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. 2011 Sep 19;589(Pt 22):5431–5442. doi: 10.1113/jphysiol.2011.215392

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

Xin Wang 1, Christopher Gorini 1, Douglas Sharp 1, Ryan Bateman 1, David Mendelowitz 1
PMCID: PMC3240882  PMID: 21930602

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.

Abstract

Abstract

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.

Introduction

The trigeminocardiac reflex (TCR) is among the most powerful autonomic reflexes (Schaller, 2004). Electrical or mechanical stimulation of the trigeminal nerve evokes pronounced bradycardia, hypotension and apnoea in many animals including humans (Kumada et al. 1977, 1978; Schaller et al. 1999; Schaller, 2005). Trigeminal afferent neurones can be activated by fluid in the nasal cavity, craniofacial pain, mechanical stimulation of the ocular and periocular structures, and during orofacial and maxillofacial surgery (Barnard & Bainton, 1990; Schaller et al. 1999; Schaller, 2004; Schaller & Buchfelder, 2006). The sensory nerve endings of the trigeminal nerve send sensory signals via the Gasserian ganglion to the trigeminal nucleus, forming the afferent pathway of the reflex arc. Second-order neurones in the ventral trigeminal nucleus that receive this sensory information have been identified as glutamatergic using both pharmacological approaches and immunohistochemistry (McCulloch et al. 1995; McCulloch & Panneton, 1997). These glutamatergic second-order neurones are mostly lateral and slightly dorsal to the nucleus ambiguus in the ventral trigeminal nucleus (McCulloch, 2005). The pathway continues from the ventral trigeminal nucleus through the short internuncial nerve fibres in the reticular formation in the brainstem to finally synapse on efferent cholinergic premotor parasympathetic cardioinhibitory neurones in the nucleus ambiguus (Schaller, 2004).

While much is known about the anatomical framework, little is known about the cellular mechanisms that mediate the TCR. Additionally, anaesthetic and analgesic modulation of the TCR is often unpredictable and variable as different anaesthetics and analgesics have been shown to depress, as well as exaggerate, the TCR. The trigeminocardiac reflex occurs less often with sevoflurane than halothane (Allison et al. 2000; Oh et al. 2007), and ketamine has been associated with a decreased incidence of the oculocardiac reflex (a subset of the TCR) compared to propofol (Hahnenkamp et al. 2000; Choi et al. 2007). The oculocardiac reflex is exaggerated with fast acting opioids (Arnold et al. 2004; Chung et al. 2008; Ghai et al. 2009). However the mechanisms responsible for this clinically relevant modulation of the TCR are unknown.

The trigeminocardiac reflex can be altered at many sites within the brainstem circuitry including the primary afferent pathway that synapses upon neurones in the sensory trigeminal nucleus, as well as the synaptic pathway from second order neurones to the CVNs in the nucleus ambiguus by acting on synaptic neurotransmission at either presynaptic or postsynaptic targets. In this study we hypothesized analgesic and anaesthetic agents alter neurotransmission at two sites in the reflex circuitry, the synapse onto trigeminal neurones as well as CVNs. To test these hypotheses, we examined the effect of ketamine, isoflurane, propofol and fentanyl, at clinically relevant concentrations, on the neurotransmission to neurones in the ventral trigeminal nucleus and to cardioinhibitory parasympathetic cardiac neurones in the nucleus ambiguus upon activation of trigeminal sensory fibres.

Methods

All animal procedures were performed with the approval of the Animal Care and Use Committee of The George Washington University in accordance with the recommendations of the panel on euthanasia of the American Veterinary Medical Association and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Labelling of CVNs in the nucleus ambiguus and trigeminal neurones in the medulla

