226 Neuroscience Letters, 115 (1990) 226-230 Elsevier Scientific Publishers Ireland Ltd.

NSL 07024

Catalepsy induced by carbachol microinjected into the pontine reticular formation of rats

Zeev Elazar and Milka Paz

Departmenl o/" Physiolo~zy and PharmacoloL,), Sackler School O~ Medicine, Tel- A viv Uni~ersily. Tel- A viv ( A'rae/

(Received 8 February 1990: Revised version received 26 March 1990: Accepted 6 April 1990)

Key wor&'. Pontine reticular forlnation: Carbachok ('alalepsy: Rat

Rals were implanted stereotaxically with permanent guide tubes aimed at the pontine reticular forma- lion. Carbachol was microinjected in the awake, freely moving rats, Catalepsy was evaluated with the hori- zontal bar test. Intense and long-lasting, dose-dependent catalepsy was observed following microinjections of 2 8 Itg of carbachol. Pretreatmenl with inlraperitoneally administered scopolamine significantly reduced the cataleptogenic effect of carbachol. These results show that the pontine reticular formation is part of the neuroanalomical substrate of catalepsy induced by cholinergic drugs,

The basal ganglia are considered to be the main anatomical substrate of catalepsy induced by neuroleptic drugs. Indeed, lesions of caudate-putamen and especially of globus pallidus were shown to reduce the catalepsy [6]. The main mechanism of neu- roleptic catalepsy was shown to depend on ascending dopaminergic systems and to be related to a blockade of dopamine receptors in the striatum and nucleus accum- bens [4, 5, 16]. The involvement of cholinergic mechanisms in the generation of cata- lepsy was demonstrated by the following findings: cholinergic drugs potentiate the effect of neuroleptic drugs and are by themselves cataleptogenic [4, 6, 7, 13, 19]; anti- cholinergic agents reduce the catalepsy induced by 6-hydroxydopamine destruction of dopaminergic neurons [18] or by neuroleptics [6, 10, 19]; destruction of the ascending dopaminergic pathway in rats caused a significant enhancement of the cataleptogenic effect of cholinergic drugs [4, 5]. The above data led to the concept of a dopaminergic - cholinergic balanced mechanism for the generation and control of catalepsy [5]. This mechanism is considered to be based on neuronal interactions between dopaminergic terminals and cholinergic interneurons intrinsic to the

striatum [! 4]. However, several reports do not easily concur with the concept of an exclusive

intrinsic striatal location of the cholinergic cataleptogenic influence. Lesions of the

Corre,wondence." Z. Elazar, Department of Physiology and Pharmacology, Sackler School of Medicine,

Tel-Aviv University, Ramat-Aviv. Tel-Aviv~ 69978, Israel.

227

caudate-putamen, of the globus pallidus and of the nucleus accumbens did not re- duce and even enhanced the catalepsy induced by cholinergic agents [4, 7, 8]. This suggests that cholinergic mechanisms in the basal ganglia and in the nucleus accum- bens are not necessary for the cataleptogenic effect of cholinergic drugs which could be produced in other brain areas. Such a possibility was indicated by the finding that acetylcholine (Ach) + eserine induced intense catalepsy when microinjected into the mesencephalic reticular formation in rats [11]. We also reported that microinjections of carbachol into the pontine reticular formation induced epileptiform seizures which were characterized by sequences of immobility and convulsions [9]. In this article we report results of microinjections into the pontine reticular formation of subseizure amounts of carbachol and their cataleptogenic effect.

Male Wistar and Charles - River rats (250 350 g) were chronically implanted under Equithesin (3 ml/kg, i.p.) anesthesia with guide tubes stereotaxically aimed at points two milimeters above the target in the pontine reticular formation [15]. The guide tubes were kept patent with stainless-steel stylets: At least one week was al- lowed for recovery from surgery and experiments were started when the animals ate, moved and looked normally. After surgery the rats were housed in individual cages in a light and temperature controlled room and were given food and water ad libi- tum. Microinjections were made unilaterally with an injection cannula (30 gauge) which protruded 2 mm from the end of the guide tube. The injection cannula was connected through a long (approx. 50 cm) polyethylene tube to a Hamiltone syringe. The animal was placed in a wooden box (25 × 25 × 40 cm) provided with a trans- parent observation wall and allowed to accustom to the box for approx. 30 min. Once the rat was quiet, the injection cannula was inserted carefully and gently and usually there was no need for handling the rat. Carbachol disolved in saline (0.5 1 pl) was injected slowly (2 min) by hand. The amount of carbachol is indicated as the free base. When injections were repeated in the same animal, an interval of one week was observed between the injections. Catalepsy was tested with a 7.5 cm high horizontal bar. The rat was gently placed with its forelimbs on the bar and the number of seconds until it descended with both limbs on the floor was counted. After conclusion of the injection experiments the animals were deeply anesthetised with nembutal and the brains were perfused through the heart with 10% formalin. The brain was uncov- ered by clipping the ventral skull bone and the head with the cannula in place was kept in formalin for at least two weeks. Forty pm slices were cut on a cryostat and stained with Neutral red. The injection point was calculated from the end of the guide tube trace. No damage to the tissue was observed at the injection site under light microscopic examination.

