Hyperpolarizing inward current?

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iamstupid's picture
Hyperpolarizing inward current?

I'm one experiment away from finishing up my graduate work, but my PI has mentally left the building and I need help!  Here is the simplified run-down of my problem:

I did a study using primarily loose-patch recordings and found that a particular drug reversibly decreases the firing rate in my cells (a particular type of neuron in brain slices).  I am now trying to identify what type of intrinsic conductances are altered by this drug to inhibit firing.  Holding cells at -60 which is close to the reported average Vrest for my cell type, application of the drug induces an inward current of ~20 pA.  Running a step protocol and generating an IV plot of steady-state current at various holding potentials indicates that the reversal potential of the drug-induced current is around -50 mV.  How on earth is this consistent with the cell's firing rate going down?

These are the possibilities I came up with:
1.) Vrest is actually greater than -50 mV since I am using brain slices from mice undergoing different treatments than those used to characterize these cells previously, and I am using a different extracellular glucose concentration (lower - though that should hyperpolarize the cells since they are glucose-excited neurons)
2.) Something about the whole-cell configuration is altering the response to the drug.  Either the concentration of ions in the internal solution (chloride, for instance) is reversing the normal direction of ion flow, or I am dialyzing out an important component (e.g. 2nd messenger) required to get the normal response
3.) Shunting inhibition?  But is any reviewer going to buy that???

Any thoughts/input on this would be appreciated.  The 7 year grad student mark is rapidly approaching, and I ned to get out of here! Thanks!!!