J Neurophysiol 113: 1165–1174, 2015. First published November 26, 2014; doi:10.1152/jn.00614.2014.

Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii Hong Xu,1 Jeffery A. Boychuk,1 Carie R. Boychuk,1 Victor V. Uteshev,2 and Bret N. Smith1 1

Department of Physiology, College of Medicine, University of Kentucky, Lexington, Kentucky; and 2Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas

Submitted 15 August 2014; accepted in final form 21 November 2014

Xu H, Boychuk JA, Boychuk CR, Uteshev VV, Smith BN. Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii. J Neurophysiol 113: 1165–1174, 2015. First published November 26, 2014; doi:10.1152/jn.00614.2014.—The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotinesensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments. GABA; nicotine receptor; nucleus tractus solitarii; parasympathetic reflex; vagal reflex THE DORSAL VAGAL COMPLEX (DVC) of the caudal brain stem consists of the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus (DMV), and the area postrema. A functional DVC is critical for maintenance of autonomic homeostasis, including gastrointestinal, cardiovascular, and respiratory reflexes. The caudal NTS is the site of the first synapse and a key integration center for autonomic visceral sensory input to the central nervous system (CNS) from the thoracic and most subdiaphragmatic viscera (Saper 2002). The caudal NTS sends excitatory (e.g., glutamatergic) and inhibitory (e.g., GABAergic) projections to preganglionic brain stem sites that modulate both parasympathetic and sympathetic reflexes (Bailey et al. 2006a; Davis et al. 2004; Glatzer et al. 2003; Mendelowitz 1999; Ruggiero et al. 1994; Talman et al. 1992), including the DMV,

Address for reprint requests and other correspondence: B. N. Smith, Dept. of Physiology, Univ. of Kentucky College of Medicine, MS508 Chandler Medical Center, 800 Rose St., Lexington, KY 40536 (e-mail: [email protected]). www.jn.org

where these projections support primarily gastrointestinal reflexes (Travagli and Rogers 2001). Glutamate is the major excitatory neurotransmitter in the brain stem, and glutamatergic neurotransmission in the DVC can be modulated by multiple presynaptic ligand-gated receptors (Bach and Smith 2012; Bailey et al. 2002, 2006b; Blessing 1990; Browning et al. 2006; Derbenev et al. 2006) including nicotinic acetylcholine (ACh) receptors (nAChRs) (Feng et al. 2012; Feng and Uteshev 2014; Kalappa et al. 2011; Smith and Uteshev 2008). The majority of DMV-projecting caudal NTS neurons exhibit robust presynaptic and somatodendritic responsiveness to nicotine (Feng et al. 2012; Feng and Uteshev 2014; Kalappa et al. 2011). Moreover, inhibitory synaptic inputs of an unidentified origin to DMV neurons are sensitive to nAChR activation in brain stem slices from rats (Bertolino et al. 1997), but functional and anatomical specificity of circuits associated with such responses has not been investigated. Nicotine is known to produce significant effects on the gastrointestinal system, which may be attributed to actions in the DVC. Specifically, nicotine can increase gastric tone by exciting neurons in the DMV or, conversely, can decrease intragastric pressure by exciting neurons in the medial NTS (mNTS) (Ferreira et al. 2001, 2002). Therefore, nicotineresponsive, DMV-projecting caudal NTS neurons, as well as their synaptic targets in the DMV, are expected to be prime contributors to central cholinergic modulation of gastrointestinal motility (Travagli et al. 2006) and therefore represent the neural circuit substrate underlying central nicotinic effects on digestion and other autonomic functions (Beleslin and Krstic 1987; Ferreira et al. 2000; Jo et al. 2002; Laffan and Borison 1957; Nagata and Osumi 1990). Inhibitory connections dominate synaptic regulation of gastric-related DMV motor neurons (Travagli et al. 2006), but there is currently no direct evidence implicating involvement of GABAergic caudal NTS neurons in gastric-related, nicotine-sensitive projections to the DMV. The present study used patch-clamp electrophysiology in acute brain stem slices from mice in which enhanced green fluorescent protein (EGFP) is expressed under a GAD67 promoter (i.e., GAD67-EGFP, “GIN” mice) to study the effect of nAChR activation in identified GABAergic neurons in the NTS and transsynaptic, retrograde pseudorabies viral labeling to unambiguously link neurons to their functional motor connection with the gastric musculature. We tested the hypothesis that nicotine excites GABAergic NTS neurons, leading to synaptic inhibition of gastric-related DMV motor neurons in the vagal complex.

