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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.

Introduction: The aim of this study was to determine the presence and distribution of nitric oxide (NO)-producing neurons in the rat corpus callosum (cc)., Material and Methods: To investigate this aspect of cc organization we used nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunocytochemistry., Results: Intense NADPH-d-positive (NADPH-d+) neurons were found along the rostrocaudal extension of the cc (sagittal sections). They were more numerous in the lateral cc and gradually decreased in the more medial regions, where they were very few or absent. The Golgi-like appearance of NADPH-d+ intracallosal neurons allowed dividing them into five morphological types: (1) bipolar; (2) fusiform; (3) round; (4) polygonal; and (5) pyramidal. The number of NADPH-d+ neurons (both hemispheres) was counted in two brains using 50-μm thick sections. In the first brain, counts involved 145 sections and neurons were 2959; in the second, 2227 neurons were counted in 130 sections. The distribution and morphology of nNOS-immunopositive (nNOSIP) neurons was identical to that of NADPH-d+neurons. Some of these neurons were observed in the cc ependymal region, where they might be in contact with cerebrospinal fluid (CSF), monitoring its composition, pH, and osmolality changes, or playing a role in regulating the synthesis and release of several peptides. The somatic, dendritic, and axonal processes of many NADPH-d+/nNOSIP neurons were closely associated with intracallosal blood vessels., Conclusions: Such close relationship raises the possibility that these neurons are a major source of NO during neural activity. As NO is a potent vasodilator, these findings strongly suggest that NO-positive neurons transduce neuronal signals into vascular responses in selected cc regions, thus giving rise to hemodynamic changes detectable by neuroimaging.

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 μm(2) (PAG-dl) to 125.13 μm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 μm(2); neurons labeled only with NADPH-d or Calb measured 89.793 μm(2) and 113.48 μm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 μm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 μm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity., (Copyright © 2011 Wiley Periodicals, Inc.)

Vesicular γ-aminobutyric acid transporter, an integral membrane protein, mediates γ-aminobutyric acid uptake into synaptic vesicles. Investigation of its expression in the rat periaqueductal gray matter using light and electron microscopic immunocytochemical techniques documents immunoreactivity in numerous puncta in all periaqueductal gray columns, with slightly denser labeling on the outer edge of the dorsolateral portion and least dense labeling in the rostral-dorsomedial portion. Electron microscopic observations show immunoreactivity over synaptic vesicles of terminals exhibiting the standard features of inhibitory synapses. Unlabeled axon terminals making symmetric synapses are also commonly observed. These data suggest the following: (i) the γ-aminobutyric acid uptake is slightly greater in the dorsolateral and less intense in the rostral-dorsomedial portion and (ii) the possible existence of an additional inhibitory amino acid transporter.

The development of intrinsic gamma-aminobutyric acid (GABA)-ergic neurons was studied in the first month of postnatal life in the rat periaqueductal gray matter (PAG) by light and electron microscopy using an anti-GABA serum. At birth (postnatal day 0: P0) GABA-immunopositive (GABA(IP)) neurons were detected only on the outer edge of dorsolateral PAG (PAG-DL) and were rare in the other PAG subdivisions. Their distribution did not change from P0 to P5, while they increased progressively from P5 to P10 in PAG-DL and began to be detected in ventrolateral PAG (PAG-VL). At the end of the second postnatal week the immunostaining pattern was nearly adult-like, and between P20 and P30 the adult pattern of GABA immunoreactivity was established. Quantitative light microscopic examination indicated that in the first postnatal month the cross-sectional area of GABA(IP) neurons gradually increased from 67.63 and 78.69 microm(2) at P0 to 122.15 and 119.16 microm(2) at P30 in PAG-DL and PAG-VL, respectively. Electron microscopic observations disclosed GABA labeling from P0 in cell bodies, dendrites, growth cones, and axon terminals. GABA(IP) terminals were few in neonatal rats and became more numerous and morphologically mature around the second week. Synapse development and maturation were examined by quantitative ultrastructural analysis. Synaptic vesicle number and size of GABA(IP) axon terminals progressively grew in the first postnatal month. In conclusion, the number and size of GABA(IP) cells progressively increase in postnatal PAG, with two populations of intrinsic neurons expressing their GABAergic nature in two different periods., (Copyright 2010 Wiley-Liss, Inc.)

