Two Conductances Mediate Thyrotropin-Releasing-Hormone-Induced Depolarization of Neonatal Rat Spinal Preganglionic and Lateral Horn Neurons

Miloslav Kolaj1, Susan J. Shefchyk2, and Leo P. Renaud1

1 Neuroscience Unit, Loeb Research Institute, Ottawa Civic Hospital and University of Ottawa, Ottawa, Ontario K1Y 4E9; and 2 Department of Physiology, University of Manitoba, Winnipeg, Manitoba R3E 3J7, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Kolaj, Miloslav, Susan J. Shefchyk, and Leo P. Renaud. Two conductances mediate thyrotropin-releasing-hormone-induced depolarization of neonatal rat spinal preganglionic and lateral horn neurons. J. Neurophysiol. 78: 1726-1729, 1997. Thyrotropin-releasing hormone (TRH) has been recognized as a neuromodulator in several CNS regions, including the thoracolumbar spinal cord where an influence on cardiovascular autonomic function has been proposed. To identify the cellular mechanisms involved in the latter, whole cell patch-clamp recordings were obtained from 52 thoracolumbar lateral horn cells, including 17 sympathetic preganglionic neurons (SPNs), in spinal cord slices from neonatal rat (11-21 days). Under current clamp, bath applications of TRH (1-20 µM) induced a slowly rising and prolonged membrane depolarization in eight of nine cells tested. Under voltage clamp (holding potential -65 mV), 33 of 37 tested cells displayed a TRH-induced, tetrodotoxin-resistant inward current that was associated with either a reduction or an increase in membrane ion conductances. Current-voltage (I-V) relationships in 28 cells suggested two conductances. In 9 cells the current reversed at about -107 mV; in 10 cells the I-V lines remained parallel, whereas in 9 cells the current reversed at around -40 mV. In three of three cells, addition of 2 mM barium was associated with an inward current, and the TRH-induced inward current was also suppressed, suggesting the presence of a resting barium- and TRH-sensitive potassium conductance. A residual barium-insensitive conductance was seen to reverse near -40 mV. Intracellular dialysis with guanosine 5'-o-(3-thiotriphosphate) significantly enhanced the duration of the TRH effect, indicating that G protein activation participates in the TRH response. These observations not only reveal a direct, G-protein-mediated depolarizing action of TRH on neonatal rat SPNs and lateral horn cells but also imply that two separate conductances may be involved in the TRH responses in some neurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Thyrotropin-releasing hormone (TRH), originally discovered as a hypothalamic neuropeptide that stimulates the release of thyroid-stimulating hormone secretion from the adenohypophysis, is also considered to have a neuromodulator or neurotransmitter role in several CNS regions. In particular, TRH alters the excitability of neurons in the brain stem (e.g., Bayliss et al. 1992; Travagli et al. 1992) and spinal cord (Fisher and Nistri 1993; Jackson and White 1988). TRH-binding sites (Prasad and Edwards 1984), receptor mRNA (Wu et al. 1992), and TRH-immunoreactive fibers and terminals have been observed in the vicinity of sympathetic preganglionic neurons (SPNs) (Appel et al. 1987; Poulet et al. 1992a,b). Spinal TRH appears to participate in autonomic regulation, because, when intrathecally administered, TRH