SLEEP L2: Yu et al. 2019. Nature - GABA & Glutamate neurons in VTA regulate sleep & wakefulness

Question 1

intro

Answer 1

• wakefulness is promoted by GABAergic and glutamatergic pathways that induce arousal & physical activity
• sleep is promoted by GABAergic/peptidergic and glutamatergic/nitrergic neurons that inhibit wake-promoting neurons
• through non-hypothesis driven chemogenetic search for circuitry controlling vigilance states, they found the VTA –> known for its regulation of goal/reward and social behaviours, containing dopamine neurons (VTADA) GABAergic, and glutamatergic neurons
• VTADA are selectively wake- and REM- active, promoting wakefulness
• VTA glutamate/NOS1 neurons increase wakefulness; GABA neurons decrease it

Question 2

chemogenetics to identify glutamatergic neurons promoting wakefulness

Answer 2

• researchers targeted the posterior hypothalamic midbrain area (PH/MB)
  -large volume injections of AAV-DIO-hM3dq-mCherry into PH/MB of Vglut2 cre mice, induced 100% wafefulness for 12 hours following intraperitoneal injection of CNO, compared to saline-injected controls –> resulted in widespread hM3Dq-mCherry receptor expression throughout various brain regions
• smaller AAV injection volumes restricted hM3dDq expression to specific brain regions, such as LH and VTA; intraperitoneal CNO injection = continous wakefulness for ~5hrs, followed by enhanced wakefulness for 7hrs
• highlights specific brain regions involved in wakefulness (involving glutamatergic neurons)
• when expression of hM3Dq was restricted to glutamatergic neurons in the mammillary area of Vglut 2 cre mice, and CNO administered, it did not induce arousal beyond baseline levels observed w/ saline controls; into the interpeduncular nucleus, did not result in arousal compared to saline
• indicates that activation of glutamatergic neurons in specific brain regions may not necessarily promote wakefulness

Question 3

glutamatergic neurons in VTA promote wakefulness

Answer 3

• hM3Dq receptor selectively expressed in VTA glutamatergic neurons in Vglut2-cre mice; injected CNO induced 5hrs of continous wakefulness, w/ elevated wakefulness for nearly the entire duration of ‘lights on’ period
• CNO-induced excitation of VTAVglut2 neurons led to increases cFos expression, confirming their activation
• EEG spectra showed higher theta activity, while EMG remained unchanged
• activation of VTAVglut2 neurons also prolonged the latency to both NREM & REMS without causing hyperactivity
• findings were further supported by optogenetic activation of VTAVglut 2 neurons w/ channelrhodopsin, which also increased wakefulness

Question 4

genetic ablation of VTAVglut2 neurons in mice

Answer 4

• using AAV-DIO-CASp3 resulted in mice w/ ~80% of VTAVglut2 neurons destroyed
• chronic lesioning of VTAVglut2 = reduced wakefulness and increaseed NREMS, specifically during “lights off” phase of 24hr sleep-wake cycle
• analysis of sleep-wake microarchitecture revealed impaired wake consolidation, w/ more frequent wake episodes w/ shorter durations, and increased transitions between wake and NREMS

Question 5

assessment of VTAVglut2 activity using VTAVglut2-GCamP6 mice

Answer 5

• enabled the recording of Ca2+ signals through fiber photometry
• Ca2+ signal increased during wake and REM sleep; lower levels during NREMS

Question 6

VTAVglut2 neurons promote wakefulness independently of dopamine

Answer 6

• immunohistochemistry confirmed that VTAVglut2 neurons are largely distinct from VTADA
• activation of VTAVglut2 neurons in mice w/ dopamine receptor antagonists still resulted in the promotion of wakefulness, indicating that wake-promoting effect of VTAVglut2 neurons is not dependent on dopamine signalling

Question 7

VTAVlgut2 neurons work through the nucleus accumbens (NAc) and LH

Answer 7

• VTAVglut2 neurons were found to promote wakefulness via projections to the NAc and LH
• activation of VTAVglut2 neurons led to increased cfos expression in various brain regions, particularly LH and NAc
• further mapping using channelrhodopsin revealed dense projections of VTAVglut2 neurons to the LH and NAc

Question 8

NOS1 marks wake- promoting VTAVglu2 neurons

Answer 8

-nitric oxide synthase

Question 9

VTAVgat neurons limit wakefulness and induce NREMS

Answer 9

• activation of VTAVgat neurons in mice induced sustained NREMS, with reduced latency to NREMS and notably increased latency to REM sleep
• chemogenetic inhibition of VTAVgat neurons resulted in sustained 100% wakefulness for 6 hrs with sustained theta frequencies in EEG and w/ increased latency to the first NREM and REM sleep bouts compared to saline-injected mice
• chemogenetic inhibition of VTAVgat neurons = prolonger NREMS for ~6hrs, resembling sedation –> suggests that targeting VTAVgat enurons could be a strategy for developing novel sedatives that promote sustained NREMS
• although subtypes like Pv and Som-expressing cells induce NREMS individually, they do not produce the full effect of activating the complete set of VTAVgat neurons; further research is needed to understand the interactions between different subtypes of VTAVgat neurons and their influence on sleep & wakefulness

Question 10

lesioning of VTAVgta neurons produce continous wakefulness

Answer 10

• chronically lesiones VTAVgat neurons using AAV-DIO-CASP3 –> destroyed ~88% of VTAVgat neurons
• mice exhibited increased wakefulness, especially during “lights off” phase, where they only slept for 40mins compared to 4hrs in non-lesioned control mice; mice also had shroter average NREM episode durations and decreased transitions between vigilance states than control mice
• during “lights on phase”, control mice slept for ~7hrs and VTAVgat-CASP3 mice for ~6hrs
• reduced sleep phenotype persisted for at least 4 months, resulting in a permanent and substantial sleep deficit in the mice

Question 11

VTAVgat neurons are selectively wake- and REM-active

Answer 11

• to investigate the activity patterns of VTAVgat neurons during different vigilance states, they used VTAVgat-GCaMP6 mice and measured their activity via fiber photometry
• found that Ca2+ signals of VTAVgat neurons increased selectively during wake and REMS; decreased during NREMS
• further investigation using micro-endoscopic calcium imaging confirmed that VTAVgat neurons are selectively active during wakefulness, reinforcing their role as wake-active neurons

Question 12

VTAVgat neurons reduce wakefulness and induce NREMS by both local inhibition and via projections to the LH

Answer 12

• VTAVgat neurons, although selectively active during wake and REMS, appear to reduce wakefulness and induce NREMS through mechanisms of local inhibition within the VTA (possibly by inhibiting VTA glutamatergic and dopaminergic neurons) and via projections to the LH (particulalry orexin neurons) –> channelrhodopsin mapping revealed dense VTAVgat fibers in VTA and LH, providing evidence for this
• inhibition of VTAVgat neurons resulted in increased cfos expression in both VTA and LH (particularly in orexin neurons)
• optogenetic activation of VTAVgat fibers in the LH promotes and maintains NREMS while reducing wakefulness and REMS

Question 13

discussion points

Answer 13

• effects of the VTAVgat and Vglut2 lesions are more pronounced during the active phase of mice, aligning w/ the circadian control of VTA neuron activity