The inhibitory effects of gamma-aminobutyric acid (GABA)ergic neurotransmission in the periaqueductal gray matter (PAG) are mediated, at least partly, by metabotropic GABA(B) receptor subtypes whose cellular and subcellular localization is still unknown. We performed immunohistochemical experiments with an antibody against GABA(B) receptor subtype 1a/b (GABA(B)R(1a/b)) by using light and electron microscopy. On light microscopy, GABA(B)R(1a/b) immunoreactivity (IR) was in all columns, defined by cytochrome oxidase histochemistry. Neuropil labeling was strongest in the lateral portion of dorsolateral PAG. Labeled neurons, albeit not numerous, were in ventrolateral, dorsal, and medial subdivisions and were sparser in dorsolateral PAG. Labeling was mostly on the soma of PAG neurons. Sometimes GABA(B)R(1a/b) IR spread along proximal dendrites; in these cases bipolar neurons were the most common type. On electron microscopy, GABA(B)R(1a/b) IR was mainly on dendrites (54.92% of labeled elements) and axon terminals (21.90%) making synapses with labeled and unlabeled postsynaptic elements. Presynaptic labeling was also on unmyelinated and myelinated axons (overall 8% of all labeled elements). Postsynaptically, GABA(B)R(1a/b) IR was at extrasynaptic sites on dendritic shafts; spines were always unlabeled. On axon terminals, GABA(B)R(1a/b) IR was on extrasynaptic membranes and sometimes on presynaptic membrane specializations. Of the labeled elements, 13.03% elements were distal astrocytic processes (dAsPs) surrounding both symmetric and asymmetric synapses whose pre- and postsynaptic elements were often labeled. Immunoreactive dAsPs were around the soma and dendrites of both labeled and unlabeled neurons. These findings provide insights into the intrinsic PAG organization and suggest that presynaptic, postsynaptic, and glial GABA(B) receptors may play crucial roles in controlling PAG neuronal activity., ((c) 2007 Wiley-Liss, Inc.)

Gamma-amino butyric acid (GABA) plasma membrane transporters (GATs) contribute to the modulation of GABA's actions and are implicated in neuropsychiatric diseases. In this study, the localization of GAT-3, the major glial GAT, was investigated in human cortex using immunocytochemical techniques. In prefrontal and temporal cortices, GAT-3 immunoreactivity (ir) was present throughout the depth of the cortex, both in puncta and in neurons. GAT-3-positive puncta were dispersed in the neuropil or closely related to cell bodies; neuronal staining was in perikarya, especially of pyramidal cells, and proximal dendrites. Electron microscopic studies showed that GAT-3 ir was in astrocytic processes as well as in neuronal elements. All GAT-3-positive neurons co-expressed heat shock protein 70. To test the possibility that the collection procedure of human samples induced the expression of GAT-3 in neurons which normally do not express it, we analyzed rat cortical tissue resected using the same procedure and found that numerous neurons are GAT-3-positive and that they co-express heat shock protein 70. Results show that in human cortex GAT-3 is expressed in astrocytic processes and in neurons and suggest that neuronal expression is related to the procedure used for collecting human samples.

SNAT1 mediates glutamine (Gln) influx into neurons and is believed to replenish the transmitters pools of glutamate (Glu) and gamma-aminobutyric acid (GABA). We investigated its distribution and cellular localization in the cerebral cortex and neighboring regions of rats and humans using light and electron microscopic immunocytochemical methods with specific antibodies. In the first somatic sensory cortex of rats and in areas 9, 10, 21 and 46 of the human cortex, numerous SNAT1-positive (+) cells were present in the cortical parenchyma and in the white matter; >95% of SNAT1+ cells were neurons, but some were astrocytes. Most SNAT1+ cells were pyramidal neurons, but numerous non-pyramidal neurons were also observed: SNAT1/GABA double-labeling studies showed that SNAT1 is expressed in all GABA+ neurons. SNAT1/synaptophysin studies showed that <0.1% of all synaptophysin+ puncta coexpressed SNAT1. SNAT1 immunoreactivity (ir) was also in leptomeninges, ependymal cells and choroid plexus. Electron microscopic studies showed that neuronal SNAT1 ir was almost exclusively observed in perikarya and dendritic profiles. SNAT1 ir was also in distal astrocytic processes, including end feet profiles, and in leptomeninges. These findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA.

