Neuroscwnce Vol 43, No 1, pp 41~-9, 1991 Printed m Great Britain

0306-4522/91 $3 00 + 0 00 Pergamon Press plc (J 1991 IBRO

THE ROLE OF THE L A T E R O D O R S A L T E G M E N T A L N U C L E U S OF T H E RAT IN E X P E R I M E N T A L SEIZURES J W MILLER,* M. E BARDGETT and B C GRAY Departments of Neurology and Neurological Surgery (Neurology), Washington Umverslty School of Medicine, St Lores, MO63110, U S A Abstract--This study determined the effects of &screte mlcromjectlons of GABA agomsts m the chohnerglc nuclei of the pontomesencephahc tegmentum on spontaneous behavior and sozures reduced by intravenous pentylenetetrazol, blcuculhne or strychnine, m the rat Injections of both the GABA A agomst plperldlne-4-sulfomc acid and the GABA B agonlst (-)baclofen m the laterodorsal tegmental nucleus produced a dose-dependent suppression of behavioral arousal and a reduction m the threshold of myoclomc and clomc but not tomc seizures induced by blcuculhne and pentylenetetrazol There were no s~gnlficant effects on any type of strychnine seizure Injections in the surrounding bralnstem structures, including the pedunculopontlne tegmental nucleus, had httle effect on spontaneous behavior and did not significantly alter the thresholds of pentylenetetrazol-mduced seizures We have prewously demonstrated that rejections of GABA agomsts m the central medial mtralammar nucleus of the thalamus have similar effects on behavior and seizures Since the central medml nucleus receives important direct chollnerg~c projections from the laterodorsal tegmental nucleus, these two nuclei form a d~screte ascending system which regulates seizure threshold

It is well k n o w n that a relation exists between epilepsy and different states o f arousal. Epileptic seizures c o m m o n l y occur during sleep and interlctal eplleptlform spikes often occur preferentially during drowsIness and light sleep. 13This p h e n o m e n o n has been most studied experimentally with electrographic seizure discharges induced in cats by parenteral penicillin s This type o f activity is increased in states where the

mlcroinjectlons o f the selective G A B A A agonlst piperldlne-4-sulfonlc acid (PSA) and the selective G A B A B agonlst ( - ) b a c l o f e n (BF) on seizures induced by continuous, timed intravenous convulsant infusion o f the indirect G A B A g antagonist pentylenetetrazol (PTZ), the direct G A B A A antagonist bicuculhne, and the glyclne antagonist strychnine

electroencephalogram is synchronized, indicating depression o f arousal, whether occurring spontaneously or as a result o f various experimental manipulations 5.6.32 Until recently the precise n e u r o a n a t o m l -

EXPERIMENTAL PROCEDURES Under halothane anesthesia, holes were drilled m the skulls of female Sprague-Dawley rats (150-180 g, Sasco), and 0 5 inch lengths of 20-gauge stainless steel grade cannulae were stereotaxlcally placed w~th the t~ps just above the surface of the brain, 6 mm above the desired target m the bralnstem at coordinates prowded by the atlas of Paxmos and Watson 23 The cannulae were fixed in place w~th acryhc and occluded w~th wire The rats were allowed to recover and placed m individual cages with food and water The next day, jugular veto catheters were put m place, under halothane anesthesm, sutured under the skin, flushed w~th heparm and occluded w~th wire The following day, the rats were gently restrained for 2 3 mm while 0 5 ~1 Hamdton syringes were threaded through the guide cannulae for undateral injection of PSA, or BF dissolved m 50 nl of 2% alcmn blue normal sahne at pH 7 4 or control rejections of 50 nl of alcmn blue sahne After an incubation t~me of 2 5 mm for the PSA rejections or 10 mm for the BF rejections, the jugular catheter was flushed w~th heparm and connected to an infusion pump for continuous mfusmn of PTZ (5 0 mg/ml), blcuculhne (0 1 mg/ml at pH 5 0), or strychnme (0 lmg/ml), m normal sahne at a rate of 1 44 ml/mm The PTZ and blcuculhne infusions resulted mmally m a series of myoclonlc jerks which revolved the face, upper hmbs and sometimes the entire body This was followed by one or more clonlc seizure episodes ("facmlforehmb clonus''t consisting of clomc forehmb movement, with writhing trunk movements often with intermixed facml or body myoclomc movements This was eventually followed by a tomc seizure which consisted of stiffening of the

cal sites responsible for these effects were undefined, We have s h o w n that injections o f G A B A agonlsts into the central medial lntralaminar nucleus o f the thalamus depress arousal and facilitate a variety o f seizure types while similar injections in m a n y other s u r r o u n d i n g thalamlc structures do not. ~718.~9 The laterodorsal tegmental nucleus (LDTg) and the p e d u n c u l o p o n t l n e tegmental nucleus (PPTn) o f the p o n t o m e s e n c e p h a h c t e g m e n t u m consist o f c h o h n erglc neurons which gave nse to projections to the thalamus and other forebraln structures This ascendlng system has been implicated in arousal. 7.3°,34 The LDTg, in particular, is the source o f the chohnerglc afferents o f the central medial nucleus. 7 The purpose o f this study is to determine the role o f the L D T g and P P T n in seizures by studying the effects o f discrete *To whom correspondence should be addressed Abbrevtattons BF, (-)baclofen, LDTg, lateral dorsal tegmental nucleus, NADPH, fl-mcotmamlde adenine dmucleotlde phosphate, reduced form, PSA, plperldme4-sulfonlc acid, PPTn, pedunculopontlne tegmental nucleus, PTZ, pentylenetetrazol 41

