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Variability in development of tolerance to repeated injections of low doses of dl-amphetamine in rats' J. A. THORNHILL, M. HIRST,AND C. b'.GOWDEY Dc>piirtine~?r o j Ph~~rr~rtrcolog~v, Ut1iz7ersit.y o j West~r.)? Ilnlnriu, Lot~do,i,ON., Cunurlrr A16A 5CI
Received March 25, 1977 THUKNHILL, J. A., HIKST,M., and GOWDEY, C'. W. 1977. Variability in devclopnlent of tolerance to repeated injections of low doses of dl-amphetamine in rats. Can. I. Physiol. Pharmacol. 55, 1170-1 178. Male albino rats received injections of saline for 5 days before and 5 days after a series of 10 daily injections of dl-amphetamine: 1 or 5 mgjkg, sc. Core tenlperatures were measured every 30 min for 4 h after each injection and feeding activity (on a CRF operant schedule) every 30 min throughout. After amphetamine at either 0800 or 2000 h, a dose-related hyperthermia, stereotypic behavior, and an initial inhibition of feeding occurred. This anorexia decreased over the 4-h post injection period only in the evening-injected rats receiiing 5 rngi'kg. Mean body weights of all groups continued to increase during amphetamine administration. Mean 24-h food intake tended to remain below that in the control period and the hyperthermic response did not change significantly in any group. Initially on withdrawal from amphetamine all groups showed 'rebound' feeding. Taken with earlier reports, these results suggest that tolerance development to amphetamine-induced anorexia, hyperthermia, and stereotypic behavior occurs at different rates and is dependent upon frequency, route, dose, time, the amphetainine used, and whether the diet was restricted. THORNHILL, J. A., HIRST,M. et GOWDEY, C. W. 1977. Variability in development of tolerance to repeated injections of low doses of dl-amphetamine in rats. Can. 1. Physiol. I'harmacol. 55, 1170-1178. Des rats albinos mgles ont r e p des injections de salin durant les 5 jours suivant et prkcedant une sQie de 10 injections quotidiennes de dl-amphetamine I ou 5 mg/kg, sc. Leur tempkrature interne a CtC mesurke toutes les 30 min durant les 4 h qui oilt suivi chaclue iiljection. I1 en a etk de m5me de la prise d9aliment(sur la base d'un horaire CRF). Suite aux i~ljectionsd'amphktamine de 0800 ou de 2000 h, il se produit une hyperthern~ierelike B la ctosc administrie, un comportelnent stCrCotypique et un debut d9inhibition de la prise d'aliment. Cet te anorexie dirninue durant la p6riode de 4 h qui suit l'injection, uniquen~entchez les rat., cher lesquels l'injection de 5 rng/kg est faite le soir. Le poids moyen de tous les groupes contlnue d'augmenter tant que se poursuit l'injection d'amphktamine. La prise moyenne de nourriture aux 24 h tend B demeurer infkrieure celle de la pkriode de contr6le et la reponse hypertherrnique ne change de f a ~ o nsignificative dans aucun des groupes. Tous les groupes ont montre un regain d'appitit dks I'arrCt des injections d'amphktamine. Cornparks I? des publications antkrieures, ces resultats suggkrent que le dCveloppement de la tolerance B une anorexie provoquke par des ampheta[nines, l'hyperthernaie et le comportement stCrCotypique se produisent ii des taux differents et dependent de la frdquence, du trajet, de la dose, de l'heure, de I'amphetantine utilisk et dc la sCv6ritk de la dikte. [Traduit par le journal]
1960; Kosinan and Unraa 1968; Kalant t.t a[. 1971; Lewander 1971 ; Giitesta~nand Lewander ~ l ~ it his generally ~ ~ ~accepted h that phetamines induce anorexia and hyperthermia, 1975; Cotestnm 1976), although tolerance to the anorexia has recently been disputed (Ghosh and the exact mechanjsnas involved are not clear Parvathy 1973, 1975). Thus, tolerance develop(Jnnes and Nickerson 1975). Tolerance to both ment to amphetamine is dependent upon many the anorexic and hyperthermic responses has been claimed after repeated administration of factors, not least of which are the particular measured amphetamine to rats ( T and ~L~~~~~~ ~ response ~ ~ ~ and ~the criterion used to define tolerance. Most of the previous studies involved measure'This work was supported by the Non Medical Use of On ments of either feeding behavior of Drugs Directorate of the Department of National Health restricted diets or of thermoregulation on only a and Welfare of Canada. Introduction
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THORNHILL ET AL.
few experimental days. The present experiment was undertaken to determine the immediate and diurnal effects induced by repeated administration of cll-amphetamine on feeding, temperature, and other bchavior in rats by measuring these parameters before, during, and aftcr a 10-day period of amphetamine administration under carefully controlled conditions. Methods Male Sprague-Dawley rats, weighing B 95-292 g at the begnrning of the experiment, were used as subjects. Mats were houscd individually in standard Gerbrands one-lever operant condrtioning chambers and learned to obtain their total food intake by barpressing for food pellets on a continuous reinforcement schedule (Ferster and Skinner 1957), a task which was quickly learned and easily performed. Close observation during day and night revealed that the rats almost invariably ate the food pellets immediately after they were acquired regardless of whether the animals were previously injected with saline or amphetamine; food consumption was, therefore, equated to pellet acquisition. All rats were kept in a shielded, temperature-controlled (24 2.0 ' C ) ,ventilated. sound-attenuated room, which had a fixed 12-h on-off lighting schedule (lights on from 0800 to 2000 h daily). The operant conditioning chambers were equipped with a water bottle and a rotary disc feeder to dispense 45-mg Noyes food pellets. Each chamber was connected by cables to electromechanical equipment in an adjacent room where print-outs of the number of food pellets eaten each h of the day. 7' days a week, were recorded. To minimize any disruption in care of power failure the feeders, clock, and all recording equipment were arranged so that they could be powered automatically by two 12-V batteries. The body weight of every rat was measured every morning, and as well at 2000 h in those rats receiving drug in the evening; these preiiljection weights were used to calculate the daily drug doses. Before the experiments core temperature measurements were taken of all rats several times each day so as to accustom them to the procedure. Core temperature was measured by mnserting a lubricated, precalibrated rectal probe (YS1 423) 6 crn beyond the rat's anus and allowing up to 20 s for the Yellow Springs 44TA Tele-thermometer to equilibrate. After all rats had become accustomed to the rectal probe and the environtment, and the diurnal pattern of daily food consunsption of each rat had stabilized, an injection of 0.9',& (w/v) saline (0.33 m1/100g body weight) was given rubcutaneously as a control once daily at either 0800 or 2000 h for 5 days. During this control period and subsequent amphetamine and withdrawal periods, the core temperatures of all rats were recorded immediately and at 15, 30, 45, 60, 90, 120, 150, 180, and 240 mill after the daily injection. Feeding activity was monitored continuously and recorded every 30 min over the whole 20-day experimental period. After the control period, groups of rats began to receive once daily subcutaneous injections of dl-amphetamine sulphate (Benzedrine Sulphate-SKF), dissolved in saline, in a volume of 0.33 inlj100 g body
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weight. Five rats were injected with I mg/kg and four with 5 mg/kg at 0800 h for 10 days just before the lights went on in the chamber. Five rats were given I nrg/kg and four received 5 mg/kg at 2000 h for 10 days just before lights went out. After this period of amphetamine administration, withdrawal was induced by substituting saline for the amphetamine injection at either 0800 or 2000 h daily for five days. During the 4-h postinjection period after each amphetamine administration the observed behavior of each rat was scored according to the method of Quinton and Halliwell (1963). Feeding activity of each amphetamine group over the 4-h postinjection period as well as the 24-h daily food consumption was compared for several experimental days (before, during, and after amphetamine administration) using Student's t test for paired data. The 4-h postinjection temperature responses of each group were compared on the same experimental days using Student's t test for paired data after the areas under the temperature-time curves for each rat were determined. The area was taken as the mean of three planimeter readings for each rat for each of the appropriate experimental daysys.
