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Department of Cell and Molecular Biology, Division of Neurobiology, Tulane University, New Orleans, Louisiana
Submitted 27 April 2004; accepted in final form 15 December 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Shapiro and Miselis 1985). The NTS is hypothesized to process visceral afferent information through activation of local excitatory and inhibitory circuits (Champagnat et al. 1985, 1986; Fortin and Champagnat 1993; Kawai and Senba 1996; Smith et al. 1998). Neurons in the NTS also transmit viscerosensory and other synaptic information to the dorsal motor nucleus of the vagus (DMV), which provides parasympathetic motor innervation to the gastric musculature. The DMV receives excitatory and inhibitory projections from the regions of the NTS that modulate preganglionic parasympathetic output (Browning et al. 2002; Davis et al. 2003; Travagli et al. 1991). Based on putative synaptic connectivity within the vagal complex, three functional groups of neurons in the NTS are therefore purported to take part in the visceral reflexes and local modulation of those reflexes. These include neurons that 1) receive primary vagal sensory input; 2) project to other NTS neurons, and 3) project to the DMV (Champagnat et al. 1986; Glatzer et al. 2003; Smith et al. 1998). Modulation of synaptic connections within the vagal complex by neuropeptides likely affects some or all of these components of vagal circuitry to modify gastrointestinal and other visceral functions.
Opioid drugs are among the oldest known pharmacologically active substances, and some of the best studied. µ-Opioid receptors (MORs) in different isoforms are abundant in the dorsal vagal complex (Abbadie et al. 2000, 2002; Ding et al. 1996) and seem to act in the area to modify parasympathetic function. MORs are present on NTS dendrites and on vagal afferent terminals (Aicher et al. 2000; Ding et al. 1996), and the MOR agonist D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) inhibits responses to stimulation of the solitary tract (ST) in rat brain slices (Rhim et al. 1993), suggesting that MOR activation affects viscerosensory synaptic input to NTS neurons. Opioid agonists applied systemically or microinjected into the NTS can cause gastric relaxation and inhibition of gastrointestinal transit and gastric secretions (Burks et al. 1987; Czapla et al. 1998, 2000; Del Tacca et al. 1987; Krowicki et al. 1999). Specific effects of MOR activation on synaptic transmission and membrane properties of gastric-projecting DMV neurons have also been described (Browning et al., 2002). Agonists of MORs act in the vagal complex to modulate motor control of visceral systems, but with the exception of effects on putative vagal afferents (Rhim et al. 1993), their cellular actions within the NTS are largely unknown.
Endomorphins 1 and 2 (EM-1, EM-2) are endogenous peptides with high affinity and selectivity for the MOR in mammals (Zadina et al. 1997). Immunohistochemistry in the rat shows robust staining in perikarya in the NTS for EM-1 and fibers throughout the DVC were immunoreactive for EM-2 (Martin-Schild et al. 1999; Pierce and Wessendorf 2000). EM-1 staining was found in cell bodies and terminal fields within the NTS, whereas EM-2 staining was located in varicose fibers, and perhaps within the solitary tract (Martin-Schild et al. 1997, 1999; Pierce and Wessendorf 2000). The distribution of EM-1 staining is closely related to the staining of MORs within the NTS (Martin-Schild et al. 1999).