0022-3077/15 Copyright © 2015 the American Physiological Society

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MATERIALS AND METHODS

Animals. Whole cell patch-clamp recordings were performed with coronal brain stem slices containing the DVC from young (3– 6 wk) mice [FVB-Tg (GadGFP) 4570Swn/J, FVB; The Jackson Laboratory, Bar Harbor, ME], which express EGFP under a GAD67 promotor in a subset of GABAergic inhibitory neurons (i.e., GIN mice) (Oliva et al. 2000). Mice were housed in a vivarium under a normal 14:10-h light-dark cycle with food and water available ad libitum. All animal procedures were approved by The University of Kentucky Animal Care and Use Committee. Neuron identification and prelabeling. A subset of somatostatincoexpressing GABAergic neurons in the NTS were identified and targeted for recording based on expression of EGFP under a GAD67 promotor in the GIN mouse used for these studies. GABA expression in EGFP-labeled neurons has been characterized thoroughly (Oliva et al. 2000). In a subset of mice, the functional connection of DMV and NTS neurons with the stomach was identified with a neuron-specific, retrogradely and transsynaptically transported, fluorescent viral vector, which is taken up by peripheral nerve terminals and transported centrally (Banfield et al. 2003; Glatzer et al. 2003; Smith et al. 2000). Briefly, under isoflurane anesthesia and laparotomy, the gastric musculature was injected with an attenuated (Bartha) strain of pseudorabies virus (PRV614, 1 ⫻ 108 PFU/ml; Banfield et al. 2003), which has been constructed to express monomeric red fluorescent protein 1 (mRFP1) (Campbell et al. 2002). Five injections (1 ␮l each) of PRV614 were made into the gastric musculature along the greater curvature. After the abdominal cavity was washed thoroughly with sterile saline the abdomen was sutured, and animals were maintained in a biosafety level 2 laboratory for up to 75 h after inoculation. Useful labeling of DMV and NTS neurons occurs after 60 –72 h, a time well before onset of cellular or functional degradation (Card et al. 1993; Glatzer et al. 2003; Smith et al. 2000). This method allows preganglionic DMV motor neurons and synaptically connected, premotor NTS neurons that are specifically related to gastric motor output to be identified and targeted for electrophysiological recording in the in vitro brain stem slice preparation (Blake and Smith 2012; Davis et al. 2003; Derbenev et al. 2004; Gao et al. 2009; Gao and Smith 2010; Glatzer et al. 2003, 2007; Glatzer and Smith 2005; Smith et al. 2000; Williams et al. 2007). Neurons expressing EGFP or mRFP1 were visualized in living tissue under epifluorescence by using fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) filter sets. Once the cell of interest was targeted, infrared-differential interference contrast (IR-DIC) optics were used to obtain recordings. Location of neurons in the NTS or DMV was determined by noting the position of the recorded neuron in relation to fiducial structures in the slice (e.g., central canal, area postrema, tractus solitarius, etc.) and compared to a map of the mouse vagal complex generated from previous studies (Glatzer et al. 2007). Brain stem slice preparation. Mice were anesthetized by isoflurane inhalation to effect (lack of tail pinch response) and decapitated while anesthetized. The brain was then rapidly removed and immediately immersed in ice-cold (0 – 4°C), oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 11 mM glucose, 1.3 CaCl2, and 1.3 MgCl2; pH 7.2–7.4, osmolality 290 –305 mosmol/kgH2O. The brain was blocked rostral to the cerebellum, the brain stem was then mounted on a metal stage, and 300-␮m coronal slices were cut with a vibratome. For consistency, slices from the caudal DVC near the level of the rostral area postrema (⫾600 ␮m rostrocaudally) were used. The slices were then transferred to a holding chamber or to the recording chamber, where they were continually perfused with warmed (32–34°C) ACSF for at least 1 h. The ACSF used for recordings was identical to that used in the dissection, except when drugs were added as described. Patch-clamp recording. After an equilibration period of 1 h, whole cell voltage-clamp recordings were obtained from NTS or DMV

neurons under visual guidance on an upright, fixed-stage microscope equipped with IR-DIC and epifluorescence optics (BX51WI, Olympus). Recording pipettes were pulled from borosilicate glass capillaries with 0.45-mm wall thickness (King Precision Glass, Claremont, CA). Open tip resistance was 2–5 M⍀, seal resistance was 1–5 G⍀, and series resistance was ⬍25 m⍀ (mean ⫽ 12.44 ⫾ 0.23 M⍀, n ⫽ 46 for NTS neurons and 16.15 ⫾ 0.62 M⍀, n ⫽ 54 for DMV neurons), uncompensated. Recordings were discarded if series resistance varied by ⬎20%. Patch pipettes were filled with (in mM) 130 K-gluconate or 130 Cs-gluconate, 1 NaCl, 5 EGTA, 1 MgCl2, 1 CaCl2, 3 KOH, 2 ATP, 0.2 biocytin; pH 7.2–7.4. Neural activity was recorded with an Axon 200B patch-clamp amplifier (Axon Instruments, Sunnyvale, CA), low-pass filtered at 5 kHz, and acquired at 20 kHz with a Digidata 1320A digitizer and pCLAMP 10.3 software. Synaptic currents were analyzed off-line on a PC-style computer with pCLAMP programs (Axon Instruments) or Mini Analysis 6.0.3 (Synaptosoft, Decatur, GA). Synaptic currents had a fast (⬍1 ms) rise time and exponential decay. A value of twice the root mean squared noise level for a given recording was used as the detection limit for synaptic currents. Inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded in voltage-clamp mode. Electrical stimulation was performed with a platinum-iridium concentric bipolar electrode (125-␮m diameter, FHC, Bowdoinham, ME) placed over the tractus solitarius. Thirty paired current pulses (2–100 ␮A, 300 ␮s; A.M.P.I., Jerusalem, Israel) at interpulse intervals of 30 ms and a cycle rate of 0.1 Hz were administered to the tractus solitarius. Synaptic responses were recorded in mNTS neurons voltage-clamped at ⫺70 mV, and EPSC amplitudes before, during, and after the application of nicotine or cytisine were analyzed. Only responses that were of relatively constant latency (latency range ⬍ 0.5 ms; “jitter” ⬍ 200 ␮s) were assessed. Amplitude ratios of evoked (e)EPSC responses (EPSC2/EPSC1) to paired-pulse stimulation of the tractus solitarius (30-ms pairing) were determined by measuring means of at least 15 responses. Drug application. Added to the ACSF for specific experiments were the following: cytisine (100 ␮M; Sigma), the Na⫹ channel blocker tetrodotoxin (TTX, 2 ␮M; Tocris Bioscience, Minneapolis, MN), mecamylamine (10 ␮M; Sigma-Aldrich, St. Louis, MO), the competitive AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 ␮M; Sigma-Aldrich), and the selective NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP-5, 100 ␮M; Sigma-Aldrich). Cytisine was bath-applied for 6 min prior to recording responses; antagonists were applied for at least 15 min prior to and during agonist application. A picospritzer (Parker Hannifin Instrumentation, Cleveland, OH) was used for rapid, localized nicotine application (200 ␮M; Sigma-Aldrich) via a pipette identical to that used for patch-clamp recordings, which was positioned within 10 ␮m of the recorded neuron. Responses to repeated application of nicotine (4-min intervals) were similar within a recording; responses to the first application are reported. Analysis. Once in whole cell configuration, cells were voltageclamped initially near the resting membrane potential (determined by measuring the voltage at which I ⫽ 0) for 10 min to allow equilibration of the intracellular milieu and recording electrode solutions. EPSCs were examined at a holding potential of ⫺70 mV with pipettes containing K-gluconate, while IPSCs were examined at 0 mV with pipettes containing Cs-gluconate to block K⫹ currents and thereby reduce noise and improve voltage-clamp quality. For spontaneous and miniature (i.e., TTX insensitive) synaptic currents, at least 2 min of continuous activity was examined to identify nicotine or cytisine effects on amplitude and frequency distributions. The nonparametric, intra-assay Kolmogorov-Smirnov (K-S) test was used to determine drug effects on PSC frequency and amplitude within a recording. Effects of drug application on mean PSC measurements for groups of cells were determined with a two-tailed, paired Student’s t-test. Statistical significance for all measures was set at P ⬍ 0.05. Statistical

