Noradrenergic influences on catalepsy

Psychopharmacology

Psychopharmacology 60, 53-57 (1978)

~ by Springer-Vertag 1978

Notadrenergic Influences on Catalepsy Stephen T. Mason*, David C. S. Roberts, and Hans C. Fibiger Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1W5, Canada

Abstract. Widespread depletion of forebrain noradre-

naline, produced by the intracerebral injection of 4 ~tg of 6-hydroxydopamine into the fibres of the dorsal noradrenergic bundle, potentiated the catalepsy induced by 20mg/kg of morphine and severely attenuated the catalepsy induced by two separate cholinergic agonists, arecoline and pilocarpine. It did not, however, affect haloperidol catalepsy at any of the four doses tested. These results suggest that cholinergic catalepsy m a y be critically dependent on an intact noradrenergic substrate, perhaps through cholinergic receptors located either presynaptically on noradrenergic terminals or on the cell bodies of origin in the locus coeruleus. Noradrenaline appears to play a modulatory role in morphine catalepsy, although other sites of action must also be involved. Ascending noradrenergic systems do not appear to influence haloperidol catalepsy. Key words: Noradrenaline - Catalepsy - Morphine - Haloperidol - Pilocarpine - Locus coeruleus -

Dorsal bundle - Arecoline

(Kuschinsky and Hornykiewicz, 1972). The neuroleptic drugs have been suggested to act through dopaminereceptor blockade in the striatum (Randrup and Munkvad, 1968), this may also be the site of the cholinergic agonists (Timsit, 1966) and perhaps morphine (Kuschinsky and Hornykiewicz, 1972). While these different classes of drugs have a similar site of action, however, there are also marked differences. Lesion to the caudate-putamen blocks haloperidol catalepsy, while potentiating the effect of arecoline (Costall and Olley, 1971) and not affecting that of morphine (Costall and Naylor, 1974a). Morphine catalepsy is, however, blocked by lesion of the amygdala (Costall and Naylor, 1974b) and of the raphe nuclei (Costall and Naylor, 1975). Pycock (1977) has recently implicated noradrenaline (NA) in haloperidol catalepsy with the finding that destruction of ascending and descending N A systems, either by electrolytic lesion to the rat locus coeruleus or by i.p. 6-hydroxydopamine (6-OHDA) given to rat neonates, potentiates haloperidol catalepsy. It was thus of interest to examine the involvement of noradrenaline in catalepsy induced by other classes of drugs.

Materials and Methods

Catalepsy, defined as a failure to correct externally imposed postures, can be elicited by a wide range of drugs. Dopamine-receptor blockers, such as haloperidol, are particularly potent cataleptic agents (Janssen et al., 1965), and centrally active cholinergic agents, such as pilocarpine, arecoline, tremorine, or nicotine, can also produce a related cataleptic state (Zetler, 1968). Other drugs, such as morphine, in which the neurochemical substrates of action are not so welldefined, are also effective in producing catalepsy * To whom requests for offprints should be sent

Subjects. Nine male albino Woodlyn rats weighing 300g were anaesthetised with Nembutal (50 mg/kg i.p.), and placed in a stereotaxic apparatus (Kopf). 6-Hydroxydopamine HBr (4 /3g, weight expressedas base) dissolvedin 2gl of 0.9 % saline with 0.3 rag/ ml ascorbic acid was injected bilaterally via a 34-gauge cannula into the fibres of the dorsal noradrenergic bundle in the mesencephalon. Injection rate was 31~tl/min,with the cannula left in foran additional minute to allow diffusion of the drug. Coordinates for the injection were AP + 2.6 mm from interaurai line, ML + i. 1mm from midline, and DV + 3.7 mm from interaural line. Ten control animals received injection of saline-ascorbate vehicle into the fibres of the dorsal bundle. All animals were maintained with food and water continuously available, and 2 weeks were allowed after the operation before testing began. It is known that no recoveryfollows 6-OHDA0033:3158/78/0060/0053/$

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Fig. 1. Left: catalepsy in response to 20 mg/kg morphine. Median latency to place one front paw on floor is plotted against postinjection time for 9 lesioned and 10 control animals. Stars: lesioned and control groups differ at that time point at the following levels of significance, * 5 % and 9* 1% (NS, not significant). Right: catalepsy in response to 100 mg/kg pilocarpine. Details as in left

induced lesions of the noradrenergic system, either biochemically (Uretsky and Iversen, 1969) or behaviorally (Mason and Iversen, 1975). This is further shown by the results of biochemical assay on the present animals (Table 1).

