Differential hemodynamic, metabolic and hormonal effects of morphine and morphine-6-glucuronide

Although the hyperglycemic effect of morphine has been previously described, it is not clear whether this is the result of increased glucose production and/or decreased glucose utilization and if this metabolic effect is lost with glucuronidation. This study assessed the hemodynamic (heart rate; HR and mean arterial blood pressure; MABP), hormonal and whole body glucose metabolic effects of morphine (MOR) and its metabolite morphine 6-glucuronide (MOR-6G) in conscious unrestrained chronically catheterized rats. Whole body glucose kinetics were assessed with a primed constant intravenous infusion of [3-³H]gluccose in rats infused i.c.v. with H₂O (Con; 5 μl/h), MOR (80 μg/h) or MOR-6G (1 μg/h) for a total of 4 h. MOR administration resulted in a significant 20% elevation in HR and no change in MABP. MOR-6G produced a 14% increase in HR and no change in MABP. A significant rise in plasma glucose (+23%), hepatic glucose production (R_a; +27–61%) and whole body glucose utilization (R_d; +31–61%) was also observed within 60 min of MOR administration. I.c.v. MOR-6G resulted in hemodynamic, metabolic and hormonal parameters of H₂O infused rats. I.c.v. MOR resulted in a significant increases in epinephrine (2-fold), norepinephrine (50%), corticosterone (97%) with no alterations in plasma insulin and glucagon. I.c.v. MOR-6G resulted in more marked elevations in norepinephrine (5-fold), epinephrine (7-fold) and similar elevation in corticosterone (99%) and modest elevation of glucagon (40%). These results indicate that (i) MOR-induced hyperglycemia is the result of direct central (CNS) mechanisms that result in increased hepatic glucose production, (ii) MOR-induced stress response is enhanced at least 80-fold with glucuronidation, and (iii) MOR inhibits the pancreatic glucose-stimulated insulin release.     

Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine

To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 μg/kg, the ED,, being 44 μg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 μg/kg, the ED₅₀ being 6 μg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20 μg the ED₅₀ is 18 μg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10 μg. The ED₅₀ of M6G i.t. is below 0.2 μg. The effects of morphine and M6G were reversed by naloxone (200 μg/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites.     

Central opiate modulation of growth hormone and insulin-like growth factor-I

The effects of central administration of morphine-sulfate (MOR:80 μg) and morphine-6-glucuronide (M6G:1 μg) on the growth hormone (GH)/insulin-like growth factor (IGF) system were assessed. MOR and M6G were injected intracerebroventricularly (ICV) in chronically catheterized 24 h fasted rats; time-matched control animals received H₂O (5 μl). MOR increased plasma GH concentrations 3-fold 2 h after ICV injection, and transiently increased the plasma concentration and liver content of IGF-I (60% and 90%, respectively) 30 min after ICV injection. M6G did not produce any significant alterations in plasma GH and IGF-I levels at the time-points measured. Both MOR and M6G increased the concentration of IGF binding protein-1 (IGFBP-1) in plasma and liver 2 h after injection. However, MOR showed 2- to 2.5-fold greater effect than M6G in stimulating plasma and liver IGFBP-1. MOR and M6G produced similar increases in plasma epinephrine (5-fold), norepinephrine (3-fold) and corticosterone (1.5-fold). Neither opiate significantly altered circulating insulin levels. These findings suggest that opiate modulation of GH and IGF may be hormone-independent and centrally modulated. We speculate that differential affinities of MOR and M6G to the different opiate receptor subtypes might be responsible for their distinct effects on GH/IGF-I system.     

