Intermittent hypoxia enhances the expression of hypoxia inducible factor HIF1A through histone demethylation.

The cellular response to hypoxia is regulated through enzymatic oxygen sensors, including the prolyl hydroxylases, which control degradation of the well-known hypoxia inducible factors (HIFs). Other enzymatic oxygen sensors have been recently identified, including members of the KDM histone demethylase family. Little is known about how different oxygen-sensing pathways interact and if this varies depending on the form of hypoxia, such as chronic or intermittent. In this study, we investigated how two proposed cellular oxygen-sensing systems, HIF-1 and KDM4A, KDM4B, and KDM4C, respond in cells exposed to rapid forms of intermittent hypoxia (minutes) and compared to chronic hypoxia (hours). We found that intermittent hypoxia increases HIF-1α protein through a pathway distinct from chronic hypoxia, involving the KDM4A, KDM4B, and KDM4C histone lysine demethylases. Intermittent hypoxia increases the quantity and activity of KDM4A, KDM4B, and KDM4C, resulting in a decrease in histone 3 lysine 9 (H3K9) trimethylation near the HIF1A locus. We demonstrate that this contrasts with chronic hypoxia, which decreases KDM4A, KDM4B, and KDM4C activity, leading to hypertrimethylation of H3K9 globally and at the HIF1A locus. Altogether, we found that demethylation of histones bound to the HIF1A gene in intermittent hypoxia increases HIF1A mRNA expression, which has the downstream effect of increasing overall HIF-1 activity and expression of HIF target genes. This study highlights how multiple oxygen-sensing pathways can interact to regulate and fine tune the cellular hypoxic response depending on the period and length of hypoxia.

Still Excited, but Less Aroused-The Effects of Nutritional Ketosis on Epinephrine Response and Hypothalamic Orexin Neuron Activation Following Recurrent Hypoglycemia in Diabetic Rats.

Hypoglycemia-associated autonomic failure (HAAF) is a serious, life-threatening complication of intensive insulin therapy, particularly in people with type 1 diabetes. The ketogenic diet is reported to beneficially affect glycemic control in people with type 1 diabetes, however its effects on the neurohormonal counterregulatory response to recurrent hypoglycemia and HAAF development are understudied. In this study we used Sprague Dawley rats to establish a HAAF model under non-diabetic and streptozotocin (STZ)-induced diabetic conditions and determined how nutritional ketosis affected the neurohormonal counterregulation and the activity of energy-sensing orexin (OX) neurons. We found that antecedent hypoglycemia diminished the sympathoexcitatory epinephrine response to subsequent hypoglycemia in chow-fed non-diabetic rats, but this did not occur in STZ-diabetic animals. In all cases a ketogenic diet preserved the epinephrine response. Contrary to expectations, STZ-diabetic keto-fed rats showed reduced OX activity in the recurrent hypoglycemia group, which did not occur in any other group. It is possible that the reduced activation of OX neurons is an adaptation aimed at energy conservation accompanied by diminished arousal and exploratory behaviour. Our data suggests that while a ketogenic diet has beneficial effects on glycemia, and epinephrine response, the reduced activation of OX neurons could be detrimental and warrants further investigation.

Slow but Steady-The Responsiveness of Sympathoadrenal System to a Hypoglycemic Challenge in Ketogenic Diet-Fed Rats.

