Time Of Behavioral Testing Research Articles

Adult Female Rats Exposed to Adolescent Chronic Restraint Stress (aCRS) Display Robust Depressive‐Like Behavior, Cognitive Impairment, and an Unexpected Correlation with Serum BDNF

IntroductionDepression is a mood disorder that disproportionately affects women, and is often predicated by childhood or adolescent stress. Symptoms of depression include persistent sadness and cognitive impairment, which have been correlated with low serum brain‐derived neurotrophic factor (BDNF), and with decreased hippocampal (HIP) volume. Our lab has previously used a novel method of adolescent chronic restraint stress (aCRS) to elicit robust depressive‐like behaviors in freely cycling female Sprague‐Dawley rats. Chronic desipramine (DES), a tricyclic antidepressant, decreased depressive‐like behaviors in aCRS rats. To estimate estrus cycle at time of behavioral testing, we removed and weighed the uteri (UTI) immediately after sacrifice, and found no differences among groups.ObjectiveThe objective of this study was to evaluate behaviors and biomarkers in female rats exposed to aCRS, DES, or aCRS+DES.Methods25adolescent female Sprague‐Dawley rats (Charles River, Kingston, NY) arrived post‐natal day (PND) 26 and habituated to the colony room for 7 days prior to the experiment. Rats were randomly assigned to Not Restrained‐Saline (NRSAL, n=6), Not Restrained Desipramine (NRDES, n=6), Restrained‐Saline (RSAL, n=6), or Restrained‐Desipramine (RDES, n=7). Beginning PND 34±1RSAL and RDES rats were restrained at random times for 60 mins/day for 14consecutive days. The final restraint session was followed by 7 days of maturation. Beginning PND 55±1, NRSAL and RSAL rats received subcutaneous (SC) saline injections (0.09% NaCl; 1 mL/kg) and NRDES and RDES rats received SC desipramine in saline solution (5 mg/kg, 1 mL/kg) daily for 14consecutive days. Behavioral testing commenced PND 70±1 and included, in order, locomotor activity (LCA), novel object recognition (NOR), and a 5‐minute forced swim test(FST). Fifteen minutes following the FST, the rats were sacrificed via rapid decapitation. Serum from trunk blood was separated using serum separating tubes, and the HIP and uteri (UTI) were harvested and weighed. Serum was evaluated for BDNF with ELISA (Boster Bio, Pleasanton, CA). All measures were evaluated with two‐way ANOVA and Holm‐Sidak post hoc, and inter‐measure relationships were evaluated with Pearson correlation analysis.ResultsNo differences were observed in locomotor activity, HIP weight, or UTI weight among groups.During NOR, RSAL rats showed significantly impaired discrimination between novel and familiar objects than NRSAL rats (p=0.011). NRDES and RDES were not different from NRSAL.In FST, RSAL rats were significantly more immobile than all other groups (p=0.011). Specifically, RSAL vs NRSAL (p=0.002), RSAL vs RDES(p=<0.001), and RSAL vs NRDES (p=0.029). NRDES and RDES were not different from NRSAL.Using Pearson correlation analysis, immobility was positively correlated with serum BDNF (r=0.54; p=0.006). No other significant correlations were observed.ConclusionsThese results indicate that aCRS elicits robust depressive‐like behavior and cognitive impairment in freely cycling female rats, effects which are ameliorated by chronic DES. Additionally, the unexpected relationship observed between serum BDNF and immobility requires further investigation.Support or Funding InformationMercer University

Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice.

Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient–rather than permanent–inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreERT2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12–30 days post-TAM displayed indices of a stress-induced anxiety phenotype–longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype–longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a depressive phenotype, and recovery of DCX+ cell number corresponds to normalization of these behaviors.

