Early life stress influences basal ganglia dopamine receptors and novel object recognition of adolescent and adult rats.

Environmental stimuli in early life are recognized to affect brain development and behavior. Mother-pup interaction constitutes a determinant stimulus during this critical period. It is known that the dopaminergic system undergoes significant reorganization during adolescence and that dopamine receptors are involved in recognition memory. Based on the above, we examined the effects of brief and prolonged maternal separation during the neonatal period (15 or 180 min daily) on basal ganglia dopamine receptors and on the behavior in the novel object recognition task of adolescent and adult male rats. Using the NOR task, we observed that the discrimination index (DI) was decreased in rats with brief maternal separations independent of age. Using receptor autoradiography, we observed that brief maternal separation induced decreases in D1, D2 and D4 receptor binding levels in adult basal ganglia nuclei, while prolonged maternal separation induced increases in D1 receptor binding levels in caudate - putamen (CPu) of adolescent rats. With immunoblotting experiments, we found decreases in D1 and increases in D2 total protein levels in CPu of adult rats with prolonged maternal separations. Α positive correlation was observed between DI and D1 binding levels in CPu, internal globus pallidus and substantia nigra, and D2 binding levels in nucleus accumbens core in adult rats, using the Pearson correlation coefficient. Our results indicate that the long-lasting effects of neonatal mother-offspring separation on dopamine receptors depend on the duration of maternal separation and age and that this early life experience impairs recognition memory in adolescent and adult rats. Furthermore, the present results suggest that modulation of striatal dopamine receptors might underlie the reduced recognition memory of adult rats with brief neonatal maternal separations.

Neonatal maternal separation affects metabotropic glutamate receptor 5 expression and anxiety-related behavior of adult rats.

Exposure to early life stress leads to long-term neurochemical and behavioral alterations. Stress-induced psychiatric disorders, such as depression, have recently been linked to dysregulation of glutamate signaling, mainly via its postsynaptic receptors. The role of metabotropic glutamate receptor 5 (mGluR5) in stress-induced psychopathology has been the target of several studies in humans. In rodents, blockade of mGluR5 produces antidepressant-like actions, whereas mice lacking mGluR5 exhibit altered anxiety-like behaviors and learning. In this study, we used well-known rodent models of early life stress based on mother-infant separation during the first 3 weeks of life in order to examine the effects of neonatal maternal separation on mGluR5 expression and on anxiety-related behavior in adulthood. We observed that brief (15 min) neonatal maternal separation, but not prolonged (3 h), induced increases in mGluR5 mRNA and protein expression levels in medial prefrontal cortex and mGluR5 protein levels in dorsal, but not ventral, hippocampus of adult rat brain. Behavioral testing using the open-field and the elevated-plus maze tasks showed that brief maternal separations resulted in increased exploratory and decreased anxiety-related behavior, whereas prolonged maternal separations resulted in increased anxiety-related behavior in adulthood. The data indicate that the long-lasting effects of neonatal mother-offspring separation on anxiety-like behavior and mGluR5 expression depend on the duration of maternal separation and suggest that the increased mGluR5 receptors in medial prefrontal cortex and hippocampus of adult rats exposed to brief neonatal maternal separations may underlie their heightened ability to cope with stress.

Effects of an early life experience on rat brain cannabinoid receptors in adolescence and adulthood.

Neonatal handling is an experimental model of early life experience associated with resilience in later life challenges, altering the ability of animals to respond to stress. The endocannabinoid system of the brain modulates the neuroendocrine and behavioral effects of stress, while this system is also capable of being modulated by stress exposure itself. The present study has addressed the question of whether neonatal handling in rats could affect cannabinoid receptors, in an age- and sex-dependent manner, using in situ hybridization and receptor binding techniques. Different effects of neonatal handling were observed in adolescent and adult brain on CB1 receptor mRNA and [3H]CP55,940 binding levels, which in some cases were sexually dimorphic. Neonatal handling interfered in the developmental trajectories of CB1 receptor mRNA levels in striatum and amygdaloid nuclei, as well as of [3H]CP55,940 binding levels in almost all regions studied. Adult handled rats showed reduced [3H]CP55,940 binding levels in the prefrontal cortex, striatum, nucleus accumbens and basolateral amygdala, while binding levels in prefrontal cortex of adolescent handled rats were increased. Finally, handling resulted in decreases in female [3H]CP55,940 binding levels in the striatum, nucleus accumbens, CA3 and DG of dorsal hippocampus and basolateral amygdala. Our results suggest that a brief and repeated maternal separation during the neonatal period induces changes on cannabinoid receptors differently manifested between adolescence and adulthood, male and female brain, which could be correlated to their stress response.