Results Figure 1 shows the mean core temperatures and food consumptioil of rats over the 4-h period immediately after the daily injection of saline or dl-amphetamine sulphate (1 mg/kg) at 0800 h (beginning of 12-h light period) on several experimental days. On the 5th saline control day, the mean core temperatures of the rats tended to decrease during the observation period and feeding and locomotor activity were slight because the rats slept most of the time. The first injection of amphetamine was followed in all five rats by increased locomotor activity, sniffing, and rearing (giving a mean behavioral score of 3) for about 2.5 h. As Fig. 1 shows, feeding was completely abolished for the first 2 h. Analysis revealed that feeding activity over the whole 4-h postinjection observation period did not significantly decline after the first injection of amphetamine, compared with that on the last control day. The initial 2 h of inhibited feeding persisted over the whole 10 days of amphetamine injections and feeding activity during the 4-h period was not significantly changed on day 10 from that on day 1. The mean core temperature reached a maximum of 38.7 _+ 0.19"C at 60 min after the initial amphetamine injection; at that time on the preceding control day the mean core temperature had been 37.6 _+ 0.05"C (P < 0.01) and the area under the temperature-time curve for the first amphetamine injection was significantly (P < 0.01) greater than that on the
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CAN. I. PEMSIOL. PPIARMACBL.
VOL. 53, 1977
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2 TIME OF DAY FIG.I. The pattern of core temperature (mean 4 SEM) and mean food pellet acquisition per 30 min are s h w n for the 4-h postil~jectionperiod on several experimental days in a group of five rats given sallne for 5 days before and 5 days after a series of 10 daily mornlllg ~njections(at 0800 h) of dl-amphetanlame sulphate (1 rmg/kg I. A is 5th saline day 1 B is 1st amphetamine clay ; C 1s 3rd amphetamine day; D is 10th amphetatnine day; E is 1st withdrawal day. Arrows dcnote time of a d m ~ n ~ s t r a t ~ o n .
preceding day, after the last saline control injection. This slight hypcrtherrzlic response, compared with the saline control was also observed afier each amphetamine injection over the 10-day period, but thc area under the d the tenth temperature-time curve p r o d ~ ~ c eby amphetamine injection %as not significantly different from that after the first injection. On the first withdrawal day all rats in this group tended to show more feeding activity than that observed in the preceding control period, and it was not significantly different from that 011 the 10th arnphetamine day during the 4-11 observation period or over the whole 24 h. The 4-h mean core temperatures tended to be solnewhat lower than those during the amphetamine period but the temperature-time areas were not statistically difTerent. Although not shown, the mean corc temperature and feeding patterns by the 5th withdrawal day closely resembled those of the saline control period. The effects on mean corc temperatures and feeding activity over the 4-h pcrlod after repeated injections of dl-amphetamine sulphate (5 mg/kg) are illustrated in Fig. 2. After the first amphetamine injection, feeding activity was minimal over the whole 4-h period, but was not statistically decreased from that of the last control day because of wide variation between rats on that saline day. Stereotypic behavior (mean behavi-
oral score of 5.0) was Inore pronounced in this group than in that receivin~the lower dose. Locomotor activity was minirnal, but sniffing, licking, biting, and bizarre head inovements were prominent. Mean core temperature seemed to follow a triphasic pattern: clear hyperthermia, follc~wcdby a decrease, and a subsequent levelling off at around 38.5"C for the last 2 h of the period. As daily injections of amphetamine continued, stereotypic behavior was still prominent and increased salivation was also noticed. Feeding activity remained minimal, only appearing near the end of the 4-h observation period, but was not statistically increased over the 10 days of amphetamine administration. The overall pattern of mean core temperatures after each amphetamine injection tended towards hyperthermia compared with that in the control period, but the area under the curve on the first amphetaminc day was not significantly different from that on the preceding control d a y ; the control was, however, significantly ( P < 0.01) less than that on the 10th arnphetamine day. On the 1st withdrawal day, the feeding activity of all rats in this group exceeded significantly (P < 0.05) that of the last amphetamine injection, and was greater than that of thc rats which had previously received the lower dose of amphetamine. The mean core temperatures remained above 38°C for some 2 h then fell by about 1°C.
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THORNHlLL ET AL.
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FIG.2. The pattern of core temperature (mean 4 SEM) and mean food pellet accluisition per 30 min are shown for the 4-h postinjection period on several experimental days in a group of b u r rats given saline for 5 days before and 5 days after a series of 10 daily morning injections (at 0800 h) of dl-amphetamine sulphate (5 mg /kg). A is 5th saline day; R is 1st amphetamine day; C is 3rd amphetamine day: D is 10th amphetamine day; E is 1st withdrawal day. Arrows denote time of administration.
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2 T I M E OF DAY FIG.3. The pattern of core temperature (mean t SEM) and mean food pellet acquisition per 30 min are shown fur the 4-h postinjection period on several experimeintal days in a group of four rats given saline for 5 days before and 5 days after a series of 10 daily evening injections (at 2000 h) of dl-amphetamine sulphate ( 5 mg/kg). A is 5th saline day; B is 1st amphetamine day; C is 3rd amphetamine day; I)is 10th amphetamine day; E is 1st withdrawal day. Arrows denote time of administration,
Although not shown, the group of rats injected with dl-amphetamine sulphate (I .O mg,'kg) in the evening underwent similar changes in temperature and feeding patterns t o those in the group which had received the same dose in the morning (Fig. 1). A period of hyperthermia occurred after eadl evening injection of am-
phetamine, but the areas of these temperature curves were not significantly changed from the 1st to 10th days of adtninistration. The usual nighttime feeding was markedly suppressed for about 2 h in this group of rats after each of the 10 amphetamine injections; and this was accsmpanied by prominent locomotor activity and
CAN. J. PHYSIOL. PHARMACOE. VOE. 55, 1977
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FIG.4. The pattern of food consumption (mean no. of pellets per 30 min) over the whole 24-h period is shown for several experimental days for the group (tl = 4) of rats given saline 5 days before and 5 days after a series of 10 daily injections of cdl-amphetamine sulphate ( 5 mg/kg) at 0800 h. (Their irnnediate responses were shown in Fig, 2.) Arrows denote the time of administration thick horizontal bars represent the 12-h period of darkness. Nlean body weight (BW) and total 24-h food intake (F1) with their respective standard deviations are also shown in grams.
stereotypic rearing and sniffing. On withdrawal, these rats again displayed marked feeding activity in the first hours after the injection of saline. Figure 3 illustrates the 4-h patterns of core temperature and feeding in rats given amphetamine (5.0 rng/:kg) at 2000 h daily. After the first evening injection of amphetamine a triphasic pattern of mean core temperature was again observed, the peak being 38.8 0.16"C at 3 h. Feeding activity was inhibited for this period and the 4-h postinjection food intake was significantly ( B < 0.001) decreased from that eaten during this time on the preceding saline corltroli evening. The rats remained stationary but showed prominent stereotypic behavior as described above (mean score 5.5). These effects all tended to persist over tile whole 10 days of amphetamine administration, although the transient pllase of decreased body temperature appeared more prominent, especially on day BO where it reached 37.2 0.18"C at 60 min. 'The area under the temperature-time curve on the 10th amphetamine day was not significantly different from that on the: first amphetamine day. Feeding activity gradually began t o return, although only in the later stages of the 4-h observation period, and the 4-h food intake was significantly (P < 0.05) higher on days 3 and 10 than on day I . On
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the 1st withdrawal day signikicant (Y < 0.05) increases in the 4-h food intake o c c ~ ~ r r eind this group comparcd with that on the 10th amphetamine day, but it also tended to exceed that on the last preamphetanmine control day. Temperatures and feeding patterns had returned towards those of the control period by the 5th day of withdrawal. Figure 4 shows the diurnal patterns of food intake as well as thc body weight and total food consumption on scveral experinlienatal days for rats given saline before and after 10 successive days of ~16-amphetanminesulphate ( 5 rng/kg) injections given at 0800 h. On the 5th saline control day the pattern of food intake was such that during thc daylight hours (0808--2000h) the raLs ate less than during the dark period, the proportion of the 24-h food consumption taken in the light period being 27.0 _t 11.0QCx,.Figure 4 shows that amphetamine caused a transient inhibition of feeding activity and an insignificant reduction in the mean 24-h food intake, cornpared with the preceding control day, and this did not change over the 10 days. Over the period of amphetamine administration, the mean body weight of the rats continued to increase; at 10 days it was statistically (P < 0.05) greater than that seen at the end of the preamphetarnine period. On the 1st day of withdrawal from
THORNHILL ET AL.
