Cell density-dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca(2+) signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca(2+) levels ([Ca(2+)](i)). The order of potency followed ATP = UTP > ADP = UDP. ATP-induced rise in [Ca(2+)](i) was suppressed by U73122 and suramin, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suggesting the functional expression of G protein-coupled P2Y(2) receptors. RT-PCR and immunohistochemical studies also showed the expression of P2Y(2) receptors. [Ca(2+)](i) response to UTP changed with cell density. The UTP-induced rise in [Ca(2+)](i) was greatest at high density. V(max) (maximum Ca(2+) response) and EC(50) (agonist concentration that evokes 50% of V(max)) suggest that the amount and property of P2Y(2) receptors were changed by cell density. Note that UTP induced Ca(2+) oscillation at only medium cell density. Pharmacological studies indicated that UTP-induced Ca(2+) oscillation required Ca(2+) influx by store-operated Ca(2+) entry. Carbenoxolone, a gap junction blocker, enhanced Ca(2+) oscillation. Immunohistochemical and quantitative real-time PCR studies revealed that proliferating cell nuclear antigen (PCNA)-positive cells declined but the mRNA expression level of the P2Y(2) receptor increased as cell density increased. Co-application of fetal calf serum with UTP induced Ca(2+) oscillation at high cell density. These results suggest that the different patterns observed for [Ca(2+)](i) mobilization with respect to cell density may be associated with cell cycle progression.

Fast cortical oscillation after thalamic degeneration: pivotal role of NMDA receptor.

We examined electrophysiological and molecular changes of the thalamocortical system after thalamic degeneration in Purkinje cell degeneration (pcd) mice. In pcd mice, neurons in specific thalamic nuclei including the ventral medial geniculate nucleus began to degenerate around postnatal day 50, whereas the visual thalamic nucleus and nonspecific thalamic nuclei remained almost intact. In association with the morphological changes, auditory evoked potentials in the primary auditory cortex (AC) began to decrease gradually. Fast Fourier transform analysis of spontaneous cortical field potentials revealed that fast oscillation (FO) around 25 Hz occurred in the AC but not in the visual cortex. Quantitative mRNA analysis demonstrated that expression of the N-methyl-D-aspartate (NMDA) receptor was up-regulated in the AC but not in the visual cortex. Systemic administration of an NMDA antagonist abolished the FO in the AC. These results indicate that increased NMDA activity may cause the FO in the AC of pcd mice.

Emergence of endoplasmic reticulum stress and activated microglia in Purkinje cell degeneration mice.

In the current studies, we characterized the molecular and cellular mechanism of cell death in Purkinje cell degeneration (pcd) mice using real-time quantitative PCR, immunohistochemistry, and Western blotting. It appears that endoplasmic reticulum (ER) stress is involved in this degeneration of Purkinje cells because ER stress-related substrates, such as CHOP and caspase 12, were strongly activated in Purkinje cells of pcd mice during the third postnatal (P) week. A significant increase in the expression of the ER-specific chaperone BiP suggested that unfolded protein responses were induced. We also found that Purkinje cells underwent apoptosis via the activation of caspase 3 and subsequent fragmentation of DNA. In addition to the activation of apoptosis in Purkinje cells, many activated microglial cells are found to be present in the molecular layer of the cerebellar cortex. In the later phase of degeneration, there was conspicuous expression of inducible nitric oxide synthase (iNOS), and some Purkinje cells were strongly labeled with an antibody to nitrotyrosine, suggesting that Purkinje cells in pcd mice are damaged by nitric oxide released from microglial cells. Administration of minocycline, which may inhibit iNOS expression, delayed the death of Purkinje cells in pcd mice and mildly improved their motor abilities. These findings suggest that ER stress participates in the degeneration of Purkinje cells and that activation of microglia accelerates Purkinje cell death in pcd mice.

Readiness potential and movement initiation in the rat.

