Neurokinin-3 receptors modulate dopamine cell function and alter the effects of 6-hydroxydopamine

and related la~~ly~~il~i1~ zed in terms of their degradation, and physiological cts mediated by multiple receptors (for reviews see I [16,24,32]). SP, neurokinin A, and the neurokinin A-derived peptides neuropeptide K and neuropeptide y are co-transmitters produced from a single gene as a result of differential RNA splicing and post-translational processing, whereas neurokinin B is produced from a distinct but related gene. Three different neurokinin receptors (NKRs) have been identified pharmacologically and cloned. The INK-1 receptor (NK-1R) exhibits an affinity for SP that is IOO-lOOOtimes higher than that for neurokinin A and B, respectively. The NM-2 receptor (NK-2R), is expressed primarily in peripheral tissues, and preferentially interacts with neurokinin A, with neurokinin B and SP possessing lo-lOOfold less affinity, respectively. The NK-3 receptor (NK-3R), perhaps the least characterized NKR, is expressed primarily (but not exclusively) in the CNS, and exhibits an

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0006-8993/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SW 0006-8993(95)0079 I- 1

re ~iffe~e~tia~~y distribute the nervous system, where they p biological processes. In addition to their potential role in normal l~euru~rans~ cant mission, and related peptides may have si iferactions. These peptides have marked neurotrop robative effects in systems ranging from planeria t lasts and smooth muscle cells [5,35] and stimulate neurite outgrowth 1341. NK-IRS are dramatically up-regulated in response to nerve transection [28]. Certain SP antagonists have neurotoxic effects [ l&17] and NK-1 R agonists reverse NK-1R antagonist-induced toxicity to hippocampal neurons in vitro [42]. lntracisternal SP infusion attenuates the biochemical and anatomical sequelae of ne~rotox~~ treatment on neonatal noradrenergic a.mdserotonergic neurons in vivo [ 19-21,331. Within the brain, the highest concentrations of S NKA are found in the substantia nigra, within the nerve terminals of striatonigral neurons. SP-immunoreactive terminals make synaptic contacts with dopamine (DA&containing neurons in the substantia nigra; in turn, DA nerve

terminals make contact with medium spiny (including presumptive SP-containing) striatal neurons 1401. These data provide the anatomical substrate for the functional interaction between DA cells and SP cells: SP and related peptides activate DA cells while, in turn. DA cells activate SP,/neurokinin A biosynthesis and release (for reviews of this extensive literature, see Refs. [16,22]). In fact, a number of lines of evidence suggest that decreased striatonigral SP expression is related to parkinsonism [6-8,161. On the other hand, the pharmacological effects of nigral taehykinin infusion, as well as the potential physiological role of the endogenous peptides, have remained enigmatic due of the paucity of traditional SP (i.e. NK-1R) binding sites in the substantia nigra. The determination of central NK-2R vs. NK-3R binding sites has historically been problematic due to poor ligand selectivity. Recently, nigral binding of a selective NK-3R ligand has been visualized [ 11,411 and NK-3R mRNA expression within DA cells directly demonstrated [44], prompting a reconsideration of the potential physiological role of nigral tachykinins and their receptors. The present study examined the acute and chronic effects of nigral NK-3R stimulation on DA turnover in nigrostriatal DA neurons. Furthermore, given the suspected neurotrophic effects of SP in other systems, the ability of NK-3R stimulation to modulate the biochemical and functional sequelae of DA neurotoxin lesions was investigated.

2, Materialsand methods

Male Sprague-Dawley rats (Hilltop; approximately ) were used in all experiments. Unilateral, parof the DA system were obtained by injection of dopamine (61OHDA; 8 pg in 2 ~1) into the caudate-putamen (+O.S mm anterior to Bregma, 3.0 mm lateral to the midline, -6.0 mm ventral to the brain surfacej of chloral hydrate-anesthetized rats, exactly as previously described [3]. For chronic intranigral infusion experiments, 28 gau&e cannulae were implanted 1 mm above the substantia nigra ( + 3.0 mm anterior to interaural, lateral - 2.7 mm, ventral -6.8 mm) and attached to Alzet osmotic minipumps (flow rate: 0.5 pi/h), as previously described [2]. Some animals simultaneously received both intrastriatal 6-OHDA injections and intranigral minipump implants. Food and water intake and we!ght gain were monitored for several days and, where significant weight loss was seen, animals were given access to a soft diet, Unless otherwise noted, animals were allowed to recover for 2 weeks before analyses. For acute nigral infusion experiments, 1 1.11of senktide was infused into the inates over a 10 min period and the rats er the initiation of infusion. Striata (excluding nucleus accumbens) were dissected as previously

