Pain and knee damage in male and female mice in the medial meniscal transection-induced osteoarthritis.

OBJECTIVE: To investigate sex effects on pain-related behaviors in the medial meniscal transection (MMT) knee osteoarthritis (OA) model. METHODS: Experiments were performed in male and female C57BL/6J mice (12/group/sex). MMT was induced by transection of the medial collateral ligament and the medial meniscus. Sham-operated and naïve mice served as controls. Mechanical and heat sensitivity in hind paws, hind limb use, and locomotor activity were measured for 3 months. Knee histology was performed on week 12. RESULTS: In males, MMT triggered a bi-phasic mechanical hypersensitivity and decreased load on OA limb, with an acute post-operative (1-5 days) and chronic (3-12 weeks) OA phases separated by a remission in the intermediate phase (1-2 weeks). Females showed a less pronounced bi-phasic pattern, with a greater mechanical hypersensitivity, but not poorer limb use, than males in the intermediate phase (maximal difference: 1.1 g, 95% confidence intervals (CI) [0.7, 1.5]). There were no major sex differences in the chronic phase. MMT did not induce heat hypersensitivity or change in locomotor activity in the chronic phase in both sexes. MMT caused more severe cartilage damage in males than in females (maximal difference: 1.1 score points, 95% CI [1.9, 0.3]), and a comparable between sexes osteophyte formation. The knee damage did not correlate with pain. CONCLUSIONS: MMT modelled human knee OA well, capturing cartilage destruction and osteophyte formation, mechanical pain, and poorer limb use in both sexes. Sex differences in pain were modality- and time-dependent, reflecting complex sex-related features of human OA.

A nontoxic pain killer designed by modeling of pathological receptor conformations.

Indiscriminate activation of opioid receptors provides pain relief but also severe central and intestinal side effects. We hypothesized that exploiting pathological (rather than physiological) conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist that, because of its low acid dissociation constant, selectively activates peripheral µ-opioid receptors at the source of pain generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, heterotrimeric guanine nucleotide-binding protein (G protein) subunit dissociation by fluorescence resonance energy transfer, and adenosine 3',5'-monophosphate inhibition in vitro. It produced injury-restricted analgesia in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation, constipation, or addiction potential.

Leukocytes in the regulation of pain and analgesia.

When tissue is destroyed or invaded by leukocytes in inflammation, numerous mediators are delivered by the circulation and/or liberated from resident and immigrated cells at the site. Proalgesic mediators include proinflammatory cytokines, chemokines, protons, nerve growth factor, and prostaglandins, which are produced by invading leukocytes or by resident cells. Less well known is that analgesic mediators, which counteract pain, are also produced in inflamed tissues. These include anti-inflammatory cytokines and opioid peptides. Interactions between leukocyte-derived opioid peptides and opioid receptors can lead to potent, clinically relevant inhibition of pain (analgesia). Opioid receptors are present on peripheral endings of sensory neurons. Opioid peptides are synthesized in circulating leukocytes, which migrate to inflamed tissues directed by chemokines and adhesion molecules. Under stressful conditions or in response to releasing agents (e.g., corticotropin-releasing factor, cytokines, noradrenaline), leukocytes can secrete opioids. They activate peripheral opioid receptors and produce analgesia by inhibiting the excitability of sensory nerves and/or the release of excitatory neuropeptides. This review presents discoveries that led to the concepts of pain generation by mediators secreted from leukocytes and of analgesia by immune-derived opioids.

Peripheral opioid analgesia.

Major recent findings in peripheral opioid analgesia include the relative lack of tolerance under inflammatory conditions, tetrapeptides as novel peripherally restricted compounds, the potent anti-inflammatory activity of mu and kappa agonists and the identification of selectins as important molecules governing the homing of opioid cells to injured tissue. Clinical studies have now moved into the field of chronic arthritic pain, a problem of major relevance and prevalence.

Opioid peptide-expressing leukocytes: identification, recruitment, and simultaneously increasing inhibition of inflammatory pain.