J Neurophysiol • doi:10.1152/jn.00614.2014 • www.jn.org

NICOTINE MODULATION OF GABA CIRCUITS

measurements were performed with Mini Analysis (Synaptosoft) or Microsoft Excel. Numbers are expressed as means ⫾ SE.

despite simultaneous large inward currents because presynaptic facilitation by nicotine was quite robust (see above), while the kinetics of mEPSCs were much faster than those of whole cell inward current responses. Nicotine therefore induced an increase in synaptic and membrane excitability in most GABAergic NTS neurons. To additionally assess the effect of nicotine on synaptic input to GABAergic NTS neurons, EPSCs were recorded in EGFP-labeled cells while applying cytisine (100 ␮M), a nicotine-like alkaloid and potent nAChR agonist that is at least 100-fold more effective in activation of ␤4-containing nAChRs than ␤2-containing nAChRs (Fenster et al. 1999; Luetje and Patrick 1991; Papke and Heinemann 1994). The effect of cytisine on mEPSC frequency and amplitude was determined in 18 GABAergic NTS neurons. In 8 of 18 neurons (44%), mEPSC frequency was significantly increased (determined by K-S test) after 6-min bath application of cytisine, from a control frequency of 2.54 ⫾ 0.89 Hz to 3.87 ⫾ 0.93 Hz in the presence of cytisine (n ⫽ 8; P ⫽ 0.013; Fig. 2). The effect was reversed after 15-min washout to normal ACSF (2.27 ⫾ 0.74 Hz). There was no effect of cytisine on mEPSC amplitude in these neurons (control ACSF 13.74 ⫾ 1.19 pA, cytisine 13.75 ⫾ 0.90 pA; P ⬎ 0.05; 13.91 ⫾ 0.88 pA wash). Cytisine therefore induced an increase in mEPSC frequency in the absence of a change in amplitude, suggesting an effect at receptors located on glutamatergic terminals. To assess the effect of cytisine on action potential-dependent glutamate release from primary viscerosensory afferents, the tractus solitarius was stimulated electrically, while recording eEPSCs in GABAergic NTS neurons (in the absence of TTX). Pairs of stimuli (30-ms separation) resulted in eEPSCs of relatively constant latency in GABAergic neurons, with the amplitude of the second response being smaller than the first,

RESULTS

Nicotine effects on EGFP-labeled, GABAergic NTS neurons. GABAergic NTS neurons were voltage-clamped at ⫺70 mV to assess effects of nicotine on excitatory synaptic transmission and holding current. Nicotine (200 ␮M) was applied with a picospritzer via a pipette positioned within 10 ␮m of the somatic membrane of the recorded cell. Currents were assessed for 25 s prior to nicotine application and in 5-s periods for 25 s after application. Local application (200 ms) of nicotine transiently increased the frequency of spontaneous (s)EPSCs in 8 of 10 GABAergic NTS neurons (80%; not shown). The sEPSC frequency prior to nicotine application (6.33 ⫾ 1.43 Hz) was increased to 13.71 ⫾ 2.96 Hz (n ⫽ 10; P ⫽ 0.011) in the first 5 s after application. This value returned to baseline values by ⬃20 s after nicotine application. Responses to repeated application of nicotine were similar within a recording. Although nicotine was applied focally, spread of the agonist in the slice could have activated local, intact glutamatergic circuits and/or synaptic terminals. To isolate nicotine effects on receptors located on synaptic terminals contacting recorded NTS neurons, TTX (2 ␮M) was used to block action potential-dependent glutamate release. In the presence of TTX nicotine increased miniature (m)EPSC frequency in 13 of 16 neurons (81%), from a mean of 3.54 ⫾ 0.31 Hz prior to nicotine application to 6.73 ⫾ 1.01 Hz after application (n ⫽ 16; P ⫽ 0.002; Fig. 1). In a subset of neurons (n ⫽ 3), nicotine application also induced a large inward current (Fig. 1). In these recordings, presynaptic responsiveness to nicotine puffs remained clearly detectable Nicotine

