Larry D. Sanford

Home cage activity and behavioral performance in inbred and hybrid mice

Locomotor activity is a key component in many behavioral tests, suggesting that genetic differences in activity levels may be a critical consideration when comparing mouse strains. In order to assess the relationship between activity and performance, we recorded home cage activity, and locomotion and defecation, a non-activity-linked behavior, in tests of anxiety in inbred (C57BL/6J (B6), n = 25; BALB/cJ (C), n = 24; DBA/2J (D2), n = 28) and hybrid (CB6F1/6J (CB6: B6 x C) n = 19) mice. Under our test conditions, the strains showed significant differences in home cage activity levels: C > B6 > D2. The CB6 mice were similar to the B6 mice in horizontal activity and were intermediate between the parental strains in vertical movement. Based on measures of locomotion and defecation in the open field, emergence and novel object tests, and the elevated zero maze, the C mice appeared to be the most anxious and the B6 were the least anxious. The D2 mice were intermediate on some measures but more similar to B6 mice on others, making ranking them more difficult. In addition, the CB6 mice displayed characteristics of both parental strains. They had greater similarity to B6 mice in measures of horizontal movement in the home cage and locomotion in the open field and emergence tests, but exhibited defecation responses similar to those of C mice in the novel object test and elevated zero maze. The results suggest that strain differences in spontaneous locomotion should be considered when interpreting strain differences in behavioral tests, and that home cage activity may be a useful interpretive aid.

Serotonin at the Laterodorsal Tegmental Nucleus Suppresses Rapid-Eye-Movement Sleep in Freely Behaving Rats

Serotonin [5-hydroxytryptamine (5-HT)] is believed to play an important inhibitory role in the regulation of rapid-eye-movement (REM) sleep. 5-HT may exert this effect on neurons of the laterodorsal tegmental (LDT) nuclei that are implicated as important in the generation of REM sleep and phasic REM events such as ponto-geniculo-occipital (PGO) waves and respiratory variability. In rat brainstem in vitro, 5-HT hyperpolarizes and inhibits the bursting properties of LDT neurons assumed to be involved in generating REM sleep and PGO waves. This study tests the hypothesis that in vivo 5-HT at the LDT nuclei suppresses REM sleep and phasic REM events. Ten rats were implanted with bilateral cannulae aimed at the LDT and with electrodes for recording the electroencephalogram, neck electromyogram, PGO waves, and diaphragm electromyogram. During REM sleep, 5-HT (100 nl; 1–1.5 mm), saline, or sham microinjections were performed; repeated microinjections were separated by ∼1 hr. After the first microinjection, REM sleep as a percent of the total sleep time was reduced with 5-HT (mean percent REM, 19.9 ± 2.5% for 5-HT vs 26.8 ± 2.4% for saline; p = 0.02). REM duration was reduced by 37% with 5-HT (p = 0.01), but REM episode frequency was changed less consistently (p = 0.21), suggesting that 5-HT mainly disrupted REM sleep maintenance. Per unit time of REM sleep, 5-HT had no effect on the amount or variability of REM PGO activity (p > 0.740) or on the mean or coefficient of variation of REM respiratory rate (p > 0.11). With subsequent microinjections, the effects of 5-HT on REM sleep were similar. A dose-dependent REM sleep suppression with 5-HT was observed in five rats tested. These data suggest that in vivo 5-HT at the LDT nuclei suppresses REM sleep expression. Although 5-HT did not disproportionately reduce the occurrence of phasic events within REM, total REM phasic activity was reduced because of less REM sleep after 5-HT.

Influence of shock training and explicit fear-conditioned cues on sleep architecture in mice: strain comparison.