The sympathoadrenal counterregulatory response to hypoglycemia is critical for individuals with type 1 diabetes due to impaired ability to produce glucagon. Ketogenic diets (KD) are an increasingly popular diabetes management tool; however, the effects of KD on the sympathoadrenal response are largely unknown. Here, we determined the effects of KD-induced ketosis on the sympathoadrenal response to a single insulin-induced hypoglycemic challenge. We investigated how a 3 week KD feeding regimen affected the main components of the sympathoadrenal counterregulatory response: adrenal sympathetic nerve activity (ASNA), adrenal gland activity, plasma epinephrine, and brainstem glucose-responsive C1 neuronal activation in anesthetized, nondiabetic male Sprague-Dawley rats. Rats on KD had similar blood glucose (BG) levels and elevated ketone body ß-hydroxybutyrate (BHB) levels compared to the control Chow diet group. All KD rats responded to hypoglycemia with a robust increase in ASNA, which was initiated at significantly lower BG levels compared to Chow-fed rats. The delay in hypoglycemia-induced ASNA increase was concurrent with rapid disappearance of BHB from cerebral and peripheral circulation. Adrenal gland activity paralleled epinephrine and ASNA response. Overall, KD-induced ketosis was associated with initiation of the sympathoadrenal response at lower blood glucose levels; however, the magnitude of the response was not diminished.

Pentobarbital Anesthesia Suppresses the Glucose Response to Acute Intermittent Hypoxia in Rat.

A key feature of sleep disordered breathing syndromes, such as obstructive sleep apnea is intermittent hypoxia. Intermittent hypoxia is well accepted to drive the sympathoexcitation that is frequently associated with hypertension and diabetes, with measurable effects after just 1 h. The aim of this study was to directly measure the glucose response to 1 h of acute intermittent hypoxia in pentobarbital anesthetized rats, compared to conscious rats. However, we found that while a glucose response is measurable in conscious rats exposed to intermittent hypoxia, it is suppressed in anesthetized rats. Intermittent hypoxia for 1, 2, or 8 h increased blood glucose by 0.7 ± 0.1 mmol/L in conscious rats but had no effect in anesthetized rats (-0.1 ± 0.2 mmol/L). These results were independent of the frequency of the hypoxia challenges, fasting state, vagotomy, or paralytic agents. A supraphysiological challenge of 3 min of hypoxia was able to induce a glycemic response indicating that the reflex response is not abolished under pentobarbital anesthesia. We conclude that pentobarbital anesthesia is unsuitable for investigations into glycemic response pathways in response to intermittent hypoxia in rats.

PACAP-PAC1 Receptor Activation Is Necessary for the Sympathetic Response to Acute Intermittent Hypoxia.

Repetitive hypoxia is a key feature of obstructive sleep apnoea (OSA), a condition characterized by intermittent airways obstruction. Patients with OSA present with persistent increases in sympathetic activity and commonly develop hypertension. The objectives of this study were to determine if the persistent increases in sympathetic nerve activity, known to be induced by acute intermittent hypoxia (AIH), are mediated through activation of the pituitary adenylate cyclase activating polypeptide (PACAP) signaling system. Here, we show that the excitatory neuropeptide PACAP, acting in the spinal cord, is important for generating the sympathetic response seen following AIH. Using PACAP receptor knockout mice, and pharmacological agents in Sprague Dawley rats, we measured blood pressure, heart rate, pH, PaCO2, and splanchnic sympathetic nerve activity, under anaesthesia, to demonstrate that the sympathetic response to AIH is mediated via the PAC1 receptor, in a cAMP-dependent manner. We also report that both intermittent microinjection of glutamate into the rostroventrolateral medulla (RVLM) and intermittent infusion of a sub-threshold dose of PACAP into the subarachnoid space can mimic the sympathetic response to AIH. All the sympathetic responses are independent of blood pressure, pH or PaCO2 changes. Our results show that in AIH, PACAP signaling in the spinal cord helps drive persistent increases in sympathetic nerve activity. This mechanism may be a precursor to the development of hypertension in conditions of chronic intermittent hypoxia, such as OSA.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density.

