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Behavioural effects of NMDA injected into the hypothalamic paraventricular nucleus of the rat T.A P. Roehng 1, A M.M van Erp 2, W Meehs 2, M . R Kruk 2 and J G. Veenlng 1 1Department of Anatomy and Embryology, Untverstty of Nqmegen, Nqmegen (The Netherlands) and 2Ethopharmacology Group, Umverslty of Letden, Letden (The Netherlands) (Accepted 18 December 1990)

Key words Hypothalamus, Paraventncular nucleus, Excitatory amino aod, N-Methyl-D-aspartate, Grooming, Feeding

Electrical stimulation of the hypothalamic paraventrlcular nucleus (PVH) and of the adjacent dorsal hypothalamlc area (DHA) evokes grooming behavlour M~crolnjectlons of low doses of kam~c aod, an agonlst of the kamate type of glutamate receptors, into the same area evokes the same behavlour To test whether other glutamate receptors are involved, mlcromjectlons w~th N-methyl-o-aspart~c acid (NMDA) were made into the PVH/DHA area and the behawour was observed From the total observation time (30 mln) up to 73% was spent on grooming, accompamed by yawning Pronounced feeding behaviour was also noticed at 3 mjecUon sites but not untd 23 mm after mjecnon Conclusions are that neurones within the PVH/DHA area are revolved m grooming behavlour, possibly wa glutamaterglc mnervatlon The interaction between grooming and feeding behawour at the level of the PVH is d~scussed

INTRODUCTION G r o o m m g behavlour has been described to have 3 separate functions to maintain the condition of an animal's fur, to regulate the body temperature and to normahze the arousal level of an ammal after stressful events 13 Studies using m t r a c e r e b r o v e n t n c u l a r (t c v ) rejections of various pepttdes have indicated, that e g adrenocortlcotroplc h o r m o n e ( A C T H ) 4 i1, bombesm27, thyrotropln-releasmg h o r m o n e ( T R H ) 34, oxytocin 3 and cortlcotropln-releasmg factor (CRF) 5'24 are involved in the neural system regulating grooming behavlour The characteristics of the displayed grooming behavlour, however, can be different as various substances are used11 34 35 Although the neural mechanism underlying the groommg response has not yet been clarified, several brain sites have been reported to be of importance in this respect The central gray m a t t e r~° and the ventral tegmental areal4 32 have been reported to be involved m peptldeInduced grooming behaviour D u n n suggested, that the site of action of t c v injected A C T H inducing excessive grooming was situated m the anteroventral recess of the third ventricle, near the o r g a n u m vasculosum of the lamina termmalls 4 Lammers et al reported grooming b e h a w o u r evoked by electrical stimulation m and near

the hypothalamlc p a r a v e n t n c u l a r nucleus ( P V H ) and the adjacent dorsal hypothalamlc a r e a ( D H A ) 18'19 Local injections into the P V H / D H A region of substances, that have been reported to evoke grooming behavlour by i c v rejection, were also capable of reducing grooming behavtour, e g A C T H 33 and C R F 16 G r o o m i n g could also be ehcited by local mtrahypothalamtc rejection of low, n o n - d a m a g i n g doses of kalmc acid into the P V H / D H A region 12'28 Kalmc acid is a k n o w n agonlst of the kamate type of glutamate receptors which is involved m fast synaptlc neurotransmlsslon2 The locahzed effect of kalmc acid on grooming behavlour therefore suggests a role of P V H / D H A n e u r o n a l cell bodies in this type of behavlour 28 To test whether this effect was specifically mediated by the kamlc acid type of glutamate receptors, we decided to investigate the effects of other excitatory amino acids In this report we present the results of mtracerebral mlcrolnJectlons with N-methyl-D-aspartlc acid ( N M D A ) N M D A is an agomst of the N M D A type of glutamate receptor which is suggested to be involved m more long-lasting neuronal effects than the kamate type of glutamate receptor 2 Since the hypothalamic paraventricular area ~s involved in other behavlours as well, e g Ieedmg behaviour 9 17 20 ~1 and yawnlng/pende erection ~ 22 2~, we

Correspondence T A P Roehng Department of Anatomy and Embryology, Umverslty of Nqmegen P O Box 911)1 65110HB Nqmegen The Netherlands

221 dec~ded to register the c o m p l e t e b e h a w o u r a l p a t t e r n that w a s d i s p l a y e d T h i s e n a b l e d us t o c o m p a r e t h e r e s u l t s o f local i n J e c t i o n o f N M D A

n o t o n l y in a q u a n t t t a t t v e w a y

as f a r as tt c o n c e r n s g r o o m m g b e h a v l o u r , b u t also m a quahtattve way MATERIALS AND METHODS The experimental design that has been used was identical to that of earher experiments33 Six male albino rats (Wistar/CPB) weighing 400-500 g were used Under Hypnorm anaesthesia they were implanted bilaterally with 13 mm stainless-steel guide cannulae (Mmitubes, Grenoble, France, o d 0 4 mm, ] d 0 3 ram) aimed at the coordinates 7 40 mm anterior, 0 5 mm lateral and 3 90 mm dorsal to the mteraural midline (coordinates of Paxmos and Watson25), at an angle of 10° In this way the tip of the guide cannula was positioned at about 2 mm dorsal to the PVH A stainless-steel stylet was placed into the cannula to prevent it from clogging After surgery the animals were individually housed m home cages (50 x 50 x 50 cm) with free access to food and water, and handled for 1 week