Long lasting effects of chronic WIN55,212-2 treatment on mesostriatal dopaminergic and cannabinoid systems in the rat brain.

Cannabinoid administration modulates dopamine transmission via an indirect, multisynaptic mechanism that includes the activation of cannabinoid type-1 receptor (CB1R). The present study evaluated in rodents, the effects of acute and chronic (20 days) WIN55,212-2 administration, a non-selective CB1R agonist, on dopamine uptake and synthesis in the mesolimbic and nigrostriatal dopaminergic pathways and associate them to its effects on the endocannabinoid system. The effect of spontaneous withdrawal, after different abstinence periods (7 days, 20 days), was also assessed. Acute and chronic administration of WIN55,212-2 decreased dopamine transporter (DAT) binding and mRNA levels, as well as tyrosine hydroxylase (TH) mRNA expression in the substantia nigra (SN) and ventral tegmental area (VTA). In the striatum, chronic WIN55,212-2 administration led to decreased protein expression of DAT and TH, whereas no alterations were observed after acute administration, suggesting a diminished dopamine uptake and synthesis after chronic agonist treatment. Furthermore, after chronic agonist treatment, we observed reduced CB1R binding and mRNA levels in SN and striatum, providing evidence for a possible regulatory role of the endocannabinoid system on dopaminergic function. Seven days after WIN55,212-2 cessation, we observed a rebound increase in mRNA, binding and total protein levels of DAT and TH in VTA, SN and striatum proposing the existence of a biphasic expression pattern, which was also observed in CB1R binding levels. Within the 20-day period of abstinence, TH mRNA and protein levels and CB1R binding levels remain increased. The above results indicate that chronic CB1R agonist treatment induces long-lasting control of the mesostriatal dopaminergic activity.

Behavioral and neurochemical changes in mesostriatal dopaminergic regions of the rat after chronic administration of the cannabinoid receptor agonist WIN55,212-2.

BACKGROUND: The endocannabinoid system interacts extensively with other neurotransmitter systems and has been implicated in a variety of functions, including regulation of basal ganglia circuits and motor behavior. The present study examined the effects of repeated administration of the nonselective cannabinoid receptor 1 agonist WIN55,212-2 on locomotor activity and on binding and mRNA levels of dopamine receptors and transporters and GABAA receptors in mesostriatal dopaminergic regions of the rat. METHODS: Rats received systemic injections of WIN55,212-2 (0, 0.1, 0.3, or 1mg/kg, intraperitoneally) for 20 consecutive days. Locomotor activity was measured on days 1, 10, and 20. Following the last measurement, rats were euthanized and prepared for in vitro binding and in situ hybridization experiments. RESULTS: Acutely, 0.3 and 1mg/kg of WIN55,212-2 produced hypolocomotion, which was sustained for the next 2 measurements, compared to vehicle. Repeated administration of WIN55,212-2 decreased the mRNA levels of the D2 autoreceptors in substantia nigra and ventral tegmental area and increased D1 receptor mRNA and binding in nucleus accumbens. Furthermore, both dopamine receptor and transporter binding and mRNA levels were decreased in substantia nigra. Moreover, repeated administration of WIN55,212-2 decreased GABAA receptor binding levels in dorsal striatum and substantia nigra. CONCLUSIONS: Our data indicate that chronic WIN55,212-2 administration results in sustained effects on locomotor activity, similar to those observed after acute administration, and modulates the dopaminergic and GABAergic systems in a region-, dose-, and neurotransmitter-selective manner.

Expression and phosphorylation of glutamate receptor subunits and CaMKII in a mouse model of Parkinsonism.

