Enhanced spinal neuronal responses as a mechanism for increased number and size of active acupoints in visceral hyperalgesia.

Acupuncture has been used to treat a variety of illness and involves the insertion and manipulation of needles into specific points on the body (termed "acupoints"). It has been suggested that acupoints are not merely discrete, static points, but can be dynamically changed according to the pathological state of internal organs. We investigated in a rat model of mustard oil (MO)-induced visceral hyperalgesia whether the number and size of acupoints were modified according to the severity of the colonic pain, and whether the changes were associated with enhanced activity of the spinal dorsal horn. In MO-treated rats, acupoints showing neurogenic inflammation (termed "neurogenic spots" or Neuro-Sps) were found both bilaterally and unilaterally on the leg. The number and size of these acupoints increased along with increasing doses of MO. Electroacupuncture of the acupoints generated analgesic effects on MO-induced visceral hypersensitivity. The MO-treated rats showed an increase in c-Fos expression in spinal dorsal horn neurons and displayed increased evoked activity and a prolonged after-discharge in spinal wide dynamic response (WDR) neurons in response to colorectal distension. Increased number and size of neurogenic inflammatory acupoints following MO treatment were reduced by inhibiting AMPA and NMDA receptors in the spinal cord. Our findings suggest that acupoints demonstrate increased number and size along with severity of visceral pain, which may be associated with enhanced neuronal responses in spinal dorsal horn neurons.

Synaptic Inputs to the Mouse Dorsal Vagal Complex and Its Resident Preproglucagon Neurons.

Stress responses are coordinated by widespread neural circuits. Homeostatic and psychogenic stressors activate preproglucagon (PPG) neurons in the caudal nucleus of the solitary tract (cNTS) that produce glucagon-like peptide-1; published work in rodents indicates that these neurons play a crucial role in stress responses. While the axonal targets of PPG neurons are well established, their afferent inputs are unknown. Here we use retrograde tracing with cholera toxin subunit b to show that the cNTS in male and female mice receives axonal inputs similar to those reported in rats. Monosynaptic and polysynaptic inputs specific to cNTS PPG neurons were revealed using Cre-conditional pseudorabies and rabies viruses. The most prominent sources of PPG monosynaptic input include the lateral (LH) and paraventricular (PVN) nuclei of the hypothalamus, parasubthalamic nucleus, lateral division of the central amygdala, and Barrington's nucleus (Bar). Additionally, PPG neurons receive monosynaptic vagal sensory input from the nodose ganglia and spinal sensory input from the dorsal horn. Sources of polysynaptic input to cNTS PPG neurons include the hippocampal formation, paraventricular thalamus, and prefrontal cortex. Finally, cNTS-projecting neurons within PVN, LH, and Bar express the activation marker cFOS in mice after restraint stress, identifying them as potential sources of neurogenic stress-induced recruitment of PPG neurons. In summary, cNTS PPG neurons in mice receive widespread monosynaptic and polysynaptic input from brain regions implicated in coordinating behavioral and physiological stress responses, as well as from vagal and spinal sensory neurons. Thus, PPG neurons are optimally positioned to integrate signals of homeostatic and psychogenic stress.SIGNIFICANCE STATEMENT Recent research has indicated a crucial role for glucagon-like peptide-1-producing preproglucagon (PPG) neurons in regulating both appetite and behavioral and autonomic responses to acute stress. Intriguingly, the central glucagon-like peptide-1 system defined in rodents is conserved in humans, highlighting the translational importance of understanding its anatomical organization. Findings reported here indicate that PPG neurons receive significant monosynaptic and polysynaptic input from brain regions implicated in autonomic and behavioral responses to stress, as well as direct input from vagal and spinal sensory neurons. Improved understanding of the neural pathways underlying the recruitment of PPG neurons may facilitate the development of novel therapies for the treatment of stress-related disorders.

Second-order spinal cord pathway contributes to cortical responses after long recoveries from dorsal column injury in squirrel monkeys.

