Hole-board Apparatus Research Articles

Modification of the Effects of Benzodiazepines on the Exploratory Behaviors of Mice on a Hole-Board by Diabetes

The effect of diabetes on the emotional behavior of mice was examined using an automatic hole-board apparatus. Changes in the emotional state of mice were evaluated in terms of changes in exploratory activity; i.e., total locomotor activity, numbers and duration of rearing and head-dipping, and latency to the first head-dipping. The number and duration of head-dipping in diabetic mice were less than those in non-diabetic mice. Diazepam (0.1-0.56 mg/kg, i.p.) dose-dependently increased the number and duration of head-dipping at doses that did not produce sedation in both diabetic and non-diabetic mice. In contrast, methyl-beta-carboline-3-carboxylate (1 and 2 mg/kg, i.p.) decreased the number and duration of head-dipping in non-diabetic mice, but not in diabetic mice. The number and duration of head-dipping in diabetic mice were increased by treatment with flumazenil (0.1 and 0.3 mg/kg, i.v.). These doses of flumazenil did not affect the number or duration of head-dipping in non-diabetic mice. The present data indicate that diabetic mice exhibited anxiety in the hole-board test and that a benzodiazepine receptor antagonist affected the attenuated number and duration of head-dipping in diabetic mice. The heightened anxiety in diabetic mice may be due to the dysfunction of the benzodiazepine receptor and/or of central inhibitory systems.

Different effects of 5-HT1A receptor agonists and benzodiazepine anxiolytics on the emotional state of naive and stressed mice: a study using the hole-board test.

The effects of 5-HT(1A) receptor agonists on the emotional behavior of naive or stressed mice were examined and compared with those of benzodiazepine anxiolytics. Changes in the emotional state of mice were evaluated in terms of changes in exploratory activity, i.e. total locomotor activity, numbers and duration of rearing and head-dipping and latency to the first head-dipping, using an automatic holeboard apparatus. The 5-HT(1A) receptor full agonists flesinoxan (0.03-1 mg/kg, IP) and 8-OH-DPAT (0.03-1 mg/kg, IP), and the partial agonist buspirone (0.3-10 mg/kg, IP) dose-dependently decreased all of the exploratory behaviors. Significant decreases in both the number and duration of head-dips, and an increase in the latency to head-dipping were observed at 30 min after exposure to acute restraint stress (60 min). These emotional changes were scarcely improved by post-stress treatment with 5-HT(1A) receptor agonists, at doses that alone did not produce a significant behavioral effect. In contrast, pretreatment with flesinoxan (0.1-1 mg/kg, IP) or 8-OH-DPAT (0.1-1 mg/kg, IP) 24 h prior to exposure to stress dose-dependently suppressed the decrease in various exploratory behaviors that was observed immediately after the exposure to acute restraint stress. Moreover, pretreatment with buspirone (1-10 mg/kg, IP) 24 h prior to exposure to stress also significantly suppressed the decrease in rearing behavior and the increase in head-dip latency. However, changes in the emotional response to stress stimuli were not observed in mice that had been pretreated with the benzodiazepine anxiolytics diazepam (0.1-1 mg/kg, IP) and chlordiazepoxide (2-8 mg/kg, IP). The present study clearly demonstrates that the behavioral effects of 5-HT(1A) receptor agonists in both naive and stressed mice were quite different from those of benzodiazepine anxiolytics, as previously reported by us. Notably, 5-HT(1A) receptor agonists but not benzodiazepine anxiolytics protect against various emotional changes produced by stress stimuli, and the results suggest that activation of 5-HT(1A) receptors may facilitate some mechanism(s) involved in the recognition of and/or ability to cope with stressful situation.

Phencyclidine-induced abnormal behaviors in rats as measured by the hole board apparatus

