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Abstinence from chronic morphine results in characteristic withdrawal symptoms in humans and experimental animals. Despite a large number of studies, the cellular mechanisms underlying opiate withdrawal symptoms are not clearly understood, in particular, the regulation of micro-opioid receptor function during this process. The present study investigated the micro-opioid receptor-stimulated G-protein activity using guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]-GTPgammaS) autoradiography. [35S]-GTPgammaS binding was performed using coronal rat brain sections (20 microm) in the presence or absence of the micro-opioid selective agonist [D-Ala(2),N-MePhe(4)Gly(5)-ol] enkephalin (DAMGO). In experiment 1, rats (male, Sprague-Dawley) were injected every 12 h with increasing doses of morphine (5-100 mg/kg, s.c.) for 12 days; a separate group of rats which received saline injections served as control. Opiate withdrawal was induced by abstinence from morphine. Thirty-six hours after the last morphine injection, spontaneous withdrawal symptoms were assessed. Rats were then decapitated and brains rapidly removed. In experiment 2, withdrawal symptoms were precipitated with the opioid receptor antagonist naloxone (1 mg/kg). Brains were taken at 5, 10, 20 and 60 min after naloxone injection. In experiment 3, morphine dependence was induced by implantation of three morphine pellets (75 mg per pellet). After 7 days, withdrawal symptoms were precipitated by naloxone (1 mg/kg) and brains were removed 30 min after naloxone injection. [35S]-GTPgammaS binding was measured in the locus coeruleus, nucleus parabrachialis, nucleus accumbens and central nucleus of amygdala. Although clear withdrawal symptoms were observed in all morphine-withdrawn rats, no significant changes in [35S]-GTPgammaS binding were detected in animals undergoing withdrawal. The present lack of differences between morphine-withdrawn and control rats indicates that micro-opioid receptor-stimulated G-protein activity is not modulated by chronic morphine administration and is not involved in the expression of opiate withdrawal.

Metabotropic glutamate receptors (mGluR) have been shown to play a role in the modulation of acute and inflammatory pain. Additionally, we have recently detected time-dependent changes in the mRNA expression of several mGluR subtypes in thalamic nuclei of monoarthritic (MA) rats. In the present study, mGluR1, -3, -4, and -7 subtype mRNA expression was analyzed by in situ hybridization with radioactively labelled oligonucleotide probes in cerebral cortical regions of normal and MA rats at 2, 4, and 14 days of the disease. The mGluR1, -4, and -7 mRNAs were at background level in normal rats and did not change in MA animals. In contrast, mGluR3 mRNA expression was abundant in normal rats and was significantly increased in cortical areas of MA rats at all time points. Higher changes were detected bilaterally at 4 days, predominantly in layers IV/V, in the motor, primary, and secondary somatosensory cortices (average increases of 50-75%), but maximum rises occurred in the contralateral cingulate cortex (+138%). No changes were detected in the auditory cortex. The present data show an up-regulation of mGluR3 mRNA expression in the motor, somatosensory, and limbic cortices of MA rats. This possibly reflects the occurrence of central mechanisms counteracting the increased transmission of nociceptive input arising from the inflamed paw and the impaired motor behavior of these rats. Changes in the cingulate cortex may be related to the motivational-affective component of nociception.

Repetitive synaptic excitation or the application of L-glutamate into the vicinity of multireceptive neurons in the dorsal horn of the spinal cord and corresponding structures of the trigeminal nucleus increases neuronal excitability, which is then reflected by an expansion of the receptive field (Fig. 1). Similar alterations of the receptive field of neurons have been observed in various other brain regions. The receptive fields of multireceptive neurons also expand their size following mechanical, chemical, inflammatory or nerve injuries. Since these multireceptive neurons are activated by converging non-nociceptive and nociceptive afferents an increased excitability of these neurons may also be the mechanism by which pain refers to distant somatic and visceral structures (Fig. 2). The increase in neuronal excitability is mediated to a great extent by the co-activation of glutamate receptors and receptors for substance P, a neuropeptide long thought to have a role in pain perception. There is evidence from recent research that this facilitatory effect on glutamatergic synaptic transmission involves membrane receptor phosphorylation, and enhances activity-dependent gene expression (Fig. 3). In order to investigate the time-dependent processing of ongoing afferent noxious stimulation in the central nervous system we recently employed the quantitative autoradiographic 14C-2-deoxyglucose technique in a model of chronic monoarthritic pain in the rat. A synopsis of these most recent experimental data and results from previous electrophysiological in vivo and in vitro studies suggests that dorsal horn neurons and probably also other neurons in pain-related structures become spontaneously active and can maintain their activity without further noxious peripheral input.