res were co described [8]. All animal use &e e to accordance with the NI the Laboratory Animals and appro ittee. University Animal Investigation 2.2. Rotational behador To assess spontaneous rotational behavior, 6 or sham-lesioned rats were placed in a velcro ha the number of ipsiversive and contraversive turns auto ically recorded over a 20 min p monitor (Coulbourn Instrume sive minus contraversive) rota 2.3. Neurochernical Levels of DA and the DA metabolite dihy lacetic acid (DOPAC), as well lite S-hydroxyindoleacetic acid by reversed phase HPLC with previously described [31]. For peptide stability studies, the NK-3R agonist senktide was quantitated using a previously described reversed phase HPLC/gradient elution protocol [26]. In situ hybridization analyses of tyrosine h and NK3-R mRNAs were conducted on 10 frozen tissue sections using %-labeled antisense c exactly as previously described [44]. Specificity of was established by comparison of hybridizations antisense vs. sense probes and by the abolition of signal upon addition of lOO-fold excess unlabelled antisense probe. Such experiments established the nonspecific nature of the weak qparent tyrosine hydroxylase mRNA si in the hippos .nrpus and the specificity of the nigral tyrosine hydroxylase and NK-3R mRNA signals. In separate experiments, RNA was extracted by the method of Chomczynski and Sacchi [lo]. NK-3R mRNA was quantitated by solution hybridization/nuclease protection analysis using a previously described protocol [44]. i3H]WIN35,428 binding to the DA transporter was carried out as previously described [9,27]. Individual striata were homogenized in 20 volumes (w/v) of ice-cold 50 mM Tris buffer (pH 7.8) and centrifuged at 39,400 x g for 10 min. The resulting pellet was washed twice with 40 volumes of Tris buffer and resuspended in 2.5 ml assay buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.8). Equal (200 ~1) aliquots of membrane suspension, [3H]WIN35,428 and assay buffer with or without cocaine were mixed and incubated on ice for 2 h. Binding was terminated by rapid filtration through glass fiber filters using a Brandel Cell Harvester; nonspecific binding was defined in the presence of 50 PM cocaine. Complete kinetic analyses were performed in initial experiments; 2 nM WIN (a concentration near the &) was used in subsequent experiments. Autoradiography was performed according to the proto-

Tyrosine hydroxy~ase

with somewhat lower label1 zona compacta and pars late recent studies [11,41,44), !W-@I~ ~uff~c,t I!W significant levels of NK-3R mRNA and protein are expressed within midbrain DA neurons. tranigral infusion of the Chronic unilateral agonist set&tide (1 p 0.5 pi/h; for up to 14 da no obvious effect on weight gain or on spontaneous behavior (not shown). Nigral senktide infusion for l-3 days significantly increased striatal concentrations of the DA metabolite DOPAC ipsilaterally (Fig. 2), indicating that nigrostriatal DA neurons were being activated via nigral NIG3Rs. The magnitude of the DOPAC effect seen after l-3 days was the same as that seen after an acute (1 h) infusion (Fig. 2). Striatal levels of the serotonin metabolite S-HIAA were unaffected by senktide infusion at any time (Fig. 2), as were DA and serotonin levels (not shown). After 7-14 days of senktide infusion, increases in stris,tal DOPAC were no longer evident (Fig. 21, suggesting that tolerance may have developed to the effects of senktide. Nigral levels of NK-3R mRNA were unchanged following 14 days of senktide infusion (control: 0.581 L!L 0.146 amol/ug RNA vs. senktide: 0.482 _I 0.0474 amol/pg RNA; n = 4), suggesting that the altered senk??

tide effect was not mediated via a down-regulation of NK-3R gene transcription. Long-term changes in the responsiveness to senktide were also not due to an instability of the peptide in solution, since after a 14 day incubation

[I:

.

140 c

d; \ *um

120

da

100

Ok a$

j 6-OHDA

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a

6-OHDA

m

Sham

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Lesion

+ Senktide

80

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Fig. 2. Effects of acute and chronic intranigral senktide infusion on striatal dopamine and serotonin metabolism. Levels of the DA metabolite DDPAC and *he S-HT metabolitc S-HIAA were determined 1 h after se&i& infusion (acute) or after 1-14 days of continuous senktide infusion (chronic). DA metabolism was increased at early times, with the apparent development of tolerance by 7 days, while serotonin metabolism was not significantly affected by senktide at any time. Data are expressed as a percentage of control values, which for DOPAC and 5-HIAA were 29.8f 1.49 and 27.6f 1.10 ng/mg protein, respectively. n = 5 for 1-7 day groups; n = 12-15 for the acute and 14 day groups. P < 0.01. ??

