Michael J. Iadarola

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding

&NA; The stimulus specificity for enhancement of dynorphin gene expression in rat spinal cord was studied by combined measurements of the peptide dynorphin A 1–8 and preprodynorphin mRNA levels during peripheral inflammation induced by several agents. The density of kappa receptors, the putative receptor for dynorphin peptides, was examined using receptor binding with autoradiographic visualization. Mu and delta receptor classes were also studied. All inflammatory agents tested (carrageenan, phorbol ester, yeast and Freunds adjuvant) rapidly induced edema and thermal hyperalgesia. All agents also induced a rapid (within 8 h) elevation in dynorphin mRNA and, in comparison, a delayed (within 2 days) elevation of dynorphin A 1–8 peptide; peak peptide levels were reached at 4 days. No alteration of kappa, mu or delta receptor binding was observed at 4 h or 4 days post inflammation. The rapid development of thermal hyperalgesia and elevation of dynorphin mRNA and peptide content indicates that the involvement of dynorphin‐containing neurons in nociceptive processing does not require a chronic abnormality and a dynamic picture of opioid modulation of sensory processing emerges. These data also demonstrate that activation of dynorphin biosynthesis in spinal cord is a feature common to hyperalgesia and peripheral inflammation and is not restricted to any one type of inflammatory agent. The lack of alteration in receptors suggests that the physiological effects of an increased biosynthesis are not accompanied by a concurrent down‐regulation of opiate receptors.

Unilateral decrease in thalamic activity observed with positron emission tomography in patients with chronic neuropathic pain

&NA; The oxygen‐15 water bolus positron emission tomography (PET) method was used to image regional brain activity in 4 patients with chronic post‐traumatic neuropathic pain confined to one lower limb and in 1 patient with post‐herpetic neuralgia. In comparison to 13 normal subjects, scans of the patients disclosed a statistically significant decrease in thalamic activity contralateral to the symptomatic side. Examination of the right/left ratio for all the subjects showed that the values for the patients fell at the extremes of the normal range, according to the side of the affected body part. These initial observations suggest that functional alterations in thalamic pain processing circuits may be an important component of chronic neuropathic pain.

Genetic influence on variability in human acute experimental pain sensitivity associated with gender, ethnicity and psychological temperament

&NA; While a variety of cultural, psychological and physiological factors contribute to variability in both clinical and experimental contexts, the role of genetic factors in human pain sensitivity is increasingly recognized as an important element. This study was performed to evaluate genetic influences on variability in human pain sensitivity associated with gender, ethnicity and temperament. Pain sensitivity in response to experimental painful thermal and cold stimuli was measured with visual analogue scale ratings and temperament dimensions of personality were evaluated. Loci in the vanilloid receptor subtype 1 gene (TRPV1), &dgr; opioid receptor subtype 1 gene (OPRD1) and catechol O‐methyltransferase gene (COMT) were genotyped using 5′ nuclease assays. A total of 500 normal participants (306 females and 194 males) were evaluated. The sample composition was 62.0% European American, 17.4% African American, 9.0% Asian American, and 8.6% Hispanic, and 3.0% individuals with mixed racial parentage. Female European Americans with the TRPV1 Val585 Val allele and males with low harm avoidance showed longer cold withdrawal times based on the classification and regression tree (CART) analysis. CART identified gender, an OPRD1 polymorphism and temperament dimensions of personality as the primary determinants of heat pain sensitivity at 49 °C. Our observations demonstrate that gender, ethnicity and temperament contribute to individual variation in thermal and cold pain sensitivity by interactions with TRPV1 and OPRD1 single nucleotide polymorphisms.

Temporal analysis of increases in c-fos, preprodynorphin and preproenkephalin mRNAs in rat spinal cord

Peripheral inflammation produces a rapid elevation (within 4 h) in preprodynorphin mRNA in neurons of the dorsal spinal cord and an even more rapid elevation in c-fos proto-oncogene mRNA (within 30 min). During this period a relatively modest increase is also observed in spinal cord preproenkephalin mRNA. Previous anatomical studies have shown that the neurons in which these transcripts increase have overlapping distributions. Assuming that these events occur in the same cells, it suggests the possibility that newly synthesized c-fos protein may participate in transcriptional regulation of opioid genes in spinal cord.

Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridization

A unilateral experimental inflammation of the hindlimb produces hyperalgesia to both mechanical and radiant thermal stimuli that is rapid in onset. During this period, parameters of dynorphin biosynthesis are elevated to a much greater degree than those of the enkephalin system. An increase in the content of the peptide dynorphin A(1-8) occurs in the spinal cord segments that receive sensory input from the affected limb. This is accompanied by a rapid (within 24 h) and pronounced increase in the levels of mRNA coding for the dynorphin protein precursor. Maximum elevations (6- to 8-fold) of preprodynorphin mRNA are observed between days 2 and 5 subsequent to the induction of inflammation. Compared to the increase in mRNA, the increase in dynorphin A(1-8) peptide was appreciably delayed and proportionately less; maximal increases in peptide (3-fold) were seen at day 5 of inflammation. Dorsal spinal cord preproenkephalin mRNA is elevated to a lesser degree (50-80%). However, the increase in preproenkephalin mRNA is apparently not enough to yield a measurable increase in the proenkephalin-derived peptide met5-enkephalin-Arg6-Gly7-Leu8, the levels of which showed no significant change during the 14-day inflammatory period. These data suggest the active participation of opioid neurons, especially those containing dynorphin, at the spinal level, in the modulation of sensory afferent input during peripheral inflammatory pain states.

Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control

Control of cancer, neuropathic, and postoperative pain is frequently inadequate or compromised by debilitating side effects. Inhibition or removal of certain nociceptive neurons, while retaining all other sensory modalities and motor function, would represent a new therapeutic approach to control severe pain. The enriched expression of transient receptor potential cation channel, subfamily V, member 1 (TRPV1; also known as the vanilloid receptor, VR1) in nociceptive neurons of the dorsal root and trigeminal ganglia allowed us to test this concept. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya induces calcium cytotoxicity by opening the TRPV1 ion channel and selectively ablates nociceptive neurons. This treatment blocks experimental inflammatory hyperalgesia and neurogenic inflammation in rats and naturally occurring cancer and debilitating arthritic pain in dogs. Sensations of touch, proprioception, and high-threshold mechanosensitive nociception, as well as locomotor function, remained intact in both species. In separate experiments directed at postoperative pain control, subcutaneous administration of RTX transiently disrupted nociceptive nerve endings, yielding reversible analgesia. In human dorsal root ganglion cultures, RTX induced a prolonged increase in intracellular calcium in vanilloid-sensitive neurons, while leaving other, adjacent neurons unaffected. The results suggest that nociceptive neuronal or nerve terminal deletion will be effective and broadly applicable as strategies for pain management.

Dynorphin expression and fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons

Fos and Fos-related proteins are increased in spinal dorsal horn neurons following noxious stimulation. The laminar location of neurons that exhibit this increase is coincident with those that exhibit an increase in dynorphin in a rat model of peripheral inflammation and hyperalgesia. In order to determine whether the increase in Fos or related proteins and dynorphin occurs in the same dorsal horn neurons, two kinds of double-labeling methods were used: in situ hybridization histochemistry to label dynorphin mRNA autoradiographically, and immunocytochemistry to label Fos and Fos-related proteins, or a double immunocytochemical method that labeled Fos and Fos-related proteins and dynorphin peptide with distinct chromagens. With both methods more than 80% of the neurons in laminae I, II, V and VI exhibiting an increase in either dynorphin mRNA or peptide following peripheral inflammation also colocalized increased nuclear Fos-like immunoreactivity. However, the number of neurons displaying increased Fos-like immunoreactivity was substantially greater than the number of neurons colocalizing increased dynorphin. These data suggest that the activation of nuclear Fos and Fos-related proteins may be related to the induction of dynorphin gene expression in a subpopulation of spinal cord neurons following peripheral inflammation and hyperalgesia.

Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: The nucleus accumbens shell as a locus of antipsychotic action.

The mechanisms by which atypical antipsychotic drugs such as clozapine exert therapeutic effects but do not induce extrapyramidal side effects are not clear. We have examined the effects of acute administration of three antipsychotic drugs on Fos protein expression in the striatal complex. The ypical neuroleptic haloperidol was compared with the atypical agent clozapine and the putative atypical antipsychotic drug remoxipride. Haloperidol increased the number of neurons expressing Fos-like immunoreactivity in both the dorsolateral and the medial striatum and increased Fos expression in the nucleus accumbens core and shell. Clozapine increased Fos in the nucleus accumbens shell, but not in the core or two neostriatal sectors. Remoxipride significantly increased the number of Fos-like immunoreactive neurons in the medial but not the dorsolateral striatum, and increased the number of cells expressing Fos protein in the nucleus accumbens shell but not core. The remoxipride-induced increase in the number of medial striatal neurons expressing Fos was entirely attributable to a selective increase in the striatal patch compartment, whereas haloperidol increased Fos protein in neurons of both striatal compartments. These data indicate that typical and atypical antipsychotic drugs exert regionally distinct effects on striatal Fos expression, and suggest that the dorsolateral striatum may be a locus involved in the genesis of extrapyramidal side effects. All three antipsychotic drugs increased Fos expression in the shell of the nucleus accumbens. The shell of the nucleus accumbens may be a site of antipsychotic action.

Spinal opioid analgesic effects are enhanced in a model of unilateral inflammation/hyperalgesia : possible involvement of noradrenergic mechanisms

We have examined the spinal analgesic activity of opioid agonists and antagonists in a model of short term, unilateral, carrageenan-induced inflammation/hyperalgesia. Rats received a single s.c. injection of carrageenan (2-6 mg in saline) 3-24 h prior to testing hindpaw withdrawal latencies to noxious thermal stimuli. Dose-response curves for intrathecally administered agonists with mu- and/or delta-opioid activity were shifted to the left for inflamed hindpaws when compared to contralateral non-inflamed paws. The selective kappa-receptor agonist U-50,488H had no activity in this analgesic assay on either inflamed or non-inflamed paws when administered intrathecally. However, systemic administration of U-50,488H did produce significant elevations of paw withdrawal latencies in inflamed paws. The alpha 2-adrenoceptor agonist clonidine also produced dose-dependent antinociception in the paw withdrawal assay after systemic or intrathecal administration. Inflamed hindpaws were significantly more sensitive to the antinociceptive effect of morphine on inflamed hindpaws was blocked by the opioid antagonist naloxone or the alpha 2-adrenoceptor antagonist idazoxan. The effect of clonidine was only blocked by idazoxan. Antagonists alone had no significant effect on withdrawal latencies. The data indicate that the analgesic action of opioids during conditions of inflammation may depend on an interaction with spinal noradrenergic pathways.