The development of chronic pain is associated with activity-dependent plastic changes in neuronal structures in the peripheral and central nervous system. In order to investigate the time-dependent processing of afferent noxious stimuli in the spinal cord we employed the quantitative autoradiographic 2-deoxyglucose technique in a model of chronic monoarthritic pain in the rat. Spinal metabolic activity was determined at various time-points (two, four and 14 days) after the injection of complete Freund's adjuvant into the left tibiotarsal joint. In addition, the effect of acute noxious mechanical stimulation of the arthritic joint was investigated at 14 days of monoarthritis. Local glucose utilization was determined in lumbar segments L2-L5, ipsi- and contralateral to the inflamed hindpaw, and compared with saline-injected controls. In general, monoarthritic animals had bilaterally increased metabolic activity in all laminae of the spinal cord. Detailing the time-course showed that in rats with two days of monoarthritis metabolic activity was significantly increased to a similar extent on both sides of all spinal laminae. In contrast, at four days, glucose utilization in deep laminae of the dorsal horn (laminae V-VI), the central gray area (laminae X) and the ventral horn (laminae VII-IX) tended to return to control levels. At 14 days of monoarthritis, however, metabolic activity showed a further increase in all laminae of the spinal cord. This increase was more pronounced on the side ipsilateral to inflammation, reaching 65% above corresponding control levels in laminae V, VI. Animals with 14 days of monoarthritis which were subjected to mechanical noxious stimulation of the arthritic joint displayed clear behavioral signs of acute pain. Although in this group metabolic activity was above control levels, it was lower than in animals with 14 days of monoarthritis that were not additionally stimulated. The data show not only a general increase of spinal cord metabolic activity during the time-course of the development of a chronic pain state, but also show a region-specific non-linear time profile. This may reflect the complexity of transducing and suppressive transmitter systems involved in the central processing of ongoing pain.

Little is known regarding opioid receptors in the human cerebellum. The present [11C]diprenorphine PET study investigated opioid receptor binding in the human cerebellum in vivo, and showed a differential binding level in cerebellar cortex, vermis and dentate nuclei. The additional study in vitro of opioid receptors in human cerebellar cortex and rat brain corroborated the presence of opioidergic mechanisms in the human cerebellum in contrast to the rat. A differential cellular distribution pattern was detected for the three major opioid receptors investigated. For the mu-receptor, and at a lower level for the kappa-receptor, mRNA expression was mainly observed over granule cells. Binding sites were most prominent in the molecular layer. For the delta-receptor no signal was detected. The consideration of cerebellar opioidergic mechanisms and the distribution patterns of the various opioid receptors may promote the understanding of cerebellar function and of opioidergic pharmacology in the human.

Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III-VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III-VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression.

This double-blind, placebo-controlled clinical trial of yohimbine hydrochloride included 86 patients with erectile dysfunction and without clearly detectable organic or psychologic causes. The patient group fulfilled all entry criteria; 85 of these could be considered for the Safety-respectively 83 for the Intention-to-treat (ITT)-analysis. Yohimbine was administered orally in a dosage of 30 mg a day (two 5 mg tablets three times daily) for eight weeks. Patients were seen for follow-up after four weeks' treatment, and for a final visit after eight weeks. Efficacy evaluation was based on both subjective and objective criteria. Subjective criteria included improvement in sexual desire, sexual satisfaction, frequency of sexual contacts, and quality of erection (penile rigidity) during sexual contact/intercourse. Objective criteria of outcome were based on improvement in penile rigidity determined by use of polysomnography in the sleep laboratory. Overall Yohimbine was found significantly more effective than placebo in terms of response rate: 71 vs 45%. Yohimbine was well-tolerated: Only 7% of patients rated tolerability fair or poor, and most adverse experiences were mild. There was no serious adverse event.

