Ryan Bateman

Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei.

Recent advances in optogenetic methods demonstrate the feasibility of selective photoactivation at the soma of neurons that express channelrhodopsin-2 (ChR2), but a comprehensive evaluation of different methods to selectively evoke transmitter release from distant synapses using optogenetic approaches is needed. Here we compared different lentiviral vectors, with sub-population-specific and strong promoters, and transgenic methods to express and photostimulate ChR2 in the long-range projections of paraventricular nucleus of the hypothalamus (PVN) neurons to brain stem cardiac vagal neurons (CVNs). Using PVN subpopulation-specific promoters for vasopressin and oxytocin, we were able to depolarize the soma of these neurons upon photostimulation, but these promoters were not strong enough to drive sufficient expression for optogenetic stimulation and synaptic release from the distal axons. However, utilizing the synapsin promoter photostimulation of distal PVN axons successfully evoked glutamatergic excitatory post-synaptic currents in CVNs. Employing the Cre/loxP system, using the Sim-1 Cre-driver mouse line, we found that the Rosa-CAG-LSL-ChR2-EYFP Cre-responder mice expressed higher levels of ChR2 than the Rosa-CAG-LSL-ChR2-tdTomato line in the PVN, judged by photo-evoked currents at the soma. However, neither was able to drive sufficient expression to observe and photostimulate the long-range projections to brainstem autonomic regions. We conclude that a viral vector approach with a strong promoter is required for successful optogenetic stimulation of distal axons to evoke transmitter release in pre-autonomic PVN neurons. This approach can be very useful to study important hypothalamus-brainstem connections, and can be easily modified to selectively activate other long-range projections within the brain.

Clonidine, an α2-receptor agonist, diminishes GABAergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus

In hypertension, there is an autonomic imbalance in which sympathetic activity dominates over parasympathetic control. Parasympathetic activity to the heart originates from cardiac vagal neurons located in the nucleus ambiguus. Presympathetic neurons that project to sympathetic neurons in the spinal cord are located in the ventral brainstem in close proximity to cardiac vagal neurons, and many of these presympathetic neurons are catecholaminergic. In addition to their projection to the spinal cord, many of these presympathetic neurons have axon collaterals that arborize into neighboring cardiorespiratory locations and likely release norepinephrine onto nearby neurons. Activation of alpha(2)-adrenergic receptors in the central nervous system evokes a diverse range of physiological effects, including reducing blood pressure. This study tests whether clonidine, an alpha(2)-adrenergic receptor agonist, alters excitatory glutamatergic, and/or inhibitory GABAergic or glycinergic synaptic neurotransmission to cardiac vagal neurons in the nucleus ambiguus. Cardiac vagal neurons were identified in an in vitro brainstem slice preparation, and synaptic events were recording using whole cell voltage clamp methodologies. Clonidine significantly inhibited GABAergic neurotransmission but had no effect on glycinergic or glutamatergic pathways to cardiac vagal neurons. This diminished inhibitory GABAergic neurotransmission to cardiac vagal neurons would increase parasympathetic activity to the heart, decreasing heart rate and blood pressure. The results presented here provide a cellular substrate for the clinical use of clonidine as a treatment for hypertension as well as a role in alleviating posttraumatic stress disorder by evoking an increase in parasympathetic cardiac vagal activity, and a decrease in heart rate and blood pressure.

Evidence that BDNF regulates heart rate by a mechanism involving increased brainstem parasympathetic neuron excitability

Autonomic control of heart rate is mediated by cardioinhibitory parasympathetic cholinergic neurons located in the brainstem and stimulatory sympathetic noradrenergic neurons. During embryonic development the survival and cholinergic phenotype of brainstem autonomic neurons is promoted by brain‐derived neurotrophic factor (BDNF). We now provide evidence that BDNF regulates heart rate by a mechanism involving increased brainstem cardioinhibitory parasympathetic activity. Mice with a BDNF haploinsufficiency exhibit elevated resting heart rate, and infusion of BDNF intracerebroventricularly reduces heart rate in both wild‐type and BDNF+/− mice. The atropine‐induced elevation of heart rate is diminished in BDNF+/− mice and is restored by BDNF infusion, whereas the atenolol‐induced decrease in heart rate is unaffected by BDNF levels, suggesting that BDNF signaling enhances parasympathetic tone which is diminished with BDNF haploinsufficiency. Whole‐cell recordings from pre‐motor cholinergic cardioinhibitory vagal neurons in the nucleus ambiguus indicate that BDNF haploinsufficiency reduces cardioinhibitory vagal neuron activity by increased inhibitory GABAergic and diminished excitatory glutamatergic neurotransmission to these neurons. Our findings reveal a previously unknown role for BDNF in the control of heart rate by a mechanism involving increased activation of brainstem cholinergic parasympathetic neurons

Endogenous Inhibition of the Trigeminally Evoked Neurotransmission to Cardiac Vagal Neurons by Muscarinic Acetylcholine Receptors

Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs.