The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat

The profound and prolonged effects of morphine in patients with renal dysfunction have been associated with high plasma levels of the opiate metabolites morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) rather than an increased concentration of morphine. We present here electrophysiological evidence to suggest that potent spinal antinociception can be produced by both M6G and normorphine, another metabolite of morphine. Extracellular recordings of Aβ- and C-fibre-evoked responses of convergent dorsal horn neuroneswere made in the halothane anaesthetised rat. M6G elicited dose-dependent, naloxone-reversible inhibitions of C-fibre-evoked responses which were completely suppressed (8% of control) by 2 μg M6G whereas Aβ-fibre-evoked responses were only reduced to 57% of controls. The ED₅₀ for the effects of M6G on C-fibre-evoked activity was calculated to be 0.53 μg. Systematic administration of M6G (2 mg/kg) also profoundly reduced noxious evoked neuronal activity. intrathecal normorphine was less potent than M6G but complete selective inhibitions of C-fibre-evoked responses could be elicited by 25 μg and the ED₅₀ was calculated to be 2.68 μg. No such inhibitions were observed following administration of M3G. A comparison with intrathecal morphine in the same preparation reveals that normorphine is equipotent with morphine whereas M6G is 13-fold more potent. These results therefore confirm that M6g and normorphine might be significant contributers to opiate analgesia after administration of morphine.     

Role of IL-1α in central nervous system immunomodulation of glucoregulation

Hyperglycemia is a hallmark of the stress response, and has been largely attributed to elevated plasma levels of catabolic hormones. Recently, various cytokines have been shown to be endogenously produced within the brain and may represent an important component of the central regulation of this metabolic response. Therefore, the aim of the present study was to determine whether the intracerebroventricular (i.c.v.) injection of one such peptide, interleukin (IL)-1, can produce hormonal and metabolic alterations comparable to those observed under stress conditions. An i.c.v. cannula and vascular catheters were placed in rats prior to the experiment. Whole body glucose flux was assessed in overnight fasted conscious unrestrained rats using [3-³H]glucose. A mild hyperglycemia was elicited 20 min after the i.c.v. injection of IL-1α (human recombinant, 100 ng) that was not detected in control rats. Glucose levels gradually increased and were 26% higher than control values during the last hour of the 3 h experimental period. The hyperglycemia resulted from a 44% increase in the rate of hepatic glucose output (HGO), which preceded a propertional rise in peripheral glucose utilization. No increase in metabolic clearance rate was observed, suggesting that the increased glucose uptake was the result of mass action. The increased glucose flux was associated with a transient hyperinsulinemia (+95%), and sustained elevations in the arterial concentrations of glucagon (56%) and corticosterone (175%). In contrast, glucose flux was not altered by intravenous administration of the same dose of IL-1α, or i.c.v. injection of IL-1β, or heat-inactivated IL-1α. Indomethacin (5 mg/kg, i.v.) blocked the hyperglycemia and increased HGO induced by i.c.v. injection of prostaglandin E₂ (100 ng) produced comparable increases in glucose flux. These results indicate that IL-1α can act centrally to enhance whole body glucose metabolism, and that this response is probably mediated by prostaglandins.     

Contribution of excitatory amino acids to morphine-induced metabolic alterations

Previous studies have indicated that excitatory amino acids are involved in the analgesic and addictive properties of morphine. However, their role in the morphine-induced alterations in glucose metabolism is not known. This study assessed the contribution of NMDA receptor activation to the morphine-induced hormonal and metabolic alterations in conscious unrestrained chronically catheterized rats. Whole body glucose flux was assessed with a primed constant intravenous infusion of [3-³H]glucose in rats pretreated with the NMDA-receptor antagonist MK-801 (0.25 mg/kg, intraarterial) or an equal volume (1.5 ml) of sterile saline (0.9% ) administered 15 min prior to i.c.v. injection of H₂O (Con; 5 μl) or morphine sulfate (80 μg). No significant alterations were noted in metabolic and hormonal parameters of H₂O injected rats. i.c.v. morphine increased the plasma glucose concentration (60%), hepatic glucose production (R_a; 60%) and whole body glucose utilization (R_d; 53%), but did not alter the glucose metabolic clearance rate (MCR). MK-801 alone resulted in transient hyperglycemia (25%), stimulation of glucose R_a (60%) and glucose R_d (53%), and a significant (30%) increase in MCR. MK-801 pretreatment blunted the morphine-induced hyperglycemia and the increased glucose R_a and R_d. Morphine increased the plasma concentration of epinephrine (4-fold), norepinephrine (2-fold) and corticosterone (67%); however, no alterations in plasma insulin and glucagon were detected. MK-801 pretreatment, blunted the morphine-induced increase in corticosterone and norepinephrine, and elicited a significant rise in insulin concentrations. These results indicate that activation of the NMDA receptors contributes to the morphine-induced hyperglycemia and hormonal alterations. Furthermore, this response appears partially mediated by activation of sympathetic outflow and suppression of insulin release, which is blunted by inhibition of NMDA receptors.     

Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate

Morphine-6-sulfate (M6S) and codeine-6-sulfate (C6S) are mu-selective opiates which have been isolated from brain. M6S is an effective analgesic, with a 30-fold greater potency than morphine in the mouse radiant heat tailflick assay and similar to the active morphine metabolite morphine-6beta-glucuronide (M6G). M6S analgesia is reversed by 3-methoxynaltrexone at low antagonist doses which are inactive against morphine, suggesting that M6S may be acting through the same mechanisms as M6G. Consistent with this possibility, antisense mapping of the MOR-1 clone revealed that M6S analgesia was lowered by probes targeting exon 2 and not by targeting exon 1, a sensitivity profile similar to that of M6G and not morphine. C6S also has analgesic activity at doses approximately 10-fold greater than M6S. However, its characterization was impeded by the appearance of seizures at doses below full analgesic activity. Thus, M6S is a potent analgesic with pharmacological properties similar to M6G. C6S has limited utility due to its high level of toxicity.     

Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats

The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5–25 μg: 5 μl) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 μg) increased MAP and HR by 32 ± 3 mmHg and 45 ± 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 μg; i.c.v.) blocked the effects of physostigmine (25 μg; i.c.v.). Also, the pre-administration of the M₂ muscarinic antagonist, methoctramine (25 μg; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M₁ muscarinic antagonist, pirenzepine (100 μg; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M₃ muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 μg; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25–30 mg/kg), physostigmine (25 μg; i.c.v.) evoked a decrease in MAP and HR of 13 ± 6 mmHg and 15 ± 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats arepossibly mediated through stimulation of central M₂ muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.     

Hormonal and metabolic effects of neuroglucopenia

We examined the role of central neuroglucopenia, induced by intracerebroventricular (i.c.v.) administration of 2-deoxyglucose (2-DG), on glucose and amino acid kinetics in conscious dogs. Group 1 received i.c.v. 2-DG at 2.5 mg·kg⁻¹·min⁻¹ for 15 min. Group 2 received an equal intravenous (i.v.) amount of 2-DG. In the i.c.v. group, plasma glucose levels rose from 106 ± 4 mg/dl to a peak of 204 ±12 mg/dl by 90 min. Blood lactate increased from 689 ± 1 to 2,812 ± 5 μ mol/1 and blood alanine did not change from basal (256 ± 41 μ mol/1). The rate of hepatic glucose production, determined isotopically, was increased 2-fold over basal (P < 0.01). Significant increases (P < 0.001) over basal were also noted in plasma epinephrine, norepinephrine, insulin, glucagon and cortisol. Leucine rate of appearance (Ra) showed a 30% decrease from basal to2.4 ± 0.05 μmol·kg⁻¹ ·min⁻¹ (P < 0.01). In group 2 plasma glucose levels were not altered but plasma cortisol and glucagon showed a modest transient increase above basal (P < 0.05). No significant changes were noted in amino acid kinetics. These findings suggest that periventricular neuroglucopenia, in the absence of peripheral glucose deprivation, is accompanied by hyperglycemia secondary to enhanced hepatic glucose production with decreased glucose utilization and by increased hepatic uptake of gluconeogenic precursors. These, however, were not accompanied by increased whole body proteolysis as was previously seen with generalized glucopenia resulting from insulin-induced hypoglycemia.     