The RVLM of spontaneously hypertensive rats (SHR) contains over-active C1 neurons, which model the pathology of essential hypertension. Hypertension involves chronic low-grade neuroinflammation. Inflammation in the brain is produced and maintained primarily by microglia. We assessed microglial gene expression (P2Y12R and CX3CR1) and morphology in the RVLM of SHR compared to normotensive Wistar-Kyoto rats (WKY). The gene expression of the metabotropic purinergic receptor P2Y12 and the fractalkine receptor CX3CR1 was downregulated in the RVLM of SHR compared to WKY (by 37.3% and 30.9% respectively). P2Y12R and CX3CR1 are required for normal microglial function, and reduced P2Y12R expression is associated with changes in microglial activity. Histological analysis showed a 22.9% reduction in microglial cell density, along with 18.7% shorter microglial processes, a phenotypic indicator of activation, in the RVLM of SHR compared to WKY. These results indicate a subtle loss of function, or a mild state of inflammation, in the RVLM microglia of SHR.

Integration of hindbrain and carotid body mechanisms that control the autonomic response to cardiorespiratory and glucoprivic insults.

Autonomic reflex responses are critical in restoring changes to circulatory factors reduced beyond the domain of homeostasis. Intermittent hypoxia triggers repeated activation of chemoreflexes, resulting in baroreflex dysfunction and widespread changes in cellular and neuronal activity regulated by sensory/motor pathways. Hypoglycaemia initiates a rapid neurally-mediated counter-regulatory response. This counter-regulatory response to hypoglycaemia increases plasma adrenaline levels, liver glycogenolysis, and thus blood glucose levels. Context-dependent activation of rostral ventral medullary neurons initiates baroreceptor unloading, peripheral chemoreflex firing and the counter-regulatory response to hypoglycaemia. In this review, we briefly focus on the functional integration between peripheral and medullary pathways comprising the sympathetic baroreflex, chemoreflexes, and the counter-regulatory response to hypoglycaemia.

Activation of µ-opioid receptors in the rostral ventrolateral medulla blocks the sympathetic counterregulatory response to glucoprivation.

Activation of neurons in the rostral ventrolateral medulla (RVLM) following glucoprivation initiates sympathoadrenal activation, adrenaline release, and increased glucose production. Here, we aimed to determine the role of RVLM µ-opioid receptors in the counterregulatory response to systemic glucoprivation. Experiments were performed in pentobarbital sodium anesthetized male Sprague-Dawley rats ( n = 30). Bilateral activation of RVLM µ-opioid receptors with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) (8 mM, 50 nl) depressed adrenal sympathetic nerve activity for ~60 min ( n = 6; Δ49.9 ± 5.8%, P < 0.05). The counterregulatory response to glucoprivation (measured by adrenal sympathetic efferent nerve activity) induced by 2-deoxyglucose (2-DG) ( n = 6; Δ63.6 ± 16.5%, P < 0.05) was completely blocked 60 min after DAMGO microinjections ( n = 6; Δ10.2 ± 3.5%, P < 0.05). Furthermore, DAMGO pretreatment attenuated the increase in blood glucose levels after 2-DG infusion ( n = 6; 6.1 ± 0.7mmol/l vs. baseline 5.2 ± 0.3mmol/l, P > 0.05) compared with 2-DG alone ( n = 6; 7.6 ± 0.4mmol/l vs. baseline 6.0 ± 0.4mmol/l, P < 0.05). Thus, activation of RVLM µ-opioid receptors attenuated the neural efferent response to glucoprivation and reduced glucose production.

Activation of µ-opioid receptors in the rostral ventrolateral medulla blocks the sympathetic counterregulatory response to glucoprivation.

