Investigations of the functional role of connexin36 in sensory and sympathetic systems in adult mice

Abstract

Electrical synapses formed by gap junctions composed of connexin36 (Cx36) occur between neurons in widespread areas of the mammalian central nervous system and enable neuron-to-neuron electrical coupling. We previously reported that dorsal root ganglia (DRG) sensory neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8, and those containing substance P in nociceptive DRG neurons express high levels of Cx36. It has also been reported that Cx36 is expressed in sympathetic preganglionic neurons (SPNs). Here we examined the functional roles of electrical synapses in primary afferent and SPN neural networks by using mice with global knockout (ko) of the Cx36 gene (Cx36 ko mice) and their wildtype (Wt) counterparts. Based on previous evidence showing that spinal neurons in Cx36 ko mice have attenuated responses to nociceptive stimulation when examined ex vivo; our hypothesis was that responses to nociceptive stimulation would be attenuated in vivo in intact Cx36 ko mice compared to Wt. To examine the role of coupling between DRG neurons, in vivo imaging was used to measure the fluorescence signal of Evans blue dye extravasation in skin of live animals after nociceptor activation. The extravasation levels relative to baseline fluorescence were 61.7±4% in Wt mice and 33.2±7% Cx36 ko mice (n=5 matched pairs, all males) after hind paw pinprick. Xylene applied topically on one of the ears induced more extravasation in the wt (255.4 ±3%) than in the Cx36 ko mice (204.6 ±1%). Using a fibre optic blood pressure monitoring system, we found that topical xylene application under isoflurane anesthesia changes the blood pressure, the heart rate, and the respiratory rate additionally to evoking the plasma extravasation. The attenuated extravasation response in the Cx36 ko mice suggests that electrical synapses known to occur between peripheral unmyelinated sensory fibers impact signal transmission from peripheral nerve endings and release other mediators that cause this response. To examine functional roles of coupling between SPNs, we compared physiological responses to colorectal distension (CRD) in Wt and Cx36 ko) mice in mice under isoflurane anesthesia. Mean arterial blood pressure during CRD increased to 108 ± 8% of baseline in Wt mice (n = 7), while this parameter did not change significantly in Cx36 ko mice (n = 12), but did show a tendency to decrease (to 97 ± 2%). Heart rate did not show significant changes, but we found that respiratory rate changes also occurred as part of the systemic response to CRD in both lines of mice. These data show differential regulation of sympathetic activity associated with electrical coupling between SPNs in Wt vs. Cx36 ko mice. In Wt mice with mid-thoracic complete spinal transection (n = 2), CRD also increased blood pressure when tested at 6-8 days after injury. The results taken together, point to the possibility that Cx36 in DRG and SPNs of adult mice has a significant functional role and warrant further research to understand how the presence of Cx36 in DRG and SPNs influences pain processing and associated autonomic responses.