Tyrosine Hydroxylase Neurons Research Articles

Abstract 15832: Optogenetic Stimulation of Intrinsic Cardiac Autonomic Neurons Reveals the Interaction Between Catecholaminergic and Cholinergic Pathways

Autonomic tone, through the interaction of catecholaminergic and cholinergic pathways, is a powerful modulator of cardiac function. Until recently, selective activation of cardiac autonomic pathways has required administration of exogeneous neurotransmitters or careful placement of electrodes to stimulate specific neural populations. Optogenetics enables broader exploration of autonomic interactions through the selective expression of channelrhodopsin (ChR2) in neural populations. Our objective was to simultaneously activate catecholaminergic and cholinergic pathways, by exogenous perfusion of neurotransmitter and photostimulation of opposing autonomic neurons, to test the hypothesis that simultaneous activation will reveal the fast kinetics of cholinergic modulation of catecholaminergic increases in heart rate (HR). Norepinephrine (NE) and acetylcholine (ACh) were administered to perfused transgenic mouse hearts that expressed ChR2 within sympathetic tyrosine hydroxylase neurons (TH+ChR2) or parasympathetic choline acetyltransferase (ChAT+ChR2) neurons. Intrinsic neurons were photostimulated by illuminating the SA node with a 465nm high power microLED. The ECG was recorded to measure beat-to-beat changes in HR. Moderate doses of exogeneous NE increased HR 26.77±5.61% (n=3) and high doses increased HR 59.74±8.77% (n=3). At all NE doses, photostimulation of ChAT+ChR2 neurons immediately reduced HR to values equivalent to, or lower than, pre-NE HR. Arrhythmias were not observed. In companion studies, moderate doses of exogeneous ACh reduced HR 19.98±4.68% (n=7) and high doses reduced HR 51.31±4.07% (n=7). HR increases caused by TH+ChR2 neuron photostimulation were diminished by exogenous ACh in a dose-dependent manner. Increased dosing of exogeneous ACh, with simultaneous TH+ChR2 neuron photostimulation, dramatically shortened the onset of 2 nd degree AV block and increased its severity. At high concentrations of exogeneous ACh, non-sustained arrhythmias were observed upon the cessation of TH+ChR2 neuron photostimulation. These results provide new insight into the kinetics of intrinsic cardiac autonomic interactions and reveal powerful modulation of catecholaminergic activity by cholinergic activation.

Chrysin supplementation mitigated neurobehavioral changes in a animal model of Parkinson’s disease: Influence on TH+ neurons

The aim of current study was investigated the effects of chrysin supplementation (10 mg/kg, per oral, p.o.) over 28 days in a model of Parkinson’s disease (PD) induced by 6-hydroxydopamine (6-OHDA), using C57BL/6 male mice. Intrastriatal 6-OHDA microinjection caused oxidative stress, impairment of antioxidant defenses, depletion of dopamine (DA) and its metabolites, loss of tyrosine hydroxylase neurons (TH+) of substantia nigra pars compacta (SNpc) and behavioral changes. Chrysin supplementation protect against oxidative stress and depletion antioxidant defenses. Moreover, supplementation with chrysin prevented the decrease of DA and its metabolites, corroborating protection against loss of TH+ neurons in SNpc, blocking behavioral changes induced by 6-OHDA.

Abstract 043: Prorenin Reduces Spontaneous Firing Rate In Cells Expressing Tyrosine Hydroxylase In The Hypothalamic Paraventricular Nucleus

