David J. Jones

THE LEVELS OF LABILE INTERMEDIARY METABOLITES IN MOUSE BRAIN FOLLOWING RAPID TISSUE FIXATION WITH MICROWAVE IRRADIATION

The levels of several labile glycolytic and organic phosphate metabolites in mouse brain were determined following rapid inactivation with 2450 MHz microwave irradiation. The levels of ATP in mouse brain following a 0·25 s exposure in a 6 kW microwave oven was found to be 2·416 ± 0·061. Whole brain levels of 8 labile intermediary metabolites in 0·4 s irradiated samples were comparable to those reported using the previously‐described methods of freeze‐blowing or whole‐animal immersion. Analysis of these same metabolites in 4 gross areas of brain did not reveal any anoxic changes betwen superficial and deeper brain areas. The advantages of the mcrowave irradiation inactivation technique for regional brain studies of labile intermediary metabolites is discussed.

Regulation of the norepinephrine transporter by chronic administration of antidepressants

BACKGROUND Downregulation of serotonin transporter was observed previously after chronic treatment with selective serotonin reuptake inhibitors (SSRIs) but not selective norepinephrine reuptake inhibitors (NRIs). This study investigated if chronic treatment of rats with selective NRIs or SSRIs also affected the norepinephrine transporter (NET). METHODS Rats were treated for 3 to 6 weeks by osmotic minipumps with either the selective NRIs, desipramine, or the SSRI paroxetine. RESULTS [(3)H]nisoxetine binding sites as well as [(3)H]norepinephrine uptake were decreased in hippocampus and cortex after treatment with desipramine. By contrast, paroxetine-treated rats showed no alteration in either [(3)H]nisoxetine binding or [(3)H]norepinephrine uptake. NET messenger RNA levels in the locus coeruleus were unchanged by desipramine treatment. CONCLUSIONS These results demonstrate that the marked decrease in NET density 1) is not a consequence of a decrease in gene expression; 2) was caused only by a selective NRI; and 3) was associated with a parallel decrease in norepinephrine uptake.

Rate of inactivation of adenyl cyclase and phosphodiesterase: Determinants of brain cyclic AMP

Abstract In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.

Adrenergic receptors in rat spinal cord

Radioligand binding assays were used to demonstrate the presence of alpha 1, alpha 2 and beta receptors in rat spinal cord. Specific and saturable binding was exhibited for [3H]-WB 4101 (alpha 1), [3H]-aminoclonidine (alpha 2) and [3H]-dihydroalprenolol (beta). Binding was of high affinity and the total number of binding sites (Bmax) were: alpha 1, 66.5 +/- 1.0 fmol/mg protein; alpha 2, 20.0 +/- 0.6 fmol/mg protein; beta, 10.2 +/- 0.3 fmol/mg protein. The data confirms the existence of adrenergic receptors in spinal cord and provides further evidence of the role of catecholaminergic neurons in regulating spinal cord physiology.

Anticonvulsant Drug Actions on In Vitro and In Vivo Levels of Cyclic AMP in the Mouse Brain

Summary: Injections of pentylenetetrazol to mice produced a rise in steady state levels of cyclic AMP in the cerebrum and cerebellum subsequent to sacrifice by focused microwave irradiation. One hour pretreatment with anticonvulsant drugs–phenytoin, phenobarbital, carbamazepine, clonazepam, and diazepam–prevented this pentylenetetrazol‐induced rise in cyclic AMP. Anticonvulsant agents which alone depressed steady state levels of cyclic AMP were phenytoin and carbamazepine in the cerebral cortex and carbamazepine in the cerebellum while elevations in the cyclic nucleotide were evoked by clonazepam in the cerebrum and cerebellum or diazepam and phenytoin in the cerebellum. Under in vitro conditions, in which incubated tissue slices of mouse cerebral cortex were used, the stimulation of cyclic AMP by norepinephrine was blocked by carbamazepine > phenobarbital > phenytoin. Inhibition of adenosine‐induced accumulation of cyclic AMP was observed with phenobarbital > carbamazepine > phenytoin. Clonazepam enhanced this response to adenosine. Ouabain stimulation of cyclic AMP was prevented by carbamazepine, phenytoin > clonazepam > phenobarbital > diazepam. Only the highest concentrations (0.5 MM) of phenytoin, phenobarbital, and carbamazepine reduced the action of KC1 on cyclic AMP elevation. Clonazepam and diazepam elevated the basal levels of the cyclic nucleotide.

Selective decreases in amphetamine self-administration and regulation of dopamine transporter function in diabetic rats.

The dopamine transporter (DAT) regulates extracellular dopamine DA levels and is an important site of action for amphetamine and cocaine. Amphetamine and cocaine increase extracellular levels of DA by acting on the DAT; thus, variations in DAT binding sites or activity might influence the action of some drugs of abuse. It was hypothesized that streptozotocin-induced diabetes decreases amphetamine self-administration and that this behavioral change is accompanied by changes in DAT function. Separate groups of male rats responded to receive either amphetamine (0.03 mg/kg/infusion), cocaine (0.25 mg/kg/infusion), or food before and for 7 days after receiving streptozotocin. Rats were sacrificed and [3H]DA uptake and [3H]WIN 35,428 binding were measured in the striatum. In a second study, rats could self-administer one of several different doses of amphetamine (0.01–0.178 mg/kg/infusion) before and after receiving streptozotocin. In streptozotocin-treated rats, a marked decrease in staining for insulin in pancreatic sections was paralleled by a more than doubling in blood glucose levels. Streptozotocin significantly decreased the number of amphetamine infusions without changing the number of cocaine infusions or food pellets received. Streptozotocin increased DA uptake (Vmax) 1.6- or 2.4-fold in rats that responded for food or amphetamine and increased 3-fold the Km for DA only in rats that responded for food; however, [3H]WIN 35,428 binding was not changed in any rat. In the second study, streptozotocin only decreased amphetamine self-administration thereby supporting the view that streptozotocin does not simply decrease the potency of amphetamine. These results demonstrate a selective decrease in amphetamine self-administration in diabetic rats that was associated with increased DAT function in the striatum. Collectively, these studies suggest that insulin pathways in the brain may play an important role in regulating DAT activity and amphetamine action.