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The experiments were performed between 13 00 and 17 30 h Animals were injected on the left side or right side with NMDA (Sigma, 0 2 nmol m 0 2 ,ul saline, pH adjusted to 7 with 0 1 NaOH) or sham injected in a randomized order with 48 h between each session The injection procedure has been described m earlier reports28 33 In short, an injection cannula (o d 0 28 ram, i d 0 18 mm), connected to a 1 /A Hamilton mlcrosyringe via waterfilled polyethylene tubing, was inserted, extending 2 mm beyond the tip of the guide cannula The NMDA solution was inJected within 30 s and after an addltmnal 30 s the injection cannula was quickly replaced by the stylet and the animal was placed back in ItS home cage A video camera connected via a video tlmeframe encoded decoder (part of a new data analysis system, PC-protocol, developed at the Ethopharmacology department and produced by IECProGamma, Gromngen, The Netherlands) to a video recorder, was placed m front of the glass front wall of the cage and video recordings of the rats' behavlour were made for a period of 30 min After completion of the testing the tapes were analyzed using PC-protocol For the present report, the total time spent on grooming behaviour is used for description of the effects of NMDA on this behaviour The following elements were considered to be part of grooming behaviour vibration of the forepaws, face washing, fur grooming, tail hcklng, hindpaw licking, anogenltal

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,'~,, / Fig 1 Injection sites plotted on the atlas of Geeraedts et al 6 7 Plates are indicated from rostral (A) to caudal (D) with intermediate distances of 150 gm The size of the black dot is an mdlcaUon of the amount of grooming behavlour that was elicited with the largest dots indicating the sites where the total amount of grooming exceeded 50% of the observation time InJection site with the largest effect (73% of observation time) is pointed out with an arrow Black dots with star indicate sites where feeding was also elicited (see text) A H A , anterior h ~ t h a l a m l c area, DHA, dorsal hypothalamlc area, fx, formx, ot, optic tract, PVH, paraventrlcular nucleus, sin, stria medullans

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C N G N Fig 2 Grooming behawour ehclted after NMDA rejection into the PVH/DHA area (N) as compared to control situation (C) A mean + S E M indicated as percentage of total observed t~me B individual responses as a percentage of total observed t~me n = 6 for control n = 12 for rejection sites

hckmg, scratching and shaking Other behavmural elements, that were observed and notated separately, were yawning, feeding/ drinking and resting/sleeping After completion of the senes of experiments, the ammals were deeply anaesthetized with ether and transcardmlly perfused with 4% paraformaldehyde and stored overmght m 0 1 M phosphate buffer/ 20% sucrose, pH 7 6 Frozen sections (40 #m) of the rejection sites were cut and counterstamed w~th Glemsa staining~° The mlect~on s=tes were plotted m a detaded cytoarch~tectonlc atlas~7 RESULTS The deepest point of the cannula tracts where some ghosts could be observed, was plotted on the atlas (Ftg 1) Four sites were located exactly wlthm the paraventrtcular nucleus Six tracts passed the p a r a v e n t n c u l a r nucleus and extended just below the ventrolateral border of the P V H and two sites were located m the dorsal part of the anterior hypothalamtc area, ventral to the dorsal hypothalamtc area The total a m o u n t of grooming was significantly mcreased in all cases (47 0 + 4 0%, control 18 9 + 4 8% of total observed ttme) with the highest response being 73% of the observation time spent on grooming (Wtlcoxon matched pairs rank test, P < 0 05) (Fig 2A,B) After N M D A rejection, the animals started to groom immediately after replacement into the home cage The a m o u n t of grooming behavlour as measured within 5-mln periods gradually dechned, but remained higher than control levels during the enttre observation Ume of 30 min (Fig 3) In the control situations, the ammals started some grooming after a short exploration of the cage and in most cases they slept d u n n g the last 5-10 mln of the observation time G r o o m i n g behavlour reduced by N M D A showed all elements of naturally occurrmg grooming behavlour (vibration of the forepaws, face washing, fur groommg, anogenltal grooming, tail licking, scratching and shaking) No excessive a m o u n t of any of these elements as compared to the a m o u n t of the other elements of grooming behaviour was seen

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F~g 3 Percentage of time spent on grooming behavlour as indicated for 5-mm penods after mlecuon Open bars are control values, filled bars are experimental values Mean values +_ S E M for n = 6 (controls) and n = 12 (experimental group) * and *** indicate slgmficancy from control for P < 0 05 and P < 0 001, respechvely (2-way ANOVA)

