Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor ␣3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.Neuropathic pain arising from peripheral or spinal nerve injury and diabetes or infection with herpes virus is a result of the final product of an altered peripheral, spinal, and supraspinal process for which the usual analgesics are not effective and novel analgesics are desired. Recent progress of research on the underlying mechanism of the pathology revealed more complexity, depending on the cause and stage of ongoing neuropathy. Among various mechanisms involved in the pathology, alterations of synaptic transmission within the spinal cord dorsal horn as well as peripheral nerves after peripheral nerve injury play key roles. In addition to the activation of stimulatory spinal neurotransmission, disinhibition of inhibitory neurotransmission has been implicated in the generation of inflammatory and neuropathic pain (Woolf and Mannion, 1999). Glycine as well as GABA serve as major inhibitory neurotransmitters in the spinal cord of vertebrates. In fact, relief from glycinergic inhibition by an inhib-

An immunohistochemical technique was employed to analyze mechanisms underlying modulation by N-methyl-D-aspartate (NMDA) receptors of proliferation of neural progenitor cells in adult mouse brain. The systemic administration of NMDA at 100 mg/kg resulted in marked expression of c-Fos, Fra-2 and c-Jun proteins in the granule cell layers of the dentate gyrus in murine hippocampus 2 h later, followed by a significant reduction of the incorporation of 5-bromo-2¢-deoxyuridine (BrdU) in a manner sensitive to the antagonist dizocilpine 2 days after administration. The administration of NMDA also suppressed constitutive expression of both nestin and proliferating cell nuclear antigen (PCNA) in the dentate granule cells 2 days later, without markedly affecting cell viability for up to 8 weeks after administration. In the subventricular zone and olfactory bulb, however, NMDA failed to affect either the incorporation of BrdU or the expression of nestin and PCNA. The NR1 subunit was highly expressed in the dentate gyrus in addition to the stratum oriens in the hippocampus, but not in the subventricular zone and olfactory bulb. These results suggest that NMDA receptors may play a role crucial for maintenance of the integrity and function of proliferative neural progenitor cells through expression of the nuclear transcription factor activator protein-1 in granule cells of the dentate gyrus in adult mouse brain.

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.

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Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors capable of self-replication and differentiation from discrete fetal rat brain structures. A reverse transcription-polymerase chain reaction analysis revealed the expression of particular GABA A receptor (GA-BA A R), GABA B R-1 and GABA C R, but not GABA B R-2, subunits in neocortical cells before commitment. Sustained exposure to the GABA A R agonist muscimol at 100 lmol/L led to significant increases in the mitochondrial activity and the total areas of neocortical neurospheres formed during the cultivation for 12 days in a manner sensitive to a GABA A R antagonist, with lactate dehydrogenase release being unchanged. Moreover, prior sustained exposure to muscimol significantly facilitated the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage, with a concomitant decrease in the neuronal marker protein expression, in an antagonist-sensitive manner on Western blotting analysis. However, muscimol failed to significantly affect the expression of both marker proteins in cells differentiated in either the presence or absence of all-trans-retinoic acid. These results suggest that prior activation of GABA A R may preferentially facilitate the commitment by CNTF of neural progenitor cells toward an astroglial lineage after simulation of the self-replication activity in the developing rat brain.

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GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.