Brain Research, 511 (1990) 71-79 Elsevier

71

BRES 15254

5-HT 2 and 5 - H T 3 receptors mediate two distinct depolarizing responses in rat dorsal root ganglion neurons Slobodan Todorovid and Edmund G. Anderson Department of Pharmacology, University of lllinois at Chicago, College of Medicine, Chicago, IL 60612 (U.S.A.) (Accepted 1 August 1989)

Key words: Serotonin receptor; Norepinephrine; Sensory afferent; Membrane resistance

The effects of serotonin (5-HT) were studied on transmembrane potentials in 188 rat dorsal root ganglion cells (150 A-type, 16 C-type and 22 unidentified neurons). 5-HT produced a concentration-dependent depolarization in 88% of these neurons. Membrane input resistance (Rin), determined from the slope of current-voltage displacement curves, was increased in 51% and decreased in 41% of the responding neurons. Both responses occurred in 8% of the neurons. No differences in these responses were observed between A- and C-type neurons. Norepinephrine (NE) depolarized 75% (n = 20) of the neurons tested while increasing the Rin. In cells where 5-HT decreased Rin, 2-methyl 5-HT, but not a-methyl 5-HT, mimicked the response. The selective 5-HT3 antagonist, ICS 205-930, blocked this response, but ketanserin and methiothepin did not affect it. The 5-HT-induced increase in Rin was blocked by 5-HT2 antagonists (ketanserin, methiothepin and spiperone); mimicked by a-methyl 5-HT, but not affected by 2-methyl 5-HT. The selective 5-HT3 antagonist, ICS 205-930, did not antagonize this response. The action of NE but not 5-HT was blocked by the selective a 1 antagonist, prazosin. These data indicate that the 5-HT induced depolarization with decreased Rin is mediated by 5-HT3 receptors and the depolarization with increased Rin is mediated by 5-HT2 receptors. Furthermore, these two receptors can occur on the same cell. INTRODUCTION

(Rin) and a fast transient depolarization with decreased Rinl7, 29.

The dorsal root ganglion ( D R G ) contains the cell bodies of primary afferent neurons whose central processes release neurotransmitters in the dorsal horn of the spinal cord (e.g. glutamate and polypeptides). The unmyelinated peripheral processes of these D R G cells release polypeptides (e.g. substance p)14, and are excited by amines and polypeptides. The receptors present on both the central and peripheral afferent terminals likely modulate sensory transmission. It is known that afferent terminals are targets of 5-HT action. Extracellular recordings from the central processes in the dorsal root reveal that systemic injection of 5-hydroxytryptophan or local application of serotonin (5-HT) will induce spontaneous discharge, or increased terminal excitability indicating primary afferent depolarization 1'5'15A6. Though this response may in part be mediated by excitation of interneurons, its persistence in the isolated spinal cord in the presence of tetrodotoxin indicates a direct action of 5-HT on central afferent terminals ~5. Stimulation of the nucleus raphe magnus (major origin of descending 5-HT neurons) also produces afferent depolarization 25,38. Intracellular studies in bullfrog D R G cells indicate that 5-HT induces a slow depolarization with increased m e m b r a n e input resistance

Peripheral afferent endings excited by 5-HT are attached to fiber groups II, III and IV; and include cutaneous mechano- and thermo-receptors 1° and muscle afferents n. This type of action probably underlies the ability of 5-HT to induce pain when applied to the human blister base. Most of the above studies were done before multiple 5-HT receptors were discovered. It now becomes important to examine the 5-HT receptor subtypes involved in mediating the various responses of primary afferents to 5-HT. We have employed the receptor responses detected in rat D R G somata as possible models of the responses to receptors located in the terminal processes of these neurons. Using intracellular recordings, and a series of agonists and antagonists selective for specific 5-HT receptor subtypes, two distinct depolarizing responses have been detected. One is mediated by a 5-HT 2 receptor and the other by a 5-HT 3 receptor. MATERIALS AND METHODS Sprague-Dawley male rats (King Sasco Madison and St. Louis colonies) weighing 100-250 g were anesthetized with diethyl ether, decapitated and the Ls-L6 DRG and attached nerves were rapidly removed and placed in cold artificial cerebrospinal fluid (aCSF). A

Correspondence: E.G. Anderson, Deparment of Pharmacology, UIC, College of Medicine m/c 868,835 S. Woicott, Chicago, IL 60612, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

