BACKGROUND CONTEXT The United States is facing an opioid crisis. Opioid use is common and widespread in the adult spinal deformity (ASD) population. Enhanced Recovery After Surgery (ERAS) is a multimodal and multidisciplinary evidence-based, perioperative approach for surgery intended to reduce surgical morbidity and improve recovery. We sought to evaluate the effects of ERAS implementation on postoperative opiate consumption, length of stay (LOS), and postoperative day of ambulation (PDA) following ASD surgery. PURPOSE The purpose of this study is to test if the implementation of ERAS protocol will help reduce in-hospital opioid use, length of stay and postoperative day of ambulation following ASD surgery. STUDY DESIGN/SETTING Single site, multisurgeon, retrospective longitudinal cohort study. PATIENT SAMPLE We sampled a total of 50 patients, 6 months pre- (n=20) and post- (n=30) ERAS implementation. METHODS A single center, multisurgeon, retrospective review identified patients with adult deformity who underwent ASD surgery 6 months pre- (n=20) and post- (n=30) ERAS implementation. The 2 cohorts were statistically similar in age, sex, BMI, diagnosis, ASA grade and levels fused. Total morphine equivalent doses (MED) in-hospital as well as 90-day postoperative prescriptions were calculated. LOS and PDA were also evaluated. Preoperative opiate prescriptions were recorded to determine baseline opioid use. RESULTS We found no difference in hospital postoperative mean total MED between the 2 cohorts (preERAS=263, postERAS=211, p=0.44). The 90-day mean total MED was also not significantly different between the cohorts (preERAS= 572, postERAS=326, p=0.12). 25 (50%) patients were taking opiates prior to surgery. Opiate naive patients required significantly less in-hospital mean total MED compared to nonopiate naive patients (124 vs 629, p=0.02, respectively). However, 90-day mean total MED was not significantly different between the 2 cohorts (naive=197 vs non-opiate naive=311, p=0.18). LOS for ERAS cohort (5.20.±73) was less when compared to preERAS cohort (5.9±0.6, p=0.22), but this was not significant. PDA was significantly decreased after ERAS implementation (1.1 vs 0.43, p=0.006). CONCLUSIONS Early adoption of ERAS did not decrease in-hospital opioid consumption or total 90-day opioid prescription. In-hospital opioid consumption was correlated with preoperative opioid use. Decreasing presurgical opioid use has a significant effect on postoperative patient opioid consumption and should be implemented as part of ERAS protocol. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs. The United States is facing an opioid crisis. Opioid use is common and widespread in the adult spinal deformity (ASD) population. Enhanced Recovery After Surgery (ERAS) is a multimodal and multidisciplinary evidence-based, perioperative approach for surgery intended to reduce surgical morbidity and improve recovery. We sought to evaluate the effects of ERAS implementation on postoperative opiate consumption, length of stay (LOS), and postoperative day of ambulation (PDA) following ASD surgery. The purpose of this study is to test if the implementation of ERAS protocol will help reduce in-hospital opioid use, length of stay and postoperative day of ambulation following ASD surgery. Single site, multisurgeon, retrospective longitudinal cohort study. We sampled a total of 50 patients, 6 months pre- (n=20) and post- (n=30) ERAS implementation. A single center, multisurgeon, retrospective review identified patients with adult deformity who underwent ASD surgery 6 months pre- (n=20) and post- (n=30) ERAS implementation. The 2 cohorts were statistically similar in age, sex, BMI, diagnosis, ASA grade and levels fused. Total morphine equivalent doses (MED) in-hospital as well as 90-day postoperative prescriptions were calculated. LOS and PDA were also evaluated. Preoperative opiate prescriptions were recorded to determine baseline opioid use. We found no difference in hospital postoperative mean total MED between the 2 cohorts (preERAS=263, postERAS=211, p=0.44). The 90-day mean total MED was also not significantly different between the cohorts (preERAS= 572, postERAS=326, p=0.12). 25 (50%) patients were taking opiates prior to surgery. Opiate naive patients required significantly less in-hospital mean total MED compared to nonopiate naive patients (124 vs 629, p=0.02, respectively). However, 90-day mean total MED was not significantly different between the 2 cohorts (naive=197 vs non-opiate naive=311, p=0.18). LOS for ERAS cohort (5.20.±73) was less when compared to preERAS cohort (5.9±0.6, p=0.22), but this was not significant. PDA was significantly decreased after ERAS implementation (1.1 vs 0.43, p=0.006). Early adoption of ERAS did not decrease in-hospital opioid consumption or total 90-day opioid prescription. In-hospital opioid consumption was correlated with preoperative opioid use. Decreasing presurgical opioid use has a significant effect on postoperative patient opioid consumption and should be implemented as part of ERAS protocol.

