GABAergic basal forebrain afferents innervate selectively GABAergic targets in the main olfactory bulb.

In this work we have analyzed the targets of the GABAergic afferents to the main olfactory bulb originating in the basal forebrain of the rat. We combined anterograde tracing of 10 kD biotinylated dextran amine (BDA) injected in the region of the horizontal limb of the diagonal band of Broca that projects to the main olfactory bulb, with immunocytochemical detection of GABA under electron microscopy or vesicular GABA transporter (vGABAt) under confocal fluorescent microscopy. GABAergic afferents were identified as double labeled BDA-GABA boutons. Their targets were identified by their ultrastructure and GABA content. We found that GABAergic afferents from the basal forebrain were distributed all over the bulbar lamination, but were more abundant in the glomerular and inframitral layers (i.e. internal plexiform layer and granule cell layer). The fibers had thick varicosities with abundant mitochondria and large perforated synaptic specializations. They contacted exclusively GABAergic cells, corresponding to type 1 periglomerular cells in the glomerular layer, and to granule cells in inframitral layers. This innervation will synchronize the bulbar inhibition and consequently the response of the principal cells to the olfactory input. The effect of the activation of this pathway will produce a disinhibition of the bulbar principal cells. This facilitation might occur at two separate levels: first in the terminal tufts of mitral and tufted cells via inhibition of type 1 periglomerular cells; second at the level of the firing of the principal cells via inhibition of granule cells. The GABAergic projection from the basal forebrain ends selectively on interneurons, specifically on type 1 periglomerular cells and granule cells, and is likely to control the activity of the olfactory bulb via disinhibition of principal cells. Possible similarities of this pathway with the septo-hippocampal loop are discussed.

Synaptic connectivity of serotonergic axons in the olfactory glomeruli of the rat olfactory bulb.

Although the major mode of transmission for serotonin in the brain is volume transmission, previous anatomical studies have demonstrated that serotonergic axons do form synaptic contacts. The olfactory glomeruli of the olfactory bulb of mammals receive a strong serotonergic innervation from the dorsal and medial raphe nuclei. In the present report, we investigate the synaptic connectivity of these serotonergic axons in the glomerular neuropil of the rat olfactory bulb. Our study shows that serotonergic axons form asymmetrical synaptic contacts on dendrites within the glomerular neuropil. Analyzing the neurochemical nature of the synaptic targets, we have found that 55% of the synapses were on GABA-immunopositive profiles and 45% on GABA-immunonegative profiles. These data indicate that barely half of the contacts were found in GABA-immunonegative profiles and half of the synapses in GABA-positive dendrites belonging to type 1 periglomerular cells. Synaptic contacts from serotonergic axons on dendrites of principal cells cannot be excluded, since some of the GABA-immunonegative postsynaptic profiles contacted by serotonergic axons had the typical ultrastructural features of bulbar principal cell dendrites. Altogether, our results suggest a complex action of the serotonergic system in the modulation of the bulbar circuitry.

Effects of chronic fluoxetine treatment on the rat somatosensory cortex: activation and induction of neuronal structural plasticity.

Recent hypotheses support the idea that disruption of normal neuronal plasticity mechanisms underlies depression and other psychiatric disorders, and that antidepressant treatment may counteract these changes. In a previous report we found that chronic fluoxetine treatment increases the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a molecule involved in neuronal structural plasticity, in the somatosensory cortex. In the present study we intended to find whether, in fact, cell activation and neuronal structural remodeling occur in parallel to changes in the expression of this molecule. Using immunohistochemistry, we found that chronic fluoxetine treatment caused an increase in the expression of the early expression gene c-fos. Golgi staining revealed that this treatment also increased spine density in the principal apical dendrite of pyramidal neurons. These results indicate that, apart from the medial prefrontal cortex or the hippocampus, other cortical regions can respond to chronic antidepressant treatment undergoing neuronal structural plasticity.

Distribution of the A3 subunit of the cyclic nucleotide-gated ion channels in the main olfactory bulb of the rat.

