Automated evaluation of sensitivity to foot shock in mice: inbred strain differences and pharmacological validation

Assessing foot shock sensitivity in rodents can be useful in identifying analgesic or hyperalgesic drugs, and phenotyping inbred or genetically altered mice. Furthermore, as foot shock is an integral part of several rodent behavioral models, sensitivity should also be assessed to accurately interpret behavioral measures from these models. To eliminate variability and increase the efficiency of manually scored shock sensitivity paradigms, we utilized a startle reflex system to automatically quantify responses to varying levels of foot shock. Eight inbred mouse strains were tested for reactivity to foot shock in this system, as well as inherent startle response activity to loud noise bursts. Strain rank order for shock reactivity differed from that for acoustic startle, suggesting that pathways activated in response to each differed. Analgesic doses of morphine and acetaminophen specifically reduced foot shock responses without affecting motor reflexive responses to loud noises in each strain tested. We also tested diazepam and scopolamine, which are often used to disrupt behavior in shock-related paradigms to illustrate the usefulness of this assay. Overall, these results demonstrate that our automated method is a quick and simple way to accurately assess potential foot shock sensitivity differences owing to strain, genotype or drug treatments.     

Genetic determinants of sensitivity to pentobarbital in inbred mice

RATIONALE: We postulated that genetic determinants of different responses to pentobarbital (PB) in mice would differ from response to response.OBJECTIVES: Mice from 14 standard inbred strains were tested for sensitivity to several effects of acute PB.METHODS: Strains were tested for sensitivity to PB-induced low-dose stimulation and high-dose depression of locomotor activity, reduced rearing, hypothermia, and ataxia assessed on a rotarod, using four doses of PB or saline.RESULTS: Strains differed in sensitivity to PB for all responses. Correlations among strain means indicated that strain sensitivity to a particular effect of PB generalized rather well across doses. Sensitivities to some of the different behavioral responses were also significantly correlated. For example, strains less sensitive to PB-induced enhanced locomotor activity were also significantly more sensitive to the drug's hypothermic effects. Some responses were genetically independent. Brain PB concentrations were also determined, and appeared to be unrelated to inbred strain drug sensitivities.CONCLUSIONS: Overall, these results suggest that there are multiple genetic determinants of behavioral sensitivity to PB effects. That is, genetically influenced sensitivity to PB is not monolithic, but is somewhat specific to the particular response variable studied, a result that also characterizes genetic control of responses to other drugs.     

Genetic variation in paraoxonase activity and sensitivity to diisopropylphosphofluoridate in inbred mice

The mechanism underlying genetic variation in the acute and chronic responses of mice to diisopropylphosphofluoridate (DFP) are unknown. We investigated whether variation in metabolism of organophosphates by A-esterase, as exemplified by the enzyme paraoxonase, was correlated to the degree of sensitivity to DFP in four inbred mouse strains. LD50s and plasma paraoxonase were measured in each strain. We observed genetic variation in both of these measures, but there was no significant correlation between the two measures. We conclude that plasma paraoxonase activity does not underlie genetic variation in sensitivity to the lethal effects of DFP in mice since it does not determine the degree of sensitivity or resistance to DFP.     

