EFFECT OF CONDITIONAL STIMULUS ON MORPHINE WITHDRAWAL SIGNS IN THE RAT

The present investigation was undertaken to demonstrate the comparisons and contrasts of the conditional stimuli's (CS's) actions with those of morphine in morphine withdrawn rats. The study shows that specific stimuli altered behavioral and physiological withdrawal signs such as: hypothermia, shakes, ptosis, piloerection, writhing and aggression. Also, one stimulus was able to effect two biochemical measures (blood glucose and striatal homovanillic acid) similar to the action of morphine. Rats were given two equally spaced injections of morphine sulfate paired with different stimuli (bell, drug, oil of anise, saccharin). The stimuli were paired with an injection for 15-25 days. Twenty-four hours after the last morphine injection the appropriate stimulus was presented. The rats learned to increase their body temperature, reduce wet shakes, increase ptosis, reduce writhing and reduce aggressive responses following the presentation of oil of anise. The bell stimulus only increased temperature. The gustatory stimulus increased temperature and the drug stimuli had no effect on withdrawal signs. The changes observed were specific only to animals that had the respective stimuli paired with morphine prior to challenge treatment. The duration of the CS in the oi l of anise study was important, the onset required a time period of greater than 2 min


ABSTRACT
The present investigation was undertaken to demonstrate the comparisons and contrasts of the conditional stimuli's (CS's) actions with those of morphine in morphine withdrawn rats.
The study shows that specific stimuli altered behavioral and physiological withdrawal signs such as: hypothermia, shakes, ptosis, piloerection, writhing and aggression. Also, one stimulus was able to effect two biochemical measures (blood glucose and striatal homovanillic acid) similar to the action of morphine.
Rats were given two equally spaced injections of morphine sulfate paired with different stimuli (bell, drug, oil of anise, saccharin). The stimuli were paired with an injection for 15-25 days. Twenty-four hours after the last morphine injection the appropriate stimulus was presented.
The rats learned to increase their body temperature, reduce wet shakes, increase ptosis, reduce writhing and reduce aggressive responses following the presentation of oil of anise. The bell stimulus only increased temperature. The gustatory stimulus increased temperature and the drug stimuli had no effect on withdrawal signs. The changes observed were specific only to animals that had the respective stimuli paired with morphine prior to challenge treatment.
The duration of the CS in the oi l of anise study was important, the onset required a time period of greater than 2 min iv but no more than 30 min for the maximal effect. When naive animals were exposed to the stimuli, no changes were observed either behaviorally or physiologically. Those animals receiving random CS or no CS during addiction demonstrated no change in observed withdrawal signs when presented the CS 24 hr or 48 hr after the last morphine injection.
In the presence of naloxone, a pure narcotic antagonist, oil of anise-morphine paired animals receiving the CS, 24 hr after the last morphine-CS pairing exhibited no change in withdrawal signs which previously had been changed by the CS. This data implies, indirectly, the release of a morphinelike substance in the production of the CS effects.
In addition to the behavioral and physiological measure brought under stimulus control, two biochemical measures (blood glucose and striatal homovanillic acid) were specif ically increased by the CS (oil of anise) in a manner similar to that seen by morphine alone, therefore showing that the conditional responses of morphine addiction are not just behavioral but also involve biochemical systems.
These data indicate that the changes resulting from morphine presentation during withdrawal can be classically conditioned. The responses are of three types -behavioral, biochemical and physiological.
v    ROffman et al. (1973) and Drawbaugh and Lal (1974) have demonstrated that hyperthermia can be conditioned during morphine abstinence by pairing a neutral stimulus (bell) with morphine administration. The procedures used in these two studies were very similar, requiring between 24-40 pairings of the bell . and morphine.
The study by Drawbaugh and Lal (1974) showed that naloxone could block the conditional phenomenon and thus concluded that the conditional stimulus, acting on the brain, may affect the same receptors that morphine affects. Understanding the conditioning factors that are present with morphine administration may be of great importance in treatment of human addicts, at both a behavorial and biochemical level.
It should be understood that treating just the physical aspects of drug addiction is not enough, but treatment of the behavioral aspects is also very important in order to increase the cure rate of narcotic addicts.
Conditional responses due to morphine administration were seen as early as 1900 by Faust in dogs injected for 3-4 weeks. Collins and Tatum (1925) reported a conditioned salivary response in dogs after repeated morphine administration.
One year later Pavlov reported that an investigator in his lab observed dogs vomiting shortly after the investigator entered the room, a response usually seen following morphine administration. Wikler and Pescor (1970) demonstrated, using the classical conditioning paradigm, that the environment associated with abstinence can act as a conditional stimulus and can elicit relapse behavior when the animal is placed in that environment for up to a year following the last morphine injection. In addition, recent evidence indicates that rats can self-administer morphine orally, in their drinking water, and show preference for the conditional stimulus (saccharin) when saccharin is given by itself (Parker et al., 1973). Kumar (1972) paired morphine drinking with an environment and observed that the rats preferred the environment where they received morphine in their drinking water.
The present investigation sought evidence to establish: 1) What is the best schedule of addiction using the bell as a conditional stimulus 2) What is the best kind of conditional stimulus in a rat analog of drug addiction (auditory, gustatory, interceptive or olfactory).

3)
Whether naloxone, a drug which is a pure narcotic antagonist (Blumberg and Day ton, 1973), can block other conditional stimuli as it was able to block the auditory CS, therefore, giving further support to the hypothesis that the CS and morph i ne work by similar mechanisms in the brain.

