Feeding following microinjection of cholinergic substances into substantia nigra

Life Sciences, Vol . 25, pp . 333-338 Printed in the U .S .A .

Pergamon Presa

FEEDING FOLLOWING MICROIN~ECTION OF CHOLINERGIC SUBSTANCES INTO SUBSTANTIA NIGRA P . Winn and P . Redgrave Department of Psychology, University of Hull, HULL, HU6 7RX, England Department of Psychology, University of Sheffield, SHEFFIELD, England (Received in final form June 7, 1979) Summary Microinfections of acetylcholine and eserine localised within the substantia nigra of the rat elicited a dose-dependant increase in feeding, but not drinking when both food and water were Freely available . When required to perform an operant response Por food, microinjections of carbachol into substantia nigra caused a dose-dependant increase in lever pressing for Food (FR5) . High doses of carbachol (1 .0 and 5 .0 elicited a behavioural stereotypy characterised by chewing, gnawing and biting . A significant negative correlation was Pound between the effectiveness of cholinergic stimulation and the distance from the site of highest feeding which was in the pars compacta region of substantia nigra . These data suggest a functional role Por acetylcholine within substantia nigra and provide indirect support for the concept of an interaction between cholinergic and dopaminergic neurons within this structure .

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The lateral hypothalamic area has been widely implicated in the control of feeding . Lesions within this region result in an aphagic syndrome (1) while electrical stimulation can induce feeding (2) . Localised injections of nor adrenaline into the lateral hypothalamus also cause feeding (3) while similar infections oP cholinomimetic drugs either increase (4) or suppress feeding (5) and potentiate drinking (6) . There is, however, evidence which suggests that several of the effects on feeding which have been attributed to hypothalamic mechanisms may resûlt from interference with fibres of passage . In particular, electrolytic or chemical lesions of the nigro-striatal dopamine (DA) system have been shown to result in an aphagic syndrome similar in many respects to that brought about by destruction of the lateral hypothalamus (7, 8) . Destruction of DA cell bodies by 6hydroxydopamine results in an aphagic syndrome which can be attenuated by systemic administration of the dopaminergic agonist apomorphine (9) . That DA is implicated in the mediation oP feeding behaviour is further suggested by experiments which show that dopaminergic antagonists suppress deprivation induced feeding and, to a greater extent, feeding elicited by infections of insulin or 2-deoxy-D-glucose (10, 11) . It is apparent though, that most of the evidence suggesting the involvement of DA in the regulation of feeding largely involves the demonstration of feeding deficits following the impairment of dopaminergic transmission . Attempts to show an increase in food intake following stimulation of DA systems are notably lacking (12, 13) while dopaminergic agonists are normally noted for their anorectic effects (14) . The purpose, therefore, of the present experiments 0024-3205/79/040333-06$02 .00/0 Copyright (c) 1979 Pergamon Presa Ltd

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Feeding Following Nigral Injections

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was to discover whether or not feeding behaviour could be elicited in satiated animals by stimulation of the nigro-striatal DA system at source . We now report that cholinergic stimulation of substantia nigra caused a dose-dependant increase in food intake in both free and operant feeding conditions . Experiment 1 :

Free Feedi~ Method

In each of nine male black-hooded rats (PVG/C strain), 230-270 g, bilateral stainless-steel guide cannulâe (23 gauge) were implanted giving access to the substantia nigra (AP -2 .4 to -4 .0 Lat . 0 .5 to 2 .0 Ho.-5 .5 ; . A further 6 animals were prepared with unilateral cannulae . After recovering from surgery the animals were trained to eat sticks of dry spaghetti from glass tubes, feeding behaviour being measured in centimetres of spaghetti consumed . At the beginning oP a test session animals were placed in individual boxes and allowed to settle for one hour, with food and water freely available . Animals were then injected with 0 .5 u 1 oP an artificial cerebrospinal fluid (pH adjusted to 7 .0) (15) containing equal quantities of acetylcholine (ACh) and eserine . Three different doses of ACh and eserine (2 .5, 5 .0 and 1U .0 ug of each) and a control injection of the vehicle alone were administered in an order which was individually randomised for each animal ; successive injections were separated by a minimum of 46 hours . On completion of all behavioural testing, the animals were killed by an overdose of barbiturate and perfused intracardially with a 0 .9% saline solution according to normal histological procedures (16) . Results Of the 24 sites at which injections were made, 19 were found to be effective . A representation of all sites at which injections were made is shown in Figure 1 . The mean amounts of food and water consumed by the animals in the 30 minutes following a microinjection of ACh and eserine are presented in Table 1 . The increase in feeding caused by cholinergic stimulation of the substantia nigra was found to be dose-dependant and statistically significant . TABLE 1 Mean amounts of food (cms spaghetti) and water (mls) consumed in the 30 minutes following microinjection of ACh and eserine, or a CSF (vehicle) control, into substantia nigra . (n = 19) r ygACh + Eserine~ Experimental conditions Free Feeding +

