Effects of (+)-bicuculline, a GABAa receptor antagonist, on auditory steady state response in free-moving rats.

Auditory steady-state responses (ASSRs) are states in which the electrical activity of the brain reacts steadily to repeated auditory stimuli. They are known to be useful for testing the functional integrity of neural circuits in the cortex, as well as for their capacity to generate synchronous activity in both human and animal models. Furthermore, abnormal gamma oscillations on ASSR are typically observed in patients with schizophrenia (SZ). Changes in neural synchrony may reflect aberrations in cortical gamma-aminobutyric acid (GABA) neurotransmission. However, GABA's impact and effects related to ASSR are still unclear. Here, we examined the effect of a GABAa receptor antagonist, (+)-bicuculline, on ASSR in free-moving rats. (+)-Bicuculline (1, 2 and 4 mg/kg, sc) markedly and dose-dependently reduced ASSR signals, consistent with current hypotheses. In particular, (+)-bicuculline significantly reduced event-related spectral perturbations (ERSPs) at 2 and 4 mg/kg between 10 and 30 minutes post-dose. Further, bicuculline (2 and 4 mg/kg) significantly and dose-dependently increased baseline gamma power. Furthermore, the occurrence of convulsions was consistent with the drug's pharmacokinetics. For example, high doses of (+)-bicuculline such as those greater than 880 ng/g in the brain induced convulsion. Additionally, time-dependent changes in ERSP with (+)-bicuculline were observed in accordance with drug concentration. This study partially unraveled the contribution of GABAa receptor signals to the generation of ASSR.

Different pathophysiology underlying animal models of fibromyalgia and neuropathic pain: comparison of reserpine-induced myalgia and chronic constriction injury rats.

The reserpine-induced myalgia (RIM) rat manifests fibromyalgia-like chronic pain symptoms. The present study explored the pathophysiology underlying the pain symptoms in the RIM rat and the chronic constriction injury (CCI) rat, an animal model of neuropathic pain as a reference. Nerve tissue samples were collected from the nociception-tested animals for pathological examinations. Additionally, the therapeutic efficacy of a sodium channel blocker mexiletine was assessed in both rats. A slight vacuolization in the substantia nigra (SN) occurred in some of the RIM rats without any other histopathological changes in the brain or peripheral neurons. All the RIM rats, with or without vacuolization, showed hypersensitivity to tactile, muscle pressure, and cold stimuli. In the CCI rat, neurodegenerative changes were apparent in the sciatic nerve and the spinal cord only. CCI rats displayed muscle hyperalgesia in addition to tactile and cold allodynia. Pharmacotherapy with mexiletine did not attenuate the pain in the RIM rat, although it was effective in the CCI rat. Taken together, it is not likely that pain symptoms in RIM rats are caused by degenerative changes at the level of primary afferents and spinal cord, as is the case for CCI rats. The significance of the vacuolization in the SN is less clear at present because of the minor extent of the change and the lack of correlation with nociceptive sensitivity. The pain symptoms in RIM rats could be associated with dysfunction of biogenic amines-mediated CNS pain control even without apparent pathologies in the nervous system.

Nociceptive cortical responses during capsaicin-induced tactile allodynia in mice with spinal dorsal column lesioning.

We investigated nociceptive cortical responses using transcranial flavoprotein fluorescence imaging in anesthetized mice with capsaicin-induced allodynia. Tactile stimuli applied to the hindpaw produced fluorescence increases in the contralateral somatosensory cortex of naïve mice. Lesioning of the ipsilateral dorsal column in the spinal cord abolished most of the cortical responses. However, the responses to the same tactile stimuli appeared again after capsaicin was injected into the hindpaw. The capsaicin treatment reduced the thresholds of the hindpaw withdrawal responses. These findings strongly suggest that the responses to tactile stimuli in the lesioned mice after capsaicin injection represented nociceptive cortical responses.

A protein-protein interaction of stress-responsive myosin VI endowed to inhibit neural progenitor self-replication with RNA binding protein, TLS, in murine hippocampus.

