Ronald M. Norton

Evidence that the Acute Behavioral and Electrophysiological Effects of Bupropion (Wellbutrin®) Are Mediated by a Noradrenergic Mechanism

Bupropion (BW 323U66) has been considered a dopaminergic antidepressant based on its ability to inhibit the uptake of dopamine (DA) somewhat more selectively than it inhibits uptake of norepinephrine (NE) or serotonin (5-HT). This report describes new evidence that bupropion selectively inhibits firing rates of NE cells in the locus coeruleus (LC) at doses significantly lower than those that inhibit activity of midbrain DA cells or dorsal raphe 5-HT cells. The IC50 dose (13 mg/kg IP) for inhibition of LC firing produced plasma concentrations that were not significantly different from those generated by the ED50 in the Porsolt test (10 mg/kg IP). The fourfold higher dose needed to inhibit DA cell firing (IC50 = 42 mg/kg IP) was similar to the dose associated with locomotor stimulation in freely moving rats. Bupropion did not change the firing rates of 5-HT cells in the dorsal raphe nucleus at any dose. In both in vitro and in vivo tests, the metabolite 306U73 (hydroxy-bupropion), a weak inhibitor of NE uptake, was approximately equipotent to bupropion with regard to inhibition of LC cells. Another metabolite, 494U73, had no effect on LC firing rates over a wide range of doses. Because of species variation in metabolism, 306U73 was not detected in plasma of rats after IV doses of bupropion that inhibited LC firing. Only trace amounts of 306U73 were detected after bupropion dosing for the Porsolt test. Pretreatment with reserpine markedly depleted catecholamines and reduced (by 30-fold) the potency of bupropion to inhibit LC firing. The effects of clonidine, a direct acting α2 agonist, were not significantly changed by reserpine. Likewise, a reduction in the effect of bupropion on LC firing was observed in vitro after depletion of catecholamines with reserpine or tetrabenazine. These results suggest that bupropion preferentially affects NE neurons in locus coeruleus at doses that are active in animal antidepressant tests. The doses of bupropion required to inhibit DA cell firing were associated with inhibition of DA uptake and behavioral stimulation and were significantly higher than those selectively producing behavioral effects in animal antidepressant tests. The acute electrophysiological actions of bupropion on NE cells require a reserpine-sensitive store of NE and occur at doses having activity in antidepressant screening tests.

Neuroprotective Properties of the Novel Antiepileptic Lamotrigine in a Gerbil Model of Global Cerebral Ischemia

BACKGROUND AND PURPOSE Elevated glutamate levels are thought to be a primary cause of neuronal death after global cerebral ischemia. The purpose of this study was to investigate the potential neuroprotective effects of lamotrigine, a novel antiepileptic drug that inhibits the release of glutamate in vitro, with both behavioral and histological measures of global ischemia in gerbils. METHODS The common carotid arteries of gerbils were occluded for either 5, 10, or 15 minutes. Twenty-one days after reperfusion, gerbils were tested for impairments in a spatial memory task (Morris water maze). After water maze testing the animals were killed, and damage to hippocampal pyramidal cells was assessed. The effect of lamotrigine on the behavioral and histological outcome of either 5 or 15 minutes of global ischemia was evaluated. RESULTS Bilateral occlusion of the common carotid arteries for 5 minutes resulted in severe degeneration of hippocampal CA1 and CA2 pyramidal cells. Lamotrigine significantly prevented loss of hippocampal CA1 neurons when administered acutely (100 mg/kg PO) immediately after reperfusion or when administered in two equal doses of 30 or 50 mg/kg 2 hours before and immediately after reperfusion. Gerbils subjected to 5 minutes of ischemic insult were not impaired in their ability to solve a spatial memory task 21 days after cerebral ischemia. However, gerbils subjected to 10 and 15 minutes of carotid artery occlusion showed significant impairment in their ability to solve a water maze task. Lamotrigine significantly protected against the cognitive deficits associated with 15 minutes of cerebral ischemia. Histologically, increased durations of cerebral ischemia resulted in a progressive loss of CA1, CA2, and CA3 pyramidal cells. Lamotrigine completely protected gerbils exposed to 15 minutes of cerebral ischemia against CA3 cell loss and greatly reduced damage to the CA1 and CA2 cell tracts of the hippocampus. Lamotrigine also reduced the mortality associated with 15 minutes of ischemia. CONCLUSIONS Lamotrigine had neuroprotective effects in a gerbil model of global cerebral ischemia. Lamotrigine protected gerbils against behavioral deficits resulting from 15 minutes of carotid occlusion and also prevented histological damage resulting from 5 and 15 minutes of global cerebral ischemia.

