Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain

Rats repeatedly exposed to restraint show a reduced hypothalamic–pituitary–adrenal axis response upon restraint re-exposure. This hypothalamic–pituitary–adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic–pituitary–adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.     

Regional adaptations in PSD-95, NGFI-A and secretogranin gene transcripts related to vulnerability to behavioral sensitization to amphetamine in the Roman rat strains

Genetically selected for high or low two-way active avoidance, Roman high-avoidance (RHA) and Roman low-avoidance (RLA) rats differ in their central dopaminergic activity, sensation/novelty- and substance-seeking profiles. These animals are, therefore, well suited to identify anatomical and neurochemical concomitants of behavioral sensitization, a phenomenon linked to addictive liability. We submitted inbred RHA (RHA-I), inbred RLA (RLA-I) and Sprague-Dawley-OFA (SD-OFA) rats to a sensitization regimen with amphetamine and studied the behavioral response to an amphetamine challenge after a 2-week withdrawal period. The expression patterns of nerve growth factor inducible clone A (NGFI-A), secretogranin, post-synaptic density protein of 95 Kd (PSD-95), prodynorphin and proenkephalin mRNA were also analyzed using in situ hybridization, after the challenge with amphetamine. RHA-I rats showed stronger sensitization than SD-OFA rats. RLA-I rats did not show sensitization but were hyper-reactive to amphetamine. Expression of behavioral sensitization in RHA-I rats activated secretogranin and PSD-95 mRNA in the nucleus accumbens core. On the other hand, high induction of NGFI-A mRNA in the central amygdala was observed in RLA-I rats when they experienced amphetamine for the first time in the challenge. Our results reveal that 1) the acute locomotor response to amphetamine does not predict vulnerability to behavioral sensitization and 2) differences in vulnerability to sensitization may involve distinctive cellular adaptations at particular brain locations which may be related to addictive vulnerability.     

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Although macrophages play an important role in lipid-induced glomerular injury, we know little of the mechanisms by which hyperlipidaemia induces monocyte recruitment. This study investigated the role of M-CSF and macrophage MIF in monocyte recruitment during the development of lipid-induced glomerular injury in the susceptible ExHC rat strain. Groups of five ExHC rats were fed a high cholesterol diet (HCD) containing 3% cholesterol, 0.6% sodium cholate and 15% olive oil, and killed after 3 days, 1, 2 or 6 weeks. Control animals were killed on day 0 or after 6 weeks on a normal diet. Animals were hypercholesterolaemic 3 days after the induction of the HCD, but showed no change in plasma triglycerides over the 6-week period. Glomerular macrophage accumulation was first evident at 1–2 weeks and increased up to week 6, when macrophage-derived foam cells were seen in almost all glomeruli, and segmental lesions and mild proteinuria were also evident. Combined in situhybridization and immunohistochemistry staining demonstrated that, coincident with the induction of hypercholesterolaemia on day 3, there was marked up-regulation of M-CSF and MIF mRNA expression by intrinsic glomerular cells (mostly mesangial cells and podocytes) which preceded monocyte recruitment. There was a highly significant correlation between the number of M-CSF and MIF-positive cells and glomerular macrophage accumulation over the 6-week period. Although some glomerular macrophages and foam cells exhibited M-CSF and MIF expression, the major source of these molecules was intrinsic glomerular cells. No local macrophage proliferation was observed during the development of glomerular lesions. In conclusion, hypercholesterolaemia caused marked up-regulation of M-CSF and MIF expression by intrinsic glomerular cells, which correlated with monocyte recruitment and the development of lipid-induced glomerular injury. This is the first study to implicate local synthesis of MIF in the pathogenesis of lipid-induced lesions.     

Expression of reelin, its receptors and its intracellular signaling protein, Disabled1 in the canary brain: relationships with the song control system

Songbirds produce learned vocalizations that are controlled by a specialized network of neural structures, the song control system. Several nuclei in this song control system demonstrate a marked degree of adult seasonal plasticity. Nucleus volume varies seasonally based on changes in cell size or spacing, and in the case of nucleus HVC and area X on the incorporation of new neurons. Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. In mammals, reelin is also expressed in the adult brain but its functions are less well characterized. We investigated the relationships between the expression of reelin and/or its receptors and the dramatic seasonal plasticity in the canary (Serinus canaria) brain. We detected a broad distribution of the reelin protein, its mRNA and the mRNAs encoding for the reelin receptors (VLDLR and ApoER2) as well as for its intracellular signaling protein, Disabled1. These different mRNAs and proteins did not display the same neuroanatomical distribution and were not clearly associated, in an exclusive manner, with telencephalic brain areas that incorporate new neurons in adulthood. Song control nuclei were associated with a particular specialized expression of reelin and its mRNA, with the reelin signal being either denser or lighter in the song nucleus than in the surrounding tissue. The density of reelin-immunoreactive structures did not seem to be affected by 4 weeks of treatment with exogenous testosterone. These observations do not provide conclusive evidence that reelin plays a prominent role in the positioning of new neurons in the adult canary brain but call for additional work on this protein analyzing its expression comparatively during development and in adulthood with a better temporal resolution at critical points in the reproductive cycle when brain plasticity is known to occur.     