Dopaminergic deficiency of the weaver mutant mouse is a valuable tool to further our understanding of Parkinson׳s disease (PD) pathogenesis since dopaminergic neurons of the nigrostriatal pathway undergo spontaneous and progressive cell death. In the present study we investigated the changes in protein expression and phosphorylation of glutamate receptor subunits and αCaMKII in weaver striatum at the end of the third and sixth postnatal month. Using immunoblotting, we found increased immunoreactivity levels of both GluN2A and GluN2B subunits of NMDA receptors and GluA1 subunit of AMPA receptors approximately from 75% to 110% in the 3-month-old weaver striatum compared to control. In the 6-month-old weaver striatum, no changes were detected in GluN2A and GluA1 immunoreactivity levels, whereas GluN2B showed a 21% statistically significant increase. Our results also indicated increased phospho-S1303 GluN2B in both 3 and 6 month-olds and increased phospho-S831 and -845 GluA1 in 3 month-old weaver striatum. However, these increases did not exceed the increases observed for total GluN2B and GluA1. Furthermore, our results showed increased immunoreactivity levels for phospho-T286 αCaMKII by approximately 180% in the 6 month-old weaver striatum, while total CaMKII immunoreactivity levels were not altered at either 3- or 6-month-old weaver. Our results suggest that distinct degrees of DA neuron degeneration differentially affect expression and phosphorylation of striatal glutamate receptors and αCaMKII. Findings on this genetic parkinsonian model suggest that striatal glutamatergic signaling may play an important role in synaptic plasticity and motor behavior that follow progressive and chronic dopamine depletion in PD with biochemical consequences beyond those seen in acute toxic models.

Lesion of the cerebellar paravermis increases dopamine D1 receptor levels in the contralateral striatum.

Anatomical and biochemical findings have long suggested that a projection from the cerebellum to the basal ganglia exists, and recent findings proposed that the cerebellum influences glutamatergic striatal activity. We have previously shown that a complete, genetic, lack of Purkinje cells induces an upregulation of dopamine D1 receptors (DRD1) in the output of the basal ganglia, the substantia nigra pars reticulata. In this study, we produced a focal unilateral lesion in the cerebellar paravermal cortex and we studied the levels and distribution of dopamine receptors and transporters, with the use of in vitro receptor autoradiography. The lesion produced a statistically significant increase in DRD1 specific binding in the contralateral medial striatum and a bilateral decrease in dopamine transporter (DAT) levels in the dorsolateral striatum. Our finding of a DRD1 increase after disruption of the cerebellar corticonuclear projection suggests that the cerebellar output modulates the basal ganglia DRD1-mediated pathway.

Neural stem cells transplanted in a mouse model of Parkinson's disease differentiate to neuronal phenotypes and reduce rotational deficit.

The most prominent pathological feature in Parkinson's disease (PD) is the progressive and selective loss of mesencephalic dopaminergic neurons of the nigrostriatal tract. The present study was conducted in order to investigate whether naive and or genetically modified neural stem/precursor cells (NPCs) can survive, differentiate and functionally integrate in the lesioned striatum. To this end, stereotaxic injections of 6-OHDA in the right ascending nigrostriatal dopaminergic pathway of mice and subsequent NPC transplantations were performed, followed by apomorphine-induced rotations and double-immunofluorescence experiments. Our results demonstrate that transplanted embryonic NPCs derived from the cortical ventricular zone of E14.5 transgenic mouse embryos expressing the green fluorescent protein (GFP) under control of the beta-actin promoter and cultured as neurospheres can survive in the host striatum for at least three weeks after transplantation. The percentage of surviving GFP-positive cells in the host striatum ranges from 0.2% to 0.6% of the total transplanted NPCs. Grafted cells functionally integrate in the striatum, as indicated by the statistically significant decrease of contralateral rotations after apomorphine treatment. Furthermore, we show that within the striatal environment GFP-positive cells differentiate into beta-III tubulin-expressing neurons, but not glial cells. Most importantly, GFP-positive cells further differentiate to dopaminergic (TH-positive) and medium size spiny (DARPP-32- positive) neuronal phenotypes. Over-expression of the cell cycle exit and neuronal differentiation protein Cend1 in NPCs enhances the generation of GABAergic, but not dopaminergic, neuronal phenotypes after grafting in the lesioned striatum. Our results encourage the development of strategies involving NPC transplantation for the treatment of neurodegenerative diseases.

Modulation of the basal ganglia dopaminergic system in a transgenic mouse exhibiting dystonia-like features.

