Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro R. A. TRAVAGLI, R. A. GILLIS, AND S. VICINI Fidia Georgetown Institute for the Neurosciences and Department of Pharmacology, Georgetown University School of Medicine, Washington, DC 20007 Travagli, R. A., R. A. Gillis, and S. Vicini. Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleusof the vagus, in vitro. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G508-G517, 1992.-We sought to characterize the excitatory effect of thyrotropin-releasing hormone (TRH) in dorsal motor nucleusof the vagus (DMV) motoneurons by using the patch-clamp technique in rat brain stem slices. In our initial studies we used the cell-attached recording configuration using concentrations of TRH from 1 to 30 PM. Exposure of DMV motoneuronsto TRH resulted in a concentration-related increase in spontaneously occurring action potential firing rate. This wasobservedin 63 of 85 DMV neurons (75%) tested and wasunrelated to their location rostra1or cauda1to the obex. Invariably, desensitization occurred to the excitatory effect of TRH. Subsequentexperiments using whole cell recordingsin the current-clamp mode confirmed that TRH excites DMV neurons located both rostra1 and caudal to the obex. In the current-clamp configuration, TRH produced depolarization; i.e., 30 PM TRH elicited a depolarization of 8.7 k 3.2 mV (P < 0.05, n = 7). Studies using whole cell current recordings in voltage-clamp mode indicated that TRH in a concentration-dependent manner producesa small inward current that is associatedwith a decreasein the input resistanceof -42.5 t 15.6 MQ (TRH 30 PM). TRH-induced inward current was also present under conditions of inhibition of synaptic transmission (i.e., in the presenceof tetrodotoxin and cobalt). We also found that TRH reduced in a concentration-dependent manner both the fast transient A-type K+ current (I*) and the Ca2+-dependent afterhyperpolarizing current &..&. Using the extracellular recording technique in the cell-attached configuration, we investigated whether any part of TRH-induced increasein firing rate was due to an increase in the synaptic releaseof Lglutamate or acetylcholine. Prior exposure of DMV neuronsto either kynurenic acid or to atropine did not antagonize any of the excitatory effect of TRH. Finally, we observedthat addition of 30 PM TRH to the perfusing solution produced an increase in spontaneously occurring excitatory postsynaptic currents (EPSCs). This occurred without any changein the amplitude of EPSCs. These results indicated that TRH-induced increasein firing of DMV neurons is due to direct postsynaptic effects to activate an inward cationic current and to counteract 1* and I AHP, aswell asa presynaptic effect to increasethe frequency of EPSCs. brain stem slices;whole cell recordingsin the,current-clamp and voltage-clamp modes; inward cationic current; excitatory postsynaptic currents; transient A-type potassiumcurrent; calcium-dependent afterhyperpolarizing current IS A GREAT DEAL of evidence that thyrotropinreleasing hormone (TRH) is a neurotransmitter and/or neuromodulator in the central vagal regulation of gastric function. This evidence is summarized in several review articles (29, 30) and in the introductory sections of several publications that present original data (13, 23, 25). Of particular note are the findings of Hernandez et al. (12) indicating that specific polyclonal TRH antibodies raised in rabbits against synthetic rat TRH infused by the intracerebroventricular route into rats 30 min before
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occlusion of the pylorus significantly decreased gastric acid secretion when compared with rats treated with normal rabbit serum. These findings indicate that endogenous brain TRH plays a physiological role in the regulation of gastric acid secretion. In a follow-up study, Hernandez and Emerick (11) reported that microinjection of TRH into the dorsal motor nucleus of the vagus of rats increased gas tric acid output and gastric mw ulcers. These investigators concluded that presynaptic “TRH fibers may modulate vagal activity at the level of the dorsal motor nucleus of the vagus and propose(d) that descending TRH pathways may play a role in experimental ulcerogenesis through acid hypersecretion.” Evidence that TRH excites DMV motoneurons in vivo in rats has been obtained by McCann and Rogers (17). These investigators identified electrophysiologitally individual DMV units by using stimulating electrodes placed on the cervical vagus. TRH was then tested on these DMV motoneurons by pressure injection of lo-40 fmol in IO-40 pl. Thirteen cells were identified, and of these, six (or 46%) were activated by TRH. Activation was reflected by an increase in the firing rate. The remaining seven DMV motoneurons were not affected by TRH; none of the DMV motoneurons were inhibited. Evidence that TRH excites DMV motoneurons in vitro in rats has been obtained by Raggenbass et al. (23). These investigators studied TRH using extracellular single-unit recordings from brain stem slices of the rat and reported that about one-third of the vagal nerves were excited by TRH. This action of TRH was present in a low-calcium, high-magnesium solution, which blocks synaptic transmission, thus suggesting that TRH was acting directly on DMV neurons. We have recently begun in vitro studies using whole cell current and voltage-clamp recordings from anatomically identified DMV neurons in rat brain stem slices (32). Our results indicate that these neurons are capable of sustained slow-frequency action potential firing probably because of the presence of a pacemaker current. Our results also indicate the presence of excitatory and inhibitory spontaneous and evoked synaptic currents in the DMV. The excitatory synaptic currents are due to activation of ionotropic glutamate receptors, and the inhibitory synaptic currents are mediated by y-aminobutyric acid (GAB A). The purpose of the present study was to use this rat brain stem slice preparation to examine the electrophysiological effects of the putative excitatory neurotransmitter at the DMV, TRH. Our goal was to confirm the findings of Raggenbass et al. (23) that TRH acts directly to increase the firing rate of these cells, and if confirmed, to determine the mechanism(s) responsible for a direct excitatory effect of TRH. An additional goal was to
0 1992 the American
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determine whether TRH induces release of glutamate, and if so, to determine whether glutamate contributes to the excitatory effect of TRH on DMV motoneurons. METHODS Brain stem slice preparation. We prepared the slices for patch-clamp recording as previously described (7, 32). Briefly, young Sprague-Dawleyrats 2-3 wk of agewere decapitated, and the brain stem was quickly removed and placed in ice-cold oxygenated physiological rat Ringer solution after excision of the cerebellum.With the useof an Oxford vibratome, 200-km-thick coronal sliceswere obtained from the medulla, both caudally and rostrally to the obex. The sliceswerethen incubated at 37°C in oxygenated physiological rat Ringer solution until used. The experimental studieswith TRH were carried out at room temperature (22-25°C). Perfusion andpatchpipette solutions and drugs. Physiological rat Ringer wascomposedof (in mM) 120 NaCl, 26 NaHCOs, 3.1 KCl, 1 MgC12, 2 CaCl,, and 5 dextrose. The bathing medium was maintained at pH 7.4 with 95% O&j% CO, bubbling. During cutting proceduresand incubation periods the Ringer solution wasmodified so that it contained 2 mM MgC12and 1 mM CaCl,. The purposefor this was to reduce glutamate-mediated neurotoxicity through excessstimulation of the N-methyl-Daspartic acid (NMDA) receptor. Cell-attached recordings of visually identified DMV neurons were performed with patch pipettes filled with Ringer solution, while whole cell recordings were performed with patch pipettes filled with (in mM) 145 potassiumgluconate, 1 MgCl,, 0.5 ethylene glycol-bis@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2 ATP, 0.65 GTP, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-KOH (pH 7.2). Drugs were addedby perfusion in the bathing medium (5 ml/min) via a system of valves controlled by a manually operated switch, the drugstaking between 40 and 60 s to equilibrate at the final concentration in the perfusion chamber. Acetylcholine, 4-aminopyridine, apamin, atropine, glutamic acid, kynurenic acid, and tetrodotoxin (TTX) were purchased from Sigma (St. Louis, MO); TRH (pGlu-His-Pro-NH,) waspurchasedfrom Peninsula (Belmont, CA). All drugsusedwere freshly preparedand dissolveddirectly in Ringer solution at the neededconcentration. Electrophysiological recordings. By means of the tight-seal whole cell recording method of Hamill et al. (lo), we measured potentials and currents from the DMV neurons. Currents and voltage signalsat the headstageof the patch-clamp amplifier (Axopatch lD, Axon Instruments, Foster City, CA) were filtered and continuously displayed on an oscilloscope.Current and voltage steps were evoked via the patch-clamp amplifier, while a double-channel stimulator (model S88 Grass Instruments, Quincy, MA) was used to control the frequency and duration of the stimuli. The pClamp data acquisition and analysis software (version 5.5.1, Axon) run on an IBM-AT personal computer wasusedfor the voltage-clamp protocols that required more complex procedures. Data collection and analysis. Data were recorded with a VCR magnetic tape (VR 10-A, Instrutech, Elmont, NY) for later