We have shown preferential expression of both mRNA and corresponding protein for myosin VI (Myo6) in the murine hippocampus within 24 h after the extreme traumatic experience, water-immersion restraint stress (WIRS), prior to a drastic decrease in neural progenitor proliferation in the dentate gyrus. Myosin (Myo6) protein levels were significantly increased in hippocampus within 24 h after flashback experience in mice previously exposed to WIRS. Myo6 protein was ubiquitously distributed in discrete mouse brain regions with exceptionally high expression in olfactory bulb, whereas Myo6 protein was expressed in cultured rat astroglia and neurons, in addition to Myo6 mRNA expression by cultured neural progenitors. In mouse embryonal carcinoma P19 cells endowed to proliferate and differentiate, Myo6 protein was expressed in line with astroglial marker protein expression. Transient over-expression of Myo6 induced a significant decrease in the size of clustered aggregates as an index of self-replication in P19 cells. Immunoprecipitation analysis revealed the interaction between Myo6 and the RNA-binding protein, translocated in liposarcoma (TLS), while TLS was predominantly expressed by neurons in the cortex, striatum, cerebellum, and hippocampus. These results suggest that Myo6 may play a pivotal role in the mechanism underlying the suppressed adult neurogenesis after traumatic stress in association with TLS.

Upregulation of Myo6 expression after traumatic stress in mouse hippocampus.

Traumatic stress has been believed to result in a variety of unusual alterations of the integrity and the functionality in the hippocampus. In this study, we searched for genes responsive to traumatic stress in the mouse hippocampus to elucidate the underlying mechanisms. Adult male mice were subjected to water-immersion restraint stress (WIRS) for 3h as an extremely stressful experience, followed by dissection of the hippocampus and subsequent extraction of RNA for differential display polymerase chain reaction (PCR) analysis. The actin-based molecular motor protein myosin VI (Myo6) was identified as a gene markedly upregulated by traumatic stress in the mouse hippocampus 24h after WIRS. Real-time PCR and Western blotting analyses clearly revealed a significant increase in the expression of both mRNA and corresponding protein for Myo6 in the hippocampus within 24h after WIRS, while WIRS failed to significantly affect the expression of Myo6 protein in the cerebral cortex, cerebellum and olfactory bulb. Immunohistochemistry analysis revealed that Myo6 protein was ubiquitously expressed throughout the mouse brain, with an extremely high level in the olfactory bulb. These results suggest that Myo6 may be selectively and rapidly upregulated to play a hitherto unidentified role in the maintenance of the integrity and functionality in the hippocampus after traumatic stress.

Transient suppression of progenitor cell proliferation through NMDA receptors in hippocampal dentate gyrus of mice with traumatic stress experience.

Post-traumatic stress disorder is a long-lasting psychiatric disease after the traumatic experience of severe fatal stress with the consequence of hippocampal atrophy. Freezing behaviors were more than quintupled on the fear-conditioning test in mice previously subjected to water immersion restrain stress (WIRS) with metronome tones when determined 1-28 days after WIRS, while these mice exhibited the increased immobility time on the forced swimming test with the increased spontaneous locomotion. Prior experience of WIRS led to a transient decrease in subsequent 5-bromo-2'-deoxyuridine (BrdU) incorporation into proliferating cells in the hippocampal dentate gyrus. These behavioral and neurochemical alterations were significantly prevented by the daily injection of the tricyclic antidepressant imipramine and the selective serotonin reuptake inhibitor fluvoxamine, respectively. Moreover, WIRS significantly decreased the number of cells holding BrdU without affecting the differentiation ratio to astroglial and neuronal lineages 28 days later. Prior administration of an NMDA receptor antagonist significantly prevented the aforementioned changes by WIRS. These results suggest that NMDA receptors may play a role in mechanisms underlying the crisis of a variety of psychiatric symptoms relevant to post-traumatic stress disorder through transient suppression of neural progenitor cell proliferation in the murine hippocampal dentate gyrus.

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Increase of AMPA receptor glutamate receptor 1 subunit and B-cell receptor-associated protein 31 gene expression in hippocampus of fatigued mice.