4)
Whether a CS can control not only behavior elicited by morphine, but also biochemical changes usually seen following morphine administration. 5 ) Whether the conditional phenomenon can be considered as indirect evidence f or the stimulus causing its effects b y inducing the release of an endogenous morphine-l ike substance.  (Pavlov, 1927).
This report, however, was not the first published report of conditioning narcotic effects even though chronologically the work was completed about 189 0 . Faust (194b ) Crisler (1928) and Kleitman (1929)  After Wikler's report another period of almost 10 years lapsed before another conditioning experiment was published (Bykov, 1957), and the concern shifted from problems involving classically conditioning morphine effects to questions about the classical conditioning of morphine withdrawal and relapse (Goldberg & Schuster, 1970;Goldberg et al. 1971;Parker et al. 19 73;Trost, 197  In the same experiment they also demonstrated that animals addicted and withdrawn in their environment will selfadminister a narcotic drug when placed back in that environment for up to 6 months after the last day of narcotic injection. Goldberg and Schuster ( 1970) utilized nalorphine, a narcotic antagonist, to demonstrate conditioned nalorphine induced abstinence changes in morphine dependent monkey s. They observed that after pairing a neutral s t imulus (ligh t) with injections, the neutral s t i mul u s alone coul d elicit a condi-tional response (emesis, salivation). These responses were normally seen only following nalorphine injection of morphine dependent animals. Goldberg et al. (1971) demonstrated that monkeys self-administer saline to overcome nalorphine's antagonistic effects if they had previously been given nalorphine and had the opportunity to self-administer morphine to overcome its effects.
Trost (1973) utilized a differential classical conditioning paradigm on morphine dependent rats and showed that the rats which had a specific environmental stimulus paired with each injection did indeed show a completely different kind of behavior when put into that situation where the stimulus or the environmental conditioner previously paired with morphine was ag?in present. He concluded from this differential training, that stimuli paired with morphine withdrawal distress play a more important role in readdiction liability than stimuli associated with withdrawal reduction. In other words the original physiological need for morphine is not present but the associated behaviors may be energized by the expectancy of an oncoming need to a greater extent than by the expectancy of an oncoming relief. Jones and Prada (1973) showed that dogs also relapse to morphine use after 1-6 months of addiction followed by 1-6 months of withdrawal.
Following this enforced abstinence, the dogs recommenced selfadrninistration of morphine whenit again was made available.
The results seem to indicate that the post-dependent dogs had maintained their dependence on morphine by self-administration. Effects other than those experienced by the initial exposure (such as nausea and vomiting), were responsible for this relapse.
Another group of experiments which bring us up to the present use either rats or primates pretreated with doses of morphine (to 200 mg/Kg/day) aimed at rendering the animal physically dependent, and subsequently testing the conditionability of withdrawal signs precipitated by either narcotic antagonists or sudden cessation of morphine injections. This period began a change in philosophy from conditioning of drug effects to concern of drug dependency as a whole (Lynch et al. 1976) . Beach (1957) began this period of experimentation by reporting that environmental stimuli we.re able to act as secondary reinforcers for morphine dependent rats. This was later substantiated by Wikler & Pescor ( 19 67) . In Beach's experiment, the rats were given a choice of either the original environment in which they were addicted or a new one whereas in the Wikler& Pescor experiment a:lJ_ were returned to their original environment. The animals preferred the environment in which they had experienced addiction and withdrawal, rather than the neutral one. Thus it was concluded that rats, when abstinent, show a preference for a distinctive environment previously associated repeatedly with withdrawal symptoms (Kumar, 1972;Thompson & Ostlund, 1965). It can further be concluded that environmental stimuli can become secondary reinforcers after repeated pairings with the effects of morphine.
There are also antecdotal reports that individual learning may contribute to the maintenance of dependent behavior in humans as well. Another experiment showing development of secondary reinforcers were that of Kumar (1972) utilizing a self-administration technique. They showed that animals given morphine in their drinking water would drink large arnounts of quinine if morphine was withdrawn from the water source.
They concluded that the bitter taste of quinine was the reason for the large intake. They further concluded that the taste had become a secondary reinforcer. Yet another example of a secondary reinforcer was done by Crowder et al. (1972).
Using both classical and operant paradigms, they showed that animals given morphine injections paired with a buzzer would bar-press for the buzzer and a saline infusion. They coneluded that the buzzer and a saline infusion had acquired secondary reinforcing properties. It was further concluded that a stimulus can become a secondary reinforcer without being a discriminative stimulus for an operant. . Roffman et al. (1973) conducted an experiment using a paradigm somewhat like Crowder et al. (1972) except that a bell was paired with morphine withdrawal and the body temperature of the animal was monitored. They showed that the bell was able to raise the temperature to normal or slightly above normal, suggesting that the animals were able to overcome the hypothermic effect of morphine withdrawal by a preparatory re-sponse to the conditional stimulus. They concluded that they were able to condition the effect of morphine; namely, the ability to alter body temperature. This experiment was later replicated by Drawbaugh and Lal (1974). In the latter study, not only were the temperature effects of morphine conditioned but the conditioned response was also blocked by naloxone.
This suggested that morphine and the conditional stimulus may be working through the same pathway(s). These experiments could also be . considered to suggest that the CS might be working by means of a morphine-like substance (endorphin).
Few systematic studies have been · done with humans dealing with conditioning of narcotic effects or withdrawal signs. Levine (1974) reported that two individuals whom he treated at NIMH Clinical Treatment Center, Lexington were true "needle freaks" and found pleasure in "sticking" themselves without heroin. He explained the "needle freak" phenomenon as a form of operant conditioning. The idea that self-injection is operant behavior that leads to the pleasurable experience of intoxication and is th~refore positively reinforced. Continued and increased self-injection in the absence of continuous reinforcement can be considered an instance of secondary reinforcement conditioning. Thus there is a behavior (selfinjection) which is directed toward a stimulus with no intrinsic utility that was previously paired with a biologically significant stimulus. Shannon et al. (1976) did a study to see if this "needle freak" phenomenon generalizes to medicinal use of hypodermic needles. They found that no generalization was observed and concluded that a chain of behavior is involved with injection of heroin which is not present in a medicinal situation.
O 'Brien et al. (1975) attempted to classical condition withdrawal signs in man as Goldberg & Schuster (1967) had earlier done in monkeys. They used naloxone as the UCS, auditory tone and oil of peppermint as the CS with each subject receiving the CS-UCS pairing three-five times per week.
On test days a saline injection was given instead of naloxone with the tone and smell. Five of eight patients showed evidence of conditiong of abstinence signs. Thompson and Pickens (1969) reviewed the conditioning literature and concluded that much of the drug self-administration can be explained by means of operant behavior. They analyzed drug dependence in terms of factors known to control acquisition, maintenance and elimination of the operant responses beliving that these same factors should also control acquisition maintenance and elimination of drug reinforced responses. They felt that looking into these specific areas would lead to a better understanding of the initial development of drug dependence and perhaps contribute to work with the problem of drug addiction. They felt that there are a number of different v ariab l es which will effect drug reinforced responses. These variabl e s include antecedent conditions (Kolb, 1962), current stimulus circumstances (Cofer & Appley, 1964) qualitati v e and quantita t ive properties of the reinforcing drug as well as a ?timuli associated with drug administration (Ausubel, 1963;Weeks & Collins, 1964).
They concluded their review of the literature by stating that drug dependence can at least be partially analyzed by using an operant paradigm and that this approach helps to provide answers to the underlying mechanisms of drug dependence. They at no time concern themselves with classical conditioning.
This lack of reference to classical experimentation may be a problem of semantics or a sincere feeling that drug addiction can best be explained and treated as an overt behavioral disorder.

