Microinjection of neurotensin into the ventral tegmental area produces hypothermia: Evaluation of dopaminergic mediation

Brain Research. 326 ( 1985 ) 210- 227 Elsevier

219

BRE 1O496

Microinjection of Neurotensin into the Ventral Tegmental Area Produces Hypothermia: Evaluation of Dopaminergic Mediation PETER W. K A L I V A S I, C H A R L E S B. N E M E R O F F -~,JANE S. MILLER t and A R I H U R . I . P R A N ( I E , . l r . :

1Department ~f Pharmacologv, LSU Medical Center, New Orb,an,s, LA and eDepartments ~/"Neurohioh)gv and I~ vchialrv, School o/ Medicine, University of North Carolina, Chapel ftill, NC t U.S.A. ) (Accepted May 8th, 1984) Key word.s, ncurotcnsin - - ventral tegmental area - - dopamine - - diagonal band of Broca - - hypothernria

micromiection

Neurotensin-producing perikarya and fibers have been identified in the ventral tegmental area of the rat, and recent microinjcction studies indicate that neurotensin may function in the ventral tegmental area to regulate body temperature. In this study, the hypothcrmic response produced by intraventral tegmental injection of neurotensin was shown to be dose-dependent, with a threshold dose between 0.25 and 0.75 ,tg. When fluphenazine, a dopamine receptor antagonist, was microinjectcd into w~rious forebrain nuclei simultaneous with neurotcnsin infusion into the ventral tegmental area, it was found to block neurotcnsin hypothermia. In contrast, injection with fluphenazinc into the nucleus accumbens, lateral septum or preoptic area did not alter thc hypothermic response. Furthermore. injection with atropine, phentolamine or diphenhydramine into the diagonal band of Broca did not block ncurotcnsin hypothermia. Neurotensin was also injected directly into the preoptic region and shown to produce hypothermia. However. concurrent infusion ol fluphenazinc with neurotensin into the preoptic region did not attenuate neurotensin hypothermia. These data are consistent with the postulate that neurotensin acts in the ventral tegmental area to enhance dopamine release in the diagonal band of Broca, thereby pro~ ducing hypothermia. However, neurotensin-induced hypothermia occurring after injection into the preoptic area does not appear to invoh,e dopamine systems. INTRODUCFION

response in the rat. This o b s e r v a t i o n is consistent with the well established role of this v e n t r o m e d i a l

N e u r o t e n s i n is an e n d o g e n o u s t r i d e c a p e p t i d e that

forebrain region in t h e r m o r e g u l a t i o n >, In addition

possesses m a n y n e u r o t r a n s m i t t e r - l i k e p r o p e r t i e s in

to the p r e o p t i c region and a d j a c e n t tissue, Kalivas el

the central n e r v o u s system L~,2s. C o n s i s t e n t with the

al. also o b s e r v e d that n e u r o t e n s i n injection into the

concept of n e u r o t e n s i n as a m o d u l a t o r of n e u r o n a l

ventral t e g m e n t a l a r e a p r o d u c e s h y p o t h e r m i a I~J.