The presence of MORs and their putative endogenous agonists, EM-1 and -2, in the NTS suggests functional roles for modulation of parasympathetic control of visceral functions by these peptides (Horvath 2000; Martin-Schild et al. 1997, 1999; Pierce and Wessendorf 2000; Venkatesan et al. 2002). In this study, we used patch-clamp electrophysiology in brain stem slices to investigate the role of EM-1 and MORs on synaptic input to NTS neurons, including those identified using a transsynaptic viral label as gastric-related premotor neurons, neurons receiving solitary tract input, and neurons receiving local excitatory and inhibitory connections. We tested the hypotheses that EM-1 1) reduces vagal afferent input to the NTS by acting at presynaptic receptors; 2) inhibits the postsynaptic membrane of NTS neurons; and 3) suppresses local circuit activity within the NTS.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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For some experiments, a fluorescently labeled viral vector, which selectively labels neurons in a transneuronal, retrograde manner, was used to identify gastric-related, putative premotor neurons for patch-clamp recordings (Davis et al. 2003; Glatzer et al. 2003; Irnaten et al. 2001; Jons and Mettenleiter 1997; Smith et al. 2000). The virus used in this study, a pseudorabies virus (PRV) isogenic with the Bartha strain (PRV-152; a gift of Dr. L. Enquist, Princeton University), was constructed to express enhanced green fluorescent protein and allow visualization of infected neurons in brain slices acutely prepared for in vitro electrophysiological analyses or in fixed tissue (Smith et al. 2000). Sprague-Dawley rats (Harlan; male; 2–6 wk old) were housed with a 12-h light/dark cycle, with food and water provided ad libitum. All the animals were treated according to the rules of the Tulane University Animal Care and Use Committee. Inoculation and labeling of vagal terminal fields in the gastric musculature was performed similar to previous descriptions (Davis et al. 2003; Glatzer et al. 2003; Rinaman et al. 1993, 1999). Briefly, under pentobarbital sodium anesthesia (50 mg/kg, ip), animals received three to four injections (5 µl total) into the ventral stomach wall of PRV-152 (2 x 108 plaque forming units/ml) over a 1-min interval using a 10-µl Hamilton syringe fitted with a 26-gauge needle. A fresh stock of virus was used for each injection. Animals recovered from surgery and were maintained in a biosafety level 2 laboratory for 60–72 h after injection. Control inoculations into the lumen of the stomach or onto the surface of the peritoneum resulted in little or no specific labeling in the vagal complex (Glatzer et al. 2003). After 72 h of infection, about 10–20% of neurons in the NTS were infected by the virus (Glatzer et al. 2003).
Slice preparation
All chemicals were purchased from Sigma-RBI (St. Louis, MO) unless otherwise noted. Both inoculated and control rats were deeply anesthetized with pentobarbital sodium (100 mg/kg, ip; Nembutal, Abbott Laboratories) or Halothane inhalation and then decapitated. The brain was removed and blocked on an ice-cold stand, and the medulla was glued to a stage and inserted into a vibrating microtome (Vibratome Series 1000, Technical Products, St. Louis, MO). Transverse brain stem slices (300–400 µm) were prepared in 0–2°C artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 3 KCl, 2 CaCl2, 1.3 MgCl2, 1.4 NaH2PO4, 26 NaHCO3, and 11 glucose. Slices were incubated for 
1 h in 35°C oxygenated (95% O2-5% CO2) ACSF. After incubation, the slice was transferred to a recording chamber on a fixed stage under an upright microscope (BX50W1, Olympus; Melville, NY). The slice was continually superfused by room temperature ACSF. NTS neurons expressing EGFP were visualized in living tissue under epifluorescence by using a fluorescein isothiocyanate (FITC) filter set and a Spot RT-slider CCD camera (Glatzer et al. 2003; Smith et al. 2000). Once a cell of interest was identified, infrared differential interference contrast (IR/DIC) optics were used to guide the recording pipette onto the cell and obtain recordings, exactly as for recordings in unlabeled neurons. In control experiments not included herein, physical properties of neurons were unaltered by fluorescent illumination of PRV-152, even with relatively long exposure times (i.e., >30 min continuous). However, for the present studies, slices were exposed to only enough fluorescent illumination to target the neurons (i.e., a few seconds).
