Blockade of cannabinoid receptors by SR141716 selectively increases Fos expression in rat mesocorticolimbic areas via reduced dopamine D2 function.

The present study investigated, in rats, whether blockade of cannabinoid CB1 receptors may alter Fos protein expression in a manner comparable to that observed with antipsychotic drugs. Intraperitoneal administration of the selective CB1 receptor antagonist, SR141716, dose-dependently (1.0, 3.0 and 10 mg/kg) increased Fos-like immunoreactivity in mesocorticolimbic areas (prefrontal cortex, ventrolateral septum, shell of the nucleus accumbens and dorsomedial caudate-putamen), while motor-related structures such as the core of the nucleus accumbens and the dorsolateral caudate-putamen were unaffected. In the ventrolateral septum, taken as a representative structure, the Fos-inducing effect of SR141716 (10 mg/kg) was maximal 2 h after injection and returned to near control levels by 4 h. Within the prefrontal cortex, SR141716 increased the number of Fos-positive cells predominantly in the infralimbic and prelimbic cortices, presumptive pyramidal cells being the major cell types in which Fos was induced. The D1-like receptor antagonist, SCH23390 (0.1 mg/kg), did not prevent the Fos-inducing effect of SR141716 in any brain region examined (prefrontal cortex, nucleus accumbens, ventrolateral septum and dorsomedial caudate-putamen), although SCH23390 significantly reduced Fos expression induced by cocaine (20 mg/kg) in all these regions. By contrast, the dopamine D2-like agonist, quinpirole (0.25 mg/ kg), counteracted SR141716-induced Fos-like immunoreactivity in the ventrolateral septum, the nucleus accumbens and the dorsomedial caudate-putamen, while no antagonism was observed in the prefrontal cortex. Microdialysis experiments in awake rats indicated that SR141716, at doses which increased Fos expression (3 and 10 mg/kg), did not alter dopamine release in the shell of the nucleus accumbens. Finally, SR141716 increased the levels of neurotensin-like immunoreactivity in the nucleus accumbens, but not in the caudate-putamen. Collectively, the present results show that blockade of cannabinoid receptors increases Fos- and neurotensin-like immunoreactivity with characteristics comparable to those reported for atypical neuroleptic drugs.

Expression and presence of septal neurokinin-2 receptors controlling hippocampal acetylcholine release during sensory stimulation in rat.

We examined the expression and presence of NK2 receptors in the septal area of rat brain, and investigated their functional role in the regulation of the septohippocampal cholinergic system. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we showed the presence of NK2 receptor mRNA expression in the septal area, and detected septal NK2 binding sites by using a fluorescent-tagged neurokinin A (NKA) derivative. In vivo microdialysis was employed to explore the functional role of NK2 receptors in the release of hippocampal acetylcholine evoked by tactile stimulation in freely moving rats. Two sessions of stroking of the neck and back of the rat for 30 min, at 90 min intervals, produced a marked and reproducible increase in hippocampal acetylcholine release. This effect was dose-dependently prevented by intraperitoneal administration of the two selective non-peptide tachykinin NK2 receptor antagonists SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3 and 1 mg/kg, i.p.), but not by the inactive enantiomer of SR48968 (SR48965, 1 mg/kg) nor by the two non-peptide NK1 receptor antagonists SR140333 (3 mg/kg, i.p.) and GR205171 (1 mg/kg, i.p.). Furthermore, the intraseptal application of SR144190 (10(-8) M) reduced the sensory response. Finally, intraseptal perfusion of neurokinin A (0.01-10 microM) in anaesthetized rats produced a concentration-dependent increase in hippocampal acetylcholine release. The response to neurokinin A (0.1 microM) was prevented by SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3-1 mg/kg, i.p.). In conclusion, this study provides direct evidence for the role of endogenous NKA/substance P, through the activation of NK2 receptors, in regulating the septohippocampal cholinergic function.

Serotoninergic control of the activity and expression of glial GABA transporters in the rat cerebellum.

