Rat CNS Research Articles

Bone morphogenetic protein receptor expressions in the adult rat brain

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β (TGF-β) superfamily. BMPs exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Although BMPR expressions have been well described in the early development of the CNS, little information is available for their expressions in the adult CNS. We, thus, investigated BMPR expressions in the adult rat CNS using immunohistochemistry. Here, we show that BMPRIA, IB and II proteins are widely expressed throughout the adult CNS. Interestingly, we observed that BMPRIA, IB and II proteins are abundantly expressed in many kinds of axons. In addition, we found that BAMRIB-IR was preferentially expressed in dendrites of many neurons throughout the CNS, while BMPRIA was mainly expressed in cell bodies, showing that BMPRIA and BMPRIB are differentially targeted in a single neuron. In addition, besides abundant BMPR expressions in neurons, we exhibited BMPR expressions in astrocytes and ependymal cells. These data indicate that BMPRs are more widely expressed throughout the adult CNS than previously reported, and their continued abundant expressions in the adult brain strongly support the idea that BMPRs play pivotal roles also in the adult brain.

Distribution of stomatin expressing in the central nervous system and its up-regulation in cerebral cortex of rat by hypoxia

Stomatin is an important membrane raft protein which can combine skeleton protein, some ion channel, and transporter to regulate their functions. However, until now no data on its expression and function in CNS are available. In this study, we examined distribution of stomatin in CNS of rat, and investigated the effects of hypoxia exposure and glucocorticoid on stomatin expression in cerebral cortex of rat. Immunofluorescence staining revealed a broad expression of stomatin protein in many areas of adult rat brain and spinal cord, including the ventral horn of spinal cord, causal magnocellular nucleus of hypothalamus, the V layer of the cerebral cortex, solitary nucleus, 10 and 12 nuclei, and so on. Hypoxia or ischemic hypoxia significantly up-regulated stomatin expression in cerebral cortex, and the up-regulation was independent on adrenocortical steroids since it also occurred in adrenalectomized (ADX) rats. Moreover, treatment of ADX or sham-operated rats with dexamethasone, a synthetic glucocorticoid alone could significantly stimulate expression of stomatin in lung and heart, but not in cerebral cortex. However, dexamethasone could enhance the hypoxia-stimulated expression of stomatin in cerebral cortex of ADX rats. These findings suggested that stomatin might be involved in various physiological functions and cellular events of neurons in CNS under physiological conditions and play a potential protective role under hypoxic conditions.

Retinoid X receptor gamma signaling accelerates CNS remyelination.

The molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increase in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS.

Brain Disorders in Fetal Alcohol Syndrome

Intrauterine effect of alcohol on the development of cytomorphological structure of CNS in rats was studied by heterogeneous enzyme-linked immunosorbent assay. The level of transforming growth factor-β1 (TGF-β1) in animals during pregnancy was analyzed. Pronounced damaging effect of alcohol on brain cell in the progeny of alcoholized animals was demonstrated: loosening of nerve cells and degenerative changes in the form of pyknosis and chromatolysis in the cortex, hypothalamus, and cerebellum; subtotal decrease (sometimes complete absence) of neuroendocrine granules. The level of TGF-β1 was significantly increased in alcoholized pregnant females, which can attest to defects of the receptor apparatus of the target cells in both females and the progeny. Thus, the observed peculiarities of TGF-β1 expression are comparable to morphological changes in the brain and can be extrapolated to similar processes in humans (fetal alcohol syndrome).

ProNGF Induces PTEN via p75NTRto Suppress Trk-Mediated Survival Signaling in Brain Neurons

Proneurotrophins and mature neurotrophins activate different signaling pathways with distinct effects on their target cells: proneurotrophins can induce apoptotic signaling via p75(NTR), whereas mature neurotrophins activate Trk receptors to influence survival and differentiation. Here, we demonstrate that the PTEN (phosphatase and tensin homolog deleted on chromosome 10) phosphatase represents a novel switch between the survival and apoptotic signaling pathways in rat CNS neurons. Simultaneous activation of p75(NTR) by proNGF and TrkB signaling by BDNF elicited apoptosis despite TrkB phosphorylation. Apoptosis induced by p75(NTR) required suppression of TrkB-induced phosphoinositide-3 kinase signaling, mediated by induction of PTEN, for apoptosis to proceed. Inhibition of PTEN restored the ability of BDNF to phosphorylate Akt and protect cultured basal forebrain neurons from proNGF-induced death. In vivo, inhibition or knockdown of PTEN after pilocarpine-induced seizures protected CNS neurons from p75(NTR)-mediated death, demonstrating that PTEN is a crucial factor mediating the balance between p75(NTR)-induced apoptotic signaling and Trk-mediated survival signaling in brain neurons.