Electrophysiology
Whole cell voltage-clamp recordings made with an Axopatch 200B amplifier (Axon Instruments, Union City, CA), digitized at 88 Hz (Neurocorder, Cygnus Instruments; Dorchester, UK), and low-pass filtered at 2–5 kHz were recorded onto videotape and a PC running Clampex 8.2 software (Axon Instruments). Patch pipettes (3-5M
) were pulled from borosilicate glass (1.65 mm OD, 0.45 mm wall thickness; Garner Glass Company, Claremont, CA) and filled with (in mM) 130–140 potassium-gluconate (or Cs-gluconate), 10 HEPES, 1 NaCl, 1 CaCl2, 3 KOH (or CsOH), 5 EGTA, 2–4 Mg-ATP, and 0.1% biocytin. Picrotoxin (50 µM; Sigma), 6-cyano-7-nitroquinoxaline-2,3-dionedisodium (CNQX; 10 µM; Sigma), D-2-amino-5-phosphonopentoic acid (AP-5; 50 µM; Sigma), TTX (2 µM; Alamone Labs; Jerusalem, Israel), Tyr-Pro-Trp-Phe-NH2 (EM-1; 0.01–10 µM), Tyr-Pro-Phe-Phe-NH2 (EM-2; 1 µM), [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO, 1 µM), naltrexone HCl (20–50 µM), D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 1 µM), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 1 µM) were applied to the bath. The criteria for detecting synaptic currents were fast rise time (<1 ms) and exponential decay. At a cycle rate of 5 s, the ST was stimulated by trains of 2–5 current pulses (2–150 µA, 300–400 µs; A.M.P.I., Jerusalem, Israel) at frequencies of 20–100 Hz through concentric bipolar platinum-iridium electrodes (125 µm diam; FHC, Bowdoinham, ME). Failure rates were calculated as the number of absences of a synaptic deflection following the first stimulus within a train divided by the total number of trains (Doyle and Andresen 2001). Two to three different concentrations of EM-1 were applied to a few neurons to assess the effect of a given concentration versus the control (ANOVA repeated measures; Bonferroni t-test). When this was done, washout of each concentration occurred before the next concentration was applied. In most cases, response to a single dose was assessed per neuron, and neurons were pooled to create a concentration-response curve (Fig. 1). The concentration–response relationship for multiple neurons was determined using Prism 4.0 (GraphPad, San Diego, CA). Changes in whole cell conductance were assessed using a ramp stimulus protocol which first stepped to –100 mV for 500 ms and then linearly depolarized the membrane to +20 mV over the course of 16 s. Responses to five such voltage ramps were averaged before, during, and after drug application (Smith et al. 2002). Resting membrane potentials were corrected for junction potential (approximately –8 mV). Frequency and amplitude of spontaneous events examined using a paired t-test or a Kolmogorov-Smirnov test (MiniAnalysis, Synaptosoft), with P < 0.05 considered significant. Group analyses were made using a paired two-tailed t-test. All values are shown as means ± SE.
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Similar to previous descriptions of glutamate photolysis in the vagal complex (Davis et al. 2003), 
-(-carboxy-2-nitrobenzyl) ester, trifluoroacetic acid salt (i.e., CNB-caged glutamate, 250 µM; Molecular Probes, Eugene, OR) was added to recirculating ACSF and uncaged using brief pulses of UV light directed into the slice. Fluorescent light (UV filter, Chroma Technology, Rockingham, VT) was directed onto the slice through the x40 objective used to obtain the recording, which was moved progressively further away from the recorded cell until a photolysis-mediated increase in synaptic events was found. Exposure time was electronically controlled using a shutter (Vincent Associates, Rochester, NY). Opening the shutter with no UV filter or with other filters in place (e.g., FITC) did not result in uncaging. Uncaging glutamate directly onto the recorded neuron (9-s interval, 20- to 30-ms exposure) resulted in a fast inward current (50–200 pA at a holding potential of –60 mV), which response was repeatable in single cells in >100 consecutive applications (n = 4). Photic stimuli were applied on the ST and at sites just outside the NTS for negative controls (i.e., no synaptic responses were observed). The effective diameter of the uncaging (
50 µm) was set by apertures in the light path and measured by moving the center of the illumination away from the cell and testing for a direct inward current after uncaging. The distance moved was analyzed posthoc by comparison with a scale slide (Microbrightfield, Williston, VT).
