GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis

Neuroscience Letters 441 (2008) 296–301

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GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis Sang-mi Han, Dong-ho Youn ∗ Department of Oral Physiology, School of Dentistry and Brain Korea 21, Brain Science and Engineering Institute, Kyungpook National University, 188-1 Samduk-2-ga, Chung-gu, Daegu 700-412, Republic of Korea

a r t i c l e

i n f o

Article history: Received 17 April 2008 Received in revised form 16 June 2008 Accepted 19 June 2008 Keywords: GABAA receptor Tonic current Spinal trigeminal nucleus Caudalis Substantia gelatinosa

a b s t r a c t In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABAA receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1 mM), SR95531 (100 ␮M) and bicuculline (100 ␮M). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5 pA) inward shift of holding currents at a holding potential (Vh) of 0 mV in 60–70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1 ␮M), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30 min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc. © 2008 Elsevier Ireland Ltd. All rights reserved.

GABA (␥-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). By exerting its distinct ionotropic type A receptors (GABAA Rs), GABA induces two types of inhibitory currents: phasic (spontaneous) inhibitory postsynaptic currents (sIPSCs) and tonic inhibitory currents [8]. The phasic IPSCs were generated by synaptic GABAA Rs on the postsynaptic membrane, whereas the tonic inhibitory currents were generated by extrasynaptic GABAA Rs [8,19,20]. The two types of inhibitory transmission may be differentially involved in many essential brain functions, including neuronal information processing [6,7,10]. GABAA Rs are pentameric ligand-gated ion channels comprising the assembly of 19 subunits, known as ␣1–6, ␤1–3, ␥1–3, ␦, ␳1–3, ␪, ␧, and ␲ [18,34]. To date, GABAA Rs containing a ␥2 subunit, in association with ␣1, ␣2 and/or ␣3 subunits, are the predominant receptor subtypes that mediate phasic IPSCs, while those containing ␣4, ␣5 and/or ␣6 subunits are exclusively extrasynaptic [8]. Particularly, ␣4 or ␣6 subunits have a higher sensitivity to GABA (EC50, ∼0.3–0.7 ␮M) than ␣1 or ␣2 subunits (EC50, ∼6–14 ␮M) [8]. This property provides the extrasynaptic GABAA Rs with the

∗ Corresponding author at: Department of Oral Physiology, School of Dentistry, Kyungpook National University, 188-1 Samduk-2-ga, Chung-gu, Daegu 700-412, Republic of Korea. Tel.: +82 53 660 6841; fax: +82 53 421 4077. E-mail address: [email protected] (D.-h. Youn). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.06.048

capability of detecting relatively low concentrations of GABA at extrasynaptic sites, thereby mediating tonic inhibitory currents. The tonic GABAergic current has been observed in various regions of the brain, including cerebellar granule cells [13], dentate gyrus granule cells [21,30], hippocampal pyramidal cells and interneurons [26], as well as thalamocortical neurons in the ventral basal complex [23]. Recently, the existence of the tonic current has also been reported in the substantia gelatinosa (SG) neurons of mouse lumbar spinal cord [2,31]. However, the existence and the physiological role of the tonic current have not yet been demonstrated in the SG region of trigeminal nucleus pars caudalis (Vc), which is the central termination site of small-diameter unmyelinated C and thinly myelinated A␦ primary afferent fibers conveying orofacial nociceptive information [15,27]. Neurosteroids can be synthesized de novo, by both glia and neurons in the CNS, independent of peripherally derived sources [5]. Some neurosteroids are selective positive allosteric modulators of GABAA R, containing anticonvulsant, anxiolytic and sedative properties [5,12]. A stress-related neurosteroid 3␣,5␣tetrahydrodeoxycorticosterone (THDOC) is one of the most powerful endogenous positive modulators of GABAA Rs, and has also been shown to directly alter the expression of ␦-subunits of GABAA R [17,28], which may contribute to the enhancement of a GABAergic tonic inhibition [29]. Therefore, in the present study the existence of a GABAergic tonic current and the modulatory effect of THDOC on the tonic current have been examined in the SG neurons of Vc.

