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γ-Aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus
Journal of the Autonomic Nervous System 75 Ž1999. 156–163
g-Aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus Tian-Le Xu b
a,b,)
a Neuropharmacology Unit, Department of Anatomy, The Fourth Military Medical UniÕersity, Xi’an 710032, China Department of Neurobiology & Biophysics, School of Life Sciences, UniÕersity of Science and Technology of China, Hefei 230027, China
Received 6 October 1998; revised 23 November 1998; accepted 30 November 1998
Abstract The electrophysiological and pharmacological properties of GABA-activated Cly currents Ž IGABA . were investigated in enzymatically dissociated rat sacral dorsal commissural nucleus ŽSDCN. neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. Exogenous application of GABA to SDCN neurons induced Cly currents which increased in a concentrationdependent manner. Bicuculline ŽBIC. and strychnine ŽSTR. antagonized the IGABA in a concentration-dependent manner. Zn2q suppressed the IGABA with an IC 50 of 2.8 = 10y5 M. Muscimol mimicked the IGABA, while baclofen evoked no response. Pentobarbital ŽPB. and 5b-pregnan-3a-ol-20-one Žpregnanolone, PGN. also induced GABA A-mimic Cly currents. Diazepam ŽDZP., PB and PGN all enhanced the IGABA by increasing the apparent affinity of the GABA A receptors to GABA. Moreover, spontaneous GABAergic inhibitory postsynaptic currents ŽIPSCs. were observed in mechanically dissociated SDCN neurons attached with synaptic boutons, so called ‘synaptic bouton preparation’. These results indicate that SDCN neurons express GABA A receptors with relatively low sensitivity to Zn2q inhibition, and that GABA may have a functional role as an inhibitory transmitter in the SDCN regulating nociceptive, analgesic, and autonomic functions. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sacral dorsal commissural nucleus; GABA A receptor; Cly current; Allosteric modulation; Cotransmission; Acutely dissociated neurons; Synaptic bouton preparation; Nystatin perforated patch clamp
1. Introduction The sacral dorsal commissural nucleus ŽSDCN. is involved in nociceptive, analgesic, and autonomic functions. Noxious chemical irritation of the bladder ŽLu¨ et al., 1995. increases the expression of c-fos-like protein ŽFOS. in the SDCN. Nicotinamide adenine dinucleotide phosphate diaphorase ŽNADPH-d. positive cell bodies and fiber networks are densely stained in the SDCN, and nitric oxide synthase immunoreactivity in this region can be upregulated by peripheral visceral nerve injury ŽVizzard et al., 1995.. Substance P ŽSP., which is generally believed to be involved in nociception, has been immunohistochemically localized in small-diameter primary afferent terminals to the SDCN. By combining SP receptor ŽSPR. with FOS ) Department of Neurobiology & Biophysics, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. Tel.: q 86-551-360-3510; fax: q 86-551-360-3142; e-m ail: [email protected]
immunohistochemistry techniques, we had demonstrated that a subpopulation of SDCN neurons receiving noxious information from the urinary bladder express SPR ŽLu¨ et al., 1995.. The SDCN receives descending projections from several regions of the brain known to modulate the spinal nociception ŽXu et al., 1996; Xu, 1997.. The SDCN also receives abundant afferent inputs from visceral organs ŽHonda, 1985; Nadelhaft and Vera, 1995; Katter et al., 1996.. Recently, a direct projection from rat SDCN to Barrington’s nucleus Žthe pontine micturition center in the pons. has been reported ŽDing et al., 1997.. g-Aminobutyric acid ŽGABA. and glycine are very important for the process of fast inhibitory synaptic transmission in the central nervous system ŽCNS.. The importance of GABA A receptors in supraspinal regions of the CNS in vertebrates has been well recognized. For inhibitory neurotransmission in the spinal cord, however, glycinergic mechanisms have generally been considered to be the major component and thus received much emphasis in the past. Recent morphological, physiological and phar-
0165-1838r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 8 7 - 8
T.-L. Xu r Journal of the Autonomic NerÕous System 75 (1999) 156–163
macological evidence indicates that GABAergic mechanisms also contribute to the inhibitory influences on the spinal cord ŽYoshimura and Nishi, 1995; Alvarez et al., 1996; Antal et al., 1996; Bohlhalter et al., 1996; Malcangio and Bowery, 1996.. GABA and its receptors are implicated in modulation of autonomic function ŽBereiter and Gann, 1989; Lewis and Coote, 1995; Barron et al., 1997; Blok et al., 1997. as well as nociception ŽMcGowan and Hammond, 1993; Lin et al., 1994; Malcangio and Bowery, 1996; Xu et al., 1998b.. However, direct current responses of SDCN neurons to GABA application have not been characterized. In addition, while modulation of GABA A receptors by benzodiazepines ŽCartmell and Mitchell, 1994., barbiturates ŽCleland et al., 1994., or neurosteroids ŽFrye and Duncan, 1994. have the potential for significant influences on spinal nociception, there is little information about the physiological effects of modulation of GABA A receptors in the SDCN. Using isolated central neurons and the voltage-clamp techniques, indirect effects via other cells or via changes in membrane potential can be excluded ŽXu et al., 1996, 1998a.. In the present study, tight-seal nystatin perforated patch recording was used to investigate the electrophysiological and pharmacological properties of GABA-induced Cly currents Ž IGA BA . in acutely dissociated SDCN neurons.