Hypocretin-1 (orexin A) prevents the effects of hypoxia/hypercapnia and enhances the GABAergic pathway from the lateral paragigantocellular nucleus to cardiac vagal neurons in the nucleus ambiguus

Hypocretins (orexins) are hypothalamic neuropeptides that play a crucial role in regulating sleep/wake states and autonomic functions including parasympathetic cardiac activity. We have recently demonstrated stimulation of the lateral paragigantocellular nucleus (LPGi), the nucleus which is thought to play a role in rapid eye movement (REM) sleep control, activates an inhibitory pathway to preganglionic cardiac vagal neurons in the nucleus ambiguus (NA). In this study we test the hypothesis that hypocretin-1 modulates the inhibitory neurotransmission to cardiac vagal neurons evoked by stimulation of the LPGi using whole-cell patch-clamp recordings in an in vitro brain slice preparation from rats. Activation of hypocretin-1 receptors produced a dose-dependent and long-term facilitation of GABAergic postsynaptic currents evoked by electrical stimulation of the LPGi. Hypoxia/hypercapnia diminished LPGi-evoked GABAergic current in cardiac vagal neurons and this inhibition by hypoxia/hypercapnia was prevented by pre-application of hypocretin-1. The action of hypocretin-1 was blocked by the hypocretin-1 receptor antagonist SB-334867. Facilitation of LPGi-evoked GABAergic current in cardiac vagal neurons under both normal condition and during hypoxia/hypercapnia could be the mechanism by which hypocretin-1 affects parasympathetic cardiac function and heart rate during REM sleep. Furthermore, our findings indicate a new potential mechanism that might be involved in the cardiac arrhythmias, bradycardia, and sudden cardiac death that can occur during sleep.

β adrenergic receptor modulation of neurotransmission to cardiac vagal neurons in the nucleus ambiguus

β-adrenergic receptors are a class of G protein-coupled receptors that have essential roles in regulating heart rate, blood pressure, and other cardiorespiratory functions. Although the role of β adrenergic receptors in the peripheral nervous system is well characterized, very little is known about their role in the central nervous system despite being localized in many brain regions involved in autonomic activity and regulation. Since parasympathetic activity to the heart is dominated by cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA), β adrenergic receptors localized in the NA represent a potential target for modulating cardiac vagal activity and heart rate. This study tests the hypothesis that activation of β adrenergic receptors alters the membrane properties and synaptic neurotransmission to CVNs. CVNs were identified in brainstem slices, and membrane properties and synaptic events were recorded using the whole-cell voltage-clamp technique. The nonselective β agonist isoproterenol significantly decreased inhibitory GABAergic and glycinergic as well as excitatory glutamatergic neurotransmission to CVNs. In addition, the β(1)-selective receptor agonist dobutamine, but not β(2) or β(3) receptor agonists, significantly decreased inhibitory GABAergic and glycinergic and excitatory glutamatergic neurotransmission to CVNs. These decreases in neurotransmission to CVNs persisted in the presence of tetrodotoxin (TTX). These results provide a mechanism by which activation of adrenergic receptors in the brainstem can alter parasympathetic activity to the heart. Likely physiological roles for this adrenergic receptor activation are coordination of parasympathetic-sympathetic activity and β receptor-mediated increases in heart rate upon arousal.

Anaesthetics differentially modulate the trigeminocardiac reflex excitatory synaptic pathway in the brainstem

Non‐Technical Summary  Activation of the trigeminal nerve during eye and head surgery often evokes a dramatic decrease in heart rate, blood pressure and breathing rate, referred to as the trigeminocardiac reflex. Different anaesthetics can depress or amplify this reflex with serious clinical consequences. In this study we focused on two populations of neurones, the neurones that receive sensory information and the neurones that control heart rate. We show that these two groups of neurones in the brain are activated in the reflex circuitry and how different anaesthetics differentially modulate the neurotransmission to these neurones. These results help us understand the mechanisms and anaesthetic modulation of the trigeminocardiac reflex and can help reduce its rate of occurrence and increase patients’ safety during surgery.

