Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents

Figure 1 Effects of CsCl on the mechenosensory activity of small intestinal afferents. A: Original recording of the mesenteric afferent nerve activity in response to distension in an ex vivo jejunum preparation; B: The pressure-afferent nerve response curves in control and in the presence of hyperpolarization-activated cyclic nucleotide-gated cation blocker, 5 mmol/L CsCl; C: Bar graph showing the low- and high- threshold mechanosensory responses with or without the presence of 5 mmol/L CsCl. ^aP < 0.05.

Figure 2 Effects of ZD-7288 on the mechanisensory responses of mesenteric afferent nerves. Note that the slopes of the average pressure-response curves in the pressure range of 10 mmHg - 60 mmHg were apparently similar with or without the hyperpolarization-activated cyclic nucleotide-gated cation channel blocker ZD-7288 (10 μmol/L). Data were pooled from 3 preparations.

Figure 3 I_h recorded under whole-cell voltage and current clamp from DiI labeled dissociated dorsal root ganglia and nodose ganglia neurons innervating the jejunum. A: DiI labeled dissociated dorsal root ganglia neurons; B: Representative current traces of I_h current (top trace); Current recorded in the presence of 5 mmol/L CsCl (middle trace); voltage clamp protocal: I_h was induced from a holding potential of -60 mV in 1 s pulses from -60 mV to -120 mV in steps of 10 mV, followed by a final step to -80 mV to record the tail current (bottom trace); C: Voltage response to test current pulse before (black) and after (grey) application of 5 mmol/L CsCl (top trace); bottom trace shows the current clamp protocol, i.e., hyperpolarizing current pulses ranging from 0 pA to -200 pA in steps of 50 pA. Note that the current elicited an instantaneous hyperpolarization that was followed by depolarization (named “sag”) of membrane potential. DRG: Dorsal root ganglia; NG: Nodose ganglia.

Figure 4 I_h steady-state parameters and activation kinetics in DiI-labeled DRG and NG neurons. A: I_h current-voltage relationship between DiI-labeled dissociated dorsal root ganglia (DRG) and nodose ganglia neurons (NG); B: I_h current density-voltage relationship between DiI-labeled DRG and NG neurons; C: The time constant of I_h current. I_h current traces were fitted with two exponentials according to the following equation: I_h(t) = Af exp(-t/τf) + As exp(-t/τs), where I_h(t) is the amplitude of the current at time t and Af and As are the initial amplitudes of the fast (τf) and slow (τs) activation time constant components, respectively. Time constants were obtained by fitting currents using pCLAMP; D: I_h current activation curves. Normalized activation curves were obtained from tail currents at -80 mV and fitted by Boltzmann function: I_h/I_h(max) =1/{1+ exp[(Vm – V_1/2)/k]}, where I_h is the peak amplitude of the tail current recorded immediately after the pre-pulse, I_h(max) is the maximal current recorded after the maximal prepulse of -120 mV, V_m is the membrane potential, V_1/2 is the membrane potential at which I_h conductance is half-activated, and k is a slope factor of the curve. Data were expressed as mean ± SE. ^aP < 0.05 vs DiI-labeled DRG neurons.

Figure 5 Effects of 8-Br-cAMP on the activation curves of I_h current in DiI-labeled dissociated dorsal root ganglia and nodose ganglia neurons. A and B: Overdraw of the I_h current traces elicited by a hyperpolarizing pulse (-120 mV) in control and in the presence of 8-Br-cAMP in DiI-labeled dissociated dorsal root ganglia (DRG) (A) and nodose neurons (B); C: Activation curves of cAMP-sensitive and cAMP-insensitive DiI-labeled DRG neurons before (black block symbols) and after treatment with 8-Br-cAMP (empty symbols); D: Activation curves of DiI-labeled nodose ganglia neurons before (black block triangle) and after treatment with 8-Br-cAMP (empty triangle). Data were expressed as mean ± SE.

Figure 6 Immunostaining for hyperpolarization-activated cyclic nucleotide-gated cation isoforms in dorsal root ganglia and nodose ganglia sections. Hyperpolarization-activated cyclic nucleotide-gated cation channel 1 (HCN₁) immunoreactivity was not detectable in dorsal root ganglia (DRG) and nodose ganglia (NG) neurons. HCN₂ immunoreactivity was prominent in DRG (cell bodies and fibers) and was also present in some NG neurons and fibers. HCN₃ immunoreactivity was profuse in NG sections but was weaker in DRG sections. HCN₄ immunoreactivity was present in a minority of DRG neurons and was absent in NG.

Figure 7 Immunostaining for hyperpolarization-activated cyclic nucleotide-gated cation channel 2 in intestinal primary afferent neurons. Spinal and vagal afferent neurons innervating the small intestine were labeled via injection of DiI (red) into the gut wall. Arrows indicate positive hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN₂) staining in DiI-labeled dorsal root ganglia neurons. DRG: Dissociated dorsal root ganglia; NG: Nodose ganglia.

Figure 8 Immunostaining for hyperpolarization-activated cyclic nucleotide-gated cation channel 3 in intestinal primary afferent neurons. Intestinal afferents were labeled via injecting DiI into the gut wall. Note that weak hyperpolarization-activated cyclic nucleotide-gated cation channel 3 (HCN₃) immunoreactivity (green) was present in some DiI-labeled (red) dorsal root ganglia neurons. In contrast, most DiI-labeled nodose ganglia neurons were moderately stained for HCN₃.