The present study was designed to demonstrate the potential effect of CGP 35348 (GABAB receptor antagonist) on the learning, memory formation, and neuromuscular coordination in albino mouse. Mice were intrapertoneally injected with 1 mg CGP 35348/mL of distilled water/Kg body weight, while the control animals were injected with equal volume of saline solution. A battery of neurological tests was applied following the intrapertoneal injections. Results of rota rod indicated that CGP 35348 had no effect on neuromuscular coordination in both male (
Glutamate and gamma-aminobutyric acid (GABA) are among the most abundant neurotransmitters in our central nervous system. Glutamate play role in excitatory responses while the GABA act as inhibitory stimuli [
Two pharmacologically and molecularly distinct GABA receptors have been identified, GABAA and GABAB. GABAB receptors are heterodimeric G protein-coupled sites, located both pre- and postsynaptically [
Eight-week old albino mice (
On the 20th day of life, mice were separated from their parents and fed on normal mouse diet until 9th week of life when they received intraperitoneal injections of GABAB receptor antagonist (CGP35348, 3-Aminopropyl diethoxymethyl phosphinic acid) or saline solution for 5 days at the rate of 1 mg/1 mL solvent/Kg body weight/day 30 minutes prior to behavioural testing.
The rota rod appartaus test balance and coordination and comprised of a rotating drum which rotated at the speed of 40 rpm. The time at which each animal fell from the drum was recorded. Each animal received three pretraining trials. Subsequently, each mouse completed three more consecutive trials and the longest time on the drum was used for analysis [
Mice were observed using a video monitoring system consisting of a video camcorder coupled to computational tracking system (Any-Maze, USA). Standard parameters for locomotors activity (i.e., total distance covered, average speed, amount of large movement, amount of local movement, resting time, and frequency of spontaneous change in direction) and exploratory behaviour (i.e., rearing, crossing the center, and time spent in the margin) recorded [
MWM consists of a circular pool (122 cm diameter, 76 cm deep) in which mice were trained to escape from water by swimming to a hidden platform (1.5 cm beneath water surface) whose location could be identified using distal extra-maze cues attached to the room walls. Visual cues had different colors and dimensions and kept constant during the whole experiment.
The pool was divided into four quadrants (compass locations, NE, NW, SW, and SE) by a computerised tracking/image analyser system coupled to computational tracking system. The platform was placed in the middle of the NE quadrant and remained at the same position during the whole experiment.
The spatial acquisition phase consisted of 16 training trials and 4 training trials per day for 4 days with an intertrial interval of 15 min. Mice were released randomly with their heads facing the pool wall from the four compass locations, and allowed to swim and search for the platform for 120 s. When mice did not locate the platform within 120 s, animals were manually placed on the platform and allowed to remain on it for another 30 s.
On day 5, after the acquisition phase, subject received a probe trial, in which the platform was removed. Mice were released from the south start point and were allowed to swim freely for 60 s. Parameters like time and path length to reach the platform area, number of times crossing the platform area, swimming strategies to reach platform area and swimming speed were recorded [
All data are expressed as mean ± standard deviation. For all the studied parameters of open field, rota rod MWM training, and probe trial parameters, two-sample
Results indicated that the CGP 35348 injection did not affect the neuromuscular coordination in male albino mice (
Comparison of rota rod test results control male/female with their respective GABAB receptor antagonist (CGP 35348) treated male and female albino mice. Data is expressed as mean ± standard deviation.
There were interesting gender specific results regarding the open field test as all the studied parameters remained insignificantly different when compared between CGP 35348 and saline treated male mice (data not shown here). On the other hand, CGP 35348 treated females had demonstrated poor exploratory behavior as compared to saline treated females for several parameters (time mobile (
Comparison of the studied open field parameters between saline and CGP 35348 treated female albino mice.
Parameters | Control female ( |
CGP 35348 treated female ( |
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Distance (m) |
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0.072 |
Mean speed (m/sec) |
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0.071 |
Time mobile (sec) |
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Time immobile (sec) |
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Mobile episodes |
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0.979 |
Immobile episodes |
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0.979 |
Max speed (m/sec) |
|
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0.158 |
Rotations |
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Clockwise rotations |
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Anticlockwise rotation |
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Acquisition phase of MWM revealed gender specific results. For male albino mouse, total distance travelled (
Comparison of total distance (m) travelled by GABAB antagonist and saline treated male albino mouse during acquisition phase of Morris water maze test. Data is expressed as mean ± standard deviation.
Comparison of time mobile (sec) by GABAB antagonist and saline treated male albino mouse during acquisition phase of Morris water maze test. Data is expressed as mean ± standard deviation.
Comparison of mean speed by GABAB antagonist and saline treated female albino mouse during acquisition phase of Morris Water Maze test. Data is expressed as mean ± standard deviation.
During probe trial, CGP 35348 treated male mice performed significantly better. They took shorter latency (
Comparison of total latency between GABAB antagonist and saline treated male albino mouse during probe trial of Morris Water Maze test. Data is expressed as mean ± standard deviation.
Comparison of time mobile between GABAB antagonist and saline treated male albino mouse during probe trial of Morris Water Maze test.
Results of swimming strategies during training days of MWM indicated that both CGP 35348 treated female and male albino mice had demonstrated improved memory formation as the direct and focal approach to the hidden platform increased, while random swimming and wall hugging decreased our the training period. Effect was more pronounced in treated male as the chaining and wall hugging strategies eliminated as the training proceeded indicating improved memory formation complementing the results of probe trial (Figures
Swimming strategies of control and CGP 35348 treated female albino mouse during acquisition phase of Morris Water Maze test.
Swimming strategies of control and CGP 35348 treated male albino mouse during acquisition phase of Morris Water Maze test.
In the central nervous system, GABAB receptor regulates cyclic AMP (cAMP) levels through adenylyl cyclase activity. Activation of the cAMP regulatory pathway is vital for long-term memory formation across a variety of species [
The rota rod test is widely used to determine the motor coordination in rodents [
While analyzing the exploratory behavior in mice through open field test, various parameters in the open field test were considered. Our results indicated gender specific effect of CGP 35348 injections as treated females had demonstrated poor exploratory behavior as compared to saline injected females for several parameters (time mobile (
In order to test hippocampal-dependent learning, including acquisition of spatial memory and learning for albino mouse, the Morris water maze (MWM) was applied. The CGP 35348 treatment led to the improvement of learning during the acquisition phase and significantly improved memory formation during probe trail in male albino mouse as they found the platform earlier and had shorter latency as compared to the saline treated male mice (Figures
Various doses of CGP 35348 were applied [
We concluded that CGP 35348 has a potential to improve the various aspects of behavior in a gender specific manner in albino mice. It has improved the memory formation of male mice during retention phase of MWM, but has negatively affected the exploratory behavior of female albino mice, while the rota rod test remained unaffected in both genders. Repetition of these tests following the application of higher doses of CGP 35348 would reveal more interesting results.
The authors declare that they do not have conflict of interests of any sort.
Quratul Aain Gillani and Shahid Iqbal contributed equally to this paper.
The Authors are thankful to Higher Education Commission of Pakistan for funding this project through indigenous Ph.D. scholarship scheme.