72 ganglion was pinned to the silicone floor of a small water bath and desheathed. The peripheral nerve was stimulated via a suction electrode. The distance from the electrode to the ganglion was measured (typically 5-20 mm) and the conduction velocity calculated. Most of the cells impaled were located near the surface of the DRG which facilitated rapid equilibration of drug with the cell surface. The bath was perfused with 32-34 °C aCSF of the following composition (in mM): NaC1 126; KCI 2.5; MgSO4 1.3; CaC12 2.4; NaH2PO 4 1.2; NaHCO 3 26; glucose 11 and saturated with 95% 02/5% CO 2 (pH 7.4). The drug composition of the bath was controlled by selecting the perfusate from a bank of gravity-fed reservoirs with a constant pressure head. Cell impalements are made with glass microelectrodes filled with 2 M KCI (20-80 MI2), or with 2 M KAcetate electrodes (50-100 MI2). All potentials were recorded using an electrometer with an active bridge circuit allowing current injection through the recording electrode. The output was amplified, digitized and displayed on a computer screen. A parallel' signal was sent to a rectilinear strip chart recorder to monitor the membrane potential. Careful bridge balance adjustments are made throughout the experiments. Data from the computer were stored on disk or film. An incremental series of 70-100 mS current pulses (which fully charged the membrane capacitance) were injected through the microelectrode, and the current and the membrane voltage displacement were recorded. Current steps were selected manually or by computer software with signal averaging (pCLAMP, Axon Instruments, Inc., Burlingame, CA). Current/voltage displacement (IN) curves were constructed, and the Rin determined from their slope. The maximal voltage deflection caused by the current injection was used to construct these curves. A single hyperpolarizing pulse was injected every 5 s, and the voltage displacement recorded, indicating the approximate Rin on a continuous basis. During peak drug effect the membrane potential was manually repolarized by injecting a DC current to neutralize voltage-activated conductances. All critical measurements of Rin are made from an I N curve. Drugs used in this study included: 5-hydroxytryptamine creatinine sulfate and norepinephrine hydrochioride (Sigma Chemical Co., St. Louis, MO); spiperone, ketanserin tartarate, 3-tropyl-indole-3carboxylate (ICS 205-930), 2-methyl 5-HT maleate and a-methyl 5-HT maleate (Research Biochemical, Inc., Wayland, MA); methiothepin (Roche Laboratories, Nutley, NJ) and prazosin hydrochloride (Pfizer, Inc., Groton, CT). Prazosin was dissolved in absolute ethanol as 1 mM stock solution; ICS 205-930 was dissolved as the hydrochloride salt; other drugs were dissolved in water. After impalement of a cell, and achievement of a stable recording, varying concentrations of 5-HT were applied and the response measured. Antagonists alone were incubated with the cell for 5 min before 5-HT or another agonist was applied in the presence of the antagonist. Longer incubation times with the antagonists were unnecessary since exposures of over 30 min did not alter their blocking efficacy. Also the effects of all antagonists used rapidly disappeared on washout. All data are expressed as mean + standard error. Statistical analyses were made using the paired Student t test (two-tailed), or the non-parametric X2-test.

RESULTS T h e response to b a t h application of 5-HT was studied in 188 rat D R G cells, including 150 A - t y p e , 16 C-type and 22 unidentified neurons. The latter group failed to r e s p o n d to nerve stimulation. The A - t y p e neurons had conduction velocities b e t w e e n 5 and 50 m/s (mean 17.5 + 0.68 m/s), Rin ranging from 4 to 50 MI2 (mean 17.8 + 1.24 MI2) and resting m e m b r a n e potentials ( R M P ) b e t w e e n - 5 0 and - 8 7 m V ( m e a n - 6 3 . 9 + 0.7 mV). C-type neurons showed conduction velocities between 0.5 and