The effects of 2,5-hexanedione on reproductive hormones and testicular enzyme activities in the F-344 rat

Abstract The chronic administration of 2,5-hexanedione (2,5-HD) to experimental animals can cause azoospermia and morphologic changes in central nervous system (CNS) areas related to visual and motor function. The present experiments were designed to determine the degree of CNS involvement in the testicular lesions seen after 2,5-HD administration. Additionally, activity measurements were made of some enzymes found in specific cell types in the testes. The 2,5-HD was administered as a 1% solution in the drinking water to adult male F-344 rats. Treated rats, pair fed controls, and ad libitum controls were killed after 1, 3, and 6 weeks of 2,5-HD treatment. The circulating levels of testosterone and the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), were not depressed at any time measured. After 6 weeks, the testes were azoospermic; this coincided with a rise in LH and FSH. After 3 weeks of 2,5-HD treatment, when the testes were morphologically normal, testicular activity of the Sertoli-cell-specific enzymes, β-glucuronidase and γ-glutamyl transpeptidase, were decreased. The testicular enzyme profile after 6 weeks was similar to that seen in the azoospermic, cryptorchid testis. Activities of hepatic β-glucuronidase and acid phosphatase were decreased at all time points. The data indicate that 2,5-HD does not act via the central gonadotropin control systems to induce azoospermia, and that demonstrable changes in Sertoli cell biochemistry occur prior to visible morphologic changes in the testis.

The effect of buthionine sulfoximine, an inhibitor of glutathione synthesis, on hepatic drug metabolism in the male mouse

Buthionine sulfoximine significantly reduced the hepatic non-protein sulfhydryl (NPSH) content of male B6C3F1 mice within 2 h after intraperitoneal (i.p.) injection. This treatment did not affect the activity of several hepatic microsomal and cytosolic enzymes responsible for xenobiotic metabolism. Pretreatment of mice with buthionine sulfoximine (2 mmol/kg) increased the hepatotoxicity of chloroform, but did not affect the hepatotoxicity of carbon tetrachloride. These findings suggest that buthionine sulfoximine can be a useful agent for studying the role of glutathione (GSH) in hepatic biotransformation of xenobiotics.

Inhibition of the acute toxicity of methyl chloride in male B6C3F1 mice by glutathione depletion

Previous data have demonstrated that methyl chloride (MeCl) is toxic to B6C3F1 mice under both acute and chronic exposure conditions, and that conjugation of MeCl with glutathione (GSH) is a key step in the metabolism of MeCl. This study examined the role of GSH in mediating the acute toxicity of MeCl to liver, kidney, and brain of male B6C3F1 mice. The lethal effects of a single 6-hr inhalation exposure of B6C3F1 males to 2500 ppm MeCl were completely prevented by pretreatment with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (4 mmol L-BSO/kg, ip 1.5 hr prior to MeCl exposure). GSH levels (measured as nonprotein sulfhydryl) in liver and kidney were depleted to 19 and 25% of control values, respectively, at the start of the exposure; the ratio of dead/exposed mice during the 18-hr postexposure declined from 14/15 mice to 0/10. Also, the LC50 for MeCl increased from 2200 to 3200 ppm in male mice pretreated with BSO. The hepatic toxicity of MeCl was detected by increased alanine aminotransferase (ALT) activities in serum 18 hr after a 6-hr exposure to 1500 ppm MeCl (2147 +/- 1327 IU/liter vs 46 +/- 6 in controls). Liver toxicity was inhibited when B6C3F1 males were depleted of GSH prior to MeCl exposure by BSO pretreatment (43 +/- 2), fasting (100 +/- 47), or injection of diethyl maleate (42 +/- 16). The effects of GSH depletion on MeCl toxicity to brain and kidney were determined in B6C3F1 males exposed to 1500 ppm MeCl 6 hr/day, 5 days/week for 2 weeks, with and without daily pretreatment with 2 mmol L-BSO/kg. This dose of BSO depleted hepatic and renal GSH by 28 and 60%, respectively, at the start of MeCl exposure. BSO-pretreated mice were protected from the central nervous system toxicity of MeCl, as assessed by microscopic examination of the granule cell layer of the cerebellum. BSO pretreatment also inhibited the renal toxicity of MeCl as measured by incorporation of [3H]thymidine ([3H]TdR) into renal DNA, an indicator of cell regeneration after cortical necrosis. [3H]TdR incorporation was 105 +/- 10,337 +/- 40, and 60 +/- 15 dpm/microgram DNA in nonexposed controls, MeCl, and MeCl + BSO treatment groups, respectively. These results indicate that GSH is an important component in the toxicity of MeCl to multiple organ systems in B6C3F1 mice. Reaction of MeCl with GSH appears to constitute a mechanism of toxication, contrary to the role usually proposed for GSH in detoxifying xenobiotics.