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Fig. 1. Excitation of GABAergic nucleus tractus solitarii (NTS) neurons by focal nicotine application. A: in the presence of tetrodotoxin (TTX; 2 ␮M), nicotine applied focally to a green fluorescent protein (GFP)-positive, GABAergic medial NTS (mNTS) neuron (arrow; 200 ␮M, 200-ms duration; 20 psi) induced an increase in miniature excitatory postsynaptic current (mEPSC) frequency. Top: continuous recording of mEPSCs before and after nicotine application. A1: expanded 5-s period prior to nicotine application (indicated by line 1, top). A2: expanded period of the first 5 s after nicotine application (indicated by line 2, top) illustrates the nicotineinduced increase in mEPSC frequency. B: response in a different GFP-positive neuron indicates increased mEPSC frequency accompanied by an inward current. B1: before nicotine. B2: after nicotine. C: plot of mean mEPSC frequency in 5-s bins prior to and after nicotine application in 16 GFP-positive, GABAergic NTS neurons. *Significant difference from the 5 s preceding nicotine application for 3 consecutive 5-s periods after nicotine application (P ⬍ 0.05). mEPSCs were recorded in neurons voltage-clamped at ⫺70 mV. Inset: example of recording targeted to a GAD-positive, GABAergic NTS neuron; arrow indicates the recorded cell, and arrowhead indicates the recording pipette.

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Fig. 2. Effects of the nicotine analog cytisine on mEPSC frequency and EPSCs evoked after tractus solitarius stimulation in GABAergic NTS neurons. A: traces showing the effect of bath-applied cytisine (100 ␮M) on mEPSCs in GFP-positive mNTS neurons. Left: mEPSCs in control ACSF containing TTX (2 ␮M). Center: the same neuron in the presence of cytisine. Right: 15 min after washout of cytisine. Arrows point to temporally expanded 1-s periods from the respective traces above. B: cumulative probability plot of mEPSC frequency in the 3 conditions (control, cytisine, wash) from the recording in A. C: plots showing mean the effect of cytisine on mEPSC frequency (left) and amplitude (right) in GABAergic NTS neurons (n ⫽ 8). D: evoked (e)EPSC responses in a GABAergic NTS neuron to paired stimulation of tractus solitarius. Left: averaged traces showing the response of a single neuron in control ACSF and in the presence of cytisine. Although cytisine tended to diminish the amplitude of the 1st eEPSC and increase the amplitude of the 2nd eEPSC, amplitude changes were not significant (P ⬎ 0.05). Right: the same responses, with the 1st EPSC amplitude normalized to that recorded in ACSF, demonstrate the larger 2nd response, relative to the 1st, in the presence of cytisine. Averages of 25–30 consecutive responses are shown for each condition. E: plot showing mean change in paired-pulse response ratio (PPr) in the presence of cytisine for the 4 GABAergic NTS neurons. *Significant increase in mEPSC frequency and PPr in the presence of cytisine (P ⬍ 0.05).

resulting in a paired-pulse response ratio (PPr) of 0.66 ⫾ 0.07 (n ⫽ 5). Addition of cytisine in these neurons significantly increased the PPr to 0.82 ⫾ 0.07 (P ⬍ 0.05; Fig. 2). The effect was reduced by 15-min wash to normal ACSF (0.81 ⫾ 0.09). Cytisine application slightly, but nonsignificantly, reduced the amplitude of the first EPSC (control ACSF 106 ⫾ 38 pA, cytisine 94 ⫾ 36 pA; P ⬎ 0.05) and increased the amplitude of the second EPSC (control ACSF 78 ⫾ 33 pA, cytisine 81 ⫾ 33 pA; P ⬎ 0.05). Application of cytisine therefore increased mEPSC frequency, but not amplitude, and also increased the PPr, suggesting that nicotine acts at receptors on presynaptic terminals to modulate glutamatergic synaptic input to GABAergic NTS neurons. Effects of nicotine on gastric-related NTS neurons. Previous studies have suggested that nicotine effects in the NTS are

differentially expressed, with neurons projecting to forebrain or parabrachial nucleus (PbN) being unaffected and neurons projecting to vagal motor neurons being excited by nicotinic receptor activation (Feng et al. 2012; Feng and Uteshev 2014). To determine whether DMV-projecting, gastric motor-related NTS neurons were affected by nicotine, NTS neurons identified after gastric musculature inoculation with PRV614 were voltage-clamped at ⫺70 mV in the presence of TTX and mEPSCs were examined for responses to focally applied nicotine. Recordings were made from eight gastric-related NTS neurons, two of which were also identified as GABAergic. Nicotine application increased the frequency of mEPSCs, from a baseline frequency of 3.81 ⫾ 0.47 Hz to 6.41 ⫾ 0.87 Hz after nicotine application (n ⫽ 6 of 8; P ⫽ 0.028; Fig. 3). Amplitude of mEPSCs was unaffected. Thus, similar to effects on iden-

