- Email: [email protected]
neuromedin N mRNA expression in topographically separate subsets of rat striatal neurons following administration of haloperidol
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The expression of the neurotensin/neuromedin N gene in the rat caudate-putamen was studied at 7 and 24 h following the administration of haloperidoi using a riboprobe and non-isotopic in hitu ~~~,~rid~~ati~~~~ histochemistry. Air has been reported by others. expression of neurotensin/neuromedin N mRNA in the dorsolaterai quadrant of the caudate-putamen was undetectable in controls. robust at 7 h and minimally detectable at 23 h foiiowin g haioperidoi administration. In the dorsomediai and ventroiaterai quadrants of the caudate-putamen. barely detectable hasal expression of neurotcnsin , ’neuromedin N mRNA observed in controls wa\ conspicuously enhanced at 24 h following administration of haloperidol both in terms of numbers of hybridized neurons. which were about 200% ol control value\, and the amount of chromogen accumulated over ilrdividuai neurons. The data are consistent with at least two subpopulations of caudate-putamen neurons in which neurotensin/neuromedin N mRNA expression is differentially regulated in response to haioperidoi administration.
ci\ en to the dorsornedial
In situ hybridization histochemistry studies have revealed that administration of typical neuroleptic drugs such as haloperidol (hall to rats produces rapid and robust, but transient, expression of the neurotensin/neuromedin N (NT/N) gene in the dorsolateral quadrant of the caudateputamen [2,8-l 31. It is peculiar then that substantial numbers of NT-inunzirzorfactir’e neurons observed in immunohistochemical studies following administration of ha1 are distributed in a manner that largely excludes the dorsolatera1 quadrant, and instead involves the dorsomedial, ventrolateral and rostra1 parts of the caudate-putamen [6,24]. In the present study, the expression of striatal NT/N mRNA has been evaluated in control brains and at two time points following administration of ha1 using non-isotopic in situ hybridization. Particular attention has been
Akhreviation\.I .
hai, haiopcridoi: NT
ratcnsin/ncuromedm N. * Corresponding author. [email protected]
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neurotensin: NT/N.
I (314) 268-5127;
ntxiE-mail:
16-7
quadrants
of the
2. Materials and methods 2. I. Probe syr thesis NT/N probes were prepared from a nearly full length cDNA clone (a gift of Dr. Paul R. Dobner. University of Massachusetts) isolated from a rat brain library as described by Alexander et al. [l]. The plasmid harboring the NT/N-encoding cDNA was provided in pGEM4 and was ca!!ed pGEM4-NT. The plasmid was electroporated into E. CO/~DI-I-5 strain celis, and a plasmid stock was prepared using standard techniques. Plasmid DNA was linearize
0169-328X/96/$15.00 Copyright 8 1996 Elsevier Science B.V. All rights reserved. PII 30169-328X(96)001
uncl ventrolateral
caudate-putamen with the expectation that the superb cellular resolution of non-isotopic hybridization histochemistry would foster insight into how detectable perikaryal NT-immunoreactivity is elicited in these striatal districts by hal.
72
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Fig. I. Photomicropraphsof the dorsomedial quadrant of the caudate-putomenin sections taken from the brains of ruts that received vehicle injections (A-C) or 2 ml/kg of haloperidol (D-F) 24 h prior to being anesthetizedand decapitated. The section\ were hybridized with neurotensin/neuromedin N cRNA probes. Hybridized neurons resemble black dots in the low magnification tigures .(A. B. D. E). and are few in number in the control section shown in panel A. The numbers of hybridized neurons were somewhnt greater in control sections if RNAse A was omitted from the rinse sequence of the hybridization protocol (B). Sections from ruts that received haloperidol contnincd many more hybridized neuroph thun their corresponding controls in normally rinsed material (compare D with A) and in material rinsed with the RNAse A-free ;rrotoco!(compare E with B). The boxed area\ in B and E are shown ut higher magnification in C and F. respectively. .Abbreviations:cc. corpus callosum: k. lateral ventricle. Scale bars: A. B. D and E. I mm: C and F. IOU pm.
some sections e preprotachykinin
y,
ybridizcd ey rals (200-250
anolamine tol-
of ethanol and finally chloroform rafter which they were air dried. SWtions from vehicle-treated controls and 24 h h&treated rats were then processed in tandem using the same hybridiL;ilowed
by rinses
in increasing
were
visualizt4
by
t.zxposing
tht:
gl
equivalent volume nol, 50% propylene ior to having been (60 mg/kgI ilnd ceived either the vehicle red for tsectioning. The brains of the rats were removed and frozen sections through on the striatum were cut at 13 pm and thaw-mounted polylysinc coated micrr)scopc hIides. The mounted section3 were fixed in 3% paraformaldehyde for 5 min followed by 2 washes in phosphate buffered saline ( SI. They were
then treated with acetic ~~~hydride in tri
neurons
were gene
dilutions
tion and rinse buffers.
Hybridization histochcmistry M’;IS carried out asaitiail> as recommended by Young [23]. Brietly, SO 0~~1ot‘ a mixture consisting of 5 p.1 of stock probe prcp;lr;~tion ( IO n&l), nucleic acids (salmon sperm DNA [ 10 t_q/mi]. yeast total RNA [25 &ml] and yeast tRNA [25 IJ-g/ml]). sodium dodecyl sulfate. sodium thiosulfate and hybridization buffer were applied per slide (two sections/slide). Hybridization buffer consisted of Tris-HCl(20 nM). EDTA (1 mM), NaCI (300 mM), formamide (SO%), dextran sulfate (10%) and Denhardt’s solution ( I X 3.DEPC-treated water was used for all solutions. Thp sections were covered with a glass coverslip and hybridized overnight at 57°C in humid chambers. The next day the coverslips were removed in 4 X SSC ( 1 X SSC = 150 mM NaCI. 15 mM sodium &rate) after which the sections were rinsed 5 times with agitation in the same solution. Some of the sections were then incubated for 10 min at 37°C in 20 y&/ml RNAse A (20 kg/ml = 1 ml of 10 mg/ml stock in 500 ml RNAse A buffer comprising 62.5 ml of 4 mM NaCI, 2.5 mi of 2M Tris-HC! pH 8.0 and 0.5 ml of 0.25M EDTA) followed by three 3-min rinses on the rotator in 0.1 X SSC and one 5-min rinse in 0.1 x SSC at 65°C. The sections were placed in 0,I x SSC to cool and then into ;i Tris-HCI
annheim, Indianapolis, IN) room temperature after CI buffer and colorir_ed ~~~~~t~ili~n~ nitrobiue tetrazolium. S-bromo--I-chloro-_3-indoyl phosphate t~)~uidi~liu~l~ and levamisoie, which were used at the conccntratior?s recommended by the supplier Laburaiories. urlingame. CA. product number
IO). The following morning the s0J1ons were SSC followed by a brief dip in 70% ET0 air dried. s 01‘labeled neurons were made only in sections the signal to background ratio allowed confident labeled neurons (see Fig. I of evidence of differential in vehicle- vs. hat-treated 5lirinkage durin(7c processing sections. the data were expressed in terms of labeled neuron densities by countin, ‘7 the labeled neurons within circular. 1-mm2 disks circumscribed in the dorsomedial quadrants of the caudate-putamen. Value\ acyuircd at comp~A~ie itx~l~ of’ tiitz caudatc-putamt‘n were compared only differentiation
of specifically
for examples). In the absence
74
vehicle-injected rats. between pair-matched hal-treated sections were teste from matched vehicle- and nerated wi:‘l tPNAse A an using a paired t-test. Data RNAse A-free protocols were compared with a t-test assuming separate variances (see Table 1). Significance was set at P < 0.05.