B. Biochemical
Perez -Cruet (1974-Cruet ( , 1976 and Lal et al. (1976) are thus far the only labs which have been able to condition biochemical changes due to morphine. Perez-Cruet utilizing a paradigm used in another lab (Roffman, et al. 1973;) paired a buzzer with a narcotic (methadone) and was able to condition morphine's effects on homovanillic acid (HVA), a dopamine metabolite. Lal and co-workers using oil of anise., paired with morphine, were able to also condition morphine's effects on HVA.
In addit ion to the use of the gustatory stimulus to control a brain metabolite, Lal and co-workers were able to also condition blood glucose levels . This same lab earlier (Roffman and Lal, 1973) was able to condition biochemical effects produced by pentobarbital, suggesting that not only overt behavior due to a drug are able to be conditioned, but biochemical effects also.

Conditioning Changes of Blood Glucose
The amount of glucose in the blood of animals is regulated by a feedback system (homeostatic). The importance of this system can be seen in the fact that small deviations of glucose levels are suggestive of serious clinical diseases.
An increase of fasting glucose was a sign of diabetes mellitus, and a decrease of fasting glucose is a sign of hypoglycemia.
The level of blood glucose is especially important because the brain, unlike the majority of other tissue, is not able to obtain energy from lipids and proteins and thus relies heavily on glucose. Therefore, if glucose levels get too low, the brain cannot function normally and if this continues for a long enough time, coma and death may ensue. A minimal level of glucose in the brain is imperative.
The level of blood glucose has been associated with certain levels of behavior. It is well-documented that the central nervous system is very sensitive to alterations in glucose levels. Changes of glucose levels at specific brain sites causes changes of electrical activity at both single unit and gross potential levels (Anand et al. 1964;Brown, 1969;Oomura et al. 1964;Oomura et al. 1969). Also, such changes cause reflex homeostatic alterations of peripheral glucose levels (i.e. adrenals, liver and pancreas) (Muller et al. 1973;Sakata et al. 1963;Szabo & Szabo, 1972), along with changes in feeding behavior (Balagura and Kanner, 1971;Miselis & Epstein, 1970) . These types of changes have been utilized as reinforcers in learning experimentation (Chambers 1956a(Chambers ,1956b (Lichko, 1959;Zakharov, 1960;Leltes & Pavlov, 1954). The neutral stimulus was able to change (decrease) blood glucose levels from 12 to 50 mg% depending upon the laboratory.
Dogs have been the ne x t most frequent choice as subjects for conditioned glucose response experiments.
In these experiments, the presentation of insulin was paired with an auditory stimulus, the stimulus was later able to decrease blood glucose levels with or without a saline injection (Savchenko, 1940;Leites and Pavlov, 1954;Alvarez-Buyllaand Carrasco-Zanini, 1960).
The remainder of experiments 'employing insulin as the unconditioned stimulus used rats as subjects. Woods, Makous and Hutton (1968) reported that rats who had repeated pairing of stimulus (auditory) with insulin, when given auditory stimulus + saline , a 12 mg% change was observed. The same laboratory under the direction of Stephen Woods completed a number of additional experiments (Hutton et al., 1970, Woods et al. 1969Woods and Shogren, 1972) showing an average conditional change in blood gluocse of 25 mg%. It must be noted that in all the experiments demonstrating conditioned hypoglycemia, the conditioned response was smaller than or equal to the unconditioned response.
The duration of the conditioned reponse was from 15 to 30 min and it declines rapidly thereafter (Alvarez-Buy.alla Woods et al., 1969). Leites and Pavlov (1954) observed a biphasic response to conditioning procedures. For about the first 30 minutes a hypoglycemic response occurred followed by hyperglycemia. This fact is extremely important and should be noted by investigators unable t~ obtain -conditional changes.
The conditioned response is a function of the number of conditioning trials. The r~sponse is able to develop over a minimal number of trials, most experimenters reporting from two to five trials are necessary (Leites & Pavlov , 1954, Woods et al, 1969. It has also been shown that too many trials cause the magnitude of the . response to lessen and may even invert and get a conditioning of hyperglycemia instead of hypoglycemia (Leites and Pavlov, 1954) .
Spontaneous recovery has been noted in rat experiments, but the stimulus effect elicited is very small and only after one week since the last test trial.
The proposed mechanism to explain the above-mentioned experiments is as follows: When insulin is administered to an animal paired with a particular stimulus or set of stimuli, the response that follows of that animal to the same stimulus but without insulin administration is a decrease of blood glucose called conditioned hypoglycemia. The conditioned response follows properties of a classically conditioned response, and the mechanism that causes this response appears to be CNS-mediated release of pancreatic insulin.

