Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions

Brain Research, 476 (1989) 183-188 Elsevier

183

BRE 23263

Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions Joseph M. Pads, Hiroshi Mitsushio and Stanley A. Lorens Laboratory of Behavioral Pharmacology, Departmentof Pharmacology, Loyola UniversityMedicalCenter, Maywood, IL 60153 (U.S.A.) (Accepted 6 September 1988) Key words: Raphe nucleus; Neurokinin; Hyperactivity;5,7-Dihydroxytryptamine;5-Hydroxytryptamineneuron

Rats were implanted with cannulae in the median raphe nucleus (MR). 5,7-Dihydroxytryptamine(5,7-DHT) or vehiclewas infused either directly through the MR cannula, or bilaterallyinto the medial forebrain bundle (MFB). The MR 5,7-DHT lesions completely blocked the hyperactivityelicited by injections into the MR of the neurokinin (NK) 3 agonists, DiMe-C7 and senktide, and the NK-2 agonist, neurokinin A. In contrast, the MFB 5,7-DHT lesionsdid not affectthe locomotorhyperactivityproduced by imra-MR administration of DiMe.C7 and senktide, but appeared to attenuate the effects of NKA. The data indicate that intra-raphe neurokinin-induced hyperactivityis mediated by 5-HT neurons, and that 5-HT projectionsto the forebrain may be involvedin the behavioral activation induced by intra-raphe neurokinin A administration, but not that induced by intra-MR NK-3 agonists.

We have been investigating the behavioral effects of the non-mammalian and mammalian tachykinins, as well as synthetic tachykinin analogues, following their infusion into the median raphe nucleus (MR) of the rat mesencephalon. We have shown that intraMR injections of these peptides elicit locomotor hyperactivity 2°'21. The increases in locomotor activity (LMA) appear to be mediated primarily by a neurokinin (NK)-3 receptor, although NK-2 receptors also may be involved (Paris and Lorens, 1988, submitted). We also have demonstrated that the hyperactivity produced by the intra-MR infusion of the metabolically stable neurokinin analogue, DiMe-C7, depends upon intact serotonin (5-HT) neurons 2°'21. The 5-HT ceil bodies located in the midbrain raphe are the principal origin of the 5-HT projections to the forebrain and are the source of serotonergic fibers which innervate areas of the mesencephalon and caudal brainstem t'4'5'9. Using the selective 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), our first objective was to determine if the LMA effects of intra-raphe infusions of neurokinin A (NKA) and the

neurokinin B agonist, senktide, depend on the integrity of MR 5-HT neurons. Our second objective was to determine if the 5-HT fibers which ascend in the medial forebrain bundle (MFB), rostral to the mesencephalic dopamine (DA) cell groups, are critical for intra-MR neurokinin-induced hyperactivity. Male Sprague-Dawley rats (Sasco-King, Orange, WI), 90-120 days old and 275-325 g at the time of surgery, were used. The animals were housed individually in an illumination (12 h light-dark cycle, fights on at 07.00 h), temperature (22 + 2 0(2), and humidity (50 + 5%) controlled facility. Food and wa~er were available ad libitum. The animals were acclimated to their new environment for at least one week prior to surgery. All surgical ~.rocedures have been detailed previously2°. In brieL the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and pretreated with the norepinephrine (NE) and DA reuptake inhibitor, nomifensine maleate (1:~ mg/kg, i.p.; Hoechst-Roussel, Somerville, NJ), 30-40 min prior to neurotoxin or vehicle infusions. One group of rats

Correspondence: S.A. Lorens, Laboratory of Behavioral Pharmacology(Bldg. 135), Department of Pharmacology,Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, U.S.A. 0006-8993/89/$03.50 (~) 1989Elsevier Science Publishers B.V. (BiomedicalDivision)