Gamma-Aminobutyric acid (GABA) transporters (GAT-1, GAT-2, and GAT-3) play a key role in the termination of GABA transmission and the regulation of extracellular GABA concentrations. In the present study, pharmacological, cellular, and molecular analyses provide evidence for a modulatory effect of serotoninergic neurons on the activity and expression of glial GABA transporters in the rat cerebellum. Degeneration of serotoninergic neurons after in vivo 5,7-dihydroxytryptamine (5,7-DHT) treatment resulted in a significant decrease (-27%) in [3H]-GABA uptake into cerebellar punches. This decrease probably occurred via inhibition of GAT-2 or GAT-3 activity since their inhibitor, beta-alanine, induced a decrease in [3H]-GABA uptake in punches of sham-operated rats (-28%), but not in punches of 5,7-DHT-treated rats, demonstrating that serotonin terminal degeneration had already impaired the beta-alanine-sensitive component of GABA uptake. In contrast, nipecotic acid, a preferential inhibitor of GAT-1, induced comparable decreases in [3H]-GABA uptake comparable in punches of 5,7-DHT (-38%) versus sham-operated rats (-37%). The decreases in GAT-1 (-16%), GAT-2 (-34%), and GAT-3 (-32%) mRNA levels after 5,7-DHT treatment (detected by quantitative RT-PCR) are consistent with a serotoninergic control of GABA transporter expression at the transcriptional level. The cellular distribution of GAT-2 and GAT-3 mRNA, shown by in situ hybridization, suggests a glial localization of these transporters in the cerebellum and demonstrated a preferential anatomical localization of GAT-2 mRNA in the granular layer and of GAT-3 mRNA in the deep cerebellar nuclei. A direct serotoninergic control of glial GABA uptake was further demonstrated in vitro since serotonin stimulated the activity and mRNA expression of the GABA transporters in cerebellar astrocyte cultures.

Specific potentialities of embryonic rat serotonergic neurons to innervate different periventricular targets in the adult brain.

During the development of the central nervous system, neurons are directed by both genetic and environmental factors to differentiate and form connections with their targets. We took advantage of the abundant homogeneous serotonergic innervations of the ependyma forming the supra- and subependymal plexuses to investigate possible commitment of embryonic neurons to innervate specific targets during axogenesis in the rat. The origin of the supraependymal innervation was determined by retrograde transport of cholera toxin (CT) from the ventricles. The supraependymal plexuses of the fourth ventricle mainly originated from neurons in the dorsocaudal region of the raphe dorsalis (DRN), while the rostral DRN and raphe centralis (CRN) contained perikarya projecting into the third ventricle. This suggested the existence, along the rostrocaudal axis of the raphe, of different neuronal subsets able to form distinct supraependymal plexuses in the third or fourth ventricle. To determine whether serotonergic neurons were committed to innervate specific areas of the ependyma, different embryonic metencephalic segments (rostral, median, or caudal) from 14-day-old rat embryos were independently grafted into the third or fourth ventricle of an adult brain in which the serotonergic neurons had been previously destroyed. The distinctive patterns of re-innervation specific to each of grafted segments indicate that subsets of embryonic serotonergic neurons are indeed committed to innervate certain restricted ependymal areas of the adult brain, presumably in response to different neurotropic and/or neurotrophic cues.

Rapid in situ hybridization using digoxigenin probe and microwave oven.

In situ hybridization has been developed with probes labelled with a non-radioactive nucleotide, especially digoxigenin-coupled nucleotides. These non-radioactive probes significantly reduce safety problems and experimentation time. In this paper, we have studied by in situ hybridization the messenger RNA (mRNA) of the neuropeptide pro-opiomelanocortin (POMC), in the rat pituitary gland using digoxigenin labelled oligonucleotide and a microwave oven. Our protocol permitted us to visualize POMC mRNA in all cells of the intermediate lobe and a few corticotroph cells in the anterior lobe, as it has been already demonstrated and to complete the experiment in less than 24 hrs.

Tropism of serotonergic neurons towards glial targets in the rat ependyma.