Activation of neurons in the rostral ventrolateral medulla (RVLM) following glucoprivation initiates sympathoadrenal activation, adrenaline release, and increased glucose production. Here, we aimed to determine the role of RVLM µ-opioid receptors in the counterregulatory response to systemic glucoprivation. Experiments were performed in pentobarbital sodium anesthetized male Sprague-Dawley rats ( n = 30). Bilateral activation of RVLM µ-opioid receptors with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) (8 mM, 50 nl) depressed adrenal sympathetic nerve activity for ~60 min ( n = 6; Δ49.9 ± 5.8%, P < 0.05). The counterregulatory response to glucoprivation (measured by adrenal sympathetic efferent nerve activity) induced by 2-deoxyglucose (2-DG) ( n = 6; Δ63.6 ± 16.5%, P < 0.05) was completely blocked 60 min after DAMGO microinjections ( n = 6; Δ10.2 ± 3.5%, P < 0.05). Furthermore, DAMGO pretreatment attenuated the increase in blood glucose levels after 2-DG infusion ( n = 6; 6.1 ± 0.7mmol/l vs. baseline 5.2 ± 0.3mmol/l, P > 0.05) compared with 2-DG alone ( n = 6; 7.6 ± 0.4mmol/l vs. baseline 6.0 ± 0.4mmol/l, P < 0.05). Thus, activation of RVLM µ-opioid receptors attenuated the neural efferent response to glucoprivation and reduced glucose production.

Glia and central cardiorespiratory pathology.

Respiration and blood pressure are primarily controlled by somatic and autonomic motor neurones, respectively. Central cardiorespiratory control is critical in moment-to-moment survival, but it also has a role in the development and maintenance of chronic pathological conditions such as hypertension. The glial cells of the brain are non-neuronal cells with metabolic, immune, and developmental functions. Recent evidence shows that glia play an active role in supporting and regulating the neuronal circuitry which drives the cardiorespiratory system. Here we will review the activities of two key types of glial cell, microglia and astrocytes, in assisting normal central cardiorespiratory control and in pathology.

Acute intermittent hypoxia with concurrent hypercapnia evokes P2X and TRPV1 receptor-dependent sensory long-term facilitation in naïve carotid bodies.

KEY POINTS: Activity-dependent plasticity can be induced in carotid body (CB) chemosensory afferents without chronic intermittent hypoxia (CIH) preconditioning by acute intermittent hypoxia coincident with bouts of hypercapnia (AIH-Hc). Several properties of this acute plasticity are shared with CIH-dependent sensory long-term facilitation (LTF) in that induction is dependent on 5-HT, angiotensin II, protein kinase C and reactive oxygen species. Several properties differ from CIH-dependent sensory LTF; H2 O2 appears to play no part in induction, whereas maintenance requires purinergic P2X2/3 receptor activation and is dependent on transient receptor potential vanilloid type 1 (TRPV1) receptor sensitization. Because P2X2/3 and TRPV1 receptors are located in carotid sinus nerve (CSN) terminals but not presynaptic glomus cells, a primary site of the acute AIH-Hc induced sensory LTF appears to be postsynaptic. Our results obtained in vivo suggest a role for TRPV1-dependent CB activity in acute sympathetic LTF. We propose that P2X-TRPV1-receptor-dependent sensory LTF may constitute an important early mechanism linking sleep apnoea with hypertension and/or cardiovascular disease. ABSTRACT: Apnoeas constitute an acute existential threat to neonates and adults. In large part, this threat is detected by the carotid bodies, which are the primary peripheral chemoreceptors, and is combatted by arousal and acute cardiorespiratory responses, including increased sympathetic output. Similar responses occur with repeated apnoeas but they continue beyond the last apnoea and can persist for hours [i.e. ventilatory and sympathetic long-term facilitation (LTF)]. These long-term effects may be adaptive during acute episodic apnoea, although they may prolong hypertension causing chronic cardiovascular impairment. We report a novel mechanism of acute carotid body (CB) plasticity (sensory LTF) induced by repeated apnoea-like stimuli [i.e. acute intermittent hypoxia coincident with bouts of hypercapnia (AIH-Hc)]. This plasticity did not require chronic intermittent hypoxia preconditioning, was dependent on P2X receptors and protein kinase C, and involved heat-sensitive transient receptor potential vanilloid type 1 (TRPV1) receptors. Reactive oxygen species (O2 ·¯) were involved in initiating plasticity only; no evidence was found for H2 O2 involvement. Angiotensin II and 5-HT receptor antagonists, losartan and ketanserin, severely reduced CB responses to individual hypoxic-hypercapnic challenges and prevented the induction of sensory LTF but, if applied after AIH-Hc, failed to reduce plasticity-associated activity. Conversely, TRPV1 receptor antagonism had no effect on responses to individual hypoxic-hypercapnic challenges but reduced plasticity-associated activity by ∼50%. Further, TRPV1 receptor antagonism in vivo reduced sympathetic LTF caused by AIH-Hc, although only if the CBs were functional. These data demonstrate a new mechanism of CB plasticity and suggest P2X-TRPV1-dependent sensory LTF as a novel target for pharmacological intervention in some forms of neurogenic hypertension associated with recurrent apnoeas.

