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In 2020, stay-at-home orders were implemented to stem the spread of SARS-CoV-2 worldwide. Social isolation can be particularly harmful to children and adolescents—during the pandemic, the prevalence of obesity increased by ∼37% in persons aged 2-19. Obesity is often comorbid with type 2 diabetes, which was not assessed in this human pandemic cohort. Here, we investigated whether male mice isolated throughout adolescence develop type 2 diabetes in a manner consistent with human obesity-induced diabetes, and explored neural changes that may underlie such an interaction. We find that isolating C57BL/6J mice throughout adolescence is sufficient to induce type 2 diabetes. We observed fasted hyperglycemia, diminished glucose clearance in response to an insulin tolerance test, decreased insulin signaling in skeletal muscle, decreased insulin staining of pancreatic islets, increased nociception, and diminished plasma cortisol levels compared to group-housed control mice. Using Promethion metabolic phenotyping chambers, we observed dysregulation of sleep and eating behaviors, as well as a time-dependent shift in respiratory exchange ratio of the adolescent-isolation mice. We profiled changes in neural gene transcription from several brain areas and found that a neural circuit between serotonin-producing and GLP-1-producing neurons is affected by this isolation paradigm. Overall, spatial transcription data suggest decreased serotonin neuron activity (via decreased GLP-1-mediated excitation) and increased GLP-1 neuron activity (via decreased serotonin-mediated inhibition). This circuit may represent an intersectional target to further investigate the relationship between social isolation and type 2 diabetes, as well as a pharmacologically-relevant circuit to explore the effects of serotonin and GLP-1 receptor agonists. ARTICLE HIGHLIGHTS: Isolating C57BL/6J mice throughout adolescence is sufficient to induce type 2 diabetes, presenting with fasted hyperglycemia. Adolescent-isolation mice have deficits in insulin responsiveness, impaired peripheral insulin signaling, and decreased pancreatic insulin production. Transcriptional changes across the brain include the endocannabinoid, serotonin, and GLP-1 neurotransmitters and associated receptors. The neural serotonin/GLP-1 circuit may represent an intersectional target to further investigate the relationship between social isolation and type 2 diabetes. Serotonin-producing neurons of adolescent-isolation mice produce fewer transcripts for the GLP-1 receptor, and GLP-1 neurons produce fewer transcripts for the 5-HT (1A) serotonin receptor. .

Social interaction is a core component of motivational behavior that is perturbed across multiple neuropsychiatric disorders, including alcohol use disorder (AUD). Positive social bonds are neuroprotective and enhance recovery from stress, so reduced social interaction in AUD may delay recovery and lead to alcohol relapse. We report that chronic intermittent ethanol (CIE) induces social avoidance in a sex-dependent manner and is associated with hyperactivity of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN). While 5-HT (DRN) neurons are generally thought to enhance social behavior, recent evidence suggests that specific 5-HT pathways can be aversive. Using chemogenetic iDISCO, the nucleus accumbens (NAcc) was identified as one of 5 regions that were activated by 5-HT (DRN) stimulation. We then employed an array of molecular genetic tools in transgenic mice to show that 5-HT (DRN) inputs to NAcc dynorphin neurons drive social avoidance in male mice after CIE by activating 5-HT (2C) receptors. NAcc dynorphin neurons also inhibit dopamine release during social interaction, reducing the motivational drive to engage with social partners. This study reveals that excessive serotonergic drive after chronic alcohol can promote social aversion by inhibiting accumbal dopamine release. Drugs that boost brain serotonin levels may be contraindicated for individuals with AUD.

