True-color 3D rendering of human anatomy using surface-guided color sampling from cadaver cryosection image data: A practical approach.

Three-dimensional computer graphics are increasingly used for scientific visualization and for communicating anatomical knowledge and data. This study presents a practical method to produce true-color 3D surface renditions of anatomical structures. The procedure involves extracting the surface geometry of the structure of interest from a stack of cadaver cryosection images, using the extracted surface as a probe to retrieve color information from cryosection data, and mapping sampled colors back onto the surface model to produce a true-color rendition. Organs and body parts can be rendered separately or in combination to create custom anatomical scenes. By editing the surface probe, structures of interest can be rendered as if they had been previously dissected or prepared for anatomical demonstration. The procedure is highly flexible and nondestructive, offering new opportunities to present and communicate anatomical information and knowledge in a visually realistic manner. The technical procedure is described, including freely available open-source software tools involved in the production process, and examples of color surface renderings of anatomical structures are provided.

Short-term improvements in the body schema can modulate pain perception in fibromyalgia syndrome.

OBJECTIVES: This study aimed to evaluate the pain perception and several aspects of disrupted body schema, in a sample of patients suffering from fibromyalgia (FM) syndrome. METHODS: Twenty-six patients were organised into two groups: the tactile discrimination group and control group (exposed to tactile stimulation alone). Outcome measures were the pain intensity in body regions commonly described as painful (visual analogue scale) and clinical status, body esteem scale (BES), interoceptive awareness. Tactile acuity was measured by the two-point discrimination test (TPD), hits in the location of the stimulus, the probe size discrimination and the graphesthesia task. RESULTS: The group exposed to tactile discrimination experienced a significant improvement in all tactile acuity outcome measures. The decrease of the Fibromyalgia Impact Questionnaire variable was relevant (81.58, SEM 3.29 vs. 72.91, SEM 6.43; p=0.07). Likewise, pain perception was lower in all of the body regions evaluated (reduction of 12.2% in the stimulated body region (cervical VAS) with a large effect size, a pain reduction of 11.3% in the wrists and 9.2% in the knees. The correlation index showed association between the cervical VAS and TPD (ρ=0.53; p<0.05). CONCLUSIONS: There was no improvement in pain scores in the control group but the TPD was decreased also. The BES scores did not show differences between groups. However, interoceptive awareness showed a slight reduction in the group exposed to tactile discrimination (3.68, SEM 0.15 vs. 3.35, SEM 0.19; p=0.01). After short-term tactile discrimination protocol, the group exposed to tactile discrimination experienced a significant improvement in all tactile acuity outcome measures: pain perception, tactile acuity and body perception, compatible with adjustments in the body schema. The tactile stimulation alone group did not show the same improvement.

External surface anatomy of the postfolding human embryo: Computer-aided, three-dimensional reconstruction of printable digital specimens.

Opportunities for clinicians, researchers, and medical students to become acquainted with the three-dimensional (3D) anatomy of the human embryo have historically been limited. This work was aimed at creating a collection of digital, printable 3D surface models demonstrating major morphogenetic changes in the embryo's external anatomy, including typical features used for external staging. Twelve models were digitally reconstructed based on optical projection tomography, high-resolution episcopic microscopy and magnetic resonance imaging datasets of formalin-fixed specimens of embryos of developmental stages 12 through 23, that is, stages following longitudinal and transverse embryo folding. The reconstructed replica reproduced the external anatomy of the actual specimens in great detail, and the progress of development over stages was recognizable in a variety of external anatomical features and bodily structures, including the general layout and curvature of the body, the pharyngeal arches and cervical sinus, the physiological gut herniation, and external genitalia. In addition, surface anatomy features commonly used for embryo staging, such as distinct steps in the morphogenesis of facial primordia and limb buds, were also apparent. These digital replica, which are all provided for 3D visualization and printing, can serve as a novel resource for teaching and learning embryology and may contribute to a better appreciation of the human embryonic development.

Embedding interactive, three-dimensional content in portable document format to deliver gross anatomy information and knowledge.