The potential and limitations of DOV 216,303 as a triple reuptake inhibitor for the treatment of major depression: A microdialyis study in olfactory bulbectomized rats

DOV 216,303 belongs to a new class of antidepressants, the triple reuptake inhibitors (TRIs), that blocks serotonin, norepinephrine and dopamine transporters and thereby increases extracellular brain monoamine concentrations.The aim of the present study was to measure extracellular monoamine concentrations both in the prefrontal cortex (PFC) and dorsal hippocampus (DH) after chronic administration of DOV 216,303 in the OBX animal model of depression and to compare the effects with acute drug treatment.OBX animals showed lower dopamine levels in PFC upon acute administration of DOV 216,303 than sham animals for up to five weeks after surgery. No such changes were observed in the DH. Unexpectedly, a DOV 216,303 challenge in chronic DOV 216,303 treated sham animals resulted in a blunted dopamine response in the PFC compared to the same challenge in vehicle treated animals. This blunted response probably reflects pharmacokinetic adaptations and/or pharmacodynamic changes, since brain and plasma concentrations of DOV 216,303 were significantly lower after chronic administration compared to acute administration.Surprisingly, and in contrast what we have reported earlier, chronic DOV 216,303 treatment was unable to normalize the hyperactivity of the OBX animals. Interestingly, by measuring the drug plasma and brain levels, it was demonstrated that at the time of behavioral testing (24h after last drug treatment) DOV 216,303 was not present anymore in either plasma or brain. This seems to indicate that this putative antidepressant drug has no lasting antidepressant-like behavioral effects in the absence of the drug in the brain.

Effects of progesterone on male-mediated infant-directed aggression

Many species that engage in parental behavior exhibit infanticide under certain circumstances. The neural signals regulating the transition from infant care giver to infant killer and back remain unclear. Previously we demonstrated that progesterone (P) and its receptor (PR) have inhibitory effects on parental behavior and increase infant-directed aggression in male mice. In the present studies we sought to elucidate the mechanisms by which the effects of P are manifested. Because the onset of parental behavior in females is associated with the withdrawal of P at the end of pregnancy we tested the hypothesis that withdrawal of P would similarly enhance parental behavior in males. Virgin male mice were implanted with P or vehicle for 21 days, replicating the duration of pregnancy in females. Tests were run for parental and infanticidal behavior 5 days after removal of the capsules. P increased the proportion of nonparental males that attacked pups. However, neither the number of males exhibiting parental care nor the quality of care was affected by P treatment. Serum P and testosterone (T) levels were not different from controls at the time of behavioral testing indicating continued elevations in peripheral hormones are not required for the expression of infanticide. In conclusion, withdrawal of P does not trigger the onset of parental behavior in males. Rather, prior exposure to P induces persistent infanticidal behavior in adult male mice.

Intact neurogenesis is required for benefits of exercise on spatial memory but not motor performance or contextual fear conditioning in C57BL/6J mice

The mammalian hippocampus continues to generate new neurons throughout life. Experiences such as exercise, anti-depressants, and stress regulate levels of neurogenesis. Exercise increases adult hippocampal neurogenesis and enhances behavioral performance on rotarod, contextual fear and water maze in rodents. To directly test whether intact neurogenesis is required for gains in behavioral performance from exercise in C57BL/6J mice, neurogenesis was reduced using focal gamma irradiation (3 sessions of 5 Gy). Two months after treatment, mice (total n=42 males and 42 females) (Irradiated or Sham), were placed with or without running wheels (Runner or Sedentary) for 54 days. The first 10 days mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. The last 14 days mice were tested on water maze (two trials per day for 5 days, then 1 h later probe test), rotarod (four trials per day for 3 days), and contextual fear conditioning (2 days), then measured for neurogenesis using immunohistochemical detection of BrdU and neuronal nuclear protein (NeuN) mature neuronal marker. Consistent with previous studies, in Sham animals, running increased neurogenesis fourfold and gains in performance were observed for the water maze (spatial learning and memory), rotarod (motor performance), and contextual fear (conditioning). These positive results provided the reference to determine whether gains in performance were blocked by irradiation. Irradiation reduced neurogenesis by 50% in both groups, Runner and Sedentary. Irradiation did not affect running or baseline performance on any task. Minimal changes in microglia associated with inflammation (using immunohistochemical detection of cd68) were detected at the time of behavioral testing. Irradiation did not reduce gains in performance on rotarod or contextual fear, however it eliminated gain in performance on the water maze. Results support the hypothesis that intact exercise-induced hippocampal neurogenesis is required for improved spatial memory, but not motor performance or contextual fear in C57BL/6J mice.