Additional file 1: Supplementary Methods: RT-PCR quantification and tau splice isoforms; Detection of tau isoforms in whole brain lysates. Supplementary Figure 1: Validation of htau mice showing presence of insoluble 3R tau isoform in whole brain lysates at 12 months of age. (A) Mouse and human tau splice isoforms in C57BL/6J, htau +/- and MAPT -/- mice. The human-specific primers only amplified products in htau +/- while mouse-specific primers only amplified products in C57BL/6J mice. (B) (i) Protein expression of human tau (HT7) in total protein extracts and (ii) 3R and 4R tau isoforms in Sarkosyl soluble and insoluble fractions from C57BL/6J, htau +/- and MAPT -/- mice. Only htau +/- mice contained human-specific tau (HT7) and the 3R tau isoform in detergent insoluble fractions. Both htau +/- and C57 mice contained the 4R isoform in soluble fractions. Supplementary Figure 2: Orthogonal images confirming hyperphosphorylated tau in 5-HT neurons in the DRN. (A) Atlas plate depicting the mid region of the DRN that was used to obtain orthogonal planes. (B-D) Representative orthogonal planes showing colocalization between 5-HT (cyan) and ptau (red) in the DRN. (E) Atlas plate depicting the mid region of the LC that was used to obtain orthogonal planes. (F-H) Representative orthogonal planes showing colocalization between TH (cyan) and ptau (red) in the LC. Supplementary Figure 3: Hyperphosphorylated tau and monoaminergic depletion in the brainstem of female htau mice at 4 months. (A) Representative confocal images of 5-HT immunostaining (20X; scale bar = 200 µm) and 5-HT/AT8 co-staining (60X; scale bar = 50 µm) in the DRN of C57BL/6J and htau +/- mice. (B) Atlas plate depicting one of the DRN regions analyzed (mid DRN) (C) Representative orthogonal image showing colocalizaton of ptau with 5-HT neurons in the DRN (100X; scale bar = 20 µm). (D) Histogram of 5-HT cell counts/mm2, (E) 5-HT immunoreactive area (%), (F) ptau (AT8) optical density in subregions of the DRN and (G) Correlation analysis between 5-HT IR area and AT8 optical density in the DRN (H) Representative confocal images of TH immunostaining (20X; scale bar = 200 µm) and TH/AT8 colocalization (60X; scale bar = 50 µm) in the LC of C57BL/6J and htau +/- mice. (I) Atlas plate depicting one of the LC regions analyzed (mid LC) (J) Representative orthogonal image showing colocalization of ptau with TH neurons in the LC (100X; scale bar = 20 µm). (K) Histogram of TH cell counts/mm2, (L) TH immunoreactive area, (M) ptau (AT8) optical density in subregions of the LC, and (N) Correlation analysis between TH immunoreactive area and AT8 optical density in the mid LC. *p < 0.05, **p < 0.01, ***p < 0.001. Supplementary Figure 4: Glial activation in the DRN and LC in htau mice at 4 months. (A) Representative confocal images of Iba-1 and GFAP immunostaining in the DRN of C57 and htau +/- mice (60X; scale bar = 50 µm). (B) Iba-1+ cell counts/mm2, (C) Iba-1 optical density, and (D) Iba-1 immunoreactive area in subregions of the DRN. (E) GFAP+ cells/mm2, (F) GFAP optical density, and (G) GFAP immunoreactive area in subregions of the DRN. (H) Representative confocal images of Iba-1 and GFAP immunostaining in the LC of C57 and htau +/- mice. (I) Iba-1+ cells/mm2, (J) Iba-1 optical density, and (K) Iba-1 immunoreactive area in subregions of the LC. (L) GFAP+ cells/mm2, (M) GFAP optical density, and (N) GFAP immunoreactive area in subregions of the LC. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. White arrows denote Iba-1 +or GFAP+ cell bodies. Supplementary Figure 5: Reduced 5-HT innervation of the entorhinal cortex in htau mice at 4 months. Representative confocal images of 5-HT fiber immunostaining (40x; scale bar = 50 um) in C57 and htau mice. (A) Atlas representation of a section of the EC used for analysis (in red). (B) Representative confocal images of 5-HT staining in the EC of C57BL/6J and htau mice at 40X. (C) Histogram of % 5-HT IR area in subregions of the EC. (D) Representative confocal images of 5-HT staining in the EC at 100X. (E) Atlas representation of CA1 region of the hippocampus used in the analysis (in red). (F) Representative confocal image of SERT staining in the CA1 region of the hippocampus in C57BL/6J and htau mice. (G) Histogram of % SERT IR area in subregions of the hippocampus (H) Representative confocal images of SERT staining in the CA1 region of the hippocampus at 100X. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01. Supplementary Table 1: List of fold change values obtained from RT-qPCR of genes tested in the dorsal raphe nucleus, locus coeruleus, dorsal and ventral hippocampus, and entorhinal cortex of C57 and htau (*p < 0.05, **p < 0.01, ***p < 0.001).

Alcoholism is frequently co-morbid with posttraumatic stress disorder (PTSD) but it is unclear how alcohol impacts neural circuits mediating recovery from trauma. We found that chronic intermittent ethanol (CIE) impaired fear extinction and remodeled the dendritic arbor of medial prefrontal cortical (mPFC) neurons in mice. CIE impaired extinction encoding by infralimbic (IL) mPFC neurons in vivo, and functionally downregulated burst-mediating NMDA GluN1 receptors. These findings suggest alcohol may increase risk for trauma-related anxiety disorders by disrupting mPFC-mediated extinction of fear.