Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

Normal development of spinal axons in early embryo stages and posterior locomotor function is independent of GAL‐1

It was recently described that Galectin‐1 (Gal‐1) promotes axonal growth after spinal cord injury. This effect depends on protein dimerization, since monomeric Gal‐1 fails to stimulate axonal re‐growth. Gal‐1 is expressed in vivo at concentrations that favor the monomeric species. The aim of the present study is to investigate whether endogenous Gal‐1 is required for spinal axon development and normal locomotor behavior in mice. In order to characterize axonal development, we used a novel combination of 3‐DISCO technique with 1‐photon microscopy and epifluorescence microscopy under high power LED illumination, followed by serial image section deconvolution and 3‐D reconstruction. Cleared whole lgals‐1^‐/‐ embryos were used to analyze the 3‐D cytoarchitecture of motor, commissural, and sensory axons. This approach allowed us to evaluate axonal development, including the number of fibers, fluorescence density of the fiber tracts, fiber length as well as the morphology of axonal sprouting, deep within the tissue. Gal‐1 deficient embryos did not show morphological/anatomical alterations in any of the axonal populations and parameters analyzed. In addition, specific guidance receptor PlexinA4 did not change its axonal localization in the absence of Gal‐1. Finally, Gal‐1 deficiency did not change normal locomotor activity in post‐natal animals. Taken together, our results show that development of spinal axons as well as the locomotor abilities observed in adult mice are independent of Gal‐1. Supporting our previous observations, the present study further validates the use of lgals‐1^‐/‐ mice to develop spinal cord‐ or traumatic brain injury models for the evaluation of the regenerative action of Gal‐1.     

Post‐crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post‐crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post‐crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post‐crossing dI1 axons in mice, using the Atoh1 enhancer‐based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post‐crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post‐crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.     

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: Are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c‐Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro‐enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2‐deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2‐deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.     

Inhibitory neuron‐specific Cre‐dependent red fluorescent labeling using VGAT BAC‐based transgenic mouse lines with identified transgene integration sites

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre‐dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)‐BAC, named VGAT‐stop‐tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron‐specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration‐related genomic disruption. Robust and inhibitory neuron‐specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT‐stop‐tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4‐CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X‐chromosome and will be used to map X‐chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well‐characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.     

An optimized dosing regimen of cimaglermin (neuregulin 1β3, glial growth factor 2) enhances molecular markers of neuroplasticity and functional recovery after permanent ischemic stroke in rats

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb‐ and hindlimb‐placing scores (proprioceptive behavioral tests), body‐swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin‐mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose‐frequency study used the dose‐ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose‐ and frequency‐dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth‐associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Parkinsonian monkeys with prior levodopa‐induced dyskinesias followed by fetal dopamine precursor grafts do not display graft‐induced dyskinesias

Clinical trials testing the hypothesis that fetal dopamine grafts would provide antiparkinsonian benefit in patients who had already developed side effects from their long‐term use of L‐dopa revealed, in some cases, the presence of dyskinesias even in the absence of L‐dopa. The form, intensity, and frequency of these dyskinesias were quite variable, but their manifestation slowed the clinical development of cell replacement therapies. Rodent models of graft‐induced dyskinesias (GIDs) have been proposed, but their accuracy in modeling GIDs has been questioned because they usually require amphetamine for their presentation. The present study attempted to model GIDs in parkinsonian monkeys and, for the first time, to test the effect of grafts on previously dyskinetic monkeys. Toward this end, monkeys were rendered parkinsonian with n‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and dyskinetic with levodopa. They then received intraputamenal grafts of fetal dopaminergic cells, control cerebellar cells, or vehicle bilaterally and were studied for 18 months. Dopaminergic cells were grafted in a manner designed to produce either “hot spot” or “widespread” striatal innervation. Although levodopa‐induced dyskinesias could be elicited postoperatively, GIDs were never observed in any animal at any time after grafting. Grafted monkeys were also challenged with levodopa but did not show any greater responses to these challenges than before grafting. These studies support the development of future dopamine neuron cell transplantation therapy‐based approaches, indicating that in relevant primate models with appropriate cell preparation methodology, with successful graft survival and putamenal dopamine innervation, there is no evidence of graft‐induced dyskinesias. J. Comp. Neurol. 525:498–512, 2017. © 2016 Wiley Periodicals, Inc.     

Sushi repeat‐containing protein X‐linked 2: A novel phylogenetically conserved hypothalamo‐pituitary protein

Sushi repeat‐containing protein X‐linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type‐specific expression was verified by double‐labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2‐ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2‐ir cells were also found in the adenohypophysis. Light SRPX2‐ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo‐pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.     

Morphology of P2X3‐immunoreactive nerve endings in the rat tracheal mucosa

Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole‐mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3‐immunoreactive nerve endings ramified into several branches that extended two‐dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3‐immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3‐immunoreactive endings were beaded, rounded, or club‐like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB‐labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3‐immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.     