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analysis. The amplitudes of the currents and voltages evoked were analyzed with a Gould DSO-405 oscilloscope(Gould, Ilford, Essex,UK) or by using the pClamp analysis software with appropriate modifications. Results are expressedas means& SE; t test for paired data was used, with P < 0.05 being the criterion for statistical significance. RESULTS
Effects of TRH on spontaneous firing rate of DMV neurons. In our initial electrophysiological study of DMV
neurons in isolated brain slices of lo- to 18-day-old rats, we employed whole-cell current-clamp recordings from anatomically identified DMV neurons and showed that these neurons are capable of sustained slow-frequency action potential firing, probably because of the presence of a pacemaker current (32). In the present study using the whole cell patch-clamp recording technique, and recording extracellular activity in the cell-attached recording configuration, we again observed that the vast majority of these neurons exhibit a spontaneous discharge. Baseline activity of DMV neurons as recorded in the cell-attached configuration ranged from 0.1 to 4.3 spikes/s and an example of their activity before exposure to TRH can be seen on the control portion of Fig. 1. This activity was recorded from 85 DMV neurons, and on exposure to TRH in concentrations ranging from 1 to 100 PM, 63 (or 75%) of the neurons exhibited an increase in spontaneous firing rate. In the 22 remaining DMV neurons, TRH had no effect. In no case did TRH produce a decrease in the spontaneous firing rate. In earlier studies, we reported that stimulation of sites in the DMV in vivo produced opposite effects on lower esophageal sphincter (LES) pressure. That is, stimulation of the DMV rostra1 to the obex elicits a rise in LES pressure, whereas stimulation of the DMV caudal to the obex elicits a fall in LES pressure (27). Hence, we examined whether TRH would exert a different effect on rostral DMV neurons vs. caudal DMV neurons. Thirty-three of the 85 DMV neurons referred to above were located rostra1 to obex, and exposure to TRH elicited only an excitatory effect (i.e., 25 of 33, or 76% exhibited an in-
crease in spontaneous firing). Fifty-two of the 85 DMV neurons were located caudal to the obex, and exposure to TRH elicited only an excitatory effect (i.e., 38 of 52 or 73% exhibited an increase in spontaneous firing). An experiment illustrating the excitatory effect of TRH on a DMV neuron appears as Fig. 1. As can be noted, adding TRH to the perfusion solution at a concentration of 30 PM produced an almost immediate (within 1 min) increase in the spontaneous firing rate. The increase became maximal at -30 s after TRH, and began to dissipate soon after washing TRH from the system. The
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Fig. 1. Effect of thyrotropin-releasing hormone (TRH) on firing of a vagal neuron using patch-clamp recording in extracellular cell-attached confirmration. TRH was added to nerfusion solution in a concentration of 30 uM and for the time corresponding to the horizontal line below the recording. Frequency of action potential firing increased from 0.55 to 1.43 spikes/s. Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 10, 2019.
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TRH effect was reversible. That is, soon after washout, the spontaneous firing rate of the DMV neuron returned to its baseline firing rate. The TRH-induced excitation appeared to be concentration related in terms of peak firing rate, and this is shown in the summarized data tabulated in Table 1. The lowest effective TRH concentration was between 1 and 3 PM. As the concentration of drug used was increased in approximate threefold steps, the percent increase in firing rate increased from approximately two- to eightfold. No indication of a “plateau effect” was indicated over the range of concentrations of TRH used. Interestingly, desensitization occurred to the excitatory effect of TRH. Initially, TRH administration produced a striking increase in the spontaneous firing of DMV neurons. However, after the initial large increase in firing rate was obtained, the firing rate began to decline and fall to a rate about halfway between the initial rate and the maximal rate obtained with TRH, even though there was continuous perfusion of the DMV neuron with TRH. The decrease in firing rate appeared -4-5 min after the peak effect of TRH was achieved, and the new firing rate stabilized at values significantly lower than the peak values (decrease from a peak firing rate of 3.5 + 0.95 to 1.8 + 0.76 spikes/s; P < 9.05) but still higher than the control values (0.6 & 0.02 spikes/s) (data obtained from 3 cells in which the phenomenon of “desensitization”;using 30 PM of TRH was carefully analyzed). After washbut of TRH and a return of the firing rate to a predrug rate, reexposure of the DMV neuron to TRH within 20 min of initial exposure did not result in the same response to TRH. That is, the response obtained on second exposure to TRH was always less than the initial response, thus showing sustained desensitization. This was noted in six DMV neurons studied. However, if the interval between initial exposure and reexposure was >20 min, TRH did produce the same response as noted with the initial exposure of the peptide (n = 5). To characterize the TRH-induced desensitization even further, additional studies were performed using potassium as a depolarizing agent. When potassium (9 mM final concn) was added to the perfusing fluid (in the form of KCl) at the time when desensitization had occurred with 30 FM TRH, potassium was found to increase the Table 1. Effect of TRH on spontaneous firing rate of DMV neurons using patch-clamp recording technique and recording either extracellular activity in cell-attached configuration or intracellular activity in current-clamp configuration
spontaneous rate of the cell to the same degree as it did when delivered to the DMV neuron not exposed to TRH (n = 13). In addition, repeated exposure of the DMV neuron to potassium at intervals 0.05) (n = 5). Finally, 30 PM TRH was added to the perfusion solution in the presence of kynurenic acid (1 mM) and elicited the usual increase in spontaneous firing rate (+140 t 30%; P < 0.05) (n = 5). For the studies on acetylcholine, the same experimental protocol was performed using 1 PM of acetylcholine. This neurotransmitter produced a 90.8 t 40.8% increase in spontaneous firing rate (n = 6), and atropine (1 PM), added to the perfusing solution, abolished the acetylcholine response (n = 4). Perfusion of DMV neurons with TRH (30 PM) in the presence of atropine (1 PM) elicited an increase in spontaneous firing rate (+227.3 t 104.8%, n =: 7) Using the whole cell recording technique in the voltageclamp mode, we observed that addition of TRH (30 PM) to the perfusing solution produced an increase in spontaneously occurring excitatory postsynaptic currents (soEPSCs). In fact, soEPSCs frequency increased 178 t 30.8% from a value of 0.55 t 0.11 to 1.45 t 0.13 Hz (n = 5). This occurred without any significant change in the amplitude of the soEPSCs in three of the five neurons tested. In the remaining two neurons, an assessment of the soEPSCs amplitude was not possible to determine because of the high baseline noise compared with the small size of the event. In contrast, addition of TRH (30 PM) to the perfusing solution did not appear to alter the frequency of spontaneously occurring inhibitory postsynaptic currents (soIPSCS). This was observed in four neurons. No discernible effect on SOIPSCS amplitude was observed. A representative experiment illustrating this effect of TRH appears as Fig. 8.