BACKGROUND: Inflammatory pain can be effectively controlled by an interaction of opioid receptors on peripheral sensory nerve terminals with opioid peptides released from immune cells upon stressful stimulation. To define the source of opioid peptide production, we sought to identify and quantify populations of opioid-containing cells during the course of Freund's complete adjuvant-induced hind paw inflammation in the rat. In parallel, we examined the development of stress-induced local analgesia in the paw. METHODS: At 2, 6, and 96 h after Freund's complete adjuvant inoculation, cells were characterized by flow cytometry using a monoclonal pan-opioid antibody (3E7) and antibodies against cell surface antigens and by immunohistochemistry using a polyclonal antibody to beta-endorphin. After magnetic cell sorting, the beta-endorphin content was quantified by radioimmunoassay. Pain responses before and after cold water swim stress were evaluated by paw pressure thresholds. RESULTS: In early inflammation, 66% of opioid peptide-producing (3E7+) leukocytes were HIS48+ granulocytes. In contrast, at later stages (96 h), the majority of 3E7+ immune cells were ED1+ monocytes or macrophages (73%). During the 4 days after Freund's complete adjuvant inoculation, the number of 3E7+ cells increased 5.6-fold (P < 0.001, Kruskal-Wallis test) and the beta-endorphin content in the paw multiplied 3.9-fold (P < 0.05, Kruskal-Wallis test). In parallel, cold water swim stress-induced analgesia increased by 160% (P < 0.01, analysis of variance). CONCLUSIONS: The degree of endogenous pain inhibition is proportional to the number of opioid peptide-producing cells, and distinct leukocyte lineages contribute to this function at different stages of inflammation. These mechanisms may be important for understanding pain in immunosuppressed states such as cancer, diabetes, or AIDS and for the design of novel therapeutic strategies in inflammatory diseases.

Analgesic and antiinflammatory effects of two novel kappa-opioid peptides.

BACKGROUND: This study investigates two new kappa-agonist tetrapeptides, FE 200665 and FE 200666, with high peripheral selectivity as a result of poor central nervous system penetration. METHODS: Four days after administration of Freund adjuvant into the hind paw of male Wistar rats, antinociceptive effects of intraplantar and subcutaneous injection of FE 200665 and FE 200666 were measured by paw pressure algesiometry and compared with the kappa-agonist U-69,593. Peripheral and kappa-receptor selectivity was assessed by the antagonists naloxone methiodide (NLXM) and nor-binaltorphimine, respectively. Antiinflammatory effects were evaluated by paw volume plethysmometry and histologic score. RESULTS: Similar to intraplantar U-69,593, intraplantar FE 200665 (3-100 microg) and FE 200666 (1-30 microg) resulted in significant and dose-related increases of paw pressure thresholds. Higher doses of FE 200665 (0.2-20 mg) and FE 200666 (0.06-6 mg) were required by subcutaneous route to produce similar antinociceptive responses, supporting a peripheral site of action. nor-Binaltorphimine dose-dependently antagonized this effect, implying kappa-opioid selectivity. Analgesic effects of subcutaneous FE 200665 and FE 200666 were abolished by intraplantar nor-binaltorphimine, and both subcutaneous and intraplantar effects were dose-dependently antagonized by subcutaneous NLXM, further demonstrating a peripheral site of action. One to 6 days after Freund adjuvant inoculation, single and repeated intraplantar injections of FE 200665, FE 200666, and U-69,593 significantly reduced paw volume and histologic scores. Both changes were reversed by intraplantar nor-binaltorphimine and subcutaneous NLXM. CONCLUSION: FE 200665 is a peripherally selective kappa-agonist with potent analgesic and antiinflammatory properties that may lead to improved analgesic-antiinflammatory therapy compared with centrally acting opioids or standard nonsteroidal antiinflammatory drugs.

Peripheral analgesic and antiinflammatory effects of opioids.

Traditionally, opioids were considered the prototype of centrally acting analgesics. In the past decade a substantial literature has emerged demonstrating that opioids can produce potent and clinically measurable analgesia by activation of opioid receptors on peripheral terminals of primary sensory neurons. In addition, endogenous ligands of these peripheral receptors were discovered in immune cells. Major recent findings in peripheral opioid analgesia include the relative lack of tolerance under inflammatory conditions, tetrapeptides as novel peripherally restricted compounds, the potent antiinflammatory activity of mu- and kappa-agonists and the identification of selectins as important molecules governing the homing of opioid cells to injured tissue. In addition to the extensively documented efficacy of locally applied morphine in post-surgical pain, clinical studies have now moved into the field of chronic arthritic pain.