3. Resulfs
Control
B
Haloperidol - 7 hours
C
Haloperidol - 24 hours
Fig. 2. Diugrums showing the distributions of hybridized neurons in the brains of rats thnt received vehicle injections (A), haloperidol at 2 mgg’kg 7 h prior to being decapituted (B) and hrdoperidol at 2 mg/kg at 24 h prior to being decapitated. The dorsomedial (dm). dorsolnterul (dl) and ventrol~teral (vi) quadrunts of the cuudnte-putamen are indicuted by urrows. Note thut il few hybridized neuronsare present in the dorsomedial and ventroluteralquadruntsin the controls (A). Mnny hybridized cells are present and centered in the dorsolaterul quadrant at 7 h after hnloperidol (B). At 24 h after haloperidol. the dorsolaterally located hybridized neurons ure no longer particularly numerous, but u conspicuous increase in the numbersof cells in the dorsomedial and ventrolateral quadruntsis observed (Cl. Note thnt the numbers of hybridized neurons in the forebrain regions beneaththe caudate-putamcn remain similur through all of the treatments (fewer cells ure pre*+entin the basal forebrain in I? becausethat .sectionis slightly more rostra1thun the others).
Sections hybridized with probe lacking WTP-digoxigenin or with hybridization buffer lacking probe produced no detectable reaction products. Sectioils processed using the preprotachykinin gene produced a pattern of hybridized neurons that was distinct from following processing with the NT/N probe. with the NT/N probe produced a pattern similar to that reported by others (see references in Sections I and 4). Within the caudate-putamen of the vehicle-treated controls, a few NT/N-hybridized neurons were present consistently only in the dorsomedial and ventrolateral quadrants (Fig. IA-C: Fig. 2A). Hybridized neurons also were observed in the rostra1 pole and medial shell of the nucleus accumbens, endopiriform nucleus, medial ventral pallidurn, preoptic region, hypothalamus, sublenticular region, septum and bed nucleus of stria terminalis (Fig. 2). (Levels caudal to the sublenticular region were not examined.) In sections from rats that received ha1 24 b prior to decapitation and were processed with the NT/N probe. more numerous hybridized neurons were observed in the dorsomedial and ventrolateral quadrants of the caudateputamen, and lesser numbers of hybridized neurons extended ventrally along the lateral ventricle and laterally into the dorsolmteral quadrant (Fig. ID-F; Fig. 2C). Counts of hybridized neurons made in the dorsomedial caudateputamen revealed that significantly more hybridized neurons were present in sections from rats that received ha1 24 h prior to sacrifice as compared to vehicle-injected controls that were hybridized in the same solutions (Table I). The average increase in the numbers of NT/N-hybridized neurons observed in sections through the dorsomedial quadrants of the caudate-putamen of hal-treated rats relative to matched sections from the vehicle-treated controls exceeded 300%. Obvious differences between controls and rats given ha1 with regard to the numbers of hybridized neurons present in extrastriatal parts of the forebrain and the patterns in which those neurons were distributed were not noted (Fig. 2A-C). If buffer containing RNAse A was omitted from the rinse sequence (see Sections 2 and 4.2) the sections exhibited substantially more hybridized neurons in all of the striatal (Fig. ID-E) and extrastriatal regions noted above, but no additional regions were observed to contain labeled neurons. In the absence of RNAse treatment, a slight to moderate labeling of apparently unhybridized cells was observed.
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Fig. 3. Photomontage illustrating the robust hybridization in the dorsolateral caudate-putamen fdl) at 7 h following administration of haloperidol at ? mg/kg, The dorsomedial qadrant of the caudate-putamen (dm) harbors few hybridized neurons. Straight arrows indicate patches of hybridked neurons located in the medial part of the caudate-putamen.The crossed arrows in A indicate patches that are enlarged in B (also indicated with crossed arrows). Abbreviations: ac. anterior commissure; cc. corpus callosum: ec. external capsule; v. lateral ventricle. Scale bars: A. I mm; B. 100 pm.
numbers of hybridized neurons in the drrrsomedial and ventrolateral quadrants of the caudate-putamen, both in controls and hal-treated rats. as compared to sections from rats that were subjected to RNAse treatment (Table 1; cases 92024 and 96026). Also, the hybridized neurons of the 24-h hal-treated rats observed following the RNAse-free protocol were considerably more strongly labeled Wig. IF) as compared to controls (Fig. 10, which was revealed as an apparent increase in the size of hybridized cells in the hal-treated rats, putatively due to the development of more chromogen (compare Fig. 1C and F). In sections from rats decapitated 7 h following ha1 administ,:ation, large numbers uf hybridized neurons were observed in a crescent shaped region that was coextensive with the dorsolateral convexity of the caudate-putamen (Fig. 2B and Fig. 3). Lesser numbers of hybridized cells extended into the rostral, dorsomedial and ventrolateral parts of the caudate-putamen. With the exception of a number of patch-like groups (Fig. 3, arrows), few hybridized neurons were observed in the ventromedial part of the caudate-putamen. However, a conspicuous increase in the numbers of hybridized neurons was observed in the nucleus accumbens rostra1 pole and shell region at 7 h following ha1 injections (not shown). Conspicuous differences with rcg,rd to the numbers and distributions of hybridized neurons in extr:astriatal forebrain regions as compared to controls were not &erved in the 7-h haltreated rats.
tectability. The other ( population has a ste increases more gradu period of time. Neu era1 quadrants arc d following
administration of hal.
