RESUMEN
Tissue damage during the neonatal period evokes long-lasting changes in nociceptive processing within the adult spinal cord which contribute to persistent alterations in pain sensitivity. However, it remains unclear if the observed modifications in neuronal activity within the mature superficial dorsal horn (SDH) following early injury reflect shifts in the intrinsic membrane properties of these cells. Therefore, the present study was undertaken to identify the effects of neonatal surgical injury on the intrinsic excitability of both GABAergic and presumed glutamatergic neurons within lamina II of the adult SDH using in vitro patch clamp recordings from spinal cord slices prepared from glutamic acid decarboxylase-green fluorescent protein (Gad-GFP) mice. The results demonstrate that hindpaw surgical incision at postnatal day (P) 3 altered the passive membrane properties of both Gad-GFP and adjacent, non-GFP neurons in the mature SDH, as evidenced by decreased membrane resistance and more negative resting potentials in comparison to naïve littermate controls. This was accompanied by a reduction in the prevalence of spontaneous activity within the GABAergic population. Both Gad-GFP and non-GFP neurons displayed a significant elevation in rheobase and decreased instantaneous firing frequency after incision, suggesting that early tissue damage lowers the intrinsic membrane excitability of adult SDH neurons. Isolation of inward-rectifying K(+) (K(ir)) currents revealed that neonatal incision significantly increased K(ir) conductance near physiological membrane potentials in GABAergic, but not glutamatergic, lamina II neurons. Overall, these findings suggest that neonatal tissue injury causes a long-term dampening of intrinsic firing across the general population of lamina II interneurons, but the underlying ionic mechanisms may be cell-type specific.
Asunto(s)
Potenciales de Acción/fisiología , Ganglios Espinales/citología , Neuronas/fisiología , Potenciales de Acción/genética , Análisis de Varianza , Animales , Animales Recién Nacidos , Biofisica , Estimulación Eléctrica , Femenino , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Médula Espinal/anatomía & histología , Regulación hacia Arriba/genéticaRESUMEN
Neurons within the superficial dorsal horn (SDH) of the rodent spinal cord exhibit distinct firing properties during early life. While this may reflect a unique combination of voltage-gated Na(+) (Na(v)) and voltage-independent (i.e. "leak'') K(+) channels which strongly influence neuronal excitability across the CNS, surprisingly little is known about which genes encoding for Na(v) and leak K(+) channels are expressed within developing spinal pain circuits. The goal of the present study was therefore to characterize the transcriptional expression of these channels within the rat SDH at postnatal days (P) 3, 10, 21 or adulthood using quantitative real-time polymerase chain reaction. The results demonstrate that Na(v) isoforms are developmentally regulated at the mRNA level in a subtype-specific manner, as Na(v)1.2 and Na(v)1.3 decreased significantly from P3 to adulthood, while Na(v)1.1 was up-regulated during this period. The data also indicate selective, age-dependent changes in the mRNA expression of two-pore domain (K(2P)) K(+) channels, as TWIK-related acid-sensitive K(+) channels TASK-1 (KCNK3) and TASK-3 (KCNK9) were down-regulated during postnatal development in the absence of any changes in the tandem of pore domains in a weak inward rectifying K(+) channel (TWIK) isoforms examined (KCNK1 and KCNK6). In addition, a developmental shift occurred within the TREK subfamily due to decreased TREK-2 (KCNK10) mRNA within the mature SDH. Meanwhile, G-protein-coupled inward rectifying K(+) channels (K(ir)3.1 and K(ir)3.2) were expressed in the SDH at mature levels from birth. Overall, the results suggest that the transcription of ion channel genes occurs in a highly age-dependent manner within the SDH, raising the possibility that manipulating the expression or function of ion channels which are preferentially expressed within immature nociceptive networks could yield novel approaches to relieving pain in infants and children.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Células del Asta Posterior/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Médula Espinal/metabolismo , Canales de Sodio Activados por Voltaje/genética , Animales , Femenino , Masculino , Células del Asta Posterior/citología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P¿Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.