Tyrosine Hydroxylase Neurons Research Articles

Locus Coeruleus to Paraventricular Thalamus Projections Facilitate Emergence From Isoflurane Anesthesia in Mice.

Locus coeruleus (LC) sends widespread outputs to many brain regions to modulate diverse functions, including sleep/wake states, attention, and the general anesthetic state. The paraventricular thalamus (PVT) is a critical thalamic area for arousal and receives dense tyrosine-hydroxylase (TH) inputs from the LC. Although anesthesia and sleep may share a common pathway, it is important to understand the processes underlying emergence from anesthesia. In this study, we hypothesize that LC TH neurons and the TH:LC-PVT circuit may be involved in regulating emergence from anesthesia. Only male mice are used in this study. Here, using c-Fos as a marker of neural activity, we identify LC TH expressing neurons are active during anesthesia emergence. Remarkably, chemogenetic activation of LC TH neurons shortens emergence time from anesthesia and promotes cortical arousal. Moreover, enhanced c-Fos expression is observed in the PVT after LC TH neurons activation. Optogenetic activation of the TH:LC-PVT projections accelerates emergence from anesthesia, whereas, chemogenetic inhibition of the TH:LC-PVT circuit prolongs time to wakefulness. Furthermore, optogenetic activation of the TH:LC-PVT projections produces electrophysiological evidence of arousal. Together, these results demonstrate that activation of the TH:LC-PVT projections is helpful in facilitating the transition from isoflurane anesthesia to an arousal state, which may provide a new strategy in shortening the emergence time after general anesthesia.

Different baseline physical activity predicts susceptibility and resilience to chronic social defeat stress in mice: Involvement of dopamine neurons

Physical inactivity, the fourth leading mortality risk factor worldwide, is associated with chronic mental illness. Identifying the mechanisms underlying different levels of baseline physical activity and the effects of these levels on the susceptibility to stress is very important. However, whether different levels of baseline physical activity influence the susceptibility and resilience to chronic social defeat stress (CSDS), and the underlying mechanisms in the brain remain unclear. The present study segregated wild-type mice into low baseline physical activity (LBPA) and high baseline physical activity (HBPA) groups based on short term voluntary wheel running (VWR). LBPA mice showed obvious susceptibility to CSDS, while HBPA mice were resilient to CSDS. In addition, the expression of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) was lower in LBPA mice than in HBPA mice. Furthermore, activation of TH neurons in the VTA of LBPA mice by chemogenetic methods increased the levels of VWR and resilience to CSDS. In contrast, inhibiting TH neurons in the VTA of HBPA mice lowered the levels of VWR and increased their susceptibility to CSDS. Thus, this study suggests that different baseline physical activities might be mediated by the dopamine system. This system also affects the susceptibility and resilience to CSDS, possibly via alteration of the baseline physical activity. This perspective on the neural control and impacts on VWR may aid the development of strategies to motivate and sustain voluntary physical activity. Furthermore, this can maximize the impacts of regular physical activity toward stress-reduction and health promotion.

Dopamine neurons gate the intersection of cocaine use, decision making, and impulsivity.

Gambling and substance use disorders are highly comorbid. Both clinical populations are impulsive and exhibit risky decision-making. Drug-associated cues have long been known to facilitate habitual drug-seeking, and the salient audiovisual cues embedded within modern gambling products may likewise encourage problem gambling. The dopamine neurons of the ventral tegmental area (VTA) are exquisitely sensitive to drugs of abuse, uncertain rewards, and reward-paired cues and may therefore be the common neural substrate mediating synergistic features of both disorders. To test this hypothesis, we first gained specific inhibitory control over VTA dopamine neurons by transducing a floxed inhibitory DREADD (AAV5-hSyn-DIO-hM4D(Gi)-mCherry) in rats expressing Cre recombinase in tyrosine hydroxylase neurons. We then trained rats in our cued rat gambling task (crGT), inhibiting dopamine neurons throughout task acquisition and performance, before allowing them to self-administer cocaine in the same diurnal period as crGT sessions. The trajectories of addiction differ in women and men, and the dopamine system may differ functionally across the sexes; therefore, we used male and female rats here. We found that inhibition of VTA dopamine neurons decreased cue-induced risky choice and reduced motor impulsivity in males, but surprisingly, enhanced risky decision making in females. Inhibiting VTA dopamine neurons also prevented cocaine-induced changes in decision making in both sexes, but nevertheless drove all animals to consume more cocaine. These findings show that chronic dampening of dopamine signalling can have both protective and deleterious effects on addiction-relevant behaviours, depending on biological sex and dependent variable of interest.

