Using Conditioned Taste avoidance to Assess Naloxone-Precipitated Withdrawal from Endogenous Opiates Released by Wheel Running in Rats.

Christopher M. DugganMemorial University of Newfoundland

Activity Anorexia

When rats are confined to activity wheels and restricted to one meal per day the activity anorexia syndrome (AA) can be observed (Epling & Pierce, 1992; Routtenberg, 1968).

The activity of the rats will increase to excessive amounts, as much as 20 km/day, yet rats will fail to compensate for the extra energy expenditure and their food intake will actually be less than that of controls (Epling & Pierce, 1992).

Activity Anorexia

This pattern of increased activity and decreased food intake eventually results in death (Routtenberg & Kuznesof, 1967).

Epling and Pierce (1992) hypothesized that addiction to the endogenous opioids produced by wheel running was responsible for the excessive running associated with the activity anorexia syndrome

Endogenous Opiates

Morris, Steinberg, Sykes, and Salmon (1990) showed that regular runners reported greater levels of depression after being deprived from running for a two week period.

Janal, Colt, Clark, & Glusman, (1983), showed that, after running, participants reported increases in mood and also observed elevated blood plasma levels of endogenous opiates and an increase in tolerance to pain or hypoalgesia that was reversible by naloxone injection.

Endogenous Opiates

Naloxone opposes the action of opiate drugs, such as morphine Effects that are reversed by naloxone injections are considered to be mediated

by mechanisms involving opiate receptors.

These results suggest that exercise, especially running, is highly rewarding and therefore may be able to support addiction.

Steinberg and Sykes, (1985) suggested that a substance can be regarded as addictive if: It produces pleasurable subjective effects that encourage

subsequent administrations Withdrawal symptoms develop following cessation of substance

administrations which can only be reversed by re-administration of the substance.

Similarities Between The Effects of Morphine and Wheel Running

It has been demonstrated that when rats are exposed periods of wheel running followed by exposure to a novel chamber they develop a conditioned place preference for the novel chamber, that was attenuated by naloxone (Lett, Grant, & Koh, 2001).

Hughes and Boakes (2008) demonstrated that when rats were given access to wheel running and then given a novel flavor they developed a conditioned flavor preference for the novel taste. Rats develop cross-tolerance to morphine when they are pre-exposed to wheel running before morphine injection (Lett, Grant, Koh, & Flynn, 2002).

Naloxone and CTA

When using CTA to assess naloxone-precipitated withdrawal, it is important to note that there is some evidence that naloxone itself can induce a CTA at certain doses (LeBlanc & Cappell, 1975).

There is a large body of research demonstrating that naloxone can precipitate withdrawal from exogenous opiates at very low doses, 0.01 – 0.05 mg/kg (Higgins and Sellers, 1992).

These doses are all much lower than those used in previous research that implicated naloxone in the development of CTA in non-addicted animals (Barrett, 2008; Vokey 2005)

Experiment 1

The Purpose of Experiment 1 was to establish a dose of naloxone that is near the threshold for producing CTA in non-addicted animals

Establishing a dose of naloxone that is just large enough to produce a weak CTA after a number of pairings in non-addicted rats would allow for the detection of additional avoidance produced by withdrawal.

Experiment 1

Hypothesis The CTA should be stronger and develop

earlier in the wheel running compared to the sedentary rats.

To this end, two doses of naloxone (0.5 and 0.15 mg/kg) were used to test for naloxone-induced CTA in sedentary rats; these doses seemed, on the basis of an earlier study (LeBlanc & Cappell, 1975), to be close to the threshold for producing CTA.

Experiment 1

Methods n=15 rats

Saline 0.50mg/kg naloxone 0.15mg/kg naloxone

Injections of saline or one of two doses of naloxone paired with a novel flavour

One pairing per day for 6 days followed by a one-bottle test

Experiment 1

Results

Figure 1. Mean (±SE) MSG-NaCl solution consumption by rats injected with saline, 0.15 mg/kg of naloxone, or 0.50 mg/kg of naloxone over six days.

Experiment 1

Discussiono The results of Experiment 1 suggest that 0.50 mg/kg of

naloxone is a weak dose for producing CTA in rats because the CTA was not evident until the fourth conditioning trial

o The dose of 0.15 mg/kg appears to be below threshold as there was no evidence of CTA after five conditioning trials.

o These results imply that a naloxone dose of 0.50 mg/kg should be suitable to measure naloxone precipitated withdrawal from endogenous opiates in rats.

o

Experiment 2o Experiment 2 was designed to test the hypothesis that the

excessive wheel running observed with the AA syndrome is the result of addiction to endogenous opiates.

o The rats were divided into two groups, Wheel and Cage. The wheel group was given daily access to activity wheels for 3 hr while the cage group acted as a sedentary control.

o If endogenous opiates are released by wheel running then animals that were exposed to 3 hr of wheel running per day for 21 days should become dependent on this release.

Experiment 2

Hypothesis If rats are made dependent on wheel running

then the 0.50 mg/kg dose of naloxone should be sufficient to precipitate withdrawal from endogenous opiates.

This would be made evident by pairing an injection of naloxone at this dose with a novel flavour and observing a CTA that develops faster in the wheel group than the cage group.

