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Changes of acetylcholinesterase activity in three major brain areas and related changes in behaviour following acute treatment with diisopropyl fluorophosphate
Nruropharmncology,
1976, 15, 291-298.
Pergamon
Press.
Punted
m Gt. Britain
CHANGES OF ACETYLCHOLINESTERASE ACTIVITY IN THREE MAJOR BRAIN AREAS AND RELATED CHANGES IN BEHAVIOUR FOLLOWING ACUTE TREATMENT WITH DIISOPROPYL FLUOROPHOSPHATE” M. D. KOZARP, D. H. OVERSTREET$,T. C. CHIPPENDAL$ and R. W. RUSSELLS Department of Psychobiology, University of California, Irvine, California 92664, U.S.A. (Accepted
23 September
1975)
Summary-In a series of experiments, the recovery of acetylcholinesterase activity in three brain regions following its depression by diisopropyl fluorophosphate was examined. Related changes in behaviour were also studied. The maximum depression in acetylcholinesterase activity in the anterior preoptic area occurred at 4 hr, about the same time as the maximum depression of deep body temperature was recorded. A significant recovery of acetylcholinesterase activity was observed at 16 hr, while temperature regulation returned to normal by 20 hr. Maximum depression of drinking behaviour and acetylcholinesterase activity in the lateral hypothalamus both occurred at approx. 1 hr following the acute diisopropyl fluorophosphate injections and both showed significant but not complete recovery by 24 hr. Single alternation performance had not completely recovered even at the final test session, i.e. 71 hr following the diisdpropyl fluorophosphate injection, at which time acetylcholinesterase activity in the caudate nucleus had reached approximately 23% of normal. These findings generally support the hypothesis that recovery of certain behaviours may be dependent upon the renewed synthesis of aeetylcholinesterase activity in brain regions which are important in mediating these behavionrs.
Previous studies from our laboratory have reported that many forms of behaviour are significantly altered during periods up to 24 hr after a single, 1.0 mg/kg injection of diisopropyl fluorophosphate (DFP), an irreversible anticholinesterase agent (RUSSELL, WARBURTONand SEGAL, 1969; RUSSELL, VASQUEZ, OVERSTREETand DALGLISH 1971a, b; OVERSTREET,KOZAR and LYNCH, 1973; OVERSTREET,RUSSELL, VASQUEZ and DALGLISH, 1974). At this time, the level of whole brain acetylcholinesterase (AChE) activity was found to be reduced to about 30% of normal (GLOW, ROSE and RICHARDSON,1966; RUSSELL,WATSONand FRANKENHAEUSER,1961; RUSSELL et al., 1969; RUSSELL, OVERSTREET,COTMAN, CARSON, CHURCHILL, DALGLISH and VASQUEZ, 1975) and the acetylcholine (ACh) content elevated to 140”/, of normal (RUSSELL et al., 1975). Recovery of behavioural measures to normal baseline levels occurred in most cases within 48 hr of the acute injection. The present investigations were
designed to examine the hypothesis that suph recovery may be dependent upon renewed synthesis of AChE in specific anatomical areas which appear to serve as substrates for these behaviours. A significant literature on the behavioural aspects of cholinergic transmission has accumulated over the past several years (CARLTON, 1968; KARCZMAR, 1970; RUSSELL, 1966, 1969). Much of the evidence for the cholinergic involvement has been established through the use of anticholinesterase agents as “tools” to vary the level of activity of the enzyme which plays an essential role in the transmission process. Among those behaviours for which cholinergic mediation has been implicated are: temperature regulation (FRIEDMANand JAFFE, 1969; LOMAX, 1970; MEETER, 1971a, b; MEETER and WOLTHUIS, 1968; OVERSTREET et al., 1973); drinking behaviour (GROSSMAN,1962; MILLER, 1965; RUSSELL, 1966); and response inhibition (CARLTON, 1963,1968; WARBURTON,1968,1969,1972; WARBURTONand RUSSELL, 1969). Other investigations suggest that the cholinergic systems mediating these behaviours may be localized in certain brain regions: drinking in the lateral hypothalamus (MILLER, GOTTESMANand EMERY, 1964); temperature regulation in the anterior preoptic area of the hypothalamus (CRAWSHAW, 1973; LOMAXand JENDEN, 1966; Lows, FOSTER and KIRKPATRICK, 1969); and, response inhibition in the caudate nucleus (DEADWYLER,MONTGOMERYand WYERS, 1972; HULL, BUCHWALDand LING, 1967; NEILL and GROSSMAN,1970) and hippocampus (GREENEand LOMAX, 1970; WARBURTON, 1969; WARBURTONand RUSSELL, 1969).
*This research was supported by Grant MH 18788 to Roger W. Russell and was based in part on a thesis submitted by the first author as partial fulfilment of the requirements for the Masters Degree in the Department of Pharmacology and Experimental Therapeutics, University of California, Irvine. t Present address: George Washington University Medical School, Washington, D.C. $ Reprint requests should be addressed to Professor R. W. Russell or to Dr. D. H. Overstreet, School of Biological Sciences, The Flinders University of South Australia, Bedford Park, South Australia 5042, Australia. 4 Present address: Department of Psychology, Princeton University, Princeton, New Jersey. 291
292
M. D. KOZAR,D. H. OVERSTREET, T. C. CHIPPENDALE and R. W. RUSSELL
To explore such relations between the choline+ system and behaviour in more detail two types of studies were carried out. First, the recovery of AChE activity in the lateral hypothalamus, the anterior preoptic area, and the caudate nucleus was determined by histochemical and neurochemical techniques following an acute injection of 1.0 mg/kg DFP. Second, this biochemical recovery was then compared with’behavioural recovery observed in other studies carried out under similar experimental conditions. METHODS Subjects A total of 147 male Sprague-Dawley (Simonsen) rats, approx 100 days old (35@450 g) at the beginning of each experiment, were used as subjects. The animals were housed in stainless steel cages under conditions of constant light and ambient temperature. Food and water were allowed ad lib. for rats used in the histochemical and biochemical studies. Any special conditions which applied to the various IXhavioural studies are included in descriptions of specific experiments. Experiment I-Histochemical
analyses
Twenty animals were given a single intramuscular injection of DFP (1.0 mg/kg) in the gastrocnemius muscle. The rats were sacrificed in pairs after 1, 4, 8, 16, 24, 48, 72, 168 or 720 hr postinjection. Two animals received only the arachis oil vehicle and served as controls. Brains were sectioned and stained for AChE activity by LANDMESSER’S (1969) modification of the Koelle thiocholine stain. Promethazine (5 x lo-‘M) was added to inhibit non-specific cholinesterases and the staining reaction was uniformly stopped by the addition of 10e4 M neostigmine. Experiment 2-Biochemical
analyses