Cardiac parasympathetic inhibitory neurones in the nucleus ambiguus were identified by the presence of a retrograde fluorescent tracer previously injected into the pericardial sac. Briefly, Sprague–Dawley rat pups (postnatal days 1–7; Hilltop, Scottsdale, PA, USA) were anaesthetized and exposed to hypothermia to slow the heart. The heart was exposed by a right thoractomy and the retrograde fluorescent tracer X-rhodamine-5-(and 6)-isothiocyanate (Molecular Probes, Eugene, OR, USA) was injected into the fat pads at the base of the heart. Parasympathetic cardioinhibitory neurones in the nucleus ambiguus were identified by the presence of the fluorescent tracer, as described previously (Mendelowitz & Kunze, 1991). Specificity of the CVN labelling was confirmed by the absence of any labelled neurones in the brainstem when rhodamine was injected into the chest cavity while keeping the pericardial sac intact, or when the injection into the pericardial sac was accompanied by section of the cardiac branch of the vagus nerve. In other control experiments intravenous injection of 10 mg of rhodamine failed to label any neurones in the medulla except for rare labelling of neurones in the area postrema, an area with a deficient blood–brain barrier.

Pathways from nasal sensory fibres were labelled using a herpes simplex virus 1 (HSV1) expressing green fluorescent protein (GFP). The nasal cavity was topically exposed to a 5 × 107 pfu HSV1.pR19EF1αGFPWPRE vector. A total of 3.5 μl of this GFP expressing vector was slowly applied to the right nasal cavity using a Hamilton fine glass syringe. The HSV1.pR19EF1αGFPWPRE vector was purchased from BioVex Ltd (London, UK).

Brainstem slice preparation, identification, and recording

Pups were anaesthetized with isoflurane 2–5 days post-injection and killed by cervical dislocation. The hindbrain was rapidly removed and placed in cold physiological saline solution composed of the following (in mm): 140 NaCl, 5 KCl, 2 CaCl2, 5 glucose, 10 Hepes, bubbled with 100% O2, pH 7.4. Slices (700 μm) were cut (Leica VT-100S, Leica Microsystems, Bannockburn, IL, USA) in a horizontal orientation. The horizontal medullary slice preparation retained the trigeminal nerve rootlet, the tract of spinal trigeminal neurones, local network pathways and CVNs. The tissue was placed in a recording chamber and perfused (4 ml min−1) with artificial cerebrospinal fluid containing (in mm): 125 NaCl, 3 KCl, 2 CaCl2, 26 NaHCO3, 5 glucose, 5 Hepes, equilibrated with 95% O2–5% CO2, pH 7.4.

Slices were viewed with infrared illumination and differential interference optics (Zeiss) and under fluorescent illumination with an infrared-sensitive cooled charged-coupled device camera (Photometrics, Tucson, AZ, USA). Neurones containing fluorescent rhodamine tracer or GFP were identified by superimposing the fluorescence and infrared images on a video monitor (Sony, Tokyo, Japan).

A group of brainstem slices were collected for confocal image examination. Briefly, the 700 μm thick brainstem slice was cut and fixed using a 10% neutral buffered formalin solution (Sigma-Aldrich, St Louis, MO, USA) for 24 h, and then washed with phosphate-buffered saline (PBS). Confocal images were collected on a Zeiss LSM 710 system (Carl Zeiss Microimaging GmbH), equipped with an Axio Examiner Z1 upright microscope and W Plan-Apo 20× (NA, 1.0) (DIC VIS-IR WD = 1.8) and Plan-Apochromat 63× (NA, 1.40) oil (DIC) objectives. The system has a 32 channel spectral-detection Quasar photomultiplier and two single channel photomultipliers to record the backward emission. The argon-488 line of a multiline 25 mW argon laser was used to excite GFP, whereas rhodamine was excited with 5 mW He–Ne emitting at 633 nm. The microscope was equipped with Prior x/y/z scanning stage, which permitted capturing tile stack images. Emission filtering was adjusted by setting the desired spectral window for recording using Zeiss Zen 2009 software. In addition, the Zen 2009 software provided an online, linear spectral unmixing algorithm, which allowed the separation of several dyes based on spectral characteristics, despite emission spectra overlap, known as online spectral fingerprinting. Emission filtering was adjusted by setting the desired spectral window for recording. Images were taken using a 20× and 63× objective.

