Brain Research, 590 (1992) 1-5 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18010

R e s e a r c h Reports

Excitatory effects of thyrotropin-releasing hormone on neurons within the nucleus ambiguus of adult guinea pigs Stephen M. Johnson and Peter A. Getting Department of Physiology and Biophysics, Unit'ersityof Iowa, Iowa Oty, IA 52242 (USA) (Accepted 31 March 1992)

Key words: Nucleus ambiguus; Thyrotropin-releasing hormone; Brain slice; Oscillation; Postinhibitory rebound

The electrophysiological effects of thyrotropin-releasing hormone (TRH) on neurons within the nucleus ambiguus (NA) of adult guinea pigs were studied using an in vitro brain stem slice preparation. In 0.01-1.0 ~m TRH, NA neurons depolarized (25/39), expressed enhanced postinhibitory rebound (8/8 tested), or exhibited oscillations of the membrane potential (17/39). Because the amplitude of postinhibitory rebound in tetrodotoxin (TTX) at various membrane potentials was not altered by TRH, it suggests that TRH enhanced postinhibitory rebound indirectly by depolarizing the cell membrane. The membrane potential oscillations in NA neurons were persistent in TTX and their frequency was dependent on the membrane potential, suggesting that these oscillations were due to intrinsic membrane properties and not to synaptic inputs. The excitation of NA neurons in vitro by TRH suggests that endogenous TRH may modulate the activity of neurons involved in the regulation of respiratory and autonomic function.

INTRODUCTION Thyrotropin-releasing hormone (TRH) has a variety of autonomic effects when injected into the central nervous system of mammals: increased respiratory rate, increased blood pressure, increased gastrointestinal motility, and altered body temperature ~''m. In addition, TRH has been localized to nerve terminals within respiratory and autonomic nuclei in the brain stem 8'17'22, suggesting that endogenous TRH might play a role in modulating respiratory and autonomic functions. Recently, TRH has been shown to have excitatory effects on neurons within the ventrolateral nucleus tractus solitarius (NTS) 3 and the dorsal motor nucleus of the vagus ~8'25, a putative respiratory center and a major parasympathetic motor nucleus, respectively. The effects of TRH on the membrane properties of respiratory and parasympathetic neurons within the NA, however, are not known. Previously, we classified NA neurons into three types based on their electrophysiological and morphological properties using an in vitro brainstem slice preparation 13. In this work, TRH was applied to these neurons in the nucleus ambiguus

(NA), a region that contains a large number of bulbospinal neurons (projecting to the phrenic motor nucleus) and vagal motoneurons t3. Our results show that TRH had three excitatory effects on most NA neurons: depolarization, enhanced postinhibitory rebound, and membrane potential oscillations. Preliminary accounts of this work have appeared ~'~2. M A T E R I A L S AND M E T H O D S

Brain slice preparation Adult female guinea pigs (300-600 g) were anesthetized with ether or halothane and decapitated. The brainstem was exposed, isolated, gently lifted out of the cranial cavity, and cooled in oxygenated Ringer solution at 3-5°C for 30-45 s. The tissue was trimmed, attached with cyanoacrylate glue to a precooled teflon block, and sliced on a vibratome (Camden Instruments) while submerged in oxygenated Ringer solution (3-5°C). Transverse brainstem slices were cut (350-400 #m thick) 0.0-1.5 mm rostral to the caudal tip of the calamus scriptorius, placed in oxygenated Ringer solution at 34°C within a standard recording chamber, and allowed to recover for 20-30 rain prior to recording. Slices subfused (i.e. not submerged) with Ringer solution (flow rate = 1-2 ml/min) could be kept alive in this chamber for 4-7 h. The standard Ringer solution contzined (in mM): 125 NaCI, 6.2 KCI, 1.3 MgSO4, 2.4 CaCI2, 2.5 NaHPO 4, 26 NaHCO3, and 10 glucose (pH 7.4 after saturation with 95% 0 2/5% CO2). Concentrated amounts of TTX (Sigma) and

Correspondence: S.M. Johnson, Systems Neurobiology Laboratory, Department of Physiological Science, University of California at Los Angeles. 405 Hilgard Ave., Los Angeles, CA 90024-9568, USA.