While non-isotopic hybridizations do not provide reliable quantitative data about absolute amounts of m being produced by individual neurons, they do show nioeiy relative differences between controls and pair-matched drug-treated sections. Thus, it was assumed during the course of this study that, in a given in situ hybridization experiment utilizing appropriate reagent ~~~~i~e~trati~)ns, a threshold range exists for the inrracellular concentration of the mRNA species being tested. above which neurons be@ to exhibit reaction product. It has been concluded in experiments in which control and test sections have been reacted with the same reagent solulions, that preparations exhibiting significantly more hybridized neurons do so because more neurons exceed the threshold for detection, from which can be reasonably inferred that mRNA synthesis is increased in those preparations. Given these considerations, non-isotopic in situ hybridizations may be regarded as a particularly valuable tool. insofar as the conspicuous nature of the nitroblue tetrazolium reaction product facilitates detection of regional and temporal differences in levels of mRNA synthesis.
4. Discussion This descriptive and quantitative account has capitalized upon the superb cellular resolution afforded by the nonisotopic approach to in sifu hybridization. It was observed that sections from 24-h hill-treated rats exhibited enhanced hybridization in the dorsomedial and ventrolateral quadrants of the caudate-putamen. when processed in tandem with the vehicle-treated controls using the same hybridization and rinse buffers. Furthermore, the amount of histochemical reaction product associated with individual cells in the dorsomedial and ventrolateral districts was noticeably greater in the 24-h hal-treated groups. These observations are regarded as evidence for increased expression of NT/N mRNA in the dorsomedial and ventrolateral quadrants of the caudate-putamen at 24 h following injection of hal. The distinct patterns of distribution of hybridized neurons observed at 7 and 24 h post-injection are consistent with at least two subpopulations of neurons in the rat striatum that express NT in response to ha1 administration, as has also been suggested by immunohisrochemical investigations using antibodies against NT peptide [4,19,24]. One (primarily dorsolateral) subpopulation comprises neurons that express NT/N mRNA rapidly and robustly, but tranisently. and fail to achieve immunohistochemical de-
It had been noted in examining material prepared preliminarily to this invesGgation that the composiiion of the rinse sequence plays a critical role in determining how many hybridized neurons are observed in a given preparation. In particular, RNAse A treatment, which sensitively regulates signal to noise ratio in isotopic in situ hybridization riboprobe preparations 1201, was observed to substantially diminish the numbers of hybridized neurons detected in the non-isotopic preparations. Although the basis for this finding has not been investigated systematically, it is hypothesized that, following hybridizations, the preparations contain a significant amount of specific labeling in which only parts of the digoxigenin containing transcripts are annealed to target mRNA in the sections. When unannealed portions of partially. but specifically. bound probe are degraded by RNAse, the histochemically detectable signal is substantially reduced, resulting in fetwer labeled neurons. It is concluded that the specificity of the hybridizations is not significantly altered by the omission of RNAse treatment, because :hz patterns of labeling both in striatal and extrastriatal tissues and the patterns of change associated with ha1 treatment were similar in the presence and absence of RNAse treatment.
77
gcnu
or
its
gem2
produ
ifferences. In this regard, it is notewor-
after injection of hal. i.e.. \vas obserked in rclatcd studies that Fos is not significantly w-L’xpreswd ;Is ;I second marker in NT-imlmunoreacti~,~ striatal neurons ~oll(~~i~i~ hal adminislration [-I. I’,]. 11‘.;LS
11)pothcsiecd, the ucuron~ (I!’ the ~radu;rl!~-cspre~sit~~
I~_-~Icc~ p2lcht3 r~!‘ men (Fig. 3, ;trrou 3) rc\cmblc Glliilarlj NT-immunoreactive neurons and fibers observed following reserpine administration [3,24]. The observed up-rcgu~~~ti~~n of the dors,r,medial/~entl-olateral response of NT/N mRNA expression is apparently more gradual to dcvetop and more enduring than the dorsoiateral response. which returns to control levels well within 24 h [8,9]. It is noteworthy in this regard that the dorsomedial and ventrolateral quadrants of the caudateputamen arc the only district., in the striatum outside of the nucleus accumbens where detectable expression of NT/N mRNA was observed, albeit minimally. in the absence of’ ha1 (see also [12]). Furthermore. it is in the dorwmedial and vcntrolatcra~ (and rostra!) parts of the cuudtitt’-putrrmc‘n
group are those that become detectably immunoreactive for NT. then the data would suggest that Fos IS not a necessary part of the regulatory apparatus for NT/N gene transcription rn neurons of the d0rsomcdiai/ventrolatera~ gradually expressin,cv subset. Also. Merchant et al. [I i j have observed that the indirect dopamine agonist methan?phetamine elicits NT/N mRNA zxprcssion, aFparentiy unpreceeded by Fos expression. in precisely the dorsomedial and icntrolateral quadrants of the caudate-putamen bvhere increrrscd NT/N mRNA was observed in Ihe present study and whcrc, ;LSnoted &ove. numerous NT-immunoreactive neurons arc obwrvcd fiillowing dopamine
as well us in the nuckus a~~~mbcns. that increased num-
s;uw
bers of NT-immunoreactive neurons are observed :bilowing administration of hal 16,241. Ensofar as their distributions and the time courses in which they appear are similar, it is hypothesized that dorsomedial/ventrotateral neurons in which NT/N mRNA accumulates in a gradual. enduring manner followin, 0 hal administration correspond to ones in which NT-immunoreactivity is detected, although this remains to be demonstrated directly. The present data confirm and extend the findings of Williams et al. [22], who also observed increases in NT/N mRNA-expressing neurons in the striatum using an oligonucleotide probe and a non-isotopic fluorescence in situ hybridization method. In that study, hai was administered in two injections at i 7 and 10 h prior to sacrificing the rats, which probably resulted in a diminished capacity to distinguish the rapidly expressing dorsolateral subset of NT/N rr?RNA-expressing neurons, in which. at 10 h following the second ha1 injection, NT/N mRNA has begun to return toward basal levels [8,9], and the gradually expressing dorsomedial/ventrolateral subsets of striatal NT/N mRNA-expressing neurons, in which NT/N mRNA
11, rwcptor antagonist administrarion [6.2-C]. Whether the subwt (11’ neurons is responding to the indirect agonist and hal by expressing NT/N mRNA remains to be determined. Much remains to be learned about how dopamine neuretransmission regulates neuronal activity and impulse conduction and transmission in the basal ganglia. The present data are consistent with at least two subpopulations of striatal neurons in which NT/N mRNA expression is differentially regulated by dopamine. Continuing refine ment of the capacity to utilize NT/N expression as a probe of dopamine effects in the basal ganglia should help to solidify a foundation upon wnich a better understanding of this complex area can be built.
Acknowledgements This work was supported by USPH Grants NIH NS23805 (D.S.Z.) and NS-21937 (J.E.K.1. The authors arc indebted to Mci Feng and Joseph Tucker for excellent technical assistance. The gift of NT/N cDNA from Dr. Paul R. Dobner is gratefully acknowledged.