J‘2000pAv m 500 msec Fig. 7. Effect of TRH on Ca 2+-dependent afterhyperpolarization due to activation of an I AHp in a vagal neuron using whole cell patch clamp in current-clamp mode. Neuron was hyperpolarized at -50 mV by a steady injection of current and then subjected to several l-s-long depolarizing stimuli of increasing intensities. This resulted in the occurrence of an afterhyperpolarizing potential. Recording in top left panel is control; recording in top right panel was taken during TRH addition to perfusing solution in a concentration of 30 PM. It can be noted that TRH decreased afterhyperpolarization despite increase in number of action potentials induced during depolarization. Recording in bottom left panel was taken during addition of 100 nM apamin. It can be noted that apamin completely inhibited afterhyperpolarization and dramatically increased number of action potentials induced by depolarizing current. Recording in bottom right panel was taken from a different cell perfused in Ringer physiological solution in which Ca2+ was substituted with cobalt (final concn 2 mM). It can be noted that perfusion with cobalt completely inhibited afterhyperpolarization as well as occurrence of action potentials induced by injection of depolarizing current.
Fig. 7. This recording was made from a neuron different from the one exposed to TRH (and apamin). The neuron was perfused with a physiological solution in which cobalt (2 mM) was substituted for calcium. As can be noted, cobalt completely inhibited the afterhyperpolarization after injection of depolarizing current. Cobalt also reduced the number of action potentials that normally occur after injection of depolarizing current. This finding with cobalt was obtained in three DMV neurons tested. As a further test of whether we were evaluating TRH against IAHP, we studied the effect of apamin (100 nM) on the afterhyperpolarization. Three DMV neurons were evaluated, and in each case, exposure of the neuron to apamin completely abolished the afterhyperpolarization. An example of the effect of apamin can be seen in the bottom left panel of Fig. 7. Studies of effect of TRH on synaptic transmission. Using the extracellular recording technique in the cell-attached configuration, we investigated whether any part of TRHinduced increase in spontaneous firing rate was due to an increase in the synaptic release of either L-glutamate or acetylcholine. We chose to look at L-glutamate release because this excitatory amino acid had been shown in our previous study to be one of the major neurotransmitters controlling the activity of DMV neurons (32). We chose to look at acetylcholine release because studies by other investigators have shown that 1) TRH releasesthis neurotransmitter from central nervous system (CNS) tissue
DISCUSSION
In a previous study, Raggenbass et al. (23) used extracellular single-unit recordings in brain stem slices and evaluated the effect of TRH on neurons in the DMV. In 365 neurons studied, 105 or 29% exhibited an increase in firing rate; no effect was noted in the remaining 260 neurons. An excitatory effect was evident with a TRH concentration of -10 nM, and a half-maximal effect occurred with a TRH concentration of -100 nM. In addition, the excitatory effect of TRH was present in a low-calcium (0.2 mM CaC12vs. 1.25 mM CaC12)and highmagnesium (6 mM MgS04 vs. 1 mM MgSO,j solution, which blocks synaptic transmission. In the present study, we confirmed the findings of Raggenbass et al. (23) by recording extracellular activity in the cell-attached configuration and adding TRH to the brain stem slice perfusing solution. In general, our results are qualitatively
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JOPA msec
200
Fig. 8. Effect of TRH on EPSCs (and IPSCs) in a vagal neuron using whole cell patch-clamp recording in voltage-clamp mode. Neuron was voltage clamped at -50 mV, and spontaneous miniature EPSCs and spontaneous miniature IPSCs were observed as inward (downward) and outward (upward) currents, respectively. A: recordings obtained during control period. B: recordings taken during TRH addition to perfusion solution in a concentration of 30 PM.