Eighty rats were given a single intramuscular injection of DFP (1.0 mg/kg) in the gastrocnemius muscle. The animals were sacrificed in groups of six at either 1, 4, 16, 24, 48, 96 or 360 hr after the injection. Eight rats were sacrificed at 168 and 720 hr and four at 1440 hr after DFP treatment. Eighteen control animals received the arachis oil vehicle only and were sacrificed 1 hr after the injection. The rats were anaesthetized with pentobarbital and sacrificed by cardiac perfusion with approx 250 ml of physiological saline to eliminate serum cholinesterases. The brains were immediately removed, frozen and uniformly blocked in the coronal plane. Upon mounting on a freezing microtome, coronal slabs of tissue were taken at two levels: the caudate nucleus and the preoptic hypothalamus (for both, slab thickness = 1000 m); and the lateral hypothalamus (1800 m). Using fine glass tubing (0.7 mm i.d.), bilateral tissue samples were taken from the caudate nucleus (AP 8.2-7.2, ML 2.0, DV 2.5), the anterior preoptic area of the hypothalamus (AP 8.2-7.2, ML 1.5, DV
-2.O), and the lateral hypothalamus (AP 6.446, ML 2.0, DV -3.0) (PELLEGRINOand CUSHMAN,1967, coordinates of DeGroot). Acetylcholinesterase activity was determined using the method described by ELLMAN,COURTNEY, ANDRES and FEATHERXNE(1961) and proteins, by the procedure of LOWRY.ROSEBROUGH, FARR and RANDALL (1951). Although the former method does not distinguish between AChE (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase cacycholine hydrolase, EC 3.1.1.8), it has been shown that the latter enzyme contributes a very small portion of the total enzyme activity when acetylthiocholine is used as substrate (HOBBIGERand LANCASTER,1971). Experimental specific activities were expressed as percentages of control activity for each particular assay session. Mean control values were expressed as ~01 of acetylthiocholine hydrolyzed per min per mg protein. Analyses of variance and t-tests were used to determine the significance of the results, which were considered significant if the difference exceeded the 0.05 level of confidence. Experiment 3-Drinking
behaviour
This study involved observations of 10 animals. They were adapted to a 22-hr water deprivation schedule and obtained their 2-hr water ration in experimental drinking chambers described by RUSSELLet al. (1971a). Following the development of stable baselines of water intake, five rats received a 1.0 mg/kg injection of DFP 1.5 hr before their next drinking session, while the five control animals received a 1.0 mg/kg injection of arachis oil. Statistical analyses were performed by means of the Mann-Whitney U test (SIEGEL,1956). Experiment #-Alternation
behaviour
The animals in this study consisted of 16 rats trained on a discrete trial single alternation task (HEISE, KELLER, KHAVARI and LAUGHLIN, 1969; OVERSTREETet al., 1974) in standard operant chambers (RUSSELLet al., 1971b). After the establishment of the baseline criterion of 90% correct alternations for three consecutive days, the eight experimental animals received a 1.0 mg/kg dose of DFP, while the eight controls received an e@ivalent dose of arathis oil. These injections occurred immediately after the last baseline session. or approximately 23 hr prior to the next behavioural session. To further examine the time course of behavioural recovery in this task, three additional animals were injected with 1.0 mg/kg DFP 0.5 hr before the behavioural session and an additional four rats received the DFP treatment 12 hr before the behavioural session. Experiment 5-Deep
body temperature
The rectal core temperatures of 11 rats were recorded on a model 2133 Rustrak continuous ternperature recorder. The animals were connected to the recorder via a YSI thermistor probe model T26Qf) in-
Cholinesterase activity and behaviour after DFP
serted 7cm into the rectum and taped to the tail loosely enough to permit normal circulation. The animals were placed in well-ventilated plexiglass restraining cages located in sound-attenuated boxes (OVERSTREET t?t d., 1973). After a 2-hr period for measuring baseline temperatures, the eight experimental rats were removed from the cages and injected intramuscularly with 1.0 mg/kg DFP. The three control rats received a 1.0 mg/l
Hour
293
of the Friedman two-way analysis of variance test (SIEGEL,1956). The Mann-Whitney U test was used to determine significance of the differences between experimental and control groups. The results were considered significant if the differences exceeded the 0.05 level of confidence. RESULTS
Experiment 1 Acetylcholinesterase staining activity at various times after the acute DFP treatment is illustrated in Figure 1. The staining reaction was extremely light
I
24
72
720
A0 Control
Fig. 1. Selected DFP-treated and control brain sections indicating the general appearance of tissue sections after thiocholine staining procedure. Enzyme activity is indicated by dark areas, density of stain being approximately proportional to activity. AO: arachis oil.
294
and R. W. RUSSELL M. D. KOZAR,D. H. OVERSTREET, ‘I C. CHIPPENDALE
LH
APO
Caudate
Fig. 2. Brain sections from two different animals with tissues samples removed. The figure illustrates the replicability of this tissue removal technique. APO: anterior preoptic hypothalamus; LH: lateral hypothalamus; NC: caudate nucleus.
at 1 hr postinjection, indicating a very significant depression of AChE activity. Recovery of enzyme activity is revealed in the gradually increasing density of staining which approaches control levels at 720 hr after the injection. Experiment 2
The technique employed for removal of the tissues allowed for very uniform and reproducible sampling,
as illustrated in Figure 2, which shows brain sections from two different rats after removal of the tissue samples. The samples were approximately 2/3 of the diameter of the hole seen in Figure 2, due to the difference between inside and outside diameters of the sterile glass tubing used to extract them. The AChF activity levels of the three brain regions at various times after the acute DFP injection are summarized in Table 1. Analyses of variance estab-
Table 1. Time course of recovery of acetylcholinesterase activity in three brain regions following acute treatment with diisopropyl fluorophosphate hr following DFP treatment
n
1 4 16 24 48 86 168 360 720 1440
6 6 6 6 6 6 8 6 8 4
Percentage of control AChE activity* (mean f S.E.M.) APO LH NC 305 + 23.1 k 33.3 * 32.7 f 30.8 f 53.5 + 60.9 f 73.7 + 79.6 f 81.6 f
4.0 2.9 2.77 4.0 1.3 4.0 4.2 6.3 5.0 2.4
28.4 k 2.1 29.4 + 2.4 435 + 48t 37.5 + 2.2 44.2 * 5.9 51.9 * 1.5 55.7 f 25 75.1 + 5.0 74.3 _+ 3.6 75.9 + 15
17.0 + 2.2 12.1 f 1.4 13.3 k 0.8 14.4 &-0.8 20.0 k O.St 28.0 f. 1.2 34.7 + 3.1 56.2 + 4.2 69.1 f 4.0 81.3 + 5.2
* The control values for each area, expressed as pm01 acetylthiocholine hydrolyzed/min per kg protein were 47.4, 33.1 and 17&8 for the anterior preoptic area (APO), lateral hypothalamus (LH) and caudate nucleus (NC), respectively. t Significant recovery (P < 0.05) from the time of maximum depression.