Experiment 2

Methods 4 groups of 8 rats (n=32)

Wheel-Naloxone (WN) Cage-Naloxone (CN) Wheel-Saline (WS) Cage-Saline (CS)

2 groups of animals were confined to activity wheels for 3hrs per day for 21 days (WN & WS)

The other 2 groups were placed in cages without food while the other groups were in the activity wheels (CN & CS)

Experiment 2

The training phase lasted 3 days. After the 3-hr running period all the animals were

given pre-weighed drinking bottles filled with MSG-NaCl solution instead of water for 15 min.

Immediately following the end of the drinking period each rat was removed and given a subcutaneous injection of either saline or 0.50 mg/kg of naloxone, 1 ml/kg, and then returned to the home cages

Experiment 2

Extinction. The extinction phase lasted for 5 days, starting the day following the last training day.

On the first extinciton day the wheel group was put in the wheels and the cage group in the cages for 3 hr.

On subsequent days they were just weighed and then returned to the home cages.

After the 3 hr elapsed, all rats were given a pre-weighed drinking bottle containing the MSG-NaCl solution for 15 min.

Experiment 2

Results

Figure 2. Mean (±SE) MSG-NaCl solution consumption by rats exposed to either wheel running (W) or cage (C) conditions and injected with either saline (S) or 0.50 mg/kg of naloxone (N) over four training days and five extinction days. Also included is the water consumption on the last pretraining day (labelled “Pre” in the figure).

Experiment 2

Results

Figure 3. Mean (±SE) Wheel Revolutions for all Groups over 21 pre-training days, 3 training days, and one extinction trial.

Experiment 2

Discussion Training. The results of Experiment 2 show that, as

predicted, the rats from condition WN developed a CTA when compared to those rats from the WS condition.

However, the control group from Experiment 2 did not mirror the trend observed in Experiment 1.

The rats from the CN condition developed a CTA after one pairing, compared to three pairings in Experiment 1. Moreover, the CTA was as strong as that for the WN condition.

Experiment 2

One factor that many account for the discrepancy in the effects of naloxone on sedentary rats from Experiments 1 and 2 is age.

Young rats tend not to develop CTA as readily as adult rats (Baker, Baker, & Kesner, 1977; Caza, Steinert, & Spear, 1980; Schramm-Sapyta, Morris, & Kuhn, 2006).

Experiment 2

Another explanation is that the rats in the first experiment were not food deprived, whereas those in the second experiment were deprived of food for 3 hr during a time when they would normally eat a considerable amount.

As food deprivation is known to release endogenous opiates, the naloxone may have precipitated withdrawal in the rats in Experiment 2.

Experiment 2

Extinction trials were carried out to determine if the WN group would extinguish more slowly than the CN, thus indicating a higher degree of CTA for the WN group.

This trend was not observed in the overall analysis. Hughes and Boakes (2008) have demonstrated

that rats exposed to wheel running paired with a novel flavour immediately after the wheel running sessions developed a preference for the novel flavour.

General Discussion• The results from Experiment 1 indicated that, in

sedentary rats, a dose of 0.50 mg/kg of naloxone would produce a CTA after three pairings. • However, in Experiment 2 the sedentary controls developed a

CTA from that dose after only one pairing.

• The second experiment provided tentative support for the notion that addiction to endogenous opiates is responsible for the excessive running associated with the AA syndrome.• The fact that the WN group took longer to extinguish than the

CN group suggests that the WN group had developed a stronger CTA to the drinking solution.

General Discussion

• Further study using a spontaneous withdrawal procedure, where drug administrations are simply ceased, may bear more fruit because the rats would not be exposed to any aversive agents other than withdrawal

• Studies have demonstrated that the adverse consequences of withdrawal are moderated by Pavlovian associative mechanisms (Poulos, Hinson, Siegel, 1981).

• A procedure to study wheel running designed to initiate the animals’ compensatory responses may allow for the measurement of endogenous opiate withdrawal by CTA.

ReferencesEpling, W. F., & Pierce, W. D. (1992). Solving the Anorexia Puzzle: A Scientific

Approach. Toronto: Hogrefe & Huber.Iversen, I. H. (1993). Techniques for establishing schedules with wheel running as

reinforcement in rats. Journal of the Experimental Analysis of Behaviour, 60, 219-238.

Higgins, G. A., & Sellers, E. M. (1994). Antagonist-precipitated opioid withdrawal in rats: Evidence for dissociations between physical and motivational signs. Pharmacology Biochemistry and Behaviour, 48, 1-8.

Lett, B. T., Grant, V. L., & Gaborko, L. L. (1996). A small amount of wheel running facilitates feeding in non-deprived rats. Appetite, 31, 351-360.

Routtenberg, A., & Kuznesof, A. W. (1967). Self-starvation of rats living in activity wheels on a restricted feeding schedule. Journal of Comparative and Physiological Psychology, 64, 414-421.

Smith, S. G., Werner, T. E., & Davis, W. M. (1976). Effect of unit dose and route of administration on self-administration of morphine. Psychopharmacology, 50, 103-105.

Lett, B. T., Grant, V. L., Koh, M. T., & Smith, J. F. (2001). Wheel running simultaneously produces conditioned taste aversion and conditioned place preference in rats. Learning and Motivation, 32, 129-136.

Reicher, M. A., & Holman, E. W. (1977). Location preference and flavour aversion reinforced by amphetamine in rats. Animal Learning and Behaviour, 5, 343-346.

LeBlanc, A. E., & Cappell, H. (1975). Antagonism of morphine-induced aversive conditioning by naloxone. Pharmacology, Biochemistry and Behaviour, 3, 185-188.