The (pro)renin receptor (PRR) plays essential roles in cardiovascular and metabolic physiology. The tyrosine hydroxylase (TH) neurons in the paraventricular nucleus of the hypothalamus (PVN) have recently emerged as a key metabolic regulator that play functional roles in the regulation of the energy expenditure and metabolism. Using RNAScope in situ hybridization, we found that 95% of TH-containing neurons in the PVN (n=872 cells/3 mice) express the PRR. In this study, we aimed to examine the effects of PRR activation on the firing activity of the TH-containing neurons in the PVN (TH PVN ). We hypothesized that prorenin activation of the PRR increases firing activity of the TH PVN neuron. To test this hypothesis, we performed electrophysiological recordings in loose cell-attached voltage-clamp configuration in PVN slices from mice expressing TdTomato reporter for TH neurons (TH-cre-TdTomato mice). These experiments were carried out to isolate and record spontaneous firing rate (SFR). We abolished all the synaptic transmission by adding a GABA A receptor antagonist (picrotoxin, 50 μM), an antagonist of AMPA/kainate receptors (CNQX, 5 μM), and an NMDA receptor antagonist (D-APV, 50 μM) throughout the recording. The cells were discarded if the access resistance was >15 MΩ or fluctuated more than 15%. At the end of the experiments, tetrodotoxin (1 μM) was added to abolish and confirm neuronal activity. The SFR in TH PVN neurons was significantly decreased after application of prorenin (PR) (4.9 ± 0.87 and 3.2 ± 1.07 Hz, before and after PR, respectively; n = 10, p = 0.006, paired t -test) indicating that PR reduces spontaneous firing activity of the TH PVN neurons. To determine if this effect was due to the prorenin activation of the PRR, we recorded SFR in the presence of PRR antagonist (PRO20). There was no difference in the SFR (5.8 ± 1.58 Hz and 5.9 ± 1.7 Hz, n = 5, p = 0.903, paired t -test) in the TH PVN neurons between PRO20 and PRO20+PR group respectively suggesting that PRO20 blocks the effect of PR in reducing SFR in TH PVN neurons. Our results showed that the prorenin acts on the PRR to reduce the SFR of the TH PVN neurons. We conclude that PRR signaling in the TH PVN neurons might play a role in cardiovascular and metabolic regulation by the modulating the TH PVN neuronal plasticity.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats

Background aimsGingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. MethodsRNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. ResultsIntravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium‐binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. ConclusionsThe authors’ research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

The Periventricular Nucleus as a Brain Center Containing Dopaminergic Neurons and Neurons Expressing Individual Enzymes of Dopamine Synthesis

Since the 1980s, the concept of dopamine-rich brain centers as clusters of only dopaminergic neurons has been fundamentally revised. It has been shown that, in addition to dopaminergic neurons, most of these centers contain neurons expressing one of the enzymes of dopamine synthesis: tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). We have obtained convincing evidence that in rats, the hypothalamic periventricular nucleus (PeVN) is one of the largest dopamine-rich centers, containing dopaminergic and monoenzymatic neurons. Indeed, using double immunostaining for TH and AADC, the PeVN was shown to contain almost three thousand dopaminergic and monoenzymatic neurons. According to high-performance liquid chromatography, PeVN contains L-DOPA and dopamine, which, apparently, are synthesized in monoenzymatic TH neurons and bienzymatic neurons, respectively. According to confocal microscopy, neurons (cell bodies, fibers), which were immunopositive only to TH, only to AADC, or both, are in close topographic relationships with each other and with the 3rd ventricle. These data suggest the mutual regulation of the neurons, as well as the delivery of dopamine and L-DOPA to the third ventricle, which is confirmed by their detection in the cerebrospinal fluid. Thus, evidence has been obtained that PeVN is one of the largest dopamine-rich centers of the brain, containing dopaminergic and monoenzymatic neurons.

196-OR: (Pro) renin Receptor Signaling in Tyrosine Hydroxylase Neurons in the Hypothalamic Paraventricular Nucleus Is Required for Obesity-Associated Glucose Metabolic Impairment

