In hypophysectomized rats, the incorporation of [3H] leucine into rat brain stem protein, measured 5 min after injection of the precursor into the diencephalon, was decreased. Chronic treatment of these rats with the N-terminal fragment of ACTH, ACTH1-10 increased the incorporation rate. Brain stem proteins were sequentially extracted with a hypotonic buffer, Triton X-100 and SDS. Analysis of these protein fractions on polyacrylamide gels revealed that hypophysectomy caused a general decrease in leucine incorporation into all protein species studied, whereas treatment of hypophysectomized rats with ACTH1-10 enhanced this incororation into all proteins. Superimposed on these general effects were minor differences found in two protein bands. Thus, removal of the pituitary and subsequent, chronic treatment of rats with ACTH1-10 interferes with overall protein metabolism rather than with certain protein species in particular.

Adrenocorticotropic Hormone↗

PubMed1974

Hypophysectomy, ACTH1-10 and in vitro protein synthesis in rat brain stem slices.

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Adrenocorticotropic Hormone↗

PubMedDate not supplied

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter.

Translocation of [3H]dopamine and binding of [3H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [3H]dopamine uptake was a one-component process; in contrast, [3H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [3H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.

Analysis of Variance↗

PubMedDate not supplied

Effects of stressful procedures as ether anesthesia and intracranial injections on amino acid incorporation into brain protein.

Ether anesthesia elevates plasma corticosterone levels considerably and interferes severely with the incorporation of centrally applied [3 H] leucine into brain protein. Only minor changes in leucine incorporation are observed in conscious rats with an implanted cannula in the third brain ventricle as compared to noncannulated controls. Injection through this cannula can be regarded as a minor stressful procedure comparable to subcutaneous injection both causing moderate elevations in plasma corticosterone levels. Injection through the cannula per se did not affect the leucine incorporation. In studies which require local application of the radioactive precursor, it is therefore recommended to avoid ether anesthesia and to use a preimplanted cannula in the brain ventricular system.

Animals↗

PubMedDate not supplied

Synaptosomal sodium channels in rat lines selected for alcohol-related behaviors.

Alcohol has been shown to inhibit veratridine- and batrachotoxin-stimulated sodium flux in rodent brain synaptosomes. In this study, binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) and uptake of [14C]guanidine in cerebrocortical synaptosomes were measured in naive rats belonging to lines with high or low acute sensitivity to ethanol (ANT, Alcohol Non-Tolerant, vs. AT, Alcohol Tolerant, lines) and to lines with high or low voluntary alcohol consumption (AA, Alko Alcohol, vs. ANA, Alko Non-Alcohol, lines). Our rat line pairs did not differ in the equilibrium binding characteristics of [3H]BTX-B nor in the properties of [14C]guanidine uptake, suggesting that the genetic selection has not modulated the genes or the expression of the genes associated with the voltage-sensitive sodium channels.

Alcohol Drinking↗

PubMedDate not supplied

Electroencephalographic characteristics of audiogenic seizures induced in metaphit-treated small rodents.

Adult male mice, rats, and guinea pigs were subjected to intense sound stimulation of an electric bell (100 dB, 12 kHz for 60 s) after a single intraperitoneal (i.p.) injection of metaphit (1-(1-(3 isothiocyanatophenyl)-cyclohexyl)piperidine) (50 mg/kg). When the animals were tested 24 h after administration of metaphit, audiogenic seizures were observed. None of the control saline-injected animals had convulsions. EEG recordings demonstrated the appearance of paroxysmal activity and spike-wave complexes in the trace from cortical and hippocampal electrodes, with frequency and amplitude increasing with time. Behaviorally, myoclonic jerks of facial muscles, ears, and neck appeared, but no correlation was noted between EEG and the motor phenomena. Auditory stimulation was necessary to elicit the full-blown sequence of seizure responses consisting of wild running followed by clonic and then tonic extension. At the time of seizures, repetitive high-amplitude spikes and waves appeared in the EEG, followed by profound EEG and behavioral depression. None of the animals died during or immediately after seizures. The seizure response to sound stimulation of mice, rats, and guinea pigs was phenomenologically similar, with minor differences in quantitative pattern of convulsive components, which suggests that all three animal species share the common property of extreme susceptibility to audiogenic stimulation caused by metaphit administration.

Acoustic Stimulation↗

PubMedDate not supplied

Metaphit-induced audiogenic seizures in mice: I. Pharmacologic characterization.

