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Faisal Khan and Kaneez Fatima ShadDr. Panjwani Center for Molecular Medicine and Drug Research, ICCBS, University of Karachi
Results
MATERIAL AND METHODS
ABSTRACTNew neurons are formed continuously throughout life in mammalian dentate gyrus region of hippocampus that contains neuronal precursor cells. These new cells are partially responsible in the generation of new memory. Many factors (such as alcohol) results in a dramatic decrease in cell proliferation in the dentate subgranular zone (SGZ) and impaired memory.We have investigated the role of A-type K currents during alcohol induced decrease in neurogenesis and memory impairment. Adult Sprague-Dawley rats weighing 15010 g were used for the experiments after getting the animal ethic approval from PCMD, University of Karachi. Animals were divided into 2 groups: the control group and the alcohol-treated group. Animals of the alcohol-treated groups were injected intraperitoneally with alcohol (2 g/kg) once a day for 3 days. Treatment with alcohol for 3 days inhibited cell proliferation. Animals were scarified right after testing their memory in T-maze. Slices (40µm) of hippocampus were labeled with proliferating cell nuclear antigen (PCNA) as to observe and compare the extent of the cell proliferation in alcohol treated group with that of control. Patch clamp technique was used in whole cell configuration mode to observe A type K+ currents in the granule cells of each group.Our initial observations indicated that alcohol increases the amplitude and frequency of A-type K+ currents and decreases the proliferation (neurogenesis) of granule cells in hippocampus and impaired the memory. We concluded that increase in A type K+ currents may results in decrease signals for cell proliferation in dentate subgranular zone and impaired memory in adult rats.
INTRODUCTION
Effect of alcohol on A-type K+ currents induced neurogenesis and resultant memory impairment in adult rats
Alcohol TreatmentAnimals were injected with alcohal 2g/kg. Once per day for 3 days and were sacrified 2 hours after last injection
Patch Clamp recordingsCells continuously perfused with ACSF containing (in mM): 140 NaCl, 5.6 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, 10 glucose, and pH 7.3, and osmolarity adjusted to 325±5 mOsml. Patch electrodes with a resistance of 3 to 5 MΩ were pulled on a Brown-Flaming P-97 electrode puller, fire-polished and filled with a solution containing (in mM): 145 KCl, 10 EGTA, 2 MgCl2, 2 CaCl2 and 5 HEPES, pH 7.2, osmolarity adjusted to 290±10mOsm. Patch clamp recordings were carried out using an HEKA amplifier.
This research was supported by a grant from Higher Education Commission, Pakistan.
For further informationPlease contact; [email protected].
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Electrophysiology Lab, Dr. Punjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi, Pakistan.
Memory TestEach rat was trained for 5 days to run to the ends of the arms for a food reward During the first day, food was liberally spread throughout the maze; each rat was allowed 20 min to explore. During each successive day, the amount of food was progressively restricted so that, by the fifth day, food was present only in the food cups. The rats were given no food in their home cages.
ImmunohistochemistryAnimals deeply anaesthetized with ketamine and transcardially perfused with 0.1 m phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. The brains were then cut 40-µm thick by cryostate.The tissue was incubated with anti-mouse PCNA (1:250, DAKO) overnight at 4oC following washes in TBS, sections were incubated in the dark for 1 h in fluorescent-coupled secondary antibodies (goat anti-mouse texas red) plus 1.5% goat serum. After sections were mounted and dried, they were coverslipped with mounting medium.
Drink Alcohol Less neuronal stimulation
Decrease neuron generation
Less neuronal integration
Problems in memory and leaning
Normal Potassium Currents
Effect of alcohol on Potassium Currents
Control Alcoholic rats showing less neurogenesis
ConclusionAlcohol increase the potassium current amplitudeIncreased potassium currents may responsible to reduce proliferation of neural stem cells.Decrease proliferation disturb the ability to learn and impair the memory.
Effect of alcohol on proliferation