Yang, SH;Yang, E;Lee, J;Kim, JY;Yoo, H;Park, HS;Jung, JT;Lee, D;Chun, S;Jo, YS;Pyeon, GH;Park, JY;Lee, HW;Kim, H;
PMID: 37105975 | DOI: 10.1038/s41467-023-38180-7
Stress management is necessary for vertebrate survival. Chronic stress drives depression by excitation of the lateral habenula (LHb), which silences dopaminergic neurons in the ventral tegmental area (VTA) via GABAergic neuronal projection from the rostromedial tegmental nucleus (RMTg). However, the effect of acute stress on this LHb-RMTg-VTA pathway is not clearly understood. Here, we used fluorescent in situ hybridisation and in vivo electrophysiology in mice to show that LHb aromatic L-amino acid decarboxylase-expressing neurons (D-neurons) are activated by acute stressors and suppress RMTg GABAergic neurons via trace aminergic signalling, thus activating VTA dopaminergic neurons. We show that the LHb regulates RMTg GABAergic neurons biphasically under acute stress. This study, carried out on male mice, has elucidated a molecular mechanism in the efferent LHb-RMTg-VTA pathway whereby trace aminergic signalling enables the brain to manage acute stress by preventing the hypoactivity of VTA dopaminergic neurons.
Hansell CAH, Love S, Pingen M, Wilson GJ, MacLeod M, Graham GJ
PMID: 32114694 | DOI: 10.1002/eji.201948374
Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa-Fluor-labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa-Fluor-labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment.
Cheadle L, Tzeng CP, Kalish BT, Harmin DA, Rivera S, Ling E, Nagy MA, Hrvatin S, Hu L, Stroud H, Burkly LC, Chen C, Greenberg ME.
PMID: 30033152 | DOI: 10.1016/j.neuron.2018.06.036
Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.
Hsu, LJ;Bertho, M;Kiehn, O;
PMID: 36797254 | DOI: 10.1038/s41467-023-36587-w
Locomotion empowers animals to move. Locomotor-initiating signals from the brain are funneled through descending neurons in the brainstem that act directly on spinal locomotor circuits. Little is known in mammals about which spinal circuits are targeted by the command and how this command is transformed into rhythmicity in the cord. Here we address these questions leveraging a mouse brainstem-spinal cord preparation from either sex that allows locating the locomotor command neurons with simultaneous Ca2+ imaging of spinal neurons. We show that a restricted brainstem area - encompassing the lateral paragigantocellular nucleus (LPGi) and caudal ventrolateral reticular nucleus (CVL) - contains glutamatergic neurons which directly initiate locomotion. Ca2+ imaging captures the direct LPGi/CVL locomotor initiating command in the spinal cord and visualizes spinal glutamatergic modules that execute the descending command and its transformation into rhythmic locomotor activity. Inhibitory spinal networks are recruited in a distinctly different pattern. Our study uncovers the principal logic of how spinal circuits implement the locomotor command using a distinct modular organization.
Lecoin, L;Dempsey, B;Garancher, A;Bourane, S;Ruffault, PL;Morin-Surun, MP;Rocques, N;Goulding, M;Eychène, A;Pouponnot, C;Fortin, G;Champagnat, J;
PMID: 35672398 | DOI: 10.1038/s41467-022-30825-3
While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.
Low, AYT;Goldstein, N;Gaunt, JR;Huang, KP;Zainolabidin, N;Yip, AKK;Carty, JRE;Choi, JY;Miller, AM;Ho, HST;Lenherr, C;Baltar, N;Azim, E;Sessions, OM;Ch'ng, TH;Bruce, AS;Martin, LE;Halko, MA;Brady, RO;Holsen, LM;Alhadeff, AL;Chen, AI;Betley, JN;
PMID: 34789878 | DOI: 10.1038/s41586-021-04143-5
The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.
