A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility, and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a novel leptin-sensing neuronal population, multiple AgRP and POMC subtypes, and an orexigenic somatostatin neuronal population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinctly responsive subtypes of AgRP and POMC neurons. Finally, integrating our data with human GWAS data implicates two previously unknown neuronal subtypes in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred

Heterosynaptic long-term potentiation at GABAergic synapses of spinal lamina I neurons

Neuronen in Lamina I des Rückenmarks spielen eine zentrale Rolle bei der Verarbeitung nozizeptiver Information. Das funktionelle Gleichgewicht der synaptischen Erregung und Hemmung dieser Neuronen ist entscheidend für eine intakte Schmerzwahrnehmung. Die synaptische Langzeitpotenzierung (LTP) zwischen nozizeptiven, erregenden afferenten C-Fasern und Neuronen in Lamina I stellt ein zelluläres Modell der Schmerzverstärkung dar. Bisher ist jedoch nicht bekannt, ob eine Langzeitplastizität auch an hemmenden Synapsen im oberflächlichen Rückenmark induziert werden kann. In verschiedenen Gehirnarealen wird die Langzeitplastizität hemmender Synapsen durch die Aktivität erregender Synapsen induziert. Wir vermuten daher, dass eine konditionierende Stimulation von nozizeptiven primären Afferenzen die Übertragungsstärke hemmender GABAerger Synapsen an Lamina I Neuronen beeinflusst. Um diese Hypothese zu untersuchen, wurden elektrophysiologische Messungen an Neuronen in Lamina I in Schnittpräparaten des Rückenmarks der Ratte mit anhängender Hinterwurzel durchgeführt. Monosynaptische, GABAerge postsynaptische Ströme wurden in Gegenwart von ionotropen Glutamat- und Glyzinrezeptor-Antagonisten fokal evoziert. Die konditionierende Stimulation der primären Afferenzen, mit einem Stimulationsprotokoll, das LTP an C-Faser Synapsen induziert, triggerte auch eine LTP der GABAergen Synapsen an Lamina I Neuronen (LTPGABA). Nach dem Auswaschen der Antagonisten am Ende der Messungen wurde der synaptische Zustrom von den stimulierten nozizeptiven Fasern untersucht. LTPGABA konnte nur bei den Neuronen induziert werden, die monosynaptischen A[delta]- oder C-Faser Input bekamen. Diese neuartige LTPGABA wurde heterosynaptisch induziert und durch metabotrope Glutamatrezeptoren der Gruppe 1 mediiert, wohingegen die Aktivierung ionotroper AMPA/KA und NMDA Rezeptoren für die Induktion nicht notwendig war. Analysen der "paired-pulse ratio", des "coefficient of variation" und der Aktionspotenzial-unabhängigen "miniature IPSCs" bestätigten, dass die LTPGABA präsynaptisch exprimiert war. Stickoxid als retrograder Botenstoff mediierte diese Erhöhung der Freisetzungswahrscheinlichkeit von GABA. Durch Blockade der Stickoxid-Synthetase wurde die LTPGABA zu einer Langzeitdepression GABAerger Synapsen an Lamina I Neuronen (LTDGABA) umgekehrt. Die Aktivierung des Typ 1 Cannabinoid-Rezeptors (CB1Rs) verringerte die Freisetzungswahrscheinlichkeit von GABA. Blockade des CB1Rs verhinderte sowohl die Induktion von LTPGABA als auch von LTDGABA. Diese Ergebnisse zeigen einen neuartigen, komplexen Wirkmechanismus zwischen Endocannabinoiden und Stickoxid im Dorsalhorn des Rückenmarks. Die neuartige LTPGABA scheint einen grundlegenden Mechanismus bei der Verarbeitung nozizeptiver Information darzustellen. Die Erregung und Hemmung wird im physiologischen Gleichgewicht gehalten und das Hintergrundrauschen in spinalen nozizeptiven Bahnen reduziert.Neurons in spinal dorsal horn lamina I play a pivotal role for nociception which critically depends on a proper balance between excitatory and inhibitory inputs. Any modification in synaptic strength may challenge this delicate balance. Long-term potentiation (LTP) at glutamatergic synapses between nociceptive C-fibers and lamina I neurons is an intensively studied cellular model of pain amplification. In contrast, nothing is presently known about long-term changes of synaptic strength at inhibitory synapses in the spinal dorsal horn. Long-term plasticity at inhibitory synapses in various brain regions is mainly mediated by activation of excitatory synapses. We propose that conditioning stimulation of primary afferents would affect GABAergic synapses impinging onto lamina I neurons. In order to test this hypothesis, electrophysiological recordings from lamina I neurons were obtained in a spinal cord-dorsal root slice preparation from rats. Monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) were evoked by a focal stimulation electrode placed ventrally to the patched neuron in the presence of ionotropic glutamate and glycine receptor antagonists. Conditioning stimulation of primary afferents with a stimulating protocol that induces LTP at C-fiber synapses also triggered LTP at GABAergic synapses (LTPGABA). Analysis of the excitatory input from attached dorsal roots after wash-out of all drugs revealed that LTPGABA could only be induced in lamina I neurons with monosynaptic A[delta]- or monosynaptic C-fiber input. This novel form of LTPGABA was heterosynaptic in nature, mediated by group I metabotropic glutamate receptors (mGluRs) whereas opening of ionotropic glutamate receptor channels of the AMPA/KA or NMDA subtype was not required. Paired-pulse ratio, coefficient of variation and miniature IPSCs analysis revealed that LTPGABA was expressed presynaptically. Nitric oxide (NO) as a retrograde messenger signal mediated this increase of GABA release at spinal inhibitory synapses. Blocking NO production converted LTPGABA to long-term depression of GABAergic synapses (LTDGABA) at spinal lamina I neurons. GABAergic input to lamina I neurons was also depressed by activation of the type 1 cannabinoid receptors (CB1Rs). Blockade of CB1Rs prevented the induction of both LTPGABA and LTDGABA suggesting a complex interaction of endocannabinoid (eCB) and NO signalling. This novel form of LTPGABA in spinal nociceptive circuits may be an essential mechanism to maintain the relative balance between excitation and inhibition and to improve the signal-to-noise ratio in nociceptive pathways.submitted by Henning FenselauAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2012OeBB(VLID)171427