Characterization and measurement of [125I]iodopindolol binding in individual rat pineal glands: existence of a 24-h rhythm in β-adrenergic receptor density

A simple procedure has been developed that permits measurement of beta-receptors in membrane preparations from individual rat pineal glands using [125I]iodopindolol ([125I]PIN). [125I]PIN binding to pineal membranes was stereospecific and saturable. Scatchard analysis of saturation isotherms yielded a Kd of 147.3 +/- 54 pM and a Bmax of 11.1 +/- 1.5 fmol/pineal gland. Binding was linear suggesting that [125I]PIN binds to a single population of pineal beta-adrenergic receptors. This procedure was used to evaluate 24-h variations in density of pineal [125I]PIN binding sites in male rats maintained in a 14:10 h light:dark cycle. Binding remained uniformly low during the daytime, increased slightly prior to lights off and peaked after 6 h of darkness decreasing abruptly 2 h later, before lights on. In animals maintained in light at night, the number of binding sites also increased, but did not exhibit the darkness-related decrease. The results demonstrate that beta-adrenergic receptors defined via [125I]PIN binding can be measured in tissue samples equivalent to less than one pineal gland. Moreover, the technique can be used in studies concerning the noradrenergic regulation of pineal function.

Fluoxetine-induced inhibition of synaptosomal [3H] 5-HT release: Possible CA2+-channel inhibition

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K(+)-induced [3H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations greater than 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K+ used to depolarize the synaptosomes and the concentration of external Ca2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [3H]5-HT release induced by the Ca(2+)-ionophore A 23187 or Ca(2+)-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K(+)-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca2+ channels and Ca2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [3H]nitrendipine to the dihydropyridine-sensitive L-type Ca2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K(+)-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K(+)-induced increase in intracellular free Ca2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K(+)-induced [3H]5-HT release by antagonizing voltage-dependent Ca2+ entry into nerve terminals.

Transfer of methohexital across the perfused human placenta

STUDY OBJECTIVES To evaluate the transfer properties of methohexital and the influence of protein binding using the in vitro human placental perfusion model. DESIGN Fresh term human placentae from healthy parturients were perfused bidirectionally via a cannulated fetal chorionic artery and vein and needles placed into the maternal intervillous space. Maternal-to-fetal (M-->F) and fetal-to-maternal (F-->M) transfer and ultimate distribution of methohexital was investigated using a closed (recirculating) placental perfusion model. SETTING Obstetric anesthesia laboratories of two university medical centers. PATIENTS No patient participation occurred as placentae were obtained after delivery. INTERVENTION M-->F and F-->M transfer of methohexital was compared in vitro in perfusates with equal protein concentrations (2 g/100 mL in both perfusates) or albumin-simulated physiologic protein binding concentrations (maternal 8 g/100 mL; fetal 4 g/100 mL). MEASUREMENTS AND MAIN RESULTS Data obtained consisted of measurements of methohexital and antipyrine concentrations by high-performance liquid chromatography. Glucose and lactate concentrations and perfusate loss were measured to assess placental viability. Methohexital protein binding was assessed at 2, 4, and 8 g/100 mL of albumin by equilibrium dialysis. The transfer index of 0.83 +/- 0.11 for the M-->F perfusions was significantly greater (p < or = 0.05) than in the F-->M direction (0.61 +/- 0.04) when albumin concentration was equal in both perfusates. This transfer asymmetry disappeared when albumin concentrations simulating maternal (8 g/100 mL) versus fetal (4 g/100 mL) protein concentrations in the perfusate were used (M-->F 0.87 +/- 0.12 and F-->M 0.95 +/- 0.11). CONCLUSION Methohexital readily crosses the placenta in both directions. Protein binding has significant effects on the degree of transfer of methohexital at any time when compared with antipyrine and its ultimate fetal/maternal distribution.

LEVELS OF CYCLIC NUCLEOTIDES IN MOUSE REGIONAL BRAIN FOLLOWING 300 ms MICROWAVE INACTIVATION

Abstract— The uniformity and speed of inactivation of mouse brain adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterase were measured after 6 kW microwave irradiation (MWR). Inactivation of enzymes was uniform throughout the brain during heating and 100% loss of activity was evident after 300 ms. MWR. For comparison of effects of inactivation times on levels of cyclic nucleotides measured in regional brain areas, cyclic AMP and cyclic GMP were estimated after 1.5 kW MWR requiring 4 s of heating and 6 kW MWR requiring 300 ms. Except for corpus striatum, uniformly lower levels of cyclic AMP were measured following 300 ms vs. 4s MWR. There was no change in cyclic GMP levels in regional brain areas after 4s vs. 300 ms MWR. Cyclic AMP and cyclic GMP were measured from the same regional brain tissue samples after 300 ms and ratios calculated. The finding of much lower cyclic AMP:cyclic GMP ratios than had previously been reported suggests that slow inactivation times provide for the measurement of regional brain cyclic nucleotide values which are not consistent with the in‐vivo state.