Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord

Abstract

Neurons in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord process nociceptive information from skin, muscle, joints and viscera. Most of what we know about the intrinsic properties of SDH neurons comes from studies in lumbar segments of the cord even though clinical evidence suggests nociceptive signals from viscera and head and neck tissues are processed differently. This ‘lumbar-centric' view of spinal pain processing mechanisms also applies to developing SDH neurons. Here we ask whether the intrinsic membrane properties of SDH neurons differ across spinal cord segments in both the developing and mature spinal cord. Whole cell recordings were made from SDH neurons in slices of upper cervical (C2-4), thoracic (T8-10) and lumbar (L3-5) segments in neonatal (P0-5) and adult (P24-45) mice. Neuronal input resistance (R(IN)), resting membrane potential, AP amplitude, half-width and AHP amplitude were similar across spinal cord regions in both neonates and adults (∼100 neurons for each region and age). In contrast, these intrinsic membrane properties differed dramatically between neonates and adults. Five types of AP discharge were observed during depolarizing current injection. In neonates, single spiking dominated (∼40%) and the proportions of each discharge category did not differ across spinal regions. In adults, initial bursting dominated in each spinal region, but was significantly more prevalent in rostral segments (49% of neurons in C2-4 vs. 29% in L3-5). During development the dominant AP discharge pattern changed from single spiking to initial bursting. The rapid A-type potassium current (I(Ar)) dominated in neonates and adults, but its prevalence decreased (∼80% vs. ∼50% of neurons) in all regions during development. I(Ar) steady state inactivation and activation also changed in upper cervical and lumbar regions during development. Together, our data show the intrinsic properties of SDH neurons are generally conserved in the three spinal cord regions examined in both neonate and adult mice. We propose the conserved intrinsic membrane properties of SDH neurons along the length of the spinal cord cannot explain the marked differences in pain experienced in the limbs, viscera, and head and neck.

Figure 1. Location of recorded SDH neurons in different spinal regions in neonate and adult mice

The location of each recorded neuron is plotted on one of three templates of the SDH for consecutive spinal segments in the upper cervical (C2–4), thoracic (T8–10) and lumbar (L3–5) cord. Note, the plots for neonatal SDH are shown at higher magnification to facilitate comparison of recorded neuron location between neonates (left) and adults (right).

Figure 2. Prevalence of AP discharge categories across spinal regions in neonate and adult SDH neurons

A, representative traces show the five types of AP discharge observed in SDH neurons. All traces are from adult upper cervical neurons, and show responses to increasing amplitude current injections (0, 40, 80 and 120 pA; 800 ms duration). B, bar plots showing the prevalence of the five discharge categories for each spinal cord region in neonates. Comparison of the distributions across segments showed they were similar (G statistic = 7.9, P = 0.4). C, bar plots showing the prevalence of the five discharge categories for each spinal cord region in adults. Comparison of the distributions across segments showed they differed (G statistic = 16.3, P < 0.05). Pairwise comparisons revealed upper cervical and lumbar distributions differed (G statistic = 14.6, P < 0.05). Comparisons between neonates and adults also revealed significant differences in the distributions in each spinal region (upper cervical G statistic = 27.6, P < 0.05; thoracic G statistic = 19.6, P < 0.05; lumbar G statistic = 43.9, P < 0.05).

Figure 3. Prevalence of responses to hyperpolarizing current injection across spinal regions in neonate and adult SDH neurons

A, representative traces show three responses to hyperpolarizing current injection in SDH neurons. All traces are from adult upper cervical neurons and show responses to increasing amplitude current injections (−20, −30 and −40 pA; 800 ms duration). Dashed line indicates membrane potential of −60 mV. B, bar plots showing the prevalence of responses to hyperpolarizing current injection for each spinal cord region in neonates. Note the rebound responses are separated for neurons that discharge an AP (filled) and those that did not (open). Comparison of the distributions across segments showed they were similar (G statistic = 11.7, P = 0.07). C, bar plots showing the prevalence of the responses to hyperpolarizing current injection for each spinal cord region in adults. Comparison of the distributions across segments showed they were similar (G statistic = 8.3, P = 0.2). Comparisons between neonates and adults revealed significant differences in the distributions in upper cervical and thoracic but not lumbar spinal regions (upper cervical G statistic = 18.4, P < 0.05; thoracic G statistic = 9.9, P < 0.05; lumbar G statistic = 2.8, P = 0.4).

Figure 4. Prevalence of subthreshold currents across spinal regions in neonate and adult SDH neurons

A, representative traces show the five subthreshold currents observed in SDH neurons. The illustrated mixed current consists of an outward A-current and an inward T-current. Bottom trace shows voltage protocol used to activate the above currents from a holding potential of −60 mV. All traces are from adult upper cervical neurons. B, bar plots showing the prevalence of subthreshold currents for each spinal cord region in neonates. Comparison of the distributions across segments showed they were similar (G statistic = 6.0, P = 0.7). C, bar plots showing the prevalence of subthreshold currents for each spinal region in adults. Comparison of the distributions across segments showed they were similar (G statistic = 8.5, P = 0.4). Comparisons between neonates and adults revealed significant differences in the distributions in each spinal region (upper cervical G statistic = 19.5, P < 0.05; thoracic G statistic = 16.3, P < 0.05; lumbar G statistic = 12.9, P < 0.05).

Figure 5. Regional and developmental comparisons of IAr activation and steady state inactivation

A-B, plots comparing activation and steady-state inactivation of IAr across spinal regions in neonates (A) and adults (B) at membrane potentials between −90 and −40 mV. C–E, plots comparing activation and steady-state inactivation of IAr between neonates and adults in upper cervical (C), thoracic (D) and lumbar spinal cord (E) at membrane potentials between −90 and −40 mV. Asterisks indicate differences (P < 0.05) in pairwise comparisons for data points at various membrane potentials.