Morphine Effect on Blood Glucose and Homovanillic Acid Levels
Morphine injected into dog (Ross, .1918;Pierce and Plant, 1928), cat (Dey et al., 1975;Feldberg and Shaligman, 1972;Borison, et al., 1962), rabbit (Araki, 1891;Sable-Amplis, 1972), mouse (Sable-Amplis, 1972) and rat (Sable-Amplis, 1972) caused hyperglycemia, the intensity of which varies according to the species and the dose. Except for Pierce and Plant (1928) and Sable-Amplis (1972) the above studies were limited to the acute effects of morphine on blood glucose levels. Sable-Amplis gave mice, rats and rabbits doses of morphine ranging from 10-40 mg/Kg for a period of up to 15 days.
In all cases tolerance developed within 7 days, 3 days for the mouse. Gradual increases of 10 mg/Kg injection was not enough to cause the increased blood glucose levels. Thus, suggesting that a rapid tolerance develops to glucose changes following morphine on all species mentioned.
If an extract (brain)' of tolerant mice is injected into naive mice the naive animals when injected with morphine will already possess altered metabolic effects due to the extract and show no change due to the morphine (Sable-Amplis, 1972) In rabbits, altered metabolism of glycogens exists for 3 weeks after withdrawal from morphine, thus the effects of morphine on glucose in vivo is dramatic and long-lasting in many types of animals.
Hornovanillic acid (HVA) levels have not been studied to any great extent following morphine administration. Therefore, the number of studies correlating HVA with morphine's effect on behavior are even fewer.
They found some degree of correlation of morphine and methadone increases in HVA with peak analgesic action. They concluded that morphine though it does not act primarily on doparninergic neuronal systems, it does affect striatal dopamine areas. This suggests that at least one and possibly other alterations in behavior due to morphine may be associated with changes in HVA levels.

Behavioral Effects of an Endogenous Morphine-Like Substance
The evidence for the presence of an endogenous ligand of the opiate receptor is now so convicing that it is important to compare known behaviorial properties of narcotics with the behavorial activ.i ty of this substance.
The substance is called endorphin and it has been shown to block electrically evoked contractions of the mouse vas deferens and the guinea pig ileum and inhibit the stereospecific receptor binding of the opiate antagonist (naloxone) in brain homogenates  Oil of anise was obtained from Merck, New Jersey.
All drugs were dissolved in distilled water. Doses are presented in terms of salts. The volume of each injection never exceeded 0.7 cc, and all saline injections were equal in volume to their corresponding drug treatment injections.  Table 1. The injections of morphine were spaced 12 hours apart beginning with 10 mg/kg/ injection, and were increased by 10 mg/kg every third injection until 100 mg/kg/injection (200 mg/kg/injection) or 200 mg/kg/day (400 mg/kg/day) was reached ( Table 1). The rats were maintained at this dose for 2-15 days and then withdrawn.
The procedure for injection during the morning session was as follows: each animal was taken out of its home cage (one animal injected at a time), placed in a plastic container and taken to a sound attenuated and temperature controlled room (21°C±2) 40 feet from the room where the animals were housed. Immediately after entering the chamber the animal was removed from the plastic container and placed into a singlepan balance to be weighed and then returned to the plastic container. The bell was turned on and after 45 seconds the animal was picked up and securely held, one hind leg and the head, so as to prevent the animal from movement and the injection was given. Then the animal was again returned to the plastic container, and after a total of 60 seconds had elpased, the bell was turned off. The rat was then immediately returned to his individual cage. Each day the order of animals going through this procedure was changed.
The identical procedure was followed during the evening session with the exception that the animal's weight was not taken at that time.
b. Taste: Conditioning consisted of giving an injection of morphine sulfate paired with saccharin (0.1%) twice daily at 0830 and 2030 ( Table 2). The injections of morphine were spaced 12 hours apart beginning with 10 mg/kg and were increased by 10 mg/kg every third injection until 100 mg/kg/ injection or 200 mg/kg/day was reached. The rats were maintained at this dose for 15 days and then withdrawn.
The procedure for injection during the morning session was as follows: each animal was taken out of its home cage (one animal injected at a time) , placed in a plastic container and taken to a single-pan balance to be weighed and then returned to the plastic container. The animal was t hen removed from the container, an intragastric tube (attached to a one ml syringe filled with 0.1% saccharin) was inserted into its mouth and 0.5 ml was slowly infused for 30 . seconds.
At this time the morphine injection was given by the same method discussed above. The animal was then placed back in its home cage. Each day the order of animals going through this procedure was changed.
The identical procedure was followed during the evening session with the exception that the animal's weight was not taken at that time. injections of morp hine were given as described in Table 1 .
The procedure for injection during the morning session was as follows: each animal was taken out of its home cage (one animal injected at a time},placed in a plastic container and taken to a single-pan balance to be weighed and then returned to the plastic container. The animal was again removed from the container and injected with one of the three abovementioned drugs. The animal was then placed back in its home cage for a specific period of time (pentobarbital and apomorphine 10 min, alcohol 5 min). After waiting a designated period of time the animal was taken from its home cage and given an injection of morphine by the same method discussed previously. The rat was placed back in its home cage immediately after being injected.
The identical procedure was followed during the evening session with the exception that the animal's weight was not taken at that time.
d. Olfactory (oil of anise): Conditioning consisted of giving an injection of morphine sulfate paired with oil of anise smell twice daily at 0830 and 2030. The injections of morphine were given as described previously (Table 2) .
The procedure for injection during the morning session was as follows: each animal was taken out of its cage, placed in a plastic container (5/ container) and taken to a single-pan balance to be weighed (each animal separately ) and then returned to the plas t ic container. The container was then taken to a room, maintained at a constant t emperature (2l+C 1), where they were placed (5/jar) into a plexiglass jar (30.5 cm x 30.5 cm).
The jar contained a 10 cm plexiglass cube filled with gauze which had 12 drops of anise oil. After the animals were placed in the jar a perforated stainless steel cover was placed over the jar to prevent the animals from escaping.
Five minutes after the animals were placed in the jar each was removed and injected with an appropriate dose of morphine. The animals were returned to the jar after the injections for the duration of the 30 min pairing session. At 30 min the animals were placed back into the plastic container and returned to their home cages.
The identical procedure was followed during the evening session with the exception that the animal's weight was not taken at that time.

