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Expression patterns of the immediate early gene c-fos and of other genes including those for the alpha-subunit of type II Ca2+/calmodulin-dependent protein kinase (CaMKII alpha), 67-kDa glutamic acid decarboxylase (GAD), and the alpha 1-, beta 2-, and gamma 2-subunits of the GABAA receptor were described in the spinal cord of normal cats and following peripheral nerve stimulation. As revealed by in situ hybridization histochemistry, CaMKII alpha messenger RNA (mRNA) is normally distributed only in cells of Rexed's laminae I-IV, whereas GAD mRNA is expressed by subpopulations of cells in all laminae, with the heaviest hybridization signal found in laminae I-III and medial parts of laminae V and VI. The three GABAA receptor subunits have varying expression patterns in the laminae. All of them are expressed by many cells located in the base of the dorsal horn and the intermediate zone, but only the gamma 2-subunit is intensely expressed by motoneurons. Single-pulse, electrical stimulation of the sciatic or median and ulnar nerve of anesthetized cats at a pulse rate of 1/s for 6-8 h failed to induce observable changes in gene expression for CaMKII alpha, GAD, or for the three subunits of the GABAA receptor; although immunoreactivity for the protein products of c-fos (or c-fos-related genes) was markedly upregulated in some neurons of the dorsal horn and the intermediate zone. Therefore, under the present experimental conditions, upregulation of the immediate early gene c-fos (or c-fos-related genes) is not associated with changes in expression of late-effector genes potentially involved in central nervous system plasticity.

Long-train tetanic stimulation of the cerebral cortex induces long-term changes in the excitability of cortical neurons, while short-train electrical stimulation does not. In the present study, we show that both forms of stimulation when applied to rat motor cortex for 4 h enhance c-fos expression, but only tetanic stimulation, when imposed upon short-train stimulation, modulates gene expression for 67-kDa glutamic acid decarboxylase (GAD) and alpha Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha). Gene expression for beta Ca2+/calmodulin-dependent protein kinase II is not affected by either stimulation mode. GAD messenger RNA (mRNA) is increased from 1 h after the end of tetanization to the longest poststimulus survival time investigated (14 h). CaMKII alpha mRNA is decreased 1-3 h after the end of tetanization but thereafter returns to prestimulus levels. These results imply not only that mechanisms underlying neocortical plasticity are stimulus-dependent but also that they involve reciprocal changes in molecules regulating the balance of excitation and inhibition.

The thalamic reticular nucleus (RTN) exerts an inhibitory influence upon the dorsal thalamus. During wakefulness and arousal, RTN neurons fire tonically, whereas during slow-wave sleep they fire rhythmic high frequency bursts. The effects produced by RTN inhibition upon the activity of dorsal thalamic neurons will therefore vary in relation to the firing mode of the RTN neurons. In the present study, we compared the effects of oscillating RTN neurons and of RTN neurons tonically activated with glutamate on the response profiles of single units reacting to controlled cutaneous stimulation in cat ventroposterior lateral thalamic nucleus (VPL). Experiments were performed under light barbiturate anesthesia and prior to the glutamate activation of the RTN, both RTN and VPL neurons showed spontaneous bursting patterns of activity consistent with the oscillatory mode. Typically, a cutaneous stimulus evoked a short latency excitatory response in VPL followed by a period of complete inhibition termed post-stimulus inhibition (PSI). In many neurons, the PSI was followed by a period of increased activity termed post-inhibitory excitation (PIE). Ejection of glutamate in the identified somatosensory division of the RTN shifted the oscillatory firing of its neurons to a high tonic mode and usually resulted in a decrease in VPL neuronal activity. Significant variations were observed in the occurrence and the magnitude of the effects among the different components of neuronal activity examined. Tonic activation of the RTN resulted in a significant reduction of ON- and OFF-PIEs in 81% of cases (30/37) and of spontaneous activity in 67% (22/33). In contrast, the response to a cutaneous stimulus was decreased in only 29% of cases (17/59) and was significantly increased in 24% (14/59). Tonic activation of the RTN by glutamate resulted in little change in the firing pattern of VPL neurons, and both short and long spike intervals were affected in a similar proportion. We conclude that the components of VPL neuronal activity most affected by switching RTN neurons from the oscillatory to the tonic mode are those normally dependent upon RTN neuronal oscillation. The present results also suggest that lowering background activity, such as occurs during the transition from sleep to wakefulness, is a factor leading to increase in the responsiveness of dorsal thalamic neurons.