1.5 m/s (mean 0.88 +_ 0.12 m/s), Rin from 20 to 100 MI2 (mean 56 _+ 16.2 M£2) and R M P from -51 to - 8 4 mV (mean -66.5 _+ 1.9 mV). Cells with R M P s less than - 5 0 mV were excluded from this study. In response to constant current injections, most A - t y p e neurons (75%) showed a t i m e - d e p e n d e n t rectification, with the maximal voltage deflection decaying over time (Fig. 1B). None of the C-type neurons exhibited this p h e n o m e n o n . Also, most A - t y p e neurons d e m o n s t r a t e d strong m e m b r a n e rectification with decreasing Rin with depolarization (Fig. 1B). 5-HT induced a d o s e - d e p e n d e n t depolarization in 88% of the neurons tested. Rin was decreased in 41% and increased in 51% of the responding neurons, while at least 8% of these neurons showed both responses. (This latter figure is p r o b a b l y an u n d e r e s t i m a t e since a d e q u a t e testing was not c o m p l e t e d in all cells to reveal a second response). H y p e r p o l a r i z i n g responses were not observed during the first application of 5-HT, but in 4 cells a hyperpolarizing response to 5 - H T a p p e a r e d after the depolarizing response was b l o c k e d by an a p p r o p r i a t e antagonist.

5-HT-induced depolarization with decreased Rin Bath application of 5-HT p r o d u c e d a d o s e - d e p e n d e n t depolarization with decreased Rin in 69 of 188 D R G neurons. A m a x i m u m depolarization was induced by 1 p M 5-HT, and averaged 4.5 + 0.4 mV. This effect of 5-HT was fast in onset (Fig. 1A), did not diminish in intensity of response during several minutes of 5-HT application and readily reversed u p o n washout. B o t h the maximal and steady-state displacement of the cell membrane potential induced by an incremental series of 100 ms constant-current pulses were r e d u c e d by bath application of 5-HT (Fig. 1B). Fig. 1C depicts the effects of 5 - H T on the slope of the I/V curves derived from a current-injection experiment. In this typical A - t y p e cell, 1 /~M 5-HT shifted the Rin from a control value of 18 MI2 to 12 MI2 (Fig. 1B and 1C). The selective 5-HT 3 agonist, 2-methyl 5-HT, qualitatively and quantitatively m i m i c k e d the effects of 5-HT in cells responding with d e p o l a r i z a t i o n and decreased Rin (n = 11, Figs. 1D and 4B). H o w e v e r , 2-Me-5-HT did not mimic 5-HT (in concentrations up to 1/zM) in cells where 5-HT p r o d u c e d depolarization with increased Rin. Bath application of the selective 5 - H T 3 antagonist, ICS 205-930 (0.5-10 nM, n = 18) did not affect the m e m b r a n e properties of the D R G cells. H o w e v e r , these concentrations caused a parallel rightward shift in the concentrat i o n - r e s p o n s e curves for 5 - H T (Fig. 1D). T h e IC5o of ICS 205-930 in antagonizing 100 nM 5-HT was nM (n = 6). ICS 205-930 (10 nM) a t t e n u a t e d the response to 1/~M

73

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Fig. 1. A: chart recording where the solid line represents RMP and the downstroke lines indicate membrane voltage displacement caused by injection of constant hyperpolarizing pulse; their length reflects Rin. One #M 5-HT depolarized this A-type neuron (RMP -52 mV, Rin 8 M£2) by 4 mV, and decreased Rin by 37.5%, measured when RMP was manually repolarized. Note that both RMP and Rin returned to control level after washout of 5-HT. B: another A-type neuron (RMP -64 mV, Rin MI2) depolarized 4 mV by 1 #M 5-HT. Depolarizing and hyperpolarizing current pulses (100 ms) were injected prior to and during application of 5-HT. Each step was the average of 3 identical pulses. This neuron showed time-dependent rectification when hyperpolarizing pulses were injected and voltage-activated rectification in a depolarizing direction (note tail-currents at the end of hyperpolarizing pulses). 5-HT depressed both the maximal and steady-state voltage deflection induced by the current pulses. C" data from 1B plotted on I/V curve, 5-HT shifted the Rin from a control value of 18 M,Q to 12 MI2. D: A-type neuron (RMP -73 mV, Rin 50.('2). On this cell 5-HT induced dose-dependent depolarization with a maximal response of 5 mV with 1 MM 5-HT. 2-Methyl-5-HT mimicked 5-HT with a maximal depolarization of 4.5 mV. ICS 205-930 (1 and 10 nM, incubated for 6 min) caused a parallel rightward shift in the concentration-response curve to 5-HT. The response to 5-HT returned to control after 5 min washout of antagonist (not shown).