The Portable Document Format (PDF) is likely the most widely used digital file format for scholarly and scientific electronic publishing. Since format specification version 1.6, three-dimensional (3D) models in Universal 3D (U3D) format can be embedded into PDF files. The present study demonstrates a repertoire of graphic strategies and modes of presentation that exploit the potentials of 3D models embedded in PDF to deliver anatomical information and knowledge. Three-dimensional models and scenes representing anatomical structures generated by 3D surface scanning or by segmentation from either clinical imaging data or cadaver sectional images were converted into U3D format and then embedded into PDF files using both freely and commercially available software. The relevant steps and required software tools are described. Built-in tools in Adobe Acrobat and JavaScript scripting both were used to pre-configure user interaction with 3D contents. Eight successive proof-of-concept examples of increasing complexity are presented and provided as supplementary material, including both unannotated and annotated 3D specimens, use of bitmap-textures, guided navigation through predetermined 3D scenes, 3D animation, and interactive navigation through tri-planar sectional human cadaver images. Three-dimensional contents embedded in PDF files are generally comparable to multimedia and dedicated 3D software in terms of quality, flexibility, and convenience, and offer new unprecedented opportunities to deliver anatomical information and knowledge.

Dopamine D1-like Receptors Regulate Constitutive, µ-Opioid Receptor-Mediated Repression of Use-Dependent Synaptic Plasticity in Dorsal Horn Neurons: More Harm than Good?

UNLABELLED: The current study reports on a synaptic mechanism through which D1-like receptors (D1LRs) modulate spinal nociception and plasticity by regulating activation of the µ-opioid receptor (MOR).D1LR stimulation with agonist SKF 38393 concentration-dependently depressed C-fiber-evoked potentials in rats receiving spinal nerve ligation (SNL), but not in uninjured rats. Depression was prevented by MOR- but not GABA-receptor blockade. Neurons expressing the D1 subtype were immunopositive for met-enkephalin and vesicular glutamate transporter VGLUT2, but not for GABAergic marker vGAT.Nerve ligation was followed by increased immunoreactivity for D1 in synaptic compartment (P3) in dorsal horn homogenates and presynaptic met-enkephalin-containing boutons. SNL led to increased immunoreactivity for met-enkephalin in dorsal horn homogenates, which was dose-dependently attenuated by selective D1LR antagonist SCH 23390. During blockade of either D1R or MOR, low-frequency (0.2 or 3 Hz) stimulation (LFS) to the sciatic nerve induced long-term potentiation (LTP) of C-fiber-evoked potentials, revealing a constituent role of both receptors in repressing afferent-induced synaptic plasticity. LFS consistently induced NMDA receptor-dependent LTP in nerve-injured rats. The ability of MOR both to prevent LTP and to modulate mechanical and thermal pain thresholds in behavioral tests was preserved in nerve-ligated rats that were postoperatively treated with SCH 23390. D1LR priming for 30 min sufficed to disrupt MOR function in otherwise naive rats via a mechanism involving receptor overuse.The current data support that, whereas D1LR-modulated MOR activation is instrumental in antinociception and endogenous repression of synaptic plasticity, this mechanism deteriorates rapidly by sustained use, generating increased vulnerability to afferent input. SIGNIFICANCE STATEMENT: The current study shows that dopamine D1-like receptors (D1LRs) and µ-opioid receptors (MOR) in the spinal dorsal horn constitutively repress the expression of synaptic long-term potentiation (LTP) of C-fiber-evoked potentials. Anatomical data are provided supporting that the D1 subtype regulates MOR function by modulating met-enkephalin release. Sustained neuropathic pain induced by spinal nerve ligation is accompanied by D1R and met-enkephalin upregulation, acquired D1LR-mediated antinociception, and a loss of endogenous repression of further synaptic plasticity. We show that the ability of MOR to oppose LTP is rapidly impaired by sustained D1LR activation via a mechanism involving sustained MOR activation.

Restoration of normal central pain processing following manual therapy in nonspecific chronic neck pain.