5HTR3A‐driven GFP labels immature olfactory sensory neurons

The ionotropic serotonin receptor, 5‐HT₃, is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5‐HT₃ is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5‐HT_3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5‐HT_3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5‐HT_3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP‐labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP‐labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5‐HT_3a is indicative of a proliferative or developmental state, regardless of age, and that the 5‐HT_3AGFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743–1755, 2017. © 2016 Wiley Periodicals, Inc.     

Adult‐specific insulin‐producing neurons in Drosophila melanogaster

Holometabolous insects undergo metamorphosis to reorganize their behavioral and morphological features into adult‐specific ones. In the central nervous system (CNS), some larval neurons undergo programmed cell death, whereas others go through remodeling of axonal and dendritic arbors to support functions of re‐established adult organs. Although there are multiple neuropeptides that have stage‐specific roles in holometabolous insects, the reorganization pattern of the entire neuropeptidergic system through metamorphosis still remains largely unclear. In this study, we conducted a mapping and lineage tracing of peptidergic neurons in the larval and adult CNS by using Drosophila genetic tools. We found that Diuretic hormone 44‐producing median neurosecretory cells start expressing Insulin‐like peptide 2 in the pharate adult stage. This neuronal cluster projects to the corpora cardiaca and dorsal vessel in both larval and adult stages, and also innervates an adult‐specific structure in the digestive tract, the crop. We propose that the adult‐specific insulin‐producing cells may regulate functions of the digestive system in a stage‐specific manner. Our study provides a neuroanatomical basis for understanding remodeling of the neuropeptidergic system during insect development and evolution.     

Sodium channel subtypes are differentially localized to pre‐ and post‐synaptic sites in rat hippocampus

Voltage‐gated Na⁺ channels (Na_v) modulate neuronal excitability, but the roles of the various Na_v subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Na_v subtypes (Na_v1.1, Na_v1.2, Na_v1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Na_v subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre‐ and post‐synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Na_v1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Na_v1.2 and Na_v1.6 were expressed both pre‐ and post‐synaptically. In contrast, in the stratum radiatum (SR) of CA1, Na_v1.1, Na_v1.2, and Na_v1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Na_v subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Na_v1.1 and Na_v1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Na_v1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Na_v subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Na_v expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.     

Parvalbumin‐expressing ependymal cells in rostral lateral ventricle wall adhesions contribute to aging‐related ventricle stenosis in mice

Aging‐associated ependymal‐cell pathologies can manifest as ventricular gliosis, ventricle enlargement, or ventricle stenosis. Ventricle stenosis and fusion of the lateral ventricle (LV) walls is associated with a massive decline of the proliferative capacities of the stem cell niche in the affected subventricular zone (SVZ) in aging mice. We examined the brains of adult C57BL/6 mice and found that ependymal cells located in the adhesions of the medial and lateral walls of the rostral LVs upregulated parvalbumin (PV) and displayed reactive phenotype, similarly to injury‐reactive ependymal cells. However, PV+ ependymal cells in the LV‐wall adhesions, unlike injury‐reactive ones, did not express glial fibrillary acidic protein. S100B+/PV+ ependymal cells found in younger mice diminished in the LV‐wall adhesions throughout aging. We found that periventricular PV‐immunofluorescence showed positive correlation to the grade of LV stenosis in nonaged mice (<10‐month‐old), and that the extent of LV‐wall adhesions and LV stenosis was significantly lower in mid‐aged (>10‐month‐old) PV‐knock out (PV‐KO) mice. This suggests an involvement of PV+ ependymal cells in aging‐associated ventricle stenosis. Additionally, we observed a time‐shift in microglial activation in the LV‐wall adhesions between age‐grouped PV‐KO and wild‐type mice, suggesting a delay in microglial activation when PV is absent from ependymal cells. Our findings implicate that compromised ependymal cells of the adhering ependymal layers upregulate PV and display phenotype shift to “reactive” ependymal cells in aging‐related ventricle stenosis; moreover, they also contribute to the progression of LV‐wall fusion associated with a decline of the affected SVZ‐stem cell niche in aged mice.     

Morphological and functional changes in TRPM8‐expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8‐expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8^BAC‐EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low‐threshold, robust responses to cooling. The remaining TRPM8⁺ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low‐firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8⁺ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low‐background activity and abnormal responsiveness to cooling pulses. These morpho‐functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age‐induced abnormal tearing and to the high incidence of DED in elderly people.     