Apparatus and Procedure. Catalepsy was measured by placing the animal's two front paws on a horizontal bar positioned 7.5 cm above the floor and measuring the time in seconds before the animal placed one or both paws on the floor. This procedure was repeated at various times after administration of drug, with the animals kept in the home cage between measurements. In the event that the animal remained on the bar for more than 5 min, it was removed and a score of 300 s assigned to that reading. Biochemistry. Following completion of behavioural testing, all animals were allowed one drug-free week and then sacrificed by cervical fracture. The brain was removed and dissected on ice into the following regions: cortex-hippocampus, hypothalamus, striatum, spinal cord, and cerebellum, as previously described (Mason and Iversen, 1975). These areas were assayed for the concentrations of noradrenaline and dopamine by the method of McGeer and McGeer (1962). Drugs~ 6-Hydroxydopamine HBr (Regis) was dissolved in saline ascorbate, as described. Morphine sulphate (Smith, Kline & French), arecoline HBr (Nutritional Biochemicals Corporation), haloperidol (MeNeill Laboratories), and methyl scopolamine bromide (Upjohn) were dissolved in distilled water. Animals received i.p. injection of the cataleptic agents, separated by a drug-free period of 1 week. In the case of pilocarpine, the peripheral anticholinergic methyl scopolamine bromide (1 mg/kg) was injected 1/2h prior to pilocarpine in order to antagonize the peripheral cholinergic effects, which severely interfere with the measurement of catalepsy (Baez et al., 1976). Since arecoline is a shorter-acting, although equally potent, cholinergic agonist, previous authors have not found pretreatment with a peripheral cholinergic blocker to be necessary with arecoline (Costall and Olley, 1971).

Results The intensity o f catalepsy i n d u c e d by 20 m g / k g o f m o r p h i n e in lesioned a n d c o n t r o l animals, p r e s e n t e d in Fig. 1 (left), is m a r k e d l y elevated in the lesioned animals at all p o s t a d m i n i s t r a t i o n test times. In contrast, the cholinergic c a t a l e p s y i n d u c e d b y p i l o c a r p i n e (Fig. 1, right) a n d b y arecoline (Fig. 2) were a l m o s t c o m p l e t e l y b l o c k e d b y the d o r s a l - b u n d l e lesion. W i t h the exception o f the 5-rain p o s t a r e c o l i n e time point, at no o t h e r time p o i n t tested d i d the lesioned animals show significant c a t a l e p s y after d r u g injection. B o t h o f the cholinergic agents used, however, p r o d u c e d significant c a t a l e p s y in control animals. A t the first dose o f h a l o p e r i d o l used (0.2 mg/kg), no p o t e n t i a t i o n o f catalepsy was seen in the lesioned animals, d e s p i t e the findings o f P y c o c k (1977) to the c o n t r a r y . A full d o s e - r e s p o n s e curve o f these animals was therefore run run to c o n f i r m this negative finding. A t n o n e o f the four doses o f h a l o p e r i d o l tested was any difference between c o n t r o l a n d lesioned animals detected (Fig. 3). C o n s i d e r a b l e c a t a l e p s y was i n d u c e d by the higher doses o f h a l o p e r i d o l , b u t this did n o t differ between the two groups.

Assay. T h e results o f the b i o c h e m i c a l assay o f those a n i m a l s p a r t i c i p a t i n g in the catalepsy e x p e r i m e n t are shown in T a b l e 1. Values are those typically f o u n d in this l a b o r a t o r y using this f l u o r o m e t r i c assay procedure. Severe a n d p e r m a n e n t d e s t r u c t i o n o f cortical-

S. T. Mason et al. : Noradrenalineand Catalepsy

55 hippocampal NA occurred, and marked but less severe loss of hypothalamic NA. A significant elevation of cerebellar and spinal cord NA was found. Some slight reduction in DA was also seen in the striatum.

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Lesions of the ascending NA projections potentiated catalepsy induced by morphine, blocked catalepsy induced by cholinergic agonists, and left the response to haloperidol unaffected. The present report - t h a t a single treatment can yield different effects on haloperidol, morphine, and cholinergic catalepsy - is n o t without precedent (see introduction). For example, Costall and Olley (1971) found that haloperidol catalepsy was blocked by lesion to the caudate-putamen, while morphine catalepsy was unaffected and arecoline catalepsy was potentiated. Contrary to Pycock's recent report (1977), haloperidol catalepsy was manifestly unaffected by destruction to the ascending NA systems. Important differences are apparent between Pycock's findings and the regional amine depletions achieved by our techniques, in that our 6-OHDA injections spared the projections from the locus coeruleus (LC) to the cerebellum and spinal cord, as shown by postmortem assay (Table 1). Pycock, on the other hand, achieved marked NA depletions in these areas by using electrolytic locus