OBJECTIVE: To determine if a 4-week manual therapy treatment restores normal functioning of central pain processing mechanisms in non-specific chronic neck pain (NSCNP), as well as the existence of a possible relationship between changes in pain processing mechanisms and clinical outcome. DESIGN: Cohort study. METHODS: Sixty-three patients with NSCNP, comprising 79% female, with a mean age of 45.8 years (standard deviation: 14.3), received four treatment sessions (once a week) of manual therapy including articular passive mobilizations, soft tissue mobilization and trigger point treatment. Pressure pain thresholds (PPTs), conditioned pain modulation (CPM) and temporal summation of pain (TSP) were evaluated at baseline and after treatment completion. Therapy outcome was measured using the Global Rating of Change Scale (GROC), the Neck disability Index (NDI), intensity of pain during the last 24 hours, Tampa Scale of Kinesiophobia (TSK) and Pain Catastrophizing Scale (PCS). Two sets of generalized linear mixed models with Gaussian response and the identity link were employed to evaluate the effect of the intervention on clinical, psychological and psychophysical measures and the association between psychophysical and clinical outcomes. RESULTS: Following treatment, an increased CPM response (Coefficient: 0.89; 95% credibility interval = 0.14 to 1.65; P = .99) and attenuated TSP (Coefficient: -0.63; 95% credibility interval = -0.82 to -0.43; P = 1.00) were found, along with amelioration of pain and improved clinical status. PPTs at trapezius muscle on the side of neck pain were increased after therapy (Coefficient: 0.22; 95% credibility interval = 0.03 to 0.42; P = .98), but not those on the contralateral trapezius and tibialis anterior muscles. Only minor associations were found between normalization of TSP/CPM and measures of clinical outcome. CONCLUSION: Clinical improvement after manual therapy is accompanied by restoration of CPM and TSP responses to normal levels in NSCNP patients. The existence of only minor associations between changes in central pain processing and clinical outcome suggests multiple mechanisms of action of manual therapy in NSCNP.

Time-dependent cross talk between spinal serotonin 5-HT2A receptor and mGluR1 subserves spinal hyperexcitability and neuropathic pain after nerve injury.

Emerging evidence implicates serotonergic descending facilitatory pathways from the brainstem to the spinal cord in the maintenance of pathologic pain. Upregulation of the serotonin receptor 2A (5-HT(2A)R) in dorsal horn neurons promotes spinal hyperexcitation and impairs spinal µ-opioid mechanisms during neuropathic pain. We investigated the involvement of spinal glutamate receptors, including metabotropic receptors (mGluRs) and NMDA, in 5-HT(2A)R-induced hyperexcitability after spinal nerve ligation (SNL) in rat. High-affinity 5-HT(2A)R agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2) enhanced C-fiber-evoked dorsal horn potentials after SNL, which was prevented by mGluR1 antagonist AIDA [(RS)-1-aminoindan-1,5-dicarboxylic acid] but not by group II mGluR antagonist LY 341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid] or NMDA antagonist d-AP5 [D-(-)-2-amino-5-phosphonopentanoic acid]. 5-HT(2A)R and mGluR1 were found to be coexpressed in postsynaptic densities in dorsal horn neurons. In the absence of SNL, pharmacological stimulation of 5-HT(2A)R with TCB-2 both induced rapid bilateral upregulation of mGluR1 expression in cytoplasmic and synaptic fractions of spinal cord homogenates, which was attenuated by PKC inhibitor chelerythrine, and enhanced evoked potentials during costimulation of mGluR1 with 3,5-DHPG [(RS)-3,5-dihydroxyphenylglycine]. SNL was followed by bilateral upregulation of mGluR1 in 5-HT(2A)R-containing postsynaptic densities. Upregulation of mGluR1 in synaptic compartments was partially prevented by chronic administration of selective 5-HT(2A)R antagonist M100907 [(R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipidinemethanol], confirming 5-HT(2A)R-mediated control of mGluR1 upregulation triggered by SNL. Changes in thermal and mechanical pain thresholds following SNL were increasingly reversed over the days after injury by chronic 5-HT(2A)R blockade. These results emphasize a role for 5-HT(2A)R in hyperexcitation and pain after nerve injury and support mGluR1 upregulation as a novel feedforward activation mechanism contributing to 5-HT(2A)R-mediated facilitation.

An artificial neural network approach for predicting functional outcome in fibromyalgia syndrome after multidisciplinary pain program.

OBJECTIVE: The objective of this study was to evaluate the ability of artificial neural networks (ANNs) to predict, on the basis of clinical variables, the response of persons with fibromyalgia syndrome (FMS) to a standard, 4-week interdisciplinary pain program. DESIGN: The design of this study is retrospective longitudinal. SETTING: Fibromyalgia outpatient clinic in a tertiary-care general hospital. SUBJECTS: The subjects of this study include outpatients with FMS. INTERVENTION: Multidisciplinary pain program including pain pharmacotherapy, cognitive-behavioral therapy, physical therapy, and occupational therapy. OUTCOME MEASURES: Reliable change (RC) of scores on the Stanford Health Assessment Questionnaire (HAQ), and accuracy of ANNs in predicting RC at discharge or at 6-month follow-up as compared to Logistic Regression. RESULTS: ANN-based models using the sensory-discriminative and affective-motivational subscales of the McGill Pain Questionnaire, the HAQ disability index, and the anxiety subscale of Hospital Anxiety and Depression Scale at baseline as input variables correctly classified 81.81% of responders at discharge and 83.33% of responders at 6-month follow-up, as well as 100% of nonresponders at either evaluation time-point. Logistic regression analysis, which was used for comparison, could predict treatment outcome with accuracies of 86.11% and 61.11% at discharge and follow-up, respectively, based on baseline scores on the HAQ and the mental summary component of the Medical Outcomes Study-Short Form 36. CONCLUSIONS: Properly trained ANNs can be a useful tool for optimal treatment selection at an early stage after diagnosis, thus contributing to minimize the lag until symptom amelioration and improving tertiary prevention in patients with FMS.