Interstrain variation in acute toxic response to caffeine among inbred mice

Acute toxic dosage-dependent behavioral effects of caffeine were compared in adult males from seven inbred mouse strains (A/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL/6J, DBA/2J, SWR/J). C57BL/6J, chosen as a "prototypic" mouse strain, was used to determine behavioral responses to a broad range (5-500 mg/kg) of caffeine doses. Five phenotypic characteristics--locomotor activity, righting ability, clonic seizure induction, stress-induced lethality, death without external stress--were scored at various caffeine doses in drug-naive animals under empirically optimized, rigidly constant experimental conditions. Mice (n = 12 for each point) received single IP injections of a fixed volume/g body weight of physiological saline carrier with or without caffeine in doses ranging from 125-500 mg/kg. Loss of righting ability was scored at 1, 3, 5 min post dosing and at 5 min intervals thereafter for 20 min. In the same animals the occurrence of clonic seizures was scored as to time of onset and severity for 20 min after drug administration. When these proceeded to tonic seizures, death occurred in less than 20 min. Animals surviving for 20 min were immediately stressed by a swim test in 25 degrees C water, and death-producing tonic seizures were scored for 2 min. In other animals locomotor activity was measured 15 or 60 min after caffeine administration. By any single behavioral criterion or a combination of these criteria, marked differences in response to toxic caffeine doses were observed between strains. These results indicate that behavioral toxicity testing of alkylxanthines in a single mouse strain may be misleading and suggest that toxic responses of the central nervous system to this class of compounds are genetically influenced in mammals.     

Genetical differences in sensitivity to tremorine and oxotremorine in mice

Male mice from 14 strains were injected i.p. with tremorine (3.0 mg/kg) or oxotremorine (0.15 or 0.1 mg/kg). Large inter-strain differences in the degree and duration of the subsequent hypothermia were noted. 2 strains, BALB/c and Simpson, were particularly sensitive to the hypothermic effect of oxotremorine. The offspring from a cross between BALB/c and Simpson were less sensitive than the parental strains, suggesting genetic complementation. A set of 7 recombinant inbred (RI) lines derived from strains C57BL and BALB/c were tested with oxotremorine. 5 RI lines resembled strain C57BL in their response and 2 RI lines resembled strain BALB/c. It was concluded that strains C57BL and BALB/c differ at a gene which has a major effect on the response to oxotremorine.     

Strain differences in amphetamine sensitivity in mice

d-Methylamphetamine (1–4 mg/kg) induced wakefulness for 3–5 h followed by paradoxical sleep (PS) rebound in C57BR, C57BL/6, and BALB/c mice. Slow-wave sleep (SWS) and PS suppression time were proportional to the given dose and different among strains. It was also dependent on the time of injection. Later PS rebound was significant in C57 strains, and in certain cases greater than PS loss, whereas rebound was small in BALB/c mice and PS loss was not completely compensated.     

Sex differences in sensitivity to seizures elicited by pentylenetetrazol in mice

Sex differences in sensitivity to seizures elicited by intraperitoneally injected pentylenetetrazol (PTZ) were studied in 240 (120 males and 120 females) adult Swiss mice. Animals were separated into four groups according to the dose that was injected: 40, 50, 60 and 70 mg/kg. Seizure severity was expressed by the following scoring scale: (0) no abnormal behavior; (1) myoclonus; (2) running bouncing (RB) clonus; (3) tonic hind limb extension (THE). The analyses of the dose-response curves indicated that females were more susceptible than males when the 50- and 60-mg/kg doses were used. Specifically, females often displayed RB clonus, while males frequently displayed only myoclonus or no abnormal behavior. No significant sex differences were demonstrated when either the 40- or the 70-mg/kg doses were used. These data indicate that, for a specific range of doses, sex differences in seizure susceptibility can be clearly demonstrated with the use of intraperitoneally injected PTZ. In this sense, this method could be used as a tool to investigate the role played by sexual hormones in regulating the sensitivity of the gamma-aminobutiric acid (GABA(A)) receptor complex (GRC).     

Contributions of dam and conceptus to differences in sensitivity to valproic acid among C57 black and SWV mice