Altered lipid metabolism in 2,5-hexanedione-induced testicular atrophy and peripheral neuropathy in the rat

2,5-Hexanedione (HD) induces testicular atrophy and peripheral neuropathy in rats. Since altered lipid metabolism is frequently associated with these disease states, lipid metabolism was investigated in vitro in testes and sciatic nerves of rats fed 1% HD in the drinking water for 6 weeks. Testes from HD-treated rats were 30–60% smaller and weighed threefold less than testes from pair-fed control (PFC) rats. Compared to testes from PFC rats, testes from HD rats exhibited increased incorporation of [14C]acetate into phospholipids (344%), triacylglycerols (281%), and cholesteryl esters + hydrocarbons (246%) but decreased incorporation into free fatty acids (25%) and sterols + diacylglycerols (65%). The increased incorporation of [14C]acetate into phospholipids induced by HD reflected an approximate 300% increase into phosphatidyl choline, lysophosphatidyl choline, phosphatidyl serine + phosphatidyl inositol, and phosphatidyl ethanolamine and a disproportionate 800% increase into sphingomyelin. HD rats exhibited clinical signs of peripheral neuropathy, including everted and flat foot placement and hindlimb weakness; similar changes were not observed in PFC rats. In sciatic nerves, the incorporation of [14C]leucine was decreased into sterols + diacylglycerols (47%), digitoninprecipitable sterols (45%), and cholesteryl esters + hydrocarbons (40%) in HD compared to PFC rats; incorporation of [14C]leucine into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. In contrast to the testis and nerve, lipid metabolism in the liver was similar in HD and PFC rats. The concentrations of 2,5-hexanedione and 2,5-dimethylfuran, respectively, were 0.6 ± 0.3 and 6.5 ± 0.9 μg/g wet weight in the testes and 3.1 ± 0.4 and 3.0 ± 0.4 μg/g wet weight in the livers of HD rats. The data indicate that altered metabolism is associated with HD-induced testicular atrophy and distal axonopathy.

Effect of 2,5-hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat☆

Abstract Distal axonopathy induced by n-hexane and methyl n-butyl ketone has been attributed to a common metabolite, 2,5-hexanedione. Since altered lipid metabolism is frequently associated with neuropathy, the effects of 2,5-hexanedione on lipid biosynthesis from [1-14C]acetate in sciatic nerve and brain of rats given 1% 2,5-hexanedione in drinking water have been studied, in vitro. Clinical signs of neuropathy appeared after 6 weeks. Loss of body weight induced by 2,5-hexanedione was similar to that observed in pair-fed control rats. Compared to nerves from pair-fed controls, nerves from rats fed 2,5-hexanedione exhibited decreased incorporation of [1-14C]acetate into triacylglycerols (32%), total sterols + diacylglycerols (54%), digitonin-precipitable sterols (55%), squalene (55%), and ubiquinone (43%). Incorporation of [1-14C]acetate into phospholipids, fatty acids, and cholesteryl esters was similar in nerves of 2,5-hexanedione-treated rats and pair-fed controls. In brain, incorporation of [1-14C]acetate into lipids was similar in 2,5-hexanedione-treated and pair-fed control rats, except into the fatty acid fraction which was significantly decreased by 11%. The data support the hypothesis that lipid metabolism, in particular sterol metabolism, is altered in hexacarbon-induced distal axonopathy.