One or 2 rain after the end of the microinjection the rat raised, oriented the body towards the experimenter and stood immobile with the legs widely separated and the head slightly bent down. No peripheral cholinergic effects (lacrimation, salivation or piloerection) were usually seen. The animal was gently placed with both forelegs on the horizontal bar. At the end of the cataleptic state the animal first moved the head from side to side, then descended from the bar and started to slowly walk around. As can be seen in Fig. 1 B, with the amounts ofcarbachoi used, intense, con-

228

I00

C

o C 50 ilml

2 4 6 8

saline carbachol ( /J ,g )

C

'~ 6o t 5 0

~ ~0 i 20 i

o~ io 1 5 u 2 4 6 8

saline corbachol (/~g)

Fig. I. A: incidence of catalcpsy at 4 different doses of carbachol microinjected into the pontine reticular lormation in 10 rats. B: catalepsy duration at different doses of carbachol (2,ug, n= 6:4 fig, n= 7; 6 pg, n = 9; 8 ,ug, n = 9)

t inuous catalepsy lasted minutes and even tens o f minutes. A high incidence o f sus- tained catalepsy was obtained with amounts o f carbachol above 2 ¢zg (Fig. 1A). With

control saline injections (1 pl) the t ime on the bar was m a x i m u m 1 second. At the histological examinat ion the injection points at which carbachol induced catalepsy

(see Fig. 2) were found to be located in the oral pontine nucleus.

One group o f rats was used for a dose response study. Different amounts o f car- bachol (2, 4, 6, 8 pg) were injected in the pontine reticular formation at intervals o f

one week. Carbachol injections were given using a crossover balanced design (4 x

4 Latin square) to min imize carry-over effects between the different doses. Fig. 1B

shows prolongat ion o f the t ime on the horizontal bar with increasing amounts o f carbachol. Statistical analysis was done with the M a n n - W h i t n e y two- ta i l ed-U test.

All differences between pairs o f dose groups were found to be significant: 2 -4 /~g ,

P < 0.01; 4 - 6 p g , P < 0.01; 6 -8 ¢tg, P < 0.05.

-8.8 m m

Fig. 2. Microinjection sites at which carbachol induced catalepsy. Coordinates indicated are from bregma, drawings are taken from the Atlas of Paxinos and Watson [15].

229

Another group of rats (n = 7) was used to test the effect of the cholinergic muscari- nic antagonist scopolamine. Carbachol alone was injected first to establish a baseline. Two weeks later carbachol was injected 15 min after an injection of scopolamine (5 mg/kg, i.p.). Scopolamine greatly reduced the cataleptogenic effect of carbachol (Fig. 3) (Mann-Whitney two-tailed-U test: P < 0.02). A second injection of carbachol alone induced again strong catalepsy (difference between first and second carbachol alone injection was not significant). Both muscarinic and nicotinic drugs were reported to induce catalepsy when injected systemically in mice [20]. In rats systemi- cally given nicotine was cataleptogenic only in toxic doses [4]. In our experience, nico- tine microinjected into the pons had a very weak cataleptogenic effect, but we did not systematically investigate the cataleptogenity of nicotinic drugs in the pons.

Our data strongly indicate a pontine locus for the cataleptogenic effect of cholinergic drugs. The pontine reticular formation is the origin of powerful descending pathways to the spinal cord. It contains neural circuits for locomotion and for postural reflexes. It was shown that lesions of the nucleus reticularis tegmenti pontis in rats released compulsory locomotion and it was proposed that this nucleus is part of a neural mechanism for inhibition of locomotion [1-3]. Since lesions or local microinjections of GABA prevented akinesia induced by systemic administration of haloperidol, it was proposed that the neuroleptic akinesia may depend on the function of this nucle- us [3]. It is thus possible that the cataleptogenic mechanism in the pontine reticular formation demonstrated by our results constitutes a necessary descending link of the basal ganglia cataleptogenic mechanism. On the other hand, the pontine reticular formation sends ascending projections directly to structures of the basal ganglia. The pedunculopontine nucleus sends projections to caudate-putamen, globus pallidus and substantia nigra [12]. The influence of the pedunculopontine nucleus on the sub- stantia nigra is mainly excitatory [17]. The second possibility is thus that cholinergic activation of the pons induces catalepsy through the intermediary of ascending influences on the basal ganglia cataleptogenic mechanism. We are currently investi- gating these two possibilities.