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.

Lesion-induced degeneration was combined with immunocytochemistry to study, with electron microscopy, the synaptic connectivity between corticothalamic axon terminals from the first and second somatosensory areas and local circuit neurons of the ipsilateral ventrobasal complex (VB), selectively labelled with an antibody raised against gamma-aminobutyric acid (GABA). Four days from the cortical ablation many degenerating axon terminals, forming asymmetric synapses, were found on dendritic trees of both labelled and unlabelled neurons of VB and occasionally on presynaptic dendrites. The main finding of the present paper is that 64.01% of degenerating axon terminals synapsed with GABA-immunopositive dendrites, suggesting that the principal target of the cortical projection to VB are local circuit neurons.

The gamma-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., (Copyright 2000 Wiley-Liss, Inc.)

Neuronal and glial high-affinity Na+/Cl(-)-dependent plasma membrane gamma-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.

The present light and electron microscopic experiments were carried out on the first somatic sensory area (SI) of cats to determine the laminar distribution of axon terminals from the ipsilateral second somatic sensory area (SII) and to identify the types of synapses between these terminals and the neuronal elements of SI. Phaseolus vulgaris-leucoagglutinin (PHA-L) was iontophoretically injected into multiple sites and at different cortical depths of the forepaw representation zone of SII. Fixed brain blocks containing the injected SII and ipsilateral SI were cut into slices and processed immunocytochemically to stain PHA-L-filled fibers and terminals. Light microscopic examination of SI revealed patches of anterograde labeling in the forepaw representation zone, concentrated mainly in supragranular layers. In these layers, thin immunolabeled fibers branched extensively and formed a dense plexus that was more prominent in layers II and I. Conversely, the infragranular layers contained fragments of vertically oriented thick fibers that rarely emitted axon collaterals. PHA-L-labeled axons had numerous swellings along their course, interpreted as boutons en passant, and stalked boutons. Of 19,661 labeled terminals (17,833 beads and 1,828 stalked boutons), 84.74% were observed in supragranular layers, with the highest concentration in layer II (33.15%) and lower in layers I (26.27%) and III (25.30%). The proportion of terminals was lower in layers IV (6.49%) and V (5.45%) and lowest in layer VI (3.32%). These counts also showed that boutons en passant were the majority (90.70%) and stalked boutons, the minority (9.30%). The ratio of these two types of presynaptic specializations was similar (9:1) in all six layers. Electron microscopic examination of the labeled regions of SI showed that both axon swellings and stalked boutons formed synapses of the asymmetric type with SI neuronal elements. The majority (85.37%) of a sample of 130 labeled terminals synapsed on SI neurons in layers I-III. The identified postsynaptic profiles were dendritic spines (61.11%) or medium-sized and small dendrites (38.89%). These results are discussed in relation to those of a companion study on the laminar pattern of the projection from SI to SII of cats (P. Barbaresi, A. Minelli, and T. Manzoni, 1994, J. Comp. Neurol. 343:582-596). Based on the anatomical organization of these reciprocal connections, there seems to be no clear hierarchicalal relationship between SI and SII in cats.