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8 O F T H E D A Y FIG.5. The pattern of food consumption (mean no. of pellets per 30 min) over the whole 24-h period is shown for several experimental days for the group (n = 4) of rats given saline 5 days before and 5 days after a series of 10 daily injections of dl-amphetamine sulphate (5 mg/kg) at ZOO0 h. (Their immediate responses were shown in Fig. j.) Arrows denote the time of administration; thick horizontal bars represent the 1 2 4 period of darkness. Mean body weight (BW) and total 24-h food intake (FI) with their respective sta~ldarddeviations are also shown in grams. H O U R
amphetamine marked feeding occurred, especially in the early daylight hours, and the mean total intake of food was higher than on any day during the amphetamine injections. Almost half (43.4 i 6.14%) of thc total food intake that day was taken during the daylight period. Similar changes in the diurnal pattern of food intake, mean total daily food intake, and mean body weight wers found in the group of rats given the smaller dose (1 mgikg) of dl-amphetamine sulphate at 0800 h, but the morning period of inhibited feeding activity was shorter and the reduction in daily intake tended to be less. On withdrawal, the changes in thc pattern of food intake tended to be similar to, but smaller than, those shown in Fig. 4. The 24-h patterns for rats receiving saline and repeated evening injections (at 2000 h j of amphetamine (5 mg, kg) are shown in Fig. 5. Normal nighttime fecding activity was inhibited after each amphetamine injection, but the duration of this inhibition of feeding tended to decrease over the 10 days of administration. The mean total 24-h food consumption of this group tended to decrease after the first administration of amphetamine and remained below control levels over the 10-day period. Calculation revealed that the average weight gained by this group was 3.70 g/day over the 10-day amgheta-
mine period, whereas for the group receiving cll-amphetamine (1 mgjkg) at 2000 h the average gain was 4.64 gjday. The mean total food intake began t o return towards control levels during the last few days of amphetamine administration. Again, the mean body weight of this group continued to increase, rising from 299,5 17.10 g on the last saline day to 336.5 4 18.80 g on the 10th day of amphetamine injection; this was a significant increase (P < 0.001). Enhanced feeding activity followed the first withdrawal injection of saline in all rats of this group, and the mean 24-h food intake tended t o increase further. This phase of vigorous feeding did not persist and within 5 days the diurnal feeding pattern closely resembled that in the preamphetaminc control period. Siillilar but less prominent changes were observed in the group receiving the smaller dose of anlphetamirle in the evening.
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Discussion The results show that in a controlled environment and with free access to food 24 h a day male rats given cll-amphetamine suiphate (1 or 5 mgjkg) in the morning or evening did not feed for 1-3 h after each amphetamine injection over a 10-day period. As expected, however (Cizek and Nocealti 1965; Thornhill et al. 1976a), rats
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CAN. J. PHYSIOL. PMAKMACOL. VOL. 53, 8979
given a free choice normally ate little during the daylight hours of the control periods in all experimental groups. For that reason, it would be difficult to detect toterance t o the anorexic effect of amphetamine during this daylight period even if it did occur. Only one of thc four experimental groups (the group given amphetamine sulphatc (5 rng/kg) at 2000 h) showed any evidence of tolerance t o the anorexic e f i c t of amphetamine: the lnunlber c~ffood pellets eaten during the 4-k postinjection period was significantly higher on the 18th amphetamine day than on the 1st amphetamine day (but was still less than that on the Bast preamphetarnine control day). This tolerance t o the short-term anorexic egects of amphetamine has been reported in other studies (Kosman and Urma 1948; Tormey and Lasagna 1960; Lewander 1971, Kalarat t.t (zl. 1971). With rats on food- or water-restricted schedrales, it was reported that tolerance occurred t o the intake-decreasing effects of comparable doses of dl- or [/-amphetamine within 7 days (Ghosh and Parvathy 1973) or within 28 days (MacPhail and Seiden 1974); yet, in both reports, as in the present experiments, food or water intake rennaained low during the first 2 h after amphetamine throughout the wlxole treatment period. It is noteworthy in the present experiments with repeated injections of dlamphetamine, unlike our previous results with heroin (Thornhill et a%. 1976h1, no apparent "riven' feeding activity occurred after the initial daily period of abolition. Amphetarnine tended to reduce the niean 24-h food intake in all groups and not once in the 10-day treatnient period were the prearnphetamine control levels surpassed in any group, but the decreases were not statistically significant. Similarly, Eawlor et cml. (1949) found in rats which received their total daily food ration within a period of 4 h each day that at the end of a 7-day testing period, dl-amphetamine-treated (8 mg; kg) rats still ate considerably less than contrrsl animals. However, Lewander (197 11, studying rats given food and water ad libitum and twice daily intraperitoneal injections of bigger doses (16 to 32 mg/kg) of dl-amphetamine sulphate, claimed that tolerance to the anorexic eEect was evident. Bn a later study, Gotestam and Lewander (1 975) reported "that complete tolerance t o tlne ar~orexigeniceffect of amphetamine developed from day 7-1 1 when amphetamine was given
cl~ro~iicalty"(16 mg,'kg, twice daily) in rats allowed food for only 5 h per day. As MacPhail and Seiden (1976) wrote: "tolerance to amphetaminc in food- and water-consumption situations is a selective process," and it may be that the duration of food access is a determining Fdctor in the rate of tolerance development to disrupted diurnal feeding patterns and anorexia. On withdrawal from amphetamine, 'rebound' feeding has been reported (Shapiro and Freedman 1957; Tormey and Lasagna 1940; Ghosh and Parvathy 1973). I n the present study this 'rebound' feeding was more marked, especially during the first few hours, in the groups of rats which had been receiving the larger doses of amphetamine, and the total meam food intake exceeded that on the last day of amphetamine administration. This observation i b in contrast with that on the 1st day of withdraws! from heroin on which rats showed sn%yslight and sporadic feeding behavior along with the typical signs of l~yperirritability(Thornhill tJt(11. 1976b). Mean body heights of all amphetamine-treated groups increased by the end of 10 days although thc mean total 24-h food intake, especially in the higher-dosed rats, tended t o be below control levels. Comnrnonly, with chronic amphetamine administration a significant loss in body weight occurs associated with the marked stimulant properties of the drug (ABphin and Ward 1969, Lawlor el d . 1969; Magour et &I[. 1974). Mowever, other studies have failed t o show significant changes in body weights after d-amphetamine in u~istarved(Abdallah and White 1970) or semistarved (Ghosh and Parvathy 1976) rats. Bt appears that cIlanges in body weight after repeated arnphetami~ne adnlinistration nnay be the result of several variables other than dose, e.g,, an adipokinetic action, which is claimed to be independent of its anorexic action (Opitz %940),and an effect on metabolisnn rather than, or in addition to, its effca on food and water intake (Ghosh and Parvathy 1976). Parenteral administration of ti-amphetamine (5-40 rng/kg) t o rats at room temperature has been shown t o cause a dose-dependent hyperthernniu, which is maximal about 601min after adnlinistration and may persist for 3-5 h (Morpugno and Theobald 1945, Simonyi and Szerntgyorgy 1949; Dolfini et al. 1969; Yehuda and Wurtnla~m 1972). Intraperitotaeal injections of &hetamine of 1 nng,/kg and of less than that
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THORNPIXLL ET AL.