Phencyclidine (PCP) and methamphetamine (MAP) are known as psychotomimetic agents. Both agents produce behavioral alterations in animals. The present study investigated the difference in behavioral alterations in rats induced by these two psychotomimetic agents using the hole board apparatus (HBA). In addition, mechanisms underlying PCP-induced behavioral changes were also investigated. After the administration of PCP (1-4 mg/kg SC) or MAP (1-4 mg/kg SC), locomotor activity and dipping behavior were assessed using HBA. Effect of selective NMDA antagonists, (+)MK801 and 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), on rat behaviors were also assessed. The effects of D-alanine (D-Ala), a coagonist of NMDA receptors, or neuroleptics, haloperidol, clozapine and risperidone, on PCP-induced behavioral changes were investigated. PCP increased locomotor activity and decreased exploratory behaviors of rats in HBA. On the other hand, MAP increased locomotor activity but did not decrease exploratory behaviors. (+)MK-801 produced hyperactivity as well as decreased exploratory behaviors, eliciting behavioral changes very similar to those of PCP. CPP decreased the exploratory behavior but failed to produce hyperactivity. D-Ala attenuated both behavioral changes induced by PCP. Three neuroleptics tested here inhibited hyperactivity but did not attenuate decreases in exploratory behavior. These results suggest that PCP-induced decrease in exploratory behavior are attributable to antagonism of NMDA receptors and may not involve dopaminergic transmission via D2 receptors.

Inbred Roman High- and Low-Avoidance Rats: Differences in Anxiety, Novelty-Seeking, and Shuttlebox Behaviors

In the present study, male inbred animals (from the 10th generation of an inbreeding program that has been carried out in parallel to that of the outbred Roman high- and low-avoidance rat lines), were compared for emotionality in different testing situations, exploratory behavior in the holeboard and two-way, active-avoidance acquisition. Compared to the inbred Roman high-avoidance (RHA-I/Verh) rats, inbred Roman low-avoidance (RLA-I-Verh) rats showed higher emotionality in the open field (reduced distance travelled and number of rearings, and increased self-grooming behavior), in the elevated plus-maze test (increased number of total and open-arm entries, reduced distance travelled in the open arms, and increased self-grooming behavior), and during the habituation period in the shuttle box (decreased number of crossings, increased self-grooming behavior and defecations). Results from the hyponeophagia test were not conclusive, probably due to the test-dependent hyperactivity shown by RHA-I/Verh rats. In the holeboard apparatus, RHA-I/Verh rats explored more than RLA-I/Verh rats, especially when novel objects were located beneath the holes. Finally, RHA-I/Verh animals rapidly acquired active, two-way (shuttlebox) avoidance, whereas RLA-I/Verh animals required four 50-trial sessions to achieve an assymptotic level of 30–40% avoidance. Thus, the behavioral patterns of the Roman inbred strains were very similar to those previously reported for the RHA/Verh outbred lines. Differences in locomotor activity, exploratory, and self-grooming behavior were actually greater between the inbred strains than between the outbred lines. Differences in defecation, however, although still significant, were not so pronounced as those noted previously at this laboratory with the outbred lines.

Effects of acute and repeated dose administration of caffeine and pentoxifylline on diazepam-induced mouse behavior in the hole-board test.

The behavioral effects of methyl xanthines and their interactions with benzodiazepines have not been clearly established in animal models of anxiety. The present study extended the previous studies to determine the effects of acute and repeated administration of caffeine, a non-specific phosphodiesterase (PDE) inhibitor and pentoxyfylline, a specific type-4 phosphodiesterase (PDE4) inhibitor on (1) baseline anxiety-like behavior and (2) the response to an acute challenge with diazepam on anxiety-like behavior in the hole-board test. Mice were observed for the number of head-dips they made into the holes of the hole-board apparatus during a 5-min period, starting 30 min after acute (20 mg/kg) and repeated oral dose (20 mg/kg, twice a day for 4 days) administration of caffeine and pentoxifylline. In separate experiments, the response to an acute challenge with graded doses of diazepam (0.375 3 mg/kg, SC) was observed in naive mice or mice on acute and repeated dose regimen with methyl xanthines. Mice on acute but not after repeated dose regimen demonstrated a significantly increased number of hole-dips, indicating an anxiolytic-like effect of methylxanthines. Diazepam at the lower doses (0.375 and 0.75 mg/kg) but not at the highest doses (1.5 and 3 mg/kg) examined produced a significant anxiolytic-like effect. After an acute dose exposure of mice to caffeine and pentoxifylline, a rightward shift in the dose-response curve of diazepam was observed and particularly at 1.5 mg/kg dose, the net effect of diazepam was significantly enhanced which was, however, impaired upon repeated administration, more so with caffeine than with pentoxifylline. It is concluded that the xanthine drugs exert anxiolytic-like activity similar to diazepam in the hole-board test. In addition, they seem to modulate the anxiolytic effects of diazepam after both acute and repeated administration, probably as a result of an endogenous adenosinergic mechanism which may have therapeutic significance.