Previous data suggest that cyclic GMP (cGMP) signaling can play key roles in the circuitry of the olfactory bulb (OB). Therefore, the expression of cGMP-selective subunits of the cyclic nucleotide-gated ion channels (CNGs) can be expected in this brain region. In the present study, we demonstrate a widespread expression of the cGMP-selective A3 subunit of the cyclic nucleotide-gated ion channels (CNGA3) in the rat OB. CNGA3 appears in principal cells, including mitral cells and internal, medium and external tufted cells. Moreover, it appears in two populations of interneurons, including a subset of periglomerular cells and a group of deep short-axon cells. In addition to neurons, CNGA3-immunoreactivity is found in the ensheathing glia of the olfactory nerve. Finally, an abundant population of CNGA3-containing cells with fusiform morphology and radial processes is found in the inframitral layers. These cells express doublecortin and have a morphology similar to that of the undifferentiated cells that leave the rostral migratory stream and migrate radially through the layers of the OB. Altogether, our results suggest that CNGA3 can play important and different roles in the OB. Channels composed of this subunit can be involved in the processing of the olfactory information taking place in the bulbar circuitry. Moreover, they can be involved in the function of the ensheathing glia and in the radial migration of immature cells through the bulbar layers.

Chronic antidepressant treatment induces contrasting patterns of synaptophysin and PSA-NCAM expression in different regions of the adult rat telencephalon.

Structural modifications occur in the brain of severely depressed patients and they can be reversed by antidepressant treatment. Some of these changes do not occur in the same direction in different regions, such as the medial prefrontal cortex, the hippocampus or the amygdala. Differential structural plasticity also occurs in animal models of depression and it is also prevented by antidepressants. In order to know whether chronic fluoxetine treatment induces differential neuronal structural plasticity in rats, we have analyzed the expression of synaptophysin, a protein considered a marker of synaptic density, and the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a molecule involved in neurite and synaptic remodeling. Chronic fluoxetine treatment increases synaptophysin and PSA-NCAM expression in the medial prefrontal cortex and decreases them in the amygdala. The expression of these molecules is also affected in the entorhinal, the visual and the somatosensory cortices.

N-methyl-d-aspartate receptor expression during adult neurogenesis in the rat dentate gyrus.

N-methyl-d-aspartate (NMDA) receptors play a crucial role in the regulation of neuronal development during embryogenesis and they also regulate the rate of neurogenesis and proliferation in the adult dentate gyrus. However, the mechanism by which they influence these processes is not fully understood. NMDA receptors seem to be functional in hippocampal precursor cells and recently generated granule neurons, although there is no anatomical correlate of these physiological observations. We have analyzed the expression of the NMDA receptor subunits NR1 and NR2B in precursor cells and recently generated granule neurons of the adult rat dentate gyrus, using 5'bromodeoxyuridine, green fluorescent protein-retrovirus and immunohistochemistry. Our results indicate that NR1 and NR2B are expressed in some proliferating cells of the adult subgranular zone. These receptors are absent from transiently amplifying progenitors (type 2-3 cells) but they are found in glial fibrillar acidic protein expressing cells in the subgranular zone, suggesting its presence in bipotential (type-1) precursor cells. NR1 and NR2B are rarely found in granule cells younger than 60 h. By contrast, many granule cells generated 14 days before killing express both NMDA receptor subunits. These results demonstrate that adult hippocampal neurogenesis may be regulated by NMDA receptors present in precursor cells and in differentiating granule neurons, although these receptors are probably not located on synapses. However, an indirect effect through NMDA receptors located in other cell types should not be excluded.

PSA-NCAM expression in the rat medial prefrontal cortex.

