Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo

https://doi.org/10.1016/j.bbrc.2019.03.051Get rights and content

Highlights

  • Rat with nerve injury or inflammation facilitate APs dependent glutamatergic transmission in the SG neurons in vitro.

  • About 20% of SG neurons in SNL and CFA groups elicited spontaneous firings of APs without changing the RMPs in vitro.

  • By using in vivo whole-cell patch-clamp recordings, SG neurons generate sAPs without affecting RMP in the SNL and CFA groups.

Abstract

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem.

The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood.

Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group.

Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Introduction

Chronic pain is characterized by abnormal sensations such as persistent unpleasantness, and afflicts people worldwide, and severely affects quality of life in patients, becoming an escalating health problem [1]. Furthermore, it is a troublesome clinical problem since current treatments are not fully effective. This is a consequence of the gradual development of resistance to conventional analgesic treatments such as anti-inflammatory drugs and opioids, even at the maximum doses that can be tolerated [2]. Animal models of chronic pain often involve nerve injury induced neuropathic pain or inflammatory pain to identify the crucial pathophysiological systems which contribute to chronic pain [3].

In the spinal dorsal horn, the substantia gelatinosa (SG; lamina II [4]) is one of the principal areas of synaptic processing of nociceptive information. The SG is considered to have sophisticated circuits to modulate synaptic transmission, and is one of the key sites for the generation of central sensitization in chronic neuropathic pain states [5]. The SG receives predominantly high-threshold inputs from thinly myelinated Aδ and unmyelinated C primary afferent fibers [6,7]. Thus far, the basic synaptic properties of SG neurons have been well-studied under physiological conditions [8,9]. Studies have used animal models of chronic pain to examine how analgesic or anesthetic drugs could impact synaptic transmission in SG neurons [10]. However, these drugs were studied on a background of pre-established chronic pain in which plastic alterations in excitatory synaptic transmission may have already occurred. Moreover, this work has been primarily conducted in vitro, where afferent communication from the injured limb has been severed.

In the present study, we analyzed excitatory synaptic transmission in the SG of rats under conditions of neuropathic pain (spinal nerve ligation; SNL), inflammatory pain (complete Freund's adjuvant; CFA) and naïve animals. Synaptic transmission was recorded using whole-cell patch-clamp from spinal cord slices and by in vivo whole-cell patch-clamp from SG neurons. We compared active and passive properties including; excitatory synaptic transmission, RMPs and sAPs in SG neurons in naïve and chronic pain model rats.

Section snippets

Materials and methods

All experimental procedures involving the use of animals were approved by the Committee of the Ethics on Animal Experiments, Kyushu University, and were in accordance with the Guidelines of the Japanese Physiological Society. All efforts were made to minimize animal suffering and the number of animals used.

The SNL and CFA group produced mechanical hypersensitivity in the injured hindpaw

To determine whether rats developed mechanical hypersensitivity after the SNL or after the CFA (Fig. 1B and C), a hindpaw withdrawal response was performed by von Frey filaments. The mechanical threshold of the ipsilateral but not the contralateral hindpaw was significantly decreased compared with pre-surgery levels. Two weeks after the SNL and 1 week after the CFA, both group of rats had a withdrawal threshold in the ipsilateral hindpaw that was significantly lower than contralateral hindpaw (

Discussion

Animal models of chronic pain have been used for assessing analgesic or anesthetic drugs in SG neurons in previous studies [10]. However these studies did not focus on changes that occur in basic electrophysiological properties as a consequence of the chronic pain models (SNL [20,21] or the CFA [10,12]). In this study, we recorded many SG neurons and compared passive and active property among naïve, SNL and CFA treated group. In the present study, we examined if major animal models of chronic

Conflicts of interest

All authors have no conflicts of interest.

Acknowledgements

We thank Mr. H. Okai for technical advice on the SNL and Dr. H. Steenland (NeuroTek Innovative Technology) for critical comments and editing the manuscript. This work was supported by JSPS KAKENHI Grant Numbers JP15K08667, JP25860431, JP23790652 to D.U, JP25460733 and JP21600005 to M.Y, JP17H05074 and JP17K19879 to K.K, and was partially supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (Grant Number S1511031 to D.U.). In part, K.K. was

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