identical to the results of Raggenbass et al., but quantitatively, we observed a higher proportion of DMV neurons (i.e., 75%) excited by TRH. On the other hand, we found that a higher concentration of TRH was required to elicit an effect in our study. A possible reason(s) for these quantitative differences is that we studied brain stem slices from lo- to l&day-old rats and at a temperature of 2225”C, whereas Raggenbass et al. studied brain stem slices from young adult rats and at a temperature of 34-35OC. One qualitative difference between the two studies is that we observed desensitization to the excitatory effect of TRH, whereas desensitization was not observed in the Raggenbass et al. study. In general, desensitization to TRH effects on neural tissue is the more common finding (18, 20, 24). Once documenting that TRH acts to increase the firing rate of DMV neurons [thus confirming Raggenbass et al. (23)], we sought to determine the mechanisms(s) whereby TRH induces DMV neurons to discharge more frequently. In pursuing this goal, we first observed that TRH produces a depolarizing action on DMV motoneurons. This was documented in our studies using whole cell recording in the current-clamp mode. We found that TRH, in addition to increasing the spontaneous firing rate of DMV neurons using this recording technique, also produced depolarization. The TRH-induced depolarization, like the TRH-induced increase in firing rate, exhibited desensitization, suggesting that TRH-induced depolarization may have caused the increase in DMV motoneuron firing. An interesting and unexplained finding was the inability of large concentrations of TRH (e.g., 30 and 100 PM) to elicit large increases in firing rate of DMV neurons studied using whole cell recordings in the current-clamp mode (Table 1). One possible reason for this is that TRH in high concentrations increases electrical excitability through an intracellular second messenger, and Drummond (6) indeed has demonstrated that TRH induces changes in phosphoinositide metabolism and intracellular calcium concentration. Whole cell patch-clamp recording could result in intracellular dialysis of putative
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second messenger components (2) and thereby interfere with TRH from evoking the large response observed in the cell-attached recording configuration. In the next series of experiments using whole cell recordings in the voltage-clamp mode, we demonstrated that TRH induces a small inward current that is associated with a decrease in the Rinp. This inward current was also induced by TRH in brain slices perfused with a medium containing TTX and cobalt (which caused an almost total inhibition of synaptic activity), suggesting that TRH is exerting its effect directly on the postsynaptic membrane of the DMV motoneuron, probably by activating an inward cationic current. Our results are similar to earlier findings by Nicoll (18, 19), who studied TRH on frog spinal motoneurons in vitro. Nicoll reported that TRH produced a direct depolarizing action on motoneurons, and this direct effect was accompanied by a fall in membrane resistance. He postulated that TRH was acting directly on the motoneuron to increase inward sodium current. Most investigators who have studied TRH on motoneurons and observed TRH-induced depolarization have noted that it was accompanied by either an increase in Ri,, or no change in Rinp (3, 16, 20, 24, 31, 33). The increase in Rinp was, in each case, postulated to be due to TRH- induced closure of potassium channels. We also obtained evidence that TRH will counteract potassium conductance. For example, we found that TRH decreases the amplitude of a calcium-dependent hyperpolarizing current as well as the fast transient Atype potassium current. In the latter case, the effect of TRH was not exerted by shifting the activation or the inactivation potential of the I*. Reductions in both of these currents could contribute to TRH-induced increase in firing of DMV neurons. Findings obtained with TRH indicating an increase in inward cationic current (and depolarization) and antagonism of 1A and I AHp in DMV neurons bathed in solution containing cobalt and TTX reflect an action of TRH at postsynaptic sites of the synapses in the DMV. In addition, we have also obtained evidence that TRH exerts an excitatory effect at a presynaptic site. For example, our data indicating that TRH increases the frequency of EPSCs suggest a presynaptic site of action of the peptide. Whether the site of action of TRH was on nerve terminals synapsing on DMV motoneurons or on perikarya of interneurons and/or projections from other brain stem nuclei in our slice preparation was not delineated in our study. Rekling (24) also reported that TRH increases the frequency of EPSPs in rat hypoglossal motoneurons. Similar to our findings, Rekling did not observe any change in the amplitude of the EPSPs. Lacey et al. (16), who studied TRH effects on frog motoneurons in vitro, reported that TRH increases both the frequency of firing and the amplitude of EPSPs, and concluded that the primary site of action of TRH on motoneurons was presynaptic. Similarly, Behbehani et al. (3), using cultured spinal cord neurons from the mouse, observed TRH-induced increases in both the frequency of firing and the amplitude
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of EPSPs. These investigators also concluded that TRHinduced excitation of motoneurons is due to a presynaptic site of action of the peptide. In terms of TRH effects of IPSPs and IPSCs, we did not examine this question in a thorough manner, but the preliminary data we have obtained indicate that TRH does not alter the frequency (or amplitude) of IPSCs. Similarly, Takahashi (31), who studied rat spinal motoneurons in vitro, did not observe any striking effects on IPSPs. It is clear from our previous study (32) of DMV motoneurons in vitro that endogenously occurring excitatory and inhibitory amino acids are the major neurotransmitters controlling the excitability of DMV motoneurons in the brain stem slice preparation. Because TRH-produced excitation of motoneurons in our preparation, we focused on the possibility that part of the excitatory effects of TRH was due to an increase in the presynaptic release of glutamate. Indeed, Lacey et al. (16) interpreted their data on TRH-induced excitation of lumbar spinal motoneurons of frog as caused by TRH enhancement of presynaptic release of glutamate. We tested this notion in our preparation by using the excitatory amino acid antagonist kynurenic acid, and found that a concentration of kynurenic acid that fully blocked the excitatory effect of exogenously administered glutamate on DMV motoneurons had no effect on TRH-induced increase in cell firing rate. Hence our data indicate that the excitatory effect of TRH on DMV motoneurons is not mediated through enhanced presynaptic release of glutamate. In addition, we also studied the role of presynaptic release of acetylcholine in the excitatory response of TRH. Acetylcholine is known to be present in the DMV and has been shown to excite DMV motoneurons (14). We found that atropine in a concentration that blocks the excitatory effect of exogenously administered acetylcholine has no effect on TRH-induced increase in firing rate. This finding is consistent with earlier results obtained in the rat in vivo by Okumo et al. (21). These investigators reported that microinjection of 3 nmol of atropine into the dorsal vagal complex does not antagonize the excitatory effects of a subsequent microinjection of 5 pmol TRH into the same area. Our experiments do not address the question as to the neurotransmitter and/or neuromodulatory role of TRH in the control of DMV motoneurons. Instead, we focused on confirming the findings of Raggenbass et al. (23) and on determining the mechanism(s) whereby TRH induces an excitatory effect on DMV motoneurons. As did Raggenbass et al., we have obtained proof that TRH acts directly on DMV motoneurons to produce excitation. We have shown that there are several mechanisms responsible for this excitatory effect. They include an antagonism of 1A and IAHP, as well as an increase in a cationic current. In addition, TRH-induced increases in ESPCs may contribute to the excitatory effect of TRH on DMV motoneurons. In terms of site of action of TRH on DMV motoneurons, Rinaman et al. (26) have demonstrated that TRH-like immunoreactive presynaptic terminals in the DMV of the rat establish contacts with dendrites. Hence we postulate that TRH may be exerting its postsynaptic effect on dendrites, suggesting a neuromod-
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MOTONEURONS
ulatory role for the peptide. In addition, Rinaman and Miselis (25) have reported that 40% of the TRH immunoreactivity in the rat dorsal vagal complex is concentrated in nonsynaptic axon varicosities. This finding also fits with the notion for a neuromodulatory role of TRH on DMV motoneurons. Rinaman and co-workers also make the point that TRH-immunoreactive presynaptic terminals establish both asymmetric (i.e., excitatory) and symmetric (i.e., inhibitory) synaptic contacts with dendrites. In our study, we only observed excitatory effects of TRH; inhibitory effects were never observed. This fits with the existence of asymmetric synaptic contacts. Finally, one important source of synaptic TRH is the medullary raphe (22). Hence an important series of experiments that need to be performed is to examine the effect of raphe stimulation on the firing rate of DMV motoneurons and assess the role of evoked release of TRH in the response. We express our thanks to Vivian F. Carter and F. Holly Travagli for assistance in the preparation of this manuscript. This work was supported by National Institutes of Health Grant POl-NS-28130. Preliminary data of this work has appeared in abstract form in Neurosci. Abstr. 17: 1099, 1991. Address for reprint requests: R. A. Travagli, F.G.I.N.-Pharmacology, Georgetown Univ. School of Medicine, 3900 Reservoir Rd. NW, Washington, DC 20007. Received
21 October
1991; accepted
in final
form
6 April
1992.
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Hornby, P. J., C. Rossiter, S. V. Pineo, K. Friedman, S. Benjamin, and R. A. releasing hormone: distribution in the vagal physiological effects upon microinjection.
