Optogenetic and chemogenetic strategies for sustained inhibition of pain.

Spatially targeted, genetically-specific strategies for sustained inhibition of nociceptors may help transform pain science and clinical management. Previous optogenetic strategies to inhibit pain have required constant illumination, and chemogenetic approaches in the periphery have not been shown to inhibit pain. Here, we show that the step-function inhibitory channelrhodopsin, SwiChR, can be used to persistently inhibit pain for long periods of time through infrequent transdermally delivered light pulses, reducing required light exposure by >98% and resolving a long-standing limitation in optogenetic inhibition. We demonstrate that the viral expression of the hM4D receptor in small-diameter primary afferent nociceptor enables chemogenetic inhibition of mechanical and thermal nociception thresholds. Finally, we develop optoPAIN, an optogenetic platform to non-invasively assess changes in pain sensitivity, and use this technique to examine pharmacological and chemogenetic inhibition of pain.

Optogenetic approaches addressing extracellular modulation of neural excitability.

The extracellular ionic environment in neural tissue has the capacity to influence, and be influenced by, natural bouts of neural activity. We employed optogenetic approaches to control and investigate these interactions within and between cells, and across spatial scales. We began by developing a temporally precise means to study microdomain-scale interactions between extracellular protons and acid-sensing ion channels (ASICs). By coupling single-component proton-transporting optogenetic tools to ASICs to create two-component optogenetic constructs (TCOs), we found that acidification of the local extracellular membrane surface by a light-activated proton pump recruited a slow inward ASIC current, which required molecular proximity of the two components on the membrane. To elicit more global effects of activity modulation on 'bystander' neurons not under direct control, we used densely-expressed depolarizing (ChR2) or hyperpolarizing (eArch3.0, eNpHR3.0) tools to create a slow non-synaptic membrane current in bystander neurons, which matched the current direction seen in the directly modulated neurons. Extracellular protons played contributory role but were insufficient to explain the entire bystander effect, suggesting the recruitment of other mechanisms. Together, these findings present a new approach to the engineering of multicomponent optogenetic tools to manipulate ionic microdomains, and probe the complex neuronal-extracellular space interactions that regulate neural excitability.

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

The trigeminocardiac reflex (TCR) occurs upon excitation of the trigeminal nerve with a resulting bradycardia and hypotension. While several anaesthetics and analgesics have been reported to alter the incidence and strength of the TCR the mechanisms for this modulation are unclear. This study examines the mechanisms of action of ketamine, isoflurane and fentanyl on the synaptic TCR responses in both neurones in the spinal trigeminal interpolaris (Sp5I) nucleus and cardiac vagal neurones (CVNs) in the Nucleus Ambiguus (NA). Stimulation of trigeminal afferent fibres evoked an excitatory postsynaptic current (EPSC) in trigeminal neurones with a latency of 1.8 ± 0.1 ms, jitter of 625 µs, and peak amplitude of 239 ± 45 pA. Synaptic responses further downstream in the reflex pathway in the CVNs occurred with a latency of 12.1 ± 1.1 ms, jitter of 0.8-2 ms and amplitude of 57.8 ± 7.5 pA. The average conduction velocity to the Sp5I neurones was 0.94 ± 0.18 mm ms(-1) indicating a mixture of A-δ and C fibres. Stimulation-evoked EPSCs in both Sp5I and CVNs were completely blocked by AMPA/kainate and NMDA glutamatergic receptor antagonists. Ketamine (10 µm) inhibited the peak amplitude and duration in Sp5I as well as more distal synapses in the CVNs. Isoflurane (300 µm) significantly inhibited, while fentanyl (1 µm) significantly enhanced, EPSC amplitude and area in CVNs but had no effect on the responses in Sp5l neurones. These findings indicate glutamatergic excitatory synaptic pathways are critical in the TCR, and ketamine, isoflurane and fentanyl differentially alter the synaptic pathways via modulation of both AMPA/kainate and NMDA receptors at different synapses in the TCR.