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Psychopharmacology60 (1978)

Table1. Regionalamine assaysin dorsal bundle lesionedanimals Substance

Region

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0.36 _+0.009 2.45 _+0.087 0.25 _+0.028 0.25 _+0.022 11.00 _+0.753

0.01 _+0.001 0.73 _+0.082 0.38 4- 0.034 0.30 _+0.024 8.71 _+0.360

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Dopamine

Values are means in microgramsof amine per gram wet weightof tissue of nine treated and eight control animals,with percentageof control amine concentrationremainingin lesionedtissues * 0.01 ** 0.001 coeruleus lesions. Both lesions destroyed the projections to cortex, hippocampus, and other telencephalic regions. We therefore suggest that the potentiation of haloperidol catalepsy reported by Pycock (1977) was due to destruction of the descending spinal or cerebellar projection, and not to ascending telencephalic projections. Morphine catalepsy was also increased in our lesioned animals. Injections of 6-OHDA into the ascending NA systems were recently shown to affect a number of morphine's actions. Price and FiNger (1975) showed that these lesions potentiate morphine analgesia. The ability of morphine to produce a conditioned taste aversion is greatly attenuated by these lesions (Roberts and Fibiger, 1977). We now add the finding that morphine catalepsy is markedly increased following these 6-OHDA injections. The pattern of NA depletion in various brain structures following this 6OHDA treatment suggests two possibilities for the observed effects: first, that depletion of forebrain NA is responsible for the increased catalepsy, or, second, that the small but significant increase in NA levels in the cerebellum and spinal cord produced this effect. A close relationship between NA and opiate agonists has been suggested by a wide variety of work. Pert and Snyder (1973) and subsequent researchers (Atweh and Kuhar, 1977) have demonstrated, by both binding studies and autoradiography, that there are high concentrations of opiate receptors in the LC. Intravenous injections of morphine selectively inhibit neuronal activity of neurons in the LC, and this effect is blocked or reversed by natoxone (Korf et al., 1974). Furthermore, microiontophoretic application of morphine or the synthetic agonist levorphanol (Bird and Kuhar, 1977), as well as the putative endogenous transmitter met-enkephalin (Young et al., 1977), inhibits the firing of LC cells. Montel et al. (1974) have demonstrated that superfusion with morphine of cerebellar or cortical slices can inhibit the release of noradrenaline produced by electrical stimulation. When taken together, these results suggest that morphine catalepsy may be associated with decreased firing of LC

units and with decreased release of NA from the terminals of these neurons. If the cataleptic action of morphine is normally associated with inhibition of NA release from the ascending projections of the LC neurons, then lesions of these projections might be expected to enhance the cataleptic action of morphine. Existing neurochemical data also permit a different interpretation of the present results. Several groups have reported that morphine can increase NA metabolites in rat brain and spinal cord (Shiomi and Takagi, 1974), increase the synthesis and turnover of NA (Bloom et al., 1976), decrease levels of NA in brain (Reis et al., 1969), and, in high concentrations, block the synaptosomal uptake of NA (Carmichael and Israel, 1973). If the cataleptic actions of morphine are in part mediated by increased synaptic concentrations of NA in the cerebellum and/or spinal cord, then the increased levels of NA found in these regions after dorsal bundle lesions (Table1) could enhance the effects of morphine. The blockage of cholinergic catalepsy suggests that cholinergic agents (arecoline and pilocarpine) are critically dependent on the presence of intact, ascending noradrenergic systems for their cataleptic action. Similar results have now been obtained on a large number of individual animals in this laboratory, many with no other drug history. This may come about as a result of cholinergic receptors on LC cells themselves, suggested by the finding that carbachol injected into the LC can induce a cataleptic state (Amatruda et al., 1975), and there may exist inputs from the putative cholinergic nucleus gigantocellularis (Papp and Bozsik, 1966; Pavlin, 1966) to the LC (Scheibel and Scheibel, 1973). On the other hand, there might be presynaptic cholinergic receptors on NA terminals, as found in the peripheral nervous system (for review see Westfall, 1977). This latter suggestion gains support from work of Reader et al. (1976), in which cholinergic agents were found to control the release of noradrenaline from superficially perfused cerebral cortex. Whatever the precise neuronal circuitry of catalepsy, the following conclusions can be drawn.

