared over a wide range of velocities in cells from areas 17 and 18 of the visual cortex in lightly-anaesthetized cats. In each area, S-cells were predominantly insensitive to motion of a field of visual noise. C-cells were more sensitive to noise motion than B-cells, but showed heterogeneity in noise sensitivity, which was associated with other response properties: strongly noise-sensitive C-cells had relatively high spontaneous activity and broad directional tuning, and were predominantly direction-selective and binocularly-driven. Frequently, directional tuning for noise was unimodal at low velocity, but became progressively more bimodal as velocity was increased: a trough of depressed response corresponding to the peak in tuning for the bar separated two progressively more widely disparate preferred directions. In area 18, cells with velocity tuned (VT) functions for bar motion developed bimodal tuning for noise well below the optimum velocity for bar or for noise motion, while velocity high-pass (VHP) cells became progressively more bimodally tuned for noise over a wide range of velocities, in parallel with a steep increase in response to bar and noise motion. A high proportion of VT and VHP cells was bimodally tuned for noise at all velocities, one VHP cell showing two discrete lobes of tuning for noise below the threshold velocity for bar motion. Among cells which remained unimodally tuned for noise, VT and VHP cells in area 18 had radically dissimilar preferred directions for noise and bar motion at all velocities. With the exception of VHP cells, velocity bandpass was higher for noise than for bar motion. These results, together with other novel observations on the modality of tuning for noise in preferred and opposite directions of motion, demonstrate that bimodality of tuning for noise cannot simply be an effect of upper cut-off velocity for bar motion (Movshon et al. 1980; Orban 1984). It is argued that the trough between the lobes of tuning arises through laterally-directed inhibitory convergence from superficial- and deep-layer, large basket cells. In 40% of noise-sensitive cells, tuning for bar motion was broader on the flank closest to the preferred direction for noise and for a moving sport, while some 25% of cells showed variations in tuning for bar motion with velocity, which were associated with velocity-dependent changes in tuning for noise.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasopressin innervation of monkey spinothalamic tract neurons

Large basket cells were identified in Golgi and horseradish peroxidase labeled material from the sensory-motor cortex of adult monkeys. Their morphology was correlated at the light and electron microscopic level with large comparable cells stained immunocytochemically for glutamate decarboxylase. In Golgi-impregnated material these cells have a very large cell body and dendrites that extend through several layers of the cortex with a predominant vertical orientation. The axon is only stained for a few micrometers. The same cells studied electron microscopically in serial sections after gold-toning show very distinctive ultrastructural characteristics: the cell bodies contain a large number of organelles, the nuclei are rounded with homogeneously dispersed chromatin and synapsing onto the somata are many axon terminals, both symmetrical and asymmetrical but the symmetrical type forms 70–80% of the total; dendrites also receive a large number of both symmetrical and asymmetrical synaptic contacts. All the axons of basket cells become myelinated and the Golgi labeling of the initial segments is interrupted at the commencement of the first myelin internode. The axon initial segments receive several symmetrical synaptic contacts in the proximal one-third of their length.The axonal arborization of a basket cell retrogradely labeled in the somatosensory cortex after intracortical injection of horseradish peroxidase was analyzed in detail. The mainly horizontal axonal collaterals of this cell are myelinated for most of their trajectory and have a preferred orientation in the anteroposterior dimension. These axonal collaterals, although very long (more than 1.8 mm), at intervals give rise to a small number of short unmyelinated terminal branches that bear a series of boutons terminaux forming a multi-terminal ending. The multi-terminal endings surround somata and proximal dendrites of pyramidal and non-pyramidal cells. Dense pericellular terminations (baskets or nests) like those drawn by Ramón y Cajal5 and Marin-Padilla31 are not formed by the axon of a single basket cell. Thus, basket formations are presumably formed by converging axons from several basket cells.Immunocytochemical material was stained for glutamate decarboxylase, the enzyme involved in the synthesis of γ-aminobutyrate (GABA). This shows that large glutamate decarboxylase-positive neurons of the same size as those positively identified as basket cells in the Golgi and horseradish peroxidase material have virtually the same morphological characteristics, at both the light and electron microscope levels, as the basket cells. Serial electron microscopic reconstructions of large glutamate decarboxylase-positive cells show the same proportions and number of synapses onto the cell bodies, dendrites and axon initial segments as for positively identified basket cells. Many of the symmetrical synaptic contacts on the cell bodies and dendrites of large glutamate decarboxylase positive cells are formed by glutamate decarboxylase positive multiterminal endings similar to those seen on pyramidal cells in both horseradish peroxidase and glutamate decarboxylase immunoreactive material. Serial electron microscopic reconstruction of these multi-terminal endings show that many of them make additional contacts on adjacent pyramidal cells. Basket cells in monkey sensory-motor cortex are, thus, revealed as GABAergic interneurons that appear to inhibit mutually one another in addition to inhibiting pyramidal neurons.

