A2 Neurons Research Articles

A linearized modeling framework for the frequency selectivity in neurons postsynaptic to vibration receptors.

Vibration is an indispensable part of the tactile perception, which is encoded to oscillatory synaptic currents by receptors and transferred to neurons in the brain. The A2 and B1 neurons in the drosophila brain postsynaptic to the vibration receptors exhibit selective preferences for oscillatory synaptic currents with different frequencies, which is caused by the specific voltage-gated Na+ and K+ currents that both oppose the variations in membrane potential. To understand the peculiar role of the Na+ and K+ currents in shaping the filtering property of A2 and B1 neurons, we develop a linearized modeling framework that allows to systematically change the activation properties of these ionic channels. A data-driven conductance-based biophysical model is used to reproduce the frequency filtering of oscillatory synaptic inputs. Then, this data-driven model is linearized at the resting potential and its frequency response is calculated based on the transfer function, which is described by the magnitude-frequency curve. When we regulate the activation properties of the Na+ and K+ channels by changing the biophysical parameters, the dominant pole of the transfer function is found to be highly correlated with the fluctuation of the active current, which represents the strength of suppression of slow voltage variation. Meanwhile, the dominant pole also shapes the magnitude-frequency curve and further qualitatively determines the filtering property of the model. The transfer function provides a parsimonious description of how the biophysical parameters in Na+ and K+ channels change the inhibition of slow variations in membrane potential by Na+ and K+ currents, and further illustrates the relationship between the filtering properties and the activation properties of Na+ and K+ channels. This computational framework with the data-driven conductance-based biophysical model and its linearized model contributes to understanding the transmission and filtering of vibration stimulus in the tactile system.

Distinct nonlinear spectrotemporal integration in primary and secondary auditory cortices

Animals sense sounds through hierarchical neural pathways that ultimately reach higher-order cortices to extract complex acoustic features, such as vocalizations. Elucidating how spectrotemporal integration varies along the hierarchy from primary to higher-order auditory cortices is a crucial step in understanding this elaborate sensory computation. Here we used two-photon calcium imaging and two-tone stimuli with various frequency-timing combinations to compare spectrotemporal integration between primary (A1) and secondary (A2) auditory cortices in mice. Individual neurons showed mixed supralinear and sublinear integration in a frequency-timing combination-specific manner, and we found unique integration patterns in these two areas. Temporally asymmetric spectrotemporal integration in A1 neurons suggested their roles in discriminating frequency-modulated sweep directions. In contrast, temporally symmetric and coincidence-preferring integration in A2 neurons made them ideal spectral integrators of concurrent multifrequency sounds. Moreover, the ensemble neural activity in A2 was sensitive to two-tone timings, and coincident two-tones evoked distinct ensemble activity patterns from the linear sum of component tones. Together, these results demonstrate distinct roles of A1 and A2 in encoding complex acoustic features, potentially suggesting parallel rather than sequential information extraction between these regions.

Noradrenergic Input from Nucleus of the Solitary Tract Regulates Parabrachial Activity in Mice.

The parabrachial complex (PB) is critically involved in aversive processes, and chronic pain is associated with amplified activity of PB neurons in rodent models of neuropathic pain. Here, we demonstrate that catecholaminergic input from the caudal nucleus of the solitary tract (cNTScat), a stress responsive region that integrates interoceptive and exteroceptive signals, causes amplification of PB activity and their sensory afferents. We used a virally mediated expression of a norepinephrine (NE) sensor, NE2h, fiber photometry, and extracellular recordings in anesthetized mice to show that noxious mechanical and thermal stimuli activate cNTS neurons. These stimuli also produce prolonged NE transients in PB that far outlast the noxious stimuli. Similar NE transients can be evoked by focal electrical stimulation of cNTS, a region that contains the noradrenergic A2 cell group that projects densely on PB. In vitro, optical stimulation of cNTScat terminals depolarized PB neurons and caused a prolonged increase the frequency of excitatory synaptic activity. A dual opsin approach showed that sensory afferents from the caudal spinal trigeminal nucleus are potentiated by cNTScat terminal activation. This potentiation was coupled with a decrease in the paired pulse ratio (PPR), consistent with an cNTScat-mediated increase in the probability of release at SpVc synapses. Together, these data suggest that A2 neurons of the cNTS generate long lasting NE transients in PB which increase excitability and potentiate responses of PB neurons to sensory inputs. These reveal a mechanism through which stressors from multiple modalities may potentiate the aversiveness of nociceptive stimuli.

