Role of the Kölliker-Fuse/Parabrachial Complex in generation of post-inspiratory vagal and sympathetic nerve activities.

In the rat, the activity of laryngeal adductor muscles, the crural diaphragm, and sympathetic vasomotor neurons is entrained to the post-inspiratory (post-I) phase of the respiratory cycle, a mechanism thought to enhance cardiorespiratory efficiency. The identity of the central neurons responsible for transmitting respiratory activity to these outputs remains unresolved. Here we explore the contribution of the Kölliker-Fuse/Parabrachial nuclei (KF-PBN) in the generation of post-I activity in vagal and sympathetic outputs under steady-state conditions and during acute hypoxemia, a condition that potently recruits post-I activity. In artificially ventilated, vagotomised and urethane-anesthetised rats, bilateral KF-PBN inhibition by microinjection of the GABAA receptor agonist isoguvacine evoked stereotypical responses on respiratory pattern, characterised by a reduction in phrenic nerve burst amplitude, a modest lengthening of inspiratory time, and an increase in breath-to-breath variability, while post-I vagal nerve activity was abolished and post-I sympathetic nerve activity diminished. During acute hypoxemia, KF-PBN inhibition attenuated tachypnoeic responses and completely abolished post-I vagal activity while preserving respiratory-sympathetic coupling. Furthermore, KF-PBN inhibition disrupted the decline in respiratory frequency that normally follows resumption of oxygenation. These findings suggest that the KF-PBN is a critical hub for the distribution of post-I activities to vagal and sympathetic outputs and is an important contributor to the dynamic adjustments to respiratory patterns that occur in response to acute hypoxia. While KF-PBN appears essential for post-I vagal activity, it only partially contributes to post-I sympathetic nerve activity, suggesting the contribution of multiple neural pathways to respiratory-sympathetic coupling.

Enhanced AAV transduction across preclinical CNS models: A comparative study in human brain organoids with cross-species evaluations.

Viral vectors based on recombinant adeno-associated virus (rAAV) have become the most widely used system for therapeutic gene delivery in the central nervous system (CNS). Despite clinical safety and efficacy in neurological applications, a barrier to adoption of the current generation of vectors lies in their limited efficiency, resulting in limited transduction of CNS target cells. To address this limitation, researchers have bioengineered fit-for-purpose AAVs with improved CNS tropism and tissue penetration. While the preclinical assessment of these novel AAVs is primarily conducted in animal models, human induced pluripotent stem cell (hiPSC)-derived organoids offer a unique opportunity to functionally evaluate novel AAV variants in a human context. In this study, we performed a comprehensive and unbiased evaluation of a large number of wild-type and bioengineered AAV capsids for their transduction efficiency in hiPSC-derived brain organoids. We demonstrate that efficient AAV transduction observed in organoids was recapitulated in vivo in both mouse and non-human primate models after cerebrospinal fluid (CSF) delivery. In summary, our study showcases the use of brain organoid systems for the pre-screening of novel AAV vectors. Additionally, we report data for novel AAV variants that exhibit improved CNS transduction efficiency when delivered via the CSF in in vivo preclinical models.

Galaninergic and hypercapnia-activated neuronal projections to the ventral respiratory column.

In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO2)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO2, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.

DeepSlice: rapid fully automatic registration of mouse brain imaging to a volumetric atlas.

Registration of data to a common frame of reference is an essential step in the analysis and integration of diverse neuroscientific data. To this end, volumetric brain atlases enable histological datasets to be spatially registered and analyzed, yet accurate registration remains expertise-dependent and slow. In order to address this limitation, we have trained a neural network, DeepSlice, to register mouse brain histological images to the Allen Brain Common Coordinate Framework, retaining registration accuracy while improving speed by >1000 fold.

