Stereological analysis of GABAergic neurons and calcium binding protein parvalbumin-containing neurons in the rat somatosensory cortex]

The number of neuronal cells in the rat somatosensory cortex (barrel area) has been estimated by a stereological method, disector, using pairs of toluidine-blue stained plastic embedded 0.5 microns thick sections, 3.0 microns distant from each other. Chemical properties of those disector-counted cells were further revealed by applying postembedding immunocytochemical methods on adjacent semithin sections. Thus we analysed quantitatively the number, distribution and proportion of three types of neurons: 1) GABA negative neurons, 2) GABA-like immunoreactive (GABA-LIR) neurons and 3) a specific calcium binding protein parvalbumin (PV) immunoreactive (PV-IR) neurons, which are a subpopulation of GABA-LIR neurons. The densities of total neurons in the rat somatosensory cortex were 55 x 10(3)/mm3. Of neurons 24% and 12% were GABA-LIR and PV-IR, respectively; almost all PV-IR neurons are GABA-LIR and thus about 50% GABA-LIR neurons are PV positive. The number of total neurons under a unit surface area 1mm2 through the thickness of the somatosensory cortex is 85 x 10(3). GABA-LIR neurons were 20 x 10(3) and PV-IR neurons and PV negative GABA-LIR neurons were 10 x 10(3) and 10 x 10(3), respectively. The laminar distribution of each group of neurons shows prominent differences, indicating that the neuronal composition was different from layer to layer. The density of GABA-LIR neuron was highest in layer IV and especially PV-IR neurons were 2-3 times more numerous in layer IV than in layer II/III, V and VI.     

The parvalbumin-containing nonpyramidal neurons in the rat hippocampus

Summary The calcium-binding protein parvalbumin is considered to be involved in the control of intracellular ion homeostasis of highly active inhibitory neurons. A review of the light and electron microscopical features as well as the identified synaptic connections of these neurons is presented. Parvalbumin-containing neurons are mostly located within or in the vicinity of the granule or pyramidal cell layer. They form a subgroup of GABAergic neurons that has a target specificity for the cell body region. Their fine structural characteristics are identical to those known for hippocampal inhibitory neurons. Parvalbumin-containing neurons are involved in several inhibitory pathways: feed-back inhibition, feed-forward inhibition and disinhibition. The functional implications of our own as well as published data are discussed. Special consideration is given to the possible physiological role of parvalbumin in these neurons.     

Axonal regeneration from GABAergic neurons in the adult rat thalamus

Summary Peripheral nerve grafts were inserted into the thalamus in 27 Sprague-Dawley rats. From 6 weeks to 15 months later, horseradish peroxidase (HRP) was applied to the extracranial end of each graft and sections of the brains reacted for peroxidase histochemistry. Of the thalamic neurons that were retrogradely labelled with HRP, more than 80% were located in the reticular nucleus of the thalamus (RNT), a distinct group of nerve cells that contain glutamic acid decarboxylase (GAD)-like immunoreactivity and are presumably GABAergic. By combining immunocytochemistry with HRP histochemistry, it was possible to confirm that the RNT neurons that had grown axons into the peripheral nerves grafts retained their GAD-like immunoreactivity. The apparent selectivity in their regenerative responses of RNT neurons to peripheral nerve grafts may relate to special properties of the neurons that did and did not grow into the grafts.     

Neuronal gap junctions between intraglomerular mitral/tufted cell dendrites in the mouse main olfactory bulb

In the mouse main olfactory bulb (MOB) gap junction-forming processes in glomeruli were analyzed by means of the serial electron microscopical reconstruction. Gap junctions were encountered between diverse types of dendritic processes and thus confirming our previous study on gap junctions in the rat MOB. Importantly, among more than 30 gap junctions examined in serial sections, we encountered 3 gap junctions made between mitral/tufted cell dendrites in the glomerulus. Then we must consider both direct coupling between mitral/tufted cells via gap junctions and indirect coupling between mitral/tufted cells via intervening interneuronal processes as suggested previously.     

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.     

GABAergic boutons establish synaptic contacts with the soma and dendrites of cuneothalamic relay neurons in the rat cuneate nucleus

This study investigates the synaptic relation between -aminobutyric acid-immunoreactive (GABA-IR) and cuneothalamic relay neurons (CTNs) in the rat cuneate nucleus. Retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase complex (WGA-HRP) was used to label CTNs while anti-GABA immunogold serum was used for the detection of GABA-IR boutons associated with CTNs. With these procedures, immunogold-labelled GABA-IR boutons were found to form axosomatic, axodendritic and axospinous synapses with the WGA-HRP-labelled but immunonegative CTNs. Quantitative estimation showed that the mean ratios of GABA-IR to GABA-immunonegative boutons making synaptic contacts with somata, proximal dendrites, and distal dendrites were 47.9%, 49.1% and 34.7%, respectively. Statistical analysis showed that the incidence of GABA-IR boutons on the somata and proximal dendrites of CTNs was significantly higher than on the distal dendrites. Our results indicate that GABA is the primary inhibitory neurotransmitter in the cuneate nucleus, thereby emphasizing the importance of postsynaptic inhibition on cuneothalamic relay neurons.     

