Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 1, 2005, pp. 20-23

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 1, 2005, pp. 20-23

Cholinergic Receptor Sensitivity Following Pharmacological Modulation of the Immune System

KURADY LAXMINARAYANA BAIRY, RAO JYOTHI, VASANTH KUMAR and K BALACHANDRA KUMAR

Department of Pharmacology (K.L.B., R.J., V.K.), and Department of Clinical Psychology (K.B.K.), Kasturba Medical College, Manipal, India.

Address correspondence to: Dr. K.L. Bairy, Professor of Pharmacology, Department of Pharmacology, Kasturba Medi-cal College, Manipal, India. E-mail: klbairy@yahoo.com

Received November 11, 2004;
Revised November 17, 2004;
Accepted February 23, 2005

Code Number: pt05005

ABSTRACT

Objective. To study the effect of immunomodulators on learning and memory and on cholinergic receptor sensitivity.
Methods. Animals were initially treated for a period of 8 days with cyclosporine/levamisole/ saline. They were then subjected to passive avoidance training and 48 hrs later, the retention test for the passive avoidance task was conducted. Twenty-four hours after the retention tests the animals were sub-jected to test for cholinergic receptor sensitivity. Oxotremorine was injected intraperitoneally in a dose of 0.46, 1.84 or 4.61 mg/kg. Rectal temperature was recorded in all the animals before administration of oxotremorine and thereafter every 15 minutes for 120 minutes.
Results. Levamisole exhibited an in-creased latency to enter the dark compartment as compared to the control rats during the retention test. This increased latency indicates a better retrieval of learned behavior and facilitation of learning and memory processes. On the other hand in the rats treated with cyclosporine, there is a decrease in pas-sive avoidance retention suggesting an adverse effect on learning and memory. The administration of oxotremorine exhibited an enhanced hypothermia in levamisole treated group as compared to control group suggesting hyperactivity of the cholinergic system. On the other hand oxotremorine administration failed to produce significant hypothermia in cyclosporine treated group as compared to control suggesting hypo activity of the cholinergic system.
Conclusion. Levamisole causes hyperactivity and cyclosporine causes hypo activity of central cholinergic system. Central cholinergic system hypo activity impairs and hyperactivity improves the learning and memory

Keywords: Cholinergic receptors, Immunomodulators, Learning, Memory

A substantial body of research indicates that the cen-tral cholinergic system plays an important role in the process of learning and memory [ 1 , 2 ]. Learning and memory process is not an independent process, but is influenced or modified by the immune system [ 3 ]. The past decades have witnessed the possible role of central nervous system (CNS) in the regulation of immune function and in turn the feedback from the immune sys-tem to the CNS. The interaction between these two sys-tems is suggested to be bi-directional influencing each other in a reciprocal manner [ 4 , 5 ]. Diminished intellec-tual functions and motor impairment are seen in patients with AIDS and psychiatric illnesses associated with immunological abnormalities [ 5 ]. Viral or cyclosporine induced immunosuppression is found to produce learn-ing and memory impairment [ 6 , 7 ]. Although a great deal of evidence supports that immunomodulators affect learning and memory, the exact mechanism that under-lies this effect is yet to be determined [ 8 , 9 ].

The present study was undertaken to determine cho-linergic receptor sensitivity following pharmacological modulation of immune system.

MATERIALS AND METHODS

Animals

Healthy male inbred albino rats of Wistar strain, weighing 300 ± 10 g, were used in the present study. The animals were housed in-groups of 3 or 4 per cage under an alternating 12-hour light: dark cycle (light on 6.00-18.00 hrs) with free access to food and water in humidity controlled environment. The study was conducted following Institutional ethical committee clearance.

Experimental Design

Animals were initially treated for a period of 8 days with cyclosporine/levamisole/saline. They were then subjected to passive avoidance training and 48 hrs later, the retention test for the passive avoidance task was conducted. Twenty-four hours after the retention tests the animals were subjected to test for cholinergic receptor sensitivity.

Assessment of Learning and Memory

Passive avoidance test. The two compartment pas-sive avoidance apparatus as described by Buresova and Bures was used [ 10 ]. Essentially the apparatus consists of a square box with a floor grid of 50 x 50 cm and wooden walls of 35 cm height. A 100 watts bulb illumi-nates this box. In the center of one of the walls is an opening of 6 × 6 cm which can be opened or closed using a transparent plexy glass sliding door. This open-ing leads to a smaller (15 × 15 cm) dark compartment provided with an electrified floor, that can be connected to a shock source (stimulator obtained from Hugo Sachs Electronics, Germany) having a maximum output of 100 mA. Ten animals each from control, cyclosporine and levamisole groups, selected at random were placed indi-vidually in an illuminated chamber facing away from the entrance to the dark compartment. The door was closed after the rat entered the dark compartment and 1mA foot shock was delivered for a period of 2 seconds. Then the animal was returned to home cage. 24 hours later each animal was placed again in the passive avoid-ance apparatus as before for a maximum period of 180 seconds. The latency time required for the animal to enter the dark compartment was measured. Animals not entering the dark compartment within this period re-ceived a latency time of 180 seconds. Absence of entry into the dark compartment indicated a positive retention.

