Central Noradrenergic Neurons Research Articles

Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for adult breathing

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders such as Sudden Infant Death Syndrome (SIDS) and Rett Syndrome. Previous studies suggest that glutamate is co-transmitted in subsets of NA neurons and may play a role in breathing control. However, the extent of glutamate co-transmission in central NA system remains uncharacterized. Furthermore, no direct evidence exists that NA-derived glutamate is required for respiratory homeostasis. Thus, our aim is to fully characterize the expression profiles of all three Vesicular Glutamate Transporters (Vglut1, Vglut2, and Vglut3) in NA neurons and to determine if Vglut2-based glutamatergic signaling in NA neurons is required for adult respiratory homeostasis. We utilized intersectional genetics to characterize the cumulative fate maps of Vglut1/2/3 co-expressing NA neurons. For each Vglut1/2/3 transporter , a corresponding Cre knock-in recombinase line was combined with DBH-p2a-Flpo mice . These Vglut1, 2, or 3-Cre; DBH-p2a-Flpo compound lines were crossed with the RC::F_TdTomato_P_GFP intersectional reporter mice. In each of the three intersectional reporter crosses, NA neurons co-expressing either Vglut1, Vglut2, or Vglut3 are labeled by green fluorescent protein (GFP) while NA neurons without any Vglut1, 2, or 3 expression are labelled by red fluorescent protein (tdTomato). We found 84.6 ± 3.75% of NA neurons express Vglut2 and 26.9 ± 3.16% posterior medullary NA neurons express Vglut3 over the lifetime of the mouse but no Vglut1 expression was observed (n=3).Functionally, to determine the requirement of NA-based Vglut2 signaling in adult breathing, we conditionally ablated Vglut2 in central NA neurons by crossing DBH-Cre/+ with Vglut2flox/flox mice. Mutants and sibling controls (6-8 weeks, n=16 per group) were assessed for respiratory function by whole-body barometric plethysmography. Ventilation and VO2 were assessed under room air (21%O2,79%N2), hypercapnia (21%O2, 74/72/69%N2, 5/7/10%CO2) and hypoxia (10%O2, 90%N2). Surprisingly, Vglut2 removal from NA neurons failed to significantly alter breathing under room air, hypercapnia and hypoxia. (A linear mixed-effects regression model was used to compare each respiratory parameter between mutants and controls.)In conclusion, anatomically, our study confirmed Vglut2 co-expression in NA neurons and identified a novel posterior NA population co-expressing Vglut3. Functionally, our data demonstrates that Vglut2-based glutamatergic signaling within the central NA system is not required for normal baseline breathing and hypercapnic, and hypoxic chemosensory reflexes, in contrast to prior studies. Our work suggests that glutamate may be not a critical target to understand NA neuron dysfunction in respiratory diseases. R01HL130249 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brainstem noradrenergic neurons: Identifying a hub at the intersection of cognition, motility, and skeletal muscle regulation.

Brainstem noradrenergic neuron clusters form a node integrating efferents projecting to distinct areas such as those regulating cognition and skeletal muscle structure and function, and receive dissimilar afferents through established circuits to coordinate organismal responses to internal and environmental challenges. Genetic lineage tracing shows the remarkable heterogeneity of brainstem noradrenergic neurons, which may explain their varied functions. They project to the locus coeruleus, the primary source of noradrenaline in the brain, which supports learning and cognition. They also project to pre-ganglionic neurons, which lie within the spinal cord and form synapses onto post-ganglionic neurons. The synapse between descending brainstem noradrenergic neurons and pre-ganglionic spinal neurons, and these in turn with post-ganglionic noradrenergic neurons located at the paravertebral sympathetic ganglia, support an anatomical hierarchy that regulates skeletal muscle innervation, neuromuscular transmission, and muscle trophism. Whether any noradrenergic neuron subpopulation is more susceptible to damaged protein deposit and death with ageing and neurodegeneration is a relevant question that answer will help us to detect neurodegeneration at an early stage, establish prognosis, and anticipate disease progression. Loss of muscle mass and strength with ageing, termed sarcopenia, may predict impaired cognition with ageing and neurodegeneration and establish an early time to start interventions aimed at reducing central noradrenergic neurons hyperactivity. Complex multidisciplinary approaches, including genetic tracing, specific circuit labelling, optogenetics and chemogenetics, electrophysiology, and single-cell transcriptomics and proteomics, are required to test this hypothesis pre-clinical.

