Brain Research, 510 (1990) 175-179

175

Elsevier BRES 23990

Excitatory effects of thyrotropin-releasing hormone (TRH) in hypoglossal motoneurons J.C.

Rekling

Institute of Neurophysiology, Copenhagen University, Copenhagen (Denmark) (Accepted 21 November 1989)

Key words: Thyrotropin-releasing hormone; Hypoglossal motoneuron; Excitatory postsynaptic potential; Guinea pig

The effect of thyrotropin-releasing hormone (TRH) was studied in 30 hypoglossal motoneurons from brainstem slices of guinea pigs. Bath application of TRH resulted in an increase of the spontaneous excitatory synaptic activity, depolarization of the neurons, increase of the input resistance and change of the duration of the falling phase of excitatory postsynaptic potentials. The depolarizing response and membrane conductance change was the result of a direct postsynaptie action of TRH, possibly mediated by a reduction of a potassium conductance.

Recently the extrapituitary effects of thyrotropinreleasing hormone (TRH) has received increasing attention. Apart from its hypophysiotrophic action, central and local administration of the tripeptide has been shown to affect the autonomic nervous system, resulting in a variety of peripheral autonomic actions 4. In addition, T R H has potent actions on the motor system resulting in increased motor activity 1'6 and increased reflex discharges 2'14. The effects of T R H on the motor system has partly been explained by a direct action of T R H on spinal motoneurons 12'15 and facilitation of presynaptic release of transmitters s. The anatomical distribution of extrahypothalamic T R H also includes several brainstem nuclei, including the hypoglossal nucleus, where the native hormone, immunohistochemicaily characterized (TRH)-containing nerve terminals and T R H binding sites have been found 5"7"9"1°. These findings suggest that T R H also has a function in cranial nerve motor nuclei and this study focuses on the effect of T R H on hypoglossal motoneurons, using intracellular recordings in current-clamp mode. Experiments were performed on acute brain slices obtained from the brainstem of guinea pigs as previously described li. Albino guinea pigs of either sex weighing 250-350 g were deeply anesthetized by intraperitoneal pentobarbital (50 mg/kg) and killed with a blow to the thorax with a metal rod. A block containing the cerebellum and the brainstem was dissected out and 3-4 frontal sections 400/~ thick were cut on a vibratome at the level of the obex. Slices were immediately transferred to a recording chamber of the interface type. It had a

volume of 2 ml, a temperature of 36.5 °C and was constantly perfused at a rate of 2 ml/min with preheated, oxygenated Ringer's solution composed of 134 mM NaCI, 2 mM KCi, 20 mM NaHCO3, 1.25 mM KH2PO4, 2 mM CaCI 2, 2 mM MgCI 2 and 9 mM glucose. The solutions were bubbled with a gas mixture composed of 95% 02, 5% CO 2, which also filled the space above the medium. Hypoglossal motoneurons were penetrated by glass microelectrodes, filled with a 2 M solution of potassium acetate, having resistances of between 30 and 60 MD. A bridge-balance amplifier (built in the laboratory) with a cut-off frequency of 8 kHz was used. The recorded signals were stored on a 4-track tape recorder with a band width of DC to 5 kHz. Off-line analysis was done using a personal computer, digitizing the signals. Current and voltage traces were plotted on a laser printer. Electrical stimulation was done using two 50 ktm thick platinum wires spaced 100/~m, applying rectangular pulses lasting 100 ~s at voltages of 25-40 V. Drugs were applied to the superfusing solution except QX-314, which was applied from the recording pipette. T R H was obtained from Diagnostika, TTX from Sigma and QX-314 was a generous gift from Astra. The findings presented here are based on recordings from 30 hypoglossai motoneurons. The response to T R H applied to the superfusing solution at a concentration of 50/~M is shown in Fig. 1A. Two to 3 min after perfusing with a T R H containing Ringer's solution the membrane potential of the hypoglossal motoneurons depolarized. All cells tested with T R H displayed depolarization and at a concentration of 50/~M the maximal depolarization was

Correspondence: J.C. Rekling, Institute of Neurophysiology, Copenhagen University, Blegdamsvej 3C, DK-2200, Copenhagen N., Denmark. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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TRH (50,pM) Fig. 1. Effect of TRH on membrane potential and input resistance in hypoglossal motoneurons. A: bath application of TRH (50 MM), resulting in depolarization and spiking. Note the desensitization of the response. B: superfusion with TRH (50 MM) applied to a Ringer solution containing TFX (1 #M) and cobalt. (Co 2÷ substituted for Ca ~~). Hyperpolarizing pulses were applied to monitor the input resistance, which is plotted above the trace, expressed in arbitrary units. Note the increase in R~np, particularly prominent during the depolarizing phase.