Temperature Measurements
All temperature measurements were taken at designated times using a digital thermistor thermometer (Digitec Model 8500-2 by United Systems Corporation, Dayton, Ohio). The rectal probe (Model 402, Yellow Springs Instrument Co., Maryland) was inserted 5 cm (Myers, 1971) into the rectum for one min (Lomax, 1970) . Each animal had his temperature taken immediately before and 30 min after the appropriate conditioning paradigm at 0830 on two successive days preceding withdrawal. These four insertions of the probe allowed the animals to adjust to the procedure. Also, the animals were handled very careful ly during both the adjustment trials mentioned above and during the experimental measurements ..
One hand was placed on the back of the animal about midline, the thumb and index finger holding the tail with a minimal amount of pressure (just enough to keep the animal still).
The other hand inserted the probe (coated with mineral oil) 5 cm and held it in place for one min.

Measurement of Withdrawal Symptoms
Rats dependent on morphine were removed from their home cages and placed in a novel environment (Wikler and Pescor, 1967) to observe their behavior. The animals were observed for 30 min prior to test treatment and at 30 min following treatment. The following bits of behavior (withdrawal symptoms) were observed: a. Shakes -These were movements of the head and/or body which resemble the behavior an animal exhibits when water is poured over him. Changes in body weight and temperature were monitored just prior to placing the animals in the observation cages.
All of the measurements were made at 0, 24 and 48 hours following the last morphine injection. These observations were always made between 0800 and 1100 hours.
e. Aggression -This was measured only in the oil of anise conditioned animals and the control groups for that experiment. This was done by observing (while the animals were in the plexiglass jars for 30 min) for vocalizations, rearing and attacking. Since no sophisticated equipment was used, aggression was considered present if vocalizations were heard and at least 20 attacks were seen accompanied by rearing behavior during the first 15 min of the test period. Thus aggression was either present or absent for each group tested.

Serum Glucose
The glucose level in the blood. was evaluated by measuring the conversion of glucose to glucose-6-phosphate by adenosine triphosphate in the presence of hexokinase, coupled with the subsequent reduction of NADP to NADPH.
Each animal was individually placed in a plexiglass restraint with only its tail outside of the restraint. The tail was submerged into a water bath (55 C) for 45 seconds to dilate the tail vein. Upon removal from the bath the tail was wiped dry and 2 mm was cut, using surgical scissors.
Blood was collected in a plastic centrifuge tube which contained .1 ml sodium fluoroacetate (anticoagulant). After blood was obtained from the animals it was taken to a clinical centrifuge and spun at 4000 rpm for 10 min. Following centrifugation serum was pipetted from each sample and placed into small (5 ml) serum vials.
The serum was added (0.02 ml) to a ~cuvet which con- spectrophotometrically (absorbance at 340 nm) (Barthelmai and Czok, 1962). The initial absorbance was taken prior to addition of the sample (mix alone) and the final absorbance was measured 5 min after the sample was added to the cuvet (mix and serum) . Serum blank was prepared by adding .02 ml serum to 3 ml/0.85% NaCl in a cuvet and its absorbance read

Statistics
The following statistical procedures were used in this study when the significance of a result was not clear.
Student's "t" tests were used for determining the significance of a difference between two correlated means (i.e., pretreatment and post-treatment temperatures) . Both independent and dependent "t" tests were used as described in Goldstein (1964).
Where data required a comparison of an experimental group with a number of control groups, chi-square test was used according to Goldstein (1964).

RESULTS
The results of this investigation are divided into four sections: effects of auditory stimulus on withdrawal signs using different schedules of addiction; alteration of withdrawal signs by gustatory and intereoceptive stimuli; effects of olfactory stimulus on withdrawal signs; and reversal of the effects of olfactory CS by a narcotic antagonist.