Histology
After recording, brain slices were fixed in 4% paraformaldehyde in 0.15 M sodium phosphate buffer overnight at 4°C (pH = 7.4) and processed as previously described (Glatzer et al. 2003). After three rinses with 0.01 M PBS, whole-mount slices were immersed in Avidin conjugated to AMCA or Texas red (1:400; Vector Laboratories) in PBS containing 0.5% Triton X-100 and incubated overnight at 4°C to verify biocytin-filled neurons were within the NTS and confirm they were labeled with EGFP. Slices were rinsed two to three times with PBS and mounted on slides to air dry for 10 min. The slices were covered in Vectashield (Vector Laboratories). Cells labeled with biocytin during a recording and/or with EGFP from PRV-152 infection were identified with a Leica DMLB microscope, and images were captured with a Spot RT CCD camera (Diagnostic Instruments) using filters for the two fluorescent dyes and EGFP. Neurons were required to be positively identified and documented by one or both of these methods to be considered EGFP-labeled NTS neurons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Sixteen recordings were made from EGFP-labeled neurons. As previously reported (Glatzer et al. 2003), none of these neurons received constant-latency input from the solitary tract. Analyses of responses in EGFP-labeled neurons were made in experiments examining effects of EM-1 on spontaneous, miniature, and photolysis-evoked synaptic inputs. Endomorphin effects on solitary tract stimulation
To establish that recorded neurons received ST input, EPSCs were monitored during electrical stimulation of the ST at a holding potential of –60 to –70 mV. Constant-latency EPSCs (<0.5 ms variability; mean latency to onset was 4.4 ± 0.3 ms; n = 10) were evoked in 10 NTS neurons and were CNQX- and TTX-sensitive. The constant-latency ST-evoked EPSCs averaged –73.8 ± 15.2 pA (n = 10) in control conditions. Bath application of the MOR agonist EM-1 (1 µM) for 2 min during ST stimulation resulted in a significant reduction of the average ST-evoked EPSC amplitude to –26.7 ± 7.0 pA (Fig. 1; P < 0.05, paired t-test). The effect of EM-1 was reversed after 15–20 min of wash in normal ACSF (Fig. 1). Application of naltrexone HCl (20–50 µM; n = 5), a broad-spectrum opioid receptor antagonist, prior to EM-1 application prevented inhibition of the evoked EPSC (Fig. 1B). Application of the opioid receptor antagonist alone was without significant effect (n = 5; 91 ± 12% of control EPSC amplitude; P > 0.05). Application of different concentrations of EM-1 (half-log increments, 10 nM to 10 µM) to different neurons resulted in a concentration-related inhibition of ST-evoked EPSCs (Fig. 1). The threshold for significant decreases in response to EM-1 was 100 nM, and maximal effect was seen at 10 µM (P < 0.05). Decreases in EPSC amplitude were not accompanied by significant changes in input resistance (average input resistance in control was 533 ± 37 M; average input resistance in EM-1 was 496 ± 36 M; n = 49; P > 0.05). However, some neurons (n = 12/49) showed a small outward current (5–30 pA) during EM-1 application at holding potentials near rest (i.e., –45 to –65 mV). Overall, average EM-1 induced current was 4.2 ± 2.4 pA (n = 49). Voltage ramps from –100 to +20 mV in the presence of TTX (2 µM) did not show significant changes in whole cell conductance (n = 7; P > 0.05).
To test the hypothesis that EM-1 reduced eEPSC amplitude by acting at receptors on vagal afferent terminals, two analyses were applied. The ratio of amplitude of two postsynaptic currents evoked in close sequence (i.e., paired-pulse ratio) was used to assess whether EM-1 acts at a presynaptic or postsynaptic site, with a change in the ratio of amplitude suggesting a presynaptic site of action. When two EPSCs were evoked at a pairing frequency of 40 Hz, paired-pulse depression resulted in the second EPSC having a smaller amplitude than the first (Chen et al. 1999, 2002; Doyle and Andresen 2001; Miles 1986; Smith et al. 1998). EM-1 reduced the amplitude of the first current to a greater degree than the second, such that the paired-pulse ratio increased from 0.51 ± 0.07 in control ACSF to 0.74 ± 0.07 in EM-1 (1 µM, n = 10; P < 0.05; Fig. 2). Second, the failure rate of ST-evoked EPSCs was analyzed. Constant-latency ST-evoked EPSCs had low initial failure rates (0–17% failures; n = 10, Fig. 2). In the presence of EM-1 (1 µM), EPSC failure rate significantly increased in all neurons tested (11–86% failure; n = 10;P < 0.05; Fig. 2). The increase in the failures was prevented by naltrexone HCl (20–50 µM; Fig. 2; n = 5). EM-1 altered both paired-pulse ratio and failure rate, suggesting a presynaptic effect on ST-evoked responses.