Co-expression of beta-endorphin with adhesion molecules in a model of inflammatory pain.

Opioid-containing immunocytes migrate to inflamed sites where they release beta-endorphin which activates peripheral opioid receptors and produces analgesia. The immigration of immunocytes to sites of inflammation is mediated by adhesion molecules. In this study, the expression of L-, P-, E-selectin and platelet-endothelial adhesion molecule-1 (PECAM-1) in relation to beta-endorphin expression was analyzed by immunohistochemistry in inflamed tissues. The proportion of immunocytes expressing L-selectin was increased in inflamed lymph nodes and subcutaneous paw tissue. P-selectin and PECAM-1 were constitutively expressed on endothelia of noninflamed lymph nodes and subcutaneous tissue and were upregulated in inflammation. beta-endorphin positive cells expressed L-selectin in lymph nodes and subcutaneous tissue. Upregulation of P-selectin and PECAM-1 and the co-localization of L-selectin and beta-endorphin in immunocytes suggest an important role of these adhesion molecules for the recruitment of immunocytes containing beta-endorphin to sites of painful inflammation.

Pain control by immune-derived opioids.

1. The nervous and immune systems communicate with each other by use of cytokines and neuropeptides. 2. Interactions between immune cell-derived opioid peptides and opioid receptors located in peripheral inflamed tissue lead to endogenous analgesia. 3. In addition to their immunological functions, immunocytes are involved in intrinsic pain inhibition. This provides new insights into pain associated with a compromised immune system, as in AIDS or in cancer. 4. The activation of opioid production and release from immune cells may be a novel approach to the development of peripherally acting analgesics. Because such drugs would be targeted towards events in peripheral injured tissue, these analgesics should lack unwanted central side effects typically associated with opioids.

Why is morphine not the ultimate analgesic and what can be done to improve it?

Although opioids are unsurpassed in the treatment of acute and cancer pain, their use in chronic noncancer pain is clearly limited. This review discusses some open and controversial issues such as the balance between pain relief and side effects, whether all types of pain can be treated with opioids, and current efforts to develop opioids with an improved efficacy-side effect ratio. Whereas respiratory depression or tolerance are usually not major issues in long-term opioid use, it seems questionable whether opioids can produce an analgesic response in certain types of pain when there is a major affective component to the pain or when learned pain behavior is the main problem. Efforts to improve opioids have traditionally aimed at enhancing the selectivity of opioid receptor ligands towards mu-, delta-, and kappa-receptors. Another major strategy has been the search for opioid analgesics acting at opioid receptors outside the central nervous system, with the prospect to avoid centrally mediated side effects.

Peripheral effects of the kappa-opioid agonist EMD 61753 on pain and inflammation in rats and humans.

The objective of the present study was to evaluate the effects of EMD 61753 (asimadoline), a kappa-opioid receptor agonist with restricted access to the central nervous system, on postoperative pain in patients who underwent knee surgery and on nociceptive thresholds and inflammation in rats treated with Freund's complete adjuvant. Patients treated with EMD 61753 (10 mg p.o.) tended to report an increase in pain, as evaluated by a visual analog scale and by the time to the first request for and the total amount of supplemental analgesic medication. The global tolerability of EMD 61753 was assessed as significantly inferior to that of a placebo by the investigator. In rats, the bilateral intraplantar (i.pl.) injection of EMD 61753 (0.1-3.2 mg) resulted in dose-dependent antinociception in both inflamed and noninflamed paws, with a peak at 5 min after injection, as evaluated by the paw pressure method. However, at later time points (1 h-4 days), a significant decrease in the paw pressure threshold was observed, confirming its tendency toward a hyperalgesic action in humans. This was accompanied by an increase in paw volume and paw temperature, with a peak at 6 h after injection. EMD 61753 (1.6 mg)-induced analgesia was blocked by the peripheral opioid receptor antagonist naloxone methiodide (2.5-10 mg/kg s.c.) and by the kappa receptor antagonist nor-binaltorphimine (0.1 mg; i.pl.). In contrast, EMD 61753 (1.6 mg)-induced hyperalgesia and increases in paw volume and paw temperature were blocked neither by naloxone methiodide (10-40 mg/kg s.c.) nor by dizocilpine maleate (0.003-0.009 mg i.pl.), a N-methyl-D-aspartic acid receptor antagonist. These data show differentially mediated peripheral actions of EMD 61753: kappa-opioid receptor-induced analgesia and nonopioid, non-N-methyl-D-aspartic acid hyperalgesic and proinflammatory effects.