Controllable fusion of human brain organoids using acoustofluidics.

The fusion of human organoids holds promising potential in modeling physiological and pathological processes of tissue genesis and organogenesis. However, current fused organoid models face challenges of high heterogeneity and variable reproducibility, which may stem from the random fusion of heterogeneous organoids. Thus, we developed a simple and versatile acoustofluidic method to improve the standardization of fused organoid models via a controllable spatial arrangement of organoids. By regulating dynamic acoustic fields within a hexagonal acoustofluidic device, we can rotate, transport, and fuse one organoid with another in a contact-free, label-free, and minimal-impact manner. As a proof-of-concept to model the development of the human midbrain-to-forebrain mesocortical pathway, we acoustically fused human forebrain organoids (hFOs) and human midbrain organoids (hMOs) with the controllable alignment of neuroepithelial buds. We found that post-assembly, hMO can successfully project tyrosine hydroxylase neurons towards hFO, accompanied by an increase of firing rates and synchrony of excitatory neurons. Moreover, we found that our controllable fusion method can regulate neuron projection (e.g., range, length, and density), projection maturation (e.g., higher firing rate and synchrony), and neural progenitor cell (NPC) division in the assembloids via the initial spatial control. Thus, our acoustofluidic method may serve as a label-free, contact-free, and highly biocompatible tool to effectively assemble organoids and facilitate the standardization and robustness of organoid-based disease models and tissue engineering.

Centrally circulating α-klotho inversely correlates with human obesity and modulates arcuate cell populations in mice

ObjectiveOur laboratory recently identified the centrally circulating α-klotho protein as a novel hypothalamic regulator of food intake and glucose metabolism in mice. The current study aimed to investigate novel molecular effectors of central α-klotho in the arcuate nucleus of the hypothalamus (ARC), while further deciphering its role regulating energy balance in both humans and mice. MethodsCerebrospinal fluid (CSF) was collected from 22 adults undergoing lower limb orthopedic surgeries, and correlations between body weight and α-klotho were determined using an α-klotho enzyme-linked immunosorbent assay (ELISA) kit. To investigate the effects of α-klotho on energy expenditure (EE), 2-day intracerebroventricular (ICV) treatment was performed in diet-induced obesity (DIO) mice housed in TSE Phenomaster indirect calorimetry metabolic cages. Immunohistochemical staining for cFOS and patch clamp electrophysiology were used to determine the effects of central α-klotho on proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) neurons. Additional stainings were performed to determine novel roles for central α-klotho to regulate non-neuronal cell populations in the ARC. Lastly, ICV pretreatment with fibroblast growth factor receptor (FGFR) or PI3kinase inhibitors was performed to determine the intracellular signaling involved in α-klotho-mediated regulation of ARC nuclei. ResultsObese/overweight human subjects had significantly lower CSF α-klotho concentrations compared to lean counterparts (1,044 ± 251 vs. 1616 ± 218 pmol/L, respectively). Additionally, 2 days of ICV α-klotho treatment increased EE in DIO mice. α-Klotho had no effects on TH neuron activity but elicited varied responses in POMC neurons, with 44% experiencing excitatory and 56% experiencing inhibitory effects. Inhibitor experiments identified an α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of ARC POMC and NPY/AgRP neurons. Acute ICV α-klotho treatment also increased phosphorylated ERK in ARC astrocytes via FGFR signaling. ConclusionOur human CSF data provide the first evidence that impaired central α-klotho function may be involved in the pathophysiology of obesity. Furthermore, results in mouse models identify ARC POMC neurons and astrocytes as novel molecular effectors of central α-klotho. Overall, the current study highlights prominent roles of α-klotho→FGFR→PI3kinase signaling in the homeostatic regulation of ARC neurons and whole-body energy balance.