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(Gastrointest. Liver Physiol. 20): G454-G462, 1989. Ilto, C., A. Fukuda, J. Nabekura, and Y. Oomura. Acetylcholine causes nicotinic depolarization in rat dorsal motor nucleus of the vagus, in vitro. Brain Res. 503: 44-48, 1989. 15. Katz, B., and R. Miledi. The timing of calcium action during neuromuscular transmission. J. Physiol. Lond. 189: 535-544, 1967. 16. Lacey, G., A. Nistri, and E. R. Rhys-Maitland. Large enhancement of excitatory postsynaptic potentials and currents by thyrotropin-releasing hormone (TRH) in frog spinal motoneurons. Brain Res. 488: 80-88, 1989. 17. McCann, M. J., and R. C. Rogers. Thyrotropin-releasing hormone effects on identified neurons of the dorsal vagal complex. J.
causes direct excitation of dorsal vagal and solitary tract neurones in rat brain stem slices. Bruin Res. 530: 85-90, 1990. 24. Rekling, J. C. Excitatory effects of thyrotropin-releasing hormone (TRH) in hypoglossal motoneurons. Bruin Res. 510: 175179, 1990. 25.
Rinaman, L., and R. R. Miselis. Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on dendrites of gastric vagal motoneurons in the rat. J. Comp. Neurol. 294: 235-
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Rinaman, L., R. R. Miselis, and M. S. Kreider. Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci. Lett. 104: 7-12,
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Rossiter, C. D., W. P. Norman, M. Jain, P. J. Hornby, S. Benjamin, and R. A. Gillis. Control of lower esophageal sphincter pressure by two sites in dorsal motor nucleus of the vagus. Am.
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Nistri, A., N. D. Fisher, and M. Gurnell. Block by the neuropeptide TRH of an apparently novel K+ conductance of rat motoneurons. Neurosci. Lett. 120: 25-30, 1990. 21. Okumo, Y., Y. Asumi, T. Ishikawa, and T. Mitsuma. Enhancement of gastric acid output and mucosal blood flow by tripeptide thyrotropin releasing hormone microinjected into the dorsal motor nucleus of the vagus in rats. J. Pharmacol. 43: 173-178, 20.
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Palkovitz, M., E. Mezey, R. L. Eskay, and M. J. Brownstein. Innervation of the nucleus of the solitary tract and dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons. Brain Res. 373: 246-251, 1986. 23. Raggenbass, M., C. Vozzi, E. Tribollet, M. Dubois-Dauphin, and J. J. Dreifuss. Thyrotropin-releasing hormone
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Sah, P., and E. M. McLachlan. Ca++-activated K+ currents underlying the afterhyperpolarization in the guinea pig vagal neurons: a role for Ca++-activated Ca++ release. Neuron 7: 257-264, 1991.
29. Tache, Y., R. L. Stephens, Jr., and T. Ishikawa. Central nervous system action of TRH to influence gastrointestinal function and ulceration. Ann. NY Acad. Sci. 533: 269-285, 1989. 30. Tache, Y., and H. Yang. Brain regulation of gastric secretion by peptides. Site and mechanism of action. Ann. NY Acad. Sci. 597: 128-145,
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Takahashi, T. Thyrotropin-releasing hormone mimics descending slow synaptic potentials in rat spinal motoneurons. Proc. R.
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Wang, M. Y., and N. Y. Dun. Direct and indirect actions of thyrotropin-releasing hormone on neonatal rat motoneurons in vitro. Neurosci. Lett. 113: 349-354, 1990.
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THERE
G508
0193~1857/92
$2.00
Copyright
occlusion of the pylorus significantly decreased gastric acid secretion when compared with rats treated with normal rabbit serum. These findings indicate that endogenous brain TRH plays a physiological role in the regulation of gastric acid secretion. In a follow-up study, Hernandez and Emerick (11) reported that microinjection of TRH into the dorsal motor nucleus of the vagus of rats increased gas tric acid output and gastric mw ulcers. These investigators concluded that presynaptic “TRH fibers may modulate vagal activity at the level of the dorsal motor nucleus of the vagus and propose(d) that descending TRH pathways may play a role in experimental ulcerogenesis through acid hypersecretion.” Evidence that TRH excites DMV motoneurons in vivo in rats has been obtained by McCann and Rogers (17). These investigators identified electrophysiologitally individual DMV units by using stimulating electrodes placed on the cervical vagus. TRH was then tested on these DMV motoneurons by pressure injection of lo-40 fmol in IO-40 pl. Thirteen cells were identified, and of these, six (or 46%) were activated by TRH. Activation was reflected by an increase in the firing rate. The remaining seven DMV motoneurons were not affected by TRH; none of the DMV motoneurons were inhibited. Evidence that TRH excites DMV motoneurons in vitro in rats has been obtained by Raggenbass et al. (23). These investigators studied TRH using extracellular single-unit recordings from brain stem slices of the rat and reported that about one-third of the vagal nerves were excited by TRH. This action of TRH was present in a low-calcium, high-magnesium solution, which blocks synaptic transmission, thus suggesting that TRH was acting directly on DMV neurons. We have recently begun in vitro studies using whole cell current and voltage-clamp recordings from anatomically identified DMV neurons in rat brain stem slices (32). Our results indicate that these neurons are capable of sustained slow-frequency action potential firing probably because of the presence of a pacemaker current. Our results also indicate the presence of excitatory and inhibitory spontaneous and evoked synaptic currents in the DMV. The excitatory synaptic currents are due to activation of ionotropic glutamate receptors, and the inhibitory synaptic currents are mediated by y-aminobutyric acid (GAB A). The purpose of the present study was to use this rat brain stem slice preparation to examine the electrophysiological effects of the putative excitatory neurotransmitter at the DMV, TRH. Our goal was to confirm the findings of Raggenbass et al. (23) that TRH acts directly to increase the firing rate of these cells, and if confirmed, to determine the mechanism(s) responsible for a direct excitatory effect of TRH. An additional goal was to
0 1992 the American
Physiological
Society
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EFFECTS
determine whether TRH induces release of glutamate, and if so, to determine whether glutamate contributes to the excitatory effect of TRH on DMV motoneurons. METHODS Brain stem slice preparation. We prepared the slices for patch-clamp recording as previously described (7, 32). Briefly, young Sprague-Dawleyrats 2-3 wk of agewere decapitated, and the brain stem was quickly removed and placed in ice-cold oxygenated physiological rat Ringer solution after excision of the cerebellum.With the useof an Oxford vibratome, 200-km-thick coronal sliceswere obtained from the medulla, both caudally and rostrally to the obex. The sliceswerethen incubated at 37°C in oxygenated physiological rat Ringer solution until used. The experimental studieswith TRH were carried out at room temperature (22-25°C). Perfusion andpatchpipette solutions and drugs. Physiological rat Ringer wascomposedof (in mM) 120 NaCl, 26 NaHCOs, 3.1 KCl, 1 MgC12, 2 CaCl,, and 5 dextrose. The bathing medium was maintained at pH 7.4 with 95% O&j% CO, bubbling. During cutting proceduresand incubation periods the Ringer solution wasmodified so that it contained 2 mM MgC12and 1 mM CaCl,. The purposefor this was to reduce glutamate-mediated neurotoxicity through excessstimulation of the N-methyl-Daspartic acid (NMDA) receptor. Cell-attached recordings of visually identified DMV neurons were performed with patch pipettes filled with Ringer solution, while whole cell recordings were performed with patch pipettes filled with (in mM) 145 potassiumgluconate, 1 MgCl,, 0.5 ethylene glycol-bis@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2 ATP, 0.65 GTP, and 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-KOH (pH 7.2). Drugs were addedby perfusion in the bathing medium (5 ml/min) via a system of valves controlled by a manually operated switch, the drugstaking between 40 and 60 s to equilibrate at the final concentration in the perfusion chamber. Acetylcholine, 4-aminopyridine, apamin, atropine, glutamic acid, kynurenic acid, and tetrodotoxin (TTX) were purchased from Sigma (St. Louis, MO); TRH (pGlu-His-Pro-NH,) waspurchasedfrom Peninsula (Belmont, CA). All drugsusedwere freshly preparedand dissolveddirectly in Ringer solution at the neededconcentration. Electrophysiological recordings. By means of the tight-seal whole cell recording method of Hamill et al. (lo), we measured potentials and currents from the DMV neurons. Currents and voltage signalsat the headstageof the patch-clamp amplifier (Axopatch lD, Axon Instruments, Foster City, CA) were filtered and continuously displayed on an oscilloscope.Current and voltage steps were evoked via the patch-clamp amplifier, while a double-channel stimulator (model S88 Grass Instruments, Quincy, MA) was used to control the frequency and duration of the stimuli. The pClamp data acquisition and analysis software (version 5.5.1, Axon) run on an IBM-AT personal computer wasusedfor the voltage-clamp protocols that required more complex procedures. Data collection and analysis. Data were recorded with a VCR magnetic tape (VR 10-A, Instrutech, Elmont, NY) for later