295
Cholinesterase activity and behaviour after DFP Table 2. Water intake of control and DFP-treated acute injection hr following DFP treatment
Water DFP
Intake (mean) Control
Baseline 1.5 25.5 49.5
21.3 3.1 15.5 24.4
19.6 22.5 19.8 21.3
rats at various times following an
‘A Baseline (median) Control DFP 1.5 79.5 114.2
U 10 0* 0* 12
108.4 100.0 112.7
* Significant difference, P < 0.05, according to the Mann-Whitney U statistic, computed on absolute water intake for baselines and on y0 baselines for the other treatments.
lished the occurrence of significant (all P < 0.001) treatment effects for each area (F = 29.70, 24.62 and 73.54 for anterior preoptic area, lateral hypothalamus, and caudate nucleus, respectively). Paired comparisons between the points of maximum AChE depression and the other time points by means of t-tests revealed that a significant recovery of AChE activity had occurred by 16, 16 and 48 hr, respectively, for these areas. Despite the significant recovery exhibited in all areas, return to normal AChE activity had not occurred in any tissue by 1440 hr. Another interesting feature in Table 1 is the inverse relationship between the pretreatment baseline AChE activity of a given area and the percentage depression of the enzyme following acute administration of DFP; e.g. the maximal depression of AChE occurred in the caudate nucleus, which had the highest control specific activity. Experiment 3 The effects of acute DFP treatment on water intake are summarized in Table 2iThere was no significant difference between the initial baselines of the two groups. The DFP-treated rats drank significantly less than the controls at both 1.5 and 25.5 hr following the acute treatment, but they drank substantially more at 25.5 than they had at 1.5 hr. Both groups exhibited suprabaseline drinking at 49.5 hr following the acute injection, but there was no statistically significant difference between the percentage baseline scores for the two groups. Experiment 4
The effects of DFP and arachis oil on three measures of the animals’ alternation performance are summarized in Table 3. There was a differential recovery of the three parameters measured following
acute DFP treatment. Although all variables were significantly affected at 23 hr following the injections, operant responding (S+) had recovered by 47 hr, while the two measures of response suppression were still impaired at 71 hr. Data for the other groups of animals treated with DFP indicated that, in fact, S+ responding had returned to normal by 36 hr. Experiment 5
The effects of DFP on deep-body temperature in the rat are summarized in Figure 3. The injection procedure produced a transient rise in temperature at the first hr. Onset of drug effects occurred after 2 hr and maximum depression of 2.65”C, 6 hr after injection. Thermoregulation gradually recovered to approach normal after approximately 24 hr. Evaluation of the data by the Friedman two-way analysis of variance test showed a significant (P < @Ol) trend in the DFP-treated rats, while no significant change (P > 0.05) with time was observed for the control animals. Differences between DFPtreated and control groups were tested by the MannWhitney U test, significant differences being noted from 2 hr consecutively to 19 hr postinjection. During hr 1 and hr 2@24 the differences were not significant. DISCUSSION
Critical enzyme JeueJs
Earlier work on the behaviourai effects of anticholinesterase agents suggested that various cholinergitally mediated behaviours would be disrupted only if the AChE activity in brain fell below some critical level, i.e. 40”/, of normal (Gmw and ROSE,1965; RUSSELL, 1964, 1966; RU~~ELLet al., 1961). Further evidence suggesting 40”/0of the normal enzyme activity
Table 3. Effects of acute DFP and arachis oil (AO) treatment on three parameters of alternation
hr following DFP treatment Baseline 23 47 71
S+ Responses (median) DFP (n) A0 (n) 191(8) 116 (8)* 172 (8) 177 (8)
196 (8) 196 (8)* 198 (8) 202 (8)
Number of animals given in brackets. * Significantly different, according to Mann-Whitney
S- Responses (median) DFP (n) A0 (n) 14 (8) 31(8)* 21(8) 20 (8)*
17 (8) 10 (8)* 16(8) 9 (8)*
behaviour in rats
% Correct alternations (median) DFP (n) AD (n) 95.8 (8) 88.9 (8)* 90.8 (8)* 93.2 (8)*
U Test, based upon the ‘A baseline scores.
94.9 (8) 94.9 (8)* 95.7 (8)* 96.3 (8)*
296
M. D. KOZAK, D. H. OVERSTIGET, T. C. CHIPPENDALE and R. W.
RUSSELL
Fig. 3. The effects of DFP (l.Om~g, ~tramuscularly) on rectal core tem~rature in the rat. Values are mean “C deviations from a 2-hr pre-injection baseline. Number of animals in experimental group = 8. Number of animals in control group = 3. as the minimum level capable of sustaining normal functions has appeared in studies of axonal conduction (WILSON and C&m, 1953), of the central respiratory reflex (Mmz, 1958) and of enzyme-substrate relationships (APRISON,1961). The results obtained in the present study are in general agreement with the above suggestions. Acetylcholinesterase activity in the various brain regions was well below 40”/, of normal when the relevant behaviours were depressed. ~ioc~em~cul recovery
2&48 hr after an acute injection of DFP is consistent with earlier reports on the recovery of these functions (MUIR and WOLTHUIS,1968; RUSSELLet al., 1969, 1971a). The two measures of the animal’s ability to suppress responding (S- and y0 correct alternations) had not recovered within 71 hr after the acute injection of DFP. Further studies must be carried out to determine the time course for recovery of this behaviour. Studies with the smaller groups of animals indicated no return to baseline at 84 hr folIowing DFP treatment, at which time the study was terminated. These findings, taken together with other reports which indicate an impaired ability of animals to suppress responding following chronic treatment with anticholinesterase agents (BANKSand RUSSELL,1967; O~ERSTREET et at., 1974; RUSSELLet at., 1961), suggest that this type of behaviour may be strongly dependent upon the integrity of the cholinergic system.