Direct optogenetic activation of upper airway muscles in an acute model of upper airway hypotonia mimicking sleep onset.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA), where the upper airway collapses repeatedly during sleep due to inadequate dilator muscle tone, is challenging to treat as current therapies are poorly tolerated or have variable and unpredictable efficacy. We propose a novel, optogenetics-based therapy, that stimulates upper airway dilator muscle contractions in response to light. To determine the feasibility of a novel optogenetics-based OSA therapy, we developed a rodent model of human sleep-related upper airway muscle atonia. Using this model, we evaluated intralingual delivery of candidate optogenetic constructs, notably a muscle-targeted approach that will likely have a favorable safety profile. METHODS: rAAV serotype 9 viral vectors expressing a channelrhodopsin-2 variant, driven by a muscle-specific or nonspecific promoter were injected into rat tongues to compare strength and specificity of opsin expression. Light-evoked electromyographic responses were recorded in an acute, rodent model of OSA. Airway dilation was captured with ultrasound. RESULTS: The muscle-specific promoter produced sufficient opsin expression for light stimulation to restore and/or enhance electromyographic signals (linear mixed model, F = 140.0, p < 0.001) and induce visible tongue contraction and airway dilation. The muscle-specific promoter induced stronger (RM-ANOVA, F(1,8) = 10.0, p = 0.013) and more specific opsin expression than the nonspecific promoter in an otherwise equivalent construct. Viral DNA and RNA were robust in the tongue, but low or absent in all other tissues. CONCLUSIONS: Significant functional responses to direct optogenetic muscle activation were achieved following muscle-specific promoter-driven rAAV-mediated transduction, providing proof-of-concept for an optogenetic therapy for patients with inadequate dilator muscle activity during sleep.

Descending pathways from the superior colliculus mediating autonomic and respiratory effects associated with orienting behaviour.

The ability to discriminate competing external stimuli and initiate contextually appropriate behaviours is a key brain function. Neurons in the deep superior colliculus (dSC) integrate multisensory inputs and activate descending projections to premotor pathways responsible for orienting, attention and defence, behaviours which involve adjustments to respiratory and cardiovascular parameters. However, the neural pathways that subserve the physiological components of orienting are poorly understood. We report that orienting responses to optogenetic dSC stimulation are accompanied by short-latency autonomic, respiratory and electroencephalographic effects in awake rats, closely mimicking those evoked by naturalistic alerting stimuli. Physiological responses were not accompanied by detectable aversion or fear, and persisted under urethane anaesthesia, indicating independence from emotional stress. Anterograde and trans-synaptic viral tracing identified a monosynaptic pathway that links the dSC to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA), a key hub for the coordination of orienting and locomotor behaviours. In urethane-anaesthetized animals, sympathoexcitatory and cardiovascular, but not respiratory, responses to dSC stimulation were replicated by optogenetic stimulation of the dSC-GiA terminals, suggesting a likely role for this pathway in mediating the autonomic components of dSC-mediated responses. Similarly, extracellular recordings from putative GiA sympathetic premotor neurons confirmed short-latency excitatory inputs from the dSC. This pathway represents a likely substrate for autonomic components of orienting responses that are mediated by dSC neurons and suggests a mechanism through which physiological and motor components of orienting behaviours may be integrated without the involvement of higher centres that mediate affective components of defensive responses. KEY POINTS: Neurons in the deep superior colliculus (dSC) integrate multimodal sensory signals to elicit context-dependent innate behaviours that are accompanied by stereotypical cardiovascular and respiratory activities. The pathways responsible for mediating the physiological components of colliculus-mediated orienting behaviours are unknown. We show that optogenetic dSC stimulation evokes transient orienting, respiratory and autonomic effects in awake rats which persist under urethane anaesthesia. Anterograde tracing from the dSC identified projections to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA). Stimulation of this pathway recapitulated autonomic effects evoked by stimulation of dSC neurons. Electrophysiological recordings from putative GiA sympathetic premotor neurons confirmed short latency excitatory input from dSC neurons. This disynaptic dSC-GiA-spinal sympathoexcitatory pathway may underlie autonomic adjustments to salient environmental cues independent of input from higher centres.

Upregulated Angiotensin Ia Receptors in the Hypothalamic Paraventricular Nucleus Sensitize Neuroendocrine Vasopressin Release and Blood Pressure in a Rodent Model of Polycystic Kidney Disease.