During development, recognition mechanisms between neurons and their targets are necessary for the formation of the neuronal network. Neural connections are synaptic or non-junctional. Both types of communication can be found between neurons and glial elements in the periventricular walls. Serotonergic fibers form synaptic contacts on the specialized ependymocytes of the subcommissural organ, a structure which forms the roof of the third ventricle at its junction with the aqueduct. A network of non-junctional fibers containing both GABA and serotonin spread between the cilia of the classical ependymocytes in the ventricles. These anatomical, morphological and biochemical features suggest a tropism and specific recognition mechanisms between glial elements and serotonergic neurons. This hypothesis can be tested by the study of the innervation of the subcommissural organ and the classical ependyma by grafted embryonic neurons after a chemical destruction of the serotonergic endogenous innervation. Solid implants or cell suspensions prepared from embryonic metencephalon were transplanted to either the third ventricle or the periventricular gray matter in 5,7-dihydroxytryptamine denervated rats. Grafted serotonergic neurons were able to reinnervate the classical ependyma and the subcommissural organ. The fibers forming the supraependymal plexus were non-junctional and contained both serotonin and GABA while those innervating the subcommissural organ formed synaptic contacts and contained only serotonin. The signals capable of inducing the ependymal innervation were specific for serotonergic neurons since catecholaminergic neurons present in the grafts were unable to innervate either classical or specialized ependymocytes. These results demonstrate that glial cells are targets for serotonergic neurons and that the morphological and biochemical characteristics of the serotonergic innervation are closely related to the target cell phenotype.

Developmental neuron-glia interactions: role of serotonin innervation upon the differentiation of the ependymocytes of the rat subcommissural organ.

The rat subcommissural organ (SCO), which forms the roof of the third ventricle is an adequate model to study certain mechanisms of neuron-glia interactions in vivo. The ependymocytes, the main component of the SCO, have a glial origin. They possess particular phenotypic characteristics: they accumulate [3H]GABA by a specific uptake mechanism, contain transitory GFAP during ontogenesis and do not express PS100; on the other hand they receive a 5HT input which forms typical synaptic contacts. This innervation is of particular interest to approach neuron-glia interactions during the differentiation. Studies of GABA uptake carriers during ontogenesis in SCO ependymocytes show a correlation between the onset of the 5HT innervation and the advent of the GABA uptake. Moreover, destruction of the 5HT innervation by a neurotoxin (5-7-dihydroxytryptamine), before its arrival at the SCO in newborn rat, inhibits the formation of the GABA uptake system and causes the expression of PS100 in adult SCO cells. On the other hand, the SCO of newborn rats transplanted to the fourth ventricle of an adult host rat had no capacity to take up GABA and expressed PS100 3 months after its transplantation. Finally, the SCO ependymocytes of species devoid of 5HT innervation (rabbit, mice) were unable to take up GABA and contain PS100. These data suggest that neuron-glia interactions are necessary for the advent of GABA uptake carriers and can control the expression of glial markers during ontogenesis in SCO ependymocytes.

Developmental expression of glial markers in ependymocytes of the rat subcommissural organ: role of the environment.

The rat subcommissural organ (SCO), principally composed of modified ependymocytes (a type of glial cell), is a suitable model for the in vivo study of glial differentiation. An immunohistochemical study of the ontogenesis of rat SCO-ependymocytes from embryonic day 13 to postnatal day 10 shows that these cells express transitory glial fibrillary acidic protein (GFAP) from embryonic day 19 until postnatal day 3. However, S100 protein (S100) is never expressed in the SCO-cells, contrasting with the ventricle-lining cells of the third ventricle, which contain S100 as early as embryonic day 17. Environmental factors could be responsible for the repression of GFAP and S100 in adult rats, because GFAP and S100 are observed in ependymocytes of SCO 3 months after being grafted from newborn rat into the fourth ventricle of an adult rat. Neuronal factors might be involved in the control of the expression of S100, since after the destruction of serotonin innervation by neurotoxin at birth, S100 can be observed in some SCO-ependymocytes of adult rats. On the other hand, GFAP expression is apparently not affected by serotonin denervation, suggesting the existence of several factors involved in the differentiation of SCO-cells.