into the lateral cerebral ventricle have caused decreases in body temperature, the maximal hypothermic effect occurring 30 min after administration (Jellinek 197%).Mantegazza et d. (1970) reported that doses of dl-amphetamine (5-40 ~ n gkg) / caused dose-related hyperthesmla in aggregated rats; whereas 2.5 mg kg caused a slight decrease followed by an increase. In the present study, amphetamine (1.0 mg/ kg), a t either injection time, regularly induced hyperthermic responses which occurred on each of the 10 days of injections. In these rats the hyperthermic responses were always associated with increased locomotor activity, which may be responsible for part of the elevated core temperature. With injections of dl-ampbetannine sulphate (5 mg,'kg) the responses in mean core temperature appeared triphasic (Figs. 2 and 3): a phase of hypertherinia (.npproaching 39°C) regularly occurred within the 4-h observation period after either morning or evening injections. Jellinek (1971) postulated that cd-amphetamine acts in the lateral cercbral ventricles of rats to cause hypotherinia and peripherally to cause hypert hermia, and Weis (1973) reported hat in decapitated rats d-aiaaphetamine retained its hyperthermic effect. From those results the dl-amphetamine used in the present experiments would be expected to cause both central (hypothermic) and peripheral (hyperthcrmic) eflects and the lack of a hypothermic response with dl-amphetamine (I mg/ kg) may be due si~alplyto the small dose. Arm additional factor which may help to explain the transient fdll in body tenlpcrature with the higher doses of dl-amphetamlne is the absence of marked locor~notoractivity, the stirnulatiorm being manifested as stereotypic sniffing, licking, and biting. The results indicate that no statistically significant changes in magnitude or duration of the hyperthermic responses appeared over the 10day period of amphetamine injections. This finding with relatively low doses of dl-amphetamine is in contrast with the tolerance to hyperthern~iareported by Eewander (1971) with twice daily injections of 16 mgikg for 12 days. Consideration of earlier reports along with the results of the present study leads to the conclusion that if tolerance to amphetamine-induced anorexia, hyperthermia, and stereotypic behavior does occur, it does so at digerent rates which are dependent upon the frequency, route and time of
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administration, dose of the drug, and whether the animals were kept on restricted diets. The criteria used for defining tolerance are, however, probably even more important.
Acknowledgments The authors thank iMr. J. KIaase for his valuable technical assistance and Mrs. Linda Watson for typing the manuscript. ABD~LLAH, A. H., and WHITE,M. D. 1910. Comparative studies of the anorectic activity of phenindamine, d-amphetamine and fenfluramine in different species. Arch. Int. Pharnnacodyn. Ther. 188, 271-283. ALPHIN,R. S., and WARD,J . W. 1969. Anorexigenic efTects of fenfluramine hydrochloride in rats, guinea pigs and dogs. Toxicol. Appl. Pharmacol. 14, 182-191. C ~ Z E Kk. , J., and NOCENTI,M. R. 1965. Relationship between water and food ingestion in the rat. Am. J. Physiol. 208, 615-620. DOLFINI, E., TANSFLLA, M., VALEELLI, L., and G A K A ~ I N I , S, 1969. Further studies on the interaction between desiprilr~ineand amphetamine. Eur. J. Pkarmacoi. 5, 185-190. FERSTEII, C . B., and SKINNER, BeF. 1957. Plan of analysis in schedules of reinforcement. Edited by 6 . B. Fzrster and B. F. Skinner. Appleton-Century-Crofts, Inc., N.Y. pp. 5-13. GHOSH, M . N., and PARVAI'HY, S. 1973. Tolerance pattern of the anorexigenic action of amphetarnine in rats. Br. J. Pharmacol. 49, 658-661. 1976. Tolerance pattern of the anorexigenic action of amphetamines, fenfuranaine, phenmetrazine and diethylproprion in rats. Br. J. Pharmacol. 57, 479-485, GOTESTAM, K. G. 1976. Acute tolerance to amphetamine in rats. Psychopharrnacology, 49, 113-1 15. GOTESTAM, K. G., and LIIWANDER, T. 1975. The duration of tolerance to the anorexigenic effect of amphetamine in rats. Psychopharmacology, 42, 41-45. INNLS,1. R., and N r c ~ e a s o M. ~ , 1975. Norepinephrine, epinephrine, and the syrnpathomimetic amines. In The pharmacological basis of therapeutics. 5th Edn. Edited by L. S. Goodman and A. Gilrnan. Macmillan Publishing Co., N.Y. p. 498. JELLINEK, P. 1971. Dual effect of dexamphetarrmine on body temperature in the rat. Eur. J. Pharmacol. 15, 389-392. KALANT,H., LEBLANC, A. E., and GIBBINS, R. J. 1971. Tolerance to and dependence on some non-opiate psychotropic drugs. Pharnnacol. Rev. 23, 135-191. KOSMAN, M. E., and UNNA,K. R. 1968. Effects of chronic administration of the amphetarniiles and other stimulants on behaviour. Clin. Pharmacol. Ther. 9, 240-254. LAWLOR, R. B., TRIVEDI, M. C., and YELNOSKY, J. 1969. A determination of the anorexigenic potential of dlamphetamine, d-amphetamine, I-amphetamine and ghentermine. Arch. Int. Pharmacodyn. Ther. 179, 401-407. LEWANDER, T. 1971. A n~echanismfor the development of
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tolerance to amphetamine in rats. Psychopharmacology, 21, 17-31. MACPHAIL, R. C., and SEIDEN,L. S. 1976. Effects of intermittent and repeated administration of d-amphetamine on restricted water intake in rats. J. Pharmacol. h p . Ther. 197, 303-310. MAGOUR,S., COPEK,H., and FACHNDRICH, C. 1974. The effects of chronic treatment with d-amphetamine on food intake, body weight, locorr~otoractivity and subcellular distribution of the drug in rat brain. Psychopharmacology, 34, 45-54. MANTEGAZZA, P., MULLER, E. E., NAIMZADA, htl. K., and RIVA, M. 1970. Studies on the lack of correlation between hyperthermia, hyperactivity and anorexia induced by amphetamine. In Amphetanlines and related compounds. Edited by E. Costa and S. Garattini. Raven Press Inc., N.Y. pp. 559-575. MOBPUGNO, C., and THEOBALD, W. 1965. Influence of imiprarnine-like compounds and chlorpromazine on the reserpine hypothermia in mice and amphetamine hyperthermia in rats. Med. Pharmacol. Exp. 12, 226-232. QUINTON,R. M., and HALLIWELL, G. 1963. Effects of a-methyl-DOPA and DOPA on the amphetamine excitatory response in reserpinized rats. Nature (London), 200, 178-179. OPITZ,K. 1970. Adipokinetic action of amphetamine-A
study in the beagle dog. ltz Amphetamines and related compounds. Edited by E. Costa and S. Garattini. Raven Press Inc., N.Y. pp. 627-639. SHAPIRO,S. L., and FREEDMAN, L. 1957. ERects of dosage level of amphetamine tartronate on weights of castrate rats. Arch. Int. Pharmacodyn. Ther. 112, 419-426. SIMONYI, J., and SZENTGY~~RGY, D. 1949. The etTect of Actedron (phenylisopropylamine) and thyroxine on the body temperature. Arch. Int. Pharmacodyn. Ther. $0, 1-14. THORNHILL, J. A., H~KST, M., and GOWDEY, C. W. 19760. Changes in diurnal temperature and feeding patterns of rats during repeated injections of heroin and withdrawal. Arch. Int . Pharmacodyn. Ther. 223, 120-1 31. 19766. Disruption of diurnal feeding patterns of rats by heroin. Pharrnacol. Bischem. Behav. 4,129-1 35. TORMEY, J., and LASAGNA, L. 1960. Relation of thyroid function to acute and chronic effects of amphetamine in the rat. J. Pharmacol. Exp. Ther. 128, 201-209. WEIS, J. 1973. On the hyperthermic response to uamphetamine in the decapitated rat. Life Sci. 13, 475-484. YEHUDA,S., and WURTMAN, K. J. 1972. The efyects of D-amphetamine and related drugs on colonic temperatures of rats kept at various ambient temperatures. Life Sci. 11, 851-859.