177 6.6 +__ 2.3 mV (mean + S.D., n = 13). During the depolarizing phase trains of EPSPs often occurred. At a concentration of 50 ,uM T R H the depolarization always resulted in spiking of the motoneurons. This is in contrast to the findings of Nicoll et al. 13, who found that frog spinal motoneurons depolarized, but never fired action potentials in response to bath application of 1 mM T R H . A consistent finding in the response to T R H was that of desensitization. All the effects of T R H reported in this study were maximal 3-4 min after entering T R H , but decreased thereafter. This p h e n o m e n o n can be appreciated in Fig. 1A, showing the decreasing firing rate and repolarization of the membrane potential during continued superfusion with T R H . Re-entering a T R H containing solution after 2 h of wash did not result in the same response to T R H , thus showing the sustained phenomenon of desensitization. One possible biochemical explanation of the p h e n o m e n o n of desensitization is a T R H induced conversion of the T R H receptor from a kinetically fast dissociating complex to a slowly dissociating complex 3 . Fig. 1B illustrates the response to T R H in a motoneu-

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rone bathed in a solution containing T T X (1 pM) and cobalt (Ca 2+ in the perfusing solution was replaced with Co 2÷ reaching a concentration of 2 mM). Hyperpolarizing pulses lasting 1 s were used to monitor the input resistance (Rinp). The experiment showed that the depolarization induced by T R H persists after synaptic input to the motoneurone has been blocked and that the depolarization is probably not carried by calcium ions. The input resistance of the cell is increased during the depolarization, which is in agreement with findings in lumbar motoneurons of neonatal rats 15, In 5 cells bathed in a T T X containing Ringer solution the input resistance was measured (a) before 50 p M T R H , (b) during the maximal depolarization and (c) shortly, at the time of maximal depolarization, at the control membrane potential using hyperpolarizing bias. The average input resistance was (a): 4.3 +_ 1.8 Mg2, (b): 7,1 +__2.0 Mr2 and (c): 6.6 + 2.0 Mr2 (mean + S.D., n = 5). The effect of T R H on cell input resistance was studied further using a stimulation current varying as a double ramp (Fig. 2A,B), at a rate so low, that the current-voltage plot corresponds to steady state (0.1 Hz). A current-voltage

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Fig. 2. I - V relationship studied with ramp stimuli and pulses. A: double ramp stimulation (0.1 Hz) of a hypoglossal motoneurone bathed in a TTX (1 pM) containing Ringer solution, r.p. = resting potential. B: ramp stimulation after addition of TRH (50 pM). C: current-voltage plot with current plotted against membrane potential - - data from A,B . Note the change of the slope of the I - V curve in response to TRH. D: depolarizing and hyperpolarizing pulse given from resting membrane potential. E: depolarizing and hyperpolarizing pulse of the same strength as in D, given from the same membrane potential (- 0.3 nA bias current), after superfusion with TRH (50 pM). F: I - V curve before (open dot) and during (filled dot) TRH (50 pM). The curve is constructed using 100 ms pulses, measuring membrane potential as voltage had relaxed (top left corner of figure, calibrations as in Fig. 2E). Linear regression of the lower segment of the two curves showed intersection at - 85.7 mV (arrow).

178 curve was generated by plotting current versus absolute m e m b r a n e potential (Fig. 2C). The I - V curve thus constructed, moved in the depolarizing direction when T R H (50 ~tM) was introduced to the superfusing solution (the test was made 3 min after entering TRH). The slope of the curve was increased dramatically indicating an increase in input resistance. The slope however decreased positive to - 3 0 mV indicating an abrupt resistance decrease. As previously shown this is probably due to activation of a Ca2+-dependent K ÷ conductance and not an effect of T R H H. Monitoring of the electrode balance during the ramp stimulation in this experiment, revealed a small electrode rectification in the hyperpolarizing direction. Thus the experiment was repeated using hyperpolarizing pulses instead of double ramp stimulation (Fig. 2F), because the bridge-balance could be adjusted continuously. Measurements were made as the voltage had relaxed to a steady state (top left, Fig. 2F). Linear regression performed on the data representing the hyperpolarizing pulses showed that the two curves intersected at - 85.7 mV (arrow), pointing to the hypothesis

