Neural correlate of reduced respiratory chemosensitivity during chronic epilepsy

While central autonomic, cardiac, and/or respiratory dysfunction underlies sudden unexpected death in epilepsy (SUDEP), the specific neural mechanisms that lead to SUDEP remain to be determined. In this study, we took advantage of single-cell neuronal Ca2+ imaging and intrahippocampal kainic acid (KA)-induced chronic epilepsy in mice to investigate progressive changes in key cardiorespiratory brainstem circuits during chronic epilepsy. Weeks after induction of status epilepticus (SE), when mice were experiencing recurrent spontaneous seizures (chronic epilepsy), we observed that the adaptive ventilatory responses to hypercapnia were reduced for 5 weeks after SE induction with its partial recovery at week 7. These changes were paralleled by alterations in the chemosensory responses of neurons in the retrotrapezoid nucleus (RTN). Neurons that displayed adapting responses to hypercapnia were less prevalent and exhibited smaller responses over weeks 3–5, whereas neurons that displayed graded responses to hypercapnia became more prevalent by week 7. Over the same period, chemosensory responses of the presympathetic rostral ventrolateral medullary (RVLM) neurons showed no change. Mice with chronic epilepsy showed enhanced sensitivity to seizures, which invade the RTN and possibly put the chemosensory circuits at further risk of impairment. Our findings establish a dysfunctional breathing phenotype with its RTN neuronal correlate in mice with chronic epilepsy and suggest that the assessment of respiratory chemosensitivity may have the potential for identifying people at risk of SUDEP

Imaging single-cell Ca2+ dynamics of brainstem neurons and glia in freely behaving mice

In vivo brain imaging, using a combination of genetically encoded Ca2+ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process

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

Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice

Regulation of systemic PCO2 is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO2 chemosensory nuclei mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pFL). Here we use GCaMP6 expression and head-mounted mini-microscopes to image Ca2+ activity in these nuclei in awake adult mice during hypercapnia. Activity in the pFL supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO2, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours

Anti-migraine effect of Areca catechu L. nut extract in bradykinin-induced plasma protein extravasation and vocalization in rats

Ethnopharmacological relevance Areca catechu Linn. (Arecaceae) nut is a popular folk remedy for the treatment of migraine in Kerala and Tamil Nadu states of India. Aim of the study This study was designed to investigate the effect of hydroalcoholic extract of A. catechu L. nut (ANE) treatment on migraine pain in rat models to strengthen its use as an anti-migraine therapy. Materials and methods Bradykinin (0.1 μmol/kg) injection in to left femoral vein of rat produced PPE which was measured with luminescence spectrometer. Vocalizations were produced in rats with 10 μg of bradykinin infusion into common carotid artery. Phonogram was recorded before, during and for 5 min after bradykinin injection and sumatriptan was used as a standard anti-migraine drug. In both models, the ANE was orally administered at doses of 250 and 500 mg/kg, 60 min before bradykinin infusion. Results The PPE was reduced in both ANE treated groups of rats. The percent fluorescein was significantly increased in positive control group (97.00±1.7%; p<0.0001) compared to negative control (63.87±1.2%). With ANE treatments (250 and 500 mg/kg) PPE was significantly decreased to 88.88±1.4% (p<0.01) and 83.55±0.1% (p<0.0001) compared to positive control group, respectively. On the other hand in the model of vocalization, with 250 and 500 mg/kg ANE treatment, vocalization was significantly reduced to 33.33% and 16.66%, respectively, compared to saline treated rats. The reduction in vocalization is comparable to the reference drug sumatriptan. Conclusion The findings provide the strong evidence for anti-migraine potential of ANE in rat models of migraine. In summary, therapeutic intervention with ANE treatment could be a promising strategy for prevention of migraine.4 page(s

Antagonism of PACAP or microglia function worsens the cardiovascular consequences of kainic-acid-induced seizures in rats

Seizures are accompanied by cardiovascular changes that are a major cause of sudden unexpected death in epilepsy (SUDEP). Seizures activate inflammatory responses in the cardiovascular nuclei of the medulla oblongata and increase neuronal excitability. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with autocrine and paracrine neuroprotective properties. Microglia are key players in inflammatory responses in the CNS. We sought to determine whether PACAP and microglia mitigate the adverse effects of seizure on cardiovascular function in a rat model of temporal lobe epilepsy. Kainic acid (KA)-induced seizures increased splanchnic sympathetic nerve activity by 97%, accompanied by increase in heart rate (HR) but not blood pressure (BP). Intrathecal infusion of the PACAP antagonist PACAP(6-38) or the microglia antagonists minocycline and doxycycline augmented sympathetic responses to KA-induced seizures. PACAP(6-38) caused a 161% increase, whereas minocycline and doxycycline caused a 225% and 215% increase, respectively. In intrathecal PACAP-antagonist-treated rats, both BP and HR increased, whereas after treatment with microglial antagonists, only BP was significantly increased compared with control. Our findings support the idea that PACAP and its action on microglia at the level of the spinal cord elicit cardioprotective effects during seizure. However, intrathecal PACAP did not show additive effects, suggesting that the agonist effect was at maximum. The protective effect of microglia may occur by adoption of an M2 phenotype and expression of factors such as TGF-β and IL-10 that promote neuronal quiescence. In summary, therapeutic interventions targeting PACAP and microglia could be a promising strategy for preventing SUDEP

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

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

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