Adenosine inhibits GABAergic and glycinergic transmission in adult rat substantia gelatinosa neurons

The effect of adenosine on inhibitory postsynaptic currents (IPSCs) was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Adenosine reversibly reduced the amplitude of GABAergic and glycinergic electrically evoked IPSCs (eIPSCs) in a dose-dependent manner (EC50 = 14.5 and 19.1 microM, respectively). The A1 adenosine-receptor agonist N6-cyclopentyladenosine also reduced the eIPSCs, whereas the A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine reversed the inhibition produced by adenosine. In paired-pulse experiments, the ratio of the second to first GABAergic or glycinergic eIPSC amplitude was increased by adenosine, whereas the response of SG neurons to exogenous GABA or glycine was unaffected. Adenosine reduced the frequency of GABAergic and glycinergic spontaneous IPSCs without changing their amplitude. This reduction in frequency disappeared in the presence of a K+ -channel blocker (4-aminopyridine) but not in the absence of Ca2+. The inhibition by adenosine disappeared in the presence of cyclic-AMP analog (8-Br-cyclic AMP) and adenylate-cyclase activator (forskolin) but not protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate). We conclude that adenosine suppresses inhibitory transmission in SG neurons by activating presynaptic A1 receptors and that this action is mediated by K+ channels and cyclic AMP but not by Ca2+ channels and PKC. This inhibitory action of adenosine probably contributes to the modulation of pain transmission in the SG.     

Ultrastructural distribution of glycinergic and GABAergic neurons and axon terminals in the rat dorsal cochlear nucleus,with emphasis on granule cell areas

A knowledge of neurotransmitters in the neurons of the rat cochlear nuclear complex is of importance in understanding the function of auditory circuits. Using post-embedding ultrastructural immunogold labelling, the distribution of glycinergic and GABAergic neurons and axonal terminals has been studied in the molecular, fusiform and polymorphic layers of the rat dorsal cochlear nucleus (DCN). This technique is not limited by the penetration of antibodies into the nervous tissue as in pre-embedding methods, and allows a fine neurochemical mapping of the nervous tissue. Numerous glycinergic and GABAergic axon terminals contain pleomorphic and flat synaptic vesicles, and are present in all layers (1, 2, 3) of the dorsal cochlear nucleus. Glycine and GABA-negative large terminals (mossy fibres) are mainly seen in granule cell areas of layer 2 (fusiform layer). Mossy fibres contact the dendrites of GABA- and glycine-negative granule cells and of the few unipolar brush cells (excitatory neurons). The least common cells in the granule cell areas are GABAergic and glycinergic Golgi-stellate neurons. In unipolar brush cells, aggregations of vesicles seem to be the origin of their characteristic ringlet-bodies. Golgi-stellate cells send their inhibitory terminals to the dendrites of granule and unipolar brush cells, occasionally directly to mossy fibres. Small or (less frequently) large GABAergic terminals contact the soma or the main dendrite of unipolar brush cells. The circuit of a hypothetical functional unit of neurons in the DCN is proposed. The inputs from auditory tonotopic or non-auditory non-tonotopic mossy fibres eventually reach pyramidal cells through axons from the granule cells or unipolar brush cells. Pyramidal cells convey an excitatory signal from the DCN to higher mesencephalic nuclei for further elaboration of the acoustic signal.     

Ultrastructural study of presumptive pro-opiocortin producing neurons in the rat hypothalamus