S. T. Mason et al. : Noradrenaline and Catalepsy

Noradrenaline, at least that contained in the ascending telencephalic and diencephalic projections, is not significantly involved in haloperidol catalepsy. Taken with the data of Pycock (1977), this focuses interest on the descending spinal or cerebellar projections for the noradrenergic modulation of haloperidol catalepsy. Noradrenaline in the CNS has at least a modulatory role in morphine catalepsy, although concomitant actions on other systems appear likely. Forebrain noradrenaline appears to be critically necessary for the catalepsy induced by the cholinergic agents arecoline and pilocarpine. An almost complete block of their cataleptic action is seen in the absence of forebrain NA. This raises the intriguing possibility that the well documented cholinergic-noradrenaline interaction seen in the peripheral nervous system may also apply to the CNS, and that a functional role in catalepsy can now be ascribed to it.

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57 Janssen~ P. A. J., Niemeegers, C. J. E., Schellekens, K. H. L. : Is it possible to predict the clinical effects of neuroleptic drugs (maj or tranquillizers) from animal data? I. Arzneim. Forsch. 15, 1 0 4 112 (1965) Korf, J., Bunney, B. S., Aghajanian, G. K. : Noradrenergic neurons : morphine inhibition of spontaneous activity. Eur. J. Pharmacol. 25, 165-169 (1974) Kuschinsky, K., Hornykiewicz, O.: Morphine catalepsy in the rat: relation to striatal dopamine metabolism. Eur. J. Pharmacol. 19, 119-122 (1972) Mason, S. T., Iversen, S. D. : Learning in the absence of forebrain noradrenaline. Nature 258, 422-424 (1975) McGeer, E. G., McGeer, P. L. : Catecholamine content of the spinal cord. Can. J. Biochem. 40, 1141-1151 (1962) MonteI, H., Starke, K., Weber, F.: Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices. Naunyn-Schmiedebergs Arch. Pharmacol. 283, 3 5 7 - 369 (1974) Papp, M., Bozsik, G. : Comparison of the cholinesterase activity in the reticular fomaation of the lower brain stem of cat and rabbit. J. Neurochem. 13, 697-703 (1966) Pavlin, R. : Cholinesterases in reticular nerve-cells. J. Neurochem. 12, 515-518 (1966) Pert, C. B., Snyder, S. H. : Opiate receptor: demonstration in nervous tissue. Science 179, 1011-1014 (1973) Price, M. T. C., Fibiger, H. C. : Ascending catecholamine systems and morphine analgesia. Brain Res. 99, 189-193 (1975) Pycock, C.: Noradrenergic involvement in dopamine-dependent stereotyped and cataleptic responses in the rat. Arch. Pharm. (Weinheim) 298, 1 5 - 2 2 (1977) Randrup, A., Munkvad, I. : Behavioural stereotypies induced by pharmacological agents. Pharmacopsychiatr. Neuropsychopharmakol. 1, 1 8 - 2 6 (1968) Reader, T. A., De Champlain, J., Jasper, H.: Catecholamines released from cerebral cortex in the cat: decrease during sensory stimulation. Brain Res. 111, 95-108 (1976) Reis, D. J., Rifkin, M., Corvelli, A. : Effects of morphine on cat brain norepinephrine in regions with daily monoamine rhythms. Eur. J. Pharmacol. 8, 149-152 (1969) Roberts, D. C. S., Fibiger, H. C. : Lesions of the dorsal noradrenergic projection attenuate morphine but not amphetamine-induced conditioned taste aversion. Psychopharmacology 55, 183-186 (1977) Scheibel, M. E., Scheibel, A. B. : Discussion in Brain Information Service Conference Report No. 32, Brain Information Service, Brain Research Institute, Los Angeles, 1 2 - 1 5 (1973) Shiomi, H., Takagi, H. : Morphine analgesia and the bulbospinal noradrenergic system: increase in the concentration of normetanephrine in the spinal cord of the rat caused by analgesics. Br. J. Pharmacol. 52, 519-526 (1974) Timsit, J. : Sur l'activite cataleptigene de quelque derives de la butyrophenone. Therapie 21, 1453-1464 (1966) Uretsky, N. J., Iversen, L. L.: Effects of 6-hydroxydopamine on noradrenaline-containing neurons in the rat brain. Nature 221, 557-559 (1969) Westfall, T. C. : Local regulation of adrenergic transmission. Physiol. Rev. 57, 659-728 (1977) Young, W. S., Bird, S. J., Kuhar, M. J. : Iontophoresis ofmethionineenkephalin in.the locus coeruleus area, Brain Res. 129,366- 370 (1977) Zetler, G.: Cataleptic state and hypothermia in mice, caused by central cholinergic stimulation and antagonized by anticholinergic and anti-depressant drugs. Int. J. Neuropharmacol. 7, 3 2 5 335 (1968)

Received January 19, 1978; Final Version April 26, 1978