Under Different Addiction Schedules
Previous work by this investigator established a bellsound as an auditory conditional stimulus (CS) capable of reversing morphine withdrawal hypothermia , 1976. It was then decided to determine if other withdrawal signs could be altered by pairing the bell sound with morphine administration. Changes in temperature were measured along with five additional withdrawal signs: weight, shakes, ptosis, piloerection, and writhing. The rats were addicted by injecting morphine paired with bell as previously described.
Following addiction they were withdrawn and exposed to either the bell sound or nothing every 24 hr and their withdrawal signs were measured at 24 and 48 hr after their last morphinebell pairing. Other animals who received morphine alone during addiction received either the bell or nothing following the same procedure mentioned above.   The data presented in Table 2 demonstrate that the bell sound paired with morphine injections during addiction did not reduce any of the withdrawal signs other than changes in body temperature at either the 24 or 48 hr observation period (p < .01). Animals not receiving bell during addiction and receiving bell during withdrawal showed no effect on any withdrawal sign measured.
In order to see if number of pairings would play a role in increasing stimulus control of withdrawal signs the number of days morphine administered was extended from 15 to 25 days (long-term -low level, Table 3) . This increase in number of days permitted 20 additional pairings of the bell with morphine. As can be seen in Table 3, 50 pairings of the bell and morphine did not reverse any of the withdrawal signs other than temperature (p < .01). However, the number of shakes were significantly increased and the variability of shakes between animals was reduced .
Since the increase in the number of pairings was ineffective in increasing the bell's effectiveness, the terminal dose of morphine was increased to 400 mg/Kg/day for 15 days ( Therefore, an attempt was made to alter withdrawal signs by using the high terminal dose ( 400 mg/Kg/day) and increasing the number of days to 25 as was previously tried at  In order to determine the effects of a gustatory stimulus paired with morphine on withdrawal signs, a saccharin solution was infused into th~ rats' mouths prior to every morphine injection and then the saccharin presented to the animals at 24 and 48 hrs after the last pairing.
In the presence of saccharin, conditioned-withdrawn rats did not show an alteration in withdrawal signs measured except for temperature at 48 hr (Table 6) . A slight, but non-significant reduction of wet shakes occurred at 48 hr of withdrawal after saccharin administration.
Since auditory and gustatory stimuli were unable to significantly alter any of the withdrawal signs other than temperature it was decided to use an interoceptive stimulus (i.e., drug). In order to determine if an interoceptive stimulus is effective, alcohol was selected to be paired with each morphine injection. Control animals were given water intragastrically with each morphine injection. During withdrawal at 24 and 48 hrs all groups had their body weight,  In the presence of alcohol at 24 hrs withdrawal, conditioned rats exhibited an increase in temperature and a non-significant reduction in shakes (Table 7) compared to the control groups. However, presentation of alcohol to the experimental group at 4B hrs of withdrawal had no effect on temperature or shakes in addition to no effect on any of the other measured symptoms.
Since the use of alcohol as an intereoceptive stimulus was unable to reliably reduce signs other than temperature, it was necessary to try another drug stimulus to check if an intereoceptive stimulus could become a conditional stimulus.
Data presented in Table 8 show that the second drug chosen, apomorphine (1.25 mg/Kg), was also ineffective in significantly altering any of the withdrawal signs at either 24 or 48 hrs after the last apomorphine-morphine pairing.
One additional drug, pentobarbital, was used as an intereoceptive stimulus. As the case with the other drugs, pentobarbital was ineffective in altering any withdrawal sign significantly except temperature at either 24 or 48 hrs of withdrawal (Table 9). Pentobarbital affected the temperature of both paired and unpaired animals; however, instead of increasing temperature as the auditory and gustatory stimuli did, pentobarbital caused a significant decrease in body temperature. This drop in temperature of drug alone    The effect of an olfactory stimulus odor is shown in Table 10 as a more effective stimulus in being able to alter not only temperature (p < 0 .01) as the bell and saccharin did, but also shakes (p <. 0. 0 5) , and ptosis (p < 0. 01) . As can be seen in Table 10, it was necessary to present the conditional stimulus (CS) for a period of time exceeding 2 min, but not more than 30 min. Also, the effectiveness of anise oil as a CS was evident at both the 24 and 48 hr withdrawal measurements.
In order to substantiate the effectiveness of anise odor as a CS, a number of control groups were done (Table 11) As shown in Table 11   """ ~ so effective in reducing specific withdrawal signs. The effect on ptosis by the CS showed an increase rather than a decrease upon observation; this effect was significant (p ' .OS) ( Table   11) .
As can be seen in Table 12 the CS was able to significantly reduce spontaneous aggression in animals which had a history of CS-morphine pairing (Chi-square analysis, p <.OS).
Anise odor, thus far, has been effective in altering physical (temperature) and behavioral withdrawal signs (aggression, ptosis, shakes and writhing). However, if anise oil were able to alter biochemical parameters of withdrawal, this would give conclusive support of its true effectiveness as a conditional stimulus.
Blood glucose and homovanillic acid measures were chosen as the biochemical parameter to be monitored in the conditioned and unconditioned animals. Data presented in Table 13 show the effect of the CS on blood glucose levels of conditioned and unconditioned rats. The CS was able to increase the glucose level in conditioned animals at 24 (p < .01) and 48 hr (p < .OS) of withdrawal. But as can be seen in Table 13, the CS groups showed no significant increase in blood gl u cose when compared to saline-treated animals.
Data summarized in Table 1 4 shows that HVA was also increased following anise oil presentation to anise oilmorphine animals during withdrawal at 24 and 72 hr (p < .OS) The increase in HVA was simi l ar to that seen fol l owing morphine   (10 mg/Kg) given to 72 hr withdrawn animals but not as great as a 10-fold high morphine (100 mg/Kg) dose given at 72 hrs (Table 14) .
D. Reversal of the Action of Olfactory Stimulus as a CS by Naloxone Data presented in Table 15 show the effects of four different doses of naloxone given to conditioned animals presented the CS during withdrawal. As can be seen, different doses of naloxone were found to reverse the CS's effect on specific withdrawal signs. Naloxone given at a dose of 0.04 mg/Kg was ineffective in blocking any of the signs measured. However, 0.16 mg/Kg of naloxone was able to block the CS reduction of writhing. At 0.64 mg/Kg of naloxone the temperature increase seen following the CS was not only blocked, but a further reduction due to the narcotic antagonist was observed. Also, the effect on shake behavior was completely blocked at a dose of 0.64 mg/Kg (naloxone). Ptosis was increased which exceeded CS's effect, but naloxone by itself in morphine dependent animals causes an increase in ptosis by itself, thus making it difficult to obtain any meaning with relation to the CS at either 0.64 or 2.5 mg/Kg. These data along with data previously reported by this lab ) using a bell sound (CS) suggest the hypothesis that the CS and morphine may indeed work on the same receptors. Present evidence that an endogenous morphinelike substance exists may be the means of the CS effect (this will be discussed in detail later) . Data presented in Table 16 show that Naloxone's (2.5 mg/Kg) effect on blood glucose levels was very unpredictable.
A slight, insignificant, decrease in blood glucose levels was observed at 24 hr and an increase at 48 hr. Thus, even though it seemed to block the CS effect as shown in this table it is very difficult to interpret because of naloxone's effects in animals not treated with the CS during withdrawal. In addition to temperature, behavioral signs such as shakes, ptosis, writhing and aggression were altered by the olfactory stimulus during withdrawal. The auditory, gustatory, and interoceptive stimuli were found not to significantly alter any of the above-mentioned behavioral signs. Also, two biochemical measures were altered by presentation of anise oil during withdrawal: 1) blood glucose, and 2) homovanillic acid.
The following discussion will be divided into four parts. The first part will deal with the auditory, gustatory and interoceptive stimuli. The second part will include evidence establishing the ability of the olfactory stimulus to alter all three types of withdrawal measurements; 1) physical, 2) behavioral and 3) biochemical changes that normally occur following a morphine injection. The third part will deal with the ability of naloxone to reverse the anise oil's conditional effects. The last part will deal with the significance of these findings.  (Puri & Lal, 1973;Gianutsos et al., 1974). Again no additional signs were altered at either 30 or 50 pairings. It is important, however, to recognize that in using the four different schedules, the temperature effect was always highly significant following such presentation during withdrawal.
The ineffectiveness of the bell to alter other signs beside temperature may have been due to a number of factors which were not tested or tested but not exhaustively anal y zed.
One possibi l ity may be that even more than 50 pairi ngs may be required to be able to bring other withdrawal signs under stimulus control. Goldberg (1971) and Lynch et al. (1973) have shown that stimulus control is indeed dependent upon the number of CS-UCS pairings. Another possible parameter which may have contributed is the length of bell presentation for each trial. This looks especially important when one considers CS strength and CS-UCS optimal interval. In addition, the fact that latter experiments using an olfactory stimulus required the CS to be presented for a time period greater than two minutes lends support to this explanation. Along with the above-mentioned classical conditioning variables is motivation. Once considered only a parameter in instrumental conditioning, it is now known to play a major role in classical conditioning (Beecroft, 1966). Wikler & Pescor (1967) conditioned their animals by presenting the morphine only once daily, thus assuring that their rats were well into withdrawal.
Another area, separate from addiction procedures, which may have limited conditionability consists of the signs that were monitored. This problem of measuring withdrawal signs has caused a number of labs to measure only one sign (body weight (Kumar, 1972), or to rate a combination of selected signs following antagonist treatment (Wei, 1973)). The concern of this _investigation was not to become too restrictive in the signs measured since it was felt that the more signs that could be shown to change following stimulus presentation the better the opportunity would be to show quantiative differences between different types of stimuli.
The behavioral and physical signs selected were easily measurable; however, many other signs exist for rats going through morphine withdrawal: salivation, rhinorrhea, restlessness, altered food and water consumption (Watanabe, 1971;Martin et al., 1963 . Since it would not be practical to monitor all the different signs, those that could be measured with ease and reliability were chosen. All of these variables must be considered in evaluating not only the auditory stimulus, but the other stimuli to be mentioned. The use of a gustatory stimulus in morphine conditioning experiments has been used by others (Wikler & Pescor, 1967) ; Kumar & Stolerman, 1972) but, not in the same manner it was used in this study.
Saccharine was chosen as a gustatory stimulus because rats have been shown to drink very large amounts when given the opportunity and show preference for it over plain water.
The results, however, did not bear out saccharine as a good choice, or that gustatory stimuli in general can serve as CS's. Again, the only sign alterable was temperature and the effect was only about 25% that of the auditory stimulus, possibly suggesting it was even weaker than the bell. However, many factors may have been involved in the ineffectiveness of saccharine's ability to become a strong conditional stimulus. One reason briefly, alluded to above was the selection of an appropriate gustatory stimulus. A bitter substance may have been a better choice. Or possibly a different concentration of saccharine may have increased its effectiveness, since it has been shown that there is a relationship between concentration and the rat's desire for the substance. Also, as was mentioned for the auditory stimulus, the schedule of addiction and the number of pairings may not have been enough to allow for saccharine to control additional morphine-like effects when presented during withdrawal.
In addition to schedule of addiction, the CS-UCS interval was very important in this experiment and may indeed have been the biggest reason for saccharine failure to elicit morphine-like effects. The saccharine was infused for only 45 seconds because the withdrawn animal became very restless; since the only restraint was the experimenter's hand, he became difficult to manage. Thus the stimulus was very short and time of UCS onset was longer than might be desired. Also, the problems of motivation and signs monitored apply to the gustatory experiment.
The third type of stimulus, interoceptive, was shown to be even less effective than either auditory or gustatory stimuli. Not even temperature was increased by any of the drugs (apomorphine, alcohol or pentobarbital) paired with morphine, when later given by themselves during withdrawal.
The use of a drug as a CS coupled with a UCS (drug) has not been shown to be effective (Lynch, et al., 1973) but few experimenters have tried such a combination.
The selection of apomorphine, alcohol and pentobarbital was based on their distinctive ability to be discriminated by the rat as being different from the UCS and thus a foundation for a CS-UCS pairing (Overton, 1972). However, combining drug effects can cause a number of problems, especially with the drugs selected. Apomorphine, it is thought, works opposite to morphine in the dopaminergic system (Kaariainen and Ahtee, 1976) and even at the dose given may cause an undesirable interaction apart from the gastrointestinal disturbances. This, unfortunately, was the case. Withdrawal signs were fewer in number without presentation of the CS and apomorphine affected temperature by itself in withdrawn animals, when it was not paired during addiction, thus confounding any temperature data that may have been obtained.
Since the experiment was completed it has been shown that very small doses (0.16 mg/Kg) of apomorphine show major endocrine effects in withdrawn rats (Lal et al., 1976)  The following section contains evidence that a conditional stimulus, anise odor, is able to elicit effects during withdrawal which normally are produced only by morphine.
In animals · addicted to anise-oil-morphine: 1. To be effective as a CS, oil of anise must be presented for more than 2 min but not to exceed 30 min to CSmorphine-paired animals during withdrawal.

