Neonatal development of the rat visual cortex: synaptic function of GABAA receptor alpha subunits

Abstract

Each GABA(A) receptor consists of two alpha and three other subunits. The spatial and temporal distribution of different alpha subunit isomeres expressed by the CNS is highly regulated. Here we study changes in functional contribution of different alpha subunits during neonatal development in rat visual cortex. First, we characterized postsynaptic alpha subunit expression in layer II-III neurons, using subunit-specific pharmacology combined with electrophysiological recordings in acutely prepared brain slices. This showed clear developmental downregulation of the effects of bretazenil (1 microm) and marked upregulation of the effect of 100 nM of zolpidem on the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the concentrations used we interpret this as downregulation of the synaptic alpha3 and upregulation of alpha1 subunit. Furthermore, the effect of furosemide, being indicative of the functional contribution of alpha4, was increased between postnatal days 6 and 21. Our second aim was to study the effects of plasticity in alpha subunit expression on decay properties of GABAergic IPSCs. We found that bretazenil-sensitive IPSCs have the longest decay time constant in juvenile neurons. In mature neurons, zolpidem- and furosemide-sensitive IPSCs have relatively fast decay kinetics, whereas bretazenil-sensitive IPSCs decay relatively slowly. Analysis of alpha1 deficient mice and alpha1 antisense oligonucleotide deletion in rat explants showed similar results to those obtained by zolpidem application. Thus, distinct alpha subunit contributions create heterogeneity in developmental acceleration of IPSC decay in neocortex.

Figure 1. Development of postsynaptic GABAergic currents

Colour coded histograms of peak current vs. τ_decay of all sIPSCs at different stages of neocortical development. The percentage of sIPSCs with a particular peak current- τ_decay combination per bin is indicated by a colour coding (black, no sIPSCs; white, highest number of sIPSCs). Number of neurons used per group: pn6, n = 60; pn11, n = 24; pn14, n = 33; pn21, n = 119 and pn35, n = 8.

Figure 2. The contribution of the zolpidem-sensitive, postsynaptic GABA_A receptors increases during neonatal development

A, example sIPSCs aligned to their rising phases. B, the same as A, but in the presence of 100 nm zolpidem. C, the effect of zolpidem increases with development. τ_decay histograms of equal numbers of sIPSCs pooled from all experiments before (♦) and after (⋄) zolpidem application. Insets: average sIPSCs, control (thick line) and in the presence of zolpidem (thin line), normalized to peak current. D, the fraction of zolpidem-sensitive neurons increases between pn6 and pn21. Some neurons showed a significant but relatively small increase in τ_decay (hatched area). * P < 0.05 increase in the fraction of zolpidem-sensitive neurons (Fisher's exact test). E, average increase of τ_decay upon zolpidem application in all neurons (open bar) and in those neurons that showed a significant τ_decay increase (filled bar). * P < 0.05 as compared to pn6 (Student's t test).

Figure 3. The contribution of the bretazenil-sensitive, postsynaptic GABA_A receptors decreases during neonatal development

A, example sIPSCs aligned to their rising phases. B, the same as A, but in the presence of 1 μm bretazenil. C, the effect of bretazenil increases with development. Histograms of equal numbers of sIPSCs pooled from all experiments before (♦) and after (⋄) bretazenil application. Insets: average sIPSCs, control (thick line) and in the presence of bretazenil (thin line), normalized to peak current. Note that at pn21 both lines overlap. D, the fraction of bretazenil-sensitive neurons decreases during neonatal development. Some pn21 neurons showed a significant but relatively small increase of τ_decay (hatched area). #P < 0.1 for the decrease in fraction of bretazenil-sensitive neurons (Fisher's exact test). E, average increase of τ_decay upon bretazenil application in all neurons (white bar) and in those neurons that showed a significant τ_decay increase (black bar). *** P < 0.001 as compared to pn6 (Student's t test).