Is psychological distress intrinsic to fibromyalgia syndrome? Cross-sectional analysis in two clinical presentations.

Clinical presentation of fibromyalgia syndrome (FMS) is heterogeneous and often involves psychological comorbidities. Clinical subgrouping of FMS patients has been proposed as a strategy to improve patients' long-term outcomes by helping identify specific treatment needs. Using the 90 Symptom Checklist Revised (SCL-90-R), we have assessed emotional distress in two FMS patient subpopulations discriminated on the basis of their differences in scores on specific items of the Fibromyalgia Impact Questionnaire (FIQ). Subjects classed as type II exhibited high emotional distress on all ten dimensions studied, which included somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, and additional items subscales, as well as on the global severity index (GSI), positive symptom total (PST), and positive symptom distress index (PDSI). T-scores in these patients were above diagnostic cutoff level of 60 on somatization, obsessive-compulsive, and depression subscales. In contrast, the profile exhibited by type I subjects fell entirely within normal values for nonpsychiatric population. Emotional status was significantly inversely correlated with present clinical pain in type I-, but not in type II-fibromyalgia patients. Regression analysis revealed a model based on phobic anxiety, paranoid ideation, and depression subscales as best contributing to classification. The present data suggest that associated psychological distress and maladaptive emotional responses that are commonly attributed to the general FMS population may be largely a distinguishing feature of one subset of patients.

Enhanced Pronociceptive and Disrupted Antinociceptive Mechanisms in Nonspecific Chronic Neck Pain.

OBJECTIVE: Evidence suggests altered pronociceptive and antinociceptive mechanisms in many chronic pain conditions. Knowledge about these mechanisms in nonspecific chronic neck pain (NSNP) would improve understanding of the causes and the design of more effective treatments. Pressure pain threshold (PPT) is often used to assess presence of altered nociceptive processing in NSNP; however, its usefulness to detect this is yet to be established. The purpose of this study was to determine the functional status of temporal summation of second pain (TSSP) and conditioned pain modulation (CPM) in NSNP and to characterize the association of both measures with PPT and clinical features of NSNP. METHODS: Thirty-two participants with NSNP (mean [SD] age = 44 [11] years; 27 female) and 32 age- and sex-matched healthy controls were recruited. TSSP was assessed using an electrical stimulus at the dorsum of the hand, and CPM was evaluated with the Cold Pressor Test. PPT was assessed bilaterally at the neck and tibialis anterior muscles. RESULTS: Participants with NSNP showed greater TSPP (mean difference = 0.23; 95% CI = 0.46-0.01; Cohen d = 0.51) and lower CPM (mean difference = 19.44; 95% CI = 10.42-28.46; Cohen d = 1.09). Pooled data from all participants showed lower PPTs at the neck than the tibialis anterior. However, PPT measures did not differ between groups at either location. PPT measures were not correlated with CPM and TSP. CONCLUSION: NSNP is associated with enhanced pronociceptive and impaired antinociceptive mechanisms, which may explain long-lasting pain and failure of some treatments to resolve symptoms. However, due to the observational nature of this study, a clear cause-effect relationship cannot be established. Normal PPT values in the clinic should not be interpreted as absence of altered nociceptive processing. IMPACT: This study fills in some gaps in knowledge. Changes in central nociceptive processing may explain persistent and recurrent symptoms in NSNP and failure of treatments to obtain long-lasting relief. Further research is required to ascertain if TSSP and CPM assessment in the clinic may help predict physical therapy treatment outcome. Whether symptomatic relief with physical therapy is mediated by an improvement in TSSP and CPM should also be explored. PPTs were unaltered in participants with NSNP despite evidence of impairment in the central pain modulatory systems. Normal PPTs should not be interpreted as evidence of unaltered central pain-related processing.