The primary auditory cortex (A1) of monkeys was investigated by immunohistochemistry, using antibodies to gamma-aminobutyric acid (GABA), to the calcium binding proteins parvalbumin and calbindin, and to certain proteoglycan epitopes. The two calcium binding proteins were found to be localized in subpopulations of GABAergic neurons. Parvalbumin immunoreactive cells were mostly found in the middle layers of the cortex. Parvalbumin immunoreactivity was found in fibres in the white matter underlying A1 and a particularly dense concentration of parvalbumin immunoreactive fibers and terminals occurred in layer IV suggesting that a significant population of geniculocortical fibers is also parvalbumin positive. Calbindin positive cells were mostly located in superficial layers and in these layers the neuropil staining was also dense. Two monoclonal antibodies (MAbs) raised against monkey brain tissue and which had previously been shown to recognize neuronal surface antigens stained overlapping subpopulations of GABAergic cells. Occasional pyramidal cells were also immunoreactive. Most of the MAb positive cells were found in the middle layers and all were parvalbumin but not calbindin immunoreactive. Although the physiological roles in the brain for calcium binding proteins and the relevant cell surface markers have not yet been clarified, the presence of these markers in selected subpopulations of cells suggests the existence of functionally distinct circuits in AI cortex.

The organization of corticocortical connections in the representation of the forepaw in cat primary somatosensory cortex (SI) was studied following injections of various tracers into different cortical cytoarchitectonic areas. Small injections of horseradish peroxidase, wheat germ agglutinin-conjugated HRP, Phaseolus vulgaris leukoagglutinin, or fast blue were placed into the representation of the forepaw in areas 3b, 1, or 2. The positions of labeled neurons in SI and the surrounding cortical areas were plotted on flattened surface reconstructions to determine the organization of the corticocortical connections within SI. A strong, reciprocal projection linked the two forepaw representations which have been described in area 3b and the part of area 2 which lies in the anterior bank of the lateral ansate sulcus (see Iwamura and Tanaka 1978a, b). Dense projections also linked these areas with SII, as previously reported (Burton and Kopf 1984a). Additional projections to area 3b arose primarily from areas 3a and 1. Projections to area 2 were more widespread than those to area 3b, and arose from all other areas of SI as well as from areas 4 and 5a. All injections into SI tended to label groups of neurons which lay in mediolateral strips. Corticocortical projection neurons which were most heavily labeled by SI injections were pyramidal cells in layer III. Additional projections from area 2 to 3b, area 5a to 2, and SII to areas 2 and 3b arose from layer VI as well. Although neurons of layers III and VI were always the most densely labeled, large injections into SI labeled neurons in layers II and V as well.

Immunocytochemical methods have been combined with serial thin section analysis to study the synaptic organization of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreactive terminals in the ventral posterior nucleus of the cat and monkey thalamus. One hundred 5-HT immunoreactive terminals from the cat and approximately forty 5-HT and TH immunoreactive terminals from the monkey were selected for analysis in serial thin sections. Only 7-10% of the immunoreactive terminals could be revealed to form conventional synaptic contacts. Most of these could be identified as the asymmetrical type. Dendritic shafts belonging to relay neurons were the major targets of these monoamine immunoreactive terminals. The remainder made intimate membrane associations with relay cell dendrites and somata or with presynaptic dendrites of interneurons, but no overt membrane specializations could be detected. The present results suggest that the modulation of thalamocortical relay function by brainstem monoamine pathways in the somatosensory thalamus may occur by release of transmitters at atypical contact sites.

