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Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

ABSTRACT: Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2⁺ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1^Tph2 ^-high versus r2-Pet1^Vglut3 ^-high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin⁺ versus VGLUT3⁺ identity. The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum. Thus r2-Pet1^Tph2- ^high and r2-Pet1^Vglut3- ^high neurons likely regulate distinct brain regions and behaviors. Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3⁺ or VGLUT3⁺/5-HT⁺ identity; this suggests that some r2-Pet1^Vglut3- ^high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling. Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation. Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerations.SIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe. The results are significant because they suggest that disparate neuronal subsystems derive from Pet1 ⁺ precursor cells of the embryonic progenitor compartment rhombomere 2 (r2). Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus. Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex. Some r2-Pet1 boutons-those that are VGLUT3⁺ or VGLUT3⁺/5-HT⁺ co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling. Results inform brain organization and circuit nodes for therapeutic consideration.

SUBMITTER: Senft RA

PROVIDER: S-EPMC8018730 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei.SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO2Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.

Project description:Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT2B-receptor stimulation by BW723C86 counteracted 5-HT1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b 5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT2B receptor expression in serotonergic neurons. In Htr2b 5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b lox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT1A-autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT2B receptor acts as a direct positive modulator of serotonin Pet1-positive neurons in an opposite way as the known 5-HT1A-negative autoreceptor.

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Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

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