activity are the findings that intracranial injection

The ventral t e g m e n t a l a r e a has b e e n shown to pos-

with n e u r o t e n s i n p r o d u c e s a variety of physiological

sess a high density of n e u r o t e n s i n - p r o d u c i n g perika-

and b e h a v i o r a l changes. A m o n g the first n e u r o t e n -

rya and fibers ];,~a, as well as n e u r o t e n s i n

sin-induced effects to be n o t e d was its capacity to

tors -~2,>. In addition to n e u r o t e n s i n , the ventral teg-

lower body t e m p e r a t u r e after intracisternal adminis-

mental area contains a high density of d o p a m i n e r g i c

tration in the r o d e n t 2. This o b s e r v a t i o n has since

perikarya with p r o j e c t i o n s to m a n y Ihnbic forebrain

recep-

been replicated in m a n y laboratories<~S23.:5 and two

regions ]],la.>. A variety of b e h a v i o r a l , n e u r o c h e m i c -

recent studies have a t t e m p t e d to locate its n e u r o a n a -

at 21-~1 and e l e c t r o p b y s i o l o g i c a l data p indicate that

tomical site of action by m i c r o i n j e c t i n g n e u r o t e n s i n

neurotensin may act in the ventral t e g m e n t a l area to

into various brain nucleil~,2 a. B o t h studies c o n c l u d e d thai m i c r o i n j e c f i o n of n e u r o t e n s i n into the diagonal

activate these d o p a m i n e n e u r o n s . An i n v o l v e l n e n l bv this d o p a m i n e system in t h c r m o r e g u l a t i o n has

band of B r o c a , medial p r e o p t i c region and medial anterior h y p o t h a l a m u s p r o d u c e s a m a r k e d h y p o t h e r m i c

shown to be a p o t e n t h y p o t h e r m i c agent w h e n m-

been suggested previously~5 and d o p a m i n e has been

Correspondence: p. Kalivas, Department of Pharmacology, LSU Medical ('enter, 19I)1 Perdido Street, New ()rlcans. LA 70112, U.SA. 0006-S993:85i$()3.3{) ('~ 1985 Elsevier Science Publishers B.V

220 jected into the preoptic region and diagonal band of the rat 7-m. The present study was designed to evaluate the involvement of dopamine in the hypothermic response after injection with neurotensin into the ventral tegmental area. MATERIALS AND METHODS

Animal housing and surgery Eighty-four S p r a g u e - D a w l e y rats (Holtzman Co., Madison, WI) were individually housed with a 12 h light-dark cycle, and food and water were made available ad libitum. When rats attained a weight of 250-300 g they were anesthetized by i.p. injection with 50 mg/kg pentobarbital plus 1.5 mg/kg methylatropine, and positioned in a stereotaxic apparatus (David Kopf, Tunjunga, CA). Each rat was then implanted with chronic 26-gauge stainless steel guide cannulae 1 mm over the desired injection site. All cannulae implanted within 0.5 mm of the midline were angled at 4°; thereby avoiding puncture of the midsagittal sinus and cerebral aqueduct or third ventricle. Rats received one of 3 types of cannulae implantation; bilateral implantation over the ventral tegmental area (n = 17), bilateral implantation over the medial preoptic area (n = 10), or bilateral implantation over both the ventral tegmental area and a desired forebrain nucleus (n = 57). All cannulac were secured in place with dental acrylic and stainless steel screws, 33-gauge stainless steel obturators placed in each cannula, and the rats returned to their home cage for a 7 - 1 0 day postoperative recovery period.

Microin]ection procedure and measurement o[" colonic temperature Either neurotensin (Bachem, Torrance, CA) or millipore-filtered 0.9% saline vehicle was bilaterally microinjected into the ventral tegmental area or preoptic area of the unrestrained rat in a total volume of 0.5 #l/side given over 60 s. In some studies, either fluphenazine hydrochloride (E. R. Squibb and Sons, Princeton, N J), atropine sulfate (Sigma Chemical Co., St. Louis, MO), diphenhydramine hydrochtoride (Sigma Chemical Co., St. Louis, MO), or phentolamine hydrochloride ( C I B A Pharmaceutical Co.. Summit, N J) was bilaterally microinjected into the forebrain, also in an injection volume of 0.5 .el saline./

side over 6{) s. Simultaneous bilateral microinjeclions were made with two 1-!~1 syringes (Hamilton. Reno, NV} connected to 33-gauge stainless steel rejection needles by PE-1(I tubing. I'.ach 33-gauge needle was inserted to a depth of 1 rain below the tip ol the guide cannulae into the desired injection locus. When rats received microm.lCCtam into both the lore-. brain and ",.entral tegmental area. the forebram mlcction was made immediately prio~ *~ lhc ventral Ic,._,mental injection. Colonic temperature was measured by inserling u YSI-402 thermistor probe n cm m~o the rectum. A measurement was obtained 20 mm prior to micron> ection, immediately prior to micl~mljection_ immediately after microiniection, and every 20 rain thereafter fl)r 120 min. Each rat received up to 4 separate experimental trials, separated by ~ mimmum 72 h intertrial period.