Utility of ethological analysis to overcome locomotor confounds in elevated maze models of anxiety

The elevated plus-maze is a commonly used model to identify putative anxiolytic and anxiogenic drugs. However, the validity of elevated plus-maze and other recently developed variants such as the elevated zero-maze has recently been questioned on the grounds that both the reference anxiolytic drug chlordiazepoxide and the psychostimulant d-amphetamine increase open arm exploration and stimulate locomotor activity. These findings suggest that measures of “anxiety” in the elevated maze cannot be adequately dissociated from simple changes in locomotor activity, which may confound the interpretation of results obtained using these models. A variety of approaches to assess drug effects on locomotor activity in the elevated maze have been suggested, including the use of total and closed arm entries, as well as supplementary tests such as exploration of the holeboard apparatus. However, all these approaches utilise the measurement of exploration in a novel environment, and as such, could potentially be influenced by either changes in anxiety or locomotor activity. Recently, it has been shown that ethological measures of “risk assessment”, such as stretched–attend postures and head-dipping, are sensitive indicators of drug-effects in the elevated maze. The present study assessed the utility of ethological analysis in dissociating locomotor activity from “anxiety” by comparing the effects of d-amphetamine to those of chlordiazepoxide in the rat elevated zero-maze. The results showed that both chlordiazepoxide and d-amphetamine increase the amount of time spent in the open arms and reduce “risk assessment” without increasing line crossing or rearing. These results confirm that under certain test conditions, psychostimulants are capable of producing “false-positives” in elevated maze models, and that both traditional methods and the ethological measures used in this study fail to unequivocally dissociate drug effects on anxiety from effects on locomotor activity. Further studies using other species and different classes of psychostimulants are warranted to determine the generality of these findings.

Changes in head-dipping behavior in the hole-board test reflect the anxiogenic and/or anxiolytic state in mice

The effects of treatment with anxiogenic or anxiolytic agents and exposure to acute restraint stress on emotional behavior in mice were examined using an automatic hole-board apparatus. Changes in the emotional state of mice were evaluated in terms of changes in exploratory activity, i.e., total locomotor activity, numbers and duration of rearing and head-dipping, and latency to the first head-dipping. The typical benzodiazepine anxiolytics diazepam (0.05–0.5 mg/kg, i.p.) and chlordiazepoxide (0.5–4 mg/kg, i.p.) dose-dependently increased the number and duration of head-dips at doses that did not produce sedation. In contrast with these anxiolytics, the typical anxiogenic drugs N-methyl- β-carboline-3-carboxamide (FG7142, 0.125–10 mg/kg, i.p.) and methyl- β-carboline-3-carboxylate ( β-CCM, 0.1–2 mg/kg, i.p.) decreased both the number and duration of head-dips, and increased the latency to head-dipping. Moreover, decreases in the number and duration of head-dips, and an increase in the latency to head-dipping, were also observed in animals that were exposed to acute restraint stress. These effects of acute restraint stress were suppressed by treatment with diazepam at a dose that alone did not produce significant behavioral effects (0.1 mg/kg, i.p.). In addition, non-benzodiazepine anxiolytic flesinoxan (0.1 mg/kg, i.p.), a 5-HT 1A receptor agonist, also had an effect on the restraint stress-induced decrease in head-dipping behavior. The present study shows that the changes in several exploratory behaviors could be objectively measured using our automatic hole-board apparatus. Therefore, this system can serve as a useful tool for evaluating the changes in various emotional states of animals. Moreover, we also found that treatment with anxiolytics or anxiogenics and exposure to acute restraint stress affected head-dipping behavior. These results suggest that changes in head-dipping behavior in the hole-board test may reflect the anxiogenic and/or anxiolytic state of animals.

A rotating holeboard procedure for testing drug effects on spatial learning and memory in mice

A procedure is described herein for evaluating drug effects on acquisition and retention of a spatial learning task in mice. The rotating holeboard apparatus is a rectangular open field containing four open holes arranged in either a four-corner or a row configuration. A mouse is trained to poke its head into a hole and retrieve a food reward from a `baited' hole which contains a reward on every trial. A massed trials protocol is used where mice are required to learn a reference (trial-independent) memory task in a single session. A retention test is administered 24 h after acquisition. Prominent distal cues are present in the testing room and reliance on proximal visual, olfactory, or tactile cues for locating the baited hole is precluded by rotating the maze on each trial.