TRH (Peninsula Laboratories. Inc.) were added to the reservoir of standard Ringer solution so that their concentrations would be fixed within the chamber. The concentrations of TTX and TRH t, sed in these experiments were 100-500 ng/ml and 0.01-1.0 gin. respectively. lntracellular recordings were made with glass microelectrodes filled with 3 M KCI (40-80 M.O). A single-electrode current clamp amplifier (Axoclamp-2, Axon Instruments) was used in all experiments. Both the bridge mode and the discontinuous current clamp mode (switching frequency = 4 - 6 kHz, 30% duty cycle) were used. In the discontinuous current clamp mode, the switching frequency was 4-6 kHz (50% duty cycle) and the headstage output was continually monitored to ensure accurate adjustment of capacitance feedback. Recordings made with TI'X in the Ringer solution were filtered at 1.0 kHz. Physiological data were accepted from neurons that had resting potentials of at least - 5 0 mV and action potential amplitudes of > 65 inV. The frequency of action potential firing was measured with a frequency-voltage converter constructed in our laboratory. All measurements are reported as means:t: S.D.

RESULTS Prior to adding TRH. all NA neurons (n = 39) received varying degrees of random synaptic input and did not have membrane potential oscillations. Most neurons fired non-rhythmically at rates of 1-5 Hz following impalement with microelectrodes. Addition of TRH to the bath resulted in depolarization of most NA neurons; many cells that depolarized in TRH also expressed either membrane potential oscillations or enhanced postinhibitory rebound (Table !). NA neurons in this study have been classified based on their repetitive firing properties (defined and char. acterized in ref. 13) as either PIR (expressing postin. hibitory rebound), DE (expressing delayed excitation), or NON cells (expressing neither postinhibitory re. bound nor delayed excitation) t,a. "Fable l shows that TRH causes depolarization and membrane potential oscillations in all three cell types. Other repetitive firing properties of NA neurons, such as spike frequency adaptation, delayed excitation, and posttetanic hyPerpolarization 13, were not affected by TRH (data not shown).

Depolarizat~an of NA neurons Approximately 1-3 min after the addition of TRH to the Ringer solution bathing the slice, most NA neurons (25/39) depolarized slowly over a period of 1-2 min and fired action potentials with increasing frequency (Fig. 1). Within 5-10 min after switching back to control Ringer solution without TRH, the membrane potential returned to the original rest potential. Measurement of the precise change in resting membrane potential was often hindered because the neuron was rapidly firing action potentials. When TTX was added to eliminate sodium-dependent action potentials, the average depolarization due to addition of TRH was 14 + 2 mV (n = 5).

TABLE I Effects of TRH on NA neurons Cell

Type *

Depolarization (mV)

1 2

PIR PIR

8 3

3

PIR

3

4 5

PIR PIR

11 6

6

PIR

+

7 8 9

PIR PIR PIR

6 + 10

10

PIR

6

11

PIR

+

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3{) 31 32 33 34 35 30

PIR PIR PIR PIR PIR PIR PIR PIR PIR PIR PIR PIR PIR DE DE DE DE DE DE NON NON NON NON NON NON

6 8 6 + 6 14 TTX 16 T r x 15 TTX 15 TTX 11 T r x

Enhanced PIR

Membrane potential

++

oscillations +++ +++ -6++

+-6 +-6 +-6

+++ +++

++ ++ ++

4.++ 4.++

4+ 4.++ +++ ++4. 4.++

+ 3 +

+4.+

4.

5 + + 5 4 5

+ .t- + 4..4-+ 4.4,+ 4.4.4.

* +

Cell tyl~eS as described previously in ref. 13 (see text). Neurons began to fire action potentials or increased action potential firing frequency due to TRH. The change in membrane potential could not be determined because of the action potentials. TTX Amplitude of depolarization measured in presence of TTX. + + Enhanced PIR demonstrated in these neurons. + + + Membrane potential oscillations observed.

To measure membrane input resistance, several small injections of negative current (2.0 s, DCC mode) were applied while at the rest potential. The resulting voltage deflections (only deflections < 25 mV were used to minimize tile effects of inward rectification) were divided by the current amplitudes to calculate the resistance. The same protocol was carried out following the addition of TRH and the calculated mean input resistances were compared. It was found that the input resistance of NA neurons was altered by TRH in an inconsistent manner. Most NA neurons (10/17) increased their input resistance while others decreased (2/17) or showed no change (5/17).