INTRODUCTION: Angiotensin (Ang) II signalling in the hypothalamic paraventricular nucleus (PVN) via Ang type-1a receptors (AT1R) regulates vasopressin release and sympathetic nerve activity - two effectors of blood pressure regulation. We determined the cellular expression and function of AT1R in the PVN of a rodent model of polycystic kidney disease (PKD), the Lewis polycystic kidney (LPK) rat, to evaluate its contribution to blood pressure regulation and augmented vasopressin release in PKD. METHODS: PVN AT1R gene expression was quantified with fluorescent in situ hybridization in LPK and control rats. PVN AT1R function was assessed with pharmacology under urethane anaesthesia in LPK and control rats instrumented to record arterial pressure and sympathetic nerve activity. RESULTS: AT1R gene expression was upregulated in the PVN, particularly in corticotrophin-releasing hormone neurons, of LPK versus control rats. PVN microinjection of Ang II produced larger increases in systolic blood pressure in LPK versus control rats (36 ± 5 vs. 17 ± 2 mm Hg; p < 0.01). Unexpectedly, Ang II produced regionally heterogeneous sympathoinhibition (renal: -33%; splanchnic: -12%; lumbar: no change) in LPK and no change in controls. PVN pre-treatment with losartan, a competitive AT1R antagonist, blocked the Ang II-mediated renal sympathoinhibition and attenuated the pressor response observed in LPK rats. The Ang II pressor effect was also blocked by systemic OPC-21268, a competitive V1A receptor antagonist, but unaffected by hexamethonium, a sympathetic ganglionic blocker. DISCUSSION/CONCLUSION: Collectively, our data suggest that upregulated AT1R expression in PVN sensitizes neuroendocrine release of vasopressin in the LPK, identifying a central mechanism for the elevated vasopressin levels present in PKD.

Changes in intrathoracic pressure, not arterial pulsations, exert the greatest effect on tracer influx in the spinal cord.

BACKGROUND: Cerebrospinal fluid (CSF) circulation in the brain has garnered considerable attention in recent times. In contrast, there have been fewer studies focused on the spine, despite the expected importance of CSF circulation in disorders specific to the spine, including syringomyelia. The driving forces that regulate spinal CSF flow are not well defined and are likely to be different to the brain given the anatomical differences and proximity to the heart and lungs. The aims of this study were to determine the effects of heart rate, blood pressure and respiration on the distribution of CSF tracers in the spinal subarachnoid space, as well as into the spinal cord interstitium. METHODS: In Sprague Dawley rats, physiological parameters were manipulated such that the effects of spontaneous breathing (generating alternating positive and negative intrathoracic pressures), mechanical ventilation (positive intrathoracic pressure only), tachy/bradycardia, as well as hyper/hypotension were separately studied. To investigate spinal CSF hydrodynamics, in vivo near-infrared imaging of intracisternally infused indocyanine green was performed. CSF tracer transport was further characterised with in vivo two-photon intravital imaging. Tracer influx at a microscopic level was quantitatively characterised by ex vivo epifluorescence imaging of fluorescent ovalbumin. RESULTS: Compared to mechanically ventilated controls, spontaneous breathing animals had significantly greater movement of tracer in the subarachnoid space. There was also greater influx into the spinal cord interstitium. Hypertension and tachycardia had no significant effect on spinal subarachnoid spinal CSF tracer flux and exerted less effect than respiration on tracer influx into the spinal cord. CONCLUSIONS: Intrathoracic pressure changes that occur over the respiratory cycle, particularly decreased intrathoracic pressures generated during inspiration, have a profound effect on tracer movement after injection into spinal CSF and increase cord parenchymal tracer influx. Arterial pulsations likely drive fluid transport from perivascular spaces into the surrounding interstitium, but their overall impact is less than that of the respiratory cycle on net tracer influx.

Do catecholaminergic TrkC DRG neurons represent a class of cardiovascular enteroceptor?

In a recent issue of Cell Reports, Morelli et al. (2021) identify a subpopulation of mechanosensitive peripheral sensory neurons that coexpress tyrosine hydroxylase (TH) and tropomyosin receptor kinase C (TrkC) and innervate cutaneous arterioles. They show that activation of TrkC sensory neurons causes cutaneous vasoconstriction and, most remarkably, that their lesion is associated with sudden death of an undetermined cause, preceded by a progressive drop in blood pressure, and conclude that TrkC+ TH+ neurons represent a baroreceptor class of homeostatic enteroceptor. This represents a radical departure from current consensus models for the central control of blood pressure. Here, we offer an alternative perspective on their findings and suggest priorities for further investigation. This Matters Arising paper is in response to Morelli et al. (2021), published in Cell Reports. See also the response by Heppenstall et al. (2022), published in this issue.

