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.     

Light-induced Fos expression in phosphate-activated glutaminase- and neurofilament protein-immunoreactive neurons in cat primary visual cortex

Previous double-stainings in the cat visual cortex [E. Van der Gucht, S. Clerens, K. Cromphout, F. Vandesande, L. Arckens, Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex, Eur. J. Neurosci. 16 (2002) 1620-1626] showed that a minority of Fos-immunoreactive nuclei was located in distinct subclasses of inhibitory neurons following sensory stimulation. This report describes double-stainings between Fos and phosphate-activated glutaminase (PAG) or Fos and neurofilament protein (SMI-32) revealing that, following a short-term visual experience, Fos is also expressed in neurochemically distinct subpopulations of non-GABAergic, pyramidal neurons in supra- and infragranular layers of cat area 17.     

Differential vulnerability of neurochemically identified subpopulations of retinal neurons in a monkey model of glaucoma

The vulnerability of subpopulations of retinal neurons delineated by their content of cytoskeletal or calcium-binding proteins was evaluated in the retinas of cynomolgus monkeys in which glaucoma was produced with an argon laser. We quantitatively compared the number of neurons containing either neurofilament (NF) protein, parvalbumin, calbindin or calretinin immunoreactivity in central and peripheral portions of the nasal and temporal quadrants of the retina from glaucomatous and fellow non-glaucomatous eyes. There was no significant difference between the proportion of amacrine, horizontal and bipolar cells labeled with antibodies to the calcium-binding proteins comparing the two eyes. NF triplet immunoreactivity was present in a subpopulation of retinal ganglion cells, many of which, but not all, likely correspond to large ganglion cells that subserve the magnocellular visual pathway. Loss of NF protein-containing retinal ganglion cells was widespread throughout the central (59–77% loss) and peripheral (96–97%) nasal and temporal quadrants and was associated with the loss of NF-immunoreactive optic nerve fibers in the glaucomatous eyes. Comparison of counts of NF-immunoreactive neurons with total cell loss evaluated by Nissl staining indicated that NF protein-immunoreactive cells represent a large proportion of the cells that degenerate in the glaucomatous eyes, particularly in the peripheral regions of the retina. Such data may be useful in determining the cellular basis for sensitivity to this pathologic process and may also be helpful in the design of diagnostic tests that may be sensitive to the loss of the subset of NF-immunoreactive ganglion cells.     

Fos induction in subtypes of cerebrocortical neurons following single picrotoxin-induced seizures

In adult rats single seizures of varying behavioural severities were caused by slow, systemic infusion of picrotoxin, an antagonist of the Cl⁻ channel at the GABA_A receptor. We used a double labelling immunohistochemical method to define the subclasses of neurons that contained Fos protein following seizures. In four cortical regions (piriform, entorhinal, motor and sensory) neuronal subclasses were defined with antibodies against the calcium-binding proteins calbindin D-28K, parvalbumin and calretinin (aspiny neurons), and neurofilament protein (spiny neurons). The remaining spiny neuron population was estimated by subtraction of defined subclasses from total neuronal numbers determined from Nissl stain. After seizures, most of the calbindin D-28K immunoreactive interneurons (> 80%) and many of the unlabelled spiny neurons (60–80%) were Fos positive. Co-localisation of Fos was found in about 30% of parvalbumin, calretinin and neurofilament protein immunoreactive neurons. Paradoxically, mild seizures were associated with induction of Fos in up to 80% of cortical cells and more severe seizures with 60%, the difference being due to different levels of Fos induction in spiny neurons. These results also demonstrate that seizures induce Fos predominantly in excitatory cortical neurons.     

Paucity of glutaminase-immunoreactive nonpyramidal neurons in the rat cerebral cortex.