2-methyl 5 - H T to 23 + 3.3% of control (n = 6, P < 0.001). T h e response of these cells to 5-HT was unaffected by 100 nM ketanserin (a selective 5-HT 2 antagonist, n --- 7) o r by 1 /zM methiothepin (a b r o a d antagonist of the 5 - H T 1 and 5 - H T 2 r e c e p t o r subtypes, n = 3). These d a t a indicate that the 5-HT induced depolarization with decreased Rin is m e d i a t e d by a 5-HT 3 receptor.

Depolarization with increased Rin In 84 of 188 D R G neurons, 5-HT induced a dosed e p e n d e n t depolarization with increased m e m b r a n e resistance. A maximal depolarization was induced by 10 g M of 5-HT, averaging 6.25 + 0.45 mV, with a 51.3 + 5%

increase in the Rin. In most A - t y p e neurons the increase in Rin was m a s k e d by the d e p o l a r i z a t i o n - i n d u c e d membrane rectification. W h e n this was corrected by injecting current to return the m e m b r a n e potential to the control level the increase in Rin was clearly a p p a r e n t (Fig. 2A). The slope of the I/V curve indicates that the Rin was increased in both depolarizing and hyperpolarizing directions (Fig. 2C). In this typical A - t y p e neuron, 1 MM 5-HT increased the Rin from 16 MI2 to 23 MS'2. This 5-HT response, like the d e p o l a r i z a t i o n with decreased membrane resistance, was fast in onset, did not fade with time and was easily reversible after washout of 5-HT. a - M e t h y l 5-HT, a preferential 5-HT 2 agonist, qualitatively and quantitatively m i m i c k e d the 5 - H T depolarization with increased Rin (Fig. 2B, n = 6). H o w e v e r , up to

74

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Fig. 2. A: this A-type neuron (RMP -65 mV, Pin 25 Mr2) was depolarized 10 mV by 1 #M 5-HT and Pin increased 31%. The increased Rin was masked by voltage-activated rectification, which was corrected by injecting a constant hyperpolarizing current. Both RMP and Pin returned to control value after washout of 5-HT. C: another A-type neuron (RMP -80 mV) depolarized 4 mV by 1 #M 5-HT. From the I/V curve the apparent Pin increased from 16 MI2 to 23 MD. B: the concentration-response curves to 5-HT and a-methyl 5-HT were practically superimposed. Both drugs had maximal effects at 10 MM, depolarizing the cell by 4.5 mV. Ketanserin at concentrations of 1 and 10 nM shifted the concentration-response curve for 5-HT to the right without reducing the Emax. The response of 5-HT returned to control level after washout of antagonist (data not shown). T h e s e l e c t i v e 5 - H T 2 a n t a g o n i s t , k e t a n s e r i n ( 1 - 1 0 0 riM)

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Fig. 3. A: 1 MM NE de .pplarized this C-type neuron RMP -72 mV) by 4 mV while 5-HT was ineffective. The slope of the I N curve was increased indicating a shift of Rin from 71 to 95 MI2. B: this A-type neuron (RMP -65 mV, Rin 19 M$3) was depolarized by both 5-HT and NE with increased Rin. Ketanserin (10 nM) after an incubation of 8 rain attenuated the response of 1 #M NE to 55.5% of control and the response of 1 #M 5-HT to 47.6% of control. Five min after wash of the antagonist the response to both NE and 5-HT returned to control. The selective al-adrenergic antagonist prazosin (10 nM), after an incubation of 10 min, attenuated the response of 1/~M NE to 40% of control but did not affect the 5-HT response even after 20 min of incubation. Note that both ketanserin and prazosin slightly depolarized this cell.

75 dose-response to 5-HT (Fig. 2B). In some cells, higher concentrations of ketanserin continued to produce further parallel shifts in the dose-response curves (n = 5). However, in other cells, an insurmountable blockade of 5-HT occurred with the higher doses of ketanserin. The ICs0 of ketanserin for antagonizing 1/zM 5-HT was 8 nM (n = 9). Ten nM ketanserin attenuated the response of 1/xM a-methyl 5-HT to 40.8 _+ 12% of control (n = 4, P < 0.05). None of these doses of ketanserin affected the responses of 5-HT on cells where 5-HT induced depolarization with decreased Rin. Methiothepin (1/xM), an antagonist for multiple 5-HT 1 and 5-HT 2 receptors 3 almost completely and insurmountably blocked the responses to 5-HT with increased Rin (n = 3), but did not affect 5-HT depolarization with decreased Rin. Spiperone (100 nM-1 gM), a neuroleptic which blocks 5-HT1A and 5-HT 2 receptors 33,35 also blocked 5-HT responses with increased Rin, showing an insurmountable blockade (n = 6). Concentrations of up to 1/zM ICS 205-930 did not affect this response (n = 7). The above data strongly indicate that the 5-HTinduced depolarization with increased Rin is mediated by a 5-HT 2 receptor.