Central fatigue is an indispensable biosignal for maintaining life, but the neuronal and molecular mechanisms involved remain unclear. In this study, we searched for genes differentially expressed in the hippocampus of fatigued mice to elucidate the mechanisms underlying fatigue. Mice were forced to swim in an adjustable-current water pool, and the maximum swimming time (endurance) until fatigue was measured thrice. Fatigued and nonfatigued mice with equal swimming capacity and body weight were compared. We found that the genes of GluR1 and B-cell receptor-associated protein 31 (Bap31), which acts as a transport molecule in the secretory pathway or as a mediator of apoptosis, were upregulated in the hippocampus of fatigued mice, and increases of GluR1 and Bap31 were confirmed by Northern blotting and real-time PCR. No change of gene expression of AMPA receptor subunits other than GluR1 was observed. These results suggest that a compositional change of AMPA receptor (increase of GluR1) and upregulation of the Bap31 gene may be implicated in fatigue in mice.

Counteraction by repetitive daily exposure to static magnetism against sustained blockade of N-methyl-D-aspartate receptor channels in cultured rat hippocampal neurons.

In rat hippocampal neurons cultured with the antagonist for N-methyl-D-aspartate (NMDA) receptors dizocilpine (MK-801) for 8 days in vitro (DIV), a significant decrease was seen in the expression of microtubule-associated protein-2 (MAP-2) as well as mRNA for both brain-derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43), in addition to decreased viability. MK-801 not only decreased the expression of the NR1 subunit of NMDA receptors but also increased NR2A expression, without affecting NR2B expression. Repetitive daily exposure to static magnetic fields at 100 mT for 15 min led to a decrease in the expression of MAP-2, without significantly affecting cell viability or the expression of neuronal nuclei (NeuN) and GAP-43. However, the repetitive magnetism prevented decreases in both BDNF mRNA and MAP-2 and additionally increased the expression of NR2A subunit, without altering NR1 expression in neurons cultured in the presence of MK-801. Repetitive magnetism was also effective in preventing the decrease by MK-801 in the ability of NMDA to increase intracellular free Ca2+ ions, without affecting the decrease in the maximal response. These results suggest that repetitive magnetism may at least in part counteract the neurotoxicity of MK-801 through modulation of the expression of particular NMDA receptor subunits in cultured rat hippocampal neurons.

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons.

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H(2)O(2)) led to a marked decrease in cell viability in a concentration-dependent manner at a concentration range of 10 microm to 1 mm irrespective of the duration between 6 and 24 h. However, H(2)O(2) was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1-3 h after the addition of H(2)O(2). Semi-quantitative RT-PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H(2)O(2) may at least in part involve expression of particular MCT isoforms responsible for the bi-directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.

Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus.

In vitro culture of neural progenitor cells isolated from adult murine hippocampus according to the Percoll density gradient method resulted in formation of round spheres not immunoreactive to microtubule-associated protein-2 (MAP-2) or glial fibrillary acidic protein in the presence of basic fibroblast growth factor within 12 days in vitro (DIV). Reverse-transcription PCR analysis revealed constitutive expression in these neurospheres of different subunits required for assembly of functional heteromeric N-methyl-D-aspartate (NMDA) receptor channels. Immunocytochemical analysis confirmed expression of NR1, NR2A, and NR2B subunits in neurospheres cultured for 4-12 DIV. Brief (5 min) exposure to NMDA induced marked expression of c-Fos, Fos-B, Fra-2, and c-Jun proteins in neurospheres cultured for 12 DIV 2 hr later. The NMDA receptor antagonist dizocilpine markedly inhibited expression of both c-Jun and c-Fos proteins in NMDA-exposed neurospheres. Sustained exposure to NMDA not only markedly inhibited neurosphere formation by 12 DIV when exposed from 4-12 DIV, but also resulted in facilitation of subsequent differentiation of neurospheres exposed to all-trans retinoic acid to cells immunoreactive to both neuron-specific enolase and neuronal nuclei, in addition to MAP-2, as revealed by Western blot and immunocytochemistry analyses. These results suggest that functional heteromeric NMDA receptors may be expressed constitutively in neural progenitor cells before differentiation to play a crucial role in commitment and differentiation to neurons in adult murine hippocampus.