Inward-rectifying K+ (Kir2) leak conductance dampens the excitability of lamina I projection neurons in the neonatal rat

Abstract

Spinal lamina I projection neurons serve as a major conduit by which noxious stimuli detected in the periphery are transmitted to nociceptive circuits in the brain, including the parabrachial nucleus (PB) and the periaqueductal gray (PAG). While neonatal spino-PB neurons are more than twice as likely to exhibit spontaneous activity compared to spino-PAG neurons, the underlying mechanisms remain unclear since nothing is known about the voltage-independent (i.e. 'leak') ion channels expressed by these distinct populations during early life. To begin identifying these key leak conductances, the present study investigated the role of classical inward-rectifying K⁺ (K_ir2) channels in the regulation of intrinsic excitability in neonatal rat spino-PB and spino-PAG neurons. The data demonstrate that a reduction in K_ir2-mediated conductance by external BaCl₂ significantly enhanced intrinsic membrane excitability in both groups. Similar results were observed in spino-PB neurons following K_ir2 channel block with the selective antagonist ML133. In addition, voltage-clamp experiments showed that spino-PB and spino-PAG neurons express similar amounts of K_ir2 current during the early postnatal period, suggesting that the differences in the prevalence of spontaneous activity between the two populations are not explained by differential expression of K_ir2 channels. Overall, the results indicate that K_ir2-mediated conductance tonically dampens the firing of multiple subpopulations of lamina I projection neurons during early life. Therefore, K_ir2 channels are positioned to tightly shape the output of the immature spinal nociceptive circuit and thus regulate the ascending flow of nociceptive information to the developing brain, which has important functional implications for pediatric pain.

Keywords: intrinsic excitability; leak conductance; projection neuron; superficial dorsal horn.

Figure 1. Neonatal rat spino-PB neurons are more likely to be spontaneously active compared to spino-PAG neurons

A, Schematic representation of the DiI back-labeling strategy; DiI injection into the periaqueductal gray or the parabrachial nucleus resulted in effective labeling of lamina I projection neurons. B, Representative micrograph of DiI back-labeled spino-PB projection neurons in an intact spinal cord preparation; scale bar, 25 µM. C, Spino-PB projection neurons exhibit a significantly higher prevalence of spontaneous activity compared to spino-PAG neurons (n = 51, 11 rats, p = 0.0008, Fischer’s exact test).

Figure 2. Spino-PB and spino-PAG projection neurons express K_ir2-mediated current during early life

A top, Representative whole-cell current traces evoked by a 100 mV (−55 mV to −155 mV) hyperpolarizing voltage ramp applied to spino-PB (left) and spino-PAG (right) projection neurons. Gray trace with arrow indicates recorded current in the presence of Ba²⁺; black trace, recorded current in the absence of Ba²⁺. A bottom, Representative Ba²⁺-sensitive components following electronic subtraction. B, comparisons of measured Ba²⁺-sensitive conductance (E_rev − 25, left; E_rev + 25, middle) and inward rectification ratio (g_{E − 25} / g_{E + 25}, right) between spino-PB and spino-PAG projection neurons revealed no significant differences between groups. n = 31, 12 rats, for all comparisons.

Figure 3. Ba²⁺ blockade of K_ir2 channels depolarizes immature lamina I projection neurons and enhances spino-PB neuron spontaneous activity

A, Representative trace showing a reversible increase in spontaneous action potential discharge in a spino-PB neuron in response to 200 µM Ba²⁺ bath application. Black bar, duration of Ba²⁺ application. Scale bars, 20 mV and 20 s. B, Representative trace of a spontaneously active spino-PB cell illustrating an increase of spontaneous action potential discharge in response to 200 µM Ba²⁺ bath application. Black bar, duration of Ba²⁺ application. Scale bars, 20 mV and 10 s. C, Ba²⁺ application significantly depolarizes the resting membrane potential (RMP) of spino-PB neurons (n = 32, 7 rats, p < 0.0001, paired t-test; left) and increases the rate of spontaneous activity (SA) (n = 32, 7 rats, p = 0.028, paired t-test; right). D, Representative trace of a spontaneously active spino-PAG neuron showing an increase of spontaneous action potential discharge in response to 200 µM Ba²⁺ bath application. Black bar, duration of Ba²⁺ application. Scale bars, 20 mV and 10 s. E, Ba²⁺ application significantly depolarizes spino-PAG RMP (n = 20, 4 rats, p < 0.0001, paired t-test; left) but did not significantly affect the frequency of spontaneous firing (n = 20, 4 rats, p = 0.09, paired t-test; right) in this population.

Figure 4. Ba²⁺ blockade of K_ir2 channels enhances the intrinsic excitability of neonatal spino-PB neurons

A, top, Representative traces of action potentials evoked via depolarizing current steps (5 pA steps, 80 ms duration) in the absence (left) and presence (right) of Ba²⁺. B, Representative traces of repetitive firing evoked by current steps (10 pA, 800 ms duration) in the absence (left) and presence (right) of Ba²⁺. C–E, Ba²⁺ application significantly reduces rheobase (p < 0.0001, paired t-test; C) and AP amplitude (p < 0.01, paired t-test; D), while enhancing membrane resistance (p < 0.05, paired t-test; E). F, Ba²⁺ application significantly enhances the firing frequency of immature spino-PB projection neurons (** p < 0.01; ^ψ p < 0.0001; two-way ANOVA, Holm-Sidak post-tests). n = 32, 7 rats, for all comparisons.

Figure 5. Ba²⁺ application increases the intrinsic excitability of neonatal spino-PAG neurons

A, Representative traces of action potentials evoked by depolarizing current steps (5 pA, 80 ms duration) in the absence (left) and presence (right) of Ba²⁺. B, Representative traces of repetitive firing evoked by intracellular current injection (10 pA steps, 800 ms duration) in the absence (left) and presence (right) of Ba²⁺. C–E, Ba²⁺ application significantly reduces rheobase (p < 0.0001, paired t-test; C) and AP amplitude (p < 0.0001, paired t-test; D) while membrane resistance was increased (p < 0.01, paired t-test; E). F, Ba²⁺ application significantly enhances the firing frequency of spino-PAG projection neurons during early life (^ψ p < 0.0001; two-way ANOVA, Holm-Sidak post-tests). n = 20, 4 rats, for all comparisons.

Figure 6. ML133 suppresses K_ir2 currents and enhances intrinsic membrane excitability in immature spino-PB neurons

A left, representative whole-cell current trace evoked by a 100 mV hyperpolarizing voltage ramp (from −55 mV to −155 mV) in the presence (arrow) and absence of the selective K_ir2 channel antagonist ML133 (100 µM); A right, representative electronic subtraction yielding the ML133-sensitive (i.e. K_ir2) current. B, Representative trace demonstrating a depolarizing shift in resting membrane potential (RMP) and increased spontaneous activity in response to 10 µM ML133 application (black bar). C, Representative action potentials evoked by depolarizing current steps (5 pA, 80 ms) in the absence (left) and presence (right) of ML133. D–G, ML133 application significantly depolarizes the RMP (p < 0.01, paired t-test; D), decreases rheobase (p < 0.01, paired t-test; E), decreases AP amplitude (p < 0.01, paired t-test; F), and has no effect on AP duration (p = 0.48, paired t-test, G). n = 11, 2 rats, for all comparisons.