To ascertain the relative contributions of genotypes of conceptus and dam to developmental toxicity occasioned by valproic acid (VPA), crosses were established between resistant C57BL/6JBk (C, C57) and susceptible SWV/Bk (S, SWV) strains of mice. These included matings of pure lines, reciprocal outcrosses, and reciprocal backcrosses with F1 hybrids. At 8 d:12 h +/- 5 h, for each mating, 0, 500, or 600 mg/kg aqueous VPA was injected ip. Fetuses were examined on gestation day (gd) 18 for exencephaly (the paradigmatic anomaly), other abnormalities, mortality, litter size, and fetal weight. At 600 mg/kg, sensitivity to exencephaly induction in all cases was that of the dam, regardless of sire. Thus exencephaly here seems to be largely a function of the uterine environment produced by the maternal genotype. This inference is confirmed in backcrosses where F1-dams x S-sires and F1-dams x C-sires produced-identical outcomes, and S-dams x F1-sires produced much higher frequencies of exencephaly than C-dams x F1-sires. For prenatal mortality, the genotypes of both dam and conceptus appear to be important determinants. Fetal contribution is inferred from the observations that S-dam x S-sire matings produced a much higher frequency of mortality than S-dams x C-sires, and C-dams x C-sires produced higher mortality than C-dams x S-sires. Therefore, heterozygosity of the conceptus was protective. Among backcrosses, fetal determination of sensitivity to mortality is also seen by the observation that F1-dams x C-sires produces the same fetal mortality as C-dams x F1-sires. The contribution of uterine environment is seen in the observation that matings of S-dams x C-sires resulted in higher fetal mortality than did those with C-dams x S-sires. Therefore, identical conceptuses in different dams showed different levels of fetal loss. Thus exencephaly response appears to be largely controlled by genes active in the dam, and mortality as a result of a multigenic outcome with contributing genes active in both conceptus and dam. The data also suggest that SWV pure-line dams make a contribution to prenatal mortality not seen in C57 or F1 dams. Mean litter size among VPA-exposed litters showed high variability in pure lines and outcrosses. In backcrosses, F1 dams produced larger litters than pure line dams, arguing for heterosis as a contributor to this parameter. Reduction in litter size occasioned by VPA exposure was great in pure line dams and nonexistent in F1 dams. The SWV dams crossed with F1 sires were the only group among the backcrosses to show reduction of litter size, providing further confirmation of the increased sensitivity of pure-line (i.e., homozygous) SWV dams to VPA exposure. Fetal weight seems to be a function of uterine environment because female SWV produced conceptuses with lower fetal weight in all crosses, and produced a greater reduction in fetal weight attributable to VPA exposure than C57 or F1 dams. Fetal weight did not correlate closely with litter size, suggesting that a lower fetal weight may be a strain characteristic, as are exencephaly induction and prenatal mortality in response to VPA. Differences in sensitivity to VPA insult are seen for all parameters investigated with SWV dams being the most sensitive, but mechanisms seem to differ for a number of the endpoints.     

Alpha-methyl-para-tyrosine effects in mice selectively bred for differences in sensitivity to ethanol

The responses of catecholamine systems in long sleep (LS) and short sleep (SS) mice to alpha-methyl-p-tyrosine (AMPT) have been examined. Marked differences were found between LS and SS mice in the dose necessary for maximal brain catecholamine depletion and in the time-course of the catecholamine depletion. Brain catecholamines in the LS mice were depleted by lower doses of AMPT and the levels remained depressed for longer periods of time in this line of mice. These differences may be explained only partially by an increased susceptibility of the LS mice to the hypothermia and toxic effects caused by AMPT administration, as they persist with non-toxic AMPT dosage regimens and under conditions where the degree of hypothermia is comparable in both lines of mice. In addition, there were no differences between the Ki values for the effect of AMPT on the tyrosine hydroxylase from striata of these mouse lines. The primary cause of the heightened response to AMPT in LS mice would appear to be pharmacokinetic in nature, as brain and plasma peak levels of AMPT in LS mice were greater and the levels remained higher for a longer time. The depletion of brain tyrosine by AMPT combined with the lower affinity of the LS striatal tyrosine hydroxylase for the substrate tyrosine may also contribute to the heightened response in LS mice.     