Variability in development of tolerance to repeated injections of low doses of dl-amphetamine in rats' J. A. THORNHILL, M. HIRST,AND C. b'.GOWDEY Dc>piirtine~?r o j Ph~~rr~rtrcolog~v, Ut1iz7ersit.y o j West~r.)? Ilnlnriu, Lot~do,i,ON., Cunurlrr A16A 5CI
Received March 25, 1977 THUKNHILL, J. A., HIKST,M., and GOWDEY, C'. W. 1977. Variability in devclopnlent of tolerance to repeated injections of low doses of dl-amphetamine in rats. Can. I. Physiol. Pharmacol. 55, 1170-1 178. Male albino rats received injections of saline for 5 days before and 5 days after a series of 10 daily injections of dl-amphetamine: 1 or 5 mgjkg, sc. Core tenlperatures were measured every 30 min for 4 h after each injection and feeding activity (on a CRF operant schedule) every 30 min throughout. After amphetamine at either 0800 or 2000 h, a dose-related hyperthermia, stereotypic behavior, and an initial inhibition of feeding occurred. This anorexia decreased over the 4-h post injection period only in the evening-injected rats receiiing 5 rngi'kg. Mean body weights of all groups continued to increase during amphetamine administration. Mean 24-h food intake tended to remain below that in the control period and the hyperthermic response did not change significantly in any group. Initially on withdrawal from amphetamine all groups showed 'rebound' feeding. Taken with earlier reports, these results suggest that tolerance development to amphetamine-induced anorexia, hyperthermia, and stereotypic behavior occurs at different rates and is dependent upon frequency, route, dose, time, the amphetainine used, and whether the diet was restricted. THORNHILL, J. A., HIRST,M. et GOWDEY, C. W. 1977. Variability in development of tolerance to repeated injections of low doses of dl-amphetamine in rats. Can. 1. Physiol. I'harmacol. 55, 1170-1178. Des rats albinos mgles ont r e p des injections de salin durant les 5 jours suivant et prkcedant une sQie de 10 injections quotidiennes de dl-amphetamine I ou 5 mg/kg, sc. Leur tempkrature interne a CtC mesurke toutes les 30 min durant les 4 h qui oilt suivi chaclue iiljection. I1 en a etk de m5me de la prise d9aliment(sur la base d'un horaire CRF). Suite aux i~ljectionsd'amphktamine de 0800 ou de 2000 h, il se produit une hyperthern~ierelike B la ctosc administrie, un comportelnent stCrCotypique et un debut d9inhibition de la prise d'aliment. Cet te anorexie dirninue durant la p6riode de 4 h qui suit l'injection, uniquen~entchez les rat., cher lesquels l'injection de 5 rng/kg est faite le soir. Le poids moyen de tous les groupes contlnue d'augmenter tant que se poursuit l'injection d'amphktamine. La prise moyenne de nourriture aux 24 h tend B demeurer infkrieure celle de la pkriode de contr6le et la reponse hypertherrnique ne change de f a ~ o nsignificative dans aucun des groupes. Tous les groupes ont montre un regain d'appitit dks I'arrCt des injections d'amphktamine. Cornparks I? des publications antkrieures, ces resultats suggkrent que le dCveloppement de la tolerance B une anorexie provoquke par des ampheta[nines, l'hyperthernaie et le comportement stCrCotypique se produisent ii des taux differents et dependent de la frdquence, du trajet, de la dose, de l'heure, de I'amphetantine utilisk et dc la sCv6ritk de la dikte. [Traduit par le journal]
1960; Kosinan and Unraa 1968; Kalant t.t a[. 1971; Lewander 1971 ; Giitesta~nand Lewander ~ l ~ it his generally ~ ~ ~accepted h that phetamines induce anorexia and hyperthermia, 1975; Cotestnm 1976), although tolerance to the anorexia has recently been disputed (Ghosh and the exact mechanjsnas involved are not clear Parvathy 1973, 1975). Thus, tolerance develop(Jnnes and Nickerson 1975). Tolerance to both ment to amphetamine is dependent upon many the anorexic and hyperthermic responses has been claimed after repeated administration of factors, not least of which are the particular measured amphetamine to rats ( T and ~L~~~~~~ ~ response ~ ~ ~ and ~the criterion used to define tolerance. Most of the previous studies involved measure'This work was supported by the Non Medical Use of On ments of either feeding behavior of Drugs Directorate of the Department of National Health restricted diets or of thermoregulation on only a and Welfare of Canada. Introduction
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few experimental days. The present experiment was undertaken to determine the immediate and diurnal effects induced by repeated administration of cll-amphetamine on feeding, temperature, and other bchavior in rats by measuring these parameters before, during, and aftcr a 10-day period of amphetamine administration under carefully controlled conditions. Methods Male Sprague-Dawley rats, weighing B 95-292 g at the begnrning of the experiment, were used as subjects. Mats were houscd individually in standard Gerbrands one-lever operant condrtioning chambers and learned to obtain their total food intake by barpressing for food pellets on a continuous reinforcement schedule (Ferster and Skinner 1957), a task which was quickly learned and easily performed. Close observation during day and night revealed that the rats almost invariably ate the food pellets immediately after they were acquired regardless of whether the animals were previously injected with saline or amphetamine; food consumption was, therefore, equated to pellet acquisition. All rats were kept in a shielded, temperature-controlled (24 2.0 ' C ) ,ventilated. sound-attenuated room, which had a fixed 12-h on-off lighting schedule (lights on from 0800 to 2000 h daily). The operant conditioning chambers were equipped with a water bottle and a rotary disc feeder to dispense 45-mg Noyes food pellets. Each chamber was connected by cables to electromechanical equipment in an adjacent room where print-outs of the number of food pellets eaten each h of the day. 7' days a week, were recorded. To minimize any disruption in care of power failure the feeders, clock, and all recording equipment were arranged so that they could be powered automatically by two 12-V batteries. The body weight of every rat was measured every morning, and as well at 2000 h in those rats receiving drug in the evening; these preiiljection weights were used to calculate the daily drug doses. Before the experiments core temperature measurements were taken of all rats several times each day so as to accustom them to the procedure. Core temperature was measured by mnserting a lubricated, precalibrated rectal probe (YS1 423) 6 crn beyond the rat's anus and allowing up to 20 s for the Yellow Springs 44TA Tele-thermometer to equilibrate. After all rats had become accustomed to the rectal probe and the environtment, and the diurnal pattern of daily food consunsption of each rat had stabilized, an injection of 0.9',& (w/v) saline (0.33 m1/100g body weight) was given rubcutaneously as a control once daily at either 0800 or 2000 h for 5 days. During this control period and subsequent amphetamine and withdrawal periods, the core temperatures of all rats were recorded immediately and at 15, 30, 45, 60, 90, 120, 150, 180, and 240 mill after the daily injection. Feeding activity was monitored continuously and recorded every 30 min over the whole 20-day experimental period. After the control period, groups of rats began to receive once daily subcutaneous injections of dl-amphetamine sulphate (Benzedrine Sulphate-SKF), dissolved in saline, in a volume of 0.33 inlj100 g body
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weight. Five rats were injected with I mg/kg and four with 5 mg/kg at 0800 h for 10 days just before the lights went on in the chamber. Five rats were given I nrg/kg and four received 5 mg/kg at 2000 h for 10 days just before lights went out. After this period of amphetamine administration, withdrawal was induced by substituting saline for the amphetamine injection at either 0800 or 2000 h daily for five days. During the 4-h postinjection period after each amphetamine administration the observed behavior of each rat was scored according to the method of Quinton and Halliwell (1963). Feeding activity of each amphetamine group over the 4-h postinjection period as well as the 24-h daily food consumption was compared for several experimental days (before, during, and after amphetamine administration) using Student's t test for paired data. The 4-h postinjection temperature responses of each group were compared on the same experimental days using Student's t test for paired data after the areas under the temperature-time curves for each rat were determined. The area was taken as the mean of three planimeter readings for each rat for each of the appropriate experimental daysys.