α1-adrenergic receptors facilitate inhibitory neurotransmission to cardiac vagal neurons in the nucleus ambiguus

The cholinergic cardiac vagal neurons (CVNs), located in the nucleus ambiguus, are the origin of cardioinhibitory parasympathetic activity. Catecholaminergic neurons in nearby regions of the brainstem, including the C1 and C2 cell groups, are thought to play a key role in both arousing from sleep and maintaining wakefulness. Because norepinephrine (NE) could play an important role in influencing the activity of CVNs, particularly in response to sleeping/waking and arousal states, the present study investigated the contribution of α(1)-adrenergic receptor activation to augment inhibitory and/or blunt excitatory neurotransmission to CVNs. To test the effects of α(1)-adrenergic receptor activation, CVNs were labeled in rats by retrograde tracing and synaptic events were recorded by whole cell voltage clamp techniques in vitro. Prazosin, an inverse agonist of α(1)-adrenergic receptor, significantly decreased the frequency of both GABAergic and glycinergic neurotransmission to CVNs. Activation of α(1)-adrenergic receptors by the α(1)-adrenergic receptor agonists NE or phenylephrine (PE) both significantly increased GABAergic and glycinergic inhibitory event frequency. This effect was prevented by the sodium channel blocker tetrodotoxin (TTX). Activation of α(1)-adrenergic receptors did not alter glutamatergic neurotransmission to CVNs. This study indicates that α(1)-adrenergic receptor activation in the brainstem can facilitate inhibitory GABAergic and glycinergic neurotransmission so as to reduce CVN activity; this synaptic modulation may play a role in the tachycardia seen during NE-dependent behavioral arousal.

Orexinergic modulation of GABAergic neurotransmission to cardiac vagal neurons in the brain stem nucleus ambiguus changes during development.

Cardiac vagal neurons (CVNs) in the nucleus ambiguus (NA) are the major determinant of parasympathetic activity to the heart. Spontaneous GABAergic neurotransmission to CVNs is modulated by hypothalamic neuropeptide orexin-A in postnatal days 2-5 (P5) rats; however, during early postnatal development, orexin expression changes, and the role of orexin-A in modulating CVN activity at other stages of development is unknown. In this study, we compared changes in GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs evoked by orexin-A in P5, P16-20 (P20), and P27-30 (P30) rats using an in vitro brain stem slice preparation. Bath-applied orexin-A enhanced GABAergic IPSCs in all CVNs tested in P5 and P30 animals and in the majority of neurons tested in P20 pups. Focal application of orexin-A ejected from a pipette positioned within 30 μm of the patched CVN did not alter GABAergic signaling in P5 pups. In contrast, in both P20 and P30 rats, focal application of orexin-A inhibited GABAergic IPSCs, and this inhibition persisted in the presence of tetrodotoxin. These results indicate orexin-A facilitates GABAergic IPSCs likely by activating preceding GABAergic neurons that project to CVNs. Orexin-A also likely acts at GABAergic presynaptic terminals surrounding CVNs within the NA to inhibit GABA release. The latter mechanism is absent in P5 pups but occurs in P20 and P30 rats. In conclusion, this study elucidates an important maturation of the parasympathetic cardiac control system. Alterations in these developmental mechanisms may play a role in pathogenesis of disorders related to a specific stage of development maturation.

Oxytocin neuron activation prevents hypertension that occurs with chronic intermittent hypoxia/hypercapnia in rats.

Hypertension is a common outcome associated with obstructive sleep apnea (OSA), a prevalent yet poorly treated cardiovascular disease. Recent studies showed oxytocin (OXT), released from hypothalamic paraventricular nucleus (PVN) neurons, activates cardiac vagal neurons in the dorsal motor nucleus of the vagus (DMNX) and may blunt cardiovascular responses to stress. This study tests whether the release of OXT from PVN fibers in the DMNX is diminished with chronic intermittent hypoxia-hypercapnia (CIH/H) exposure, an animal model of OSA, and whether activation of PVN OXT neurons restores OXT release in the DMNX and prevents the hypertension resulting from CIH/H. To assess OXT release from PVN fibers, Chinese hamster ovarian (CHO) cells were engineered to be highly sensitive to OXT by stable expression of the human recombinant OXT receptor and the calcium indicator R-GECO1. PVN fibers in the DMNX were selectively photoactivated in vitro by expression of channelrhodopsin. The release of OXT onto CHO cells in the DMNX was blunted in rats exposed to 21 days of CIH/H. Chronic activation of PVN OXT neurons in vivo, using designer receptors exclusively activated by designer drugs, restored the release of OXT onto CHO cells in the DMNX. Chronic PVN OXT neuron activation in vivo also prevented the hypertension that occurred in conscious unrestrained telemetry-equipped sham rats exposed to 3 wk of CIH/H. These results demonstrate that chronic activation of OXT neurons restores the release of OXT from PVN fibers in the DMNX and prevents the hypertension that occurs with 3 wk of CIH/H exposure.