References Dl Alexander, M.J.. Miller, M.A.. Dorsa. D.M.. Bullock. B.P.. Melloni. R.H., Dobner, P.R. and Leeman. S.E., Distribution of neurotensin/neuromedin N mRNA in rat forebrain. unexpected abundance in hippocampus and subiculum. Proc. Nctrl. Acc~l. Sci. USA. 86 (1989) 5202-5206.
El Augood. S.J.. Kiyama, H.. Faull. R.M. and Emson. PC.. Differential effects of acute dopaminergic D, and D, receptor antagonists on proneurotensinmRNA expression in rat striatum. MO/. Brcritl Res., 9 (1991) 341-346.
131 Bean. A.J., During. M.J., Deutch, A.Y. and Roth, R.H.. Effects of dopamine depletion on striatal neurotensuf: biochemical and immunohistochemicalstudies, J. Netrrosci., 9 ( 1989) 4430-4438. [41 Brog. J.S. and Zahm, D.S.. Morphology and Fos immunoreactivity reveal two subpopulationsof striatal neurotensin neurons rollowing acute 6-hydroxydopamine lesions and reserpineadministration, Nellroscierrce, 65 ( 1995) 7 I-86. 151 Deutch. A.Y. and Zahm, D.S.. The current status of neurotensindopamine interactions: issues and speculations. In P. Kitagbi and C.B. Nemeroff (Eds.). The Neurobiology of Neurotensin. Am. NY AC&. Sci.. 668 (1992) 232-252. [6l Eggerman. K.W. and Zahm. D.S., Numbers of neurotensin-immunoreactive neurons selectively increased in rat ventral striatum 1I ( I9881 following acute haloperidol administration, N~‘fr~~)pt~pri’lc~.\. 125-132. t71 Krause, J.E., Chirgwin, J.M., Carter, M.S.. Xu, Z.S. and Hershey. A.D.. Identification of three rat preprotachykinins encoding both substance P and neurokinin A. Proc. Nutl. Acad. Sci. USA. 84 ( 1987) 9881-9885. 181 Merchant. K.M.. Dobie. D.J. and Dorsa, D.M., Expression of the proncurotensingene in the rat brain and its regulation by antipsychotic drugs. AWL NY Acad. Sci., 668 (1992) 652-653. 191 Merchant, K.M.. Dobner. P.R. and Dorsa. D.M.. Differential effects of haloperidol and clozapine on neurotensingene transcription in rat ncostriatum. J. Nctrrmci.. I2 (1992) 652-663. [I()1 Merchant. K.M. and Dorsa. D.M.. Differential induction of ncurotensin and c-&s gene expression by typical versus atypical antipsychotics. Pm. Nurl. Awd. Sci. USA. 90 ( 109.3) 3447-345 I. 11II Merchant, K.M.. Hanson, G.R. and Dorsa, D.M.. Induction of ncurotcnsinand C$~J.V mRNA in distinct subregionsof rat ncostriaturn after acute methamphetamine: comparison with acute haloperido1 cffCcts, J. Phmtrrrcol. Exp. The,:, 269 (1994) 806-812. [I21 Mcrchnnt, KM. und Miller, M.A., Cocxprcssionof neurotcnsinand C-~H mRNAs in rat neostriatal neurons following acute haloperidoi. Mol. Brctiti Res.. 23 ( 1994) 27 I-277.
WI Merchant. K.M.. Miller. M.A..
Ashleigh, A. ,md Dor\a. D. Hnloperidol rapidly incrcascs the II~III~W of ncurotcnain mRNA-cxpressing neurons in neo\triatum of the rat braon, Hrrrrrr Rts\.. .kl-!O (1991) 31 I-313. WI Morgan. J.I. and Currim. T.. Stimulus-tran~cril~tlon couphng in the nervous system: involvement of the inducible proto-oncogcnc\ /o.\ and ,ilul. Atrrrrr.Rcr~. Nemm~i.. 13 ( 191j114.?I-45 1 Roberkon. G.S. and Fibiger. C4.C.. Ncurckpr~c~ tncwLw c,-ftjt cripression in the forebrain: contrastmg effects o! halop~r~dol and clozapine, Newosciww, 46 ( 1992)3 I S-328. iI61 Robertson. G.S., Vincent. S.R. and Fibigcr, H.C., Striatonigal projection neurons contain D, dopamine recept<)r-activatedc,/i),s. l~rcrii~ Ras., 52.3 ( 1990) 288-290. [I71 Robertson. G.S.. Vincent, S.R. and Fibigcr. H.C.. Dl and D2 dopamine receptors differentially regulate c-ji>.s expression in striatonigral and striatopallidal neurons, Newosciettc ;‘, -10 ( IO02128S-296.
[1N Robertson. H.A.. Peterson. M.R.. Murphy, K. and Robertson, G.S.. D, dopaminc receptor agonists selectively aclivatc \triatal c-fir.\ independentof rolationid beha\ ictr, &.tun I?(*.\..SO3( I!Sc))346-3-W. 1191 Senger, B.. Brog. J.S. and Zahm. D.S.. Stihscrs of ncurotensin irnmunoreactive neurons in the rat striatid complex following i!tltilfonism of the dopamine D-2 receptor: an immunohistochclnicnl double-labeling study using antihodics against Fo\. N~~III.~).\(./~‘II(.(‘. 57 ( I’~%) 649-660. [20] Valentino. K.L.. Eberwine. J.H. and Barchns. J.D. (Eds.). Irr .Wr Hhridimim. Applictrtiom to Nerrrohiokq,v. Oxford University Press. New York. 1987. D11 Wan. X.S.T.. Liang. F.. Moret. V.. Wiesendanger. M. and Rouiller. E.M.. Mapping of the motor pathways in rats: c+\ induction by intracortical microstimulation of the motor cortex correlated with cfferent connectivity of the site of cortical strmulation. Nc>frro.sc*iertw,40 ( I992 1749-76 I .
[El Williams, F.G., Murtaugh, M.P. and Beit/. A.J.. The cffcct of acute haloperiol treatment on brain proneurotensin mRNA: in situ hybridization analysis using a novel Iluorcscence detection procedure. Mol. Rrh Res.. 7 (1990) 3-17-358.
[23] Young 111.W.S.. In situ hybridi/.ation hisrochomistry. In A. Bjiirkland, T. Hiikfelt. F.G. Wouterlood and A,N. van den Pol (Eds.), cd N1~1r~c~~1trcrri~t~r~ I \irl. S, Artlrhi.~ r!j N~vrroturl Mkrwitwit~ lttd .Swrtrp~ic Itt/errrc~iorrs. Elscvier. Ams,terdam. 1990. pp 4x1 -51 I.
Chrcltwohr!/ ~‘l~cwrrt
1241 ZaPrm.D.S.. Distinct subsetsof neurotensinimmunoreactive neurons revealed following antagonism of the dopamine-mediated suppression of neurotensin immunoreactivity in the rat striatum, Newasciemx;. 46 I19921 335-350.