2. Materials and methods 2.1. Preparation The sacral dorsal commissural nucleus ŽSDCN. neurons were acutely dissociated as described elsewhere ŽXu et al., 1998a.. Briefly, 2-week-old Wistar rats were decapitated under pentobarbitone-sodium anesthesia Ž50 mg kgy1 i.p... A segment of lumbosacral ŽL 5 –S 3 . spinal cord was dissected out and sectioned with a vibratome tissue slicer ŽDTK-1000, Dosaka. to yield several transverse slices Ž400 mm thick. containing the SDCN region. The slices were preincubated in oxygenated incubation solution Žsee below. for 50 min at room temperature Ž22–258C.. Thereafter, slices were treated enzymatically in oxygenated incubation solution containing 1 mgr5 ml pronase for 20 min at 318C followed by exposure to 1 mgr5 ml thermolysin for another 15 min in the same conditions. After the enzyme treatment, the slices were kept in enzyme-free incubation solution for 1 h. Then a portion of SDCN region was micro-punched out with an electrolytically polished injection needle and transferred into a culture dish filled with standard external solution Žsee below.. Neurons were mechanically dissociated with fire-polished Pasteur pipettes under visual guidance under a phase contrast microscope ŽIX 70, Olympus.. For recording spontaneous synaptic currents, the neurons were mechanically dissociated in some experiments to
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obtain SDCN neurons with synaptic boutons attached ŽDrewe et al., 1988; Xu and Li, 1997., so called ‘synaptic bouton preparation’. Briefly, the slices were transferred into the dish and a fire-polished glass pipette was touched lightly onto the surface of the SDCN. The pipette was vibrated horizontally at about 3–6 Hz for about 1 min, and then slices were removed. The mechanically andror enzymatically dissociated neurons adhered to the bottom of the dish within 20 min, allowing the electrophysiological studies to be conducted. 2.2. Solutions The composition of incubation solution was ŽmM.: NaCl 124, NaHCO 3 24, KCl 5, KH 2 PO4 1.2, CaCl 2 2.4, MgSO4 1.3 and glucose 10, aerated with 95% O 2 –5% CO 2 to a final pH of 7.4. The normal external standard solution was ŽmM.: NaCl 150, KCl 5, CaCl 2 2, MgCl 2 1, N-2-hydroxyethylpiperazine-N X-2-ethanesulphonic acid ŽHEPES. 10 and glucose 10. The pH was adjusted to 7.4 with tris–hydroxymethyl aminomethane ŽTris–base.. The patch-pipette solution for nystatin perforated patch recording was ŽmM.: CsCl 150 and HEPES 10. The pH was adjusted to 7.2 with Tris–base. A nystatin stock solution dissolved in acidified methanol at concentration of 10 mgrml was prepared and stored at y208C. The stock solution was added to the patch-pipette solution in a final nystatin concentration of 200 mgrml just before use. Voltage-activated Naq, Ca2q and Kq channels were blocked by adding 3 = 10y7 M tetrodotoxin ŽTTX. and 10y5 M CdCl 2 in the standard external solution and by using Csq-containing internal solution, when the current– voltage Ž I–V . relationships for GABA-and glycine-induced Cly currents were examined. CdCl 2 had no noticeable effect on the GABA- and glycine-induced currents at the concentration used. 2.3. Perforated patch recording Electrical measurements were carried out by using nystatin perforated patch recording configuration under voltage-clamp condition at room temperature Ž22–258C.. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller ŽPB-7, Narishige.. The resistance between the recording electrode filled with a pipette solution and the reference electrode was 4–6 M V. Series resistance checked every 10 min was 10–30 M V. The change in series resistance through recording was less than 10%. The electrode was connected to a patch-clamp amplifier ŽCEZ-2300, Nihon Koden., and filtered at 1 kHz. The current and voltage signals were monitored with a pen recorder ŽOmniace RT 3108, San-ei., sampled and analyzed using a DigiData 1200A interface and a computer with pCLAMP 6.0.2 program ŽAxon Instruments.. Except when examining I–V relationships, the membrane potential was held at y40 mV throughout the
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experiment. This potential relates to the resting potentials of the isolated SDCN neurons recorded with the Csq-containing pipette solution Žsee Section 3..
The equation for concentration–inhibition curves of bicuculline, strychnine and Zn2q is the mirror image of the Michaelis–Menten equation:
2.4. Drugs and their application
IrImax s 1 y C nr Ž C n q K dn . ,
The following drugs were used in the present experiments: pronase ŽCalbiochem.; GABA, glycine, diazepam and pentobarbital ŽKasei, Tokyo.; muscimol, thermolysin, nystatin, bicuculline, strychnine, 5b-pregnan-3a-ol-20-one Žpregnanolone., CNQX, D-AP5 and TTX ŽSigma.. Diazepam and pregnanolone were first dissolved in dimethyl sulfoxide ŽDMSO. at 10y2 M and then diluted to the respective final concentrations in the normal standard solution just before use. The maximal concentration of DMSO was less than 0.3%, at which concentration it did not affect the GABA-induced current. Rapid application of the drugs was performed via a ‘Y-tube’ ŽXu et al., 1996.. This system allows a complete exchange of external solution surrounding a neuron within 20 ms. 2.5. Statistical analysis Data were calculated as mean " standard error ŽS.E.M.. and the S.E.M. is indicated by a vertical bar in the figures. For evaluation of the half-maximal effective concentration ŽEC 50 . and Hill coefficient Ž n. of concentration–response curves, data were fitted to the Michaelis–Menten Eq. Ž1. using a least-squares fitting: I s Imax C nr Ž C n q K dn . ,
Ž 1.
where I is current, Imax is maximum response, n is Hill coefficient, C is the corresponding agonist concentration and K d is dissociation constant.
Ž 2.
where C is the concentration of the antagonists. Assuming that the value of Imax is 1, then I s 1 y C nr Ž C n q K dn . ,
Ž 3.
and I s K dnr Ž C n q K dn . ,
Ž 4.
where I represents the fraction of a current that remains after bicuculline, strychnine or Zn2q treatment. The data for the concentration–inhibition curves were fitted to Eq. Ž4. by using a least-squares fitting to obtain the half-maximal inhibition concentration ŽIC 50 ..
3. Results 3.1. GABA- and glycine(Gly)-induced responses in SDCN neurons In the present experimental conditions with pipette solution containing 150 mM CsCl, the isolated SDCN neurons had resting potentials in the range of y35 to y55 mV Ž n s 120. under a whole-cell current-clamp condition. When voltage clamped at y40 mV, all neurons tested responded to both GABA and glycine ŽGly. with inward currents. Both the inward currents recorded with the nystatin technique were stable in amplitude and time course for more than 1 h as long as the intervals between repeated
Fig. 1. GABA and glycine ŽGly. responses in SDCN neurons. ŽA. concentration–response curve for IGA BA . Inset shows actual currents induced by GABA at various concentrations. All currents were normalized to the peak response induced by 10y5 M GABA Ž).. Each point is the average of 6–8 neurons. Here and in subsequent figures the vertical bars show the "S.E.M. ŽB. Concentration dependence of IGly . All responses were normalized to the peak current obtained with 3 = 10y5 M Gly Ž).; six neurons were tested.
T.-L. Xu r Journal of the Autonomic NerÕous System 75 (1999) 156–163
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tude and GABA concentration is shown in Fig. 1A. The EC 50 and Hill coefficient Ž n. of the IGABA were 6.9 = 10y6 M and 1.21, respectively. Gly-evoked inward currents Ž IGly . became detectable at a concentration of about 3 = 10y6 M and increased in a sigmoidal fashion with increasing Gly concentration ŽFig. 1B.. The EC 50 and n of the IGly were 4.0 = 10y5 M and 1.27, respectively. 3.2. Current–Õoltage (I–V) relationships for IG A B A and IG l y
Fig. 2. Current–voltage Ž I – V . relationships for IGA BA and IGly . ECl represents the Cly equilibrium potential. Both curves were obtained from the same cell.