HLftology and data analysis All rats were examined histologically for cannulae tract placement. Rats were killed under ether anesthesia via intracardiac perfusion with IJ. 1 M phosphate buffer (pH = 7.51, followed by 300 ml o f t 4c.,> paraformaldehyde-phosphate buffer solution. Brains were removed and stored overnight in fixative. Coronal sections (ll)0 uml were made with a vibratome (Ted Pella. Tujunga. C A / . mounted on gelatin-coated slides and stained with cresvl violet. Using the atlas of Pelligrino et al. > the forebrain injection Ioct were determined, and ventral tegmental area cannulae placement was evaluated as described in detail elsewhere :-~. In this study, all ventral tegmental cannulae were placed within 0.5 mm of the midline, which includes the nucleus linearis ~nd the medial portion of the nucleus paranigralis and nucleus parabrachialis pigmentosus ~2 Data were analyzed as difference from baseline colonic temperature, and the baseline temperature was considered the average of the measuremenls made immediatelv before and after micromjection In most studies each rat received an experimental trial with control rejection. Thus. the data were paired and statistical estimates made with a two-tailed paired Student's t-test. Since each rat received 3 experimental trials, the probability values were mcreased according to the method ot Bonferoni > When data were not paired, statisucal differences be-

221

EFFECT OF INTRA-VTA INJECTION OF NT

Fig. 1 shows that the decrease in cohmic temperature produced by ncurotensin injection into the ventral tegmental area of the rat is dose-related. The

+08 0 •

+06

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•

120 rain. This may have restlltcd [ro111 :.l marked in-

* p
o_.. + 0 4 b

L~

threshold dose for producing a significant hypothermic response ~\ras between 0.25 and (). 75 Hg of ncurotensin/side. Interestingly, rats receiving 2.5 #g neurotensin/sidc tended to be slightly hyperthermic at

Saline (N =8) 0 2 5 ,ug NT (N : 6 ) 0 7 5 Ng N T ( N : 7 ) 250 jugN (N:IO)

crease in motor activity that of t0n oc'ctirred after injection with this dose of neurotensiil into some sub-

I

nuclei of the ventral tegmenlal area m,:_'

i i

Fig. 2 shows the cannulae tracks from a rat implanted in the ventral tegmental area. In general it was observed that canilulae tips located within 0.5 mm of the midline, not including the intcrpeduncular nucleus, wcrc most effective in producing hypothernlia 22.

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120

TIME A F T E R MICROINJECTION (Min) Fig. 1. l)o,c response curve lor hyp<)lhcrmia produccd b\ m iccihm el i]ctirolcn~,Jll imo the \ cntral tC.~lllCnl~llarea. [)ata ~lit.' sho\~,ll tl~, recall .~ ~. E. M . challgC in c o l o n i c l c m p c r a l u r c c o m p a r e d wid~ b u s c l i n c lllc'dstlrelllC.'ilis [st_'c Methods). :' f' < (I.(15. t)llt.'-\i+u ~, A N ( ) V : \ l o l l o w c d b\ ii D u n l l c t ' s tcs[ c o m p a r i n g till ilcurolCil,,Jil IrcatmL'nts v, itl~ , < l i m e +" [ ' < (i.(t0.>.

tweei1 groups \yore cslinlaled with a one-wa\ ailalvsJs of x ariancc followed bv a lwo-tailed Dunnctt's test. RESt'I_IS