Neurochemical phenotype of growth hormone-responsive cells in the mouse paraventricular nucleus of the hypothalamus.

Multiple neuroendocrine, autonomic and behavioral responses are regulated by the paraventricular nucleus of the hypothalamus (PVH). Previous studies have shown that PVH neurons express the growth hormone (GH) receptor (GHR), although the role of GH signaling on PVH neurons is still unknown. Given the great heterogeneity of cell types located in the PVH, we performed a detailed analysis of the neurochemical identity of GH-responsive cells to understand the possible physiological importance of GH action on PVH neurons. GH-responsive cells were detected via the phosphorylated form of the signal transducer and activator of transcription-5 (pSTAT5) in adult male mice that received an intraperitoneal GH injection. Approximately 51% of GH-responsive cells in the PVH co-localized with the vesicular glutamate transporter 2. Rare co-localizations between pSTAT5 and vesicular GABA transporter or vasopressin were observed, whereas approximately 20% and 38% of oxytocin and tyrosine hydroxylase (TH) cells, respectively, were responsive to GH in the PVH. Approximately 55%, 35% and 63% of somatostatin, thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons expressed GH-induced pSTAT5, respectively. Additionally, 8%, 49% and 75% of neuroendocrine TH, TRH and CRH neurons, and 67%, 32% and 74% of nonneuroendocrine TH, TRH and CRH neurons were responsive to GH in the PVH of Fluoro-Gold-injected mice. Our findings suggest that GH action on PVH neurons is involved in the regulation of the thyroid, somatotropic and adrenal endocrine axes, possibly influencing homeostatic and stress responses.

Medullary tyrosine hydroxylase catecholaminergic neuronal populations in sudden unexpected death in epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is mechanistically complex and one probable cause is seizure‐related respiratory dysfunction. Medullary respiratory regulatory nuclei include the pre‐Bötzinger complex (pre‐BötC) in the ventrolateral medulla (VLM), the medullary raphé nuclei (MR) and nucleus of solitary tract in the dorsomedial medulla (DMM). The region of the VLM also contains intermingled tyrosine hydroxylase (TH) catecholaminergic neurones which directly project to the pre‐BötC and regulate breathing under hypoxic conditions and our aim was to evaluate these neurones in SUDEP cases. In post‐mortem cases from three groups [SUDEP (18), epilepsy controls (8) and non‐epilepsy controls (16)] serial sections of medulla (obex + 2 to + 13 mm) were immunolabeled for TH. Three regions of interest (ROI) were outlined (VLM, DMM and MR) and TH‐immunoreactive (TH‐IR) neurones were evaluated using automated detection for overall labeling index (neurones and processes) and neuronal densities and compared between groups and relative to obex level. C‐fos immunoreactivity was also semi‐quantitatively evaluated in these regions. We found no significant difference in the density of TH‐IR neurones or labeling index between the groups in all regions. Significantly more TH‐IR neurones were present in the DMM region than VLM in non‐epilepsy cases only (P < 0.01). Regional variations in TH‐IR neurones with obex level were seen in all groups except SUDEP. We also identified occasional TH neurones in the MR region in all groups. There was significantly less c‐fos labeling in the VLM and MR in SUDEP than non‐epilepsy controls but no difference with epilepsy controls. In conclusion, in this series we found no evidence for alteration of total medullary TH‐IR neuronal numbers in SUDEP but noted some differences in their relative distribution in the medulla and c‐fos neurones compared to control groups which may be relevant to the mechanism of death.