Modification of morphine-induced locomotor activity by pertussis toxin: biochemical and behavioral studies in mice

The effect of pertussis toxin (PTX) on the locomotor-enhancing action of systemic and intracerebroventricular (i.c.v.) morphine was investigated in mice. Mice were i.c.v. injected with either PTX (0.25 and 0.5 μg) or saline as a control. The s.c. (5–20 mg/kg) and i.c.v. (7–30 nmol) administration of morphine produced a dose-related locomotor-enhancing action in control mice. The peak effect of morphine (30 nmol, i.c.v.)-induced hyperlocomotion was observed 90 min after the morphine injection. At the same time, morphine significantly increased dopamine (DA) metabolism in the limbic forebrain (nucleus accumbens and olfactory tubercle). Similarly, the selective μ-opioid receptor agonist[d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAGO, 4 nmol, i.c.v.) also significantly increased locomotor activity and DA metabolism in the limbic forebrain. Both morphine- and DAGO-induced hyperlocomotion and elevation of DA turnover were antagonized by pretreatment with the μ antagonist β-funaltrexamine (β-FNA). These results suggest that the locomotor-enhancing action of morphine results from the activation of central μ-opioid receptors, and that the activation of the mesolimbic DA system may be involved in the expression of morphine-induced hyperlocomotion in mice. Furthermore, pretreatment with PTX (0.5 μg, i.c.v., 6 days prior to the testing) significantly reduced hyperlocomotion and elevation of DA turnover in the limbic forebrain which had been induced by administrations of morphine (30 nmol, i.c.v.) and DAGO (4 nmol, i.c.v.). These findings suggest that the central PTX-sensitive GTP-binding protein (G-protein) mechanism may play an important role in opioids-induced locomotor-enhancing action. Furthermore, the activation of mesolimbic DA transmission by μ-opioid agonists may also be mediated by a PTX-sensitive G-protein mechanism in mice.     

Effects of central injection of glucose on thermogenesis in normal, VMH-lesioned and genetically obese rats

Intra-cerebroventricular (i.c.v.) injection of glucose (0.1–1.0 μmol) caused dose-dependent increases in resting oxygen consumption (V_O2) of conscious rats (maximum increase of 15.4 ± 2% at 0.5 μmol). These effects were significantly attenuated by peripheral (i.p.) pretreatment with the β-adrenoceptor antagonist propranolol, indicating the importance of the sympathetic nervous system (SNS) in the response. Plasma glucose concentrations were elevated (11%) 30 min after central injection of glucose, but intravenous glucose (0.5 μmol) did not affect resting V_O2. Animals which had been fasted for 12 h prior to V_O2 measurements exhibited reduced basal V_O2 values, but the nutritional state of the animal did not affect the metabolic response to central injections of glucose (0.5 μmol). Rats exhibiting genetic (fa/fa Zucker rats) and hypothalamic (VMH-lesioned) obesity showed similar thermogenic responses to centrally administered glucose, to their lean counterparts. These data suggest a dual action of central glucose in the regulation of energy balance, involving stimulation of energy expenditure in addition to its reported inhibition of energy intake. The defective diet-induced thermogenesis associated with VMH and genetic obesities does not appear to result from an inability to respond to changes in intracerebroventricular glucose concentrations.     