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The effects of EM-1 on spontaneous EPSCs (sEPSCs) were studied in 17 NTS neurons including 4 EGFP-labeled neurons and 5 neurons receiving solitary tract input. The frequency and amplitude of sEPSCs was measured while voltage-clamping the cell near –65 mV, and the sEPSCs were blocked by application of CNQX (10 µM; n = 16). In 14 of 17 NTS neurons tested (including 4/4 EGFP-labeled neurons and 4/5 neurons receiving ST input), EM-1 significantly reduced the frequency of sEPSCs (P < 0.05; Fig. 3). The remaining three cells showed a nonsignificant decrease or no change in sEPSC frequency. The mean sEPSC frequency for all neurons measured at –65 mV was 6.99 ± 1.28 Hz (n = 17). In the presence of EM-1, the mean sEPSC frequency was reduced to 3.11 ± 0.57 Hz (n = 17; P < 0.05). The amplitude of sEPSCs was also significantly reduced in 9 of 17 neurons, with amplitude being unchanged in the remaining eight cells. Overall sEPSC amplitude was reduced from –23.6 ± 3.1 pA in control to –19.2 ± 2.5 pA in EM-1 (1 µM; n = 17; P < 0.05; paired t-test). CTAP had no significant effect on sEPSC frequency or amplitude (1–2 µM; 99 ± 3% of control sEPSC frequency; 96 ± 8% of control sEPSC amplitude; n = 4; P > 0.05), but the antagonist completely prevented the effects of EM-1.
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To determine whether the reduction in EPSC frequency and amplitude by EM-1 could occur independently of Na+-dependent synaptic release (i.e., action potentials in afferent intact neurons), the effects of EM-1 on miniature EPSCs (mEPSCs) were measured in the presence of TTX (2 µM) in 10 neurons (including 2 EGFP-labeled neurons). In 8 of these 10 NTS neurons, including 2/2 EGFP-labeled neurons, EM-1 (1 µM) significantly reduced the frequency of mEPSCs compared with control (P < 0.05; Fig. 4), with no significant reduction in mEPSC amplitude (P > 0.05). The mean frequency of mEPSCs was 4.5 ± 1.3 Hz and amplitude was –13.2 ± 2.0 pA in control ACSF. The overall mEPSC frequency was reduced to 3.4 ± 1.0 Hz in EM-1 (1 µM; n = 10), whereas amplitude was unaltered (–13.3 ± 2.3 pA; n = 10; P > 0.05, paired t-test). The reduction in mEPSC frequency was blocked by CTOP or CTAP, MOR-selective antagonists (1–2 µM; 96 ± 2% of control frequency; n = 4; P > 0.05).
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To determine the effects of EM-1 on GABAergic synaptic inputs, Cs+ was used as the primary cation in the electrode solution and sIPSCs were examined at a holding potential of –10 to 0 mV. The sIPSCs appeared as outward chloride currents, sensitive to picrotoxin (50 µM; n = 19) or bicuculline methiodide (30 µM; n = 4). Spontaneous IPSCs were significantly reduced in frequency by EM-1 in six of eight neurons tested, including two of two EGFP-labeled neurons (1.5 ± 0.5 in control vs. 0.8 ± 0.2 in EM-1; P < 0.05). Although EM-1 reduced sIPSC amplitude in two neurons, overall amplitude was unchanged (P > 0.05). To test whether decreases in sIPSCs were caused by the MOR-induced reduction in glutamatergic synaptic excitation of local GABA neurons, we examined the effects of EM-1 on IPSCs in the presence of AP-5 (50 µM) and CNQX (10 µM). In the presence of glutamate receptor antagonists, sIPSCs were significantly reduced to 60 ± 8% of control frequency (2.3 ± 0.9 Hz in control, 1.3 ± 0.6 Hz in EM-1; n = 5; P < 0.05, paired t-test) in this set of neurons with no change in mean sIPSC amplitude (20.2 ± 4.3 pA in control, 18.6 ± 3.9 pA in EM-1; 92 ± 4% of control; P > 0.05; n = 5).
To test whether the effects of EM-1 on IPSCs were action potential-dependent, we applied EM-1 in the presence of TTX (2 µM). The average frequency of mIPSCs was 1.43 ± 0.58 Hz in control ACSF and 1.17 ± 0.57 Hz after addition of EM-1 (1 µM; n = 6; P > 0.05). The average amplitude of mIPSCs was 20.2 ± 4.0 pA in control ACSF and 19.9 ± 3.6 pA in the presence of EM-1 (1 µM; n = 6; P > 0.05). Overall there was no effect of EM-1 on mIPSC frequency or amplitude (Fig. 5).