Chlorpyrifos Induced Region Specific Vulnerability in Rat CNS and Modulation by Age and Cold Stress: An Interactive Study

Chlorpyrifos (CPF), an organophosphorus insecticide is known to cause ill health in non-target animals by inducing oxidative stress. In this study influence of cold stress (15°C and 20°C) and age as modulating factors on CPF induced oxidative stress was addressed to assess age-related differences and vulnerability in central nervous system of rats. The results indicated an interaction with age and cold exposure resulting in marked decreased activity levels of SOD (P < 0.05), CAT (P < 0.05), GPx (P < 0.05), GST (P < 0.05) followed by increased MDA (P < 0.05) and decreased GSH levels (P < 0.05). The ANOVA and Post-hoc analysis showed that antioxidant enzymes decreased significantly (P < 0.05) on CPF exposure. Moreover synergistic action of CPF and cold stress at 15°C caused higher inhibition on comparison with CPF and cold stress alone and together at 20°C indicating the extent of peroxidative damage in discrete regions of CNS. Further this study showed young individuals to be more sensitive than adults.

Differential localization of neuregulin-1 type III in the central and peripheral nervous system

In the developing PNS, axonal neuregulin-1 (NRG1) type III is the key determinant for myelination. However, the specific role for NRG1 (III) in the CNS has not been established. To address this issue, isotype-specific antibodies were generated, characterized, and used for the immunofluorescent localization of NRG1 (III) in the developing and adult CNS of rat. In contrast to adult peripheral nerve, which showed robust axonal staining, no immunoreactivity was observed in CNS myelinated tracts during the period of active myelination or in the adult CNS. Surprisingly, NRG1 (III) was prominently expressed on dendrites and soma in both the developing and adult CNS. These findings were corroborated through the subcellular fractionation of adult rat brain combined with an immunoblotting analysis. The immunolocalization of NRG1 (III) suggests that it plays a novel role in the myelination fate of CNS axons possibly through undetermined roles in neuronal maturation, or dendritic development and activation.

Intrathecal eugenol administration alleviates neuropathic pain in male Sprague‐Dawley rats

The main objective of this study was to determine the central effect of eugenol on neuropathic pain when injected intrathecally at the level of the lumbar spinal cord. In a preliminary study the penetrability of eugenol was evaluated in the CNS of rats. Blood, brain and spinal cord samples were collected at selected time points following eugenol administration and concentrations were determined by tandem liquid chromatography-mass spectrometry. Brain-to-plasma and spinal cord-to-plasma ratios (3.3 and 6.7, respectively) suggest that eugenol penetrates relatively well the CNS of rats, with a preferential distribution in the spinal cord. Following the induction of neuropathic pain in rats using the sciatic nerve ligation model, intrathecal injections of eugenol were done to evaluate the central effect of eugenol. Treatment with 50 μg of eugenol significantly decreased secondary mechanical allodynia after 15 min, 2 h and 4 h (p < 0.05; <0.005; <0.05, respectively) and improved thermal hyperalgesia after 2 h and 4 h (p < 0.001 and p < 0.05). The results support the hypothesis that eugenol may alleviate neuropathic pain, both allodynia and hyperalgesia, by acting centrally most probably at the level of the dorsal horn of the spinal cord where vanilloid receptors can be found.