Fgf15 neurons of the dorsomedial hypothalamus control glucagon secretion and hepatic gluconeogenesis
Picard, A;Metref, S;Tarussio, D;Dolci, W;Berney, X;Croizier, S;Labouebe, G;Thorens, B;
PMID: 33883213 | DOI: 10.2337/db20-1121
The counterregulatory response to hypoglycemia is an essential survival function. It is controlled by an integrated network of glucose responsive neurons, which trigger endogenous glucose production to restore normoglycemia. The complexity of this gluco-regulatory network is, however, only partly characterized. In a genetic screen of a panel of recombinant inbred mice we previously identified Fgf15, expressed in neurons of the dorsomedial hypothalamus, as a negative regulator of glucagon secretion. Here, we report on the generation of Fgf15CretdTomato mice and their use to further characterize these neurons. We showed that they were glutamatergic and comprised glucose inhibited and glucose excited neurons. When activated by chemogenetics, Fgf15 neurons prevented the increase in vagal nerve firing and the secretion of glucagon normally triggered by insulin-induced hypoglycemia. On the other hand, they increased the activity of the sympathetic nerve in the basal state and prevented its silencing by glucose overload. Higher sympathetic tone increased hepatic Creb1 phosphorylation, Pck1 mRNA expression, and hepatic glucose production leading to glucose intolerance. Thus, Fgf15 neurons of the dorsomedial hypothalamus participate in the counterregulatory response to hypoglycemia by a direct adrenergic stimulation of hepatic glucose production while suppressing vagally-induced glucagon secretion. This study provides new insights into the complex neuronal network that prevents the development of hypoglycemia.
Fluri F, Mützel T, Schuhmanna MK, Krstić M, Endres H, Volkmann J.
PMID: 28842194 | DOI: 10.1016/j.jneumeth.2017.08.024
Abstract
BACKGROUND:
Commercial neurostimulators for clinical use are effective in patients; however they are too large and prohibitively expensive for preclinical studies. Thus, there is an urgent need of a small inexpensive and wireless microstimulator which is fully programmable in frequency, pulse width and amplitude for rodent experiments.
NEW METHODS:
Rats were subjected to a photothrombotic stroke of the right sensorimotor cortex and a microelectrode was implanted in the right mesencephalic locomotor region. The microstimulator was connected with the head plug of the rat. Three different stimulation frequencies were applied and different stimulating amplitudes were chosen. Under these conditions, gait velocity and locomotor behavior of six rats were examined on a beam.
RESULTS:
The head-mounted microstimulator allowed freedom in all motor activities performed spontaneously by the tested rats. Increasing either the frequency or the stimulating amplitude increased gait velocity and ameliorated locomotor behavior after stroke.
COMPARISON WITH EXISTING METHODS:
Other devices for DBS in rodents must be implanted under the skin or worn in an animal jacket on the back by the tested rat. Some available systems require even a tethering of the tested animal via a cable to an external stimulation system, which limits the freedom of movement.
CONCLUSION:
Here, we present a freely programmable microstimulator including DBS-typical stimulating parameters. The lightweight device is connected by a simple plug to the head allowing full freedom of movement and exchange of batteries for long-term experiments. The design of this stimulator is suitable for sophisticated behavior tests requiring balance and skilled walking.
Liu, X;Wang, Y;Zeng, Y;Wang, D;Wen, Y;Fan, L;He, Y;Zhang, J;Sun, W;Liu, Y;Tao, A;
PMID: 36876522 | DOI: 10.1111/all.15699
Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR+ neurons and promote chronic itch.RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR+ neuron interactions.We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR+ neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β+ microglia are localized in close proximity to GRPR+ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR+ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR+ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.
Shi Y, Stornetta RL, Stornetta DS, Onengut-Gumuscu S, Farber EA, Turner SD, Guyenet PG, Bayliss DA.
PMID: 29066557 | DOI: 10.1523/JNEUROSCI.2055-17.2017
The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, non-cholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Nmb mRNA. Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, Kcnk5 and Gpr4 These Nmb-low RTN neurons also express varying levels of transcripts for Gal, Penk and Adcyap1,and receptors for substance P, orexin, serotonin and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb-high RTN neurons do not express Fos after hypercapnia, have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1, but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb-low neurons, the vast majority, are central respiratory chemoreceptors whereas Nmb-high neurons likely have other functions.SIGNIFICANCE STATEMENTCentral respiratory chemoreceptors regulate arterial PCO2 by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed in situ hybridization, we show here that a single marker, Neuromedin B mRNA (Nmb), identifies RTN neurons in rodents. We also suggest that >75% of these Nmb neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (Kcnk5 and Gpr4). The other RTN neurons express very high levels of Nmb, but low levels of Kcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin, and do not respond to hypercapnia. Their function is unknown.
Cell metabolism, 18(6), 860–870.
Xu, Y, Wu Z, Sun H, Zhu Y, Kim ER, Lowell BB, Arenkiel BR, Xu Y, Tong Q (2013).
PMID: 24315371 | DOI: 10.1016/j.cmet.2013.11.003.
The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.
Frezel, N;Ranucci, M;Foster, E;Wende, H;Pelczar, P;Mendes, R;Ganley, RP;Werynska, K;d'Aquin, S;Beccarini, C;Birchmeier, C;Zeilhofer, HU;Wildner, H;
PMID: 36947543 | DOI: 10.1016/j.celrep.2023.112295
Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.