C-

E v

5O C o

--~ 40 o D 50

~D

20 D_ q~

O 10.

0 o 0

m saline r7-21 ca rbacho l

ca rbacho l + scopo lam ine

Fig. 3. Effect of pretreatment with scopolamine on the cataleptogenic effect of carbachol in the pontine reticular formation. One ktl of saline was injected as control. Six ag of carbachol alone induced catalepsy. Two weeks later scopolamine (5 mg/kg, i.p.) was injected 15 min before carbachol. Scopolamine greatly reduced the cataleptogenic effect of carbachol. Two weeks after scopolamine carbachol was injected again.

230

I Brudzynski, S.M. and Mogenson, G.J., The role of the nucleus reticularis pontis in locomotion: a lesion study in the rat, Brain Res. Bull., 12 (1984) 513 520.

2 Cheng, J.T., Schallert, T., De Ryck. M. and Teitelbaum, P., Galoping induced by pontine tegmentum damage in rats: a form of 'parkinsonian festination' not blocked by haloperidol, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 3279 3283.

3 Chesire, R.M., Cheng, J.T. and Teitelbaum, P., The inhibition of movement by morphine or haloperi- dol depends on an intact nucleus reticularis tegmenti pontis, Physiol. Behav., 30 (19831 809 818.

4 Costall, B. and Naylor, R.J., Neuroleptic and non-neuroleptic catalepsy, Arzneim. Forsch., 23 (1973) 674 683.

5 Costall, B. and Naylor, R.J., The importance of the ascending dopaminergic systems to the extrapyra- midal and mesolimbic brain areas for the cataleptic action of the neuroleptic and cholinergic agents, Neuropharmacology, 13 (1974) 353 364.

6 Costall, B. and Naylor, R.J., On catalepsy and catatonia and the predictability of the catalepsy test for neuroleptic activity, Psychopharmacologia, 34 (1974) 233 241.

7 Costall, B.. Naylor, R.J. and Olley, J.E., Catalepsy and circling behavior after intracerebral injections of neuroleptic, cholinergic and anticholinergic agents into the caudate-putamen, globus pallidus and substantia nigra of the rat brain, Neuropharmacology, 1 l (19721 645 663. Costall, B. and Olley, J.E., Cholinergic and neuroleptic induced catalepsy: modification by lesions in the caudate-putamen, Neuropharmacology, 10 (1971) 297 306.

9 Elazar, Z. and Feldman, Z., Brainstem experimental seizures produced by microinjections of carba- chol, Epilepsia, 28 (19871 463 47(I.

l0 Erzin-Waters, C., Muller, P. and Seeman, P., Catalepsy induced by morphine or haloperidol: effects of apomorphine and anticholinergic drugs, Can. J. Physiol. Pharmacol., 54 (1976) 516 519.

11 Hartgraves, S.L. and Kelly, P.H., Role of mesencephalic reticular formation in cholinergic induced catalepsy and anticholinergic reversal of neuroleptic induced catalepsy, Brain Res., 307 (1984) 47-54.

12 Jones, B.E. and Yang, T.Z., The efferent projections from the reticular formation and the locus coeru- leus studied by anterograde and retrograde axonal transport in the rat, J. Comp. Neurol., 242 (1985)

56 92. 13 Klemm, W.R., Evidence for a cholinergic role in haloperidol induced catalepsy, Psychopharmacology,

85 (1985) 139- 142. 14 McGeer, E.G., McGeer, P.L., Grewnal, D.S. and Singh, V.K., Cholinergic interneurons and their rela-

tion to dopaminergic nerve endings, J. Pharmacol., 2 (19751 t43 152. 15 Paxinos, G. and Watson. C.. The rat brain in stereotaxic coordinates, Academic Press, New York,

1982. 16 Sanberg, P.R., Haloperidol-induced catalepsy is mediated by postsynaptic dopamine receptors, Na-

lure, 284 (1980) 472 473. 17 Scarnati, E., Proia, A.. Di Loreto. S. and Pacitti, C., The reciprocal electrophysiological influence

between the nucleus pedunculopontinus and the substantia nigra in normal and decorticated rats,

Brain Res., 423 ( 19871 116 124. 18 Schallert, T., De Rick, M., Wishaw, I.Q., Ramirez, V.D. and Teitelbaum, P., Excessive bracing reac-

tions and their control by atropine and L-DOPA in an animal analog of parkinsonism, Exp. Neurol.,

64(19791 33 43. 19 Zetler, G., Cataleptic state and hypothermia in mice, caused by central cholinergic stimulation and

antagonized by anticholinergic and antidepressant drugs, Int. J. Neuropharmacol., 7 (1968) 325 335. 20 Zetler, G., Pharmacological differentiation of 'nicotinic" and "muscarinic' catalepsy, Neuropharmaco-

logy, 10 (1971) 289-296.