Paolo Barbaresi

Neuronal, glial, and epithelial localization of γ-aminobutyric acid transporter 2, a high-affinity γ-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures

Neuronal and glial high‐affinity Na+/Cl −‐dependent plasma membrane γ‐aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT‐2 by immunocytochemistry with affinity‐purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser‐scanning light microscopic studies revealed intense GAT‐2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT‐2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double‐labeled with GAT‐2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT‐2‐positive profiles also were GFAP positive. Ultrastructural studies showed GAT‐2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT‐2‐positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT‐2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT‐2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain. J. Comp. Neurol. 409:482–494, 1999.

Callosal mechanism for the interhemispheric transfer of hand somatosensory information in the monkey.

The retrograde transport of horseradish peroxidase (HRP) was combined with extracellular microelectrode recording from single and multiple-neurones to study the anatomical and functional organization of the callosal connections of the hand sensory projection field in the parietal operculum of monkeys (Macaca Irus). In 3 animals anaesthetized with ketamine, a single injection of HRP (0.5 microliter) was delivered into the cortex forming the upper bank of the sylvian sulcus at a site where neuronal responses to somatic sensory stimulation of the hand were recorded. In the ipsilateral hemisphere, retrogradely HRP-labelled cells were found in the cortex of the post-central gyrus and in the thalamic nuclei ventralis posteroinferior and pulvinar oralis. In the contralateral hemisphere HRP-labelled neurones were present in the opercular cortex lying dorsal, and slightly caudal, to the posterior pole of the insula. Few scattered callosal neurones were also found in the post-central gyrus. In 3 other animals, multiple injections (5-8; 0.5 microliter each) of HRP were performed in the parietal operculum. In the ipsilateral hemisphere, retrogradely labelled cells were present in the post-central gyrus and in the following thalamic nuclei: ventralis posteroinferior, pulvinar oralis and medialis, ventralis posteromedialis and posterior complex. Few labelled cells were also present in the ventral part of the nucleus ventralis posterolateralis. In the contralateral hemisphere, numerous callosal cells were labelled with HRP. These cells were found, with regional variations in density, in wide regions of the buried and exposed cortex of the parietal operculum and in the post-central gyrus. These 3 monkeys were subjected to microelectrode mapping experiments (N2O and halothane anaesthesia) to explore the peripheral receptive fields of neurones in the parietal operculum and post-central gyrus contralateral to the injected side. HRP labelled callosal neurones were found in regions of the second and first somatosensory cortical areas which also contained units driven from the contralateral hand.

Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in approximately 90% of VGLUT1+, approximately 30% of VGLUT2+ and 30-50% of VGAT+ puncta; SYPI was expressed in approximately 95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in approximately 7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in approximately 94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in approximately 3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that approximately 30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats.

Postnatal development of high-affinity plasma membrane GABA transporters GAT-2 and GAT-3 in the rat cerebral cortex.

We investigated the developmental profile of plasma membrane gamma-aminobutyric acid (GABA) transporters (GATs) GAT-2 and GAT-3 expression by immunocytochemistry with affinity-purified polyclonal antibodies in the rat neocortex. At all developmental ages investigated, GAT-2 ir was prominent in the arachnoid and in the trabeculae of the subarachnoid space, whereas it was weak within the cortical parenchyma; the adult pattern was reached during the third week of postnatal life. GAT-3 ir was present at birth and increased rapidly in the first week, when numerous positive cells were present throughout the cortical layers; at P10, GAT-3-positive cells became less numerous and GAT-3 ir switched to the adult pattern, which was expressed at P20. Confocal and electron microscopic investigations showed that GAT-3 positive cells were both neurons and astrocytes. The present evidence indicates that early in development GAT-3 is abundantly expressed in the cerebral cortex, where its expression appears to correlate with developmental variations in GABA levels, and suggests that it accounts for the largest fraction of GABA transport observed in the neonatal cerebral cortex.

Cytochrome oxidase histochemistry reveals regional subdivisions in the rat periaqueductal gray matter

The identification of different anatomical regions of the periaqueductal gray matter of rats was addressed in the present study by using the histochemical staining for the mitochondrial enzyme cytochrome oxidase. At caudal and middle levels, cytochrome oxidase histochemistry clearly demonstrates the existence of four subdivisions: dorsal, dorsolateral, ventrolateral and medial, whereas in sections from the rostral periaqueductal gray matter only two concentric bands are identifiable on the basis of the degree of cytochrome oxidase activity.

HETEROGENEITY OF GLUTAMATERGIC AND GABAergic RELEASE MACHINERY IN CEREBRAL CORTEX: ANALYSIS OF SYNAPTOGYRIN, VESICLE-ASSOCIATED MEMBRANE PROTEIN, AND SYNTAXIN

To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles.

Functional topography of the corpus callosum investigated by DTI and fMRI.

This short review examines the most recent functional studies of the topographic organization of the human corpus callosum, the main interhemispheric commissure. After a brief description of its anatomy, development, microstructure, and function, it examines and discusses the latest findings obtained using diffusion tensor imaging (DTI) and tractography (DTT) and functional magnetic resonance imaging (fMRI), three recently developed imaging techniques that have significantly expanded and refined our knowledge of the commissure. While DTI and DTT have been providing insights into its microstructure, integrity and level of myelination, fMRI has been the key technique in documenting the activation of white matter fibers, particularly in the corpus callosum. By combining DTT and fMRI it has been possible to describe the trajectory of the callosal fibers interconnecting the primary olfactory, gustatory, motor, somatic sensory, auditory and visual cortices at sites where the activation elicited by peripheral stimulation was detected by fMRI. These studies have demonstrated the presence of callosal fiber tracts that cross the commissure at the level of the genu, body, and splenium, at sites showing fMRI activation. Altogether such findings lend further support to the notion that the corpus callosum displays a functional topographic organization that can be explored with fMRI.