Results Figure 1 shows the mean core temperatures and food consumptioil of rats over the 4-h period immediately after the daily injection of saline or dl-amphetamine sulphate (1 mg/kg) at 0800 h (beginning of 12-h light period) on several experimental days. On the 5th saline control day, the mean core temperatures of the rats tended to decrease during the observation period and feeding and locomotor activity were slight because the rats slept most of the time. The first injection of amphetamine was followed in all five rats by increased locomotor activity, sniffing, and rearing (giving a mean behavioral score of 3) for about 2.5 h. As Fig. 1 shows, feeding was completely abolished for the first 2 h. Analysis revealed that feeding activity over the whole 4-h postinjection observation period did not significantly decline after the first injection of amphetamine, compared with that on the last control day. The initial 2 h of inhibited feeding persisted over the whole 10 days of amphetamine injections and feeding activity during the 4-h period was not significantly changed on day 10 from that on day 1. The mean core temperature reached a maximum of 38.7 _+ 0.19"C at 60 min after the initial amphetamine injection; at that time on the preceding control day the mean core temperature had been 37.6 _+ 0.05"C (P < 0.01) and the area under the temperature-time curve for the first amphetamine injection was significantly (P < 0.01) greater than that on the
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2 TIME OF DAY FIG.I. The pattern of core temperature (mean 4 SEM) and mean food pellet acquisition per 30 min are s h w n for the 4-h postil~jectionperiod on several experimental days in a group of five rats given sallne for 5 days before and 5 days after a series of 10 daily mornlllg ~njections(at 0800 h) of dl-amphetanlame sulphate (1 rmg/kg I. A is 5th saline day 1 B is 1st amphetamine clay ; C 1s 3rd amphetamine day; D is 10th amphetatnine day; E is 1st withdrawal day. Arrows dcnote time of a d m ~ n ~ s t r a t ~ o n .
preceding day, after the last saline control injection. This slight hypcrtherrzlic response, compared with the saline control was also observed afier each amphetamine injection over the 10-day period, but thc area under the d the tenth temperature-time curve p r o d ~ ~ c eby amphetamine injection %as not significantly different from that after the first injection. On the first withdrawal day all rats in this group tended to show more feeding activity than that observed in the preceding control period, and it was not significantly different from that 011 the 10th arnphetamine day during the 4-11 observation period or over the whole 24 h. The 4-h mean core temperatures tended to be solnewhat lower than those during the amphetamine period but the temperature-time areas were not statistically difTerent. Although not shown, the mean corc temperature and feeding patterns by the 5th withdrawal day closely resembled those of the saline control period. The effects on mean corc temperatures and feeding activity over the 4-h pcrlod after repeated injections of dl-amphetamine sulphate (5 mg/kg) are illustrated in Fig. 2. After the first amphetamine injection, feeding activity was minimal over the whole 4-h period, but was not statistically decreased from that of the last control day because of wide variation between rats on that saline day. Stereotypic behavior (mean behavi-
oral score of 5.0) was Inore pronounced in this group than in that receivin~the lower dose. Locomotor activity was minirnal, but sniffing, licking, biting, and bizarre head inovements were prominent. Mean core temperature seemed to follow a triphasic pattern: clear hyperthermia, follc~wcdby a decrease, and a subsequent levelling off at around 38.5"C for the last 2 h of the period. As daily injections of amphetamine continued, stereotypic behavior was still prominent and increased salivation was also noticed. Feeding activity remained minimal, only appearing near the end of the 4-h observation period, but was not statistically increased over the 10 days of amphetamine administration. The overall pattern of mean core temperatures after each amphetamine injection tended towards hyperthermia compared with that in the control period, but the area under the curve on the first amphetaminc day was not significantly different from that on the preceding control d a y ; the control was, however, significantly ( P < 0.01) less than that on the 10th arnphetamine day. On the 1st withdrawal day, the feeding activity of all rats in this group exceeded significantly (P < 0.05) that of the last amphetamine injection, and was greater than that of thc rats which had previously received the lower dose of amphetamine. The mean core temperatures remained above 38°C for some 2 h then fell by about 1°C.
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FIG.2. The pattern of core temperature (mean 4 SEM) and mean food pellet accluisition per 30 min are shown for the 4-h postinjection period on several experimental days in a group of b u r rats given saline for 5 days before and 5 days after a series of 10 daily morning injections (at 0800 h) of dl-amphetamine sulphate (5 mg /kg). A is 5th saline day; R is 1st amphetamine day; C is 3rd amphetamine day: D is 10th amphetamine day; E is 1st withdrawal day. Arrows denote time of administration.
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2 T I M E OF DAY FIG.3. The pattern of core temperature (mean t SEM) and mean food pellet acquisition per 30 min are shown fur the 4-h postinjection period on several experimeintal days in a group of four rats given saline for 5 days before and 5 days after a series of 10 daily evening injections (at 2000 h) of dl-amphetamine sulphate ( 5 mg/kg). A is 5th saline day; B is 1st amphetamine day; C is 3rd amphetamine day; I)is 10th amphetamine day; E is 1st withdrawal day. Arrows denote time of administration,
Although not shown, the group of rats injected with dl-amphetamine sulphate (I .O mg,'kg) in the evening underwent similar changes in temperature and feeding patterns t o those in the group which had received the same dose in the morning (Fig. 1). A period of hyperthermia occurred after eadl evening injection of am-
phetamine, but the areas of these temperature curves were not significantly changed from the 1st to 10th days of adtninistration. The usual nighttime feeding was markedly suppressed for about 2 h in this group of rats after each of the 10 amphetamine injections; and this was accsmpanied by prominent locomotor activity and
CAN. J. PHYSIOL. PHARMACOE. VOE. 55, 1977
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5th saline day
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FIG.4. The pattern of food consumption (mean no. of pellets per 30 min) over the whole 24-h period is shown for several experimental days for the group (tl = 4) of rats given saline 5 days before and 5 days after a series of 10 daily injections of cdl-amphetamine sulphate ( 5 mg/kg) at 0800 h. (Their irnnediate responses were shown in Fig, 2.) Arrows denote the time of administration thick horizontal bars represent the 12-h period of darkness. Nlean body weight (BW) and total 24-h food intake (F1) with their respective standard deviations are also shown in grams.
stereotypic rearing and sniffing. On withdrawal, these rats again displayed marked feeding activity in the first hours after the injection of saline. Figure 3 illustrates the 4-h patterns of core temperature and feeding in rats given amphetamine (5.0 rng/:kg) at 2000 h daily. After the first evening injection of amphetamine a triphasic pattern of mean core temperature was again observed, the peak being 38.8 0.16"C at 3 h. Feeding activity was inhibited for this period and the 4-h postinjection food intake was significantly ( B < 0.001) decreased from that eaten during this time on the preceding saline corltroli evening. The rats remained stationary but showed prominent stereotypic behavior as described above (mean score 5.5). These effects all tended to persist over tile whole 10 days of amphetamine administration, although the transient pllase of decreased body temperature appeared more prominent, especially on day BO where it reached 37.2 0.18"C at 60 min. 'The area under the temperature-time curve on the 10th amphetamine day was not significantly different from that on the: first amphetamine day. Feeding activity gradually began t o return, although only in the later stages of the 4-h observation period, and the 4-h food intake was significantly (P < 0.05) higher on days 3 and 10 than on day I . On
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the 1st withdrawal day signikicant (Y < 0.05) increases in the 4-h food intake o c c ~ ~ r r eind this group comparcd with that on the 10th amphetamine day, but it also tended to exceed that on the last preamphetanmine control day. Temperatures and feeding patterns had returned towards those of the control period by the 5th day of withdrawal. Figure 4 shows the diurnal patterns of food intake as well as thc body weight and total food consumption on scveral experinlienatal days for rats given saline before and after 10 successive days of ~16-amphetanminesulphate ( 5 rng/kg) injections given at 0800 h. On the 5th saline control day the pattern of food intake was such that during thc daylight hours (0808--2000h) the raLs ate less than during the dark period, the proportion of the 24-h food consumption taken in the light period being 27.0 _t 11.0QCx,.Figure 4 shows that amphetamine caused a transient inhibition of feeding activity and an insignificant reduction in the mean 24-h food intake, cornpared with the preceding control day, and this did not change over the 10 days. Over the period of amphetamine administration, the mean body weight of the rats continued to increase; at 10 days it was statistically (P < 0.05) greater than that seen at the end of the preamphetarnine period. On the 1st day of withdrawal from
THORNHILL ET AL.