Test for cholinergic receptor sensitivity. The ac-tivity of cholinergic receptor system was studied using oxotremorine (a synthetic, centrally active cholinomi-metic drug that stimulates central muscarinic receptors and used as an experimental tool) [ 11 ]. Animals in sa-line and drugs treated (cyclosporine and levamisole) group were given intraperitoneally 0.46, 1.84 or 4.61 mg/kg of oxotremorine salt (in a volume of 1mg/ml). Thirty minutes before the administration of oxot-remorine, methylscopolamine nitrate (Sigma Chemicals, St. Louis, US), and well known to be devoid of central effects was injected to all animals in the dose of 1 mg/kg in order to block the peripheral effects of oxot-remorine. Rectal temperature was recorded in all ani-mals just before the administration of oxotremorine (t₀ ) and thereafter at every 15 min for 120 min. the results were expressed as the mean difference in temperature between (t₀ ) and other time points.

Drug Schedule

The drugs, dose and number of animals per treat-ment are given in Table 1 . The dose for levamisole and cyclosporine was selected based on previous study [ 12 , 13 ].

Statistical Analysis

Statistical analysis of the variables of latency to enter the dark compartment (among cyclosporine, levami-sole and saline treated groups) was done by One-Way Analysis of Variance (ANOVA) and followed by post hoc Scheffé test using the SPSS computer package.

The mean difference in the rectal temperature be-tween time-zero (t₀) and other time points in drug and saline treated groups was subjected to Student's 't' test.

RESULTS

Passive Avoidance Test

There was no significant difference between the dif-ferent treatment groups (saline, cyclosporine and le-vamisole) in the initial latency to enter the dark com-partment in the passive avoidance paradigm. Therefore any differences seen subsequently are a reflection of differences in the retrieval and are not related to the initial baseline activities. Cyclosporine exhibited a sig-nificant reduction and levamisole exhibited a significant increased latency to enter the dark compartment as compared to control (saline) during the retention test (F(2, 27) = 10.70 at p < 0.01). Longer latency indicates a better retrieval of learned behavior ( Table 2 ).

Test for Cholinergic Receptor Sensitivity

The analysis of the effect of oxotremorine induced hypothermia indicated that levamisole treated animals exhibited an enhanced cholinomimetic-induced hypo-thermia as compared to control animals. Throughout the period of measurement isolated animals manifested a high sensitivity to all the 3 doses (0.46, 1.84, 4.61 mg/kg), whereas cyclosporine treated animals exhibited a decreased cholinomimetic-induced hypothermia as compared to control animals ( Table 3 , Table 4 , Table 5 ).

DISCUSSION

The present work was undertaken to study the sensi-tivity of cholinergic receptors in animals treated with immunostimulant and immunosuppressant drugs. To this end we measured the hypothermia induced by the muscarinic agonist, oxotremorine. We also assessed the passive avoidance memory in immunostimulant and immunosuppressant treated rats, since studies have shown that hypo activity of central cholinergic system impairs and hyperactivity improves passive avoidance memory [ 13].

In our study we observed that levamisole, a known immunostimulant exhibited an increased latency to enter the dark compartment as compared to the control rats during the retention test. The increased latency indicates a better retrieval of learned behavior and suggests facili-tation of learning and memory processes. On the other hand in the rats treated with cyclosporine, there is a de-crease in passive avoidance retention suggesting an ad-verse effect of immunosuppressant on learning and memory.

The administration of oxotremorine, a centrally ac-tive cholinomimetic drug exhibited an enhanced hypo-thermia in levamisole treated group as compared to con-trol group suggesting hyperactivity of the cholinergic system. This supports the earlier finding that hypother-mia is related to hyperactivity of cholinergic system [ 14 ].

On the other hand oxotremorine administration failed to produce significant hypothermia in cyc-losporine treated group as compared to control group suggesting hypo activity of the cholinergic system.

Several studies have suggested that increased cho-linergic activity results in improved passive avoidance memory and a decreased cholinergic activity with down regulation of the muscarinic receptors results in deficit in passive avoidance memory [ 13 , 15 ]. The human and animal observations suggest that the relation of the cho-linergic system to memory is due to the plasticity of cortical cholinergic synapses and the cholinergic de-pendence of limbic structures involved in the memory processes [ 16 ].

From the above discussion it is clear that immu-nostimulants (levamisole) increase cholinergic sensitiv-ity and enhance learning and memory. On the other hand immunosuppressants (cyclosporine) decrease cho-linergic sensitivity and adversely affect the cognitive functions. Therefore the altered cholinergic sensitivity observed in this study suggests a possible interaction between the cholinergic system, immune system and cognitive function.

The mnemonic effects of immunomodulators, medi-ated by central cholinergic system have an important role in the neurocognitive disturbances seen in-patients with Alzheimer's disease, AIDS and such condition re-lated to altered immunological states. However, the im-plication of the finding is yet to be assessed and has to be validated by a well-defined clinical trial.

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