Differences in anxiety-related behaviours and in sensitivity to diazepam in inbred and outbred strains of mice

Rationale: Natural strain differences exist in mice for behavioural traits such as emotional reactivity. Objective: The present experiments compared the behavioural profiles of nine strains of mice (BALB/c, C57BL/6, C3H, CBA, DBA/2, NMRI, NZB, SJL, Swiss) in two models of anxiety after the administration of the benzodiazepine diazepam. Methods: The tests used were the light/dark choice task and the elevated plus-maze, two well-validated anxiolytic screening tests. Results: In vehicle-treated animals, differences on variables designed to measure anxiety-related behaviours were observed in both tests. In the light/dark test, the strains could be divided into three distinct groups: two non-reactive strains (NZB and SJL), an intermediate-reactive group (C3H, CBA, DBA/2, NMRI, C57BL/6 and Swiss), and one highly reactive strain (BALB/c). In the elevated plus-maze, SJL, NMRI, CBA and, to a lesser extent, C3H strains of mice, consistently showed low levels of anxiety-related behaviours. Intermediate levels were seen in the Swiss and BALB/c strains, and high levels of emotional reactivity were seen in C57BL/6, DBA/2 and NZB. The strain distribution between the light/dark and the elevated plus-maze tests shows similarities and differences, suggesting that each of these experimental procedures represents a different set of behaviours. Marked differences between a number of strains of mice in their sensitivity to the anxiolytic-like action of diazepam were observed in both the light/dark and the elevated plus-maze tests. Mice of the BALB/c, Swiss and, to a lesser extent, CBA and C3H strains were responsive to diazepam in both tests, although in the case of CBA mice, effects may have been contaminated by behavioural suppression. SJL mice were largely unresponsive to the action of the benzodiazepine in both tests, whereas in C57, DBA/2, NMRI and NZB mice, diazepam produced positive effects only in the elevated plus-maze. Conclusion: The finding of differential strain distributions both with and without diazepam treatment in the light/dark and the elevated plus-maze tests, indicates that not all strains of mice are suitable for investigating the effects of GABA/BZ receptor ligands. This study may thus provide a useful guide for choosing the best strain of mice for studying the pharmacology of fear-related behaviours. Received: 24 March 1999 / Final version: 10 July 1999     

Chromosomal mapping of loci influencing sensitivity to cocaine-induced seizures in BXD recombinant inbred strains of mice

Among inbred mice, genetic factors mediate differences in sensitivity to the convulsant properties of cocaine; however, the gene(s) underlying cocaine's effects have not been identified. To help elucidate the gene(s) responsible for cocaine seizure susceptibility, we used recombinant inbred-quantitative trait loci (RI-QTL) analyses to identify chromosomal loci associated with cocaine-induced seizures. RI-QTL analyses seek to identify associations between a quantitative measure of a particular phenotype and one or more previously mapped marker genes across a panel of RI strains. This report describes an RI-QTL analysis of cocaine seizure susceptibility among 26 BXD RI strains. These strains showed a skewed, bimodal range of seizure susceptibility which could be the result of one or more modifying genes acting in concert with a major gene to influence cocaine sensitivity. Correlating the percent seizures displayed by each strain following 60 mg/kg cocaine with chromosomal marker data for these strains revealed a number of significant correlations clustered in two regions on chromosomes 12 and 6. This is the first identification of putative chromosomal loci associated with a cocaine-related phenotype and should facilitate identification of the gene(s) underlying cocaine toxicity and other cocaine-related phenotypes.     

Genetic differences in daily rhythms of pain sensitivity in mice

A dark phase increase in pain sensitivity was evident in C57BL/6 inbred mice. On the contrary Swiss mice are characterized by decreased nocturnal pain sensitivity. The latter finding is in agreement with a number of previous studies based on albino mice. However, our findings indicate that (1) nocturnal decreased pain sensitivity is not the rule in the mouse and (2) large differences are evident when the onset in peak nocturnal analgesia is considered.     