Rapid Effects of Selection on Brain-wide Activity and Behavior

Interindividual variation in behavior and brain activity is universal and provides substrates for natural selection [1-9]. Selective pressures shift the expression of behavioral traits at the population level [10, 11], but the accompanying changes of the underlying neural circuitry have rarely been identified [12, 13]. Selection likely acts through the genetic and/or epigenetic underpinnings of neural activity controlling the selected behavior [14]. Endocrine and neuromodulatory systems participate in behavioral diversity and could provide the substrate for evolutionary modifications [15-21]. Here, we examined brain-wide patterns of activity in larval zebrafish selectively bred over two generations for extreme differences in habituation of the acoustic startle response (ASR) [22]. The ASR is an evolutionarily conserved defensive behavior induced by strong acoustic/vibrational stimuli. ASR habituation shows great individual variability that is stable over days and heritable [4, 22]. Selection for high ASR habituation leads to stronger sound-evoked activation of ASR-processing brain areas. In contrast, animals selected for low habituation displayed stronger spontaneous activity in ASR-processing centers. Ablation of dopaminergic tyrosine hydroxylase (TH) neurons decreased ASR sensitivity. Independently selected ASR habituation lineages link the effect of behavioral selection to dopaminergic caudal hypothalamus (HC) neurons [23]. High ASR habituation co-segregated with decreased spontaneous swimming phenotypes, but visual startle responses were unaffected. Furthermore, high- and low-habituation larvae differed in stress responses as adults. Thus, selective pressure over a couple of generations on ASR habituation behavior is able to induce substantial differences in brain activity, carrying along additional behaviors as piggyback traits that might further affect fitness in the wild. VIDEO ABSTRACT.

The antiparkinson drug ropinirole inhibits movement in a Parkinson's disease mouse model with residual dopamine neurons

The dopamine (DA) D2-like receptor (D2R) agonist ropinirole is often used for early and middle stage Parkinson's disease (PD). However, this D2-like agonism-based strategy has a complicating problem: D2-like agonism may activate D2 autoreceptors on the residual DA neurons in the PD brain, potentially inhibiting these residual DA neurons and motor function. We have examined this possibility by using systemic and local drug administration in transcription factor Pitx3 null mutant (Pitx3Null) mice that mimic the DA denervation in early and middle stage PD and in DA neuron tyrosine hydroxylase (TH) gene knockout (KO) mice that mimic the severe DA loss in late stage PD. We found that in Pitx3Null mice with residual DA neurons and normal mice with normal DA system, systemically injected ropinirole inhibited locomotion, whereas bilateral dorsal striatal-microinjected ropinirole stimulated movement in Pitx3Null mice; bilateral microinjection of ropinirole into the ventral tegmental area also inhibited movement in Pitx3Null mice; we further determined that ropinirole inhibited nigral DA neuron spike firing in WT mice. In contrast, both systemically and striatum-locally administered ropinirole increased movements in TH KO mice, but produced relatively more dyskinesia than L-dopa. Although requiring confirmation in non-human primates and PD patients, these data suggest that while activating D2-like receptors in striatal projection neurons and hence stimulating movements, D2-like agonists can inhibit residual DA neurons and cause akinesia when the residual DA neurons and motor functions are still substantial, and this motor-inhibitory effect disappears when almost all DA neurons are lost such as in late stage PD.

Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice

ABSTRACT Microglial activation-induced neuroinflammation is closely associated with the development of Parkinson disease (PD). Macroautophagy/autophagy regulates many biological processes, but the role of autophagy in microglial activation during PD development remains largely unclear. In this study, we showed that deletion of microglial Atg5 caused PD-like symptoms in mice, characterized by impairment in motor coordination and cognitive learning, loss of tyrosine hydroxylase (TH) neurons, enhancement of neuroinflammation and reduction in dopamine levels in the striatum. Mechanistically, we found that inhibition of autophagy led to NLRP3 (NLR family pyrin domain containing 3) inflammasome activation via PDE10A (phosphodiesterase 10A)–cyclic adenosine monophosphate (cAMP) signaling in microglia, and the sequential upregulation of downstream IL1B/IL-1β in turn increased the expression of MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]), a pro-inflammatory cytokine. Inhibition of NLRP3 inflammasome activation by administration of MCC950, a specific inhibitor for NLRP3, decreased MIF expression and neuroinflammatory levels, and rescued the loss of TH neurons in the substantial nigra (SN). Interestingly, we found that serum MIF levels in PD patients were significantly elevated. Taken together, our results reveal an important role of autophagy in microglial activation-driven PD-like symptoms, thus providing potential targets for the clinical treatment of PD. Abbreviations: ATG: autophagy related; cAMP: cyclic adenosine monophosphate; cKO: conditional knockout; NOS2/INOS: nitric oxide synthase 2, inducible; IL1B: interleukin 1 beta; ITGAM/CD-11b: integrin alpha M/cluster of differentiation molecule 11B; MAP1LC3: microtubule-associated protein 1 light chain 3; MIF: macrophage migration inhibitory factor (glycosylation-inhibiting factor); NLRP3: NLR family pyrin domain containing 3; PBS: phosphate-buffered saline; PD: parkinson disease; PDE10A: phosphodiesterase 10A; SN: substantial nigra; TH: tyrosine hydroxylase; TNF: tumor necrosis factor; WT: wild type.

Dopamine-Producing Neurons in Rat Ontogeny: Phenotypic Features Underlying Molecular Mechanisms of Secretion and Regulation

Dopamine (DA), which is synthesized in DA-ergic neurons and so-called monoenzymatic neurons, is involved in the regulation of neuronal differentiation during the critical period of morphogenesis. The aim of the present study was to examine phenotypic features of DA-producing neurons in several brain regions, including the striatum, suprachiasmatic nucleus, and arcuate nucleus, during rat ontogeny starting from embryonic day 18 (E18) to postnatal day 60 (P60). The enzymes of DA synthesis tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AAAD), vesicular monoamine transporters (VMAT), and D1 and D2 DA receptors were used as markers for functional activity of DA neurons. Expression of these markers was studied using double immunohistochemical labeling of TH and AAAD or PCR assay of TH, AAAD, VMAT1, VMAT2, and D1 and D2 DA receptors. In the striatum, mRNAs of all mentioned markers were revealed within the whole period of ontogeny studied, bienzymatic TH and AAAD-immunopositive neurons were observed at E18, whereas monoenzymatic TH- or AAAD-containing neurons were observed at P60 only. In the suprachiasmatic nucleus, monoenzymatic AAAD neurons coexist with monoenzymatic TH-immunoreactive fibers, which innervate the ventral part of the nucleus, in the early postnatal period (P10). At P10 in the suprachiasmatic nucleus, the TH, VMAT1, and VMAT2 genes are not expressed; however, transcripts of the D1 and D2 genes were detected. The arcuate nucleus of rat embryos contained only monoenzymatic TH neurons in the ventrolateral area and AAAD neurons in the dorsomedial area in the late prenatal period (E21). At the same time TH, AAAD, VMAT2, D1, and D2 mRNAs were expressed in both parts of the arcuate nucleus. Thus, during rat ontogeny, nondopaminergic neurons containing one of the enzymes, TH or AAAD, are present in the striatum, suprachiasmatic nucleus, and arcuate nucleus, which allows for suggesting the cooperative synthesis of dopamine in these brain structures.

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones.

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague-Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90minutes of myocardial ischaemia, brains were collected. Dual-label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin- or oxytocin-labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90minutes post-MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure.

Dual Sympathetic Input into Developing Salivary Glands

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

Comparative study of environmental pollutants bisphenol A and bisphenol S on sexual differentiation of anteroventral periventricular nucleus and spermatogenesis