SE

Free Drinking +

(cms)

St

(mls)

10 .0

Control

2 .5

5 .0

11 .17

32 .55

45 .16*

22 .41

3 .92

8 .41

6 .34

6 .31

0 .21

0 .24

0 .16

0 .22

0 .09

0 .13

0 .06

0 .15

sig . difP . to control, p < 0 .01 sig . diff . to 5 .0 Ug dose, p < 0 .01 (H = 15 .4816, p < 0 .01 ; Kruskal-Wallis one way non-parametric analysis of variance (17) . Differences between doses and the control injection weretested using the Wilcoxon matched-pairs signed rank test (17 .) Apart from one animal,

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no significant drinking was observed . It is interesting to note that the one animal which both ate and drank Following cholinergic stimulation w as found to have the most anteriorly placed infection site . (a) A measure For each animal the value of two Factors was determined . oP the effectiveness of cholinergic stimulation was made by determining the difference between the amount of food consumed under each of the three drug conditions and the amount consumed Following the control injection : these values were then added and the mean taken for each animal . (b) A measure of the physical distance between the site of injection and the site from which most feeding was observed, which was in the pars compacts region of substantia nigra (see Figure 1), was taken for each animal . A statistically significant (p < 0 .025) negative correlation of -0 .501 between these two measures Por the group as a whole was obtained, indicating that the cholinergic Feeding response became weaker with increasing distance oP the infection site Prom the pars compacts region .

A P-1 "6

AP 2 " 8 AP-3" 0

A P2 " 0

AP~ß A P-2"2 A P-~4ß AP-2 "4

A P~4 " 4 A P-+48

AP-2 " 6

FIG . 1 Representative Frontal sections of the rat brain, modified from the stereotaxic atlas of Pellegrino and Cushman (30) indicating the placement of injection sites . The left side shows the infection sites Por animals in the free feeding condition, the right side those oP animals in the operant condition . " : effective site . O : ineffective site . * : represents the site of infection in the animal with the greatest overall food intake elicited by cholinergic stimulation in the Pree-feeding condition . (Abbreviations : arh, arcuate nucleus oP hypothalamus . cl, nucleus subthalamicus . dtd, decussation of Meynert . hp, Pasciculus retroPlexus . ip, interpeduncular nucleus . lm, medial lemniscus . mp, posterior mamillary nucleus . ot, optic tract . pc, cerebral peduncle . ph posterior nucleus oP hypothalamus . pvg, central grey substance . re, nucleus reuniens thalami . s n substantia nigra . vLn, nucleus ventralis tegmenti . zi, zona incerta .)

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Experiment 2 : - Op erant Fee d Method The fact that feeding but not drinking was induced by the microinjections of Experiment 1 suggests a degree of behavioural specificity is associated with cholinergic stimulation of substantia nigra . Additional behavioural character isation of cholinergically induced feeding was attempted by requiring animals to perform an operatt response to obtain food . lnto each of six male Wistar rats, bilateral cannulae (23 gauge) were implanted (AP -2 .5, Lat . 1 .5, Ho ., -5 .0) . After recovering from surgery the rats were trained to operate a lever in a standard conditioning apparatus (Campden Instruments) in order to receive Noyes food pellets on a fixed ratio scale â reinforcement . A second lever which, if pressed, provided no reinforcement was also present in the apparatus . Animals were placed in the experimental apparatus and allowed to settle, after which a 0 .5 ul microinjection of 0 .9% saline containing one of four doses (0 .1, 0 .5, 1 .0 and 5 .0 ul) of the cholinomimetic carbachol was made into the area of substantia nigra of each rat . Lever pressing was then monitored for 60 minutes . Every animal received each of the four doses oP carbachol and a vehicle control injection, in an order which was individually randomised ; successive injections were separated by a minimum of 24 hours . Results Uf the 18 sites at which injections were made, 13 were found to be effective ; a representation of sites from five of the six animals is shown in Figure 1. Table 2 shows the mean number of lever presses made on both the reinforced and non-reinforced levers following injection oP various doses of carbachol . TABLE 2 Mean number of lever presses on food reinforced (FR5) and non-reinforced levers in the 60 minutes following microinjections of carbachol or a saline (vehicle) (N = 13) control, into substantia nigra . yg Carbachol Experimental condition FR5 Reinforced lever +