Activation of the trigeminal sensory nerve rootlet

A concentric bipolar stimulating electrode (200 μm outer diameter, WPI, Sarasota, FL, USA) was used to stimulate the trigeminal nerve rootlet. Stimuli were delivered from an isolated programmable stimulator (Master 08; A.M.P.I., Jerusalam, Israel) at 0.1 Hz frequency with a pulse duration of 1 ms. The current intensity was increased until the threshold for excitation was found in which an excitatory postsynaptic current (EPSC) was evoked with every stimulation, typically ∼30 μA, and the stimulating intensity was increased to twice the event threshold and maintained at this intensity throughout the experiment to maintain consistent evoked responses. Control periods were 10 min, and each drug application condition lasted between 10 and 20 min to insure a steady state response to the drugs.

Whole-cell patch clamp and electrophysiological recording

Patch pipettes (2.5–4.5 MΩ) were visually guided to the surface of individual CVNs or spinal trigeminal neurones using differential interference optics and infrared illumination (Zeiss). In voltage-clamp experiments pipettes were filled with a solution containing 130 mm potassium gluconate, 10 mm Hepes, 10 mm EGTA, 1 mm CaCl2, and 1 mm MgCl2. Voltage clamp recordings were made with Axopatch 200B and pCLAMP 8 software (Axon Instruments, Union City, CA, USA). All synaptic activity in spinal trigeminal neurones as well as parasympathetic cardioinhibitory neurones was recorded at –80 mV. No more than one anaesthetic was applied to any neurone, and only one experiment was performed in each slice of tissue. Drugs were applied by focal application using a micropipette positioned within 50 μm of the patched neurone using a PV830 Pneumatic PicoPump pressure delivery system (WPI). The maximum range of drug application was determined previously to be 100–120 μm downstream from the drug pipette and was considerably less behind the drug pipette (Wang et al. 2002). All drugs and compounds were purchased from Sigma-Aldrich.

Latency variability – synaptic jitter

The latency to event onset and its variation across sweeps are fundamental indicators of the synaptic transmission process. Latency of stimulating-evoked EPSC was calculated as the time from the end of stimulus artifact to the onset of each EPSC. Variability in latency (jitter) was calculated as the standard deviation (SD) of latency. Synaptic jitter calculations included as least 20 individual latency values for each neurone. Conduction velocity (CV) was calculated as the distance between stimulation electrode and patched neurone divided by EPSC latency (mm ms−1).

Electrical stimulations did not always evoke a synaptic response. Serially connected, polyneuronal pathways, in addition to having high-jitter latencies, are particularly prone to synaptic failures.

Data analysis

All electrophysiological data were digitized and collected via Clampex (10.2) and analysed using Clampfit (10.2). Stimulus-evoked EPSCs were analysed with respect to maximum initial amplitude (the first ∼20 ms) and charge (area under the baseline holding current) of the response. Baseline was defined in the stimulation-evoked EPSC studies as the mean of five pretreatment responses. Analysis of stimulation evoked synaptic currents was performed using Clampfit (v. 10.2, Molecular Devices, Sunnyvale, CA, USA). Results are presented as means ± SEM. Statistical analysis was performed using GraphPad Prism 4 software. Student's t test for paired or unpaired data as appropriate and one-way ANOVA with Dunnett's post hoc test were performed where indicated. Significant difference was set at P < 0.05.

Results

Trigeminal sensory nerve fibres transfected with HSV1-GFP permitted visualization of nasal sensory fibres and putative synaptic terminals in the brainstem, shown in Fig. 1. This HSV1-GFP labelling was essential for success in subsequent electrophysiological experiments to identify the trigeminal fibres and target the trigeminal neurones that receive this sensory input. Figure 1A illustrates HSV1-GFP labelled trigeminal fibres extending from rostral to caudal in a horizontal orientation. HSV1-GFP labelled trigeminal fibres were densely localized to the spinal trigeminal tract (lateral at the spinal trigeminal interpolaris nucleus (Sp5I) level (Fig. 1B). Co-localization of parasympathetic CVNs in the brainstem was accomplished by the presence of the fluorescent tracer rhodamine previously applied to the synaptic endings of these neurones in cardiac ganglia located at the base of the heart (Huang, 1989; Viana et al. 1990; Christian et al. 1993). Cardiac parasympathetic neurones in the nucleus ambiguus from the same animal with labelling of trigeminal afferent neurones with GFP is shown in Fig. 1C.