The subfornical organ drives hypertension in polycystic kidney disease via the hypothalamic paraventricular nucleus.

AIMS: Hypertension is a prevalent yet poorly understood feature of polycystic kidney disease. Previously, we demonstrated that increased glutamatergic neurotransmission within the hypothalamic paraventricular nucleus produces hypertension in the Lewis Polycystic Kidney (LPK) rat model of polycystic kidney disease. Here, we tested the hypothesis that augmented glutamatergic drive to the paraventricular nucleus in Lewis polycystic kidney rats originates from the forebrain lamina terminalis, a sensory structure that relays blood-borne information throughout the brain. METHODS AND RESULTS: Anatomical experiments revealed that 38% of paraventricular nucleus-projecting neurons in the subfornical organ of the lamina terminalis expressed Fos/Fra, an activation marker, in LPK rats while <1% of neurons were Fos/Fra+ in Lewis control rats (P = 0.01, n = 8). In anaesthetized rats, subfornical organ neuronal inhibition using isoguvacine produced a greater reduction in systolic blood pressure in LPK vs. Lewis rats (-21±4 vs. -7±2 mmHg, P < 0.01; n = 10), which could be prevented by prior blockade of paraventricular nucleus ionotropic glutamate receptors using kynurenic acid. Blockade of ionotropic glutamate receptors in the paraventricular nucleus produced an exaggerated depressor response in LPK relative to Lewis rats (-23±4 vs. -2±3 mmHg, P < 0.001; n = 13), which was corrected by prior inhibition of the subfornical organ with muscimol but unaffected by chronic systemic angiotensin II type I receptor antagonism or lowering of plasma hyperosmolality through high-water intake (P > 0.05); treatments that both nevertheless lowered blood pressure in LPK rats (P < 0.0001). CONCLUSION: Our data reveal multiple independent mechanisms contribute to hypertension in polycystic kidney disease, and identify high plasma osmolality, angiotensin II type I receptor activation and, importantly, a hyperactive subfornical organ to paraventricular nucleus glutamatergic pathway as potential therapeutic targets.

A medullary centre for lapping in mice.

It has long been known that orofacial movements for feeding can be triggered, coordinated, and often rhythmically organized at the level of the brainstem, without input from higher centers. We uncover two nuclei that can organize the movements for ingesting fluids in mice. These neuronal groups, IRtPhox2b and Peri5Atoh1, are marked by expression of the pan-autonomic homeobox gene Phox2b and are located, respectively, in the intermediate reticular formation of the medulla and around the motor nucleus of the trigeminal nerve. They are premotor to all jaw-opening and tongue muscles. Stimulation of either, in awake animals, opens the jaw, while IRtPhox2b alone also protracts the tongue. Moreover, stationary stimulation of IRtPhox2b entrains a rhythmic alternation of tongue protraction and retraction, synchronized with jaw opening and closing, that mimics lapping. Finally, fiber photometric recordings show that IRtPhox2b is active during volitional lapping. Our study identifies one of the subcortical nuclei underpinning a stereotyped feeding behavior.

Polysialic acid in the rat brainstem and thoracolumbar spinal cord: Distribution, cellular location, and comparison with mouse.

Polysialic acid (polySia), a homopolymer of α2,8-linked glycans, is a posttranslational modification on a few glycoproteins, most commonly in the brain, on the neural cell adhesion molecule. Most research in the adult central nervous system has focused on its expression in higher brain regions, where its distribution coincides with regions known to exhibit high levels of synaptic plasticity. In contrast, scant attention has been paid to the expression of polySia in the hindbrain. The main aims of the study were to examine the distribution of polySia immunoreactivity in the brainstem and thoracolumbar spinal cord, to compare the distribution of polySia revealed by two commercial antibodies commonly used for its investigation, and to compare labeling in the rat and mouse. We present a comprehensive atlas of polySia immunoreactivity: we report that polySia labeling is particularly dense in the dorsal tegmentum, medial vestibular nuclei and lateral parabrachial nucleus, and in brainstem regions associated with autonomic function, including the dorsal vagal complex, A5, rostral ventral medulla, A1, and midline raphe, as well as sympathetic preganglionic neurons in the spinal cord and central targets of primary sensory afferents (nucleus of the solitary tract, spinal trigeminal nucleus, and dorsal horn [DH]). Ultrastructural examination showed labeling was present predominantly on the plasma membrane/within the extracellular space/in or on astrocytes. Labeling throughout the brainstem and spinal cord were very similar for the two antibodies and was eliminated by the polySia-specific sialidase, Endo-NF. Similar patterns of distribution were found in rat and mouse brainstem with differences evident in DH.

PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate.

Heart rate and blood pressure oscillate in phase with respiratory activity. A component of these oscillations is generated centrally, with respiratory neurons entraining the activity of pre-sympathetic and parasympathetic cardiovascular neurons. Using a combination of optogenetic inhibition and excitation in vivo and in situ in rats, as well as neuronal tracing, we demonstrate that preBötzinger Complex (preBötC) neurons, which form the kernel for inspiratory rhythm generation, directly modulate cardiovascular activity. Specifically, inhibitory preBötC neurons modulate cardiac parasympathetic neuron activity whilst excitatory preBötC neurons modulate sympathetic vasomotor neuron activity, generating heart rate and blood pressure oscillations in phase with respiration. Our data reveal yet more functions entrained to the activity of the preBötC, with a role in generating cardiorespiratory oscillations. The findings have implications for cardiovascular pathologies, such as hypertension and heart failure, where respiratory entrainment of heart rate is diminished and respiratory entrainment of blood pressure exaggerated.

Corrigendum: A Student's Guide to Neural Circuit Tracing.

[This corrects the article DOI: 10.3389/fnins.2019.00897.].

Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat.

Breathing results from sequential recruitment of muscles in the expiratory, inspiratory, and postinspiratory (post-I) phases of the respiratory cycle. Here we investigate whether neurons in the medullary intermediate reticular nucleus (IRt) are components of a central pattern generator (CPG) that generates post-I activity in laryngeal adductors and vasomotor sympathetic nerves and interacts with other members of the central respiratory network to terminate inspiration. We first identified the region of the (male) rat IRt that contains the highest density of lightly cholinergic neurons, many of which are glutamatergic, which aligns well with the putative postinspiratory complex in the mouse (Anderson et al., 2016). Acute bilateral inhibition of this region reduced the amplitudes of post-I vagal and sympathetic nerve activities. However, although associated with reduced expiratory duration and increased respiratory frequency, IRt inhibition did not affect inspiratory duration or abolish the recruitment of post-I activity during acute hypoxemia as predicted. Rather than representing an independent CPG for post-I activity, we hypothesized that IRt neurons may instead function as a relay that distributes post-I activity generated elsewhere, and wondered whether they could be a site of integration for para-respiratory CPGs that drive the same outputs. Consistent with this idea, IRt inhibition blocked rhythmic motor and autonomic components of fictive swallow but not swallow-related apnea. Our data support a role for IRt neurons in the transmission of post-I and swallowing activity to motor and sympathetic outputs, but suggest that other mechanisms also contribute to the generation of post-I activity.SIGNIFICANCE STATEMENT Interactions between multiple coupled oscillators underlie a three-part respiratory cycle composed from inspiratory, postinspiratory (post-I), and late-expiratory phases. Central post-I activity terminates inspiration and activates laryngeal motoneurons. We investigate whether neurons in the intermediate reticular nucleus (IRt) form the central pattern generator (CPG) responsible for post-I activity. We confirm that IRt activity contributes to post-I motor and autonomic outputs, and find that IRt neurons are necessary for activation of the same outputs during swallow, but that they are not required for termination of inspiration or recruitment of post-I activity during hypoxemia. We conclude that this population may not represent a distinct CPG, but instead may function as a premotor relay that integrates activity generated by diverse respiratory and nonrespiratory CPGs.

A Student's Guide to Neural Circuit Tracing.

The mammalian nervous system is comprised of a seemingly infinitely complex network of specialized synaptic connections that coordinate the flow of information through it. The field of connectomics seeks to map the structure that underlies brain function at resolutions that range from the ultrastructural, which examines the organization of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, distant and local connections between neuronal populations are identified, providing insights into circuit-level architecture. Although neural tract tracing techniques have been available to experimental neuroscientists for many decades, considerable methodological advances have been made in the last 20 years due to synergies between the fields of molecular biology, virology, microscopy, computer science and genetics. As a consequence, investigators now enjoy an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to identify their efferent and afferent connectomes. Unfortunately, the intersectional nature of this progress presents newcomers to the field with a daunting array of technologies that have emerged from disciplines they may not be familiar with. This review outlines the current state of mesoscale connectomic approaches, from data collection to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used by contemporary neuroscientists to resolve mesoscale organization, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. We consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing data and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.

