Neuroscience
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We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in approximately 90% of VGLUT1+, approximately 30% of VGLUT2+ and 30-50% of VGAT+ puncta; SYPI was expressed in approximately 95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in approximately 7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in approximately 94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in approximately 3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that approximately 30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats.
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Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. ⋯ Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.
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Sleep fragmentation, a feature of sleep apnea as well as other sleep and medical/psychiatric disorders, is thought to lead to excessive daytime sleepiness. A rodent model of sleep fragmentation was developed (termed sleep interruption, SI), where rats were awakened every 2 min by the movement of an automated treadmill for either 6 or 24 h of exposure. The sleep pattern of rats exposed to 24 h of SI resembled sleep of the apneic patient in the following ways: sleep was fragmented (up to 30 awakening/h), total rapid eye movement (REM) sleep time was greatly reduced, non-rapid eye movement (NREM) sleep episode duration was reduced (from 2 min, 5 s baseline to 58 s during SI), whereas the total amount of NREM sleep time per 24 h approached basal levels. ⋯ BF AD levels were significantly elevated during SI, peaking at 220% of baseline during 30 h of SI exposure. These combined findings imply an elevation of the homeostatic sleep drive following either 6 or 24 h of SI, and BF AD levels appear to correlate more with sleepiness than with the cumulative amount of prior wakefulness, since total NREM sleep time declined only slightly. SI may be partially responsible for the symptom of daytime sleepiness observed in a number of clinical disorders, and this may be mediated by mechanisms involving BF AD.
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Recent evidence suggests that human immunodeficiency virus (HIV)-induced pathogenesis is exacerbated by opioid abuse and that the synergistic toxicity may result from direct actions of opioids in immature glia or glial precursors. To assess whether opioids and HIV proteins are directly toxic to glial-restricted precursors (GRPs), we isolated neural stem cells from the incipient spinal cord of embryonic day 10.5 ICR mice. GRPs were characterized immunocytochemically and by reverse transcriptase-polymerase chain reaction (RT-PCR). ⋯ Moreover, MOR and KOR are widely expressed by Sox2 and/or Nkx2.2-positive GRPs in vitro and the pattern of receptor expression appears to be developmentally regulated. The temporal requirement for prolonged morphine and HIV-1 Tat exposure to evoke toxicity in glia may coincide with the attainment of a particular stage of maturation and/or the development of particular apoptotic effector pathways and may be unique to spinal cord GRPs. Should similar patterns occur in vivo then we predict that immature astroglia and oligodendroglia may be preferentially vulnerable to HIV-1 infection or chronic opiate exposure.
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Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric alpha7 nAChRs, neuroprotection to heteromeric alpha4beta2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. ⋯ CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.