The role of 5-HT3 and other excitatory receptors in central cardiorespiratory responses to hypoxia: implications for sudden infant death syndrome.

Although brainstem serotonergic (5-HT) systems are involved in the protective responses to hypoxia, abnormalities of 5-HT function are strongly implicated in SIDS, and the neurochemical mechanisms by which 5-HT receptors influence brainstem cardiorespiratory responses to hypoxia remains unclear. This study focuses on the role of excitatory neurotransmission, including 5-HT3 signaling, to cardiac vagal neurons (CVNs) that dominate the control of heart rate. Excitatory synaptic inputs to CVNs, located in the nucleus ambiguus (NA), were recorded simultaneously with respiratory activity in in vitro brainstem slices. During control conditions excitatory inputs to CVNs were blocked by application of NMDA and AMPA/kainate glutamatergic receptor antagonists, whereas the 5-HT3 and purinergic receptor antagonists ondansetron and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), respectively, had no effect. However, during hypoxia ondansetron inhibited excitatory neurotransmission to CVNs. In recovery from hypoxia, spontaneous and respiratory-related excitatory events were blocked by glutamatergic and purinergic receptor blockers, respectively, whereas ondancetron had no effect. These results demonstrate that hypoxia recruits a 5-HT pathway to CVNs that activates 5-HT3 receptors on CVNs to maintain parasympathetic cardiac activity during hypoxia. Exaggeration of this 5-HT neurotransmission could increase the incidence of bradycardia and risk of sudden infant death during hypoxia.

5HT2 receptor activation facilitates P2X receptor mediated excitatory neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

Parasympathetic preganglionic cardiac vagal neurons (CVNs) which dominate the control of heart rate are located within the nucleus ambiguus (NA). Serotonin (5HT), and in particular 5HT2 receptors, play an important role in cardiovascular function in the brainstem. However, there is a lack of information on the mechanisms of action of 5HT2 receptors in modulating parasympathetic cardiac activity. This study tests whether activation of 5HT2 receptors alters excitatory glutamatergic and purinergic neurotransmission to CVNs. Application of alpha-methyl-5-hydroxytryptamine (alpha-Me-5HT), a 5HT2 agonist, reversibly increased both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in CVNs. Similar responses were obtained with alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride (BW723C86), and m-chlorophenylpiperazine (m-CPP), 5HT2B and 5HT2B/C receptor agonists, respectively. The facilitation evoked by alpha-Me-5HT was prevented by the 5HT2B/C receptor antagonist SB206553 hydrochloride (SB206553). Interestingly, the blockage of both NMDA and non-NMDA glutamatergic receptors did not prevent alpha-Me-5HT-evoked facilitation of mEPSCs, however, the responses were blocked by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The responses evoked by alpha-Me-5HT were mimicked by application of alpha,beta-methylene ATP (alpha,beta-Me-ATP), a P2X receptor agonist, which were also blocked by PPADS. In summary, these results indicate activation of 5HT2 receptors facilitates excitatory purinergic, but not glutamatergic, neurotransmission to CVNs.

5-Hydroxytryptamine 1A/7 and 4alpha receptors differentially prevent opioid-induced inhibition of brain stem cardiorespiratory function.