Acetylcholinesterase activity recovered_gradually in all tissues examined. However, control levels had not been fully reached even after 60 days. This finding confirms the results of other workers (BLABERand CREASEY,1960; GLOW et al., 1966; KOELLEand GELMAN, 1946) indicating a slow rate of synthesis for AChE. Of special interest was the discovery that the pattern of recovery of AChE differed in the three regions examined. One possible explanation of this Biochemical and behavtiural comparisons finding is that the areas contain isoenzymes of AChE The notion that certain behaviours may be depenwhich differ in their rates of synthesis, as has been demonstrated in the retina (DAVIS and AGRANOFF, dent upon the integrity of particul~ anatomical areas 1968). An alternative explanation arises from two has been challenged in recent years by a number of investigators. For example, a water-satiated rat will facts: first, that the areas differ in the relative amounts of nerve cell bodies and axon terminals, i.e. the drink when cholinergically stimulated in several different regions of the brain other than the lateral caudate nucleus contains a preponderance of axon hypothalamus (FISHER and COURY, 1962; LEVIT& terminals while the hypotha~us contains many choW~rrn and SANDER,1970). There is evidence that the linergic nuclei and, second, that AChE is synthesized in nuclei and translocated to axon terminals by axo- caudate nucleus may be divided into several parts responding plasmic flow (AUSTINand Jahl~s, 1970; CHIPPENDALE, which differentially affect inhibitory 1970). It has also COTMAN,KOZARand LYNCH,1974; KASAand CSILIK, (DIVAC,1968; NEILLand CROSSMAN, been suggested that inhibitory responding may be 1968; LUBINSKA,1964). Thus, the more rapid initial dependent upon a redundant, noncholinergic system recovery of AChE in the anterior preoptic area and as well as a cholinergic system in the hip~mpus the. lateral hypothal~us may be a reflection of re1968). Finally, the work of BUSS (1971) synthesis of AChE in cholinergic nuclei which pre- ~ARB~~N, and HULST (1972) indicates that regions other than cedes its appearance in axon terminals. the anterior preoptic area may also play a role in Behavioural recovery temperature regulation in rats. Thus, it seems more The recovery of temperature regulation, operant likely that particular behaviours are dependent upon responding, and drinking behaviour to normal within the complex interaction between functional circuits
297
Cholinesterase activity and behaviour after DFP which include a number of different anatomical
areas than upon the integrity of any one specific area (RusSELL, 1966). The general position outlined above does not preclude the possibility that one or more areas within these functional circuits may serve as “pace maker(s)“. Thus, it is well known that temperature regulation is severely disrupted following lesions in the anterior preoptic area (e.g. RANWN and MAGOUN, 1939) as is drinking behaviour following lesions in the lateral hypothalamus (e.g. TEITELBAUM and EPSTEIN, 1962). The present results are consistent with this latter viewpoint, for they suggest that the recovery of particular behaviours may be dependent upon renewed synthesis of AChE in specific brain regions. In general, significant recovery of AChE preceded the return of the relevant behaviours to baseline levels. For example, AChE in the lateral hypothalamus and the anterior preoptic area showed significant recovery by 16 hr, while the corresponding behaviours did not recover to normal until 48 and 20 hr, respectively. In the caudate nucleus, there was no significant recovery of AChE until 48 hr, while the measures of alternation performance had still not returned to baseline levels by 71 hr. In a recent investigation it was found that AChE activity in the septum and hippocampus, two other areas which have been implicated as chohnergic systems mediating inhibitory responding, showed significant recovery by 24 hr (CHIPPENDALE et al., 1974). That the recovery of AChE activity in the particular brain regions precedes the recovery of the relevant behaviours is to be expected because the behaviours are contingent upon an adequately functioning nervous system. Although the present findings generally indicate that the recovery of the behaviours may be dependent upon renewed synthesis of AChE, they do not completely support the hypothesis of 40% of normal as the critical level of enzyme activity. Temperature regulation had recovered (Fig. 3) long before AChE activity in the anterior preoptic area had reached 40% of normal (Table 1). On the other hand, the fact that the return of AChE activity in the lateral hypothalamus to above 40% of normal preceded the recovery of drinking behaviour is consistent with the hypothesis. Furthermore, the failure of AChE activity to reach 40% of normal in the caudate nucleus until 96 hr (Table 1) or in the hippocampus until 360 hr (CHIPPENDALE et al., 1974) may account for the failure of parameters of response suppression to recover during the periods of observation in the present studies. The finding that at least some functions may recover before AChE activity reaches 40% of normal in a relevant anatomical area suggests that other factors may be important in influencing the recovery of specific behaviours following acute treatment with DFP. These factors may include adaptation of postsynaptic cells to AChE (e.g. KIM and KARCZMAR, 1967; MEETER,1969), resynthesis of an isozyme of AChE which is critical for synaptic transmission but is only
a small portion of the total enzyme @AVIS and AGRANOFF,1968), or increased activity of redundant noncholinergic systems (MARTIN, 1970; WARBURTON, 1968). In conclusion, the bulk of the data in the present experiments support the hypotheses that AChE activity must be lowered below 40”/, of normal by acute treatment before behavioural effects are observed and that recovery of particular behaviours are at least partially dependent on the renewed synthesis of AChE in specific brain regions.
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MILLER,N. E. (1965). Chemical coding of behaviour in of alterthe brain. Science, N.f: 14% 328-338. nation learning in rats following mi~oinjection of carbaMILLER,N. W., GOTTESMAN, K. S. and EMERY,N. (1964). choi into the hippocampus. Bruin Res. 1% 3.55-359. Doseresponse to carbachol and norepinephrine in rat GROSSMAN, S. P. (1962). Direct adrenergic and cholinergic hypothalamus. Am. J. Pkysiol. 206~ 1384-1388. stimulation of hypothalamic mechanisms. Am. J, Pbysiof. NEILL, D. B. and GROSS~XAN,S. P. (1970). Behavioural 202: 872-882. effects of lesions or cholinei& blockade of the dorsal HEISE,G. A., KELLER,C., KHAVARI,K. and LAUGHLIN,N. and ventral caudate of rats. 1. camp. physiol. Psyckof. (1969). Discrete-trial alternation in the rat. J. exp. An+ 71: 311-317. sis Bekav. 12: 609-622. OVWSTREET, D. I%., KOZAR, M. D. and LYNCH, G. S. HO~~IC+ER, F. and LAN~ZASTE~, R. (1971). The determination (1973). Reduced hypothermic effects of cholinomjmet~c of a#tyl~holinesterase activity of brain slices and its sigagents following chronic anti~hoiinesterase t~atment. nificance in studies of extracellular a~tyi~holinesterase. ~europhar~coiogy 12 1017-1032. J. Neurochem. 18: 1741-1749. D. II,, RUSSELL,R. W., VASQZIEZ, B. J. and HULL, C. D., BUCHWALD,A. N. and LING, G. M. (1967). OVERSTREET, DALGLISH,F. W. (1974). Involvement of muscarinic and Effect of direct cholinergic stimulation of forebrain strucnicotinic receptors in behavioural tolerance to DFP. tures. Bruin Res. 6: 22-35. Pharmac. B&hem. Behan. 2: 45-54. HULST,S. T. H. (1972). Intracerebral imp~ntation of carbaPELLEGRINO,L. J. and CUSHMAN, A. J. (1967). A Stereot~ic chol in the rat: its effect on water intake and body temAthS 0f the Rat &Q&L AppIeto~-~ntury-Crofts, &W perature. ~~ys~o~.Behav, 8: 865-872. York. KARCXAR, A. G. (1970). Central ~~oii~er~~‘pathways and RANSON,S. W. and MAGOUN,H. W. (1939). The hypotheir ~havioural impIi~tions. In: P~~nc~p~esof Psychothalamus. Ergebn. Physiof. 