Bulbospinal Neurons Research Articles

Molecular organization of adrenergic and noradrenergic neurons in the mouse ventrolateral medulla

Catecholaminergic neurons in the ventrolateral medulla (VLM) regulate many important aspects of physiology, including the sympathetic control of blood pressure, glucose homeostasis, immune function, and the neuroendocrine responses to inflammation and stress. Previous studies have parsed catecholaminergic VLM neurons into distinct sub-populations based on neurochemical content, connectivity, and distinct response to the activation of autonomic reflexes. However, there is no consensus for the molecular organization of catecholaminergic neurons. In this study, we used high-throughput single nuclei RNA sequencing combined with anatomically- and genetically-targeted cell-sorting of spinally-projecting and catecholaminergic neurons to systematically characterize C1 and A1 neurons in the mouse VLM. Our results, based on an analysis of 894 catecholaminergic neurons derived from a dataset of 114,805 single-nuclei transcriptomes, indicate that the VLM contains 7 molecularly distinct subpopulations of catecholaminergic neurons, including 5 adrenergic subtypes and 2 noradrenergic subtypes identified by the expression of enzymes involved in the production of catecholamine ( Th, Dbh) and the vesicular transporters for glutamate ( Slc6a17) and noradrenaline ( Slc6a2). Neurons with projections to the spinal cord were present in 4 of 5 adrenergic cell clusters, and 1 noradrenergic cell cluster. We histologically verified two novel markers of the adrenergic ( Hk2) and noradrenergic ( Eya1) neurons using RNA fluorescent in situ hybridization. Hk2 expression was found primarily in Dbh+ neurons in the rostral VLM, including both bulbospinal and non-bulbospinal neurons, as well as majority of Pnmt+ neurons in the rostral VLM and C2/C3 regions. Eya1 co-localized with Dbh and Slc6a2 consistently in the caudal VLM, aligning with the location of the A1 neurons, whereas Eya1 signal was diffuse in the rostral VLM. Collectively, this shows that Hk2 expression is enriched in adrenergic neurons as judged by conventional criteria irrespective of the cell's location in the medulla, whereas Eya1 is enriched in brainstem noradrenergic neurons and provides a marker for A1 neurons. These data show molecularly distinct adrenergic and noradrenergic neurons are spatially intermixed in the VLM, and provides novel gene markers for distinct subtypes of catecholaminergic neurons that can be used to dissect the function in future studies. This work was supported by National Institute of Health (NIH) R01 HL148004 to SBGA; and R01 HL153916, Pathway to Stop Diabetes Initiator Award 1-18-INI-14, and UVA 3Cavaliers grant to JNC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SGLT2 and SGLT1 inhibitors suppress the activities of the RVLM neurons in newborn Wistar rats.

Hypertension is well-known to often coexist with diabetes mellitus (DM) in humans. Treatment with sodium-glucose cotransporter 2 (SGLT2) inhibitors has been shown to decrease both the blood glucose and the blood pressure (BP) in such patients. Some reports show that SGLT2 inhibitors improve the BP by decreasing the activities of the sympathetic nervous system. Therefore, we hypothesized that SGLT2 inhibitors might alleviate hypertension via attenuating sympathetic nervous activity. Combined SGLT2/SGLT1 inhibitor therapy is also reported as being rather effective for decreasing the BP. In this study, we examined the effects of SGLT2 and SGLT1 inhibitors on the bulbospinal neurons of the rostral ventrolateral medulla (RVLM). To investigate whether bulbospinal RVLM neurons are sensitive to SGLT2 and SGLT1 inhibitors, we examined the changes in the neuronal membrane potentials (MPs) of these neurons using the whole-cell patch-clamp technique during superfusion of the cells with the SGLT2 and SGLT1 inhibitors. A brainstem-spinal cord preparation was used for the experiments. Our results showed that superfusion of the RVLM neurons with SGLT2 and SGLT1 inhibitor solutions induced hyperpolarization of the neurons. Histological examination revealed the presence of SGLT2s and SGLT1s in the RVLM neurons, and also colocalization of SGLT2s with SGLT1s. These results suggest the involvement of SGLT2s and SGLT1s in regulating the activities of the RVLM neurons, so that SGLT2 and SGLT1 inhibitors may inactivate the RVLM neurons hyperpolarized by empagliflozin. SGLT2 and SGLT1 inhibitors suppressed the activities of the bulbospinal RVLM neurons in the brainstem-spinal preparations, suggesting the possibilities of lowering BP by decreasing the sympathetic nerve activities. RVLM, rostral ventrolateral medulla. IML, intralateral cell column. aCSF, artificial cerebrospinal fluid.