applications of GABA or Gly were longer than 2 min. The mean current amplitudes induced by 10y5 M GABA and 10y4 M Gly were 887 " 59 pA Ž n s 12. and 904 " 41 pA Ž n s 12., respectively. GABA-induced inward currents Ž IGA BA . increased steeply in amplitude as the concentration increased to 3 = 10y4 M, which gave a nearly maximal response. A further increase to 10y3 M gave little additional increase in the maximal response. The IGA BA was always followed by a rapid decline Ždesensitization. during a continuous application at concentrations above 10y5 M. The relationship between the peak current ampli-
The current–voltage Ž I–V . relationships for IGA BA and IGly were linear at membrane potentials between y60 and q60 mV ŽFig. 2.. The reversal potentials of IGA BA and IGly were y2.7 " 1.2 mV Ž n s 6. and y1.4 " 0.9 mV Ž n s 6., respectively. They were close to the Cly equilibrium potential Ž ECl . of y1.8 mV calculated from the Nernst equation, given the external and internal Cly concentration Ž161 and 150 mM, respectively.. These results indicated that the IGA BA and IGly in the SDCN neurons are passing through the Cly channels. 3.3. Effect of antagonists The above data show that the acutely dissociated SDCN neurons responded to both GABA and Gly to operate Cly channels. Since previous studies have characterized in detail the pharmacology of IGly in the SDCN neurons ŽXu et al., 1996; Wang et al., 1998., the following experiments were made to examine the pharmacological char-acteristics of IGA BA . The effects of bicuculline ŽBIC., strychnine
Fig. 3. Inhibition of IGA BA by bicuculline ŽBIC., strychnine ŽSTR. and Zn2q. ŽAa. IGABA was inhibited by BIC and STR. All antagonists were perfused for 30 s before simultaneous application with GABA. ŽAb. concentration–response curves for inhibition of IGA BA by BIC and STR. Each points represents the mean of five experiments. ŽBa. IGA BA in the presence of different concentrations of Zn2q. Zn2q was applied for 15 s before simultaneous application of GABA. ŽBb. concentration dependence of Zn2q inhibition on IGA BA . Each point is the mean from five neurons.
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blocks the Cly channels coupling to the GABA A receptors ŽPang et al., 1998.. In the presence of 10y4 M picrotoxin, the currents activated by 3 = 10y4 M PB and 3 = 10y6 M PGN decreased by 95 " 3% Ž n s 4. and 94 " 4% Ž n s 4., respectively. The concentration–response curve of I PB was sigmoidal with EC 50 of 3.5 = 10y4 M and n of 1.5 ŽFig. 4B.. At PB concentrations ) 10y3 M, the washout was often accompanied by a tail ‘hump’ current ŽFig. 4Ab., as previously described in the same preparation ŽPang et al., 1998.. The I PGN increased slowly to its peak, and it recovered slowly after wash. This was prominent at 10y5 M, where the current showed only a gradual decrease even with extensive washout ŽFig. 4Ac.. The concentration–response curve of I PGN was sigmoidal with EC 50 of 4.6 = 10y7 M and n of 1.3 ŽFig. 4B.. The reversal potentials of I PB and I PGN were also close to ECl , being y1.8 " 1.5 mV Ž n s 4. and 0.38 " 1.2 mV Ž n s 4., respectively. Baclofen, which is a GABA B receptor agonist, evoked no response at concentrations up to 10y4 M. 3.5. Allosteric potentiation of GABA A receptors Fig. 4. Currents activated by GABA A receptor agonists. ŽA. Inward currents elicited by muscimol Ža., pentobarbital ŽPB, b. and 5b-pregnan3a-ol-20-one Žpregnanolone, PGN, c.. The arrow in b indicates the tail ‘hump’ current after the washout of PB. ŽB. Concentration–response curves of muscimol-, PB- and PGN-activated responses. Peak amplitudes were normalized to that evoked by 10y5 M muscimol Ž).. Each point is the average of 5–7 neurons.
ŽSTR. and Zn2q were examined on the IGA BA of SDCN neurons. BIC, the selective GABA A receptor antagonist, inhibited 10y5 M IGA BA in a concentration-dependent manner, in which IC 50 was 3.1 = 10y6 M. STR, which is a specific Gly receptor antagonist, also suppressed IGA BA in a concentration dependent manner. STR was effective at concentrations higher than 10y7 M and the IC 50 was 9.9 = 10y6 M. BIC was three times more effective on the IGA BA than STR ŽFig. 3A.. On the other hand, Zn2q inhibited IGA BA with an IC 50 of 2.8 = 10y5 M ŽFig. 3B.. 3.4. GABA A receptor agonist-actiÕated responses Muscimol, a specific agonist of GABA A receptorP Cly channel complex, also induced the inward current in a concentration-dependent manner ŽFig. 4Aa and B.. The EC 50 and n of the muscimol response were 8.0 = 10y6 M and 0.76, respectively. The reversal potential of the muscimol induced current was y1.8 " 2.2 mV Ž n s 4.. Both pentobarbital ŽPB. and 5b-pregnan-3a-ol-one Žpregnanolone, PGN. induced inward currents in a concentration-dependent manner, with different properties from those of GABA and muscimol-induced currents ŽFig. 4Ab and Ac.. In contrast, diazepam ŽDZP. did not induce any current. Both PB-induced current Ž I PB . and PGN-induced current Ž I PGN . were reversibly blocked by picrotoxin, that
Because GABA A receptors are allosterically modulated by benzodiazepines, barbiturates and neurosteroids ŽXu et al., 1997., the effects of DZP, PB and PGN were examined on IGA BA of the SDCN neurons. All these modulators potentiated the IGA BA in a concentration-dependent manner, resulting in a parallel shift of the GABA concentration–response curves to the left without affecting the maximal response. As shown in Table 1, the EC 50 values decreased significantly, while the Hill coefficient Ž n. and the reversal potentials of the IGA BA Ž EGABA . in the presence of 3 = 10y8 M DZP, 3 = 10y5 M PB or 10y7 M PGN were not affected. The results indicated that the enhancement of IGA BA was mediated by the increase of the apparent affinity of the GABA A receptors to GABA. 3.6. Spontaneous GABAergic IPSCs in SDCN neurons In the presence of 3 = 10y7 M TTX, after ionotropic glutamate receptors were blocked with CNQX Ž3 = 10y6 M. and D-AP5 Ž10y5 M., spontaneous inhibitory postsynaptic currents ŽsIPSCs. were recorded in the mechanically dissociated SDCN neurons under whole-cell voltageclamp at a holding potential Ž V h . of y40 mV. Typical sIPSCs, inward currents under these recording conditions, Table 1 Allosteric modulatory properties of GABA A receptors in SDCN neurons EC 50 ŽM. Control DZP 3=10y8 M PB 3=10y5 M PGN 10y7 M
y6
9.0"1.3=10 2.8"0.6=10y6 2.5"1.3=10y6 2.3"1.7=10y6
Ž12. Ž4.) Ž4.) Ž4.)
n
EGABA ŽmV.
1.2"0.2 Ž12. 1.0"0.1 Ž4. 1.1"0.2 Ž4. 1.2"0.1 Ž4.
y2.3"2.2 Ž12. y2.7"1.8 Ž4. y1.9"0.9 Ž4. y0.3"1.1 Ž4.
The number of experiments is shown in the parenthesis. ) P - 0.05 compared with control ŽANOVA test..
T.-L. Xu r Journal of the Autonomic NerÕous System 75 (1999) 156–163
Fig. 5. Spontaneous GABAergic and glycinergic IPSCs recorded in the presence of 3=10y7 M TTX, 3=10y6 M CNQX and 10y5 M D-AP5. ŽA. BIC completely blocked the STR-resistant sIPSCs. The representative results were obtained from other nine neurons. ŽB. In this neuron, 10y6 M STR abolished all the sIPSCs.
are shown in Fig. 5. Bath perfusion with the glycine receptor antagonist STR partially blocked the sIPSCs. After adding 10y6 M BIC, the GABA A receptor antagonist, the remained sIPSCs were completely abolished, indicating that they are GABAergic sIPSCs ŽFig. 5A.. In a small population of neurons Ž3r13., application of 10y6 M STR abolished all the sIPSCs ŽFig. 5B.. We attribute that, at least in part, to the reduction of GABAergic sIPSCs by STR ŽFig. 3A. to a level below our threshold of detection.