and decreased Rin while N E depolarized and increased Rin, in 2 cells (11.7%) 5-HT depolarized and decreased Rin while NE was ineffective and in 4 cells (23%) NE depolarized and increased Rin while 5-HT was ineffec-

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Norepinephrine The parallel distribution of norepinephrine-(NE) and 5-HT-containing fibers in the spinal cord makes it important to compare the actions of 5-HT with those of NE to make certain that the actions of 5-HT on the D R G can be distinguished from those of NE. This is especially important, since ketanserin blocks the effects of NE as well as 5-HT in some vascular tissue, an action attributed to its a 1 adrenergic receptor antago~nism 19,23,45. Like 5-HT, NE induced a concentration-dependent depolarization in 75% of the D R G cells tested (13 of 17 A-type and 2 of 3 C-type neurons). This response was always accompanied by an increase in Rin. One /zM NE produced an average depolarization of 4.31 + 0.4 mV (n = 11) and increased Rin by 22.6 + 4.7% (n = 10). The I/V curve illustrated in Fig. 3A shows that, in a C-type neuron, NE increased the slope indicating a shift in Rin from 71 to 95 MQ. Ketanserin (10 riM) blocked the depolarizing responses to NE as well as 5-HT (n = 3). However, the responses to NE were blocked by the selective a I receptor antagonist, prazosin, without affecting the responses to 5-HT (n = 6) (Fig. 3B). Both ketanserin and prazosin, in some neurons, tended to depolarize the membrane potential (Fig. 3B) which might suggest a partial agonist action. A comparison of the effects of both 5-HT and NE applied to the same cell (n = 17), showed that in 9 cells (52.9%) both 5-HT and NE depolarized the membrane and increased Rin, in 2 cells (11.7%) 5-HT depolarized

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5-HT 10.6 2 IVE5-HT 10.6 Fig. 4. A: this A-type neuron (RMP -60 mV) showed both types of depolarizing responses to 5-HT. Initially 1/~M 5-HT depolarized this cell by 4 mV and it increased Rin by 29%. After 10 nM ketanserin was applied for 8 rain, 1 ~M 5-HT depolarized the cell by 4 mV but decreased the Rin by 25%. After washout of ketanserin Rin returned to control value (not shown) and 1 nM ICS 205-930 was applied for 8 rain. In the presence of this antagonist, 1/xM 5-HT depolarized the cell and increased Rin by 50% which was much greater than the increase when 5-HT was applied alone. B: another A-type neuron (RMP -70 mV, Rin 8.5 M•) that responded in both ways. Initially 1 /~M 5-HT depolarized the cell by 7 mV and decreased Rin by 26%, while 1/~M 2-methyl 5-HT depolarized the cell by 4 mV and decreased Rin by 18%. After 10 nM ICS 205-930 was applied for 8 rain the same dose of 5-HT depolarized the cell by 5 mV but now increased Rin by 26% while the response to 2-methyl 5-HT was blocked. Note that ICS 205-930 alone changed neither RMP nor Rin.

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Fig. 5. A: averaged concentration-response curves to 5-HT induced depolarizations from cells with decreased and cells with increased Rin. Both curves are the average of 15 cells where full concentration-response curves were obtained and where only one type of response was detected. Depolarization with increased Rin had maximal effect with 10/~M 5-HT and an ECs0 of 65 nM. Depolarization with decreased Rin had maximal effect with 1 pM 5-HT and an ECs0 of 8 nM. In lower concentrations both curves overlap. B: concentration-response curves to change in Rin obtained from I/V curves (number of ceils are in brackets). Depolarization with increased Rin had maximal effects with 10 /tM 5-HT and an ECs0 of 650 nM. Depolarization with decreased Rin had maximal effects with 1/~M 5-HT and an ECs0 of 67 nM. There is striking similarity between the curves in Part A and B, but the ECs0's are larger when the Rin is measured.

tive. This leads to the conclusion that NE and 5-HT 2 or 5-HT 3 receptors may appear together on the same cell or to be exclusive to a cell. Furthermore, when both monoamines act on the same cell, they may mimic each other or they may have opposing effects on the Rin.