The expression of Fos, Jun and Krox-24 proteins was investigated in spinal cord neurons of the rat 2, 4 and 8 h following noxious thermal stimulation of one hind-paw and pre-treatment with morphine. The number of neurons expressing c-Fos, c-Jun, Jun B and Krox-24 were maximal after 2 h and thereafter declined. The number of Fos B and Jun D immunoreactive neurons increased constantly for up to 8 h with Jun D showing expression above baseline only after 4 h following stimulation. Intravenous application of morphine (5 and 10 mg/kg) 20 min before noxious heat stimulation decreased the expression of all six proteins at any time-point with a predilective effect on neurons of deeper laminae of the dorsal horn. The suppressive effects of morphine were more pronounced with the higher dose of morphine and completely reversed by intravenous naloxone (1 and 10 mg/kg). The temporospatial patterns of expression following morphine were similar to those seen without morphine, but in a much smaller number of neurons and with a shorter time-course. However, despite the high dose of morphine and continuous halothane anaesthesia during the whole experimental procedures, a considerable number of neurons expressing the various genes remained in all laminae of the spinal cord. At 2 h following noxious heat stimulation morphine had decreased the number of labelled neurons for c-Fos, Fos B, Krox-24, c-Jun and Jun B to 30-60% of control levels in laminae I-II and to 10-30% in laminae III-VII,X of the spinal cord. At 4 h the level of reduction had further increased while Jun D was only moderately reduced to 75% in all laminae of the spinal cord. Eight hours following noxious heat plus morphine application we did not detect noxious evoked immunoreactivity for c-Fos, Krox-24, c-Jun and Jun B, while there was residual labelling for Fos B in the superficial dorsal horn and for Jun D in laminae I-VII and X of the spinal cord. The different temporospatial pattern of immediate early gene expression in neurons of the spinal cord dorsal horn following noxious stimulation suggest that variable transcription complexes may interact with DNA regulatory sequences and could thus activate alternative secondary response genes, even under protection of a high dosage of morphine applied before noxious stimulation.

Following the cloning of the novel nociceptin opioid receptor (NOP 1 ) and the identification of its endogenous ligand orphanin FQ/nociceptin the distribution and functional role of the NOP 1 receptor system have been studied mainly in the rodent CNS. In the present study the regional distribution and splice variant expression of the NOP 1 receptor was investigated in the adult human brain using [ 3 H]-nociceptin autoradiography, NOP 1 reverse transcriptase PCR and mRNA in situ hybridization. Ligand binding revealed strong expression of functional NOP 1 receptors in the cerebral cortex and moderate signals in hippocampus and cerebellum. Interestingly, the NOP 1 receptor specific ligand was also strongly bound in the human striatum. A matching pattern of mRNA expression was observed with high amounts of NOP 1 mRNA in the prefrontal and cingulate cortex as well as in the dentate gyrus of the hippocampus. mRNA levels in the Ammon's horn and cerebellar cortex were moderate and low in the striatum. A considerable expression of N-terminal NOP 1 splice variant mRNAs was not detectable in the human brain by means of in situ hybridization. This suggests that functional NOP 1 receptors in the human brain are encoded by N-terminal full length NOP 1 transcripts. The present data on the anatomical distribution of nociceptin binding sites and NOP 1 receptor mRNA contribute to the knowledge about opioid receptor systems in the human brain and may promote the understanding of function and pharmacology of the orphanin FQ/nociceptin receptor system in the human CNS.

The efficacy and tolerability of entacapone was investigated in a randomized, double-blind, placebo-controlled, 3-month study of 162 patients with Parkinson's disease (PD) treated with levodopa and a dopamine agonist and experiencing wearing-off motor fluctuations. Patients were randomized in a 3 : 2 ratio to entacapone 200 mg or placebo, administered with each dose of levodopa. Efficacy was judged on the improvement of “on” and “off” time while awake (Patient Diary and UPDRS part IV Item 39), Investigators' Global Assessment, the SF-36 Health Survey, and changes in levodopa dosages. Patients were monitored for adverse events, laboratory safety and vital signs throughout the study. Improvements in “on” time as assessed using patient diary data showed a trend in favour of entacapone, however these did not reach statistical significance. “Off” time while awake (UPDRS part IV Item 39) showed an improvement of at least one category in 36% of entacapone-treated patients, compared with 22% in the control group (p = 0.0038). The proportion of patients showing an improvement at the Investigators' Global Assessment was significantly higher (p = 0.0006) in the entacapone-treated group of patients. Also, the proportion of patients with a reduction in their daily levodopa dose was significantly higher (p = 0.02) in the entacapone group (28%) compared with placebo (13%). As expected, the most frequent adverse events were dopamine-mediated (dyskinesia: entacapone 31% versus placebo 13%), and harmless urinary discoloration. The modest increase in dyskinesias could be readily managed by levodopa down-adjustment, and, at study end there was no significant difference for the UPDRS “overall dyskinesia score” between entacapone and placebo. In conclusion, although the primary efficacy variable did not reach statistical significance, the present results demonstrate that entacapone provides additional antiparkinsonian benefits to levodopa therapy and is well tolerated in levodopa-treated PD patients experiencing wearing-off motor fluctuations despite adjunct dopamine agonist therapy.

