PharmacologyBiochemistryand Behavior,Vol. 48, No. 4, pp. 1041-1045, 1994 Copyright©1994ElsevierScienceLtd Printed in the USA.All rights reserved Pergamon 0091-3057/94 $6.00 + .00 0091-3057(94)E0051-I BRIEF C O M M U N I C A T I O N Electrical Stimulation of the Dorsal Raphe Nucleus as a Discriminative Stimulus: Generalization to ( +_)-DOI D A V I D J. M O K L E R , l M A R K D I X O N A N D L Y N D A S T A M B A U G H Department o f Pharmacology, University o f N e w England, College o f Osteopathic Medicine, Biddeford, M E 04005 R e c e i v e d 6 J u l y 1993 MOKLER, D. J., M. DIXON AND L. STAMBAUGH. Electrical stimulation of the dorsal raphe nucleus as a discriminative stimulus: Generalization to (+)-DOI. PHARMACOL BIOCHEM BEHAV 48(4) 1041-1045, 1994.- Electrical stimulation of the dorsal raphe nucleus of Sprague-Dawley rats was used as the cue for discrimination using a taste aversion paradigm. Rats were trained to associate saccharin drinking during electrical stimulation of the dorsal raphe nucleus with LiC1 injection after the session as the aversive unconditioned stimulus. In sessions without stimulation, rats were allowed to consume saccharin and received a saline injection after the session. Suppression of saccharin consumption during electrical stimulation was learned within 12 trials. Rats trained in the reverse discrimination, i.e., sessions with no electrical stimulation paired with LiC1 injection, showed a similar learning curve. Animals injected prior to the session with the hallucinogenic 5-HT2 agonist ( + )-DOI associated DOI with electrical stimulation of the dorsal raphe nucleus. Thus, animals may be trained to discriminate electrical stimulation of the dorsal raphe nucleus. Furthermore, animals generalize from activation of 5-HT2 receptors to electrical stimulation of the dorsal raphe nucleus. Electrical stimulation Dorsal raphe nucleus Discrimination Rats DOI 5-HT2agonist C U R R E N T hypotheses for the actions of indolealkylamine and phenylalkylamine hallucinogens suggest an interaction with 5-hydroxytryptamine (5-HT) systems of the brain. Data supporting this hypothesis are a) binding o f hallucinogenic drugs to 5-HT receptors particularly the 5-HT 2 subtype (14, 17,22,28,33), and b) antagonism of the behavioral effects of these hallucinogenic drugs with antagonists such as ketanserin and pirenpirone which are selective for the 5-HT 2 receptors (16,26,27,32,33,40). The dorsal raphe nucleus of the midbrain is one of the major nuclei of 5-hydroxytryptamine (5-HT) cells that project to the forebrain. Electrical stimulation of the dorsal raphe nucleus (DRN) activates ascending 5-hydroxytryptamine (5HT) neurons. This stimulation has been shown to increase the tissue levels o f the 5-HT metabolite 5-HIAA (1,20), increase the turnover of 5-HT in forebrain regions (9,35,37), and in- 5-Hydroxytryptamine crease the levels of extracellular 5-HT as determined by in vivo microdialysis (36). Animals can be trained to discriminate electrical stimulation of the DRN. Using a classical operant drug discrimination paradigm, Hirschhorn et al. (18) trained rats to discriminate nonaversive stimulation of the DRN. Animals were trained to a stimulus of 200-300 #A biphasic stimulation. After learning this discrimination, rats were tested for generalization to LSD or morphine. Rats generalized from the hallucinogen LSD (100 #g/kg) to electrical stimulation of the DRN. The purpose of this investigation was to further examine the discrimination of DRN stimulation and to extend these results to the 5-HT 2 agonist DOI. DOI is an agonist at 5-HT 2 receptor (2,8,10,12,13,22,31,40,41). DOI binds to 5-HT2A and 5-HT2c receptors in the forebrain, produces behavioral effects that are blocked by the 5-HT2A antagonist ketanserin, and To whom requests for reprints should be addressed. 