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To identify the effects of EM-1 on input from intact neurons within the slice, we used caged glutamate, which is activated by UV light, to depolarize local NTS neurons. Focal, short-duration exposures to UV light directed through the microscope objective into the NTS (<50 µm diam; 10–30 ms) resulted in an increase in synaptic activity in NTS neurons (n = 12). Stimuli directed onto the ST or to areas adjacent to the NTS were ineffective, suggesting that responses to NTS stimulation were due to activation of neurons within the NTS. Application of TTX (2 µM) blocked the evoked IPSCs (n = 3), indicating that they were due to actions potentials generated in intact, afferent neurons (Callaway and Katz 1993; Dalva and Katz 1994; Davis et al. 2003; Katz and Dalva 1994). At a holding potential of 0 mV, uncaging glutamate in nearby areas of the NTS resulted in an average increase in the peak IPSC frequency of 373 ± 84% (500-ms periods before and after uncaging; Fig. 6; n = 7). EM-1 (1 µM) reduced the glutamate-evoked IPSC frequency increase to 227 ± 24% over the prephotolysis period (n = 7; P < 0.05). This effect of EM-1 was prevented by application of CTAP (1 µM; n = 3; P > 0.05; Fig. 6F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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, 1999). Effects of endomorphin were observed in most NTS neurons tested, but there was heterogeneity in the magnitude and type of response, and in some cases no response at all, which is consistent with the complex effects of MOR agonists previously shown in the NTS (Rhim and Miller 1993, 1994) and DMV (Browning et al. 2002, 2004). Our data suggest that EM-1 causes a reduction in excitatory glutamatergic transmission to the NTS via interactions with viscerosensory afferents, as well as a reduction in locally generated excitatory inputs. The effects of EM-1 on excitatory synaptic input to NTS neurons seem to occur mainly by activation of presynaptic receptors. To the contrary, local and spontaneous inhibitory input seems to be reduced via a reduction in action potential frequency in putative local GABA neurons. Identification of neurons in the NTS that connect to gastric-projecting DMV neurons via retrograde viral labeling with PRV-152 was previously used to study the synaptic input to these neurons (Glatzer et al. 2003). Since neurons were labeled by virtue of their association with motor neurons innervating the stomach (i.e., probably 1 or 2 synapses upstream), they were considered to be gastric-related premotor neurons. Both excitatory and inhibitory synaptic input to putative premotor neurons, identified using PRV-152, were inhibited by EM-1 to about the same degree as in the overall population, which probably included neurons associated with any of several visceral functions. Neurons related to other viscera may also be affected, because systemic application or microinjection of endomorphins into the NTS has been shown to elicit either increases or decreases in heart rate and blood pressure, perhaps indicating a general alteration of parasympathetic output (Czapla et al. 2000; Sapru and Chitravanshi 2002; Shah et al. 2003). The pre- versus postsynaptic specificity of the effects reported herein therefore supports a role for EM-1 in modulating vagally mediated processes, including gastrointestinal function. Endomorphins reduce excitatory synaptic transmission in the NTS
Agonists of MORs, but not kappa or delta opioid receptor agonists, reduced excitatory solitary tract evoked neurotransmission in the NTS (Rhim et al. 1993). The discovery of EM-1 and -2, and the robust labeling for EM-1 producing neurons and EM-2 fibers within the NTS are consistent with the hypothesis that endomorphins are endogenous MOR agonists in the NTS (Martin-Schild et al. 1999; Zadina et al. 1997). This study indicates that endomorphins have similar functions to previously studied synthetic agonists on ST input (Rhim et al. 1993). In addition to a reduction in primary afferent input to NTS neurons, we found effects on excitatory and inhibitory input arising from within the NTS. These effects were seen on presumed second-order viscerosensory neurons as well as on putative premotor NTS neurons that regulate output to the gastric musculature via their putative connection with preganglionic motor neurons in the DMV.