Glutaminase has been considered to be a synthesizing enzyme of transmitter glutamate in pyramidal neurons of the cerebral cortex. In the present study, an attempt was made to examine with a double immunofluorescence method whether or not nonpyramidal neurons of the cerebral cortex are immunoreactive for glutaminase. Glutaminase was stained with mouse anti-glutaminase IgM and FITC-labeled anti-[mouse IgM] antibody. In the same section, parvalbumin (PA), calbindin (CB), choline acetyltransferase (CAT), vasoactive intestinal polypeptide (VIP), corticotropin releasing factor (CRF), cholecystokinin (CCK), somatostatin (SS), or neuropeptide Y (NPY) was visualized as a marker for nonpyramidal neurons with an antibody to each substance, biotinylated secondary antibody and Texas Red-labeled avidin. Virtually no glutaminase immunoreactivity was seen in PA-, CB-, CAT-, VIP-, CRF-, CCK-, SS-, or NPY-immunoreactive neuronal perikarya in the neocortex and mesocortex (cingulate and retrosplenial cortices), although it was detected in a few PA-, CB-, VIP-, CCK-, SS-, or NPY-immunoreactive nonpyramidal neurons in the piriform, entorhinal, and hippocampal cortices. PA- and CB-positive neurons have been reported to constitute the major population of GABAergic neurons in the cerebral cortex. Thus, the present results, together with the previous reports, suggest that most GABAergic, cholinergic and peptidergic nonpyramidal neurons in the neo- and mesocortex do not contain glutaminase.     

Interneurons in the rat striatum: relationships between parvalbumin neurons and cholinergic neurons

A pre-embedding double-labeling immunocytochemical method was used to examine the synaptic relationships between cholinergic neurons and the parvalbumin immunoreactive (PV+) neurons. The PV+ neurons were labeled by silver-intensified colloidal gold particles, and the cholinergic neurons by immunoperoxidase reaction products. Cholinergic and PV+ axon terminals form synapses with both the somata and dendrites of PV+ neurons, as well as unlabeled medium-sized somata with round un-indented nuclei, a typical characteristic of the medium spiny projection neurons. These observations suggest that the PV+ and the cholinergic neurons have converging influences on both the projection neurons and the PV+ interneurons in the striatum.     

The laminar distributions and postnatal development of neurotransmitter and neuromodulator receptors in cat visual cortex

We review efforts to further understand the development and nature of sensory processing mechanisms in the cat visual cortex. In vitro autoradiographic and homogenate assay techniques have been employed to determine the laminar distribution and characteristics of various neurotransmitter and neuromodulator receptor populations during postnatal development. Each receptor population shows a distinct laminar-specific pattern of binding, which, in most cases, is age-dependent. Changes in receptor number and affinity are also observed during postnatal development. These findings indicate that major alterations in the basic chemical circuitry of cat visual cortex are a normal feature of postnatal maturation and may play a role in plasticity mechanisms.     

Sets of neurons in somatic cerebral cortex of the cat and their ontogeny

The process of set formation is briefly reviewed and five monothetic schemes for classification of neurons in the somatic cerebral cortex are described. Criteria for evaluation of neuronal sets are presented and applied to the five different monothetic classification schemes. Classification by size and distribution of peripheral receptive fields orders existing data on cortical neurons better than classification by possession of an axon in the pyramidal tract, by modality, by lability of receptive field, or by ‘lemniscal properties’; however, no monothetic scheme orders all the data. A useful polythetic scheme, using s and m terminology is suggested. The ontogeny of the cerebral cortex is reviewed in detail. It is suggested that sa. neurons are Golgi type II neurons while m neurons are Golgi type I neurons. The hypothesis is presented that wide-field or m neurons develop and are recognizable before small-field or sa. neurons in ontogeny. Evidence regarding this hypothesis is indirect, often conflicting, but suggestive that the hypothesis may be correct. The idea that m neurons may also be phylogenetically older than sa neurons is presented and shown to be consistent with ontogenetic data and interpretations.     