The LectinVicia VillosaLabels a Distinct Subset of GABAergic Cells in Macaque Visual Cortex

The morphology and distribution of neurons labeled specifically by the lectin, Vicia villosa (VVA), were examined in striate cortex of adult macaque monkeys. Following incubation with VVA conjugated to histochemical markers, fine punctate reaction product appears to cover the surface of the soma and proximal dendrites of a population of cortical neurons. Although a small number of VVA-labeled cells are located in layers 2, 3A, 5, and 6, approximately 75% are located in a strip of cortex overlying layers 3B through 4Ca. Layers 1 and 4C beta are virtually devoid of labeled cells. The morphology of labeled cells varies throughout the layers. In the supragranular layers, the labeled cells generally display a round or multipolar soma with a small number of radially disposed dendrites. In deeper layers, labeled cells are multipolar or horizontal, and their proximal dendrites are often more densely labeled. There is no clear correlation between the distribution of labeled cells and the pattern of cytochrome oxidase staining in supragranular layers. Double labeling of single sections for VVA and for GABA (gamma-aminobutyric acid) immunoreactivity revealed that most VVA-labeled cells are also immunoreactive for GABA. The double-labeled cells comprise approximately 30% of all GABA immunoreactive cells. Soma size analysis of double-labeled cells shows that medium-to-large GABA cells in each layer are labeled by VVA. The soma size, laminar distribution, and morphology of the VVA-labeled GABA cells suggest that they include the large basket cells originally observed in Golgi preparations.

γ-Aminobutyric acid (GABA) immunoreactivity in mouse and rat first somatosensory (SI) cortex: description and comparison

The location and morphological characteristics of γ-aminobutyric acid (GABA)-immunopositive cells and their processes were studied in rat and mouse first somatosensory (SI) cortex (including ‘barrels’) in layer IV, and layers above (I–III), and below (V and VI). In coronal sections of SI cortex of both species GABA-immunopositive cells and punctate profiles were found in each of layers I–VI. The cells were of various sizes: the largest, located in layers III and V of each species, resemble the large basket cells seen in Golgi-impregnated material. Most of the immunopositive cells were multipolar and circular or ellipsoidal in shape, but occasionally bipolar cells with fusiform perikarya were also seen. In coronal sections, immunopositive cells did not form a characteristic pattern. GABA-immunopositive cells were observed to be most numerous in the supragranular layers whereas GABA-positive punctate profiles were more numerous in layer IV. In tangential sections from layer IV of SI cortex of both species. GABA-immunopositive cells, processes and punctate profiles were visible throughout the entire barrel field. The pattern of distribution of immunopositive cells was similar (a) in two different morphological groups — i.e. the posteromedial barrel subfield (PMBSF) and the anterolateral barrel subfield (ALBSF) in rat barrel field, and (b) in PMBSF barrels of both rat and mouse (excluding differences due to structural dissimilarities between rat and mouse barrels). GABA-immunopositive neurons were grouped mainly in the barrel side and septum and were visible frequently in small clusters. In barrels of both species GABA-immunopositive cells were of a variety of sizes and ranged in shape from ellipsoidal to circular.

Immunogold demonstration of GABA in synaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat's visual cortex