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1st amphetamine day
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8 O F T H E D A Y FIG.5. The pattern of food consumption (mean no. of pellets per 30 min) over the whole 24-h period is shown for several experimental days for the group (n = 4) of rats given saline 5 days before and 5 days after a series of 10 daily injections of dl-amphetamine sulphate (5 mg/kg) at ZOO0 h. (Their immediate responses were shown in Fig. j.) Arrows denote the time of administration; thick horizontal bars represent the 1 2 4 period of darkness. Mean body weight (BW) and total 24-h food intake (FI) with their respective sta~ldarddeviations are also shown in grams. H O U R
amphetamine marked feeding occurred, especially in the early daylight hours, and the mean total intake of food was higher than on any day during the amphetamine injections. Almost half (43.4 i 6.14%) of thc total food intake that day was taken during the daylight period. Similar changes in the diurnal pattern of food intake, mean total daily food intake, and mean body weight wers found in the group of rats given the smaller dose (1 mgikg) of dl-amphetamine sulphate at 0800 h, but the morning period of inhibited feeding activity was shorter and the reduction in daily intake tended to be less. On withdrawal, the changes in thc pattern of food intake tended to be similar to, but smaller than, those shown in Fig. 4. The 24-h patterns for rats receiving saline and repeated evening injections (at 2000 h j of amphetamine (5 mg, kg) are shown in Fig. 5. Normal nighttime fecding activity was inhibited after each amphetamine injection, but the duration of this inhibition of feeding tended to decrease over the 10 days of administration. The mean total 24-h food consumption of this group tended to decrease after the first administration of amphetamine and remained below control levels over the 10-day period. Calculation revealed that the average weight gained by this group was 3.70 g/day over the 10-day amgheta-
mine period, whereas for the group receiving cll-amphetamine (1 mgjkg) at 2000 h the average gain was 4.64 gjday. The mean total food intake began t o return towards control levels during the last few days of amphetamine administration. Again, the mean body weight of this group continued to increase, rising from 299,5 17.10 g on the last saline day to 336.5 4 18.80 g on the 10th day of amphetamine injection; this was a significant increase (P < 0.001). Enhanced feeding activity followed the first withdrawal injection of saline in all rats of this group, and the mean 24-h food intake tended t o increase further. This phase of vigorous feeding did not persist and within 5 days the diurnal feeding pattern closely resembled that in the preamphetaminc control period. Siillilar but less prominent changes were observed in the group receiving the smaller dose of anlphetamirle in the evening.
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Discussion The results show that in a controlled environment and with free access to food 24 h a day male rats given cll-amphetamine suiphate (1 or 5 mgjkg) in the morning or evening did not feed for 1-3 h after each amphetamine injection over a 10-day period. As expected, however (Cizek and Nocealti 1965; Thornhill et al. 1976a), rats
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given a free choice normally ate little during the daylight hours of the control periods in all experimental groups. For that reason, it would be difficult to detect toterance t o the anorexic effect of amphetamine during this daylight period even if it did occur. Only one of thc four experimental groups (the group given amphetamine sulphatc (5 rng/kg) at 2000 h) showed any evidence of tolerance t o the anorexic e f i c t of amphetamine: the lnunlber c~ffood pellets eaten during the 4-k postinjection period was significantly higher on the 18th amphetamine day than on the 1st amphetamine day (but was still less than that on the Bast preamphetarnine control day). This tolerance t o the short-term anorexic egects of amphetamine has been reported in other studies (Kosman and Urma 1948; Tormey and Lasagna 1960; Lewander 1971, Kalarat t.t (zl. 1971). With rats on food- or water-restricted schedrales, it was reported that tolerance occurred t o the intake-decreasing effects of comparable doses of dl- or [/-amphetamine within 7 days (Ghosh and Parvathy 1973) or within 28 days (MacPhail and Seiden 1974); yet, in both reports, as in the present experiments, food or water intake rennaained low during the first 2 h after amphetamine throughout the wlxole treatment period. It is noteworthy in the present experiments with repeated injections of dlamphetamine, unlike our previous results with heroin (Thornhill et a%. 1976h1, no apparent "riven' feeding activity occurred after the initial daily period of abolition. Amphetarnine tended to reduce the niean 24-h food intake in all groups and not once in the 10-day treatnient period were the prearnphetamine control levels surpassed in any group, but the decreases were not statistically significant. Similarly, Eawlor et cml. (1949) found in rats which received their total daily food ration within a period of 4 h each day that at the end of a 7-day testing period, dl-amphetamine-treated (8 mg; kg) rats still ate considerably less than contrrsl animals. However, Lewander (197 11, studying rats given food and water ad libitum and twice daily intraperitoneal injections of bigger doses (16 to 32 mg/kg) of dl-amphetamine sulphate, claimed that tolerance to the anorexic eEect was evident. Bn a later study, Gotestam and Lewander (1 975) reported "that complete tolerance t o tlne ar~orexigeniceffect of amphetamine developed from day 7-1 1 when amphetamine was given
cl~ro~iicalty"(16 mg,'kg, twice daily) in rats allowed food for only 5 h per day. As MacPhail and Seiden (1976) wrote: "tolerance to amphetaminc in food- and water-consumption situations is a selective process," and it may be that the duration of food access is a determining Fdctor in the rate of tolerance development to disrupted diurnal feeding patterns and anorexia. On withdrawal from amphetamine, 'rebound' feeding has been reported (Shapiro and Freedman 1957; Tormey and Lasagna 1940; Ghosh and Parvathy 1973). I n the present study this 'rebound' feeding was more marked, especially during the first few hours, in the groups of rats which had been receiving the larger doses of amphetamine, and the total meam food intake exceeded that on the last day of amphetamine administration. This observation i b in contrast with that on the 1st day of withdraws! from heroin on which rats showed sn%yslight and sporadic feeding behavior along with the typical signs of l~yperirritability(Thornhill tJt(11. 1976b). Mean body heights of all amphetamine-treated groups increased by the end of 10 days although thc mean total 24-h food intake, especially in the higher-dosed rats, tended t o be below control levels. Comnrnonly, with chronic amphetamine administration a significant loss in body weight occurs associated with the marked stimulant properties of the drug (ABphin and Ward 1969, Lawlor el d . 1969; Magour et &I[. 1974). Mowever, other studies have failed t o show significant changes in body weights after d-amphetamine in u~istarved(Abdallah and White 1970) or semistarved (Ghosh and Parvathy 1976) rats. Bt appears that cIlanges in body weight after repeated arnphetami~ne adnlinistration nnay be the result of several variables other than dose, e.g,, an adipokinetic action, which is claimed to be independent of its anorexic action (Opitz %940),and an effect on metabolisnn rather than, or in addition to, its effca on food and water intake (Ghosh and Parvathy 1976). Parenteral administration of ti-amphetamine (5-40 rng/kg) t o rats at room temperature has been shown t o cause a dose-dependent hyperthernniu, which is maximal about 601min after adnlinistration and may persist for 3-5 h (Morpugno and Theobald 1945, Simonyi and Szerntgyorgy 1949; Dolfini et al. 1969; Yehuda and Wurtnla~m 1972). Intraperitotaeal injections of &hetamine of 1 nng,/kg and of less than that
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THORNPIXLL ET AL.