Lamina-specific differences of visual latencies following photic stimulation in the cat striate cortex

Vertical penetrations were made in the part of the cat's area 17 subserving central vision. Single units were recorded every 100-200 micron and the recording sites histologically reconstructed. Visual latencies of complex cells following whole-field ON/OFF stimulation were determined as a function of cortical depth. The shortest latencies were found for cells in layers IV and VI (mean ON response 44 ms/OFF response 51 ms for IV and 43 ms/42 ms for VI). In contrast, mean latencies in layer V were 54 ms/57 ms, while cells of the supragranular layers had the longest mean latencies of 62 ms/73 ms. Furthermore, this analysis revealed significant differences of between 10 and 30 ms on the average between the individual layers (the differences between individual cells can even be in the range of more than 60 ms). These differences cannot easily be explained by conduction time and synaptic delays, which both can account for a few ms only. One possible explanation is based on a dynamic model of intracortical information processing with multiple positive feedback loops.     

Immunocytochemical and ultrastructural evidence of dendritic degeneration in motor neurons of aluminum-intoxicated rabbits

Summary Using immunocytochemical and ultrastructural methods, we observed extensive and characteristic dendritic changes in motor neurons of rabbits inoculated intracisternally with aluminum phosphate. Anti-microtubule-associated protein 2 immunostaining revealed markedly reduced immunoreactivity in motor neuron dendrites and a reduced number of dendritic trees in aluminum phosphate-intoxicated rabbits. These dendritic changes were confirmed at the ultrastructural level; neurofilamentous accumulations, membranous inclusions and disrupted microtubules were common features of motor neuron dendrites, but less prominent in motor neuron axons. These observations suggest that dendrites are characteristically involved in aluminum intoxication in addition to the widely reported accumulation of phosphorylated neurofilament in perikarya and axons.     

GABAergic neurons in the rabbit visual cortex: percentage, layer distribution and cortical projections

6250 neurons yielding either callosal or inter-areal ipsilateral projections extrinsic to area 17 was GABAergic. Comparing these findings with those reported for other mammals, it seems that the incidence and distribution of GABAergic neurons in the visual cortex is similar in rabbits and rats. In contrast to rats but akin to higher mammals, no GABAergic neuron was found to furnish cortico-cortical connections to area 17 other than intrinsic connections.     

Polyneuronal innervation of spiny stellate neurons in cat visual cortex

Our hypothesis was that spiny stellate neurons in layer 4 of cat visual cortex receive polyneuronal innervation. We characterised the synapses of four likely sources of innervation by three simple criteria: the type of synapse, the target (spine, dendritic shaft), and the area of the presynaptic bouton. The layer 6 pyramids had the smallest boutons and formed asymmetric synapses mainly with the dendritic shaft. The thalamic afferents had the largest boutons and formed asymmetric synapses mainly with spines. The spiny stellates had medium-sized boutons and formed asymmetric synapses mainly with spines. We used these to make a “template” to match against the boutons forming synapses with the spiny stellate dendrite. Of the asymmetric synapses, 45% could have come from layer 6 pyramidal neurons, 28% from spiny stellate neurons, and 6% from thalamic afferents. The remaining 21% of asymmetric synapses could not be accounted for without assuming some additional selectivity of the presynaptic axons. Additional asymmetric synapses may come from a variety of sources, including other cortical neurons and subcortical nuclei such as the claustrum. Of the symmetric synapses, 84% could have been provided by clutch cells, which form large boutons. The remainder, formed by small boutons, probably come from other smooth neurons in layer 4, e.g., neurogliaform and bitufted neurons. Our analysis supports the hypothesis that the spiny stellate receives polyneuronal innervation, perhaps from all the sources of boutons in layer 4. Although layer 4 is the major recipient of thalamic afferents, our results show that they form only a few percent of the synapses of layer 4 spiny stellate neurons.     