2.
In the presence of the CS, 24 and 48 hours after the last CS-morphine pairing, the rats showed respectively a 30 % Even though the changes in HVA levels has been defined as a conditional phenomenon, in terms of neurotransmitter function, this change may represent a much more complex activity because the stimulus-reflex association most likely occurs cortically, as well as striatally. Also, the drug may act at a receptor whereas the stimulus may cause something to be released which will then affect the receptor.
Considering all of the many possibilities discussed above in explaining how the conditional stimulus affects neurotransmitter function the fact remains that the process does occur with pairing an auditory stimulu· s with methadone (Perez-Cruet, 1976) and pairing an olfactory stimulus with morphine. Thus it seems that the brain functions can be conditioned like those of other visceral organs.
A second biochemical change was brought under stimulus control, blood glucose. The changes due t o CS presentation could be seen at 24 and 48 hr withdrawal in animals which had 50 pairings of CS-morphine. This conditional increase is · difficult to explain as it has been shown by Sable-Amplis (1972) that tolerance develops to the increase in blood glucose levels following morphine administration. Thus after only 3 or 4 injections the injection of morphine and CS are no longer paired with the biochemical change. Thus one explanation for this increase in blood glucose level during withdrawal following CS presentation may be that another system was conditioned and the resultant effect was expressed as increased blood glucose. There is no doubt that the glucose level increased, just that the system or systems causing this change remain to be elucidated.