We recently reported that neuron-specific (pro) renin receptor (PRR) deletion reduces fasting blood glucose (BG) levels and improves glucose tolerance in high fat diet (HFD) -treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (THPVN neurons) , a largely GABAergic population that expresses PRR and projects to autonomic centers important for BG regulation. To delete PRR from THPVN neurons, we injected into the PVN of PRR-loxp mice either an adeno-associated virus expressing cre recombinase under the control of the rat TH promoter (AAV2-TH-Cre) or AAV2-TH-eGFP as a control. After placing animals on a HFD for 6 wk, we found that the obesity-induced increase of fasting BG levels was blocked by PRR deletion from THPVN neurons (118.0 ± 3.7 vs. 139.3 ± 6.6 mg/dL, n=6/group, p= 0.04) , accompanied by a trend towards improved i.p. glucose tolerance (AUC: 12990 ± 1136 vs. 9467 ± 1298, n=6, p=0.07) . To further investigate the role of THPVN neurons in glucose homeostasis, we employed a Designer Receptors Exclusively Activated by Designer Drugs (DREADD) approach whereby AAV2-TH-Cre was injected bilaterally into the PVN of either hM3Dq-loxp (excitatory) or hM4Di-loxp (inhibitory) mice, and continuous glucose monitoring was performed in conscious, freely moving mice. Following i.p. injection of clozapine-N-oxide (CNO, 1 mg/Kg) or vehicle, we found that whereas basal BG levels were not altered by THPVN neuron activation, THPVN neuron inhibition significantly increased basal BG levels compared to vehicle in male (ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03) but not female mice. These findings establish a role for THPVN neuron PRR signaling in the pathogenesis of obesity-associated glucose metabolic impairment, and show that in male mice, basal THPVN neuron activity is required for normal glucose homeostasis. Disclosure L.Ac. souza: None. C.J.Worker: None. A.Gayban: None. M.W.Schwartz: None. Y.Feng earley: None. Funding National Institutes of Health (R01HL122770, R35HL155008, RDK83042, RDK124238)

The activity of ventral tegmental area dopamine neurons during shock omission predicts safety learning.

We studied the role of dopamine [tyrosine hydroxylase (TH)] neurons in the rat ventral tegmental area (VTA) in safety learning. First, we used an AX +/BX-discrimination procedure to establish conditioned stimulus (CS) B as a learned safety signal that passed both summation and retardation tests of conditioned inhibition. Then, we combined this procedure with fiber photometry in TH-Cre rats to study the activity of VTA dopamine neurons during safety learning. We show that whereas footshock is associated with calcium transients in TH neurons across the VTA, shock omission during safety learning is selectively associated with calcium transients in dopamine neurons in the medial but not lateral VTA. Moreover, the magnitude of medial VTA calcium transients during shock omission accurately predicts the amount of safety that is learned and expressed during summation testing. Our findings are consistent with a common medial VTA dopamine mechanism contributing to the learned inhibition of fear in extinction and safety. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Focused Ultrasound Promotes the Delivery of Gastrodin and Enhances the Protective Effect on Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease globally; however, it lacks effective treatment at present. Focused ultrasound (FUS) combined with microbubbles could increase the efficacy of drug delivery to specific brain regions and is becoming a promising technology for the treatment of central nervous system diseases. In this study, we explored the therapeutic potential of FUS-mediated blood–brain barrier (BBB) opening of the left striatum to deliver gastrodin (GAS) in a subacute PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration of GAS in the left hemisphere was detected by ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap) and the distribution of tyrosine hydroxylase (TH) neurons was detected by immunohistochemical staining. The expression of TH, Dopamine transporter (DAT), cleaved-caspase-3, B-cell lymphoma 2 (Bcl-2), brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), and synaptophysin (SYN) protein were detected by western blotting. Analysis showed that the concentration of GAS in the left hemisphere of PD mice increased by approximately 1.8-fold after the BBB was opened. FUS-mediated GAS delivery provided optimal neuroprotective effects and was superior to the GAS or FUS control group. In addition, FUS enhanced GAS delivery significantly increased the expression of Bcl-2, BDNF, PSD-95, and SYN protein in the left striatum (P < 0.05) and reduced the levels of cleaved-caspase-3 remarkably (P = 0.001). In conclusion, the enhanced delivery by FUS effectively strengthened the protective effect of GAS on dopaminergic neurons which may be related to the reinforcement of the anti-apoptotic activity and the expression of synaptic-related proteins in the striatum. Data suggests that FUS-enhanced GAS delivery may represent a new strategy for PD treatment.