In order to analyse secretory mechanisms in presumptive pro-opiocortin neurons, an ultrastructural study of their perikarya was performed in the hypothalamic arcuate nucleus of adult rats intraventricularly injected with colchicine. This study was carried out both in tissue fixed with aldehydes alone and in tissue fixed with aldehydes and osmium tetroxide.In tissue not exposed to osmium tetroxide, pro-opiocortin neurons can be adequately identified by immunostaining in semi-thin sections with both ACTH 17–39 and β-endorphin antisera. But in adjacent ultra-thin sections of the same neurons, their ultra-structural aspect cannot be studied in detail since, without fixation in osmium tetroxide. the cellular membranes are not preserved. Among the organelles, only lysosomes and elementary granules can be unambiguously identified in the cytoplasm; the latter are selectively immunostained with ACTH antiserum.In semi-thin sections of tissue fixed with aldehydes and then with osmium tetroxide, the pro-opiocortin neurons can be stained only with ACTH 17–39 antiserum and then very poorly when compared with aldehyde-fixed tissue. A detailed ultra-structural study of the presumptive pro-opiocortin perikarya was possible in adjacent ultra-thin sections stained with uranyl acetate and lead citrate. They have the ultrastructural characteristics of active secretory neurons: their cytoplasm is filled with many organelles; it contains an abundant rough endoplasmic reticulum, numerous lysosomes and secretory granules probably accumulated in perikarya by the colchicine-induced blockage of the axonal flow. The Golgi complexes are conspicuously enlarged. They are probably the source of lysosomes and granules. Images of crinophagy and autophagic vacuoles suggest an autoregulatory mechanism in pro-opiocortin neurons to reabsorb organelles accumulated by colchicine.These results show that neurons containing pro-opiocortin have organelles necessary for a synthesis of neuropeptides and their packaging in granules. This supports the hypothesis that the presence of pro-opiocortin-derived peptides in brain is, at least partly, due to their neural synthesis by hypothalamic secretory neurons.     

Spatiotemporally graded NMDA spike/plateau potentials in basal dendrites of neocortical pyramidal neurons

Glutamatergic inputs clustered over approximately 20-40 microm can elicit local N-methyl-D-aspartate (NMDA) spike/plateau potentials in terminal dendrites of cortical pyramidal neurons, inspiring the notion that a single terminal dendrite can function as a decision-making computational subunit. A typical terminal basal dendrite is approximately 100-200 microm long: could it function as multiple decision-making subunits? We test this by sequential focal stimulation of multiple sites along terminal basal dendrites of layer 5 pyramidal neurons in rat somatosensory cortical brain slices, using iontophoresis or uncaging of brief glutamate pulses. There was an approximately sevenfold spatial gradient in average spike/plateau amplitude measured at the soma, from approximately 3 mV for distal inputs to approximately 23 mV for proximal inputs. Spike/plateaus were NMDA receptor (NMDAR) conductance-dominated at all locations. Large Ca(2+) transients accompanied spike/plateaus over a approximately 10- to 40-microm zone around the input site; smaller Ca(2+) transients extended approximately uniformly to the dendritic tip. Spike/plateau duration grew with increasing glutamate and depolarization; high Ca(2+) zone size grew with spike/plateau duration. The minimum high Ca(2+) zone half-width (just above NMDA spike threshold) increased from distal (approximately 10 microm) to proximal locations (approximately 25 microm), as did the NMDA spike glutamate threshold. Depolarization reduced glutamate thresholds. Simulations exploring multi-site interactions based on this demonstrate that if appropriately timed and localized inputs occur in vivo, a single basal dendrite could correspond to a cascade of multiple co-operating dynamic decision-making subunits able to retain information for hundreds of milliseconds, with increasing influence on neural output from distal to proximal. Dendritic NMDA spike/plateaus are thus well-suited to support graded persistent firing.     

A light and electron microscopic study of NADPH-diaphorase-, calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.     

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 m (SD ± 0.18; range 0.37–1.29 m) and their mean area 0.26 m² (SD ± 0.145; range 0.075–0.93 m²); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.     

Effects of GABAergic inhibition on neocortical long-term potentiation in the chronically prepared rat

In this study we investigated the hypothesis whether P2-related differences tested in a visual priming paradigm are associated with theta phase-locking. We recorded the EEG from 31 electrodes and calculated phase-locking index and total power differences for frequencies between 2 and 20 Hz. ERPs (event-related potentials) were analyzed for P1, N1 and P2 components. P2 showed strongest task-related amplitude differences between congruent and incongruent targets. A source analyses was performed for the P2 component using sLoreta that revealed local generators of the P2 in parieto-occipital regions. Phase-locking analyses showed specific effects in the theta range (4-6 Hz) appearing in time windows at around the P2 component. We draw the conclusion that phase-locked theta reflect top-down regulation processes mediating information between memory systems and is in part involved in the modulation of the P2 component.     

Annulated gap junctions in the rat neural tube.

Annulated gap junctions have been studied electron microscopically in normal rat embryo neural tube on day 12 of gestation. Localization, size and content of annulated gap junctions were characterized. Like in other tissues annulated gap junctions are thought to be involved in a degradation cycle of gap junctions.     

Astrocytic gap junctions in the rat lateral hypothalamic area

An extensive system of gap or nexus junctions has been found between astrocytic processes in the neuropil of the lateral hypothalamic area in the albino rat. These specialized interastrocytic junctions occur in regions of high synaptic density where neural processes are separated by the interconnected glial system. In this study, 90% of the gap junctions observed in the lateral hypothalamic neuropil are in the immediate proximity of synaptic terminals. The close morphological relationship of these glial gap junctions with synaptic contacts suggests that they may play a significant role in the process of synaptic transmission.