41: 56-163. phurmacolog~. (CLARK,W. G. and D~LGIUDICE,J., Eds.) RUSSELL,R. W. (1964). Neurophysiological and biochemipp. 57-86, Academic Press, New York. cal correlates of effects of drugs on behavior: the acetylKASA, P. and CSILLIK,B. H. (1968). AChE synthesis in choline system. In: CIBA Foundation Symposium: Animal cholinergic neurons: electron histochemistry of enzyme Behuuiour and Drug Action. (STEINBERG,H., Ed.) pp. translocation. Histochemie 12: 175-183. 144-159, J. and A. Churchill, London. KIM, K. G. and KARCZMAR,A. G. (1967). Adaptation of RUSSELL,R. W. (1966). Biochemical substrates of behavthe ne~omuscular junction to constant concentrations ionr. In: Frontiers of P~ys~~~gjca~ Psychology. (RUSSELL, of acetylcholine. 1%. J. ~e~ophar~c. 6: 51-61. R. W., Ed.) pp. 185-246, Academic Press, New York. KOELLE,G. B. and GILMAN,A. (1946). The chronic toxicity RUSSELL,R. W. (1969). ~havioral aspects of choliner~c of diisopropyl fluorophosphate (DFP) in dogs, monkeys, transmission. Fedn Proc. Fedn Am. Sots exp. Biot. 2% and rats. J. Pkarmac. exp. Ther. 87: 435447. 121-131. LAN~~SSER, L. (1969). Properties of the frog sartorius RUSSELL,R. W., OVERSI?IEET, D. H., COTMAN,C. W., CARmuscle re-innervated by preganghonic autonomic nerve SON,V. G., CHURCHILL, L., DALGLISH,F. W. and VASfibers. Doctoral Dissertation, University of California, QUEZ, B. J. (1975). Experimental tests of hypotheses Los Angeles. about neurochemical mechanisms underlying behavLEVITT,R. A., WHITE, C. S. and SANDER,D. M. (1970). Doseresponse analysis of carbachol”elicited drinking in , ioural tolerance to the anticholineste~~~ DFP. J. Phurthe rat Iimbic system. J. camp. pkysioi. PsvckoE. 72: mat. exp. Ther. x92: 73-85, - _345-350. RUSSELL,R. W., VAFQLJEZ, B. J., OVERSTREET, D. W. and LOMAX,P. (1970). Drugs and body tem~rature. int. Rev. DALGLISH,F. W. (1971af. Consummatory behaviour dur~eurob~o~. 12~ l-43. ing tolerance to and withdrawa from chronic depression Lo?&x, P., FOSTER,R. S. and KIR~A~IC~ W. E. (1969). of cholinesterase activity. Physiol. Behav. 7: 523-528. Cholinergic and adrenergic interactions in the thermoRUSSELL,R. W., VASQ~XZ,B. J., OVEXSTREET, D. H. and regulatory centers of the rat. Brain Res. 1% 431-438. DALGLISH,F. W. (1971b). Effects of cholinolytic agents LOMAX,P. and JENDEN,D. J. (1966). Hypothermia followon behaviour following development of tolerance to low ing systematic and intracerebral injection of oxotremorcholinesterase activity. Psy~h~phar~ologia 20: 3241. me in the rat. Int. J. ~e~op~r~c. S: 353-359. RUSSELL R. W.. WARBURTON.D. M. and SEGAL D. S. (1969): Behavioral tolerance’ during chronic changes in LOWRY,0. H., RO~BROU~~ H. 1, FARR, A. L. and RANDALL,R. J. (1951). Protein measnrement with the Fohnthe ~~ol~ner~c system. Comm. be&v. Biol. 4: 121-m. RUSSELL.R. W.. WATSON.R. H. J. and FRA~~~~us~. phenol reagent. J. biul. Chem. 193: 265-275. LU~INSKA,C. (1964). Axoplasmic stream~g in regenerating M. (1961). Elects of chronic reduction in brain choline& and in normal nerve fibres. Prog. Brain Res. 13~ l-71. terase activity on acquisition and extinction of a condiMARTIN,W. R. (1970). Pharmacological redundancy as an tioned avoidance response. Stand. J. Psychol. 2: 21-29. SIEGEL,S. (1956). Nonparametric Statistics for the Behavadaptive mechanism in the central nervous system. Fedn ioral Sciences. McGraw-Hill, New York. Proc. Fedn Am. Sots exp. Biol. 29: 13-18. MEETER,E. (1969). Desensitization of end-plate membrane TEITELBAUM, P. and EPSTEIN,A. N. (1962). The lateral hypotha~mic syndrome. Psyekol. Reu. 6% X-90. following cholinesterase inhibition, an adjustment to a D. M. (1968). Effects of atropine sulfate on new working situation. Acta. Physiol. Phar~c. nderi. 1% W~~~~N, 243-258. single alternation in hippo~~ctomized rats. ~~ys~o~. MEETER,E. (1971a). Some new effects of anticholinesterases Behau. 4: 641-644. W~B~~ON, D. M. (1969). Behavioral effects of central in the whole animals, with special emphasis on the and peripheral changes in a~tyIcholine system. J. camp. b~othermia inducing action in the rat. In: ~~echanisms physiol. ~s~cko~. 68: 56-M of Toxicitp (ALDRIDGE, W. N., Ed.) pp. 29-41, Ma~illan, London. W~R&SR~$D. M. {19?2). The choline& control of internal inhtbition, In: Inhibition and Learnin& @OAKI?? MEETER,E. (1971b). The effects of atropine on the hypoR. and HALLIRAY, M. S., Eds.) pp. 431-460,~A~demic thermia and the shift in set point for heat release evoked Press, London. by a chohnesterase inhibitor in the rat. Proc. R. N&h. Acad. W~BURTON, D. M. and RUSSELL,R. W. (1969). Some besci. 74: 105-112. MEETER,E. and WOL’IHUIS,0. L. (196X). The effects of havioral effects of cholinergiic stimulation in the hippocholmesterase inhibitors on the body tem~rat~e of the campus. Li& Sci. 8: 617-627. rat. Eur. J. P~ur~c. 4: 18-24. WILSON, 1. B. and COHEN, M. (1953). The essentiality of a~tylchoiinester~e in conduction. 3~ock~. b~op~ys. ii&z, B. (1958). Brain a~tyI~holi~estera~ and a respiraActa 11: 147-156. tory reflex. Am. J. Pkpsiol. 192: 101. GREENE,E. G. and J.BMAX,P. (1970). Impairment
1976, 15, 291-298.
Pergamon
Press.
Punted
m Gt. Britain
CHANGES OF ACETYLCHOLINESTERASE ACTIVITY IN THREE MAJOR BRAIN AREAS AND RELATED CHANGES IN BEHAVIOUR FOLLOWING ACUTE TREATMENT WITH DIISOPROPYL FLUOROPHOSPHATE” M. D. KOZARP, D. H. OVERSTREET$,T. C. CHIPPENDAL$ and R. W. RUSSELLS Department of Psychobiology, University of California, Irvine, California 92664, U.S.A. (Accepted
23 September
1975)
Summary-In a series of experiments, the recovery of acetylcholinesterase activity in three brain regions following its depression by diisopropyl fluorophosphate was examined. Related changes in behaviour were also studied. The maximum depression in acetylcholinesterase activity in the anterior preoptic area occurred at 4 hr, about the same time as the maximum depression of deep body temperature was recorded. A significant recovery of acetylcholinesterase activity was observed at 16 hr, while temperature regulation returned to normal by 20 hr. Maximum depression of drinking behaviour and acetylcholinesterase activity in the lateral hypothalamus both occurred at approx. 1 hr following the acute diisopropyl fluorophosphate injections and both showed significant but not complete recovery by 24 hr. Single alternation performance had not completely recovered even at the final test session, i.e. 71 hr following the diisdpropyl fluorophosphate injection, at which time acetylcholinesterase activity in the caudate nucleus had reached approximately 23% of normal. These findings generally support the hypothesis that recovery of certain behaviours may be dependent upon the renewed synthesis of aeetylcholinesterase activity in brain regions which are important in mediating these behavionrs.