J Neurophysiol • doi:10.1152/jn.00614.2014 • www.jn.org

NICOTINE MODULATION OF GABA CIRCUITS

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the presence of cytisine (P ⫽ 0.041; Fig. 4, B and C). Amplitude returned to 38.34 ⫾ 6.81 pA after 20-min wash to normal ACSF. These data suggested that activation by cytisine of nAChRs located in the slice preparation modulated synaptic inhibition of DMV neurons. To determine whether effects on synaptic input were due to activation of receptors on synaptic terminals, the effect of cytisine on mIPSCs in DMV neurons was examined. In the presence of TTX, mIPSC frequency was 1.68 ⫾ 0.47 Hz and was not significantly altered by addition of cytisine (1.65 ⫾ 0.48 Hz; n ⫽ 10; P ⫽ 0.57; Fig. 5). The amplitude of mIPSCs was likewise unaffected by cytisine application (control 22.89 ⫾ 1.66 pA, cytisine 22.33 ⫾ 1.65 pA; P ⫽ 0.39). These data were not consistent with the hypothesis that cytisine acted at receptors on presynaptic terminals to increase GABA release in the

tified GABAergic neurons, nicotine increased excitatory synaptic input to gastric-related NTS neurons. Nicotine effects on synaptic input to DMV neurons. GABAergic NTS neurons make abundant, intact connections with DMV neurons in coronal slices (Davis et al. 2004). To determine the relationship of nicotine-induced activity in the NTS to synaptic regulation of DMV neurons, effects of cytisine on sIPSCs recorded in DMV cells were determined in the presence of CNQX (10 ␮M) and AP-5 (100 ␮M). In control ACSF, sIPSC frequency in DMV neurons was 4.07 ⫾ 0.76 Hz, was increased to 8.96 ⫾ 1.11 Hz in the presence of cytisine (n ⫽ 7; P ⫽ 0.0018; Fig. 4), and returned to values near baseline after 20-min wash to normal ACSF (3.59 ⫾ 0.68 Hz). There was also a significant increase in sIPSC amplitude, being changed from 45.54 ⫾ 6.89 pA in control ACSF to 52.29 ⫾ 8.93 pA in

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Fig. 3. Excitation of premotor gastric-related NTS neurons by focal nicotine application. A: focal application of nicotine (arrow; 200 ␮M, 200-ms duration, 20 psi) induced an increase in mEPSC frequency in a gastricrelated NTS neuron. Portions of the trace indicated above lines 1 and 2 are expanded in A1 and A2 to demonstrate the increase in miniature inhibitory postsynaptic current (mIPSC) frequency in the 5 s after nicotine application. B: mean mEPSC frequency for 5-s bins prior to and after nicotine application. Control ACSF contained 2 ␮M TTX. *Significant difference in frequency vs. the 5 s immediately preceding nicotine application (P ⬍ 0.05). Inset: example of recording targeted to a PRV614-labeled NTS neuron; arrow indicates the recorded cell, and arrowhead indicates the recording pipette.

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Fig. 4. Effect of cytisine on spontaneous (s)IPSCs in a recorded dorsal motor nucleus of the vagus (DMV) neuron. A: recording of sIPSCs and response to bath-applied cytisine (100 ␮M) in a DMV neuron. Left: trace showing continuous recording in control ACSF. Center: the same cell in the presence of cytisine. Right: the same recording after 15-min washout to control ACSF. Arrows point to temporally expanded portions of each trace. B: cumulative probability plots for the traces shown in A. Cytisine increased both amplitude and frequency of sIPSCs in this recording (P ⬍ 0.05; Kolmogorov-Smirnov test). C: plots of mean amplitude (left) and frequency (right) for sIPSCs recorded for 7 DMV neurons, respectively. *Significant differences from control ACSF (P ⬍ 0.05). J Neurophysiol • doi:10.1152/jn.00614.2014 • www.jn.org

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gested that nicotine acts to excite GABAergic NTS neurons that provide synaptic inhibition of DMV cells. Identified gastric-related DMV cells were also examined for responses to nicotine application in the NTS. Brief (200 ms), focal application of nicotine (200 ␮M) in the NTS significantly increased the frequency of sIPSCs in 12 of 18 PRV614-labeled DMV neurons (67% of cells; response determined by K-S analysis), from a background frequency of 6.14 ⫾ 0.70 Hz to 9.50 ⫾ 1.14 Hz 5 s after nicotine application (n ⫽ 12; P ⬍ 0.01; Fig. 6). In seven neurons that initially responded to nicotine application in the NTS, treatment for 15 min with mecamylamine [10 ␮M; a nonselective nAChR antagonist with somewhat greater potency for ␣3␤4* nAChRs (Papke et al. 2010)] prevented the effects of subsequent nicotine application on sIPSC frequency (n ⫽ 7; P ⫽ 0.4; Fig. 6), supporting the involvement of nAChRs. To assess possible effects of nicotine on excitatory synaptic transmission from NTS to DMV, nicotine was applied in the NTS while sEPSCs were recorded in a separate group of PRV614-labeled, gastric-related DMV neurons voltage-clamped at ⫺70 mV. Application of nicotine in the NTS was without effect on sEPSC frequency in any recording (control 6.53 ⫾ 2.15 Hz, nicotine 6.54 ⫾ 2.25 Hz; n ⫽ 4, P ⫽ 0.29; Fig. 7). Nicotine application in the NTS resulted in enhancement of inhibitory but not excitatory synaptic signaling in gastricrelated DMV neurons.