CONTROL

10-e M TRH

CONTROL

A

I~ V

--~---- :-

:'-:

......~ ~

.......~

POST-INHIBITORY REBOUND

~ ~ l j ~ |

o

,.v

'--1_1 ' --1_1

•

> 20 mV 5 S Fig. 1. Depolarization of NA neuron in TRH. The membrane potential of a quiescent NA neurnn is shown in control Ringer solution (left). Two minutes after the addition of TRH, the neuron depolarized and fired action potentials (middle). After several minutes in control Ringer solution (right), the neuron hyperpolarized back to the original resting potential (dotted line).

v

E

=

0:

10-

~

0,2

CONTROL TRN

tO mV

1.0 nA •

o

Oo~ GO ~o

coo ~ 08o

0

I

m ~ 00

I

-80

I

I

-60

PULSE VOLTAGE (mY)

Posti,hibitory reboundenhancedby TRH Most NA neurons with postinhibitory rebound that were tested with TRH (n = 24) were depolarized (n = 21). In all NA neurons whose postinhibitory rebound could be examined before and after TRH (n = 8), the expression of postinhibitory rebound was increased. This enhanced postinhibitory rebound was observed as an increase in action potential frequency following release from the same amount of prior hyperpolarization (Fig. 2). Postinhibitory rebound in NA neurons, however, is a voltage-dependent process and increases if the neuron is released to more depolarized voltages following hyperpolarization 13. Therefore it is possible that TRH enhanced the expression of postinhibitory rebound in NA neurons indirectly by depolarizing the cell membrane.

CONTROL

10 -TM

TRH -0

v

mV

To test this hypothesis, the amplitude of postinhibitory rebound was determined under control conditions and with TRH. In discontinuous current clamp mode with TTX in the Ringer solution, NA neurons with postinhibitory rebound (n = 3) were hyperpolarized (0.2-1.0 s) to allow for the maximal amount of postinhibitory rebound to be expressed when the membrane potential was subsequently released to more positive membrane potentials (Fig. 3A). Immediately following the negative current pulse, a rapid depolarization of the membrane was observed that decayed back to the original holding voltage. Postinhibitory rebound was measured as the difference between the peak and steady state voltages following the pulse (Fig. 3B). It was found that postinhibitory rebound increased in a roughly linear fashion fron, about - 9 0 mV to - 6 3 mV. In the presence of TRH, however, the same curve was obtained (Fig. 3C).

Membranepotentialoscillations

-

u

Fig. 3. Postinhibitory rebound was not directly affected by TRH. A: current pulse paradigm in discontinuous current clamp mode. B: sample voltage traces for an NA neuron with postinhibitory rebound in TTX. C: relationship between postinhibitory rebound magnitude and pulse voltage in control Ringer solution and with TRH added. The data are similar no matter if the duration of the hyperpolarizing pulse is 0.2 or 1.0 s (see Fig. 3C of ref. 13).

U

U

._jlO mV 2 nA 0.2 S Fig. 2. Enhanced postinhibitory rebound. In the control condition (left), a transient depolarization was evoked upon release from each negative current pulse. In TRH (right), several ~.ction potentials were evoked from the same current pulses.

In the NA neurons that were depolarized, TRH also induced oscillations of the membrane potential (n = 16) (Fig. 4). Only one NA neuron did not depolarize and yet still expressed oscillations (cell 22 in Table I). In the absence of TRH, oscillations could not be induced with injections of positive current in any of the NA neurons. Likewise, in the four NA neurons that were

tion, small depolarizing perturbations of the membrane potential could induce another oscillation immediately and reset the timing of subsequent oscillations (Fig. 5).

"I, / lil/I i /

DISCUSSION

Fig. 4. A: membrane potential oscillations for an NA neuron induced

by TRH (100 riM). Baseline membranepotential was -74 inV. B: action potentialfrequencj for the voltagetrace above.

exposed to TRH and did not depolarize or express oscillations, no oscillations were observed with positive current injection. This suggests that depolarization alone was insufficient to produce oscillations. Furthermore, no depolarizing afterpotentials were observed in oscillating NA neurons. Depolarizing afterpotentials are transient depolarizations immediately following action potentials and are characteristic of endogenous oscillating neurons in invertebrates and mammals ~4'''7. Membrane potential oscillations induced by TRH were characterized by a rapid depolarization that reached a peak within 100-200 ms, followed by a gradual hyperpolarization to rest within 0.5-3.0 s (Fig. 4). When TTX was added (n = 3), the rhythmic membrane potential oscillations were 10-15 mV in amplitude and characterized by a rapid rise to a peak, followed by a gradual return to rest. The frequency of the oscillations was dependent on the membrane potential; if the membrane was hyperpolarized, the frequency of the oscillations decreased (Fig. 5). In addi-

-45

mV

I/

-

-

'

I

-50

mV

r-i-

I

t

-52

mV

V

. . . . . . . . . . . . . . . . . . . . . . . . .

I 10 m V ._.l , 2 5 n A 1 s

Fig. 5. Membrane potential oscillations induced by TRH (100 nM) were resistant to .'vrx and their frequency was voltage-dependent.

Small injectionsof positivecurrent(arrow) induced another oscillation and reset the timingof subsequentoscillations.