NMDA-receptor-mediated mechanisms may be crucial in addictive states, e.g. alcoholism, and provide a target for the novel anti-craving compound acamprosate. Here, the pharmacological effects of acamprosate on NMDA-receptors were studied using electrophysiological techniques in different cell lines in vitro. Additionally, a possible modulation of brain NMDA-receptor subunit expression was examined in vivo in rats, and compared to two effective non-competitive NMDA-receptor antagonists, memantine and MK-801. Electrophysiology in cultured hippocampal neurons (IC(50) approx. 5.5mM) and Xenopus oocytes (NR1-1a/NR2A assemblies: IC(50) approx. 350 microM, NR1-1a/NR2B: IC(50) approx. 250 microM) consistently revealed only a weak antagonism of acamprosate on native or recombinant NMDA-receptors. In HEK-293 cells, acamprosate showed almost no effect on NR1-1a/NR2A or NR1-1a/NR2B recombinants (IC(50)s not calculated). Protein blotting demonstrated an up-regulation of NMDA-receptor subunits after acamprosate as well as after memantine or MK-801, in comparison to controls. After acamprosate, protein levels were increased in the cortex (NR1-3/1-4: 190+/-11% of controls) and hippocampus (NR1-1/1-2: 163+/-11%). The up-regulations observed after memantine (cortex, NR2B: 172+/-17%; hippocampus, NR1-1/1-2: 156+/-8%) or MK-801 (cortex, NR2B: 174+/-22%; hippocampus, NR1-1/1-2: 140+/-3%) were almost identical. No changes were detected in the brainstem. The present data indicate an extremely weak antagonism of NMDA-receptors by acamprosate. However, its ability to modulate the expression of NMDA-receptor subunits in specific brain regions - shared with the well established NMDA-antagonists memantine and MK-801 - may be of relevance for its therapeutic profile, especially considering the growing importance of NMDA-receptor plasticity in the research of ethanol addiction.

Metabotropic glutamate receptors (mGluR) have been shown to play a role in the modulation of acute and inflammatory pain. Additionally, we have recently detected time-dependent changes in the mRNA expression of several mGluR subtypes in thalamic nuclei of monoarthritic (MA) rats. In the present study, mGluR1, -3, -4, and -7 subtype mRNA expression was analyzed by in situ hybridization with radioactively labelled oligonucleotide probes in cerebral cortical regions of normal and MA rats at 2, 4, and 14 days of the disease. The mGluR1, -4, and -7 mRNAs were at background level in normal rats and did not change in MA animals. In contrast, mGluR3 mRNA expression was abundant in normal rats and was significantly increased in cortical areas of MA rats at all time points. Higher changes were detected bilaterally at 4 days, predominantly in layers IV/V, in the motor, primary, and secondary somatosensory cortices (average increases of 50-75%), but maximum rises occurred in the contralateral cingulate cortex (+138%). No changes were detected in the auditory cortex. The present data show an up-regulation of mGluR3 mRNA expression in the motor, somatosensory, and limbic cortices of MA rats. This possibly reflects the occurrence of central mechanisms counteracting the increased transmission of nociceptive input arising from the inflamed paw and the impaired motor behavior of these rats. Changes in the cingulate cortex may be related to the motivational-affective component of nociception.

Background: The restless legs syndrome (RLS) is a common sensomotor disorder associated with severe sleep disturbances. Symptoms clearly improve following treatment with dopaminergic or opioidergic agonists.Objective: To further elucidate the involvement of opioidergic and dopaminergic mechanisms in the pathophysiology of RLS, the effects of specific antagonists on motor (periodic leg movements) and subjective (sensory) RLS-symptoms during daytime were assessed.Methods: A modified suggested immobilization test was performed in eight drug-naive RLS-patients. An infusion of either naloxone, metoclopramide or placebo was administered. In addition, the hormonal levels of adrenocorticotropic hormone, cortisol, prolactin and growth hormone were determined, to elucidate a possible involvement of the hypothalamic-pituitary-adrenocortical (HPA) system in RLS.Results: RLS sensory or motor symptoms could not be provoked. Hormonal responses showed no abnormal profiles.Conclusions: Rather than a general alteration of the opioidergic/dopaminergic tone and an involvement of the HPA system, it is more likely that specific neuronal dopaminergic or opioidergic pathways are altered in the pathophysiology of RLS.

