European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology
Ngoc, KH;Kecskés, A;Kepe, E;Nabi, L;Keeble, J;Borbély, É;Helyes, Z;
PMID: 37156112 | DOI: 10.1016/j.euroneuro.2023.04.017
The Transient Receptor Potential Vanilloid 1 (TRPV1) non-selective cation channel predominantly expressed in primary sensory neurons of the dorsal root and trigeminal ganglia mediates pain and neurogenic inflammation. TRPV1 mRNA and immunoreactivity were described in the central nervous system (CNS), but its precise expression pattern and function have not been clarified. Here we investigated Trpv1 mRNA expression in the mouse brain using ultrasensitive RNAScope in situ hybridization. The role of TRPV1 in anxiety, depression-like behaviors and memory functions was investigated by TRPV1-deficient mice and pharmacological antagonism by AMG9810. Trpv1 mRNA is selectively expressed in the supramammillary nucleus (SuM) co-localized with Vglut2 mRNA, but not with tyrosine hydroxylase immunopositivity demonstrating its presence in glutamatergic, but not dopaminergic neurons. TRPV1-deleted mice exhibited significantly reduced anxiety in the Light-Dark box and depression-like behaviors in the Forced Swim Test, but their performance in the Elevated Plus Maze as well as their spontaneous locomotor activity, memory and learning function in the Radial Arm Maze, Y-maze and Novel Object Recognition test were not different from WTs. AMG9810 (intraperitoneal injection 50 mg/kg) induced anti-depressant, but not anxiolytic effects. It is concluded that TRPV1 in the SuM might have functional relevance in mood regulation and TRPV1 antagonism could be a novel perspective for anti-depressant drugs.
The Journal of clinical investigation
Chen, O;He, Q;Han, Q;Furutani, K;Gu, Y;Olexa, M;Ji, RR;
PMID: 36520531 | DOI: 10.1172/JCI160807
Our understanding of neuropathic itch is limited, due to the lack of relevant animal models. Patients with cutaneous T-cell lymphoma (CTCL) suffer from severe itching. Here we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produces time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early-phase (20 days), CTCL causes hyper-innervations in the epidermis. However, chronic itch is associated with loss of epidermal nerve fibers in the late-phases (40 and 60 days). CTCL is also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early- and late-phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal gabapentin injection reduced late-phase but not early-phase pruritus. IL-31 is upregulated in mouse lymphoma, while its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in CTCL mice. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, intrathecal administration of TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.
Papalampropoulou-Tsiridou, M;Shiers, S;Wang, F;Godin, AG;Price, TJ;De Koninck, Y;
PMID: 36337346 | DOI: 10.1093/braincomms/fcac256
Acid-sensing ion channels (ASICs) play a critical role in nociception in human sensory neurons. Four genes (ASIC1, ASIC2, ASIC3, and ASIC4) encoding multiple subunits through alternative splicing have been identified in humans. Real time-PCR experiments showed strong expression of three subunits ASIC1, ASIC2, and ASIC3 in human dorsal root ganglia; however, their detailed expression pattern in different neuronal populations has not been investigated yet. In the current study, using an in situ hybridization approach (RNAscope), we examined the presence of ASIC1, ASIC2, and ASIC3 mRNA in three subpopulations of human dorsal root ganglia neurons. Our results revealed that ASIC1 and ASIC3 were present in the vast majority of dorsal root ganglia neurons, while ASIC2 was only expressed in less than half of dorsal root ganglia neurons. The distribution pattern of the three ASIC subunits was the same across the three populations of dorsal root ganglia neurons examined, including neurons expressing the REarranged during Transfection (RET) receptor tyrosine kinase, calcitonin gene-related peptide, and a subpopulation of nociceptors expressing Transient Receptor Potential Cation Channel Subfamily V Member 1. These results strongly contrast the expression pattern of Asics in mice since our previous study demonstrated differential distribution of Asics among the various subpopulation of dorsal root ganglia neurons. Given the distinct acid-sensitivity and activity dynamics among different ASIC channels, the expression differences between human and rodents should be taken under consideration when evaluating the translational potential and efficiency of drugs targeting ASICs in rodent studies.
