David R. Schneider

Ethanol effects on harmaline-induced tremor and increase of cerebellar cyclic GMP

The spectra of pharmacological effects of ethanol and the benzodiazepine show a degree of overlap. Neurophysiological and neurochemical evidence indicates that both ethanol and benzodiazepines facilitate inhibitory neurotransmission mediated by GABA. Diazepam has been reported to inhibit both the tremor and mechanism of cerebellar cyclic GMP caused by harmaline by a neurotransmission in the cerebellum. Because of the similarities between ethanol and benzodiazepines, the effects of ethanol on harmaline-induced tremor and increase of cerebellar cyclic GMP were studied. Ethanol inhibited harmaline-induced tremor at doses as low as 0.1 g/kg. At this low dose, however, a dissociation between inhibition of harmaline tremor and inhibition of the harmaline-induced increase of cerebellar cyclic GMP was observed.

On the Use of Microwave Radiation Energy for Brain Tissue Fixation

Abstract: Focused microwave irradiation (MWR) is an increasingly accepted method of sacrifice of laboratory animals such as the mouse or rat. By fixing the brain within a fraction of a second with heat inactivation, the investigation of fast neurochemical events may be obtained. Even though the technique is widely utilized, its application is inconsistent. This report illustrates some of the requirements necessary for the proper application of MWR for the sacrifice of animals, particularly those related to the length of time MWR is applied and the efficiency with which generated MWR power is coupled to the brain tissue. Studies were performed on the mouse, using either a 2.5 KW or 6.3 KW generator with a focused, closed system waveguide at time intervals of 350 or 500 ms or 1.4 s. During each of these intervals MWR was varied so that core brain temperatures for all groups were held between 83 and 95°C. In contrast with reported studies that used full animal restraint, all animals were minimally restrained for less than 1 s before sacrifice. Tissue content of cyclic AMP, an index of neuronal activity grossly affected by subtle changes in the activity of adenylate cyclase and/or phosphodiesterases, was monitored. No differences in tissue cyclic AMP content in any of 12 brain regions were detected after MWR, either at 350 or 500 ms. A substantial increase in cyclic AMP content occurred in 8 of 12 brain regions examined following microwave irradiation for 1.4 s. On the basis of these experiments, accurate determination of cyclic AMP in rodent brain requires that the maximum time interval of MWR exposure should not exceed 500 ms.

Desglycyl-8-arginine vasopressin affects regional mouse brain cyclic AMP content

The content of cyclic AMP in various regions of the brains of young, adult male Swiss ICR mice, were altered at 60 minutes and 24 hours after a single intraperitoneal administration of desglycyl-8-arginine vasopressin (DGAVP). These time periods are well beyond the reported plasma half-life for this behaviorally active peptide. At 60 minutes, 5 brain regions showed significant elevations. The most notable response was in the septal area, (225%), followed by the thalamus (121%), the olfactory bulb, (144%), and hippocampus, (122%). After 24 hours, cyclic AMP content was significantly elevated in 10 brain regions. Maximal response was found in the hippocampus, (167%), the parietal cortex, (194%), and the occipital cortex, (190%). Other responsive regions included the olfactory bulb, (197%), medulla-pons, (153%), hypothalamus, (152%), septal area, (154%), striatum, (135%), and the frontal cortex, (133%). A substantial response to the injection of a placebo was noted in control animals. In each of the 12 regions examined, the content of cyclic AMP was significantly lower (p<0.01 or greater) at 24 hours compared to 1 hour after administration of the placebo. This is consistent with our previous findings of a late effect of the placebo injection. The regional cyclic AMP responses were qualitatively different from those observed after a single injection of ORG 2766, a tri-substituted ACTH (4–9) peptide fragment with different effects on memory. On the basis of the marked increase of cyclic AMP in the septal area, thalamus and olfactory bulb, at 60 minutes, and the prolonged response to DGAVP in multiple brain regions which were found at 24 hours, we conclude that DGAVP triggers serially related biochemical mechanisms which continue to affect the metabolism and function of these areas for long time periods after a single injection.

Microwave radiation energy: A probe for the neurobiologist

Abstract Microwave energy radiation is widely used as a method for rapidly sacrificing small laboratory animals so that measurements of endogenous levels of labile neurochemical substances can be assessed after various drug treatments or pharmacological maneuvers. Several factors are important to insure that microwave energy is efficiently coupled to the rodent brain, including: the frequency of microwave radiation, the size of the waveguide for the propagation of the energy, the tuning of the waveguide system, the placement of the animal at the point of maximum power within the waveguide, the amount of power which is delivered, the time during which the power is delivered, and whether the animal is restrained during the microwave protocol.