Presynaptic effects of EM-1 on EPSCs
Several lines of evidence suggest that endomorphin effects on EPSCs involve activation of receptors on glutamatergic terminals. Frequency-dependent paired-pulse depression is characteristic of the primary afferent synapse in the caudal NTS (Andresen and Mendelowitz 1996; Champagnat et al. 1986; Chen et al. 2002; Doyle and Andresen 2001; Fortin and Champagnat 1993; Kline et al. 2002; Miles 1986; Smith et al. 1998). To examine pre- versus postsynaptic sites of action of EM-1, we used the model that a reduction in the presynaptic probability of release in response to ST stimulation in the presence of EM-1 would result in a greater reduction in the first EPSC of the pair, while a change in postsynaptic response would result in equal reduction of both EPSCs (Kline et al. 2002; Regehr and Stevens 2001). Our results indicate that the first EPSC was reduced to a greater extent than the second during EM-1 application, resulting in an increase in the paired-pulse ratio. The EM-1 induced increase in the failure rate also supports the hypothesis, because it indicates a reduction in the probability of glutamate release from terminals. At the electron microscopic level, MORs in the NTS have been isolated to vagal afferent terminals (Aicher et al. 2000), and these data support those of Rhim et al. (1993), which also suggested a MOR-dependent effect on ST-mediated input to second-order viscerosensory neurons in the NTS. However, the reduction in the amplitude of the EPSCs may also indicate a change in quantal content, which could result in pre- or postsynaptic effects (Regehr and Stevens 2001). In the presence of TTX, EM-1 reduced the frequency but not the amplitude of mEPSCs. This effect further suggests that activation of MORs on glutamatergic terminals reduces the probability of glutamate release, rather than reducing amplitude of glutamate-dependent synaptic currents in the postsynaptic neuron.
Local NTS circuit alterations by EM-1
Electrical stimulation during slice recordings can recruit cut axons of passage in addition to intact cell bodies and processes to activate an assortment of inputs to the recorded neuron. In addition to glutamate released from cut axons in the ST, many sEPSCs and sIPSCs received by NTS neurons are likely to be of local origin (Champagnat et al. 1986; Fortin and Champagnat 1993; Kawai and Senba 1996; Smith et al. 1998). Inputs arising from various central locations also impinge on these neurons (Agarwal and Calaresu 1990; Rogers and McCann 1993; Sawchenko and Swanson 1982; Spyer et al. 1984), and activation of such fibers of passage is also possible during electrical stimulation in the NTS. Studies aimed at identifying local circuits have used application of glutamate to stimulate connections between intact neurons without activating fibers of passage, which is critical for understanding modulation of local circuits (Boudaba et al. 1996; Callaway and Katz 1993; Christian and Dudek 1988; Katz and Dalva 1994; Smith and Dudek 2002). In this study, we used focal photolysis of caged glutamate to stimulate local NTS neurons and determine the effect of EM-1 on the connections between activated neurons and the recorded cell. Synaptic responses to stimulation of a given NTS region were consistent and repeatable within an individual recording. Moving the stimulus slightly (<50 µm) away from an effective stimulation site typically eliminated the response, implying that evoked PSCs originated from activity in individual neurons or, at most, small groups of cells. In theory, it is possible that glutamate-induced release of endogenous neuroactive substances (e.g., retrograde release of peptides, nitric oxide) could indirectly change PSC frequency after a stimulus without necessarily activating local interneurons. However, because of the focal nature of the stimulus, the lack of responses from nearby extranuclear stimulation sites, and the sensitivity of evoked responses to blockade of action potentials, it seems most likely that glutamate photolysis-evoked PSCs were due to activation of local circuits in the NTS. EM-1 reduced the increase in frequency of EPSCs and IPSCs evoked by glutamate photolysis activation of putative local neurons, suggesting an inhibitory effect on the stimulated action potential firing and/or the neurotransmitter release from the axon terminals of local neurons synapsing onto the recorded neuron. These results are consistent with the hypothesis that EM-1 modulates local NTS circuits in addition to viscerosensory inputs.
Postsynaptic effects on GABA neurons
Previous studies found MOR agonists produced a hyperpolarization in NTS neurons and reduced calcium conductances associated with N, P/Q/R channels in NTS neurons in slices and acute cultures (Rhim and Miller 1994; Rhim et al. 1993, 1996). The effects of EM-1 on holding current in some neurons (25%) may indicate changes in resting or voltage-gated K+-channel conductances, similar to Rhim et al. (1993) and Rhim and Miller (1994), but changes to fast calcium conductances may have been masked by calcium buffer EGTA in the intracellular solution. The effects of MOR agonists on membrane potential may also contribute to the heterogeneity in effects on spontaneous PSC amplitude due to membrane hyperpolarization of afferent neurons or decreased input resistance in recorded cells. Postsynaptic changes in firing properties of local GABA neurons were suggested by IPSC frequency decreases during EM-1 application. The decreased IPSC frequency was not seen in the presence of TTX, suggesting that EM-1 acted to modify the action potential firing behavior of intact GABA neurons that projected to the recorded cell. This was probably not simply due to an EM-1–induced decrease in synaptic excitation of GABA neurons because the decrease in sIPSC frequency by EM-1 was observed in the presence of AMPA/kainate and NMDA receptor antagonists. If MORs were found on GABA cells, but not on afferent GABAergic terminals, EM-1 would be expected to decrease the frequency of sIPSCs but not affect mIPSCs, which is what we observed. Based on these data and on those of Rhim et al. (1993), we predict that EM-1 reduces calcium currents and action potential firing in GABAergic NTS neurons.
