Optogenetic Modulation of Primary Afferent Neurons in Vivo Produces Mixed Effects in Spinal Neuronal Responses During Urinary Bladder Distention

Abstract

This ongoing study characterizes the responses of dorsal horn neurons to graded urinary bladder distention (UBD) during optogenetic modulation of bladder-innervating primary afferent neurons. Ai32 and Ai39 mice were crossed with Scn10atm3(cre/ERT2)Jwo mice to generate offspring expressing ChR2(H134R)/EYFP or NpHR3.0/EYFP, respectively, in Nav1.8+ primary afferents. At 6-8 weeks of age, female heterozygous mice were anesthetized and instrumented with a transurethral catheter. A fiber optic was passed through the catheter and coupled to a diode-pumped solid-state laser for photomodulation. Mice were prepared for in vivo spinal recordings. Single-unit extracellular recordings were discriminated from background, converted into uniform pulses, and saved to computer as peristimulus-time histograms. UBD (20, 40, and 60 mmHg) was administered without and with concurrent photomodulation. Evoked activity was calculated as total activity during 10-sec UBD minus spontaneous activity in 10-sec period prior to UBD. Responses to cutaneous non-noxious mechanical input were used to determine whether neurons were wide dynamic range (WDR) or nociceptive specific (NS). Preliminary data from Scn10aCre;Ai39 mice demonstrate that 2/9 spinal neurons were uniformly inhibited by afferent photoinhibition (589 nm) during UBD and 7/9 exhibited mixed responses (facilitation at 20 mmHg, inhibition at 40 and 60 mmHg). 8/9 of these neurons were WDR and 1/9 was NS. Preliminary data from Scn10aCre;Ai32 mice show that 1/4 UBD-responsive spinal neurons was uniformly inhibited, 1/4 was uniformly excited, and 2/4 exhibited mixed responses to afferent photoexcitation (473 nm) during UBD. 2/2 neurons were WDR and 2/2 were NS. Application of a heterotopic noxious conditioning stimulus (HCNS) during 60 mmHg UBD revealed no effect on neurons with a mixed phenotype, but HCNS produced >20% inhibition of neurons that were uniformly excited or inhibited during photoexcitation. In sum, these results reflect the complexity of viscerosensory spinal neuronal processing through excitation, inhibition, and disinhibition of second order neurons via multiple mechanisms. Grant support from K01DK101681.