4. Discussion GABA receptors in the CNS are divided into three types: Ž1. GABA A receptors, which are BIC-sensitive, directly coupled with an integral Cly channel, and are potentiated by allosteric modulators, such as benzodiazepines, barbiturates and neurosteroids ŽXu et al., 1997.; Ž2. GABA B receptors, which are BIC-insensitive and indirectly coupled with Kq andror Ca2q channels through GTP-binding proteins ŽBormann, 1988.; and Ž3. GABA C receptors, which operate as BIC-insensitive Cly channel, and are not potentiated by allosteric modulators of GABA A receptors. The IGA BA in the acutely dissociated SDCN neurons was mimicked by muscimol, a selective GABA A receptor agonist, and antagonized by BIC in a concentration-dependent manner. The IGABA was carried by Cly and allosterically potentiated by diazepam ŽDZP., pento-
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barbital ŽPB. and 5b-pregnan-3a-ol-20-one Žpregnanolone, PGN.. On the other hand, the specific GABA B receptor agonist, baclofen, evoked no response in the present preparation. Although the lack of the response evoked by baclofen under the present experimental conditions is not indicative that the cells lack GABA B receptors, the results clearly show that the IGA BA is mediated by GABA A receptors. Further evidence in favor of this conclusion is provided by recent immunohistochemical studies ŽAlvarez et al., 1996; Bohlhalter et al., 1996; Todd et al., 1996.. We also recorded spontaneous synaptic currents mediated by GABA A receptors from neurons isolated from the SDCN ŽFig. 5.. Since response of an isolated neuron to an applied transmitter does not confirm its role as a natural transmitter, the ‘synaptic bouton preparation’ Žsee Section 2. allows this determination. It is proposed that the STR-sensitive current fluctuations are caused by a spontaneous release of Gly from presynaptic boutons that have remained attached to a neuron during its isolation, and that the BIC-sensitive activity is caused by inhibitory GABAergic inputs to these neurons. STR has been widely used as a specific blocker of Gly receptors, and BIC as a specific blocker of GABA A receptors. However, there is evidence indicating that these assignments are not absolute. For example, STR has much greater affinity than BIC for Gly receptor ŽWang et al., 1998., but both STR and BIC can block certain classes of neuronal acetylcholine receptors ŽZhang and Feltz, 1991.. Fig. 3A shows that STR also suppressed IGA BA in a concentration dependent manner, though BIC was three times more effective on the IGA BA than STR. Therefore, while the compounds retain diagnostic value, none of them alone should be considered definitive for determining the class of receptors mediating transmission at a synapse. Previous reports propose that Zn2q-sensitivity is a marker for a g subunit being present in GABA A receptors. In this study, Zn2q blocked the IGA BA with an IC 50 of 28 mM. The IC 50 for Zn2q inhibition of GABA currents induced by the activation of reconstituted GABA A receptors lacking g2 subunit was much lower compared with the value obtained here. For the subunit composition of a1b1 the IC 50 was 1.5 mM ŽSmart et al., 1991., whereas for the composition of a1b2 the IC 50 was 0.56 mM ŽDraguhn et al., 1990.. The low sensitivity to Zn2q inhibition observed here probably reflects the presence of the g2 subunit in the SDCN neurons. However, the study by White and Gurley Ž1995. indicated that the type of a subunit has strong influences on the Zn2q sensitivity. To elucidate definitely which subtypes of GABA A receptors present in the SDCN still needs further experiments. In the SDCN neurons, the anesthetic agents PB and PGN induced GABA A -mimic currents, which increased in a concentration-dependent manner. Moreover, PB, PGN and DZP acted on the GABA A -receptor Cly channel complex to augment the IGA BA by increasing the apparent affinity of GABA A receptors to GABA. The present re-
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sults together with other observations ŽCartmell and Mitchell, 1994; Cleland et al., 1994; Frye and Duncan, 1994; Collins et al., 1995; Pang et al., 1998. support the idea that GABA A receptors are the common targets for numerous analgesic and anesthetic agents throughout the CNS and anesthetic-induced changes could happen not only at the supraspinal sites but also at the spinal level ŽCollins et al., 1995.. In addition to extracellular allosteric modulation, GABA A receptors are modulated by a lot of intracellular substances such as Ca2q, ATP, cAMP-protein kinase ŽPKA., protein kinase C ŽPKC., Ca2q-calmodulin dependent protein kinase II, protein phosphatases and tyrosine protein kinases ŽSmart, 1997.. GABA A receptors are in situ thus under extensive modulation both extracellularly and intracellularly. The spinal dorsal horn receives serotonergic and GABAergic innervation form brainstem descending pathways ŽMillhorn et al., 1987; Sorkin et al., 1993; Antal et al., 1996., raising the possibility that the GABA A receptors are functionally modulated by serotonin Ž5-HT.. Indeed, we have recently found that the stimulation of 5-HT2 receptors by 5-HT facilitates GABA A responses via intracellular PKC in the SDCN neurons ŽXu et al., 1998b.. Still more modulations of GABA A receptor in the SDCN neurons are expected to be revealed, since a number of endogenous substances such as noradrenaline, substance P ŽSP., dopamine, acetylcholine, dynorphin, enkaphalin, somatostatin and cholecystokinin ŽSasek et al., 1984; Xu, 1997., which may modulate the intracellular signal transductions, are also widely distributed in the SDCN region. Many spinal neurons are inhibited by GABA and glycine, and certain types of evoked inhibition can be blocked by both BIC and STR ŽYoshimura and Nishi, 1995., which indicates that the inhibition is mediated through GABA A and glycine receptors. In the present study, we have shown that both glycinergic and GABAergic sIPSCs are present in SDCN neurons ŽFig. 5. and that SDCN neurons responded to both exogenously applied GABA and Gly, which activate GABA A and STR-sensitive Gly receptors, respectively. The results extend those from immunohistochemical studies of the colocalization of GABA and Gly as well as their receptors in the synapses of spinal cord ŽTodd et al., 1996., suggesting that functional GABAergic and glycinergic cotransmission could occur in the SDCN. Recent work has shown that spinal interneurons release both Gly and GABA to activate functionally distinct receptors in their postsynaptic target cells ŽJonas et al., 1998.. In summary, the present study demonstrated that the acutely dissociated SDCN neurons responded directly to both exogenously applied and endogenously released GABA to operate GABA A receptorP Cly channel complexes. The pharmacological properties of the GABA A receptors resemble those described in supraspinal central areas, suggesting that not only Gly but also GABA has a functional role as inhibitory transmitter in the spinal cord
even in the lowest area such as SDCN. Given that SDCN receives GABAergic innervation from focal circuits andror the descending pathway ŽYoshimura and Nishi, 1995; Antal et al., 1996., the GABA A receptor-mediated transmission to the SDCN neurons may have important functional roles in regulating nociceptive, analgesic, and autonomic functions.
Acknowledgements I thank Ms Min Li for critical reading of the manuscript and Dr Zhi-Ping Pang for assistance in figure and table preparations. This work was supported in part by Grant-inAid from the National Natural Science Foundation of China ŽNo. 39770248..