PACAP-(6-38) or kynurenate microinjections in the RVLM prevent the development of sympathetic long-term facilitation after acute intermittent hypoxia.

Intermittent hypoxia causes a persistent increase in sympathetic activity that progresses to hypertension in chronic conditions such as obstructive sleep apnea. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neurotransmitter that causes long-lasting sympathetic excitation. We aimed to determine if intermittent activation of the rostral ventrolateral medulla (RVLM) causes PACAP-mediated elevation of sympathetic nerve activity, termed sympathetic long-term facilitation (sLTF). The role of PACAP in mediating sLTF in response to intermittent activation of the RVLM was investigated in urethane-anaesthetized and artificially ventilated rats ( n = 65, Sprague-Dawley). Bilateral RVLM microinjections of the PACAP type 1 receptor/vasoactive intestinal polypeptide receptor type 2 receptor antagonist PACAP-(6-38) [ n = 6, change (Δ): -16.4 ± 6.5%) or an ionotropic glutamate antagonist, kynurenate ( n = 6, Δ:-7.2 ± 2.3%), blocked the development of acute intermittent hypoxia-induced sLTF ( n = 6, Δ: 49.2 ± 14.2%). Intermittent RVLM microinjections of glutamate caused sLTF ( n = 5, Δ: 56.9 ± 14.7%) that was abolished by PACAP-(6-38) pretreatment ( n = 5, Δ:-1.2 ± 4.7%). Conversely, intermittent microinjections of PACAP in the RVLM did not elicit sLTF. Intermittent bilateral disinhibition of the RVLM by microinjection of γ-aminobutyric acid in the caudal ventrolateral medulla did not elicit sLTF. Direct activation of RVLM neurons is crucial for the development of sLTF. PACAP and glutamate act synergistically in the RVLM, with both being necessary for the sLTF response. We found that activation of glutamate but not PACAP receptors is necessary and sufficient to generate sLTF, even in the absence of intermittent hypoxia. Our results demonstrate that PACAP within the RVLM may contribute to the development of obstructive sleep apnea -induced hypertension. NEW & NOTEWORTHY Pharmacological blockade of either pituitary adenylate cyclase-activating polypeptide (PACAP) or ionotropic glutamate receptors in the rostral ventrolateral medulla prevents development of sympathetic long-term facilitation. PACAP receptor inhibition prevents the occurrence of hypoxia-induced peripheral chemoreflex sensitization. Thus, PACAP receptors may be a potential therapeutic target serving to reduce heightened sympathetic tone and hypersensitized cardiovascular reflexes.

Inhibition of microglial activation with minocycline at the intrathecal level attenuates sympathoexcitatory and proarrhythmogenic changes in rats with chronic temporal lobe epilepsy.