1041 1042 MOKLER, DIXON AND STAMBAUGH acts in a manner similar to 5-HT to stimulate phosphoinositol hydrolysis in forebrain regions, an action of 5-HT attributed to activation of 5-HTEA receptors. An inherent problem with training animals to discriminate electrical stimulation using classical operant behavioral techniques is the time required for training may be long enough to produce tissue changes around the electrode, thus reducing the efficacy of the stimulation. To facilitate learning of the stimulus cue, a rapid method of discrimination training using a conditioned taste aversion paradigm was utilized (19,21). METHOD Male Sprague-Dawley rats (250-300 g, Charles River Laboratories, Wilmington, MA) were trained to drink a 0.25010 saccharin solution in dally 30-min sessions. Animals were water deprived for 22 h prior to the session. Animals were given access to water for 1 h after the end of the session, as well as continuous access to food. Once stable levels of saccharin were being consumed, dally sessions were reduced to 15 min. Animals were then implanted with stainless steel bipolar electrodes (Plastics One, Roanoke, VA) into the DRN at the stereotaxic coordinates of A 1.2; L 0.0; V 3.5, with reference to intraural zero according to the atlas of Paxinos and Watson (29). Anesthesia was induced by pentobarbital sodium (50 rag/ kg, Sigma Chemical) and supplemented with additional pentobarbital sodium or halothane as needed. Electrical stimulation (ES; 100 #A, 100 #s biphasic pulse pairs at 20 Hz) was delivered on alternate days using a Grass S11 Stimulator with Grass Constant Current Stimulus Isolation Units (Grass Instruments, Quincy, MA). Animals were placed in a standard Plexiglas animal cage without bedding. An electrode lead was attached to the electrode while the animal was under gentle hand restraint. The electrode lead was attached to a single channel commutator (both from Plastics One, Roanoke, VA) mounted on a Plexiglas cover for the cage. The animal was given access for 15 min to a 100 ml graduated drinking tube containing saccharin and the number of ml drank during the session recorded. In the first group of animals, electrical stimulation of the DRN was followed by an injection of 1 ml/kg LiC1 (1.8 mEq/ml, 76.32 mg/ml, Sigma Chemicals) immediately after the session. This dose of LiCl makes the rat sick for approximately 1-2 min. The animals show a characteristic behavior that includes licking the sight of injection, hindlimb extension, and dorsiflexion. After injection, animals were placed back in their home cages and returned to the animal quarters. On nonstimulation days, the animals were connected to the electrode lead and placed in the cage without stimulation. Immediately after the session, the animals were injected with 1 ml/kg 0.9010 NaCI. In the second group of animals, electrical stimulation of the DRN was followed by an injection of 0.9010 NaCl, whereas nonstimulation was followed by an injection of LiCl. Thus, half of the animals were trained to associate electrical stimulation with LiCl injection, whereas the other half learned to associate nonstimulation with LiC1 injection. The amount of saccharin consumed in milliliters was measured after each session. For generalization testing, animals were injected IP with ( + ) - 1-(2,5 -dimethoxy-4-iodophenyl)-2-aminopropane HCI (DOI HC1, D101, Research Biochemicals, Inc., Nadick, MA) or saline 5 rain prior to the beginning of the session. DOI was dissolved in distilled water. Animals were then allowed access to a saccharin solution for 15 min. No stimulation was administered nor were any injections given after the session. In order to determine if DOI alone had an effect on the consumption of saccharin, a separate group of 10 animals was trained to drink saccharin. These animals were deprived of access to water in their home cages in the same manner described above. DOI (0.25, 0.5, and 1.0 mg/kg) was administered 5 min before the beginning of the session. Saccharin was then offered to the animals for 15 min and the amount of saccharin (ml) consumed was recorded. After the behavioral testing was complete, animals were anesthetized with an overdose of Na pentobarbital. After the animal had reached a surgical level of anesthesia, the animal was decapitated and the brain removed and frozen. The brain was then mounted on a freezing stage microtome and sliced to determine electrode placement. Data was analyzed as the amount of saccharin consumed" during the test session. A two-way analysis of variance with repeated measures was used to compare stimulation vs. nonstimulation during training. A one-way analysis of variance with repeated measures was used to examine the effects of DOI. A Student-Newman-Keuls test was used for post hoc comparisons. A probability of less than 5010 was used in all tests. Statistics were performed using SigmaStat v. 1.01 (Jandel Scientific, San Rafael, CA). RESULTS Animals learned to drink a steady level of saccharin within 12 sessions with consumption of 