The rat medial prefrontal cortex, an area considered homologous to the human prefrontal cortex, is a region in which neuronal structural plasticity has been described during adulthood. Some plastic processes such as neurite outgrowth and synaptogenesis are known to be regulated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). Since PSA-NCAM is present in regions of the adult CNS which are undergoing structural remodeling, such as the hypothalamus or the hippocampus, we have analyzed the expression of this molecule in the medial prefrontal cortex of adult rats using immunohistochemistry. PSA-NCAM immunoreactivity was found both in cell bodies and in the neuropil of the three divisions of the medial prefrontal cortex. All cell somata expressing PSA-NCAM corresponded to neurons and 5' bromodeoxyuridine labeling after long survival times demonstrated that these neurons were not recently generated. Many of these PSA-NCAM immunoreactive neurons in the medial prefrontal cortex could be classified as interneurons on the basis of their morphology and glutamate decarboxylase, isoform 67 expression. Some of the PSA-NCAM immunoreactive neurons also expressed somatostatin, neuropeptide Y and calbindin-D28K. By contrast, pyramidal neurons in this cortical region did not appear to express PSA-NCAM. However, some of these principal neurons appeared surrounded by PSA-NCAM immunoreactive puncta. Some of these puncta co-expressed synaptophysin, suggesting the presence of synapses. Since the etiology of some psychiatric disorders has been related to alterations in medial prefrontal cortex structural plasticity, the study of PSA-NCAM expression in this region may open a new approach to the pathophysiology of these mental disorders.

Zinc chelation during non-lesioning overexcitation results in neuronal death in the mouse hippocampus.

In the hippocampus, chelatable zinc is accumulated in vesicles of glutamatergic presynaptic terminals, abounding specially in the mossy fibers, from where it is released with activity and can exert a powerful inhibitory action upon N-methyl-D-aspartate receptors. Zinc is therefore in a strategic situation to control overexcitation at the zinc-rich excitatory synapses, and consequently zinc removal during high activity might result in excitotoxic neuronal damage. We analyzed the effect of zinc chelation with sodium dietyldithiocarbamate under overexcitation conditions induced by non-lesioning doses of kainic acid in the mouse hippocampus, to get insight into the role of zinc under overexcitation. Swiss male mice were injected with kainic acid (15 mg/kg, i.p.) 15 min prior to sodium dietyldithiocarbamate (150 mg/kg, i.p.), and left to survive for 6 h, 1 day, 4 days, or 7 days after the treatment. Cell damage was analyzed with the hematoxylin-eosin and acid fuchsin stainings. Neither control animals treated only with kainic acid nor those treated only with sodium dietyldithiocarbamate suffered seizures or neuronal damage. By contrast, the kainic acid+sodium dietyldithiocarbamate-treated animals showed convulsive behavior and cell death involving the hilus, CA3, and CA1 regions. Pretreatment with the N-methyl-D-aspartate receptor antagonist MK801 (1 mg/kg, i.p.) completely prevented neuronal damage. Experiments combining different doses of sodium dietyldithiocarbamate and kainic acid with different administration schedules demonstrated that the overlap of zinc chelation and overexcitation is necessary to trigger the observed effects. Moreover, the treatment with a high dose of sodium dietyldithiocarbamate (1000 mg/kg), which produced a complete bleaching of the Timm staining for approximately 12 h, highly increased the sensitivity of animals to kainic acid. Altogether, our results indicate that the actions of sodium dietyldithiocarbamate are based on a reduction of zinc levels, which under overexcitation conditions induce seizures and neuronal damage. These findings fully support a protective role for synaptically released zinc during high neuronal activity, most probably mediated by its inhibitory actions on N-methyl-D-aspartate receptors, and argue against a direct action of synaptic zinc on the observed neuronal damage.

Parvalbumin-containing interneurons do not innervate granule cells in the olfactory bulb.

Combining pre-embedding parvalbumin immunostaining and post-embedding immunogold detection of GABA in the olfactory bulb, we investigated whether the parvalbumin-containing GABAergic interneurons of the external plexiform layer exclusively innervate principal cells, or whether they also establish inhibitory synapses upon GABAergic local neurons such as granule cells. Our results demonstrate that the parvalbumin-containing cells do not contact GABAergic interneurons in the neuropil of the external plexiform layer. On the contrary, their postsynaptic elements were always non-GABAergic principal cells. Although classically it has been accepted that the interneurons of the external plexiform layer could exert a disinhibitory action upon principal cells, via inhibition of GABAergic granule cells, we conclude that they exert a feedback inhibitory action directly and exclusively upon principal cells.