(Pro)renin Receptor Knockdown in Tyrosine Hydroxylase neurons in the PVN Protects Against the Development of HFD‐Induced Hyperglycemia and Increases Energy Expenditure in Mice

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin‐angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy‐expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)‐induced metabolic physiology is not well understood. We recently showed that neuron‐specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)‐containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD‐induced hyperglycemia. To test this hypothesis, we injected both male and female TdTomato‐PRR‐LoxP mice with adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre) to ablate PRR in the THPVN neurons (THPVNPRRKO). The Tdtomato‐LoxP mice received the same virus were used as control. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following 6‐week HFD (60% calories from fat). Mouse metabolic parameters were measured over 48 hours in Sable Promethion metabolic cages with ad libitum access to HFD. In male mice, PRR‐LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, P= 0.0059) at the end of the six week HFD treatment. Interestingly, PRR ablation in the THPVN did not improve FBG and glucose handling in female mice. Metabolic cage measurements revealed that THPVN PRRKO mice had elevated oxygen consumption (VO2, ml/minute/g body weight) (0.047 ± 0.009 vs. 0.043 ± 0.0008 P= 0.027) and carbon dioxide emission (VCO2, ml/minute/g body weight) (0.035 ± 0.0005 vs. 0.032 ± 0.0005, P=0.0125) versus control HFD‐fed mice over 48 hours, indicating enhanced metabolic rate. The energy expenditure was significantly higher (0.0125 ± 0.00017 vs 0.0116 ± 0.00014, kCal/Hr/g body weight P=0.0026) in the THPVN PRRKO mice compared with their control counterparts. Locomotor activity over 48 hours was similar between the two groups (59.94 ± 19.6 vs. 103.5 ± 42.42 total meters traveled, P=0.204), and no significant difference in food intake (0.254 ± 0.011 vs. 0.274 ± 0.012 g, P=0.090) or body weight (42.7 ± 3.65 vs. 43.6 ± 1.41 g, P=0.842) was observed. This data suggests that PRR knockout in THPVN neurons protects against glucose dysregulation and elevates energy expenditure in mice following HFD. We conclude that PRR THPVN neurons play a key regulatory role in glucose and metabolic regulation.

Castration mediated schwann cell dedifferentiation leads to slower nerve conduction, decreased neuritogenesis, and nitrergic neuron loss

ABSTRACT Introduction/Objectives Low levels of androgens due to androgen deprivation therapy for prostate cancer treatment or due to aging lead to erectile dysfunction (ED). Castration, a preclinical model of low androgens, induces ED due to damage to pelvic nerves and a loss of erectogenic nitrergic nerves. Schwann cells (SC) release neurotrophic factors to promote pelvic nerve growth and survival. It is unknown how testosterone deficiency impacts SC function. This study determines how testosterone deficiency influences SC phenotype and trophic support in neuronal regeneration. We hypothesize testosterone deficiency will decrease myelinated pelvic SC populations, delay penile nerve conduction and reduce pelvic nitrergic neurons. Methods Male Sprague Dawley rats (12 wks) were divided into control (CON) and surgical castration (CAST) groups. All experiments were performed six weeks following castration. Dorsal penile nerves (DPN; 15-16 mm) were excised, placed in a sylgard dish containing oxygenated buffer and conduction velocity of myelinated fast fibers was assessed (n=8 per group). The major pelvic ganglia (MPG) were collected and dissociated into SC or neurons. Dissociated neurons were grown on laminin coated coverslips. To determine whether SCs could provide trophic support, we co-cultured neurons on top of confluent SCs. We assessed the following co-culture groups: CON SC + CON MPG, CON SC + CAST MPG, CAST SC + CON MPG (n=4 per group, cultured in triplicate). After 72 hours, all cultures were fixed and stained with beta tubulin class III, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH) to assess neurite length and branching, nitrergic and sympathetic neuron populations, respectively. Dissociated SC were characterized via qPCR for markers of all SC (Sox10), myelinated SC (Krox20), and dedifferentiated SC (glial fibrillary acidic protein; GFAP). Results CAST slows DPN myelinated fast fiber conduction velocity. In dissociated cultured MPG neurons, castration reduces both neurite length and number of branches. Interestingly, CAST MPG neurons co-cultured with CON SC restore neurite length and branching to same level as CON MPG neurons co-cultured with CON SC; however, CAST SC do not impact CON MPG neurite length. CAST MPG neurons co-cultured with CON SC and CON MPG neurons co-cultured with CAST SC both have less nitrergic neurons than CON SC + CON MPG neurons. CAST does not impact the number of TH neurons in single or co-cultured conditions. CAST reduces gene expression of Sox10, myelinated SC, and dedifferentiated SC. Conclusions CAST markedly impairs penile nerve conduction, reduces neurite outgrowth and the proportion of pro-erectile nitrergic neurons. Healthy SC are able to rescue neuritogenesis but not the survival of nitrergic neurons. Additionally, healthy MPG neurons plated on CAST SC have a reduced number of nitrergic neurons. We believe CAST SC are undergoing a phenotypic switch from a myelinated neurotrophic form into dedifferentiated SC promoting neuritogenesis and loss of nitrergic neurons. Preserving the myelinated SC phenotype may prevent impaired nerve damage and subsequent ED in patients with testosterone deficiency. Disclosure Work supported by industry: no.

