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ATP P2X Receptors Mediate Fast Synaptic Transmission in the Dorsal Horn of the Rat Spinal Cord

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ATP P2X Receptors Mediate Fast Synaptic Transmission in the Dorsal Horn of the Rat Spinal Cord The Journal of Neuroscience, July 15, 1997, 17(14):5297–5304 ATP P2X Receptors Mediate Fast Synaptic Transmission in the Dorsal Horn of the Rat Spinal Cord Rita Bardoni,1,3 Peter A. Goldstein,2 C. Justin Lee,1,3 Jianguo G. Gu,1,3 and Amy B. MacDermott1,3 Departments of 1Physiology and Cellular Biophysics, 2Anesthesiology, and the 3Center for Neurobiology and Behavior, Columbia University, New York, New York 10032 ATP has been proposed to mediate synaptic transmission in the antagonists suramin and pyridoxal-phosphate-6-azophenyl- spinal cord dorsal horn, particularly in the pathway carrying 29,49-disulfonic acid (PPADS). These characteristics are consis- nociceptive information. Using transverse spinal cord slices tent with a heterogeneous population of P2X receptors, the from postnatal rats, we show that EPSCs mediated by P2X composition of which includes P2X2 ,P2X4 , and P2X6 receptor receptors, and presumably activated by synaptically released subtypes. Our results suggest that ATP-activated P2X receptors ATP, are evoked in a subpopulation of spinal cord lamina II in lamina II of the rat spinal cord may play a role in transmitting neurons, a region known to receive strong input from nocicep- or modulating nociceptive information. tive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurons are nondesensitizing, insensitive to ab Key words: ATP; purinergic receptors; synaptic transmission; methylene ATP, and show strong but variable sensitivity to the spinal cord; rat; patch-clamp technique; slice preparation Holton and Holton (1954) first proposed ATP as a possible in homomeric form, they all mediate a nondesensitizing response neurotransmitter in the dorsal horn over 40 years ago. Since then, to ATP and are insensitive to the agonist ab methylene ATP its role as a fast neurotransmitter in the peripheral nervous system (Brake et al., 1994; Collo et al., 1996; Se´gue´la et al., 1996), making has been demonstrated (Burnstock et al., 1972; Evans et al., 1992; them pharmacologically distinguishable from the other P2X sub- Silinsky and Gerzanich, 1993; Galligan and Bertrand, 1994). In units. A further pharmacological distinction among subunits is the CNS only one study, performed on neurons in the medial that, whereas responses mediated by the P2X2 subunit are sensi- habenula, has demonstrated that ATP can act as a fast neuro- tive to the antagonists suramin and pyridoxal-phosphate-6- transmitter (Edwards et al., 1992). Within the spinal cord dorsal azophenyl-29,49-disulfonic acid (PPADS), P2X4 and P2X6 subunits horn, despite strong evidence implicating ATP as a putative are relatively insensitive (Buell et al., 1996; Collo et al., 1996) (but neurotransmitter (Jahr and Jessell, 1983; Fyffe and Perl, 1984; see Se´gue´la et al., 1996). Using spinal cord slices prepared from Salter and Henry, 1985; Salter and Hicks, 1994), its role in this rat pups 1–2 weeks after birth, we investigated whether excitatory regard has not been demonstrated (Li and Perl, 1995). synaptic transmission in the spinal cord is mediated by the ATP- Several ATP receptor subunits have been cloned from different sensitive P2X receptors. We found that a subset of lamina II tissues (Brake et al., 1994; Valera et al., 1994; Chen et al., 1995; neurons receives synaptic inputs mediated by those receptors. Lewis et al., 1995; Buell et al., 1996; Collo et al., 1996; Vulch- However, the subpopulation was ,5% of the neurons tested, anova et al., 1996). Of those cloned, the ATP P2X2 ,P2X4 , and making detailed pharmacological studies of these synapses diffi- P2X6 receptor subunit RNAs have been shown to be expressed in cult. Therefore, we used pharmacological and physiological the spinal cord dorsal horn, particularly in the superficial laminae means to characterize the P2X receptor subunits expressed on of the dorsal horn (Brake et al., 1994; Buell et al., 1996; Collo et acutely dissociated postnatal dorsal horn neurons. Our results al., 1996; Vulchanova et al., 1996), lending further support to the provide evidence for the synaptic release of ATP and for postsyn- idea that ATP receptors may participate in synaptic function aptically localized P2X receptors in the spinal cord dorsal horn, there. Recently, P2X7 mRNA also was detected in both brain and indicating that ATP and P2X receptors have a role in mediating spinal cord (and elsewhere), but its exact distribution was not and possibly modulating sensory transmission there. described (Surprenant et al., 1996). When P2X2 ,P2X4 , and P2X6 receptor subunits are expressed in heterologous cellular systems MATERIALS AND METHODS Tissue preparation. Acutely prepared transverse cervical spinal cord sec- Received Feb. 24, 1997; revised April 29, 1997; accepted May 1, 1997. tions from postnatal (P2–P13) rats were prepared by following the This work was supported by Human Frontier Science Program (R.B.), the White- method of Takahashi (1990). Briefly, postnatal rats were anesthetized hall Foundation, and National Institutes of Health (A.B.M.). We thank Steven with isoflurane and decapitated. The cervical spinal column was removed Roper, Arnold Kriegstein, Pier Cosimo Magherini, Detlev Schild, and Cristo´va˜o de rapidly and placed in ice-cold oxygenated (95% O /5% CO ) Krebs’ Albuquerque for their thoughtful comments on this manuscript. We also thank 2 2 Frances Edwards and Megumu Yoshimura for helpful discussions on experimental solution. Ventral and dorsal laminectomies were performed and the protocols. ventral roots cut as close to the cord as possible. The meninges were Correspondence should be addressed to Dr. Rita Bardoni at her present address: removed, except around the roots as they enter the cord, anda1cm Dipartimento di Scienze Biomediche, Sezione di Fisiologia, Universita` di Modena, segment of cervical spinal cord with the dorsal roots attached was Via Campi 287, I-41100 Modena, Italy. isolated. Then the spinal cord was embedded in low-melting-point aga- Copyright © 1997 Society for Neuroscience 0270-6474/97/175297-08$05.00/0 rose 2.5% w/v dissolved in Krebs’ at 35–38°C. The agarose block was 5298 J. Neurosci., July 15, 1997, 17(14):5297–5304 Bardoni et al. • Purinergic Fast Synaptic Transmission in the Dorsal Horn affixed to a vibratome stage with cyanoacrylate glue and immersed in from visually identified neurons 50–100 mm below the slice surface. The ice-cold oxygenated Krebs’. Transverse slices of 350–400 mm were cut recording electrode was advanced while positive pressure was applied, and incubated in oxygenated Krebs’ at 37°C for 1 hr. Then a slice was andaGVseal was obtained. In some cells the ATP-mediated EPSC was transferred to a recording chamber, the agarose removed, and the slice evoked by stimulating the ipsilateral white matter, using a concentric held in the chamber by a platinum grid with nylon wires. The chamber bipolar stimulating electrode (Rhodes Medical Instruments, Woodland was placed on the stage of an upright microscope (Zeiss Axioskop, Hills, CA); in other cases, focal stimulation of the nearby tissue was Oberkochen, Germany) for recording. performed with a glass pipette (;3 mm tip size) filled with Krebs’ Preparation of acutely dissociated dorsal horn neurons. The preparation solution. In both conditions the identification of the stimulated presyn- of acutely dissociated dorsal horn neurons is described in Kyrozis et al. aptic element (fiber or interneuron) was not possible. Stimuli of 1–15 mA (1996). Postnatal rats (age 7–12 d) were anesthetized with isoflurane and (constant current) and 0.1–0.2 msec usually were required to evoke the decapitated, and the spinal cord was removed rapidly and submerged in ATP-mediated EPSCs in lamina II neurons; stimulus frequencies ranged ice-cold Krebs’ solution saturated with 95% O2 /5% CO2. The dorsal between 0.1 and 0.5 Hz. one-third of the spinal cord slice was excised and digested with 1 mg/ml Acutely dissociated neurons were voltage-clamped at 270 mV in the trypsin (Type III, Sigma, St. Louis, MO) in saturated Krebs’ for 30 min whole-cell configuration, and 100 mM ATP was fast-applied onto the at 37°C, washed three times with Krebs’, and then placed in 1 mg/ml neurons for 4 sec. A 2 sec voltage ramp (from 285 to 120 mV) was trypsin inhibitor (Type II-O, Sigma) in Krebs’ at room temperature. started 1 sec after ATP application and terminated 1 sec before the end Several dorsal horn segments were placed in Krebs’ in a 35 mm dish of ATP application. I–V curves of ATP-evoked whole-cell currents were containing a coverslip previously coated with 1 mg/ml poly-D-lysine and obtained by subtracting a control voltage ramp from the test ramp. In washed thoroughly. Then the segments were triturated gently with an some experiments recordings were made with cultured dorsal horn ultramicropipette and allowed to adhere to the coverslip for at least 30 neurons in the perforated-patch configuration (Gu et al., 1996). min before use. Infrared imaging. Video-enhanced infrared microscopy was performed by using a modification of a previously described technique (Stuart et al., RESULTS 1993). Briefly, a spinal cord slice was transferred to a recording chamber and placed on the stage of an upright microscope fit with a 103 [numer- Fast excitatory synaptic currents mediated by P2X ical aperture (NA) 0.25] lens, 403 water immersion lens (NA 0.75), and receptors in a subset of lamina II neurons a 775 nm infrared bypass filter. Using the 103 lens, we identified lamina II, although we identified individual neurons in lamina II by using the Whole-cell patch-clamp recordings were obtained from lamina II 403 objective and an infrared filter after offsetting the Ko¨hler illumina- neurons in postnatal rat spinal cord slices. Using video-enhanced tion by 50% of the visual field under low magnification. The image was infrared microscopy, we chose individual lamina II neurons for enhanced further with a VE-1000 CCD camera (Dage–MTI, Michigan recording (Stuart et al., 1993; Bardoni et al., 1995).
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