184 (n = 11) was first implanted with chronically indwelling guide cannulae (equipped with removable stylets) aimed at the MR. They then received an infusion of 5,7-DHT creatinine sulfate (Sigma, St. Louis, MO; 8.0pg in 2.0~1 of 0.1% ascorbate in 0.9% saline, as the base) directly through the cannula. The drug was infused over 10 min (0.2/d/min), and the internal cannula was left in situ an additional 3-5 min to allow adequate spread of the solution. The stylet then was replaced and the animals allowed to recover. The second group of rats (n = 11) received bilateral infusions of 5,7-DHT (8.0/~g in 3.0gl/side in the ascorbate vehicle, as the base) via 28-gauge stainlesssteel tubing into the MFB at the level of the premamillary nuclei. With the incisor bar set 3.3 mm below the interaural plane, the coordinates were: anteroposterior, bregma ~ ) -3.8 mm; mediolateral, ~ _+ 1.7 mm; dorsoventral,/~ -8.7 mm. The drug was infused over 10 min (0.3/~l/min) as described above. Immediately following the 5,7-DHT infusion, an MR cannula was implanted. Two separate groups of control rats were prepared. One group (n = 5) was implanted with MR cannulae, and infused with 2.0~1 of ascorbate vehicle in a manner identical to the MR 5,7-DHT lesion group. The second group of controls (n = 5) received bilateral infusions of ascorbate vehicle (3.0 pl/side) into the MFB as described above, prior to MR cannula implantation. All activity measurements were made in darkened, enclosed, cylindrical photocell chambers (46 cm diameter x 42 cm high) as previously described~. Beginning 14 days postoperatively, and following two days of hank rig, the animals were habituated to the t~gtina an .... t . , ~ ¢,,~~ days, 60 min per day. Drug testing was initiated on the day immediately following the last day of habituation. At 48-h intervals, each rat (n = 32) received intra-MR injections of vehicle (1.0/~l), DiMe-C7 (1140 pmol), NKA (230 pmol) and senktide (30 pmol). These doses were selected on the basis of our previous dose-response analysis which showed that they produced submaxireal but equivalent increases in LMA (Paris and Lotens, submitted; also see Fig. 1). Each drug was administered only once, according to a randomized design. Following the 30-rain poe-drug habituation period, the animals were removed h o m the appara-

tus, injected with drug or vehicle solution, then returned to the chamber for 30 min. The drugs and vehicle were administered at a rate of 1.0 ~l/min in a volume of 1.0/~l. On drug days, total activity counts were recorded for 30 min postinjection. Seventy-two hours following the last drug injection, the animals were instantaneously sacrificed with a guillotine. Their brains were removed and dissected on a cold brass plate. The brainstems were placed in buffered formalin for subsequent histological analysis. The amygdala, hippocampus, hypothalamus, medial frontal cortex, nucleus accumbens and striatum were obtained using a modification of the method described by Heffner et al.m°. The tissue samples were immediately frozen on dry ice, then stored at -80 °C. Tissue levels of 5-HT, 5-hydroxyindole acetic acid (5-HIAA), DA, 3,4-dihydroxyphenylacetic acid (DOPAC), NE, and homovanillic acid (HVA) were determined within 1-2 weeks in each of the brain areas by high performance liquid chromatography (HPLC) with electrochemical detection, using a modification of ~ method previously described 2°. DiMe-C7 ((pGlu 5, MePhe s, Sar9)substance P(5-ll); Peninsula Laboratories, Belmont, CA), NKA (Peninsula), and senktide ((succinyl-Asp6-MePheS)substance Pt6-11); Bachem, Torrance, CA) were dissolved in 0.01 M acetic acid, diluted to a stock concentration with phosphate-buffered saline (PBS; pH 7.4), aliquoted, and kept frozen (-20 °C). On each drug day, an aliquot was thawed and diluted with vehicle (PBS containing 0.01 M acetic acid, pH 7.2). All peptide solutions were kept dark and cold until used. Care was taken throughout to avoid the use of glass or p~lystyrene which can dramatically alter the potency ot tire peptide. Frozen coronal sections (50 pro) through the cannula pla~:ements and MFB 5,7-DHT injection sites were cut on a sliding microtome. Every 4th section was floa~-,nounted from cold PBS ovto gelatinchrome alum-coated slides, and stained with Cresyl violet acetate. All data were subjected to an ~nalysis of variance (ANOVA), and post-hoc comparisons of means were made with Duncan's multiple range test 24. For the biochemical data, separate analyses (either ANOn9, or Student's t-test) were performed for each brain area and substance determined.