Use of Naloxone to Reverse the Effects of a CS
The ability of naloxone to block the CS effects on temperature and shakes in conditioned animals as it does in morphine addicted animals given morphine has some very important implications. Since an opiate receptor was found by Pert and Snyder (1973) and a number of substances isolated which may affect this receptor (one endorphin associated with a reduction in pain) these bits of information may indeed be valuable in explaining how naloxone blocks a conditional stimulus from mimicking morphine's effects. Given that an endorphin exists (Goldstein, 1976;Hughes et al., 1975;Terenius and Wahlstrom, 1975;Simantov and Snyder, 1976), then the CS may be activating this substance which in turn acts on the opiate receptor and the resultant behaviorial changes which were previously attributed to morphine, now occur following CS presentation.

71
To explain in detail the last statement it is important to understand how endorphin may be working. Normally, enkephalin may be assumed to control certain inhibitory mechanisms determining the level of neurotransmitter release.
If an opiate is administered to increase this level of inhibition, to cause analgesia, then the control will be under the exogenous narcotic and not the endogenous enkephalin. A negative feedback may slow down or even stop the synthesis of the endogenous substance now that an exogenous opiate has taken control (Kosterlitz and Hughes, 1975). Now the central nervous system will be completely dependent on the injected narcotic to maintain the inhibitory mechanism.
When the narcotic is withdrawn suddenly, those inhibitory mechanisms become inactive, because what endorphin exists will not be able to stimulate the opiate receptor until the receptors regain their sensitivity or until enough enkephalin can be synthesized or both. Thus the withdrawal syndrome develops which may be attributed· to the lack of control of the inhibitory system. Now if the CS were presented to conditioned animals it may cause any one or more of the following: 1) A transient alteration in receptor sensitivity; 2) release the peptide substance (enkephalin) i n large enough quantities to partially alter a number of withdrawal signs, or 3) mobilize morphine which still can be found in tissue to affect opiate receptors.
The above mentioned phenomenon of the CS altering opiate receptors or releasing enkephalin can be borne out by the fact that naloxone was able to reverse the CS effect as it reverses morphin effects; thus, providing strong indirect evidence for a mechanism of how the CS works. Unfortunately, the attempt to block the CS's effect on blood glucose levels was very confusing, and little can be said. It looked as though naloxone was affecting baseline levels of blood glucose thus making it difficult to interpret the results, But, it must be remembered that the effect seen may not be a direct effect of the CS as hepatic glycogenolysis which occurs following morphine initially disappears after 3-4 injections.
Also, glucose metabolism remains difficult to assess even after a month of withdrawal (Sable-Amplis, 1972). Thus the system itself is rather complex and its function not fully understood following narcotic administration.
It does look from Table 16 that the conditioned changes in blood glucose levels were blocked, but further work must be done before such a conclusion can be drawn. A better understanding of glucose metabolism following narcotics is necessary and naloxone's effects on glucose levels of both addicted and naive rats should be studied.

Significance of the Findings
Many researchers are approaching conditional aspects of narcotic addiction from the standpoint that human addicts take heroin in order to relieve withdrawal. However, this study suggests that neutral stimuli which are paired frequently enough with morphine injections will come to elicit the same or similar physiological reactions as the drugs themselves. For example, when the addict sees a syringe and any other environmental factors associated with "shooting-up," it will begin to elicit physiological reactions to morphine before it is ingested. This can be seen more vividly by "needle freaks" who will stick themselves and shoot anything in order to postpone the onset of withdrawal.
What may be happening in the above situation of the "needle freak" is that he is able to release the morphine-like substance and relieve (temporarily) some withdrawal signs by This study has provided additional groundwork for future work by: 1) neurochemists to determine if the condtioning of other neurotransmitter systems will be useful in establishing whether there is a general pattern of conditioned neurotransmitter functions in brain or specific conditioning of monoaminergic functions; 2) physiological psychologists to determine which a~ea of the brain has the major responsibility for narcotic conditioning behavior; 3) psychiatrist to develop experiments utilizing human subjects to see if the major concern of the addict is to prevent withdrawal or the conditioning is of the drug effects as eluted to above; and 4) sociologist to better characterize the environmental cues that may be best associated with narcotic administration given that a better understanding of the conditional phenomenon is forthcoming.
This study also supports the use of narcotic antagonists in treatment of narcotic addicts. The problem that arises now is what part of drug taking behavior can be classified as operant and what part as classical conditioning. Thus all that really can be said from this study is that a narcotic antagonist would be useful in treatment of human addicts, as seen from the animal analog, but the extent of its usefulness may be just of limited value.
Many more systematic studies must be done to evaluate the components of classical conditioning such as CS-UCS interval, motivation, acquisition and extinction in drug conditioning experiments.

1)
The conditional stimulus found best in development of an animal analog of narcotic addiction was oil of anise (olfactory) (see Table 17).
2) The olfactory conditional stinulus was able to alter both physical and psychological temperature withdrawal signs which hclude: temperature ~.inilar to an auditory stimulus (bell)), shakes, writhing and aggression.
3) The olfactory conditional stimulus was able to alter two biochemical systems: 1) dopamine metabolism (HVA levels) and 2) blood glucose levels similar to the action of morphine.

4)
Naloxone was able to block both physical and behavorial properties of the CS supporting the possibility that the CS may work by releasing an endogenous morphine-like substance.