BackgroundBisphenol A is well known endocrine-disrupting chemical while Bisphenol S was considered a safe alternative. The present study aims to examine the comparative effects of xenobiotic bisphenol-A (BPA) and its substitute bisphenol-S (BPS) on spermatogenesis and development of sexually dimorphic nucleus population of dopaminergic neurons in the anteroventral periventricular nucleus (AVPV) of the hypothalamus in male pups.MethodsSprague Dawley rat’s pups were administered subcutaneously at the neonatal stage from postnatal day PND1 to PND 27. Thirty animals were divided into six experimental groups (6 animals/group). The first group served as control and was provided with normal olive oil. The four groups were treated with 2 μg/kg and 200 μg/kg of BPA and BPS, respectively. The sixth group was given with 50 μg/kg of estradiol dissolved in olive oil as a standard to find the development of dopaminergic tyrosine hydroxylase neurons in AVPV regions. Histological analysis for testicular tissues and immunohistochemistry for brain tissues was performed.ResultsThe results revealed adverse histopathological changes in testis after administration of different doses of BPA and BPS. These degenerative changes were marked by highly significant (p < 0.001) decrease in tubular and luminal diameters of seminiferous tubule and epithelial height among bisphenols treated groups as compared to control. Furthermore, significantly increased (p < 0.001) TH-ir cell bodies in the AVPV region of the brain with 200 μg/kg dose of BPA and BPS was evident.ConclusionIt is concluded that exposure of BPA and BPS during a critical developmental period can structural impairments in testes and affects sexual differentiation of a dimorphic dopaminergic population of AVPV region of hypothalamus in the male brain.

RhoA in tyrosine hydroxylase neurones regulates food intake and body weight via altered sensitivity to peripheral hormones.

Dopamine-producing tyrosine hydroxylase (TH) neurones in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signalling and body weight homeostasis. In the present study, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurones in response to peripheral hormones. Diet-induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity-associated increased activity of ARC TH neurones. Mice in which RhoA was specifically knocked out in TH neurones (TH-RhoA-/- mice) were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH-RhoA-/- mice were challenged with a high-fat high-sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wild-type control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wild-type mice after long-term HFHS diet feeding. Patch-clamp electrophysiological analysis demonstrated increased ghrelin-induced excitability of TH neurones in lean TH-RhoA-/- mice compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides agouti-related peptide and neuropeptide Y was observed in TH-RhoA-/- mice. Overall, our data indicate that TH neurones in ARC are important for the regulation of body weight homeostasis and that RhoA is both a central effector in these neurones and important for the development of obesity-induced ghrelin resistance. The obese phenotype of TH-RhoA-/- mice may be a result of increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake.

Both neural and global androgen receptor overexpression affect sexual dimorphism in the mouse brain.

Testosterone is the main endocrine mechanism mediating sexual differentiation of the mammalian brain, although testosterone signalling is complex and important mechanistic questions remain. Notably, the extent to which testosterone acts via androgen receptors (AR) in this process remains unknown and it is also not clear where testosterone acts in the body to produce sexual dimorphisms in neuroanatomy. To address these questions, we used a transgenic mouse model of Cre/loxP-driven AR overexpression in which AR was induced selectively in neural tissue (Nestin-cre) or in all tissues (CMV-cre). We then studied sexually dimorphic features of several well-characterised sexual dimorphisms: calbindin-immunoreactive neurones in the medial preoptic area (CALB-SDN), tyrosine hydroxylase neurones in the anteroventral periventricular nucleus, and vasopressin-immunoreactive neurones originating in the bed nucleus of the stria terminalis and their projections in the lateral septum. We additionally evaluated oestrogen receptor α immunoreactivity in these nuclei. Briefly, we found that global but not neural overexpression of AR resulted in masculinisation of CALB-SDN nucleus volume, cell number and cell size in transgenic females. Furthermore, neural AR overexpression resulted in increased oestrogen receptor α staining in females compared to males in the medial preoptic area. AR overexpression did not affect other measures. Overall, the results of the present study provide support for the hypothesis that androgenic mechanisms external to the nervous system can affect sexual differentiation of the brain.