Hypollldrtllia ~(/Idr inieclion q/ nettrotensi/t into t/w I'{'#I[YEI[ [f'<~IIH.'I1H.I/ Elr(',(l

<~/dopamineneHrrmN

Fig. 3 smnmarizes the slereotaxic location of cannulae tips in the forebram of the rat. Iniection with fluphenazine into the region of the diagonal band of Broca markedly attenuated the h_~pothcrmia produccd by ncurotensin injection into tile ventral togmental area. By grouping the data sho\\,r|l ill Fig. 3 according to cannulae placement in specific brain nuclei, statistical cvalualion of regi~mal scnsiti\ritv to fluphcnazinc block~ldc was possible. [:ig. 4 shows that only in the diagonal band of Broc'a did fluphenazinc significantly attenuate neUl-Otcnsin-induced hvpothermia. Injection of fluphenazmc into two adjaCelll mcsolimbic dopamine terminal fields, the nudetlS aCCUlllbells and lateral septum, did not block the hypothcrmia produced by inlravcntr'41 tegmcntal in.ieclion with ncurotensin. Likewise. fluphcnazin¢ in.jCCtion .jl_lSt caudal to lhe diagonal lxmd of Broca rote the medial prcoptic area did not signilicantly attentlatC the hypothermic response of neurotensin. To c~aluatc tile specificity of the I)A receptor blockade by fluphenazine, neurotensin-induccd h_~pothermia was measured after inicction ~ri|h LIn cquimolar dose of atropine (muscarmic antagonist t, diphenhydramine ( Hi histamine antagonist ) or phcnt(q'Jminc {+/-adrenergic antagonist) into the diagonal band of Broca. I a b l c I shows thal in lhc presence el these drugs neurotensin still produced a signilicanl hypothermic I-CSp()nsc.

Fig. 5 shows cannulae track placement in the fore-

222

Fig. 2. Representative micrograph showing location of bilatcral cannulac tips in the ventral tegmentaJ area, All cannulac tips ~crc 1o. cated within 0.5 mm of the midline, in or near the nucleus linearis 3~,>. Note the lesion produced by the chronic guide cannutae above the injection site to the left of the cerebral aqueduct. This illustrates the maximum size of tissue displacement produced by chronic implantation of the 26-gauge cannulae. FR, fasciculus retroflexus; IF, nucleus interfascicularis: L, nucleus lineari~; PN. nucleus paranb gralis. Bar = I ram.

brain from two rats used in this study. The rat m Fig. 5 (top) was implanted into the diagonal band of Broca, and fluphenazine produced a marked attenuation of neurotensin hypothermia. In contrast.

Fig. 5 (bottoml shows cannulae placement in the preoptic area where fluphenazinc was not effective lntection q/ neurorensin mto the preopttc region

TABLE 1

Effect o f various drugs injected into the diagonal band o~ Broca on neurotensin-mduced hypothermia All doses are equimolar to 5.0/*g fluphenazine/side. Rats were given one injection with treatment + neurotensin 12.5/,g/side) and one with treatment + saline. The data are shown as the mean _+ S.E.M. difference between these two treatments for each measurement of colonic temperature after injection.

Treatment

Atropine Phentolamine Diphenhydramine Fluphenazine

~

10 7 7 10

Time 1rain; 20

4o

60

~o

100

-0.8 +_ 0.2* -0.7 _+ 0.2* -0.8 __+0.2* -0.4_+0.3

- 1.2 _+ 0.2 =' -1.0 _+ 0.3" - 1.5 ± 0.3* -0.4±0.3

- 1.0 + 0.2* -1.0 +_ 0.3 ~ - 1.3 _+ 0.54 -0.4_+0.3

1. l ± 0.2* -11.9 ± 0.3" --1.1 _+ (1.5 0.2_+0.3

-tJ.8 -I).5 1.4 -11.1

* P < 0.05, two-tailed paired Studem's t-test.