The Role of Glutamatergic Signaling in Central Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS) and Rett Syndrome, in which abnormal chemosensory function is thought to play a role. However, which NA populations modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Histological evidence suggests that glutamate is co‐transmitted in subsets of brainstem NA neurons, some of which may contribute to regulating chemoreflexes. However, the extent of glutamate co‐transmission in the central NA system remains uncharacterized. Further, it remains unclear what role NA‐based glutamatergic signaling plays in various aspects of respiratory homeostasis.To anatomically map potential glutamate‐expressing NA neurons, we characterized the expression profile of all three known of Vesicular Glutamate Transporters (Vglut1, Vglut2 and Vglut3) in NA neurons by intersectional genetic fate mapping. For each Vglut1/2/3 transporter, acorresponding Cre knock‐in recombinase line was combined with DBH‐p2a_Flpomice. These Vglut1/2/3_Cre; DBH‐p2a_Flpocompound lines were crossed with the RC::F_TdTomato_P_GFPintersectional reporter mice. In each of the three intersectional reporter crosses, NA neurons co‐expressing either Vglut1, Vglut2, or Vglut3 are labeled by green fluorescent protein (GFP) while NA neurons without any Vglut1/2/3 expression are labelled by red fluorescent protein (tdTomato). Our preliminary fate maps, which reflect the cumulative expression profiles of glutamate markers in NA neurons throughout the development and lifetime of the mouse, found that about 80% NA neurons express Vglut2 at some point during their development and posterior medullary NA neurons express Vglut3 as well.Prior studies suggest that Vglut2‐based glutamatergic signaling may play a role in respiratory control. To determine what physiological role, if any, Vglut2‐based glutamatergic signaling may play in adult respiratory homeostasis, we conditionally ablated Vglut2 in central NA neurons by crossing DBH‐Cre/+with Vglut2flox/flox mice. Mutants and sibling controls (6‐8 weeks) were assessed for respiratory function by whole‐body barometric plethysmography. Ventilation and VO2 were assessed under room air (21%O2,79%N2), hypercapnia (21%O2, 74/72/69%N2, 5/7/10%CO2) and hypoxia (10% O2, 90% N2). Our preliminary data indicates that loss of Vglut2 in NA neurons does not significantly impact breathing under room air, hypercapnia, or hypoxia. Thus, our data suggests that Vglut2‐based glutamatergic signaling within the central NA system is dispensable for normal baseline breathing and hypercapnic, and hypoxic chemosensory reflexes.

The many faces of the E3 ubiquitin ligase, RNF220, in neural development and beyond.

Ubiquitin modification plays important roles in many cellular processes that are fundamental for vertebrate embryo development, such as cell division, differentiation, and migration. Aberrant function or deregulation of ubiquitination enzymes can cause developmental disorders, cancer progression, and neurodegenerative diseases in humans. RING finger protein 220 (RNF220) is an evolutionarily conserved RING-type ubiquitin E3 ligase. Recent studies have revealed the roles and mechanisms of RNF220 and its partner protein, zinc finger C4H2-type containing protein (ZC4H2), in embryonic development and human diseases. Using mouse and zebrafish models, it has been shown that RNF220 regulates sonic hedgehog (Shh) signaling via Gli and embryonic ectoderm development (EED), a polycomb repressive complex 2 (PRC2) component, during ventral neural patterning and cerebellum development. In addition, RNF220 also regulates the development and functions of central noradrenergic and motor neurons in mice. By stabilizing β-catenin and signal transducer and activator of transcription 1 (STAT1), RNF220 is also involved in Wnt and interferon (IFN)-STAT1 signaling and thus the regulation of tumorigenesis and immune response, respectively. In humans, both RNF220 and ZC4H2 mutations have been reported to be associated with diseases accompanied by complicated neural defects. In this review, we summarize the current knowledge of RNF220 with special emphasis on its roles and mechanisms of action in signal transduction, vertebrate neural development, and related human disorders.

