Control of feeding by a bottom-up midbrain-subthalamic pathway.

Investigative exploration and foraging leading to food consumption have vital importance, but are not well-understood. Since GABAergic inputs to the lateral and ventrolateral periaqueductal gray (l/vlPAG) control such behaviors, we dissected the role of vgat-expressing GABAergic l/vlPAG cells in exploration, foraging and hunting. Here, we show that in mice vgat l/vlPAG cells encode approach to food and consumption of both live prey and non-prey foods. The activity of these cells is necessary and sufficient for inducing food-seeking leading to subsequent consumption. Activation of vgat l/vlPAG cells produces exploratory foraging and compulsive eating without altering defensive behaviors. Moreover, l/vlPAG vgat cells are bidirectionally interconnected to several feeding, exploration and investigation nodes, including the zona incerta. Remarkably, the vgat l/vlPAG projection to the zona incerta bidirectionally controls approach towards food leading to consumption. These data indicate the PAG is not only a final downstream target of top-down exploration and foraging-related inputs, but that it also influences these behaviors through a bottom-up pathway.

Characterization of ventromedial hypothalamus activity during exposure to innate and conditioned threats.

In the face of imminent predatory danger, animals quickly detect the threat and mobilize key survival defensive actions, such as escape and freezing. The dorsomedial portion of the ventromedial hypothalamus (VMH) is a central node in innate and conditioned predator-induced defensive behaviours. Prior studies have shown that activity of steroidogenic factor 1 (sf1)-expressing VMH cells is necessary for such defensive behaviours. However, sf1-VMH neural activity during exposure to predatory threats has not been well characterized. Here, we use single-cell recordings of calcium transients from VMH cells in male and female mice. We show this region is activated by threat proximity and that it encodes future occurrence of escape but not freezing. Our data also show that VMH cells encoded proximity of an innate predatory threat but not a fear-conditioned shock grid. Furthermore, chemogenetic activation of the VMH increases avoidance of innate threats, such as open spaces and a live predator. This manipulation also increased freezing towards the predator, without altering defensive behaviours induced by a shock grid. Lastly, we show that optogenetic VMH activation recruited a broad swath of regions, suggestive of widespread changes in neural defensive state. Taken together, these data reveal the neural dynamics of the VMH during predator exposure and further highlight its role as a critical component of the hypothalamic predator defense system.

Confirmation of Decreased Rates of Cerebral Protein Synthesis In Vivo in a Mouse Model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that results in intellectual disability and, in ∼50% of patients, autism spectrum disorder. The protein products that are altered in TSC (TSC1 and TSC2) form a complex to inhibit the mammalian target of rapamycin [mTOR; mTOR complex 1 (mTORC1)] pathway. This pathway has been shown to affect the process of mRNA translation through its action on ribosomal protein S6 and 4-elongation binding protein 1. It is thought that mutations in the TSC proteins lead to upregulation of the mTORC1 pathway and consequently an increase in protein synthesis. Unexpectedly, our previous study of a mouse model of TSC (Tsc2Djk +/) demonstrated decreased in vivo rates of protein synthesis throughout the brain. In the present study, we confirm those results in another Tsc2+/- mouse model, one with a different mutation locus and on a mixed background (Tsc2Mjg +/-). We also examine mTORC1 signaling and possible effects of prior isoflurane anesthesia. Because measurements of protein synthesis rates in vivo require surgical preparation of the animal and anesthesia, we examine mTORC1 signaling pathways both under baseline conditions and following recovery from anesthesia. Our results demonstrate regionally selective effects of prior anesthesia. Overall, our results in both in vivo models suggest differences to the central hypothesis regarding TSC and show the importance of studying protein synthesis in vivo Significance StatementProtein synthesis is an important process for brain function. In the disorder, tuberous sclerosis complex (TSC), the inhibition of the mammalian target of rapamycin (mTOR) pathway is reduced and this is thought to lead to excessive protein synthesis. Most studies of protein synthesis in models of TSC have been conducted in vitro We report here confirmation of our previous in vivo study showing decreased brain protein synthesis rates in a second mouse model of TSC, results counter to the central hypothesis regarding TSC. We also explore the possible influence of prior isoflurane exposure on signaling pathways involved in regulation of protein synthesis. This study highlights a novel aspect of TSC and the importance of studying cellular processes in vivo.