120 + 0.2* ± tl.3 2 tl.5" ~ {l.3

-0,6 ± 0.2* -0.2 z_+0.3 4/.8 ~ 1).5 0.0±0.3

0.4

I)iogo~l Bond

""°

0

Preol~ic Arlo N-6

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Nucklu$ Ac,cumben$ N-6

o -0.4.

-0,8

-12

- 1.6 40

i 80

|

i 120

o

: Xo'

8o

'~o

TIME (min) Pi~..], ,'~UIllI/I{U\ e l cailnl.ilac tip piaccmClll ill coronal di~igralnS ot the i-~it i'orc'brahl (:. Plupl3cnazhlo did not block ilcLirotonsin hypothcrmia: Ih. 'at cittlor 2(t or 60 mm after microm ieclion fluphcmlzhlc produced at least a I °(_" rcvcrsn] e l i1cui-ol e n s e h)pothcrmia. 0 . l]upt'~ellazino produced a ~> 1 (" rcvu'r~ul ~1 nourotcrlnill h)p~>thcrilli:l ~ii both 21) aild 60 mhl.

Fig. h shows that bilateral microinjection of neurotensin (2.5 ,ug/side) into the rostral preoptic area of diagonal band of Broca produced hypothermia. However, this hypothermic response was not significantly attenwited [~r simultaneous injection of fluphenazinc ( 2.5 ,. g,'side ). DIS( t'SSI()N Neurotensin injection into the ventral tegmental area of the rat has been shown previously to produce ~t decrease in colonic temperature I
Fig. 4. El'loci e l t'lul~honazmc injection into 4 hlrcbraitl nuclei on hypothc'rnlia produced by in.icction el ncuiotcnsin iluo tile ventral togmelllal ~lrca. All Fats rcc~:i~ cd .~ ~,up < (i.(i~,. lwo-l~lilc'd paired Student's t-test with probabilil,+ valtios increased I])r muliiplc trcatlllont ~lS described by Bonlcroni i~. compared v, iih l luphc, nazinc plus saline. *~ P < ()(10f,. : P < IllLq coil~purmd ~ilh lluphonazine plus nou fotoilsin.

significantly attenuates the hypotht,,rmia produced by neurotensin injection into the \cntral tegmental area. In contrast, no blockade was observed after fluphenazine injection into adiacen! mesolimbic terminal areas, including the nucleus accumbcns and lateral septum, Likewise, injection of fluphenazine c:ludal to the diagonal band in the preoplic ~ire~ was also ineffective m blocking neurotensin-induccd hypothermia. All these data are consistent ~ith lhe postulate that neurotensin produces hypothermia after injection into the ventral tegmental area b} increasing dop~lmine release m the diagonal band ot t}roca. However. these data do not indicate whether neurotensin

224

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.~

39.

o_. 38"

L~ 3r

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120

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.

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80

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!

120

TIME (rain) Fig. 5. Representative cannulae placement in the forebrain oI the raL Top: cannulae placed in the diagonal band of Broca. Fluphcnazine markedly attenuated neurotensin hypothermia. Bottom: cannulae placed in the preoptic/anterior hypc~lhalamus. Fluphenazine did not markedly alter neurotensin hypothermia. D. fluphenazme plus saline: %. saline plus neurotensm: O, fluphenazine plus neurotensin.

