Restless Leg Syndrome (RLS) is a chronic sensorimotor disorder that has recently been linked to a downregulation in adenosine A1 receptors (A1R), leading to decreased formation of adenosine-dopamine heteromers and a disinhibited or hyperdopaminergic transmission pathway within the central nervous system. Dopamine D1 receptor (D1R) activation has been shown to modulate HCN1 channels via its stimulatory effect on cAMP production to increase the hyperpolarization-activated cation current (Ih). Ih has been related to the regulation of bursting activity in rhythmic motor networks, specifically in controlling the firing frequency of the system. Our study aims to establish the importance of Ih in mammalian locomotion under hyperdopaminergic conditions mimicking those seen in RLS patients. We hypothesized that blocking Ih would increase the cycle period and destabilize the rhythm.
Methods: We isolated the spinal cords of neonatal (P0-P5; n = 6) Swiss Webster mice via ventral laminectomy and recorded the extracellular activity of ventral roots L2 and L5 using suction electrodes to assess burst amplitude, burst duration, and cycle period. Fictive locomotion was induced using serotonin (5HT; 9-15μM) and N-methyl-D-aspartate (NMDA; 6 μM) before adding dopamine (DA; 50μM) to determine the effect of DA on the system. We then used ZD7288 (1μM) block HCN1 channels to investigate the role of Ih in motor-like activity.
Results: Blocking Ih caused a significant increase in all three parameters relative to the 5HT/NMDA control in the L2 roots, but only cycle period was significantly increased relative to the high dopamine condition. There were no significant changes in any of the parameters in the L5 roots.
Conclusions: Our results suggest that Ih primarily plays an excitatory role in locomotor regulation under hyperdopaminergic conditions, such as those seen in RLS. The differential effects between roots may suggest that one is more susceptible to the blockade of Ih by ZD7288; however, given the large degree of variability in the L5 data, a larger sample size is needed to confirm this hypothesis. Future experiments include collecting more data on L5 to confirm if there are differential effects, as well as pharmacological approaches to confirm that D1R is upregulating Ih via the cAMP pathway.
This work was supported by the Student Faculty Research Grant Fellowship and the NIH Comparative Functional Genomics INBRE in Maine grant.