Figure 1. Confocal image evidence of primary afferent trigeminal nerve terminals and efferent cardiac vagal neurones in the brainstem.

Figure 1

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Typical afferent trigeminal sensory nerve fibres labelled with a HSV1 expressing GFP were clearly seen under 2 photon confocal microscopy in horizontal (A) and coronal (B) brainstem slices. The HSV vector labelled trigeminal fibres at the Sp5I level and the fibres transverse from lateral to medial. Cardiac parasympathetic neurones labelled with rhodamine (red) in the nucleus ambiguus are shown in C from the same animal. Scale bars: A: 40 μm, B: 100 μm, C: 21 μm.

Electrical stimulation of trigeminal afferent fibres in the nerve rootlet evoked an excitatory postsynaptic current in trigeminal sensory neurones in the interpolaris nucleus of the spinal trigeminal (Sp5I) area (Fig. 2). Incrementally increasing the stimulus intensity recruited these synaptic responses in an all-or-none manner that is consistent with activation of a single afferent axon. Every GFP positive trigeminal neurone received an evoked excitatory postsynaptic current with a constant latency. The average latency was 1.78 ± 0.1 ms with a jitter/standard deviation of latency of 625 μs, and average peak amplitude of 239.4 ± 45 pA.

Figure 2. Electrical stimulation of trigeminal afferent fibres evoked an excitatory EPSC in trigeminal sensory neurones in the interpolaris nucleus of the spinal trigeminal area.

Figure 2

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Electrical stimulation of trigeminal afferent nerve ending evoked an excitatory postsynaptic current in trigeminal sensory neurones in the Sp5I area. The average conduction velocity from the trigeminal nerve ending to the trigeminal subnucleus interpolaris was 0.94 ± 0.18 mm ms−1. The average latency was 1.78 ± 0.1 ms with a jitter of 625 μs, and average peak amplitude of 239.4 ± 45 pA. The evoked EPSC was inhibited by the selective NMDA receptor antagonist AP-5 (50 μm) and blocked by the selective AMPA/kainate receptor antagonist CNQX (50 μm). The inhibition of EPSC is reversible. After 20 min wash out, the stimulation evoked EPSC is gradually recovered. **P < 0.01, ***P < 0.001.

Stimulation of trigeminal nerve endings evoked EPSCs that activated postsynaptic NMDA as well as non-NMDA receptors. NMDA receptors were responsible for a slow component (long decay phase) and were blocked by the selective NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5). Non-NMDA receptor activation evoked a fast and large EPSC current response which was completely blocked by the selective AMPA/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione (CNQX; Fig. 2).

There are two groups of trigeminal afferent sensory fibres, capsaicin-sensitive unmyelinated C-type, and capsaicin-resistant myelinated A-type. Two-thirds of the trigeminal sensory fibres are unmyelinated C fibres. Myelinated A fibres include small diameter A-δ fibres and large diameter A-β axons. Identification of unmyelinated C-fibre and myelinated A-fibre responses was conducted by application of capsacin and analysis of conduction velocity. The conduction velocities for A-β, A-δ and C fibres are reported as 9–27 m s−1, 1–9 m s−1 and 0.3–0.7 m s−1, respectively. In our results, as shown in Fig. 2, the average conduction velocity from the trigeminal nerve ending to the trigeminal subnucleus interpolaris was 0.94 ± 0.18 mm ms−1, indicating a mixture of A-δ and C fibres mediate the first synaptic neurotransmission in the TCR.

Electrical stimulation of the trigeminal nerve rootlet also induced excitatory currents further downstream in the reflex pathway in the CVNs. However, the stimulation induced EPSCs in CVNs have a reduced peak amplitude (57.8 ± 7.5 pA), and a more inconsistent and longer latency (with jitter between 0.8 and 2.0 ms, Fig. 3). The average latency of the evoked EPSC in CVNs was 12.1 ± 1.1 ms, suggesting there is likely to be more than one synapse in the brainstem circuitry from trigeminal sensory fibres to CVNs, probably within the trigeminal sensory nucleus. The stimulation evoked EPSCs were completely blocked by the AMPA/kainate and NMDA glutamergic receptor antagonists CNQX (50 μm) and AP-5 (50 μm), respectively (Fig. 3).

