Cortical development of AMPA receptor trafficking proteins

Abstract

AMPA-receptor trafficking plays a central role in excitatory plasticity, especially during development. Changes in the number of AMPA receptors and time spent at the synaptic surface are important factors of plasticity that directly affect long-term potentiation (LTP), long-term depression (LTD), synaptic scaling, and the excitatory-inhibitory (E/I) balance in the developing cortex. Experience-dependent changes in synaptic strength in visual cortex (V1) use a molecularly distinct AMPA trafficking pathway that includes the GluA2 subunit. We studied developmental changes in AMPA receptor trafficking proteins by quantifying expression of GluA2, pGluA2 (GluA2serine880), GRIP1, and PICK1 in rat visual and frontal cortex. We used Western Blot analysis of synaptoneurosome preparations of rat visual and frontal cortex from animals ranging in age from P0 to P105. GluA2 and pGluA2 followed different developmental trajectories in visual and frontal cortex, with a brief period of over expression in frontal cortex. The over expression of GluA2 and pGluA2 in immature frontal cortex raises the possibility that there may be a period of GluA2-dependent vulnerability in frontal cortex that is not found in V1. In contrast, GRIP1 and PICK1 had the same developmental trajectories and were expressed very early in development of both cortical areas. This suggests that the AMPA-interacting proteins are available to begin trafficking receptors as soon as GluA2-containing receptors are expressed. Finally, we used all four proteins to analyze the surface-to-internalization balance and found that this balance was roughly equal across both cortical regions, and throughout development. Our finding of an exquisite surface-to-internalization balance highlights that these AMPA receptor trafficking proteins function as a tightly controlled system in the developing cortex.

Keywords: AMPA receptor; GRIP; PICK1; critical period; frontal cortex; synaptic plasticity; trafficking; visual cortex.

Figure 1

Development of AMPA receptor subunits GluA2 and pGluA2 in visual and frontal cortex. There were significant differences in the developmental trajectories of GluA2 and pGluA2 in visual versus frontal cortex. All of the results were plotted for each run (light gray symbols) as well as the averages for each run (dark symbols). Exponential decay curves were fit to the data for visual cortex. (A) For GluA2 expression (R² = 0.72, p < 0.0001) adult expression levels were reached at P38 (3τ); (B) pGluA2 expression (R² = 0.68, p < 0.0001) reached adult levels by P36 (3τ). The results for frontal cortex were fit with a membrane transport function. (C) In frontal cortex: GluA2 expression (R² = 0.77, p < 0.0001) had the maximum level at P24 (range P18–P35); (D) pGluA2 expression (R² = 0.79, p < 0.0001) had the maximum level at P24 (range P19 and P32).

Figure 2

Developmental changes in AMPA receptor subunit composition in visual and frontal cortex. (A) GluA2 and pGluA2 expression in both visual (r = 0.88, p < 0.0001) and (C) frontal (r = 0.94, p < 0.0001) cortex was highly correlated during development. The index of GluA2:pGluA2 expression during development showed that (B) in visual cortex, there was higher GluA2 expression initially, but a balance was reached by P33 (3τ, R² = 0.37, p < 0.0005). (D) In frontal cortex, the index remained in favor of relatively more GluA2 throughout development, except for a brief period of balance between P18 and P30 (membrane transport curve, R² = 0.30, p = 0.04).

Figure 3

Development of GRIP and PICK1 in visual and frontal cortex. The developmental trajectories for GRIP and PICK1 were similar in both cortical areas. Exponential decay curves were fit to all the data. (A) In visual cortex, GRIP expression levels increased by 1.7 times and reached adult levels by P60 (3τ, R² = 0.33, p = 0.002). (B) PICK1 expression levels increased three-fold and reached adult values by P69 (3τ, R² = 0.32, p = 0.002). (C) In frontal cortex, GRIP levels increased three-fold and reached adult levels by P56 (3τ, R² = 0.61, p < 0.0001). (D) Similarly, PICK1 expression increased three-fold during development and adult level was attained by P58 (3τ, R² = 0.63, p < 0.0001).

Figure 4

Developmental changes in GRIP and PICK1 in visual and frontal cortex. (A) GRIP and PICK1 expression in visual cortex was highly correlated during development (r = 0.72, p < 0.0001). (B) The index of GRIP:PICK1 expression showed that the two proteins were balanced during postnatal development. (C) GRIP and PICK1 expression in frontal cortex was correlated during development (r = 0.48, p < 0.0001). (D) The index showed that GRIP and PICK1 develop in balance in frontal cortex.

Figure 5

Development of surface and internalized components in visual and frontal cortex. (A) Index of GluA2:GRIP during development in visual cortex showed that initially more GRIP was present, but by P13, a balance with slightly more GluA2 was reached (3τ, R² = 0.62, p < 0.0001). (B) Index of pGluA2:PICK1 in visual cortex showed that more PICK1 was present early in development, but a balance was reached by P22 (3τ, R² = 0.69, p < 0.0001). (C) In frontal cortex, index of GluA2:GRIP was in favor of GRIP before P10, followed by an increase in GluA2 expression between P10 and P40, and then another shift to relatively more GRIP (membrane transport curve: R² = 0.74, p < 0.0001). (D) Index of pGluA2:PICK1 was in favor of more PICK1 before P13, more pGluA2 between P13 and P34, and then another shift to relatively more PICK1 that persists throughout development.

Figure 6

Development of AMPA receptor in visual and frontal cortex. (A) There was a strong correlation between the indices for GluA2:GRIP and pGluA2:PICK1 in both visual (green symbols) (r = 0.61, p < 0.0001) and frontal cortex (red symbols) (r = 0.81, p < 0.0001). (B) The difference between the indices for GluA2:GRIP and pGluA2:PICK1 as a function of age showed a tight surface-to-internalization balance throughout development.