Rnf220/Zc4h2-mediated monoubiquitylation of Phox2 is required for noradrenergic neuron development.

Noradrenaline belongs to the monoamine system and is involved in cognition and emotional behaviors. Phox2a and Phox2b play essential but non-redundant roles during development of the locus coeruleus (LC), the main noradrenergic (NA) neuron center in the mammalian brain. The ubiquitin E3 ligase Rnf220 and its cofactor Zc4h2 participate in ventral neural tube patterning by modulating Shh/Gli signaling, and ZC4H2 mutation is associated with intellectual disability, although the mechanisms for this remain poorly understood. Here, we report that Zc4h2 and Rnf220 are required for the development of central NA neurons in the mouse brain. Both Zc4h2 and Rnf220 are expressed in developing LC-NA neurons. Although properly initiated at E10.5, the expression of genes associated with LC-NA neurons is not maintained at the later embryonic stages in mice with a deficiency of either Rnf220 or Zc4h2 In addition, we show that the Rnf220/Zc4h2 complex monoubiquitylates Phox2a/Phox2b, a process required for the full transcriptional activity of Phox2a/Phox2b. Our work reveals a role for Rnf220/Zc4h2 in regulating LC-NA neuron development, and this finding may be helpful for understanding the pathogenesis of ZC4H2 mutation-associated intellectual disability.

Locus Coeruleus-Noradrenergic Neurons Regulate Stress Coping During Subchronic Exposure to Social Threats: A Characteristic Feature in Postpartum Female Mice.

Stress-coping strategies have been implicated in depression. The control of stress coping may improve the symptom and higher prevalence of depression during the postpartum period in women. However, the neuronal mechanisms underlying stress coping remain to be fully elucidated in postpartum women. In this study, we examined how locus coeruleus-noradrenergic (LC-NA) neurons, which have been associated with both stress coping and depression, regulate changes in coping style induced by subchronic exposure to unfamiliar male mice as a social threat in postpartum female mice. In contrast to virgin females, dams exposed to unfamiliar males daily for four consecutive days showed reduced immobility duration in the forced swim test, indicating that exposure to unfamiliar males decreased passive stress coping in dams. Exposure to unfamiliar males also decreased sucrose palatability in the sucrose preference test and suppressed the crouching behavior in the maternal care test but did not affect anxiety-like behavior in the hole-board test in dams. In fiber photometry analyses, LC-NA neurons showed differential activity between dams and virgin females in response to unfamiliar males. Chemogenetic inhibition of LC-NA neurons during exposure to unfamiliar males prevented the social threat-induced decrease in immobility duration in the forced swim test in dams. Furthermore, inhibition or activation of LC-NA neurons exacerbated crouching behavior in dams. These results indicate that LC-NA neurons regulate the social threat-induced decrease in passive stress coping and relieve social threat-induced inhibition of maternal care in postpartum female mice.

A5 noradrenergic-projecting C1 neurons activate sympathetic and breathing outputs in anaesthetized rats.

NEW FINDINGS: What is the central question of this study? C1 neurons innervate pontine noradrenergic cell groups, including the A5 region: do A5 noradrenergic neurons contribute to the activation of sympathetic and respiratory responses produced by selective activation of the C1 group of neurons. What is the main finding and its importance? The increase in sympathetic and respiratory activities elicited by selective stimulation of C1 neurons is reduced after blockade of excitatory amino acid within the A5 region, suggesting that the C1-A5 pathway might be important for sympathetic-respiratory control. ABSTRACT: Adrenergic C1 neurons innervate and excite pontine noradrenergic cell groups, including the ventrolateral pontine noradrenergic region (A5). Here, we tested the hypothesis that C1 activates A5 neurons through the release of glutamate and this effect is important for sympathetic and respiratory control. Using selective tools, we restricted the expression of channelrhodopsin2 under the control of the artificial promoter PRSx8 to C1 neurons (69%). Transduced catecholaminergic terminals within the A5 region are in contact with noradrenergic A5 neurons and the C1 terminals within the A5 region are predominantly glutamatergic. In a different group of animals, we performed retrograde lesion of C1 adrenergic neurons projecting to the A5 region with unilateral injection of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP) directly into the A5 region during the hypoxic condition. As expected, hypoxia (8% O2 , 3 h) induced a robust increase in fos expression within the catecholaminergic C1 and A5 regions of the brainstem. Depletion of C1 cells projecting to the A5 regions reduced fos immunoreactivity induced by hypoxia within the C1 region. Physiological experiments showed that bilateral injection of kynurenic acid (100 mM) into the A5 region reduced the rise in mean arterial pressure, and sympathetic and phrenic nerve activities produced by optogenetic stimulation of C1 cells. In conclusion, the C1 neurons activate the ventrolateral pontine noradrenergic neurons (A5 region) possibly via the release of glutamate and might be important for sympathetic and respiratory outputs in anaesthetized rats.