Cardiovascular effects of central administration of clonidine in conscious cats

The effects on arterial blood pressure and heart rate after an intracerebroventricular (i.c.v.) administration of clonidine were investigated using conscious normotensive cats. Injection of clonidine (5–10 μg; 5 μl; i.c.v.) elicited a decrease in mean arterial pressure (MAP) and heart rate (HR) in a dose-dependent manner. The highest dose of 10 μg of clonidine decreased MAP and HR by 39 ± 3 mmHg and 74 ± 5 b.p.m., respectively (n = 7). Pretreatment with yohimbine, the α₂-adrenoceptor antagonist (8 μg; 5 μl; i.c.v.) blocked the cardiovascular responses to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). Furthermore, preadministration of cimetidine (100 μg; 5 μl; i.c.v.), the H₂ histamine receptor antagonist with imidazoline receptor activating properties, prevented the decreases in MAP and HR to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). By contrast, pretreatment with the specific I₁ imidazoline receptor blocker, efaroxan (100–500 μg; 5 μl; i.c.v.), failed to inhibit the cardiovascular effects of an i.c.v. administration of 10 μg clonidine (n = 7). These results suggest that the effects of centrally administered clonidine on MAP and HR are probably not mediated through activation of the I₁ subtype of imidazoline receptors in conscious cats. However, the cardiovascular effects elicited by i.c.v. administration of clonidine appear to result from stimulation of central α₂-adrenergic or the H₂ histaminergic-like receptors.     

Intracerebroventricular injection of choline increases plasma oxytocin levels in conscious rats

In the present study, we examined the effect of intracerebroventricularly (i.c.v.) injected choline on both basal and stimulated oxytocin release in conscious rats. I.c.v. injection of choline (50–150 μg) caused time- and dose-dependent increases in plasma oxytocin levels under normal conditions. The increase in plasma oxytocin levels in response to i.c.v. choline (150 μg) was greatly attenuated by the pretreatment of rats with atropine (10 μg; i.c.v.), muscarinic receptor antagonist. Mecamylamine (50 μg; i.c.v.), a nicotinic receptor antagonist, failed to suppress the effect of 150 μg choline on oxytocin levels. Pretreatment of rats with 20 μg of hemicholinium-3 (HC-3), a specific inhibitor of choline uptake into nerve terminals, greatly attenuated the increase in plasma oxytocin levels in response to i.c.v. choline injection. Osmotic stimuli induced by either oral administration of 1 ml hypertonic saline (3 M) following 24-h dehydration of rats (type 1) or an i.c.v. injection of hypertonic saline (1 M) (type 2) increased plasma oxytocin levels significantly, but hemorrhage did not alter basal oxytocin concentrations. The i.c.v. injection of choline (50, 150 μg) under these conditions caused an additional and significant increase in plasma oxytocin concentrations beyond that produced by choline in normal conditions. These data show that choline can increase plasma oxytocin concentrations through the stimulation of central cholinergic muscarinic receptors by presynaptic mechanisms and enhance the stimulated oxytocin release.     

Selective immunotoxin-induced cholinergic deafferentation alters blood flow distribution in the cerebral cortex

Adult rats received intracerebroventricular (i.c.v.) administration of either phosphate buffer (PBS) or 192 IgG-saporin (Toxin), 3.6 μg rat⁻¹, a cholinergic immunotoxin. Six to eight weeks later, the animals received a continuous intravenous (i.v.) infusion of either physostigmine (4.2 μg kg⁻¹ min⁻¹) or saline, followed by measurement of cerebral cortical blood flow (CBF) with the autoradiographic Iodo-¹⁴C-antipyrine methodology in four groups of animals: Toxin i.c.v.+saline i.v. (n=9), Toxin i.c.v.+physostigmine i.v. (n=6), PBS i.c.v.+saline i.v. (n=6) and PBS i.c.v.+physostigmine i.v. (n=6). Choline acetyltransferase activity (ChAT) was assessed with Fonnum's method in samples of cortical tissue adjacent to the sites of CBF measurement. ChAT decreased in all regions of the Toxin groups when compared to PBS (% decrease: hippocampus=93%, neocortex=80–84%, entorhinal-piriform cortex=42%, amygdala=28%). CBF decreased globally in Toxin+SAL, most severely in posterior parietal and temporal regions (24–40% decrease from PBS+saline). Physostigmine enhanced CBF predominantly in these same areas both in PBS and Toxin animals although to a lesser extent in the latter. Our results demonstrate the importance of cholinergic mechanisms in the control of CBF. The similarity between the topography of CBF decrease following administration of the immunotoxin to that observed in Alzheimer's disease suggests that the CBF pattern observed in this disease may be the result of cholinergic deafferentation.     