Periaqueductal grey projection to the ventrobasal complex in the cat: An horseradish peroxidase study

Horseradish peroxidase (HRP) was injected within the thalamic ventrobasal complex of 14 cats. The aim was to ascertain whether the periaqueductal grey matter (PAG) sends fibres to this complex. Retrogradely labelled cells were found within the PAG following HRP delivery either in the nucleus ventralis posterolateralis (VPL) or ventralis posteromedialis (VPM). PAG-VPL projection is only ipsilateral and arises mainly from lateral PAG, PAG-VPM projection is bilateral and originates from latero-ventral regions of the central grey. The hypothesis that PAG might control the activity of ventrobasal nociceptive neurones is proposed.

gamma-Aminobutyric acid transporters in the cat periaqueductal gray: a light and electron microscopic immunocytochemical study.

The γ‐aminobutyric acid (GABA) plasma membrane transporters (GATs) mediate GABA uptake into presynaptic axon terminals and glial processes, thus contributing to the regulation of the magnitude and duration of the action of GABA at the synaptic cleft. The aim of the present study was to investigate the expression of three high‐affinity GABA transporters (GAT‐1, GAT‐2, and GAT‐3) in the periaqueductal gray matter (PAG) of adult cats by using immunocytochemistry with affinity‐purified antibodies. Light microscopic observations revealed GAT‐1 immunoreactivity in punctate structures, particularly dense in the lateral portion of the dorsolateral PAG column. Weak GAT‐2‐immunopositive puncta were homogeneously distributed in the PAG. GAT‐3 immunoreactivity was detected in each column of the PAG but was more intense in the dorsolateral PAG column and around the aqueduct. Electron microscopic studies showed GAT‐1 immunoreactivity in distal astroglial processes, in unmyelinated and small myelinated axons, and in axon terminals making symmetric synapses on both PAG neurons and dendrites. GAT‐2 immunoreactivity was present mostly in the form of patches of different sizes in the cytoplasm of neuronal elements like the perikarya and dendrites of PAG neurons, in myelinated and unmyelinated axons, and in the axon terminals forming both symmetric and asymmetric synapses. Labeling was also observed in nonneuronal elements. Astrocytic cell bodies and their distal processes as well as the ependymal cells lining the wall of the aqueduct showed patches of GAT‐2 immunoreactivity. Electron microscopic observation revealed GAT‐3 immunoreactivity exclusively in distal astrocytic processes adjacent to the somata of PAG neurons and in axon terminals making both symmetric and asymmetric synapses. The present results suggest that three types of termination systems of GABAergic transmission are present in the cat periaqueductal gray matter. J. Comp. Neurol. 429:337–354, 2001.

Thalamic connections of the second somatic sensory area in cats studied with anterograde and retrograde tract-tracing techniques

The thalamic connections of the second somatosensory area in the anterior ectosylvian gyrus of cats have been investigated using the retrograde tracer horseradish peroxidase and the anterograde tracer Phaseolus vulgaris leucoagglutinin. Horseradish peroxidase was injected iontophoretically in several somatotopic zones of the second somatosensory area map of six cats. Sites of horseradish peroxidase delivery were identified preliminarily by recording with microelectrodes the responses of neurons to skin stimulation. Phaseolus vulgaris leucoagglutinin was iontophoretically injected within the ventrobasal complex (one cat) or in the posterior complex (one cat). Horseradish peroxidase injections into cytoarchitectonic area SII retrogradely labeled neurons in the ipsilateral ventrobasal complex and in the posterior complex. Counts of labeled neurons from the ipsilateral thalamus showed that the overwhelming majority of horseradish peroxidase-labeled neurons were in the ventrobasal complex (96.3-96.9%) and few were in the posterior complex (3.1-3.7%). Neurons labeled in the ventrobasal complex were observed throughout the anteroposterior extent of the nucleus, while their mediolateral distribution varied with the site of horseradish peroxidase delivery in the body map of the second somatosensory area, which indicates that the projections from the ventrobasal complex to the second somatosensory area are somatotopically organized. In the cat in which the horseradish peroxidase injection involved both the second somatosensory area proper and the second somatosensory area medial, which lies in the lower bank of suprasylvian sulcus, labeled neurons were almost as numerous in the ventrobasal complex as in the posterior complex. Phaseolus vulgaris leucoagglutinin injected in the ventrobasal complex anterogradely labeled thalamocortical fibers in the ipsilateral anterior ectosylvian gyrus. In this case, patches of labeled fibers and terminals were distributed exclusively within the cytoarchitectonic borders of the second somatosensory area proper. Labeled terminals were numerous in layer IV and lower layer III, but terminal boutons and fibers with axonal swellings, probably forming synapses en passant, were frequently observed also in layers VI and I. Injection of Phaseolus vulgaris leucoagglutinin in the posterior complex labeled thalamocortical fibers in two distinct regions in the ipsilateral anterior ectosylvian gyrus, one lying laterally and the other medially, which correspond, respectively, to the fourth somatosensory area and the second somatosensory area medial. In both areas the densest plexus of labeled fibers and axon terminals was in layer IV and lower layer III, but numerous labeled fibers and terminals were also observed in layer I. In this case, only rare fragments of labeled fibers were present in second somatosensory area proper, but no labeled terminals could be observed.