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Fig. 1. GABAA R-mediated tonic currents in SG neurons of Vc. Bath-application of picrotoxin (1 mM; (A) left) inwardly shifted the baseline holding current in a SG neuron of Vc, whereas that of SR95531 (100 ␮M; (B) left) did not (<5 pA). Both antagonists completely blocked spontaneous IPSCs. The traces at an expanded scale show before and during the application of picrotoxin (A) or SR95531 (B). All-point histograms ((A) and (B), right) illustrate amplitudes of the tonic currents, and noise levels of holding currents before (control) and during antagonists. Bicuculline (100 ␮M; (C)) also generated large inward shift of the holding current, and completely blocked spontaneous IPSCs. The histogram (D) summarizes the average amplitudes of tonic currents estimated by using the GABAA R antagonists: picrotoxin (1 mM, n = 5), SR95531 (100 ␮M, n = 5), bicuculline (100 ␮M, n = 9; 5 ␮M, n = 6). * P < 0.05, t-test.

All experiments were approved by the Institutional Animal Care and Use Committee of the School of Dentistry, Kyungpook National University. Sprague Dawley rats (5–18 days-old; male or female) were deeply anaesthetized by an intraperitoneal injection of urethane (1.5 g/kg), and decapitated. In order to prepare live slices, the brainstem, with a portion of spinal cord, was promptly removed and immersed in an ice-cold Krebs’ solution (composition in mM: NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2 PO4 1.2, NaHCO3 25, and glucose 11; pre-oxygenated with 95% O2 /5% CO2 at pH 7.4). Horizontal slices (400 ␮m) of the brainstem [11] were cut by using a Vibratome 1000+ (Vibratome, St. Louis, MO, USA). Usually two slices were prepared and transferred to a glass beaker containing oxygenated Krebs’ solution. After an incubation period lasting 60 min at room temperature (23–25 ◦ C), one side (either the right or the left) of the horizontal brainstem slice was transferred to a recording chamber, where it was submerged and immobilized with nylon strands drawn taut across a C-shaped silver wire (∼0.5 cm o.d.), and was continuously perfused (2–3 ml/min) with oxygenated Krebs’ solution. Blind whole-cell voltage-clamp recordings at room temperature were performed by using patch pipettes (borosilicate glass, TW150F; WPI, Sarasota, Fl, USA). The resistance of patch pipettes

was measured at 8–12 M when filled with an internal solution (composition in mM: CS2 SO4 110, CaCl2 ·2H2 O 0.5, MgCl2 ·6H2 O 2, TEA.Cl 5, EGTA 5, HEPES 5, and ATP-Mg 5, adjusted to pH 7.3 with 1N CsOH). Vc was identified in the slices under an upright BX51WI microscope (Olympus, Tokyo, Japan) with low-power magnification of the objective (4×) [11]. Pipette electrodes were positioned to the center of ‘translucent’ SG (lamina II) area, using a motorized micromanipulator (MP-285; Sutter instruments, Novato, CA, USA). To monitor seal formation, voltage pulses of −5 mV were applied. Once a seal was established on a cell (typically a seal resistance of more than 3 G), negative pressure pulses to the pipette were applied to rupture the cell membrane. Pipette series resistance (typically below 25 M) was not compensated for. Whole-cell formation was identified by the appearance of capacitance transients upon voltage pulses and by spontaneous events at a holding potential (Vh) of −70 mV. Recordings (5–10 kHz sampling; 1–2 kHz filtering) were performed by using a Multiclamp 700 A amplifier (Molecular Devices, Sunnyvale, CA, USA) enhanced with pClamp acquisition software (version 9). To record spontaneous IPSCs and estimate tonic GABAergic currents in SG neurons, membrane potentials were held at 0 mV where spontaneous excitatory postsynaptic currents

