Substance K and substance P in the ventral tegmental area

Neuroscience Letters, 57 (1985) 241-246

241

Elsevier Scientific Publishers Ireland Ltd.

NSL 03361

S U B S T A N C E K A N D S U B S T A N C E P IN TIlE V E N T R A L T E G M E N T A L AREA

P E T E R W. K A L I V A S ~,*, A R I E L Y. DEUTCH% J O H N E. M A G G I O 2, P A T R I C K W. M A N T Y H 3 and R O B E R T H. R O T H 2

1Department of Pharmacology, Louisiana State University Medical Center, New Orleans, LA 70112; ZDepartments of Psychiatry and Pharmacology, Yale University Medical School, New Haven, CT 06510; and 3Center for Ulcer Research and Education, Veterans Administration Center, Los Angeles, CA 90073 (U.S.A.) (Received March 12th, 1985; Accepted March 21st, 1985)

Key words" substance P - substance K - dopamine - ventral tegmental area - receptor - immunocytoc h e m i s t r y - autoradiography

motor activity

A comparison was made between substance P (SP) and substance K (SK) in the ventral tegmental area (VTA) (A10 dopamine cell group) of the rat. Approximately equal densities o f SP and SK-immunoreactive neuronal fibers were observed. However, while previous reports demonstrate negligible density of autoradiographically defined SP receptors in the VTA, we observed a high density of SK receptors. SK or SP was microinjected into the VTA, and changes in spontaneous motor activity were measured using a photocell apparatus. SK was found to be at least 10 times more potent than SP in producing an increase in motor activity. These data suggest that while both SK and SP are present in the VTA, SK m a y have a more significant physiological role in modulating dopamine neurons in the ventromedial mesencephalon.

Substance P (SP) was isolated and characterized in 1970 [1, 2] and is the best known member of a family of peptides which share a conserved carboxyl terminus (-Phe-X-Gly-Leu-Met-NH2) termed the tachykinins [3]. Differences in the biological activities of various'SP analogues and fragments led to the suggestion that endogenous tachykinin ligands other than SP might be present in the central nervous system (CNS) of mammals [11, 24]. Subsequently, two novel tachykinins (X=valine) were isolated in the mammalian CNS: substance K (SK; neurokinin alpha) [10, 14, 17] and neuromedin K [8, 10]. Both SP and SK are derived from a common prohormone in the CNS [17]. While numerous data support a neurotransmitter-like role for SP in the CNS [19], only recently have data been advanced supporting a similar central function by SK. SK is heterogeneously distributed within the CNS [13, 14, 22], and SK release has been demonstrated to occur in striatal and cortical slices in a calcium-dependent fashion [18]. Since in bovine striatum the gene coding for SP also contains a sequence coding for SK, it is not surprising that these two tachykinins are colocalized in both peripheral and central tissues [13, 22]. Thus, SK and SP in the spinal cord are both depleted by capsaicin treatment [13]. *Author for correspondence at present address: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, W A 99164-6520, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.

