Regulatory role of cotransporters KCC2 and NKCC1 on postnatal maturation of the vestibular system

Abstract

The neurotransmitter γ-αminobutyric acid (GABA) elicits depolarizing and hyperpolarizing synaptic response in immature and mature neurons, respectively. The polarity of GABA-mediated response is known to be mediated by two chloride co-transporters, namely NKCC1 and KCC2, which exhibit spatiotemporal shift in expression, phosphorylation and oligomerization levels. In the cerebral cortex and hippocampus, immature neurons contain primarily NKCC1, whereas mature neurons express KCC2 predominantly. We hypothesize that NKCC1 and KCC2 modulate the maturation of vestibular functions at the behavioral level. With the use of immunohistochemistry, we showed that NKCC1 expression in the rat vestibular nucleus (VN) was restricted to early postnatal stage (P1, P4 and P7) whereas KCC2 was expressed throughout postnatal development (P1, P4, P7, P14 and P21) in the VN.

Previous experiments conducted in our laboratory using gramicidinperforated patch-clamp technique indicated that (1) GABAA receptormediated response of neurons in the medial VN changed from depolarizing to hyperpolarizing at around P12, and (2) reversal potential of GABA shifted with acute application of either NKCC1 blocker at early postnatal stage or KCC2 blocker at late postnatal stage. In the present study, we investigated the roles of NKCC1 and KCC2 on vestibular-dependent behaviors by delivery of their blocker to VN with the use of Elvax slices (an inert ethylene-vinyl acetate polymer) that allowed prolonged and controlled release of drugs. Implantation of Elvax slice loaded with either NKCC1 blocker or KCC2 blocker over the dorsal surfaces of VN in neonatal rats resulted in delay in postnatal emergence of graviception, as indicated by the negative geotaxis test.

Rats pretreated with KCC2 blocker when raised to adulthood showed impairment in sensorimotor coordination during beam-walking test and spatial navigation during dead reckoning test. In dark, the rats displayed chaotic homeward paths and high heading angle toward the home base, suggesting poor ability to use self-movement cues during pathfinding. Severe impairment in spatial navigation was also observed if the rats were released from a new home base that was 180° opposite to the previous home base, suggesting that they relied on environmental cues to compensate for impairments in using self-movement cues. Moreover, normal response was observed in open-field test, indicating that the impairment in spatial reckoning was not due to anxiety or impaired locomotory function. Likewise, motor learning was not affected, as indicated by rota-rod test.

On the other hand, rats pretreated with NKCC1 blocker showed impairment in the acquisition of motor function during rota-rod test, but not the ability to navigate in the absence of environmental cues during dead reckoning test. Interestingly, motor coordination of these NKCC1-blocked rats was enhanced during beam walking test.

To conclude, postnatal development of vestibular-related behaviors is dependent on sequential activities of two chloride co-transporters, which modulate the polarity of GABA-mediated response in VN neurons. These results add to our understanding of GABA-mediated synaptic responses that regulate the maturation of vestibular circuit for behavioral expression of vestibular function.