| Baclofen
induced activation of GABAB receptors facilitate calcium current in low
concentrations and inhibit calcium currents in high concentrations
Kathryn
A. Gniotczynski
Marquette
University
Milwaukee,
WI
Mentor:
Dr. Michelle Mynlieff
GABA
is the main inhibitory neurotransmitter in the vertebrate central nervous
system, and are receptors involved in modulation. It has been previously
shown that activation of GABAB receptors has an inhibitory effect on synaptic
activity, and there is growing evidence that GABAB receptors are also involved
in a facilitory effect by up-regulating high-voltage activated calcium
channels. Since previous studies have shown two opposing effects of GABAB
receptor activation, there is the possibility that there could be two GABAB
receptor subtypes. Prior experiments in our lab showing facilitation and
inhibition were carried out using the GABAB agonist baclofen at a single
concentration of 10mM. Shen & Slaughter (1999) demonstrated that salamander
retinal cells also responded with both an inhibition and a facilitation
of calcium currents with the activation of GABAB receptors using baclofen.
However in their experiments a single cell had both effects which produced
a dose response curve showing inhibition of calcium currents at high concentrations
of baclofen (300mM) and at low concentrations (500nM) facilitation. The
objective of the present study was to determine whether a dual dose response
curve is also seen in hippocampal cells in the response to baclofen. Prior
to attempting a full dose response curve, it was necessary to address the
problem of rundown. Creatine an ATP regenerator was added to the internal
solution in an attempt to decrease rundown of calcium currents.
Whole-cell
voltage clamp recording was used in all experiments. The cultured hippocampal
neurons of 5 to 7 day old pups were kept at a holding potential of –80mV.
Depending on the experiment the cells were given depolarizing pulses in
the range of –50mV to +50mV to elicit calcium currents. Six cells were
tested for rundown, three were recorded from using the original solution,
and the other three were recorded from using the mixture of the original
and the new internal solution with creatine. Two components, peak and sustained,
of the calcium currents were analyzed and a rundown rate was calculated.
The original solution had a peak rundown rate of –11.1%/min ± 7.41
and a sustained of -6.54 ± 3.56. The currents of the cells using
the mixture of internal solutions had a peak of -7.53 ± 7.12 and
a sustained of -1.93 ± 5.28. There was no significant difference
in the rundown of either the peak or the sustained components between the
two internal solutions. The baclofen cell showed peak inhibition of 12.4%
at 1mM and peak facilitation of 18.6% at 500nM. The sustained component
only showed facilitation of 40.5% at a baclofen concentration of 500nM.
Although
the rundown data showed no significant difference between solutions the
standard deviation was too large to conclude anything. The baclofen data
is promising and follows the suspected hypothesis that there is a dual
dose response curve in hippocampal neurons. |