Immunohistochemical localization of the neuropeptide S receptor in the rat central nervous system

The neuropeptide S receptor (NPSR) is a G-protein coupled receptor that is potently activated by the linear 20 amino acid peptide, neuropeptide S (NPS). Central administration of NPS promotes arousal and anxiolytic-like effects in rodents, and fails to promote such effects in NPSR knockout animals or in the presence of NPSR-selective antagonists. In situ hybridization (ISH) studies in rat brain have revealed that the mRNAs encoding the NPS precursor and the NPS receptor are expressed at high levels in discrete regions of the rat CNS. The distribution of the NPSR protein in brain has not been reported due to a lack of available antibodies. We have generated and validated a NPSR-specific antibody and used it to determine the distribution of the NPSR in male Sprague–Dawley (SD) rat brain. The anti-NPSR antibody identified a single protein by Western blot with an estimated molecular weight of 65 kD, which was prevented by pre-incubation of the antibody with the immunizing peptide. The protein distribution identified with this antibody in rat brain was consistent both with the mRNA distribution identified by in situ hybridization, and to the localization pattern identified by a second NPSR-specific antibody against a distinct NPSR epitope. NPSR protein was identified in the medial amygdala (MeA), substantia nigra pars compacta, subiculum, dorsal raphe, and several hypothalamic and thalamic regions. Additionally, NPSR protein was localized in the pyramidal cell layer of the ventral hippocampus, the medial habenula (MHb), and was widely distributed in the cortex. The distribution of NPSR protein provides further insight into the organization of the NPS system and may guide future studies on the role of the NPSR in brain.

Activation of PPAR‐γ and PTEN cascade participates in lovastatin‐mediated accelerated differentiation of oligodendrocyte progenitor cells

Previously, we and others documented that statins including-lovastatin (LOV) promote the differentiation of oligodendrocyte progenitor cells (OPCs) and remyelination in experimental autoimmune encephalomyelitis (EAE), an multiple sclerosis (MS) model. Conversely, some recent studies demonstrated that statins negatively influence oligodendrocyte (OL) differentiation in vitro and remyelination in a cuprizone-CNS demyelinating model. Therefore, herein, we first investigated the cause of impaired differentiation of OLs by statins in vitro settings. Our observations indicated that the depletion of cholesterol was detrimental to LOV treated OPCs under cholesterol/serum-deprived culture conditions similar to that were used in conflicting studies. However, the depletion of geranylgeranyl-pp under normal cholesterol homeostasis conditions enhanced the phenotypic commitment and differentiation of LOV-treated OPCs ascribed to inhibition of RhoA-Rho kinase. Interestingly, this effect of LOV was associated with increased activation and expression of both PPAR-γ and PTEN in OPCs as confirmed by various pharmacological and molecular based approaches. Furthermore, PTEN was involved in an inhibition of OPCs proliferation via PI3K-Akt inhibition and induction of cell cycle arrest at G1 phase, but without affecting their cell survival. These effects of LOV on OPCs in vitro were absent in the CNS of normal rats chronically treated with LOV concentrations used in EAE indicating that PPAR-γ induction in normal brain may be tightly regulated-providing evidences that statins are therapeutically safe for humans. Collectively, these data provide initial evidence that statin-mediated activation of the PPAR-γ-PTEN cascade participates in OL differentiation, thus suggesting new therapeutic-interventions for MS or related CNS-demyelinating diseases.

Ventilatory impairment in the dysmyelinated Long Evans shaker rat

Although respiratory complications significantly contribute to morbidity/mortality in advanced myelin disorders, little is known concerning mechanisms whereby dysmyelination impairs ventilation, or how patients compensate (i.e. plasticity). To establish a model for studies concerning mechanisms of ventilatory impairment/compensation, we tested the hypotheses that respiratory function progressively declines in a model of CNS dysmyelination, the Long Evans shaker rat (les). The observed impairment is associated with abnormal inspiratory neural output. Minimal myelin staining was found throughout the CNS of les rats, including the brainstem and cervical bulbospinal tracts. Ventilation (via whole-body plethysmography) and phrenic motor output were assessed in les and wild-type (WT) rats during baseline, hypoxia (11% O2) and hypercapnia (7% CO2). Hypercapnic ventilatory responses were similar in young adult les and WT rats (2 months old); in hypoxia, rats exhibited seizure-like activity with sustained apneas. However, 5–6 month old les rats exhibited decreased breathing frequencies, mean inspiratory flow (VT/TI) and ventilation (V˙E) during baseline and hypercapnia. Although phrenic motor output exhibited normal burst frequency and amplitude in 5–6 month old les rats, intra-burst activity was abnormal. In WT rats, phrenic activity was progressive and augmenting; in les rats, phrenic activity was decrementing with asynchronized, multipeaked activity. Thus, although ventilatory capacity is maintained in young, dysmyelinated rats, ventilatory impairment develops with age, possibly through discoordination in respiratory motor output. This study is the first reporting age-related breathing abnormalities in a rodent dysmyelination model, and provides the foundation for mechanistic studies of respiratory insufficiency and therapeutic interventions.

Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel‐dependent glutamate and GABA release in rat CNS

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

Neurotensin modulates central muscarinic receptors, an effect which does not involve the high-affinity neurotensin receptor (NTS1)

Neurotensin (NT) is a tridecapeptide distributed in central and peripheral nervous systems, which can behave as a neurotransmitter or neuromodulator at central and peripheral levels. Herein we tested the potential effect of this peptide on quinuclidinyl benzilate ([ 3H]-QNB) binding to muscarinic receptor in rat CNS membranes. It was observed that NT decreased up to 50–70% ligand binding at 1 × 10 − 7 M–1 × 10 − 5 M concentration in cerebral cortex, cerebellum and striatum. In the hippocampus, NT exerted a biphasic effect, behaving as a stimulator in the presence of 1 × 10 − 12 M–1 × 10 − 10 M concentration but as an inhibitor at 1 × 10 − 8 M–1 × 10 − 5 M concentration. In order to test the involvement of high-affinity NT receptor (NTS1) in NT inhibitory effect, assays were carried out in the presence of 1 × 10 − 6 M NT and/or SR 48692 (Sanofi-Aventis, U.S., Inc.), a specific antagonist for this receptor, dissolved in dimethylsulfoxide (DMSO) 10% v/v. As controls, membranes incubated with DMSO and/or NT 1 × 10 − 6 M plus DMSO were processed. It was found that NT + DMSO decreased [ 3H]-QNB binding to cerebral cortex, cerebellum and hippocampal membranes by 49%, 32% and 53%, respectively. This inhibition was not observed with the DMSO control group. Membrane preincubation with 1 × 10 − 6 M SR 48692 failed to alter NT effect on binding. SR 48692 at 1 × 10 − 6 M concentration decreased the binding by 50% only in cerebral cortex membranes, suggesting a possible direct effect of the antagonist on muscarinic receptors in this area. It was therefore concluded that the high-affinity NT receptor may not be involved in ligand binding inhibition to muscarinic receptor by NT.

Functional Magnetic Resonance Imaging of Neural Activity in Rat CNS in Response to Chromatic Stimuli

Functional Magnetic Resonance Imaging of Neural Activity in Rat CNS in Response to Chromatic Stimuli

Morphofunctional Analysis of the Effects of Total Sleep Deprivation on the CNS in Rats

The aim of the present work was to perform a morphofunctional analysis of the effects of total sleep deprivation of different durations on neuron and synapse ultrastructure in the somnogenic formations (layers III-V of the anterior limbic cortex, field CA1 of the dorsal hippocampus, the pontine reticular formation, the dorsal cervical nucleus, and the locus ceruleus) in rats, with assessment of study investigative, comfort, and sexual behavior, as well as food and water consumption. Reparative changes in neurons and synapses after total sleep deprivation lasting 12-24 h were accompanied by increases in all types of behavioral reactions. Repair processes in neurons and synapses were somewhat weakened after total sleep deprivation lasting 36 h, while dystrophic changes affected greater proportions of neurons and synapses, and the numbers of behavioral reactions decreased.

Expression profile of multiple secretory phospholipase A 2 isoforms in the rat CNS: Enriched expression of sPLA 2-IIA in brainstem and spinal cord

Phospholipases A 2 (PLA 2) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A 2 (sPLA 2) isoforms in the normal rat CNS with focus on sPLA 2-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA 2-IB expression was low throughout the CNS, sPLA 2-IIA expression was high in the brainstem and spinal cord, sPLA 2-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA 2-V expression was high in the olfactory bulb and cerebellum, and sPLA 2-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA 2-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA 2-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA 2-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA 2-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA 2 in nociceptive transmission.