Metaphit [an analogue of phencyclidine (PCP) with an acylating isothiocyanate group] induced audiogenic clonic to clonic-tonic seizures in mice exposed to audio stimulation 24 h after metaphit administration. The incidence of seizures was reduced by treatment 30 min before audio stimulation with specific PCP-like compounds [5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801), and PCP itself], competitive N-methyl-D-aspartate antagonists 2-amino-5-phosphonopentanoic acid (AP-5 and NPC-12626), antiepileptic drugs [phenobarbital (PB), phenytoin (PHT)], and gamma-aminobutyric acid (GABA) agonists (muscimol and diazepam). In contrast, when given in conjunction with metaphit, most of these drugs were ineffective in protecting animals from audiogenic seizures 24 h later. Only compounds with long half-lives (t1/2) such as MK-801, PB, and PHT had a protective effect. High-performance liquid chromatography (HPLC) determination of [3H]MK-801 showed its long-term presence in the brain after intraperitoneal (i.p.) administration of [3H]MK-801. Audiogenic seizures observed 24 h after metaphit administration were potentiated by administration of the GABA antagonist picrotoxin 15 min before audio stimulation, and picrotoxin-induced spontaneous seizures were enhanced by pretreatment (24 h earlier) with a dose of metaphit that in itself did not produce spontaneous seizures at the time of the picrotoxin test. Similar observations were made with N-methyl D-aspartic acid (NMDA) instead of picrotoxin. Thus, an interplay exists between excitatory glutaminergic and inhibitory GABAergic circuitries in the metaphit seizure model.

Acoustic Stimulation↗

PubMedDate not supplied

Metaphit-induced audiogenic seizures in mice: II. Studies on N-methyl-D-aspartic acid, GABA, and sodium channel receptors and on the disposition of metaphit in the brain.

We previously demonstrated that metaphit (a phencyclidine analogue with an acylating isothiocyanate group) induces occurrence of audiogenic seizures in mice exposed to audio stimulation 24 h after metaphit administration. We have studied various receptor systems associated with excitatory and inhibitory networks: sites for competitive and noncompetitive antagonists of the N-methyl D-aspartic acid (NMDA) receptor complex, for [3H]muscimol on the gamma-aminobutyric acid (GABA) receptor complex, and for [3H]batrachotoxinin A20-alpha-benzoate on the voltage-dependent sodium channel. Mice were examined for neurochemical changes at 24 h after pretreatment with metaphit, when susceptibility to audiogenic seizures is greatest. Ex vivo receptor binding studies detected no changes; in vivo labeling of the phencyclidine site in the NMDA receptor complex was reduced by 20% in cortical and midbrain regions. A separate group of experiments was aimed at measuring brain levels of metaphit. One minute after retroorbital administration of [3H]metaphit at a dose sufficient to produce susceptibility to audiogenic seizures 24 h later, the brain level of [3H]metaphit (determined by high-performance liquid chromatography, HPLC) was 49 pmol/mg tissue; at 1, 4, and 24 h, the level was 12, 6, and 1.4 pmol/mg tissue or microM if metaphit was evenly distributed throughout the brain. Although the observed metaphit concentrations during the first 4 h are high enough to acylate receptors, no firm evidence for acylation was found for most of the examined receptors. Finally, the time course of the brain level of metaphit showing a continuous decrease is entirely different from that of development of the seizure susceptibility, which peaks at 18-24 h.

Acoustic Stimulation↗

PubMedDate not supplied

Facilitation of amygdala kindling development and kindled seizures by metaphit.

The effect of metaphit (a phencyclidine analogue with an acylating isothiocyanate) on kindling development and kindled seizures from amygdala was investigated in rats pretreated once with metaphit. Administration of a single dose of metaphit (10 or 20 mg/kg intraperitoneally i.p.) 4 h before the first electrical stimulation of the amygdala did not in itself induce seizures, but greatly facilitated development of behavioral seizures during kindling. This effect persisted throughout the whole process of electrical amygdala kindling without further dosing. In contrast, metaphit only transiently and modestly increased the growth of afterdischarge (AD) duration. In kindled rats, pretreatment with a single dose of metaphit (20 mg/kg) 8 h before the test stimulation reduced the threshold current required to elicit a stage 5 seizure and shortened the latency for bilateral forelimb clonus (BFC) without changing AD duration or BFC duration. The facilitation of kindling development and kindled seizures may be due to an excessive excitatory transmission by metaphit in the limbic seizure circuitry.

Amygdala↗