On the presence and functional significance of sympathetic premotor neurons with collateralized spinal axons in the rat.

KEY POINTS: Spinally-projecting neurons of the rostral ventrolateral medulla (RVLM) determine sympathetic outflow to different territories of the body. Previous studies suggest the existence of RVLM neurons with distinct functional classes, such as neurons that target sympathetic nerves bound for functionally-similar tissue types (e.g. muscle vasculature). The existence of RVLM neurons with more general actions had not been critically tested. Using viral tracing, we show that a significant minority of RVLM neurons send axon collaterals to disparate spinal segments (T2 and T10 ). Furthermore, optogenetic activation of sympathetic premotor neurons projecting to lumbar spinal segments also produced activation of sympathetic nerves from rostral spinal segments that innervate functionally diverse tissues (heart and forelimb muscle). These findings suggest the existence of individual RVLM neurons for which the axons branch to drive sympathetic preganglionic neurons of more than one functional class and may be able to produce global changes in sympathetic activity. ABSTRACT: We investigate the extent of spinal axon collateralization of rat rostral ventrolateral medulla (RVLM) sympathetic premotor neurons and its functional consequences. In anatomical tracing experiments, two recombinant herpes viral vectors with retrograde tropism and expressing different fluorophores were injected into the intermediolateral column at upper thoracic and lower thoracic levels. Histological analysis revealed that ∼21% of RVLM bulbospinal neurons were retrogradely labelled by both vectors, indicating substantial axonal collateralization to disparate spinal segments. In functional experiments, another virus with retrograde tropism, a canine adenovirus expressing Cre recombinase, was injected into the left intermediolateral horn around the thoracolumbar junction, whereas a Cre-dependent viral vector encoding Channelrhodopsin2 under LoxP control was injected into the ipsilateral RVLM. In subsequent terminal experiments, blue laser light (473 nm × 20 ms pulses at 10 mW) was used to activate RVLM neurons that had been transduced by both vectors. Stimulus-locked activation, at appropriate latencies, was recorded in the following pairs of sympathetic nerves: forelimb and hindlimb muscle sympathetic fibres, as well as cardiac and either hindlimb muscle or lumbar sympathetic nerves. The latter result demonstrates that axon collaterals of lumbar-projecting RVLM neurons project to, and excite, both functionally similar (forelimb and hindlimb muscle) and functionally dissimilar (lumbar and cardiac) preganglionic neurons. Taken together, these findings show that the axons of a significant proportion of RVLM neurons collateralise widely within the spinal cord, and that they may excite preganglionic neurons of more than one functional class.

Somatostatin 2 Receptors in the Spinal Cord Tonically Restrain Thermogenic, Cardiac and Other Sympathetic Outflows.

The anatomical and functional characterization of somatostatin (SST) and somatostatin receptors (SSTRs) within the spinal cord have been focused in the dorsal horn, specifically in relation to sensory afferent processing. However, SST is also present within the intermediolateral cell column (IML), which contains sympathetic preganglionic neurons (SPN). We investigated the distribution of SSTR2 within the thoracic spinal cord and show that SSTR2A and SSTR2B are expressed in the dorsal horn and on SPN and non-SPN in or near the IML. The effects of activating spinal SSTR and SSTR2 were sympathoinhibition, hypotension, bradycardia, as well as decreases in interscapular brown adipose tissue temperature and expired CO2, in keeping with the well-described inhibitory effects of activating SSTR receptors. These data indicate that spinal SST can decrease sympathetic, cardiovascular and thermogenic activities. Unexpectedly blockade of SSTR2 revealed that SST tonically mantains sympathetic, cardiovascular and thermogenic functions, as activity in all measured parameters increased. In addition, high doses of two antagonists evoked biphasic responses in sympathetic and cardiovascular outflows where the initial excitatory effects were followed by profound but transient falls in sympathetic nerve activity, heart rate and blood pressure. These latter effects, together with our findings that SSTR2A are expressed on GABAergic, presumed interneurons, are consistent with the idea that SST2R tonically influence a diffuse spinal GABAergic network that regulates the sympathetic cardiovascular outflow. As described here and elsewhere the source of tonically released spinal SST may be of intra- and/or supra-spinal origin.