The Role of Noradrenergic and Glutamatergic Signaling in Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are a critical component of the brainstem respiratory network that maintains blood CO2 and O2 levels through respiratory chemoreflexes. NA dysfunction is implicated in numerous developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS), Rett Syndrome, and Congenital Central Hypoventilation Syndrome (CCHS), in which abnormal chemosensory function is implicated. However, which NA neurons modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Previous studies in our lab utilizing intersectional chemogenetic (hM4D and hM3D DREADD mediated) inhibition and excitation identified multiple rhombomere (transient genetically-defined hindbrain segments arising during early development) specific NA populations that play a role in the adult hypercapnic chemoreflex. NA neurons are typically thought to communicate with synaptic partners through noradrenaline as their primary neurotransmitter. However, the excitatory neurotransmitter, glutamate, is also expressed in subpopulations of NA neurons including those derived from rhombomeres playing a role in the hypercapnic reflex. Prior studies have implicated NA derived glutamatergic signaling in respiratory control. Thus, we seek to determine the relative roles of noradrenergic and glutamatergic signaling in rhombomeric NA populations that are involved in adult respiratory chemoreflexes. To determine the requirement of noradrenergic signaling in the hypercapnic reflex, we aim to conditionally ablate noradrenergic signaling in rhombomeric NA sub-populations by crossing rhombomere specific Credrivers with the THflox/flox(tyrosine hydroxylase) allele to remove dopamine and downstream (nor) adrenaline production in targeted neurons. We also aim to determine if the glutamate producing NA neurons represented a larger functional cohort in breathing control. We therefore used our intersectional strategy to target and chemogenetically activate (hM3D) or inhibite (hM4De) glutamate co-expressing NA neurons (defined by partially overlapping Vglut2Cre; DBHFLPo activity) which are expected to comprise a super-set of rhombomere specific NA neurons that play a role in respiratory chemoreflexes. Whole-body barometric plethysmography was used to measure the respiratory function of the mice under room air (21%O2,79%N2) and hypercapnia (21%O2, 74%N2, 5% CO2). Our preliminary data suggests that removing noradrenergic signaling in a key rhombomeric NA subpopulation does not inhibit the hypercapnic reflex while DREADD mediated inhibition does reduce the hypercapnic reflex. Acute inhibition of glutamate-expressing NA neurons shows a reduction in the hypercapnic ventilatory reflex while acute activation of these same neurons shows a significantly enhanced reflex. Altogether, these preliminary data suggest that glutamate production may delineate a functionally specific NA subpopulation in the hypercapnic reflex and offers additional clues to the respective roles of noradrenaline and glutamatergic signaling in the noradrenergic system.

Oleoylethanolamide decreases frustration stress-induced binge-like eating in female rats: a novel potential treatment for binge\xa0eating disorder

Binge eating disorder (BED) is the most frequent eating disorder, for which current pharmacotherapies show poor response rates and safety concerns, thus highlighting the need for novel treatment options. The lipid-derived messenger oleoylethanolamide (OEA) acts as a satiety signal inhibiting food intake through the involvement of central noradrenergic and oxytocinergic neurons. We investigated the anti-binge effects of OEA in a rat model of binge-like eating, in which, after cycles of intermittent food restrictions/refeeding and palatable food consumptions, female rats show a binge-like intake of palatable food, following a 15-min exposure to their sight and smell (“frustration stress”). Systemically administered OEA dose-dependently (2.5, 5, and 10 mg kg−1) prevented binge-like eating. This behavioral effect was associated with a decreased activation (measured by mapping the expression of c-fos, an early gene widely used as a marker of cellular activation) of brain areas responding to stress (such as the nucleus accumbens and amygdala) and to a stimulation of areas involved in the control of food intake, such as the VTA and the PVN. These effects were paralleled, also, to the modulation of monoamine transmission in key brain areas involved in both homeostatic and hedonic control of eating. In particular, a decreased dopaminergic response to stress was observed by measuring dopamine extracellular concentrations in microdialysates from the nucleus accumbens shell, whereas an increased serotonergic and noradrenergic tone was detected in tissue homogenates of selected brain areas. Finally, a decrease in corticotropin-releasing factor (CRF) mRNA levels was induced by OEA in the central amygdala, while an increase in oxytocin mRNA levels was induced in the PVN. The restoration of a normal oxytocin receptor density in the striatum paralleled the oxytocinergic stimulation produced by OEA. In conclusion, we provide evidence suggesting that OEA might represent a novel potential pharmacological target for the treatment of binge-like eating behavior.