Opioids evoke respiratory depression, bradycardia, and reduced respiratory sinus arrhythmia, whereas serotonin (5-HT) agonists stimulate respiration and cardiorespiratory interactions. This study tested whether serotonin agonists can prevent the inhibitory effects of opioids on cardiorespiratory function. Spontaneous and rhythmic inspiratory-related activity and gamma-aminobutyric acid (GABA) neurotransmission to premotor parasympathetic cardioinhibitory neurons in the nucleus ambiguus were recorded simultaneously in an in vitro thick slice preparation. The mu-opioid agonist fentanyl inhibited respiratory frequency. The 5-hydroxytryptamine 1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin increased respiratory frequency by itself and also prevented the fentanyl-induced respiratory depression. The 5-hydroxytryptamine 4alpha agonist BIMU-8 did not by itself change inspiratory activity but prevented the mu-opioid-mediated respiratory depression. Both spontaneous and inspiratory-evoked GABAergic neurotransmission to cardiac vagal neurons were inhibited by fentanyl. 8-Hydroxy-2-(di-n-propylamino)tetralin inhibited spontaneous but not inspiratory-evoked GABAergic activity to parasympathetic cardiac neurons. However, 8-hydroxy-2-(di-n-propylamino)tetralin differentially altered the opioid-mediated depression of inspiratory-evoked GABAergic activity but did not change the opioid-induced reduction in spontaneous GABAergic neurotransmission. In contrast, BIMU-8 did not alter GABAergic neurotransmission to cardiac vagal neurons by itself but prevented the fentanyl depression of both spontaneous and inspiratory-elicited GABAergic neurotransmission to cardiac vagal neurons. In the presence of tetrodotoxin, the inhibition of GABAergic inhibitory postsynaptic currents with fentanyl is prevented by coapplication of BIMU-8, indicating that BIMU-8 acts at presynaptic GABAergic terminals to prevent fentanyl-induced depression. These results suggest that activation of 5-hydroxytryptamine receptors, particularly 5-hydroxytryptamine 4alpha agonists, may be a useful therapeutic approach in preventing opioid-evoked cardiorespiratory depression.

Purinergic P2X receptors mediate excitatory transmission to cardiac vagal neurons in the nucleus ambiguus after hypoxia.

Challenges such as hypoxia elicit a powerful response from both the central cardiovascular and respiratory neuronal networks. Recent work indicates that purinergic neurotransmission in the brain stem is an important modulator of central respiratory network responses to hypoxia. This study tests whether alterations in purinergic neurotransmission extend beyond respiratory responses to hypoxia and also mediates respiratory inputs to cardiac vagal neurons. To examine central cardiorespiratory responses to hypoxia, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and synaptic neurotransmission to cardioinhibitory vagal neurons. Here we show that P2X receptor activation mediates respiratory-related excitatory neurotransmission to parasympathetic cardiac vagal neurons, the dominant control of heart rate. These data demonstrate a critical functional role for adenosine 5'-triphosphate-mediated purinergic signaling in facilitating respiratory-related excitatory neurotransmission to cardiac vagal neurons after hypoxia.

Reactive oxygen species mediate central cardiorespiratory network responses to acute intermittent hypoxia.

Although oxidative stress and reactive oxygen species generation is typically associated with localized neuronal injury, reactive oxygen species have also recently been shown to act as a physiological signal in neuronal plasticity. Here we define an essential role for reactive oxygen species as a critical stimulus for cardiorespiratory reflex responses to acute episodic hypoxia in the brain stem. To examine central cardiorespiratory responses to episodic hypoxia, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and synaptic neurotransmission to cardioinhibitory vagal neurons. We show that whereas continuous hypoxia does not stimulate excitatory neurotransmission to cardioinhibitory vagal neurons, acute intermittent hypoxia of equivalent duration incrementally recruits an inspiratory-evoked excitatory neurotransmission to cardioinhibitory vagal neurons during intermittent hypoxia. This recruitment was dependent on the generation of reactive oxygen species. Further, we demonstrate that reactive oxygen species are incrementally generated in glutamatergic neurons in the ventrolateral medulla during intermittent hypoxia. These results suggest a neurochemical basis for the pronounced bradycardia that protects the heart against injury during intermittent hypoxia and demonstrates a novel role of reactive oxygen species in the brain stem.

NO differentially regulates neurotransmission to premotor cardiac vagal neurons in the nucleus ambiguus.