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Fig. 2. Properties of GABAA R-mediated tonic currents in the SG of Vc. A sample trace ((A) left) shows inward shift of baseline holding current upon the application of bicuculline (100 ␮M), in the presence of strychnine (1 ␮M), TTX (1 ␮M) and kynurenic acid (1 mM), recorded from a SG neuron of Vc. The lower traces show at an expanded scale. An all-point histogram ((A) right) illustrates amplitude of the tonic current, and noise levels of holding currents before (control) and during bicuculline. A histogram (B) compares average amplitudes of tonic currents after application of bicuculline (100 ␮M) in Krebs’s solution (n = 9), and Krebs’s solution containing strychnine (1 ␮M; n = 5) or strychnine (1 ␮M) + TTX (1 ␮M) + kynurenic acid (1 mM) (n = 3). A histogram (C) shows the relative contribution of charge transfers of phasic and tonic inhibitory currents to the total charge transfer in the presence of strychnine (1 ␮M; n = 5; ** P < 0.01, t-test). A diagram (inset) indicates the measurement of charge transfers for phasic and tonic currents.

(EPSCs) are minimal because their reversal potentials are close to 0 mV [3,36]. In this condition, spontaneous IPSCs were recorded in an outward direction, and spontaneous EPSCs disappeared. To determine the magnitude of tonic current, the GABAA R antagonists, picrotoxin, 2-(3-carboxypropyl)-3-amino-6(4-methoxyphenyl)pyridazinium bromide (SR95531 or gabazine) or bicuculline ((+)-bicuculline), were applied by changing the bath solution. The magnitude of the tonic current was calculated as the difference in holding currents before and after the application of GABAA R antagonists. The isolation of GABAergic spontaneous IPSCs and tonic currents was further accomplished by adding 1 ␮M strychnine, the glycine receptor antagonist, in the Krebs’ solution. In some experiments, the bath solution additionally contained 1 mM of kynurenic acid and 0.5 ␮M of tetrodotoxin (TTX) to block all the glutamate receptors and the sodium channels, respectively. Due to the fact that the noise levels were typically the root of the mean square of 2–4 pA, neurons were chosen with a holding current change of more than 5 pA as the neurons supplemented with the tonic current. A charge transfer was calculated by measuring areas of spontaneous IPSCs, or by multiplying the tonic current amplitude by the time interval used to measure the area of spontaneous IPSCs [2]. Chemicals were purchased either from Sigma (St. Louis, USA) or Tocris Cookson (Ellisville, MO, USA). The statistical significance of means and probabilities between the groups was assessed by unpaired Student’s t-test and 2 -test, respectively (significance, * P < 0.05 or ** P < 0.01). Data are represented as mean ± the standard error of the mean (S.E.M.). GABAA R-mediated tonic currents were identified by determining the inward shift of the baseline holding current upon bath-application of the GABAA R antagonists, picrotoxin, SR95531

and bicuculline (Fig. 1). The criterion for the existence of a tonic current in an individual SG neuron was the inward shift of more than 5 pA. Under this criterion, tonic currents were identified in five out of eight SG neurons (62.5%) by 1 mM picrotoxin (10.0 ± 2.7 pA, n = 5; Fig. 1A and D), and in nine out of thirteen SG neurons (69.2%) by 100 ␮M bicuculline (15.9 ± 5.1 pA, n = 9; Fig. 1C and D). However, the application of 100 ␮M SR95531 could not detect a significant tonic current in all five SG neurons (2.7 ± 0.8 pA, n = 5; Fig. 1B and D, P < 0.05 vs. 100 ␮M bicuculline). On the other hand, the application of 5 ␮M of bicuculline revealed tonic current existence only in one (5.5 pA) out of six SG neurons (16.7%). The average tonic current amplitude from all six SG neurons significantly decreased (5 ␮M bicuculline, 2.6 ± 1.4 pA, n = 6, P < 0.05; Fig. 1D), when compared to that of 100 ␮M bicuculline. In most Vc SG neurons, all the antagonists blocked phasic (spontaneous) IPSCs, with a decrease in background noise level (Figs 1A and B, right) leaving only few events in some neurons which are presumably glycinergic IPSCs [2]. Together, these results indicate that the tonic inhibitory current exists in a subpopulation of Vc SG neurons and has different sensitivities to GABAA R antagonists, although all the conditions are sufficient to block synaptic GABAergic transmissions. On the other hand, it was assumed in this study that 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711), the specific GABA transporter inhibitor, would raise extracellular GABA levels and consequently increase the amplitude of tonic GABAA Rmediated currents. However, the NO-711 (5–10 ␮M) solution did not significantly cause an outward shift in the baseline holding current (0.6 ± 1.9 pA, n = 5), indicating that the slice preparations have adequate concentrations of ambient GABA to activate GABAA Rs mediating tonic currents.