The Role of Noradrenergic and Glutamatergic Signaling in Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are a critical component of the brainstem respiratory network that maintains blood CO2 and O2 levels through respiratory chemoreflexes. NA dysfunction is implicated in numerous developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS), Rett Syndrome, and Congenital Central Hypoventilation Syndrome (CCHS), in which abnormal chemosensory function is implicated. However, which NA neurons modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Previous studies in our lab utilizing intersectional chemogenetic (hM4D and hM3D DREADD mediated) inhibition and excitation identified multiple rhombomere (transient genetically‐defined hindbrain segments arising during early development) specific NA populations that play a role in the adult hypercapnic chemoreflex. NA neurons are typically thought to communicate with synaptic partners through noradrenaline and adrenaline as their primary neurotransmitter. However, the excitatory neurotransmitter, glutamate, is also expressed in subpopulations of NA neurons including rhombomeric NA neurons playing a role in the hypercapnic reflex. Prior studies have implicated NA derived glutamatergic signaling in respiratory control. Thus, we seek to determine the relative roles of noradrenergic and glutamatergic signaling in rhombomeric NA populations that are involved in adult respiratory chemoreflexes. To determine the requirement of noradrenergic signaling in the hypercapnic reflex, we aim to conditionally ablate noradrenergic signaling in rhombomeric NA sub‐populations by crossing rhombomere specific Credrivers with the THflox/flox(tyrosine hydroxylase) allele to remove dopamine and downstream (nor) adrenaline production in targeted neurons. We also aim to determine if the glutamate producing NA neurons represent a larger functional cohort in breathing control. We therefore used our intersectional strategy to target and chemogenetically activate (hM3D) or inhibite (hM4De) glutamate co‐expressing NA neurons (defined by partially overlapping Vglut2Cre; DBHFLPo activity) which are expected to comprise a super‐set of rhombomere specific NA neurons that play a role in respiratory chemoreflexes. Whole‐body barometric plethysmography was used to measure the respiratory function of the mice under room air (21%O2, 79%N2) and hypercapnia (21%O2, 74%N2, 5% CO2). Our preliminary data suggests that removing noradrenergic signaling in a key rhombomeric NA subpopulation does not inhibit the hypercapnic reflex while DREADD mediated inhibition does reduce the hypercapnic reflex. Acute inhibition of glutamate‐expressing NA neurons shows a reduction in the hypercapnic ventilatory reflex while acute activation of these same neurons shows a significantly enhanced reflex. Altogether, these preliminary data suggest that glutamate production may delineate a functionally specific NA subpopulation in the hypercapnic reflex and offers additional clues to the respective roles of noradrenaline and glutamatergic signaling in the noradrenergic system.Support or Funding InformationR01HL153505

Axonal projection-specific differences in somatodendritic α2 autoreceptor function in locus coeruleus neurons.

