Discharge and Role of GABA Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recordings in Mice.

The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-(ChR2)-EYFP in vesicular GABA transporter (VGAT)-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep.SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states.

Discharge and Role of Acetylcholine Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recording in Mice.

Acetylcholine (ACh) neurons in the pontomesencephalic tegmentum (PMT) are thought to play an important role in promoting cortical activation with waking (W) and paradoxical sleep [PS; or rapid eye movement (REM)], but have yet to be proven to do so by selective stimulation and simultaneous recording of identified ACh neurons. Here, we employed optogenetics combined with juxtacellular recording and labeling of neurons in transgenic (TG) mice expressing ChR2 in choline acetyltransferase (ChAT)-synthesizing neurons. We established in vitro then in vivo in anesthetized (A) and unanesthetized (UA), head-fixed mice that photostimulation elicited a spike with short latency in neurons which could be identified by immunohistochemical staining as ACh neurons within the laterodorsal (LDT)/sublaterodorsal (SubLDT) and pedunculopontine tegmental (PPT) nuclei. Continuous light pulse stimulation during sleep evoked tonic spiking by ACh neurons that elicited a shift from irregular slow wave activity to rhythmic θ and enhanced γ activity on the cortex without behavioral arousal. With θ frequency rhythmic light pulse stimulation, ACh neurons discharged in bursts that occurred in synchrony with evoked cortical θ. During natural sleep-wake states, they were virtually silent during slow wave sleep (SWS), discharged in bursts during PS and discharged tonically during W. Yet, their bursting during PS was not rhythmic or synchronized with cortical θ but associated with phasic whisker movements. We conclude that ACh PMT neurons promote θ and γ cortical activity during W and PS by their tonic or phasic discharge through release of ACh onto local neurons within the PMT and/or more distant targets in the hypothalamus and thalamus.

Homeostatic regulation through GABA and acetylcholine muscarinic receptors of motor trigeminal neurons following sleep deprivation.

Muscle tone is regulated across sleep-wake states, being maximal in waking, reduced in slow wave sleep (SWS) and absent in paradoxical or REM sleep (PS or REMS). Such changes in tone have been recorded in the masseter muscles and shown to correspond to changes in activity and polarization of the trigeminal motor 5 (Mo5) neurons. The muscle hypotonia and atonia during sleep depend in part on GABA acting upon both GABAA and GABAB receptors (Rs) and acetylcholine (ACh) acting upon muscarinic 2 (AChM2) Rs. Here, we examined whether Mo5 neurons undergo homeostatic regulation through changes in these inhibitory receptors following prolonged activity with enforced waking. By immunofluorescence, we assessed that the proportion of Mo5 neurons positively stained for GABAARs was significantly higher after sleep deprivation (SD, ~65%) than sleep control (SC, ~32%) and that the luminance of the GABAAR fluorescence was significantly higher after SD than SC and sleep recovery (SR). Although, all Mo5 neurons were positively stained for GABABRs and AChM2Rs (100%) in all groups, the luminance of these receptors was significantly higher following SD as compared to SC and SR. We conclude that the density of GABAA, GABAB and AChM2 receptors increases on Mo5 neurons during SD. The increase in these receptors would be associated with increased inhibition in the presence of GABA and ACh and thus a homeostatic down-scaling in the excitability of the Mo5 neurons after prolonged waking and resulting increased susceptibility to muscle hypotonia or atonia along with sleep.

Homeostatic Changes in GABA and Acetylcholine Muscarinic Receptors on GABAergic Neurons in the Mesencephalic Reticular Formation following Sleep Deprivation.