Technical Note: Modulation of fMRI brainstem responses by transcutaneous vagus nerve stimulation

Our increasing knowledge about gut-brain interaction is revolutionising the understanding of the links between digestion, mood, health, and even decision making in our everyday lives. In support of this interaction, the vagus nerve is a crucial pathway transmitting diverse gut-derived signals to the brain to monitor of metabolic status, digestive processes, or immune control to adapt behavioural and autonomic responses. Hence, neuromodulation methods targeting the vagus nerve are currently explored as a treatment option in a number of clinical disorders, including diabetes, chronic pain, and depression. The non-invasive variant of vagus nerve stimulation (VNS), transcutaneous auricular VNS (taVNS), has been implicated in both acute and long-lasting effects by modulating afferent vagus nerve target areas in the brain. The physiology of neither of those effects is, however, well understood, and evidence for neuronal response upon taVNS in vagal afferent projection regions in the brainstem and its downstream targets remain to be established. Therefore, to examine time-dependent effects of taVNS on brainstem neuronal responses in healthy human subjects, we applied taVNS during task-free fMRI in a single-blinded crossover design. During fMRI data acquisition, we either stimulated the left earlobe (sham), or the target zone of the auricular branch of the vagus nerve in the outer ear (cymba conchae, verum) for several minutes, both followed by a short ‘stimulation OFF’ period. Time-dependent effects were assessed by averaging the BOLD response for consecutive 1-minute periods in an ROI-based analysis of the brainstem. We found a significant response to acute taVNS stimulation, relative to the control condition, in downstream targets of vagal afferents, including the nucleus of the solitary tract, the substantia nigra, and the subthalamic nucleus. Most of these brainstem regions remarkably showed increased activity in response to taVNS, and these effect sustained during the post-stimulation period. These data demonstrate that taVNS activates key brainstem regions, and highlight the potential of this approach to modulate vagal afferent signalling. Furthermore, we show that carry-over effects need to be considered when interpreting fMRI data in the context of general vagal neurophysiology and its modulation by taVNS

Technical Note: Modulation of fMRI brainstem responses by transcutaneous vagus nerve stimulation

Our increasing knowledge about gut-brain interaction is revolutionising the understanding of the links between digestion, mood, health, and even decision making in our everyday lives. In support of this interaction, the vagus nerve is a crucial pathway transmitting diverse gut-derived signals to the brain to monitor of metabolic status, digestive processes, or immune control to adapt behavioural and autonomic responses. Hence, neuromodulation methods targeting the vagus nerve are currently explored as a treatment option in a number of clinical disorders, including diabetes, chronic pain, and depression. The non-invasive variant of vagus nerve stimulation (VNS), transcutaneous auricular VNS (taVNS), has been implicated in both acute and long-lasting effects by modulating afferent vagus nerve target areas in the brain. The physiology of neither of those effects is, however, well understood, and evidence for neuronal response upon taVNS in vagal afferent projection regions in the brainstem and its downstream targets remain to be established. Therefore, to examine time-dependent effects of taVNS on brainstem neuronal responses in healthy human subjects, we applied taVNS during task-free fMRI in a single-blinded crossover design. During fMRI data acquisition, we either stimulated the left earlobe (sham), or the target zone of the auricular branch of the vagus nerve in the outer ear (cymba conchae, verum) for several minutes, both followed by a short 'stimulation OFF' period. Time-dependent effects were assessed by averaging the BOLD response for consecutive 1-minute periods in an ROI-based analysis of the brainstem. We found a significant response to acute taVNS stimulation, relative to the control condition, in downstream targets of vagal afferents, including the nucleus of the solitary tract, the substantia nigra, and the subthalamic nucleus. Most of these brainstem regions remarkably showed increased activity in response to taVNS, and these effect sustained during the post-stimulation period. These data demonstrate that taVNS activates key brainstem regions, and highlight the potential of this approach to modulate vagal afferent signalling. Furthermore, we show that carry-over effects need to be considered when interpreting fMRI data in the context of general vagal neurophysiology and its modulation by taVNS