Months after the occurrence of spinal cord dorsal column lesions (DCLs) at the cervical level, neural responses in the hand representation of somatosensory area 3b hand cortex recover, along with hand use. To examine whether the second-order spinal cord pathway contributes to this functional recovery, we injected cholera toxin subunit B (CTB) into the hand representation in the cuneate nucleus (Cu) to label the spinal cord neurons, and related results to cortical reactivation in four squirrel monkeys (Saimiri boliviensis) at least 7 months after DCL. In two monkeys with complete DCLs, few CTB-labeled neurons were present below the lesion, and few neurons in the affected hand region in area 3b responded to touch on the hand. In two other cases with large but incomplete DCLs, CTB-labeled neurons were abundant below the lesion, and the area 3b hand cortex responded well to tactile stimulation in a roughly somatotopic organization. The proportions of labeled neurons in the spinal cord hand region reflected the extent of cortical reactivation to the hand. Comparing monkeys with short and long recovery times suggests that the numbers of labeled neurons below the lesion increase with time following incomplete DCLs (<95%) but decrease with time after nearly complete DCLs (≥95%). Taken together, these results suggest that the second-order spinal cord pathway facilitates cortical reactivation, likely through the potentiation of persisting tactile inputs from the hand to the Cu over months of postlesion recovery.

Inhibition Mediated by Glycinergic and GABAergic Receptors on Excitatory Neurons in Mouse Superficial Dorsal Horn Is Location-Specific but Modified by Inflammation.

The superficial dorsal horn is the synaptic termination site for many peripheral sensory fibers of the somatosensory system. A wide range of sensory modalities are represented by these fibers, including pain, itch, and temperature. Because the involvement of local inhibition in the dorsal horn, specifically that mediated by the inhibitory amino acids GABA and glycine, is so important in signal processing, we investigated regional inhibitory control of excitatory interneurons under control conditions and peripheral inflammation-induced mechanical allodynia. We found that excitatory interneurons and projection neurons in lamina I and IIo are dominantly inhibited by GABA while those in lamina IIi and III are dominantly inhibited by glycine. This was true of identified neuronal subpopulations: neurokinin 1 receptor-expressing (NK1R+) neurons in lamina I were GABA-dominant while protein kinase C gamma-expressing (PKCγ+) neurons at the lamina IIi-III border were glycine-dominant. We found this pattern of synaptic inhibition to be consistent with the distribution of GABAergic and glycinergic neurons identified by immunohistochemistry. Following complete Freund's adjuvant injection into mouse hindpaw, the frequency of spontaneous excitatory synaptic activity increased and inhibitory synaptic activity decreased. Surprisingly, these changes were accompanied by an increase in GABA dominance in lamina IIi. Because this shift in inhibitory dominance was not accompanied by a change in the number of inhibitory synapses or the overall postsynaptic expression of glycine receptor α1 subunits, we propose that the dominance shift is due to glycine receptor modulation and the depressed function of glycine receptors is partially compensated by GABAergic inhibition.SIGNIFICANCE STATEMENT Pain associated with inflammation is a sensation we would all like to minimize. Persistent inflammation leads to cellular and molecular changes in the spinal cord dorsal horn, including diminished inhibition, which may be responsible for enhance excitability. Investigating inhibition in the dorsal horn following peripheral inflammation is essential for development of improved ways to control the associated pain. In this study, we have elucidated regional differences in inhibition of excitatory interneurons in mouse dorsal horn. We have also discovered that the dominating inhibitory neurotransmission within specific regions of dorsal horn switches following peripheral inflammation and the accompanying hypersensitivity to thermal and mechanical stimuli. Our novel findings contribute to a more complete understanding of inflammatory pain.

Pioglitazone Inhibits the Development of Hyperalgesia and Sensitization of Spinal Nociresponsive Neurons in Type 2 Diabetes.