NO is involved in the neural control of heart rate, and NO synthase expressing neurons and terminals have been localized in the nucleus ambiguus where parasympathetic cardiac vagal preganglionic neurons are located; however, little is known about the mechanisms by which NO alters the activity of premotor cardiac vagal neurons. This study examines whether the NO donor sodium nitroprusside ([SNP] 100 micromol/L) and precursor, l-arginine (10 mmol/L), modulate excitatory and inhibitory synaptic neurotransmission to cardiac vagal preganglionic neurons. Glutamatergic, GABAergic, and glycinergic activity to cardiac vagal neurons was examined using whole-cell patch-clamp recordings in an in vitro brain slice preparation in rats. Both SNP, as well as l-arginine, increased the frequency of GABAergic neurotransmission to cardiac vagal preganglionic neurons but decreased the amplitude of GABAergic inhibitory postsynaptic currents. In contrast, both l-arginine and SNP inhibited the frequency of glutamatergic and glycinergic synaptic events in cardiac vagal preganglionic neurons. SNP and l-arginine also decreased glycinergic inhibitory postsynaptic current amplitude, and this response persisted in the presence of tetrodotoxin. Inclusion of the NO synthase inhibitor 7-nitroindazole (100 mumol/L) prevented the l-arginine-evoked responses. These results demonstrate that NO differentially regulates excitatory and inhibitory neurotransmission, facilitating GABAergic and diminishing glutamatergic and glycinergic neurotransmission to cardiac vagal neurons.

Multiple types of GABAA receptors mediate inhibition in brain stem parasympathetic cardiac neurons in the nucleus ambiguus.

Recent work suggests neurons can have different types of gamma-aminobutyric acid type A (GABA(A)) receptors that mediate phasic inhibitory postsynaptic currents (IPSCs) and tonic currents. This study examines the diversity of GABAergic synaptic currents in parasympathetic cardioinhibitory neurons that receive rhythmic bursts of GABAergic neurotransmission. Focal application of gabazine (25 microM) to cardiac vagal neurons in vitro did not change the frequency of firing in spontaneously active neurons or the resting membrane potential; however, picrotoxin (100 microM) significantly depolarized cardiac vagal neurons and increased their firing. Similarly, gabazine (25 microM) selectively blocked GABAergic IPSCs but did not change holding current in cardiac vagal neurons, whereas picrotoxin (100 microM) not only blocked GABAergic IPSCs but also rapidly decreased the tonic current. Because the tonic current could be attributable to activation of GABA receptors by ambient GABA or, alternatively, spontaneous opening of constitutively active GABA channels, an antagonist for the GAT-1 GABA transporter NO-711 (10 microM) was applied to distinguish between these possibilities. NO-711 did not significantly alter the holding current in these neurons. The benzodiazepine flunitrazepam (1 microM) significantly increased the tonic current and GABAergic IPSC decay time; surprisingly, however, in the presence of gabazine flunitrazepam failed to elicit any change. These results suggest cardiac vagal neurons possess gabazine-sensitive GABA(A) receptors that mediate phasic synaptic currents, a gabazine-insensitive but picrotoxin-sensitive extrasynaptic tonic current that when blocked depolarizes and increases the firing rate of cardiac vagal neurons, and benzodiazepines recruit a third type of GABA(A) receptor that is sensitive to gabazine and augments the extrasynaptic tonic current.

Prenatal nicotine exposure alters the nicotinic receptor subtypes that modulate excitation of parasympathetic cardiac neurons in the nucleus ambiguus from primarily alpha3beta2 and/or alpha6betaX to alpha3beta4.

Nicotinic receptors play an essential role in central cardiorespiratory function, however, the types of nicotinic receptors responsible for activating cardiac vagal neurons in the nucleus ambiguus that control heart rate are unknown. This study tests whether alpha-conotoxin MII and alpha-conotoxin AuIB sensitive nicotinic receptors are involved in augmentation of glutamatergic neurotransmission and changes in holding current in cardiac vagal neurons, and whether exposure to nicotine in the prenatal period alters these responses. The nicotinic agonist cytisine significantly increased the holding current and amplitude of glutamatergic mEPSCs. In unexposed animals alpha-conotoxin MII (100nM) significantly reduced the increase in mEPSC amplitude and change in holding current evoked by cytisine. However, in animals prenatally exposed to nicotine, alpha-conotoxin MII blunted but did not block the increase in mEPSC amplitude but blocked the increase in holding current evoked by cytisine. In unexposed animals, alpha-conotoxin AuIB (10microM) blocked the cytisine evoked increase in mEPSC amplitude and inhibited but did not abolish the increase in holding current. In contrast, in animals exposed to nicotine, alpha-conotoxin AuIB blunted the increase in mEPSC amplitude, and completely abolished the cytisine evoked increase in holding current. These data demonstrate that the prenatal nicotine exposure alters the nicotinic receptors involved in excitation of cardiac vagal neurons.