Nucleus of the solitary tract A2 neurons control feeding behaviors via projections to the paraventricular hypothalamus

Hindbrain NTS neurons are highly attuned to internal physiological and external environmental factors that contribute to the control of food intake but the relevant neural phenotypes and pathways remain elusive. Here, we investigated the role of NTS A2 neurons and their projections in the control of feeding behaviors. In male TH Cre rats, we first confirmed selective targeting of NTS A2 neurons and showed that chemogenetic stimulation of these neurons significantly suppressed dark cycle food intake, deprivation re-feed and high fat diet intake. Despite reducing intake, activation of NTS A2 neurons had no effect on food approach, anxiety-like behaviors, locomotor activity, blood glucose levels nor did it induce nausea/malaise, thus revealing a selective role for these neurons in the consummatory aspect of food intake control. Pathway-specific mapping and manipulation of NTS A2 neurons showed that these effects were mediated by NTS A2 neurons projecting to the paraventricular nucleus of the hypothalamus (PVH) because chemogenetic activation of these projections, but not projections to bed nucleus of the stria terminalis (BNST), reduced food intake. Cell-type specific analyses demonstrated that activation of NTS A2 neurons recruited both PVH oxytocin (OT)- and corticotropin-releasing factor (CRF)-expressing neurons, and plasma analyses showed increased plasma corticosterone following NTS A2 stimulation. While we also showed that chemogenetic inhibition of NTS A2 neurons attenuated the intake inhibitory effects of CCK, the specificity of transgene expression was low. Together, these findings showed that NTS A2 neurons are sufficient to control the consummatory aspects of feeding, regardless of energy status or food palatability and identified their projections to PVH, but not BNST, in food intake control.

Effects of short-term food deprivation on catecholamine and metabolic-sensory biomarker gene expression in hindbrain A2 noradrenergic neurons projecting to the forebrain rostral preoptic area: Impact of negative versus positive estradiol feedback.

Hindbrain A2 noradrenergic neurons assimilate estrogenic and metabolic cues. In female mammals, negative- versus positive-feedback patterns of estradiol (E) secretion impose divergent regulation of the gonadotropin-releasing hormone (GnRH)-pituitary-gonadal (HPG) neuroendocrine axis. Current research used retrograde tracing, dual-label immunocytochemistry, single-cell laser-microdissection, and multiplex qPCR methods to address the premise that E feedback modes uniquely affect metabolic regulation of A2 neurons involved in HPG control. Ovariectomized female rats were given E replacement to replicate plasma hormone levels characteristic of positive (high-E dose) or negative (low-E dose) feedback. Animals were either full-fed (FF) or subjected to short-term, e.g., 18-h food deprivation (FD). After FF or FD, rostral preoptic area (rPO)-projecting A2 neurons were characterized by the presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunostaining. FD augmented or suppressed mRNAs encoding the catecholamine enzyme dopamine-beta-hydroxylase (DβH) and the metabolic-sensory biomarker glucokinase (GCK), relative to FF controls, in nGKRP-immunoreactive (ir)-positive A2 neurons from low-E or high-E animals, respectively. Yet, these transcript profiles were unaffected by FD in nGKRP-ir-negative A2 neurons at either E dosage level. FD altered estrogen receptor (ER)-alpha and ATP-sensitive potassium channel subunit sulfonylurea receptor-1 gene expression in nGKRP-ir-positive neurons from low-E, but not high-E animals. Results provide novel evidence that distinct hindbrain A2 neuron populations exhibit altered versus unaffected transmission to the rPO during FD-associated metabolic imbalance, and that the direction of change in this noradrenergic input is controlled by E feedback mode. These A2 cell types are correspondingly distinguished by FD-sensitive or -insensitive GCK, which correlates with the presence versus absence of nGKRP-ir. Further studies are needed to determine how E signal volume regulates neurotransmitter and metabolic sensor responses to FD in GKRP-expressing A2 neurons.