Molecular heterogeneity of aggrecan‐based perineuronal nets around five subclasses of parvalbumin‐expressing neurons in the mouse hippocampus

Subsets of GABAergic neurons are surrounded by perineuronal nets (PNNs), which play a critical role in the regulation of neural plasticity and neuroprotection. Although the plant lectin Wisteria floribunda agglutinin (WFA) has been commonly used to label PNNs, WFA only detects N‐acetyl‐d ‐galactosamine on aggrecan, a member of the lectican family. In this study, we used WFA and the antibody against the core protein of aggrecan (ACAN) to investigate the molecular heterogeneity of aggrecan‐based PNNs around five subclasses of parvalbumin‐expressing (PV+) γ‐aminobutyric acid (GABA)ergic neurons in the CA1 and CA3 regions of the mouse hippocampus. The vast majority of ACAN+ PNNs were colocalized with WFA in the stratum pyramidale, whereas a substantial population of ACAN+ PNNs lacked WFA labeling in the stratum oriens. We then defined the subclasses of PV+ neurons based on their cellular locations, molecular expression, and septal projection. Like the WFA+ PNNs, ACAN+ PNNs surrounded PV+ basket cells and bistratified cells but not axo‐axonic cells. Unlike the WFA+ PNNs, ACAN+ PNNs frequently surrounded PV+ oriens‐lacunosum moleculare cells and hippocampo‐septal cells. Interestingly, the relative densities of GABAergic synapses were higher around PV+ neurons with ACAN+ PNNs than around those without ACAN+ PNNs. Degradation of WFA+ PNNs by chondroitinase ABC did not affect the GABAergic synaptic densities around PV+ neurons. Our findings suggest that the molecular composition of aggrecan‐based PNNs around PV+ neurons may differ in a subclass‐specific manner, and also might help determine the functional involvement of PNNs in the regulation of GABAergic synapses around PV+ neurons in the hippocampus. J. Comp. Neurol. 525:1234–1249, 2017. © 2016 Wiley Periodicals, Inc.     

Neurotransmitter diversity in pre‐synaptic terminals located in the parvicellular neuroendocrine paraventricular nucleus of the rat and mouse hypothalamus

Virtually all rodent neuroendocrine corticotropin‐releasing‐hormone (CRH) neurons are in the dorsal medial parvicellular (mpd) part of the paraventricular nucleus of the hypothalamus (PVH). They form the final common pathway for adrenocortical stress responses. Their activity is controlled by sets of GABA‐, glutamate‐, and catecholamine‐containing inputs arranged in an interactive pre‐motor network. Defining the nature and arrangement of these inputs can help clarify how stressor type and intensity information is conveyed to neuroendocrine neurons. Here we use immunohistochemistry with high‐resolution 3‐dimensional image analyses to examine the arrangement of single‐ and co‐occurring GABA, glutamate, and catecholamine markers in synaptophysin‐defined pre‐synaptic terminals in the PVHmpd of unstressed rats and Crh‐IRES‐Cre;Ai14 transgenic mice: respectively, vesicular glutamate transporter 2 (VGluT2), vesicular GABA transporter (VGAT), dopamine β‐hydroxylase (DBH), and phenylethanolamine n ‐methyltransferase (PNMT). Just over half of all PVHmpd pre‐synaptic terminals contain VGAT, with slightly less containing VGluT2. The vast majority of terminal appositions with mouse CRH neurons occur non‐somatically. However, there are significantly more somatic VGAT than VGluT2 appositions. In the rat PVHmpd, about five times as many pre‐synaptic terminals contain PNMT than DBH only. However, because epinephrine release has never been detected in the PVH, PNMT terminals may functionally be noradrenergic not adrenergic. PNMT and VGluT2 co‐occur in some pre‐synaptic terminals indicating the potential for co‐transmission of glutamate and norepinephrine. Collectively, these results provide a structural basis for how GABA/glutamate/catecholamine interactions enable adrenocortical responses to fast‐onset interosensory stimuli, and more broadly, how combinations of PVH neurotransmitters and neuromodulators interact dynamically to control adrenocortical activity.     

Kölliker–Fuse GABAergic and glutamatergic neurons project to distinct targets

The Kölliker‐Fuse nucleus (KF) is known primarily for its respiratory function as the “pneumotaxic center” or “pontine respiratory group.” Considered part of the parabrachial (PB) complex, KF contains glutamatergic neurons that project to respiratory‐related targets in the medulla and spinal cord (Yokota, Oka, Tsumori, Nakamura, & Yasui, 2007). Here we describe an unexpected population of neurons in the caudal KF and adjacent lateral crescent subnucleus (PBlc), which are γ‐aminobutyric acid (GABA)ergic and have an entirely different pattern of projections than glutamatergic KF neurons. First, immunofluorescence, in situ hybridization, and Cre‐reporter labeling revealed that many of these GABAergic neurons express FoxP2 in both rats and mice. Next, using Cre‐dependent axonal tracing in Vgat‐IRES‐Cre and Vglut2‐IRES‐Cre mice, we identified different projection patterns from GABAergic and glutamatergic neurons in this region. GABAergic neurons in KF and PBlc project heavily and almost exclusively to trigeminal sensory nuclei, with minimal projections to cardiorespiratory nuclei in the brainstem, and none to the spinal cord. In contrast, glutamatergic KF neurons project heavily to the autonomic, respiratory, and motor regions of the medulla and spinal cord previously identified as efferent targets mediating KF cardiorespiratory effects. These findings identify a novel, GABAergic subpopulation of KF/PB neurons with a distinct efferent projection pattern targeting the brainstem trigeminal sensory system. Rather than regulating breathing, we propose that these neurons influence vibrissal sensorimotor function.