Probes for INS

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood and their potential contribution to pain-related behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in GRP::cre and GRP::eGFP transgenic male mice. We combined behavioral tests with neuronal circuit tracing, morphology, chemogenetics, optogenetics, and electrophysiology to obtain a more comprehensive picture. We found that GRP neurons form a rather homogenous population of central cell-like excitatory neurons located in lamina II of the superficial dorsal horn. Multicolor high-resolution confocal microscopy and optogenetic experiments demonstrated that GRP neurons receive direct input from MrgprA3-positive pruritoceptors. Anterograde herpes simplex virus-based neuronal tracing initiated from GRP neurons revealed ascending polysynaptic projections to distinct areas and nuclei in the brainstem, midbrain, thalamus, and the somatosensory cortex. Spinally restricted ablation of GRP neurons reduced itch-related behaviors to different pruritogens while their chemogenetic excitation elicited itch-like behaviors and facilitated responses to several pruritogens. By contrast, responses to painful stimuli remained unaltered. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission, but do not support a role in pain.Significance statement: Dorsal horn GRP neurons serve a well-established function in the spinal transmission of pruritic (itch) signals. A potential role in the transmission of nociceptive (pain) signals has remained controversial. Our results provide further support for a critical role of dorsal horn GRP neurons in itch circuits, but we failed to find evidence supporting a role in pain.

Pre-existing Alzheimer's disease is a risk factor for severe/fatal COVID-19 and infection by SARS-CoV2 virus has been associated with an increased incidence of un-masked Alzheimer's disease. The molecular basis whereby SARS-CoV2 may amplify Alzheimer's disease is not well understood. This study analyzed the molecular changes in autopsy brain tissues from people with pre-existing dementia who died of COVID-19 (n = 5) which was compared to equivalent tissues of people who died of COVID-19 with no history of dementia (n = 8), Alzheimer's disease pre-COVID-19 (n = 10) and aged matched controls (n = 10) in a blinded fashion. Immunohistochemistry analyses for hyperphosphorylated tau protein, α-synuclein, and β-amyloid-42 confirmed the diagnoses of Alzheimer's disease (n = 4), and Lewy body dementia (n = 1) in the COVID-19 group. The brain tissues from patients who died of COVID-19 with no history of dementia showed a diffuse microangiopathy marked by endocytosis of spike subunit S1 and S2 in primarily CD31+ endothelia with strong co-localization with ACE2, Caspase-3, IL6, TNFα, and Complement component 6 that was not associated with SARS-CoV2 RNA. Microglial activation marked by increased TMEM119 and MCP1 protein expression closely paralleled the endocytosed spike protein. The COVID-19 tissues from people with no pre-existing dementia showed, compared to controls, 5-10× fold increases in expression of neuronal NOS and NMDAR2 as well as a marked decrease in the expression of proteins whose loss is associated with worsening Alzheimer's disease: MFSD2a, SHIP1, BCL6, BCL10, and BACH1. In COVID-19 tissues from people with dementia the widespread spike-induced microencephalitis with the concomitant microglial activation co-existed in the same areas where neurons had hyperphosphorylated tau protein suggesting that the already dysfunctional neurons were additionally stressed by the SARS-CoV2 induced microangiopathy. ACE2+ human brain endothelial cells treated with high dose (but not vaccine equivalent low dose) spike S1 protein demonstrated each of the molecular changes noted in the in vivo COVID-19 and COVID-19/Alzheimer's disease brain tissues. It is concluded that fatal COVID-19 induces a diffuse microencephalitis and microglial activation in the brain due to endocytosis of circulating viral spike protein that amplifies pre-existing dementia in at least two ways: 1) modulates the expression of proteins that may worsen Alzheimer's disease and 2) stresses the already dysfunctional neurons by causing an acute proinflammatory/hypercoagulable/hypoxic microenvironment in areas with abundant hyperphosphorylated tau protein and/or βA-42.

Although glucose is the basic fuel essential to maintain the viability and functions of all cells, some neurons, namely glucose-inhibited (GI) neurons, paradoxically increase their firing activities when glucose falls and are inhibited by high glucose. The ionic mechanisms mediating electric responses of GI neurons to glucose fluctuations remain unclear. Here we showed that currents mediated by anoctamin 4 (Ano4) channel are only detected in GI neurons in the ventromedial hypothalamic nucleus (VMH) and are functionally required for their activation in response to low glucose. Genetic disruption of the Ano4 gene in VMH neurons reduced blood glucose and impaired counterregulatory responses during hypoglycemia in mice. Activation of VMHAno4 neurons increased food intake and blood glucose, while chronic inhibition of VMHAno4 neurons ameliorated hyperglycemia in a type 1 diabetic mouse model. Finally, we showed that VMHAno4 neurons represent a unique orexigenic VMH population and transmit a positive valence, while stimulation of non-Ano4 neurons in the VMH suppress feeding and transmit a negative valence. Together, our results indicate that the Ano4 channel and VMHAno4 neurons are potential therapeutic targets for human diseases with abnormal feeding behavior or glucose imbalance.