Somatostatin 2 Receptor Activation in the Rostral Ventrolateral Medulla Does Not Mediate the Decompensatory Phase of Haemorrhage.

Decompensation, a critical phase in the response to hemorrhage, is characterized by profound sympathoinhibition and the overriding of baroreflex mediated compensation. As sympathoexcitatory neurons of the rostral ventrolateral medulla (RVLM) maintain vasomotor tone and are essential for sympathetic baroreceptor reflex function, the RVLM is the likely mediator. However, how decompensation occurs is a mystery. Our previous work demonstrated that the inhibitory neuropeptide somatostatin (SST), evokes potent sympathoinhibition. Here we test the hypothesis that, in response to hypovolemia, SST in the RVLM evokes sympathoinhibition, driving decompensation and suppressing baroreflex compensation. We evaluated neuronal activation at sites that contain SST mRNA and project to the RVLM and, in SST2A expressing neurons in the RVLM. We determined the effects on cardiovascular and sympathetic responses to haemorrhage, of bilateral blockade of SST2 receptors in both the RVLM and A1 regions. Haemorrhage in conscious rats evoked c-Fos immunoreactivity in the amygdala, periaqueductal gray, and parabrachial nuclei, regions previously associated with hemorrhage, shown to contain SST and project to the RVLM. Although c-Fos labeling was found throughout the ventrolateral medulla, only a small subset of RVLM SST2A receptor expressing neurons were activated, consistent with the idea that these neurons are inhibited during hemorrhage. However, SST2 receptor antagonists bilaterally injected in the RVLM or the A1 region did not affect the decompensation response to hemorrhage. Thus somatostatin in the RVLM does not mediate decompensation. The physiological role associated with somatostatin-induced sympathoinhibition in the RVLM together with the central mechanisms responsible for decompensation remain elusive.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract.

Expression of the large extracellular glycan, polysialic acid (polySia), is restricted in the adult, to brain regions exhibiting high levels of plasticity or remodeling, including the hippocampus, prefrontal cortex, and the nucleus of the solitary tract (NTS). The NTS, located in the dorsal brainstem, receives constant viscerosensory afferent traffic as well as input from central regions controlling sympathetic nerve activity, respiration, gastrointestinal functions, hormonal release, and behavior. Our aims were to determine the ultrastructural location of polySia in the NTS and the functional effects of enzymatic removal of polySia, both in vitro and in vivo polySia immunoreactivity was found throughout the adult rat NTS. Electron microscopy demonstrated polySia at sites that influence neurotransmission: the extracellular space, fine astrocytic processes, and neuronal terminals. Removing polySia from the NTS had functional consequences. Whole-cell electrophysiological recordings revealed altered intrinsic membrane properties, enhancing voltage-gated K+ currents and increasing intracellular Ca2+ Viscerosensory afferent processing was also disrupted, dampening low-frequency excitatory input and potentiating high-frequency sustained currents at second-order neurons. Removal of polySia in the NTS of anesthetized rats increased sympathetic nerve activity, whereas functionally related enzymes that do not alter polySia expression had little effect. These data indicate that polySia is required for the normal transmission of information through the NTS and that changes in its expression alter sympathetic outflow. polySia is abundant in multiple but discrete brain regions, including sensory nuclei, in both the adult rat and human, where it may regulate neuronal function by mechanisms identified here.SIGNIFICANCE STATEMENT All cells are coated in glycans (sugars) existing predominantly as glycolipids, proteoglycans, or glycoproteins formed by the most complex form of posttranslational modification, glycosylation. How these glycans influence brain function is only now beginning to be elucidated. The adult nucleus of the solitary tract has abundant polysialic acid (polySia) and is a major site of integration, receiving viscerosensory information which controls critical homeostatic functions. Our data reveal that polySia is a determinant of neuronal behavior and excitatory transmission in the nucleus of the solitary tract, regulating sympathetic nerve activity. polySia is abundantly expressed at distinct brain sites in adult, including major sensory nuclei, suggesting that sensory transmission may also be influenced via mechanisms described here. These findings hint at the importance of elucidating how other glycans influence neural function.