Comparison of the two 5-HT depolarizing responses In at least 8% of the cells tested we noticed both types of depolarization responses to 5-HT (i.e. an increase and a decrease in Rin) to be present. When both types of responses occurred together, either the increase or decrease in Rin could dominate. However, the presence of an opposing change in Rin was unmasked by selective antagonists or agonists. Fig. 4 illustrates this phenomenon. The I/V curves in Fig. 4A are from an A-type cell in which 1 /~M 5-HT increased the Rin by 29%. In the presence of 10 nM ketanserin, 1/~M 5-HT now decreased the Rin. Furthermore, in the presence of 1 nM ICS 205-930, 5-HT increased the Rin by 50%, which was much greater than the increase when 5-HT was given alone. These data demonstrate that the 5-HT 2 and 5-HT 3 receptor subtypes occur on the same cell with the 5-HT 2 response dominating. Fig. 4B illustrates the opposite interaction. Both 5-HT and 2-methyl 5-HT depolarized this A-type cell with a decrease in Rin. In the presence of 10 nM ICS 205-930, the effects of 2-methyl 5-HT were blocked, but the response to 5-HT was converted to depolarization with increased Rin. These data also indicate that both receptor subtypes are present, but that the 5-HT 3 response was dominant.

The presence of two receptor systems on D R G neurons activated by the same transmitter raised the question of whether these systems differed in sensitivity or persistence of action. Figs. 5A and B present composite concentration-response curves for 5-HT on membrane depolarization and percent change in Rin for these two responses. The 5-HT 2 response has a higher Emax whether membrane depolarization or change in Rin is measured. Consequently, the ECs0s for the 5-HT2 responses are higher (65 nN for membrane depolarization and 650 nM for change in Rin) than for the 5-HTs responses ( 8 nM for membrane depolarization and 67 nM for change in Rin). However, both the 5-HT2 receptors respond to 5-HT with similar intensities in the lower concentration ranges. An important point established by Fig. 5 is that though 5-HT only depolarized cells by 4-8 mV, this was accompanied by Rin changes of 30-50%. Neither the 5-HT2 nor the 5-HT 3 response showed any tendency toward desensitization. As is apparent from the chart recordings in Figs. 1-4, both responses had similar onset and offset rates, and these responses could be repeated every 3-5 min without diminution of response. When supramaximal concentrations of 5-HT were left in the bath, for periods of up to 15 min, no fade in the intensity of the response occurred. Neither the 5-HT 2 nor the 5-HT 3 response correlated with neuron conduction velocity, initial Rin or the presence or absence of time-dependent rectification. Neither receptor was preferentially associated with A- or C-type neurons. Neither response was affected by the use

77 of KAcetate electrodes, suggesting that CI- concentration was unimportant to the membrane response. A difference in the incidence of occurrence of the receptor subtypes was observed between two colonies of Sprague-Dawley rats. In D R G cells from rats of the St. Louis colony of King Sasco Laboratories, 60% showed a 5-HT 2 response and 40% a 5-HT 3 response. However, in D R G cells from rats of their Madison colony, only 36% exhibited a 5-HT 2 response while 64% showed a 5-HT 3 response. This difference was statistically significant (P < 0.05, ;~2-test). DISCUSSION The pharmacological responses of the 5-HT-induced depolarization with decreased Rin meet the proposed criteria for a 5-HT 3 receptor 3. This response was blocked by low nM concentrations of ICS 205-930, while much higher concentrations of ketanserin or methiothepin did not affect it. The IC5o for ICS 205-930 for antagonizing this response was 3 nM, which is close to the IC50 of 0.5 nM for the displacement of [3H]5-HT from apparent 5-HT3 binding sites in rat spinal cord ~2. The ability of the selective 5-HT 3 antagonist, 2-methyl 5-HT, but not the selective 5-HT 2 agonist, a-methyl 5-HT, to mimic the 5-HT depolarization with decreased Rin further strenthens the conclusion that this is a 5-HT 3 receptor-mediated response. Extracellular studies have revealed that 5-HT3 receptors mediate depolarization of rabbit and rat isolated vagus nerve 2°,39 and the rabbit superior cervical and nodose ganglia 42. Intracellular studies have reported that 5-HT 3 receptors mediate a depolarizing response with decreased Rin in guinea pig enteric neurons 4,z6 and neuroblastoma cells3°. However, the D R G response demonstrated an important difference from enteric neurons and neuroblastoma cells, in that it did not show desensitization. It is not without precedence that a receptor subtype may show desensitization in some tissues and not in others. The 5-HT 3 receptor system has been reported to show desensitization in rabbit nodose and superior cervical ganglia 42 but not in rat isolated vagus nerve 2°. The 5-HT2 system shows desensitization in guinea pig trachea 6, but not in vascular tissue 45 or rat frontal cortex TM. The pharmacological profile of the 5-HT induced depolarization with increased Rin meets the proposed criteria for a 5-HT 2 receptor response 3. This response was potently blocked by ketanserin, methiothepin and spiperone, but not by ICS 205-930 or prazosin. In addition, it was mimicked by a-methyl 5-HT but not by 2-methyl 5-HT. a-Methyl 5-HT has a high affinity for 5-HT 2 and 5-HTlc receptors ~8,24, but'since the response