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Fig. 3. Effect of THDOC on GABAA R-mediated tonic current. A sample trace (A) shows that THDOC (0.1 ␮M) causes outward shift of holding current in the presence of strychnine (1 ␮M). Further application of bicuculline (100 ␮M) in the presence of THDOC as well as strychnine, induces a bigger inward holding current than that before the application of THDOC. The lower traces show at an expanded scale. Histograms compare probability of SG neurons with tonic current ((B); * P < 0.05) and the average amplitudes of tonic currents (C) after incubating slices for 30 min with 0.1 ␮M of THDOC.

The application of 100 ␮M of bicuculline also caused an inward shift of the holding current in the presence of 1 ␮M strychnine, the glycine receptor antagonist, in five out of ten SG neurons (50%), or in the presence of 1 ␮M strychnine, 1 ␮M TTX, the sodium channel blocker, and 1 mM kynurenic acid, the glutamate receptor blocker, in three out of six SG neurons (50%). The average amplitudes under both conditions (strych, 16.7 ± 5.6 pA, n = 5; strych + TTX + kyn, 8.0 ± 1.5 pA, n = 3; Fig. 2A and B) were not significantly different when compared to that of Krebs’ condition. Apparently, the smaller average amplitude in the presence of strychnine, TTX and kynurenic acid, may reflect a possible contamination of glutamate receptors-mediated currents in our recording conditions, due to the lower holding potentials in comparison to the reversal potentials of the glutamate receptors, or a contribution of GABA, released by action potential-dependent mechanism, to the tonic current. In the five SG neurons with significant tonic currents in the presence of strychnine, a relative GABAergic charge transfer ratio between phasic and tonic inhibitory currents was determined (Fig. 2C). The phasic current charge transfer mediated by GABAA R occupied only 2.3% of the total (phase + tonic) inhibitory charge transfer, while giving rise to 97.7% for a proportion of the tonic charge transfer (n = 5, Fig. 2C), indicating the predominant role of tonic currents in regards to the GABAA R-mediated inhibition in the SG region of Vc. A test was also conducted whether the tonic current existing in SG neurons of Vc is sensitive to an acute treatment of the neurosteroid THDOC [17]. The application of 0.1 ␮M of THDOC in the presence of strychnine (1 ␮M) caused a small outward tonic current in two out of six neurons. Further application of 100 ␮M of bicuculline in the presence of THDOC produced a larger inward baseline holding current than that estimated in the absence of THDOC