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The expression of the neurotensin/neuromedin N gene in the rat caudate-putamen was studied at 7 and 24 h following the administration of haloperidoi using a riboprobe and non-isotopic in hitu ~~~,~rid~~ati~~~~ histochemistry. Air has been reported by others. expression of neurotensin/neuromedin N mRNA in the dorsolaterai quadrant of the caudate-putamen was undetectable in controls. robust at 7 h and minimally detectable at 23 h foiiowin g haioperidoi administration. In the dorsomediai and ventroiaterai quadrants of the caudate-putamen. barely detectable hasal expression of neurotcnsin , ’neuromedin N mRNA observed in controls wa\ conspicuously enhanced at 24 h following administration of haloperidol both in terms of numbers of hybridized neurons. which were about 200% ol control value\, and the amount of chromogen accumulated over ilrdividuai neurons. The data are consistent with at least two subpopulations of caudate-putamen neurons in which neurotensin/neuromedin N mRNA expression is differentially regulated in response to haioperidoi administration.
ci\ en to the dorsornedial
In situ hybridization histochemistry studies have revealed that administration of typical neuroleptic drugs such as haloperidol (hall to rats produces rapid and robust, but transient, expression of the neurotensin/neuromedin N (NT/N) gene in the dorsolateral quadrant of the caudateputamen [2,8-l 31. It is peculiar then that substantial numbers of NT-inunzirzorfactir’e neurons observed in immunohistochemical studies following administration of ha1 are distributed in a manner that largely excludes the dorsolatera1 quadrant, and instead involves the dorsomedial, ventrolateral and rostra1 parts of the caudate-putamen [6,24]. In the present study, the expression of striatal NT/N mRNA has been evaluated in control brains and at two time points following administration of ha1 using non-isotopic in situ hybridization. Particular attention has been
Akhreviation\.I .
hai, haiopcridoi: NT
ratcnsin/ncuromedm N. * Corresponding author. [email protected]
Fax:
+
neurotensin: NT/N.
I (314) 268-5127;
ntxiE-mail:
16-7
quadrants
of the
2. Materials and methods 2. I. Probe syr thesis NT/N probes were prepared from a nearly full length cDNA clone (a gift of Dr. Paul R. Dobner. University of Massachusetts) isolated from a rat brain library as described by Alexander et al. [l]. The plasmid harboring the NT/N-encoding cDNA was provided in pGEM4 and was ca!!ed pGEM4-NT. The plasmid was electroporated into E. CO/~DI-I-5 strain celis, and a plasmid stock was prepared using standard techniques. Plasmid DNA was linearize
0169-328X/96/$15.00 Copyright 8 1996 Elsevier Science B.V. All rights reserved. PII 30169-328X(96)001
uncl ventrolateral
caudate-putamen with the expectation that the superb cellular resolution of non-isotopic hybridization histochemistry would foster insight into how detectable perikaryal NT-immunoreactivity is elicited in these striatal districts by hal.
72
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Fig. I. Photomicropraphsof the dorsomedial quadrant of the caudate-putomenin sections taken from the brains of ruts that received vehicle injections (A-C) or 2 ml/kg of haloperidol (D-F) 24 h prior to being anesthetizedand decapitated. The section\ were hybridized with neurotensin/neuromedin N cRNA probes. Hybridized neurons resemble black dots in the low magnification tigures .(A. B. D. E). and are few in number in the control section shown in panel A. The numbers of hybridized neurons were somewhnt greater in control sections if RNAse A was omitted from the rinse sequence of the hybridization protocol (B). Sections from ruts that received haloperidol contnincd many more hybridized neuroph thun their corresponding controls in normally rinsed material (compare D with A) and in material rinsed with the RNAse A-free ;rrotoco!(compare E with B). The boxed area\ in B and E are shown ut higher magnification in C and F. respectively. .Abbreviations:cc. corpus callosum: k. lateral ventricle. Scale bars: A. B. D and E. I mm: C and F. IOU pm.
some sections e preprotachykinin
y,
ybridizcd ey rals (200-250
anolamine tol-
of ethanol and finally chloroform rafter which they were air dried. SWtions from vehicle-treated controls and 24 h h&treated rats were then processed in tandem using the same hybridiL;ilowed
by rinses
in increasing
were
visualizt4
by
t.zxposing
tht:
gl
equivalent volume nol, 50% propylene ior to having been (60 mg/kgI ilnd ceived either the vehicle red for tsectioning. The brains of the rats were removed and frozen sections through on the striatum were cut at 13 pm and thaw-mounted polylysinc coated micrr)scopc hIides. The mounted section3 were fixed in 3% paraformaldehyde for 5 min followed by 2 washes in phosphate buffered saline ( SI. They were
then treated with acetic ~~~hydride in tri
neurons
were gene
dilutions
tion and rinse buffers.
Hybridization histochcmistry M’;IS carried out asaitiail> as recommended by Young [23]. Brietly, SO 0~~1ot‘ a mixture consisting of 5 p.1 of stock probe prcp;lr;~tion ( IO n&l), nucleic acids (salmon sperm DNA [ 10 t_q/mi]. yeast total RNA [25 &ml] and yeast tRNA [25 IJ-g/ml]). sodium dodecyl sulfate. sodium thiosulfate and hybridization buffer were applied per slide (two sections/slide). Hybridization buffer consisted of Tris-HCl(20 nM). EDTA (1 mM), NaCI (300 mM), formamide (SO%), dextran sulfate (10%) and Denhardt’s solution ( I X 3.DEPC-treated water was used for all solutions. Thp sections were covered with a glass coverslip and hybridized overnight at 57°C in humid chambers. The next day the coverslips were removed in 4 X SSC ( 1 X SSC = 150 mM NaCI. 15 mM sodium &rate) after which the sections were rinsed 5 times with agitation in the same solution. Some of the sections were then incubated for 10 min at 37°C in 20 y&/ml RNAse A (20 kg/ml = 1 ml of 10 mg/ml stock in 500 ml RNAse A buffer comprising 62.5 ml of 4 mM NaCI, 2.5 mi of 2M Tris-HC! pH 8.0 and 0.5 ml of 0.25M EDTA) followed by three 3-min rinses on the rotator in 0.1 X SSC and one 5-min rinse in 0.1 x SSC at 65°C. The sections were placed in 0,I x SSC to cool and then into ;i Tris-HCI
annheim, Indianapolis, IN) room temperature after CI buffer and colorir_ed ~~~~~t~ili~n~ nitrobiue tetrazolium. S-bromo--I-chloro-_3-indoyl phosphate t~)~uidi~liu~l~ and levamisoie, which were used at the conccntratior?s recommended by the supplier Laburaiories. urlingame. CA. product number
IO). The following morning the s0J1ons were SSC followed by a brief dip in 70% ET0 air dried. s 01‘labeled neurons were made only in sections the signal to background ratio allowed confident labeled neurons (see Fig. I of evidence of differential in vehicle- vs. hat-treated 5lirinkage durin(7c processing sections. the data were expressed in terms of labeled neuron densities by countin, ‘7 the labeled neurons within circular. 1-mm2 disks circumscribed in the dorsomedial quadrants of the caudate-putamen. Value\ acyuircd at comp~A~ie itx~l~ of’ tiitz caudatc-putamt‘n were compared only differentiation
of specifically
for examples). In the absence
74
vehicle-injected rats. between pair-matched hal-treated sections were teste from matched vehicle- and nerated wi:‘l tPNAse A an using a paired t-test. Data RNAse A-free protocols were compared with a t-test assuming separate variances (see Table 1). Significance was set at P < 0.05.