Bilateral olfactory deprivation reveals a selective noradrenergic regulatory input to the olfactory bulb.

Unilateral olfactory deprivation in the rat induces changes in the catecholaminergic system of the olfactory bulb. Nevertheless, evidence suggests that unilateral deprivation does not fully prevent stimulation of the deprived bulb. The present report analyses the response of the catecholaminergic system of the olfactory bulb in fully deprived rats obtained by bilateral naris occlusion. The complete deprivation produces more rapid and dramatic changes in both the intrinsic and extrinsic catecholaminergic systems of the olfactory bulb. Intrinsic responses involve a rapid decrease in dopamine-containing cells to about 25% of controls, correlated with a decreased Fos expression in juxtaglomerular cells of all olfactory glomeruli, with the only exception of those of the atypical glomeruli which maintain unaltered expression of both markers. In parallel with these events, there is a progressive increase in the density of extrinsic noradrenergic axons arising from neurons in the locus coeruleus, which shows, in parallel, a progressive increase in Fos expression. This model demonstrates plastic changes in the catecholaminergic system of the olfactory bulb forming a valid morphological substrate for lowering thresholds in the processing of olfactory information. In addition to this generalized response, there is another one, directed to a specific subset of olfactory glomeruli (atypical glomeruli) involved in the processing of odor pheromone-like cues related to behavioral responses, that could be responsible for keeping active this reduced and selected group of glomeruli carrying crucial olfactory information. These results indicate the existence of adaptive changes in the catecholaminergic system of the olfactory bulb as a response to the lack of afferent peripheral stimulation. These changes involve dopamine- and noradrenaline-immunoreactive elements, in a strategy presumably directed at maintaining to the highest possible level the ability to detect olfactory signals.

Subcellular localization of m2 muscarinic receptors in GABAergic interneurons of the olfactory bulb.

We analysed the ultrastructural distribution of the m2 muscarinic receptor (m2R) in the rat olfactory bulb (OB) using immunohistochemical techniques and light and electron microscopy. m2R was differentially distributed within the cellular compartments of gamma-aminobutyric acid (GABA)ergic bulbar interneurons. It is located in the gemmules of granule cells and in the synaptic loci of the interneurons of the external plexiform layer, suggesting that m2R activation could modulate the release of GABA from these interneurons onto principal cells by a presynaptic mechanism. By contrast, the receptor appears in the somata and dendritic trunks of second-order short-axon interneurons located in the inframitral layers, suggesting that postsynaptic muscarinic activation in these cells could elicit the inhibition of granule cells, leading to a disinhibition of principal cells. We also detail the anatomical substrate for a new putative muscarinic modulation that has not been previously described, and that could influence the reception of sensory information within the olfactory glomeruli. m2R appears in a subset of GABAergic/dopaminergic juxtaglomerular cells innervated by olfactory axons but is absent in juxtaglomerular cells that do not receive sensory inputs. This finding suggests that m2R activation could modify, through dopaminergic local circuits, the strength of olfactory nerve inputs onto principal cells. Activation of the muscarinic receptor may modulate the olfactory information encoding within olfactory glomeruli and may facilitate the bulbar transmission to superior centres influencing the GABA release by presynaptic and postsynaptic mechanisms. Taken together, our data provide the neuroanatomical basis for a complex action of m2R at different levels in the mammalian OB.

Recurrent mossy fibers preferentially innervate parvalbumin-immunoreactive interneurons in the granule cell layer of the rat dentate gyrus.

Detection of vesicular zinc and immunohistochemistry against markers for different interneuron subsets were combined to study the postsynaptic target selection of zinc-containing recurrent mossy fiber collaterals in the dentate gyrus. Mossy fiber collaterals in the granule cell layer selectively innervated parvalbumin-containing cells, with numerous contacts per cell, whereas the granule cells were avoided. Under the electron microscope, those boutons made asymmetrical contacts on dendrites and somata. These findings suggest that, in addition to the hilar perforant path-associated (HIPP) interneurons, the basket and chandelier cells also receive a powerful feed-back drive from the granule cells, and thereby are able to control population synchrony in the dentate gyrus. On the other hand, the amount of monosynaptic excitatory feed-back among granule cells is shown to be negligible.