Depression of C fibre-evoked spinal field potentials by the spinal delta opioid receptor is enhanced in the spinal nerve ligation model of neuropathic pain: involvement of the mu-subtype.

The depression rate of C fibre-evoked spinal field potentials by spinally applied morphine is increased in two states of spinal hyperexcitation, namely the spinal ligation model (SNL) of neuropathic pain and long-term potentiation (LTP) of C fibre-evoked spinal field potentials. This present work sought to determine opioid receptor subtypes involved in such increase in the SNL model. We recorded spinal field potentials during spinal superfusion with increasing, cumulative concentrations of selective subtype-specific agonists in rats subjected to SNL, as well as in non-ligated animals. The mu opioid receptor (MOR) agonist DAMGO significantly depressed field potentials evoked by C (100 nM) or Adelta fibres (1 microM) both in neuropathic and non-ligated rats, whereas the kappa receptor opioid (KOR) agonist +/-U-50488 was ineffective. The delta opioid receptor (DOR) (D-Ala2)-Deltorphin II was more effective in reducing C fibre-evoked spinal field potentials in rats subjected to SNL (100 nM) than in non-ligated rats (100 microM). Subclinical MOR activation (10 nM DAMGO) produced a leftward shift in (D-Ala2)-Deltorphin II dose-response curve in non-ligated rats (IC50 16.59 +/- 0.99 microM vs 120.3 +/- 1.0 microM in the absence of DAMGO), and isobolar analysis revealed synergistic interaction (interaction index 0.25). MOR blockade (100 microM CTOP) disinhibited C fibre-evoked potentials in neuropathic, but not in basal animals, and partially impeded DOR depression in both groups. DOR blockade (1 mM naltrindole) was ineffective in either group. We show that DOR-mediated depression of spinal responses to peripheral unmyelinated fibre-input is increased in the SNL model, an increase that is contributed to by positive interaction with the spinal MOR.

Embodied pain in fibromyalgia: Disturbed somatorepresentations and increased plasticity of the body schema.

Fibromyalgia syndrome (FMS) is a highly prevalent, chronic musculoskeletal condition characterized by widespread pain and evoked pain at tender points. This study evaluated various aspects of body awareness in a sample of 14 women with FMS and 13 healthy controls, such as plasticity of the body schema, body esteem, and interoceptive awareness. To this end, the Rubber Hand Illusion (RHI), the Body Esteem Scale (BES), and the Body Perception Questionnaire (BPQ) were used, respectively. Consistent with increased plasticity of the body schema, FMS patients scored higher, with large or very large effect sizes, across all three domains evaluated in the RHI paradigm, namely proprioceptive drift and perceived ownership and motor control over the rubber hand. Scores on all items addressed by the BES were consistently lower among FMS subjects (2.52, SEM .19 vs 3.89, SEM .16, respectively, p < .01, Cohen's d = .38-.66). In the FMS sample, BES scores assigned to most painful regions also were lower than those assigned to the remaining body sites (1.58, SEM .19 vs 2.87, SEM .18, respectively, p < .01). Significantly higher scores (p < .01, Cohen's d = .51-.87) were found in the FMS sample across awareness (3.57 SEM .15 vs 1.87 SEM .11), stress response (3.76 SEM .11 vs 1.78 SEM .11), autonomic nervous system reactivity (2.59 SEM .17 vs 1.35 SEM .07), and stress style 2 (2.73 SEM .27 vs 1.13 SEM .04) subscales of the BPQ. Intensity of ongoing clinical pain was found to be strongly correlated with interoceptive awareness (r = .75, p = .002). The results suggest a disturbed embodiment in FMS, characterized by instability of the body schema, negatively biased cognitions regarding one's own body, and increased vigilance to internal bodily cues. These manifestations may be interpreted as related with the inability of incoming sensory inputs to adequately update negatively biased off-line somatorepresentations stored as long-term memory.

Characterization of functional µ opioid receptor turnover in rat locus coeruleus: an electrophysiological and immunocytochemical study.