Small iontophoretic injections of the lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), were made into different layers of the primary auditory cortex (AI) of cats. Injections in layer I labeled two types of morphologically distinct fibers in layer I as well as a smaller number of axons in layers II and III. Layer II injections labeled descending axons that produced a dense plexus of terminal fibers in layers I-III of both AI and adjacent auditory fields. Injections in layer III also labeled a dense plexus of axon collaterals at the junction of layers V and VI and labeled patches of terminal fibers in both AI and adjacent auditory fields. These were densest in layers I-III but usually extended into layers IV and V as well. The patches were partly formed by axon collaterals of layer III pyramidal cells that traveled for over 4 mm in the gray matter. Injections confined to layer IV labeled axons in all layers of the cortex but none of these axons appeared to reach the white matter. The axons spread laterally in layer IV and up into the superficial layers and ramified especially layer I. Injections in layers V and VI labeled axons in all layers' of the cortex but these were densest in the deep layers where labeling was fairly homogeneous. In the upper layers the labeling was arranged in semi-discrete patches. Large injections involving layers I-III were studied in tangential sections. Between 3 and 8 patches of terminal labeling were observed in AI and these were mainly arranged in a band with its long axis aligned approximately in the dorsoventral direction. However dense patches of terminal labeling also occurred both anterior and posterior to the injection site. In selected experiments portions of the tonotopic map in AI were mapped by single unit recording and subsequently the map was related to patches of anterogradely labeled fibers that surrounded injections of PHA-L. Rows of dorsoventrally oriented patches were among cells with a similar best frequency to those in the injection site. However patches located anterior or posterior to the injection site were among cells with higher or lower best frequencies. Two injections of PHA-L close together produce different patterns of labeling. One of the injections usually produces one or more patches that has no correlate among the patches of fibers labeled by the adjacent injection. This is clearest when one of the injections is made with biotinylated PHA-L that can be visualized directly without the use of primary antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase (AChE) activity, demonstrated histochemically, defines an area of cortex on the middle ectosylvian gyrus that appears to correspond to the cytoarchitectonically defined area 41 and the physiologically defined primary auditory area (AI). In this area there are high levels of AChE in layers III, IV and VI while in the surrounding areas there are comparatively low levels of enzyme in these layers. The monoclonal antibody CAT 301, which was raised against a cell surface proteoglycan, also defines this area. There are high levels of CAT 301 immunoreactivity in cell bodies and the neuropil of layer III and an absence of very large immunoreactive neurons in layer V. Furthermore there are higher levels of the calcium binding protein, parvalbumin and the metabolic enzyme, cytochrome oxidase, in layers III and IV of AI, than in most of the surrounding cortex. By contrast the distribution of the calcium binding protein, calbindin and the distribution of myelinated fibers are similar in area 41 and the surrounding areas.

The areal and laminar distribution of GABAA receptor immunoreactivity was examined in fetal, early postnatal and adult monkey sensory-motor cortex by using a monoclonal antibody to the purified GABAA receptor complex (Vitorica et al. 1988). GABAA receptor immunoreactivity was distributed throughout the neuropil, often outlining the unstained somata of pyramidal and non-pyramidal cells. In all areas of the adult sensory-motor cortex, layers I-IIIA exhibited the most intense immunostaining. In deeper layers of the four cytoarchitectonic fields of the first somatic sensory area (SI), layers IIIB and V were lightly stained and alternated with somewhat more intensely stained layers IV and VI. In deeper layers of area 4, the deeper half of layer IIIA through layer VA was lightly immunostained, but layers VB and VI were slightly more intensely immunoreactive. A variable number of nonpyramidal cell somata in the cortex and underlying white matter showed immunoreactive staining. GABAA receptor immunoreactivity was present throughout the sensory-motor cortex from the youngest fetal age examined (E121), but the pattern of immunostaining differed from that in the adult. In all areas, the densest immunoreactivity was found in a diffuse band in layers III and IV and in the subplate zone. Within the subplate zone, the presence of receptor immunoreactivity and some intensely stained neuronal somata at all fetal ages suggests the presence of a synaptic neuropil. With increasing age, gradual changes in the distribution of receptor immunoreactivity occurred, resulting in an adult-like pattern of immunostaining by postnatal day 1.5. These results show that laminar pattern of GABAA receptor distribution closely follows the major concentrations of GABA immunoreactive neurons in adults and it is suggested that laminar changes seen in development are associated with the establishment of afferent connections and inhibitory circuits in the sensory-motor cortex.