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

Oxytocin (OXT) and oxytocin receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current- and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity. Activation of OXTRs inhibits an outward potassium current mediated by inward rectifier potassium channels (_I_Kir) and thus favoring membrane depolarization. Concomitantly, OXT signaling also diminishes inward current mediated by hyperpolarization-activated cyclic-nucleotide-gated channels (_I_h), providing a hyperpolarizing drive. The combined reduction in both _I_Kir and _I_h synergistically elevate the membrane resistance and favor dendritic integration while the membrane potential is restrained from quickly depolarizing from rest. As a result, the responsiveness of CA2 PYRs to synaptic inputs is highly sharpened during OXTR activation. Unexpectedly, OXTR signaling also strongly enhances a tetrodotoxin-resistant, voltage-gated sodium current that helps drive the membrane potential to spike threshold and thus promote rhythmic firing. This novel array of OXTR-stimulated ionic mechanisms operates in close coordination and underpins OXT-induced burst firing, a key step in CA2 PYRs’ contribution to hippocampal information processing and broader influence on brain circuitry. Our study deepens our understanding of underpinnings of OXT-promoted social memory and general neuropeptidergic control of cognitive states.

The growth hormone receptor (GHR) is expressed in brain regions that are known to participate in the regulation of energy homeostasis and glucose metabolism. We generated a novel transgenic mouse line (GHRcre) to characterize GHR-expressing neurons specifically in the arcuate nucleus of the hypothalamus (ARC). Here, we demonstrate that ARCGHR+ neurons are co-localized with agouti-related peptide (AgRP), growth hormone releasing hormone (GHRH), and somatostatin neurons, which are activated by GH stimulation. Using the designer receptors exclusively activated by designer drugs (DREADD) technique to control the ARCGHR+ neuronal activity, we demonstrate that the activation of ARCGHR+ neurons elevates a respiratory exchange ratio (RER) under both fed and fasted conditions. However, while the activation of ARCGHR+ promotes feeding, under fasting conditions, the activation of ARCGHR+ neurons promotes glucose over fat utilization in the body. This effect was accompanied by significant improvements in glucose tolerance, and was specific to GHR+ versus GHRH+ neurons. The activation of ARCGHR+ neurons increased glucose turnover and whole-body glycolysis, as revealed by hyperinsulinemic-euglycemic clamp studies. Remarkably, the increased insulin sensitivity upon the activation of ARCGHR+ neurons was tissue-specific, as the insulin-stimulated glucose uptake was specifically elevated in the skeletal muscle, in parallel with the increased expression of muscle glycolytic genes. Overall, our results identify the GHR-expressing neuronal population in the ARC as a major regulator of glycolysis and muscle insulin sensitivity in vivo.

Neurons in the inferior colliculus (IC), the midbrain hub of the central auditory pathway, send ascending and descending projections to other auditory brain regions, as well as projections to other sensory and non-sensory brain regions. However, the axonal projection patterns of individual classes of IC neurons remain largely unknown. Vasoactive intestinal polypeptide (VIP) is a neuropeptide expressed by subsets of neurons in many brain regions. We recently identified a class of IC stellate neurons that we called VIP neurons because they are labeled by tdTomato (tdT) expression in VIP-IRES-Cre x Ai14 mice. Here, using fluorescence in situ hybridization, we found that tdT+ neurons in VIP-IRES-Cre x Ai14 mice express Vglut2, a marker of glutamatergic neurons, and VIP, suggesting that VIP neurons use both glutamatergic and VIPergic signaling to influence their postsynaptic targets. Next, using viral transfections with a Cre-dependent eGFP construct, we labeled the axonal projections of VIP neurons. As a group, VIP neurons project intrinsically, within the ipsilateral and contralateral IC, and extrinsically to all the major targets of the IC. Within the auditory system, VIP neurons sent axons and formed axonal boutons in higher centers, including the medial geniculate nucleus and the nucleus of the brachium of the IC. Less dense projections terminated in lower centers, including the nuclei of the lateral lemniscus, superior olivary complex, and dorsal cochlear nucleus. VIP neurons also project to several non-auditory brain regions, including the superior colliculus, periaqueductal gray, and cuneiform nucleus. The diversity of VIP projections compared to the homogeneity of VIP neuron intrinsic properties suggests that VIP neurons play a conserved role at the microcircuit level, likely involving neuromodulation through glutamatergic and VIPergic signaling, but support diverse functions at the systems level through their participation in different projection pathways.

The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized.We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo.S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected.Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity.A full list of funding bodies that contributed to this study can be found under Acknowledgments.

The D2 dopamine receptor (Drd2) is implicated in several brain disorders such as schizophrenia, Parkinson's disease, and drug addiction. Drd2 is also the primary target of both antipsychotics and Parkinson's disease medications. Although the expression pattern of Drd2 is relatively well known in mouse brain, the temporal and spatial distribution of Drd2 is lesser clear in rat brain due to the lack of Drd2 reporter rat lines. Here, we used CRISPR/Cas9 techniques to generate two knockin rat lines: Drd2::Cre and Rosa26::loxp-stop-loxp-tdTomato. By crossing these two lines, we produced Drd2 reporter rats expressing the fluorescence protein tdTomato under the control of the endogenous Drd2 promoter. Using fluorescence imaging and unbiased stereology, we revealed the cellular expression pattern of Drd2 in adult and postnatal rat forebrain. Strikingly, the Drd2 expression pattern differs between Drd2 reporter rats and Drd2 reporter mice generated by BAC transgene in prefrontal cortex and hippocampus. These results provide fundamental information needed for the study of Drd2 function in rat forebrain. The Drd2::Cre rats generated here may represent a useful tool to study the function of neuronal populations expressing Drd2.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.