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Fig. 5. Cytisine did not alter mIPSC frequency or amplitude in DMV neurons. A: traces showing mIPSCs in control ACSF, with cytisine (100 ␮M), and after 15-min wash. B: cumulative probability plots of mIPSC frequency (left) and amplitude (right) did not reveal an effect of cytisine in the recordings illustrated in A. C: plots showing mIPSC mean frequency (left) and amplitude (right) for recordings from DMV neurons. There was no significant effect of cytisine on mIPSC frequency or amplitude in DMV neurons (n ⫽ 10; P ⬎ 0.05).

DMV. Combined with the effects on sIPSCs, however, they suggested that nAChR activation on upstream neurons with intact projections to the DMV resulted in increased GABA release onto DMV neurons. Since inhibitory synaptic transmission was enhanced by cytisine but mIPSCs were unaffected, and activity of intact GABAergic NTS neurons is known to significantly alter synaptic inhibition of DMV neurons (Davis et al. 2004; Derbenev et al. 2004; Glatzer et al. 2007), the hypothesis that enhanced GABAergic input to DMV neurons is due to nicotine actions in the NTS was tested. To assess nicotine effects on inhibitory synaptic transmission from NTS to DMV, DMV neurons were voltage-clamped at 0 mV and nicotine (200 ␮M) was applied focally in the NTS with a picospritzer while IPSCs were recorded in the DMV. Flow of ACSF was directed dorsally, away from the DMV. Focal, brief nicotine application (200 ms) in the NTS significantly increased the frequency of sIPSCs in 15 of 28 DMV neurons (54% of cells), from a background frequency of 4.83 ⫾ 0.54 Hz to 6.69 ⫾ 0.69 Hz in the 5 s after nicotine application (n ⫽ 15; P ⫽ 0.0099). These data sug-

Results of this study demonstrate that activation of presynaptic and somatodendritic nAChRs in the NTS results in enhanced glutamatergic neurotransmission within the NTS and increased synaptic excitation of GABAergic NTS neurons. The excitation of NTS neurons translates into an increase in synaptic inhibition of DMV motor neurons. In particular, nicotinic modulation of NTS-to-DMV synaptic inhibition was evident in gastric-related circuitry, indicating a role for nicotinic receptor activation in the NTS in regulating gastric function. This is consistent with nicotine effects identified previously in intact animals, demonstrating that the NTS-DMV circuitry may serve as a substrate for some of the centrally mediated effects of nicotine on gastric motility and secretion (Ferreira et al. 2000, 2002; Nagata and Osumi 1990, 1991). Here we demonstrate that the ability of nicotinic agonists to enhance inhibition of the DMV by activation of presynaptic and/or somatodendritic nAChRs within the NTS depends on intact synaptic connections because facilitation of GABAergic synaptic release in the DMV by nicotine applied to the NTS required action potentialdependent neurotransmitter release. The cytisine-induced increase in sIPSC frequency in the DMV is consistent with increased action potential firing of upstream, premotor neurons that are connected synaptically with DMV motor neurons. Increased activity of GABA neurons could also manifest in summated IPSCs, reflecting the increased IPSC amplitude in the presence of cytisine. Furthermore, facilitation of miniature glutamatergic or GABAergic synaptic activity (i.e., mPSCs) in the DMV by nicotine agonists was not detected. It is evident that the effects of nAChR activation in the NTS result in enhanced synaptic inhibition, but not excitation, of most gastric-related DMV neurons. Thus activation of nAChRs in DMV-projecting, GABAergic NTS neurons might effectively

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increase the gain of vagal circuits involved in reflexive inhibition of vagal motor output to the gut (Ferreira et al. 2000). Effects of excess, exogenous nicotine might also alter normal brain-gut interactions, impairing autonomic homeostasis and ultimately contributing to symptoms of nicotine toxicity: emesis, dysphagia, gastric stasis, and irritable bowel syndrome (Aggarwal et al. 1994; Camilleri 1990; Camilleri and Bharucha 1996; van Orshoven et al. 2006). In humans, smoking cessation leads to significant gains in body weight, affecting the efficacy of smoking cessation programs (Nordstrom et al. 1999; Pomerleau et al. 2000). Our findings are also consistent with a potential brain stem component of anorexigenic effects of nicotine, since nicotine decreases food intake per meal size without significant changes in meal frequency (Blaha et al. 1998; Grunberg et al. 1986; Jo et al. 2002; Miyata et al. 2001).

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GABAergic neurons are densely distributed throughout the NTS (Blessing 1990; Fong et al. 2005; Gao et al. 2009; Glatzer et al. 2007), and most can be activated after stimulation of primary visceral afferents (Bailey et al. 2008; Glatzer et al. 2007). Here we used a transgenic mouse that expresses EGFP in the somatostatinergic subset of GABA neurons in the NTS. As previously described, at least a subset of these neurons were also gastric related, as demonstrated by expression of mRFP1 subsequent to retrograde, transsynaptic labeling from the gastric wall (Glatzer et al. 2007). GABAergic signaling in the mNTS is an important factor in regulating functional activity within the vagovagal reflex circuitry (Herman et al. 2009; Travagli et al. 2006). In particular, GABAergic connections between the NTS and DMV exert important control over vagal motor neuron activity and gastric function (Davis et al. 2004;

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Fig. 6. Increased synaptic inhibition of gastric-related DMV neurons after focal application of nicotine in the NTS. A: increased sIPSC frequency in a gastric-related DMV neuron after focal nicotine application (200 ␮M, 200 ms, 20 psi) in the adjacent NTS. Responses to 2 separate applications (arrows) are shown. Areas marked with lines 1 and 2 in the 2nd application are expanded below. A1: before nicotine. A2: after nicotine. B: mean sIPSC frequency in DMV neurons (n ⫽ 18) for 5-s periods prior to and after nicotine application. *Significant difference in frequency vs. the 5 s immediately preceding nicotine application (P ⬍ 0.05). Inset: example of recording targeted to a PRV614-labeled DMV neuron; arrow indicates the recorded cell, and arrowhead indicates the recording pipette. C: traces illustrating the effect of focal nicotine application on sIPSCs in a gastric-related DMV neuron recorded in the presence of mecamylamine (10 ␮M). D: mecamylamine prevented the increase in sIPSC frequency in gastric-related DMV neurons after nicotine application in the NTS (n ⫽ 7; P ⬎ 0.05).

