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

National Institute On Alcohol Abuse And Alcoholism

Investigators

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Abstract

FY2025 was the fourth year of this project. We have continued following up on findings from the first publication related to this research program (See: Kesner et. al., 2022. PMID: 35478010). That publication describes our work developing pre-clinical sleep and behavioral models of cannabis withdrawal symptoms. This research 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. We have previously observed that removing CB1 from specific presynaptic terminals in the MS caused changes in sleep-wake architecture, confirming that both eCB release and its action on CB1 receptors in the MS is important for sleep-wake processes. We are currently working on methodology to measure endocannabinoid (eCB) release in-vivo within the MS to assess the natural function of these neuromodulators during sleep-wake transitions. Specifically, we are using a custom built spectrally resolved fiber photometry system developed by researchers at NIEHS to measure fluorescent signals from a genetically encoded eCB sensor, GRABeCB. By combining this technique with EEG/EMG recordings we can detect changes in eCB dynamics during vigilance state transitions. There is one technical limitation we are currently overcoming, in that blood pressure drops during REM sleep, and this artifact makes interpretation of eCB dynamics difficult since it is seen in non-dynamic, “control” recordings. Beyond its role in sleep, the MS has also been implicated in reward seeking behaviors, making it an interesting region to study in relation to 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. 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. Over the past year we have added more interesting findings building off our main discovery that augmenting MS-GLU activity plays a role in enhancing cognitive flexibility. In particular we found that inhibition of MS-GLU greatly disrupts general acquisition of operant behavior, where mice fail to reliably associate a lever with reward. Importantly, when glutamate neurons outside of the MS are manipulated in a similar fashion, this effect is no longer observed, providing an additional level of specificity to the findings related to MS-GLU in reward seeking. We then used fiber photometry techniques to measure calcium activity in MS-GLU neurons. Calcium activity is a correlate of neuronal activity, so increased calcium levels indicate action potentials and general neuronal activity. In mice performing the same strategy switching 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 for the same lever press depending on whether it was before or after a lever-reversal procedure. In addition, we have begun studying the interactions between MS-GLU neurons and canonical reward circuitry, specifically dopamine (DA) activity in the nucleus accumbens (NAc). We examined NAc-DA responses while the animals performed a Pavlovian strategy switch paradigm, all while MS-GLU neurons were modulated via DREADDs. Our results indicate NAc-DA changes in response to the two cues in this paradigm differ depending on MS-GLU modulation via DREADDs. Of particular note, we see a far smaller NAc-DA response upon a stimulus that used to predict a reward (but now does not) in Gq mice compared to Gi or mCherry groups on the first session of the strategy switch paradigm. We also observed potentially larger increase in NAc-DA upon reward retrieval after a new stimulus that predicts a reward in the Gq group. These observations are intriguing as they suggest increasing MS-GLU activity during the first day of a strategy switch paradigm helps the animal better disregard the old reward predictive cue, and better associate the new predictive cue with reward. In essence, increasing MS-GLU activity via Gq DREADDs seems to enhance the timeline in which reward prediction error may factor into the animal learning the new cue-reward pairing, and extinguish the old cue-reward pairing. These data are being prepared for article submission and will hopefully be published in a peer reviewed journal by the next annual report. Over the past year we have also incorporated sophisticated neural recording paradigms to study local field potentials simultaneously across several brain sites while mice perform various behavioral tasks. We have begun using machine learning and neural networks to model and predict brain networks associated with attention, reward seeking, and uncertainty. We have also used these approaches to investigate the effects of ethanol in pathways with known roles in exploratory behavior and cognition. These ethanol studies have elucidated very interesting modes of communication within septohippocampal circuitry, which are disrupted when mice are intoxicated. We are nearing submission of a manuscript reporting these findings. 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.