To identify the putative transmitter of large basket and clutch cells in ihe cat's visual cortex, an antiserum raised against GABA coupled to bovine serum albumen by glutaraldehyde and a postembedding, electron microscopic immunogold procedure were used. Two basket and four clutch cells were revealed by intracellular injection of horseradish peroxidase. They were identified on the basis of Ihe distribution of their processes and their synaptic connections. Large basket cells terminale mainly in layer III, while clulch cells which have a more restricted axon, terminate mainly in layer IV. Both types of neuron have a small radial projection. They establish type II synaptic contacts and about 20–30% of their synapses are made with the somata of other neurons, the rest with dendrites and dendritic spines. Altogether 112 identified, HRP-filled boutons, the dendrites of three clutch cells and myelinated axons of both basket and clutch cells were tested for the presence of GABA. They were all immunopositive. The postsynaptic neurons received synapses from numerous other GABA-positive boutons in addition to the horseradish peroxidase-filled ones. Dendritic spines that received a synapse from a GABA-positive basket or clutch cell bouton also received a type I synaptic contact from a GABA-negative bouton. A few of the postsynaptic dendrites, but none of the postsynaptic somata. were immunoreactive for GABA. The fine slructural characteristics of the majority of postsynaptic targets suggested that they were pyramidal and spiny stellate cells. These results provide direct evidence for the presence of immunoreactive GABA in identified basket and clutch cells and strongly suggest that GABA is a neurotransmitter at their synapses. The laminar dislribulion of the synaplic terminals of basket and clutch cells demonstrates that some GABAergic neurons with similar target specificity segregate into different laminae, and that the same GABAergic cells can lake part in both horizontal and radial interactions.

A correlative electron microscopic study of basket cells and large gabaergic neurons in the monkey sensory-motor cortex

Large basket cells were identified in Golgi and horseradish peroxidase labeled material from the sensory-motor cortex of adult monkeys. Their morphology was correlated at the light and electron microscopic level with large comparable cells stained immunocytochemically for glutamate decarboxylase. In Golgi-impregnated material these cells have a very large cell body and dendrites that extend through several layers of the cortex with a predominant vertical orientation. The axon is only stained for a few micrometers. The same cells studied electron microscopically in serial sections after gold-toning show very distinctive ultrastructural characteristics: the cell bodies contain a large number of organelles, the nuclei are rounded with homogeneously dispersed chromatin and synapsing onto the somata are many axon terminals, both symmetrical and asymmetrical but the symmetrical type forms 70–80% of the total; dendrites also receive a large number of both symmetrical and asymmetrical synaptic contacts. All the axons of basket cells become myelinated and the Golgi labeling of the initial segments is interrupted at the commencement of the first myelin internode. The axon initial segments receive several symmetrical synaptic contacts in the proximal one-third of their length. The axonal arborization of a basket cell retrogradely labeled in the somatosensory cortex after intracortical injection of horseradish peroxidase was analyzed in detail. The mainly horizontal axonal collaterals of this cell are myelinated for most of their trajectory and have a preferred orientation in the anteroposterior dimension. These axonal collaterals, although very long (more than 1.8 mm), at intervals give rise to a small number of short unmyelinated terminal branches that bear a series of boutons terminaux forming a multi-terminal ending. The multi-terminal endings surround somata and proximal dendrites of pyramidal and non-pyramidal cells. Dense pericellular terminations (baskets or nests) like those drawn by Ramón y Cajal 5 and Marin-Padilla 31 are not formed by the axon of a single basket cell. Thus, basket formations are presumably formed by converging axons from several basket cells. Immunocytochemical material was stained for glutamate decarboxylase, the enzyme involved in the synthesis of γ-aminobutyrate (GABA). This shows that large glutamate decarboxylase-positive neurons of the same size as those positively identified as basket cells in the Golgi and horseradish peroxidase material have virtually the same morphological characteristics, at both the light and electron microscope levels, as the basket cells. Serial electron microscopic reconstructions of large glutamate decarboxylase-positive cells show the same proportions and number of synapses onto the cell bodies, dendrites and axon initial segments as for positively identified basket cells. Many of the symmetrical synaptic contacts on the cell bodies and dendrites of large glutamate decarboxylase positive cells are formed by glutamate decarboxylase positive multiterminal endings similar to those seen on pyramidal cells in both horseradish peroxidase and glutamate decarboxylase immunoreactive material. Serial electron microscopic reconstruction of these multi-terminal endings show that many of them make additional contacts on adjacent pyramidal cells. Basket cells in monkey sensory-motor cortex are, thus, revealed as GABAergic interneurons that appear to inhibit mutually one another in addition to inhibiting pyramidal neurons.

