Understanding interactions between brain reward and sleep systems in driving maladaptive behaviors

National Institute On Alcohol Abuse And Alcoholism

Investigators

Linked publications & trials

Abstract

FY2022 was the first year of this project. Firstly, we published a series of studies from prior work performed within the Laboratory for Integrative Neuroscience (See: Kesner et. al., 2022. PMID: 35478010). This publication forms the basis of the work within the newly established Unit on Motivation and Arousal, which is dedicated to advancing this research project. This publication describes our work developing pre-clinical sleep and behavioral models of cannabis withdrawal symptoms. Prior to this work, there was controversy in our field as to whether rodents experience cannabis withdrawal symptoms purely upon cessation of this drug. It had previously been reported that withdrawal symptoms could be precipitated in rodents by administration of drugs that block the action of delta-9-tetrahydroannabinol (THC; the main driver of cannabis use and misuse), but this does not appropriately mimic the human condition where withdrawal symptoms occur after abrupt cessation (i.e spontaneous withdrawal) of cannabis/THC use. We found that, indeed, mice do experience withdrawal symptoms that have face validity to those experienced by humans: sleep disruption, irritability, and alterations in reward-seeking behaviors. In addition, we found sex differences in the strength of several of these mouse THC-withdrawal symptoms, which is consistent with the existence of sex-differences in the endocannabinoid (eCB) system (which THC directly acts on) and cannabis withdrawal symptoms in humans. The research summarized above suggests that brain regions that express cellular machinery related to eCB activity and are associated with both sleep and motivated behaviors may be important loci to assess the neural mechanisms governing withdrawal phenomena associated with cessation of cannabis, and potentially other misused substances. One such brain area that we feel is understudied is the medial septum (MS). The MS expresses cannabinoid receptor type 1 (CB1), which predominantly occupies presynaptic neuron terminals, and is the main target for the psychoactive properties and misuse liability of THC. Release of eCBs from neurons in the MS would act in a retrograde manner to reduce the release of neurotransmitters from neuron terminals in the MS. To assess whether eCB release within the MS correlates to vigilance state (e.g. wake, NREM sleep, or REM sleep), we used the newly developed genetically encoded eCB sensor, GRABeCB2.0. This is essentially a CB1 receptor genetically modified to contain a fluorescent protein whose fluorescence will increase when eCBs bind to the GRABeCB2.0 protein. Using viral strategies, we expressed this sensor in the MS of mice and implanted an optic fiber into the MS to record the changes in fluorescence from this sensor while simultaneously recording sleep physiology. We then aligned the fluorescence signals to changes in vigilance state (i.e. sleep stage) and found that MS eCB activity is reduced during NREM to REM transitions and comes back to baseline levels just before the transition from REM to wake. There is no change in eCB activity during Wake to NREM or NREM to wake transitions. These data are the first to show real time correlation of eCB activity with changes in vigilance state. We then reasoned that presynaptic CB1 on terminals within the MS would be influenced by this vigilance-state specific change in eCB tone. There are several brain regions that have neurons that make CB1 and project to the MS. One region, the supramammillary nucleus (SuM), is known to be involved in sleep-wake processes, heavily expresses mRNA for CB1, and densely projects to the septal complex. We used an intersectional viral strategy to express Cre-recombinase in SuM neurons projecting to the MS in mice that have the gene encoding for CB1 flanked by loxp sites. This results in a Cre-mediated recombination-mediated deletion of CB1 on SuM neurons that project to MS. We compared sleep in these mice to wildtype littermates that received the same intracranial viral injections and found that SuM to MS CB1 knockout increases NREM sleep during the active phase of the light cycle, while decreasing REM sleep in the inactive phase. These data confirm that both eCB release and its action on CB1 receptors in the MS is important for sleep-wake processes. Beyond its role in sleep, the MS has also been implicated in reward seeking behaviors, making it an interesting region to study synergy of sleep and reward related brain circuitry. The MS is comprised of neurons that primarily make and release the inhibitory neurotransmitter gamma-Aminobutyric acid, the excitatory neurotransmitter glutamate (GLU), or the modulatory neurotransmitter acetylcholine. Of these distinct subpopulations of neurons within the MS, the GLU neurons (MS-GLU) are particularly understudied and have recently been implicated in reward processes. We know that selective stimulation of these neurons using optogentics is reinforcing in mice, and they will actively press a lever to earn stimulation specifically of MS-GLU neurons. These neurons in turn project to the ventral tegmental area, a brain region highly implicated in motivated behavior, and influence dopamine neuron activity here and dopamine release in the ventral striatum. Beyond these recent findings, little is known about how MS-GLU neurons respond during natural reward seeking behaviors or how modulation of their activity can influence these behaviors. Our group has performed a set of studies to elucidate the role of these neurons in these processes. We expressed either the excitatory designer receptor exclusively activated by designer drug (DREADD) hM3D-Gq, the inhibitory DREADD hM4D-Gi, or control mCherry protein exclusively on MS-GLU neurons. We trained mice to perform a basic reward seeking procedure where one lever press on an active lever (of two levers) resulted in delivery of a sucrose reward. We found that administration of the DREADD ligand, clozapine-n-oxide (CNO) resulted in reduced lever pressing in hM4D-Gi-expressing mice, but not in hM3D-Gq or control mice. Additionally, consummatory behaviors were increased in the hM4D-Gi group but not in other groups. These data suggest that taking MS-GLU neurons offline alters the appetitive and consummatory processes associated with goal directed behaviors in a converse fashion. When we reversed the lever assignments, where the previously active lever did nothing, but pressing the other lever now delivers reward, the hM3D-Gq group adapted their behavior to this new contingency much faster than the control mice, and the hM4D-Gi mice never appropriately adapted. These findings suggest augmenting MS-GLU activity plays a role in enhancing cognitive flexibility. Finally, we used fiber photometry techniques to measure calcium activity in MS-GLU neurons. Calcium activity is a correlate of neuron activity, so increased calcium levels indicate action potentials and general neuronal activity. In mice performing the same reward-seeking behaviors we found profoundly reduced activity in MS-GLU neurons during reward consumption. MS-GLU neuron activity also appears to encode some valence of different components of the appetitive behaviors; as activity dynamics are different depending on which lever the mouse presses and whether reward was present during consummatory behaviors. These data are being prepared for article submission. Overall, we have begun homing in on brain circuitry that is involved in both motivation and arousal processes to assess the interaction between these processes in sleep and goal directed behavior.