Antinociception after both peripheral and intrathecal injection of oxotremorine is modulated by spinal nitric oxide.

The present study investigated the role of spinal nitric oxide (NO) in the antinociception induced by intraperitoneal (i.p.) and intrathecal (i.th.) injection of oxotremorine. The experiments were carried out on male Wistar rats, which had cannulas chronically implanted in the lumbar enlargement of the spinal cord. Antinociceptive effects were evaluated using a tail-flick and a paw pressure test. To raise the spinal NO level, the rats received the NO donor, 3-morpholino-sydnonimine (SIN-1, 10 and 100 microg/5 microl); to lower the NO level, the inhibitor of NO synthase, N-nitro-L-arginine methyl ester (L-NAME, 50 and 400 microg/5 microl), was administered. Both those substances were injected i.th. Systemic injections of oxotremorine (0.02 and 0.1 mg/kg) produced a significant increase in the thermal nociceptive threshold, while the mechanical threshold was affected only by the higher dose (0.1 mg/kg) of the muscarinic agonist. I.th. injections of oxotremorine (0.1 ng, 1 ng, 1 microg/5 microl) produced significant antinociception in both those tests. I.th. administration of SIN-1 in doses which themselves did not affect the nociceptive threshold antagonized both the peripheral and central oxotremorine antinociception. I.th. administration of L-NAME (50 and 400 microg/5 microl) did not change the nociceptive threshold, but dose-dependently potentiated the effects of oxotremorine injected i.p. in both tests; however, the effect of i.th. administration of oxotremorine was potentiated only in the tail-flick test. Our results demonstrate that irrespective of the way of its injection, the antinociceptive effect of oxotremorine is modulated by activity of the spinal NO. Moreover, our results further support the hypothesis that NO present in the spinal cord exerts pronociceptive effects.

Intrathecal oxotremorine affects formalin-induced behavior and spinal nitric oxide synthase immunoreactivity in rats.

The present research was undertaken to investigate, by behavioral and immunohistochemical methods, the effects of intrathecal (i.th.) injection of the muscarinic agonist oxotremorine on the response to the long-lasting nociceptive stimulus induced by injection of formalin into the rat hind paw. Formalin injection induced a biphasic, pain-induced behavioral response (paw jerks), as well as an increase in the number of nitric oxide (NO) synthase-labeled neurons in laminae I-III, IV, and X, but not in laminae V-VI. Oxotremorine (0.1-10 ng, i.th.) inhibited paw-jerk frequency in both phases of formalin-induced behavior. The immunohistochemical results showed that i.th.-injected oxotremorine differently affected the level of NO synthase in lumbar part of the spinal cord: no change or increase after the dose of 1 ng, and a significant reduction of nitric oxide synthase neurons after the higher dose (10 ng). These results evidenced a role of cholinergic system in the modulation of tonic pain and in nitric oxide synthase expression at the spinal cord level, which further suggests that these two systems could be involved in phenomena induced by long-lasting nociceptive stimulation.

Pain control in inflammation governed by selectins.

Opioid-containing immune cells migrate preferentially to inflamed sites, where they release beta-endorphin which activates peripheral opioid receptors to inhibit pain. Immunocyte recruitment is a multistep, sequential engagement of various adhesion molecules located on immune cells and vascular endothelium. Selectins mediate the initial phase of immunoctye extravasation into inflamed sites. Here we show that anti-selectin treatment abolishes peripheral opioid analgesia elicited either endogenously (by stress) or by corticotropin-releasing factor. This results from a blockade of the infiltration of immunocytes containing beta-endorphin and the consequent decrease of the beta-endorphin content in the inflamed tissue. These findings indicate that the immune system uses mechanisms of cell migration not only to fight pathogens but also to control pain in injured tissue. Thus, pain is exacerbated by measures inhibiting the immigration of opioid-producing cells or, conversely, analgesia might be conveyed by adhesive interactions that recruit those cells to injured tissue.