GABAergic CA1 neurons are more stable following context changes than glutamatergic cells.

The CA1 region of the hippocampus contains both glutamatergic pyramidal cells and GABAergic interneurons. Numerous reports have characterized glutamatergic CAMK2A cell activity, showing how these cells respond to environmental changes such as local cue rotation and context re-sizing. Additionally, the long-term stability of spatial encoding and turnover of these cells across days is also well-characterized. In contrast, these classic hippocampal experiments have never been conducted with CA1 GABAergic cells. Here, we use chronic calcium imaging of male and female mice to compare the neural activity of VGAT and CAMK2A cells during exploration of unaltered environments and also during exposure to contexts before and after rotating and changing the length of the context across multiple recording days. Intriguingly, compared to CAMK2A cells, VGAT cells showed decreased remapping induced by environmental changes, such as context rotations and contextual length resizing. However, GABAergic neurons were also less likely than glutamatergic neurons to remain active and exhibit consistent place coding across recording days. Interestingly, despite showing significant spatial remapping across days, GABAergic cells had stable speed encoding between days. Thus, compared to glutamatergic cells, spatial encoding of GABAergic cells is more stable during within-session environmental perturbations, but is less stable across days. These insights may be crucial in accurately modeling the features and constraints of hippocampal dynamics in spatial coding.

Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization.

During threat exposure, survival depends on defensive reactions. Prior works linked large glutamatergic populations in the midbrain periaqueductal gray (PAG) to defensive freezing and flight, and established that the overarching functional organization axis of the PAG is along anatomically-defined columns. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains diverse cell types that vary in neurochemistry. How these cell types contribute to defense remains unknown, indicating that targeting sparse, genetically-defined populations may reveal how the PAG generates diverse behaviors. Though prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found in mice that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (CCK) cells selectively caused flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-CCK cells reduced predator avoidance without altering other defensive behaviors like freezing. Lastly, l/vlPAG-CCK activity decreased when approaching threat and increased during movement to safer locations. These results suggest CCK cells drive threat avoidance states, which are epochs during which mice increase distance from threat and perform evasive escape. Conversely, l/vlPAG pan-neuronal activation promoted freezing, and these cells were activated near threat. Thus, CCK l/vlPAG cells have opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar boundaries. In addition to the anatomical columnar architecture of the PAG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats.

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.

Increased rates of cerebral protein synthesis in Shank3 knockout mice: Implications for a link between synaptic protein deficit and dysregulated protein synthesis in autism spectrum disorder/intellectual disability.

SHANK3 is a postsynaptic scaffolding protein that plays a critical role in synaptic development and brain function. Mutations in SHANK3 are implicated in Phelan-McDermid syndrome (PMS), a neurodevelopmental disorder characterized by autistic-like behavior, delayed speech, hypotonia, and intellectual disability (ID). Moreover, mutations in SHANK3 occur in 1-2% of cases of idiopathic autism spectrum disorder (ASD). In fragile X syndrome (FXS), a syndromic form of autism, SHANK3 is one of the 842 targets of fragile X mental retardation protein (FMRP), the protein product of the silenced FMR1 gene. FXS is likely a primary disorder of the regulation of translation, whereas other syndromic forms of ASD/ID, e.g. PMS, appear to be primary disorders of synaptic structure. In this study, we asked if a knockout of the synaptic protein, Shank3, is linked to an effect on translation. Specifically, we measured the effect of Shank3 loss on rates of cerebral protein synthesis (rCPS) in vivo by means of the L-[1-14C]leucine quantitative autoradiographic method. We found that Shank3 knockout mice had significantly increased rCPS in every brain region examined. Our results suggest a link in ASD/ID between synaptic structure and regulation of translation.