185 Histological analysis revealed that of the 32 rats studied, 5 had to be eliminated because their MR cannula placements were greater than 1.0 mm lateral or ventrolateral to the midline. Their data were dropped prior to all subsequent analyses. A typical MR cannula placement, and the histological damage produced by intra-MR 5,7-DHT injections, have been illustrated in an earlier publication2°. The 5,7DHT injections in the present study produced some cell loss (presumably 5-HT neurons) with minimal glial scarring in the region surrounding the cannula tip. No extensive scarring or cavitation was ob~orved. Analysis of the MFB injection sites revealed that the 5,7-DHT and vehicle infusions did n o t cause any destruction of perikarya in the region, cavitation at the tip of the injection needle, or discernible retrograde degeneration of neurons in the MR. An initial analysis of the data (not shown) indicated that the two groups of control animals did n o t differ either in their response to the neurokinin infusions or neurochemically, therefore thdr data were combined for subsequent analysis. Fig. 1 shows the total activity counts for ~il~ 30 min following the injection of DiMe-C7, NKA, ar~ senktide for the 3 groups of rats. An ANOVA revealed significant lesion (F(2,24~ = 6.18; P < 0.01) and drug (F(3,72) = 24.91; P < 0.001) effects. The lesion x t~rug interaction also was significant (Ft6,72) = 4.63; P <

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Fig. 1. Effects of intra-median raphe (MRI in.fusions of senktide (30 pmol), neurokinin A (NKA; 230 pmol), DiMe-C7 (1140 pmol) and vehicle (l.0ld PBS with acetic zcid, pH 7.2) in rats which had sustained either MR or MFB 5,7-dihydroxytryptamine lesions or vehicle control injections two ,•eeks previously (n = 9 per group). Data represent mean -+ S.E.M. total activity counts for 30 min postinjection. Significant diffcrence from corresponding vehicle infusion: *P < 0.05; significant difference from corresponding infusion in control group: +P < 0.05.

0.001). The posthoc analysis indicated that for the control group, administration of all 3 peptides significantly elevated LMA compared to vehicle, and that the activity levels produced by these infusions did not differ significantly from each other. Further analysis indicated that the LMA was equivalent following the vehicle infusions in all 3 groups. The activity counts produced by infusions of DiMe-C7 and senktide into the control and MFB lesion groups were virtually identical. The MR 5,7-DHT lesions completely blocked the LMA elicited by the infusions of DiMe-C7, NKA and senktide. In contrast, the MFB 5,7-DHT lesions did not affect the locomotor hyperactivity produced by intra-MR administration of scnktide and DiMe-C7, but appeared to attenuate the effects of NKA. In rats with MFB lesions, the hyperactivity produced by infusions of NKA was significantly lower than that produced by DiMe-C7 and senktide. In addition, the hyperactivity elicited by intra-MR infusions of NKA into the MFB lesion group was intermediate to that produced by similar infusions in the control and MR lesion groups. The MR 5,7-DHT injections significantly reduced the 5-FIT and 5-HIAA in all 5 brain regions assayed (Table I). The MFB 5,7-DHT injections produced virtual depletion of 5-HT and 5-HIAA concentrations in all the brain regions. In all cases, the MFB le~,iens produced reductions greater than 86% from comrol levels. "Iable II shows the effects of the MR and MFB 5,7DHT lesions on forebrain DA, NE and metabolite levels. The MFB lesions produced significant reductions in NE levels in both the medial frontal cortex and hipFocampus. These lesions also produced significant reductions in DA levels in all brain regions analyzed, except the amygdala. DOPAC levels were affected by the MFB lesions only in the striatum and nucleus accumbens. HVA concentrations were significantly reduced in the nucleus accumbens of rats with MFB lesions. The only effect of the MR lesions on DA levels was an increase i~. the amygdala. Similarly, DOPAC levels were significantly increased in both the amygdala and medial frontal c6rtex in rats with MR 5,7-DHT lesions. We have shown previously that infusion of the substance P anaiogue, DiMe-C7, into the MR produces