Brain neurotransmitter receptor systems in mice genetically selected for differences in sensitivity to ethanol

The density of receptors for neurotransmitters and receptor-mediated cellular responsiveness were determined in tissues from two lines of mice selectively bred for differences in sleep time after ethanol administration. The studies were undertaken to ascertain in differences in neurotransmitter receptors between the lines might explain any of the differences seen in initial sensitivity to ethanol. The results were compared with those obtained in studies using the parent heterogeneous stock (HS). The animals used in the current experiments did not receive ethanol. β-Adrenergic receptor density, as measured by the binding of (¹²⁵I)-iodohydroxybenzylpindolol, was lower in cortices of alcohol-sensitive long-sleep mice (LS) as compared to the short-sleep (SS) animals or to HS controls. The densities of β₁ and β₂-adrenergic receptors were lower by approximately the same amount. The results were tissue specific in that there was no difference in the density of β-adrenergic receptors in the cerebellum. Cyclic AMP accumulation in response to isoproterenol was not significantly different in the cortex of LS mice despite the decrease in β-adrenergic receptors. No differences were found in ³H-spiroperidol binding to dopamine receptors in the striatum or in ³H-quinuclidinylbenzilate binding to muscarinic cholinergic receptors in the cortex, striatum or hippocampus. Dopamine-stimulated adenylate cyclase activity was, how- ever, lower in striata of SS mice. The affinities of the receptors for the various ligands studied were the same in the three lines of mice.     

Strain differences in sensitivity to the hypothermic effects of benzodiazepine receptor ligands in mice

The hypothermic effects of intraperitoneal (IP) administration of the full benzodiazepine agonist lop razolam (1, 10 mg/kg); the partial agonist Ro 17-1812 (1, 10 mg/kg); the benzodiazepine receptor antagonist flumazenil (10, 20 mg/kg); the benzodiazepine inverse agonists Ro 15-4513 (1, 3, 10 mg/kg) and Ro 19-4603 (0.03, 0.1, 0.3 mg/kg) and the-carboline inverse agonists FG 7142 (10, 30 mg/kg) and DMCM (1, 3, 10 mg/kg) were investigated in three strains of mice. TO mice were less sensitive than CBA/cA and DBA/2 mice, since only loprazolam and the partial and full-carboline inverse agonists FG 7142 and DMCM lowered body temperature in these animals. CBA/cA mice were particularly sensitive to the hypothermic effects of loprazolam and Ro 17-1812, and also responded to the-carboline but not the benzo diazepine inverse agonists. In contrast, DBA/2 mice responded with moderate hypothermia to loprazolam, Ro 17-1812, and to the partial inverse agonist Ro 15-4513, and exhibited marked hypothermia in response to the more efficacious benzodiazepine inverse agonist Ro 19-4603 and to FG 7142 and DMCM. Flumazenil did not alter body temperature. DBA/2 mice were also more sensitive to the convulsant activity of inverse agonists than TO mice. CBA/cA mice exhibited enhanced sensitivity to the convulsant, but not the hypothermic, effects of Ro 19-4603, showing dissociation of these responses. The mechanisms underlying the genetic differences in sensitivity of mice to the hypothermic and convulsant action of the different ligands are unknown and warrant further investigation.     

Intergeneric behavioral differences among methamphetamine treated mice

Methamphetamine sulfate was administered in acute and chronic experiments, either at constant or increasing doses, to mouse-like rodents belonging to various genera and strains; the behavioral effects arising in a pseudo-natural habitat are described. The animals studied were Onychomys leucogaster, Peromyscus maniculatus Bairdii, Microtus ochrogaster and four Mus strains, Mus musculus ICR, Mus musculus Mo., Mus musculus C57BL/6J, and Mus musculus CF-1. Aggression, grooming, digging, contactual, sexual, sleeping, ingestive, exploratory and stereotypic behaviors were quantitated. Both similarities and differences in the behavioral responses to methamphetamine of the various mice types were noted. These responses are discussed as to their modification by tolerance or increasing doses. Certain neurological and behavioral aberrations due to methamphetamine were noted and correllations with the parameters of mice neurochemistry established in these laboratories are presented.Supported in part by the Research Grant No. 1-RO1 NB 06455 and the Training Grant ST 1 GM77 from the National Institutes of Health, and by the Illinois Mental Health Grant No. 17-176.     

