Fluoxetine increased adult neurogenesis is mediated by 5-HT3 receptor.

Adult neurogenesis is an aspect of structural plasticity that remains active during adulthood in some brain regions. One of them is the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. Adult neurogenesis is reduced by different factors and in disorders of the CNS, including major depression. Antidepressant treatments, such as chronic fluoxetine administration, recover the normal level of adult neurogenesis. Fluoxetine treatment increases the free concentration of the neurotransmitter serotonin and this monoamine is implicated in the regulation of the neurogenic process; however, the target of the action of this neurotransmitter has not been fully elucidated. In this study, we have tried to determine the relevance of the serotonin receptor 3 (5-HT3) in the hippocampal neurogenesis of adult rats. We have used fluorescent immunohistochemistry to study the expression of the 5-HT3 receptor in different neurogenesis stages in the SGZ, identifying its expression in stem cells, amplifying neural progenitors and immature neurons. Moreover, we have studied the impact of a 5-HT3 antagonist (ondansetron) in the fluoxetine-induced adult neurogenesis. We observed that fluoxetine alone increases the number of both proliferating cells (ki67 positive) and immature neurons (DCX positive) in the SGZ. By contrast, co-treatment with ondansetron blocked the increase in proliferation and neurogenesis. This study demonstrates that the activation of 5-HT3 receptors is necessary for the increase of adult neurogenesis induced by fluoxetine.

Characterization of a population of tyrosine hydroxylase-containing interneurons in the external plexiform layer of the rat olfactory bulb.

The olfactory bulb (OB) of mammals contains the major endogenous dopamine-producing system in the forebrain. The vast majority of dopaminergic neurons consists of juxtaglomerular cells, which innervate the olfactory glomeruli and modulate the entrance of sensory information to the OB. Although dopaminergic juxtaglomerular cells have been widely investigated, the presence of dopaminergic interneurons other than juxtaglomerular cells has been largely unexplored. In this study, we analyze a population of tyrosine hydroxylase (TH)-containing interneurons located in the external plexiform layer (EPL) of the rat OB. These interneurons are GABAergic and morphologically heterogeneous. They have an axon and two to four dendrites running throughout the EPL. Frequently, they have appendages similar to spines in the dendrites and, sometimes, the distal portions of the dendritic branches show enlargements or swellings similar to varicosities. Contrary to other interneurons of the EPL, the TH-containing ones do not form dendro-dendritic synapses on principal cells and do not receive dendro-dendritic synapses from them. In fact, no synapses were found from the dendrites of these interneurons. When their dendrites are involved in synaptic contacts, they are always the postsynaptic element. They receive symmetrical and asymmetrical synapses from GABAergic and non-GABAergic axons of unidentified origin. Our data indicate that the local circuits of the EPL are more complex than previously thought. Although most of the interneurons of this layer establish dendro-dendritic synaptic relationships with principal cells, the TH-containing interneurons constitute an exception to this rule, resembling interneurons from other cortical areas.

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.

Alteration of inhibitory circuits in the somatosensory cortex of Ts65Dn mice, a model for Down's syndrome.

Down's syndrome (DS), with an incidence of one in 800 live births, is the most common genetic disorder associated with mental retardation. This trisomy on chromosome 21 induces a variable phenotype in which the only common feature is the presence of mental retardation. The neural mechanisms underlying mental retardation might include defects in the formation of neuronal networks and neural plasticity. DS patients have alterations in the morphology, the density and the distribution of dendritic spines in the pyramidal neurons of the cortex. Our hypothesis is that the deficits in dendritic arborization observed in the principal neurons of DS patients and Ts65Dn mice (a model for DS that mimics most of the structural alterations observed in humans) may be mediated to some extent by changes in their inhibitory inputs. Different types of interneurons control different types of inhibition. Therefore, to understand well the changes in inhibition in DS, it is necessary to study the different types of interneurons separately. We have studied the expression of synaptophysin, Glutamic acid decarboxylase-67 (GAD-67) and calcium-binding protein-expressing cells in the primary somatosensory cortex of 4-5 month old Ts65Dn mice. We have observed an increment of GAD67 immunoreactivity that is related mainly to an increment of calretinin-immunoreactive cells and among them the ones with bipolar morphology. Since there is a propensity for epilepsy in DS patients, this increase in interneurons might reflect an attempt by the system to block overexcitation rather than an increment in total inhibition and could explain the deficit in interneurons and principal cells observed in elderly DS patients.

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.

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.

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.

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.

Distribution, ultrastructure, and connectivity of calretinin-immunoreactive mossy cells of the mouse dentate gyrus.