into the lateral cerebral ventricle have caused decreases in body temperature, the maximal hypothermic effect occurring 30 min after administration (Jellinek 197%).Mantegazza et d. (1970) reported that doses of dl-amphetamine (5-40 ~ n gkg) / caused dose-related hyperthesmla in aggregated rats; whereas 2.5 mg kg caused a slight decrease followed by an increase. In the present study, amphetamine (1.0 mg/ kg), a t either injection time, regularly induced hyperthermic responses which occurred on each of the 10 days of injections. In these rats the hyperthermic responses were always associated with increased locomotor activity, which may be responsible for part of the elevated core temperature. With injections of dl-ampbetannine sulphate (5 mg,'kg) the responses in mean core temperature appeared triphasic (Figs. 2 and 3): a phase of hypertherinia (.npproaching 39°C) regularly occurred within the 4-h observation period after either morning or evening injections. Jellinek (1971) postulated that cd-amphetamine acts in the lateral cercbral ventricles of rats to cause hypotherinia and peripherally to cause hypert hermia, and Weis (1973) reported hat in decapitated rats d-aiaaphetamine retained its hyperthermic effect. From those results the dl-amphetamine used in the present experiments would be expected to cause both central (hypothermic) and peripheral (hyperthcrmic) eflects and the lack of a hypothermic response with dl-amphetamine (I mg/ kg) may be due si~alplyto the small dose. Arm additional factor which may help to explain the transient fdll in body tenlpcrature with the higher doses of dl-amphetamlne is the absence of marked locor~notoractivity, the stirnulatiorm being manifested as stereotypic sniffing, licking, and biting. The results indicate that no statistically significant changes in magnitude or duration of the hyperthermic responses appeared over the 10day period of amphetamine injections. This finding with relatively low doses of dl-amphetamine is in contrast with the tolerance to hyperthern~iareported by Eewander (1971) with twice daily injections of 16 mgikg for 12 days. Consideration of earlier reports along with the results of the present study leads to the conclusion that if tolerance to amphetamine-induced anorexia, hyperthermia, and stereotypic behavior does occur, it does so at digerent rates which are dependent upon the frequency, route and time of
I177
administration, dose of the drug, and whether the animals were kept on restricted diets. The criteria used for defining tolerance are, however, probably even more important.
Acknowledgments The authors thank iMr. J. KIaase for his valuable technical assistance and Mrs. Linda Watson for typing the manuscript. ABD~LLAH, A. H., and WHITE,M. D. 1910. Comparative studies of the anorectic activity of phenindamine, d-amphetamine and fenfluramine in different species. Arch. Int. Pharnnacodyn. Ther. 188, 271-283. ALPHIN,R. S., and WARD,J . W. 1969. Anorexigenic efTects of fenfluramine hydrochloride in rats, guinea pigs and dogs. Toxicol. Appl. Pharmacol. 14, 182-191. C ~ Z E Kk. , J., and NOCENTI,M. R. 1965. Relationship between water and food ingestion in the rat. Am. J. Physiol. 208, 615-620. DOLFINI, E., TANSFLLA, M., VALEELLI, L., and G A K A ~ I N I , S, 1969. Further studies on the interaction between desiprilr~ineand amphetamine. Eur. J. Pkarmacoi. 5, 185-190. FERSTEII, C . B., and SKINNER, BeF. 1957. Plan of analysis in schedules of reinforcement. Edited by 6 . B. Fzrster and B. F. Skinner. Appleton-Century-Crofts, Inc., N.Y. pp. 5-13. GHOSH, M . N., and PARVAI'HY, S. 1973. Tolerance pattern of the anorexigenic action of amphetarnine in rats. Br. J. Pharmacol. 49, 658-661. 1976. Tolerance pattern of the anorexigenic action of amphetamines, fenfuranaine, phenmetrazine and diethylproprion in rats. Br. J. Pharmacol. 57, 479-485, GOTESTAM, K. G. 1976. Acute tolerance to amphetamine in rats. Psychopharrnacology, 49, 113-1 15. GOTESTAM, K. G., and LIIWANDER, T. 1975. The duration of tolerance to the anorexigenic effect of amphetamine in rats. Psychopharmacology, 42, 41-45. INNLS,1. R., and N r c ~ e a s o M. ~ , 1975. Norepinephrine, epinephrine, and the syrnpathomimetic amines. In The pharmacological basis of therapeutics. 5th Edn. Edited by L. S. Goodman and A. Gilrnan. Macmillan Publishing Co., N.Y. p. 498. JELLINEK, P. 1971. Dual effect of dexamphetarrmine on body temperature in the rat. Eur. J. Pharmacol. 15, 389-392. KALANT,H., LEBLANC, A. E., and GIBBINS, R. J. 1971. Tolerance to and dependence on some non-opiate psychotropic drugs. Pharnnacol. Rev. 23, 135-191. KOSMAN, M. E., and UNNA,K. R. 1968. Effects of chronic administration of the amphetarniiles and other stimulants on behaviour. Clin. Pharmacol. Ther. 9, 240-254. LAWLOR, R. B., TRIVEDI, M. C., and YELNOSKY, J. 1969. A determination of the anorexigenic potential of dlamphetamine, d-amphetamine, I-amphetamine and ghentermine. Arch. Int. Pharmacodyn. Ther. 179, 401-407. LEWANDER, T. 1971. A n~echanismfor the development of
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tolerance to amphetamine in rats. Psychopharmacology, 21, 17-31. MACPHAIL, R. C., and SEIDEN,L. S. 1976. Effects of intermittent and repeated administration of d-amphetamine on restricted water intake in rats. J. Pharmacol. h p . Ther. 197, 303-310. MAGOUR,S., COPEK,H., and FACHNDRICH, C. 1974. The effects of chronic treatment with d-amphetamine on food intake, body weight, locorr~otoractivity and subcellular distribution of the drug in rat brain. Psychopharmacology, 34, 45-54. MANTEGAZZA, P., MULLER, E. E., NAIMZADA, htl. K., and RIVA, M. 1970. Studies on the lack of correlation between hyperthermia, hyperactivity and anorexia induced by amphetamine. In Amphetanlines and related compounds. Edited by E. Costa and S. Garattini. Raven Press Inc., N.Y. pp. 559-575. MOBPUGNO, C., and THEOBALD, W. 1965. Influence of imiprarnine-like compounds and chlorpromazine on the reserpine hypothermia in mice and amphetamine hyperthermia in rats. Med. Pharmacol. Exp. 12, 226-232. QUINTON,R. M., and HALLIWELL, G. 1963. Effects of a-methyl-DOPA and DOPA on the amphetamine excitatory response in reserpinized rats. Nature (London), 200, 178-179. OPITZ,K. 1970. Adipokinetic action of amphetamine-A
study in the beagle dog. ltz Amphetamines and related compounds. Edited by E. Costa and S. Garattini. Raven Press Inc., N.Y. pp. 627-639. SHAPIRO,S. L., and FREEDMAN, L. 1957. ERects of dosage level of amphetamine tartronate on weights of castrate rats. Arch. Int. Pharmacodyn. Ther. 112, 419-426. SIMONYI, J., and SZENTGY~~RGY, D. 1949. The etTect of Actedron (phenylisopropylamine) and thyroxine on the body temperature. Arch. Int. Pharmacodyn. Ther. $0, 1-14. THORNHILL, J. A., H~KST, M., and GOWDEY, C. W. 19760. Changes in diurnal temperature and feeding patterns of rats during repeated injections of heroin and withdrawal. Arch. Int . Pharmacodyn. Ther. 223, 120-1 31. 19766. Disruption of diurnal feeding patterns of rats by heroin. Pharrnacol. Bischem. Behav. 4,129-1 35. TORMEY, J., and LASAGNA, L. 1960. Relation of thyroid function to acute and chronic effects of amphetamine in the rat. J. Pharmacol. Exp. Ther. 128, 201-209. WEIS, J. 1973. On the hyperthermic response to uamphetamine in the decapitated rat. Life Sci. 13, 475-484. YEHUDA,S., and WURTMAN, K. J. 1972. The efyects of D-amphetamine and related drugs on colonic temperatures of rats kept at various ambient temperatures. Life Sci. 11, 851-859.