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g-Aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus Tian-Le Xu b
a,b,)
a Neuropharmacology Unit, Department of Anatomy, The Fourth Military Medical UniÕersity, Xi’an 710032, China Department of Neurobiology & Biophysics, School of Life Sciences, UniÕersity of Science and Technology of China, Hefei 230027, China
Received 6 October 1998; revised 23 November 1998; accepted 30 November 1998
Abstract The electrophysiological and pharmacological properties of GABA-activated Cly currents Ž IGABA . were investigated in enzymatically dissociated rat sacral dorsal commissural nucleus ŽSDCN. neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. Exogenous application of GABA to SDCN neurons induced Cly currents which increased in a concentrationdependent manner. Bicuculline ŽBIC. and strychnine ŽSTR. antagonized the IGABA in a concentration-dependent manner. Zn2q suppressed the IGABA with an IC 50 of 2.8 = 10y5 M. Muscimol mimicked the IGABA, while baclofen evoked no response. Pentobarbital ŽPB. and 5b-pregnan-3a-ol-20-one Žpregnanolone, PGN. also induced GABA A-mimic Cly currents. Diazepam ŽDZP., PB and PGN all enhanced the IGABA by increasing the apparent affinity of the GABA A receptors to GABA. Moreover, spontaneous GABAergic inhibitory postsynaptic currents ŽIPSCs. were observed in mechanically dissociated SDCN neurons attached with synaptic boutons, so called ‘synaptic bouton preparation’. These results indicate that SDCN neurons express GABA A receptors with relatively low sensitivity to Zn2q inhibition, and that GABA may have a functional role as an inhibitory transmitter in the SDCN regulating nociceptive, analgesic, and autonomic functions. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sacral dorsal commissural nucleus; GABA A receptor; Cly current; Allosteric modulation; Cotransmission; Acutely dissociated neurons; Synaptic bouton preparation; Nystatin perforated patch clamp
1. Introduction The sacral dorsal commissural nucleus ŽSDCN. is involved in nociceptive, analgesic, and autonomic functions. Noxious chemical irritation of the bladder ŽLu¨ et al., 1995. increases the expression of c-fos-like protein ŽFOS. in the SDCN. Nicotinamide adenine dinucleotide phosphate diaphorase ŽNADPH-d. positive cell bodies and fiber networks are densely stained in the SDCN, and nitric oxide synthase immunoreactivity in this region can be upregulated by peripheral visceral nerve injury ŽVizzard et al., 1995.. Substance P ŽSP., which is generally believed to be involved in nociception, has been immunohistochemically localized in small-diameter primary afferent terminals to the SDCN. By combining SP receptor ŽSPR. with FOS ) Department of Neurobiology & Biophysics, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. Tel.: q 86-551-360-3510; fax: q 86-551-360-3142; e-m ail: [email protected]
immunohistochemistry techniques, we had demonstrated that a subpopulation of SDCN neurons receiving noxious information from the urinary bladder express SPR ŽLu¨ et al., 1995.. The SDCN receives descending projections from several regions of the brain known to modulate the spinal nociception ŽXu et al., 1996; Xu, 1997.. The SDCN also receives abundant afferent inputs from visceral organs ŽHonda, 1985; Nadelhaft and Vera, 1995; Katter et al., 1996.. Recently, a direct projection from rat SDCN to Barrington’s nucleus Žthe pontine micturition center in the pons. has been reported ŽDing et al., 1997.. g-Aminobutyric acid ŽGABA. and glycine are very important for the process of fast inhibitory synaptic transmission in the central nervous system ŽCNS.. The importance of GABA A receptors in supraspinal regions of the CNS in vertebrates has been well recognized. For inhibitory neurotransmission in the spinal cord, however, glycinergic mechanisms have generally been considered to be the major component and thus received much emphasis in the past. Recent morphological, physiological and phar-
0165-1838r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 8 7 - 8
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macological evidence indicates that GABAergic mechanisms also contribute to the inhibitory influences on the spinal cord ŽYoshimura and Nishi, 1995; Alvarez et al., 1996; Antal et al., 1996; Bohlhalter et al., 1996; Malcangio and Bowery, 1996.. GABA and its receptors are implicated in modulation of autonomic function ŽBereiter and Gann, 1989; Lewis and Coote, 1995; Barron et al., 1997; Blok et al., 1997. as well as nociception ŽMcGowan and Hammond, 1993; Lin et al., 1994; Malcangio and Bowery, 1996; Xu et al., 1998b.. However, direct current responses of SDCN neurons to GABA application have not been characterized. In addition, while modulation of GABA A receptors by benzodiazepines ŽCartmell and Mitchell, 1994., barbiturates ŽCleland et al., 1994., or neurosteroids ŽFrye and Duncan, 1994. have the potential for significant influences on spinal nociception, there is little information about the physiological effects of modulation of GABA A receptors in the SDCN. Using isolated central neurons and the voltage-clamp techniques, indirect effects via other cells or via changes in membrane potential can be excluded ŽXu et al., 1996, 1998a.. In the present study, tight-seal nystatin perforated patch recording was used to investigate the electrophysiological and pharmacological properties of GABA-induced Cly currents Ž IGA BA . in acutely dissociated SDCN neurons.