Fear conditioning is thought to model anticipatory anxiety. Inbred mouse strains exhibit different levels of reactivity to aversive environmental stimuli, which may reflect anxiety. We examined the effects of fear conditioning on sleep in mouse strains that differ on behavioral measures of anxiety. Mice (BALB/cJ [C], C57BL/6J [B6], CB6F1/J [CB6], n = 7–10 per strain) were implanted with transmitters for recording sleep by telemetry. Baseline sleep was recorded, and the mice were trained to associate a cue (tone) with footshock (15 cue–shock pairings on 4 consecutive days). Sleep was recorded after shock training and again 4 to 5 days later after presentation of the cue alone. Shock training produced a relatively selective suppression of rapid eye movement sleep (REM) that was greater in the C strain compared to the B6 and CB6 mice. Post-training exposure to the cue alone suppressed REM in all strains. The C strain exhibited a relatively greater immediate suppression of REM, and the CB6 hybrid mice showed the greatest overall suppression of REM. These data demonstrate that stimuli associated with an aversive event can alter sleep and suppress REM in much the same way as exposure to the event itself. Fear conditioning may provide a model for examining genetic and neural mechanisms underlying the influence of anxiety on sleep.

Activation of a distinct arousal state immediately after spontaneous awakening from sleep.

In contrast to the many neural studies into the mechanisms of sleep onset and maintenance, few studies have focused specifically on awakening from sleep. However, the abrupt electrographic changes and large brief cardio-respiratory activation at awakening suggest that a distinct, transiently aroused, awake state may exist compared to later wakefulness. To test this hypothesis we utilized the acoustic startle reflex, a standard un-conditioned reflex elicited by a sudden loud noise. This reflex is modulated under specific conditions, one being a diminution of startle when a quieter pre-stimulus is presented immediately before the loud stimulus. This pre-pulse inhibition (PPI) is used as a measure of sensorimotor gating, with smaller PPI indicating less filtering of sensory inputs and increased responsiveness to external stimuli. Eight rats with electrodes for recording sleep-wake state were studied. An accelerometer measured startle responses. The startle reflex was elicited by 115 dB, 40 ms tones. PPI was produced by 74 dB, 20 ms tones preceding the 115 dB tone by 100 ms. Responses within 100 ms were measured. Stimuli were applied either 3-10 s after spontaneous awakenings, or in established wakefulness (> 30 s). Responses to the startle stimuli alone were similar in the different awake states (P = 0.821). However, PPI was smaller at awakening from non-REM sleep compared to established wakefulness (45.4 +/- 7.5% vs. 74.3 +/- 6.1%, P = 0.0002). PPI after awakening from REM sleep (52.8 +/- 17.9%) was not significantly different than established wakefulness (P = 0.297). Reduced PPI of the startle reflex at awakening from non-REM sleep supports the hypothesis that wakefulness immediately after spontaneous sleep episodes is neurophysiologically distinct from later wakefulness and associated with reduced gating of motor responses to sensory inputs. Spontaneous activation of this distinct, transiently aroused, state upon awakening may serve a protective function, preparing an animal to respond immediately to potentially threatening stimuli.

Central administration of two 5-HT receptor agonists: Effect on REM sleep initiation and PGO waves

Cholinergic neurons in the pedunculopontine tegmental (PPT) and the laterodorsal tegmental (LDT) nuclei are implicated in the generation of rapid eye movement sleep (REM) and ponto-geniculo-occipital (PGO) waves. Serotonin (5-HT) has a role in sleep-wake regulation and appears to inhibit PGO wave generation. We studied the effects of the central infusion of the relatively specific 5-HT1A receptor agonist 8-hydroxy-2-(n-dipropylamino)tetralin (DPAT) and the less specific 5-HT1 receptor agonist 1(3-chlorophenyl)piperazine (mCPP) on the regulation of REM and on PGO wave generation. DPAT (0.0, 0.002, 0.01, 0.08, and 0.8 microgram/0.5 microliter normal saline) and mCPP (0.0, 0.02, 0.2, 2.0, and 20.0 micrograms/0.5 microliter normal saline) were infused unilaterally into the peribrachial region of PPT (PB) in cats. Additionally, DPAT (0.01 microgram/0.5 microliter) was infused bilaterally into PB in a separate experiment. Low dosages of DPAT (unilateral or bilateral) decreased successful entrances into REM (0.01 microgram) and time spent asleep (0.002 microgram and 0.01 microgram) without affecting outward behavior. No dosage of mCPP significantly decreased the number of REM episodes, and neither drug decreased REM episode duration once REM had been entered. Neither drug affected the rate of PGO waves independently of modulating behavioral state. We propose that 5-HT1A receptor mechanisms have an inhibitory role in actual REM initiation, possibly by facilitating endogenously generated excitation of brainstem startle mechanisms at the onset of REM.