Harris NA, Isaac AT, Günther A, Merkel K, Melchior J, Xu M, Eguakun E, Perez R, Nabit BP, Flavin S, Gilsbach R, Shonesy B, Hein L, Abel T, Baumann A, Matthews R, Centanni SW, Winder DG.
PMID: 30150361 | DOI: 10.1523/JNEUROSCI.0963-18.2018
Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress-responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress yet minimally affects relapse, potentially due to competing actions in the brain. Here we show that heteroceptor α2A-ARs postsynaptically enhance dorsal BNST (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, as inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons, and its activation elicits anxiety-like behavior in the elevated plus maze. Together, this data provides a framework for elucidating cell-specific actions of GPCR signaling and provides a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENTStress impacts the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here we show that guanfacine increases dBNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation (HCN) channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest 1) that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons, and 2) these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting care must be taken regarding interpretation of data obtained with these tools.
Yu, B;Zhang, Q;Lin, L;Zhou, X;Ma, W;Wen, S;Li, C;Wang, W;Wu, Q;Wang, X;Li, XM;
PMID: 36788214 | DOI: 10.1038/s41421-022-00506-y
The amygdala, or an amygdala-like structure, is found in the brains of all vertebrates and plays a critical role in survival and reproduction. However, the cellular architecture of the amygdala and how it has evolved remain elusive. Here, we generated single-nucleus RNA-sequencing data for more than 200,000 cells in the amygdala of humans, macaques, mice, and chickens. Abundant neuronal cell types from different amygdala subnuclei were identified in all datasets. Cross-species analysis revealed that inhibitory neurons and inhibitory neuron-enriched subnuclei of the amygdala were well-conserved in cellular composition and marker gene expression, whereas excitatory neuron-enriched subnuclei were relatively divergent. Furthermore, LAMP5+ interneurons were much more abundant in primates, while DRD2+ inhibitory neurons and LAMP5+SATB2+ excitatory neurons were dominant in the human central amygdalar nucleus (CEA) and basolateral amygdalar complex (BLA), respectively. We also identified CEA-like neurons and their species-specific distribution patterns in chickens. This study highlights the extreme cell-type diversity in the amygdala and reveals the conservation and divergence of cell types and gene expression patterns across species that may contribute to species-specific adaptations.
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
Domi, E;Xu, L;Toivainen, S;Wiskerke, J;Coppola, A;Holm, L;Augier, E;Petrella, M;Heilig, M;
PMID: 36739350 | DOI: 10.1038/s41386-023-01543-1
Alcohol use despite negative consequences is a core phenomenon of alcohol addiction. We recently used alcohol self-administration that is resistant to footshock punishment as a model of this behavior, and found that activity of PKCδ + GABAergic neurons in the central amygdala (CeA) is a determinant of individual susceptibility for punishment resistance. In the present study, we examined whether activation of GABAB receptors in CeA can attenuate the activity of PKCδ + neurons in this region, and whether this will result in suppression of punishment- resistant alcohol self-administration in the minority of rats that show this behavior. Systemic administration of the clinically approved GABAB agonist baclofen (1 and 3 mg/kg) dose- dependently reduced punishment-resistant alcohol self-administration. Bilateral microinjections of baclofen into CeA (64 ng in 0.3 µl/side) reduced the activity of PKCδ + neurons, as measured by Fos expression. This manipulation also selectively suppressed punished alcohol self-administration in punishment-resistant rats. Expression analysis indicated that virtually all CeA PKCδ + neurons express the GABAB receptor. Using in vitro electrophysiology, we found that baclofen induced hyperpolarization of CeA neurons, reducing their firing rate in response to depolarizing current injections. Together, our findings provide a potential mechanism that contributes to the clinical efficacy of baclofen in alcohol addiction. Therapeutic use of baclofen itself is limited by problems of tolerance and need for dose escalation. Our findings support a mechanistic rationale for developing novel, improved alcohol addiction medications that target GABAB receptors, and that lack these limitations, such as e.g., GABAB positive allosteric modulators (PAM:s).