The locus coeruleus (LC) contains the majority of central noradrenergic neurons sending wide projections throughout the entire CNS. The LC is considered to be essential for multiple key brain functions including arousal, attention and adaptive stress responses as well as higher cognitive functions and memory. Electrophysiological studies of LC neurons have identified several characteristic functional features such as low-frequency pacemaker activity with broad action potentials, transient high-frequency burst discharges in response to salient stimuli and an apparently homogeneous inhibition of firing by activation of somatodendritic α2 autoreceptors (α2AR). While stress-mediated plasticity of the α2AR response has been described, it is currently unclear whether different LC neurons projecting to distinct axonal targets display differences in α2AR function. Using fluorescent beads-mediated retrograde tracing in adult C57Bl6/N mice, we compared the anatomical distributions and functional in vitro properties of identified LC neurons projecting either to medial prefrontal cortex, hippocampus or cerebellum. The functional in vitro analysis of LC neurons confirmed their mostly uniform functional properties regarding action potential generation and pacemaker firing. However, we identified significant differences in tonic and evoked α2AR-mediated responses. While hippocampal-projecting LC neurons were partially inhibited by endogenous levels of norepinephrine and almost completely silenced by application of saturating concentrations of the α2 agonist clonidine, prefrontal-projecting LC neurons were not affected by endogenous levels of norepinephrine and only partially inhibited by saturating concentrations of clonidine. Thus, we identified a limited α2AR control of electrical activity for prefrontal-projecting LC neurons indicative of functional heterogeneity in the LC-noradrenergic system.

Central noradrenergic activity affects analgesic effect of Neuropeptide S.

Neuropeptide S (NPS) is an endogenous neuropeptide controlling anxiolysis, wakefulness, and analgesia. NPS containing neurons exist near to the locus coeruleus (LC) involved in the descending anti-nociceptive system. NPS interacts with central noradrenergic neurons; thus brain noradrenergic signaling may be involved in NPS-induced analgesia. We tested NPS analgesia in noradrenergic neuron-lesioned rats using a selective LC noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). A total 66 male Sprague-Dawley rats weighing 350-450g were used. Analgesic effects of NPS were evaluated using hot-plate and tail-flick test with or without DSP-4. The animal allocated into 3 groups; hot-plate with NPS alone intracerebroventricular (icv) (0.0, 1.0, 3.3, and 10.0nmol), tail-flick NPS alone icv (0.0 and 10.0nmol), and hot-plate with NPS and DSP-4 (0 or 50mg/kg ip). In hot-plate with NPS and DSP-4 group, noradrenaline content in the cerebral cortex, pons, hypothalamus, were measured. NPS 10nmol icv prolonged hot plate (%MPE) but not tail flick latency at 30 and 40min after administration. DSP-4 50mg/kg decreased noradrenaline content in the all 3 regions. The NA depletion inhibited NPS analgesic effect in the hot plate test but not tail flick test. There was a significant correlation between hot plate latency (percentage of maximum possible effect: %MPE) with NPS 10nmol and NA content in the cerebral cortex (p=0.017, r 2=0.346) which noradrenergic innervation arisen mainly from the LC. No other regions had the correlation. NPS analgesia interacts with LC noradrenergic neuronal activity.

Two Subpopulations of Noradrenergic Neurons in the Locus Coeruleus Complex Distinguished by Expression of the Dorsal Neural Tube Marker Pax7

Central noradrenergic neurons, collectively defined by synthesis of the neurotransmitter norepinephrine, are a diverse collection of cells in the hindbrain, differing in their anatomy, physiological and behavioral functions, and susceptibility to disease and environmental insult. To investigate the developmental basis of this heterogeneity, we have used an intersectional genetic fate mapping strategy in mice to study the dorsoventral origins of the En1-derived locus coeruleus (LC) complex which encompasses virtually all of the anatomically defined LC proper, as well as a portion of the A7 and subcoeruleus (SubC) noradrenergic nuclei. We show that the noradrenergic neurons of the LC complex originate in two different territories of the En1 expression domain in the embryonic hindbrain. Consistent with prior studies, we confirm that the majority of the LC proper arises from the alar plate, the dorsal domain of the neural tube, as defined by expression of Pax7Cre. In addition, our analysis shows that a large proportion of the En1-derived A7 and SubC nuclei also originate in the Pax7Cre-defined alar plate. Surprisingly, however, we identify a smaller subpopulation of the LC complex that arises from outside the Pax7Cre expression domain. We characterize the distribution of these neurons within the LC complex, their cell morphology, and their axonal projection pattern. Compared to the broader LC complex, the newly identified Pax7Cre-negative noradrenergic subpopulation has very sparse projections to thalamic nuclei, suggestive of distinct functions. This developmental genetic analysis opens new avenues of investigation into the functional diversity of the LC complex.