Evidence for the involvement of metabotropic glutamate receptors (mGluR) in sensory processing has been emerging. Additionally, the differential distribution of distinct mGluR subtypes mRNA in particular thalamic nuclei of normal rats suggests that they could be involved in the processing of somatosensory information. In the present study, mGluR1, 3, 4 and 7 mRNAs expression was investigated by in situ hybridisation in selected brainstem and thalamic nuclei of adult monoarthritic rats at different time points of the disease (2, 4 and 14 days). Monoarthritic rats displayed behavioural and physical signs of painful arthritis at all time points. At 2 days of monoarthritis the mGluR1 mRNA expression was decreased mainly in the ventrobasal complex (VB) and in the posterior thalamic nuclei (Po) contralateral to the inflamed joint. The mGluR4 mRNA expression was also reduced, but minimum values were found at 4 days of monoarthritis, when no changes could be found in mGluR1 mRNA expression. At 14 days, mGluR4 mRNA expression was similar to controls, while mGluR1 mRNA was again reduced. Similar decreases of mGluR7 mRNA expression in the VB and Po were found at all time points, while mGluR3 mRNA expression was bilaterally increased in the reticular thalamic nucleus (Rt). In the brainstem no changes could be found in the expression of any mGluR subtype mRNA. The reduced expression of mGluR1, 4 and 7 transcripts in VB and Po, and the increases of mGluR3 mRNA in the Rt may contribute to counteract the increased noxious input arising from the periphery.

6-O(2-[18F]fluoroethyl)-6- -desmethyldiprenorphine ([18F]DPN) was developed and biologically evaluated. Results of animal experiments, binding studies in vivo, and a human PET study are reported and compared with those of [11C]DPN.[18F]DPN was obtained by 18F-fluoroethylation of 3-O-trityl-6-O-desmethyldiprenorphine and subsequent deprotection in good radiochemical yields (23% +/- 7%; 100 min; 37 TBq/mmol). Binding of [18F]DPN to mu, kappa, and delta opioid receptors was shown by autoradiography studies on rat brain slices. Quantification of cerebral opioid receptor binding in men was performed by spectral analysis of a dynamic PET scan (25 frames, 90 min) after intravenous application of 63 MBq [18F]DPN (36 GBq/micromol) and correction for metabolites.[18F]DPN shows high affinity to opioid receptors. Parametric images (impulse response function at 60 min) of this human study showed a binding pattern of [18F]DPN equal to that of a control group (n = 9 healthy volunteers) after administration of [11C]DPN.The advantage of the longer half-life of 18F will allow extended scanning periods, more flexible interventions (e.g., displacement studies), and DPN to be available to PET centers without an on-site cyclotron.

L-Glutamate (L-Glu) is present in most excitatory synapses of the mammalian brain, acting on several receptor subtypes. Height different genes encoding metabotropic glutamate receptors (mGluRs) subtypes have been described (mGluR1-8), having a distinct distribution in the brain. In the present study, the distribution of mGluR1, 3, 4, 5 and 7 mRNAs was determined in 20 thalamic nuclei of adult rats by performing in situ hybridisation with subtype-specific 35S-labelled oligonucleotide probes. High expression of mGluR1 mRNA mainly occurred in midline nuclei such as the centromedial/centrolateral (CM/CL) nuclei, parafascicular and submedius nuclei, and in the ventroposteromedial (VPM) and posterior (Po) nuclei. In contrast, mGluR5 mRNA was more uniformly distributed at weak to moderate levels, except in the reuniens nucleus where a strong signal was detected. The mGluR3 mRNA was highly expressed in the reticular thalamic nucleus and almost not detectable in any other thalamic region. Additionally, mGluR3 mRNA was found not only in neurones but also in putative glial cells. The mGluR4 mRNA was abundant in most thalamic nuclei, with prominent expression in the CM/CL, Po and ventrobasal complex (VPM and ventroposterolateral, VPL). Finally, mGluR7 transcripts were found evenly distributed throughout the thalamus at moderate levels, the highest signal being detected in the paraventricular thalamic nucleus, VPM, VPL and Po. This differential distribution of mGluR subtypes in the rat thalamus may contribute to the heterogeneity of glutamate effects on thalamic neurones. The mGluR1, mGluR4 and mGluR7 receptors may be involved in the processing of somatosensory information because they are expressed in nuclei that receive direct sensory input.