Achieving reversible, temporally specific inhibition of motor neurons has the potential to revolutionize treatment of disorders marked by muscle hyperactivity. Current treatment strategies are inadequate – surgical interventions are irreversible and pharmaceutical interventions have off-target effects. Optogenetic strategies for inhibiting muscle activity have theoretical promise; however, trafficking and expression problems have prevented translatable optogenetic suppression of muscle activity. Here, we exploit recent innovations in opsin engineering to demonstrate virally mediated, temporally specific optogenetic inhibition of motor neurons and muscle activity in vivo . We show that intra-muscular injection of adeno-associated virus serotype 6 can drive expression of the inhibitory channelrhodopsin mutant iC++ in both immunodeficient and wild-type mice. Illumination of the sciatic nerve in wild-type mice resulted in 64.8% inhibition of evoked twitch force. Optical excitation during tetanic stimulation in wild-type mice resulted in 59.1% inhibition at 10 Hz and 55.4% at 25 Hz. The extent of optogenetic inhibition was titratable, and ranged from 0% to 78.4% as illumination intensity was changed from 1 mW to 20 mW/mm 2 . These results could have therapeutic applicability to disorders such as spasticity, hypertonia and urinary incontinence, and provide a new tool to neuroscientists and muscle physiologists wishing to reversibly inhibit motor neuron activity in vivo .

Neurological disorders and injuries can result in paralysis and involuntary muscle contractions that interfere with speech, movement and activities of daily living. Current approaches to reduce these dysfunctions, such as selective surgical ablation, oral medications, or electrical stimulation, have important limitations including lack of action or behavioral specificity. There is currently no treatment for spasticity or paralysis that provides targeted, tunable and rapidly reversible control of fine muscle activity. Recently, implantable cortical electrodes have been targeted as a potential therapy for fine control of robotic prosthesis. Implanting these electrodes in the motor cortex has shown promise, however development of long-term systems has yet to be fully understood and the effects of the broad electrical stimulation produced by these devices is still unclear. Optogenetics, a technique that uses light to modulate cells, may provide some relief in producing a stable, long-term intracortical implant with high specificity. Although optogenetic technology has proven to be effective as a research tool and shown promise acutely as a therapeutic intervention in preclinical work, few studies have utilized optogenetics for chronic applications. There is a paucity of knowledge regarding the stability of opsin expression or the neuronal network response to repeated optical stimulation over time. In order to contribute to the understanding of the long-term effects of intracortical devices and optogenetic gene therapy, we performed bilateral injections of an Adeno-associated viral delivery system to transfect the excitatory opsin, channelrhodopsin-2 (ChR2), into the motor cortex of mice. We then unilaterally implanted a 16 channel shank recording optrode into one of the previously injected cortical hemispheres. Following recovery from surgery, spontaneous cortical activity was recorded in freely moving mice. During recording sessions the cortex was illuminated with pulsed 473nm laser light at a variety of frequencies to excite ChR2+ motor neurons. After 8-12 months of weekly recordings, animals were sacrificed and tissue analyzed for changes in microglia response, neuroinflammation, or morphology.

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.

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.

In this study we used a rat model for prenatal nicotine exposure to test whether clinically relevant concentrations of brain nicotine and cotinine are passed from dams exposed to nicotine to her pups, whether this changes the trigeminocardiac reflex (TCR), and whether serotonergic function in the TCR brainstem circuitry is altered. Pregnant Sprague-Dawley dams were exposed to 6 mg·kg(-1)·day(-1) of nicotine via osmotic minipumps for the duration of pregnancy. Following birth dams and pups were killed, blood was collected, and brain nicotine and cotinine levels were measured. A separate group of prenatal nicotine-exposed pups was used for electrophysiological recordings. A horizontal brainstem slice was obtained by carefully preserving the trigeminal nerve with fluorescent identification of cardiac vagal neurons (CVNs) in the nucleus ambiguus. Stimulation of the trigeminal nerve evoked excitatory postsynaptic current in CVNs. Our data demonstrate that prenatal nicotine exposure significantly exaggerates both the TCR-evoked changes in heart rate in conscious unrestrained pups, and the excitatory neurotransmission to CVNs upon trigeminal afferent nerve stimulation within this brainstem reflex circuit. Application of the 5-HT1A receptor antagonist WAY 100635 (100 μM) and 5-HT2A/C receptor antagonist ketanserin (10 μM)significantly decreased neurotransmission, indicating an increased facilitation of 5-HT function in prenatal nicotine-exposed animals. Prenatal nicotine exposure enhances activation of 5-HT receptors and exaggerates the trigeminocardiac reflex.

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.