> 90% of the starting peptide aP 1 NM senktide at 3X, was recovered as authentic senktide as determined by HPLC analysis (not shown). Another series of experiments examined the effects of senktide administration on the sequelae of 6-OHDA letal DA system, Intrastriatal unilateral sions of the ions 0 consistently produced moderate ( y loss of atriatalDA levels by 14 days post-lesion (Fig. 3). DA transporter levels are thought to reflect DA terminal density [3]. [3H]WIN35,428 binding to the DA transporter, measured in membrane aliquots from the same striata, was decreased to the same extent as DA after 6-OHDA treatment (Fig. 3). Sham lesions affected neither DA nor DA transporter levels (Fig. 3). Chronic intranigral infusion of senktide, beginning at the time of intrastriatal 6-OHDA injection, did not alter the extent of loss of DA nerve terminals (DA or DA transporter levels; Fig. 3). Indices of DA utilization, including the DOPAC/DA ratio, are affected only after DA depletions exceeding 80-90% [45]. In the present study, the more moderate DA lesions obtained did not significantly alter basal DOPAC/DA (Fig. 3). On the other hand, the DOPAC/DA ratio was increased significantly in 6-OHDA-lesionedanimals receiving intranigral senktide infusions (Fig. 3), indicating that chronic nigral NIG3R stimulation selectively increased the output of DA neurons surviving neurotoxin treatment. Chronic intranigral senktide infusion did not alter DA utilization in animals receiving sham lesions (Fig.

DOPAC/DA

Fig. 3. Measures of dopamine function after nigral senktide infusion in 6-hydroxydopamine lesioned animals. Animals received intrastriatal injections of 6-OHDA or vehicle concomitant with the initiation of intranigral infusions of senktide or vehicle for 14 days. Senktide infusion did not alter the extent of 6-QHDA-induced lesion (as reflected by DA and [3H]WIN35,428 binding levels) but did increase DA turnover (DOPAC/DA levels) in the surviving DA cells. Sham lesions were without significant effect on any measure. Data are expressed as a percentage of control values (shown as means~S.E.M.s with n in parentheses). Basal values were: WIN binding: 302+ 13.4 (5) and 171& 21.5 (5) fmol/mg protein in two separate experiments; DA levels: 185 rt 5.43 (51, 265 + 10.2 (5) and 261 f 7.01 (7) ng/mg protein in three separate experiments;DOPAC levels: 18.6 + 0.87 (51, 39.3 + 3.05 (5) and 31.0f 1.01 (71 in three separate experiments. * Different from 6-OHDA plus vehicle, P < 0.0 1.

3), in keeping with the lack of chronic senktide effect in unlesioned animals (Fig. 2). In some cxpcrimcnts, several hours prior to biochemical determinations, animals were tested for functiona%asym-

Fig. 4. Spontaneous rotation in 6-hydroxydopamine-lesionedanimals and the effects of senktide. Some of the animals whose biochemical indices of DA function reported in Fig. 3 were tested behaviorally for 20 min several hours before death. Unilateral DA lesions elicited spontaneous ipsiversive rotations. Lesion-induced rotations were significantly attenuated in senktide-infused animals to a level not different from senktide-infused, sham-lesioned animals. n = 10. * Different from lesion plus vehicle, P < 0.0005.

agonist a~~i~i~~ration on any neuronal system. EspeciaPly interest the effects of se&tide infusion on nigrostriatal surviving DA neurotoxin 6-OHDA treatment. intranigral senktide infusion did not alter the 6duced loss of DA nerve terminals, it did significantly increase DA output (DOPAC/DA ratio) from the remaining DA neurons, an effect which was present after 2 weeks (Fig. 3). Thus, unlike unlesioned animals, no apparent tolerance to senktide’s effects developed in 6-OHDA lesioned rats. The persistent increase in DA turnover was correlated with the elimination of DA lesion-induced spontaneous rotation (Fig. 4), suggesting that the increase in DA turnover was sufficient to compensate for the loss of most striatal DA nerve terminals. The compensatory capacity of the DA system following lesions is well-known [45]. The present behavioral data are in keeping with the report of Huston and colleagues that repeated SP injections facilitated recovery from 6-OHDA-induced asymmetries in turning and thigmotactic scanning [30]. The effects of senktide reported here are reminiscent of the effects of brain-derived neurotrophic factor and neurotrophin-3 on DA cells. These neurotrophins are generally

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