2. Materials and methods 2.1. Preparation The sacral dorsal commissural nucleus ŽSDCN. neurons were acutely dissociated as described elsewhere ŽXu et al., 1998a.. Briefly, 2-week-old Wistar rats were decapitated under pentobarbitone-sodium anesthesia Ž50 mg kgy1 i.p... A segment of lumbosacral ŽL 5 –S 3 . spinal cord was dissected out and sectioned with a vibratome tissue slicer ŽDTK-1000, Dosaka. to yield several transverse slices Ž400 mm thick. containing the SDCN region. The slices were preincubated in oxygenated incubation solution Žsee below. for 50 min at room temperature Ž22–258C.. Thereafter, slices were treated enzymatically in oxygenated incubation solution containing 1 mgr5 ml pronase for 20 min at 318C followed by exposure to 1 mgr5 ml thermolysin for another 15 min in the same conditions. After the enzyme treatment, the slices were kept in enzyme-free incubation solution for 1 h. Then a portion of SDCN region was micro-punched out with an electrolytically polished injection needle and transferred into a culture dish filled with standard external solution Žsee below.. Neurons were mechanically dissociated with fire-polished Pasteur pipettes under visual guidance under a phase contrast microscope ŽIX 70, Olympus.. For recording spontaneous synaptic currents, the neurons were mechanically dissociated in some experiments to
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obtain SDCN neurons with synaptic boutons attached ŽDrewe et al., 1988; Xu and Li, 1997., so called ‘synaptic bouton preparation’. Briefly, the slices were transferred into the dish and a fire-polished glass pipette was touched lightly onto the surface of the SDCN. The pipette was vibrated horizontally at about 3–6 Hz for about 1 min, and then slices were removed. The mechanically andror enzymatically dissociated neurons adhered to the bottom of the dish within 20 min, allowing the electrophysiological studies to be conducted. 2.2. Solutions The composition of incubation solution was ŽmM.: NaCl 124, NaHCO 3 24, KCl 5, KH 2 PO4 1.2, CaCl 2 2.4, MgSO4 1.3 and glucose 10, aerated with 95% O 2 –5% CO 2 to a final pH of 7.4. The normal external standard solution was ŽmM.: NaCl 150, KCl 5, CaCl 2 2, MgCl 2 1, N-2-hydroxyethylpiperazine-N X-2-ethanesulphonic acid ŽHEPES. 10 and glucose 10. The pH was adjusted to 7.4 with tris–hydroxymethyl aminomethane ŽTris–base.. The patch-pipette solution for nystatin perforated patch recording was ŽmM.: CsCl 150 and HEPES 10. The pH was adjusted to 7.2 with Tris–base. A nystatin stock solution dissolved in acidified methanol at concentration of 10 mgrml was prepared and stored at y208C. The stock solution was added to the patch-pipette solution in a final nystatin concentration of 200 mgrml just before use. Voltage-activated Naq, Ca2q and Kq channels were blocked by adding 3 = 10y7 M tetrodotoxin ŽTTX. and 10y5 M CdCl 2 in the standard external solution and by using Csq-containing internal solution, when the current– voltage Ž I–V . relationships for GABA-and glycine-induced Cly currents were examined. CdCl 2 had no noticeable effect on the GABA- and glycine-induced currents at the concentration used. 2.3. Perforated patch recording Electrical measurements were carried out by using nystatin perforated patch recording configuration under voltage-clamp condition at room temperature Ž22–258C.. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller ŽPB-7, Narishige.. The resistance between the recording electrode filled with a pipette solution and the reference electrode was 4–6 M V. Series resistance checked every 10 min was 10–30 M V. The change in series resistance through recording was less than 10%. The electrode was connected to a patch-clamp amplifier ŽCEZ-2300, Nihon Koden., and filtered at 1 kHz. The current and voltage signals were monitored with a pen recorder ŽOmniace RT 3108, San-ei., sampled and analyzed using a DigiData 1200A interface and a computer with pCLAMP 6.0.2 program ŽAxon Instruments.. Except when examining I–V relationships, the membrane potential was held at y40 mV throughout the
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experiment. This potential relates to the resting potentials of the isolated SDCN neurons recorded with the Csq-containing pipette solution Žsee Section 3..
The equation for concentration–inhibition curves of bicuculline, strychnine and Zn2q is the mirror image of the Michaelis–Menten equation:
2.4. Drugs and their application
IrImax s 1 y C nr Ž C n q K dn . ,
The following drugs were used in the present experiments: pronase ŽCalbiochem.; GABA, glycine, diazepam and pentobarbital ŽKasei, Tokyo.; muscimol, thermolysin, nystatin, bicuculline, strychnine, 5b-pregnan-3a-ol-20-one Žpregnanolone., CNQX, D-AP5 and TTX ŽSigma.. Diazepam and pregnanolone were first dissolved in dimethyl sulfoxide ŽDMSO. at 10y2 M and then diluted to the respective final concentrations in the normal standard solution just before use. The maximal concentration of DMSO was less than 0.3%, at which concentration it did not affect the GABA-induced current. Rapid application of the drugs was performed via a ‘Y-tube’ ŽXu et al., 1996.. This system allows a complete exchange of external solution surrounding a neuron within 20 ms. 2.5. Statistical analysis Data were calculated as mean " standard error ŽS.E.M.. and the S.E.M. is indicated by a vertical bar in the figures. For evaluation of the half-maximal effective concentration ŽEC 50 . and Hill coefficient Ž n. of concentration–response curves, data were fitted to the Michaelis–Menten Eq. Ž1. using a least-squares fitting: I s Imax C nr Ž C n q K dn . ,
Ž 1.
where I is current, Imax is maximum response, n is Hill coefficient, C is the corresponding agonist concentration and K d is dissociation constant.
Ž 2.
where C is the concentration of the antagonists. Assuming that the value of Imax is 1, then I s 1 y C nr Ž C n q K dn . ,
Ž 3.
and I s K dnr Ž C n q K dn . ,
Ž 4.
where I represents the fraction of a current that remains after bicuculline, strychnine or Zn2q treatment. The data for the concentration–inhibition curves were fitted to Eq. Ž4. by using a least-squares fitting to obtain the half-maximal inhibition concentration ŽIC 50 ..
3. Results 3.1. GABA- and glycine(Gly)-induced responses in SDCN neurons In the present experimental conditions with pipette solution containing 150 mM CsCl, the isolated SDCN neurons had resting potentials in the range of y35 to y55 mV Ž n s 120. under a whole-cell current-clamp condition. When voltage clamped at y40 mV, all neurons tested responded to both GABA and glycine ŽGly. with inward currents. Both the inward currents recorded with the nystatin technique were stable in amplitude and time course for more than 1 h as long as the intervals between repeated
Fig. 1. GABA and glycine ŽGly. responses in SDCN neurons. ŽA. concentration–response curve for IGA BA . Inset shows actual currents induced by GABA at various concentrations. All currents were normalized to the peak response induced by 10y5 M GABA Ž).. Each point is the average of 6–8 neurons. Here and in subsequent figures the vertical bars show the "S.E.M. ŽB. Concentration dependence of IGly . All responses were normalized to the peak current obtained with 3 = 10y5 M Gly Ž).; six neurons were tested.
T.-L. Xu r Journal of the Autonomic NerÕous System 75 (1999) 156–163
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tude and GABA concentration is shown in Fig. 1A. The EC 50 and Hill coefficient Ž n. of the IGABA were 6.9 = 10y6 M and 1.21, respectively. Gly-evoked inward currents Ž IGly . became detectable at a concentration of about 3 = 10y6 M and increased in a sigmoidal fashion with increasing Gly concentration ŽFig. 1B.. The EC 50 and n of the IGly were 4.0 = 10y5 M and 1.27, respectively. 3.2. Current–Õoltage (I–V) relationships for IG A B A and IG l y
Fig. 2. Current–voltage Ž I – V . relationships for IGA BA and IGly . ECl represents the Cly equilibrium potential. Both curves were obtained from the same cell.
applications of GABA or Gly were longer than 2 min. The mean current amplitudes induced by 10y5 M GABA and 10y4 M Gly were 887 " 59 pA Ž n s 12. and 904 " 41 pA Ž n s 12., respectively. GABA-induced inward currents Ž IGA BA . increased steeply in amplitude as the concentration increased to 3 = 10y4 M, which gave a nearly maximal response. A further increase to 10y3 M gave little additional increase in the maximal response. The IGA BA was always followed by a rapid decline Ždesensitization. during a continuous application at concentrations above 10y5 M. The relationship between the peak current ampli-
The current–voltage Ž I–V . relationships for IGA BA and IGly were linear at membrane potentials between y60 and q60 mV ŽFig. 2.. The reversal potentials of IGA BA and IGly were y2.7 " 1.2 mV Ž n s 6. and y1.4 " 0.9 mV Ž n s 6., respectively. They were close to the Cly equilibrium potential Ž ECl . of y1.8 mV calculated from the Nernst equation, given the external and internal Cly concentration Ž161 and 150 mM, respectively.. These results indicated that the IGA BA and IGly in the SDCN neurons are passing through the Cly channels. 3.3. Effect of antagonists The above data show that the acutely dissociated SDCN neurons responded to both GABA and Gly to operate Cly channels. Since previous studies have characterized in detail the pharmacology of IGly in the SDCN neurons ŽXu et al., 1996; Wang et al., 1998., the following experiments were made to examine the pharmacological char-acteristics of IGA BA . The effects of bicuculline ŽBIC., strychnine
Fig. 3. Inhibition of IGA BA by bicuculline ŽBIC., strychnine ŽSTR. and Zn2q. ŽAa. IGABA was inhibited by BIC and STR. All antagonists were perfused for 30 s before simultaneous application with GABA. ŽAb. concentration–response curves for inhibition of IGA BA by BIC and STR. Each points represents the mean of five experiments. ŽBa. IGA BA in the presence of different concentrations of Zn2q. Zn2q was applied for 15 s before simultaneous application of GABA. ŽBb. concentration dependence of Zn2q inhibition on IGA BA . Each point is the mean from five neurons.