BACKGROUND AND PURPOSE: Regulation of µ receptor dynamics such as its trafficking is a possible mechanism underlying opioid tolerance that contributes to inefficient recycling of opioid responses. We aimed to characterize the functional turnover of µ receptors in the noradrenergic nucleus locus coeruleus (LC). EXPERIMENTAL APPROACH: We measured opioid effect by single-unit extracellular recordings of LC neurons from rat brain slices. Immunocytochemical techniques were used to evaluate µ receptor trafficking. KEY RESULTS: After near-complete, irreversible µ receptor inactivation with ß-funaltrexamine (ß-FNA), opioid effect spontaneously recovered in a rapid and efficacious manner. In contrast, α2 -adrenoceptor-mediated effect hardly recovered after receptor inactivation with the irreversible antagonist EEDQ. When the recovery of opioid effect was tested after various inactivating time schedules, we found that the longer the ß-FNA pre-exposure, the less efficient and slower the functional µ receptor turnover became. Interestingly, µ receptor turnover was slower when ß-FNA challenge was repeated in the same cell, indicating constitutive µ receptor recycling by trafficking from a depletable pool. Double immunocytochemistry confirmed the constitutive nature of µ receptor trafficking from a cytoplasmic compartment. The µ receptor turnover was slowed down when LC neuron calcium- or firing-dependent processes were prevented or vesicular protein trafficking was blocked by a low temperature or transport inhibitor. CONCLUSIONS AND IMPLICATIONS: Constitutive trafficking of µ receptors from a depletable intracellular pool (endosome) may account for its rapid and efficient functional turnover in the LC. A finely-tuned regulation of µ receptor trafficking and endosomes could explain neuroadaptive plasticity to opioids in the LC.

Disinhibition of spinal responses to primary afferent input by antagonism at GABA receptors in urethane-anaesthetised rats is dependent on NMDA and metabotropic glutamate receptors.

Disruption of spinal GABAergic circuits, which regulate the conveyance of sensory information to spinal cord neurones from the primary afferent system, leads to miscoding of afferent input and often results in hyperresponsiveness states. In the present work, extracellular field potentials elicited by electrical peripheral nerve activation were recorded in the urethane-anaesthetised rat following spinal administration of GABA(A) or GABA(B) receptor-antagonists, and the involvement of glutamate receptors of the NMDA and metabotropic types in changes induced by altered GABAergic function was examined by pre-treating the spinal dorsal horn with appropriate antagonist drugs. Spinal administration of the GABA(A) receptor antagonist bicuculline (BIC) dose-dependently augmented poly- but not monosynaptic field potentials elicited by activation of A fibres or potentials elicited by activation of C fibres, whereas application of the GABA(B) receptor antagonist CGP35348 significantly increased the amplitudes of C- but not A fibre-evoked potentials. BIC-induced augmentation was blocked by pre-treatment with the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) or the group I or II metabotropic glutamate receptor (mGluR)-antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) or (2S)-alpha-ethylglutamic acid (EGLU), respectively, but not by the group III mGluR-antagonist (RS)-alpha-methylserine-O-phosphate (MSOP). Augmentation of spinal field potentials induced by CGP35348 was prevented by pre-treatment with D-AP5 but not with mGluR-antagonists. The present findings provide novel evidence that disparate synaptic mechanisms subserved by metabotropic and NMDA glutamate receptors may be involved in spinal hyperresponsiveness states secondary to decreased GABA(A) or GABA(B) receptor activity.

Regulation of Nociceptive Plasticity Threshold and DARPP-32 Phosphorylation in Spinal Dorsal Horn Neurons by Convergent Dopamine and Glutamate Inputs.

Dopamine can influence NMDA receptor function and regulate glutamate-triggered long-term changes in synaptic strength in several regions of the CNS. In spinal cord, regulation of the threshold of synaptic plasticity may determine the proneness to undergo sensitization and hyperresponsiveness to noxious input. In the current study, we increased endogenous dopamine levels in the dorsal horn by using re-uptake inhibitor GBR 12935. During the so-induced hyperdopaminergic transmission, conditioning low-frequency (1 Hz) stimulation (LFS) to the sciatic nerve induced long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn neurons. The magnitude of LTP was attenuated by blockade of either dopamine D1-like receptors (D1LRs) by with SCH 23390 or NMDA receptor subunit NR2B with antagonist Ro25-6981. Conditioning LFS during GBR 12935 administration increased phosphorylation of dopamine- and cAMP-regulated phosphoprotein of Mr 32kDa (DARPP-32) at threonine 34 residue in synaptosomal (P3) fraction of dorsal horn homogenates, as assessed by Western blot analysis, which was partially prevented by NR2B blockade prior to conditioning stimulation. Conditioning LFS also was followed by higher co-localization of phosphorylated form of NR2B at tyrosine 1472 and pDARPP-32Thr34- with postsynaptic marker PSD-95 in transverse L5 dorsal horn sections. Such increase could be significantly attenuated by D1LR blockade with SCH 23390. The current results support that coincidental endogenous recruitment of D1LRs and NR2B in dorsal horn synapses plays a role in regulating afferent-induced nociceptive plasticity. Parallel increases in DARPP-32 phosphorylation upon LTP induction suggests a role for this phosphoprotein as intracellular detector of convergent D1L- and NMDA receptor activation.