Experiments were carried out on the second somatic sensory area (SII) of cats to study 1) the laminar distribution of axon terminals from the ipsilateral first somatic sensory cortex (SI); and 2) the topographical relations between their terminal field and the callosal neurons projecting to the contralateral homotopic cortex. To label simultaneously in SII both ipsilateral cortical afferents and callosal cells, cats were given iontophoretic injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the forepaw zone of ipsilateral SI, and pressure injections of horseradish peroxidase (HRP) in the same zone of contralateral SII. The possibility that ipsilateral cortical axon terminals synapse callosal neurons was investigated with the electron microscope by combining lesion-induced degeneration with retrograde HRP labelling. Fibers and terminations immunolabelled with PHA-L from ipsilateral SI were distributed in SII in a typical patchy pattern and were mostly concentrated in supragranular layers. Labelled fibers formed a very dense plexus in layer III and ramified densely also in layers I and II. Labelled axon terminals were both en passant and single-stalked boutons. Counts of 8,303 PHA-L-labelled terminals of either type showed that 82.40% were in supragranular layers. The highest concentration was in layer III (43.99%), followed by layers II (30.32%) and I (8.09%). The remaining terminals were distributed among layers IV (6.96%), V (4.93%), and VI (5.68%). The same region of SII containing anterogradely labelled axons and terminals also contained numerous neurons retrogradely labelled with HRP from contralateral SII. Callosal projection neurons were pyramidal, dwelt mainly in layer III, and were distributed tangentially in periodic patches. Patches of anterograde and retrograde labelling either interdigitated or overlapped both areally and laminarly. In the zones of overlap, numerous PHA-L-labelled axon terminals were seen in close apposition to HRP-labelled pyramidal cell dendrites. Combined HRP-electron microscopic degeneration experiments showed that in SII axon terminals from ipsilateral SI form asymmetric synapses with HRP-labelled dendrites and dendritic spines pertaining to callosal projection neurons. These results are discussed in relation to the layering and function of the SI to SII projection, and to the evidence that SII neurons projecting to the homotopic area of the contralateral hemisphere have direct access to the sensory information transmitted from ipsilateral SI.

Different fluorescent dyes were injected in the face (S1fa) and hand (S1hn) representations of the primary somatosensory cortex, involving both areas 3b and 1. Claustral neurons labeled by either S1fa or S1hn were divided in two populations. One population was located in the dorsal part of the nucleus: neurons labeled from S1fa were placed laterally to those labeled from S1hn. The second population was located more ventrally, with a rostro-caudal distribution of S1fa vs S1hn neurons. These findings demonstrate the existence of ordered and possibly multiple somatosensory representations in the monkey claustrum.

Anatomical and electrophysiological experiments were performed on cats to investigate the pattern of divergence and convergence in the association projections from the first (SI) to the second (SII) somatic sensory cortex and to ascertain whether diverging and converging fibre components from SI have receptive fields (RFs) matching those of target neurons in SII. In the first group of six cats, a single deposit of horseradish peroxidase (HRP) was iontophoretically placed (2-4 microA for 20 minutes) into an electrophysiologically identified site of the SII map: the digit (3 cats), forepaw (2 cats), and arm (1 cat) zones. The forelimb representation in ipsilateral SI was subsequently explored with microelectrodes and RFs from small clusters of neurons systematically mapped. Planar maps of this area were reconstructed with the aid of a computer from serial sections, to correlate on the tangential plane the topographical distribution of retrogradely labelled association neurons with the physiological map of the forelimb. Since diverging projections were observed from a zone of SI to multiple zones of SII, double-labelling experiments were carried out in a second group of three cats, in which two retrograde fluorescent dyes (diamidino yellow and fast blue) were injected by pressure into two different sites of the SII map, to ascertain whether SI sends diverging projections by branching axons. HRP injections in SII retrogradely labelled a discrete number of association neurons in SI. Their distribution area was several tens of times wider than that covered by the injection site. This suggests that a remarkable amount of divergence and convergence exists in the association projections from SI to SII. Despite the substantial difference in the extent of the injected and labelled areas, RFs of afferent and target neurons corresponded closely. Injections covering a small region within a single digit zone of SII labelled neurons throughout the entire representation of the same digit in SI, while neurons labelled in somatotopically inappropriate zones were rare. RFs mapped from several sites of the labelled region in SI were individually smaller than the RF mapped from the injection site in SII, but the overall size of afferent RFs encompassed that of target neurons. Divergence and convergence in the SI projections to SII zones representing more proximal portions of the forelimb may be even greater since HRP injections in the forepaw and arm zones of SII labelled a number of neurons also in the digit zone of SI, providing the RFs mapped from the injection sites were sufficiently wide to include the digits.(ABSTRACT TRUNCATED AT 400 WORDS)

With the aim to study the detailed topography of the thalamo-cortical neurones projecting to the trunk representation zone of the first somatosensory area (SI), punctate injections of three different fluorescent tracers (Evans Blue, Nuclear Yellow and Fast Blue) were performed in the three physiologically defined subareas forming the trunk region of SI. These injections resulted in the labelling of three different cell aggregates, narrow in dorsoventral and mediolateral extent but elongated rostrocaudally, located in topographically distinct regions of the nucleus ventralis posterio-lateralis. The results suggest that the highly organized topography of the trunk representation of area SI is imposed by the thalamo-cortical input from VPL.