Opioid receptors are known to couple to G-proteins and to inhibit adenylyl cyclase. Receptor activation of G-proteins can be measured by agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S-) binding in brain sections to localize neuroanatomically functional coupling of receptors to intracellular signal transduction mechanisms. In the present study the selective mu-, delta- and kappa 1-opioid agonists DAMGO ([D-Ala2,N-Me-Phe4, Gly-ol5]-enkephalin), DPDPE ([D-Pen2,5]-enkephalin) and enadoline (CI-977) were used to stimulate [35S]GTP gamma S-binding in human brain sections of frontal cortex and cerebellum. In human frontal cortex mu- and delta- opioid stimulated [35S]GTP gamma S-binding was evenly distributed throughout the gray matter, while kappa(1)-opioid stimulated [35S]GTP gamma S-binding was detected predominantly in lamina V and VI. In the cerebellar cortex stimulated [35S]GTP gamma S-binding revealed functional coupling of mu- and kappa 1-opioid receptors in the molecular layer.

Publisher Summary This chapter discusses the neuroplasticity in the spinal cord of monoarthritic rats. In animal studies, the use of the quantitative autoradiographic 2-deoxyglucase technique (2DG) enables to investigate changes in metabolic activity. The tracing of metabolic activity helps to understand neuroplastic changes in such as it characterizes the regional distribution and time course of dynamic alterations in central nervous system (CNS) structures involved in the processing of noxious information. Doing this it may guide other experimental approaches, such as molecular biology techniques to those CNS structures where the evaluation of functional and structural changes especially changes in gene expression can give new insights into the understanding of the neurobiology of chronic pain. Following this approach, the 2-deoxyglucase and mRNA differential display techniques is used to investigate neuroplastic changes in the spinal cord following induction of chronic monoarthritis (MA).

Pain is a multi-dimensional experience including sensory-discriminative and affective-motivational components. The attribution of such components to a corresponding cerebral neuronal substrate in the brain refers to conclusions drawn from electrical brain stimulation, lesion studies, topographic mappings and metabolic imaging. Increases in neuronal metabolic activity in supraspinal brain regions, suggested to be involved in the central processing of pain, have previously been shown in various animal studies. The present investigation is the first to describe supraspinal structures which show increased metabolic activity during ongoing monoarthritic pain at multiple time-points. Experimental chronic monoarthritis of a hindlimb induced by complete Freund's adjuvant is one of the most used models in studies of neuronal plasticity associated with chronic pain. Such animals show typical symptoms of hyperalgesia and allodynia for a prolonged period. Metabolic activity changes in supraspinal brain regions during monoarthritis were assessed using the quantitative [14C]-2deoxyglucose technique at two, four, 14 days of the disease and, furthermore, in a group of 14-day monoarthritic rats which were mechanically stimulated by repeated extensions of the inflamed joint. Local glucose utilization was determined ipsi- and contralateral to the arthritic hindpaw in more than 50 brain regions at various supraspinal levels, and compared with saline-injected controls. At two and 14 days of monoarthritis significant bilateral increases in glucose utilization were seen in many brain structures, including brainstem, thalamic, limbic and cortical regions. Within the brainstem, animals with 14-day monoarthritis showed a higher number of regions with increased metabolic activity compared with two days. No differences between ipsi- and contralateral sides were detected in any of the experimental groups. Average increases ranged from 20 to 40% compared with controls and maximum values were detected in specific brain regions, such as the anterior pretectal nucleus, the anterior cingulate cortex and the nucleus accumbens. Interestingly, at four days of monoarthritis, the glucose utilization values were in the control range in almost all regions studied. Moreover, in monoarthritic rats receiving an additional noxious mechanical stimulation, the rates of glucose utilization were also comparable to controls in all brain areas investigated. Such patterns of brain metabolic activity agreed with concomitant changes in the lumbar spinal cord, described in the accompanying report. The present data show that a large array of supraspinal structures displays elevated metabolic activity during painful monoarthritis, with a non-linear profile for the time-points investigated. This observation most probably reflects mechanisms of transmission and modulation of nociceptive input arising from the monoarthritis and accompanying its development.