Retrograde axoplasmic transport of differently colored fluorescent dyes was used to determine the distributions and relative proportions of cells in the ventrobasal complex of the monkey thalamus that project to each of the architectonic fields of the first somatic sensory cortex. Fast blue was injected into portions of area 3a identified by first recording short latency, multiunit responses to electrical stimulation of Group I afferents in a muscle nerve of the forelimb or hindlimb. Nuclear Yellow was later injected into a part of area 2 responding to maximal electrical stimulation of the same nerve. In experiments that served as controls, Fast Blue was injected into area 3b and Nuclear Yellow into area 1. The results confirm the division of the ventrobasal complex into: (i) a central core with an inner part projecting to area 3b and a surrounding part projecting area 1; (ii) a peripheral shell projecting to areas 3a and 2 (Jones et al. 1982). A far greater proportion of VB cells projects to areas 3b or 3a than to areas 1 or 2. In portions of the ventrobasal complex projecting to two areas, no cells were double labelled provided that the injections of blue and yellow dyes did not overlap. The results, thus, show a lack of collateral thalamocortical projections to the fields of the postcentral gyrus and, when taken in conjunction with other data, imply the independent relay of modality specific information through the thalamus.

Immunocytochemistry of fetal and adult monkey thalamus reveals a dense concentration of tachykinin immunoreactive fibers and terminals in the dorsolateral part of the VPM nucleus in which the contralateral side of the head, face and mouth is represented. The immunoreactive fibers enter the VPM nucleus from the thalamic fasciculus and electron microscopy reveals that they form large terminals resembling those of lemniscal axons and terminating in VPM on dendrites of relay neurons and on presynaptic dendrites of interneurons. Double labeling strategies involving immunostaining for tachykinins after retrograde labeling of brainstem neurons projecting to the VPM failed to reveal the origin of the fibers. The brainstem trigeminal nuclei, however, are regarded as the most likely sources of the VPM-projecting, tachykinin positive fibers.

Single and multiunit mapping was used to determine the extent of the representation of ipsilateral structures in the ventral posterior medial (VPM) nucleus of the thalamus in cynomolgus monkeys. The extent of the VPM occupied by terminations of afferent fibers arising in the ipsilateral principal trigeminal nucleus was also determined by anterograde transport of horseradish peroxidase. Both methods indicate that most of the medial half of VPM is occupied by the ipsilateral representation. This is much larger than previously suspected. Units in the medial half of VPM have small, well localized receptive fields on the ipsilateral side of the lower lip, tongue and palate, in the ipsilateral cheek pouch and on the ipsilateral teeth. The representation is largest for the ipsilateral side of the tongue and the cheek pouch. Most units in the lateral half of VPM have small, contralateral receptive fields. Few units in VPM have bilateral receptive fields. VPM is clearly distinguishable by cytochrome oxidase (CO) staining. Anteroposteriorly elongated, CO-positive aggregations correspond to elongated aggregations of units with the same or closely similar receptive fields, especially in the medial, ipsilateral representation.

The morphology of single neurons in area 3b of cat primary somatosensory (SI) cortex was examined after horseradish peroxidase (HRP) injections. Neurons were labeled either by intracellular injection of HRP following intracellular recording or by small extracellular iontophoretic HRP injections. Both pyramidal and nonpyramidal neurons were labeled and reconstructed from serial sections. Their axons had local, interlaminar and interareal patterns of termination. Most neurons formed local axonal fields around their cell bodies and dendrites. Pyramidal neurons in cortical layer IV sent axons up into layers II and III, neurons in layers II and III sent axons down to layer V, and layer V neurons sent axons to layer VI as well as back to the upper layers. Layer VI neurons sent axons back to the upper cortical layers in a unique bowl-shaped pattern. The horizontal distribution of axons of pyramidal cells in layer III was extremely widespread. Axons of layer III neurons in area 3b terminated within 3b and area 1, but not in other areas of SI. Layer III neurons in area 1 distributed axon collaterals to all fields of SI as well as projecting a main axon to motor cortex. In general, the axon collaterals of area 3b pyramidal cells outside layer III remained confined to area 3b. Most of the nonpyramidal neurons labeled were basket cells in layers III and VI. These neurons formed dense axonal fields around their cell bodies, and none of their axons could be followed into the underlying white matter. The results of the present study demonstrate that area 3b somatosensory cortical neurons and their axons are vertically organized in a manner similar to that reported for other sensory cortical areas. They also show that widespread horizontal connections are formed by pyramidal neurons of layer III, and that these horizontal axons can travel for great distances in the cortical grey matter.