Activation of the trigeminocardiac reflex by airborne irritants or water, stimulates nasotrigeminal sensory fibers eliciting a pronounced bradycardia and increase in parasympathetic cardiac activity. Exaggeration of this response can be fatal, and has been implicated in cardiorespiratory diseases such as sudden infant death syndrome (SIDS). Parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus (NA) play an integral role in mediating this reflex. Stimulation of trigeminal sensory afferents elicits a polysynaptic excitatory glutamatergic neurotransmission to CVNs. We use a UV photo uncaging system to identify the neurons in the spinal trigeminal nucleus (sp5) that project to CVNs by sequential photostimulation of different neuron clusters as well as individual neurons that elicit excitatory glutamatergic neurotransmission to CVNs. Herpes simplex virus 1 expressing GFP was injected into the nasal mucosa of neonatal rats, and, using 2 photon confocal microscopy, areas of the brainstem expressing GFP+ trigeminal sensory afferent fibers within sp5 were identified. Preliminary results indicate scattered neurons in the sp5 that receive sensory afferent synaptic terminals project to and excite CVNs. Further work is necessary to determine if these neurons are located diffusely or in discrete clusters within the sp5.

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.

Stimulation of the trigeminal nerve evokes a dramatic decrease in heart rate and blood pressure, and this reflex has generally been termed the trigeminocardiac reflex. A subset of the trigeminocardiac reflex is the diving reflex in which the nasal mucosa is stimulated with water or air-borne chemical irritants. Activation of the diving reflex evokes a pronounced bradycardia, mediated by increased parasympathetic cardiac activity, and is the most powerful autonomic reflex. However, exaggeration of this protective response could be detrimental and has been implicated in Sudden Infant Death Syndrome (SIDS). Despite the importance and strength of the trigeminocardiac reflex, there is little information about the cellular mechanisms and brain stem pathways that constitute this reflex. To address these issues, stimulation of trigeminal afferent fibers and the evoked excitatory postsynaptic currents were recorded in cardiac vagal neurons (CVNs) in an in vitro brain stem slice preparation. This synaptic pathway is robust and activation of the trigeminal pathway often evoked action potentials in CVNs. Application of the serotonin (5-HT) reuptake inhibitor citalopram significantly enhanced these responses. Consistent with the hypothesis this pathway is endogenously modulated by 5-HT receptors the 5-HT1A receptor antagonist, WAY 100635 inhibited, whereas the 5-HT2A/C receptor antagonist, ketanserin facilitated the excitatory neurotransmission to CVNs. The 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide increased, whereas the 5-HT2 receptor agonist, alpha-methylserotonin maleate salt inhibited this reflex pathway. These results indicate stimulation of trigeminal fibers evokes a powerful excitatory and polysynaptic pathway to CVNs, and this pathway is endogenously modulated and differentially enhanced and depressed, by 5-HT1A and 5-HT2 receptors, respectively.

The neural control of heart rate is determined primarily by the activity of preganglionic parasympathetic cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA) in the brain stem. GABAergic inputs to CVNs play an essential role in determining the activity of these neurons including a robust inhibition during each inspiratory burst. The origin of GABAergic innervation has yet to be determined however. A transgenic mouse line expressing green florescent protein (GFP) in GABAergic cells was used in conjunction with caged glutamate to identify both clusters and individual GABAergic neurons that evoke inhibitory GABAergic synaptic responses in CVNs. Transverse slices were taken with CVNs patch-clamped in the whole cell configuration. Sections containing both the pre-Botzinger complex as well as the calamus scriptorius were divided into approximately 90 quadrants, each 200 x 200 microm and were sequentially photostimulated. Inhibitory post synaptic currents (IPSCs) were recorded in CVNs after a 5-ms photostimulation of 50 microM caged glutamate. The four areas that contained GABAergic cells projecting to CVNs were 200 microm medial, 400 microm medial, 200 microm ventral, and 1,200 microm dorsal and 1,000 microm medial to patched CVNs. Once foci of GABAergic cells projecting to CVNs were determined, photostimulation of individual GABAergic neurons was conducted. The results from this study suggest that GABAergic cells located in four specific areas project to CVNs, and that these cells can be individually identified and stimulated using photouncaging to recruit GABAergic neurotransmission to CVNs.