242 SP appears to exert an excitatory influence on the midbrain dopamine (DA) neurons in the ventral tegmental area (VTA), as evidenced by an increase in locomotor activity and enhanced DA metabolism in the nucleus accumbens after intra-VTA injection with SP [6, 9, 23]. Therefore, we have compared the locomotor response of intra-VTA injection with SK to that of SP. Autoradiographic evaluation of central receptor binding sites for SP and SK or the SK analogue, kassinin (which is not found in mammals), has revealed a marked divergence in receptor density in certain brain areas [16]. Thus, we have also compared the receptor binding density and distribution of immunoreactive nerve fibers for SK and SP in the VTA using immunocytochemistry. Male Sprague-Dawley rats were housed individually with food and water made available ad libitum. Behavioral testing was performed in a photocell apparatus [6]. When rats attained a weight of 300-350 g they were anesthetized with ketamine plus pentobarbital and stereotaxically implanted with chronic bilateral 26-gauge stainlesssteel injection cannulae I mm over the VTA [7]. One week after surgery, behavioral testing was begun, and each rat received up to 4 behavioral trials separated by a minimum 72-h interval. In each trial, rats were adapted to the photocell cage for 60 min, after which they were removed from the cage and microinjected with either SK, SP or vehicle in a volume of 0.5 iLl/side infused over 60 s [7]. During the injection the rats were unrestrained and immediately following the injection they were returned to the same photocell cage where photocell beam disruptions were counted for 120 min. After the last behavioral trial, the rats were killed, brains sliced in the coronal plane, and the slices prepared for histological evaluation of cannulae placement in the ventral mesencephalon [7]. All rats used for data analysis (n = 27) had cannulae placed in the nucleus paranigralis or nucleus parabrachialis pigmentosus rostral to the nucleus interpeduncularis. For the behavioral study, SK and SP were dissolved in sterile distilled water containing 0.010,~, (v/v) glacial acetic acid pH 3.4. Thus, control injections consisted of a 0.01°~; glacial acetic acid solution. Both SK and SP were aliquoted in 10-/A volumes into polypropylene tubes and stored at - 7 0 C until use. Following injection the remainder of the aliquot was returned t o - 70'C and stored until radioimmunoassay, and amino acid analysis for SP and SK was performed to verify the content of each aliquot [13]. In all samples, the content determined before and after centrifugation (20,000 g, 2 h) was found to be unchanged. Peptide purity was further confirmed using high-performance liquid chromatography (HPLC) [13]. Immunohistochemical examination of SK and SP distribution was performed by the peroxidase-immunoperoxidase technique [7]. Antiserum was raised in rabbit against kassinin and exhibited less than 0.5°~ cross-reactivity with SP [13]. However, full cross-reactivity was observed with neuromedin K, which has the same C-terminal sequence as kassinin and SK. Polyclonal antiserum to SP was purchased from Immunoclear Corp. and exhibited negligible cross-reactivity with SK. Frozen sections were cut and incubated in 1:2000 dilution of either SK or SP antisera. For each tachykinin, adsorption and cross-adsorption controls were performed by adding either 5 /Lg/ml of peptide towards which the primary antiserum was directed or 50 ~g/ml of the other

243

peptide. Times in the sheep anti-rabbit IgG, peroxidase-anti-peroxidase complex and DAB-H202 solution were held constant across the various trials. Autoradiographic localization of SK binding sites was assessed using 125I-labelled Bolton-Hunter SK [16]. Slide-mounted coronal tissue sections (20 #m) were allowed to come to room temperature and placed in a preincubation medium (25°C for 10 min) of 50 mM Tris-HC1, pH 7.4, containing bovine serum albumin (200 mg/l), chymostatin (2 mg/l), leupeptin (4 mg/l) and bacitracin (40 mg/1). The slide-mounted tissue sections were then incubated at 25c~C for 2 h in the incubation medium which consisted of the same solution as described above except that 100 pmol of 125Ilabelled Bolton-Hunter SK was added to the incubation solution. To estimate the non-specific binding, paired serial sections were incubated as described above except that 1 #mol SK (Bachem) was added to the incubation solution. From this analysis, specific binding was estimated to be > 95~o of the total binding. Following this incubation the slides were rinsed in 3 washes of pre-incubation solution (4°C, 5 min each)

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D Fig. 1. Dark-field micrographs showing the distribution of kassinin-immunoreactive fibers in the VTA and adjacent nuclei (A), and the elimination of stain after adsorption of the kassinin-directed antiserum with 5.0 ~g,'ml SK (B). Dark-field (C) and light-field (D) photomicrograph of the same coronal section ofmidbrain that has been incubated in 100 pM [~zsI]SKand then Nissl-stained. In the dark-field photomicrograph, [~25I[SK binding sites appear as white silver grains. Note that the VTA has a relatively high concentration of [125I]SK binding sites, whereas the SNR has very low concentrations. Abbreviations are the same as those in Paxinos and Watson and are: CA3, field CA3 of Ammon's horn; CG, central grey; fr, fasciculus retroflexus; IP, interpeduncular nucleus; L, nucleus linearis; ML, medial lemniscus; MP, posterior medial mammillary nucleus; RLi, rostral linear raphe nucleus; S, subiculum; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. Bar = 1.2 mm.