Genetic Ablation of Prorenin Receptor in the Rostral Ventrolateral Medulla Influences Blood Pressure and Hydromineral Balance in Deoxycorticosterone-Salt Hypertension.

Non-enzymatic activation of renin via its interaction with prorenin receptor (PRR) has been proposed as a key mechanism of local renin-angiotensin system (RAS) activation. The presence of renin and angiotensinogen has been reported in the rostral ventrolateral medulla (RVLM). Overactivation of bulbospinal neurons in the RVLM is linked to hypertension (HTN). Previous studies have shown that the brain RAS plays a role in the pathogenesis of the deoxycorticosterone (DOCA)-salt HTN model. Thus, we hypothesized that PRR in the RVLM is involved in the local activation of the RAS, facilitating the development of DOCA-salt HTN. Selective PRR ablation targeting the RVLM (PRRRVLM-Null mice) resulted in an unexpected sex-dependent and biphasic phenotype in DOCA-salt HTN. That is, PRRRVLM-Null females (but not males) exhibited a significant delay in achieving maximal pressor responses during the initial stage of DOCA-salt HTN. Female PRRRVLM-Null subsequently showed exacerbated DOCA-salt-induced pressor responses during the "maintenance" phase with a maximal peak at 13 d on DOCA-salt. This exacerbated response was associated with an increased sympathetic drive to the resistance arterioles and the kidney, exacerbated fluid and sodium intake and output in response to DOCA-salt, and induced mobilization of fluids from the intracellular to extracellular space concomitant with elevated vasopressin. Ablation of PRR suppressed genes involved in RAS activation and catecholamine synthesis in the RVLM but also induced expression of genes involved in inflammatory responses. This study illustrates complex and sex-dependent roles of PRR in the neural control of BP and hydromineral balance through autonomic and neuroendocrine systems. Graphical abstract.

Motor neuron involvement threatens survival in spinocerebellar ataxia type 1.

Motor neuron involvement threatens survival in spinocerebellar ataxia type 1.

Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance.

The rostral ventromedial medulla (RVM) exerts bidirectional descending modulation of pain attributable to the activity of electrophysiologically identified pronociceptive ON and antinociceptive OFF neurons. Here, we report that GABAergic ON neurons specifically express G protein-coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, but their ablation abrogated, pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain, and ameliorated morphine analgesia through desensitizing μ-type opioid receptor-mediated (MOR-mediated) activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia, and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly indicate that GPER is a marker for GABAergic ON cells and illuminate the mechanisms underlying hormonal regulation of pain and analgesia, thus highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

Potential presynaptic effects of opioids on medullary inspiratory circuits: New insights from a joint neural biomechanical model

Administration of opioids can lead to respiratory depression and eventually death. Opioids can alter the frequency and magnitude of inspiratory motor drive by binding to mu‐opioid receptors on inspiratory neurons located in several areas of the brainstem. Lalley (Am J Physiol: Regul. 285:R1287‐1304, 2003) suggested that some of the effects of fentanyl on bulbospinal inspiratory neuron activity were presynaptic. We tested the feasibility of pre‐ vs post‐synaptic effects of opioids with an integrate‐and‐fire computational neural biomechanical model of the brainstem respiratory network. Our previous simulations utilizing this model involved separately reducing conductance at groups of synapses that excited inspiratory‐augmenting (I‐Aug) or inspiratory‐decrementing (I‐Dec) neuron populations to simulate opioid‐mediated decreases in the presynaptic excitability of excitatory axons to these neurons from inspiratory driver neurons (I‐driver). We found that the presynaptic hypothesis was best supported by a mechanism involving depression of the excitatory synapse between I‐driver and I‐decrementing neurons. The current modeling investigation extends that work by focusing on three inspiratory neuron populations: I‐Aug, I‐Dec, and late inspiratory (late‐I). We separately decreased the conductance of each excitatory synapse to examine how these changes would affect the respiratory pattern. Simulations revealed that systematically decreasing the conductance of synapses from the I‐Aug neuron population to late‐I and I‐ Aug bulbospinal populations by 50% resulted in a decrease in frequency, duration, and amplitude of inspiratory drive. Decreasing the conductance of excitatory synapses from I‐Dec neurons to I‐Aug, and late‐I populations had a limited effect on inspiratory activity. Similar results were observed when the conductance of excitatory synapses was reduced from the late‐I population to its I‐Dec and I‐Aug targets. Overall, these results indicate that a plausible mechanism for respiratory suppression by opioids could include depression of excitatory synaptic actions between selected medullary inspiratory neuron populations.