Cerebral blood flow after treatment with ORG-2766, a potent analog of ACTH 4–9

Regional cerebral blood flows (rCBF) were measured in conscious, male rats at 10, 30, 60 min and 24 hr after intravenous administration of a potent, behaviorally active analog of ACTH/MSH 4--9 (ORG-2766). Flows in the basal ganglia, hippocampus, septal area and frontal cortex were depressed significantly throughout the 60 min postinjection period. HYpothalamic and parietal flows were depressed at 10 and 30 min, but recovered by 60 min, whereas flow to the cerebellum was depressed between 30 and 60 min postinjection. The least changed and therefore relatively better perfused area throughout the first 60 min period was the occipital cortex. By contrast, at 24 hr, when perfusion of all brain regions had returned to near control levels, flow to the occipital cortex was elevated. During the first hour after treatment with either ORG-2766 or alphaMSH the patterns of regional circulation in the brain were qualitatively the same. The data suggest that ORG-2766 and, probably, alpha MSH trigger serially linked neurophysiologic changes in the brain lasting at least 24 hr, which organize the behavioral actions of this class of peptides on memory and attentional processes.

The effect of αMSH on β-adrenergic receptor mechanisms in the rat pineal

Abstract The effects of αMSH on beta adrenergic receptor response of rat pineal cells were studied in vitro. Responses measured were membrane hyperpolarization, measured with a micro-electrode, and cyclic AMP formation, measured by radio immunoassay. Normal resting membrane potential of pineal cells is approximately -40mV. Addition of NE produces a dose-dependent hyperpolarization of these cells. The addition of αMSH in vitro produces a very slight, but significant, depolarization, and markedly attenuates subsequent NE responses. αMSH has no effect on the cyclic AMP content of pineal glands, but again attenuates the action of NE in producing increased cellular cyclic AMP. These results suggest that αMSH may modulate pineal responsiveness.

Cyclic AMP in Female Mouse Brain is Altered by the Adrenocorticotropic Hormone(4–9) Analogue Organon 2766

Abstract: Cyclic AMP content was determined in 12 brain regions of young adult female mice at 30 min and at 24 h following an intraperitoneal injection of the tri‐substituted adrenocorticotropic hormone(4–9) [ACTH(4–9)] analogue Organon 2766 [ORG 2766]. Animals were killed by focused 3.5 kW microwave radiation applied for 350 ms. Unlike previously reported responses in male mice, at 30 min post‐injection there were no detectable differences in cyclic AMP content between the placebo and ORG 2766‐treated animals. By contrast, 24 h after injection, the content of cyclic AMP was changed significantly in 8 of the 12 brain regions examined: medulla‐pons, septal area, thalamus, hypothalamus, hippocampus, olfactory bulb, and parietal and occipital cortices. In most of the regions examined, differences consisted of 50% or greater reductions of tissue cyclic AMP content. The changes were unrelated to the estrus cycle of these animals

An analog of ACTH/MSH (4-9), ORG-2766, reduces cerebral uptake of morphine.

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4-9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.

Development and use of a nonrestraining waveguide chamber for rapid microwave radiation killing of the mouse and neonate rat

The use of microwave energy for rapid killing of small rodents such as the mouse or rat has become a standard pharmacologic technique since approximately 1975. This method allows investigation of rapidly modulated neurochemical indices, neuromodulatory substances, and some neurotransmitters to be determined at basal concentrations in brain regions and microregions. Previously described devices for use with microwave generators have relied on total body restraining holders in order to properly position rodents and neonates within a closed waveguide during microwave energy exposures. The present information describes two alternate chamber designs which do not require restraint of the rodent. A positioning device is described which must be used with the waveguide chambers. The animal chambers are designed to be used with 2450 MHz energy.

An automated microprocessor-controlled data collection device for use with the Technicon AutoAnalyzer system.

An automated microprocessor-controlled data collection system for use with a Technicon AutoAnalyzer in a research or clinical laboratory is described. The collection system can digitize an analog output from the AutoAnalyzer, perform mathematical operations on this digital data, detect and record peak-trough data, and perform these data acquisition and mathematical data processing operations in parallel. The data collection system can be interfaced to a variety of host computers for further data analysis. The accuracy of the data collection system was tested and compared to existing procedures using an analysis of bovine serum albumin protein. The correlation coefficient for the comparison of the results was 0.99998. Hence, the automated system is as accurate as previous methods, but is considerably faster, more efficient, and, in comparison to existing devices, less expensive.