(Fig. 3A). To further determine whether GABAA R-mediated tonic currents can be changed by a prolonged exposure of THDOC, the effect of a 30 min brain slice exposure to THDOC solution (0.1 ␮M) was analyzed in regards to a GABAergic tonic inhibition. The incubation of slices with THDOC significantly increased the probability of the existence of SG neurons with significant tonic current upon the application of 100 ␮M of bicuculline in the presence of strychnine (1 ␮M) (no incubation, 50%, 5/10; 30 min THDOC incubation, 89%, 8/9; P < 0.05, 2 -test; Fig. 3B), although the average amplitudes of tonic currents were not significantly different between two groups (Fig. 3C). The tonic activation of GABAA R was first identified in voltageclamp recordings from rat cerebellar granule cells [13]. The application of the GABAA R antagonists, bicuculline and SR95531, not only blocked phasic IPSCs, but also decreased the holding current, required to keep the cell at a given holding membrane potential. Subsequent studies have indicated that GABAergic tonic currents exist in dentate gyrus granule cells [21,30], pyramidal cells and interneurons in the CA1 of hippocampus [26], as well as thalamocortical neurons in the ventral basal complex [23]. Recently, the tonic current was also identified in SG neurons obtained from the mouse lumbar spinal cord [2,31]. Since many GABAergic inhibitory interneurons are found in the SG region of the lumbar spinal cord (approximately 30%) [32] and have a high release probability of GABA [14], GABA may overflow and diffuse out of synapses, and tonically inhibit SG neurons if they contain GABAA Rs with high affinity. Hence, it was assumed in this study that the ambient GABA-induced tonic inhibition also exists in the SG region of Vc in view of the fact that Vc has an anatomically similar structure to the lumber spinal cord. This tonic mode of inhibition is expected to contribute to the modulation and the integration of sensory information in the SG

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of Vc, together with the mode performed by a fast and precisely timed inhibitory synaptic transmission. Therefore, in the present study we attempted to identify GABAA R-mediated tonic currents in SG neurons of Vc. One feature resulting from this study is that a high percentage of Vc SG neurons (60–70%) exhibited variable but significant (more than 5 pA) tonic currents, which were estimated based on the change of holding currents upon the bath-application of GABAA R antagonists. The variable amplitudes of tonic currents may be due to a heterogeneous population of Vc SG neurons with differing membrane properties and firing patterns [16]. Another noteworthy feature is that the tonic currents, recorded in SG neurons, were less sensitive to SR95531. This result is in accordance with the result obtained from hippocampal interneurons [26], and can be explained by comprehending the competitive nature of SR95531 as an aryl-aminopyridazine derivative of GABA [9]. Other explanations are also likely, such as different binding properties of two antagonists [33] as well as differing sensitivity levels of GABAA R subunits to the antagonists [1]. On the other hand, the present findings noting that the GABA uptake inhibitor NO-711 failed to enhance the GABAergic tonic current suggest that our slice preparation condition already contains a saturated concentration of ambient GABA to induce tonic currents. It has been proven that neurosteroids specifically enhance a tonic inhibitory current in central neurons [4]. Among these neurosteroids, THDOC is one of the most potent positive modulators as it binds GABAA Rs [5,12]. THDOC at the low concentration potentiated GABA responses in human embryonic kidney cells [24], and produced a small but significant increase in GABA currents recorded in cultured hippocampal neurons [25]. Based on these results, the modulation of GABAA R-mediated inhibition by the neurosteroid THDOC in SG neurons of rat Vc was examined. Consequently, the acute application of THDOC (0.1 ␮M) enhanced the tonic current in two out of six SG neurons, and incubating slices with THDOC for 30 min increased the probability of neurons with significant tonic current, although the average amplitude did not significantly change. These results contrast with the report in which the tonic conductance in dentate gyrus and cerebellar granule cells was approximately doubled by THDOC [29]. Since GABAA Rs containing ␦-subunits are highly sensitive to THDOC [29], its minimal effect in the present study may reflect a low level of expression of ␦-subunits in this brain region [22]. Otherwise, this result is attributable to the distribution of THDOC into cell membranes of surrounding neuropil, due to its highly lipophilic property [35]. The present study demonstrates the presence of GABAA Rmediated tonic inhibition and its adjustment by the neurosteroid THDOC in SG neurons of rat Vc. In future studies, it will be relevant to analyze the functional role regarding tonic inhibition in spinal trigeminal nucleus under various physiological or pathological conditions.

Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-331-E00311).

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