can alter blood pressure and sympathetic tone (Helke and Phillips 1988; Yashpal et al. 1989; Yusof and Coote 1988) and antisense to TRH receptors can reduce arterial blood pressure in hypertensive rats (Suzuki et al. 1995). However, at the cellular level, little is known of the mechanisms mediating the actions of TRH on spinal autonomic circuits. In the present investigation we used whole cell patch-clamp technique to examine TRH-induced actions on neurons, including SPNs, within the thoracolumbar lateral horn.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Preparations consisted of 400-µm transverse thoracolumbar spinal cord slices from 12- to 21-day-old Sprague-Dawley rats (Pickering et al. 1991). Briefly, animals were anesthetized with methoxyflurane and the spinal cord was exposed, removed, placed in ice-cold artificial cerebrospinal fluid (ACSF), and sectioned on a vibratome. Slices were placed in a recording chamber at room temperature (20-23°C) and continuously superfused (3-6 ml/min) with ACSF containing (in mM) 127 NaCl, 26 NaHCO3, 3.1 KCl, 1.2 MgCl2, 2.4 CaCl2, and 10 D-glucose saturated with 95% O2-5% CO2. Neurons in the lateral column were recorded with the "blind" whole cell patch-clamp technique with the use of an Axopatch-1D amplifier. The intracellular solution in the patch pipettes contained (in mM) 130 potassium gluconate, 10 KCl, 10 NaCl, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 Mg-ATP, and 0.3 guanosine 5'-triphosphate (GTP), pH adjusted to 7.4 with NaOH. Lucifer yellow (1 mg/ml) was included in the pipette solution for later visualization and morphological identification of neurons. In some experiments, instead of GTP, cells were loaded (by passive diffusion from patch pipette) with 0.2 mM guanosine 5'-o-(3-thiotriphosphate) (GTP-gamma -S). Final electrode resistance ranged from 3 to 7 MOmega . Correction of the liquid junction potential was applied to recorded membrane currents and voltages. Input resistance was determined from the linear slope (i.e., between -50 and -80 mV) of current-voltage (I-V) relationships. Drugs were dissolved in ACSF at known concentrations and applied by bath or by a local pressure application system (DAD-12, Adams & List Associates). Slices containing Lucifer-yellow-filled neurons were transferred to a 4% formaldehyde in 0.1 mM phosphate buffer solution, stored overnight at 4°C, cleared for 45-60 min with dimethyl sulfoxide, and viewed under epifluorescence. Statistical comparisons between control and experimental values were determined with the use of paired or unpaired Student's t-test. Results are expressed as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Stable whole cell recordings obtained from 52 neurons within the intermediolateral horn of the thoracolumbar spinal cord included 17 neurons recognized as SPNs on the basis of their morphology (cf. Pickering et al. 1991; Shen and Dun 1990). For this neuron sample, resting membrane potential was -62.7 ± 0.8 mV (range -48 to -81 mV) and membrane input conductance was 4.4 ± 0.35 nS. Injection of depolarizing current pulses elicited action potentials (amplitude 77 ± 2 mV, width at half amplitude 1.55 ± 0.1 ms) that were followed by a fast and a slow afterhyperpolarization with amplitudes of 17.4 ± 1.1 mV (n = 47) and12.5 ± 0.6 mV (n = 48), respectively.