3. Resulfs
Control
B
Haloperidol - 7 hours
C
Haloperidol - 24 hours
Fig. 2. Diugrums showing the distributions of hybridized neurons in the brains of rats thnt received vehicle injections (A), haloperidol at 2 mgg’kg 7 h prior to being decapituted (B) and hrdoperidol at 2 mg/kg at 24 h prior to being decapitated. The dorsomedial (dm). dorsolnterul (dl) and ventrol~teral (vi) quadrunts of the cuudnte-putamen are indicuted by urrows. Note thut il few hybridized neuronsare present in the dorsomedial and ventroluteralquadruntsin the controls (A). Mnny hybridized cells are present and centered in the dorsolaterul quadrant at 7 h after hnloperidol (B). At 24 h after haloperidol. the dorsolaterally located hybridized neurons ure no longer particularly numerous, but u conspicuous increase in the numbersof cells in the dorsomedial and ventrolateral quadruntsis observed (Cl. Note thnt the numbers of hybridized neurons in the forebrain regions beneaththe caudate-putamcn remain similur through all of the treatments (fewer cells ure pre*+entin the basal forebrain in I? becausethat .sectionis slightly more rostra1thun the others).
Sections hybridized with probe lacking WTP-digoxigenin or with hybridization buffer lacking probe produced no detectable reaction products. Sectioils processed using the preprotachykinin gene produced a pattern of hybridized neurons that was distinct from following processing with the NT/N probe. with the NT/N probe produced a pattern similar to that reported by others (see references in Sections I and 4). Within the caudate-putamen of the vehicle-treated controls, a few NT/N-hybridized neurons were present consistently only in the dorsomedial and ventrolateral quadrants (Fig. IA-C: Fig. 2A). Hybridized neurons also were observed in the rostra1 pole and medial shell of the nucleus accumbens, endopiriform nucleus, medial ventral pallidurn, preoptic region, hypothalamus, sublenticular region, septum and bed nucleus of stria terminalis (Fig. 2). (Levels caudal to the sublenticular region were not examined.) In sections from rats that received ha1 24 b prior to decapitation and were processed with the NT/N probe. more numerous hybridized neurons were observed in the dorsomedial and ventrolateral quadrants of the caudateputamen, and lesser numbers of hybridized neurons extended ventrally along the lateral ventricle and laterally into the dorsolmteral quadrant (Fig. ID-F; Fig. 2C). Counts of hybridized neurons made in the dorsomedial caudateputamen revealed that significantly more hybridized neurons were present in sections from rats that received ha1 24 h prior to sacrifice as compared to vehicle-injected controls that were hybridized in the same solutions (Table I). The average increase in the numbers of NT/N-hybridized neurons observed in sections through the dorsomedial quadrants of the caudate-putamen of hal-treated rats relative to matched sections from the vehicle-treated controls exceeded 300%. Obvious differences between controls and rats given ha1 with regard to the numbers of hybridized neurons present in extrastriatal parts of the forebrain and the patterns in which those neurons were distributed were not noted (Fig. 2A-C). If buffer containing RNAse A was omitted from the rinse sequence (see Sections 2 and 4.2) the sections exhibited substantially more hybridized neurons in all of the striatal (Fig. ID-E) and extrastriatal regions noted above, but no additional regions were observed to contain labeled neurons. In the absence of RNAse treatment, a slight to moderate labeling of apparently unhybridized cells was observed.
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Fig. 3. Photomontage illustrating the robust hybridization in the dorsolateral caudate-putamen fdl) at 7 h following administration of haloperidol at ? mg/kg, The dorsomedial qadrant of the caudate-putamen (dm) harbors few hybridized neurons. Straight arrows indicate patches of hybridked neurons located in the medial part of the caudate-putamen.The crossed arrows in A indicate patches that are enlarged in B (also indicated with crossed arrows). Abbreviations: ac. anterior commissure; cc. corpus callosum: ec. external capsule; v. lateral ventricle. Scale bars: A. I mm; B. 100 pm.
numbers of hybridized neurons in the drrrsomedial and ventrolateral quadrants of the caudate-putamen, both in controls and hal-treated rats. as compared to sections from rats that were subjected to RNAse treatment (Table 1; cases 92024 and 96026). Also, the hybridized neurons of the 24-h hal-treated rats observed following the RNAse-free protocol were considerably more strongly labeled Wig. IF) as compared to controls (Fig. 10, which was revealed as an apparent increase in the size of hybridized cells in the hal-treated rats, putatively due to the development of more chromogen (compare Fig. 1C and F). In sections from rats decapitated 7 h following ha1 administ,:ation, large numbers uf hybridized neurons were observed in a crescent shaped region that was coextensive with the dorsolateral convexity of the caudate-putamen (Fig. 2B and Fig. 3). Lesser numbers of hybridized cells extended into the rostral, dorsomedial and ventrolateral parts of the caudate-putamen. With the exception of a number of patch-like groups (Fig. 3, arrows), few hybridized neurons were observed in the ventromedial part of the caudate-putamen. However, a conspicuous increase in the numbers of hybridized neurons was observed in the nucleus accumbens rostra1 pole and shell region at 7 h following ha1 injections (not shown). Conspicuous differences with rcg,rd to the numbers and distributions of hybridized neurons in extr:astriatal forebrain regions as compared to controls were not &erved in the 7-h haltreated rats.
tectability. The other ( population has a ste increases more gradu period of time. Neu era1 quadrants arc d following
administration of hal.
While non-isotopic hybridizations do not provide reliable quantitative data about absolute amounts of m being produced by individual neurons, they do show nioeiy relative differences between controls and pair-matched drug-treated sections. Thus, it was assumed during the course of this study that, in a given in situ hybridization experiment utilizing appropriate reagent ~~~~i~e~trati~)ns, a threshold range exists for the inrracellular concentration of the mRNA species being tested. above which neurons be@ to exhibit reaction product. It has been concluded in experiments in which control and test sections have been reacted with the same reagent solulions, that preparations exhibiting significantly more hybridized neurons do so because more neurons exceed the threshold for detection, from which can be reasonably inferred that mRNA synthesis is increased in those preparations. Given these considerations, non-isotopic in situ hybridizations may be regarded as a particularly valuable tool. insofar as the conspicuous nature of the nitroblue tetrazolium reaction product facilitates detection of regional and temporal differences in levels of mRNA synthesis.