An Anxiolytic Action of Oxytocin is Enhanced by Estrogen in the Mouse

The established role of oxytocin (OT) in facilitation of steroid-modulated reproductive and affiliative behaviors led to the speculation that it may have anxiolytic actions under certain hormonal conditions. NIH-Swiss mice were tested for responsiveness to OT in two behavioral tests of anxiety, the holeboard apparatus and elevated plus-maze. Dose-response assessment indicated that 3 mg/kg was the optimal dose for peripherally administered (IP) OT on the elevated plus-maze. There were no consistent effects at any dose on the holeboard apparatus. In ovariectomized mice pretreated with estradiol (E2), peripherally administered OT increased the number of entrances onto the open arms, as well as the amount of time on the open arms compared to other groups (ANOVA; p < 0.05). There was little to no effect of OT in ovariectomized animals not pretreated with E2. When OT was administered intracerebroventricularly (ICV), there was an increase in entrances and time on the open arms compared to that of females infused with arginine vasopressin (AVP). This increase was apparent in ovariectomized females, but was further enhanced in those pretreated with E2 (ANOVA; p < 0.05). In contrast, the combination of E2 pretreatment and ICV AVP decreased the number of entrances and time spent on the open arms of the elevated plus-maze compared to those receiving OT, suggesting an estrogen-modulated anxiogenic action of AVP. Analyses of [125]I-OVTA binding density indicated a significant increase in binding density in the lateral septum of E2-treated females compared to OIL-treated controls (ANOVA; p < 0.05). There was no effect of E2 treatment on [125]I-OVTA binding density in the amygdala or ventromedial nucleus of the hypothalamus. Taken together, these data indicate that OT exerts an anxiolytic action that is enhanced in the presence of circulating estrogen. This behavioral effect may be mediated by estrogen-induced increases in OT binding density in the lateral septum and may be important to the facilitation of social interactions.

Reproductive effects in F1-generation rats perinatally exposed to PCB126, PCB 118, PCB 153 or 2,3,4,7,8-PnCDF

Neonatal lipopolysaccharide (LPS) exposure increases anxiety-like behaviour and alters neuroendocrine responses to stress in adult rats. The current study assessed whether this anxiety-related phenotype observed in rats neonatally exposed to LPS is transferable to subsequent generations. Wistar rats were exposed to LPS (0.05 mg/kg, Salmonella enteritidis) or non-pyrogenic saline (equivolume) on postnatal days 3 and 5. In adulthood, animals were subjected to restraint and isolation stress or no stress, and subsequently evaluated for anxiety-like behaviours on the elevated plus maze, acoustic startle response, and holeboard apparatus. Blood was collected to examine corticosterone responses to stress and behavioural testing in adulthood. Animals from both treatment groups which exhibited the anxiety-like phenotype were bred with untreated partners. Maternal care of the second generation (F2) was monitored over the first week of life. In adulthood, the F2 generation underwent identical testing procedures as the parental (F1) generation. The F2 offspring of females exposed to LPS as neonates exhibited an anxiety-like phenotype in adulthood and a potentiated corticosterone response to stress (p < .05). F2 offspring of males exposed to LPS as neonates also exhibited an anxiety-like phenotype (p < .05), however, no differences in corticosterone responses were observed. To determine the impact of maternal care on the anxiety-like phenotype, a cross-fostering study was conducted in which offspring of LPS-treated females were fostered to saline-treated mothers and vice versa, which was found to reverse the behavioural and endocrine phenotypes of the F2 generation. These data indicate that a neonatally bacterially induced anxiety phenotype is transferable across generations in both sexes. Maternal care is the mediating mechanism along the maternal line. We suggest that transmission may be dependent upon heritable epigenetic phenomena for the paternal line. The implications of this study apply to potential neuroimmune pathways through which psychopathology may be transmitted along filial lines.