Hindbrain lactate regulation of hypoglycemia-associated patterns of catecholamine and metabolic-sensory biomarker gene expression in A2 noradrenergic neurons innervating the male versus female ventromedial hypothalamic nucleus

Caudal hindbrain A2 noradrenergic neurons provide critical metabolic-sensory input to the brain glucostatic circuitry. In males, insulin-induced hypoglycemia (IIH)-associated patterns of A2 cell dopamine-beta-hydroxylase (DβH) protein expression reflect diminution of the oxidizable fuel L-lactate, yet DβH exhibits sex-dimorphic responses to IIH. Here, retrograde tracing and combinatory single-cell laser-microdissection/multiplex qPCR techniques were used to examine whether lactate imposes sex-specific control of hypoglycemia-associated metabolic-sensory function and noradrenergic neurotransmission in A2 neurons that innervate the ventromedial hypothalamic nucleus (VMN), a key glucose-regulatory structure. VMN-projecting A2 neurons from each sex were characterized by presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunoreactivity (-ir). IIH caused lactate-reversible up- or down-regulation of DβH mRNA in male and female nGKRP-ir-positive A2 neurons, respectively, and stimulated glucokinase (GCK) and sulfonylurea receptor-1 (SUR-1) gene expression in these cells in each sex. Hypoglycemia did not alter DβH, GCK, and SUR-1 transcript profiles in nGKRP-ir-negative male or female A2 neurons innervating the VMN. Estrogen receptor (ER) gene profiles in nGKRP-ir-positive neurons showed sex-specific [ER-alpha; G-protein-coupled estrogen-receptor-1 (GPER)] or sex-monomorphic (ER-beta) transcriptional responses to IIH. Fewer ER gene profiles were affected by IIH in nGKRP-ir-negative A2 neurons from male or female rats. Results show that during IIH, VMN-projecting A2 neurons may deliver altered, sex-dependent (nGKRP-positive) or unaffected (nGKRP-negative) noradrenergic input to the VMN. In each sex, metabolic-sensory gene profiles were reactive to hypoglycemia in nGKRP-ir-positive, not -negative A2 cells. Further studies are needed to elucidate the role of GKRP in transduction of metabolic imbalance into noradrenergic signaling, and to determine if input by one or more ER variants establishes sex differences in DβH transcriptional sensitivity to IIH.

Acute stress anticipates and amplifies the luteinizing hormone pre-ovulatory surge in rats: Role of noradrenergic neurons

The stress experienced during rape seems to facilitate ovulation since the pregnancy rate in raped women is higher than that resulting from consensual intercourse. Adrenal progesterone, as well as central norepinephrine, is released in stressful situations. At adequate estrogenic levels, one of the main actions of progesterone is to anticipate the preovulatory LH surge through noradrenaline release. We aimed to investigate whether acute stresses that mimic those of rape (exposure to predator, restraint and cervix stimulation) applied on the proestrus morning in female rats could release progesterone, activate the noradrenergic neurons and facilitate the occurrence of the LH surge. Female rats were submitted to jugular vein cannulation immediately following acute stress: restraint (R), exposure to cat (P), uterine cervix stimulation (CS) applied individually or in association (SA). Non-stressed rats were used as control. Blood samples were collected from 11:00–18:00 h for LH, progesterone, corticosterone and estradiol measurements. Double labeling for c-Fos and tyrosine hydroxylase (TH) was examined in A1, A2 and A6 noradrenergic neurons after stresses. The SA group showed a greater stress-induced increase in progesterone compared to the other groups and the preovulatory LH surge was anticipated and amplified. This effect of SA seems to be related to the higher number of c-Fos/TH + neurons in the A1 and A2. The effect of anticipating the preovulatory surge of LH could in part elucidate why, in raped women, conception can occur in phases of the menstrual cycle other than the ovulatory phase facilitating the occurrence of pregnancies.

Communication sound recognition and response modification in the secondary auditory cortex of female mice

