Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3097-3101, April 1994 Neurobiology Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes ANDRAS MORALES*, QUOC-THANG NGUYEN, AND RICARDO MILEDI Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717 Contributed by Ricardo Miledi, December 30, 1993 ABSTRACT The properties of glycine receptors (GlyRs) quickly frozen in liquid nitrogen and stored at -800C. New- from newborn and adult rat spinal cord were studied in born rats (postnatal days 0-3) were deeply anaesthetized with Xenopus oocytes injected with whole mRNA or the heavy (H) ether and decapitated and spinal cords were dissected as or light (L) mRNA fractions encig their respective GlyRs. above. Mean open times and conductances of channels gated by H- or mINA Isolation and Size Fractionation. mRNA preparation L-GlyRs were determined by noise analysis or voltage jumps. and size separation on sucrose gradients were as described We found that adult H- and L-GlyRs opened channels that (2). Briefly, tissues were homogenized in phenol-saturated differed in their mean open time but had the same channel homogenization buffer (200 mM Tris-HCl/50 mM NaCl/10 conductance. Both H- and L-GlyRs gated Cl currents that mM EDTA/0.5% SDS, pH 9) supplemented with heparin (1 displayed a mir strong outward rectification. Neverthe- mg/ml). The mixture was shaken for 5 min and centrifuged less, single chanls ofadult H-and L-GlyRs did not rectify and for 10 min at 15,300 x g. Nucleic acids were isolated in the their mean open times were only slightiy altered by voltage. It aqueous phase by several extractions with chloroform and follows that the outward rectification of adult GlyRs is due phenol/chloroform, 1:1 (wt/vol). Total RNA was separated mainly to a reduction in the number of open channels. in by selective precipitation in 3 M NaCl and washed three times contrast to H-GlyRs, whose characteristics seem to remain with 3 M sodium acetate (pH 6). Poly(A)+ mRNA was essentiafly unchanged with age, L-GlyRs from newborn and purified by oligo(dT) chromatography, precipitated, washed adult rats have different properties. Channels of newborn in 70% ethanol, and concentrated to 1 pg/gl in diethyl L-GlyRs have a higher conductance, longer open time, and pyrocarbonate-treated water. About 100 gg of adult or new- greater voltage dependency than those from the adult. Inter- born mRNA was size fractionated on a sucrose gradient as estingly, properties of newborn GlyRs expressed by whole described (8). A rRNA gradient was run in parallel to give the mRNA were markedly different from those encoded by new- positions of the 18S and 28S rRNAs. Thirty fractions were born or adult L or H mRNA. These results demonstrate that the collected, ethanol-precipitated, dried in vacuum, and resus- functional heterogeneity of GlyRs is developmentally regu- pended in 10-12 ;4 of diethyl pyrocarbonate-treated water. lated. Oocyte Injection and Recording. Oocytes were injected with 50 nl of whole (W) mRNA or the H- or L-GlyR fraction Glycine is the major inhibitory neurotransmitter in the spinal and kept at 15-16'C in a modified Barth's solution (with cord and acts via glycine receptors (GlyRs) that differ in their Nystatin at 50 units/ml and gentamicin at 0.1 mg/ml). Two molecular structure and functional properties and which are days after injection, oocytes were treated with collagenase developmentally regulated (1-4). These two features of (0.5 mg/ml) to remove the surrounding layers (9) and record- GlyRs, heterogeneity and developmental regulation, have ings were performed at room temperature (20-240C) 4-10 been demonstrated in the Xenopus oocyte expression sys- days after injection. tem. For example, both the adult and the neonatal spinal cord To determine the mean open time and unitary currents of contain heavy (H) and light (L) mRNAs that code for distinct channels gated by H- or L-GlyRs, responses to glycine were GlyRs, all of which gate Cl- channels. Furthermore, in the subjected to noise analysis (5) or voltage jumps (10). Cells adult spinal cord the preponderant GlyR mRNA is of the H were voltage clamped with a conventional two-electrode type, whereas in the neonatal spinal cord the main GlyR voltage-clamp amplifier and exposed to glycine at a concen- mRNA is of the L type (2-4). The present study was tration (100-200 ;LM) well below the EC50 (2). Currents were undertaken, several years ago, to determine the character- ac-filtered (high-pass filter, one-pole, -3 decibels; fre- istics of the single channels opened by H- or L-GlyRs and to quency, 2 Hz or 0.015 Hz) and the resulting fluctuations, as see whether those characteristics were developmentally reg- well as the dc