UNLABELLED: Thiazolidinedione drugs (TZDs) such as pioglitazone are approved by the U.S. Food and Drug Administration for the treatment of insulin resistance in type 2 diabetes. However, whether TZDs reduce painful diabetic neuropathy (PDN) remains unknown. Therefore, we tested the hypothesis that chronic administration of pioglitazone would reduce PDN in Zucker Diabetic Fatty (ZDF(fa/fa) [ZDF]) rats. Compared with Zucker Lean (ZL(fa/+)) controls, ZDF rats developed: (1) increased blood glucose, hemoglobin A1c, methylglyoxal, and insulin levels; (2) mechanical and thermal hyperalgesia in the hind paw; (3) increased avoidance of noxious mechanical probes in a mechanical conflict avoidance behavioral assay, to our knowledge, the first report of a measure of affective-motivational pain-like behavior in ZDF rats; and (4) exaggerated lumbar dorsal horn immunohistochemical expression of pressure-evoked phosphorylated extracellular signal-regulated kinase. Seven weeks of pioglitazone (30 mg/kg/d in food) reduced blood glucose, hemoglobin A1c, hyperalgesia, and phosphorylated extracellular signal-regulated kinase expression in ZDF. To our knowledge, this is the first report to reveal hyperalgesia and spinal sensitization in the same ZDF animals, both evoked by a noxious mechanical stimulus that reflects pressure pain frequently associated with clinical PDN. Because pioglitazone provides the combined benefit of reducing hyperglycemia, hyperalgesia, and central sensitization, we suggest that TZDs represent an attractive pharmacotherapy in patients with type 2 diabetes-associated pain. PERSPECTIVE: To our knowledge, this is the first preclinical report to show that: (1) ZDF rats exhibit hyperalgesia and affective-motivational pain concurrent with central sensitization; and (2) pioglitazone reduces hyperalgesia and spinal sensitization to noxious mechanical stimulation within the same subjects. Further studies are needed to determine the anti-PDN effect of TZDs in humans.

Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.

Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.

The lidocaine metabolite N-ethylglycine has antinociceptive effects in experimental inflammatory and neuropathic pain.

Glycine transporter 1 (GlyT1) plays a crucial role in regulating extracellular glycine concentrations and might thereby constitute a new drug target for the modulation of glycinergic inhibition in pain signaling. Consistent with this view, inhibition of GlyT1 has been found to induce antinociceptive effects in various animal pain models. We have shown previously that the lidocaine metabolite N-ethylglycine (EG) reduces GlyT1-dependent glycine uptake by functioning as an artificial substrate for this transporter. Here, we show that EG is specific for GlyT1 and that in rodent models of inflammatory and neuropathic pain, systemic treatment with EG results in an efficient amelioration of hyperalgesia and allodynia without affecting acute pain. There was no effect on motor coordination or the development of inflammatory edema. No adverse neurological effects were observed after repeated high-dose application of EG. EG concentrations both in blood and spinal fluid correlated with an increase of glycine concentration in spinal fluid. The time courses of the EG and glycine concentrations corresponded well with the antinociceptive effect. Additionally, we found that EG reduced the increase in neuronal firing of wide-dynamic-range neurons caused by inflammatory pain induction. These findings suggest that systemically applied lidocaine exerts antihyperalgesic effects through its metabolite EG in vivo, by enhancing spinal inhibition of pain processing through GlyT1 modulation and subsequent increase of glycine concentrations at glycinergic inhibitory synapses. EG and other substrates of GlyT1, therefore, may be a useful therapeutic agent in chronic pain states involving spinal disinhibition.

In vivo two-photon imaging of structural dynamics in the spinal dorsal horn in an inflammatory pain model.

Two-photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two-photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine-like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple-site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N-methyl-D-aspartate receptor antagonists, and gabapentin, a presynaptic Ca(2+) channel blocker, completely suppressed the inflammation-induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two-photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation-associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.

In vivo electrophysiological recording techniques for the study of neuropathic pain in rodent models.

Neuropathic pain develops following nerve injury, and is a chronic pain syndrome that can persist long after repair of a wound or removal of the neurological insult. This condition remains poorly treated, not least because of a lack of mechanism-based therapeutics. Clinically, neuropathic pain is characterized by three major symptoms: thermal or mechanical allodynia (pain sensation in response to previously non-noxious stimuli); hyperalgesia (enhanced pain sensation to noxious stimulation); and spontaneous, ongoing pain. These clinical symptoms can be modeled in rodent neuropathic pain models using behavioral and electrophysiological readouts. This unit describes techniques designed to record pathophysiological electrical activity associated with neuropathic pain at the level of the periphery, in single fibers of primary sensory neurons, and from wide dynamic range (WDR) neurons of the dorsal horn of the spinal cord. These techniques can be employed in both naïve animals and in animal models of neuropathy to investigate fundamental mechanisms contributing to the neuropathic pain state and the site, mode, and mechanism of action of putative analgesics.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge.

The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p<.01), and at PND 22, increased flinching in response to formalin injection (p<.05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p<.001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p<.05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis.

Analysis of interspike interval of dorsal horn neurons evoked by different needle manipulations at ST36.