Figure 4. The contribution of furosemide-sensitive, postsynaptic GABA_A receptor increases during neonatal development

A, example sIPSCs aligned to their rising phases. B, the same as A, but in the presence of 100 μm furosemide. C, the effect of furosemide increases with development. Peak current histograms of equal numbers of sIPSCs pooled from all experiments before (♦) and after (⋄) furosemide application. Insets:average sIPSCs, control (thick line) and in the presence of furosemide (thin line). D, the fraction of neurons displaying a decrease of peak current upon furosemide application increases between pn6 and pn21. Some neurons show a significant but relatively small decrease of peak current (hatched area). * P < 0.05 for increase in fraction of furosemide-sensitive neurons (Fisher's exact test). E, average decrease of peak current upon furosemide application in all neurons (open bar) and in those neurons that showed a significant peak current decrease (filled bar). ** P < 0.01 as compared to pn6 (Student's t test). F, there is no difference in the fraction of neurons that show a decrease of sIPSC frequency upon furosemide application (Fisher's exact test). In order to calculate the sIPSC frequency, dendritically filtered sIPSCs were also used. G, average decrease in sIPSC frequency upon furosemide application in all neurons (open bar) and in those neurons that showed a significant decrease in sIPSCfrequency (filled bar). The small increases in frequency upon furosemide application are not statistically different from no effect (Student's t test). There were no significant changes between pn6 and pn21 (Student's t test).

Figure 5. The contribution of the different, pharmacologically classified GABA_A receptor types to the overall sIPSC kinetics of neurons

Aa-Ac, the average (± s.d.) τ_decay of all neurons tested (filled symbols: pn6; open symbols: pn21) before ligand application versus the pharmacogical effect (as a percentage of control; average ± s.d.). The parameters used for the pharmacological effects were: τ_decay (for zolpidem to assess the role of α1 and bretazenil for α3) and peak current (for furosemide (α4)). A regression line was fitted (continuous line) with its 95 % confidence interval (dotted lines). This resulted in a negative correlation for zolpidem (P < 0.05, Pearson test) and a positive correlation for bretazenil (P < 0.01, Pearson test). The negative correlation found between τ_decay and furosemide-induced peak current decrease was not significant (P = 0.2, Pearson test). The correlation coefficients (R) are indicated. Ba-Cc, averaged τ_decay histogram constructed from all histograms from individual neurons before (filled symbols) and after (open symbols) ligand application. Histograms were from pn6 (Ba-Bc) and pn21 (Ca-Cc). It can be seen that all α subunits contribute to faster τ_decay values at pn21 than at pn6.

Figure 6. Deletion of the GABAA receptor α1 subunit results in sIPSCs with a longer τ_decay

A, example sIPSCs aligned to their rising phases from adult wild-type mice (left) and adult α 1 −/− mice (right). E, example sIPSCs aligned to their rising phases from untreated organotypical slice cultures from rat visual cortex (left) and from cultures that were grown in the presence of α1 subunit antisense (AS) oligonucleotide for one week (right). Both groups were measured after 15 days in vitro. B and F, τ_decay histograms made from equal number of sIPSCs from control (filled symbols) and α1 knockout (B) and α1 AS-treated cultures (F) (open symbols). Insets: averaged sIPSCs from control (thick line) and knockout (B)/antisense-treated cultures (F) (thin line), normalized to peak current. C andG, box plots of average τ_decay values. NS: non-sense sequence oligonucleotide, as mentioned in Methods. D and H, averaged histograms, calculated from the τ_decay histograms of all individual neurons. Number of neurons used per group: wild-type (WT), n = 17; knockout (KO), n = 21; control cultures, n = 26; AS-treated cultures, n = 7; NS-treated cultures, n = 10.

Figure 7. Putative subunit composition of GABA_A receptors before and after neonatal development of the visual cortex

In immature synapses, the most abundant α subunit is α3, although α1 also occurs. In mature synapses, α1 is the dominant α subunit, but α4 also occurs. In addition, low levels of α3 may also be present.