SE

Non-Reinforced lever +

SE

0 .1

0 .5

4 .38

43 .15

91 .46*

43 .08* }

83 .08*

j .59

19 .13

21 .99

13 .66

21 .46

0 .38

0 .69

1 .54

9 .77

18 .15

0 .29

0 .50

3 .68

4 .97

Control

0 .24

1 .0

5 .0

* sig . diff . t o control, p < 0 .01 sig . diff . to 0 .5 yg dose, p<

0 .05

Cholinergic stimulation produced a statistically significant, (H = 21 .58 p< 0 .001, Kruskal-Wallis one way non-parametric analysis of variance (17)) dose-dependant increase in lever pressing which at low doses was accompanied by normal feeding . At the higher doses (1 .0 and 5 .0 ug) the animals were clearly stereotypic, exhibiting the non-specific biting and gnawing commonly associated with this phenomenon . Much of the stereotyped behaviour was directed towards the two levers present in the box giving a spurious increase in lever pressing at the highest dose . This in Pact reflects increased biting and gnawing of the levers

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with no attempt made by the animals to consume the pellets arriving in the food tray . Discussion The results presented here show that feeding behaviour in both free and operant conditions can be elicited in a dose-dependant fashion by cholinergic stimulation of substantia nigra . Within the range of doses used it is clear that normal Feeding was elicited only by lower doses of cholinergic stimulation . The high doses were probably less effective because of the emergence of stereotyped behaviour . These data suggest that the low level of cholinergic stimulation motivated animals to acquire food rather than simply perform non-specific chewing, gnawing and biting movements . However, additional experiments are in progress to further analyse the behavioural specificity oP cholinergic stimulation of substantia nigra . The present data provide behavioural evidence Por the concept oP cholinergic transmission within the substantia nigra . There are also several biochemical and anatomical findings which lend further support for the idea . oP cholinergic synapses within the substantia nigra . (a) The enzyme responsible for the synthesis of ACh, chdine~acetyltransferase, has been Pound both within the substantia nigra and the structures which profect to it (18, 19 ) . (b) ACh to both the pars reticulata and pars comItself has recently been localised pact~ regions of substantia nigra (20) . (c) The enzyme responsible for the breakdown of ACh, acetylcholinesterase, appears to be present within the DA containing cell bodies of the pars compacts (21) ; presumably this, in part at least, may serve to inactivate ACh released from afferent terminals (d) The administration of cholinergic agonists has been shown to increase both the frequency of firing and the Fluorescence intensity of nigral DA cells (22) . Finally, the data presented in the present paper confirm that direct application oP cholinergic substances to the substantia nigra may have a functional significance in behavioural terms . Though no direct evidence is presented, aspects of the present results would seem to suggest that cholinergic nerve terminals in substantia nigra may interact with the dopaminergic system originating in the pars compacts . The data presented here indicate that the sites Prom which most cholinergically induced feeding was observed were located in the pars compacts and that cholinergic stimulation became less effective with increasing distance from this region . This study also confirms previous reports (23, 24) that high doses of carbachol also elicit stereotyped behaviour when injected into substantia nigra ; dopaminergic systems have previously been implicated in the mediation of these effects (25 ) . The involvement oP DA systems in the control of food intake has recently been supported by several studies, Firstly, there are the experiments, referred to in the introduction, showing that either lesions oP DA systems or the blocking of dopaminergic neurotransmission inhibit feeding (see introduction) (7, 8, 9, 10, 11) . Secondly, it has been reported recently that systemic administration oP low doses of amphetamine to satiated animals causes small but reliable increases in food intake (26) . Thirdly, Kelly et al (27) report that local infections oP gamma aminobutyric acid (GAGA) into substantia nigra produced a significant suppression of feeding, while infections into the adjacent ventral . tegmental area were without effect . It is suggested that GAGA mediates an inhibitory input to substantia nigra from the caudate nucleus and globus pallidus (18, 29) . These data, together with the results of the present experiment indirectly suggest that mild potentiation of dopaminergic transmission can elicit feeding béhaviour in satiated rats, while higher levels of dopaminergic activation cause animals to exhibit the intense but behaviourally restricted repertoires characteristics of the dopaminergic stereotypies .

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