Response of abducens internuclear neurons to axotomy in the adult cat.

The highly specific projection of abducens internuclear neurons on the medial rectus motoneurons of the oculomotor nucleus constitutes an optimal model for investigating the effects of axotomy in the central nervous system. We have analyzed the morphological changes induced by this lesion on both the cell bodies and the transected axons of abducens internuclear neurons in the adult cat. Axotomy was performed by the transection of the medial longitudinal fascicle. Cell counts of Nissl-stained material and calretinin-immunostained abducens internuclear neurons revealed no cell death by 3 months postaxotomy. Ultrastructural examination of these cells at 6, 14, 24, and 90 days postaxotomy showed normal cytological features. However, the surface membrane of axotomized neurons appeared contacted by very few synaptic boutons compared to controls. This change was quantified by measuring the percentage of synaptic coverage of the cell bodies and the linear density of boutons. Both parameters decreased significantly after axotomy, with the lowest values at 90 days postlesion ( approximately 70% reduction). We also explored axonal regrowth and the possibility of reinnervation of a new target by means of anterograde labeling with biocytin. At all time intervals analyzed, labeled axons were observed to be interrupted at the caudal limit of the lesion; in no case did they cross the scar tissue to reach the distal part of the tract. Nonetheless, a conspicuous axonal sprouting was present at the caudal aspect of the lesion site. Structures suggestive of axonal growth were found, such as large terminal clubs, from which short filopodium-like branches frequently emerged. Similar findings were obtained after parvalbumin and calretinin immunostaining. At the electron microscopy level, biocytin-labeled boutons originating from the sprouts appeared surrounded by either extracellular space, which was extremely dilated at the lesion site, or by glial processes. The great majority of labeled boutons examined were, thus, devoid of neuronal contact, indicating absence of reinnervation of a new target. Altogether, these data indicate that abducens internuclear neurons survive axotomy in the adult cat and show some form of axonal regrowth, even in the absence of target connection.

Unusual target selectivity of perisomatic inhibitory cells in the hilar region of the rat hippocampus.

Perisomatic inhibitory innervation of all neuron types profoundly affects their firing characteristics and vulnerability. In this study we examined the postsynaptic targets of perisomatic inhibitory cells in the hilar region of the dentate gyrus where the proportion of potential target cells (excitatory mossy cells and inhibitory interneurons) is approximately equal. Both cholecystokinin (CCK)- and parvalbumin-immunoreactive basket cells formed multiple contacts on the somata and proximal dendrites of mossy cells. Unexpectedly, however, perisomatic inhibitory terminals arriving from these cell types largely ignored hilar GABAergic cell populations. Eighty-ninety percent of various GABAergic neurons including other CCK-containing basket cells received no input from CCK-positive terminals. Parvalbumin-containing cells sometimes innervated each other but avoided 75% of other GABAergic cells. Overall, a single mossy cell received 40 times more CCK-immunoreactive terminals and 15 times more parvalbumin-positive terminals onto its soma than the cell body of an average hilar GABAergic cell. In contrast to the pronounced target selectivity in the hilar region, CCK- and parvalbumin-positive neurons innervated each other via collaterals in stratum granulosum and moleculare. Our observations indicate that the inhibitory control in the hilar region is qualitatively different from other cortical areas at both the network level and the level of single neurons. The paucity of perisomatic innervation of hilar interneurons should have profound consequences on their action potential generation and on their ensemble behavior. These findings may help explain the unique physiological patterns observed in the hilus and the selective vulnerability of the hilar cell population in various pathophysiological conditions.

Coexpression of neurocalcin with other calcium-binding proteins in the rat main olfactory bulb.

