John M. Carney

An unanesthetized-gerbil model of cerebral ischemia-induced behavioral changes

A surgical procedure has been developed to study the effects of cerebral ischemia in the unanesthetized Mongolian gerbil. The methodology is based upon the surgical isolation and instrumentation of both common carotid arteries. A loop of dental floss is placed around each carotid artery and passed through double lumen catheter material; this allows for later occlusion of the carotid arteries and their release in unanesthetized subjects. Functional changes following transient carotid artery occlusion are readily demonstrated by the occurrence of altered spontaneous locomotor activity at various times postischemia. This model should be useful in the evaluation of potential therapeutic agents in the treatment of cerebral ischemia.

Effects of caffeine, theophylline and theobromine on scheduled controlled responding in rats.

1 Rats were trained to respond under a variable interval 30 s (VI 30) schedule of food reinforcement. 2 Caffeine (0.32–32 mg/kg), theophylline (1.0–56 mg/kg) and theobromine (10–320 mg/kg) in general produced dose‐related decreases in operant responding. At relatively low doses, caffeine (1.0 mg/kg) and theophylline (3.2 mg/kg) produced slight but nonsignificant increases in VI 30 responding. 3 The rank order of potency for producing decreases in responding was caffeine > theophylline > theobromine. 4 Daily caffeine injections (32 mg/kg, i.p.) resulted in the development of caffeine tolerance. This tolerance was characterized by a 6 fold shift to the right in the caffeine dose‐effect curve Saline substitution for the 32.0 mg/kg caffeine maintenance dose resulted in a substantial decrease in responding.

Systematic comparison of apomorphine-induced behavioral changes in two mouse strains with inherited differences in brain dopamine receptors

Dosage and time dependencies of apomorphine-induced changes in stereotyped behaviors (climbing, gnawing and sniffing), locomotor activity and rearing activity were compared in young adult male mice of two inbred strains, DBA/2 and C57BL/6. These two strains are known to differ in their genetically specified brain dopamine receptor number. Apomorphine administered intraperitoneally at dosages of 0.5-20 mg/kg failed to induced stereotyped climbing in DBA/2 at any of the doses tested but markedly increased climbing in C57BL/6 at higher dosages. Apomorphine-induced stereotyped gnawing occurred in both strains at higher dosages although the latency was shorter and maximal effect greater in C57BL/6. Stereotyped sniffing was induced in both strains to a comparable degree at doses greater than or equal to 2.0 mg/kg, and the duration of this stereotypy was indistinguishable between strains. Locomotor activity was inhibited maximally in DBA/2 at an apomorphine dosage of 2 mg/kg and was inhibited to a greater extent than was C57BL/6. Rearing was inhibited in both strains by doses of apomorphine greater than or equal to 0.5 mg/kg; however the duration of the effect was considerably greater in DBA/2 than in C57BL/6. These data suggest that genetically determined differences in central dopamine receptors may have profound and selective effects on behaviors mediated by dopamine pathways; that complex behavioral patterns, e.g., apomorphine-induced stereotypy, may be dissected in to individual components by identifying neuropharmaco genetic differences between strains; that marked strain-specific, inherited differences in dopamine agonist-induced behavioral changes do occur among inbred, non-mutant mouse strains and that their occurrence in other mammalian species including man should be considered.

Caffeine discrimination in the rat.

Rats were trained to discriminate 32 mg/kg caffeine from saline in a two-lever appetitive task. Across a range of caffeine test doses (1-32 mg/kg) rats showed a dose related generalization to the training cue. At intermediate caffeine dose levels, caffeine appeared to produce a more potent cue on tests following saline-training days than after drug-training days. Several psychomotor stimulants (d-amphetamine, methylphenidate, nicotine and TRH) failed to generalize to the caffeine cue. In contrast, theophylline did generalize to caffeine at a dose roughly twice that of the caffeine training dose.

Inherent differences in sensitivity to methylxanthines among inbred mice

The behavioral effects of caffeine, theophylline, paraxanthine, and theobromine on locomotor activity were analyzed in four strains of inbred mice that were previously shown to differ in their acute toxic responses to caffeine administered at high dosages. Dose response curves for the effects of caffeine, theophylline, paraxanthine and theobromine on locomotor activity were established in CBA/J, C57BL/6J, DBA/2J and SWR/J strains of inbred mice. Paraxanthine was the maximally effective methylxanthine in the CBA/J, DBA/2J and SWR/J strains, while in the C57BL/6J strain, caffeine was the maximally effective methylxanthine. Theophylline failed to stimulate locomotor activity in the C57BL/6J strain and theobromine failed to stimulate activity in all of the strains tested. Decreases in locomotor activity were seen at the 100 mg/kg dose of caffeine in the C57BL/6J mice and at the 100 mg/kg dose of theophylline in the C57BL/6J, DBA/2J and SWR/J strains. Theobromine produced decreases in locomotor activity in the C57BL/6J, DBA/2J and SWR/J strains of mice. In contrast to the other methylxanthines, paraxanthine failed to decrease activity across the range of doses tested (1.0-150 mg/kg). These data suggest that the methylxanthines have genetically specified multiple modes of action upon locomotor activity and that the use of genetically distinct strains of mice may have important value in the neurochemical and pharmacological dissection of methylxanthine-induced behavioral effects.