Exercise increases NPY/AgRP and TH neuron activity in the hypothalamus of female mice.

Recent evidence identifies a potent role for aerobic exercise to modulate the activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low- (10 m/min), moderate- (14 m/min), and high (18 m/min)-intensity treadmill exercise in a randomized crossover design. Mice were fasted 10 h before exercise, and food intake was monitored for 48 h after bouts. Immunohistochemical detection of cFOS was performed 3 h post-exercise to determine the changes in hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), tyrosine hydroxylase (TH), and SIM1-expressing neuron activity concurrent with the changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate- and high-intensity exercises increased 24-h food intake by 5.9 and 19%, respectively, while low-intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and TH neuron activity were observed 3 h after high-intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3 h post-exercise. Overall, aerobic exercise increased the activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.

Microglia Activation in the Midbrain of the Human Neonate: The Effect of Perinatal Hypoxic-Ischemic Injury.

Perinatal hypoxia-ischemia (PHI) is a major risk factor for the development of neuropsychiatric deficits later in life. We previously reported that after prolonged PHI, the dopaminergic neurons of the human neonate showed a dramatic reduction of tyrosine hydroxylase (TH) in the substantia nigra, without important signs of neuronal degeneration despite the significant reduction in their cell size. Since microglia activation could precede neuronal death, we now investigated 2 microglia activation markers, ionized calcium-binding adapter molecule 1 (Iba1), and the phagocytosis marker Cd68. The highest Iba1 immunoreactivity was found in neonates with neuropathological lesions of severe/abrupt PHI, while the lowest in subjects with moderate/prolonged or older PHI. Subjects with very severe/prolonged or chronic PHI showed an increased Iba1 expression and very activated microglial morphology. Heavy attachment of microglia on TH neurons and remarkable expression of Cd68 were also observed indicating phagocytosis in this group. Females appear to express more Iba1 than males, suggesting a gender difference in microglia maturation and immune reactivity after PHI insult. PHI-induced microglial "priming" during the sensitive for brain development perinatal/neonatal period, in combination with genetic or other epigenetic factors, could predispose the survivors to neuropsychiatric disorders later in life, possibly through a sexually dimorphic way.