Uncovering diversity in the development of central noradrenergic neurons and their efferents

Uncovering the mechanisms that underlie central noradrenergic neuron heterogeneity is essential to understanding selective subtype vulnerability to disease and environmental insult. Using recombinase-based intersectional genetic fate mapping we have previously demonstrated that molecularly distinct progenitor populations give rise to mature noradrenergic neurons differing in their anatomical location, axon morphology and efferent projection pattern. Here we review the findings from our previous study and extend our analysis of the noradrenergic subpopulation defined by transient developmental expression of Hoxb1. Using a combination of intersectional genetic fate mapping and analysis of a targeted loss of function mutation in Hoxb1, we have now uncovered additional heterogeneity based on the requirement of some noradrenergic neurons for Hoxb1 expression. By comparing the distribution of noradrenergic neurons derived from the Hoxb1 expression domain in wild-type and mutant mice, we demonstrate that Hoxb1 expression is required by a subset of neurons in the pons. Additional fate mapping, using a Hoxb1 enhancer element that drives Cre recombinase expression exclusively in rhombomere 4 of the hindbrain, reveals the existence of a subpopulation of noradrenergic neurons in the pons with more restricted axonal targets than the full Hoxb1-derived subpopulation. The unique projection profile of this newly defined subpopulation suggests that it may be functionally distinct. These analyses shed new light on the molecular determinants of noradrenergic identity in the pons and the overall complexity of the central noradrenergic system.This article is part of a Special Issue entitled SI: Noradrenergic System.

Shine bright: considerations on the use of fluorescent substrates in living monoaminergic neurons in vitro.

The biogenic monoamines dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) are major neuromodulators in the mammalian central nervous system (CNS). DA containing neurons are found in i) the mesolimbic system in which cell bodies in the ventral tegmental area (VTA) project axons into the amygdala, cortex, hippocampus and the nucleus accumbens; and ii) the nigrostriatal system in which cell bodies located in the substantia nigra pars compacta send their axons into the dorsolateral parts of the striatum (Bjorklund and Dunnett, 2007). The central noradrenergic neurons are concentrated in distinct brainstem nuclei with the locus coeruleus (LC) being the most prominent nucleus which projects a diffusely arborizing axonal network to most areas of the CNS (Szabadi, 2013). Serotonergic neurons are located in the raphe nuclei in the brain stem with widespread efferent axonal trajectories with a high number of collateral arborizations into many brain regions such as cortical areas, the hippocampus, the basal ganglia and the spinal cord (Sur et al., 1996a, b). Malfunctions of the three monoaminergic systems are associated with different psychiatric and neurological diseases such as depression, anxiety, chronic pain, sleep disorders, schizophrenia, various aspects of drug abuse, Parkinson's disease and Alzheimer's disease.

Monosynaptic Glutamatergic Activation of Locus Coeruleus and Other Lower Brainstem Noradrenergic Neurons by the C1 Cells in Mice

The C1 neurons, located in the rostral ventrolateral medulla (VLM), are activated by pain, hypotension, hypoglycemia, hypoxia, and infection, as well as by psychological stress. Prior work has highlighted the ability of these neurons to increase sympathetic tone, hence peripheral catecholamine release, probably via their direct excitatory projections to sympathetic preganglionic neurons. In this study, we use channelrhodopsin-2 (ChR2) optogenetics to test whether the C1 cells are also capable of broadly activating the brain's noradrenergic system. We selectively expressed ChR2(H134R) in rostral VLM catecholaminergic neurons by injecting Cre-dependent adeno-associated viral vectors into the brain of adult dopamine-β-hydroxylase (DβH)(Cre/0) mice. Most ChR2-expressing VLM neurons (75%) were immunoreactive for phenylethanolamine N-methyl transferease, thus were C1 cells, and most of the ChR2-positive axonal varicosities were immunoreactive for vesicular glutamate transporter-2 (78%). We produced light microscopic evidence that the axons of rostral VLM (RVLM) catecholaminergic neurons contact locus coeruleus, A1, and A2 noradrenergic neurons, and ultrastructural evidence that these contacts represent asymmetric synapses. Using optogenetics in tissue slices, we show that RVLM catecholaminergic neurons activate the locus coeruleus as well as A1 and A2 noradrenergic neurons monosynaptically by releasing glutamate. In conclusion, activation of RVLM catecholaminergic neurons, predominantly C1 cells, by somatic or psychological stresses has the potential to increase the firing of both peripheral and central noradrenergic neurons.