Figure 3. Electrical stimulation of trigeminal afferent fibres induced EPSCs in cardiac vagal neurones in the nucleus ambiguus.

Figure 3

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Electrical stimulation of the trigeminal nerve rootlet also induced excitatory currents further downstream in the reflex pathway in the CVNs. The average conduction velocity from the trigeminal nerve ending to the nucleus ambiguus was 0.14 ± 0.009 mm ms−1. The stimulation induced EPSCs in CVNs have a reduced peak amplitude (57.8 ± 7.5 pA), a more inconsistent and longer latency (12.1 ± 1.1 ms) with jitter of 0.8–2 ms. The stimulation evoked EPSCs were completely blocked by the AMPA/kainate and NMDA glutamergic receptor antagonists CNQX (50 μm) and AP-5 (50 μm), respectively. ***P < 0.001.

Several general anaesthetics and analgesics have been reported to alter the magnitude and incidence of the TCR, but the mechanisms for this clinically relevant modulation are unclear. In this study, we tested the effects of the intravenous anaesthetics ketamine and propofol, the inhalational agent isoflurane, as well as the μ-opioid receptor agonist fentanyl on the TCR in the brainstem slice.

None of the anaesthetics and analgesics tested in the study significantly changed the latency or conduction velocity of stimulation evoked excitatory postsynaptic currents in either trigeminal sensory nuclei or CVNs. The intravenous anaesthetic ketamine (10 μm) inhibited both the EPSC peak amplitude and EPSC area in the synaptic responses both in trigeminal neurons (n = 7) and CVNs (n = 10), as shown in Fig. 4A and B, respectively. This indicates ketamine probably inhibited the NMDA receptor and AMPA/kainate receptor mediated synaptic responses in both trigeminal neurones and CVNs. Surprisingly, propofol (3 μm) did not significantly change (n = 7, P > 0.05) trigeminal nerve stimulation induced EPSCs recorded from the CVNs in the nucleus ambiguus (data not shown).

Figure 4. Effects of the anaesthetic ketamine on the evoked EPSC in trigeminal sensory neurones in the Sp5I area as well as in cardiac vagal neurones in the nucleus ambiguus.

Figure 4

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Ketamine inhibited evoked EPSC in trigeminal neurones in the Sp5I area (A). The peak amplitude of EPSC was depressed from an average of –199.6 ± 41 pA to –141 ± 31 pA (P = 0.04, n = 7), and the area was reduced from –23693 ± 5589 pA ms to –9390 ± 1708 pA ms (P = 0.008, n = 7). B shows that ketamine also attenuated the evoked EPSC in cardiac vagal neurones in the nucleus ambiguus. The peak amplitude of EPSC was depressed from an average of –52 ± 6.7 pA to –35.4 ± 5.8 pA (P = 0.002, n = 10), and the area was reduced from –825 ± 296 pA ms in control to –679 ± 277 pA ms (P = 0.03, n = 10). *P < 0.05, **P < 0.01.

The inhalational anaesthetics, isoflurane, (at the dose of 300 μm, an equivalent to 1MAC) significantly inhibited evoked EPSC peak amplitude and area in the CVNs in the nucleus ambiguus (Fig. 5B, n = 6); however, at the same concentration, isoflurane failed to change stimulating evoked EPSCs in trigeminal neurones (Fig. 5A, n = 5). In contrast, the μ-opioid receptor agonist fentanyl (1 μm) enhanced the stimulation evoked EPSC peak amplitude and area (Fig. 6B, n = 10) in the CVNs but did not alter EPSC in the trigeminal terminals (Fig. 6A, n = 5).

Figure 5. Effects of the anaesthetic isoflurane on the evoked EPSC in trigeminal sensory neurones in the Sp5I area as well as in cardiac vagal neurones in the nucleus ambiguus.