186 dose-dependent increases in LMA which can be blocked by pretreatment with the tryptophan hydroxylase inhibitor, parachlorophenylalanine (PCPA), and by MR 5,7-DHT lesions :°'21. In the present study, we sought to determine if 5,7-DHT lesions of the MR would similarly block the effects of intraraphe infusions of NKA, and NK-2 receptor agonist, and of senktide, an NK-3 receptor agonist 15. Doses which previously were determined to produce equivalent increases in LMA (Paris and Lorens, submitted) were administered to rats two weeks following 5,7-DHT lesions of the MR. These lesions completely blocked the hyperactivity produced by intraraphe neurokinin infusions. In order to test whether the 5-HT fiber projections to the forebrain mediate the LMA effects of intra-MR infused neurokinins, 5,7-DHT was injected bilaterally into the MFB, anterior to the D A cell bodies of the rostroventral midbrain tegmentum. These lesions, in contrast to the 5,7-DHT lesions of the MR, did not block the hyper-

TABLE I Effects of intra-median raphe (MR) and medial forebrain bun. die (MFB) 5, 7-dihydroxytryptamine lesions on forebrain sero. ionin ~.evels Region

5-HT (ng/g)

Group Amygdala Control 1114 +_58 MR-lesion 292 + 74* (-74%) MFB-lesion 60 4- 29* (-95%) Medial frontal cortex Control 792 4- 44 MR-lefion 222___71" (-72%) MFB-lesion 42+ 16" (-95%) Striatum Ccntroi 794 _-247 MR-les'on 429 4- 68* (-46%) MFB-lesion 114 _+8* (-86%) Nucleus accumbens Control 1157 _ 83 MR-lesion 652 +_155+ (-44%) MFB-lesion N.D. (->98%) Hippocampus Control 583 _+37 MR-lesion 67 4- 13" (-89%) MFB-lesion 53 4- 16" (-91%)

5-HIAA (ng/g)

255 + 17 89 + 19+ (-65%) N.D. (->96%) 192 + I 1 49+17" (-74%) N.D. (->97%) 319 4- 23 158 + 32* (-50%) N.D. (->94%) 327 _+18 167 4- 38* (-49%) N.D. (->94%) 278 4- 17 61 4- 9* (-78%) 30_+9* (-89%)

n = 7-9 per group; data represent mean + S.E.M.; numbers in parentheses show % change from corresponding Control; significant difference from corresponding Control: +, P < 0(15; *P < 0.01. N.D., not detectable; less than limits of sensitivity: amygdala: <10 ng/g; medial frontal cortex: <5 ng/g; striaWm: <20 ng/g; and nucleus accumbens: <20 ng/g wet wt. of tissue.

activity produced by intra-raphe injections of DiMeC7 and senktide, although they appeared to attenuate the hyperactivity produced by NKA. These results indicate that intra-raphe neurokinin induced hypgractivity depends on the integrity of 5-HT neurons in the MR. In addition, the hyperactivity produced by activation of MR NK-2, but not NK-3 receptors, depends, in part, on the 5-HT fibers which course through the MFB. Since our MFB 5,7-DHT lesions were placed rostral to the DA cell bodies in the ventral tegmental area (VTA) and substantia nigra (SN), it is possible that a 5-HT projection to one of these regions is critical for MR neurokinin-induced effects on LMA. 5HT neurons have been reported to make synaptic contacts with DA neurons in the VTA and SN n,17. Although the 5-HT innervation of the SN has been reported to originate in the MR 8. recent anatomical evidence suggests that the dorsal raphe nucleus (DR) is the more likely source 2s. The DA neurons of the VTA, on the other hand, are innervated by 5-HT fibers which arise in the MR TM.These D A neurons, and the forebrain regions they innervate, moreover, have been implicated in the regulation of LMA 2a9. Thus, it can be postulated that a MR neurokinin --+ 5-HT ---) D A circuit is involved in the regulation of behavioral arousal. In support of this view, we recently have found that low doses of the DA antagonist, haloperidol (200/~g/kg, i.p.; unpub!ished data), can block the LMA effects of intra-MP. DiMe-C7, but not that of the GABA A agonist muscimol. Studies are in progress using 6-hydroxydopamine lesions and more selective DA antagonists to further test this hypothesis. Alternatively, it is possible that intra-MR neurokinin-induced hyperactivity depends on a descending or intra-brainstem 5-HT projection. The MR has been shown to send 5-HT as well as non-5-HT projections to the nuclei raphe magnus and obscuras in the rat, and the reticular formation and nucleus raphe pallidus in the cat 4,5,9. No direct spinal projections from the MR have been observed, although a projection to cervical levels originating in the reticular formation, just lateral to the MR, has been described 6. The MR 5,7-DHT lesions led to increases in amygdaloid DA and DOPAC levels, and to increased DOPAC levels in the medial frontal cortex (Table I1). No changes in DA or DOPAC levels were found