Impact of Adolescent Intermittent Ethanol on Noradrenergic Development

In clinical populations, adolescent alcohol drinking has been shown to increase stress sensitivity and the likelihood of developing alcohol use disorders (AUDs) in adulthood. These findings suggest pathways mediating stress‐relating behaviors are impacted by developmental exposure to alcohol, contributing to a greater risk for alcoholism and potentially stress‐induced relapse following treatment. Animal models indicate tyrosine hydroxylase (TH) neurons in the A2 region of the nucleus of the tractus solitarius (NTS) contribute to stress‐induced relapse through enhanced norepinephrine signaling, subsequently increasing neuronal activity in the bed nucleus of the stria terminalus (BNST). Previous research suggests NTS TH expression undergoes developmental changes during adolescence that may make this neuronal population susceptible to modulation by alcohol during this age. We hypothesized adolescent intermittent ethanol (AIE) exposure in mice, a standard preclinical model of adolescent binge drinking, will impact NTS TH neuron development, ultimately altering the NTS‐BNST circuitry resulting in a higher risk of AUDs in adulthood. NTS TH mRNA expression in male and female C57Bl6/J mice exposed to AIE (postnatal day [PND] 28–42) or used as air controls was quantified via qRT‐PCR at different time points (PND29, PND36, PND43, PND49, PND70). Immunohistochemistry methods were implemented to determine the impact of AIE on NTS TH neuronal activity following an ethanol challenge in adulthood (PND70). Results showed a significant increase in TH mRNA in males one day after beginning AIE (PND29) relative to air controls, while females exhibited the greatest TH expression after their first week of exposure to ethanol (PND36). In both sexes, heightened TH mRNA expression occurred earlier than expected during normal development. Surprisingly, compared to adult ethanol naïve mice, AIE decreased the ability of ethanol at doses of 2g/kg and 4g/kg to alter NTS TH neuron cFos expression at PND70 compared to control. Together, these findings suggest an enhancement of NTS TH activity early in development and reduction of ethanol sensitivity in adulthood. Studies are in progress to further delineate the mechanisms by which AIE reduces adult ethanol sensitivity and how this may alter stress‐related behaviors.Support or Funding InformationAA22937AA017823This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Do Sedentary Conditions Affect the Number of C1 Neurons in Rat Rostral Ventrolateral Medulla?

Cardiovascular disease (CVD) is the leading cause of death in the United States. One of the primary risk factors for CVD is a lack of regular exercise. The brain controls the activity of the autonomic nervous system, which regulates many physiological functions; including blood pressure. The rostral ventrolateral medulla (RVLM) is an important region in the brainstem for blood pressure control because it contains a critical subset of adrenergic (C1) neurons that project to sympathetic preganglionic neurons in the spinal cord. C1 neurons from sedentary rats show more dendritic branching than in active rats, suggesting a novel mechanism by which inactivity could increase sympathetic activity and contribute to hypertension. However, it is also possible that the absolute number of C1 neurons changes along with the number of dendritic branches in sedentary rats. Therefore, the purpose of this study was to test the hypothesis that there are more C1 neurons in the RVLM of rats living under sedentary conditions compared to freely exercising rats. Aiming to understand the mechanisms by which inactivity leads to hypertension, we maintained four‐week old, male Sprague‐Dawley rats for 10–12 weeks under either physically active conditions (running wheels; 410±5.6 km total per rat) or sedentary conditions (no running wheels). Following perfusion and post‐fixation, sections of brainstem were first immunoperoxidase‐stained black for phenylethanolamine N‐methyltransferase (PNMT), a synthetic enzyme and marker of C1 neurons, and then brown for tyrosine hydroxylase (TH), the enzyme that all catecholamine neurons contain. PNMT+TH and TH only neurons in each rat were counted in the 6 sections containing the 600 μm caudal to the caudal pole of the facial nucleus (FN). Sedentary conditions did not produce a difference in the number, or rostrocaudal pattern, of C1 neurons compared to the active group nor an interaction between the two (two way RM ANOVA, p&gt;0.05 ea). Sedentary conditions also did not affect the low number of TH neurons (likely A1 neurons) in the RVLM compared to active rats (2±1 vs. 2±1, respectively). Because the number of RVLM C1 neurons is the same in active and sedentary rats, we can exclude the possibility that an increase in the number of C1 neurons is a cause of increased activation of vasomotor spinal neurons and that structural neuroplasticity in C1 neurons may underlie increased functional excitability of the RVLM in our other studies. Future functional neuroanatomical studies using Fos protein should show whether the overall activation of the population of C1 neurons differs between sedentary and active rats.Support or Funding Information(HL096787‐09)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of adenosine A1 receptor agonist induced‐hibernation on the arousal pathway in the Arctic Ground Squirrel

Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN in AGS treated with CHA in both summer and winter seasons.Here we test the hypothesis that seasonal response to CHA is associated with difference in activation in another nucleus of the ascending arousal pathway, the locus coeruleus (LC).AGS implanted with body temperature (Tb) transmitter, were treated with CHA or vehicle (0.5mg/kg, IP). AGS were perfused with 4% paraformaldehyde and brains were dissected and cryoprotected in 5%, 10%, 15%, 20%, 30% sucrose. We performed free‐floating immunohistochemistry on 40μm brain slices in four experimental groups: winter CHA, winter control, summer CHA and summer control. We sectioned brains from eight AGS per group for a total of 32 animals and identified active nuclei with cFos immunoreactivity (mouse anti‐cFos 1:20,000, Santa Cruz) and active tyrosine hydroxylase neurons (rabbit anti‐TH). Blinded analysis of cFos immunoreactive neurons was done using bright field microscopy and Metamorph software. Statistical analysis was performed on R.CHA produced a hibernation‐like response measured by a decrease in Tb in winter AGS but not in the summer. We did not observe any significant difference in neuronal activation in the LC, thus CHA is associated with a decreased neuronal activity in the TMN, resulting in the TMN being the major wakefulness center affected by CHA but not by seasons.Support or Funding InformationResearch was supported by NIH R03 NS081637, NSF IOS‐1258179, RL5GM118990 and Alaska INBRE P20GM103395. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Respiratory disturbances in a mouse model of Parkinson's disease.

What is the central question of this study? Clinical reports have described and suggested central and peripheral respiratory abnormalities in Parkinson's disease (PD) patients; however, these reports have never addressed the occurrence of these abnormalities in an animal model. What is the main finding and its importance? A mouse model of PD has reduced neurokinin-1 receptor immunoreactivity in the pre-Bӧtzinger complex and Phox2b-expressing neurons in the retrotrapezoid nucleus. The PD mouse has impairments of respiratory frequency and the hypercapnic ventilatory response. Lung collagen deposition and ribcage stiffness appear in PD mice. Parkinson's disease (PD) is a neurodegenerative motor disorder characterized by dopaminergic deficits in the brain. Parkinson's disease patients may experience shortness of breath, dyspnoea, breathing difficulties and pneumonia, which can be linked as a cause of morbidity and mortality of those patients. The aim of the present study was to clarify whether a mouse model of PD could develop central brainstem and lung respiratory abnormalities. Adult male C57BL/6 mice received bilateral injections of 6-hydroxydopamine (10μgμl-1 ; 0.5μl) or vehicle into the striatum. Ventilatory parameters were assessed in the 40days after induction of PD, by whole-body plethysmography. In addition, measurements of respiratory input impedance (closed and opened thorax) were performed. 6-Hydroxydopamine reduced the number of tyrosine hydroxylase neurons in the substantia nigra pars compacta, the density of neurokinin-1 receptor immunoreactivity in the pre-Bӧtzinger complex and the number of Phox2b neurons in the retrotrapezoid nucleus. Physiological experiments revealed a reduction in resting respiratory frequency in PD animals, owing to an increase in expiratory time and a blunted hypercapnic ventilatory response. Measurements of respiratory input impedance showed that only PD animals with the thorax preserved had increased viscance, indicating that the ribcage could be stiff in this animal model of PD. Consistent with stiffened ribcage mechanics, abnormal collagen deposits in alveolar septa and airways were observed in PD animals. Our data showed that our mouse model of PD presented with neurodegeneration in respiratory brainstem centres and disruption of lung mechanical properties, suggesting that both central and peripheral deficiencies contribute to PD-related respiratory pathologies.