Experiments were carried out on cats to ascertain whether corticocortical neurones of somatosensory areas I (SI) and II (SII) could be labelled by retrograde axonal transport of D-[3H]aspartate (D-[3H]Asp). This tritiated enantiomer of the amino acid aspartate is (1) taken up selectively by axon terminals of neurones releasing aspartate and/or glutamate as excitatory neurotransmitter, (2) retrogradely transported and accumulated in perikarya, (3) not metabolized, and (4) visualized by autoradiography. A solution of D-[3H]Asp was injected in eight cats in the trunk and forelimb zones of SI (two cats) or in the forelimb zone of SII (six cats). In order to compare the labelling patterns obtained with D-[3H]Asp with those resulting after injection of a nonselective neuronal tracer, horseradish peroxidase (HRP) was delivered mixed with the radioactive tracer in seven of the eight cats. Furthermore, six additional animals received HRP injections in SI (three cats; trunk and forelimb zones) or SII (three cats; forelimb zone). D-[3H]Asp retrograde labelling of perikarya was absent from the ipsilateral thalamus of all cats injected with the radioactive tracer but a dense terminal plexus of anterogradely labelled corticothalamic fibres from SI and SII was observed, overlapping the distribution area of thalamocortical neurones retrogradely labelled with HRP from the same areas. D-[3H]Asp-labelled neurones were present in ipsilateral SII (SII-SI association neurones) in cats injected in SI. In these animals a bundle of radioactive fibres was observed in the rostral portion of the corpus callosum entering the contralateral hemisphere. There, neurones retrogradely labelled with silver grains were present in SI (SI-SI callosal neurones). Association and callosal neurones labelled from SI showed a topographical distribution similar to that of neurones retrogradely labelled with HRP. The laminar patterns of corticocortical neurones labelled with D-[3H]Asp or with HRP were also similar, with one exception. In the inner half of layer II, SII-SI association neurones and SI-SI callosal neurones labelled with the radioactive marker were much less numerous than those labelled with HRP. In cats injected in SII, D-[3H]Asp retrogradely labelled cells were present in ipsilateral SI (SI-SII association neurones). Their topographical and laminar distribution overlapped that of neurones labelled with HRP but, as in cats injected in SI, association neurones labelled with silver grains were unusually rare in the inner layer III.(ABSTRACT TRUNCATED AT 400 WORDS)

In 4 adult cats the left and right thalamic nucleus VPM have been injected with fluorescent substances, Evans Blue and Fast Blue in order to ascertain whether PAG cells send bifurcated axons to the thalamic nuclei of both sides. The results confirm previous studies on the PAG-VPM connections performed in this laboratory by using HRP technique, and demonstrate, within the PAG, the presence of a good number of double-labeled cells (stained by both E.B. and F.B.) intermingled with single-labeled cells, stained either by E.B. or F.B. The double-labeled cells might represent the anatomical substrate of a possible bilateral analgesic mechanism in the trigeminal region.

Neurons of cat cerebral cortex, area S1, labelled by retrograde transport of HRP, have been studied by electron microscopy; the animals were sacrificed 48 h after HRP injection. The cells studied are those in which the enzyme is found both in granular and in diffused form in the cytoplasm (diffuse-granular HRP neurons, DGN), or in diffused form in the cytoplasm (Golgi-like labelled neurons, GLN). The observations have shown that the certain recognition of DGN through electron microscopy, requires a comparison with a semithin section adjacent to the ultrathin section since the labelling granules are light-scattering in dark field. Instead the GLN are not light-scattering in dark field but are directly and unmistakably identifiable even by electron microscopy. The DGN are well preserved on the whole in all their organelles; the GLN show a highly damaged aspect in relation to both the Golgi apparatus and the smooth reticulum, to the cytoplasmic organelles and to the nucleus. Our hypothesis is that the latter neurons take on this appearance because of the combined action of: a) the peripheral mechanical lesions caused by the needle and b) the enormous amount of HRP that the cell is forced to take up (through these pathways) because of its lost integrity and because of the large quantity of marker around the axons. In conclusion intracellular HRP is identifiable by electron microscopy only when it is found in diffused form in the cytoplasm.

