Involvement of PACAP receptor in primary afferent fibre-evoked responses of ventral roots in the neonatal rat spinal cord

Abstract

The role of PACAP receptor in nociceptive transmission was investigated in vitro using maxadilan, a PACAP receptor selective agonist and max.d.4, a PACAP receptor selective antagonist. Potentials, from a ventral root (L3 - L5) of an isolated spinal cord preparation or a spinal cord - saphenous nerve - skin preparation from 0 - 3-day-old rats, were recorded extracellularly. In the isolated spinal cord preparation, single shock stimulation of a dorsal root at C-fibre strength induced a slow depolarizing response lasting about 30 s (slow ventral root potential; slow VRP) in the ipsilateral ventral root of the same segment. Bath-application of max. d.4 (0.01 - 3 microM) inhibited the slow VRP in a concentration-dependent manner. In the spinal cord - saphenous nerve - skin preparation, application of capsaicin (0.1 microM) to the skin evoked a depolarization of the ventral root. This response was also depressed by max.d.4 (1 microM). Application of maxadilan evoked a long-lasting depolarization in a concentration-dependent manner in the spinal cord preparation. In the presence of max.d.4 (0.3 microM), the concentration response curve of maxadilan was shifted to the right. Reverse transcription-polymerase chain reaction (RT - PCR) experiments demonstrated the existence of PACAP receptor and VPAC(2) receptor in the neonatal rat spinal cord and [(125)I]-PACAP27 binding was displaced almost completely by maxadilan and max.d.4, but not by vasoactive intestinal peptide (VIP). These data indicate that PACAP receptor is dominantly distributed in the neonatal rat spinal cord. The present study suggests that PACAP receptor may play an excitatory role in nociceptive transmission in the neonatal rat spinal cord.

Figure 1

Effect of max.d.4 on the primary afferent evoked potential in the neonatal rat spinal cord. A single-shock stimulus (200 μs 20 V) was given to the dorsal root (L4) at the time indicated by ▴ and the potential was recorded extracellularly from the ispilateral ventral root. Max.d.4 was applied to the spinal cord in a cumulative manner. (A) Sample records of the fast time course monosynaptic and polysynaptic reflexes that were stored in a transient memory device and recorded by a pen recorder with a 500 fold expansion of the time base. (B) Sample records of slow ventral root potential (VRP) recorded by a pen recorder.

Figure 2

Concentration-inhibition curves showing the effects of max.d.4 and PACAP(6-38) on the slow VRP. Ordinate scale: integrated area of slow VRP expressed as a percentage of the averaged three consecutive control responses just before the drug application. Abscissa scale: logarithmic concentration of max.d.4 and PACAP(6-38). Each point and vertical bar represent mean±s.e.mean (n=5) ^*P<0.05.

Figure 3

Effect of max.d.4 on the ventral root depolarization evoked by noxious skin stimulation. Potentials were recorded extracellularly from the L3 ventral root of a spinal cord-saphenous nerve-skin preparation prepared from a 1-day-old rat. Capsaicin (0.1 μM) was applied to the skin during the period (30 s) indicated by the bars. (A) Sample records of capsaicin-induced depolarization. (a) Control response. (b) After pretreatment of max.d.4 (1 μM) to the medium superfusing the spinal cord for 15 min. (c) Forty-five minutes after the removal of max.d.4. (B) Each column and vertical bar represent mean±s.e.mean of the magnitude of capsaicin-induced depolarization (n=5) ^*P<0.05.

Figure 4

Ventral root depolarization evoked by maxadilan. Potential was recorded extracellularly from the L4 ventral root of an isolated spinal cord from a 1-day-old rat. Maxadilan was applied to the medium superfusing the spinal cord during the period (60 s) indicated by the bar. (a) 0.01 μM (b) 0.1 μM (c) 1 μM.

Figure 5

Effects of max.d.4 (0.3 μM) and tetrodotoxin (TTX 0.3 μM) on the concentration-response curve for maxadilan. Maxadilan was applied to the medium superfusing the spinal cord for 60 s. Max.d.4 and TTX were pre-applied for 15 min before the application of maxadilan. Ordinate scale: peak amplitude (mV) of ventral root depolarization. Abscissa scale: logarithmic concentration of maxadilan. Each point and vertical bar represent mean±s.e.mean (n=3 – 4) ^*P<0.05.

Figure 6

Displacement curves of [¹²⁵I]-PACAP27 binding to neonatal spinal cord preparations by related peptides. Ordinate scale: [¹²⁵I]-PACAP27 binding expressed as a percentage of control. Abscissa scale: logarithmic concentration of peptides. Each point and vertical bar represent mean±s.e.mean (n=3).

Figure 7

The mRNA expression of PACAP/VIP family receptors in neonatal rat spinal cord using RT – PCR. Total RNA was isolated by the guanidinium isothiocyanate/CsCl procedure from neonatal rat spinal cord (1-day-old). RT – PCR was performed using RNA PVR Kit (TaKaRa, Ohtsu, Japan) as described in ‘Methods'.