To study the response characteristics of the secondary auditory cortex (A2) to wriggling calls (WC) and the mechanism of response modulation in female mice. We used patch-clamp and immunofluorescence labeling technique to mark and record the action potential and cell type of A2 neurons. Female C57 mice were stimulated with pure tone and white noise (control), 4.5 kHz, 9 kHz, or 13.5 kHz sound waves extracted from WC (single-frequency simulation group), the combinations of every two of the 3 single-frequency sound waves (two-tone frequency simulation group), or the combinations of 4.5 kHz+7.7 kHz+13.5 kHz and 4.5 kHz+9 kHz+13.5 kHz sound waves (three-frequency simulation group). The firing pattern, firing number, threshold, and latency of the action potential of the A2 neurons were recorded in response to the stimulations. By comparison of the spikes elicited by different sound stimulations, we identified 3 types of neurons with different sensitivities to WC. The WC-sensitive neurons had a significantly greater number of spikes in response to WC than to other sounds and noise (P < 0.001). Comparison of the latency and threshold revealed significantly longer latencies of the WC-sensitive neurons and WC-insensitive neurons in response to WC stimulation than to pure tone stimulation (P=0.002), but their latencies to WC and noise stimulation were similar (P=0.093). The WC-sensitive neurons also had lower threshold to WC than to pure tone and noise stimulations (P=0.02). Analysis of the firing patterns of action potentials showed that the WCsensitive neurons consisted mainly of parvalbumin interneurons. The results of immunofluorescence labeling indicated that inhibitory interneurons were present in the A2 neurons that responded to WC. A2 contains 3 types of neurons with different sensitivities to WC. Among them, the WC-sensitive neurons is mainly PV neurons, whose response characteristics to different types of sounds can help to explain the mechanism of communication sound recognition and response modification in A2.

Activation of Norepinephrine Neurons in the NTS (A2 population) is Sufficient to Suppress Pulsatile LH Secretion in Female Mice

The overarching goal of this work is to identify neural pathways underlying inhibition of pulsatile luteinizing hormone (LH) secretion during stress. Stress-induced suppression of LH secretion is mediated, at least in part, by suppression of arcuate kisspeptin (ARCKiss1) neurons. The mechanisms by which acute stress suppresses ARCKiss1 cell activity are largely unknown; however, several lines of evidence support the hypothesis that A2 neurons (norepinephrine [NE] neurons in the nucleus of the solitary tract [NTS] of the brainstem) are involved. First, A2 cells are activated during several reactive stress paradigms. Second, NE administered into the paraventricular nucleus, which is innervated by A2 neurons, suppressed pulsatile LH secretion. Finally, ablation of brainstem NE neurons restored estrous cyclicity following chronic glucoprivation (chronic metabolic stress model). The present study employed chemogenetics to test the hypothesis that A2 neurons are sufficient to suppress pulsatile LH secretion in ovariectomized female dopamine beta-hydroxylase (DBH) Cre positive and negative (wild type) mice. Mice received bilateral injections of either a Cre-dependent stimulatory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) virus (AAV1-DIO-hM3Dq-mCherry) or a control virus (AAV1-DIO-mCherry) into the NTS. Mice were randomly assigned to receive either clozapine N-oxide (CNO, specific DREADD agonist; 1mg/kg, i.p.) or saline and blood samples were collected at 6-min intervals for 60 min before and 90 min after injection. Two weeks later, mice received the alternate treatment in a cross-over design (n= 5-10/grp). During the pre-injection period, all mice had clear LH pulses (mean: 6.0 ± 0.2 ng/mL, pulses/60 min: 3.4 ± 1.5). In DBH Cre- (wild type) mice with hM3D virus and DBH Cre+ with mCherry virus (both control groups), neither CNO nor saline altered mean LH or LH pulse frequency. However, DBH Cre+ mice with hM3D virus had a 54% reduction in mean LH (p < 0.05) and 59% reduction in pulse frequency (p < 0.05) following CNO; neither LH metric was altered in response to saline. To assess transduction efficiency, fixed neural tissue was collected. In tissue analyzed thus far, DBH Cre+ mice have mCherry labeling in ~70% of DBH-immunoreactive neurons in the NTS and >90% of mCherry neurons contained DBH immunoreactivity. Three DBH Cre+ mice with hM3D virus mice had no LH response to CNO and may represent missed viral injections, which will be determined when tissue is analyzed. These data demonstrate that activation of A2 neurons is sufficient to impair pulsatile LH secretion in female mice. Moreover, these data support the broad hypothesis that the A2 population of neurons is critical for modulating neuroendocrine function during stress and raises the possibility that A2 neurons directly or indirectly influence ARCKiss1 cell activity.

Medullary Noradrenergic Neurons Mediate Hemodynamic Responses to Osmotic and Volume Challenges.

