Michiko Okamoto

Destruction of mammalian motor nerve terminals by black widow spider venom

Black widow spider venom selectively poisons motor nerve endings. A progressive and irreversible failure of neuromuscular transmission occurs in the cat. Electron microscopy of the poisoned nerve-muscle junction shows a sequence of motor nerve ending damage that culminates in disruption of the prejunctional membrane and loss of all organelles, including synaptic vesicles. The postjunctional membrane was morphologically unaffected. After complete poisoning, the contractile response to exogenous acetylcholine was severely impaired, an indication that the prejunctional site is chiefly involved in the contractile response produced by exogenous acetylcholine and that the pre- and postjunctional effects of acetylcholine were separated.

Determination of diazepam and its pharmacologically active metabolites in blood by bond elut™ column extraction and reversed-phase high-performance liquid chromatography

A rapid and quantitative analytical micro method for the determination of diazepam and its major pharmacologically active metabolites utilizing high-performance liquid chromatography (HPLC) is reported. The drug and its metabolites were extracted from 50-100 microliter samples of whole blood, serum or plasma using Bond Elut C 18 column and quantitated by high-performance liquid chromatography, using Technician Fast-LC-C-8 (RP 5 micrometers) bonded column and a mobile phase consisting of 53% methanol, 1% acetonitrile in KH2PO4 buffer and 10 microliters/l triethylamine. Methyl nitrazepam and medazepam were used as internal and external standards respectively. The extraction and recovery of diazepam and its major pharmacologically active metabolites, i.e., 3-hydroxydiazepam, desmethyldiazepam and oxazepam from blood were higher than 88% for all compounds. The minimum detection range of each compound was approximately 2.5 ng per 100-microliter sample. This micro method of simultaneous quantitation of diazepam and its major pharmacologically active metabolites provides a valuable technique for the study of diazepam pharmacokinetics in a small animal model without disturbance of normal hemodynamics from excess blood loss, as well as in clinical evaluation of pediatric patients.

Procaine Action: Antagonism by Adenosine Triphosphate and Other Nucleotides

Appropriate concentrations of adenosine tri-, di-, and monophosphate antagonize the depressant action of procaine on isolated nerve. Some calcium ion is required in the external solution in order for these nucleotides to produce their maximum effects. When applied either to normal or sodium deficient nerves, the nucleotides do not increase action-potential amplitude.

Withdrawal characteristics following chronic pentobarbital dosing in cat

Abstract Experiments were performed to provide a quantitative description of the barbiturate withdrawal syndrome. Physical dependence was produced in 63 cats by ‘maximally tolerable’ dosing with sodium pentobarbital. After 5 weeks of chronic treatment each animal was placed in an activity monitoring cage and observed closely for signs of barbiturate abstinence. Electroencephalographic monitoring of sleep—wake cycles was performed in 5 of these cats. Most withdrawal signs appeared on 12–18 h and rapidly intensified. These included signs of neural hyper-excitability that involved motor, autonomic and behavioral function. Data are presented describing the incidence, severity and time course of many withdrawal signs. 26 animals (41%) died during abstinence, usually during or immediately following grand mal type convulsions. The importance of quantitating withdrawal phenomena is discussed with respect to investigation of the requirements for physical dependency production, comparison of different drug dependencies, and pre-clinical evaluation of potential treatments of sedative-hypnotic dependence.

Neuromuscular pharmacology in rat neonates: development of responsiveness to prototypic blocking and reversal drugs.

The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine. The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incomplete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc.

Effect of chronic pentobarbital treatment on blood-cerebrospinal fluid kinetics☆

Cats were made tolerant and physically dependent by twice daily equieffective anesthetic dosing with sodium pentobarbital intragastrically for 5 weeks. Serial blood and CSF samples were simultaneously collected on day 1 and day 35 and analyzed for pentobarbital by a gas chromatographic method. Pentobarbital penetration into CSF was rapid and unchanged by chronic treatment. The concentration in blood and CSF increased significantly with chronic treatment; however, the relative CSF/blood concentration ratios were unchanged.