Bulbospinal connections to intercostal motoneurones following a chronic lateral spinal cord lesion

Previous evidence from electrophysiological experiments in anaesthetized cats with a chronic lateral lesion of the lower thoracic spinal cord indicated an expansion of the functional projections of expiratory bulbospinal neurones (EBSNs) in the segment above the lesion, measured at 16 weeks post-lesion. Here we investigate connections made by the same EBSNs to motoneurones in that segment, using cross-correlations between their discharges. The connections to the internal intercostal nerve motoneurones were found to be no different from controls. However, a significant increase was found in the number of connections between EBSNs and γ motoneurones of the external intercostal nerve (8/24, compared to 1/16) with possibly additional connections to the α motoneurones of the same nerve. Increased connections to the γ motoneurones of the internal intercostal nerve could not be ruled out. The expanded functional projections are thus likely to include new connections to γ motoneurones. We suggest that γ motoneurones may be inherently more receptive to new inputs. If so, the previously discounted role of abnormal fusimotor discharges in motor disorders would be worth reconsideration.

Essential Role of the cVRG in the Generation of Both the Expiratory and Inspiratory Components of the Cough Reflex.

As stated by Korpáš and Tomori (1979), cough is the most important airway protective reflex which provides airway defensive responses to nociceptive stimuli. They recognized that active expiratory efforts, due to the activation of caudal ventral respiratory group (cVRG) expiratory premotoneurons, are the prominent component of coughs. Here, we discuss data suggesting that neurons located in the cVRG have an essential role in the generation of both the inspiratory and expiratory components of the cough reflex. Some lines of evidence indicate that cVRG expiratory neurons, when strongly activated, may subserve the alternation of inspiratory and expiratory cough bursts, possibly owing to the presence of axon collaterals. Of note, experimental findings such as blockade or impairment of glutamatergic transmission to the cVRG neurons lead to the view that neurons located in the cVRG are crucial for the production of the complete cough motor pattern. The involvement of bulbospinal expiratory neurons seems unlikely since their activation affects differentially expiratory and inspiratory muscles, while their blockade does not affect baseline inspiratory activity. Thus, other types of cVRG neurons with their medullary projections should have a role and possibly contribute to the fine tuning of the intensity of inspiratory and expiratory efforts.

Rostral ventrolateral medulla neuron activity is suppressed by Klotho and stimulated by FGF23 in newborn Wistar rats.

Hypertension often occurs in patients with chronic kidney disease (CKD). Considering the decrease in serum Klotho and increase in serum FGF23 levels in such patients, decreased Klotho and increased FGF23 levels were thought to be associated with hypertension. Presympathetic neurons at the rostral ventrolateral medulla (RVLM) contribute to sympathetic activity and regulation of blood pressure. Therefore, we hypothesized that Klotho would reduce the activities of RVLM neurons and FGF23 would stimulate them. Accordingly, this study examined the effects of Klotho and FGF23 on bulbospinal neurons in the RVLM. We used a brainstem-spinal cord preparation to record from RVLM presympathetic neurons and to evaluate the effects of Klotho and FGF23 on firing rate and membrane potentials of these neurons. Our results showed that Klotho-induced RVLM neuron hyperpolarization, while ouabain, a Na+/K+-ATPase inhibitor, suppressed the effects of Klotho on such neurons. Moreover, FGF23 induced RVLM neuron depolarization, while SU5402, an FGF23 receptor (FGFR1) antagonist, induced RVLM neuron hyperpolarization. Histological examinations revealed that Klotho, Na+/K+-ATPase, FGF23, and FGFR1 were present in RVLM neurons and that Klotho was localized in the same neurons as FGFR1. These results suggest that Klotho and FG23 regulate the activity of RVLM neurons. Klotho may reduce the activity of RVLM neurons via stimulating Na+/K+-ATPase on those neurons while FGF23 may activate those neurons via FGFR1.