Injection of D-[3H]aspartate into area SII of cats retrogradely labelled association cells in area SI. Numerous intensely labelled cells were found in layer II and in the upper layer III but labelling was scanty in other layers. In contrast, association neurones labelled by horseradish peroxidase injected in area SII mixed with the radioactive marker were also numerous in the other sublaminae of layer III and in infragranular layers of SI. Association neurones of the outer laminae of this area are likely to use aspartate and/or glutamate as neurotransmitter(s).

The double-labeling technique based on the retrograde axonal transport of fluorescent tracers (Evans blue, EB; Fast blue, FB; Nuclear yellow, NY) was used in the cat in order to investigate the occurrence of axonal branching in the periaqueductal gray (PAG) projections to some thalamic nuclei (n. ventralis postero-lateralis, VPL; n. ventralis postero-medialis, VPM; n. parafascicularis, Pf). In a first group of cats, FB and EB were injected, respectively, within the right and left VPM. In another two groups of cats, FB injections into Pf were combined with either EB or NY injections within VPL or VPM. Double-labeled neurons were found within the PAG only in the animals of the first group. The present results show that some PAG neurons project bilaterally to VPM by means of axons collaterals.

The present experiments are based upon evidence that neurons may selectively take up at their terminals, and retrogradely transport, the same chemical they use as a neurotransmitter or its analogues. In an attempt to identify dorsal root ganglion (DRG) neurons that use glutamic acid as a neurotransmitter, [3H]D-aspartate ([3H]D-Asp) was chosen as a marker, since it is a metabolically inert amino acid known to be taken up by the same affinity mechanism as L-aspartate and L-glutamate. Adult rats and cats received injections of 50 nl to 1.5 microliter of [3H]D-Asp (500 microCi/microliter) in the dorsal horn of cervical segments (C3 to C6). At 9 to 48 hr after injection, all animals were perfused with 5% glutaraldehyde. After sections were processed for autoradiography, the DRG neurons situated most closely to the injection site were chosen from representative cases, and the number and cross-sectional area of labeled and unlabeled perikarya with a nucleolus in the plane of the section were calculated. In rats, about 4% of the sampled DRG neurons were autoradiographically labeled, and the mean perikaryal area of these neurons was about twice that of unlabeled perikarya. In cats, the percentage of labeled perikarya ranged between 6.5% and 13.27% of the sampled population. The ratio of the mean perikaryal area of labeled neurons to that of unlabeled neurons ranged between 1.6 and 2.5. In a control cat injected with [3H]proline at C7, all perikarya in the C7 DRG were autoradiographically labeled. However, with injection of [3H]gamma-aminobutyric acid ([3H]GABA) selective retrograde labeling was observed. Quantitative data in rat showed that perikarya labeled at the C6 level after injection of this amino acid constituted about 8% of the sample population in C6 DRG. The ratio of the size of labeled to unlabeled perikarya was 2.02. In one cat injected with [3H]GABA at caudal C3, the largest number of labeled perikarya were in C4 DRG and comprised up to 5.32% of the sampled population. The ratio of the size of labeled to unlabeled perikarya was 1.57. The results in cases of injection with [3H]D-Asp may be interpreted as consistent with the idea that a fraction of DRG neurons use glutamate and/or aspartate as neurotransmitter(s).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

In order to study the callosal connections of the hand sensory field of the second somatosensory area of the monkey, experiments were carried out by combining the method of retrograde neuronal tracing with microelectrode recording. In six monkeys, Macaca Irus, single or multiple (5-8) injections of horseradish peroxidase (HRP) were performed into the cortex of the parietal operculum of one side. Neurones retrogradely labelled with HRP (callosal neurones) were found in the post-central gyrus and in the parietal operculum of the contralateral hemisphere. Microelectrode recording from this hemisphere showed that the cortical zones of both the first and the second somatosensory area containing neurones excited by sensory stimulation of the contralateral hand also contained HRP-positive neurones.