Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.     

Acute ethanol induces Fos in GABAergic and non-GABAergic forebrain neurons: a double-labeling study in the medial prefrontal cortex and extended amygdala

The purpose of this study was to further address the hypothesis that ethanol activates GABAergic neurons in specific brain neurocircuits that mediate motivated behavior and control of action, such as the central extended amygdala and medial prefrontal cortex. Male Sprague-Dawley rats received habituation to 7 days of daily intragastric administration of water (5 ml/kg) followed by a single acute intragastric dose of ethanol (2.5 g/kg) or water then, 2 h later, by paraformaldehyde perfusion. Rats left undisturbed in the animal room throughout the experiment were also perfused (naive group). Brain sections were processed for single Fos immunohistochemistry or dual Fos immunohistochemistry/glutamic acid decarboxylase (GAD) mRNA in situ hybridization. Intragastric water administration increased the number of Fos-immunoreactive cells in the infralimbic cortex and lateral part of the central nucleus of the amygdala compared with the naive group. Ethanol administration increased the number of Fos-immunoreactive cells in the infralimbic (+57.5%) and prelimbic (+105.3%) cortices, nucleus accumbens shell region (+88.2%), medial part of the central nucleus of the amygdala (+160%), and lateral part of the bed nucleus of the stria terminalis (+198.8%) compared with the water-treated group. In the nucleus accumbens shell region, central nucleus of the amygdala, and bed nucleus of the stria terminalis, more than 80% of Fos-immunoreactive neurons were GABAergic after ethanol administration. In contrast, in the prelimbic cortex, 75% of Fos-immunoreactive neurons were not GABAergic. These results constitute new evidence for region-specific functional interactions between ethanol and GABAergic neurons.     

Effects of aging on the electrophysiological properties of layer 5 pyramidal cells in the monkey prefrontal cortex

A significant decline in executive system function mediated by the prefrontal cortex (PFC) often occurs with normal aging. In vitro slice studies have shown that layer 2/3 pyramidal cells in the monkey PFC exhibit increased action potential (AP) firing rates which may, in part, contribute to this decline. Given that layer 5 cells also play a role in executive system function, it is important to determine if similar age-related changes occur in these cells. Whole-cell patch-clamp recordings in in vitro slices prepared from the PFC of young and aged behaviorally characterized rhesus monkeys were employed to answer this question. Basic membrane and repetitive AP firing properties were unaltered with age. Aged cells exhibited significantly decreased single AP amplitude, duration and fall time and increased slow afterhyperpolarization (sAHP) amplitude, but these changes were not associated with cognitive performance. This study demonstrates that layer 5 pyramidal cells, unlike layer 2/3 pyramidal cells, undergo only modest electrophysiological changes with aging, and that these changes are unlikely to contribute to age-related cognitive decline.     

Growth hormone and its receptor in projection neurons of the chick visual system: retinofugal and tectobulbar tracts

Recent studies have shown the presence of growth hormone (GH) in the retinal ganglion cells (RGCs) of the neural retina in chick embryos at the end of the first trimester [embryonic day (E) 7] of the 21 day incubation period. In this study the presence of GH in fascicles of the optic fiber layer (OFL), formed by axons derived from the underlying RGCs, is shown. Immunoreactivity for GH is also traced through the optic nerve head, at the back of the eye, into the optic nerve, through the optic chiasm, into the optic tract and into the stratum opticum and the retinorecipient layer of the optic tectum, where the RGC axons synapse. The presence of GH immunoreactivity in the tectum occurs prior to synaptogenesis with RGC axons and thus reflects the local expression of the GH gene, especially as GH mRNA is also distributed within this tissue. The distribution of GH-immunoreactivity in the visual system of the E7 embryo is consistent with the distribution of the GH receptor (GHR), which is also expressed in the neural retina and tectum. The presence of a GH-responsive gene (GHRG-1) in these tissues also suggests that the visual system is not just a site of GH production but a site of GH action. These results support the possibility that GH acts as a local growth factor during early embryonic development of the visual system.     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

Expression of melanocortin-4 receptor by rat parabrachial neurons responsive to immune and aversive stimuli