Prolonged exposure to stressors suppresses exploratory behavior in zebrafish larvae.

Environmental stressors induce rapid physiological and behavioral shifts in vertebrate animals. However, the neurobiological mechanisms responsible for stress-induced changes in behavior are complex and not well understood. Similar to mammalian vertebrates, zebrafish adults display a preference for dark environments that is associated with predator avoidance, enhanced by stressors, and broadly used in assays for anxiety-like behavior. Although the larvae of zebrafish are a prominent model organism for understanding neural circuits, few studies have examined the effects of stressors on their behavior. This study examines the effects of noxious chemical and electric shock stressors on locomotion and light preference in zebrafish larvae. We found that both stressors elicited similar changes in behavior. Acute exposure induced increased swimming activity, while prolonged exposure depressed activity. Neither stressor produced a consistent shift in light-dark preference, but prolonged exposure to these stressors resulted in a pronounced decrease in exploration of different visual environments. We also examined the effects of exposure to a noxious chemical cue using whole-brain calcium imaging, and identified neural correlates in the area postrema, an area of the hindbrain containing noradrenergic and dopaminergic neurons. Pharmaceutical blockade experiments showed that α-adrenergic receptors contribute to the behavioral response to an acute stressor but are not necessary for the response to a prolonged stressor. These results indicate that zebrafish larvae have complex behavioral responses to stressors comparable to those of adult animals, and also suggest that these responses are mediated by similar neural pathways.

Lack of Cannabinoid Receptor Type-1 Leads to Enhanced Age-Related Neuronal Loss in the Locus Coeruleus.

Our laboratory and others have previously shown that cannabinoid receptor type-1 (CB1r) activity is neuroprotective and a modulator of brain ageing; a genetic disruption of CB1r signaling accelerates brain ageing, whereas the pharmacological stimulation of CB1r activity had the opposite effect. In this study, we have investigated if the lack of CB1r affects noradrenergic neurons in the locus coeruleus (LC), which are vulnerable to age-related changes; their numbers are reduced in patients with neurodegenerative diseases and probably also in healthy aged individuals. Thus, we compared LC neuronal numbers between cannabinoid 1 receptor knockout (Cnr1-/-) mice and their wild-type littermates. Our results reveal that old Cnr1-/- mice have less noradrenergic neurons compared to their age-matched wild-type controls. This result was also confirmed by the analysis of the density of noradrenergic terminals which proved that Cnr1-/- mice had less compared to the wild-type controls. Additionally, we assessed pro-inflammatory glial activity in the LC. Although the density of microglia in Cnr1-/- mice was enhanced, they did not show enhanced inflammatory profile. We hypothesize that CB1r activity is necessary for the protection of noradrenergic neurons, but its anti-inflammatory effect probably only plays a minor role in it.

Integration of the Proprioceptive and Central Inspiratory Inhibitory Afferent Inputs by Pontine Noradrenergic A5 Neurons in Rats.

Inhibitory afferent inputs to pontine A5 noradrenergic neurons (A5 NN) are not known, except partial baroreceptor input. In spontaneously breathing pentobarbital-anesthetized rats, we registered 35 A5 NN that were activated by hypoxia (100% N2, 10 sec) by more than 5 times in comparison with the background. Cooling of retrotrapezoid nucleus (15°C, 6 sec) completely blocked the motor inspiratory output and A5 NN discharge frequency increased (23/23) by more than 7 times in comparison with the background values. The beginning of A5 NN activation coincided with cessation of inspiratory activity. Short-term passive stretching of the shin muscles (1 sec, 100 g) caused BP drop and complete inhibition of A5 NN (12/12) activated by hypoxia. Inhibitory afferent inputs from proprioceptors and central inspiratory neurons that can limit A5 NN activity were demonstrated.