Figure 5

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Isoflurane (at a dose of 300 μm, an equivalent to 1MAC) did not alter EPSC amplitude and area charges in the trigeminal terminals (A, n = 5). However, at the same concentration, isoflurane inhibited stimulation evoked EPSCs in CVNs (B). The peak amplitude of EPSC was reduced from an average of control of –60 ± 8.8 pA to –28.3 ± 8.3 (P = 0.002, n = 6), and the EPSC area was decreased from –271.5 ± 44 pA ms in control to −148.8 ± 58 pA ms (P = 0.005, n = 6). **P < 0.01.

Figure 6. Effects of the analgesic fentanyl on the evoked EPSC in trigeminal sensory neurones in the Sp5I area as well as in cardiac vagal neurones in the nucleus ambiguus.

Figure 6

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Fentanyl (at a concentration of 1 μm) had no effect on EPSC in the trigeminal sensory neurones (A, n = 5). But, as shown in B, fentanyl did increase stimulation induced EPSC amplitude and area charges in CVNs. The peak amplitude of EPSC was enhanced from an average of control of –41.8 ± 3.4 pA to –95.3 ± 10.3 (P = 0.04, n = 10), and the EPSC area was increased from –2261 ± 584 pA ms in control to –2643 ± 695 pA ms (P = 0.0016, n = 10). *P < 0.05, **P < 0.01.

Discussion

There are several important and clinically relevant findings in this study. First, neurotransmission to the trigeminal sensory neurones in the spinal trigeminal nucleus interpolaris area (Sp5I) is mediated by a glutamatergic synaptic pathway which activates both NMDA and AMPA/kainate receptors in neurones within the trigeminal nucleus. Second, electrical stimulation of trigeminal sensory fibres in the trigeminal nerve rootlet activated polysynaptic pathways to premotor parasympathetic CVNs in the nucleus ambiguus that also acted via activation of both NMDA and AMPA/kainate receptors in CVNs. Thirdly, whereas ketamine inhibited the NMDA receptor mediated responses in both spinal trigeminal neurones and CVNs, the synaptic responses in spinal trigeminal neurones were unaltered by isoflurane and fentanyl, but synaptic activation of CVNs was differentially inhibited and enhanced by isoflurane and fentanyl, respectively.

The pathways from the sensory neurones to the trigeminal nucleus and further downstream in the reflex pathway from the trigeminal nucleus to CVNs are two indispensable bridges between peripheral afferent input and autonomic responses in the trigeminocardiac reflex. The spinal trigeminal nucleus is the initial brainstem afferent relay in the TCR. The sensory trigeminal nuclei receive inhibitory GABAergic, glycinergic (Avendano et al. 2005) and excitatory glutamatergic neurotransmission (McCulloch et al. 1995; McCulloch, 2005). Our study has confirmed that glutamate is the dominant neurotransmitter in trigeminal sensory neurones in the Sp5I. Upon electrical stimulation, the characteristics of evoked EPSCs were typical of second-order neurones in Sp5I. The all-or-none characteristics of trigeminal sensory fibres’ intensity–recruitment relations indicates a reliance on activation of single axons impinging on individual trigeminal neurones, and a high-release probability glutamatergic synapse. Application of the NMDA receptor antagonist AP-5 reduced the area of the evoked EPSC but did not significantly decrease the peak amplitude. The AMPA/kainate receptor antagonist CNQX completely blocked stimulation evoked EPSCs in the trigeminal neurones. The combined anatomical, electrophysiological and pharmacological results in this study indicate the spinal trigeminal subnucleus interpolaris neurones are coupled to TCR afferent inputs via direct, monosynaptic glutamatergic synapses that activate both NMDA and AMDA/kainate receptors in trigeminal nucleus neurones.

Although application of AP-5 and CNQX also completely blocked the induced response in the premotor parasympathetic CVNs upon electrical stimulation of trigeminal nerve rootlet, the neurotransmission in the CVNs is likely to be mediated by more than one excitatory synaptic pathway because the stimulation evoked EPSCs have a long latency and variable waveforms, as well as a high failure rate. Previous studies from our laboratory have reported that serotonergic as well as muscarinic acetylcholinergic pathways are involved in modulation of the trigeminocardiac reflex (Gorini et al. 2009, 2010). The present results are consistent with these studies and suggest there are many potential sites for modulation of CVN activation in the TCR.