Role of Propriospinal Neurons in Control of Respiratory Muscles and Recovery of Breathing Following Injury.

Respiratory motor failure is the leading cause of death in spinal cord injury (SCI). Cervical injuries disrupt connections between brainstem neurons that are the primary source of excitatory drive to respiratory motor neurons in the spinal cord and their targets. In addition to direct connections from bulbospinal neurons, respiratory motor neurons also receive excitatory and inhibitory inputs from propriospinal neurons, yet their role in the control of breathing is often overlooked. In this review, we will present evidence that propriospinal neurons play important roles in patterning muscle activity for breathing. These roles likely include shaping the pattern of respiratory motor output, processing and transmitting sensory afferent information, coordinating ventilation with motor activity, and regulating accessory and respiratory muscle activity. In addition, we discuss recent studies that have highlighted the importance of propriospinal neurons for recovery of respiratory muscle function following SCI. We propose that molecular genetic approaches to target specific developmental neuron classes in the spinal cord would help investigators resolve the many roles of propriospinal neurons in the control of breathing. A better understanding of how spinal circuits pattern breathing could lead to new treatments to improve breathing following injury or disease.

Subregional differences in GABAA receptor subunit expression in the rostral ventrolateral medulla of sedentary versus physically active rats.

Neurons in the rostral ventrolateral medulla (RVLM) regulate blood pressure through direct projections to spinal sympathetic preganglionic neurons. Only some RVLM neurons are active under resting conditions due to significant, tonic inhibition by gamma-aminobutyric acid (GABA). Withdrawal of GABAA receptor-mediated inhibition of the RVLM increases sympathetic outflow and blood pressure substantially, providing a mechanism by which the RVLM could contribute chronically to cardiovascular disease (CVD). Here, we tested the hypothesis that sedentary conditions, a major risk factor for CVD, increase GABAA receptors in RVLM, including its rostral extension (RVLMRE ), both of which contain bulbospinal catecholamine (C1) and non-C1 neurons. We examined GABAA receptor subunits GABAAα1 and GABAAα2 in the RVLM/RVLMRE of sedentary or physically active (10-12 weeks of wheel running) rats. Western blot analyses indicated that sedentary rats had lower expression of GABAAα1 and GABAAα2 subunits in RVLM but only GABAAα2 was lower in the RVLMRE of sedentary rats. Sedentary rats had significantly reduced expression of the chloride transporter, KCC2, suggesting less effective GABA-mediated inhibition compared to active rats. Retrograde tracing plus triple-label immunofluorescence identified fewer bulbospinal non-C1 neurons immunoreactive for GABAAα1 but a higher percentage of bulbospinal C1 neurons immunoreactive for GABAAα1 in sedentary animals. Sedentary conditions did not significantly affect the number of bulbospinal C1 or non-C1 neurons immunoreactive for GABAAα2 . These results suggest a complex interplay between GABAA receptor expression by spinally projecting C1 and non-C1 neurons and sedentary versus physically active conditions. They also provide plausible mechanisms for both enhanced sympathoexcitatory and sympathoinhibitory responses following sedentary conditions.

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

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. 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 (473nm×20ms pulses at 10mW) 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.