18.3 + 1.0 ml of saccharin in 30 min. On the first day of 15 rain sessions, animals consumed 19.2 + 0.6 ml Stimulation of the DRN prior to the beginning of discrimination training decreased saccharin consumption, but by four exposures the animals were drinking equivalent amounts on stimulated and nonstimulated days (12.4 + 1.2 ml and 14.6 + 1.0 ml, respectively). Pairing of the stimulation or nonstimulation of the DRN with LiCI produced a clear discrimination in 6 reversals or 12 sessions (Fig. 1). Pairing of stimulation with LiCI injection resulted in a significant effect on stimulation [stimulation vs. 2O 15 0~ zo lo ~D z "~ ~D u rn 5 o 2 3 4 6 5 TRAINING SESSION O NONSTIM - • STIM - Lie1 NaCI A STIM - • NONSTIM - NaC1 LiC1 FIG. 1. The training curves for discrimination of ES of the DRN. Graph shows two groups trained under two conditions. One group was trained with ES associated with LiCl injection (circles). The other group was trained with nonstimulation associated with LiCl injection (triangles). Points represent mean ± SEM, n = 4. *Significantlydifferent from session 1; + Significantly different from corresponding NaC! session, two-way ANOVA, Student-Newman-Keuls post hoc test, p < 0.05. DISCRIMINATION OF DORSAL R A P H E STIMULATION 1043 criminated taste aversion paradigm. The training time in the current study was similar to the rapid training of drug discrimination using this paradigm reported by Lucki (21) and Riley and co-workers (24,34). Hirschhorn et al. (18), obtained the same discrimination using a classical operant paradigm with higher stimulus intensity. The discriminated taste aversion paradigm allows for rapid training of animals as compared to a classical two-lever operant paradigm which requires more than twice as many sessions to reach a minimal discrimination [Mokler, unpublished data; (18)]. A reversal of the discrimination conditions was used to determine the effects of electrical stimulation of the dorsal raphe nucleus on the ability of the animals to perform the behavioral task. One group was trained to suppress drinking of saccharin during ES and the other to suppress drinking during nonstimulation sessions. The two conditions yielded almost identical results, indicating that the electrical stimulation did not influence the ability of the animals to perform the behavior or, indeed, to acquire the discrimination. Thus, it is the aversive conditions the rats associated with stimulation of the DRN that affected saccharin drinking behavior, not the ES itself. The hallucinogenic 5-HT 2 agonist DOI generalized to ES of the DRN. Following DOI administration, animals trained to suppress saccharin consumption during electrical stimulation suppressed saccharin consumption. Conversely, animals trained to drink saccharin under ES continued to drink at normal levels with DOI administration. At the highest dose of DOI, saccharin consumption was suppressed in both groups. This, and independent findings that 1.0 mg/kg DOI suppresses saccharin consumption, indicates that this dose suppresses behavior generally. DOI has been shown to suppress milk consumption at a dose of 1/~mol/kg (.36 mg/kg) (38). In the present study, doses of .25 or .5 mg/kg DOI did not suppress saccharin consumption. The differences between the results of Simansky and Valdya (38) and our results may relate to the state of deprivation of the animals. DISCUSSION The present results also suggest that activation of ascending Male Sprague-Dawley rats were trained to discriminate 5-HT neurons of the dorsal raphe nucleus may have stimulus electrical stimulation of the dorsal raphe nucleus using a disproperties similar to activation of 5-HT 2 receptors. This is similar to the findings of Hirschhorn et al. (18) that LSD also generalizes to electrical stimulation of the DRN. In a number of behavioral and neurochemical experiments LSD has been 20 20 shown to interact with 5-HT 2 receptors (4-6,11,14,15,25,27, 30,40). This adds to considerable data to suggest that the halv lucinogens act to stimulate postsynaptic receptors. Presum15 15 ably the 5-HT2 receptor plays a major role in these actions. Also of interest in this regard is the findings that projections o f the DRN terminate in forebraln regions rich in 5-HT2 recep5'3 tors (3,23). Thus, stimulation of the DRN may preferential 10 10 C.) stimulate 5-HT2 receptors. z Using a two-lever operant discrimination task, Glennon (13) showed that