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T.-L. Xu r Journal of the Autonomic NerÕous System 75 (1999) 156–163
blocks the Cly channels coupling to the GABA A receptors ŽPang et al., 1998.. In the presence of 10y4 M picrotoxin, the currents activated by 3 = 10y4 M PB and 3 = 10y6 M PGN decreased by 95 " 3% Ž n s 4. and 94 " 4% Ž n s 4., respectively. The concentration–response curve of I PB was sigmoidal with EC 50 of 3.5 = 10y4 M and n of 1.5 ŽFig. 4B.. At PB concentrations ) 10y3 M, the washout was often accompanied by a tail ‘hump’ current ŽFig. 4Ab., as previously described in the same preparation ŽPang et al., 1998.. The I PGN increased slowly to its peak, and it recovered slowly after wash. This was prominent at 10y5 M, where the current showed only a gradual decrease even with extensive washout ŽFig. 4Ac.. The concentration–response curve of I PGN was sigmoidal with EC 50 of 4.6 = 10y7 M and n of 1.3 ŽFig. 4B.. The reversal potentials of I PB and I PGN were also close to ECl , being y1.8 " 1.5 mV Ž n s 4. and 0.38 " 1.2 mV Ž n s 4., respectively. Baclofen, which is a GABA B receptor agonist, evoked no response at concentrations up to 10y4 M. 3.5. Allosteric potentiation of GABA A receptors Fig. 4. Currents activated by GABA A receptor agonists. ŽA. Inward currents elicited by muscimol Ža., pentobarbital ŽPB, b. and 5b-pregnan3a-ol-20-one Žpregnanolone, PGN, c.. The arrow in b indicates the tail ‘hump’ current after the washout of PB. ŽB. Concentration–response curves of muscimol-, PB- and PGN-activated responses. Peak amplitudes were normalized to that evoked by 10y5 M muscimol Ž).. Each point is the average of 5–7 neurons.
ŽSTR. and Zn2q were examined on the IGA BA of SDCN neurons. BIC, the selective GABA A receptor antagonist, inhibited 10y5 M IGA BA in a concentration-dependent manner, in which IC 50 was 3.1 = 10y6 M. STR, which is a specific Gly receptor antagonist, also suppressed IGA BA in a concentration dependent manner. STR was effective at concentrations higher than 10y7 M and the IC 50 was 9.9 = 10y6 M. BIC was three times more effective on the IGA BA than STR ŽFig. 3A.. On the other hand, Zn2q inhibited IGA BA with an IC 50 of 2.8 = 10y5 M ŽFig. 3B.. 3.4. GABA A receptor agonist-actiÕated responses Muscimol, a specific agonist of GABA A receptorP Cly channel complex, also induced the inward current in a concentration-dependent manner ŽFig. 4Aa and B.. The EC 50 and n of the muscimol response were 8.0 = 10y6 M and 0.76, respectively. The reversal potential of the muscimol induced current was y1.8 " 2.2 mV Ž n s 4.. Both pentobarbital ŽPB. and 5b-pregnan-3a-ol-one Žpregnanolone, PGN. induced inward currents in a concentration-dependent manner, with different properties from those of GABA and muscimol-induced currents ŽFig. 4Ab and Ac.. In contrast, diazepam ŽDZP. did not induce any current. Both PB-induced current Ž I PB . and PGN-induced current Ž I PGN . were reversibly blocked by picrotoxin, that
Because GABA A receptors are allosterically modulated by benzodiazepines, barbiturates and neurosteroids ŽXu et al., 1997., the effects of DZP, PB and PGN were examined on IGA BA of the SDCN neurons. All these modulators potentiated the IGA BA in a concentration-dependent manner, resulting in a parallel shift of the GABA concentration–response curves to the left without affecting the maximal response. As shown in Table 1, the EC 50 values decreased significantly, while the Hill coefficient Ž n. and the reversal potentials of the IGA BA Ž EGABA . in the presence of 3 = 10y8 M DZP, 3 = 10y5 M PB or 10y7 M PGN were not affected. The results indicated that the enhancement of IGA BA was mediated by the increase of the apparent affinity of the GABA A receptors to GABA. 3.6. Spontaneous GABAergic IPSCs in SDCN neurons In the presence of 3 = 10y7 M TTX, after ionotropic glutamate receptors were blocked with CNQX Ž3 = 10y6 M. and D-AP5 Ž10y5 M., spontaneous inhibitory postsynaptic currents ŽsIPSCs. were recorded in the mechanically dissociated SDCN neurons under whole-cell voltageclamp at a holding potential Ž V h . of y40 mV. Typical sIPSCs, inward currents under these recording conditions, Table 1 Allosteric modulatory properties of GABA A receptors in SDCN neurons EC 50 ŽM. Control DZP 3=10y8 M PB 3=10y5 M PGN 10y7 M
y6
9.0"1.3=10 2.8"0.6=10y6 2.5"1.3=10y6 2.3"1.7=10y6
Ž12. Ž4.) Ž4.) Ž4.)
n
EGABA ŽmV.
1.2"0.2 Ž12. 1.0"0.1 Ž4. 1.1"0.2 Ž4. 1.2"0.1 Ž4.
y2.3"2.2 Ž12. y2.7"1.8 Ž4. y1.9"0.9 Ž4. y0.3"1.1 Ž4.
The number of experiments is shown in the parenthesis. ) P - 0.05 compared with control ŽANOVA test..
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Fig. 5. Spontaneous GABAergic and glycinergic IPSCs recorded in the presence of 3=10y7 M TTX, 3=10y6 M CNQX and 10y5 M D-AP5. ŽA. BIC completely blocked the STR-resistant sIPSCs. The representative results were obtained from other nine neurons. ŽB. In this neuron, 10y6 M STR abolished all the sIPSCs.
are shown in Fig. 5. Bath perfusion with the glycine receptor antagonist STR partially blocked the sIPSCs. After adding 10y6 M BIC, the GABA A receptor antagonist, the remained sIPSCs were completely abolished, indicating that they are GABAergic sIPSCs ŽFig. 5A.. In a small population of neurons Ž3r13., application of 10y6 M STR abolished all the sIPSCs ŽFig. 5B.. We attribute that, at least in part, to the reduction of GABAergic sIPSCs by STR ŽFig. 3A. to a level below our threshold of detection.