The neural control of arterial pressure by non‐catecholaminergic RVLM

Tonic sympathetic nerve activity (SNA) is necessary for the maintenance of arterial pressure (AP) under anesthesia, and this tone is dependent on supraspinal input. Over the last two centuries, numerous studies have collectively identified the rostral ventrolateral medulla (RVLM) as a critical region for neural control of AP and SNA in anesthetized animals, however the relevance of these results to conscious animals and the nature of the RVLM neurons responsible remain conjectural. The RVLM has direct projections onto sympathetic preganglionic neurons, but the evidence that this is the critical pathway for AP regulation and SNA in conscious animals is not definitive—and even if this is the principal BP regulatory pathway of the RVLM, the bulbospinal neurons that produce these effects are still debated.Historically, the most prominent pressor candidate within the RVLM has been the rostral portion of the catecholaminergic C1 group. Rostral C1 neurons are glutamatergic, occupy a ventral region of the medulla that is coextensive with the vasomotor RVLM region, are barosensitive, directly innervate sympathetic preganglionic neurons, and activate sympathetic nerve discharge upon stimulation. Despite these data, optogenetic stimulation of C1 in conscious and anesthetized rodents has thus far failed to achieve the magnitude of increases in arterial pressure predicted by pharmacological stimulation of the RVLM.Here, we show that stimulation of a non‐catecholaminergic population within the RVLM is sufficient to increase AP in conscious mice, even after C1 lesion. We then targeted bulbospinal neurons using a retrograde virus and demonstrated that optogenetic activation of only bulbospinal RVLM neurons increases AP in conscious mice, and in anesthetized mice, stimulation increases AP and renal sympathetic nerve activity. Unlike optogenetic stimulation of C1 neurons, stimulation of non‐catecholaminergic bulbospinal RVLM causes robust and sustained increases in AP in both conscious and anesthetized states, and sustained increases in SNA under anesthesia. This data suggests non‐catecholaminergic bulbospinal neurons are powerful activators of AP and SNA and may play an important role along with C1 in arterial pressure maintenance.Support or Funding InformationAmerican Heart Association Predoctoral Fellowship; University of Virginia Wagner FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Sedentary Conditions on the Rostrocaudal Expression of the NMDA NR1 Receptor Subunit in Bulbospinal C1 and non‐C1 Neurons of the Rat Rostral Ventrolateral Medulla (RVLM)

Bulbospinal RVLM neurons directly innervate sympathetic preganglionic cells and therefore play important roles in the regulation of sympathetic nerve activity and blood pressure. RVLM neurons are divided phenotypically into catecholaminergic (C1) and non‐catecholaminergic (non‐C1).Previous studies from our laboratory reported that the RVLM of sedentary compared to physically active rats demonstrates greater structural and functional neuroplasticity in a subregion‐specific manner. For example, RVLM neurons from sedentary versus physically active rats exhibit greater dendritic branching and elicit greater sympathoexcitation upon direct stimulation with glutamate in rostral versus caudal RVLM. Bulbospinal RVLM neurons also express NMDA receptors and could contribute to inactivity‐induced neuroplasticity and enhanced sympathetic activation.The purpose of the present study was to test the hypothesis that sedentary conditions increase NMDA NR1 subunit expression in the RVLM in a subregion‐dependent manner. Male Sprague‐Dawley rats were sedentary (no running wheels) or physically active (free access to running wheels) for 10–12 wks (n=6 ea). Spinal cord injections of cholera toxin B (CTB)‐labeled bulbospinal RVLM neurons. We examined coronal sections for triple‐label immunofluorescence by laser confocal microscopy, and quantified CTB‐positive C1 and non‐C1 neurons expressing the NR1 receptor subunit.As expected from previous work, significantly more bulbospinal C1 neurons were positive for NR1 in the caudal RVLM compared to rostral RVLM in both experimental groups (p<0.001). Also as expected, more non‐C1 neurons immunoreactive for NR1 were in the rostral compared to caudal RVLM (p<0.001). Interestingly, sedentary rats demonstrated a strong trend for a higher percentage of C1 cells and significantly lower percentage of non‐C1 cells positive for NR1 in rostral RVLM when compared to active animals (p=0.067 and p=0.0048, respectively). In contrast, there were no overt differences between sedentary versus active rats in the percentage of C1 and non‐C1 cells positive for NR1 in caudal RVLM.Preliminary western blotting data showed lower expression of PSD95 (an excitatory post‐synaptic density protein) in sedentary versus active animals (0.89±0.15 vs. 1.64±0.24, PSD95/GAPDH, respectively), suggesting differential synaptic/extrasynaptic localization of glutamate receptors, including NR1, in sedentary and active groups.The greater number of NR1‐positive, bulbospinal C1 neurons in rostral RVLM of sedentary rats is consistent with our hypothesis and previous data showing enhanced SNA following activation of glutamate receptors in sedentary vs. active rats. Sedentary conditions may also enhance excitation of RVLM neurons by altering the composition of NMDAR subunits, their synaptic/extrasynaptic localization, trafficking, or expression of synapse‐related proteins and/or transporters.Support or Funding InformationHL096787‐08; AHA25810010This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Sympathoexcitatory Pathway between The Deep Superior Colliculus and The Rostral Ventromedial Medulla