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-ß-hydroxylase (DßH) 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 DßH 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.

Corticotropin releasing factor up-regulates the expression and function of norepinephrine transporter in SK-N-BE (2) M17 cells.

Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [(3) H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-ß in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.

Hindbrain lactostasis regulates hypothalamic AMPK activity and metabolic neurotransmitter mRNA and protein responses to hypoglycemia.

Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolosensory loci operate autonomously or interact to coordinate central nervous system (CNS) reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite l-lactate during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal AMPK activity, coincident with exacerbation of hypoglycemia. Hindbrain lactate repletion also blunted hypoglycemic upregulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 phosphoAMPK (pAMPK), elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus.

Aging disrupts locus coeruleus-driven norepinephrine transmission in the prefrontal cortex: Implications for cognitive and motor decline.

The locus coeruleus (LC)-prefrontal cortex (PFC) circuitry is crucial for cognition, planning, posture and mobility. This study examines the role of norepinephrine (NE) in elucidating the neurobiological basis of age-related cognitive and motor declines. Aged mice exhibited reduced spatial learning, impaired memory, decreased physical endurance, and notable changes in locomotor behavior. The neurochemical foundations of these deficits were investigated through fast-scan cyclic voltammetry to measure NE release in the PFC and LC, both in vivo and in brain slices. Additionally, oxygen levels were monitored as a proxy for PFC neuronal function, and NE levels were analyzed in the extracellular space via microdialysis and total content in the PFC. Aged mice exhibited a frequency-dependent increase in NE release in the PFC upon LC stimulation, suggesting alterations in neural responsiveness due to aging. We also recorded slower NE reuptake rates and increased NE content and neuronal activity, indicated by higher oxygen levels and facilitated neuron activation due to membrane depolarization recorded via whole-cell patch-clamp. To understand the basis for LC-driven NE surges in the PFC with aging, we examined the expression levels of two proteins critical for presynaptic NE release and NE reuptake: the α2a-adrenergic receptor and the NE transporter. Both showed a significant decrease in the PFC with aging. These findings support the concept that aging significantly alters the structural and functional dynamics within the LC-PFC neural circuit, impacting NE modulation and neuronal activity, which may underlie the observed declines in cognitive and motor functions in aging populations.

Sex differences in single neuron function and proteomics profiles examined by patch-clamp and mass spectrometry in the locus coeruleus of the adult mouse.

AIMS: This study aimed to characterize the properties of locus coeruleus (LC) noradrenergic neurons in male and female mice. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. METHODS: Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal functional properties were assessed using patch-clamp recordings. Proteomic analyses of individual LC neuron soma was conducted using mass spectrometry to discern protein expression profiles. Data are available via ProteomeXchange with identifier PXD045844. RESULTS: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. CONCLUSIONS: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

Chronic intermittent hypoxia attenuates noradrenergic innervation of hypoglossal motor nucleus.

The state-dependent noradrenergic activation of hypoglossal motoneurons plays an important role in the maintenance of upper airway patency and pathophysiology of obstructive sleep apnea (OSA). Chronic intermittent hypoxia (CIH), a major pathogenic factor of OSA, contributes to the risk for developing neurodegenerative disorders in OSA patients. Using anterograde tracer, channelrhodopsin-2, we mapped axonal projections from noradrenergic A7 and SubCoeruleus neurons to hypoglossal nucleus in DBH-cre mice and assessed the effect of CIH on these projections. We found that CIH significantly reduced the number of axonal projections from SubCoeruleus neurons to both dorsal (by 68%) and to ventral (by73%) subregions of the hypoglossal motor nucleus compared to sham-treated animals. The animals' body weight was also negatively affected by CIH. Both effects, the decrease in axonal projections and body weight, were more pronounced in male than female mice, which was likely caused by less sensitivity of female mice to CIH as compared to males. The A7 neurons appeared to have limited projections to the hypoglossal nucleus. Our findings suggest that CIH-induced reduction of noradrenergic innervation of hypoglossal motoneurons may exacerbate progression of OSA, especially in men.

Alzheimer's Disease: An Attempt of Total Recall.