Dystonia is a movement disorder characterized by involuntary excessive muscle activity and abnormal postures. There are data supporting the hypothesis that basal ganglia dysfunction, and specifically dopaminergic system dysfunction, plays a role in dystonia. In the present study, we used hyperkinetic transgenic mice generated as a model of DYT1 dystonia and compared the basal ganglia dopaminergic system between transgenic mice exhibiting hyperkinesia (affected), transgenic mice not showing movement abnormalities (unaffected), and non-transgenic littermates. A decrease in the density of striatal D2 binding sites, measured by [³H]raclopride binding, and D2 mRNA expression in substantia nigra pars compacta (SNpc) was revealed in affected and unaffected transgenic mice when compared with non-transgenic. No difference in D1 receptor binding and DAT binding, measured by [³H]SCH23390 and [³H]WIN35428 binding, respectively, was found in striatum of transgenic animals. In SNpc, increased levels of DAT binding sites were observed in affected and unaffected animals compared to non-transgenic, whereas no change in DAT mRNA expression was found. Our results show selective neurochemical changes in the basal ganglia dopaminergic system, suggesting a possible involvement in the pathophysiology of dystonia-like motor hyperactivity.

Experience-dependent regulation of NMDA receptor subunit composition and phosphorylation in the retina and visual cortex.

Purpose. Experimental manipulation of experience during development can have profound effects on the functioning of the resulting circuits. N-methyl-d-aspartate glutamate receptor (NMDAR) activity is required for the establishment and refinement of neural circuits during development. In the present study, the authors addressed the issue of experience-dependent regulation of NMDARs by examining the effects of visual experience and deprivation on subunit composition and subunit phosphorylation of NMDAR in the retina and visual cortex. Methods. Total homogenates were prepared from retinas and visual cortices of 30-day-old (P30) Wistar rats, raised either in a normal 12-hour light/12-hour dark cycle (normal-reared [NR]) or in complete darkness from birth (dark-reared [DR]). Some of the DR animals were exposed to light for 6 hours at P30 (DR+6h). Immunoblotting was performed for the NMDAR subunits, NR2A and NR2B, and for the phosphorylated NR2B subunit protein at serine 1303 (pNR2B-Ser1303). Results. Dark rearing for 1 month decreased the NR2A/NR2B ratio and increased the level of phosphorylation of NR2B subunit at Ser1303 in the retina and visual cortex. Light exposure at P30 reversed the effects of visual deprivation on NMDAR composition and NR2B phosphorylation in both regions. Conclusions. These results indicated that NMDAR subunit composition and NR2B phosphorylation at Ser1303 is regulated bidirectionally by visual experience and deprivation in rat retina and visual cortex.

Expression of GluR6 kainate receptor subunit in granular layer of weaver mouse cerebellum.

The Girk2 ( wv ) (weaver) mutation impairs migration of cerebellar granule cells from external to internal granular layer and induces neuronal death during the first 2 weeks of postnatal life. Kainate receptors are heteromeric ionotropic receptors of glutamate consisting of five subunits termed GluR5, GluR6, GluR7, KA1 and KA2. In order to investigate whether the weaver gene affects the expression of kainate receptors in weaver cerebellum, we determined mRNA expression levels of GluR6 kainate receptor subunit and [(3)H]kainic acid specific binding in the developing cerebellum, using in situ hybridization and receptor film autoradiography, respectively. In the weaver postnatal day 10 (P10) cerebellum, our data indicated lower levels of GluR6 mRNA expression and lower [(3)H]kainic acid specific binding in external granular layer (EGL) compared to normal EGL. Our results are indicative of either down-regulation of kainate receptors or modulation of their functional characteristics in weaver granule cells.

Pharmacological characterization and anatomical distribution of the dopamine transporter in the mouse cerebellum.

We studied the binding parameters, the pharmacological profile and the anatomical distribution of the dopamine transporter in the mouse cerebellum by using the specific dopamine uptake antagonist [(3)H]GBR12935 and an antidopamine transporter monoclonal antibody. Competition experiments in cerebellar and striatal membrane preparations showed that [(3)H]GBR12935 binds to a specific binding site, sensitive to dopamine and low concentrations of mazindol. The affinity of dopamine for the cerebellar binding site was one order of magnitude lower than the affinity for the striatal binding site. Saturation experiments in cerebellar membrane preparations and thin frozen sections showed that the affinity of [(3)H]GBR12935 for this binding site is similar to its affinity for the striatal dopamine transporter. Saturable binding was lobule specific and in general was higher in the molecular layer compared to the granule cell layer. The immunohistochemical signal was mostly concentrated in the Purkinje cell layer and the cerebellar nuclei. The results suggest that the cerebellar dopamine transporter is similar but not identical to the striatal dopamine transporter and that it is present in the mouse cerebellum in a lobule and lamina specific pattern.

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression.

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [(35)S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

BACKGROUND: The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. METHODS: [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. RESULTS: In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). CONCLUSIONS: The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.