Hypoxia modulates cholinergic but not opioid activation of G proteins in rat hippocampus

Intermittent hypoxia, such as that associated with obstructive sleep apnea, can cause neuronal death and neurobehavioral dysfunction. The cellular and molecular mechanisms through which hypoxia alter hippocampal function are incompletely understood. This study used in vitro [ 35 S]guanylyl-5′- O -(Γ-thio)-triphosphate ([ 35 S]GTPΓS) autoradiography to test the hypothesis that carbachol and DAMGO activate hippocampal G proteins. In addition, this study tested the hypothesis that in vivo exposure to different oxygen (O 2 ) concentrations causes a differential activation of G proteins in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. G protein activation was quantified as nCi/g tissue in CA1, CA3, and DG from rats housed for 14 days under one of three different oxygen conditions: normoxic (21% O 2 ) room air, or hypoxia (10% O 2 ) that was intermittent or sustained. Across all regions of the hippocampus, activation of G proteins by the cholinergic agonist carbachol and the mu opioid agonist [D-Ala 2 , N-Met-Phe 4 , Gly 5 ] enkephalin (DAMGO) was ordered by the degree of hypoxia such that sustained hypoxia > intermittent hypoxia > room air. Carbachol increased G protein activation during sustained hypoxia (38%), intermittent hypoxia (29%), and room air (27%). DAMGO also activated G proteins during sustained hypoxia (52%), intermittent hypoxia (48%), and room air (43%). Region-specific comparisons of G protein activation revealed that the DG showed significantly less activation by carbachol following intermittent hypoxia and sustained hypoxia than the CA1. Considered together, the results suggest the potential for hypoxia to alter hippocampal function by blunting the cholinergic activation of G proteins within the DG. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57386/1/20312_ftp.pd

Metabolomic analysis of mouse prefrontal cortex reveals upregulated analytes during wakefulness compared to sleep

By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80–100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes

Muscarinic and GABA A receptors modulate acetylcholine release in feline basal forebrain

Acetylcholine (ACh) release within the basal forebrain changes significantly as a function of sleep and wakefulness, hence identifying the neurochemical modulators of basal forebrain ACh release will contribute to a mechanistic understanding of sleep cycle regulation. This study tested the hypothesis that muscarinic and gamma aminobutyric acid A (GABA A ) receptors modulate basal forebrain ACh release. Cats were anaesthetized with halothane to hold arousal state constant and a microdialysis probe was aimed stereotaxically for the substantia innominata region of the basal forebrain. Four concentrations of the muscarinic antagonist scopolamine (0.1, 0.3, 1.0, and 10 nm) and five concentrations of the GABA A antagonist bicuculline (3, 10, 30, 100, and 300 µm) were delivered by reverse dialysis from the same probes used to collect ACh. These results are based on 27 experiments in nine animals. Scopolamine and bicuculline each caused a concentration dependent enhancement of ACh release. Scopolamine increased ACh by 118% above control levels whereas bicuculline was more effective, causing a 287% increase in ACh release. Scopolamine was more potent (EC 50  = 0.16 nm) than bicuculline (EC 50  ≥ 90 µm) for increasing ACh release. The results support the hypothesis that substantia innominata ACh release is modulated by muscarinic autoreceptors and inhibited by GABA A receptors. These findings are consistent with the interpretation that inhibition of basal forebrain cholinergic neurotransmission by GABA contributes to the generation of sleep.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75037/1/j.1460-9568.2003.02451.x.pd

Hypocretin-1 causes G protein activation and increases ACh release in rat pons

The effects of the arousal-promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague-Dawley rats. In vitro [ 35 S]GTPγS autoradiography was used to test the hypothesis that hypocretin-1-stimulated G protein activation is concentration-dependent and blocked by the hypocretin receptor antagonist SB-334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration–response data revealed a significant ( P  < 0.001) effect of hypocretin-1 (2–2000 nm) in all brain regions examined. Maximal increases over control levels of [ 35 S]GTPγS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB-334867 (2 µm) significantly ( P  < 0.002) blocked hypocretin-1 (200 nm)-stimulated [ 35 S]GTPγS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin-1 activates G proteins in arousal-related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin-1 increases ACh release in PnO. Dialysis delivery of hypocretin-1 (100 µm) significantly ( P  < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75751/1/j.1460-9568.2003.02905.x.pd