Contrasting alterations to synaptic and intrinsic properties in upper-cervical superficial dorsal horn neurons following acute neck muscle inflammation

Molecular Pain Contrasting alterations to synaptic and intrinsic properties in upper-cervical superficial dorsal horn neurons following acute neck muscle inflammation Belinda M Harris 0 David I Hughes 1 Philip S Bolton 0 Melissa A Tadros 0 Robert J Callister 0 Brett A Graham 0 0 School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle and Hunter Medical Research Institute, Room 411 Medical Sciences Building, University Drive , Newcastle, NSW 2308 , Australia 1 Institute of Neuroscience & Psychology, College of Medical, Veterinary & Life Sciences, University of Glasgow , Glasgow , UK Background: Acute and chronic pain in axial structures, like the back and neck, are difficult to treat, and have incidence as high as 15%. Surprisingly, most preclinical work on pain mechanisms focuses on cutaneous structures in the limbs and animal models of axial pain are not widely available. Accordingly, we developed a mouse model of acute cervical muscle inflammation and assessed the functional properties of superficial dorsal horn (SDH) neurons. Results: Male C57/Bl6 mice (P24-P40) were deeply anaesthetised (urethane 2.2 g/kg i.p) and the rectus capitis major muscle (RCM) injected with 40 l of 2% carrageenan. Sham animals received vehicle injection and controls remained anaesthetised for 2 hrs. Mice in each group were sacrificed at 2 hrs for analysis. c-Fos staining was used to determine the location of activated neurons. c-Fos labelling in carrageenan-injected mice was concentrated within ipsilateral (87% and 63% of labelled neurons in C1 and C2 segments, respectively) and contralateral laminae I - II with some expression in lateral lamina V. c-Fos expression remained below detectable levels in control and sham animals. In additional experiments, whole cell recordings were obtained from visualised SDH neurons in transverse slices in the ipsilateral C1 and C2 spinal segments. Resting membrane potential and input resistance were not altered. Mean spontaneous EPSC amplitude was reduced by ~20% in neurons from carrageenan-injected mice versus control and sham animals (20.63 1.05 vs. 24.64 0.91 and 25.87 1.32 pA, respectively). The amplitude (238 33 vs. 494 96 and 593 167 pA) and inactivation time constant (12.9 1.5 vs. 22.1 3.6 and 15.3 1.4 ms) of the rapid A type potassium current (IAr), the dominant subthreshold current in SDH neurons, were reduced in carrageenan-injected mice. Conclusions: Excitatory synaptic drive onto, and important intrinsic properties (i.e., IAr) within SDH neurons are reduced two hours after acute muscle inflammation. We propose this time point represents an important transition period between peripheral and central sensitisation with reduced excitatory drive providing an initial neuroprotective mechanism during the early stages of the progression towards central sensitisation. Mice; A-current; EPSC; Carrageenan; Action potential - Introduction Acute inflammation in peripheral structures, such as muscle, results in nociceptor activation and pain. Unfortunately, pain sometimes outlasts the initial inflammatory insult and persists in a more chronic form. Longterm changes in components of the pain neuroaxis such as altered synapses and neuronal properties are thought to underlie this process [1]. Understanding the mechanisms involved in the progression from the acute to chronic pain state, however, remains a major challenge in pain neurobiology. Regardless of the site involved in chronic pain states, the central processing of nociceptive signals begins in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord [2]. Here, the passage of information to be relayed from primary afferent fibres to higher brain regions can be gated by synaptic inputs from local segmental interneurons and descending brainstem centres. Such a process greatly influences whether nociceptor activation can ultimately be perceived as pain [3]. Since the initial discovery that the electrophysiological properties of neurons in the lumbar spinal cord could be altered after repeated noxious peripheral input [4], plasticity in spinal cord pain circuits, termed central sensitisation, has been considered a key element in the development of chronic pain [5,6]. Surprisingly, most of our understanding of the spinal mechanisms underlying plasticity in pain circuits comes from studies on the rodent hindlimb following nerve injury or inflammation, even though there is both clinical and pre-clinical evidence suggesting that such mechanisms may differ for axial and limb pain [7]. Patients with neck pain often complain of a range of symptoms not obviously associated with damage to neck structures. These include dizziness, visual disturbances, general weakness, numbness or parathesis, cutaneous hyperalgesia, as well as psychological symptoms such as disturbances in concentration and memory [8]. These signs and symptoms are not normally associated with limb pain where sensory disturbances tend to be more localised [9]. At the cellular level we know that upper cervical SDH neurons receive input from a unique combination of tissues including cutaneous and deep structures in the neck, head and cranial vault [10,11]. This afferent convergence presumably plays an important role in the complex presentation of pain originating in neck tissues [12]. Together these data suggest the mechanisms underlying processing of nociceptive signals originating in neck structures differ from those in the limbs, and may be important for the development of neck pain and its symptoms. In order to begin to understand the spinal mechanisms that underlie the development of chronic neck pain, we assessed the impact of acute muscle inflammation on the synaptic and intrinsic properties of SDH neurons in the mouse upper cervical spinal cord. After two hours of acute inflammation of the rectus capitus major (RCM) muscle we find excitatory synaptic drive to SDH neurons and the rapid A-type potassium current are reduced in SDH neurons. We hypothesise that two hours post inflammation is an important epoch during the cascade of events where aberrant nociceptive signaling transitions from a peripheral to central locus. Methods The University of Newcastle Animal Care & Ethics Committee approved all experimental procedures. Male C57/ Bl6 mice (P24 - P40) were deeply anaesthetised with urethane (2.2 g/kg i.p) and underwent the following treatments: control animals remained anaesthetised for 2 hrs; sham animals received a unilateral injection of 40 l phosphate-buffered saline (PBS, pH 7.4) via a 26 gauge needle into the RCM muscle; experimental animals received an injection of 2% carrageenan (in phosphate buffered saline - PBS) into the RCM. This muscle is particularly large in rodents and runs from the spinous process of C2 to the skull. It is easily located by palpating the external occipital protuberance (inion) and the spinous process of the C7 vertebrae. The location of th (...truncated)