In most cases an mcrease in yawning was seen (frequency 8 9 +_ 1 5, control 1 5 + 0 7), frequently, but not always, accompamed by stretchmg However, a clear clustermg of rejection sites that gave the highest frequency m yawnmg responses within the total area that was covered by the mlectlon sites could not be nottced At 3 most medially sttuated Injection sites, a constderable a m o u n t of feeding behavtour was elicited, that consisted of more than 10% of the total observatton time (Fig 1) Thts behavlour consisted of the full repertoire of grasping food out of the storage box, using the upper and lower lnctsors, gnawing on the pellets, using the forepaws and chewing, alternated with drinking This behaviour was not seen unttl about 23 mln after the beginning of the observation ttme Since thts a m o u n t of feeding behaviour was never seen m the control sttuatton, we tend to consider tt as a late-appearing effect of the N M D A injection In these sites grooming behavtour was also greatly increased and preceded the feeding response (Fig 1) DISCUSSION The mlectlon sites as shown in Fig 1 are all m such close proximity to the area indicated m earher reports TM 28 33, that we consider the injection sites to be located within the 'grooming area' By Injecting N M D A into the P V H and adjacent D H A , we mduced an increase in total a m o u n t of grooming behavlour up to 73% of the total observation time of 30 mln (Fig 2B) In a previous report, we have shown that mtcromjecttons of kamlc acid ( K A ) evoked the same

223 response 28 T a k e n together, the results strongly suggest,

behavlour that can be mduced by injections of norad-

that n e u r o n a l cell bodies m this area are responsible for

r e n a h n e in this area 2° This effect was suggested to be

the observed behavloural effect and that a direct involvem e n t of glutamate-hke actw~ty seems evident The results described m the present report on groommg behavlour are comparable w~th the results of local

induced by inhibition of the s p o n t a n e o u s activity of PVH neurones, m e d m t e d through postsynaptlc a2-receptors 9' ~5,29 Feeding behavlour after N M D A injection was seen only after about 23 m m observation t~me m which the rats

rejections of other substances m this region C R F and A C T H - ( 1 - 2 4 ) are also capable of inducing groommg behavlour when rejected Into the P V H / D H A region ~6 33

groomed most of the u m e Intracellular currents evoked

However, there have been no indications for the presence of receptors of e,ther of these substances in the P V H / D H A region

Therefore the explanation of the

by N M D A stimulation have been reported to fade out m m wtro experiments 21 It ts very possible that such a decrease m act,wty was apparent in our experiments, although the time latency of the effects differed (150 s m vitro, 23 mln in VlVO) This decrease m local n e u r o n a l

locahzed effects of these substances upon groommg behav~our ~s difficult Since differences in the structure of grooming responses might implicate differences in neu-

achvtty may have been effectwe in starting the feeding response W h e t h e r or not these n e u r o n e s have been

ronal circuitry that ~s activated, a comparison of the characteristics of grooming behav~our ehclted by both N M D A and A C T H into the P V H / D H A area might be useful m this respect, as well as a closer study on the (co-)locahzatlon of both glutamaterglc and A C T H - t m m u n o r e a c t w e fibres w~thln th~s area

u n k n o w n The seemingly close connection between grooming and feeding is dlustrated by the observations

The ehcltatlon of yawning and penile erection has been reported to be m e d m t e d by oxytocmerglc n e u r o n s in the p a r a v e n t n c u l a r nucleus 122 W h e t h e r th~s response is orgamzed m the P V H i n d e p e n d e n t l y from grooming behavlour or must be regarded as part of P V H reduced g r o o m m g behavlour ~s as yet u n k n o w n Van Erp et al have already reported, that groommg wtth yawning e h o t e d only after A C T H injection mto the P V H and whale after mjecUon into the adjacent D H A region only g r o o m m g could be ehctted 33 P r o n o u n c e d feeding behavlour was observed only after mjectlons into the most medial sites m the P V H and with a long latency Feedmg b e h a w o u r is well known as a

involved m the

as well is still

that ~ c v injections of several substances increase grooming while decreasing feeding, e g C R F 1624, A C T H 36 and b o m b e s m 26 Local injections of b o m b e s m mto various brain areas, among which the PVH, showed the same effect 17 These opposite effects were found to be speofic and not merely a shift in time expenditure 36 Such a correlation could very well be explained by the involvement of at least partmlly the same n e u r o n a l substrate for both b e h a w o u r a l systems A t the level of the PVN th~s could be a single p o p u l a t i o n of neurones, the excitation of which results m grooming behavlour and the m h l b m o n of which results m feeding More conclusive ewdence ~s needed to settle this ~ssue

Acknowledgements This study was supported by the Foundation for Biological Research (BION), which is subsidized by the Netherlands Orgamzatlon for Soentlfic Research (NWO)

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