that the response to T R H is mediate by a reduction of a K + conductance, as originally suggested by Takahasi L~. The effect of T R H on cell excitability is evident from Fig. 2D,E. When stimulated by depolarizing and hyperpolarizing pulses of constant strength the voltage response is increased in both directions, resulting in spikes and increased hyperpolarization. The cell was stimulated from the same membrane potential using hyperpolarizing bias current (- 0.3 hA, Fig. 2E). Recently Lacey et al. ~ reported large enhancement of the size of EPSPs in response to T R H in frog spinal motoneurons. These findings could not be replicated in hypoglossal motoneurons as shown in Fig. 3. Motoneurons were penetrated by electrodes containing QX-314 (200 mM) dissolved in 2 M potassium acetate, blocking sodium spikes, without affecting synaptic activation. Membrane potential was kept constant by hyperpolarizing bias current. Electrical stimulation with an electrode placed dorsolaterally to the hypoglossal nucleus elicited an EPSP and a short (50 ms) hyperpolarizing pulse was applied after the synaptic potential had relaxed, in order

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ls Fig. 3. Effect of TRH on synaptic potentials. A: average of 10 EPSPs elicited by electrical stimulation with an electrode placed dorsolaterally to the hypoglossal nucleus. The recording electrode contained 200 mM QX-314. A conductance measuring pulse was given after the EPSP had relaxed and the membrane potential was held constant using bias current. Note the change of the shape of the falling phase of the EPSP in response to TRH (40gM). B: average of 10 quadruple Stimulations (stimuli spaced 10 ms), generating summating EPSPs, before and during TRH (40 ~M). The recording electrode contained 200 mM QX-314. In the lower part of the figure the control record has been subtracted from the TRH record illustrating the net increase in cell excitability. C: spontaneous synaptic potentials in a hypoglossal motoneurone recorded with an electrode containing QX-314. D,E: recordings show the spontaneous synaptic activity during and after superfusion with TRH (40gM). Above the traces, plots of averaged (20 sweeps) EPSPs above 4 mV is shown. (Dotted line = normal Ringer). Note the increase of the number of EPSPs and the change of the shape of the EPSPs in response to TRH.

179 to m o n i t o r the input resistance. Superfusing with T R H (40 ~ M ) did not increase the amplitude of the EPSP, but p r o l o n g e d the falling phase of the EPSP (Fig. 3A). The average increase in decay time of the EPSPs (time from p e a k to resting potential) was 7.2 ms (control: 22.9 + 6.6 ms. T R H : 30.1 _+ 9.6 ms, m e a n _+ S . D . , n = 5). A n effect on the time course of synaptic potentials is to be e x p e c t e d , because an increase in input resistance also m e a n s an increase in m e m b r a n e time constant (r = R x C). Q u a d r u p l e stimulation (stim. s e p a r a t e d by 10 ms) p r o d u c e d 4 EPSPs displaying s u m m a t i o n (Fig. 3B). The 4 EPSPs were followed by a hyperpolarizing potential, which is p r o b a b l y the result of a previously described Q-like conductance in the hypoglossal m o t o n e u r o n s H and not s u m m a t i n g IPSPs. W h e n T R H (40 /~M) was a d d e d to the m e d i u m the prolongation of the falling phase of the EPSPs resulted in summation reaching a larger final a m p l i t u d e of the response and an increased net d e p o l a r i z a t i o n ( T R H - - Control, Fig. 3B). Thus the effect of T R H is a net increase in cell excitability when activated by repetitive synaptic activation, brought about by the change in input resistance. The effect of T R H on stimulus elicited EPSPs was confirmed on spontaneous EPSPs, which occasionally are seen in hypoglossal mo-