4. Discussion GABA receptors in the CNS are divided into three types: Ž1. GABA A receptors, which are BIC-sensitive, directly coupled with an integral Cly channel, and are potentiated by allosteric modulators, such as benzodiazepines, barbiturates and neurosteroids ŽXu et al., 1997.; Ž2. GABA B receptors, which are BIC-insensitive and indirectly coupled with Kq andror Ca2q channels through GTP-binding proteins ŽBormann, 1988.; and Ž3. GABA C receptors, which operate as BIC-insensitive Cly channel, and are not potentiated by allosteric modulators of GABA A receptors. The IGA BA in the acutely dissociated SDCN neurons was mimicked by muscimol, a selective GABA A receptor agonist, and antagonized by BIC in a concentration-dependent manner. The IGABA was carried by Cly and allosterically potentiated by diazepam ŽDZP., pento-
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barbital ŽPB. and 5b-pregnan-3a-ol-20-one Žpregnanolone, PGN.. On the other hand, the specific GABA B receptor agonist, baclofen, evoked no response in the present preparation. Although the lack of the response evoked by baclofen under the present experimental conditions is not indicative that the cells lack GABA B receptors, the results clearly show that the IGA BA is mediated by GABA A receptors. Further evidence in favor of this conclusion is provided by recent immunohistochemical studies ŽAlvarez et al., 1996; Bohlhalter et al., 1996; Todd et al., 1996.. We also recorded spontaneous synaptic currents mediated by GABA A receptors from neurons isolated from the SDCN ŽFig. 5.. Since response of an isolated neuron to an applied transmitter does not confirm its role as a natural transmitter, the ‘synaptic bouton preparation’ Žsee Section 2. allows this determination. It is proposed that the STR-sensitive current fluctuations are caused by a spontaneous release of Gly from presynaptic boutons that have remained attached to a neuron during its isolation, and that the BIC-sensitive activity is caused by inhibitory GABAergic inputs to these neurons. STR has been widely used as a specific blocker of Gly receptors, and BIC as a specific blocker of GABA A receptors. However, there is evidence indicating that these assignments are not absolute. For example, STR has much greater affinity than BIC for Gly receptor ŽWang et al., 1998., but both STR and BIC can block certain classes of neuronal acetylcholine receptors ŽZhang and Feltz, 1991.. Fig. 3A shows that STR also suppressed IGA BA in a concentration dependent manner, though BIC was three times more effective on the IGA BA than STR. Therefore, while the compounds retain diagnostic value, none of them alone should be considered definitive for determining the class of receptors mediating transmission at a synapse. Previous reports propose that Zn2q-sensitivity is a marker for a g subunit being present in GABA A receptors. In this study, Zn2q blocked the IGA BA with an IC 50 of 28 mM. The IC 50 for Zn2q inhibition of GABA currents induced by the activation of reconstituted GABA A receptors lacking g2 subunit was much lower compared with the value obtained here. For the subunit composition of a1b1 the IC 50 was 1.5 mM ŽSmart et al., 1991., whereas for the composition of a1b2 the IC 50 was 0.56 mM ŽDraguhn et al., 1990.. The low sensitivity to Zn2q inhibition observed here probably reflects the presence of the g2 subunit in the SDCN neurons. However, the study by White and Gurley Ž1995. indicated that the type of a subunit has strong influences on the Zn2q sensitivity. To elucidate definitely which subtypes of GABA A receptors present in the SDCN still needs further experiments. In the SDCN neurons, the anesthetic agents PB and PGN induced GABA A -mimic currents, which increased in a concentration-dependent manner. Moreover, PB, PGN and DZP acted on the GABA A -receptor Cly channel complex to augment the IGA BA by increasing the apparent affinity of GABA A receptors to GABA. The present re-
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sults together with other observations ŽCartmell and Mitchell, 1994; Cleland et al., 1994; Frye and Duncan, 1994; Collins et al., 1995; Pang et al., 1998. support the idea that GABA A receptors are the common targets for numerous analgesic and anesthetic agents throughout the CNS and anesthetic-induced changes could happen not only at the supraspinal sites but also at the spinal level ŽCollins et al., 1995.. In addition to extracellular allosteric modulation, GABA A receptors are modulated by a lot of intracellular substances such as Ca2q, ATP, cAMP-protein kinase ŽPKA., protein kinase C ŽPKC., Ca2q-calmodulin dependent protein kinase II, protein phosphatases and tyrosine protein kinases ŽSmart, 1997.. GABA A receptors are in situ thus under extensive modulation both extracellularly and intracellularly. The spinal dorsal horn receives serotonergic and GABAergic innervation form brainstem descending pathways ŽMillhorn et al., 1987; Sorkin et al., 1993; Antal et al., 1996., raising the possibility that the GABA A receptors are functionally modulated by serotonin Ž5-HT.. Indeed, we have recently found that the stimulation of 5-HT2 receptors by 5-HT facilitates GABA A responses via intracellular PKC in the SDCN neurons ŽXu et al., 1998b.. Still more modulations of GABA A receptor in the SDCN neurons are expected to be revealed, since a number of endogenous substances such as noradrenaline, substance P ŽSP., dopamine, acetylcholine, dynorphin, enkaphalin, somatostatin and cholecystokinin ŽSasek et al., 1984; Xu, 1997., which may modulate the intracellular signal transductions, are also widely distributed in the SDCN region. Many spinal neurons are inhibited by GABA and glycine, and certain types of evoked inhibition can be blocked by both BIC and STR ŽYoshimura and Nishi, 1995., which indicates that the inhibition is mediated through GABA A and glycine receptors. In the present study, we have shown that both glycinergic and GABAergic sIPSCs are present in SDCN neurons ŽFig. 5. and that SDCN neurons responded to both exogenously applied GABA and Gly, which activate GABA A and STR-sensitive Gly receptors, respectively. The results extend those from immunohistochemical studies of the colocalization of GABA and Gly as well as their receptors in the synapses of spinal cord ŽTodd et al., 1996., suggesting that functional GABAergic and glycinergic cotransmission could occur in the SDCN. Recent work has shown that spinal interneurons release both Gly and GABA to activate functionally distinct receptors in their postsynaptic target cells ŽJonas et al., 1998.. In summary, the present study demonstrated that the acutely dissociated SDCN neurons responded directly to both exogenously applied and endogenously released GABA to operate GABA A receptorP Cly channel complexes. The pharmacological properties of the GABA A receptors resemble those described in supraspinal central areas, suggesting that not only Gly but also GABA has a functional role as inhibitory transmitter in the spinal cord
even in the lowest area such as SDCN. Given that SDCN receives GABAergic innervation from focal circuits andror the descending pathway ŽYoshimura and Nishi, 1995; Antal et al., 1996., the GABA A receptor-mediated transmission to the SDCN neurons may have important functional roles in regulating nociceptive, analgesic, and autonomic functions.
Acknowledgements I thank Ms Min Li for critical reading of the manuscript and Dr Zhi-Ping Pang for assistance in figure and table preparations. This work was supported in part by Grant-inAid from the National Natural Science Foundation of China ŽNo. 39770248..
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