Electrical synapses and the development of inhibitory circuits in the thalamus

Abstract

Key points: The thalamus is a structure critical for information processing and transfer to the cortex. Thalamic reticular neurons are inhibitory cells interconnected by electrical synapses, most of which require the gap junction protein connexin36 (Cx36). We investigated whether electrical synapses play a role in the maturation of thalamic networks by studying neurons in mice with and without Cx36. When Cx36 was deleted, inhibitory synapses were more numerous, although both divergent inhibitory connectivity and dendritic complexity were reduced. Surprisingly, we observed non-Cx36-dependent electrical synapses with unusual biophysical properties interconnecting some reticular neurons in mice lacking Cx36. The results of the present study suggest an important role for Cx36-dependent electrical synapses in the development of thalamic circuits.

Abstract: Neurons within the mature thalamic reticular nucleus (TRN) powerfully inhibit ventrobasal (VB) thalamic relay neurons via GABAergic synapses. TRN neurons are also coupled to one another by electrical synapses that depend strongly on the gap junction protein connexin36 (Cx36). Electrical synapses in the TRN precede the postnatal development of TRN-to-VB inhibition. We investigated how the deletion of Cx36 affects the maturation of TRN and VB neurons, electrical coupling and GABAergic synapses by studying wild-type (WT) and Cx36 knockout (KO) mice. The incidence and strength of electrical coupling in TRN was sharply reduced, but not abolished, in KO mice. Surprisingly, electrical synapses between Cx36-KO neurons had faster voltage-dependent decay kinetics and conductance asymmetry (rectification) than did electrical synapses between WT neurons. The properties of TRN-mediated inhibition in VB also depended on the Cx36 genotype. Deletion of Cx36 increased the frequency and shifted the amplitude distributions of miniature IPSCs, whereas the paired-pulse ratio of evoked IPSCs was unaffected, suggesting that the absence of Cx36 led to an increase in GABAergic synaptic contacts. VB neurons from Cx36-KO mice also tended to have simpler dendritic trees and fewer divergent inputs from the TRN compared to WT cells. The findings obtained in the present study suggest that proper development of thalamic inhibitory circuitry, neuronal morphology, TRN cell function and electrical coupling requires Cx36. In the absence of Cx36, some TRN neurons express asymmetric electrical coupling mediated by other unidentified connexin subtypes.

Figure 1. Electrical coupling in the TRN

A, current clamp recording of electrical coupling between two TRN neurons aged P8. The incidence (B) and mean ± SEM conductance (C) of electrical coupling between adjacent neuron pairs during development. Data in (B) and (C) are taken from 105 WT and 129 KO pairs.

Figure 2. Effect of Cx36 genotype on voltage‐dependent kinetics of electrical coupling in the TRN

A, protocol for measuring electrical coupling in voltage clamp (see Methods). B, overlay of the junctional current from WT (black) and Cx36 KO (grey) neuron pairs. Current amplitudes are normalized.

Figure 3. Effect of Cx36 genotype on the asymmetry of electrical coupling in TRN

A, junctional currents induced by +80 mV and –80 mV transjunctional voltage steps (black and red traces, respectively). Junctional currents in the WT pair are similar in both directions. Junctional currents in the KO pair are direction‐dependent (i.e. asymmetric). B, relationship between junctional coupling asymmetry (G _j ratio) and input resistance (R _in ratio) among WT and KO neuron pairs. C, coupling asymmetry vs. mean coupling strength (pA) between WT and KO neuron pairs. Each symbol in (B) and (C) represents data from a neuron pair of the type as specified in the insets (VC, voltage clamp; CC, current clamp measurements).

Figure 4. Morphology of TRN neurons by Cx36 genotype and age

A, photomicrographs of example neurons used for morphological analysis. B, counts of Sholl intersections by distance from the soma (mean ± SEM) by genotype and age group. P values are from a Kolmogorov–Smirnov test. C, morphological characteristics determined from Sholl intersections and 3‐D reconstruction of somata. Numbers of sampled neurons for each group are indicated on the bars. n.s., not significant.

Figure 5. Passive membrane properties of TRN neurons as a function of age and Cx36 genotype

A, input resistance declined with age and was significantly higher in KO neurons (P < 0.001, r = 0.74). B, cell capacitance increased with age and was unaffected by genotype (P = 0.31, r = 0.57). P values were determined based on linear regression. A total of 305 neurons was sampled (159 WT and 146 KO neurons); n for each genotype per 2 day age group ranged from 12 to 39 neurons.

Figure 6. Effects of Cx36 genotype on the properties of IPSCs in VB relay neurons

A, examples of mIPSCs and sensitivity to picrotoxin, a GABA_A receptor antagonist. B, mIPSC properties as a function of age and Cx36 genotype. mIPSC frequency is significantly higher in the KO compared to WT (P = 0.006, r = 0.53) (n = 175 neurons; 84 KO, 91 WT). The mIPSC decay rate (τ_IPSC, P = 0.14, r = 0.72) and amplitude (P = 0.96, r = 0.46) were not affected by genotype. Bars plot the mean ± SEM for each age and genotype group; P values are based on linear regression. C, examples of WT and KO mIPSCs compared at P2 and P13. Each trace is the average of 40 mIPSCs, with amplitudes normalized. D, mIPSC amplitude frequency histogram for P2–5 (n = 12 neurons for each genotype) and P12–13 (n = 4 neurons for each genotype). P values from the Kolmogorov–Smirnov test. E, paired‐pulse ratio of evoked IPSCs. Bar graph shows the mean ± SEM, with sample numbers specified on each bar. Inset: example trace of IPSCs from a WT cell.

Figure 7. Coincident IPSCs from VB neuron pairs

A, example of cIPSCs; asterisks indicate IPSCs that are coincident (i.e. peaks within ±1 ms of each other). The box indicates the cIPSCs shown at a fast time scale below. B, prevalence of neuron pairs with statistically significant occurrences of cIPSCs by age range (see Methods).

Figure 8. Morphology of VB neurons by Cx36 genotpye and age

A, photomicrographs of example neurons used for morphological analysis. B, counts of Sholl intersections by distance from the soma (mean ± SEM) by genotype and age group. P values are from a Kolmogorov–Smirnov test. C, morphological characteristics determined from Sholl intersections and 3‐D reconstruction of somata. Numbers of sampled neurons for each group are indicated on the bars. n.s., not significant.

Figure 9. Passive membrane properties of VB neurons as a function of age and Cx36 genotype

A, input resistance declined with age but was unaffected by genotype (P = 0.66, r = 0.73). B, cell capacitance increased with age but was unaffected by genotype (P = 0.17, r = 0.61). P values were determined based on linear regression. A total of 138 neurons were sampled (71 WT and 67 KO neurons); n for each genotype per 2‐day age group ranged from four to 15 neurons.