Histopathological changes of rat brain after direct injection of Hb-vesicles (artificial oxygen carriers) and neurological impact in an intracerebral hemorrhage model

For use as artificial oxygen carriers during transfusion, the safety and efficacy of Hb-vesicles (HbV, 250 nm), have been investigated extensively. Nevertheless, their neurotoxicity remains unknown. We explored potential adverse effects of HbV in the brain using a rat intracerebral hemorrhage model. Male Wistar rats were anesthetized with sevoflurane and fixed on a stereotaxic frame. Then HbV or homologous RBC suspension ([Hb] = 8.6 g/dL, 20 microL) was injected into the right caudate nucleus. All animals survived, gained weight, and maintained their well-being until the time of sacrifice; except during the first few days after surgery. However, both groups showed slight weakness in hind leg retraction, occasional ataxia/gait, and piloerection. Neutrophils accumulated at the onset of injury in perihematomal tissues in both groups at 1st day, but had disappeared by 3 days. Infiltration of small HbV in the perihematomal tissue was prominent at 1st day; phagocytized HbV were detected in macrophages. Hemeoxygenase-1 and hemosiderin deposition appeared after 3 days, reflecting the degradation of both HbV and RBC. The HbVs were detectable even after 28 days in the HbV group, but no residual RBCs were detected in the RBC group. Both groups showed proliferation of astrocytes, named gliosis, for tissue reconstruction after 3 days. This study revealed no notable differences in adverse effects between the intra-cerebral injection of HbV and the RBC control on behavioral functions and brain tissue responses.

The interactions of ouabain with post-tetanic and facilitatory drug potentiations at cat soleus neuromuscular junctions in vivo

Cat soleus motor nerve terminals, after high frequency conditioning, generate a post-tetanic repetition (PTR) which leads to a post-tetanic (PTP) of the muscle response. This property enables quantitative assessment of enhancement or depression of this nerve terminal excitability in vivo. The present study focuses on ionic mechanisms underlying the PTRs produced in this neuromuscular system either by high frequency stimulation or edrophonium. Ouabain was used as a specific probe for inhibition of Na+−K+ ATPase and its known consequences on Na+ and Ca2+ translocation. Ouabain pretreatment doubled the duration over which single stimuli, following either high frequency or edrophonium conditioning produced PTR. Ouabain in the doses used had no effectper se but as a function of dose augmented the frequency dependent responses. This pointed to Na+ loading of nerve terminals via high frequency stimulation plus ouabain inhibition of Na+−K+ ATPase. Ouabain potentiation of PTR responses evidently depends on exchange of intra-terminal sodium for external calcium. Thus, calcium entry blockers, Mn2+, and Co2+ suppressed or abolished the potentiations both before and after ouabain. Diphenylhydantoin, a Na+ and Ca2+ blocker, acted similarly. The effects of stimulation frequency, ouabain and the sequence of events leading to PTR in the soleus neuromuscular system appeared in general no different from those derived from the many in vitro microphysiologic studies of this phenomenon. Thus, EPPs were augmented and prolonged. It was concluded that intracellular Ca2+ is critical for regulating the stability of systems in which repetitive firing is both a normal and abnormal function.

Further observations on subacute denervation and mammalian motor nerve terminal function

a In a previous study of neuromuscular function in the cat soleus, a slow nerve-muscle system, it was learned that 48 hours after nerve section (subacute denervation), disorders in motor nerve terminal function precede transmission loss.’ Companion studies explored the pharmacology of the 48 hour denervated junction and revealed that functional integrity of the unmyelinated terminals is essential for the a c t i o n of the neostigminelike facilitatory drugs.* This finding confirmed a number of earlier studies showing that the mammalian motor nerve terminals are the site of this facilitatory drug a ~ t i o n . ~ Because of the known functional differences between motor nerves t o tonic and phasic musc l e ~ , ~ ~ it was decided t o ascertain the physiologic and pharmacologic losses that might be exhibited by a phasic system 4 8 hours after nerve section. For this purpose, a gastrocnemius nerve-muscle preparation of the cat was chosen. The results established that the same pattern of losses occurs in this preparation as in the soleus. However, certain quantitative differences were noted.

Effect of chronic pre- and post-natal low-dose ethanol exposure on brain enolase isoenzyme activities

Sprague-Dawley dams were treated with 3 v/v % ethanol in liquid diet from the 8th day gestation through 3 weeks nursing period. Offsprings, ages 1, 2 and 3 weeks, and their dams were studied. Brain weights and their total proteins were not affected by this ethanol treatment. Total enolase activity/mg protein and its isoenzymes, i.e. non-neuron-specific, hybrid and neuron-specific, (mumol/min/mg protein) in 100,000 g supernate, were significantly lower in the treated. Additionally, enolase isoenzyme transformation was delayed.