is acting directly on d o p a m i n e neurons m the ventral tegmental area that project to the diagonal band of Broca, or facilitating d o p a m i n e release indirectly wa a polysynaptic mechanism. A series of papers by Cox and his associates demonstrate that microinjection of d o p a m i n e into the preoptic area, and, to a lesser extent, the diagonal band of Broca produces h y p o t h e r m i a 8,9,m. This observation has since been replicated by others 7. In the present report, injection of fluphenazine only into the diagonal b a n d blocked neurotensin hypothermia. The lack of response in the preoptic region may result from the fact that this brain region is not it terminal

field for ascending dopaminergm eflerents from the ventral tegmental area 4 L _~. In comrast, the diagonal band of Broca is innervated from the ventral tegmental area3--'{L and contains a h i g h concentration of d o p a m i n e 5. Further m~croinjection of neurotensm into the ventral tegmental area increases the level of d o p a m i n e metabolites in the diagonal band of Broca -'0. Thus. two separate d o p a m i n e systems may pla}. a t h e r m o r e g u l a t o r y role in the r a t . d o p a m i n e neurons innervating the diagonal band from the ventral tegmental area. and d o p a m i n e terminals in the preoptic area perhaps arising from the A I 4 d o p a m i n e cell group in the hypothalamu~ L"

33S

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0

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I

4'0

'

8 0:

"

~"ImO

TIME (rain) Fig f~ Etlcct el fluphcnazinc on hypothcrnml produced h} ncuroicnshl in cotton into the prcoptic at-ca, t l , tTluphcnazmu plus ilcurotunsin ejected shllululncol.lsl\ into tt~c prcoptic arcci, -:', nt_,urotcllsin, .~c,t_,Fig. 4 lor ~rcatcr dct
In contrast with the intravcntral tegmental ~trca injcctitm, the hyt~othernlic response following direct injcctitm of neuroteilsin into the preoptic tirea or diagon'
injection \vittl netir()tensil] in the mouse

or tat2~`-~'.

When administered into tile ccrebroventricular s\.sten1. iletlrotcnsiil probably ~ictf, at periventricular situs shown to be" rusl3onsive to direct injection \vith ilCUrtHeilSill; including the medial preoptic at-ca alld the t3ontine periventricul'c,;urring aflcr intruvcntricular adnlinistration m~lv not invohc ~111action in the ventral icgmental area to tq-oi31ote dopaminc release in tile diagonal b~lnd of Broc'
these data, it was postulated that ncurolellsin in the ventral tegmenta] ~lleti itcti\.'illC~> doparnine neurons

projecting to the nucleus accunlbens, thereby promoring behavioral hypcructivity 21. "[his raises interesting spccuhitions regarding the significance o1: a single putative transmitter initiating two independent physiological responses sinlultaneoush'. V~'lletl one conskiers thllt thc incrcased muscle :ictivitv produced during exloration of the en~.ironmcnt will increase helit production, it seems reasonable ti3ttt a simultaI1COLIS mcchanisnl lor I~och. hclii loss would I~c initiated. While phni-nlacoh)Tical adnlinistration with neurotcnsin produces non-ph)'siological cxtrctllcs in both systems, i.e. anlphctanfinc-likc h_vpcr
226 t h e f u n c t i o n a l d a t a p r e s e n t e d in this r e p o r t a n d else-

t e g m e n t a l a r e a 3~. a n d t h e e x i s t e n c e o f n e u r o t e n s m

w h e r e ~6,21.22, e l e c t r o p h y s i o l o g i c a l

p r o d u c i n g p e r i k a r y a a n d f i b e r s in t h e v e n t r a l t e g m e n tal a r e a 17.34

iontophoretic neurotensin

studies

showing

to b e p o t e n t e x c i t a t o r y

a g e n t in t h e v e n t r a l t e g m e n t a l area~, n e u r o c h e m i c a l data demonstrating increased dopamine metabolite

ACKNOWLEDGEMENTS

p r o d u c t i o n in t h e n u c l e u s a c c u m b e n s a n d d i a g o n a l band after microinjection of neurotensin

into the

T h i s r e s e a r c h was f u n d e d b y U S P H S G r a n t s M H -

v e n t r a l t e g m e n t a l area 2o,21, t h e e x i s t e n c e of n e u r o -

08935.

t e n s i n r e c e p t o r s o n d o p a m i n e n e u r o n s in t h e v e n t r a l

22536 a n d N I C H H D

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