Evidence against oppositional and pharmacokinetic mechanisms of tolerance to diazepam's sedative effects

Acute administration of diazepam (2 mg/kg i.p.) to rats decreased the number of head-dips, locomotor activity and the number of rears made in the holeboard apparatus, indicating sedative effects. After daily treatment for 7 days with diazepam (2 mg/kg) tolerance developed to all these behavioural effects, despite serum concentrations of diazepam and N -desmethyldiazepam significantly higher than those following acute treatment. After 7 drug-free recovery days the rats were quite unresponsive to a probe dose of diazepam (2 mg/kg) and although there was a gradual recovery of responsiveness to diazepam, the reduction in rears still did not reach the level of the acute group even after 21 drug-free days. There was evidence for pharmacokinetic changes when probe doses of diazepam were given after 7, 14 or 21 recovery days. Lower levels of diazepam and higher levels of N -desmethyldiazepam than following an acute dose to the drug-naive group were detected, indicating that the chronic treatment had resulted in a persistently enhanced rate of N -demethylation. It is argued that these changes do not fully account for the reduced responsiveness to the probe doses, and nor can they account for the gradual return of response over the 3-week recovery period. There were no detectable serum concentrations of either compound 24 h after the end of the chronic treatment. However, no rebound increases in behavioural responses were detected at any time-point in withdrawal. Thus, the mechanism underlying this behavioural tolerance was not oppositional in nature. It is suggested that a situation-independent learned behavioural strategy is the most likely mechanism for the observed tolerance.

Evidence against oppositional and pharmacokinetic mechanisms of tolerance to diazepam's sedative effects

Acute administration of diazepam (2 mg/kg i.p.) to rats decreased the number of head-dips, locomotor activity and the number of rears made in the holeboard apparatus, indicating sedative effects. After daily treatment for 7 days with diazepam (2 mg/kg) tolerance developed to all these behavioural effects, despite serum concentrations of diazepam andN-desmethyldiazepam significantly higher than those following acute treatment. After 7 drug-free recovery days the rats were quite unresponsive to a probe dose of diazepam (2 mg/kg) and although there was a gradual recovery of responsiveness to diazepam, the reduction in rears still did not reach the level of the acute group even after 21 drug-free days. There was evidence for pharmacokinetic changes when probe doses of diazepam were given after 7, 14 or 21 recovery days. Lower levels of diazepam and higher levels ofN-desmethyldiazepam than following an acute dose to the drug-naive group were detected, indicating that the chronic treatment had resulted in a persistently enhanced rate ofN-demethylation. It is argued that these changes do not fully account for the reduced responsiveness to the probe doses, and nor can they account for the gradual return of response over the 3-week recovery period. There were no detectable serum concentrations of either compound 24 h after the end of the chronic treatment. However, no rebound increases in behavioural responses were detected at any time-point in withdrawal. Thus, the mechanism underlying this behavioural tolerance was not oppositional in nature. It is suggested that a situation-independent learned behavioural strategy is the most likely mechanism for the observed tolerance.

Behavioral effects of the putative anxiolytic (±)-1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane (ALEPH-2) in rats and mice

Behavioral effects of the phenethylamine derivative (±)-1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane (ALEPH-2) were studied in mice and rats. Murine locomotor activity, measured with a photocell actometer, was markedly depressed following IP injection of 2 and 6 mg/kg of the drug. The same doses of the drug also decreased frequency and duration of head dipping and the number of rearings in the hole board apparatus. In the murine elevated plus maze 2 and 6 mg/kg of ALEPH-2 increased the percentage of both open arm entries and time. The total number of entries into the enclosed arms was not significantly affected by the drug. In the rat, 2–12 mg/kg ALEPH-2, IP, decreased photobeam counts in the actometer in a dose-dependent fashion. Both 2 and 4 mg/kg of the drug increased the percentage of open arm entries, but only the highest dose significantly increased the percentage of time spent on the open arms. The dose of 4 mg/kg ALEPH-2 also significantly decreased the total number of enclosed arm entries. Finally, in a recently developed model of anxiety and memory, the elevated T-maze, the doses of 2 and 4 mg/kg ALEPH-2 did not change inhibitory avoidance of the open arms. Nevertheless, the highest dose had an amnestic effect on this task, repeated 72 h later in the absence of drug. In addition, this dose significantly increased the latency to escape from the open arms and had an amnestic effect measured 72 h later. Overall, these results indicate that ALEPH-2 possesses anxiolytic, amnestic as well as sedative and/or motor depressant actions.

Neonatal exposure to anti-nerve growth factor antibodies affects exploratory behavior of developing mice in the hole board.