Spinal D1-like dopamine receptors modulate NMDA receptor-induced hyperexcitability and NR1 subunit phosphorylation at serine 889.

Activation of the N-methyl-d-aspartate receptor (NMDAR) in dorsal horn neurons is recognized as a fundamental mechanism of central sensitization and pathologic pain. This study assessed the influence of dopaminergic, D1-like receptor-mediated input to the spinal dorsal horn on NMDAR function. Spinal superfusion with selective NMDAR agonist cis-ACPD significantly increased C-fiber-evoked field potentials in rats subjected to spinal nerve ligation (SNL), but not in sham-operated rats. Simultaneous application of D1LR antagonist SCH 23390 dramatically reduced hyperexcitability induced by cis-ACPD. Furthermore, cis-ACPD-induced hyperexcitability seen in nerve-ligated rats could be mimicked in unin-jured rats during stimulation of D1LRs by agonist SKF 38393 at subthreshold concentration. Phosphorylation of NMDAR subunit NR1 at serine 889 at postsynaptic sites was found to be increased in dorsal horn neurons 90 min after SNL, as assessed by increased co-localization with postsynaptic marker PSD-95. Increased NR1 phosphorylation was attenuated in the presence of SCH 23390 in the spinal superfusate. The present results support that D1LRs regulate most basic determinants of NMDAR function in dorsal horn neurons, suggesting a potential mechanism whereby dopaminergic input to the dorsal horn can modulate central sensitization and pathologic pain.

Plasticity of α2-adrenergic spinal antinociception following nerve injury: selective, bidirectional interaction with the delta opioid receptor.

Interactions of opioid receptors with other receptor families can be made use of to improve analgesia and reduce adverse effects of opioid analgesics. We investigated interactions of the α2-adrenergic receptor (α2AR) with opioid receptors of the mu (MOR) and delta (DOR) types in the spinal dorsal horn in an animal model of neuropathic pain induced by spinal nerve ligation. Nine days after nerve injury, immunoreactivity for the α2AR subtype A (α2AAR) was increased both in tissue homogenates and at pre- and post-synaptic sites in transverse sections. The efficacy of spinally administered α2AAR agonist guanfacine at reducing C-fiber-evoked field potentials was increased in nerve-ligated rats. This reducing effect was impaired by simultaneous administration of DOR antagonist naltrindole, but not MOR antagonist CTOP, suggesting that concurrent DOR activation was required for α2AAR-mediated inhibition. While DOR agonist deltorphin II and MOR agonist DAMGO both effectively depressed C-fiber-evoked spinal field potentials, DOR- but not MOR-mediated depression was enhanced by subclinical guanfacine. In conscious, nerve-ligated rats, chronically administered deltorphin II produced stable thermal and mechanical antinociception over the 9 following days after nerve injury without apparent signs of habituation. Such an effect was dramatically enhanced by co-administration of a low dose of guanfacine, which reversed thermal and mechanical thresholds to levels near those prior to injury. The results suggest that spinal, α2AAR-mediated antinociception is increased after nerve injury and based on DOR co-activation. We demonstrate in vivo that α2AAR/DOR interaction can be exploited to provide effective behavioral antinociception during neuropathic pain.

Induction of long-term potentiation of C fibre-evoked spinal field potentials requires recruitment of group I, but not group II/III metabotropic glutamate receptors.

In superficial layers of the lumbar spinal dorsal horn, N-methyl-D-aspartate-dependent long-term potentiation (LTP) of C fibre-evoked field potentials, a synaptic model of central sensitisation and hyperalgesia, ensues the application of electrical high-frequency, high-intensity conditioning stimulation to the sciatic nerve. In order to investigate the putative involvement of the G protein-coupled metabotropic glutamate receptors (mGluRs) in the induction of this form of LTP, we applied a series of mGluR antagonists exhibiting distinct group-specific activity profiles to the spinal lumbar enlargement, prior to conditioning stimulation. The group I (mGluR1/5) and group II (mGluR2/3) mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine or the selective mGluR1/5 antagonist (S)-4-carboxyphenylglycine consistently impaired the development of spinal LTP. However, potentiation occurred in the presence of the inactive enantiomer (R)-alpha-methyl-4-carboxyphenylglycine. LTP proved insensitive to the selective mGluR2/3 antagonists (2S)-alpha-ethylglutamic acid and LY341495, either spinally or intravenously delivered. LTP could also be induced in the presence of the selective group III (mGluR4/mGluR6-mGluR8) mGluR antagonist (RS)-alpha-methylserine-O-phosphate. However, none of the mGluR-active compounds alone noticeably altered the amplitudes of C fibre-evoked field potentials in the absence of conditioning stimulation. These findings suggest that the induction of LTP of C fibre-evoked field potentials in the spinal dorsal horn by high-frequency, high-intensity stimulation of afferent C fibres requires a group-specific mGluR recruitment, activation of mGluR1/5 but not that of mGluR4/6-8 and mGluR2/3 being a requisite step.