4. Discussion This descriptive and quantitative account has capitalized upon the superb cellular resolution afforded by the nonisotopic approach to in sifu hybridization. It was observed that sections from 24-h hill-treated rats exhibited enhanced hybridization in the dorsomedial and ventrolateral quadrants of the caudate-putamen. when processed in tandem with the vehicle-treated controls using the same hybridization and rinse buffers. Furthermore, the amount of histochemical reaction product associated with individual cells in the dorsomedial and ventrolateral districts was noticeably greater in the 24-h hal-treated groups. These observations are regarded as evidence for increased expression of NT/N mRNA in the dorsomedial and ventrolateral quadrants of the caudate-putamen at 24 h following injection of hal. The distinct patterns of distribution of hybridized neurons observed at 7 and 24 h post-injection are consistent with at least two subpopulations of neurons in the rat striatum that express NT in response to ha1 administration, as has also been suggested by immunohisrochemical investigations using antibodies against NT peptide [4,19,24]. One (primarily dorsolateral) subpopulation comprises neurons that express NT/N mRNA rapidly and robustly, but tranisently. and fail to achieve immunohistochemical de-
It had been noted in examining material prepared preliminarily to this invesGgation that the composiiion of the rinse sequence plays a critical role in determining how many hybridized neurons are observed in a given preparation. In particular, RNAse A treatment, which sensitively regulates signal to noise ratio in isotopic in situ hybridization riboprobe preparations 1201, was observed to substantially diminish the numbers of hybridized neurons detected in the non-isotopic preparations. Although the basis for this finding has not been investigated systematically, it is hypothesized that, following hybridizations, the preparations contain a significant amount of specific labeling in which only parts of the digoxigenin containing transcripts are annealed to target mRNA in the sections. When unannealed portions of partially. but specifically. bound probe are degraded by RNAse, the histochemically detectable signal is substantially reduced, resulting in fetwer labeled neurons. It is concluded that the specificity of the hybridizations is not significantly altered by the omission of RNAse treatment, because :hz patterns of labeling both in striatal and extrastriatal tissues and the patterns of change associated with ha1 treatment were similar in the presence and absence of RNAse treatment.
77
gcnu
or
its
gem2
produ
ifferences. In this regard, it is notewor-
after injection of hal. i.e.. \vas obserked in rclatcd studies that Fos is not significantly w-L’xpreswd ;Is ;I second marker in NT-imlmunoreacti~,~ striatal neurons ~oll(~~i~i~ hal adminislration [-I. I’,]. 11‘.;LS
11)pothcsiecd, the ucuron~ (I!’ the ~radu;rl!~-cspre~sit~~
I~_-~Icc~ p2lcht3 r~!‘ men (Fig. 3, ;trrou 3) rc\cmblc Glliilarlj NT-immunoreactive neurons and fibers observed following reserpine administration [3,24]. The observed up-rcgu~~~ti~~n of the dors,r,medial/~entl-olateral response of NT/N mRNA expression is apparently more gradual to dcvetop and more enduring than the dorsoiateral response. which returns to control levels well within 24 h [8,9]. It is noteworthy in this regard that the dorsomedial and ventrolateral quadrants of the caudateputamen arc the only district., in the striatum outside of the nucleus accumbens where detectable expression of NT/N mRNA was observed, albeit minimally. in the absence of’ ha1 (see also [12]). Furthermore. it is in the dorwmedial and vcntrolatcra~ (and rostra!) parts of the cuudtitt’-putrrmc‘n
group are those that become detectably immunoreactive for NT. then the data would suggest that Fos IS not a necessary part of the regulatory apparatus for NT/N gene transcription rn neurons of the d0rsomcdiai/ventrolatera~ gradually expressin,cv subset. Also. Merchant et al. [I i j have observed that the indirect dopamine agonist methan?phetamine elicits NT/N mRNA zxprcssion, aFparentiy unpreceeded by Fos expression. in precisely the dorsomedial and icntrolateral quadrants of the caudate-putamen bvhere increrrscd NT/N mRNA was observed in Ihe present study and whcrc, ;LSnoted &ove. numerous NT-immunoreactive neurons arc obwrvcd fiillowing dopamine
as well us in the nuckus a~~~mbcns. that increased num-
s;uw
bers of NT-immunoreactive neurons are observed :bilowing administration of hal 16,241. Ensofar as their distributions and the time courses in which they appear are similar, it is hypothesized that dorsomedial/ventrotateral neurons in which NT/N mRNA accumulates in a gradual. enduring manner followin, 0 hal administration correspond to ones in which NT-immunoreactivity is detected, although this remains to be demonstrated directly. The present data confirm and extend the findings of Williams et al. [22], who also observed increases in NT/N mRNA-expressing neurons in the striatum using an oligonucleotide probe and a non-isotopic fluorescence in situ hybridization method. In that study, hai was administered in two injections at i 7 and 10 h prior to sacrificing the rats, which probably resulted in a diminished capacity to distinguish the rapidly expressing dorsolateral subset of NT/N rr?RNA-expressing neurons, in which. at 10 h following the second ha1 injection, NT/N mRNA has begun to return toward basal levels [8,9], and the gradually expressing dorsomedial/ventrolateral subsets of striatal NT/N mRNA-expressing neurons, in which NT/N mRNA
11, rwcptor antagonist administrarion [6.2-C]. Whether the subwt (11’ neurons is responding to the indirect agonist and hal by expressing NT/N mRNA remains to be determined. Much remains to be learned about how dopamine neuretransmission regulates neuronal activity and impulse conduction and transmission in the basal ganglia. The present data are consistent with at least two subpopulations of striatal neurons in which NT/N mRNA expression is differentially regulated by dopamine. Continuing refine ment of the capacity to utilize NT/N expression as a probe of dopamine effects in the basal ganglia should help to solidify a foundation upon wnich a better understanding of this complex area can be built.
Acknowledgements This work was supported by USPH Grants NIH NS23805 (D.S.Z.) and NS-21937 (J.E.K.1. The authors arc indebted to Mci Feng and Joseph Tucker for excellent technical assistance. The gift of NT/N cDNA from Dr. Paul R. Dobner is gratefully acknowledged.
References Dl Alexander, M.J.. Miller, M.A.. Dorsa. D.M.. Bullock. B.P.. Melloni. R.H., Dobner, P.R. and Leeman. S.E., Distribution of neurotensin/neuromedin N mRNA in rat forebrain. unexpected abundance in hippocampus and subiculum. Proc. Nctrl. Acc~l. Sci. USA. 86 (1989) 5202-5206.