Despite being involved in homeostatic control and hydro-electrolyte balance, the contribution of medullary (A1 and A2) noradrenergic neurons to the hypertonic saline infusion (HSI)-induced cardiovascular response after hypotensive hemorrhage (HH) remains to be clarified. Hence, the present study sought to determine the role of noradrenergic neurons in HSI-induced hemodynamic recovery in male Wistar rats (290–320 g) with HH. Medullary catecholaminergic neurons were lesioned by nanoinjection of antidopamine-β-hydroxylase–saporin (0.105 ng·nl−1) into A1, A2, or both (LES A1; LES A2; or LES A1+A2, respectively). Sham rats received nanoinjections of free saporin in the same regions (SHAM A1; SHAM A2; or SHAM A1+A2, respectively). After 15 days, rats were anesthetized and instrumented for cardiovascular recordings. Following 10 min of stabilization, HH was performed by withdrawing arterial blood until mean arterial pressure (MAP) reaches 60 mmHg. Subsequently, HSI was performed (NaCl 3 M; 1.8 ml·kg−1, i.v.). The HH procedure caused hypotension and bradycardia and reduced renal, aortic, and hind limb blood flows (RBF, ABF, and HBF). The HSI restored MAP, heart rate (HR), and RBF to baseline values in the SHAM, LES A1, and LES A2 groups. However, concomitant A1 and A2 lesions impaired this recovery, as demonstrated by the abolishment of MAP, RBF, and ABF responses. Although lesioning of only a group of neurons (A1 or A2) was unable to prevent HSI-induced recovery of cardiovascular parameters after hemorrhage, lesions of both A1 and A2 made this response unfeasible. These findings show that together the A1 and A2 neurons are essential to HSI-induced cardiovascular recovery in hypovolemia. By implication, simultaneous A1 and A2 dysfunctions could impair the efficacy of HSI-induced recovery during hemorrhage.

Sex-specific acclimation of A2 noradrenergic neuron dopamine-β-hydroxylase and estrogen receptor variant protein and 5’-AMP-Activated protein kinase reactivity to recurring hypoglycemia in rat

Hindbrain estrogen receptors (ER) impose sex-dimorphic control of counter-regulatory hormone and hypothalamic glucoregulatory transmitter and glycogen metabolic responses to hypoglycemia. A2 noradrenergic neurons are estradiol- and metabolic-sensitive. Estradiol controls dopamine-beta-hydroxylase (DBH) protein habituation to recurrent insulin-induced hypoglycemia (RIIH) in females. Current research investigated the premise that sex-dimorphic patterns of A2 ER variant acclimation to RIIH correlate with differential A2 DBH and 5’-AMP-activated protein kinase (AMPK) adaptation to RIIH. A2 neurons were laser-catapult-microdissected from male and female rats after one or four insulin injections for Western blot analysis. A2 pAMPK and DBH levels were increased in males, but suppressed in females after single insulin dosing. ER-alpha (ERα) and -beta (ERβ) protein profiles were unaffected or decreased by acute hypoglycemia in each sex, whereas G protein-linked ER-1 (GPER) reactivity varied by sex. Antecedent hypoglycemia diminished basal A2 ERα/GPER and elevated ERβ content in each sex, yet reduced pAMPK and DBH levels in female rats only. Reintroduced hypoglycemia suppressed A2 ERβ levels in each sex, but altered DBH (↓), ERα (↓), and GPER (↑) levels in males only. Data document sex differences in A2 DBH adaptation to RIIH, e.g. a shift from positive-to-negative response in males versus loss of negative reactivity in females, as well as attenuated AMPK activation in both sexes. Between hypoglycemic episodes, A2 neurons in each sex likely exhibit diminished sensitivity to ERα/GPER signaling, but heightened receptivity to ERβ input. RIIH-induced changes in ERα and GPER expression in male but not female may contribute to DBH suppression (males) versus no change (females) relative to adapted baseline expression.

