Developmental changes in GABAergic mechanisms in human visual cortex across the lifespan

Abstract

Functional maturation of visual cortex is linked with dynamic changes in synaptic expression of GABAergic mechanisms. These include setting the excitation-inhibition balance required for experience-dependent plasticity, as well as, intracortical inhibition underlying development and aging of receptive field properties. Animal studies have shown that there is developmental regulation of GABAergic mechanisms in visual cortex. In this study, we show for the first time how these mechanisms develop in the human visual cortex across the lifespan. We used Western blot analysis of postmortem tissue from human primary visual cortex (n = 30, range: 20 days to 80 years) to quantify expression of eight pre- and post-synaptic GABAergic markers. We quantified the inhibitory modulating cannabinoid receptor (CB1), GABA vesicular transporter (VGAT), GABA synthesizing enzymes (GAD65/GAD67), GABA(A) receptor anchoring protein (Gephyrin), and GABA(A) receptor subunits (GABA(A)alpha1, GABA(A)alpha2, GABA(A)alpha3). We found a complex pattern of different developmental trajectories, many of which were prolonged and continued well into the teen, young adult, and even older adult years. These included a monotonic increase or decrease (GABA(A)alpha1, GABA(A)alpha2), a biphasic increase then decrease (GAD65, Gephyrin), or multiple increases and decreases (VGAT, CB1) across the lifespan. Comparing the balances between the pre- and post-synaptic markers we found three main transition stages (early childhood, early teen years, aging) when there were rapid switches in the composition of the GABAergic signaling system, indicating that functioning of the GABAergic system must change as the visual cortex develops and ages. Furthermore, these results provide key information for translating therapies developed in animal models into effective treatments for amblyopia in humans.

Keywords: GABA; aging; development; human; inhibition; plasticity; visual cortex.

Figure 1

Developmental changes in GAD65 (A,B) and GAD67 (C,D) expression in human visual cortex across the lifespan plotted relative to the youngest age group (<1 year) for the grouped means and scattergrams (● average expression for each case; all runs). Example blots for each case are presented above the scattergrams (B,D). The arrow indicates the band that was quantified. A weighted average curve was fit to each scatter plots using the locally weighted least squares method at 50% (dotted line). GAD65 showed a significant change across the lifespan (Kruskal–Wallis, p < 0.0001) with progressive increase in expression levels into the teenage and adult years then a loss with aging (A,B). There was no change in expression levels of GAD67 across the lifespan (C,D). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001).

Figure 2

Quantification of changes in expression levels of CB1 (A,B) and VGAT (C,D) in human visual cortex across the lifespan. The graphs follow the same conventions as described in Figure 1. CB1 levels are high in infants (<1 year) then fall into young children (1–2 years) before rising again in pre-teens (5–11 years) and falling again in teens (12–20 years) and leveling off (Kruskal–Wallis, p < 0.0001) (A,B). For VGAT 2 bands were quantified (arrows at 50 and 57 kDa). VGAT levels are highest in infants (<1 year) then gradually fall into children (3–4 years) before rising again into pre-teens (5–11 years) then falling into adults (21–55 years) before finally rising again in older adults (>55 years) (Kruskal–Wallis, p < 0.005) (C,D). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001) (● average expression for each case; all runs; dotted line is the weighted average).

Figure 3

The index of GAD65:VGAT (A,B) gives insight into the relative balance between production and trafficking of GABA. The balance is in favor of VGAT early (<11 years) and late (>55 years) in development, but there is much more GAD65 during the teenage to adult years (12–55 years) (Kruskal–Wallis, p < 0.0002) (A,B). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001) (● average expression for each case; all runs; dotted line is the weighted average).

Figure 4

Changes in expression of GABA_A receptor subunits GABA_Aα1 (A,B), GABA_Aα2 (C,D), and GABA_Aα3 (E,F) during postnatal development. An exponential decay function (solid line) was fit to the expression levels for GABA_Aα1 and GABA_Aα2 and the time constants (τ) were calculated with adult levels defined as 3τ. GABA_Aα1 expression increased until early teens when it leveled off (3τ = 13.5 years, τ = 4.50 years, R = 0.67; p < 0.0001; B). GABA_Aα2 expression decreased until older childhood when it leveled off (3τ = 10 years of age, τ = 3.34 years, R = 0.96; p < 0.0001; D). GABA_Aα3 expression levels did not change significantly throughout the lifespan (E,F) (dotted line is the weighted average). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001) (● average expression for each case; all runs).

Figure 5

The indices of GABA_Aα1:GABA_Aα2 (A,B) and GABA_Aα1:GABA_Aα3 (C,D) are important for determining the kinetics and maturation of the GABA_A receptor. The GABA_Aα1:GABA_Aα2 index starts in favor of GABA_Aα2, then shifts to more GABA_Aα1 in children (3–4 years), reaching the adult balance in younger childhood (3τ = 4.5 years of age, τ = 1.50 years, R = 0.84; p < 0.0001; B). The GABA_Aα1: GABA_Aα3 index starts out with more GABA_Aα3 very early, then quickly shifts to slightly more GABA_Aα1, reaching adult levels within the first year (3τ = 0.69 years of age, τ = 0.23 years, R = 0.65; p < 0.0001; D). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001) (● average expression for each case; all runs; solid lines are exponential decay functions).

Figure 6

Changes in expression of Gephyrin (A,B) and the index of Gephyrin:GAD65 (C,D) during postnatal development. Expression levels of Gephyrin are lowest in infants (<1 year) and older adults (<55 years) and higher for the rest of the lifespan (Kruskal–Wallis, p < 0.005) (A). The Gephyrin:GAD65 index shows a shift from more GAD65 in infants (<1 year) to more Gephyrin in children (1–11 years) to a balance from the teenage to adult years (12–55) then a switch to much more GAD65 in aging (>55 years) (Kruskal–Wallis, p < 0.008) (C). The lines above the histograms identify the groups that were significantly different (Tukey's post hoc HSD, *p < 0.05, **p < 0.02, ***p < 0.001) (● average expression for each case; all runs; dotted line is the weighted average).