Antinociceptive effect of ketanserin in mice: involvement of supraspinal 5-HT2 receptors in nociceptive transmission

The involvement of 5-HT₂ receptors in pain transmission was investigated in mice. Subcutaneous administration of the selective 5-HT₂ receptor antagonist ketanserin produced dose-dependent antinociception in the hot-plate and acetic acid-induced writhing tests withED₅₀ values (95% confidence limit) of 1.51 (1.13–1.89) and 0.62 (0.10–1.40) mg/kg, respectively, but was without any significant effect on the tail-flick test. Pretreatment with the catecholamine depletors 6-hydroxydopamine (2.5 μg, i.c.v.) orα-methyl-p-tyrosine (200 mg/kg, s.c.), or the serotonin synthesis inhibitorp-chlorophenylalanine methylester (200 mg/kg, s.c.), resulted in a significant decrease in the antinociceptive effect of ketanserin. Likewise, intrathecal (i.t.) administration of 1 μg/mouse of idazoxan (anα₂-antagonist), methysergide (mixed 5-HT₁, and 5-HT₂ antagonist) or ketanserin also reversed the antinociceptive effect of s.c. administered ketanserin. The results of this work indicate that 5-HT₂ receptors located supraspinally may inhibit descending nociceptive neurotransmission. In addition, these studies suggest that 5-HT₂ receptors located at the spinal level modulate nociception.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Potency ratios of morphine and morphine-6β-glucuronide analgesia elicited from the periaqueductal gray, locus coeruleus or rostral ventromedial medulla of rats

The present study examined whether morphine and morphine-6β-glucuronide (M6G) analgesia on the tail-flick and jump tests differed in potency in the periaqueductal gray, the locus coeruleus or the rostral ventromedial medulla. Morphine and M6G significantly and dose-dependently elicited analgesia on both nociceptive tests from each site. Site-specific differences were observed in the potency of M6G, but not morphine analgesia on both tests. Periaqueductal gray placements displayed analgesic ED_50s on the tail-flick (morphine: 2.1 μg, M6G: 0.2 μg) and jump (morphine: 2.2 μg, M6G: 0.4 μg) tests with respective potency ratios of 12.9 and 6.5. Locus coeruleus placements displayed analgesic ED_50s on the tail-flick (morphine: 1.7 μg, M6G: 0.1 μg) and jump (morphine: 3.4 μg, M6G: 0.2 μg) tests with respective potency ratios of 15.9 and 15.1. Rostral ventromedial placements displayed analgesic ED_50s on the tail-flick (morphine: 1.4 μg, M6G: 0.06 μg) and jump (morphine: 1.9 μg, M6G: 0.08 μg) tests with potency ratios of 21.9 on both tests. The greater analgesic sensitivity of the rostral ventromedial medulla to M6G may be due to either pharmacodynamic (splice variants of the MOR-1 gene) and/or pharmacokinetic (lipid solubility) factors.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Intracerebroventricular infusion of CRF increases extracellular concentrations of norepinephrine in the hippocampus and cortex as determined by in vivo voltammetry