El Augood. S.J.. Kiyama, H.. Faull. R.M. and Emson. PC.. Differential effects of acute dopaminergic D, and D, receptor antagonists on proneurotensinmRNA expression in rat striatum. MO/. Brcritl Res., 9 (1991) 341-346.
131 Bean. A.J., During. M.J., Deutch, A.Y. and Roth, R.H.. Effects of dopamine depletion on striatal neurotensuf: biochemical and immunohistochemicalstudies, J. Netrrosci., 9 ( 1989) 4430-4438. [41 Brog. J.S. and Zahm, D.S.. Morphology and Fos immunoreactivity reveal two subpopulationsof striatal neurotensin neurons rollowing acute 6-hydroxydopamine lesions and reserpineadministration, Nellroscierrce, 65 ( 1995) 7 I-86. 151 Deutch. A.Y. and Zahm, D.S.. The current status of neurotensindopamine interactions: issues and speculations. In P. Kitagbi and C.B. Nemeroff (Eds.). The Neurobiology of Neurotensin. Am. NY AC&. Sci.. 668 (1992) 232-252. [6l Eggerman. K.W. and Zahm. D.S., Numbers of neurotensin-immunoreactive neurons selectively increased in rat ventral striatum 1I ( I9881 following acute haloperidol administration, N~‘fr~~)pt~pri’lc~.\. 125-132. t71 Krause, J.E., Chirgwin, J.M., Carter, M.S.. Xu, Z.S. and Hershey. A.D.. Identification of three rat preprotachykinins encoding both substance P and neurokinin A. Proc. Nutl. Acad. Sci. USA. 84 ( 1987) 9881-9885. 181 Merchant. K.M.. Dobie. D.J. and Dorsa, D.M., Expression of the proncurotensingene in the rat brain and its regulation by antipsychotic drugs. AWL NY Acad. Sci., 668 (1992) 652-653. 191 Merchant, K.M.. Dobner. P.R. and Dorsa. D.M.. Differential effects of haloperidol and clozapine on neurotensingene transcription in rat ncostriatum. J. Nctrrmci.. I2 (1992) 652-663. [I()1 Merchant. K.M. and Dorsa. D.M.. Differential induction of ncurotensin and c-&s gene expression by typical versus atypical antipsychotics. Pm. Nurl. Awd. Sci. USA. 90 ( 109.3) 3447-345 I. 11II Merchant, K.M.. Hanson, G.R. and Dorsa, D.M.. Induction of ncurotcnsinand C$~J.V mRNA in distinct subregionsof rat ncostriaturn after acute methamphetamine: comparison with acute haloperido1 cffCcts, J. Phmtrrrcol. Exp. The,:, 269 (1994) 806-812. [I21 Mcrchnnt, KM. und Miller, M.A., Cocxprcssionof neurotcnsinand C-~H mRNAs in rat neostriatal neurons following acute haloperidoi. Mol. Brctiti Res.. 23 ( 1994) 27 I-277.
WI Merchant. K.M.. Miller. M.A..
Ashleigh, A. ,md Dor\a. D. Hnloperidol rapidly incrcascs the II~III~W of ncurotcnain mRNA-cxpressing neurons in neo\triatum of the rat braon, Hrrrrrr Rts\.. .kl-!O (1991) 31 I-313. WI Morgan. J.I. and Currim. T.. Stimulus-tran~cril~tlon couphng in the nervous system: involvement of the inducible proto-oncogcnc\ /o.\ and ,ilul. Atrrrrr.Rcr~. Nemm~i.. 13 ( 191j114.?I-45 1 Roberkon. G.S. and Fibiger. C4.C.. Ncurckpr~c~ tncwLw c,-ftjt cripression in the forebrain: contrastmg effects o! halop~r~dol and clozapine, Newosciww, 46 ( 1992)3 I S-328. iI61 Robertson. G.S., Vincent. S.R. and Fibigcr, H.C., Striatonigal projection neurons contain D, dopamine recept<)r-activatedc,/i),s. l~rcrii~ Ras., 52.3 ( 1990) 288-290. [I71 Robertson. G.S.. Vincent, S.R. and Fibigcr. H.C.. Dl and D2 dopamine receptors differentially regulate c-ji>.s expression in striatonigral and striatopallidal neurons, Newosciettc ;‘, -10 ( IO02128S-296.
[1N Robertson. H.A.. Peterson. M.R.. Murphy, K. and Robertson, G.S.. D, dopaminc receptor agonists selectively aclivatc \triatal c-fir.\ independentof rolationid beha\ ictr, &.tun I?(*.\..SO3( I!Sc))346-3-W. 1191 Senger, B.. Brog. J.S. and Zahm. D.S.. Stihscrs of ncurotensin irnmunoreactive neurons in the rat striatid complex following i!tltilfonism of the dopamine D-2 receptor: an immunohistochclnicnl double-labeling study using antihodics against Fo\. N~~III.~).\(./~‘II(.(‘. 57 ( I’~%) 649-660. [20] Valentino. K.L.. Eberwine. J.H. and Barchns. J.D. (Eds.). Irr .Wr Hhridimim. Applictrtiom to Nerrrohiokq,v. Oxford University Press. New York. 1987. D11 Wan. X.S.T.. Liang. F.. Moret. V.. Wiesendanger. M. and Rouiller. E.M.. Mapping of the motor pathways in rats: c+\ induction by intracortical microstimulation of the motor cortex correlated with cfferent connectivity of the site of cortical strmulation. Nc>frro.sc*iertw,40 ( I992 1749-76 I .
[El Williams, F.G., Murtaugh, M.P. and Beit/. A.J.. The cffcct of acute haloperiol treatment on brain proneurotensin mRNA: in situ hybridization analysis using a novel Iluorcscence detection procedure. Mol. Rrh Res.. 7 (1990) 3-17-358.
[23] Young 111.W.S.. In situ hybridi/.ation hisrochomistry. In A. Bjiirkland, T. Hiikfelt. F.G. Wouterlood and A,N. van den Pol (Eds.), cd N1~1r~c~~1trcrri~t~r~ I \irl. S, Artlrhi.~ r!j N~vrroturl Mkrwitwit~ lttd .Swrtrp~ic Itt/errrc~iorrs. Elscvier. Ams,terdam. 1990. pp 4x1 -51 I.
Chrcltwohr!/ ~‘l~cwrrt
1241 ZaPrm.D.S.. Distinct subsetsof neurotensinimmunoreactive neurons revealed following antagonism of the dopamine-mediated suppression of neurotensin immunoreactivity in the rat striatum, Newasciemx;. 46 I19921 335-350.