The Role of Mediobasal Hypothalamic PACAP in the Control of Body Weight and Metabolism

Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus.This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, adeno-associated virus (AAV(Cre)) injections to ablate Adcyapl (gene coding for PACAP) in mice (Adcyap1(MBH)KO mice). Adcyap1(MBH)KO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1(MBH)KO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to brown adipose tissue (BAT) dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis.While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1(MBH)KO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of functions for PACAP in overall energy homeostasis

Gut-brain communication by distinct sensory neurons differently controls feeding and glucose metabolism

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control

Hypothalamic Pomc Neurons Innervate the Spinal Cord and Modulate the Excitability of Premotor Circuits

Locomotion requires energy, yet animals need to increase locomotion in order to find and consume food in energy-deprived states. While such energy homeostatic coordination suggests brain origin, whether the central melanocortin 4 receptor (Mc4r) system directly modulates locomotion through motor circuits is unknown. Here, we report that hypothalamic Pomc neurons in zebrafish andmice have long-range projections into spinal cord regions harboring Mc4r-expressing V2a interneurons, crucial components of the premotor networks. Furthermore, in zebrafish, Mc4r activation decreases the excitability of spinal V2a neurons as well as swimming and foraging, while systemic or V2a neuron-specific blockage of Mc4r promotes locomotion. In contrast, in mice, electrophysiological recordings revealed that two-thirds of V2a neurons in lamina X are excited by the Mc4r agonist alpha-MSH, and acute inhibition of Mc4r signaling reduces locomotor activity. In addition, we found other Mc4r neurons in spinal lamina X that are inhibited by alpha-MSH, which is in line with previous studies in rodents where Mc4r agonists reduced locomotor activity. Collectively, our studies identify spinal V2a interneurons as evolutionary conserved second-order neurons of the central Mc4r system, providing a direct anatomical and functional link between energy homeostasis and locomotor control systems. The net effects of this modulatory system on locomotor activity can vary between different vertebrate species and, possibly, even within one species. We discuss the biological sense of this phenomenon in light of the ambiguity of locomotion on energy balance and the different living conditions of the different species

A rapidly-acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by α-MSH

Arcuate nucleus (ARC) neurons sense the fed/fasted state and regulate hunger. Agouti-related protein (ARCAgRP) neurons are stimulated by fasting, and once activated, they rapidly (within minutes) drive hunger. Pro-opiomelanocortin (ARCPOMC) neurons are viewed as the counterpoint to ARCAgRP neurons. They are regulated in an opposite fashion and decrease hunger. However, unlike ARCAgRP neurons, ARCPOMC neurons are extremely slow in affecting hunger (many hours). Thus, a temporally analogous, rapid ARC satiety pathway does not exist or is presently unidentified. Here, we show that glutamate-releasing ARC neurons expressing oxytocin receptor, unlike ARCPOMC neurons, rapidly cause satiety when chemo- or optogenetically manipulated. These glutamatergic ARC projections synaptically converge with GABAergic ARCAgRP projections on melanocortin-4 receptor (MC4R)-expressing satiety neurons in the paraventricular hypothalamus (PVHMC4R neurons). Importantly, transmission across the ARCGlutamatergic→PVHMC4R synapse is potentiated by the ARCPOMC neuron-derived MC4R agonist, α-MSH. This excitatory ARC→PVH satiety circuit, and its modulation by α-MSH, provides new insight into regulation of hunger/satiety

A hypothalamic circuit for the circadian control of aggression

'Sundowning' in dementia and Alzheimer's disease is characterized by early-evening agitation and aggression. While such periodicity suggests a circadian origin, whether the circadian clock directly regulates aggressive behavior is unknown. We demonstrate that a daily rhythm in aggression propensity in male mice is gated by GABAergic subparaventricular zone (SPZ(GABA)) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZ(GABA) neurons receive input from vasoactive intestinal polypeptide suprachiasmatic nucleus neurons and innervate neurons in the ventrolateral part of the ventromedial hypothalamus (VMH), which is known to regulate aggression. Additionally, VMH-projecting dorsal SPZ neurons are more active during early day than early night, and acute chemogenetic inhibition of SPZ(GABA) transmission phase-dependently increases aggression. Finally, SPZ(GABA)-recipient central VMH neurons directly innervate ventrolateral VMH neurons, and activation of this intra-VMH circuit drove attack behavior. Altogether, we reveal a functional polysynaptic circuit by which the suprachiasmatic nucleus clock regulates aggression