The paraventricular nucleus of the thalamus (PVT) is increasingly being recognized as a critical node linking stress detection to the emergence of adaptive behavioral responses to stress. However, despite growing evidence implicating the PVT in stress processing, the neural mechanisms by which stress impacts PVT neurocircuitry and promotes stressed states remain unknown. Here we show that stress exposure drives a rapid and persistent reduction of inhibitory transmission onto projection neurons of the posterior PVT (pPVT). This stress-induced disinhibition of the pPVT was associated with a locus coeruleus-mediated rise in the extracellular concentration of dopamine in the midline thalamus, required the function of dopamine D2 receptors on PVT neurons, and increased sensitivity to stress. Our findings define the locus coeruleus as an important modulator of PVT function: by controlling the inhibitory tone of the pPVT, it modulates the excitability of pPVT projection neurons and controls stress responsivity.

The ability to directly measure acetylcholine (ACh) release is an essential step towards understanding its physiological function. Here, we optimized the GRAB ACh ( G PC R - A ctivation‒ B ased- ACh ) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the sub-second response kinetics, physiological-relevant affinity and precise molecular specificity to ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice in a variety of behaviors.

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as “AP-4 deficiency syndrome”. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.

Summary Obesity is associated with physical inactivity, which exacerbates the health consequences of weight gain. However, the mechanisms that mediate this association are unknown. We hypothesized that deficits in dopamine signaling contribute to physical inactivity in obesity. To investigate this, we quantified multiple aspects of dopamine signaling in lean and obese mice. We found that D2-type receptor (D2R) binding in the striatum, but not D1-type receptor binding or dopamine levels, was reduced in obese mice. Genetically removing D2Rs from striatal medium spiny neurons was sufficient to reduce motor activity in lean mice, while restoring Gi signaling in these neurons increased activity in obese mice. Surprisingly, while mice with low D2Rs were less active, they were not more vulnerable to diet-induced weight gain than control mice. We conclude that deficits in striatal D2R signaling contribute to physical inactivity in obesity, but inactivity is more a consequence than a cause of obesity.

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRABACh (GPCR-Activation-Based-ACh) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors 2 / 37 Cholinergic signals mediated by the neurotransmitter ACh are involved in a wide range of physiological processes, including muscle contraction, cardiovascular function, neural plasticity, attention and memory 1-3 .Previously, cholinergic activity was mainly measured using either electrophysiology to record nicotinic receptormediated currents 4, 5 or microdialysis followed by biochemical purification and identification 6 . However, these methods generally lack both cell-type specificity and the spatial-temporal resolution needed to precisely dissect cholinergic signals in vivo. Combining the type 3 muscarinic ACh receptor (M3R) with the conformationalsensitive circular permutated GFP (cpGFP), we recently developed GACh2.0 (short as ACh2.0), a genetically encoded GRAB (GPCR-Activation Based) ACh sensor that can convert the ACh-induced conformational change on M3R into a sensitive fluorescence response 7 . The ACh2.0 sensor responds selectively to physiological concentration of ACh with an EC50 of 2 μM and has been used in several model organisms to detect the endogenous release and regulation of cholinergic signals. Here, we optimized the GRABACh sensor using sitedirected mutagenesis and cell-based screening to further increase the sensitivity.To improve the performance of the GRABACh sensor, we focused on the interface between M3R and cpGFP, including the receptor's third intracellular loop (ICL3) and linker peptides, as well as critical residues in cpGFP that contribute to its fluorescence intensity (Figs. 1A and S1A-D). Our initial screening based on mediumthroughput imaging identified several variants with improved performance; these variants were subsequently verified using confocal microscopy (see Methods for details). The sensor with the largest ACh-induced fluorescence response was selected for further study and is named as GRABACh3.0 or ACh3.0 (Fig. 1A). We also generated a ligand-insensitive form of ACh3.0 by introducing the W200A mutation into the receptor 8 (Figs. 1A and S1E). When expressed in HEK293T cells or cultured neurons, the ACh3.0 sensor localized to the plasma membrane of the soma, and trafficked to dendrites and axons in neurons ( Fig. 1B-D). Moreover, compared to ACh2.0, the ACh3.0 sensor had a significantly larger fluorescence change (ΔF/F0~280%) in response to 100 μM ACh (Figs. 1B-D and S2A-E); in contrast, the ligand-insensitive ACh3.0-mut sensor had no detectable 4 coverslips for ACh2.0, ACh3.0, and ACh3.0-mut, respectively, with an average of >20 cells per co...

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit a dual imbalance of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/f mice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that NRG/ErbB4 signaling directly in DAergic axonal projections contributes to the modulation of DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors with relevance to psychiatric disorders.

Numerous genetic and functional studies implicate variants of Neuregulin-1 and its neuronal receptor ErbB4 in schizophrenia and many of its endophenotypes. While the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, practically nothing is known about the function of NRG2, the closest NRG1 homologue. We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior on the elevated plus-maze and deficits on the T-maze alteration reward test - a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with NMDAR antagonists, we demonstrate that NMDA receptor synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDA receptors. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders.

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