This review is an attempt to compile existing hypotheses on the mechanisms underlying the initiation and progression of Alzheimer's disease (AD), starting from sensory impairments observed in AD and concluding with molecular events that are typically associated with the disease. These events include spreading of amyloid plaques and tangles of hyperphosphorylated tau and formation of Hirano and Biondi bodies as well as the development of oxidative stress. We have detailed the degenerative changes that occur in several neuronal populations, including the cholinergic neurons in the nucleus basalis of Meynert, the histaminergic neurons in the tuberomammillary nucleus, the serotonergic neurons in the raphe nuclei, and the noradrenergic neurons in the locus coeruleus. Furthermore, we discuss the potential role of iron accumulation in the brains of subjects with AD in the disease progression which served as a basis for the idea that iron chelation in the brain may mitigate oxidative stress and decelerate disease development. We also draw attention to possible role of sympathetic system and, more specifically, noradrenergic neurons of the superior cervical ganglion in triggering of the disease. We also explore the alternative possibility of compensatory protective changes that may occur in these neurons to support cholinergic function in the forebrain of subjects with AD.

Cell-specific expression of Cre recombinase in rat noradrenergic neurons via CRISPR-Cas9 system.

Noradrenergic neurons play a crucial role in the functioning of the nervous system. They formed compact small clusters in the central nervous system. To target noradrenergic neurons in combination with viral tracing and achieve cell-type specific functional manipulation using chemogenetic or optogenetic tools, new transgenic animal lines are needed, especially rat models for their advantages in large body size with facilitating easy operation, physiological parameter monitoring, and accommodating complex behavioral and cognitive studies. In this study, we successfully generated a transgenic rat strain capable of expressing Cre recombinase under the control of the dopamine beta-hydroxylase (DBH) gene promoter using the CRISPR-Cas9 system. Our validation process included co-immunostaining with Cre and DBH antibodies, confirming the specific expression of Cre recombinase. Furthermore, stereotaxic injection of a fluorescence-labeled AAV-DIO virus illustrated the precise Cre-loxP-mediated recombination activity in noradrenergic neurons within the locus coeruleus (LC). Through crossbreeding with the LSL-fluorescence reporter rat line, DBH-Cre rats proved instrumental in delineating the position and structure of noradrenergic neuron clusters A1, A2, A6 (LC), and A7 in rats. Additionally, our specific activation of the LC noradrenergic neurons showed effective behavioral readout using chemogenetics of this rat line. Our results underscore the effectiveness and specificity of Cre recombinase in noradrenergic neurons, serving as a robust tool for cell-type specific targeting of small-sized noradrenergic nuclei. This approach enhances our understanding of their anatomical, physiological, and pathological roles, contributing to a more profound comprehension of noradrenergic neuron function in the nervous system.

Caudal fourth ventricular administration of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside regulates glucose and counterregulatory hormone profiles, dorsal vagal complex metabolosensory neuron function, and hypothalamic Fos expression.

This study investigated the hypothesis that estrogen controls hindbrain AMP-activated protein kinase (AMPK) activity and regulation of blood glucose, counterregulatory hormone secretion, and hypothalamic nerve cell transcriptional status. Dorsal vagal complex A2 noradrenergic neurons were laser microdissected from estradiol benzoate (E)- or oil (O)-implanted ovariectomized female rats after caudal fourth ventricular (CV4) delivery of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR), for Western blot analysis. E advanced AICAR-induced increases in A2 phospho-AMPK (pAMPK) expression and in blood glucose levels and was required for augmentation of Fos, estrogen receptor-α (ERα), monocarboxylate transporter-2, and glucose transporter-3 protein in A2 neurons and enhancement of corticosterone secretion by this treatment paradigm. CV4 AICAR also resulted in site-specific modifications in Fos immunolabeling of hypothalamic metabolic structures, including the paraventricular, ventromedial, and arcuate nuclei. The current studies demonstrate that estrogen regulates AMPK activation in caudal hindbrain A2 noradrenergic neurons during pharmacological replication of energy shortage in this area of the brain, and that this sensor is involved in neural regulation of glucostasis, in part, through control of corticosterone secretion. The data provide unique evidence that A2 neurons express both ERα and -ß proteins and that AMPK upregulates cellular sensitivity to ERα-mediated signaling during simulated energy insufficiency. The results also imply that estrogen promotes glucose and lactate uptake by these cells under those conditions. Evidence for correlation between hindbrain AMPK and hypothalamic nerve cell genomic activation provides novel proof for functional connectivity between this hindbrain sensor and higher order metabolic brain loci while demonstrating a modulatory role for estrogen in this interaction.