Impact of caudal hindbrain glycogen metabolism on A2 noradrenergic neuron AMPK activation and ventromedial hypothalamic nucleus norepinephrine activity and glucoregulatory neurotransmitter marker protein expression

The brain glycogen reserve is a source of oxidizable substrate fuel. Lactoprivic-sensitive hindbrain A2 noradrenergic neurons provide crucial metabolic-sensory input to downstream hypothalamic glucose-regulatory structures. Current research examined whether hindbrain glycogen fuel supply impacts A2 energy stability and governance of ventromedial hypothalamic nucleus (VMN) metabolic transmitter signaling. Male rats were injected into the caudal fourth ventricle (CV4) with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prior to continuous intra-CV4 infusion of L-lactate or vehicle. Lactate reversed DAB suppression of A2 neuron AMPK protein and up-regulated phosphoAMPK profiles. A2 dopamine-β-hydroxylase expression was refractory to DAB, but elevated by DAB/lactate. Lactate normalized A2 estrogen receptor-alpha and GPER proteins and up-regulated estrogen receptor-beta levels in DAB-treated rats. VMN norepinephrine content was decreased by DAB, but partially restored by lactate. DAB caused lactate-reversible or -irreversible augmentation of VMN glycogen phosphorylase-brain (GPbb) and -muscle type (GPmm) variant profiles, and correspondingly up- or down-regulated VMN protein markers of glucose-stimulatory nitrergic and glucose-inhibitory γ-aminobutyric acid transmission. DAB did not alter plasma glucose, but suppressed or elevated circulating glucagon and corticosterone in that order. Results show that diminished hindbrain glycogen breakdown is communicated to the VMN, in part by NE signaling, to up-regulate VMN glycogen breakdown and trigger neurochemical signaling of energy imbalance in that site. DAB effects on GPmm, VMN glycogen content, and counter-regulatory hormone secretion were unabated by lactate infusion, suggesting that aside from substrate fuel provision rate, additional indicators of glycogen metabolism such as turnover rate may be monitored in the hindbrain.

Ghrelin signaling contributes to fasting-induced attenuation of hindbrain neural activation and hypophagic responses to systemic cholecystokinin in rats.

In rats, overnight fasting reduces the ability of systemic cholecystokinin-8 (CCK) to suppress food intake and to activate cFos in the caudal nucleus of the solitary tract (cNTS), specifically within glucagon-like peptide-1 (GLP-1) and noradrenergic (NA) neurons of the A2 cell group. Systemic CCK increases vagal sensory signaling to the cNTS, an effect that is amplified by leptin and reduced by ghrelin. Since fasting reduces plasma leptin and increases plasma ghrelin levels, we hypothesized that peripheral leptin administration and/or antagonism of ghrelin receptors in fasted rats would rescue the ability of CCK to activate GLP-1 neurons and a caudal subset of A2 neurons that coexpress prolactin-releasing peptide (PrRP). To test this, cFos expression was examined in ad libitum-fed and overnight food-deprived (DEP) rats after intraperitoneal CCK, after coadministration of leptin and CCK, or after intraperitoneal injection of a ghrelin receptor antagonist (GRA) before CCK. In fed rats, CCK activated cFos in ~60% of GLP-1 and PrRP neurons. Few or no GLP-1 or PrRP neurons expressed cFos in DEP rats treated with CCK alone, CCK combined with leptin, or GRA alone. However, GRA pretreatment increased the ability of CCK to activate GLP-1 and PrRP neurons and also enhanced the hypophagic effect of CCK in DEP rats. Considered together, these new findings suggest that reduced behavioral sensitivity to CCK in fasted rats is at least partially due to ghrelin-mediated suppression of hindbrain GLP-1 and PrRP neural responsiveness to CCK.

GLP-1 neurons form a local synaptic circuit within the rodent nucleus of the solitary tract.

Glutamatergic neurons that express pre‐proglucagon (PPG) and are immunopositive (+) for glucagon‐like peptide‐1 (i.e., GLP‐1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP‐1 neurons give rise to an extensive central network in which GLP‐1 receptor (GLP‐1R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via GLP‐1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP‐1R, whereas PPG neurons do not. In this study, confocal microscopy in rats confirmed that prolactin‐releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP‐1+ axonal varicosities. Surprisingly, GLP‐1+ appositions were also observed on dendrites of PPG/GLP‐1+ neurons in both species, and electron microscopy in rats revealed that GLP‐1+ boutons form asymmetric synaptic contacts with GLP‐1+ dendrites. However, RNAscope confirmed that rat GLP‐1 neurons do not express GLP‐1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP‐1, and a mouse crossbreeding strategy revealed that <1% of PPG neurons co‐express GLP‐1R. Collectively, these data suggest that GLP‐1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local “feed‐forward” synaptic network among GLP‐1 neurons that apparently does not use GLP‐1R signaling. This local GLP‐1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP‐1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Involvement of median preoptic nucleus and medullary noradrenergic neurons in cardiovascular and sympathetic responses of hemorrhagic rats