A double labeling method that permits accurate delineation of the terminals of medial lemniscal fibers was used to determine whether thalamic neurons projecting to motor cortex in the cat are in a position to be contacted by such terminals. Thalamic neurons in the VL nucleus were retrogradely labeled by injections of fluorogold placed in the cytoarchitectonically defined area 4, while lemniscal axons and their terminal boutons were anterogradely labeled, in a Golgi-like manner, from injections of Fast Blue placed under physiological control in different parts of the contralateral dorsal column nuclei. In additional experiments, spinothalamic fibers were similarly labeled by injections of Fast Blue in the spinal cord. The results reveal that there is no significant overlap in the distributions of lemniscal terminals and motor cortex-projecting neurons and that no somata or proximal dendrites of motor cortex-projecting neurons are in a position to receive lemniscal terminals. Spinothalamic terminals, on the other hand, end in clusters around motor cortex-projecting neurons in the VL nucleus as well as in other nuclei and are a more likely route for short latency somatosensory inputs to the motor cortex.

Immunocytochemical methods were used to study the distribution and ultrastructure of serotonin (5-hydroxytryptamine; 5-HT) immunoreactive fibers innervating the monkey sensory-motor cortex. Beaded 5-HT positive fibers were found in all cortical layers of both areas but with relatively fewer in middle cortical layers. Examination of 2 micron-thick plastic sections at the light microscope level, revealed that the vast majority of the bouton-like structures on the fibers lay in the neuropil and not adjacent to neuronal somata. A few beaded immunoreactive fibers were seen around certain pyramidal and non-pyramidal cell somata, very occasionally forming modest pericellular ramifications. Serial reconstructions made from electron micrographs after resectioning the 2 micron-thick sections, revealed that the dilatations of the fibers are 5-HT positive boutons but the boutons examined rarely formed morphologically identifiable synaptic contacts. Of 191 reconstructed boutons only 5 made contacts with obvious membrane specializations, all of which were of the asymmetrical type. No immunoreactive synaptic contacts were seen on pyramidal cell somata in the cortex, nor on dendrites or somata in the white matter underlying the cortex, although 5-HT positive boutons commonly lay closely adjacent to neuronal profiles in both sites. 5-HT fibers in the cortex and white matter have a similar morphological appearance and both myelinated and unmyelinated types are seen.

Neurons responding to deep or cutaneous stimuli are situated in different parts of the ventrobasal complex. Within the cutaneous region elongated clusters of cells with common place and modality properties project to single columns in the somatic sensory cortex. The present study sought to determine to what extent single lemniscal axons contribute terminals to different regions and to different cell clusters. Lemniscal axons, anterogradely labelled by horseradish peroxidase injected into the medial lemniscus of cats were examined light and electron microscopically. Labelled axons bore one or two, mainly anteroposteriorly oriented, terminal ramifications. These ramifications were relatively small when compared to the length of the complex. Some of the axons bore one or two collaterals that ascended towards the dorsal edge of the complex and formed an additional small ramification there. Electron microscopic analyses of labelled lemniscal axons provided further evidence to that already available that most of their boutons synapse on proximal dendrites of relay neurons and on presynaptic dendrites, presumably belonging to interneurons. A concurrent study of Golgi-impregnated ventrobasal neurons showed three morphological types all with dendritic fields of similar extent. From measurements of the lemniscal terminal ramifications and the counting of counterstained cells it was calculated that 50-120 neurons may receive input from a single terminal ramification. However, because of the restricted extent of the ramifications, the elongated clusters of cells projecting to a single cortical column probably receive input from multiple lemniscal axons and not all members of the cluster receive inputs from the same axons.

Calcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurons show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Histochemical staining for a mitochondrial enzyme, cytochrome oxidase demonstrates elongated, rod-like configurations of probable axon terminals in the trigeminal representation of the monkey somatic sensory thalamus. The stained rods are colocalized with similar aggregations of immunocytochemically stained GABAergic thalamic interneurons. Other data suggest the rods also contain clusters of relay neurons projecting to cortical columns, and they are here demonstrated as somatotopic units by micro-electrode mapping and the distribution of afferent fibers. Similar somatotopic rods can be revealed in the rest of the thalamic body representation by the reduction in cytochrome oxidase staining ensuing from the cutting of selected peripheral nerves.