α‐Adrenoceptor Assays

α-Adrenoceptors mediate responses to activation of both peripheral sympathetic nerves and central noradrenergic neurons. They also serve as autoreceptors that modulate the release of norepinephrine (NE) and other neurotransmitters. There are two major classes of α-adrenoceptors, the α(1)- and α(2). Each class is subdivided into three subtypes: α(1A), α(1B), α(1D), and α(2A), α(2B), α(2C). Described in this unit are in vitro isolated tissue methods used to study α-adrenoceptor functions and to identify novel ligands for these receptors. Detailed protocols describing use of isolated tissues to study the various α(1)- and α(2)-adrenoceptor subtypes are provided.

Role of coerulean noradrenergic neurones in general anaesthesia in rats

Central noradrenergic neurones have a role in alertness, analgesia, and thermoregulation; these neurones are also involved in the mechanism of anaesthesia. Locus coeruleus neurones innervate various central nervous regions including the cerebral cortex and hippocampus, and are responsible for wakefulness and analgesia. We hypothesized that these neurones are also involved in both activation of the γ-aminobutyric acid type A (GABA(A)) receptor and inhibition of N-methyl-d-aspartate (NMDA) receptor-mediated anaesthesia. Forty-seven male rats were used to compare duration of anaesthesia before and 10 days after noradrenergic neurone depletion after intraperitoneal (i.p.) administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 0, 5, and 50 mg kg(-1)). The animals received i.p. thiopental (GABA anaesthetic, 45 mg kg(-1)) or ketamine (NMDA anaesthetic, 100 mg kg(-1)). We also tested the effects of coerulean noradrenergic neurone depletion on i.p. ketamine analgesia (15 mg kg(-1)) using the hot-plate test in a further 21 male rats. At the end of each experiment, norepinephrine contents in the cerebral cortex and hippocampus were measured. I.P. DSP-4 5 and 50 mg kg(-1) significantly decreased ketamine anaesthesia duration by 12.7% and 22.4%, increased thiopental anaesthesia duration by 10.8% and 24.5%, and reduced ketamine-increased hot-plate latency by 55.2% and 68.1%, respectively. In addition, i.p. DSP-4 5 and 50 mg kg(-1) significantly reduced norepinephrine contents in coerulean brain regions by ∼20% and ∼75%, respectively. There were significant correlations between norepinephrine contents in coerulean brain regions and anaesthesia durations and ketamine analgesia. The present data indicate that coerulean noradrenergic neurones may be responsible for both GABA- and NMDA-mediated anaesthetic actions.

Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine β-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Evidence for a central α-sympathomimetic action of Clonidine in the rat

The antihypertensive effects of clonidine (0.15 mg kg-1, i.p.) were studied in conscious DOCA/saline hypertensive rats having chronically implanted arterial cannulae. The response to clonidine was markedly reduced by simultaneously administered desipramine (3 mg kg-1, i.p.), antagonized dose-dependently by piperoxan (2-10 mg kg-1, i.v.) and prevented by pretreatment with phentolamine (0.2 mg, i.c.v.). Pretreatment with 6-hydroxydopamine (3 x 250 mu-g, i.c.v.), haloperidol (1 mg kg-1, i.p.), p-chloro-N-methylamphetamine (3.5 mg kg-1, i.p.) or 5,6-dihydroxytryptamine (50 mu-g and 25 mu-g, i.c.v.) did not significantly modify the antihypertensive response. It is concluded that the antihypertensive response to clonidine is mediated via stimulation of central alpha-adrenoceptors and is independent of central dopaminergic receptors and intact central serotoninergic neurons. The necessity for intact central noradrenergic neurons remains uncertain.