current, were recorded separately on an FM ulated. For that purpose we chose to use noise analysis (5) tape recorder or on a digital videotape recorder, for off-line rather than the patch-clamp method because some of the analysis. Current relaxations were obtained by stepping the responses were rather small due to the small amounts of holding potential from -60 mV to the desired voltage before mRNAs in some fractions (see ref. 2). Preliminary results and during the application of glycine (100-200 ,AM). Re- have been published elsewhere (6, 7). sponses to voltage jumps were recorded on a digital oscillo- scope and stored on floppy disks for further analysis. Cur- METHODS rent-voltage relations were obtained by applying 2-s pulses to different potentials before and during glycine application. Tissue Collection. Adult rats were anaesthetized with Nem- Data Analysis. Mean open times and elementary currents butal and decapitated. Spinal cords were immediately dis- were determined from current fluctuations by using a pro- sected out from the upper cervical to the lower lumbar gram written Rico Miledi or the SPAN segments. During the operation, the cords were kept moist by program (J. Demp- with a physiological solution. After removal, the tissues were Abbreviations: GlyR, glycine receptor; W-, H-, or L-GlyRs, glycine receptors expressed by whole (W), heavy (H), or light (L) mRNA The publication costs of this article were defrayed in part by page charge fractions. payment. This article must therefore be hereby marked "advertisement" *Present address: Departamento de Fisiologfa, Facultad de Medic- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ina, Universidad de Alicante, Alicante, Spain. 3097 Downloaded by guest on September 24, 2021 3098 Neurobiology: Morales et al. Proc. NatL Acad. Sci. USA 91 (1994) ster, University of Strathclyde, Glasgow, U.K.). Prior to potential in oocytes (11). As shown by the graph in Fig. 1, the sampling, ac currents were low-pass filtered in an anti- power spectra of current fluctuations induced by glycine aliasing filter (Butterworth, four poles) set to half the sam- acting on both types of GlyRs could be well fitted by a single plingfrequency. After sampling ac and dc currents, variances Lorentzian curve. However, the half-power frequencies of and power spectra were calculated. Mean open times (X) were the spectra derived from H- and L-GlyRs were significantly derived from the corner frequency (fj) of the power spectra different, indicating a difference in the mean open time ofthe by the formula X = 1/(2irf,). Elementary currents were corresponding membrane channels. At -60 mV the mean determined from the total variance of fluctuations and, as a open time (X) for H-GlyRs was 7.2 ± 1.3 ms (n = 22), while control, they were also calculated from the power spectrum. for L-GlyRs it was 18.1 ± 4.3 ms (n = 18); P < 0.01. Values given by the two methods were in good agreement. We also studied glycine current fluctuations in oocytes Fitting of current relaxations was performed by another injected with the W mRNA from adult rat spinal cord. As program written by Rico Miledi. Responses to voltagejumps expected, these GlyRs showed elementary currents similar to before glycine application were subtracted from those re- H- and L-GlyRs (-1.51 ± 0.46 pA; n = 13). The power sponses during drug application. The resulting decays were spectrum of these current fluctuations was well fitted by a usually well fitted by one or two exponentials. Unless oth- single Lorentzian, with a T of 8.36 ± 1.74 ms (n = 13). This values in the text correspond to the value is very similar to that obtained for receptors expressed erwise specified, given by H-GlyR mRNA (7.21 ± 1.25 ms, n = 22), which is by far mean and standard deviation. Means were compared by the predominant type in the adult rat spinal cord (2-4). Student's t test. Voltage Dependence of Adult W-, H-, and L-GIyR Unitary Conductance and Mean Open Time. As already described for RESULTS various GlyRs (12-16), the current-voltage relation of chan- nels gated by H- or L-GlyRs showed a marked rectification Unitary Conductances and Mean Open Times of Adult W-, at hyperpolarizing potentials (Fig. 2). To identify the mech- H-, and L-GlyRs. Oocytes were injected with W, H, or L anism underlying this outward rectification, we determined mRNA from the spinal cord of the adult rat and only those the mean elementary currents and open times of GlyR oocytes in which glycine (100-200 p;M) generated currents channels at various membrane potentials. Fig. 3A shows an over 50 nA at a holding potential of -60 mV were included example of the membrane current fluctuations elicited by for noise analysis.