TRH 30 FM
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analysis. The amplitudes of the currents and voltages evoked were analyzed with a Gould DSO-405 oscilloscope(Gould, Ilford, Essex,UK) or by using the pClamp analysis software with appropriate modifications. Results are expressedas means& SE; t test for paired data was used, with P < 0.05 being the criterion for statistical significance. RESULTS
Effects of TRH on spontaneous firing rate of DMV neurons. In our initial electrophysiological study of DMV
neurons in isolated brain slices of lo- to 18-day-old rats, we employed whole-cell current-clamp recordings from anatomically identified DMV neurons and showed that these neurons are capable of sustained slow-frequency action potential firing, probably because of the presence of a pacemaker current (32). In the present study using the whole cell patch-clamp recording technique, and recording extracellular activity in the cell-attached recording configuration, we again observed that the vast majority of these neurons exhibit a spontaneous discharge. Baseline activity of DMV neurons as recorded in the cell-attached configuration ranged from 0.1 to 4.3 spikes/s and an example of their activity before exposure to TRH can be seen on the control portion of Fig. 1. This activity was recorded from 85 DMV neurons, and on exposure to TRH in concentrations ranging from 1 to 100 PM, 63 (or 75%) of the neurons exhibited an increase in spontaneous firing rate. In the 22 remaining DMV neurons, TRH had no effect. In no case did TRH produce a decrease in the spontaneous firing rate. In earlier studies, we reported that stimulation of sites in the DMV in vivo produced opposite effects on lower esophageal sphincter (LES) pressure. That is, stimulation of the DMV rostra1 to the obex elicits a rise in LES pressure, whereas stimulation of the DMV caudal to the obex elicits a fall in LES pressure (27). Hence, we examined whether TRH would exert a different effect on rostral DMV neurons vs. caudal DMV neurons. Thirty-three of the 85 DMV neurons referred to above were located rostra1 to obex, and exposure to TRH elicited only an excitatory effect (i.e., 25 of 33, or 76% exhibited an in-
crease in spontaneous firing). Fifty-two of the 85 DMV neurons were located caudal to the obex, and exposure to TRH elicited only an excitatory effect (i.e., 38 of 52 or 73% exhibited an increase in spontaneous firing). An experiment illustrating the excitatory effect of TRH on a DMV neuron appears as Fig. 1. As can be noted, adding TRH to the perfusion solution at a concentration of 30 PM produced an almost immediate (within 1 min) increase in the spontaneous firing rate. The increase became maximal at -30 s after TRH, and began to dissipate soon after washing TRH from the system. The
--A 10 see
1SOpA
Fig. 1. Effect of thyrotropin-releasing hormone (TRH) on firing of a vagal neuron using patch-clamp recording in extracellular cell-attached confirmration. TRH was added to nerfusion solution in a concentration of 30 uM and for the time corresponding to the horizontal line below the recording. Frequency of action potential firing increased from 0.55 to 1.43 spikes/s. Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 10, 2019.
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TRH effect was reversible. That is, soon after washout, the spontaneous firing rate of the DMV neuron returned to its baseline firing rate. The TRH-induced excitation appeared to be concentration related in terms of peak firing rate, and this is shown in the summarized data tabulated in Table 1. The lowest effective TRH concentration was between 1 and 3 PM. As the concentration of drug used was increased in approximate threefold steps, the percent increase in firing rate increased from approximately two- to eightfold. No indication of a “plateau effect” was indicated over the range of concentrations of TRH used. Interestingly, desensitization occurred to the excitatory effect of TRH. Initially, TRH administration produced a striking increase in the spontaneous firing of DMV neurons. However, after the initial large increase in firing rate was obtained, the firing rate began to decline and fall to a rate about halfway between the initial rate and the maximal rate obtained with TRH, even though there was continuous perfusion of the DMV neuron with TRH. The decrease in firing rate appeared -4-5 min after the peak effect of TRH was achieved, and the new firing rate stabilized at values significantly lower than the peak values (decrease from a peak firing rate of 3.5 + 0.95 to 1.8 + 0.76 spikes/s; P < 9.05) but still higher than the control values (0.6 & 0.02 spikes/s) (data obtained from 3 cells in which the phenomenon of “desensitization”;using 30 PM of TRH was carefully analyzed). After washbut of TRH and a return of the firing rate to a predrug rate, reexposure of the DMV neuron to TRH within 20 min of initial exposure did not result in the same response to TRH. That is, the response obtained on second exposure to TRH was always less than the initial response, thus showing sustained desensitization. This was noted in six DMV neurons studied. However, if the interval between initial exposure and reexposure was >20 min, TRH did produce the same response as noted with the initial exposure of the peptide (n = 5). To characterize the TRH-induced desensitization even further, additional studies were performed using potassium as a depolarizing agent. When potassium (9 mM final concn) was added to the perfusing fluid (in the form of KCl) at the time when desensitization had occurred with 30 FM TRH, potassium was found to increase the Table 1. Effect of TRH on spontaneous firing rate of DMV neurons using patch-clamp recording technique and recording either extracellular activity in cell-attached configuration or intracellular activity in current-clamp configuration
spontaneous rate of the cell to the same degree as it did when delivered to the DMV neuron not exposed to TRH (n = 13). In addition, repeated exposure of the DMV neuron to potassium at intervals 0.05) (n = 5). Finally, 30 PM TRH was added to the perfusion solution in the presence of kynurenic acid (1 mM) and elicited the usual increase in spontaneous firing rate (+140 t 30%; P < 0.05) (n = 5). For the studies on acetylcholine, the same experimental protocol was performed using 1 PM of acetylcholine. This neurotransmitter produced a 90.8 t 40.8% increase in spontaneous firing rate (n = 6), and atropine (1 PM), added to the perfusing solution, abolished the acetylcholine response (n = 4). Perfusion of DMV neurons with TRH (30 PM) in the presence of atropine (1 PM) elicited an increase in spontaneous firing rate (+227.3 t 104.8%, n =: 7) Using the whole cell recording technique in the voltageclamp mode, we observed that addition of TRH (30 PM) to the perfusing solution produced an increase in spontaneously occurring excitatory postsynaptic currents (soEPSCs). In fact, soEPSCs frequency increased 178 t 30.8% from a value of 0.55 t 0.11 to 1.45 t 0.13 Hz (n = 5). This occurred without any significant change in the amplitude of the soEPSCs in three of the five neurons tested. In the remaining two neurons, an assessment of the soEPSCs amplitude was not possible to determine because of the high baseline noise compared with the small size of the event. In contrast, addition of TRH (30 PM) to the perfusing solution did not appear to alter the frequency of spontaneously occurring inhibitory postsynaptic currents (soIPSCS). This was observed in four neurons. No discernible effect on SOIPSCS amplitude was observed. A representative experiment illustrating this effect of TRH appears as Fig. 8.