CRISPR/Cas9-based QF2 knock-in at the tyrosine hydroxylase (th) locus reveals novel th-expressing neuron populations in the zebrafish mid- and hindbrain.

Catecholaminergic neuron clusters are among the most conserved neuromodulatory systems in vertebrates, yet some clusters show significant evolutionary dynamics. Because of their disease relevance, special attention has been paid to mammalian midbrain dopaminergic systems, which have important functions in motor control, reward, motivation, and cognitive function. In contrast, midbrain dopaminergic neurons in teleosts were thought to be lost secondarily. Here, we generated a CRISPR/Cas9-based knock-in transgene at the th locus, which allows the expression of the Q-system transcription factor QF2 linked to the Tyrosine hydroxylase open reading frame by an E2A peptide. The QF2 knock-in allele still expresses Tyrosine hydroxylase in catecholaminergic neurons. Coexpression analysis of QF2 driven expression of QUAS fluorescent reporter transgenes and of th mRNA and Th protein revealed that essentially all reporter expressing cells also express Th/th. We also observed a small group of previously unidentified cells expressing the reporter gene in the midbrain and a larger group close to the midbrain-hindbrain boundary. However, we detected no expression of the catecholaminergic markers ddc, slc6a3, or dbh in these neurons, suggesting that they are not actively transmitting catecholamines. The identified neurons in the midbrain are located in a GABAergic territory. A coexpression analysis with anatomical markers revealed that Th-expressing neurons in the midbrain are located in the tegmentum and those close to the midbrain-hindbrain boundary are located in the hindbrain. Our data suggest that zebrafish may still have some evolutionary remnants of midbrain dopaminergic neurons.

Dual action of serotonin on local excitatory and inhibitory neural circuits regulating the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus.

Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate- and γ-aminobutyric acid (GABA)-containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5-hydroxytryptamine [5-HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch-clamp, using the CRF-modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential-dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%-30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5-HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin-induced decrease in sIPSC frequency was inhibited by a selective 5-HT2C antagonist RS102221. 8-OH-DPAT (5-HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin-induced increase in sEPSC frequency was inhibited by a selective 5-HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic- and downregulating GABAergic interneurons; the former may partly be mediated by 5-HT7 receptors, whereas the latter by 5-HT2C receptors. The CRF-Venus ΔNeo mouse was useful for the electrophysiological examination.

Possible role of locus coeruleus neuronal loss in age-related memory and attention deficits.

Introduction: Aging is associated with a decline in cognitive abilities, including memory and attention. It is generally accepted that age-related histological changes such as increased neuroinflammatory glial activity and a reduction in the number of specific neuronal populations contribute to cognitive aging. Noradrenergic neurons in the locus coeruleus (LC) undergo an approximately 20 % loss during ageing both in humans and mice, but whether this change contributes to cognitive deficits is not known. To address this issue, we asked whether a similar loss of LC neurons in young animals as observed in aged animals impairs memory and attention, cognitive domains that are both influenced by the noradrenergic system and impaired in aging. Methods: For that, we treated young healthy mice with DSP-4, a toxin that specifically kills LC noradrenergic neurons. We compared the performance of DSP-4 treated young mice with the performance of aged mice in models of attention and memory. To do this, we first determined the dose of DSP-4, which causes a similar 20 % neuronal loss as is typical in aged animals. Results: Young mice treated with DSP-4 showed impaired attention in the presence of distractor and memory deficits in the 5-choice serial reaction time test (5-CSRTT). Old, untreated mice showed severe deficits in both the 5-CSRTT and in fear extinction tests. Discussion: Our data now suggest that a reduction in the number of LC neurons contributes to impaired working memory and greater distractibility in attentional tasks but not to deficits in fear extinction. We hypothesize that the moderate loss of LC noradrenergic neurons during aging contributes to attention deficits and working memory impairments.

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.