Effect of Coffee against MPTP-Induced Motor Deficits and Neurodegeneration in Mice Via Regulating Gut Microbiota

The mechanisms of coffee against Parkinson disease (PD) remained incompletely elucidated. Numerous studies suggested that gut microbiota played a crucial role in the pathogenesis of PD. Here, we explored the further mechanisms of coffee against PD via regulating gut microbiota. C57BL/6 mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce a PD mouse model, then treated with coffee for 4 consecutive weeks. Behavioral tests consisting of the pole test and beam-walking test were conducted to evaluate the motor function of mice. The levels of tyrosine hydroxylase (TH) and α-synuclein (α-syn) were assessed for dopaminergic neuronal loss. The levels of occludin, glial fibrillary acidic protein (GFAP), Bcl-2, Bax, cleaved caspase-3, and cytochrome c (Cyt c) were detected. Moreover, microbial components were measured by 16s rRNA sequencing. Our results showed that coffee significantly improved the motor deficits and TH neuron loss, and reduced the level of α-syn in the MPTP-induced mice. Moreover, coffee increased the level of BBB tight junction protein occludin and reduced the level of astrocyte activation marker GFAP in the MPTP-induced mice. Furthermore, coffee significantly decreased the levels of proapoptotic proteins, including Bax, cleaved caspase-3, and cytochrome c, while it increased the level of antiapoptotic protein Bcl-2, consequently preventing MPTP-induced apoptotic cascade. Moreover, coffee improved MPTP-induced gut microbiota dysbiosis. These findings suggested that the neuroprotective effects of coffee on PD were involved in the regulation of gut microbiota, which might provide a novel option to elucidate the effects of coffee on PD.

Arcuate Angiotensin II Increases Arterial Pressure via Coordinated Increases in Sympathetic Nerve Activity and Vasopressin Secretion.

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here, we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aRs): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrous cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory neuropeptide Y (NPY) inputs, and activation of proopiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e., GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.

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 dor-somedial 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 postmortem 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 neu-rones were present in the DMM region than VLM in non-epilepsy cases only (P &lt; 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.

Adenovirus-induced Reactive Astrogliosis Exacerbates the Pathology of Parkinson's Disease.

Parkinson’s disease (PD) is the most prevalent neurodegenerative motor disorder. While PD has been attributed to dopaminergic neuronal death in substantia nigra pars compacta (SNpc), accumulating lines of evidence have suggested that reactive astrogliosis is critically involved in PD pathology. These pathological changes are associated with α-synuclein aggregation, which is more prone to be induced by an A53T mutation. Therefore, the overexpression of A53T-mutated α-synuclein (A53T-α-syn) has been utilized as a popular animal model of PD. However, this animal model only shows marginal-to-moderate extents of reactive astrogliosis and astrocytic α-synuclein accumulation, while these phenomena are prominent in human PD brains. Here we show that Adeno-GFAP-GFP virus injection into SNpc causes severe reactive astrogliosis and exacerbates the A53T-α-syn-mediated PD pathology. In particular, we demonstrate that AAV-CMV-A53T-α-syn injection, when combined with Adeno-GFAP-GFP, causes more significant loss of dopaminergic neuronal tyrosine hydroxylase level and gain of astrocytic GFAP and GABA levels. Moreover, the combination of AAV-CMV-A53T-α-syn and Adeno-GFAP-GFP causes an extensive astrocytic α-syn expression, just as in human PD brains. These results are in marked contrast to previous reports that AAV-CMV-A53T-α-syn alone causes α-syn expression mostly in neurons but rarely in astrocytes. Furthermore, the combination causes a severe PD-like motor dysfunction as assessed by rotarod and cylinder tests within three weeks from the virus injection, whereas Adeno-GFAP-GFP alone or AAV-CMV-A53T-α-syn alone does not. Our findings implicate that inducing reactive astrogliosis exacerbates PD-like pathologies and propose the virus combination as an advanced strategy for developing a new animal model of PD.

Sex-specific Disruption of the Prairie Vole Hypothalamus by Developmental Exposure to a Flame Retardant Mixture.