Low (20 cGy) Doses of 1 GeV/u 56Fe-Particle Radiation Lead to a Persistent Reduction in the Spatial Learning Ability of Rats

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. 56Fe. In previous ground-based experiments, exposure to high doses of HZE-particle radiation induced pronounced deficits in hippocampus-dependent learning and memory in rodents. Recent data suggest that glutamatergic transmission in hippocampal synaptosomes is impaired after low (60 cGy) doses of 1 GeV/u 56Fe particles, which could lead to impairment of hippocampus-dependent spatial memory. To assess the effects of mission-relevant (20–60 cGy) doses of 1 GeV/u 56Fe particles on hippocampus-dependent spatial memory, male Wistar rats either received sham treatment or were irradiated and tested 3 months later in the Barnes maze test. Compared to the controls, rats that received 20, 40 and 60 cGy 1 GeV/u 56Fe particles showed significant impairments in their ability to locate the escape box in the Barnes maze, which was manifested by progressively increasing escape latency times over the 3 days of testing. However, this increase was not due to a lack of motivation of the rats to escape, because the total number of head pokes (and especially incorrect head pokes) remained constant over the test period. Given that rats exposed to X rays did not exhibit spatial memory impairments until >10 Gy was delivered, the RBE for 1 GeV/u 56Fe-particle-induced hippocampal spatial memory impairment is ∼50. These data demonstrate that mission-relevant doses of 1 GeV/u 56Fe particles can result in severe deficits in hippocampus-dependent neurocognitive tasks, and the extreme sensitivity of these processes to 1 GeV/u 56Fe particles must arise due to the perturbation of multiple processes in addition to killing neuronal cells.

Home cage activity and activity-based measures of anxiety in 129P3/J, 129X1/SvJ and C57BL/6J mice.

We investigated the home cage activity and emotional behavior in mouse strains used as background for many studies of altered genes [C57BL/6J (B6, n=20), 129X1/SvJ (X1, n=20) and 129P3/J (P3, n=19)]. In their home cages, X1 and P3 mice exhibited less locomotion than did B6 mice, and the X1 mice showed significantly greater rearing than B6 and P3 mice did. A battery of three tests conducted in an open field (open field, emergence and novel object) revealed strain rankings of B6>X1>P3 or B6>X1=P3 in most activity variables. Significant correlations were found between home cage activity and activity in each of three tests, but not in all observation periods. Strain rankings on the elevated zero maze test were B6=X1>P3 in the number of stretched-attend body postures (SAPS) during the initial 6-min exposure for naive mice. Naive and nonnaive mice showed significantly different behaviors on the elevated zero maze. The results suggest that rankings on anxiety are P3>X1>B6 and that B6 mice have greater exploration in a novel environment compared with X1 and P3 mice. However, anxiety-like behaviors differed among strains in open-field-based tests and in the zero maze, and testing experience impacted performance on the zero maze. The findings illustrate that test variations and experience can influence performance and suggest the need for the consideration of how these factors interact with background strains in assessing gene-altered mice.

Sleep after differing amounts of conditioned fear training in BALB/cJ mice.