to this agonist was blocked by low concentrations of ketanserin, a 5-HTz receptor is probably involved. The IC50 for ketanserin blockade of the depolarization with increased Rin (8 nM) was close to its Ki value (2.1 nM) obtained from binding studies in brain cortex23 and IC50's (1.7, 3.2 and 30 nM) in various arteries 45 where ketanserin inhibits 5-HT-induced contractions. The same IC50 for ketanserin (8 nM) was reported in rat aorta where 5-HT 2 receptors stimulate PI turnover 41. We observed that the 5-HT 2 response in D R G cells was elicited by low concentrations of 5-HT (ECso 65 nM), whereas binding data suggest that 5-HT has low/~M affinity for 5-HT: sites 34. However, some functional studies measuring vascular contraction 45 and PI turnover in brain cortex36 have shown responsiveness of 5-HTz receptors in the nM range. Recently, Pierce and Peroutka 37 have identified high and low affinity 5-HT: receptors. Thus, the receptor that we observe in rat D R G could be a high affinity 5-HT2 receptor. NE elicits excitatory responses in brainstem 8 and facial motoneurons 44 similar to 5-HT. Interestingly, these two amines have parallel anatomical projections to the brainstem and spinal cord. On D R G cells we observed that NE-induced depolarization with an increased Rin was strikingly similar to the 5-HT 2 receptor response. However, the response to NE appeared to be mediated by a I adrenergic receptors since it was selectively blocked by low nM concentrations of prazosin that did not affect the 5-HT response. The excitatory effect of NE on lateral geniculate neurons is also blocked by both ketanserin and prazosin 22,4°, The question arises whether the receptors present on the DRG cell bodies are also present on the afferent terminals in the spinal cord. Binding data report relatively few 5-HT 2 receptors in rat spinal cord zs. However, quantitative autoradiography revealed the existence of 5-HTz receptors in both ventral and dorsal horns of the spinal cord 9,3z. 5-HT3 binding sites occur in the brain and the spinal cord 12,zl,31. The spinal cord 5-HT3 binding sites are localized to synaptosomes prepared from the dorsal horn lz. Autoradiography 13 has shown that 5-HT3 receptors are heavily localized in the substantia gelatinosa of the spinal cord, and appear on some afferent terminals since 5-HT 3 binding was reduced by 52% in the dorsal but not in the ventral horn after capsaicin treatment. Both a 1 and a2 adrenoreceptors are also present in the spinal cord with high density in lumbar dorsal horn 43. The results of this study suggest that extracellular recording methods would fail to distinguish between the 5-HT3 and the 5-HT2 receptor-mediated responses in the DRG. If this situation attains in other tissues, it could be a source of inconsistent data regarding the selectivity and efficacy of various pharmacological probes. Fortunately

78 the r a p i d desensitization of 5-HT 3 receptors in many tissues p r o b a b l y m o d e r a t e s such confusion. H o w e v e r , since these two receptors not only can occur in the same tissue, but also on the same cell, it is important that the contributions of one of the receptors be eliminated by use of a selective antagonist before the other system is quantitatively studied. In considering the possible functional significance of the 5 - H T 2 and 5 - H T 3 r e c e p t o r systems on the D R G , it is i m p o r t a n t to recognize that in most tissues these recep-

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