Interrelation among properties of visual cortex neurons in the cat

In acute experiments on immobilized cats 13 functional characteristics of 96 visual cortex neurons were investigated. Regressional and cluster analyses were used to divide these neurons into two subgroups with different density and degree of connections between characteristics. The receptive fields of cells of the first subgroup were localized relatively centrally in the visual field, those of the second subgroup were localized more often on the periphery. A valuable correlation was found in the half of the studied characteristics. In each subgroup the more centrally localized cells with small receptive fields had relatively shorter latencies, lower thresholds, shorter temporal summation, wider intensity range and greater differential sensitivity; their responses were phasic, with high-frequency discharges. The density of valuable correlation of the characteristics varied from 0.21 to 0.99. The amount of these correlations in the first subgroup was two times higher than in the second one. The possible mechanisms of the correlation between the properties of the visual cortex neurons are discussed, as well as their differences in two subgroups and in the cortex and LGB.     

Response properties and morphological identification of neurons in the cat motor cortex

Intracellular recordings and morphological identification of neurons using intracellular HRP staining were performed in the cat motor cortex. By thalamic ventrolateral (VL) or cerebellar nucleus stimulation, pyramidal cells in layer III, fast pyramidal tract neurons (PTNs) and stellate cells in layers II and III were activated with short latency and fast rising EPSPs, while pyramidal cells in layer II and slow PTNs showed longer latency and slow rising EPSPs. This difference may be related to activation through the deep and superficial thalamocortical projections. Although pyramidal cells in layer VI did not respond orthodromically to VL or cerebellar stimulation, some of them proved to receive the recurrent action of PTNs because of the response to stimulation of the cerebral peduncle (CP). One aspinous stellate cell in layer III was activated by CP as well as VL stimulation. This cell was supposed to be an inhibitory interneuron responsible for both recurrent and VL-evoked inhibition.     

Somatodendritic minicolumns of output neurons in the rat visual cortex

The apical dendrites of the pyramidal neurons of the cerebral cortex form radial bundles in all species and areas. Using microtubule-associated protein (MAP)2 immunostaining and Voronoi tessellation analysis in the rat visual cortex, we obtained objective criteria to define dendritic bundles in tangential sections: in supragranular layers of the rat visual cortex we found bundles of 6-6.4 dendrites, at a density of 1929 bundles/mm(2) and a centre-to-centre distance of 27 micro m. Using lipophilic tracers to label different pyramidal cell populations, based on the same criteria as in MAP2-immunostained material, we found that in the rat visual cortex the bundles consist of neurons with specific targets. Neurons projecting to the ipsi- or contralateral cortex form bundles together and with neurons projecting to the striatum, but not with those projecting to the superior colliculus, dorsal division of the lateral geniculate nucleus or through the cerebral peduncle. The latter neurons form bundles with neurons projecting to the striatum. Thus, the cerebral cortex is organized in minicolumns of output neurons visible at the earliest ages studied (P3), which might have a higher probability of being interconnected than those outside.     

Intracortical microstimulation of neurons in the visual cortex of the cat

The response of visual cortex neurons to local intracortical microstimulation was measured in the anesthetized cat. When the recording microelectrode was very close (about 20 micrometers) to the tip of the stimulating electrode, threshold currents as low as 10 micro A were capable of firing neurons. Over a 20-fold range in distance from the site of stimulation, an 80-fold increase in threshold current was observed. The mean latency of activation for 30 neurons tested with intracortical stimulation was 2.88 +/- 0.45 msec. The majority of these cells were probably synaptically activated. The mean threshold current for these neurons was 0.55 +/- 0.12 mA (N = 30). These values were significantly smaller than the thresholds found previously when stimulating electrodes were located on the pia-arachnoid surface of the visual cortex.     