OBJECTIVES: Previous research has suggested that different manual acupuncture (MA) manipulations may have different physiological effects. Recent studies have demonstrated that neural electrical signals are generated or changed when acupuncture is administered. In order to explore the effects of different MA manipulations on the neural system, an experiment was designed to record the discharges of wide dynamic range (WDR) neurons in the spinal dorsal horn evoked by MA at different frequencies (0.5, 1, 2 and 3 Hz) at ST36. METHODS: Microelectrode extracellular recordings were used to record the discharges of WDR neurons evoked by different MA manipulations. Approximate firing rate and coefficient of variation of interspike interval (ISI) were used to extract the characteristic parameters of the neural electrical signals after spike sorting, and the neural coding of the evoked discharges by different MA manipulations was obtained. RESULTS: Our results indicated that the neuronal firing rate and time sequences of ISI showed distinct clustering properties for different MA manipulations, which could distinguish them effectively. CONCLUSIONS: The combination of firing rate and ISI codes carries information about the acupuncture stimulus frequency. Different MA manipulations appear to change the neural coding of electrical signals in the spinal dorsal horn through WDR neurons.

PKCα is required for inflammation-induced trafficking of extrasynaptic AMPA receptors in tonically firing lamina II dorsal horn neurons during the maintenance of persistent inflammatory pain.

UNLABELLED: Persistent inflammation promotes internalization of synaptic GluR2-containing, Ca(2+)-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing, Ca(2+)-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are unclear. Here, using antisense (AS) oligodeoxynucleotides (ODN) that specifically knock down PKCα, we found that a decrease in dorsal horn PKCα expression prevents complete Freund's adjuvant (CFA)-induced increase in functional expression of extrasynaptic Ca(2+)-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. Augmented AMPA-induced currents and associated [Ca(2+)](i) transients were abolished, and the current rectification 1 day post-CFA was reversed. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Finally, dorsal horn PKCα knockdown produced an antinociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Our results indicate that inflammation-induced trafficking of extrasynaptic Ca(2+)-permeable AMPARs in tonically firing SG neurons depends on PKCα, and that this PKCα-dependent trafficking may contribute to persistent inflammatory pain maintenance. PERSPECTIVE: This study shows that PKCα knockdown blocks inflammation-induced upregulation of extrasynaptic Ca(2+)-permeable AMPARs in dorsal horn neurons and produces an antinociceptive effect during the maintenance period of inflammatory pain. These findings have potential implications for use of PKCα gene-silencing therapy to prevent and/or treat persistent inflammatory pain.

Extrasynaptic AMPA receptors in the dorsal horn: evidence and functional significance.

Extrasynaptic AMPA receptors (AMPARs) are widely expressed in the brain, spinal cord and periphery. These receptors are critically involved in activity-dependent synaptic transmission and changes in their functioning are causally linked to multiple neuropathologies in the central nervous system (CNS). However, most studies in this field have been concentrated on elucidating synaptic AMPAR functioning, leaving a possible involvement of an extrasynaptic pool of AMPARs in normal and pathological signaling open for consideration. Here, we review the present evidence for extrasynaptic AMPAR function in the dorsal horn neurons of the spinal cord, linking these receptors to neurotransmission and non-synaptic signaling in this part of the CNS. In addition, we summarize current knowledge about the role of extrasynaptic AMPARs in the development and maintenance of pain states during inflammation. This knowledge potentially suggests the development of alternative therapies to prevent and/or treat inflammatory pain.

[Nonlinear dynamic analysis of electrical signals of wide dynamic range neurons in the spinal dorsal horn evoked by acupuncture manipulation at different frequencies].

OBJECTIVE: To study the encoding information of electrical signals of wide dynamic range (WDR) neurons in the spinal dorsal horn evoked by acupuncture manipulation at different frequencies using nonlinear dynamics analysis. METHODS: Microelectrode extracellular recordings were used to observe the WDR neuron discharge evoked by acupuncture manipulation at Zusanli point (ST36) with different frequencies (0.5, 1, 2, and 3 Hz) in SD rats. The nonlinear dynamics analysis method was used to extract the nonlinear characteristic parameters, such as interspike interval, the Lyapunov exponent, Lempel-Ziv complexity, and the neural coding of the electrical signal evoked by acupuncture manipulations at different frequencies. RESULTS: Different characteristics were manifested with acupuncture manipulations at 4 different frequencies. More than a simple linear correlation was shown between the firing rate of the WDR neurons and the frequency of the acupuncture manipulation. The electrical signals evoked by acupuncture manipulation at Zusanli point (ST36) showed distinguished chaotic features. CONCLUSIONS: It is applicable and feasible to describe and summarize the rhythm of the acupuncture electrical signal using the concepts and terminology of the nonlinear dynamics. Different acupuncture manipulation methods could interfere the transmission, coding, and processing of electrical signals in the spinal dorsal horn.