Synaptic connections of morphologically identified and physiologically characterized large basket cells in the striate cortex of cat

Neurons were studied in the striate cortex of the cat following intracellular recording and iontophoresis of horseradish peroxidase. The three selected neurons were identified as large basket cells on the basis that (i) the horizontal extent of their axonal arborization was three times or more than the extent of the dendritic arborization; (ii) some of their varicose terminal segments surrounded the perikarya of other neurons. The large elongated perikarya of the first two basket cells were located around the border of layers III and IV. The radially-elongated dendritic field, composed of beaded dendrites without spines, had a long axis of 300–350 μm, extending into layers III and IV, and a short axis of 200 μm. Only the axon, however, was recovered from the third basket cell. The lateral spread of the axons of the first two basket cells was 900 μm or more in layer III and, for the third cell, was over 1500 μm in the antero-posterior dimension, a value indicating that the latter neuron probably fulfills the first criterion above. The axon collaterals of all three cells often branched at approximately 90° to the parent axon. The first two cells also had axon collaterals which descended to layers IV and V and had less extensive lateral spreads. The axons of all three cells formed clusters of boutons which could extend up a radial column of their target cells. Electron microscopic examination of the second basket cell showed a large lobulated nucleus and a high density of mitochondria in both the perikarya and dendrites. The soma and dendrites were densely covered by synaptic terminals. The axons of the second and third cells were myelinated up to the terminal segments. A total of 177 postsynaptic elements was analysed, involving 66 boutons of the second cell and 89 boutons of the third cell. The terminals contained pleomorphic vesicles and established symmetrical synapses with their postsynaptic targets. The basket cell axons formed synapses principally on pyramidal cell perikarya (approximately 33% of synapses), spines (20% of synapses) and the apical and basal dendrites of pyramidal cells (24% of synapses). Also contacted were the perikarya and dendrites of non-pyramidal cells, an axon, and an axon initial segment. A single pyramidal cell may receive input on its soma, apical and basal dendrites and spines from the same large basket cell. Seven pyramidal neurons that were postsynaptic to the second and third identified basket cells received synaptic contacts on their soma (average of 4.4 from one basket cell) and proximal dendrites (average 2.3). The results provide evidence that large basket cells establish multiple synaptic contacts with the neurons they seem to select as postsynaptic targets. At least 30% or more of their postsynaptic targets are the perikarya of other neurons. In the light of recent immunocytochemical studies on the visual cortex of the cat, large basket cells may use γ-aminobutyrate as their transmitter and they are probably inhibitory in function. Since large basket cells also contact dendrites and spines, their effect cannot be explained by a simple somatic inhibitory mechanism and their functional role must also be considered in relation to other inputs to different parts of the same neuron. The structural features of large basket cells are compared with those of other putative inhibitory interneurons and their role in the functioning of the visual cortex is discussed.

Morphological diversity of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex.

GABAergic neurons have been identified in monkey sensory-motor cerebral cortex by light microscopic, immunocytochemical localization of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD). All GAD-positive neurons are non-pyramidal cells. Their somata are present in all layers and are evenly distributed across layers II-VI of the motor cortex (area 4), but are found in greater concentrations in layers II, IV and VI of all areas of first somatic sensory cortex (SI; areas 3a, 3b and 1-2). GAD-positive puncta (putative axon terminals) are present throughout the sensory-motor cortex, and they are found immediately adjacent to the somata, dendrites and presumptive axon initial segments of GAD-negative pyramidal cells. In addition, they are observed in close approximation to the somata of both large and small GAD-positive neurons. In area 4, the density of puncta is highest in the superficial cortical layers (layers I-III) and gradually declines throughout the deeper layers. In SI, the highest densities of puncta are present in layer IV, while moderately high densities are found in layers I-III and VI. In areas 3a and 3b, the puncta in layers IV and VI are particularly numerous and form foci that exhibit greater density than adjacent regions. GAD-positive neurons with large somata, 15-33 micron in diameter, are present in layers IIIB-VI of all areas. Such cells have many primary dendrites that radiate in all directions. In addition they have axons that ascend either from the superficial aspect of the somata or from primary dendrites, and that exhibit horizontal collateral branches. These neurons closely resemble the large basket cells (Marin-Padilla, 1969; Jones, 1975), and they may give rise to many of the GAD-positive endings surrounding the somata and proximal dendrites of pyramidal cells in layers III-VI. In addition, small GAD-positive somata are present in all layers, but they are most numerous in layers II and IIIA of all areas and in layer IV of SI. The somata and proximal dendrites of these cells vary from a multipolar shape with small, beaded dendrites, found primarily in layer IV, to bitufted and multipolar shapes with larger, smooth dendrites. The diversity of somal sizes and locations, the variety of dendritic patterns, and the different distributions of GAD-positive puncta, all combine to suggest that several different morphological classes of intrinsic neurons comprise the GABA neurons of monkey cerebral cortex.