rats could be trained to discriminate DOI from saline. LSD and DOM, but not the 5-HTIA agonists 8-OHDPAT or TFMPP, generalized to the stimulus properties of DOI. The present data suggests that activation of 5-HT2 0") o • 0 receptors with DOI has discriminative stimulus properties simSTIM 0.5 i. 0 SALINE 0. I ilar to the electrical stimulation of the DRN. Numerous studies have shown that electrical stimulation of DOI ( m g / k g ) the DRN increases the release of 5-HT in forebrain regions • STIMULATION - LiE1 • NON-STIMULATION - LiC1 (1,7,9,20,35-37). Recently, however, other studies have shown that the administration of DOI produces a decreased release FIG. 2. Effects of administration of DOI, IP before the session in of 5-HT in forebrain regions (41). Wright et al. (1990) further rats trained to discriminate ES of the DRN. Animals were trained to showed that microinjection of DOI into the frontal cortex did associate ES with injection of LiCI (circles) or NaCI (triangles). Points not produce a decrease in release of 5-HT as determined by in represent mean + SEM, n = 4. *Significantly different from saline. vivo microdialysis. This suggests that DOI may produce One-way ANOVA, Student-Newman-Keuls post hoc test, p < 0.05. nonstimulation, F ( I , 15) = 22.685] and the interaction between session number and stimulation, F(5, 15) = 5.608, without a significant effect on session number, F(5, 15) = 2.039. Pairing of nonstimulation with LiC1 injection resulted in a significant effect of stimulation, F(1, 15) = 29.251, session, F(5, 15) = 7.701, and the interaction between stimulation and session, F(5, 15) = 6.99. No differences were seen between animals with stimulation of the DRN paired with LiCl and nonstimulation paired with LiCI (Fig. 1). Thus, stimulation of the DRN alone does not affect the animal's ability to drink saccharin or to associate LiCl with stimulation. In order to determine the effects of lower levels of stimulation, the current was lowered to 20 #A. Under this condition, animals with stimulation associated with LiCl drank 11.3 _+ 2.2 ml during stimulation (data not shown). Likewise, rats with nonstimulation associated with LiC1 drank 9.25 + 1.3 ml saccharin during stimulation. These data suggest that animals do not generalize completely from stimulation at 100 # A to stimulation at 20 #A. Injection of the hallucinogenic 5-HT2 agonist DOI before the session resulted in stimulation appropriate behavior (Fig. 2). Animals that had been conditioned to suppress saccharin consumption during stimulation showed a suppression of saccharin drinking, F(3, 9) = 88.47, with generalization occurring at all doses of DOI. Animals conditioned to suppress saccharin consumption during nonstimulation also showed stimulation appropriate responding at doses of 0.1 and 0.5 mg/kg DOI, F(3, 9 = 28.203. At a dose of 1.0 m g / k g DOI, both groups showed a suppression o f drinking, suggesting that at this dose DOI suppresses behavior (Fig. 2). Thus, DOI generalizes to stimulation of the DRN in this paradigm. In a separate experiment, rats drinking saccharin without training in the discrimination paradigm showed a significant decrease in saccharin consumption after 1.0 m g / k g DOI, but not 0.25 or 0.5 m g / k g (data not shown). 1044 MOKLER, DIXON AND S T A M B A U G H changes in the release of 5-HT in forebraln regions independent of, or secondary to stimulation of postsynaptic 5-HT2 receptors. Lower levels of ES of the DRN should serve as discrimination cues. Although Hirschhorn et al. (18) used higher levels o f stimulation (200-300 #A) than in the current study, Wheeling et al. (39) have determined that the threshold of stimulus is lower than the levels employed in this work (20-30/~A). Data using a stimulation current of 20 #A, however, suggest this level of stimulation is not similar to the effects of stimulation at 100/~A. Further experiments are necessary to determine if animals can be trained to lower levels of stimulation. These experiments have suggested a similarity between stimulation of one of the major nuclei o f 5-HT cells projecting to the forebrain and the stimulus properties of a drug that stimulates postsynaptic 5-HT2 receptors. These are preliminary findings that suggest a number of experiments to further explore the relationship between the hallucinogens and stimulation of the dorsal raphe nucleus. 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