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Fig. 7. Focal application of nicotine in the NTS did not increase excitatory synaptic input to gastric-related DMV neurons. A: traces illustrating sEPSCs in a gastric-related DMV neuron before and after focal nicotine administration in the NTS (arrows). Two applications are shown. A1 and A2 are expanded traces corresponding to the areas under lines 1 and 2, respectively, in the 2nd application. B: graph of mean sEPSC frequency before and after nicotine application in the NTS. There was no significant effect on sEPSC frequency in any neuron (n ⫽ 4; P ⬎ 0.05).

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Gao and Smith 2010; Travagli et al. 1991, 2006). In rats, DMV-projecting NTS neurons form the most populous nicotine-responsive group of neurons in the nucleus (Feng et al. 2012; Feng and Uteshev 2014). Our data demonstrate that GABAergic NTS neurons in particular act as nicotine-sensitive, second-order viscerosensory neurons and that nicotine enhances synaptic excitation in premotor GABA neurons related to gastric projections. Nicotine application in the NTS resulted in increased GABA release in the DMV, suggesting that nicotine enhances excitability of NTS GABA neurons, which inhibits gastric vagal motor neurons. Gastric-related circuits were almost universally responsive, implying a consistent nicotine effect on parasympathetic gastric motor control. Whereas most DMV cells responded to nicotine application in the NTS, some were nonresponsive to nicotine application at a single site in the NTS. It is possible that applications at other positions in the NTS may have resulted in responses in these DMV neurons. Conversely, not all GABAergic or gastric-related premotor NTS neurons responded to nicotine. It is possible that nAChR distribution on glutamatergic terminals contacting NTS neurons is not ubiquitous, being present in functionally or anatomically specified circuits, including a large portion of those regulating gastric motor function. Focal nicotine application increased EPSCs in ⬃80% of GABAergic NTS neurons, whereas bath-applied cytisine enhanced excitatory synaptic input in only 44% of cells. This discrepancy between agonist effects could indicate that cytisine and nicotine activate different pools of nAChRs (i.e., ␣3␤4 and ␣7 vs. ␣4␤2 containing). Mecamylamine (10 ␮M) is relatively nonselective for nAChRs of different subunit composition at this concentration and should block all these receptor types. Previous experiments, however, indicated that the majority of presynaptic nAChRs in the NTS are ␣3␤4 or ␣7 because RJR-2403 (a potent agonist of ␣4␤2 nAChRs) did not potentiate while DH␤E (a potent antagonist of ␣4* nAChRs) did not inhibit mEPSC frequency (Smith and Uteshev 2008); GTS-21 (a selective agonist of ␣7 nAChRs) potentiated glutamate release in ⬃26% of tested caudal NTS neurons (Kalappa et al. 2011). Thus there is little evidence for presynaptic ␣4␤2 nAChRs in the caudal NTS. Alternatively, it is also possible that responses to prolonged (i.e., ⬃6 min) bath application of cytisine caused a partial desensitization of nAChRs, reducing the ability to detect agonist-induced changes in EPSC frequency. Conversely, little or no desensitization would be expected after rapid, focal application of nicotine. While further investigation of specific nAChR subtypes responsible for the responses may be informative, it seems likely that the diminished response to bath-applied cytisine reflects a partial desensitization of the receptors. The source of endogenous ACh that might induce nAChRmediated activity in the NTS is uncertain, but a DMV-to-NTS feedback mechanism represents an intriguing hypothesis. Because of the close proximity of the NTS and the DMV, cholinergic DMV neurons may not need to establish synaptic connections with the NTS to exert their action: cholinergic signaling may be transmitted from the DMV to the NTS via dendritic release of ACh, similar to other neurotransmitters (Bergquist and Ludwig 2008) and/or volume transmission of ACh (Lendvai and Vizi 2008). Once in the NTS, ACh and/or its main metabolite, choline, are expected to activate presyn-