Under current-clamp conditions, eight of nine cells tested were responsive to applications of TRH (1-10 µM for 20-60 s) and displayed a slowly rising 14 ± 2.5 mV membrane depolarization (resting membrane potentials of -64 ± 2.6 mV) that was sufficient to initiate action potentials in six cells (Fig. 1A), with recovery after 5-25 min. At the peak of the response, several cells demonstrated baseline thickening that was interpreted to be an indirect (presynaptic) effect (cf. Wang and Dun 1990) because this was never observed in ACSF containing 1 µM tetrodotoxin, which blocked action potentials.


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FIG. 1. A: whole cell current-clamp recording from a sympathetic preganglionic neuron (SPN) with a resting membrane potential of -65 mV illustrates a thyrotropin-releasing-hormone (TRH)-induced slowly rising, prolonged, and reversible membrane depolarization with action potentials occurring during the peak of the response. All TRH-induced responses were reproducible assuming >= 30 min of washout. B: voltage-clamp trace (holding potential -65 mV) recorded from another lateral horn cell [in artificial cerebrospinal fluid (ACSF) containing 1µM tetrodotoxin] illustrates a TRH-induced slow inward current.

Under voltage clamp (holding potential -65 mV) and in the presence of 1µM tetrodotoxin, 33 (including 13 SPNs) of 37 cells tested responded to local applications of TRH (10-20 µM, 10-50 s; Fig. 1B) with a reversible inward current (mean 46.6 ± 6.4 pA) that peaked by 52.5 ± 6.2 s and recovered after 7.7 ± 1 min. Detailed comparison ofI-V relationships in 28 cells revealed in 9 cells (including 3 SPNs) a TRH-induced reduction in membrane conductance from 4.3 ± 0.4 nS to 3.7 ± 0.5 nS (Fig. 2A). In these cells, the net current had an amplitude of 38.9 ± 8.3 pA and reversed at -107.6 ± 6.7 mV, close to the estimated potassium reversal potential (EK+) of - 97 mV (Fig. 2A4). In another 10 cells (including 4 SPNs), the response to TRH was also associated with a reduction in membrane conductance from 4.3 ± 0.7 nS to 3.9 ± 0.7 nS. The net current was 67.9 ± 12.6 pA and I-V relationships did not cross within the tested voltage range of -30 to -130 mV (Fig. 2B4). By contrast, in the remaining nine cells (including 6 SPNs), the TRH net current of 30.9 ± 7.5 pA was associated with an actual increase in membrane conductance from 3.7 ± 0.7 nS to 4.1 ± 0.8 nS, and this response was associated with reversal potentials close to -40 mV (Fig. 2C4).


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FIG. 2. Typical examples of individual current-voltage (I-V) relationships observed during TRH-induced responses in cells expressing a reversal potential close to potassium reversal potential (EK+, A), no reversal potential within the tested voltage range (B), or a reversal close to -40 mV (C), respectively. Data for I-V plots were taken at the end of the voltage pulse. There was no significant difference between measurements taken at the beginning of the time-dependent rectification or at the end of the voltage pulse, suggesting minimal contribution of these conductances to the results. A4, B4, and C4: pooled data of the net TRH-induced current, calculated as a difference between I-V plots obtained before and during the peak of the TRH responses, for the corresponding I-V patterns shown in the 3rd column. Data are expressed as means ± SE.

Because barium ions are known to inhibit various K+ currents, including leak K+ currents, cells were tested for a barium-sensitive resting K+ conductance and for the effects of barium on TRH-induced inward currents. In ACSF containing 2 mM barium, each of three cells (including 2 SPNs) displayed a persistent inward current (33.3 ± 14.5 pA) and a decrease in membrane conductance (from 2.7 ± 0.8 nS to 2.2 ± 0.6 nS). The barium-induced inward current decreased with membrane hyperpolarization and actually reversed at -110 ± 16 mV, leading to the conclusion that barium acted by decreasing a resting K+ conductance. In addition, the magnitude of the TRH-induced current in these cells was reduced from 43.3 ± 4.4 pA (2 cells showed no reversal and 1 reversed at -103 mV) in control conditions to 24 ± 1 pA in barium. A residual barium-resistant component of the net TRH current was seen to reverse at -40 ± 10.5 mV and was accompanied by an increase in membrane conductance from 2.2 ± 0.6 nS to 2.6 ± 0.4 nS.

TRH mediates its effects through G-protein-coupled receptors. At 5 min after the establishment of whole cell voltage clamp with the use of pipettes containing GTP-gamma -S, a gradual inward current of 14 ± 1 pA was measured in three (including 2 SPNs) of five cells. Under these recording conditions, recovery from a TRH-induced response was not obvious even after 25 min of wash (Fig. 3). The average TRH-induced current was ~42 pA in the presence of GTP-gamma -S as compared with ~39 pA in the cells recorded with controlpipette solutions containing GTP. In the presence of GTP-gamma -S,membrane conductance near resting membrane potential levels (from -50 to -80 mV) decreased in two cells, increased in one cell, and was unchanged in the remaining two cells. The net TRH-induced current reversed close to EK+ in one cell, with no reversal detected in the other four cells.


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FIG. 3. A: top trace depicts an irreversibly enhanced current produced by 10 µM TRH recorded 10 min after establishment of whole cell voltage clamp with the use of a pipette containing guanosine 5'-(thio)triphosphate (GTP-gamma -S; 0.2 mM). B: pooled data from 17 cells reveal that application of 10 µM TRH in control cells (open circle ) produces inward current that reaches a maximum during the 1st min and then slowly declines to resting values. Filled circles: data from cells loaded with GTP-gamma -S (0.2 mM for 10 min) in which the TRH-induced current reaches a maximum and then declines to ~60% of maximum current and remains there. This effect was present even in the cells displaying no gradual inward current after establishment of whole cell recordings. Data are expressed as means ± SE. P < 0.006 when control and GTP-gamma -S containing cells are compared at the 6-min mark.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