Previous studies from our laboratory have reported that many forms of behaviour are significantly altered during periods up to 24 hr after a single, 1.0 mg/kg injection of diisopropyl fluorophosphate (DFP), an irreversible anticholinesterase agent (RUSSELL, WARBURTONand SEGAL, 1969; RUSSELL, VASQUEZ, OVERSTREETand DALGLISH 1971a, b; OVERSTREET,KOZAR and LYNCH, 1973; OVERSTREET,RUSSELL, VASQUEZ and DALGLISH, 1974). At this time, the level of whole brain acetylcholinesterase (AChE) activity was found to be reduced to about 30% of normal (GLOW, ROSE and RICHARDSON,1966; RUSSELL,WATSONand FRANKENHAEUSER,1961; RUSSELL et al., 1969; RUSSELL, OVERSTREET,COTMAN, CARSON, CHURCHILL, DALGLISH and VASQUEZ, 1975) and the acetylcholine (ACh) content elevated to 140”/, of normal (RUSSELL et al., 1975). Recovery of behavioural measures to normal baseline levels occurred in most cases within 48 hr of the acute injection. The present investigations were
designed to examine the hypothesis that suph recovery may be dependent upon renewed synthesis of AChE in specific anatomical areas which appear to serve as substrates for these behaviours. A significant literature on the behavioural aspects of cholinergic transmission has accumulated over the past several years (CARLTON, 1968; KARCZMAR, 1970; RUSSELL, 1966, 1969). Much of the evidence for the cholinergic involvement has been established through the use of anticholinesterase agents as “tools” to vary the level of activity of the enzyme which plays an essential role in the transmission process. Among those behaviours for which cholinergic mediation has been implicated are: temperature regulation (FRIEDMANand JAFFE, 1969; LOMAX, 1970; MEETER, 1971a, b; MEETER and WOLTHUIS, 1968; OVERSTREET et al., 1973); drinking behaviour (GROSSMAN,1962; MILLER, 1965; RUSSELL, 1966); and response inhibition (CARLTON, 1963,1968; WARBURTON,1968,1969,1972; WARBURTONand RUSSELL, 1969). Other investigations suggest that the cholinergic systems mediating these behaviours may be localized in certain brain regions: drinking in the lateral hypothalamus (MILLER, GOTTESMANand EMERY, 1964); temperature regulation in the anterior preoptic area of the hypothalamus (CRAWSHAW, 1973; LOMAXand JENDEN, 1966; Lows, FOSTER and KIRKPATRICK, 1969); and, response inhibition in the caudate nucleus (DEADWYLER,MONTGOMERYand WYERS, 1972; HULL, BUCHWALDand LING, 1967; NEILL and GROSSMAN,1970) and hippocampus (GREENEand LOMAX, 1970; WARBURTON, 1969; WARBURTONand RUSSELL, 1969).
*This research was supported by Grant MH 18788 to Roger W. Russell and was based in part on a thesis submitted by the first author as partial fulfilment of the requirements for the Masters Degree in the Department of Pharmacology and Experimental Therapeutics, University of California, Irvine. t Present address: George Washington University Medical School, Washington, D.C. $ Reprint requests should be addressed to Professor R. W. Russell or to Dr. D. H. Overstreet, School of Biological Sciences, The Flinders University of South Australia, Bedford Park, South Australia 5042, Australia. 4 Present address: Department of Psychology, Princeton University, Princeton, New Jersey. 291
292
M. D. KOZAR,D. H. OVERSTREET, T. C. CHIPPENDALE and R. W. RUSSELL
To explore such relations between the choline+ system and behaviour in more detail two types of studies were carried out. First, the recovery of AChE activity in the lateral hypothalamus, the anterior preoptic area, and the caudate nucleus was determined by histochemical and neurochemical techniques following an acute injection of 1.0 mg/kg DFP. Second, this biochemical recovery was then compared with’behavioural recovery observed in other studies carried out under similar experimental conditions. METHODS Subjects A total of 147 male Sprague-Dawley (Simonsen) rats, approx 100 days old (35@450 g) at the beginning of each experiment, were used as subjects. The animals were housed in stainless steel cages under conditions of constant light and ambient temperature. Food and water were allowed ad lib. for rats used in the histochemical and biochemical studies. Any special conditions which applied to the various IXhavioural studies are included in descriptions of specific experiments. Experiment I-Histochemical
analyses
Twenty animals were given a single intramuscular injection of DFP (1.0 mg/kg) in the gastrocnemius muscle. The rats were sacrificed in pairs after 1, 4, 8, 16, 24, 48, 72, 168 or 720 hr postinjection. Two animals received only the arachis oil vehicle and served as controls. Brains were sectioned and stained for AChE activity by LANDMESSER’S (1969) modification of the Koelle thiocholine stain. Promethazine (5 x lo-‘M) was added to inhibit non-specific cholinesterases and the staining reaction was uniformly stopped by the addition of 10e4 M neostigmine. Experiment 2-Biochemical
analyses
Eighty rats were given a single intramuscular injection of DFP (1.0 mg/kg) in the gastrocnemius muscle. The animals were sacrificed in groups of six at either 1, 4, 16, 24, 48, 96 or 360 hr after the injection. Eight rats were sacrificed at 168 and 720 hr and four at 1440 hr after DFP treatment. Eighteen control animals received the arachis oil vehicle only and were sacrificed 1 hr after the injection. The rats were anaesthetized with pentobarbital and sacrificed by cardiac perfusion with approx 250 ml of physiological saline to eliminate serum cholinesterases. The brains were immediately removed, frozen and uniformly blocked in the coronal plane. Upon mounting on a freezing microtome, coronal slabs of tissue were taken at two levels: the caudate nucleus and the preoptic hypothalamus (for both, slab thickness = 1000 m); and the lateral hypothalamus (1800 m). Using fine glass tubing (0.7 mm i.d.), bilateral tissue samples were taken from the caudate nucleus (AP 8.2-7.2, ML 2.0, DV 2.5), the anterior preoptic area of the hypothalamus (AP 8.2-7.2, ML 1.5, DV
-2.O), and the lateral hypothalamus (AP 6.446, ML 2.0, DV -3.0) (PELLEGRINOand CUSHMAN,1967, coordinates of DeGroot). Acetylcholinesterase activity was determined using the method described by ELLMAN,COURTNEY, ANDRES and FEATHERXNE(1961) and proteins, by the procedure of LOWRY.ROSEBROUGH, FARR and RANDALL (1951). Although the former method does not distinguish between AChE (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase cacycholine hydrolase, EC 3.1.1.8), it has been shown that the latter enzyme contributes a very small portion of the total enzyme activity when acetylthiocholine is used as substrate (HOBBIGERand LANCASTER,1971). Experimental specific activities were expressed as percentages of control activity for each particular assay session. Mean control values were expressed as ~01 of acetylthiocholine hydrolyzed per min per mg protein. Analyses of variance and t-tests were used to determine the significance of the results, which were considered significant if the difference exceeded the 0.05 level of confidence. Experiment 3-Drinking
behaviour
This study involved observations of 10 animals. They were adapted to a 22-hr water deprivation schedule and obtained their 2-hr water ration in experimental drinking chambers described by RUSSELLet al. (1971a). Following the development of stable baselines of water intake, five rats received a 1.0 mg/kg injection of DFP 1.5 hr before their next drinking session, while the five control animals received a 1.0 mg/kg injection of arachis oil. Statistical analyses were performed by means of the Mann-Whitney U test (SIEGEL,1956). Experiment #-Alternation
behaviour
The animals in this study consisted of 16 rats trained on a discrete trial single alternation task (HEISE, KELLER, KHAVARI and LAUGHLIN, 1969; OVERSTREETet al., 1974) in standard operant chambers (RUSSELLet al., 1971b). After the establishment of the baseline criterion of 90% correct alternations for three consecutive days, the eight experimental animals received a 1.0 mg/kg dose of DFP, while the eight controls received an e@ivalent dose of arathis oil. These injections occurred immediately after the last baseline session. or approximately 23 hr prior to the next behavioural session. To further examine the time course of behavioural recovery in this task, three additional animals were injected with 1.0 mg/kg DFP 0.5 hr before the behavioural session and an additional four rats received the DFP treatment 12 hr before the behavioural session. Experiment 5-Deep
body temperature
The rectal core temperatures of 11 rats were recorded on a model 2133 Rustrak continuous ternperature recorder. The animals were connected to the recorder via a YSI thermistor probe model T26Qf) in-
Cholinesterase activity and behaviour after DFP
serted 7cm into the rectum and taped to the tail loosely enough to permit normal circulation. The animals were placed in well-ventilated plexiglass restraining cages located in sound-attenuated boxes (OVERSTREET t?t d., 1973). After a 2-hr period for measuring baseline temperatures, the eight experimental rats were removed from the cages and injected intramuscularly with 1.0 mg/kg DFP. The three control rats received a 1.0 mg/l
Hour
293
of the Friedman two-way analysis of variance test (SIEGEL,1956). The Mann-Whitney U test was used to determine significance of the differences between experimental and control groups. The results were considered significant if the differences exceeded the 0.05 level of confidence. RESULTS
Experiment 1 Acetylcholinesterase staining activity at various times after the acute DFP treatment is illustrated in Figure 1. The staining reaction was extremely light
I
24
72
720
A0 Control
Fig. 1. Selected DFP-treated and control brain sections indicating the general appearance of tissue sections after thiocholine staining procedure. Enzyme activity is indicated by dark areas, density of stain being approximately proportional to activity. AO: arachis oil.
