Presynaptic maturation of inhibitory connections onto vasoactive intestinal polypeptide-expressing GABAergic interneurons in the mouse barrel field

Abstract

Vasoactive intestinal polypeptide-expressing inhibitory interneurons (VIP-INs) in the adult barrel cortex are crucial for mediating active whisking (AW) by disinhibiting pyramidal neurons. Past studies have investigated the development of VIP-IN network integration, focusing mainly on the excitatory network or the postsynaptic side of the inhibitory network. Hence, we aimed to explore the inhibitory network integration of VIP-INs, concentrating on the presynaptic side. We addressed this by investigating VIP-INs in three different age groups (postnatal day (P)8-P10, P14-P16, and P30-P36) in Vip-IRES-cre x tdTomato mice with whole-cell patch clamp recordings. By placing a stimulation electrode into L4 of the barrel field, we elicited electrically-evoked inhibitory postsynaptic currents (eIPSCs) in L2/3 VIP-INs following a high-frequency stimulation. We then analysed recorded eIPSCs by applying the binomial model of synaptic transmission. Our results show significant increases in both the number of readily-releasable vesicles and the presynaptic release probability between P9 and P15, suggesting that the inhibitory network integration is at least partially conducted via a presynaptic functional maturation. Despite an increase in the release probability, synaptic depression is decreased at P30-P36 due to an accelerated vesicle replenishment rate within the same time window. Lastly, asynchronous vesicle release decreases in favour of a stimulus-locked signal transmission by P30-P36. Our results suggest a maturation of the inhibitory projections towards a strong, precise, and stimulus-locked inhibition. This can be physiologically relevant to define the temporal precision of AW at the relevant frequencies.

Keywords: Development; Inhibition; Interneuron; Quantal analysis; Somatosensory; VIP.

Fig. 1

Increase in mIPSC rate indeveloping VIP-INs. Left: representative traces of mIPSCs recorded at P9, P15, and P30 +. Bath application of 50 µM PTX completely abolished mIPSCs. Right: box plots indicating a significant increase in mIPSC rate between P9 and P15. Corresponding p-values: p = 0.0439 (P9 and P15) and p = 0.0009 (P9 and P30 +), Kruskal–Wallis. Number of cells: 10–12. *p < 0.05, ***p < 0.001. Box plots indicating the minimum and maximum value, median, and 25% and 75% percentiles

Fig. 2

Functional presynaptic maturation of inhibitory connections onto VIP-INs. A Left: representative eIPSC traces elicited by a high-frequency electrical stimulation (20 pulses applied at 20 Hz) at P9, P15 and P30 +. Incision at the P30 + trace shows delayed IPSCs (dIPSCs) selected for the calculation of the quantal size for each cell. Middle: cumulative eIPSC amplitudes during a 20-Hz train. Lines indicate a linear fit of the last 10 events back-extrapolated to the onset of stimulation. The y-intercept serves as an estimation for the RRP. Right: box plots indicating that the RRP [nA] significantly increases between P9 and P15. Corresponding p-values: p = 0.0359 (P9 and P15) and p < 0.0001 (P9 and P30 +), Kruskal–Wallis. B Box plots indicating developmental changes in the presynaptic release probability. Corresponding p-values: p = 0.0051 (P9 and P15) and p < 0.0001 (P9 and P30 +), one-way ANOVA F(2,32) = 13.14. C Box plots indicating developmental changes in the quantal size (mean dIPSC amplitude) between P9 and P30 +; one-way ANOVA, F(2,32) = 4.727, p = 0.0132. D Box plots indicating developmental changes in the RRP (vesicles) between P9 and P30 +; Kruskal–Wallis, p = 0.0007. Number of cells: 11–12. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Box plots indicating the minimum and maximum value, median, and 25% and 75% percentile

Fig. 3

Decrease in presynaptic depression is mediated by an accelerated vesicle replenishment. A Vesicle replenishment rate becomes significantly accelerated between P9 and P30 +, p = 0.0013, Kruskal–Wallis. Vesicle replenishment rate was normalised to the RRP [nA] per cell. B Dependence of stimulus-locked eIPSC amplitude on the pulse number. eIPSCs were normalised to the first eIPSC of each cell. For determining the steady state, the average of the last 5 normalised eIPSCs was calculated (grey box). C Box plots showing a significant increase of the steady state between P9 and P30 +, p = 0.0115, Kruskal–Wallis. Number of cells: 11–12 cells. *p < 0.05, **p < 0.01. Box plots indicating the minimum and maximum value, median, and 25% and 75% percentiles

Fig. 4

Inhibitory connections mature from an asynchronous towards a stimulus-locked release. A Visual representation of eIPSCs in response to a 20-Hz stimulation. Insets show the asynchronous release as grey highlighted areas at P9, P15, and P30 +. The areas for asynchronous release were normalised to the quantal size and RRP. B Box plots showing a significant reduction in the normalised asynchronous release between P9 and P30 +, p = 0.0007, Kruskal–Wallis. C Ratio of asynchronous release to the total area of the last evoked response gradually shifts towards synchronous release by P30 +, p = 0.0006, Kruskal–Wallis. Number of cells: 10–12. ***p < 0.001. Box plots indicating the minimum and maximum value, median, and 25% and 75% percentiles

Fig. 5

Summarising cartoon of maturing presynaptic inhibitory connections onto VIP-INs. The direction and length of the arrows indicate the direction and strength of parameter change at the respective time points. Between P9 and P15, the presynaptic release probability (p_r) increases. The size of the readily releasable pool (RRP) increases gradually between P9 and P30 +. The vesicle refilling rate accelerates in parallel to a decrease in the synaptic depression when reaching P30 +. The mode of vesicle release is dominated by an asynchronous vesicle release (ASR) at P9 and P15, while it becomes more synchronous (SR) in the P30 + age group. These developmental changes are beneficial for mediating a strong and temporally precise inhibitory signal transmission towards VIP-INs