Abstract
The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.
Highlights
We have addressed the following questions: (1) whether equilibrium potential for GABA-A receptor mediated currents (EGABA) is shifted following Theta-burst stimulation (TBS) in neonatal hippocampal CA1 neurons and, if any, whether KCC2 or NKCC1 is responsible for this shift; (2) are Metabotropic glutamate receptors (mGluRs) involved in TBS-induced changes in KCC2 or NKCC1 activity and EGABA in both juvenile and neonatal hippocampal neurons; (3) which mGluR subtype is involved in regulation of KCC2 and/or NKCC1 activity following TBS in rat hippocampal neurons?
When the IPSC is evoked at 0.05 Hz, its amplitude as well as EGABA were stable during a 45 min recording period (n = 6; Fig 1); the records taken at 5–45 min were not significantly different from the first one (0 time)
EGABA was not changed in the presence of gramicidin D during the observation period (0 min: -56.1±0.8, 30 min: -56.6±1.0 mV, n = 6; p>0.05) in another set of experiment, suggesting that shifts in EGABA were not caused by gramicidin but TBS conditioning
Summary
Hippocampal acute slices were prepared from postnatal day 9–12 or 3–5 day old male Wistar rats (Animal Care Centre, The University of British Columbia) following a protocol approved by Canadian Council on Animal Care, and maintained as previously described [2]. Electrodes (glass capillaries) with a resistance of ~5 MO were tip filled with intracellular solution (ICS) and backfilled with the same pipette solution used in whole cell patch clamp recordings but containing 50 μg/ml gramicidin D (diluted from a stock solution of 50 mg/ml in DMSO). After getting the whole-cell configuration, control responses were recorded for 10 min following a 10–20 min initial stabilization period. Perforated patch recordings is generally thought to clamp the intracellular Clgradient and yield a more accurate calculation of EGABA, this technique does not allow the intracellular application of drugs or agents (such as NKCC1 sense or antisense mRNA, used in the current study). Since EGABA and the amplitude of GABA-ergic IPSC are stable when the IPSC is evoked at 0.05 Hz using the whole-cell patch clamp recording method ([3]; Fig 1), it is employed in the current study. A total of 93 slices from 62 animals were used for this study
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