These observations indicate that TRH acts on postsynaptic receptors to induce membrane depolarization and thereby increase the excitability of thoracolumbar lateral column neurons, including SPNs, in neonatal rat spinal cord. Interestingly, this depolarization may involve at least two mechanisms, because the TRH-induced inward current was accompanied by either a decrease (68% of cells) or an increase (32% of cells) in a resting membrane conductance, as reflected in the I-V relationships illustrated in Fig. 2.

For SPNs and other unidentified cells displaying a decrease in membrane conductance at resting levels, the dominant component of the TRH-induced inward current likely results from blockade of a barium-sensitive resting K+ conductance: 1) this current is decreased with hyperpolarizing potentials, presumably by virtue of the reduced driving force for K+ ions at potentials closer to EK+ and is reversed at a potential close to the estimated EK+; 2) this current is associated with a decrease in apparent membrane conductance; and 3) barium ions mimic the TRH effect and suppress the TRH-induced inward current.

The data reveal that a second barium-resistant component of the TRH-induced current is associated with an increase, rather than a decrease, in membrane conductance. Although the charge carrier for this component remains to be defined, the observation that the net current reversed around -40 mV is consistent with opening of nonselective cationic channels. Furthermore, in 10 neurons, control and test I-V curves did not cross and/or displayed voltage rectification at potentials positive to -50 mV, and the amplitude of their TRH-induced current was notably larger (Fig. 2B). Additionally, the TRH-induced inward current reversed at about -107 mV, which is about 10 mV more hyperpolarized than the estimated EK+ of -97 mV. This may indicate that TRH alters both a barium-sensitive potassium conductance and a barium-insensitive nonselective cationic conductance in the same neurons. The downward shift, the voltage rectification of the net TRH-induced I-V relationship, and the more hyperpolarized reversal potential could be due to the distinct contribution of these two conductances in the same neuron.

TRH has been reported to reduce a resting potassium conductance in spinal (Fisher and Nistri 1993) and hypoglossal motoneurons (Bayliss et al. 1992). Although these investigators also noted a barium-insensitive component of the net TRH current, this was reported to be essentially voltage independent, with no obvious conductance change (Bayliss et al. 1992), or calcium dependent (Fisher and Nistri 1993). Perhaps these differences reflect regional and functional variability in the CNS and/or differences in the maturity of the preparation, although a mechanism that is similar to that proposed here (i.e., block of resting K+ conductances and activation of nonselective cationic conductances) has been suggested for neurotransmitter actions in other neurons (Benson et al. 1988; Dong et al. 1996; Larkman and Kelly 1992).

The inward current modulated by TRH has a specific action of increasing neuronal excitability. Because this current contributes to the resting membrane potential, TRH can facilitate initial responsiveness to excitatory neurotransmission through membrane depolarization and an increase in input resistance. The additional conductance may serve to depolarize spinal lateral horn neurons at a stage when blockade of resting potassium conductance would be an inefficient means of increasing cellular activity, as for example during sustained hyperpolarization.

The results with GTP-gamma -S support molecular evidence that TRH receptors belong to the family of G-protein-coupled receptors (Gershengorn and Osman 1996). The observation that TRH-induced responses involve increases and/or decreases in membrane conductances after the treatment with GTP-gamma -S is consistent with involvement of G proteins in both conductances.

In summary, these novel observations support the notion that endogenous TRH can affect autonomic functions in vivo by indicating that exogenous TRH acts at postsynaptic G-protein-coupled receptors to depolarize SPNs and other lateral horn neurons by an action that can involve two different conductances that, in certain instances, may be present in the same neuron.

    ACKNOWLEDGEMENTS

  This work was supported by the Medical Research Council and the Heart and Stroke Foundation of Canada.

    FOOTNOTES

  Address for reprint requests: L. P. Renaud, Neuroscience Unit, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario K1Y 4E9, Canada.

  Received 12 May 1997; accepted in final form 12 June 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society