IntroductionThe superior colliculus plays key roles in the immediate processing of threatening sensory stimuli and generates rapid behavioural responses that are critical for survival. We have previously found that disinhibition of the deep superior colliculus (dSC) unmasks coordinated respiratory, sympathetic and somatomotor outputs that are independent of processing in higher centres. We postulate that these effects are a result of direct innervation of autonomic and respiratory medullary neurons based on the results of neuroanatomical tracing studies that identified dSC synaptic contacts on spinally projecting neurons within the rostral ventromedial medulla (RVMM). In the present study we investigate the physiological significance of this relay and compare effects of dSC stimulation to activation of dSC‐RVMM terminals.MethodsWe used an AAV vector to express Channelrhodopsin2 (ChR2) in the dSC and chronically implanted a fibre optic cannula into the dSC. After transgene expression electrophysiology experiments were conducted under urethane anesthesia with rats instrumented to record blood pressure, diaphragmatic EMG and splanchnic sympathetic nerve activity (SNA). One subset of experiments then positioned the optrode in the RVMM to deliver light to the terminal projections from the dSC. For the other subset of experiments, extracellular recordings were made of spinally projecting RVMM neurons.ResultsdSC photoactivation evoked an increase in respiratory frequency (35±8.7%, P=<0.01, N=7) and heart rate (11±2.6 bpm, P=<0.01, N=7), and a modest change in blood pressure when compared to controls. Stimulus‐triggered averaging of SNA revealed short‐latency excitatory potentials in most cases. Photoactivation of ChR2‐expressing dSC‐RVMM terminals evoked similar effects on cardiorespiratory outputs as dSC stimulation, with an increase in respiratory frequency (14 ±9.2%, P<0.001, N=6) and blood pressure (13 ±2 mmHg P<0.01, N=7), as well as qualitatively similar sympathoexcitation. In preliminary experiments that recorded extracellular action potentials in bulbospinal RVMM neurons we recorded excitatory effects of dSC stimulation in 6/8 spontaneously active neurons.ConclusionWe conclude that optogenetic activation of dSC neurons that project to the RVMM can drive cardiorespiratory effects that are consistent with our previous findings and that dSC stimulation evokes excitatory effects in some RVMM bulbospinal neurons. This confirms our hypothesis of the existence of a previously uncharacterized excitatory pathway between the dSC and RVMM. Ongoing experiments will functionally characterize RVMM neurons that receive dSC input.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apolipoprotein E modulates respiratory motor plasticity following cervical spinal cord injury

Each year, 17,700 Americans suffer a spinal cord injury, over half of which occur at the cervical level. These high level injuries can interrupt bulbospinal neurons that innervate the phrenic motornucleus, the origin of the phrenic nerve. Loss of these descending inputs to the phrenic nerve paralyzes the ipsilateral diaphragm, leading to breathing impairments. One approach to promote recovery of breathing function is by enhancing plasticity through strengthening of synapses or activating spared but latent pathways in the spinal cord. Activation of the latent crossed phrenic pathway can lead to a form of respiratory motor plasticity known as long term facilitation (LTF), which is characterized by a prolonged increase in breathing motor output. LTF can be induced through exposure to intermittent hypoxia (IH) or by intermittently dosing the spinal cord with serotonin (5‐HT). While a portion of the spinal cord injured population responds to IH therapy with the expected increase in respiratory output, others remain non‐responders. This inconsistency indicates that variability in the human population may influence how individuals respond to treatments that aim to enhance plasticity. Therefore, we propose that genetic diversity among the SCI population could be a key factor in determining an individual's propensity for plasticity. Apolipoprotein E (apoE) is a promising candidate gene that could be responsible for this variability because one of the apoE alleles, E4, has previously been shown to reduce synaptic plasticity by decreasing expression of glutamate receptors when compared to the E2 or E3 alleles. The present study investigates the influence of human apoE4 on respiratory motor plasticity in rats following C2 hemisection. 20 weeks after injury, rats were dosed with one isoform of the human apoE protein, E3 or E4, prior to receiving intermittent 5‐HT to induce LTF. Diaphragmatic EMG recordings demonstrated that animals exposed to human apoE3 protein exhibited an increase in diaphragmatic activity ipsilateral to the injury, but this increase was abolished in E4 dosed animals. Analysis of tissue dosed with human apoE protein indicated that apoE also modulates synaptic expression of glutamate receptors, a crucial component of LTF induction. Collectively, these experiments demonstrate ApoE4's potential to inhibit plasticity following spinal cord injury, emphasizing the importance of considering genetic diversity while developing SCI therapeutics for the human population.Support or Funding InformationNational Science Foundation Graduate Research Fellowship, University of Kentucky College of Medicine Fellowship for Excellence in Graduate Research, University of Kentucky Startup FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Further observations on cardiac modulation of thoracic motoneuron discharges