Hilar mossy cells of the mouse were shown recently to display calretinin immunoreactivity (Liu et al. [1996] Exp Brain Res 108:389-403). The morphological and connectional characteristics of these cells are poorly understood. In the present study, we used immunohistochemical, electron microscopic, and neuronal tracing techniques to describe their distribution, morphology, and connectivity. The distribution of calretinin-immunoreactive mossy cells varied significantly along the dorsoventral axis of the hilus. At dorsal levels, calretinin immunoreactivity was limited largely to a subpopulation of interneurons. At mid-dorsoventral and ventral levels, however, most if not all mossy cells displayed calretinin immunoreactivity. We found that most hilar mossy cells are calretinin immunoreactive but lack gamma-aminobutyric acid, as demonstrated by postembedding immunostaining of alternate semithin sections. Calretinin-immunoreactive mossy cells typically had two to three thick dendrites covered with complex spines (thorny excrescences). Electron microscopy revealed that these spines received multiple asymmetric contacts from mossy fibres. Axons arising from these cells formed a strong belt of calretinin immunoreactivity restricted to the inner third of the dentate molecular layer. This immunoreactivity was equally dense throughout the dorsoventral length of the dentate gyrus, suggesting that axons of calretinin-immunoreactive mossy cells located in the ventral levels diverge greatly and are capable of innervating distant regions of the dentate gyrus. Ultrastructural examination showed that calretinin-immunoreactive boutons made asymmetric synaptic contacts primarily on spines and, occasionally, on dendritic shafts of granule cells and accounted for the majority of asymmetrical synapses in the inner molecular layer. Injections of the retrograde tracer wheatgerm agglutinin-gold into the dentate gyrus demonstrated that calretinin-immunoreactive mossy cells concentrated in the ventral hilus project massively to both the dorsal and ventral aspect of the contralateral dentate gyrus. A small proportion of retrogradely labelled cells showed immunoreactivity for neuropeptide Y or somatostatin. If mossy cells of the ventral hilus receive the majority of their input from ventral granule cells, one may expect ventral granule cells to be more efficient in recruiting large numbers of granule cells during synchronous activity patterns than dorsal granule cells. Spontaneous activity originating from granule cells in the ventral dentate gyrus can be propagated throughout the dorsoventral length of the dentate gyrus bilaterally via the dorsoventrally divergent and contralaterally projecting axons of the mossy cells. This organization may explain why the ventral dentate gyrus is frequently involved in pathological phenomena.

Synaptic input of horizontal interneurons in stratum oriens of the hippocampal CA1 subfield: structural basis of feed-back activation.

The synaptic input of interneurons with horizontal dendrites in stratum oriens of the CA1 region was investigated, with particular attention to the portion of synapses originating from local pyramidal cells. Most of these GABAergic interneurons are known to contain somatostatin, and terminate on pyramidal dendrites in conjunction with entorhinal afferents in stratum lacunosum-moleculare. A smaller number of horizontal cells in this layer are immunoreactive for calbindin, and project to the medial septum. Selective ischaemic degeneration was used to label local axon collaterals of CA1 pyramidal cells, and immunostaining for mGluR1 or calbindin to visualise somatostatin- and calbindin-containing horizontal interneurons, respectively, at the stratum oriens-alveus border. The number of degenerating and intact synaptic boutons was counted on mGluR1- as well as on calbindin-positive dendrites and somata, whereas in another group of animals the proportion of GABA-immunoreactive synapses was estimated on calbindin-positive dendrites. On average, > 60% of the total presynaptic elements of both cell types were degenerating, i.e. originated from CA1 pyramidal cells, whereas GABA-positive boutons, which are known to survive ischaemia, are likely to account for a large proportion of non-degenerating boutons. Thus the vast majority of presumed excitatory synapses on somatostatin- and calbindin-containing horizontal neurons derives from local collaterals of CA1 pyramidal cells. The remaining GABA-negative synapses surviving ischaemia may also originate from CA1 pyramidal cells, e.g. from those in the ventral hippocampus, which are rarely damaged by global forebrain ischaemia. Alternative sources may include subcortical afferents known to innervate interneurons, or ipsi- and contralateral CA3 pyramidal cells, which, according to the present results, may account only for a negligible number of synapses on these interneurons types. We conclude that somatostatin-containing neurons at the oriens-alveus border of CA1, which are likely to mediate an inhibitory control of the efficacy and/or plasticity of entorhinal synapses on pyramidal cell dendrites, are driven primarily in a feed-back manner. The source of afferent excitation for calbindin-containing horizontal neurons in this region is very similar, suggesting that the GABAergic hippocamposeptal feed-back is also activated by local pyramidal cell collaterals.

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.

Serotoninergic innervation of nonprincipal cells in the cerebral cortex of the lizard Podarcis hispanica.

The mechanism of serotoninergic transmission in the neo- and archicortex of mammals is complex, including both synaptic and nonsynaptic components, direct actions on principal cells, and indirect effects mediated by GABAergic interneurons. Here we studied the termination pattern and synaptic organization of the serotoninergic afferents in the cerebral cortex of the lizard, Podarcis hispanica, which is considered to correspond in part to the mammalian hippocampal formation, with the aim of unraveling basic, phylogenetically preserved rules in the connectivity of this pathway. We demonstrate that serotoninergic afferents, visualized by immunostaining for serotonin itself, establish multiple synaptic contacts with different subpopulations of nonprincipal cells containing parvalbumin, neuropeptide Y, and opioid peptides. The former two subpopulations contain GABA, whereas the opioid-immunoreactive neurons are most likely GABA-negative cells. Evidence is provided at the electron microscopic level that serotonin-immunoreactive varicosities establish conventional asymmetric synaptic contacts with their nonprincipal targets, but nonsynaptic varicosities also exist. We conclude that, similarly to mammals, a selective synaptic innervation of nonprincipal, possibly inhibitory, neurons is among the mechanisms of serotoninergic modulation of cerebral cortical activity in the lizard.

Postnatal increase of GABA- and PV-IR cells in the cerebral cortex of the lizard Podarcis hispanica.

The number and distribution of GABA- and parvalbumin (PV)-immunoreactive (IR) cells have been studied by immunocytochemistry in the cerebral cortex of newborn and adult lizards. The distribution of GABA-IR cells as well as that of PV-IR cells were similar in newborn and adult lizards, and PV-IR cells were GABA-IR in all cases. However, the absolute number of GABA- and PV-IR cells increased significantly during development. In addition, the rate of of GABA-IR cells also displaying PV immunoreactivity also increased after birth. Moreover, dendrites were rarely found to be PV-IR in newborn lizards, whereas they appeared stained in a Golgi-like manner in adult animals. These results suggest that the GABAergic neuronal population of the cerebral cortex of lizards experiments a significant increment in number and neurochemical maturation after birth.