Long-Term Characterization of Hippocampal Remapping during Contextual Fear Acquisition and Extinction.

Hippocampal CA1 place cell spatial maps are known to alter their firing properties in response to contextual fear conditioning, a process called "remapping." In the present study, we use chronic calcium imaging to examine remapping during fear retrieval and extinction of an inhibitory avoidance task in mice of both sexes over an extended period of time and with thousands of neurons. We demonstrate that hippocampal ensembles encode space at a finer scale following fear memory acquisition. This effect is strongest near the shock grid. We also characterize the long-term effects of shock on place cell ensemble stability, demonstrating that shock delivery induces several days of high fear and low between-session place field stability, followed by a new, stable spatial representation that appears after fear extinction. Finally, we identify a novel group of CA1 neurons that robustly encode freeze behavior independently from spatial location. Thus, following fear acquisition, hippocampal CA1 place cells sharpen their spatial tuning and dynamically change spatial encoding stability throughout fear learning and extinction.SIGNIFICANCE STATEMENT The hippocampus contains place cells that encode an animal's location. This spatial code updates, or remaps, in response to environmental change. It is known that contextual fear can induce such remapping; in the present study, we use chronic calcium imaging to examine inhibitory avoidance-induced remapping over an extended period of time and with thousands of neurons and demonstrate that hippocampal ensembles encode space at a finer scale following electric shock, an effect which is enhanced by threat proximity. We also identify a novel group of freeze behavior-activated neurons. These results suggest that, more than merely shuffling their spatial code following threat exposure, place cells enhance their spatial coding with the possible benefit of improved threat localization.

Lincoln center moments: integrating accessibility and enhancement through expanding performing arts experiences.

BACKGROUND: Exposure to the arts is vital to our quality of life, providing opportunities for social connection, engagement with past hobbies and perhaps most essentially in providing the potential for enhanced means of expression. For individuals with dementia and their care-givers, access to the arts can be challenging. METHOD: This article describes a pilot program designed to enhance access by people with dementia and to provide deeper engagement with artists, therapist s and caregivers. Participants attended music and theater performances with caregivers, then attended workshops designed to deepen the experience. Workshops grouped participants by severity of dementia and measurements of engagement during and after the program were developed to assess the efficacy of the pilot program. RESULTS: Enhanced quality of life was noted for people with dementia and the caregivers who attended the programs with them. CONCLUSION: This project, the first of its kind, is a starting point. There has not been another to integrate professional musicians of varying genres, with people in the community with Alzheimer' s, and most particularly, by providing after-performance groups that assisted members' processing. Designing programs incorporating the arts as applicable to community-based institutions may expand their impact.

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Noninvasive, High-throughput Determination of Sleep Duration in Rodents.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

Deficient Sleep in Mouse Models of Fragile X Syndrome.

In patients with fragile X syndrome (FXS), sleep problems are commonly observed but are not well characterized. In animal models of FXS (dfmr1 and Fmr1 knockout (KO)/Fxr2 heterozygote) circadian rhythmicity is affected, but sleep per se has not been examined. We used a home-cage monitoring system to assess total sleep time in both light and dark phases in Fmr1 KO mice at different developmental stages. Fmr1 KOs at P21 do not differ from controls, but genotype × phase interactions in both adult (P70 and P180) groups are statistically significant indicating that sleep in Fmr1 KOs is reduced selectively in the light phase compared to controls. Our results show the emergence of abnormal sleep in Fmr1 KOs during the later stages of brain maturation. Treatment of adult Fmr1 KO mice with a GABAB agonist, R-baclofen, did not restore sleep duration in the light phase. In adult (P70) Fmr1 KO/Fxr2 heterozygote animals, total sleep time was further reduced, once again in the light phase. Our data highlight the importance of the fragile X genes (Fmr1 and Fxr2) in sleep physiology and confirm the utility of these mouse models in enhancing our understanding of sleep disorders in FXS.