The pontine parabrachial nucleus is a major relay area for visceral and other interoceptive information, and has been implicated in mechanisms underlying anorexia and food aversion during disease. Thus, physiological studies have shown that peripheral immune stimuli, as well as the administration of aversive substances such as lithium chloride, evoke a prominent Fos-expression in the lateral parabrachial nucleus and behavioral experiments have demonstrated that this structure is critical for the acquisition of conditioned taste aversion. The present study examined in rats the relationship between parabrachial neurons activated by systemic administration of bacterial cell-wall lipopolysaccharide or lithium chloride and the melanocortin system, a major regulator of feeding and energy homeostasis that also has been implicated in aversive behavior. Dual-labeling in situ hybridization showed melanocortin-4 receptor expression on neurons in the external lateral parabrachial subnucleus that displayed lipopolysaccharide- or lithium chloride-induced expression of c-fos mRNA. Melanocortin-4 receptor mRNA was also co-expressed with mRNA for calcitonin gene-related peptide in this subnucleus. Taken together with previous observations showing that calcitonin gene-related peptide expressing neurons in the external lateral parabrachial subnucleus are activated by peripheral immune challenge, that lipopolysaccharide-activated external lateral parabrachial subnucleus neurons project to the amygdala, and that the amygdala-projecting neurons in the external lateral parabrachial subnucleus are calcitonin gene-related peptide-positive, the present findings suggest the presence of a melanocortin-regulated calcitonin gene-related peptide-positive pathway from the external lateral parabrachial subnucleus to the amygdala that relays information of importance to forebrain responses to certain aspects of sickness behavior. These observations may thus help explain how melanocortins can reduce feeding and influence conditioned taste aversion during inflammation and other disease conditions.     

GABAergic actions on cholinergic laterodorsal tegmental neurons: implications for control of behavioral state

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl(-) -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABA(A) receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca(2+) solutions. THIP, a selective GABA(A) receptor agonist with a preference for δ-subunit containing GABA(A) receptors, induced inward currents, suggesting the existence of extrasynaptic GABA(A) receptors. LDT cells also possess GABA(B) receptors as baclofen-activated a TTX- and low Ca(2+)-resistant outward current that was attenuated by the GABA(B) antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a G(IRK) mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABA(B) receptors linked to the same G(IRK) activated in these cells by muscarinic receptor stimulation. Activation of GABA(B) receptors inhibited Ca(2+) increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca(2+) were unaltered by prior emptying of intracellular Ca(2+) stores, but were abolished by low extracellular Ca(2+) and pre-application of nifedipine, indicating that activation of GABA(B) receptors inhibits influx of Ca(2+) involving L-type Ca(2+) channels. Presence of GABA(C) receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABA(C) receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABA(A, B and C) receptors, including extrasynaptically located GABA(A) receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT.     

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen

Summary In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarily, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudateputamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.     

Neurotrophin-3 administration alters neurotrophin, neurotrophin receptor and nestin mRNA expression in rat dorsal root ganglia following axotomy

In the months following transection of adult rat peripheral nerve some sensory neurons undergo apoptosis. Two weeks after sciatic nerve transection some neurons in the L4 and L5 dorsal root ganglia begin to show immunoreactivity for nestin, a filament protein expressed by neuronal precursors and immature neurons, which is stimulated by neurotrophin-3 (NT-3) administration. The aim of this study was to examine whether NT-3 administration could be compensating for decreased production of neurotrophins or their receptors after axotomy, and to determine the effect on nestin synthesis. The levels of mRNA in the ipsilateral and contralateral L4 and L5 dorsal root ganglia were analyzed using real-time polymerase chain reaction, 1 day, 1, 2 and 4 weeks after unilateral sciatic nerve transection and NT-3 or vehicle administration via s.c. micro-osmotic pumps. In situ hybridization was used to identify which cells and neurons expressed mRNAs of interest, and the expression of full-length trkC and p75NTR protein was investigated using immunohistochemistry. Systemic NT-3 treatment increased the expression of brain-derived neurotrophic factor, nestin, trkA, trkB and trkC mRNA in ipsilateral ganglia compared with vehicle-treated animals. Some satellite cells surrounding neurons expressed trkA and trkC mRNA and trkC immunoreactivity. NT-3 administration did not affect neurotrophin mRNA levels in the contralateral ganglia, but decreased the expression of trkA mRNA and increased the expression of trkB mRNA and p75NTR mRNA and protein. These data suggest that systemically administered NT-3 may counteract the decrease, or even increase, neurotrophin responsiveness in both ipsi- and contralateral ganglia after nerve injury.     