We have examined whether GABAergic neurons in the mesencephalic reticular formation (RFMes), which are believed to inhibit the neurons in the pons that generate paradoxical sleep (PS or REMS), are submitted to homeostatic regulation under conditions of sleep deprivation (SD) by enforced waking during the day in mice. Using immunofluorescence, we investigated first, by staining for c-Fos, whether GABAergic RFMes neurons are active during SD and then, by staining for receptors, whether their activity is associated with homeostatic changes in GABAA or acetylcholine muscarinic type 2 (AChM2) receptors (Rs), which evoke inhibition. We found that a significantly greater proportion of the GABAergic neurons were positively stained for c-Fos after SD (∼27%) as compared to sleep control (SC; ∼1%) and sleep recovery (SR; ∼6%), suggesting that they were more active during waking with SD and less active or inactive during sleep with SC and SR. The density of GABAARs and AChM2Rs on the plasma membrane of the GABAergic neurons was significantly increased after SD and restored to control levels after SR. We conclude that the density of these receptors is increased on RFMes GABAergic neurons during presumed enhanced activity with SD and is restored to control levels during presumed lesser or inactivity with SR. Such increases in GABAAR and AChM2R with sleep deficits would be associated with increased susceptibility of the wake-active GABAergic neurons to inhibition from GABAergic and cholinergic sleep-active neurons and to thus permitting the onset of sleep and PS with muscle atonia.

GABA Receptors on Orexin and Melanin-Concentrating Hormone Neurons Are Differentially Homeostatically Regulated Following Sleep Deprivation.

Though overlapping in distribution through the hypothalamus, orexin (Orx) and melanin-concentrating hormone (MCH) neurons play opposite roles in the regulation of sleep-wake states. Orx neurons discharge during waking, whereas MCH neurons discharge during sleep. In the present study, we examined in mice whether GABAA and GABAB receptors (Rs) are present on Orx and MCH neurons and might undergo differential changes as a function of their different activities following sleep deprivation (SD) and sleep recovery (SR). Applying quantitative stereological image analysis to dual-immunofluorescent stained sections, we determined that the proportion of Orx neurons positively immunostained for GABAARs was significantly higher following SD (∼48%) compared with sleep control (SC; ∼24%) and SR (∼27%), and that the luminance of the GABAARs was significantly greater. In contrast, the average proportion of the MCH neurons immunostained for GABAARs was insignificantly lower following SD (∼43%) compared with SC (∼54%) and SR (56%), and the luminance of the GABAARs was significantly less. Although, GABABRs were observed in all Orx and MCH neurons (100%), the luminance of these receptors was differentially altered following SD. The intensity of GABABRs in the Orx neurons was significantly greater after SD than after SC and SR, whereas that in the MCH neurons was significantly less. The present results indicate that GABA receptors undergo dynamic and differential changes in the wake-active Orx neurons and the sleep-active MCH neurons as a function of and homeostatic adjustment to their preceding activity and sleep-wake state.

Somatostatin varicosities contain the vesicular GABA transporter and contact orexin neurons in the hypothalamus.

Somatostatin (SST) is a neuropeptide with known inhibitory actions in the hypothalamus, where it inhibits release of growth hormone-releasing hormone (GHRH), while also influencing the sleep-wake cycle. Here we investigated in the rat whether SST neurons might additionally release GABA (gamma-aminobutyric acid) or glutamate in different regions and whether they might contact orexin neurons that play an important role in the maintenance of wakefulness. In dual-immunostained sections viewed by epifluorescence microscopy, we examined if SST varicosities were immunopositive for the vesicular transporter for GABA (VGAT) or glutamate (VGLUT2) in the posterolateral hypothalamus and neighboring arcuate nucleus and median eminence. Of the SST varicosities in the posterolateral hypothalamus, 18% were immunopositive for VGAT, whereas ≤ 1% were immunopositive for VGLUT2. In the arcuate and median eminence, 26 and 64% were VGAT+ and < 3% VGLUT2 + , respectively. In triple-immunostained sections viewed by epifluorescence and confocal microscopy, SST varicosities were seen in contact with orexin somata, and of these varicosities, a significant proportion (23%) contained VGAT along with synaptophysin, the presynaptic marker for small synaptic vesicles, and a similar proportion (25%) abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. Our results indicate that a significant proportion of SST varicosities in the hypothalamus have the capacity to release GABA, to form inhibitory synapses upon orexin neurons, and accordingly through their peptide and/or amino acid, to inhibit orexin neurons, as well as GHRH neurons. Thus while regulating GHRH release, SST neurons could serve to attenuate arousal and permit progression through the sleep cycle.