TRH caused the majority of NA neurons to depolarize and express membrane potential oscillations or enhanced postinhibitory rebound. In other types of cells, however, TRH has caused only membrane depolarization 2'~6'1s'24-26 or membrane potential oscillations 3, but not both effects within the same population of neurons. Furthermore, to our knowledge, there are no other examples of TRH altering postinhibitory rebound (directly or indirectly). Thus the NA represents a site where TRH mediates complex changes in the membrane properties of neurons. The voltage dependence of postinhibitory rebound in NA neurons has been more fully characterized elsewhere ~a. In this study, the enhancement of postinhibitory rebound by TRH appeared to be a secondary consequence of the depolarization produced by TRH because: (1) it is known that depolarization of the membrane causes increased postinhibitory rebound, (2) the enhancement of postinhibitory rebound was always preceeded by membrane depolarization, and (3) the amplitude of postinhibitory rebound in TTX was not altered by TRH. The membrane potential oscillations induced by TRH were TTX-resistant and voltage-dependent, properties characteristic of endogenously oscillating cells ~'~''~'~. Furthermore, NA neurons can be considered as 'conditional bursters '2"~ because the membrane potential oscillations could not be expressed without TRH. TRH can also induce oscillations in ventrolateral NTS neurons 3, but there are differences in the nature of the oscillations. In NTS neurons, the membrane depolarizes and hyperpolarizes in slow waves, whereas in NA neurons there is a rapid depolarization and gradual hyperpolarization. Second, the membrane potential oscillations in NTS neurons are related to the induction of depolarizing afterpotentials, a property not observed in NA neurons. The explanation for these differences is not clear but is probably due to the interaction of TRH with the different ionic conductances within the membranes of NA and NTS neurons. The presence of TRH in nerve terminals within the NA s'!7'22 suggests that TRH may play a physiologic role in modulating the firing properties of NA neurons. In support of this view, centrally administered TRH increases the respiratory rate in mammals with variable changes in tidal volume7,9,~5 In contrast, however, no changes in respiratory parameters are observed when

TRH is injected directly into the NA ~9'2°, suggesting that TRH may mediate its respiratory effects at a location other than the NA. It is difficult to reconcile these findings because TRH has been shown to have profound effects on the membrane properties of neurons in the NA and NTS. Perhaps TRH plays a subtle role in the regulation of respiratory motor drive and parasympathetic function. Alternatively, TRH may be released only during certain behaviors such as exercise or fright. Further studies are required to test these hypotheses. Acknowledgments. We thank Drs. Corey Cleland and Michael Dekin for critical review of the manuscript, and David Lawrence and Prvan Nielsen for their excelleat technical assistance. This work was supported by National Institutes of Health Grants NS15350 and HL32336 to P.A.G. and a Lutheran Brotherhood Scholarship to S,M.J. REFERENCES 1 Andrew, R.D. and Dudek, F.E., Burst discharge in mammalian neuroendocrine cells involves an intrinsic regenerative mechanism, Science, 221 (1983) 1050-1052. 2 Clarke, K.A. and Stirk, G., Motoneurone excitability after administration of a thyrotropin releasing hormone analogue, Br. J. PlaarmacoP., 80 (1983) 561-565. 3 Dekin, M.S., Richerson, G.B. and Getting, P.A., Thyrotropin-releasing hormone induces rhythmic bursting in neurons of the nucleus tractus solitarius, Science, 229 (1985) 67-69. 4 Gahwiler, B.H. ~t,~,t Dreifuss, J.J., Phasically firing neurons in long-term cultures of th~ ~at hypothalamus supraoptic area: pacemaker and follower cells, Bra#~ Res., 177 (1979) 95-103. 5 Gainer, H., Electrophysiological activity of an endogenously active neumsecretory cell, Brain Res., 39 (1972) 403-418. 6 Griffiths, E.C., Thyrotropin releasing hormone: endocrine and central effects, Psychoneuroemlocrinok~gy, 10 (1985) 225-235. 7 Hedner, J., tledner. T., Wessberg, P., Lundberg, D. and Jonason, J,, Effects of TRlt and TRH analogues on the central regulation of breathing in the rat, Acta PhysioL Stand., 117 (1983) 427-437. 8 Hokfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, NeuroscL Lett., 1 (1975) 133-139. 9 Holtman, J.R., Bullet, A.L., ltamosh, P. and Gillis, R.A., Central respiratory stimulation produced by thyrotropin-releasing hormone in the cat, Peptides, 7 (1986) 207-212. 10 Horita, A., Carino, M.A. and Lai, H., Pharmacology of thyrotropin-releasing hormone, Annu. Ret'. Pharmacol. Toxicol., 26 (1986) 311-332. II Johnson, S.M. and Getting, P.A., Repetitive firing properties of

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