The distribution patterns of four calcium-binding proteins (CaBPs)-calbindin D-28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)-in the rat main olfactory bulb were compared, and the degrees ofcolocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques. All investigated CaBPs were detected in groups of periglomerular cells and Van Gehuchten cells, whereas other cell types expressed some of the investigated proteins but not all four. Double-labeling techniques demonstrated the colocalization of NC with CB, CR, or PV in periglomerular cells, whereas each neurochemical group constituted entirely segregated populations in the remaining neuronal types. This is evident in granule cells that demonstrated large but segregated populations immunoreactive to either NC or CR. This study provides a further biochemical characterization of interneuronal types in the rat main olfactory bulb. On the basis of the distinct calcium-binding affinities, each neurochemically defined population may have different responses to calcium influx that would result in the existence of distinct functional subgroups within morphologically defined neuronal types. The expression of the investigated CaBPs in periglomerular cells with both single and colocalized patterns suggests that the local circuits in the glomerular layer are constituted by a complex network of elements with particular calcium requirements.

Enkephalin-containing interneurons are specialized to innervate other interneurons in the hippocampal CA1 region of the rat and guinea-pig.

Enkephalins are known to have a profound effect on hippocampal inhibition, but the possible endogenous source of these neuropeptides, and their relationship to inhibitory interneurons is still to be identified. In the present study we analysed the morphological characteristics of met-enkephalin-immunoreactive cells in the CA1 region of the rat and guinea-pig hippocampus, their coexistence with other neuronal markers and their target selectivity at the light and electron microscopic levels. Several interneurons in all subfields of the hippocampus were found to be immunoreactive for met-enkephalin. In the guinea-pig, fibres arising from immunoreactive interneurons were seen to form a plexus in the stratum oriens/alveus border zone, and basket-like arrays of boutons on both enkephalin-immunoreactive and immunonegative cell bodies in all strata. Immunoreactive boutons always established symmetric synaptic contacts on somata and dendritic shafts. Enkephalin-immunoreactive cells co-localized GABA, vasoactive intestinal polypeptide and calretinin. Postembedding immunogold staining for GABA showed that all the analysed enkephalin-immunoreactive boutons contacted GABAergic postsynaptic structures. In double-immunostained sections, enkephalin-positive axons were seen to innervate calbindin D28k-, somatostatin-, calretinin- and vasoactive intestinal polypeptideimmunoreactive cells with multiple contacts. Based on these characteristics, enkephalin-containing cells in the hippocampus are classified as interneurons specialized to innervate other interneurons, and represent a subset of vasoactive intestinal polypeptide- and calretinin-containing cells. The striking match of ligand and receptor distribution in the case of enkephalin-mediated interneuronal communication suggests that this neuropeptide may play an important role in the synchronization and timing of inhibition involved in rhythmic network activities of the hippocampus.

Neurocalcin-immunoreactive cells in the rat hippocampus are GABAergic interneurons.