Effects of maternal intravenous nicotine administration on locomotor behavior in pre-weanling rats

Maternal tobacco use is associated with adverse developmental outcomes in offspring, including hyperactivity. Animal studies attempting to model this phenomenon have primarily used continuous s.c. nicotine infusion as the method of nicotine administration, which does not model the intermittent bolus delivery of nicotine associated with smoking in humans. The purpose of the present experiment was to examine the locomotor activity of pre-weanling offspring of pregnant rats exposed to an i.v. nicotine dosing protocol that approximates the pattern of nicotine exposure in moderate to heavy smokers. Pregnant rats were administered an i.v. bolus of 0.03 mg/kg nicotine ( N = 13) or saline ( N = 10) every 14 min for 16 h/day, resulting in a total daily dose of 2 mg/kg (base), from gestational day 4 to delivery. Pups from each litter were tested for spontaneous locomotor activity on postnatal days (PND) 19–21 and nicotine-induced locomotor activity on PND 22. Mean birth weight was significantly lower in nicotine-exposed pups compared to controls, but body weights were equivalent between groups by the time of behavioral testing. Mean total distance traveled, vertical counts, and stereotypy counts were lower on PND 19 in nicotine-exposed pups compared to controls, but only the difference in mean stereotypy counts was statistically significant. Within-session analysis revealed that both distance traveled and stereotypy were significantly decreased in nicotine-exposed pups in the first 5 min of the session on PND 19. Total time spent in the center of the field was also lower in nicotine-exposed pups. Nicotine-induced increases in activity on PND 22 did not differ according to gestational exposure. These findings demonstrate that prenatal nicotine exposure in a model that mimics the pattern of nicotine exposure from cigarette smoking in humans results in offspring that exhibit low birth weight and hypoactivity in a novel environment.

Neuroanatomic contributions to slowed orienting of attention in children with autism

Previous research has demonstrated that adult autistic patients are abnormally slow to orient attention, with degree of slowed orienting associated with severity of cerebellar hypoplasia. This research was extended to children who, at ages two through six, met diagnostic criteria for autism and underwent magnetic resonance imaging (MRI). An average of 3 years later, when old enough to participate in behavioral experiments, the children returned to the laboratory and completed a spatial attention paradigm. Degree of slowed attentional orienting to visual cues was significantly correlated with degree of cerebellar hypoplasia, but not with size of other neuroanatomic regions. Additionally, there was a trend for orienting speed to differ between diagnostic outcome subgroups; children with confirmed diagnoses of autism at time of behavioral testing had larger orienting deficits than those who no longer met diagnostic criteria for autism. This research is among the first to establish a specific brain-behavior link in autistic children.

Genetic Determinants of Morphine Activity and Thermal Responses in 15 Inbred Mouse Strains

Mice from 15 standard inbred strains were tested for sensitivity to two effects of acute morphine administration, open-field activity, and body temperature changes, at doses of 0, 4, 8, 16, and 32 mg/kg, IP. Large strain differences were consistently observed, indicating a substantial degree of genetic determination of these traits. For morphine-induced activity, some strains were markedly insensitive to all doses (e.g., C3H/He, CE), while others showed increases and some decreases at the same morphine dose. For thermal responses, one strain was insensitive to all doses employed (C3H/He), while others showed marked hypothermia and some hyperthermia at the same dose. Although strains differed in brain morphine concentrations at time of behavioral testing, pharmacokinetic differences were unrelated to both measures of morphine sensitivity. Correlations among strain means (estimates of genetic correlations) were rather high across doses within each measure, indicating that strain differences to a given effect of morphine were rather stable across doses. This suggests substantial commonality in genetically mediated mechanisms across the dose range used for activity, and also for thermal responses. In contrast, genetic correlations between activity and thermal responses were not significant at any dose, indicating that these two traits are largely genetically independent.