Previous studies have indicated that intracerebroventricular (i.c.v.) infusions of corticotropin-releasing factor (CRF) activate locus coeruleus (LC) noradrenergic neurons and increase the metabolism and extracellular concentrations of norepinephrine (NE) in several brain regions, suggesting increased release. To examine the temporal aspects and mechanism of the presumed release of NE, CRF was infused i.c.v. and the oxidation current was recorded using carbon fiber voltammetric electrodes placed in rat hippocampus or cortex. The CRF (1 μg, i.c.v.) caused a significant increase of oxidation current with a delay of approximately 5 min, and a peak at approximately 35 min. Similar responses were observed in the medial prefrontal cortex. The hippocampal response was markedly attenuated when CRF was infused into rats pretreated with DSP-4 to deplete NE, suggesting that the observed changes in current resulted from oxidation of NE. The increase of NE-like current did not occur when 25 μg alpha-helical CRF_9–41 (ahCRF) was injected immediately before 1 μg CRF, suggesting that the response was mediated by cerebral CRF-receptors. Subcutaneous pretreatment with the ganglionic blocker, chlorisondamine, at a dose of 3 mg/kg had no effect on the voltammetric response to CRF, but a 6 mg/kg dose completely prevented the response. The β-adrenoceptor antagonists, S-propranolol (5 mg/kg), nadolol (5 and 10 mg/kg), and timolol (5 mg/kg) attenuated the NE response to i.c.v. CRF to varying degrees. When chlorisondamine (3 μg) or nadolol (5 μg) were given i.c.v. before the CRF, the hippocampal responses were not blocked. These results suggest peripheral actions of ganglionic and β-adrenergic blockers. We conclude that peripheral autonomic mechanisms, and probably both central and peripheral β-adrenoceptors, contribute to the increased secretion of hippocampal NE in response to i.c.v. CRF.     

The peripheral nociceptive actions of intravenously administered 5-HT in the rat requires dual activation of both 5-HT2 and 5-HT3 receptor subtypes

In 16-week-old Sprague-Dawley rats lightly anesthetized with pentobarbital, 5-HT (3–96 μg/kg, i.v.;n = 6) produced distinct pseudaffective responses and a dose-dependent (slope= 17.2 ± 6.8s/log₁₀dose) inhibition of the tail-flick (TF) reflex (ED₅₀ = 32.6 ± 9.2 μg/kg). In the same rats, a 1:1 combination of α-methyl 5-HT (a 5-HT₂ receptor selective agonist) and 2-methyl 5-HT (a 5-HT₃ receptor selective agonist) (3–192 μg/kg, i.v.), produced the same profile of pseudaffective responses and also resulted in a dose-dependent (slope= 34.0± 7.0s/log₂dose) inhibition of the TF reflex (ED₅₀ = 88.4 ± 20.5 μg/kg). In contrast, administration of α-methyl 5-HT (3–192 μg/kg, i.v.) or 2-methyl 5-HT (3–192 μg/kg, i.v.) alone did not produce any pseudaffective responses or any change in TF latency from baseline. In conscious 16-week-old male Sprague-Dawley rats, administration of 5-HT (48 μg/kg, i.v.;n = 5), or a 1:1 combination of α-methyl 5-HT and 2-methyl 5-HT (total dose= 120 μg/kg, i.v.;mn = 5), resulted in a passive avoidance behavior assessed in a step-down paradigm (slopes= 139.7 ± 58.2and154.9 ± 63.9s/trial, respectively), and the same profile of distinct pseudaffective responses exhibited by the lightly pentobarbital-anesthetized rats. However, administration of either α-methyl 5-HT (96 μg/kg, i.v.;n = 4) or 2-methyl 5-HT (96 μg/kg, i.v.;n = 4), while producing significant 5-HT receptor-mediated cardiovascular responses, produced a learned behavior not different from saline (0.25 ml, i.v.;n = 6) (slopes= 7.6 ± 2.5, 6.3 ± 1.8and7.4 ± 3.6s/trial, respectively). These results are consistent with the hypothesis that the peripheral nociceptive responses to i.v. 5-HT requires dual activation of 5-HT₂ and 5-HT₃ receptor subtypes.