Prevalence of neurodevelopmental disorders (NDDs) with social deficits is conspicuously rising, particularly in boys. Flame retardants (FRs) have long been associated with increased risk, and prior work by us and others in multiple species has shown that developmental exposure to the common FR mixture Firemaster 550 (FM 550) sex-specifically alters socioemotional behaviors including anxiety and pair bond formation. In rats, FRs have also been shown to impair aspects of osmoregulation. Because vasopressin (AVP) plays a role in both socioemotional behavior and osmotic balance we hypothesized that AVP and its related nonapeptide oxytocin (OT) would be vulnerable to developmental FM 550 exposure. We used the prairie vole (Microtus ochrogaste) to test this because it is spontaneously prosocial. Using siblings of prairie voles used in a prior study that assessed behavioral deficits resulting from developmental FM 550 exposure across 3 doses, here we tested the hypothesis that FM 550 sex-specifically alters AVP and OT neuronal populations in critical nuclei, such as the paraventricular nucleus (PVN), that coordinate those behaviors, as well as related dopaminergic (determined by tyrosine hydroxylase (TH) immunolabeling) populations. Exposed females had fewer AVP neurons in the anterior PVN and more A13 TH neurons in the zona incerta than controls. By contrast, in FM 550 males, A13 TH neuron numbers in the zona incerta were decreased but only in 1 dose group. These results expand on previous work showing evidence of endocrine disruption of OT/AVP pathways, including to subpopulations of PVN AVP neurons that coordinate osmoregulatory functions in the periphery.

Effect of n-3 polyunsaturated fatty acids on dopaminergic neurons in substantia nigra, brain inflammatory response and behavior in mice with Parkinson’s disease

Purpose: To examine the effect of n-3 polyunsaturated fatty acids (PUFAs) on dopaminergic neurons in substantia nigra, intracerebral inflammatory response and ethology in mice with Parkinson’s disease (PD).&#x0D; Methods: Four groups of male C57BL/6 mice (n = 48) were used: normal control, negative control, n3PUFA, and Madopa groups. Except for normal control group, all groups were given 6- hydroxydopamine hydrochloride (6-OHDA) to establish Parkinson’s mice model. The expressions of tyrosine hydroxylase (TH) and calcium-binding protein (CB) in substantia nigra dopaminergic neurons were determined with immunohistochemistry and Western blot. The contents of nitric oxide (NO), tumor necrosis factor (TNF-α) and interferon γ (IFN-γ) (indices of intracerebral inflammatory response) were measured. Tremor paralysis, moving grid number, standing times, swimming ability, and the number of rollers in each group were observed as indices of ethology.&#x0D; Results: The number of TH and CB-positive neurons in the substantia nigra of n-3PUFA-treated mice was significantly increased, relative to those in Madopa-treated mice (p &lt; 0.05). The expressions of TH and CB proteins in substantia nigra in n-3PUFA group were markedly higher than the corresponding expressions in Madopa-treated mice (p &lt; 0.05). Decreased levels of NO, TNF-α and IFN-γ levels were seen in 3PUFA group, when compared to mice in Madopa group, but higher behavioral scores were obtained in n-3PUFA-treated mice, relative to Madopa-treated mice (p &lt; 0.05).&#x0D; Conclusion: The n-3PUFAs protect substantia nigra compact dopaminergic neurons against Parkinson’s disease, alleviate immune inflammation, and improve the coordination of limb movement. Thus, n-3PUFAs have potential therapeutic application in the management of Parkinson’s disease.

(Pro)Renin Receptor in the Hypothalamic Tyrosine Hydroxylase‐Containing Neurons Contributes to High Fat Diet Induced Hyperglycemia