What Is the Optimal Value of the g-Ratio for Myelinated Fibers in the Rat CNS? A Theoretical Approach

BackgroundThe biological process underlying axonal myelination is complex and often prone to injury and disease. The ratio of the inner axonal diameter to the total outer diameter or g-ratio is widely utilized as a functional and structural index of optimal axonal myelination. Based on the speed of fiber conduction, Rushton was the first to derive a theoretical estimate of the optimal g-ratio of 0.6 [1]. This theoretical limit nicely explains the experimental data for myelinated axons obtained for some peripheral fibers but appears significantly lower than that found for CNS fibers. This is, however, hardly surprising given that in the CNS, axonal myelination must achieve multiple goals including reducing conduction delays, promoting conduction fidelity, lowering energy costs, and saving space.Methodology/Principal FindingsIn this study we explore the notion that a balanced set-point can be achieved at a functional level as the micro-structure of individual axons becomes optimized, particularly for the central system where axons tend to be smaller and their myelin sheath thinner. We used an intuitive yet novel theoretical approach based on the fundamental biophysical properties describing axonal structure and function to show that an optimal g-ratio can be defined for the central nervous system (≈0.77). Furthermore, by reducing the influence of volume constraints on structural design by about 40%, this approach can also predict the g-ratio observed in some peripheral fibers (≈0.6).Conclusions/SignificanceThese results support the notion of optimization theory in nervous system design and construction and may also help explain why the central and peripheral systems have evolved different g-ratios as a result of volume constraints.

Dissociation of AGAT, GAMT and SLC6A8 in CNS: Relevance to creatine deficiency syndromes

AGAT and GAMT, the two enzymes of the creatine synthesis pathway, are well expressed within CNS, suggesting autonomous brain creatine synthesis. This contradicts SLC6A8 deficiency, which causes creatine deficiency despite CNS expression of AGAT and GAMT. We hypothesized that AGAT and GAMT were not co-expressed by brain cells, and that guanidinoacetate must be transported between cells to allow creatine synthesis. We finely analyzed the cell-to-cell co-expression of AGAT, GAMT and SLC6A8 in various regions of rat CNS, and showed that in most structures, cells co-expressing AGAT + GAMT (equipped for autonomous creatine synthesis) were in low proportions (< 20%). Using reaggregating brain cell cultures, we also showed that brain cells take up guanidinoacetate and convert it to creatine. Guanidinoacetate uptake was competed by creatine. This suggests that in most brain regions, guanidinoacetate is transported from AGAT- to GAMT-expressing cells through SLC6A8 to allow creatine synthesis, thereby explaining creatine deficiency in SLC6A8-deficient CNS.

Characterization of CLP36/Elfin/PDLIM1 in the nervous system

CLP36, one of the alpha-Actinin Associated LIM Protein (ALP)/Enigma family proteins, has a wide tissue distribution, but little is known about its expression and role in the nervous system. We show here that CLP36 is expressed in sensory ganglia but not in the CNS of adult rats. In primary dorsal root ganglion (DRG) neurons, CLP36 is distributed in the soma and neurites with enrichment in the growth cones. CLP36 forms a complex with alpha-actinin and is localized to actin cytoskeleton. To examine the role of CLP36 in neuronal cells, we transfected PC12 cells with a series of CLP36 deletion mutants and found that over-expression of CLP36 PDZ domain affects neurite outgrowth. Reduction of CLP36 function in PC12 cells by RNA interference (RNAi) induced lamellipodial protrusions around cell periphery and activated growth-cone movements, resulting in an increase in the length and number of neurites. Similarly, inhibition of CLP36 in primary DRG neurons increased the rate of neurite-bearing cells. We also found that CLP36 is up-regulated in DRG neurons and facial motoneurons after nerve injury. These findings suggest that CLP36 serves as a scaffold to form a multiprotein complex that regulates actin cytoskeleton dynamics and plays a role in controlling neurite outgrowth.

Light and electron microscopy study of the neuropathologic effects of different doses of acrylamide monomer on CNS of rat

Light and electron microscopy study of the neuropathologic effects of different doses of acrylamide monomer on CNS of rat