Subacute Pb Exposure During Development and Unbaited Tunnel Maze Performance in Mice

Although research has linked chronic, low-level Pb exposure to behavioral and cognitive changes in humans and animals, far less is known about the effects of transient, subchronic Pb exposure during early postnatal development. The need to understand such effects is underscored by the possibility that subchronic Pb exposure may not produce chronically elevated blood–Pb levels, but may produce long-term behavioral changes. To test this hypothesis, we investigated the effect of low-level Pb exposure on unbaited tunnel maze performance in Binghamton Heterogeneous Stock mice. Mice were either nontreated or given subchronic sodium acetate, 5, 10, or 25 mg/kg Pb acetate intragastrically on postnatal (PN) days 6, 9, 12, 15, and 18. No further Pb exposures were given after postnatal day 18. Blood–Pb measurements were taken from selected mice on PN 18, 19, 23, 28, and 38. On PN 38–42, all mice were individually tested in an unbaited tunnel maze under nondeprived conditions. Locomotor activity, exploration, and experience-dependent changes in cul-de-sac entries were recorded. Although Pb did not affect bodyweight and blood–Pb levels were below 10 μg/dl at the time of behavioral testing, a history of low-level preweaning Pb exposure caused a dose-dependent increase in cul-de-sac entries. This behavioral change was dissociable from changes in bodyweight, degree of exploration or an a priori bias to enter cul-de-sacs. The current results support the hypothesis that brief, subchronic Pb exposure during development produces behavioral changes that last well beyond the exposure period, even when blood–Pb declines to within “acceptable” levels (10 μg/dl).

A quantitative assessment of the effects of malnutrition on behavioral development in laboratory mice

The aim of this study was to compare the effects of malnutrition on behavioral development in mice with those due to heredity obtained in a previous study. To this end behavioral development was assessed using a developmental time scale which allowed comparison of the two studies. Pregnant B6D2F1/J mice were maintained on either a protein restricted or normal protein diet from Day 7 to Day 24 postconception, at which time the low protein group was returned to the normal diet until behavioral testing at Day 32. Behavioral development in the control group replicated the values obtained in previous studies, whereas the malnourished pups were retarded by 1.4 days. This was of the same order of magnitude as that reported previously for maternal inbreeding. Measures of maternal and pup body weight at birth, Day 24 and Day 32 indicated that although the malnourished animals were initially significantly lighter in weight they had recovered by Day 32. Litter size however, declined between birth and time of behavioral testing in the malnourished group.

Failure to replicate the dorsal bundle extinction effect: Telencephalic norepinephrine depletion does not reliably increase resistance to extinction but does augment gustatory neophobia

Depletion of telencephalic noradrenaline (Ne), caused by lesion of the dorsal tegmental bundle, has been reported to increase persistance of non-reinforced responding in various operant tasks. This has been referred to as the dorsal bundle extinction effect (DBEE). In an effort to reproduce this effect, rats receiving 6-hydroxydopamine lesions of the dorsal Ne bundle (DB-OHDA) were compared to controls during extinction of a continous food rewarded (CRF) lever-response. While the lesion group showed an increase in responding during initial extinction, no significant difference in resistance to extinction using a 2-min non-response criterion was obtained. Moreover, no differences in reinforced response rates were observed with CRF, fixed ratio (FR-15, FR-30, FR-60) or variable interval (VI-30, VI-60, VI-20 s) schedules of reinforcement. In order to test the hypothesis that the DBEE is dependent on time of behavioral testing after surgery, subsequent experiments were performed where rats began CRF operant training 5, 17, 31 or 110 days post-lesion. No differences in resistance to extinction were observed between lesion and control rats at any post-lesion interval. Neonatal treatment with 6-OHDA which permanently lesions forebrain Ne terminals also failed to prolong extinction. Finally, when both DB-OHDA and neonatal rats were given a choice between water and saccharine the lesioned animals exhibited a neophobic reaction whereby they drank significantly less saccharine. We conclude that while the DBEE is not a reliably reproducible phenomenon other effects of forebrain Ne lesions, such as neophobia, appear to be robust.