Previous analyses of recordings of alpha motoneuron discharges from branches of the intercostal and abdominal nerves in anesthetized cats under neuromuscular blockade demonstrated modulation with the cardiac cycle. This modulation was interpreted as evidence that thoracic somatosensory afferents, most likely muscle spindles, provide a signal to the CNS that could contribute to cardiac interoception. Here, two aspects of these observations have been extended. First, new measurements of thoracic and abdominal EMG activity in spontaneously breathing dogs show that a very similar modulation exists in these rather different circumstances. Second, further analysis of the cat recordings shows that cardiac modulation of the discharges of bulbospinal neurons that transmit the expiratory drive to thoracic motoneurons is weak and of an inappropriate time-course to be a contributor to the effect seen in the motoneurons.

C4BP-mediated complement evasion in streptococcal infections: How the good guys turn bad

Previous analyses of recordings of alpha motoneuron discharges from branches of the intercostal and abdominal nerves in anesthetized cats under neuromuscular blockade demonstrated modulation with the cardiac cycle. This modulation was interpreted as evidence that thoracic somatosensory afferents, most likely muscle spindles, provide a signal to the CNS that could contribute to cardiac interoception. Here, two aspects of these observations have been extended. First, new measurements of thoracic and abdominal EMG activity in spontaneously breathing dogs show that a very similar modulation exists in these rather different circumstances. Second, further analysis of the cat recordings shows that cardiac modulation of the discharges of bulbospinal neurons that transmit the expiratory drive to thoracic motoneurons is weak and of an inappropriate time-course to be a contributor to the effect seen in the motoneurons.

Contribution of non‐catecholaminergic rostral ventrolateral medulla neurons to blood pressure control

Previous work identified the rostral ventrolateral medulla (RVLM) as a critical location for the sympathetic regulation of circulation. C1 neurons are a group of catecholaminergic neurons that are co‐extensive with this region, contain VGLUT2 and release glutamate, project to the intermediolateral cell column of the spinal cord, increase renal sympathetic tone, are barosensitive, and are activated by psychological and physiological stress. Despite these findings, selective stimulation of C1 neurons produced complex cardiovascular responses with no distinct increase in BP in conscious mice. C1 neurons are likely made up of several sub‐groups with unique roles, and stimulation of C1 neurons en masse may mask the cardiovascular effect of a pressor subgroup. In rats, selective stimulation of the rostral C1 neurons—the bulbospinal subgroup—increases BP, and selective inhibition of this subgroup decreases BP under hypoxia and anesthesia. Due to differences in the distribution of C1 subgroups in mouse and rat, selective stimulation of bulbospinal C1 neurons in mouse using previous techniques is not possible. Therefore, we sought to determine whether the RVLM BP regulatory neurons in mouse were a bulbospinal subgroup of C1 neurons or a separate non‐catecholaminergic group. All experiments were carried out using dopamine β‐hydoxylase Cre mice, which, in the rostral ventrolateral medulla, exclusively express Cre in C1 neurons. For selective stimulation, we injected an AAV to express the light‐driven ChR2 protein in either C1 or non‐C1 neurons. C1 neurons were targeted using a Cre‐dependent AAV to deliver ChR2, while non‐C1 RVLM neurons were targeted using an AAV where ChR2 was driven by the CaMKIIα promoter. First, we verified that our CaMKIIα‐ChR2 AAV injections labeled few C1 neurons using immunohistochemistry. We then tested whether stimulation of C1 or non‐C1 RVLM neurons increased BP in conscious and anesthetized mice. Next, we asked whether stimulation of the spinally projecting axons of either C1 or non‐C1 RVLM neurons increased BP or sympathetic nerve activity (SNA) in anesthetized mice. We conclude that in dopamine β‐hydoxylase Cre mice, non‐C1 RVLM neurons contribute to BP regulation, while we found no physiological evidence to support the role of bulbospinal C1 neurons in BP control. Our conclusions are based on the following results: (1.) Our CaMKIIα‐ChR2 AAV injections were selective for non‐C1 neurons in the RVLM. (2.) Stimulation of CaMKIIα RVLM neurons in conscious mice increases BP while stimulation of C1 does not. (3.) Stimulation of CaMKIIα bulbospinal axons from RVLM neurons increases BP in anesthetized mice while stimulation of C1 axons does not. (4.) Continued high‐frequency stimulation of CaMKIIα RVLM neurons causes robust and sustained increases in BP, while continued high‐frequency stimulation of C1 causes a depressor effect.Support or Funding InformationAmerican Heart Association National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ROSTROCAUDAL‐DEPENDENT DECREASES IN BRAIN‐DERIVED NEUROTROPHIC FACTOR IN THE ROSTRAL VENTROLATERAL MEDULLA OF SEDENTARY VERSUS PHYSICALLY ACTIVE RATS