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and 2 washes of deuterated H20 (4°C, 5 s each) and then quickly dried in the cold room using a stream of cold air. Sections were then left for 3 h to dry in the cold and stored in desiccant filled boxes overnight at -20~'C. For estimating the specific: non-specific binding ratios, tissue sections were scraped from the slide with a razor blade and then placed in tubes and the radioactivity measured by a gamma counter. For autoradiographic analysis of the [125I]SK binding sites, the slides were placed in opposition to LKB Ultrafilm. After 10 days the LKB film was developed in Kodak D-19 developer, washed and fixed. Sections were then defatted in xylene, hydrated, Nissl-stained, dehydrated to xylenes and mounted with Permount (Fisher). Darkfield and lightfield photomicrographs were taken of the silver grains and Nissl stain, respectively. Fig. 1 shows the distribution of kassinin-like-immunoreactive fibers in the caudal ventromedial mesencephalon. No SK neuronal perikarya were observed in the A10 dopamine (DA) region, and the fiber density was higher in the nucleus linearis than in the VTA. High fiber densities were also observed in the lateral nucleus interpeduncularis and pars reticulata of the substantia nigra. In the rostral A10 D A area the density of kassinin-like-immunoreactive fibers was greater in the nucleus interfascicularis than in the VTA. This distribution of kassinin-like immunoreactivity was qualitatively similar to that of SP (data not shown; see ref. 12), supporting the recent observation that levels of kassinin-like material and SP are equivalent in the VTA when measured with radioimmunoassy [22]. Fig. 1C shows the distribution of autoradiographically defined SK binding sites in the rostral ventromedial mesencephalon. The density of SK binding sites was high in the VTA, lower in the nucleus linearis, nucleus interfascicularis and substantia nigra, and absent in the nucleus ruber. This B

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Fig. 2. Effect of SK and SP microinjection into the VTA on motor activity. Data shown as mean _+ S.E.M. photocell counts, and the number of determinations at each dose is shown in parentheses. *P < 0.05, comparing SP and SK with vehicle using a two-tailed paired Student's t-test with probability values increased for multiple comparisons using the Bonferoni method, f P <0.05, comparing SK with SP.

245

high density of SK receptors is in contrast to the lack of SP receptors in the VTA reported by others [15, 20, 21]. In concordance with the anatomical data indicating a greater density of SK receptors relative to SP receptors, microinjection of SK into the VTA was at least 10 times more potent than SP in eliciting an increase in spontaneous motor activity (Fig. 2A). While the threshold dose for SP-induced motor activity was 1.0-3.3 nmol, the threshold dose for the SK response was 0.01-0.1 nmol. Fig. 2B illustrates the time-course of the behavioral response following microinjection of 1.0 nmol of SK or SP. Note that the motor response to SK was of greater intensity and duration than that of SP. SP microinjection into the VTA of the rat produces an increase in spontaneous motor activity that is blocked by DA antagonists [9] and elevates the level of DA metabolites in the nucleus accumbens [6]. These data have resulted in speculation that SP may act in the VTA to activate DA neurons projecting to the nucleus accumbens [6, 9, 23]. The fact that SK and SP immunoreactivity are present in the VTA indicates a potential function for either of these tachykinins as a neurotransmitter or neuromodulator in this brain region. However, the observation that SK receptors exist in higher density than SP receptors indicates that SK may represent the biologically significant tachykinin ligand. This possibility was supported by the fact that SK microinjection into the VTA was more effective than SP an increasing motor activity. Further, iontophoresis of SK adjacent to the VTA in the pars compacta of the substantia nigra consistently produced an increase in firing frequency of DA neurons, while iontophoretic SP was much less effective [5]. In summary, our data plus the recent electrophysiological evidence indicate that SK may function in the VTA in a neurotransmitter-like capacity to directly or indirectly modulate the activity of DA neurons. Further, the greater density of SK receptors and greater efficacy of intra-VTA injection with SK to produce behavioral hyperactivity supports a postulate that SK may be of greater physiological significance in modulating the mesolimbic DA system than SP. It is important to note that these data do not exclude the possibility that SK may be acting at the neuromedin K receptor in the VTA. Nevertheless, they clearly show that aliphatic tachykinins are more likely candidates than SP as modulators of the mesolimbic DA system. We are grateful to Jan Ho and Diana Armstrong for typing and editing this manuscript. The research was supported by: NIH Grants DA-03906, MH-40817, MH-09156 and MH-14092; the Scottish Rite Schizophrenia Research Program; a Smith, Kline and Beckman Award; the Muscular Dystrophy Association; and the State of Connecticut.

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