Parvalbumin in the human anterior cingulate cortex: morphological heterogeneity of inhibitory interneurons

The calcium-binding protein parvalbumin (PV) is a marker for a certain subset of GABAergic cortical interneurons. In the present study, indirect immunocytochemistry with an antibody against PV was performed on serial sections of human anterior cingulate cortex (Brodmann's area 24), an important relay centre of the limbic system. PV-positive structures are distributed in a layer- and cell type-specific manner. Based on morphological features and laminar distribution pattern, PV-immunoreactive interneurons are subdivided into eight different classes. PV immunoreactivity within the neuropil comprises dendritic and axonal processes. Area 24 contains two densely immmunolabelled neuropil bands in layers III and Vb. Axon cartridges are preferably located in layers V and VI. The results provide a 'PV immunoarchitecture' as a basis for further studies of PV immunoreactivity under pathological conditions. PV is assumed to play a role in maintaining calcium homeostasis in nerve cells, and to modulate neuronal excitability and resistance to biochemical damage. On the other hand, PV immunoreactivity has recently been shown to undergo characteristic changes during different stages of brain maturation. Therefore, examination of PV-positive structures will provide new insights into cortical circuitry in neurodegenerative as well as neurodevelopmental disorders.     

Distribution of neurons in the pigmented rabbit''s visual cortex

Past observations revealed a relationship between mammalian retinal ganglion cell distribution and cortical and subcortical projection of the rabbit's visual field. High ganglion cell density sectors of the retina claimed larger areas in their cortical and subcortical projections (with a high magnification factor) than retinal areas with low ganglion cell density. In this study, it was observed that distribution of nonpyramidal neurons in laminae IV and VI of the pigmented rabbit's visual cortex was not uniform. The nonuniformity in layer IV roughly followed the variation in the magnification factor in the cortical projection of the animal's visual field. The highest density of nonpyramidal neurons in layer IV was observed in the cortical area with the highest magnification factor.     

A subpopulation of primate corticocortical neurons is distinguished by somatodendritic distribution of neurofilament protein

In recent immunohistochemical studies of human and monkey neocortex we observed that the somatodendritic distribution of neurofilament protein appears to be restricted to a subpopulation of pyramidal neurons. To further characterize this apparent specificity in cytoskeletal organization, combined retrograde tract tracing and immunohistochemical methods were used to examine the extent to which neurons from different cortical areas providing a projection to prefrontal cortex have a somatodendritic distribution of neurofilament proteins. These studies revealed that the proportion of neurons providing a projection from different cortical areas to prefrontal cortex varied from nearly 30% to 90%, and appeared to be related to the functional nature of the projection.     

Neurofilament protein: a selective marker for the architectonic parcellation of the visual cortex in adult cat brain.

In this immunocytochemical study, we examined the expression profile of neurofilament protein in the cat visual system. We have used SMI-32, a monoclonal antibody that recognizes a nonphosphorylated epitope on the medium- and high-molecular-weight subunits of neurofilament proteins. This antibody labels primarily the cell body and dendrites of pyramidal neurons in cortical layers III, V, and VI. Neurofilament protein-immunoreactive neurons were prominent in 20 visual cortical areas (areas 17, 18, 19, 20a, 20b, 21a, 21b, and 7; posteromedial lateral, posterolateral lateral, anteromedial lateral, anterolateral lateral, dorsal lateral, ventral lateral, and posterior suprasylvian areas; anterior ectosylvian, the splenial, the cingulate, and insular visual areas; and the anterolateral gyrus area). In addition, we have also found strong immunopositive cells in the A laminae of the dorsal part of the lateral geniculate nucleus (dLGN) and in the medial interlaminar nucleus, but no immunoreactive cells were present in the parvocellular C (1-3) laminae of the dLGN, in the ventral part of the LGN and in the perigeniculate nucleus. This SMI-32 antibody against neurofilament protein revealed a characteristic pattern of immunostaining in each visual area. The size, shape, intensity, and density of neurofilament protein-immunoreactive neurons and their dendritic arborization differed substantially across all visual areas. Moreover, it was also obvious that several visual areas showed differences in laminar distribution and that such profiles may be used to delineate various cortical areas. Therefore, the expression of neurofilament protein can be used as a specific marker to define areal patterns and topographic boundaries in the cat visual system.