The rostral ventrolateral medulla (RVLM) contains bulbospinal neurons that contribute to normal and pathologically‐related increases in sympathetic nerve activity. Both structural and functional forms of neuroplasticity occur in the RVLM following sedentary versus physical active conditions, and are subregion specific. For example, in the rostral subregion of the RVLM, sedentary rats exhibit greater sympathoexcitation in response to glutamate microinjections (functional neuroplasticity) as well as more extensive dendritic branching (structural neuroplasticity), both consistent with increased excitability. Although these findings suggest a link between subregion‐specific neuroplasticity in the RVLM and inactivity‐related cardiovascular diseases, the mechanisms mediating these alterations remain unknown. The role of brain‐derived neurotrophic factor (BDNF) is well established in the field of neuroplasticity in terms of neuronal growth and remodeling. It has also been proposed that BDNF serves as a neurotransmitter in brainstem regions involved in sympathetic outflow. Whether sedentary conditions alter levels of the mature form of BDNF (mBDNF, which promotes dendritic branching) or the pro‐form of BDNF (proBDNF, which inhibits dendritic branching) in the RVLM is unknown. The purpose of this study was to test the hypothesis that mBDNF is increased and proBDNF is decreased in more rostral regions of the RVLM, particularly in sedentary rats. To test this hypothesis, male Sprague‐Dawley rats were divided into two groups: physically active (running wheels) and sedentary (without wheels, n=6 ea). Animals were housed for 10–12 weeks before sacrificing for fresh tissue removal. Frozen brainstems were cryosectioned at 80 μm, from which bilateral tissue punches were obtained for Western blotting. Punches were pooled based on their location relative to the caudal pole of the facial nucleus (FN+480; FN+240; FN‐240; FN‐480 μm), as determined in cresyl violet‐stained, post‐punched sections. The RVLMs of sedentary and active rats exhibited rostrocaudal‐dependent levels of mBDNF, with rostral regions exhibiting significant increases compared to caudal regions (main effect; p=0.039). However, sedentary rats exhibited significantly less mBDNF at FN+240 to FN‐480 (p<0.05 vs active). proBDNF levels were also lower in sedentary rats (p<0.05) as were TrkB receptors, the primary receptor for mBDNF (p<0.001; full length‐TrkB; p=0.002; truncated‐TrkB), suggesting an overall decrease in mBDNF signaling in sedentary rats. These results further demonstrate activity‐dependent neuroplasticity in subregions of the RVLM, but do not support a contribution of mBDNF towards the increased dendritic branching in rostral regions of the RVLM following sedentary conditions. In contrast, reductions in the pro‐form of BDNF and loss of its inhibitory effect on dendritic branching may contribute to increased dendritic branching in sedentary animals. Future studies could also examine BDNF‐induced alterations in synaptic transmission as well as other neurotrophic growth factors, all of which may contribute to increased cardiovascular disease in sedentary individuals via influences on sympathetic outflow.Support or Funding InformationHL096787‐07; AHA25810010This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.