J‘2000pAv m 500 msec Fig. 7. Effect of TRH on Ca 2+-dependent afterhyperpolarization due to activation of an I AHp in a vagal neuron using whole cell patch clamp in current-clamp mode. Neuron was hyperpolarized at -50 mV by a steady injection of current and then subjected to several l-s-long depolarizing stimuli of increasing intensities. This resulted in the occurrence of an afterhyperpolarizing potential. Recording in top left panel is control; recording in top right panel was taken during TRH addition to perfusing solution in a concentration of 30 PM. It can be noted that TRH decreased afterhyperpolarization despite increase in number of action potentials induced during depolarization. Recording in bottom left panel was taken during addition of 100 nM apamin. It can be noted that apamin completely inhibited afterhyperpolarization and dramatically increased number of action potentials induced by depolarizing current. Recording in bottom right panel was taken from a different cell perfused in Ringer physiological solution in which Ca2+ was substituted with cobalt (final concn 2 mM). It can be noted that perfusion with cobalt completely inhibited afterhyperpolarization as well as occurrence of action potentials induced by injection of depolarizing current.
Fig. 7. This recording was made from a neuron different from the one exposed to TRH (and apamin). The neuron was perfused with a physiological solution in which cobalt (2 mM) was substituted for calcium. As can be noted, cobalt completely inhibited the afterhyperpolarization after injection of depolarizing current. Cobalt also reduced the number of action potentials that normally occur after injection of depolarizing current. This finding with cobalt was obtained in three DMV neurons tested. As a further test of whether we were evaluating TRH against IAHP, we studied the effect of apamin (100 nM) on the afterhyperpolarization. Three DMV neurons were evaluated, and in each case, exposure of the neuron to apamin completely abolished the afterhyperpolarization. An example of the effect of apamin can be seen in the bottom left panel of Fig. 7. Studies of effect of TRH on synaptic transmission. Using the extracellular recording technique in the cell-attached configuration, we investigated whether any part of TRHinduced increase in spontaneous firing rate was due to an increase in the synaptic release of either L-glutamate or acetylcholine. We chose to look at L-glutamate release because this excitatory amino acid had been shown in our previous study to be one of the major neurotransmitters controlling the activity of DMV neurons (32). We chose to look at acetylcholine release because studies by other investigators have shown that 1) TRH releasesthis neurotransmitter from central nervous system (CNS) tissue
DISCUSSION
In a previous study, Raggenbass et al. (23) used extracellular single-unit recordings in brain stem slices and evaluated the effect of TRH on neurons in the DMV. In 365 neurons studied, 105 or 29% exhibited an increase in firing rate; no effect was noted in the remaining 260 neurons. An excitatory effect was evident with a TRH concentration of -10 nM, and a half-maximal effect occurred with a TRH concentration of -100 nM. In addition, the excitatory effect of TRH was present in a low-calcium (0.2 mM CaC12vs. 1.25 mM CaC12)and highmagnesium (6 mM MgS04 vs. 1 mM MgSO,j solution, which blocks synaptic transmission. In the present study, we confirmed the findings of Raggenbass et al. (23) by recording extracellular activity in the cell-attached configuration and adding TRH to the brain stem slice perfusing solution. In general, our results are qualitatively
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EFFECTS
OF TRH
ON DMV
JOPA msec
200
Fig. 8. Effect of TRH on EPSCs (and IPSCs) in a vagal neuron using whole cell patch-clamp recording in voltage-clamp mode. Neuron was voltage clamped at -50 mV, and spontaneous miniature EPSCs and spontaneous miniature IPSCs were observed as inward (downward) and outward (upward) currents, respectively. A: recordings obtained during control period. B: recordings taken during TRH addition to perfusion solution in a concentration of 30 PM.