Type 2 diabetes mellitus (T2D) is the major form of human diabetes, accounting for approximately 90–95% of diagnosed diabetes cases in the United States. The brain renin-angiotensin system (RAS), traditionally viewed as a cardiovascular regulatory system, has recently emerged as a critical part of metabolic and energy-expenditure signaling systems in the central nervous system (CNS). The (pro)renin receptor (PRR) is a key component of the brain RAS and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)-induced metabolic physiology is not well understood. We recently showed that neuron-specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the specific neuronal cell types and synaptic mechanisms by which PRR acts is largely unknown. Tyrosine Hydroxylase (TH)-containing neurons are catecholaminergic neurons found in multiple regulatory nuclei throughout the brain. The TH neurons in the paraventricular nucleus (PVN, THPVN) have been previously implicated in metabolic and cardiovascular control via inhibition of brainstem regulatory nuclei. Accordingly, we hypothesize that PRR in THPVN neurons plays a critical role in the regulation of HFD-induced hyperglycemia. To test this hypothesis we injected male PRR-LoxP mice with adeno-associated virus serotype 2 (AAV2)-mediated Cre-recombinase driven by a rat TH promoter (AAV2-TH-Cre) to ablate PRR in the THPVN neurons (THPVN PRRKO) or AAV2-eGFP (Control) prior to 6 week HFD (60% calories from fat) treatment. Fasting blood glucose (FBG), glucose and insulin tolerance tests were performed to assess glucose handling and development of hyperglycemia following HFD. PRR-LoxP mice had significantly higher FBG when compared with the THPVN PRRKO mice (139.3 ± 6.551 vs. 118.0 ±3.715 mg/dl, P= 0.0409) and trended towards improved glucose tolerance when compared with the control mice following the treatment period (Area under the curve-AUC: 12990 ± 1494 vs. 9035 ± 1354, P=0.0782), suggesting that THPVN PRRKO protects against hyperglycemia and may improve glucose tolerance after 6 weeks HFD treatment. No significant difference between the two groups was observed in the insulin tolerance test (AUC: 10833 ± 727.7 vs. 10135 ± 729.5, P=0.7142), suggesting a possible mechanism of THPVN PRR glycemia regulation independent of insulin resistance. These data indicate a critical role of THPVN PRR in the regulation of HFD-induced hyperglycemia and a potential pathway for understanding the mechanism behind the metabolic action of neuronal PRR.

Characterization of Tyrosine Hydroxylase‐Positive Neurons Projections from the Paraventricular Nucleus of Hypothalamus to Hindbrain Autonomic Regulatory Centers

Hypothalamus is a key brain region for cardiovascular, behavioral, and metabolic regulation that contains complex type of excitatory or inhibitory neurons. Tyrosine hydroxylase (TH)-positive neurons in the hypothalamus play an important role in cardiometabolic regulation; however, the anatomical projections of TH neurons in the paraventricular nucleus hypothalamus (PVN) to key parasympathetic and sympathetic premotor nuclei in preautonomic regions of the brain and their connections to the liver remains undefined. This study aims to characterize the projections from THPVN neurons to ventral preautonomic regulating centers and their connection to liver neuronal projections. To achieve our goal, Cre recombinase-dependent TIT2L-eGFP reporter mice were microinjected with adeno-associated virus encoding the Cre recombinase driven by a rat TH promoter (AAV2-TH-Cre, 6.8 x 108 vg/site) bilaterally into the PVN using a stereotaxic apparatus. A retrograde pseudorabies virus with red fluorescence reporter (PRV-614-RFP) was injected into the liver (3 sites, 3ul/site) to identify liver-related neurons in the brainstem preautonotmic centers. One month after injection, mice were transcardially perfused and brain tissues were processed for confocal imaging. The AAV2-TH-Cre induced a robust expression of GFP mostly in the PVN neuronal cell bodies but not in other brain regions. In the brainstem preautonomic centers, we found dense projections in the nucleus tractus solitarius (NTS) and some projections to the dorsal motor nucleus of the vagus (DMV) and rostral ventrolateral medulla (RVLM). Interestingly, there was also dense projects into the median eminence (ME) that regulates neural hormones release. Importantly, the PRV-RFP retrograded from liver was found throughout the DMV and ventrolateral NTS . Particularly in the NTS, there was dense synaptic contact of the THPVN neuronal projections with the liver related neurons (PRV-RFP+ cell). In addition, we also observed that some PRV-RFP+ cells colocalized with TH-positive neurons in the PVN. In sum, the THPVN neurons project anatomically to the autonomic and neurosecretory regions of brain and may modulate the cardiometabolic activity and liver metabolism through these projections.