Regional distribution of importin subtype mRNA expression in the nervous system: study of early postnatal and adult mouse

Importin-alpha and beta1 mediate the translocation of macromolecules bearing nuclear localization signals across the nuclear pore complex. Five importin-alpha isoforms have been identified in mice and six in human. Some of these importins play an important role in neural activity such as long term potentiation, but the functional differences of each isoform in the CNS are still unclear. We performed in situ hybridization (ISH) using non-isotopic probes to clarify the expression patterns of importin-alpha subtypes (alpha5, alpha7, alpha1, alpha4, alpha3) and importin-beta1 in the mouse CNS of adult and early postnatal stages. The mRNAs of the importin-alpha subtypes and importin beta1 were expressed throughout the CNS with specific patterns; importin-alpha5, alpha7, alpha3, and beta1 showed moderate to high expression levels throughout the brain and spinal cord; importin-alpha4 showed a lack of expression in limited regions; and importin-alpha1 showed a low expression level throughout the brain and spinal cord but with a moderate expression level in the olfactory bulb and reticular system. We also demonstrated that importin-alphas and beta1 mRNAs were predominantly expressed in neurons in the adult mouse brain by using double-labeling fluorescence ISH and immunohistochemistry. Moreover, importin-alphas and beta1 mRNAs were detected throughout the CNS of postnatal mice and were highly expressed in the external granule layer of the cerebellar cortex on postnatal days 0, 4, and 10. This is the first report of importin-alphas and beta1 expression throughout the CNS of adult mice, as well as in the developing brain, including cell type specific localization.     

Developmental expression and distribution of opioid receptors in zebrafish

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Involvement of glutamate neurotransmission and N-methyl-d-aspartate receptor in the activation of midbrain dopamine neurons by 5-HT1A receptor agonists: an electrophysiological study in the rat

Systemic administration of selective 5-HT1A agonists, such as 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT), stimulates the electrical activity of ventral tegmental area (VTA) dopamine neurons by a mechanism which remains unknown. We have examined if this activation is dependent on glutamatergic, serotonergic and GABAergic neurotransmission and if 5-HT1A receptors located within the VTA or within the prefrontal cortex (PFC) could contribute. In vivo electrophysiological recordings were obtained from VTA dopamine neurons from anesthetized rats. The i.v. administration of the 5-HT1A agonist 8-OHDPAT induced a strong stimulation of burst and firing activity of dopamine neurons. This activation remained unchanged in rats pre-treated with the 5-HT depleting agent parachlorophenylalanine. However, pre-administration of the GABAB receptor antagonist phaclophen, but not of the GABAA antagonist picrotoxin, significantly reduced the 8-OHDPAT-induced activation. The N-methyl-d-aspartate (NMDA) antagonist MK 801 (dizocilpine), but not the AMPA/kainate antagonist [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)quinoxalin-1-yl] methyl-phosphonate (ZK 200775), partially prevented or reversed the effects of 8-OHDPAT. However, only the combined pre-administration of the two glutamate antagonists did completely prevent the activatory response to 8-OHDPAT and even converted the effect of 8-OHDPAT into an inhibition, in half of the dopamine neurons tested. Inactivation of the local 5-HT1A receptors by the microinfusion within the VTA of the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100,635), or of pertussis toxin, reduced the ability of 8-OHDPAT to stimulate the firing of dopamine neurons but not their burst activity. On the other hand, burst activation elicited by 8-OHDPAT was strongly reduced following the inactivation of prefrontal 5-HT1A receptors achieved by the microinfusion of WAY 100,635 within the PFC. These results show that activation of midbrain dopamine neurons by the systemic administration of 5-HT1A agonists does involve the inactivation of a tonic GABAergic tone, involving mainly the GABAB receptors, probably leading to the stimulation of a glutamatergic excitatory drive from the PFC to the VTA and an increase in glutamate release. This will excite dopamine neurons, preferentially through NMDA receptors. Furthermore, our results suggest that some 5-HT1A receptors located within the VTA may also participate in this activation.     

