0270.6474/84/0403-0732$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 4, No. 3, pp. 732-7480 Printed in U.S.A. March 1984 EFFECTS OF IONTOPHORETICALLY RELEASED AMINO ACIDS AND AMINES ON PRIMATE SPINOTHALAMIC TRACT CELLS’ W. S. WILLCOCKSON, J. M. CHUNG, Y. HORI, K. H. LEE, AND W. D. WILLIS’ Marine Biomedical Institute, University of Texas Medical Branch, Galveston, Texas 77550 Received June 10, 1983; Revised September 28, 1983; Accepted September 29, 1983 Abstract The effects of glutamate (Glu), y-aminobutyric acid (GABA), glycine (Gly), serotonin (5HT), norepinephrine (NE), dopamine (DA), and acetylcholine (ACh) were examined in this study by iontophoretic application onto primate spinothalamic tract (STT) neurons identified antidromically by stimulation in the contralateral thalamus. Drugs were tested for effects on background activity, Glu-induced firing, and activity evoked by pinching of the skin, Whereas Glu excited STT cells and was thus used for tests of the other compounds, the amino acids GABA and Gly inhibited Glu- and pinch-induced activity in all STT cells examined. STT cells were also inhibited by 5-HT, NE, and DA. Only two cases of excitation by 5-HT were seen (of 58 cells tested). ACh also had inhibitory actions on STT cells, although 3 of 21 cells exhibited some enhancement of activity. The effects of these compounds on identified STT cells resemble previous demonstrations of the effects of these drugs on dorsal horn interneurons. The results suggest that GABA, Gly, 5-HT, NE, and DA may be inhibitory neurotransmitters on nociceptive STT cells. A key problem to be solved concerning the processing fecting the activity of STT cells. We have demonstrated of somatosensory information is the identification of the that serotonin (5-HT) released iontophoretically near neurotransmitters that are used in the sensory pathways STT cells usually causes a depression of the background originating in the periphery, synapsing in the spinal cord discharges, the responses to noxious stimuli, and gluta- and brainstem and projecting to the thalamus and cere- mate (Glu)-evoked activity (Jordan et al., 1978, 1979). bral cortex. Of equal interest are those transmitters that However, some STT cells that have subcutaneous recep- modify transmission along the somatosensory pathways. tive fields can be excited by iontophoretically applied 5- For example, there may be practical uses of agents that HT (Jordan et al., 1979). Recently, it has been shown modulate nociceptive transmission in the spinal cord. that primate STT and trigeminothalamic tract cells la- Our laboratory has been investigating the responses of beled with retrogradely transported horseradish peroxi- spinothalamic tract neurons in the monkey (Willis et al., dase receive synaptic contacts containing enkephalin- 1974; Chung et al., 1979). The spinothalamic tract (STT) like immunoreactivity (Ruda, 1982). Thus, it would be is a major somatosensory pathway in primates, including predicted that microiontophoretically applied enkepha- humans, and it is crucial for pain sensation (White and lin might have a postsynaptic action on primate STT Sweet, 1955; Noordenbos and Wall, 1976; Vierck and cells. Luck, 1979). In addition, the STT is likely to contribute A number of putative neurotransmitters are present to temperature and tactile sensations (White and Sweet, in the spinal cord dorsal horn and have an action on 1955). dorsal horn interneurons following microiontophoretic Very little is known about the neurotransmitters af- application. These substances include the amino acids glutamic acid, glycine (Gly), and y-aminobutyric acid (GABA) (Curtis et al., 1959, 1960; 1968; Graham et al., 1 This work was supported by Grants NS 09743, NS 11255, and NS 1967; Zieglgansberger and Puil, 1973; McLaughlin et al., 18830 from the National Institutes of Health and by a grant from the 1975), the catecholamines norepinephrine (NE) and do- Moody Foundation. We wish to thank Helen Willcockson for her expert pamine (DA) (Engberg and Ryall, 1966; Zivin et al., 1975; technical assistance in the surgical preparation of the animals and in Headley et al., 1978), acetylcholine (ACh) (Engberg and the art work. We thank also G. Gonzales for her help with the histology and Phyllis Waldrop for typing the manuscript. Ryall, 1966; Zieglgansberger and Reiter, 1974; Houser et ’ To whom correspondence should be addressed, at Marine Biomed- al., 1983; however, cf., Curtis et al., 1961), and several ical Institute, 200 University Boulevard, Galveston, TX 77550. peptides, including substance P and somatostatin (Tak- 732 The Journal of Neuroscience Amino Acids and Amines on STT Cells 733 ahashi and Otsuka, 1975; Henry, 1976; Hokfelt et al., Drug concentrations and pH were as follows: L-mon- 1976), in addition to 5-HT and enkephalin. osodium-glutamate salt (Sigma), 0.2 M, pH 8.5; GABA The present study is a survey of the effects of a number (Sigma), 0.2 M, pH 3.5; Gly (Sigma), 0.2 M, pH 3.5; 5- of candidate neurotransmitters released by