Shock training and auditory cues associated with shock produce alterations in sleep that can be long-lasting in BALB/cJ (C) mice. We examined sleep in C mice after different amounts of shock training, and after cues with different strength cue-shock associations. Mice were implanted with transmitters for determining sleep via telemetry. After baseline sleep recording, the mice were trained (between 08:00 and 09:00 h) to associate a cue (tone) with footshock in either single shock training (SST: a single tone-shock pairing) or multiple shock training (MST: 15 tone-shock pairings) conditions. For testing, the mice were presented 15 cues (tone only) in their home cage between 10:45 and 11:00 h on post-training days 6, 13, 20, 27, and 34 (Cue 1 to Cue 5) following shock training. Sleep was recorded for two days after shock training or cue presentation. A separate group of mice received 15 tone-shock pairings and had their sleep recorded for 10 consecutive uninterrupted days. Both SST and MST mice showed decreases in rapid eye movement sleep (REM) after shock training, with the larger effect in the MST mice. Only MST mice showed significant reductions in REM in response to the fearful cues, and longer-term alterations in sleep could be observed even on the day after cue presentation. These results indicate that reminders of an aversive event can impact sleep for prolonged periods, and that the degree of the impact varies with amount of training.

Effects of tetrodotoxin (TTX) inactivation of the central nucleus of the amygdala (CNA) on dark period sleep and activity

The amygdala has been implicated in emotional arousal and in the regulation of sleep. Previously, we demonstrated that tetrodotoxin (TTX), a sodium channel blocker that temporarily inactivates neurons and tracts, microinjected into the central nucleus of the amygdala (CNA) during the light period significantly reduced REM, shortened sleep latency, and increased EEG delta power in rats. TTX inactivation of CNA also reduced activity in the open field. These findings suggest that the amygdala modulates arousal in a variety of situations. To test the hypothesis that the amygdala may influence spontaneous arousal, we examined the effects of TTX inactivation of CNA on sleep and activity during the dark period when rats show higher arousal and less sleep. EEG and activity were recorded via telemetry in Wistar rats (n = 8). Bilateral microinjections of TTX (L: 2.5 ng/0.1; H: 5.0 ng/0.2 microl) or SAL (saline, 0.2 microl) were administered before lights off followed by recording throughout the 12-h dark period and following 12-h light period. Microinjections were given at 5-day intervals and were counterbalanced across condition. TTX significantly shortened sleep latency, increased NREM time, decreased REM time, and decreased activity. TTX increased NREM episode duration, whereas the number and duration of REM episodes were decreased. The present results indicate that TTX inactivation of CNA can increase NREM time when spontaneous arousal is high, suggesting a broad role for the amygdala in regulating arousal. The results suggest that understanding the ways in which the amygdala modulates arousal may provide insight into the mechanisms underlying altered sleep in mood and anxiety disorders.

GABAergic regulation of the central nucleus of the amygdala: implications for sleep control

It is becoming established that the amygdala has a strong influence on arousal state, with most evidence indicating a role in the regulation of rapid eye movement sleep (REM). Electrically activating the central nucleus of the amygdala (CNA) can increase subsequent REM and enhance REM-related phenomena. However, drugs that may be inhibitory to CNA have been typically reported to reduce REM. This suggests that enhancing activity in CNA could promote REM, and that inhibiting activity in CNA could suppress REM. We reversibly inactivated CNA using the GABA(A) agonist, muscimol, or blocked GABAergic inhibition with the GABA(A) antagonist, bicuculline, and examined the effects on sleep and wakefulness. Rats (90-day-old male Sprague-Dawley) were implanted with electrodes for recording EEG and EMG. Cannulae were aimed into CNA for microinjecting muscimol (0.001, 0.3 and 1.0 microM/0.2 microl saline) or bicuculline (56 and 333 pM/0.2 microl saline). Each animal received bilateral microinjections of muscimol, bicuculine or saline alone followed by 6-h sleep recordings. Microinjections of low concentrations of muscimol into CNA produced relatively selective decreases in total REM and number of REM episodes that lasted up to 6 h. In contrast, microinjections of bicuculline into CNA produced significant increases in REM. There were no significant reductions in NREM or wakefulness. These findings demonstrate that inactivating CNA can produce a relatively selective suppression of REM. The possible role that spontaneous activity in CNA may play in REM initiation and/or maintenance is discussed.