D(1) dopaminergic control of G protein-dependent inward rectifier K(+) (GIRK)-like channel current in pyramidal neurons of the medial prefrontal cortex

Pyramidal neurons of the medial prefrontal cortex (mPFC) exhibit dopamine-dependent prolonged depolarization, which may lead to persistent activity. Persistent activation of prefrontal cortex neurons has been proposed to underlie the working memory process. The purpose of our study was to test the hypothesis that activation of D(1) dopamine receptors leads to inhibition of G protein-dependent inward rectifier K(+) (GIRK) channels, thereby supporting the prolonged depolarization of mPFC pyramidal neurons. Experiments were performed on 3-week-old rats. GIRK-like channel currents recorded from pyramidal neurons showed the following properties at -75 mV: open probability (NPo), 2.5+/-0.3 x 10(-3); mean open time, 0.53+/-0.05 ms; and conductance, 29.9+/-1.6 pS (n=60). The channel currents were strongly inward-rectified. GIRK channel currents were reversibly inhibited by the D(1) agonists SKF 38393 (10 microM) and SKF 81297 (10 microM). This inhibition was abolished by prior application of a dopamine receptor antagonist and by application of the membrane-permeable protein kinase C inhibitors chelerythrine chloride (3 microM) and calphostin C (10 microM). It was also found that the application of D(1) dopamine receptor agonists or GIRK channel inhibitors evoked depolarization of mPFC pyramidal neurons in rats. Moreover, prior application of a GIRK channel blocker eliminated the depolarizing effect of D(1) agonists. We conclude that activation of D(1) dopamine receptors may lead to inhibition of GIRK channel currents that may, in turn, lead to the prolonged depolarization of mPFC pyramidal neurons in juvenile rats.     

Effects of environmental and pharmacological stressors on c-fos and corticotropin-releasing factor mRNA in rat brain: Relationship to the reinstatement of alcohol seeking

We have observed marked heterogeneity among different stressors in their ability to reinstate alcohol seeking in rats. Of the stressors we have tested, only the environmental stressor footshock and the pharmacological stressor yohimbine induce reinstatement. The reasons for such differences among stressors are not known. The purpose of the experiments presented here is to determine the neuroanatomical substrates that underlie these behavioral differences. To this end, we assessed whether stressors effective in inducing reinstatement of alcohol seeking activate a different set of neuronal pathways than do those that are ineffective, using the technique of in situ hybridization of the mRNAs for c-fos, a marker of neuronal activation, and corticotropin-releasing factor (CRF), a stress-related peptide we have shown to be critical to footshock-induced reinstatement of alcohol seeking. Exposure of rats to the environmental stressors footshock, restraint or social defeat, or the pharmacological stressors yohimbine or FG-7142 increased levels of the mRNAs for c-fos and CRF in the brain in a number of areas previously shown to be responsive to stressors. We found regionally specific effects of the stressors on c-fos and CRF mRNA in brain regions associated with the rewarding effects of alcohol and other abused drugs. The two stressors we have previously shown to be effective in inducing reinstatement of alcohol seeking, footshock and yohimbine, induced c-fos mRNA in the shell of the nucleus accumbens, and the basolateral and central amygdalar nuclei. These two stressors also induced CRF mRNA in the dorsal region of the bed nucleus of the stria terminalis. Taken together, these results provide evidence that activity in these regions may be involved in the reinstatement of alcohol seeking induced by these stressors. These results are also in keeping with the previously demonstrated role of CRF neurons in the dorsal bed nucleus of the stria terminalis in the reinstatement of alcohol seeking induced by stress.     

The influence of group III metabotropic glutamate receptor stimulation by (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid on the parkinsonian-like akinesia and striatal proenkephalin and prodynorphin mRNA expression in rats

Group III metabotropic glutamate receptors (mGluRs) are widely distributed in the basal ganglia, especially on the terminals of pathways which seem to be overactive in Parkinson's disease. The aim of the present study was to determine whether (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-1), an agonist of group III mGluRs, injected bilaterally into the globus pallidus (GP), striatum or substantia nigra pars reticulata (SNr), can attenuate the haloperidol-induced catalepsy in rats, and whether that effect was related to modulation of proenkephalin (PENK) or prodynorphin (PDYN) mRNA expression in the striatum. Administration of ACPT-1 (0.05-1.6 microg/0.5 microl/side) caused a dose-and-structure-dependent decrease in the haloperidol (0.5 mg/kg i.p. or 1.5 mg/kg s.c.)-induced catalepsy whose order was as follows: GP>striatum>SNr. ACPT-1, given alone to any of those structures, induced no catalepsy in rats. Haloperidol (3 x 1.5 mg/kg s.c.) significantly increased PENK mRNA expression in the striatum, while PDYN mRNA levels were not affected by that treatment. ACPT-1 (3 x 1.6 microg/0.5 microl/side) injected into the striatum significantly attenuated the haloperidol-increased PENK mRNA expression, whereas administration of that compound into the GP or SNr did not influence the haloperidol-increased striatal PENK mRNA levels. Our results demonstrate that stimulation of group III mGluRs in the striatum, GP or SNr exerts antiparkinsonian-like effects in rats. The anticataleptic effect of intrastriatally injected ACPT-1 seems to correlate with diminished striatal PENK mRNA expression. However, since the anticataleptic effect produced by intrapallidal and intranigral injection of ACPT-1 is not related to a simultaneous decrease in striatal PENK mRNA levels, it is likely that a decrease in enkephalin biosynthesis is not a necessary condition to obtain an antiparkinsonian effect.     