Elucidation of pathophysiology and treatment of neuropathic pain.

Neuropathic pain, pain arising as a direct consequence of a lesion or disease affecting the somatosensory system, is relatively common, occurring in about 1% of the population. Studies in animal models describe a number of peripheral and central pathophysiological processes after nerve injury that would be the basis of underlying neuropathic pain mechanism. Additionally, neuro-imaging (positron emission tomography and functional magnetic resonance imaging) provides insights in brain mechanisms corresponding with mechanistic processes including allodynia, hyperalgesia, altered sensation, and spontaneous pain. A change in function, chemistry, and structures of neurons (neural plasticity) underlie the production of the altered sensitivity characteristics of neuropathic pain. Peripheral processes in neuropathic pain involve production of mediators (cytokines, protons, nerve growth factor), alterations in calcium channels, sodium channels, hyperpolarisation-activated nucleotide-gated ion channels, and potassium channels, phenotypic switches and sprouting of nerves endings, and involvement of the sympathetic nervous system. Stimulation of the N-Methyl-D-Aspartate receptor, activation of microglia, oligodendrocytes, and astrocytes, increased production of nerve growth factor and brain-derived neurotrophic factor together with loss of spinal inhibitory control are responsible for central neuron hyperexcitability and maintenance of neuropathic pain. Recent advances, including functional imaging techniques, in identification of peripheral and central sensitization mechanisms related to nervous system injury have increased potential for affecting pain research from both diagnostic as well as therapeutic view. Key brain regions involved in generating pharmacologically induced analgesia may be identified. Despite the progress in pain research, neuropathic pain is challenge to manage. Although numerous treatment options are available for relieving neuropathic pain, there is no consensus on the most appropriate treatment. However, recommendations can be proposed for first-line, second-line, and third-line pharmacological treatments based on the level of evidence for the different treatment strategies. Available therapies shown to be effective in managing neuropathic pain include opioids and tramadol, anticonvulsants, antidepressants, topical treatments (lidocaine patch, capsaicin), and ketamine. Tricyclic antidepressants are often the first drugs selected to alleviate neuropathic pain (first-line pharmacological treatment). Although they are very effective in reducing pain in several neuropathic pain disorders, treatment may be compromised (and outweighed) by their side effects. In patients with a history of cardiovascular disorders, glaucoma, and urine retention, pregabalin and gabapentine are emerging as first-line treatment for neuropathic pain. In addition these anti-epileptic drugs have a favourable safety profile with minimal concerns regarding drug interactions and showing no interference with hepatic enzymes. Alternatively, opioids (oxycodone and methadone) and tramadol may alleviate nociceptive and neuropathic pain. Despite the numerous treatment options available for relieving neuropathic pain, no more than half of patients experience clinically meaningful pain relief, which is almost always partial but not complete relief. In addition, patients frequently experience burdensome adverse effects and as a consequence are often unable to tolerate the treatment. In the remaining patients, combination therapies using two or more analgesics with different mechanisms of action may also offer adequate pain relief. Although combination treatment is clinical practice and may result in greater pain relief, trials regarding different combinations of analgesics (which combination to use, occurrence of additive or supra-additive effects, sequential or concurrent treatment, adverse-event profiles of these analgesics, alone and in combination) are scarce. If medical treatments have failed, invasive therapies such as intrathecal drug administration and neurosurgical stimulation techniques (spinal cord stimulation, deep brain stimulation, and motor cortex stimulation) may be considered.

Sex differences in spinal processing of transient and inflammatory colorectal stimuli in the rat.

Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil-treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats.

CGRPα-expressing sensory neurons respond to stimuli that evoke sensations of pain and itch.

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.