microionto- HT creatinine sulfate (Sigma), 50 IIIM, pH 5.0; L-nor- phoresis in the vicinity of primate STT cells. The exper- epinephrine hydrochloride (NE) (Sigma), 0.1 M, pH 4.0; iments are the initial stage of an analysis of the phar- dopamine hydrochloride (DA) (Sigma), 0.1 M, pH 4.0; macology of synaptic excitation and inhibition of these acetylcholine iodide (ACh) (Sigma), 1.0 M, pH 5.0. All neurons that are thought to play an important role in drugs were dissolved in sterile, deionized water (dH,O), pain mechanisms. The actions of amino acids and amines except NE and DA which were made up in 5 x lop5 M are described in this paper, and the actions of peptides ascorbic acid in dHzO to prevent oxidation. Glu and 5- are described in the companion paper (Willcockson et HT were dissolved 4 to 5 hr before use; all other drugs al., 1984). A preliminary report has been made (Will- were made up immediately before filling the electrodes. cockson et al., 1983a). With the exception of Glu, all compounds were applied with cationic current. Retaining currents were always Materials and Methods less than 7 nA. Current neutralization was used during all drug applications through a balancing barrel filled Experiments were performed on 19 monkeys (Mucaca with 0.2 M NaCl. fascicularis) weighing 1.9 to 3.2 kg. Animals were initially Drugs were tested on STT neurons for effects on anesthetized with a mixture of halothane, nitrous oxide, background activity, Glu-induced firing, and activity and oxygen, followed by a single dose of a-chloralose (60 evoked by innocuous (brush) and noxious (pinch with a mg/kg, i.v.). Gallamine triethiodide (Flaxedil) was used small clip) mechanical stimuli. Glu activation was to immobilize the animal and artificial ventilation was achieved by pulsed release of Glu ions (5 to 25 nA) for 5 instituted. End-tidal CO* was maintained between 3.5 set at lo-set intervals. Drugs were iontophoresed contin- and 4.5%. A supplemental infusion of sodium pentobar- uously over a 40-set period. bital (5 mg/kg/hr) and gallamine triethiodide (4 mg/kg/ Extracellular spikes were used to trigger a window hr) was given throughout the duration of the experiment. discriminator. Peristimulus time histograms were com- Rectal temperature was regulated near 37°C. piled by computer. Control responses to background, Glu, The lumbosacral spinal cord was exposed by laminec- or mechanically evoked activity were compared to re- tomy at vertebral levels L2 to L6 to allow recordings sponses during the test drug application. Percentage of from STT cells. A craniotomy at the vertex of the skull inhibition or excitation was calculated as [number of provided access to the thalamus. In three experiments impulses during test period/number of impulses during decorticate spinalized animals were used. Decortication control period) x lOO%] - 100%. was accomplished by bilateral common carotid artery At the conclusion of each experiment stimulating and ligation and spinalization by compression of the cord at recording sites were marked by passing direct current the C2 spinal level. No chloralose was given, and supple- through the electrodes. Steel electrode marks in the mental pentobarbital was omitted from the infusion in thalamus were enhanced histologically by the Prussian these animals. blue reaction. Carbon-fiber marks at STT cell locations The caudal part of the ventral posterior lateral nucleus were visible as microlesions. of the right contralateral thalamus (VPLc nucleus of Olszewski, 1952) was found by recording potentials Results evoked by electrical stimulation of the dorsal column The locations of marks where STT cells were recorded and by mechanical stimulation of the hindlimb. The are shown in Figure 1. Because most of the cells were thalamic electrode was then used to activate STT cells tested with both amines and peptides, the recording spots antidromically (Trevino et al., 1973). In spinalized ani- from all experiments described in this and the following mals, lumbar dorsal horn neurons were activated anti- paper (Willcockson et al., 1984) are condensed into one dromically by surface stimulation of the contralateral figure. Not every recording site was marked, often be- lateral column at a high cervical level. The dorsal column cause several cells were in close proximity to each other. was interrupted caudal to this stimulation site to prevent Most of the recordings were made in laminae I and V, volleys in the dorsal columns from activating STT cells where STT cells projecting to the VPLc nucleus are most orthodromically (cf. Foreman et al., 1976). STT cells concentrated (Willis et al., 1974). were classified according to their responses to innocuous A total of 48 STT cells in 16 different animals were or noxious mechanical stimuli applied to the receptive tested with biogenic amines. Cell depths ranged from 400 field (Chung et al., 1979). In experiments with spinalized to 2070 pm, and antidromic thresholds were 20 to 600 animals, antidromically activated dorsal horn cells were PA.