The incidence of sudden unexpected death in epilepsy (SUDEP) is highest in people with chronic and drug-resistant epilepsy. Chronic spontaneous recurrent seizures cause cardiorespiratory autonomic dysfunctions. Pituitary adenylate cyclase-activating polypeptide (PACAP) is neuroprotective, whereas microglia produce both pro- and anti-inflammatory effects in the CNS. During acute seizures in rats, PACAP and microglia produce sympathoprotective effect at the intermediolateral cell column (IML), whereas their action on the presympathetic rostral ventrolateral medulla (RVLM) neurons mediates proarrhythmogenic changes. We evaluated the effect of PACAP and microglia at the IML on sympathetic nerve activity (SNA), cardiovascular reflex responses, and electrocardiographic changes in the post-status epilepticus (SE) model of acquired epilepsy, and control rats. Chronic spontaneous seizures in rats produced tachycardia with profound proarrhythmogenic effects (prolongation of QT interval). Antagonism of microglia, but not PACAP, significantly reduced the SNA and the corrected QT interval in post-SE rats. PACAP and microglia antagonists did not change baroreflex and peripheral or central chemoreflex responses with varied effect on somatosympathetic responses in post-SE and control rats. We did not notice changes in microglial morphology or changes in a number of M2 phenotype in epileptic nor control rats in the vicinity of RVLM neurons. Our findings establish that microglial activation, and not PACAP, at the IML accounts for higher SNA and proarrhythmogenic changes during chronic epilepsy in rats. This is the first experimental evidence to support a neurotoxic effect of microglia during chronic epilepsy, in contrast to their neuroprotective action during acute seizures.

Intrathecal Intermittent Orexin-A Causes Sympathetic Long-Term Facilitation and Sensitizes the Peripheral Chemoreceptor Response to Hypoxia in Rats.

Intermittent hypoxia causes a persistent increase in sympathetic nerve activity (SNA), which progresses to hypertension in conditions such as obstructive sleep apnea. Orexins (A and B) are hypothalamic neurotransmitters with arousal-promoting and sympathoexcitatory effects. We investigated whether the sustained elevation of SNA, termed sympathetic long-term facilitation, after acute intermittent hypoxia (AIH) is caused by endogenous orexin acting on spinal sympathetic preganglionic neurons. The role of orexin in the increased SNA response to AIH was investigated in urethane-anesthetized, vagotomized, and artificially ventilated Sprague-Dawley rats (n = 58). A spinally infused subthreshold dose of orexin-A (intermittent; 0.1 nmol × 10) produced long-term enhancement in SNA (41.4% ± 6.9%) from baseline. This phenomenon was not produced by the same dose of orexin-A administered as a bolus intrathecal infusion (1 nmol; 7.3% ± 2.3%). The dual orexin receptor blocker, Almorexant, attenuated the effect of sympathetic long-term facilitation generated by intermittent orexin-A (20.7% ± 4.5% for Almorexant at 30 mg∙kg(-1) and 18.5% ± 1.2% for 75 mg∙kg(-1)), but not in AIH. The peripheral chemoreflex sympathoexcitatory response to hypoxia was greatly enhanced by intermittent orexin-A and AIH. In both cases, the sympathetic chemoreflex sensitization was reduced by Almorexant. Taken together, spinally acting orexin-A is mechanistically sufficient to evoke sympathetic long-term facilitation. However, AIH-induced sympathetic long-term facilitation appears to rely on mechanisms that are independent of orexin neurotransmission. Our findings further reveal that the activation of spinal orexin receptors is critical to enhance peripheral chemoreceptor responses to hypoxia after AIH.

Microglial number is related to the number of tyrosine hydroxylase neurons in SHR and normotensive rats.

Microglia are ubiquitously distributed throughout the central nervous system (CNS) and play a critical role in the maintenance of neuronal homeostasis. Recent advances have shown that microglia, never resting cells of the CNS, continuously monitor and influence neuronal/synaptic activity levels, by communicating with neurons with the aid of their dynamic processes. The brainstem contains many catecholaminergic nuclei that are key to many aspects of brain function. This includes C1 neurons of the ventrolateral medulla that are thought to play a critical role in control of the circulation. Despite the role of catecholaminergic brainstem neurons in normal physiology, the presence of microglia that surrounds them is poorly understood. Here, we investigate the spatial distribution and morphology of microglia in catecholaminergic nuclei of the brainstem in 3 strains of rat: Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our data reveal that microglia are heterogeneously distributed within and across different strains of rats. Interestingly, intra-strain comparison of tyrosine hydroxylase-immunoreactive (TH-ir) neuronal and microglial number reveals that microglial number varies with the TH-ir neuronal number in the brainstem. Even though microglial spatial distribution varies across brainstem nuclei, microglial morphology (% area covered, number of end point processes and branch length) does not differ significantly. This work provides the first evidence that even though microglia, in their surveilling state, do not vary appreciably in their morphology across brainstem areas, they do have a heterogeneous pattern of distribution that may be influenced by their local environment.