Intergration of nociceptive signaling comprises peripheral, spinal, and supraspinal sites of the nervous system. Various excitatory or inhibitory neurotransmitter and -modulator systems participate in pain processing and modulation. Chronic pain states are associated with functional and structural alterations of nociceptive pathways. The numerous dynamic changes in the pharmacologically distinct systems offer novel targets for selective pharmacotherapy.

Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III-VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III-VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression.

Publisher Summary This chapter focuses on the study that induces monoarthritis in rats and investigates the expression patterns of ionotropic glutamate receptor subunit messenger RNA (mRNA), as well as the binding intensity and the laminar distribution of protein kinase C (PKC) in the spinal cord dorsal horn. Molecular cloning studies have demonstrated that ionotropic glutamate receptor constituents belong to the same gene family. Recombinant expression studies have showed that native glutamate receptors are supposed to be heterooligomeric assemblies of five subunits. Protein phosphorylation of ligand-gated ion channels appears to be another major mechanism to regulate the properties of the diverse types of glutamate activated receptor channels. As the functional properties of neurons are critically determined by their individual receptor subunit composition, the chapter investigates the distribution of ionotropic glutamate receptor subunit mRNA in spinal cord neurons. It may be assumed that a differential distribution of glutamate receptor subunits may be tailored to fit the neurons individual response characteristics that are required to participate in particular spinal neuronal circuits.

The expression of the nuclear immediate-early gene-encoded protein c-Fos in spinal cord dorsal horn neurons of the rat following noxious thermal stimulation was compared in carbamazepine-, valproate- and phenytoine-treated animals. Single intraperitoneal injection of carbamazepine (50 mg/kg), valproate (300 mg/kg) or intravenous injection of phenytoine (20 mg/kg) before noxious stimulation reduced the number of c-Fos immunoreactive neurons to 65–80% of control levels in superficial laminae and to 30–60% in deep laminae of the dorsal horn. Pretreatment with carbamazepine or valproate for 4 or 8 days combined with an injection immediately before noxious stimulation further significantly decreased the number of c-Fos neurons in the deep dorsal horn only in animals treated with valproate. The observation that activity-dependent gene expression in the spinal cord is effectively modulated by anticonvulsants discloses a novel therapeutic potential of these compounds. Presumably via an acute suppression of high-frequency repetitive firing and/or altered synaptic transmission of intraspinal or descending neurotransmitter systems these drugs gain access to neuroplastic mechanisms which might be relevant for the restoration of physiological levels of neuronal excitability in the central nervous system.

Cellular immediate-early genes (IEGs) share a close structural homology with some viral oncogenes. In the eukaryotic genome these counterparts of oncogenes have been termed proto-oncogenes. Recent advances in cellular biology have identified the activation and deactivation of IEGs as molecular mechanisms to control regulated and deregulated growth, cellular differentiation and development. In neurobiology IEGs are believed to be involved in the neuron’s ability to convert short-term synaptic stimulation into long-lasting responses and thus contributing to the adaptive alterations involved in neuronal plasticity (Evan 1991; Goelet et al. 1986; for review see Morgan and Curran 1991). IEGs may be viewed as “third messengers” in a stimulus-transcription cascade transferring extracellular information via second messenger systems, such as calcium, into changes in target-gene transcription, thereby changing the phenotype of neurons. IEGs which encode proteins with a leucine zipper structure have to dimerize to function. While Jun proteins can form homo- as well as heterodimers, members of the Fos family require dimerization with Jun proteins. The various IEG complexes possess different binding affinities for DNA consensus sequences (AP-1 site) and variable transcriptional activities. The zinc-finger protein Krox-24, also termed NGFI-A, Egr-1, Zif/268, can achieve transcriptional activation in a monomeric fashion.

[This corrects the article DOI: 10.2196/14863.]., (©Luciana Midaglia, Patricia Mulero, Xavier Montalban, Jennifer Graves, Stephen L Hauser, Laura Julian, Michael Baker, Jan Schadrack, Christian Gossens, Alf Scotland, Florian Lipsmeier, Johan van Beek, Corrado Bernasconi, Shibeshih Belachew, Michael Lindemann. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 08.10.2019.)