Differential control of central cardiorespiratory interactions by hypercapnia and the effect of prenatal nicotine.

Hypercapnia evokes a strong cardiorespiratory response including gasping and a pronounced bradycardia; however, the mechanism responsible for these survival responses initiated in the brainstem is unknown. To examine the effects of hypercapnia on the central cardiorespiratory network, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and inhibitory synaptic neurotransmission to cardioinhibitory vagal neurons (CVNs). Hypercapnia differentially modulated inhibitory neurotransmission to CVNs; whereas hypercapnia selectively depressed spontaneous glycinergic IPSCs in CVNs without altering respiratory-related increases in glycinergic neurotransmission, it decreased both spontaneous and inspiratory-associated GABAergic IPSCs. Because maternal smoking is the highest risk factor for sudden infant death syndrome (SIDS) and prenatal nicotine exposure is proposed to be the link between maternal smoking and SIDS, we examined the cardiorespiratory responses to hypercapnia in animals exposed to nicotine in the prenatal and perinatal period. In animals exposed to prenatal nicotine, hypercapnia evoked an exaggerated depression of GABAergic IPSCs in CVNs with no significant change in glycinergic neurotransmission. Hypercapnia altered inhibitory neurotransmission to CVNs at both presynaptic and postsynaptic sites. Although the results obtained in this study in vitro cannot be extrapolated with certainty to in vivo responses, the results of this study provide a likely neurochemical mechanism for hypercapnia-evoked bradycardia and the dysregulation of this response with exposure to prenatal nicotine, creating a higher risk for SIDS.

Ketamine inhibits inspiratory-evoked gamma-aminobutyric acid and glycine neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

BACKGROUND: Ketamine can be used for perioperative pain management as well as a dissociative anesthetic agent in emergency situations. However, ketamine can induce both cardiovascular and respiratory depression, especially in pediatric patients. Although ketamine has usually been regarded as sympathoexcitatory, recent work has demonstrated that ketamine has important actions on parasympathetic cardiac vagal efferent activity. The current study tests the hypothesis that ketamine, at clinical relevant concentrations, alters central cardiorespiratory interactions in the brainstem and, in particular, the inspiration-evoked increase in gamma-aminobutyric acid-mediated and glycinergic neurotransmission to parasympathetic cardiac efferent neurons. METHODS: Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer. Respiratory evoked gamma-aminobutyric acid-mediated and glycinergic synaptic currents were recorded in cardiac vagal neurons using whole cell patch clamp techniques while spontaneous rhythmic respiratory activity was recorded simultaneously. RESULTS: : Ketamine, at concentrations from 0.1 to 10 microM, evoked a concentration-dependent inhibition of inspiratory burst frequency. Inspiration-evoked gamma-aminobutyric acid-mediated neurotransmission to cardiac vagal neurons was inhibited at ketamine concentrations of 0.5 and 1 microM. The increase in glycinergic activity to cardiac vagal neurons during inspiration was also inhibited at ketamine concentrations of 0.5 and 1 microM. CONCLUSIONS: At clinically relevant concentrations (0.5 and 1 microM), ketamine alters central respiratory activity and diminishes both inspiration-evoked gamma-aminobutyric acid-mediated and glycinergic neurotransmission to parasympathetic cardiac efferent neurons. This reduction in inhibitory neurotransmission to cardiac vagal neurons is likely responsible for the compromised respiratory sinus arrhythmia that occurs with ketamine anesthesia.