040 Colour-dependent modulation of the pupillary light reflex: interaction with clonidine

AimsShort-wave length (blue) light is more effective in evoking the pupillary light reflex than long-wave length (red/orange) light, due to the stimulation of melanopsin-containing photoreceptors on retinal ganglion cells by...

A functional dopamine-β-hydroxylase gene promoter polymorphism is associated with impulsive personality styles, but not with affective disorders

Dopamine-beta-hydroxylase (DbetaH) catalyzes the conversion of dopamine to norepinephrine in central noradrenergic and adrenergic neurons and thus is critically involved in the biosynthesis of catecholamines. There are equivocal findings concerning the question whether or not DssH activity levels are altered in affective disorders or in subtypes of affective disorders. Moreover, information about the role of dopamine beta-hydroxylase (DBH) genotype, which explains a large part of the variance of enzymatic activity, in affective disorders and personality dimensions is limited. To resolve these inconsistencies, association tests were performed using four independent samples, healthy volunteers (N = 387), patients with affective disorders (N = 182), adult attention deficit hyperactivity disorder (ADHD) patients (N = 407), and patients with personality disorders (N = 637). In the latter two samples, the revised NEO personality inventory (NEO-PI-R) was administered. All participants were genotyped for a putatively functional single nucleotide polymorphism (C-1021T, rs1611115). No differences in DBH C-1021T genotype distribution were observed between patients with affective disorders and healthy control subjects. Also when the patient sample was divided into uni- and bipolar patients versus controls, no significant differences emerged. Furthermore, no clear-cut association was detected between the TT genotype and personality disorder clusters while there was a significant association with adult ADHD. However, personality disorder patients carrying the DBH TT genotype exhibited higher neuroticism and novelty seeking scores as compared to individuals with the CC or CT genotype. Analyses on the level of the neuroticism and novelty seeking subscales revealed that the DBH TT genotype was primarily associated with personality features related to impulsiveness and aggressive hostility. Also adult ADHD patients carrying the homozygous TT genotypes displayed by significantly increased neuroticism scores; when both personality disorder and adult ADHD patient were analyzed together, TT carriers also displayed by significantly lower conscientiousness levels. Our results thus do not implicate the DBH C-1021T polymorphism in the pathophysiology of depressive disorders or personality disorders, yet homozygosity at this locus appears to increase the risk towards personality traits related to impulsiveness, aggression and related disease states, namely adult ADHD. These data argue for a dimensional rather than categorical effect of genetic variance in DBH activity; accordingly, the inconsistency of previous findings concerning DbetaH levels in affective disorders might be caused by the underlying association of the TT genotype at DBH-1021 with impulsive personality traits.

Chronic cold exposure increases RGS7 expression and decreases α2‐autoreceptor‐mediated inhibition of noradrenergic locus coeruleus neurons

Chronic stress exposure alters the central noradrenergic neurons originating from the locus coeruleus (LC). Previously, we demonstrated that evoked increases in the firing rate of LC neurons and their release of norepinephrine are enhanced following chronic cold exposure. In the present studies, we tested the hypothesis that reduced feedback inhibition of LC neurons might underlie these alterations in LC activity by examining the effect of alpha(2)-autoreceptor stimulation on LC activity in chronically stressed rats using in vivo and in vitro single unit recordings. Given that regulators of G-protein signaling (RGS) proteins can impact the coupling of alpha(2)-autoreceptors to downstream signaling cascades, we also explored the expression of several RGS proteins following chronic stress exposure. We observed that the alpha(2)-autoreceptor-evoked inhibition of LC neurons was reduced and that the expression of RGS7 was increased following chronic stress exposure. Finally, we demonstrated that intracellular administration of RGS7 via patch clamp electrodes mimicked the stress-induced decrease in clonidine-evoked autoreceptor-mediated inhibition. These novel data provide a mechanism to explain how chronic stress-induced alterations in receptor coupling can result in changes in alpha(2)-autoreceptor control of noradrenergic function throughout the central nervous system, potentially leading to alterations in anxiety-related behaviors, and may suggest novel therapeutic targets for the treatment of mood and anxiety disorders.