aptic and/or somatodendritic nAChRs (i.e., ␣7, ␣3␤4, ␣4␤2 subtypes), including those expressed on paraventricular nucleus of hypothalamus (PVN)- and caudal ventrolateral medulla (CVLM)/nucleus ambiguus (NAmb)-projecting NTS neurons involved in modulation of neuroendocrine (Srinivasan et al. 2013) and baroreflex (Ashworth-Preece et al. 1998; Ferreira et al. 2000) functions. Therefore, this scenario also predicts the possibility of central cholinergic interactions between gastrointestinal and cardiovascular autonomic pathways at the level of the NTS (Ferreira et al. 2000). There is considerable diversity among NTS neurons in the expression of presynaptic and somatodendritic nAChRs, which is consistent with the multimodal role of the NTS in controlling autonomic homeostasis (Feng et al. 2012; Feng and Uteshev 2014; Kalappa et al. 2011). In rats, the majority of glutamatergic connections with caudal NTS neurons projecting to the PbN are unresponsive to nicotine (Feng et al. 2012). By contrast, the majority of phenotypically unidentified caudal NTS neurons with projections to the DMV exhibit robust responsiveness to nicotine (Feng et al. 2012), which was confirmed here for GABAergic and gastric-related DMV projections. A more comprehensive analysis of neuron targetdependent sensitivity to nicotine in the caudal NTS demonstrated significant correlations between the percentage of caudal NTS neurons responsive to nicotine and neuronal projection targets (Feng and Uteshev 2014). DMV-projecting caudal NTS neurons are the most responsive to nicotine among the projection groups tested, while PbN-, CVLM-, and NAmbprojecting cells are the least responsive to nicotine. Although the exact reason for this preferential expression of nAChRs in DMV-projecting, GABAergic caudal NTS neurons is not known, the finding is consistent with a predominant nicotinic effect on parasympathetic regulatory circuits controlling the digestive viscera. In this study, facilitation of excitatory synaptic input to GABAergic NTS neurons was found after brief nicotine application and activation of nAChRs by cytisine resulted in enhanced glutamate release onto GABAergic NTS neurons. Synaptic release of excitatory neurotransmitter was enhanced by activation of nAChRs on presynaptic terminals, as demonstrated by an increase in mEPSC frequency by nicotine and cytisine. Receptors located on presynaptic terminals of primary viscerosensory afferents were probably also affected, since the PPr of EPSCs evoked after tractus solitarius stimulation was significantly changed. Curiously, eEPSC amplitude, which might be expected to increase with nAChR-dependent enhancement of glutamate release probability at these terminals, was not significantly altered. Several possibilities may underlie this apparent dichotomy of effect. For example, cytisine could affect a pool of vesicles with agonist sensitivities different from the primary vesicle pool released after stimulation of primary afferents (Fawley et al. 2014). The drug could also affect different mechanisms of release at vagal afferent terminals versus terminals of other neurons, as can occur with TRPV1 activation (Peters et al. 2010). A similar increase in PPr, in combination with increased mEPSC frequency, has also been reported after positive modulation of AMPA receptors in the calyx of Held (Bellingham and Walmsley 1999), which was attributed to a Ca2⫹-independent mechanism of enhanced glutamate release. Finally, the duration of the eEPSC analysis necessitated prolonged cytisine application (i.e., ⬃10 min),

J Neurophysiol • doi:10.1152/jn.00614.2014 • www.jn.org

NICOTINE MODULATION OF GABA CIRCUITS

which could have partially desensitized nAChRs in this experiment. Cytisine effects on mEPSC frequency and PPr are consistent with the interpretation that the agonist acts at nAChRs located on glutamatergic terminals, but focused experiments to identify subcellular mechanisms of synaptic release by nAChR may be informative. Our data suggest the intriguing possibility that primary viscerosensory afferent terminals express functional nAChRs capable of enhancing the efficacy of these inputs to the NTS at the level of the first synapse. This possibility is supported by observations that nodose ganglionic neurons express nAChRs, while ganglionectomy reduces binding in the mNTS (Ashworth-Preece et al. 1998). While the default failure rate of primary sensory synapses is relatively low (McDougall et al. 2009), especially compared with failure rates observed in cortical regions (Rosenmund et al. 1993), presynaptic modulation by cytisine reveals a reduction in paired-pulse depression, which would be expected to effectively increase glutamate release at the first afferent synapse with GABAergic, second-order sensory neurons in the NTS, especially during sustained vagal afferent activity. Because primary afferents may innervate other neuronal phenotypes (e.g., glutamatergic, adrenergic), and it is possible that nAChR activation could also affect primary afferent input to those neurons, the outcome of exogenously applied nAChR agonists acting globally in the NTS may include a number of physiological effects in addition to enhanced inhibition of gastric-related DMV motor neurons, depending on the network organization and cytochemistry. The results of this study further help define neuronal properties and nAChR expression in the NTS and suggest that activation of functional nAChRs expressed on presynaptic glutamatergic terminals in the caudal NTS may enhance the efficacy of glutamatergic neurotransmission in GABAergic NTS neurons to modulate autonomic gastrointestinal functions and reflexes. High-fidelity synapses from viscerosensory afferents could draw physiological benefit from expressing presynaptic nAChRs and presynaptic facilitation by nicotinic agonists, as may lower-fidelity glutamatergic synapses originating from within the CNS. Our data indicate that GABAergic inhibition of DMV neurons is potentiated by nicotine. Therefore, nicotine and other nicotine agonists affect the inhibition of DMV neurons by enhancing synaptic activation of GABAergic NTS neurons, which in turn project to neurons that control gastric function. Understanding pharmacological properties of nAChRs and nicotinic effects in the DVC may benefit development of therapeutic approaches for selective targeting of gastric visceral reflexes. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK-056132 and R01 DK-080901 (B. N. Smith) and R01 DK-082625 (V. V. Uteshev). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: H.X., V.V.U., and B.N.S. conception and design of research; H.X. performed experiments; H.X., J.A.B., C.R.B., and B.N.S. analyzed data; H.X., V.V.U., and B.N.S. interpreted results of experiments;

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H.X., J.A.B., C.R.B., and B.N.S. prepared figures; H.X., V.V.U., and B.N.S. drafted manuscript; H.X., J.A.B., C.R.B., V.V.U., and B.N.S. edited and revised manuscript; H.X., J.A.B., C.R.B., V.V.U., and B.N.S. approved final version of manuscript.

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