t o n e u r o n s (Fig. 3C). In the t o p traces of Fig. 3 A - C averages of 20 EPSPs a b o v e 4 m V are shown. The effect on the falling phase is evident (Fig. 3D) and in addition Fig. 3D shows the increase in synaptic b o m b a r d m e n t during T R H superfusion. In conclusion, the results p r e s e n t e d here d e m o n s t r a t e that T R H increases the excitability of hypoglossal motoneurons. T h e effect of T R H is m e d i a t e d by postsynaptic mechanisms resulting in d e p o l a r i z a t i o n , increased input resistance and change in the shape of EPSPs, possibly m e d i a t e d by a reduction of a potassium conductance. The increase in synaptic b o m b a r d m e n t on the m o t o n e u r o n s also suggests presynaptic actions of T R H in the hypoglossal nucleus, but w h e t h e r this effect was a result of an action on presynaptic terminals or interneurons was not d e t e r m i n e d in this study. The actions of T R H on hypoglossal m o t o n e u r o n s were consistent with findings in neonatal rat spinal neurons 15, but was different from the effects on amphibian spinal m o t o n e u r o n s 8'13. Thus the species difference should not be ignored when assessing the effect of T R H on m o t o n e u r o n s .

1 Clarke, K.A. and Parker, A.J., Further studies on the effects of a thyrotropin releasing hormone analogue on locomotor activity in the rat, Neuropeptides, 8 (1986) 99-109. 2 Clarke, K.A. and Stirk, G., Motoneurone excitability after administration of a thyrotropin releasing hormone analogue, Br. J. Pharmaeol., 80 (1983) 561-565. 3 Hawkins, E.E and Engel, W.K., Kinetic analysis of thyrotropin releasing hormone binding in the central nervous system: evidence for receptor desensitization, Neurosci. Lett., 79 (1987) 1577-162. 4 Horita, A., Carino, M.A. and Lai, H., Pharmacology of thyrotropin-releasing hormone, Annu. Rev. Pharmacol. Toxicol., 26 (1986) 311-332. 5 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Distribution of thyrotropin-releasing hormone (TRH) in the central nervous system as revealed with immunohistochemistry, Eur. J. Pharrnacol., 344 (1975) 389-392. 6 Johnson, J.V., Fone, K.C.E, Havler, M.E., Tulloek, I.F., Bennett, G.W. and Marsden, C.A., A comparison of the motor behaviors produced by the intrathecal administration of thyrotropin-releasing hormone and thyrotropin-releasing hormone analogues in the conscious rat, Neuroscience, 29 (1989) 463-47[). 7 Kubek, M.J., Rea, M.A., Hodes, Z.I. and Aprison, M.H., Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat, J. Neurochem., 40 (1983) 1307-1313.

8 Lacey, G., Nistri, A. and Rhys-Maitlaand, E.R., Large enhancement of excitatory postsynaptic potentials and currents by thyrotropin-releasing hormone (TRH) in frog spinal motoneutones, Brain Research, 488 (1989) 80-89. 9 Manaker, S., Winokur, A., Rostene, W.H. and Rainbow, T.C., Autoradiographic localization of thyrotropin-releasing hormone receptors in the rat central nervous system, J. Neurosci., 5 (1985) 167-174. 10 Mantyh, P.W. and Hunt S.P., Tbyrotropin-releasing hormone (TRH) receptors. Localization by light microscopic autoradiography in rat brain using [3H][3-Me-His2]TRH as the radioligand, J. Neurosci., 5 (1985) 551-561. 11 Mosfeldt Laursen, A. and Rekling, J.C., Electrophysiological properties of hypoglossal motoneurones of guinea pigs studied in vitro, Neuroscience, 30(3) (1989) 619-637. 12 Nicoll, R.A., Excitatory action of TRH on spinal motoneurons, Nature (Lond.), 265 (1977) 242-243. 13 Nicolk R.A., Alger, B.E. and Jahr, C.E., Peptides as putative excitatory neurotransmitters: Carnosine, enkephalin, substance P and TRH, Proc. R. Soc. Lond., 210 (1980) 133-149. 14 Ono, H. and Fukuda, H., Ventral root depolarization and spinal reflex augmentation by a TRH analog in rat spinal cord, Neuropharmacology, 21 (1982) 739-744. 15 Takahashi, T., Thyrotropin-releasing hormone mimics descending slow sytlaptic potentials in rat spinal motoneurons, Proc. R. Soc. Lond., 225 (1985) 391-398.

This work was supported by Dir. Ib Henriksens Fond, Foundation for Experimental Research in Neurology and The Danish Medical Research council (SLF 12-8279)

Excitatory effects of thyrotropin-releasing hormone (TRH) in hypoglossal motoneurons.

The effect of thyrotropin-releasing hormone (TRH) was studied in 30 hypoglossal motoneurons from brainstem slices of guinea pigs. Bath application of ...
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