Age-related differences in sensitivity to organophosphorus pesticides

Organophosphorus (OP) pesticides are used extensively throughout the world to control undesirable pest species. The primary mechanism of action for OP insecticides is inhibition of acetylcholinesterase (AChE), an enzyme dynamically involved in cholinergic neurotransmission. Extensive inhibition of AChE leads to accumulation of acetylcholine in the synapse, disruption of normal impulse flow and subsequent signs of toxicity, including autonomic dysfunction, involuntary movements, muscle fasciculations and a host of others. It is generally believed that young individuals are more sensitive to the neurotoxic effects of these agents relative to adults. Essentially all studies addressing age-related differences in sensitivity to these toxicants have examined responses to acute exposures, however, using acute toxicity (lethality) as the endpoint. As the biochemical mechanism of toxicity for this class of toxicants (inhibition of AChE) is well known and considering that low level, repeated exposures are of great concern to the general public, we propose that evidence of neurochemical alterations, especially when exposures occur during development and maturation, is a more relevant endpoint of toxicity than lethality for estimating susceptibility. This report briefly summarizes previous and ongoing work in our laboratory which examines the relative sensitivity to these toxicants between young and adult rats.     

Sex differences in the sensitivity of CBA mice to convulsions induced by GABA antagonists are age-dependent

The administration of the GABA-blocking agents picrotoxin and bicuculline to adult (2.5–3 months old) CBA/HZgr mice resulted in the appearance of convulsions, the occurrence and/or lethality of which was greater in males than in females. The latency of picrotoxin-induced convulsions was also shorter in male mice. Strchnine, a drug which induces convulsions by blocking glycine receptors was equally effective in producing convulsions in both male and female adult mice. Unlike adult mice, young (20 days old) or old (2 years old) mice fail to display sex differences following the picrotoxin administration. Accordingly, the observed sex differences in the sensitivity of CBA mice to administration of convulsive agents are specific for the GABA system and present only in sexually mature, but not in immature or old animals.     

p-Chlorophenylalanine-mediated decrease in susceptibility to audiogenic seizures in inbred mice.

p-Chlorophenylalanine caused a marked reduction in the susceptibility of inbred mice, O'Grady strain, to audiogenic convulsive seizures. The effect was maximal at 2 hr following injection, with gradual return to full responsiveness by 72 hr. The relationship between dose and effect at 2 hr was linear from 100 mg/kg to 400 mg/kg. As mice matured and their susceptibility to audiogenic seizures diminished, the protective effect of p-chlorophenylalanine became less evident.     

Ethanol-induced changes in tyrosine hydroxylase activity in brains of mice selectively bred for differences in sensitivity to ethanol

The effects of ethanol on tyrosine hydroxylase (TH) activity in five brain areas were analyzed in two lines of mice selectively bred for their differences in sensitivity to ethanol. Following a 4.1 g/kg dose of ethanol, intraperitoneally, short sleep (SS) mice lose their righting reflex for a duration of 20 minutes and long sleep (LS) mice fail to regain their righting reflex until 120 minutes. A significant increase in TH activity occurred in the striatum, locus coeruleus and frontal cortex in both lines of mice approximately 25 minutes following ethanol administration. A decrease in TH activity occurred in the substantia nigra of SS mice at 5 minutes following ethanol administration. However, there was no significant difference in TH activity in any of these four brain regions between LS and SS mice at any time following ethanol administration. In contrast, hypothalamic TH activity was significantly increased at 25 minutes in the SS mice and at 125 minutes in the LS mice following the administration of ethanol, times which coincided with the regaining of the righting reflex. These data suggest that activation of TH in the hypothalamus of LS and SS mice in response to ethanol is associated with arousal from ethanol induced narcosis.