The infusion of hypertonic saline solution (HSS) is known to be beneficial to the treatment of hypovolemic hemorrhage (HH). The central mechanism of HSS-induced cardiovascular and autonomic recovery of animals subjected to HH remains unclear. Hence, the present study evaluated the involvement of median preoptic nucleus (MnPO) and medullary noradrenergic neurons (A1 and A2) in HSS-induced cardiovascular and sympathetic responses in hemorrhagic rats. The wistar rats were subjected to specific lesion of noradrenergic neurons through the nanoinjections of anti-DβH-saporin into caudal ventrolateral medulla (A1 neurons) and nucleus of the solitary tract (A2 neurons). After recovery, mean arterial pressure (MAP) and renal sympathetic nervous activity were recorded. The HH was performed through blood withdrawal until a MAP of 60 mmHg was attained. In sham rats, HSS infusion (3M NaCl) reestablished MAP without change in HH-induced sympathoinhibition. The muscimol (agonist of GABAA receptor) was nanoinjected in MnPO during HH and MnPO inhibition abolished the recovery of MAP and HSS-induced sympathoinhibition. Simultaneous lesions of A1 and A2 abolished MAP restoration and sympathoinhibition after HSS infusion. These results suggest that the recovery of MAP and HSS-induced sympathoinhibition in hemorrhaged rats depend on intact neural projections from A1 and A2 to MnPO.

Activation of catecholamine neurons in the ventral medulla reduces CCK-induced hypophagia and c-Fos activation in dorsal medullary catecholamine neurons.

Catecholamine (CA) neurons within the A1 and C1 cell groups in the ventrolateral medulla (VLM) potently increase food intake when activated by glucose deficit. In contrast, CA neurons in the A2 cell group of the dorsomedial medulla are required for reduction of food intake by cholecystokinin (CCK), a peptide that promotes satiation. Thus dorsal and ventral medullary CA neurons are activated by divergent metabolic conditions and mediate opposing behavioral responses. Acute glucose deficit is a life-threatening condition, and increased feeding is a key response that facilitates survival of this emergency. Thus, during glucose deficit, responses to satiation signals, like CCK, must be suppressed to ensure glucorestoration. Here we test the hypothesis that activation of VLM CA neurons inhibits dorsomedial CA neurons that participate in satiation. We found that glucose deficit produced by the antiglycolytic glucose analog, 2-deoxy-d-glucose, attenuated reduction of food intake by CCK. Moreover, glucose deficit increased c-Fos expression by A1 and C1 neurons while reducing CCK-induced c-Fos expression in A2 neurons. We also selectively activated A1/C1 neurons in TH-Cre+ transgenic rats in which A1/C1 neurons were transfected with a Cre-dependent designer receptor exclusively activated by a designer drug (DREADD). Selective activation of A1/C1 neurons using the DREADD agonist, clozapine- N-oxide, attenuated reduction of food intake by CCK and prevented CCK-induced c-Fos expression in A2 CA neurons, even under normoglycemic conditions. Results support the hypothesis that activation of ventral CA neurons attenuates satiety by inhibiting dorsal medullary A2 CA neurons. This mechanism may ensure that satiation does not terminate feeding before restoration of normoglycemia.

Vagally mediated gastric effects of brain stem α2-adrenoceptor activation in stressed rats.

Chronic stress exerts vagally dependent effects to disrupt gastric motility; previous studies have shown that, among other nuclei, A2 neurons are involved in mediating these effects. Several studies have also shown robust in vitro and in vivo effects of α2-adrenoceptor agonists on vagal motoneurons. We have demonstrated previously that brainstem vagal neurocircuits undergo remodeling following acute stress; however, the effects following brief periods of chronic stress have not been investigated. Our aim, therefore, was to test the hypothesis that different types of chronic stress influence gastric tone and motility by inducing plasticity in the response of vagal neurocircuits to α2-adrenoreceptor agonists. In rats that underwent 5 days of either homotypic or heterotypic stress loading, we applied the α2-adrenoceptor agonist, UK14304, either by in vitro brainstem perfusion to examine its ability to modulate GABAergic synaptic inputs to vagal motoneurons or in vivo brainstem microinjection to observe actions to modulate antral tone and motility. In neurons from naïve rats, GABAergic currents were unresponsive to exogenous application of UK14304. In contrast, GABAergic currents were inhibited by UK14304 in all neurons from homotypic and, in a subpopulation of neurons, heterotypic stressed rats. In control rats, UK14304 microinjection inhibited gastric tone and motility via withdrawal of vagal cholinergic tone; in heterotypic stressed rats, the larger inhibition of antrum tone was due to a concomitant activation of peripheral nonadrenergic, noncholinergic pathways. These data suggest that stress induces plasticity in brainstem vagal neurocircuits, leading to an upregulation of α2-mediated responses. NEW & NOTEWORTHY Catecholaminergic neurons of the A2 area play a relevant role in stress-related dysfunction of the gastric antrum. Brief periods of chronic stress load induce plastic changes in the actions of adrenoceptors on vagal brainstem neurocircuits.