Autonomic regulation by EM-1
Gastric regulation involves a balance between sympathetic and parasympathetic inputs to the gut. Modulation of viscerosensory inputs to the NTS by EM-1 suggests that endogenous MOR ligands are involved in suppression of sensory input from the viscera. Gastric-related premotor neurons have qualitative and quantitative similarities in synaptic inputs and electrical properties to unidentified NTS neurons (Glatzer et al. 2003). EM-1 had effects on synaptic input to these gastric-related, putative premotor neurons that were identical to effects on unidentified NTS neurons. Whether these neurons were retrogradely labeled one or more synapses from the DMV motor neurons, their connection with the efferent limb of gastric vagal innervation imparts a functional relevance that is different from that of second-order sensory neurons. The consistent EM-1–induced reduction of ST input to viscerosensory neurons and local circuit control of premotor neurons within the NTS suggests that EM-1 modifies vagal reflexes at multiple levels within the NTS to alter parasympathetic gastric control. These findings are consistent with the hypothesis that EM-1 acts at MORs on presynaptic terminals to inhibit glutamate release and on postsynaptic MORs to reduce activity of local GABA neurons. A similar pattern of MOR activation has been shown in the DMV, where MORs were proposed to be located on glutamatergic, but not GABAergic synaptic terminals contacting gastric motor neurons (Browning et al. 2002).
These experiments examined the effects of EM-1 on responses of functionally diverse groups of NTS neurons. The solitary tract input to the NTS is the major drive for visceral sensation. Neurons that respond to solitary tract stimulation in the slice are assumed to be part of the visceral sensory processing system. Interneurons within the NTS may mediate integration and processing of primary afferent and other sources of synaptic input. The effects of EM-1 on putative local inputs were examined using glutamate photolysis, which activates afferent neurons with soma and/or dendrites, but not axons, near the photolysis site. We also tested the effects of EM-1 on synaptic inputs to neurons infected transneuronally from the stomach, which represent a component of the motor output of the vagal complex. We found similar effects of EM-1 on all of these types of neurons, which suggest several conclusions. One possibility is that individual neurons can participate in all of these functions (i.e., some neurons concomitantly receive solitary tract input, send collaterals to other NTS neurons, and project to vagal motor neurons), and therefore respond similarly to MOR agonists. Another is that three or more separate functional neural groups exist in the NTS, each of which is affected similarly by MOR activation, but mediated by temporally and/or spatially relevant opioid release at specific glutamatergic and GABAergic connections in the NTS. A more complex circuit model of the dorsal vagal complex is needed to decide between these hypotheses.
These data are consistent with the hypothesis that endomorphins modulate both viscerosensory input to the NTS and information processed within the NTS. This may include satiety signals from the stomach, but could also affect information from cardiorespiratory or a number of other visceral systems. EM-1 has primarily inhibitory effects on sensory afferent neurotransmission to the NTS, on local synaptic processing within the NTS, and on NTS neurons that regulate the activity of DMV neurons. Understanding how EM-1 and other MOR agonists affect visceral sensory-motor processing is essential to analyzing the effects of some of the most commonly used drugs in medicine and addiction.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: B. N. Smith, Dept. of Cell and Molecular Biology, Tulane Univ., 6400 Freret St., New Orleans, LA 70118 (E-mail: BNSmith{at}tulane.edu)
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) and EM-1 (
) are shown on the graph. *Significantly lower frequency within the individual bins in the presence of EM-1 (P < 0.05). Uncaging occurred at time 0 (arrow). F: normalized effects of EM-1 (1 µM; n = 7) and µ-opioid receptor (MOR) antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 1 µM; n = 3) on pre- and postphotolysis IPSC frequency (500 ms) and on the evoked currents. *Significant effect of the agonist vs. preagonist response (P < 0.05). Inset: location of the recorded neuron (