Dilly, GA;Kittleman, CW;Kerr, TM;Messing, RO;Mayfield, RD;
PMID: 35859068 | DOI: 10.1038/s41398-022-02063-0
The central amygdala (CeA) contains a diverse population of cells, including multiple subtypes of GABAergic neurons, along with glia and epithelial cells. Specific CeA cell types have been shown to affect alcohol consumption in animal models of dependence and may be involved in negative affect during alcohol withdrawal. We used single-nuclei RNA sequencing to determine cell-type specificity of differential gene expression in the CeA induced by alcohol withdrawal. Cells within the CeA were classified using unbiased clustering analyses and identified based on the expression of known marker genes. Differential gene expression analysis was performed on each identified CeA cell-type. It revealed differential gene expression in astrocytes and GABAergic neurons associated with alcohol withdrawal. GABAergic neurons were further subclassified into 13 clusters of cells. Analyzing transcriptomic responses in these subclusters revealed that alcohol exposure induced multiple differentially expressed genes in one subtype of CeA GABAergic neurons, the protein kinase C delta (PKCδ) expressing neurons. These results suggest that PKCδ neurons in the CeA may be uniquely sensitive to the effects of alcohol exposure and identify a novel population of cells in CeA associated with alcohol withdrawal.
International Journal of Molecular Sciences
Vanneste, M;Mulier, M;Nogueira Freitas, A;Van Ranst, N;Kerstens, A;Voets, T;Everaerts, W;
| DOI: 10.3390/ijms23010107
The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.
Capsazepine decreases corneal pain syndrome in severe dry eye disease
Journal of neuroinflammation
Fakih, D;Guerrero-Moreno, A;Baudouin, C;Goazigo, AR;Parsadaniantz, SM;
PMID: 33975636 | DOI: 10.1186/s12974-021-02162-7
Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.
Nguyen MQ, Wu Y, Bonilla LS, von Buchholtz LJ, Ryba NJP.
PMID: 28957441 | DOI: 10.1371/journal.pone.0185543
The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system.
Dohnalová, L;Lundgren, P;Carty, JRE;Goldstein, N;Wenski, SL;Nanudorn, P;Thiengmag, S;Huang, KP;Litichevskiy, L;Descamps, HC;Chellappa, K;Glassman, A;Kessler, S;Kim, J;Cox, TO;Dmitrieva-Posocco, O;Wong, AC;Allman, EL;Ghosh, S;Sharma, N;Sengupta, K;Cornes, B;Dean, N;Churchill, GA;Khurana, TS;Sellmyer, MA;FitzGerald, GA;Patterson, AD;Baur, JA;Alhadeff, AL;Helfrich, EJN;Levy, M;Betley, JN;Thaiss, CA;
PMID: 36517598 | DOI: 10.1038/s41586-022-05525-z
Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.
Biological Psychiatry Global Open Science
Jiang, S;Zhang, H;Eiden, L;
| DOI: 10.1016/j.bpsgos.2023.04.001
Background The neuropeptide PACAP is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. Methods We used AAV neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57Bl6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. Results PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex (mPFC) to hypothalamus, impairs c-fos activation and CRH mRNA elevation in PVN after 2 hr of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in non-hypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala (EA), on the other hand, attenuates ARS-induced hypophagia, along with EA fos induction, without affecting ARS-induced CRH mRNA elevation in PVN. PACAP projections to EA terminate at PKCδ neurons in both central amygdala (CeA) and oval nuclei of bed nucleus of stria terminalis (BNSTov). Silencing of PKCδ neurons in CeA, but not in BNSTov, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n>5 per group. Conclusions A frontocortical descending PACAP projection controls PVN CRH mRNA production, to maintain hypothalamo-pituitary adrenal (HPA) axis activation, and regulate the endocrine response to stress. An ascending PACAPergic projection from eLPBn to PKCδ neurons in central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.