187 in the nucleus accumbens or neostriatum. These observations suggest that damage to M R 5-HT neurons results in enhanced D A utilization in select forebrain regions and supports the notion of 5 - H T - D A interactions in the mesencephalon. The increases in D O P A C levels in the medial frontal cortex are in contrast to those observed following electrolytic lesions of the M R 12. This discrepancy, however, may be due to the non-specificity of electrolytic lesions as a technique to deplete 5-HT concentrationst6. Although 5-HT neurons are known to interact with basal ganglia structures22, the M R 5,7-DHT lesions did not affect D A - D O P A C levels in the striatum, which is in agreement with results following electrolytic lesions7'22. In fact, only electrolytic lesions of both the dorsal and median raphe nuclei have been observed to produce transient changes in striatal D O P A C levels 13. The MFB 5,7-DHT lesions produced falls in D A , D O P A C , and NE levels in all areas assayed, except the amygdala, suggesting that the nomifensine pretreatment did not completely protect cateeholamine neurons. The decreases in catecholamine and metab-

olite concentrations (20-40%) were not as ~ e a t as the falls in 5-HT and 5 - H I A A levels. The decreases in catecholamine levels, however~ were not as extensive as those observed following MFB 5,7-DHT lesions without pretreatment 23. Baumgarten3 has suggested that a nomifensine and desmethylimipramine combination may provide optimum selectivity of 5,7D H T for 5-HT neurons. Alternatively, it is possible that the MFB 5 , 7 - D H T lesion effects on forebrain catecholamines could be due to mechanical damage to fibers of passage within the MFB. However, our histological data do not support this view. It is important to note, furthermore, that although the MFB 5,7-DHT lesions did produce 20-40% falls in forebrain eatecholamine and metabolite concentrations, these lesions did not block the LMA effects produced by activation of M R NK-3 receptors, although they did attenuate the hyperkinesis produced by stimulation of M R NK-2 receptors. Data from the present study indicate that intraraphe neurokinin-induced hyperactivity is mediated by 5-HT neurons. Ascending fibers from the MR to the forebrain do not appear to play a major role in

TABLE II

Effects of intra-median raphe (MR) and medial forebrain bundle (MI'B) 5, 7-dihydroxytryptamine lesions on forebrain dopamine and norepinephrine levels Region Group

NE (ng/g)

DA

DO PA C

HV,4

Amygdala Control 597 ±_34 381 + 42 30 + 3 MR-lesioti 659 + 36 503 + 21" (+32%) 43 + 4* (+43%~ MFB-lesion 476 + 22 299 + 22 29 + 2 Medial frontal cortex Control 353 +_ 17 105 _+9 18 + 1 MR-lesion 351 ~. -~ 18 130 _ 11 29 + 3* (+61%~ MFB-lesion 268 ± 16" (-24%) 83 + 9* (-21%) 16 + 2 Striatum Control 7678 - 346 887 + 67 352 + 26 MR-lesion 7543 ± 358 1003 + 29 412 + 26 MFB-lesion 5737 + 406* (-25%) 533 + 60* (-40% I 264 + 40 Nucleus accumbens Control 7222 - 332 1109 + 49 378 + 27 ~" 6251 + 371 1088 + 101 372 + 45 MR-lesion MFB-lesion 5682 + 141" (-21%) 760 + 78* (-31%) 231 ± 31" (-39%) Hippocampus Control 808 + 31 MR-lesion 831 +_53 MFB-lesion 590 + 45* (-27%) n = 7-9 per group; data represent mean ± S.E.M.; numbers in parentheses show % change from correspondingcontrol; significant difference from corresponding control: *P < 0.01.

188 mediating the L M A effects of NK-3 agonists (DiMeC7 a n d senktide), but may be involved, at least in part, in m e d i a t i n g the effects of NK-2 agonists ( N K A ) . T h e results imply, m o r e o v e r , that the locomotor effects of i n t r a - M R injections of NK-2 and -3 agonists m a y d e p e n d o n a 5 - H T projection to D A cell bodies in the V T A . Alternatively, 5 - H T projections

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