Electroacupuncture reduces the evoked responses of the spinal dorsal horn neurons in ankle-sprained rats.

Acupuncture is shown to be effective in producing analgesia in ankle sprain pain in humans and animals. To examine the underlying mechanisms of the acupuncture-induced analgesia, the effects of electroacupuncture (EA) on weight-bearing forces (WBR) of the affected foot and dorsal horn neuron activities were examined in a rat model of ankle sprain. Ankle sprain was induced manually by overextending ligaments of the left ankle in the rat. Dorsal horn neuron responses to ankle movements or compression were recorded from the lumbar spinal cord using an in vivo extracellular single unit recording setup 1 day after ankle sprain. EA was applied to the SI-6 acupoint on the right forelimb (contralateral to the sprained ankle) by trains of electrical pulses (10 Hz, 1-ms pulse width, 2-mA intensity) for 30 min. After EA, WBR of the sprained foot significantly recovered and dorsal horn neuron activities were significantly suppressed in ankle-sprained rats. However, EA produced no effect in normal rats. The inhibitory effect of EA on hyperactivities of dorsal horn neurons of ankle-sprained rats was blocked by the α-adrenoceptor antagonist phentolamine (5 mg/kg ip) but not by the opioid receptor antagonist naltrexone (10 mg/kg ip). These data suggest that EA-induced analgesia in ankle sprain pain is mediated mainly by suppressing dorsal horn neuron activities through α-adrenergic descending inhibitory systems at the spinal level.

Septal stimulation inhibits spinal cord dorsal horn neuronal activity.

Deep brain stimulation (DBS) has been used for relieving chronic pain in patients that have been through other existing options. The septum has been one of the targets for such treatment. The purpose of this study was to determine the inhibitory effect of electrical stimulation in the medial septum diagonal band of broca (MSDB) on neuronal activity in the spinal cord of rats anesthetized with sodium pentobarbital. While unilaterally stimulating the MSDB, wide dynamic range neurons in the lumbar region of the spinal cord were recorded in response to graded mechanical stimulation of the hind paws (brush, pressure, and pinch). Stimulation was at 1, 5, 10, and 20V, at 100Hz, and 0.1ms duration. Significant bilateral reduction was observed in response to pressure (ipsilaterally: 0.90±0.05, 0.48±0.06*, 0.55±0.05*, 0.40±0.05*; and contralaterally: 0.70±0.06*, 0.59±0.08*, 0.75±0.05*, 0.49±0.07*) and pinch (ipsilaterally: 0.89±0.08, 0.46±0.05*, 0.54±0.04*, 0.50±0.05*; and contralaterally: 0.78±0.05, 0.61±0.07*, 0.64±0.04*, 0.53±0.06*). Data were expressed as a fraction of control. Significant changes were also found in responses to brush in certain groups (ipsilaterally: 1.08±0.08, 0.72±0.06*, 1.00±0.12, 0.65±0.06*; and contralaterally: 0.93±0.05, 0.77±0.07*, 0.98±0.05, 0.84±0.07). Further analysis suggested that 5V was adequate for achieving optimal inhibition. It is concluded that the MSDB can be used as alternative target for DBS in the treatment of pain.

Central neural pathways for thermoregulation.

Central neural circuits orchestrate a homeostatic repertoire to maintain body temperature during environmental temperature challenges and to alter body temperature during the inflammatory response. This review summarizes the functional organization of the neural pathways through which cutaneous thermal receptors alter thermoregulatory effectors: the cutaneous circulation for heat loss, the brown adipose tissue, skeletal muscle and heart for thermogenesis and species-dependent mechanisms (sweating, panting and saliva spreading) for evaporative heat loss. These effectors are regulated by parallel but distinct, effector-specific neural pathways that share a common peripheral thermal sensory input. The thermal afferent circuits include cutaneous thermal receptors, spinal dorsal horn neurons and lateral parabrachial nucleus neurons projecting to the preoptic area to influence warm-sensitive, inhibitory output neurons which control thermogenesis-promoting neurons in the dorsomedial hypothalamus that project to premotor neurons in the rostral ventromedial medulla, including the raphe pallidus, that descend to provide the excitation necessary to drive thermogenic thermal effectors. A distinct population of warm-sensitive preoptic neurons controls heat loss through an inhibitory input to raphe pallidus neurons controlling cutaneous vasoconstriction.