Neurocalcin (NC) is a recently described calcium-binding protein isolated and characterized from bovine brain. NC belongs to the neural calcium-sensor proteins defined by the photoreceptor cell-specific protein recoverin that have been proposed to be involved in the regulation of calcium-dependent phosphorylation in signal transduction pathways. We analyzed the distribution and morphology of the NC-immunoreactive (IR) neurons in the rat dorsal hippocampus and the coexistence of NC with GABA and different neurochemical markers which label perisomatic inhibitory cells [parvalbumin (PV) and cholecystokinin (CCK)], mid-proximal dendritic inhibitory cells [calbindin D28k (CB)], distal dendritic inhibitory cells [somatostatin (SOM) and neuropeptide Y (NPY)], and interneurons specialized to innervate other interneurons [calretinin (CR) and vasoactive intestinal polypeptide (VIP)]. NC-IR cells were present in all layers of the dentate gyrus and hippocampal fields. In the dentate gyrus, NC-IR cells were concentrated in the granule cell layer, especially in the hilar border, whereas in the CA fields they were most frequently found in the stratum radiatum. NC-IR cells were morphologically heterogeneous and exhibited distinctive features of non-principal cells. In the dentate gyrus, pyramidal-like, multipolar and fusiform (horizontal and vertical) cells were found. In the CA3 region most NC-IR cells were multipolar, but vertical and horizontal fusiform cells also appeared. In the CA1 region, where NC-IR cells showed most frequently vertically arranged dendrites, multipolar, bitufted and fusiform (vertical and horizontal) cells could be distinguished. All the NC-IR cells were found to be GABA-IR in all hippocampal layers and regions, and they represented about 19% of the GABA-positive cells. NC/CB, NC/CR and NC/VIP double-labeled cells were found in all hippocampal regions, and represented 29%, 24% and 18% of the NC-IR cells, respectively. NC and CCK did not coexist in the dentate gyrus; however, 9% of the NC-IR cells in the CA fields also contained CCK. No coexistence of NC with PV, SOM or NPY was found in any hippocampal region. We conclude that NC is exclusively expressed by interneurons in the rat hippocampus. NC-IR cells are a morphologically and neurochemically heterogeneous subset of GABAergic non-principal cells, which, on the basis of the known termination pattern of the colocalizing markers, are also functionally heterogeneous and are mainly involved in feed-forward dendritic inhibition in the commissural-associational and Schaffer collateral termination zones (CB containing cells), in innervation of other interneurons (CR- and VIP-containing cells), and in perisomatic inhibition (CCK-containing cells). NC is never present in perisomatic inhibitory PV-containing cells, or in feed-back distal dendritic inhibitory SOM/NPY-containing cells.

Localization of parvalbumin, calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat.

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15-20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions.

Changes in GABA and parvalbumin immunoreactivities in the cerebral cortex of lizards after narine occlusion.

Olfactory deprivation produced by narine occlusion has been suggested to reduce the activity in the cerebral cortex of lizards. Here we analyzed the short-term changes in GABA and parvalbumin (PV) immunoreactivities in the cerebral cortex of lizards after narine occlusion. The number and distribution of GABA- and parvalbumin-immunoreactive (IR) cells have been studied by immunocytochemistry in the cerebral cortex of control and olfactory-deprived lizards. The distribution of GABA-IR cells as well as that of PV-IR cells was similar in control and deprived animals, and PV-IR cells were GABA-IR in all cases. However, significant changes were observed in the absolute number of GABA- and PV-IR cells. GABA-IR cells were more abundant in deprived animals than in control ones. In contrast, the number of PV-IR cells decreased significantly and PV immunoreactivity in dendrites and boutons was lower in deprived animals. These results suggest that the reduction in the number of PV-IR cells in olfactory-deprived lizards occurs without loss of GABA cells, and that PV expression is under the control of olfactory activity and remains plastic in the cerebral cortex of adult lizards.

An axosomatic and axodendritic multipolar neuron in the lizard cerebral cortex.

The morphology and synaptic organisation of a type of multipolar neuron of the lizard cerebral cortex were studied by Golgi impregnation, intracellular injection of horseradish peroxidase, electron microscopy, and immunocytochemistry. It is a GABA-immunoreactive interneuron and most likely parvalbumin-immunoreactive. Its conspicuous axonal arbor is characterised by an initial segment arising from the soma or from a juxtasomatic dendritic segment. The initial axon segment ramifies and gives rise to thick myelinated segments that terminate in short unmyelinated branches studded with thick boutons 'en passant' that (1) make axosomatic synapses on bipyramidal neuronal somata and (2) synapse on initial apical dendritic segments of bipyramidal neurons forming climbing-like cartridges. The dendrites extend throughout the thickness of the cortex, receiving synaptic input from a variety of sources of which the most prominent is that of zinc-positive boutons coming from granule cells of the medial cortex. According to its synaptology, this interneuron may play a role in regulating the activity of bipyramidal neurons by both feed-forward and feed-back inhibition mechanisms. From a comparative standpoint, it may be related to the sparsely spiny or nonspiny multipolar neurons of the stratum oriens of the mammalian hippocampus.