Dynamic changes in the relationship of microglia to cardiovascular neurons in response to increases and decreases in blood pressure.

Microglia are present throughout the central nervous system (CNS) and express receptors for every known neurotransmitter. During inflammation, microglia change into a state that either promotes removal of debris (M1), or into a state that promotes soothing (M2). Caudal- and rostral- ventrolateral medullary regions (CVLM and RVLM, respectively) of the brainstem are key nuclei involved in all aspects of the cardiovascular system. In this study, we investigate a novel role for microglia in cardiovascular control in the brainstem of adult male Sprague-Dawley (SD) rat. Here we show, that increases and decreases in blood pressure (BP) triggers alertness in the physiology of microglia in the brainstem region; inducing changes in microglial spatial distribution and the number of synapses in contact with microglial end processes. Following 6h of acute hypertension, the number of synapses in contact with microglia increased by ≈30% in both regions of the brainstem, CVLM and RVLM. Induction of acute hypotension for 6h causes microglia to reduce the number of synaptic contacts by >20% in both, CVLM and RVLM, nuclei of the brainstem. Our analysis of the morphological characteristics of microglia, and expression levels of M1 and M2, reveals that the changes induced in microglial behavior do not require any obvious dramatic changes in their morphology. Taken together, our findings suggest that microglia play a novel, unexpected, physiological role in the uninjured autonomic nuclei of CNS; we therefore speculate that microglia act cooperatively with brainstem cardiovascular neurons to maintain them in a physiologically receptive state.

Intermittent hypoxia-induced cardiorespiratory long-term facilitation: A new role for microglia.

Intermittent hypoxia induces plasticity in neural networks controlling breathing and cardiovascular function. Studies demonstrate that mechanisms causing cardiorespiratory plasticity rely on intracellular signalling pathways that are activated by specific neurotransmitters. Peptides such as serotonin, PACAP and orexin are well-known for their physiological significance in regulating the cardiorespiratory system. Their receptor counterparts are present in cardiorespiratory centres of the brainstem medulla and spinal cord. Microglial cells are also important players in inducing plasticity. The phenotype and function of microglial cells can change based on the physiological state of the central nervous system. Here, we propose that in the autonomic nuclei of the ventral brainstem the relationship between neurotransmitters and neurokines, neurons and microglia determines the overall neural function of the central cardiorespiratory system.

Microglia PACAP and glutamate: Friends or foes in seizure-induced autonomic dysfunction and SUDEP?

Seizure-induced cardiorespiratory autonomic dysfunction is a major cause of sudden unexpected death in epilepsy (SUDEP), and the underlying mechanism is unclear. Seizures lead to increased synthesis, and release of glutamate, pituitary adenylate cyclase activating polypeptide (PACAP), and other neurotransmitters, and cause extensive activation of microglia at multiple regions in the brain including central autonomic cardiorespiratory brainstem nuclei. Glutamate contributes to neurodegeneration, and inflammation in epilepsy. PACAP has neuroprotective, and anti-inflammatory properties, whereas microglia are key players in inflammatory responses in CNS. Seizure-induced increase in PACAP is neuroprotective. PACAP produces neuroprotective effects acting on microglial PAC1 and VPAC1 receptors. Microglia also express glutamate transporters, and their expression can be increased by PACAP in response to harmful or stressful situations such as seizures. Here we discuss the mechanism of autonomic cardiorespiratory dysfunction in seizure, and the role of PACAP, glutamate and microglia in regulating cardiorespiratory brainstem neurons in their physiological state that could provide future therapeutic options for SUDEP.