Synaptic upregulation and superadditive interaction of dopamine D2- and µ-opioid receptors after peripheral nerve injury.

A sound strategy for improving the clinical efficacy of opioids involves exploiting positive interactions with drugs directed at other targets in pain pathways. The current study investigated the role of dopamine receptor D2 (D2R) in modulation of spinal dorsal horn excitability to noxious input, and interactions therein with µ-opioid receptor (MOR) in an animal model of neuropathic pain induced by spinal nerve ligation (SNL). C-fiber-evoked field potentials in the spinal dorsal horn were depressed concentration dependently by spinal superfusion with the D2R agonist quinpirole both in nerve-injured and sham-operated (control) rats. However, quinpirole-induced depression was significant at 10 µmol/L after SNL but only at 100 µmol/L in control rats. This quinpirole effect was completely abolished by MOR antagonist CTOP at subclinical concentration (1 µmol/L) in nerve-injured rats, but was unaltered in sham-operated rats. Nine days after SNL, D2R was upregulated to both presynaptic and postsynaptic locations in dorsal horn neurons, as revealed by double confocal immunofluorescence stainings for synaptophysin and PSD-95. In addition, D2R/MOR co-localization was increased after SNL. Co-administration of 1 µmol/L quinpirole, insufficient per se to alter evoked potentials, dramatically enhanced inhibition of evoked potentials by MOR agonist DAMGO, reducing the IC50 value of DAMGO by 2 orders of magnitude. The present data provide evidence of profound functional and subcellular changes in D2R-mediated modulation of noxious input after nerve injury, including positive interactions with spinal MOR. These results suggest D2R co-stimulation as a potential avenue to improve MOR analgesia in sustained pain states involving peripheral nerve injury.

Coupling of serotonergic input to NMDA receptor-phosphorylation following peripheral nerve injury via rapid, synaptic up-regulation of ND2.

Evidence implicates serotonergic input to spinal dorsal horn neurons in shifting the NMDA receptor (NMDAR) into a high functional output profile after spinal nerve ligation (SNL). We investigated the involvement of adaptor protein NADH dehydrogenase subunit 2 (ND2) in NMDAR-phosphorylation and spinal hyperexcitability secondary to peripheral nerve injury. Immunofluorescence for ND2 was found in dorsal horn neurons immunopositive for NMDAR subunit NR1. Co-localization of ND2 with postsynaptic marker PSD-95 was significantly increased 60min after SNL (Rr 0.77 vs Rr 0.06 in sham controls; z=-242.85; p<0.01 at Fisher's exact test). Western blot analyses confirmed ND2 up-regulation both in cytoplasmic (S2) and synaptic (P3) compartments (p<0.01 at the Student's t test). SNL was followed by increased co-localization of ND2 with the phosphorylated form (serine 896) of NR1 (pNMDA). Spinal superfusion with ND2 inhibitor rotenone prevented up-regulation of ND2 (Rr 0.06 after rotenone vs Rr 0.78 in vehicle-treated controls, z=-253.22, p<0.01) and pNR1 in P3. C fiber-evoked dorsal horn field potentials were increased 60min after SNL by superfusion with NMDA agonist cis-ACPD at 100nM (p<0.01 at the Bonferroni test), however cis-ACPD was effective only at 10µM following prior administration of rotenone. Rotenone also abolished enhancement of evoked potentials induced by simultaneous stimulation of NMDA and 5-HR2B receptors in uninjured rats. Increased postsynaptic up-regulation of ND2/pNMDAR 60min after SNL was prevented by prior administration of selective 5-HT2B antagonist SB204741. These results support a pivotal role for ND2 in coupling serotonergic input to NMDAR-activation during neuropathic pain.