Impact of recurrent hypoglycemic stress on hindbrain A2 nerve cell energy metabolism and catecholamine biosynthesis: modulation by estradiol.

It is unclear if habituation of hindbrain A2 metabolo‑sensory neurons to recurrent insulin-induced hypoglycemia (RIIH) correlates with estradiol-dependent adjustments in energy metabolism that favor positive energy balance. Laser-microdissected A2 cells from estradiolor oil-implanted ovariectomized female rats were analyzed by Western blot to assess effects of three prior daily insulin injections on basal and hypoglycemic patterns of catecholamine biosynthetic enzyme dopamine-beta-hydroxylase (DβH) and rate-limiting energy pathway enzyme protein expression. Precedent hypoglycemia respectively decreased or increased baseline DβH expression in estradiol- (E) vs. oil (O)-treated rats; this protein profile was further suppressed or augmented in those animals at 2 hr after re-induction of hypoglycemia. These data suggest that estradiol may curtail A2 noradrenergic‑controlled functions both in the midst of and between hypoglycemic bouts. Results also show that prior hypoglycemia exposure upregulated A2 neuron glycolytic enzyme protein levels when E was present, and exerted differential effects on basal and hypoglycemia-associated respiratory chain and fatty acid synthetic pathway enzyme expression. E may thus accordingly amplify glycolysis-derived metabolites/energy, coupled with reduced reliance on oxidative phosphorylation, and activate the fatty acid synthetic pathway during RIIH. E may also be of benefit by preventing maladaptive reductions in A2 neuron Krebs cycle/electron transport enzyme expression during re-exposure to hypoglycemia. Augmentation of negative energy balance during this recurring metabolic stress in the absence of E is a likely impetus for augmented vs. decreased A2 signaling of energy imbalance by DβH in O vs. E rats during RIIH.

Cardiovascular dysfunction associated with neurodegeneration in an experimental model of Parkinson’s disease

Patients with Parkinson’s disease (PD) exhibit both motor and non-motor symptoms. Among the non-motor symptoms, cardiovascular autonomic dysfunction is frequently observed. Here, we evaluated baroreflex function, vascular reactivity and neuroanatomical changes in brainstem regions involved in the neural control of circulation in the 6-hydroxydopamine (6-OHDA) model of PD. Male Wistar rats received a bilateral injection of 6-OHDA or vehicle into the striatum. After 61days, baroreflex function and vascular reactivity were assessed. The 6-OHDA and vehicle groups showed similar increases in mean arterial pressure (MAP) in response to phenylephrine (PE). However, the bradycardia observed in the vehicle group was blunted in the 6-OHDA-treated rats. Injection of sodium nitroprusside (SNP) decreased hypotension, tachycardia and vascular relaxation in 6-OHDA-treated rats. Bilateral intrastriatal 6-OHDA led to massive degeneration of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra and to reductions in the numbers of A1/C1 and A5 catecholaminergic neurons while sparing A2 neurons within the nucleus of the solitary tract (NTS). 6-OHDA-treated rats also showed decreases in Phox2b-expressing neurons in the NTS and in choline acetyltransferase (ChAT) immunoreactivity in the nucleus ambiguus. Altogether, our data suggest that this model of PD includes neuroanatomical and functional changes that lead to cardiovascular impairment.

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the absence vs. presence of E. Mechanisms underlying translation of E-contingent A2 neuron responses to FD into regulatory signaling remain to be determined. © 2016 Wiley Periodicals, Inc.