The aim of this study was to assess in developing mice whether the neutralization of endogenous NGF following ICV administration of anti-NGF antibodies (50 micrograms/2 microliters) on postnatal days 3, 6, 9, and 12 affected locomotor activity, exploratory behavior, and response to the cholinergic blocker scopolamine. In Experiments 1 and 2 activity and age-typical scopolamine effects were evaluated on PND 13 or 17 in an automated apparatus. No significant main effect of anti-NGF treatment was found at either age. On day 13 scopolamine (0.2, 1, or 2 mg/kg) decreased locomotion in both anti-NGF and control animals. In Experiment 3, locomotion and exploratory behavior were analyzed in an open field arena or in a hole board apparatus on PND 16. No significant effects of anti-NGF treatment on general motor activity and investigation of a novel object in the open field was found, though anti-NGF animals tended to be less active than controls. In the hole board anti-NGF pups showed a different pattern of head dipping behavior from controls, exploring mainly the holes located in the periphery of the apparatus.

Sex-related effects on behaviour and β-endorphin of different intensities of formalin pain in rats

The effects of two intensities of formalin on behaviour and β-Endorphin (β-EP) concentration in the brain and pituitary were studied in male and female rats. The animals were familiarized with the Hole-Board apparatus for 3 days, and then, after a subcutaneous injection of formalin (50 μl, 0.1 or 10%) or Sham-injection (Control) in the hindpaw, they were tested in the Hole-Board for 60 min. Licking, Flexing and Paw-Jerk of the injected limb were recorded. β-EP concentration was determined in the hypothalamus (HYP), the periaqueductal gray matter (PAG), the anterior pituitary (AP) and the neurointermediate lobe (NIL). Licking and Flexing durations were greater in females than males only with formalin 10%. Sex differences in β-EP concentration between the Control groups were found in all tissues except the HYP; β-EP levels were higher in females in the PAG and NIL, but greater in the AP in males. β-EP concentration increased in males in the HYP and NIL with formalin 10%; in females, a decrease was found in the HYP with formalin 0.1%. The present results suggest that: (a) there are differences between males and females in the responses to formalin pain, and the nature (pattern and duration) of the sex differences varies according to the pain intensity; (b) there are differences in β-EP concentration between the two sexes in control animals, and male and female rats also exhibit differences in the modifications of β-EP in response to formalin-induced pain.

Antisense oligonucleotide inhibition of tryptophan hydroxylase activity in mouse brain

To examine in vivo effectiveness of antisense oligonucleotides against tryptophan hydroxylase (TPH) mRNA, adult male swiss-NIH mice were implanted with in-dwelling cannula into the 4th ventricle and after recovery infused with either antisense oligonucleotide to TPH, scrambled control oligo or saline vehicle for four consecutive days. An additional group of animals bearing cannula were injected a single time i.p. with the TPH inhibitor para-chlorophenylalanine (PCPA; 300 mg/kg). All animals were sacrificed on the afternoon of the 4th day of treatment. TPH activity was measured by enzymatic assay and HPLC quantification of end-product synthesis. There was a significant decrease (> 50%) in TPH activity in both the PCPA-treated and antisense-oligo infused animals compared to either scrambled-oligo or saline-infused subjects (ANOVA; P < 0.05). There was no difference between saline and scrambled oligo-infusion. In a separate group of animals treated in the same way, behavioral tests were conducted on the afternoon of the 4th day. Two tests of anxiety, the hole-board apparatus and the elevated plus-maze, indicated some significant effects of PCPA treatment and/or antisense oligo-infusion but confounding effects due to alterations in locomotion could not be ruled out. However, tests on a rotorod apparatus indicated that antisense oligo-infused animals retained good balance and coordination in that their performance significantly improved on the second test, as did that of scrambled-oligo infused animals. In contrast, PCPA-treated animals did not improve, suggesting that locomotor performance had been impaired. These data support the notion that antisense oligo blockade may offer advantages over pharmacological manipulations of enzyme activity.

Dietary Iron and Exposure to Lead Influence Susceptibility to Seizures

Previous work in this laboratory showed that concurrent consumption of an iron-deficient diet and exposure to lead caused seizure activity in Albany heterogeneous (HET) stock mice. In the present investigation, 26 Albany HET mice (ages 35 to 57 days of age) ate either an American Institute of Nutrition approved iron-sufficient (30 ppm) diet or an iron-deficient (less than 3 ppm) diet and drank either a 0.5% lead solution or distilled water for 12 weeks. We measured several activities in an open-field, hole-board apparatus, and spontaneous seizures which occurred during testing, changes in body weight, and hemoglobin levels. Replicating previous findings, mice fed the iron-deficient diet and treated with lead had more seizures with longer durations and longer postictal periods than animals given the iron-sufficient diet but also treated with lead. Mice not exposed to lead did not seize. Both lead-treated groups had lower rates of body-weight gain over the 3-mo. period and lower hemoglobin values than nonlead-treated animals. Changes in activity were observed in the open field as a function of diet and exposure to lead.