Controlling behavior experiments with BASIC on 6502-based microcomputers

A combination of high-level BASIC and transparent machine language routines forms an inexpensive (about

Discriminative stimulus properties of methylxanthines and their metabolites in rats

800), powerful, and easy-to-use microcomputer system for behavior research, based on the AIM-65. The interrupt-driven machine language routines keep track of real time, poll inputs, and are not typically modified for different experiments. Expandable machine language listings that support eight input lines and eight output lines on the AIM-65 are presented. The user-written BASIC program controls experiments and records data. BASIC programs for simple reinforcement schedules are used as examples. The software is readily transferred to other 6602-based microcomputers with BASIC.

Schedule-controlled behavior as an index of the development and loss of ethanol tolerance in the rat

Rats were trained to discriminate methylxanthines from saline under a two-lever concurrent variable ratio schedule of reinforcement. One group was trained to discriminate between saline and 32 mg/kg caffeine. A second group was trained to discriminate between 56 mg/kg theophylline and saline. Rats reliably discriminated between saline and the training methylxanthine, displaying graded generalization curves across training-drug doses. Caffeine-trained rats demonstrated caffeine-appropriate responding when tested with theophylline, paraxanthine, and 3-methylxanthine. Theobromine failed to generalize to the caffeine cue at test doses up to 75 g/kg. In contrast to the caffeine group, rats trained to discriminate theophylline from saline were less sensitive (higher ED50) to the effects of caffeine and paraxanthine test doses. Only partial generalization to the theophylline cue occurred at paraxanthine doses up to 100 mg/kg. Based upon these data, it is suggested that the underlying substrate(s) for the caffeine cue is in some respects different from the substrate(s) for the theophylline cue.

Increased in vitro lipid peroxidation of gerbil cerebral cortex as compared with rat

Twelve male Sprague-Dawley rats, following training on one of two food-motivated operant schedules (Fixed-Ratio 30 or Variable Interval 30 s), were exposed to an escalating regimen of daily ethanol (1.125–3.0 g/kg, IP) administration. This increasing dose regimen continued until the maximally tolerable dose for each subject was reached. Tolerance was then monitored for approximately 6 months by periodic ethanol challenge doses (1.5 g/kg). Dose-effect curves (DECs) were obtained prior to chronic ethanol (DEC1), immediately after ethanol tolerance development (DEC2), and 6 months (DEC3) following termination of ethanol exposure. At DEC1, ethanol produced dose-dependent decreases in rate on both schedules with no significant schedule differences in ED50 (the dose effective at reducing the maximal response rate by one-half) values. Maximal tolerance was achieved in means of 46 and 55 days on the VI and FR schedules, respectively. Differences in rate of tolerance acquisition on the initial dose of the chronic regimen (1.125 g/kg) account for most of the difference in the overall rate of acquisition. Comparison of the ED50 data from DECs 1 and 2 indicated that daily ethanol exposure resulted in a 2-fold decrease in ethanol sensitivity (i.e., tolerance) on both operant schedules. The ED50 data from DECs 1 and 3 demonstrated a 1.7-fold decrease in ethanol potency on DEC3. This duration of tolerance was considerably longer than that generally reported, and possibly related to the extended ethanol exposure and the sensitivity of operant schedules to drug effects.

Sensitivity of inbred mice to methylxanthines is not determined by plasma xanthine concentration

The in vitro thiobarbituric acid test was used as a measure of lipid peroxidation in the gerbil and rat. Synaptosomal preparations were isolated from the cerebral cortex of each species and incubated with a free radical generating system. Varying concentrations of ADP-Fe3+, with ascorbate and oxygenated incubation medium were used to generate hydroxy-radicals. Peroxidation of the synaptosomal membrane lipids was determined using malondialdehyde (MDA) accumulation. Both the gerbil and rat demonstrated significant increases in MDA in the presence of the generating system, while the gerbil P2 fraction consistently showed an increased level of MDA accumulation as compared with rat at each of the concentrations of ADP-Fe3+. Across a range of concentrations, there was a 2-2.6-fold greater increase in MDA accumulation in gerbil as compared with rat. Free radical generation is currently thought to be involved in the associated damage following cerebral ischemia. An in vitro model capable of producing biochemically similar damage to membrane systems by means of a controlled free-radical generating system may prove useful in studying possible mechanisms of ischemic damage.