Korean J Physiol Pharmacol.  2017 Nov;21(6):695-702. 10.4196/kjpp.2017.21.6.695.

Developmental changes in GABAA tonic inhibition are compromised by multiple mechanisms in preadolescent dentate gyrus granule cells

• Pandit S
• Lee GS
• Park JB

• KMID: 2395265
• DOI: http://doi.org/10.4196/kjpp.2017.21.6.695

Abstract

The sustained tonic currents (I(tonic)) generated by γ-aminobutyric acid A receptors (GABA(A)Rs) are implicated in diverse age-dependent brain functions. While various mechanisms regulating I(tonic) in the hippocampus are known, their combined role in I(tonic) regulation is not well understood in different age groups. In this study, we demonstrated that a developmental increase in GABA transporter (GAT) expression, combined with gradual decrease in GABA(A)R α₅ subunit, resulted in various I(tonic) in the dentate gyrus granule cells (DGGCs) of preadolescent rats. Both GAT-1 and GAT-3 expression gradually increased at infantile (P₆₋₈ and P₁₃₋₁₅) and juvenile (P₂₀₋₂₂ and P₂₇₋₂₉) stages, with stabilization observed thereafter in adolescents (P₃₄₋₃₆) and young adults (P₄₁₋₄₃). I(tonic) facilitation of a selective GAT-1 blocker (NO-711) was significantly less at P₆₋₈ than after P₁₃₋₁₅. The facilitation of I(tonic) by SNAP-5114, a GAT-3 inhibitor, was negligible in the absence of exogenous GABA at all tested ages. In contrast, I(tonic) in the presence of a nonselective GAT blocker (nipecotic acid, NPA) gradually decreased with age during the preadolescent period, which was mimicked by I(tonic) changes in the presence of exogenous GABA. I(tonic) sensitivity to L-655,708, a GABA(A)R α₅ subunit inverse agonist, gradually decreased during the preadolescent period in the presence of NPA or exogenous GABA. Finally, Western blot analysis showed that the expression of the GABA(A)R α₅ subunit in the dentate gyrus gradually decreased with age. Collectively, our results suggested that the I(tonic) regulation of altered GATs is under the final tune of GABA(A)R α₅ subunit activation in DGGCs at different ages.

Keyword

Ages; Dentate gyrus granule cells; Extrasynaptic GABAA receptors; GABA transporter

MeSH Terms

Adolescent
Animals
Blotting, Western
Brain
Dentate Gyrus*
gamma-Aminobutyric Acid
Hippocampus
Humans
Rats
Young Adult
gamma-Aminobutyric Acid

Figure

• 

  Fig. 1 Expression of GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3) in the dentate gyrus at different ages. Western blot analysis showing neuronal GAT-1 (A) and glial GAT-3 expression (B) in the dentate gyrus at different postnatal days (P). The expression was normalized to the level detected at P7 and compared with the expression at each age group (n=5), shown by the bar graphs. **p<0.01 compared with P7 expression.

• 

  Fig. 2 Effects of NO-711 and SNAP-5114 on Itonic at different ages. (A) Representative current traces of dentate gyrus granule cell (DGGC) showing the effect of NO-711 (5 mM), a GAT-1 blocker. Note that the effects of NO-711, which were blocked by bicuculline (BIC), were smaller at P6-8 than in older age. Mean changes in the holding current (Iholding) after NO-711 application are summarized in the bar graph. (B) Representative current traces showing the effect of NO-711 in the presence of GABA (1 µM). Mean Iholding changes after the application of GABA and GABA+NO-711 are summarized in the bar graph. (C) Representative current traces showing the effect of SNAP-5114 (300 µM), a GAT-3 blocker, in the presence of NO-711 (5 µM). Note that the effects of SNAP-5114 were smaller at P6-8 than in older age. Mean changes in Iholding after by the sequential application of NO-711 and NO-711+SNAP-5114 (100 or 300 µM) are summarized. (D) Representative current traces showing the effect of SNAP-5114 (100 µM) in the presence of GABA (1 µM). Mean changes in Iholding after the application of GABA and GABA+SNAP-5114 (100 µM) are shown in the bar graph. Summarized data are shown as means±standard error of the mean (SEM) (n=5~6). *p<0.05, **p<0.01 compared with P6–8.

• 

  Fig. 3 Effects of nipecotic acid (NPA) on Itonic according to age. (A) Representative current traces before and during the sequential application of NPA (100 µM), a nonselective GAT blocker, and NPA+L-655,708, an inverse agonist of the GABAA receptor α5 subunit. Note that the effects of NPA, which were blocked by BIC, were larger at P6–8 than in older age. (B) Mean changes in the inward Iholding shift after NPA (INPA), and the outward Iholding shift following additional application of L-655,708 (IL,655–708), are summarized. Summarized data are shown as means±SEM (n=6~7). *p<0.05, **p<0.01 compared with P6–8.

• 

  Fig. 4 Age-dependent decrease of GABAAR α5 subunit expression in preadolescent rats. (A) Representative current traces before and after the sequential application of GABA (5 µM) and GABA+L-655,708, an inverse agonist of the GABAA receptor α5 subunit. (B) Mean changes in the inward Iholding shift by GABA and outward Iholding shift following additional application of L-655,708 (IL,655–708) are summarized. Summarized data are shown as means±SEM (n=6~7). *p<0.05, **p<0.01 **, ***p<0.001 compared with P6–8. (C) Representative Western blot analysis showing GABAAR α5 subunit expression at different ages. (D) Summarized GABAAR α5 subunit expression at different ages. The protein expression was normalized to the level detected at P21. Summarized data are shown as means±SEM (n=4).*p<0.05, **p<0.01, ***p<0.001 compared with P21.

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