Chronic administration of flumazenil increases life span and protects rats from age-related loss of cognitive functions: A benzodiazepine/GABAergic hypothesis of brain aging

Under barrier condition and with ad lib access to food and water, 20 Fischer-344 rats were chronically treated for 10 months with the benzodiazepine (BDZ) antagonist, flumazenil (FL; 4 mg/kg/day in drinking water acidified to pH = 3.0), beginning at the age of 13 months, while the group of 20 control age-matched rats received plain acidified water. The life span of the first 8 deceased rats treated with FL was significantly longer than that of the first 8 deceased rats in the age-matched control group. In tests for spontaneous ambulation and exploratory behavior in the Holeboard apparatus, conducted during the 3rd and the 8th month of treatment, the FL group, relative to controls, had significantly higher scores for the ambulation and exploratory behavior. In tests for unrewarded spontaneous alternation in the T maze, conducted at days 7, 39, 42, and 47 through 54 after drug withdrawal, i.e., at the age of 24–25 months, the FL-exposed group, compared to age-matched controls, showed a significantly higher percent of alternating choices, a behavior that was statistically comparable to that of the “young” 6-month-old controls. In the Radial Maze tests conducted 2 months after drug withdrawal, the FL group made significantly less “working memory” errors and “reference memory” errors, relative to the age-matched 25-month-old control group, a performance that was comparable to that of the young 7-month-old control group. In conclusion, chronic FL significantly protected rats from age-related loss of cognitive functions. It is postulated that the age-related alterations in brain function may be attributable to the negative metabolic/trophic influences of the “endogenous” benzodiazepine (BDZ) ligands and/or those ingested with food. A BDZ/GABAergic hypothesis of brain aging has been formulated which assumes that age-related and abnormally strong BDZ/GABAergic influences promote neurodegeneration by suppressing trophic functions of the aminergic and peptidergic neurons through opening of chloride channels in soma membrane and axon terminals, causing excessive hyperpolarizing and depolarizing inhibition, respectively. The review of human clinical and animal data indicates that FL has nootropic actions by enhancing vigilance cognitive and habituation processes.

Decreased platelet aggregability in rats after immobilization stress

AbstractIn August rats, a strain highly susceptible to emotional stress, 24 h immobilization stress (IS) decreased the rate and amplitude of platelet aggregation induced by collagen (2–4 m̈g/ml) or ADP (0.25–5.0 m̈M) in platelet‐rich plasma, but had no effect on ATP release reaction and calculated peripheral blood platelet counts. Platelet aggregability was unchanged in groups deprived of food and water for 24 h or subjected to a 6 h IS. Only after 24 h IS were the effects of collagen and low concentrations of ADP potentiated by preincubation with epinephrine (1 m̈M). The IS‐induced decrease of platelet aggregation significantly correlated with the thymus involution, and both effects were most pronounced in rats displaying low scores of activity in the hole–board apparatus. The data indicate that behavioural variables should be taken into account in the analysis of stress‐induced changes in platelet functions, and possibly of any other stress response.

Peripheral olfactory deafferentation of the primary olfactory system in rats using ZnSO 4 nasal spray with special reference to maternal behavior

A modified method of applying ZnSO 4 to the olfactory mucosa is described. Treated rats experienced severe nasal congestion that cleared within 24 h; more persistent morbidity did not occur. Nonpregnant females observed with male intruders 24 h following ZnSO 4 showed no alterations in behavior other than a reduction in anogenital sniffing, indicating that they were not hypoactive or irritable. In other experiments, lactating females were observed in a hole-board apparatus; 2 days posttreatment anosmia was confirmed in 80% of bilaterally ZnSO 4-treated females by the absence of preference for pup odors. After bilateral but not unilateral ZnSO 4 treatment, initially activity scores and nose pokes were equivalent in all groups, but later they both were lower than in controls, probably due to a more rapid habituation to the novel apparatus. We conclude that intranasal ZnSO 4 by small-volume spray is a useful experimental tool.