3,4-Methylenedioxymethamphetamine induces differential regulation of tryptophan hydroxylase 2 protein and mRNA levels in the rat dorsal raphe nucleus

Previous investigations with 3,4-methylenedioxymethamphetamine (MDMA) have suggested that administration of this drug results in a degeneration of 5-HT nerve terminals and subsequent alterations in 5-HT neurotransmission. However, only limited investigations have examined the effects of MDMA on the dorsal raphe nucleus. The present study was designed to assess the effect of MDMA on the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), by measuring TPH2 protein and mRNA levels in rat dorsal raphe (DR) nucleus. Rats were administered MDMA (20 mg/kg, s.c.) or saline twice daily for 4 days and killed 14 days later. Tissue sections of the DR were processed for quantitative immunoautoradiography and in situ hybridization histochemistry for measurements of the levels of TPH2-immunoreactivity (IR) and TPH2 mRNA. To assess 5-HT axon terminal integrity after MDMA treatment, the density of 5-HT transporter (SERT) binding sites was measured by quantitative autoradiography using [125I]RTI-55 ((-)-2beta-carbomethoxy-3 beta-(4-iodophenyl) tropane) ((125)I-RTI-55) as a ligand. TPH2-IR levels were significantly decreased by 45% in the mid DR and by 40% in the caudal DR in the MDMA-treated rats compared with saline-injected rats. In contrast, TPH2 mRNA levels were significantly increased by 24% in the mid DR and by 12% in the caudal DR. MDMA treatment significantly decreased (125)I-RTI-55 labeled SERT binding sites in the striatum, nucleus accumbens and cingulate cortex demonstrating a loss of 5-HT terminals. The increase in TPH2 mRNA levels in both the mid DR and caudal DR of MDMA-treated rats may reflect a compensatory mechanism in the injured 5-HT neurons to increase TPH2 protein synthesis. Taken together, our results suggest that a serious defect occurs in the biosynthesis of TPH2 in the DR following MDMA administration.     

Long-term evaluation of cytoarchitectonic characteristics of prefrontal cortex pyramidal neurons, following global cerebral ischemia and neuroprotective melatonin treatment, in rats

Global cerebral ischemia induces alterations of working memory, as evidenced in the eight-arm radial maze, in the absence of significant changes of pyramidal neuron population in the prefrontal cortex. These alterations can be prevented by a neuroprotective melatonin treatment. Thus, the cytoarchitectonic characteristics of the pyramidal neurons located at layers III and V in the prefrontal cortex of rats that had been submitted 120 days earlier to acute global cerebral ischemia (15 min four-vessel occlusion), and melatonin (10 mg/(kgh) for 6h, i.v.) or vehicle administration, starting 30min after the end of cerebral blood flow interruption, were evaluated in order to gain information on the changes of the neural substrate underlying disruption of prefrontocortical functioning. Soma size, rough length and number of bifurcations of basilar and apical dendrites, as well as spine density and proportions of the different types of spines in a 50 microm length segment of a secondary dendrite branching from the apical and the basilar dendrites, of pyramidal neurons of the dorsal medial prefrontal cortex, were evaluated in Golgi material. A significant reduction of soma size, apical and basilar dendrite length, number of dendritic bifurcations, and spine density were observed in pyramidal neurons at layers III and V after cerebral ischemia, while these alterations were prevented by melatonin treatment. These cytoarchitectural differences between groups seem to underlie the observed alterations in spatial working memory of ischemic, vehicle-treated rats in the absence of pyramidal neuron loss, as well as the better display of these functions long after ischemia and melatonin neuroprotection.