Effects of pentylenetetrazol kindling on glutamate receptor genes expression in the rat hippocampus.

It has been hypothesized that changes in the excitatory amino acid receptor biosynthesis may be involved in the mechanism of kindling-an animal model of epileptogenesis. In order to test this hypothesis, we investigated the effects of pentylenetetrazol kindling on the expression of genes coding for NMDAR1 and GluR2 in the rat hippocampal formation. Pentylenetetrazol kindling decreased the hippocampal NMDAR1 mRNA level after 3 and 24 h; lowered the GluR2 flip level and elevated the flop mRNA one in the CA1 field and dentate gyrus after 3 and 24 h, respectively. A receptor autoradiography showed an increase in the [3H]MK-801 binding density in the hippocampus following both acute and repeated pentylenetetrazol administration. We conclude that an early occurrence of downregulation of the glutamate receptor gene expression may be an adaptive response of glutamate receptors to an oversupply of excitatory amino acids during repeated seizures.

Antinociceptive effects of isoleucine derivatives of deltorphin I and deltorphin II in rat spinal cord: a search for selectivity of delta receptor subtypes.

Deltorphins show a high affinity and selectivity for delta opioid receptors. Analogs of deltorphins with substitution of Val residues with more hydrophobic Ile appear to have a higher in vitro activity and selectivity than parent deltorphins. In our study, changes in the nociceptive threshold after intrathecally injected deltorphin I (DELT I), deltorphin II (DELT II) and their Ile - derivatives (ILE-DELT I and ILE-DELT II, respectively) were investigated in a tail-flick (TF) and a paw pressure (PP) tests. Male Wistars rats (260-350 g) with a chronically implanted catheter in the lumbar enlargement of the spinal cord were used. DELT I and DELT II, injected i.th. in doses of 0.15, 1.5 and 15 microg, increased the TF latency in a dose-dependent manner. The effect of their derivatives was similar, but the action of ILE-DELT II was shorter than that of the parent peptide. In the PP test, the antinociceptive effects of DELT I and their derivative ILE-DELT I were similar, but the effect of a higher dose of ILE-DELT I lasted longer in comparison with the parent peptide. Both DELT II and ILE-DELT II exhibited a low and short-lasting antinociceptive potency in the PP test. The effect of DELT I (1.5 microg) was antagonized by pretreatment with NTI (30 microg), a non-selective delta opioid receptor antagonist, as well as by the delta2 receptor antagonist NTB (3 microg) and the delta1 antagonist BNTX (1 microg) in both those tests used. The antinociceptive effect of DELT II (1.5 microg) was antagonized by pretreatment with NTI (30 microg) and NTB (3 microg) in the TF test, but not in the PP test. In the latter test, the antinociceptive effect of DELT II was potentiated by pretreatment with BNTX (1 microg). The effects of both the derivatives ILE-DELT I and ILE-DELT II were antagonized by NTI (30 microg) in the TF test, and by NTI (30 microg) and NTB (3 microg) in the PP test. Like in the case of the parent peptide, the effect of ILE-DELT II was potentiated by pretreatment with the delta1 antagonist BNTX (1 microg). Summing up, modification of the DELT I and II by substituting Ile for Val residues appears to influence the delta selectivity rather then the potency of the peptides at spinal delta receptors.

Differential effects of intrathecally and intracerebroventricularly administered nitric oxide donors on noxious mechanical and thermal stimulation.