It is interesting that whereas the synaptic transmission onto neurones in the trigeminal nucleus was unaffected by isoflurane or fentanyl, isoflurane inhibited, whereas fentanyl enhanced, the synaptic responses in CVNs. This suggests that both isoflurane and ketamine act on receptors that are more densely localized to the synapses surrounding CVNs, or the subunit composition of these receptors involved in neurotransmission to CVNs are more readily modulated by these agents than receptors involved in the neurotransmission to spinal trigeminal neurones. It is likely fentanyl acted via activation of μ-opioid receptors to enhance glutamate release and/or post-synaptic glutamate receptor activation; however while it is unknown which receptors were responsible for the inhibition of responses that occurred with isoflurane, one likely target of both isoflurane and ketamine are the NMDA receptors in CVNs.

A range of anaesthetics produce negative effects on NMDA receptors. The intravenous anaesthetic ketamine is a prototypical non-competitive blocker of NMDA receptors (Lodge & Johnson, 1990); ketamine antagonizes NMDA receptors by both open channel blockade and closed channel blockade associated with decreased opening frequency. Ketamine must access at least one transmembrane domain binding site via the cell membrane (Orser et al. 1997; Hollmann et al. 2001). Such a specific selectivity of ketamine for glutamatergic receptors is further confirmed in our experiments. Application of ketamine, similar to AP-5, blocked the long decay phase component and decreased the duration of evoked EPSC in both trigeminal sensory neurones and CVNs. This result may explain why ketamine anaesthesia may reduce the incidence and strength of the TCR during surgeries involving trigeminal nerve stimulation. The results in this study also indicate a likely mechanism for the exaggeration of the oculocardiac reflex with fast acting opioids (Arnold et al. 2004; Chung et al. 2008; Ghai et al. 2009).

NMDA receptors are tetramers composed of two NR1 and two NR2 subunits, which bind glycine and glutamate, respectively. The inhalational anaesthetic isoflurane is reported to be a competitive antagonist at the NMDA receptor glycine binding site (Dickinson et al. 2007). At a clinically relevant concentration, isoflurane significantly inhibited evoked EPSC peak amplitude in CVNs but not in trigeminal neurones. Isoflurane has been reported to depress inhibitory GABAergic neurotransmission via endogenous nicotinic receptor to CVNs (Wang, 2009). The divergent responses of trigeminal neurones and CVNs may be due to the differing sensitivity/selectivity of the receptors involved in glutamatergic neurotransmission to trigeminal neurones and CVNs, and the results in this study suggest isoflurane is likely to be able to inhibit the NMDA receptors in CVNs, but not those NMDA receptors in neurones in the trigeminal nucleus.

In conclusion, stimulation of trigeminal nerve endings, through a mixture of A-δ and C fibres, evoked the glutamatergic neurotransmission to trigeminal afferent neurones and polysynaptic glutamatergic inputs to CVNs in the nucleus ambiguus. The intravenous anaesthetic ketamine inhibited the synaptic transmission at both of these sites in the reflex circuitry. In contrast, fentanyl enhanced, whereas isoflurane inhibited, the reflex activation of CVNs, but neither of these agents altered the neurotransmission to neurones in SP5I. These actions are likely to constitute mechanisms by which these anaesthetics and analgesics, at clinically relevant concentrations, differentially regulate the receptors involved at each site within the TCR.

Acknowledgments

This work was supported by the American Heart Association award (10BGIA3720042) to X.W. and NIH grants HL 49965, HL72006 and HL59895 to D.M. Confocal images were generated in part with a grant from National Centre for Research Resources 1S10RR025565-01.

Glossary

Abbreviations

CV

conduction velocity

CVN

cardiac vagal neurone

EPSC

excitatory postsynaptic current

HSV

herpes simplex virus

MAC

minimum alveolar concentration

Sp5I

spinal trigeminal interpolaris nucleus

TCR

trigeminocardiac reflex

Author contributions

X.W. and D.M. designed the research and wrote the paper. X.W., C.G., D.S., and R.B. conducted the experiments. X.W. and C.G. analyzed the data. All authors approved the final version for publication, and the work was performed at The George Washington University.

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