294
and R. W. RUSSELL M. D. KOZAR,D. H. OVERSTREET, ‘I C. CHIPPENDALE
LH
APO
Caudate
Fig. 2. Brain sections from two different animals with tissues samples removed. The figure illustrates the replicability of this tissue removal technique. APO: anterior preoptic hypothalamus; LH: lateral hypothalamus; NC: caudate nucleus.
at 1 hr postinjection, indicating a very significant depression of AChE activity. Recovery of enzyme activity is revealed in the gradually increasing density of staining which approaches control levels at 720 hr after the injection. Experiment 2
The technique employed for removal of the tissues allowed for very uniform and reproducible sampling,
as illustrated in Figure 2, which shows brain sections from two different rats after removal of the tissue samples. The samples were approximately 2/3 of the diameter of the hole seen in Figure 2, due to the difference between inside and outside diameters of the sterile glass tubing used to extract them. The AChF activity levels of the three brain regions at various times after the acute DFP injection are summarized in Table 1. Analyses of variance estab-
Table 1. Time course of recovery of acetylcholinesterase activity in three brain regions following acute treatment with diisopropyl fluorophosphate hr following DFP treatment
n
1 4 16 24 48 86 168 360 720 1440
6 6 6 6 6 6 8 6 8 4
Percentage of control AChE activity* (mean f S.E.M.) APO LH NC 305 + 23.1 k 33.3 * 32.7 f 30.8 f 53.5 + 60.9 f 73.7 + 79.6 f 81.6 f
4.0 2.9 2.77 4.0 1.3 4.0 4.2 6.3 5.0 2.4
28.4 k 2.1 29.4 + 2.4 435 + 48t 37.5 + 2.2 44.2 * 5.9 51.9 * 1.5 55.7 f 25 75.1 + 5.0 74.3 _+ 3.6 75.9 + 15
17.0 + 2.2 12.1 f 1.4 13.3 k 0.8 14.4 &-0.8 20.0 k O.St 28.0 f. 1.2 34.7 + 3.1 56.2 + 4.2 69.1 f 4.0 81.3 + 5.2
* The control values for each area, expressed as pm01 acetylthiocholine hydrolyzed/min per kg protein were 47.4, 33.1 and 17&8 for the anterior preoptic area (APO), lateral hypothalamus (LH) and caudate nucleus (NC), respectively. t Significant recovery (P < 0.05) from the time of maximum depression.
295
Cholinesterase activity and behaviour after DFP Table 2. Water intake of control and DFP-treated acute injection hr following DFP treatment
Water DFP
Intake (mean) Control
Baseline 1.5 25.5 49.5
21.3 3.1 15.5 24.4
19.6 22.5 19.8 21.3
rats at various times following an
‘A Baseline (median) Control DFP 1.5 79.5 114.2
U 10 0* 0* 12
108.4 100.0 112.7
* Significant difference, P < 0.05, according to the Mann-Whitney U statistic, computed on absolute water intake for baselines and on y0 baselines for the other treatments.
lished the occurrence of significant (all P < 0.001) treatment effects for each area (F = 29.70, 24.62 and 73.54 for anterior preoptic area, lateral hypothalamus, and caudate nucleus, respectively). Paired comparisons between the points of maximum AChE depression and the other time points by means of t-tests revealed that a significant recovery of AChE activity had occurred by 16, 16 and 48 hr, respectively, for these areas. Despite the significant recovery exhibited in all areas, return to normal AChE activity had not occurred in any tissue by 1440 hr. Another interesting feature in Table 1 is the inverse relationship between the pretreatment baseline AChE activity of a given area and the percentage depression of the enzyme following acute administration of DFP; e.g. the maximal depression of AChE occurred in the caudate nucleus, which had the highest control specific activity. Experiment 3 The effects of acute DFP treatment on water intake are summarized in Table 2iThere was no significant difference between the initial baselines of the two groups. The DFP-treated rats drank significantly less than the controls at both 1.5 and 25.5 hr following the acute treatment, but they drank substantially more at 25.5 than they had at 1.5 hr. Both groups exhibited suprabaseline drinking at 49.5 hr following the acute injection, but there was no statistically significant difference between the percentage baseline scores for the two groups. Experiment 4
The effects of DFP and arachis oil on three measures of the animals’ alternation performance are summarized in Table 3. There was a differential recovery of the three parameters measured following
acute DFP treatment. Although all variables were significantly affected at 23 hr following the injections, operant responding (S+) had recovered by 47 hr, while the two measures of response suppression were still impaired at 71 hr. Data for the other groups of animals treated with DFP indicated that, in fact, S+ responding had returned to normal by 36 hr. Experiment 5
The effects of DFP on deep-body temperature in the rat are summarized in Figure 3. The injection procedure produced a transient rise in temperature at the first hr. Onset of drug effects occurred after 2 hr and maximum depression of 2.65”C, 6 hr after injection. Thermoregulation gradually recovered to approach normal after approximately 24 hr. Evaluation of the data by the Friedman two-way analysis of variance test showed a significant (P < @Ol) trend in the DFP-treated rats, while no significant change (P > 0.05) with time was observed for the control animals. Differences between DFPtreated and control groups were tested by the MannWhitney U test, significant differences being noted from 2 hr consecutively to 19 hr postinjection. During hr 1 and hr 2@24 the differences were not significant. DISCUSSION
Critical enzyme JeueJs
Earlier work on the behaviourai effects of anticholinesterase agents suggested that various cholinergitally mediated behaviours would be disrupted only if the AChE activity in brain fell below some critical level, i.e. 40”/, of normal (Gmw and ROSE,1965; RUSSELL, 1964, 1966; RU~~ELLet al., 1961). Further evidence suggesting 40”/0of the normal enzyme activity
Table 3. Effects of acute DFP and arachis oil (AO) treatment on three parameters of alternation
hr following DFP treatment Baseline 23 47 71
S+ Responses (median) DFP (n) A0 (n) 191(8) 116 (8)* 172 (8) 177 (8)
196 (8) 196 (8)* 198 (8) 202 (8)
Number of animals given in brackets. * Significantly different, according to Mann-Whitney
S- Responses (median) DFP (n) A0 (n) 14 (8) 31(8)* 21(8) 20 (8)*
17 (8) 10 (8)* 16(8) 9 (8)*
behaviour in rats
% Correct alternations (median) DFP (n) AD (n) 95.8 (8) 88.9 (8)* 90.8 (8)* 93.2 (8)*
U Test, based upon the ‘A baseline scores.