To examine the role of 5-HT2 receptors in the central cardiorespiratory network, and in particular the respiratory modulation of parasympathetic activity to the heart, we used an in vitro medullary slice that allowed simultaneous examination of rhythmic inspiratory-related activity recorded from hypoglossal rootlet and excitatory inspiratory-related neurotransmission to cardioinhibitory vagal neurons (CVNs) within the nucleus ambiguus (NA). Focal application of ketanserin, a 5-HT2 receptor antagonist, did not significantly alter the frequency of spontaneous excitatory postsynaptic excitatory currents (EPSCs) in CVNs in control conditions. However, ketanserin diminished spontaneous excitatory neurotransmission to CVNs during hypoxia. The inhibitory action of ketanserin was on 5-HT3 mediated EPSCs during hypoxia since these responses were blocked by the 5-HT3 receptor antagonist ondansetron. In addition, a robust inspiratory-related excitatory neurotransmission was recruited during recovery from hypoxia. Focal application of ketanserin during this posthypoxia period evoked a significant augmentation of the frequency of inspiratory-related, but not spontaneous EPSCs in CVNs. This excitatory effect of ketanserin was prevented by application of the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These results demonstrate 5-HT2 receptors differentially modulate excitatory neurotransmission to CVNs during and after hypoxia. Activation of 5-HT2 receptors acts to maintain excitatory neurotransmission to CVNs during hypoxia, likely via presynaptic facilitation of 5-HT3 receptor-mediated neurotransmission to CVNs. However, activation of 5HT2 receptors diminishes the subsequent inspiratory-related excitatory neurotransmission to CVNs that is recruited during the recovery from hypoxia likely exerting an inhibitory action on inspiratory-related purinergic signaling.

Cardioinhibitory cardiac vagal neurons (CVNs) do not receive inspiratory-related excitatory inputs under normal conditions. However, excitatory purinergic and serotonergic pathways are recruited during inspiratory activity after episodes of hypoxia and hypercapnia (H/H). Prenatal nicotine (PNN) exposure is known to dramatically change cardiorespiratory responses and decrease the ability to resuscitate from H/H. This study tested whether PNN exposure alters excitatory neurotransmission to CVNs in the nucleus ambiguus during and after H/H. Spontaneous and inspiratory evoked excitatory postsynaptic currents were recorded in CVNs from rats that were exposed to nicotine (6 mg x kg(-1) x d(-1)) throughout the prenatal period. In contrast to unexposed animals, in PNN animals H/H recruited excitatory neurotransmission to CVNs during inspiratory-related activity that was blocked by the alpha3beta4 nicotinic acetylcholine receptor (nAChR) blocker alpha-conotoxin AuIB (alpha-CTX AuIB, 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM) and d(-)-2-amino-5-phosphonopentanoic acid (AP5, 50 microM), selective AMPA/kainate and N-methyl-d-aspartate receptor blockers, respectively. Following H/H, there was a significant increase in inspiratory-related excitatory postsynaptic currents that were unaltered by alpha-CTX AuIB or ondansetron, a 5-HT3 receptor blocker, but were subsequently inhibited by pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (100 microM), a purinergic receptor blocker and CNQX and AP5. The results from this study demonstrate that with PNN exposure, an excitatory neurotransmission to CVNs is recruited during H/H that is glutamatergic and dependent on activation of alpha3beta4-containing nAChRs. Furthermore, exposure to PNN abolishes a serotonergic long-lasting inspiratory-related excitation of CVNs that is replaced by recruitment of a glutamatergic pathway to CVNs post H/H.

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.

Inhibitory GABAergic and glycinergic neurotransmission to cardioinhibitory cardiac vagal neurons ( CVNs) increase during inspiratory activity and likely mediate respiratory sinus arrhythmia, while the frequency of excitatory postsynaptic currents ( EPSCs) in CVNs are unaltered during the different phases of respiration. However, following hypoxia and hypercapnia ( H/ H), the parasympathetic activity to the heart increases and thus far, identification of the pathways and neurotransmitters that are responsible for exciting CVNs post H/ H are unclear. This study identifies different excitatory pathways to CVNs recruited post H/ H. Spontaneous and inspiratory-related EPSCs were recorded in CVNs before, during, and after 10 min of H/ H in an in vitro slice preparation that retains rhythmic respiratory activity. Before and during H/ H, EPSCs in CVNs were completely blocked by 6-cyano-7-nitroquinoxaline-2,3- dione ( CNQX) and D(-)-2-amino-5-phosphonopentanoic acid ( AP5), selective AMPA/ kainate and N-methyl-D-apartate ( NMDA) receptor blockers, respectively. However, after H/ H, there was a significant increase in EPSCs during each inspiratory burst. While some of the inspiratory-related EPSCs were blocked by the broad purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid ( PPADS) and the specific P2X receptor antagonist 2', 3'-O-( 2,4,6-trinitrophenyl) adenosine 5'-triphosphate monolithium trisodium salt ( TNP-ATP) a P2X receptor blocker, most of the recruited excitatory neurotransmission to CVNs is serotonergic because odansetron, a selective 5-HT3 antagonist, abolished the majority of the spontaneous and inspiratory-related EPSCs evoked during recovery from H/ H. The results from this study suggest that following episodes of H/ H, two nonglutamatergic excitatory pathways, purinergic and serotonergic, activating P2X and 5-HT3 receptors, respectively, are recruited to excite CVNs in the post H/ H recovery period.

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.

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.