Seizure-Induced Sympathoexcitation Is Caused by Activation of Glutamatergic Receptors in RVLM That Also Causes Proarrhythmogenic Changes Mediated by PACAP and Microglia in Rats.

Cardiovascular autonomic dysfunction in seizure is a major cause of sudden unexpected death in epilepsy. The catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) maintain sympathetic vasomotor tone and blood pressure through their direct excitatory projections to the intermediolateral (IML) cell column. Glutamate, the principal excitatory neurotransmitter in brain, is increased in seizures. Pituitary adenylate cyclase activating polypeptide (PACAP) is an excitatory neuropeptide with neuroprotective properties, whereas microglia are key players in inflammatory responses in CNS. We investigated the roles of glutamate, PACAP, and microglia on RVLM catecholaminergic neurons during the cardiovascular responses to 2 mg/kg kainic acid (KA)-induced seizures in urethane anesthetized, male Sprague Dawley rats. Microinjection of the glutamate antagonist, kynurenic acid (50 nl; 100 mM) into RVLM, blocked the seizure-induced 43.2 ± 12.6% sympathoexcitation (p ≤ 0.05), and abolished the pressor responses, tachycardia, and QT interval prolongation. PACAP or microglia antagonists (50 nl) (PACAP(6-38), 15 pmol; minocycline 10 mg/ml) microinjected bilaterally into RVLM had no effect on seizure-induced sympathoexcitation, pressor responses, or tachycardia but abolished the prolongation of QT interval. The actions of PACAP or microglia on RVLM neurons do not cause sympathoexcitation, but they do elicit proarrhythmogenic changes. An immunohistochemical analysis in 2 and 10 mg/kg KA-induced seizure rats revealed that microglia surrounding catecholaminergic neurons are in a "surveillance" state with no change in the number of M2 microglia (anti-inflammatory). In conclusion, seizure-induced sympathoexcitation is caused by activation of glutamatergic receptors in RVLM that also cause proarrhythmogenic changes mediated by PACAP and microglia. SIGNIFICANCE STATEMENT: Sudden unexpected death in epilepsy is a major cause of death in epilepsy. Generally, seizures are accompanied by changes in brain function leading to uncontrolled nerve activity causing high blood pressure, rapid heart rate, and abnormal heart rhythm. Nevertheless, the brain chemicals causing these cardiovascular changes are unknown. Chemicals, such as glutamate and pituitary adenylate cyclase activating polypeptide, whose expression is increased after seizures, act on specific cardiovascular nuclei in the brain and influence the activity of the heart, and blood vessels. Microglia, which manage excitation in the brain, are commonly activated after seizure and produce pro- and/or anti-inflammatory factors. Hence, we aimed to determine the effects of blocking glutamate, pituitary adenylate cyclase activating polypeptide, and microglia in the RVLM and their contribution to cardiovascular autonomic dysfunction in seizure.

Alerted microglia and the sympathetic nervous system: A novel form of microglia in the development of hypertension.

Microglia, commonly known as the tissue resident macrophages of the central nervous system (CNS), are ubiquitously expressed in the CNS. Microglia, in their resting, or surveilling, stage, play a critical role in the maintenance of normal neuronal physiology and homeostasis. On activation, microglia can acquire either a neurotoxic (M1) or a neuroprotective (M2) phenotype. Prior to development of the M1 or M2 phenotype, little was known about changes in microglial activity, when subjected to stimuli. It is postulated, that an inability of microglia to maintain neuronal physiology within a normal working range can contribute to the development of cardiovascular disorders (CVDs) such as hypertension, but clear evidence supporting this hypothesis is missing. Even though our understanding of microglial function in a state of CNS injury/inflammation is extensive, the literature concerning role of microglia in the healthy CNS, is limited. Involvement of microglia in the pathophysiology of CVDs, in a neuroprotective/neurotoxic manner, is a key area that requires further investigation.