Preclinical neurochemical and electrophysiological profile of 1192U90, a potential antipsychotic

11192U90 was submitted to receptor binding and monoamine uptake assays. It bound potently at serotonin 5-HT2, dopaminergic D2, serotonin 5-HT1A, and adrenergic α1 and α2 receptors. It also bound to dopaminergic D1, serotonin 5-HT3, serotonin 5-HT4, and sigma sites, albeit with lower affinity. It was essentially inactive at 22 other sites, including those for cholinergic M1 and M2. It weakly inhibited uptake of 3H-norepinephrine, 3H-serotonin and 3H-dopamine. Acute doses of 1192U90 (5 and 20 mg/kg P.O.) increased whole-brain levels of dopamine metabolites but did not affect levels of norepinephrine, dopamine, and serotonin.Subcutaneous injection of 1192U90 (0.8 mg/kg/day) and clozapine (20 mg/kg/day) for 28 days preferentially decreased the number of spontaneously active dopamine cells in the ventral tegmental area (VTA) but not the substantia nigra (SN) of rats, as measured by population sampling. This outcome is characteristics of atypical antipsychotics like clozapine. Acute injections of 1192U90 reversed the rate-inhibiting effects of microiontophoretically applied dopamine and intravenously injected apomorphine and d-amphetamine on dopamine cell firing. Intravenous injection or iontophoretic application of 1192U90 or the 5-HT1A agonist (±)8-OH-DPAT inhibited the firing rates of dorsal raphe nucleus (DRN) neurons in rats, and the effects of both compounds were blocked by iontophoretically applied S(−) propranolol, a 5-HT1A antagonist. The results suggest that 1192U90 is a preferential dopamine D2 antagonist as well as a 5-HT1A agonist that may prove to be an atypical antipsychotic.

Inhibition of sciatic nerve sterologenesis in hexacarbon-induced distal axonopathy in the rat

Abstract Sterologenesis from [ 14 C]acetate and [ 3 H]mevalonolactone has been studied, in vitro , in sciatic nerves of rats fed 1% 2,5-hexanedione in the drinking water for 6 weeks. Nerves from rats given 2,5-hexanedione, in comparison with those from pair-fed control rats, exhibited reduced incorporation of [ 14 C]acetate, but not of [ 3 H]mevalonolactone, into the sterol precursor squalene (38%), C 30 sterols (53%), C 27 sterols (46%), and digitonin-precipitable sterols (41%). Sterologenesis was not inhibited in nerves obtained from untreated rats and incubated in vitro with 1 m m 2,5-hexanedione, nor in nerves of rats that had inhaled 1000 ppm n -hexane for 6 hr. Incorporation of [ 14 C]acetate into triacylglycerols of nerves from rats exposed to n -hexane was increased by 50%; [ 14 C]acetate incorporation into other lipid fractions was similar in n -hexane-exposed and control rats. The concentration of 2,5-hexanedione in nerves of rats receiving 1% 2,5-hexanedione in drinking water for 6 weeks or exposed to 1000 ppm n -hexane for 6 hr was determined by gas chromatography-mass spectrometry and found to be 0.8 ± 0.2 and 5.2 ± 0.6 μg/g tissue wet weight, respectively.

Inhibition of sterologenesis but not glycolysis in 2,5-hexanedione-induced distal axonopathy in the rat

If glycolysis is inhibited in distal axonopathy, there should be a concomitant inhibition of lipogenesis from glucose. To investigate this possibility, lipogenesis from [14C]glucose and [3H]acetate was studied in sciatic nerves incubated with iodoacetate, a known inhibitor of glycolysis, in sciatic nerves incubated with 2,5-hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2,5-hexanedione. Nerves incubated with 1.0 mm iodoacetate, in comparison with untreated nerves, exhibited decreased incorporation of [14C]glucose into sterols + diacylglycerols (33-fold), free fatty acids (14-fold), triacylglycerols (27-fold), and phospholipids (21-fold). In addition, these nerves exhibited decreased incorporation of [3H]acetate into sterols + diacylglycerols (30-fold), free fatty acids (2-fold), triacylglycerols (23-fold), and phospholipids (12-fold). In contrast, the incorporation of [14C]glucose into sterols + diacylglycerols, free fatty acids, and triacylglycerols was not affected by 1.0 mm 2,5-hexanedione. Compared to untreated nerves, nerves incubated with 1.0 mm 2,5-hexanedione exhibited a small decrease (15%) in the incorporation of [14C]glucose into phospholipids. Nerves from rats given 1% 2,5-hexanedione in the drinking water for 6 weeks, in comparison with those from pair-fed control rats, exhibited decreased (45%) incorporation of [14C]glucose and [3H]acetate into digitonin-precipitable sterols. Nerves from 2,5-hexanedione-treated and pair-fed control rats exhibited similar incorporation of [14C]glucose and [3C]acetate into free fatty acids, triacylglycerols, and phospholipids. The data indicate that while sterologenesis is inhibited in distal axonopathy, glycolysis is not.