Changes in Laminin Chain Expression in Pre- and Postnatal Rat Pituitary Gland

Cell–matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5–15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.     

Postnatal changes in expression of vesicular glutamate transporters in the main olfactory bulb of the rat

Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization–immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events.     

Expression of P2X5 receptors in the mouse CNS

P2X receptors are ATP-gated cationic channels composed of seven known subunits (P2X1-7) which are involved in different functions in neural tissue. The present study investigates the P2X5 receptor expression pattern in the mouse CNS using immunohistochemistry and in situ hybridization histochemistry. The specificity of the immunostaining has been verified by pre-absorption, Western blot and in situ hybridization methods. Heavy P2X5 receptor immunostaining was observed in the mitral cells of the olfactory bulb; cerebral cortex; globus pallidum, anterior cortical amygdaloid nucleus, amygdalohippocampal area of subcortical telencephalon; anterior nuclei, anteroventral nucleus, ventrolateral nucleus of thalamus; supraoptic nucleus, ventromedial nucleus, arcuate nucleus of hypothalamus; substantia nigra of midbrain; pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, dorsal motor vagus nucleus, area postrema of hindbrain; Purkinje cells of cerebellum; and spinal cord. The identification of extensive P2X5 receptor immunoreactivity and mRNA distribution within the CNS of the mouse demonstrated here is consistent with a role for extracellular ATP acting as a fast neurotransmitter.     

微管相关蛋白Doublecortin在成年大鼠神经元前体细胞发生中的表达

目的:探索神经元前体细胞的微管相关蛋白Doublecortin(DCX)在成年大鼠神经元前体细胞发生中的表达,为神经元前体细胞的增殖和迁移研究提供理论及实验基础。方法:将大鼠脑组织行冰冻切片,采用免疫组化在光镜下观察DCX免疫阳性细胞的表达部位和形态特点。结果:神经元前体细胞的标志物DCX主要分布在4个神经发生相关区域:室下区、齿状回的粒下层、吻侧迁移流和嗅球。然而在皮质等无神经发生的区域只有极少量的DCX免疫阳性细胞。DCX免疫阳性细胞在形态上均呈梭形的胞体和单个的前导突起。结论:DCX免疫阳性细胞的形态符合神经元前体细胞的形态特征。DCX作为神经元前体细胞的标志物可以用来研究神经元前体细胞的增殖和迁移。     

Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain

Protocadherins (PCDHs) consist of the largest subgroup of the cadherin superfamily, and most PCDHs are expressed dominantly in the CNS. Because PCDHs are involved in the homophilic cell-cell adhesion, PCDHs in the nervous system have been suggested to play roles in the formation and maintenance of the synaptic connections. Although many PCDHs (>50) are in tandem arranged as a cluster in a specific chromosome locus, there are also considerable numbers of non-clustered PCDH members (approximately 20). In this study, we examined the spatiotemporal distribution of mRNAs for 12 non-clustered PCDHs in rat brain using in situ hybridization. Some of them (PCDH1, PCDH7, PCDH9, PCDH10, PCDH11, PCDH17, and PCDH20) exhibited region-dependent expression pattern in the cerebral cortex during the early postnatal stage (P3), which is a critical period for the establishment of specific synaptic connections: PCDH7 and PCDH20 mRNAs were predominantly expressed in the somatosensory (parietal) and visual (occipital) cortices, whereas PCDH11 and PCDH17 mRNAs were preferentially expressed in the motor (forelimb and hindlimb areas) and auditory (temporal) cortices, and PCDH9 mRNA was highly expressed in the motor and main somatosensory cortices. These PCDHs were also expressed in the specific regions of the connecting thalamic nuclei. These cortical regionalization and thalamic nuclei-specificity appeared to be most distinct in P3 compared with those of embryonic and adult stages. Taken together, these results suggest that PCDHs may play specific roles in the establishment of selective synaptic connections of specific modality of cerebral cortex with other communicating brain regions such as the thalamus.     

Temporal profile of connexin 43 expression after photothrombotic lesion in rat brain

Following focal ischemic injury, several mechanisms lead to secondary expansion of the affected area and therefore increase the initial damage. We thoroughly investigated the expression of astrocytic connexin 43 (Cx43) after photothrombosis in rat brain. The temporal profile of Cx43 mRNA as well as protein expression was studied in remote, structurally uninjured cortical and hippocampal areas. The hippocampal formation revealed an increased number of Cx43 mRNA positive astrocytes and an up-regulated protein expression exclusively in the ipsilateral stratum oriens. We assume a participation of this region in glia scar formation. While Cx43 mRNA positive cells were transiently increased, immunoreactivity was reduced in the somatosensory cortex of injured hemispheres. The observed decrease of Cx43 protein in the post-ischemic cerebral cortex implies an impairment of gap junctional intercellular communication which might be detrimental to the brain.     

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.     

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.     

Stable expression of the vesicular GABA transporter following photothrombotic infarct in rat brain

Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.