J W MILLER et al

42

trunk with forehmh extension which m~ght or might not be accompanied by tonic hmdhmb extensmn, depending on the convulsant mfused The threshold for myoclomc seizures was defined as the dose of convulsant per kg, calculated from the ttme at wtuch the first myoclomc seizure occurred, with a correction for the dead space of the catheter The threshold for clomc and tonic seizures was determined m a similar fashion from the time to the onset of their first occurrence Clomc seizures often occurred as the initial event after strychnme infusion, and usually conststed of clomc limb movements, with a variable loss of posture, without intermixed facml movements or myoclonus Tonic seizures consisted of stiffening of the trunk with forehmb extension which might or might not be accompanied by tomc hmdhmb extension, depending on the convulsant infused, After the tonic seizure, the ammals were killed with 175mg/kg i.v sodium pentobarbltal. The brains were removed, frozen, and secttoned on a cryostat at 32#m Different sectmns were stamed with neutral red, a varmnt of the Koelle ~4 acetylchohnesterase stare, s and NADPHdlaphorase histochemlstry29 for localization of the mjectlons A variable degree of t~ssue damage, or small haemorrhages, were seen at some injection sttes (Fig 4). with no relationship to the agent injected or its concentration We attribute this variable tissue damage to the mechamcal effects of the somewhat raptd mject~on rate reqmred m these unanesthesized, restramed ammals

Matertals BF was a gift of Ctha-Geigy PSA, PTZ, blcuculhne, and strychnine were purchased from Sigma (St Louts, MO) RESULTS

Laterodorsal tegmental nucleus dose response studws on pentylenetetrazol seizures T h e effects o f a senes o f injections in the L D T g o f different a m o u n t s o f B F a n d P S A o n P T Z seizures were determined. Only results from animals in which histological e x a m i n a t i o n showed the center of the injection site to lie in the L D T g were included in d a t a analysis. Injections with leakage o f blue dye into the ventricles were excluded, a l t h o u g h we have previously s h o w n t h a t l n t r a v e n t r i c u l a r injection o f similar doses o f G A B A agonists have little effect o n behaxqor or seizures? 7 A n i m a l s where the d i a m e t e r o f tissue d a m a g e or h e m o r r h a g e exceeded 300 # m at its

greatest extent were also excluded from all data analysis Applying these criteria, there was no slgmficant &fference in P T Z seizure thresholds between animals receiving control reJections of alclan blue saline m the L D T g a n d animals receiving no brain rejections (Table 1), d e m o n s t r a t i n g that the mjectmn procedure itself does n o t alter seizure threshold InJections of five to 50 n m o l of PSA or of five to 30 nmol of B F resulted in identical, p r o f o u n d effects on the a m m a l s ' b e h a v i o r (Fig 1). This progressed from a mild unsteadiness a n d reduction of spont a n e o u s m o v e m e n t at lower doses to, at the highest doses, a state in which the a m m a l s would he motionless o n their sides with eyes closed a n d httle spontaneous m o v e m e n t o t h e r t h a n n o r m a l respiration. These effects were indistinguishable from those we have prewously observed with reJections of m a n y different direct a n d redirect G A B A agomsts into the central medial i n t r a l a m l n a r nucleus but not o t h e r thalamlc regions, 17-19 a n d were graded on a scale defined m the legend to Fig. 1. Since these effects reached a m a x i m u m by 2.5 m m after PSA mject~ons a n d 1 0 m l n after B F injection, seizure testing was begun at those t~mes These experiments d e m o n s t r a t e d a significant dosed e p e n d e n t reduction in the threshold of P T Z myoclonlc a n d clomc seizures (Fig. 1), after b o t h PSA (myoclomc, m a x i m u m effect, - - 4 7 % , clomc, - - 4 4 % ) a n d B F (myoclonlc, - 4 2 % ; clomc, - 3 7 % ) mjectlons in the L D T g T o n i c seizures were unaffected except for a slight ( - 1 6 % ) , statistically msigmficant reduction in threshold with very large mject~ons (50 nmol) o f BF

Defimtton of the site o f action F o r this purpose, the a n a t o m i c a l resolutmn o f the rejection technique was maximized by using mject m n s of 5 nmol o f B F or PSA, the lowest effective dose o f these substances (Fig. 2). A n i m a l s with injections centered in the L D T g were c o m p a r e d with the animals with injections m n e a r b y structures. W i t h PSA injections myoclonlc a n d clomc seizures

Table 1. Comparison of effects on pentylenetetrazol seizures of injections of 5 nmol of plpendine-4-sulfomc aod or (-)baclofen m the lateral dorsal tegmental nucleus vath rejections centered m nearby structures (Misses) and control injections of aleian blue saline Semure threshold (mg/kg) Clomc Tonic

Agent

Group

n

Myoclonic

None

No injection

13

24 3 + 2 9

28 9 + 3 5

73 0 + 8 7

Behavior score 00

Controls PSA, 5 nmol PSA, 5 nmol

LDTg LDTg Misses

13 23 20

22 3 + 1.6 15 7 _+ 1 l**t 19 8 _+ 1 4

27.6 + 2 0 20 3 __+1 4**t 26 2 _+ 1 9

73 5 + 5 4 74.5 _ 5 2 70 0 _ 5 0

00 2 2"**~ 0 8***

Controls BF, 5 nmol BF, 5 nmol

LDTg LDTg Misses

18 19 26

21.5 + 1 5 16.2 + 1 2* 18 3 + 1.4

26 6 + 1 9 21 9 _ 1.6 21 7 +__1 6

71 2 _+ 5 0 69 6 _ 5 0 69.6 + 4.9

00 2 0"**:~ 0 8***

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