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E 2) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E 2, as was the combination of E 2 and DHT. The relative efficacy of a single dose of T, DHT, and E 2 plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E 2B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E 2, and E 2B suggests that an appreciable, but not necessarily sustained, elevation of E 2 levels in the brain may be critical in the facilitation of male copulatory behavior in mice.

P-Chloroamphetamine: Evidence against a serotonin mediated learning deficit in PKU

Rats were injected with 5.0 mg/kg of p-chloroamphetamine (PCA) or saline from 1–28 days of age. Groups treated with PCA showed hypoactivity in the open field at 29, 50 and 75 days of age when compared to rats treated with saline and facilitated avoidance in Y-maze acquisition at 57–61 days of age. At 36 and 68 days of age, at the time of behavioral testing, brain serotonin (5HT) was reduced slightly or not at all. However, in separate groups, brain 5HT was reduced 23% and 35% in 14 and 26 day-old rats after only a single treatment with PCA, suggesting that 5HT was reduced during the treatment period. At 66–68 days of age, 5HT turnover was also unaltered. These results suggest that the behavioral effects were due to neonatal 5HT depletion during a critical period of development rather than a concurrent 5HT depletion at the time of behavioral testing. Moreover, these data are at variance with the concept that neonatal 5HT depletion impairs learning ability and is the underlying CNS defect in phenylketonuria.

Brain cell number and motor activity in rats subjected to neonatal undernutrition

Rat pups were undernourished until weaning by raising them in oversized litters. After weaning they were fed ad lib . Control subjects were raised in normal-sized litters and fed ad lib . After weaning. They were all examined between 4 and 5 weeks of age in 3 behavioral tests, measuring various aspects of motor capabilities: orienting locomotor activity, rotarod performance, and activity wheel running. Following the completion of behavioral studies the animals were sacrificed and cerebral and cerebellar DNA (cell number) were assayed. Comparisons between control and undernourished groups revealed significant reduction in body weights in the undernourished subjects, both at weaning and at the time of behavioral testing. Cerebral and cerebellar DNA were reduced in the undernourished rats as compared to controls. No difference was noted between the groups in rotarod performance or activity wheel running. The orienting locomotor activity, however, was greater in controls than in undernourished subjects. When these one-hour activity counts were divided into periods, the early counts did not differ between groups, while the counts late in the hour were significantly less in the undernourished animals. Thus, while the high counts of the initial orienting response were unaffected by the state of nutrition, the decay with habituation occurred at a more rapid rate in the rats that were poorly nourished. Cerebellar levels of DNA were found to correlate with locomotor activity counts, but cerebral DNA levels did not. The findings suggest that cerebellar underdevelopment can influence behavior in subtle ways not clearly associated with deficiencies in control of skeletal muscle tone or coordination.

Septal lesions enhance shock avoidance behavior in the rat

King reported that rats with septal lesions showed quicker learning of a conditioned avoidance response than did control subjects. Our study extends these findings to Long-Evans hooded rats and a training procedure using a fixed intertrial interval of 1 min, a guillotine door separating the two compartments of the avoidance apparatus and a light instead of a buzzer as the CS. Also, subjects were trained 70 days after the septal lesions had been made. Because eight of the fifteen rats with septal lesions did not demonstrate the hyperemotionality syndrome at the time of behavioral testing but performed the conditioned avoidance response significantly better than the controls, it is concluded that the septal syndrome as here measured is not a necessary condition for the improved learning. These results can be accounted for if one assumes that septal lesions weaken fear-elicited, unconditioned responses, which usually compete with the instrumental avoidance response.