Cortical field potentials were recorded by electrodes implanted chronically on the surface and at a 2.0 mm depth in various cortical areas in the left hemisphere in the rat during self-paced movements of the right forelimb. A surface-negative (s-N), depth-positive (d-P) cortical field potential appeared about 1.0 s (range: 0.5-1.5 s) before movement onset in the rostral (RFA) and caudal (CFA) forelimb areas of the motor cortex, and the somatosensory cortex, but not in the occipital cortex. Bipolar recording of electromyographic activities induced by the electrical stimulation of various cortical loci was also performed by pairs of steel electrodes inserted in the face, trunk, forelimb and hindlimb muscles on both sides. The stimulation of the forelimb motor cortex activated the face and/or forelimb muscles, while that of the somatosensory cortex generally activated several body part muscles including the forelimb muscle. Stronger stimulus intensity was requested to elicit the activities of most of the ipsilateral muscles to the cortex stimulated than the contralateral ones. The minimum intensity for inducing the forelimb muscle activity was lowest in the CFA among cortical areas producing the activity. The stimulation of cortical loci in which the s-N, d-P potential was recorded could induce muscle activities in the forelimb contralateral to the stimulation. It is suggested that the s-N, d-P potential is the readiness potential for activating muscles to initiate movement in the rat forelimb.

Preparative activities in posterior parietal cortex for self-paced movement in monkeys.

Cortical field potentials were recorded by electrodes implanted chronically on the surface and at a 2.0-3.0 mm depth in various cortices in monkeys performing self-paced finger, toe, mouth, hand or trunk movements. Surface-negative, depth-positive potentials (readiness potential) appeared in the posterior parietal cortex about 1.0 s before onset of every self-paced movement, as well as in the premotor, motor and somatosensory cortices. Somatotopical distribution was seen in the readiness potential in the posterior parietal cortex, although it was not so distinct as that in the motor or somatosensory cortex. This suggests that the posterior parietal cortex is involved in preparation for self-paced movement of any body part. This study contributes to the investigation of central nervous mechanisms of voluntary movements initiated by internal stimulus.

Thalamo-cortical projections to the posterior parietal cortex in the monkey.

Thalamo-cortical projections to the posterior parietal cortex (PPC) were investigated electrophysiologically in the monkey. Cortical field potentials evoked by the thalamic stimulation were recorded with electrodes chronically implanted on the cortical surface and at a 2.0-3.0 mm cortical depth in the PPC. The stimulation of the nucleus lateralis posterior (LP), nucleus ventralis posterior lateralis pars caudalis (VPLc), and nucleus pulvinaris lateralis (Pul.l) and medialis (Pul.m) induced surface-negative, depth-positive potentials in the PPC. The LP and VPLc projected mainly to the superior parietal lobule (SPL) and the anterior bank of the intraparietal sulcus (IPS), and the Pul.m mainly to the inferior parietal lobule (IPL) and the posterior bank of the IPS. The Pul.l had projections to all of the SPL, the IPL and both the banks. The significance of the projections is discussed in connection with motor functions.

Perirhinal cortex relays auditory information to the frontal motor cortices in the rat.

Auditory evoked potentials (AEPs) were recorded in the motor cortices (MC) with chronically implanted electrodes in the rat. Some of the AEPs in the MC, namely negative potentials on the surface and positive ones at a depth of 2 mm at latencies of about 50-150 ms, were abolished by limited bilateral lesions of the anterior perirhinal cortex (PERa) which was responsive to auditory stimulus, indicating that the AEPs in the MC were at least partially relayed in the PERa. The auditory response in the MC was prominently enhanced when water was supplied or the medial forebrain bundle was stimulated after auditory stimulus. These results indicate that the MC receives the reward associated auditory information from the PERa.

Cortical field potentials preceding self-paced forelimb movements and influences of cerebellectomy upon them in rats.

Seven rats were well trained to move lever to the left by right forelimb at self-pace (self-paced forelimb movements). Cortical field potentials associated with self-paced forelimb movements were recorded by electrodes implanted chronically on the surface and at a 2.0 mm depth in the forelimb motor cortex on the left side. A surface-negative, depth-positive potential starting about 1.0 s prior to the movement was recorded in the rostral part of the forelimb motor cortex. Further we found that the premovement potential was eliminated by the cerebellar hemispherectomy on the right side. This suggests the participation of the cerebellar hemisphere in preparing the activity of the motor cortex before self-paced forelimb movements in rats, by cerebello-thalamo-cortical projections.

Projection from the perirhinal cortex to the frontal motor cortex in the rat.

Stimulation of the anterior perirhinal cortex (PERa) induced marked surface-negative and depth-positive field potentials in the rat frontal motor cortex (MC) including the rostral and caudal forelimb areas. Injection of biotinylated dextran into the PERa densely labeled axon terminals in the superficial layers of the MC, where vigorous unit responses were evoked after PERa stimulation, indicated that the perirhinal-frontal projection preferentially activates the superficial layer neurons of the MC.