Involvement of nitric oxide (NO) in nociceptive transmission is well documented. However, there is controversy concerning the exact role of NO in mediation of nociception at different levels of the nervous system. Most studies agree that NO promotes hyperalgesia at the level of the spinal cord. Conversely, at supraspinal sites exogenously applied NO has been found to be both pro- and antinociceptive. In light of this discrepancy, the aim of the present study was to compare the effects of NO donors on nociceptive transmission at spinal and supraspinal sites of the central nervous system using mechanical (paw pressure; PP) and thermal (tail-flick; TF) noxious stimulation. Four NO donors which release NO through different mechanisms were used: S-nitrosoglutathione (SNOG; 3-600 nmol), S-nitroso-N-acetylpenicillamine (SNAP; 0.18-4.5 nmol), hydroxylamine (HYD; 60-1200 nmol) and 3-morpholino-sydnonimine (SIN-1; 490-970 nmol). They were injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) to male Wistar rats and nociceptive thresholds were evaluated in TF and PP tests. It was found that NO donors administered i.t. or i.c.v. produced a dose-dependent hyperalgesia in the PP test. The hyperalgesia induced by mechanical stimuli was stronger after i.t. than after i.c.v. administration of NO donors. The SIN-1-induced hyperalgesia, as evaluated by teh PP test, was reversed by i.t. pretreatment with haemoglobin (1.5-4 nmol) a NO scavenger, and methylene blue (267-1070 nmol) a guanylate cyclase and NO synthase inhibitor, suggesting that NO exerts its action by facilitating cyclic guanosine 3',5'-monophosphate (GMP) formation. Unlike in the PP test, SNAP and SNOG had no effect on the nociceptive threshold in the TF test, and only SIN-1 administered i.t. produced a weak hyperalgesia in that test, while HYD caused a mild but significant prolongation of the TF reflex. The above data show that NO produces hyperalgesia principally in response to noxious mechanical stimuli. This effect seems to be predominantly mediated in the spinal cord, however, it occurs at both levels of the central nervous system.

Inhibition of nitric oxide synthase enhances antinociception mediated by mu, delta and kappa opioid receptors in acute and prolonged pain in the rat spinal cord.

Our study was designed to determine involvement of nitric oxide (NO) in the antinociception mediated by mu, delta and kappa opioid receptors in acute and prolonged pain in the rat spinal cord. The effect of intrathecally (i.t.) injected NO synthase inhibitors and opioid receptor agonists was evaluated in acute pain using a tail-flick and a paw pressure tests, and in prolonged pain by quantification the pain-related behavior after peripheral formalin injection. It was found that the neuronal NO synthase inhibitor 7-nitroindazole (50-400 microg), used in inactive doses, dose-dependently enhanced antinociception induced by morphine (0.5 microg) in the tail-flick and paw pressure. Moreover, coadministration of N(G)-nitro-L-arginine methyl ester (50 microg) another NO synthase inhibitor, with morphine (0.05-0.5 microg) as well as with specific agonists of mu ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin 0.1-2.5 ng) and delta ([D-Pen(2,5)]enkephalin 0.02-0.5 microg) opioid receptors, enhanced dose-dependent antinociception in the tail-flick and paw pressure. Coadministration of N(G)-nitro-L-arginine methyl ester with specific kappa opioid receptor agonist 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg), produced antinociception in the paw pressure only. Additionally, N(G)-nitro-L-arginine methyl ester (100 microg) profoundly potentiated the antinociception induced by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (0.5, 15 ng) and [D-Pen(2,5)]enkephalin (2, 10 microg) in the dose-related manner in the formalin test. N(G)-nitro-L-arginine methyl ester (100 microg) also enhanced the antinociception induced by 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzenacetamid e (10-100 microg) but only at the last two time points of the second phase of the formalin test. These data show that inhibition of the spinal NO synthase potentiates the mu-, delta- and to a lesser extent, kappa-mediated spinal antinociception in both acute and prolonged pain.

Chronic morphine increases biosynthesis of nitric oxide synthase in the rat spinal cord.

The present study was undertaken to determine the influence of chronic morphine treatment on the biosynthesis of nitric oxide synthase (NOS) in the rat spinal cord using in situ hybridization and immunohistochemical methods. Repeated administration of morphine (20-100 mg/kg/day; 10 days) increased the NOS mRNA level in laminae I-IV and X 3 h after the last injection. That effect was accompanied by an increase in both the number of NOS-positive cells (24 h) and the optical density of NOS-immunoreactivity (3 and 24 h). The results indicate that repeated morphine administration increases NOS biosynthesis in the rat spinal cord, which may reflect adaptive changes accounting for development of opiate tolerance and dependence.

Effects of pilocarpine and kainate-induced seizures on N-methyl-D-aspartate receptor gene expression in the rat hippocampus.

The effects of pilocarpine- and kainate-induced seizures on N-methyl-D-aspartate receptor subunit-1 messenger RNA and [3H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-D-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-D-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [3H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [3H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-D-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-D-aspartate receptor subunit-1 and [3H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate. These data point to some differences in hippocampal N-methyl-D-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-D-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [3H]dizocilpine maleate binding in the rat hippocampal formation.