94.9 (8) 94.9 (8)* 95.7 (8)* 96.3 (8)*
296
M. D. KOZAK, D. H. OVERSTIGET, T. C. CHIPPENDALE and R. W.
RUSSELL
Fig. 3. The effects of DFP (l.Om~g, ~tramuscularly) on rectal core tem~rature in the rat. Values are mean “C deviations from a 2-hr pre-injection baseline. Number of animals in experimental group = 8. Number of animals in control group = 3. as the minimum level capable of sustaining normal functions has appeared in studies of axonal conduction (WILSON and C&m, 1953), of the central respiratory reflex (Mmz, 1958) and of enzyme-substrate relationships (APRISON,1961). The results obtained in the present study are in general agreement with the above suggestions. Acetylcholinesterase activity in the various brain regions was well below 40”/, of normal when the relevant behaviours were depressed. ~ioc~em~cul recovery
2&48 hr after an acute injection of DFP is consistent with earlier reports on the recovery of these functions (MUIR and WOLTHUIS,1968; RUSSELLet al., 1969, 1971a). The two measures of the animal’s ability to suppress responding (S- and y0 correct alternations) had not recovered within 71 hr after the acute injection of DFP. Further studies must be carried out to determine the time course for recovery of this behaviour. Studies with the smaller groups of animals indicated no return to baseline at 84 hr folIowing DFP treatment, at which time the study was terminated. These findings, taken together with other reports which indicate an impaired ability of animals to suppress responding following chronic treatment with anticholinesterase agents (BANKSand RUSSELL,1967; O~ERSTREET et at., 1974; RUSSELLet at., 1961), suggest that this type of behaviour may be strongly dependent upon the integrity of the cholinergic system.
Acetylcholinesterase activity recovered_gradually in all tissues examined. However, control levels had not been fully reached even after 60 days. This finding confirms the results of other workers (BLABERand CREASEY,1960; GLOW et al., 1966; KOELLEand GELMAN, 1946) indicating a slow rate of synthesis for AChE. Of special interest was the discovery that the pattern of recovery of AChE differed in the three regions examined. One possible explanation of this Biochemical and behavtiural comparisons finding is that the areas contain isoenzymes of AChE The notion that certain behaviours may be depenwhich differ in their rates of synthesis, as has been demonstrated in the retina (DAVIS and AGRANOFF, dent upon the integrity of particul~ anatomical areas 1968). An alternative explanation arises from two has been challenged in recent years by a number of investigators. For example, a water-satiated rat will facts: first, that the areas differ in the relative amounts of nerve cell bodies and axon terminals, i.e. the drink when cholinergically stimulated in several different regions of the brain other than the lateral caudate nucleus contains a preponderance of axon hypothalamus (FISHER and COURY, 1962; LEVIT& terminals while the hypotha~us contains many choW~rrn and SANDER,1970). There is evidence that the linergic nuclei and, second, that AChE is synthesized in nuclei and translocated to axon terminals by axo- caudate nucleus may be divided into several parts responding plasmic flow (AUSTINand Jahl~s, 1970; CHIPPENDALE, which differentially affect inhibitory 1970). It has also COTMAN,KOZARand LYNCH,1974; KASAand CSILIK, (DIVAC,1968; NEILLand CROSSMAN, been suggested that inhibitory responding may be 1968; LUBINSKA,1964). Thus, the more rapid initial dependent upon a redundant, noncholinergic system recovery of AChE in the anterior preoptic area and as well as a cholinergic system in the hip~mpus the. lateral hypothal~us may be a reflection of re1968). Finally, the work of BUSS (1971) synthesis of AChE in cholinergic nuclei which pre- ~ARB~~N, and HULST (1972) indicates that regions other than cedes its appearance in axon terminals. the anterior preoptic area may also play a role in Behavioural recovery temperature regulation in rats. Thus, it seems more The recovery of temperature regulation, operant likely that particular behaviours are dependent upon responding, and drinking behaviour to normal within the complex interaction between functional circuits
297
Cholinesterase activity and behaviour after DFP which include a number of different anatomical
areas than upon the integrity of any one specific area (RusSELL, 1966). The general position outlined above does not preclude the possibility that one or more areas within these functional circuits may serve as “pace maker(s)“. Thus, it is well known that temperature regulation is severely disrupted following lesions in the anterior preoptic area (e.g. RANWN and MAGOUN, 1939) as is drinking behaviour following lesions in the lateral hypothalamus (e.g. TEITELBAUM and EPSTEIN, 1962). The present results are consistent with this latter viewpoint, for they suggest that the recovery of particular behaviours may be dependent upon renewed synthesis of AChE in specific brain regions. In general, significant recovery of AChE preceded the return of the relevant behaviours to baseline levels. For example, AChE in the lateral hypothalamus and the anterior preoptic area showed significant recovery by 16 hr, while the corresponding behaviours did not recover to normal until 48 and 20 hr, respectively. In the caudate nucleus, there was no significant recovery of AChE until 48 hr, while the measures of alternation performance had still not returned to baseline levels by 71 hr. In a recent investigation it was found that AChE activity in the septum and hippocampus, two other areas which have been implicated as chohnergic systems mediating inhibitory responding, showed significant recovery by 24 hr (CHIPPENDALE et al., 1974). That the recovery of AChE activity in the particular brain regions precedes the recovery of the relevant behaviours is to be expected because the behaviours are contingent upon an adequately functioning nervous system. Although the present findings generally indicate that the recovery of the behaviours may be dependent upon renewed synthesis of AChE, they do not completely support the hypothesis of 40% of normal as the critical level of enzyme activity. Temperature regulation had recovered (Fig. 3) long before AChE activity in the anterior preoptic area had reached 40% of normal (Table 1). On the other hand, the fact that the return of AChE activity in the lateral hypothalamus to above 40% of normal preceded the recovery of drinking behaviour is consistent with the hypothesis. Furthermore, the failure of AChE activity to reach 40% of normal in the caudate nucleus until 96 hr (Table 1) or in the hippocampus until 360 hr (CHIPPENDALE et al., 1974) may account for the failure of parameters of response suppression to recover during the periods of observation in the present studies. The finding that at least some functions may recover before AChE activity reaches 40% of normal in a relevant anatomical area suggests that other factors may be important in influencing the recovery of specific behaviours following acute treatment with DFP. These factors may include adaptation of postsynaptic cells to AChE (e.g. KIM and KARCZMAR, 1967; MEETER,1969), resynthesis of an isozyme of AChE which is critical for synaptic transmission but is only
a small portion of the total enzyme @AVIS and AGRANOFF,1968), or increased activity of redundant noncholinergic systems (MARTIN, 1970; WARBURTON, 1968). In conclusion, the bulk of the data in the present experiments support the hypotheses that AChE activity must be lowered below 40”/, of normal by acute treatment before behavioural effects are observed and that recovery of particular behaviours are at least partially dependent on the renewed synthesis of AChE in specific brain regions.
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