identical to the results of Raggenbass et al., but quantitatively, we observed a higher proportion of DMV neurons (i.e., 75%) excited by TRH. On the other hand, we found that a higher concentration of TRH was required to elicit an effect in our study. A possible reason(s) for these quantitative differences is that we studied brain stem slices from lo- to l&day-old rats and at a temperature of 2225”C, whereas Raggenbass et al. studied brain stem slices from young adult rats and at a temperature of 34-35OC. One qualitative difference between the two studies is that we observed desensitization to the excitatory effect of TRH, whereas desensitization was not observed in the Raggenbass et al. study. In general, desensitization to TRH effects on neural tissue is the more common finding (18, 20, 24). Once documenting that TRH acts to increase the firing rate of DMV neurons [thus confirming Raggenbass et al. (23)], we sought to determine the mechanisms(s) whereby TRH induces DMV neurons to discharge more frequently. In pursuing this goal, we first observed that TRH produces a depolarizing action on DMV motoneurons. This was documented in our studies using whole cell recording in the current-clamp mode. We found that TRH, in addition to increasing the spontaneous firing rate of DMV neurons using this recording technique, also produced depolarization. The TRH-induced depolarization, like the TRH-induced increase in firing rate, exhibited desensitization, suggesting that TRH-induced depolarization may have caused the increase in DMV motoneuron firing. An interesting and unexplained finding was the inability of large concentrations of TRH (e.g., 30 and 100 PM) to elicit large increases in firing rate of DMV neurons studied using whole cell recordings in the current-clamp mode (Table 1). One possible reason for this is that TRH in high concentrations increases electrical excitability through an intracellular second messenger, and Drummond (6) indeed has demonstrated that TRH induces changes in phosphoinositide metabolism and intracellular calcium concentration. Whole cell patch-clamp recording could result in intracellular dialysis of putative
MOTONEURONS
G515
second messenger components (2) and thereby interfere with TRH from evoking the large response observed in the cell-attached recording configuration. In the next series of experiments using whole cell recordings in the voltage-clamp mode, we demonstrated that TRH induces a small inward current that is associated with a decrease in the Rinp. This inward current was also induced by TRH in brain slices perfused with a medium containing TTX and cobalt (which caused an almost total inhibition of synaptic activity), suggesting that TRH is exerting its effect directly on the postsynaptic membrane of the DMV motoneuron, probably by activating an inward cationic current. Our results are similar to earlier findings by Nicoll (18, 19), who studied TRH on frog spinal motoneurons in vitro. Nicoll reported that TRH produced a direct depolarizing action on motoneurons, and this direct effect was accompanied by a fall in membrane resistance. He postulated that TRH was acting directly on the motoneuron to increase inward sodium current. Most investigators who have studied TRH on motoneurons and observed TRH-induced depolarization have noted that it was accompanied by either an increase in Ri,, or no change in Rinp (3, 16, 20, 24, 31, 33). The increase in Rinp was, in each case, postulated to be due to TRH- induced closure of potassium channels. We also obtained evidence that TRH will counteract potassium conductance. For example, we found that TRH decreases the amplitude of a calcium-dependent hyperpolarizing current as well as the fast transient Atype potassium current. In the latter case, the effect of TRH was not exerted by shifting the activation or the inactivation potential of the I*. Reductions in both of these currents could contribute to TRH-induced increase in firing of DMV neurons. Findings obtained with TRH indicating an increase in inward cationic current (and depolarization) and antagonism of 1A and I AHp in DMV neurons bathed in solution containing cobalt and TTX reflect an action of TRH at postsynaptic sites of the synapses in the DMV. In addition, we have also obtained evidence that TRH exerts an excitatory effect at a presynaptic site. For example, our data indicating that TRH increases the frequency of EPSCs suggest a presynaptic site of action of the peptide. Whether the site of action of TRH was on nerve terminals synapsing on DMV motoneurons or on perikarya of interneurons and/or projections from other brain stem nuclei in our slice preparation was not delineated in our study. Rekling (24) also reported that TRH increases the frequency of EPSPs in rat hypoglossal motoneurons. Similar to our findings, Rekling did not observe any change in the amplitude of the EPSPs. Lacey et al. (16), who studied TRH effects on frog motoneurons in vitro, reported that TRH increases both the frequency of firing and the amplitude of EPSPs, and concluded that the primary site of action of TRH on motoneurons was presynaptic. Similarly, Behbehani et al. (3), using cultured spinal cord neurons from the mouse, observed TRH-induced increases in both the frequency of firing and the amplitude
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of EPSPs. These investigators also concluded that TRHinduced excitation of motoneurons is due to a presynaptic site of action of the peptide. In terms of TRH effects of IPSPs and IPSCs, we did not examine this question in a thorough manner, but the preliminary data we have obtained indicate that TRH does not alter the frequency (or amplitude) of IPSCs. Similarly, Takahashi (31), who studied rat spinal motoneurons in vitro, did not observe any striking effects on IPSPs. It is clear from our previous study (32) of DMV motoneurons in vitro that endogenously occurring excitatory and inhibitory amino acids are the major neurotransmitters controlling the excitability of DMV motoneurons in the brain stem slice preparation. Because TRH-produced excitation of motoneurons in our preparation, we focused on the possibility that part of the excitatory effects of TRH was due to an increase in the presynaptic release of glutamate. Indeed, Lacey et al. (16) interpreted their data on TRH-induced excitation of lumbar spinal motoneurons of frog as caused by TRH enhancement of presynaptic release of glutamate. We tested this notion in our preparation by using the excitatory amino acid antagonist kynurenic acid, and found that a concentration of kynurenic acid that fully blocked the excitatory effect of exogenously administered glutamate on DMV motoneurons had no effect on TRH-induced increase in cell firing rate. Hence our data indicate that the excitatory effect of TRH on DMV motoneurons is not mediated through enhanced presynaptic release of glutamate. In addition, we also studied the role of presynaptic release of acetylcholine in the excitatory response of TRH. Acetylcholine is known to be present in the DMV and has been shown to excite DMV motoneurons (14). We found that atropine in a concentration that blocks the excitatory effect of exogenously administered acetylcholine has no effect on TRH-induced increase in firing rate. This finding is consistent with earlier results obtained in the rat in vivo by Okumo et al. (21). These investigators reported that microinjection of 3 nmol of atropine into the dorsal vagal complex does not antagonize the excitatory effects of a subsequent microinjection of 5 pmol TRH into the same area. Our experiments do not address the question as to the neurotransmitter and/or neuromodulatory role of TRH in the control of DMV motoneurons. Instead, we focused on confirming the findings of Raggenbass et al. (23) and on determining the mechanism(s) whereby TRH induces an excitatory effect on DMV motoneurons. As did Raggenbass et al., we have obtained proof that TRH acts directly on DMV motoneurons to produce excitation. We have shown that there are several mechanisms responsible for this excitatory effect. They include an antagonism of 1A and IAHP, as well as an increase in a cationic current. In addition, TRH-induced increases in ESPCs may contribute to the excitatory effect of TRH on DMV motoneurons. In terms of site of action of TRH on DMV motoneurons, Rinaman et al. (26) have demonstrated that TRH-like immunoreactive presynaptic terminals in the DMV of the rat establish contacts with dendrites. Hence we postulate that TRH may be exerting its postsynaptic effect on dendrites, suggesting a neuromod-
DMV
MOTONEURONS
ulatory role for the peptide. In addition, Rinaman and Miselis (25) have reported that 40% of the TRH immunoreactivity in the rat dorsal vagal complex is concentrated in nonsynaptic axon varicosities. This finding also fits with the notion for a neuromodulatory role of TRH on DMV motoneurons. Rinaman and co-workers also make the point that TRH-immunoreactive presynaptic terminals establish both asymmetric (i.e., excitatory) and symmetric (i.e., inhibitory) synaptic contacts with dendrites. In our study, we only observed excitatory effects of TRH; inhibitory effects were never observed. This fits with the existence of asymmetric synaptic contacts. Finally, one important source of synaptic TRH is the medullary raphe (22). Hence an important series of experiments that need to be performed is to examine the effect of raphe stimulation on the firing rate of DMV motoneurons and assess the role of evoked release of TRH in the response. We express our thanks to Vivian F. Carter and F. Holly Travagli for assistance in the preparation of this manuscript. This work was supported by National Institutes of Health Grant POl-NS-28130. Preliminary data of this work has appeared in abstract form in Neurosci. Abstr. 17: 1099, 1991. Address for reprint requests: R. A. Travagli, F.G.I.N.-Pharmacology, Georgetown Univ. School of Medicine, 3900 Reservoir Rd. NW, Washington, DC 20007. Received
21 October
1991; accepted
in final
form
6 April
1992.
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