Startle Test Research Articles

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal's response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal's response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal's overall temperament.

Adolescent behavioral abnormalities in a Scn1a+/− mouse model of Dravet syndrome

Dravet syndrome is an intractable pediatric epilepsy associated with SCN1A mutations. In addition to having a large seizure burden and a reduced lifespan, patients with Dravet syndrome also exhibit delays in reaching normal developmental milestones in attentional, emotional, and cognitive function. These developmental delays manifest in autistic-like social withdrawal and compulsive behavior. Additionally, cognitive impairments including deficits in sensorimotor processing and memory function are present. Several mouse models utilizing heterozygous deletion of Scn1a (Scn1a+/− mice) have been generated that recapitulate many aspects of Dravet syndrome. Studies in these mouse models of Dravet syndrome have characterized behavioral phenotypes in adult mice. In the present study, we characterized the behavioral phenotype of Scn1a+/− mice generated by targeted deletion of Scn1a exon 1 (Scn1atm1Kea) during adolescence. Identifying behavioral deficits in adolescent mice would more closely model the early onset of attentional, emotional, and cognitive delays observed in patients with Dravet syndrome. The behaviors of adolescent Scn1a+/− and wildtype (WT) mice were compared across several behavioral domains. We assessed motor function (open-field test), sociability and social recognition memory (three-chambered social preference and social interaction tests), memory function (novel object recognition, Barnes maze, fear conditioning paradigm), anxiety-related behavior (elevated plus maze and open-field thigmotaxis), startle reflex and sensorimotor gating (prepulse inhibition of startle (PPI) tests), and repetitive compulsive behavior (marble burying test). Adolescent Scn1a+/− mice exhibited normal locomotor activity, marble burying behavior, sociability, and sensorimotor gating. However, adolescent Scn1a+/− mice displayed increased anxiety-related thigmotactic behavior, atypical fear expression, blunted acoustic startle responses, and impaired social recognition and spatial memory. Our results show that Scn1a+/− mice display various behavioral impairments during adolescence, which provides a foundation for testing early intervention therapies targeting developmental delays modeled in Dravet syndrome mice.

Relationship of prolonged acoustic startle latency to diagnosis and biotype in the bipolar-schizophrenia network on intermediate phenotypes (B–SNIP) cohort

BackgroundLatency of the acoustic startle reflex is the time from presentation of the startling stimulus until the response and provides an index of neural processing speed. Latency is prolonged in schizophrenia, is 90% heritable, and predicts conversion to schizophrenia in a high-risk population. The Bipolar-Schizophrenia Network for Intermediate Phenotypes (B–SNIP) consortium investigates neurobiological features found in psychotic disorders spanning diagnostic criteria for schizophrenia (SCZ), schizoaffective disorder (SAD), and psychotic bipolar disorder (BP). We investigated whether differences in startle latency and prepulse inhibition (PPI) occur in probands, their first-degree relatives, and neurobiologically defined subgroups of the probands (Biotypes). Methods1143 subjects were included from the B–SNIP cohort: 143 with SCZ, 178 SCZ relatives (SCZ-Fam), 123 with SAD, 152 SAD relatives (SAD-Fam), 138 BP, 183 BP relatives (BP-Fam), and 226 controls (CON). A Biopac system recorded the eyeblink component of the startle reflex during startle testing. ResultsLatency differed by diagnosis (F(3,620) = 5.10, p = 0.002): SCZ, SAD, and BP probands had slower latency than CON, with relatives intermediate. Biotypes 1 and 2 had slower latency than CON (p < 0.031) but Biotype 3 did not differ from CON. PPI did not separate CON from other subjects when analyzed by diagnoses nor when analyzed by biotype. Biotype 1 relatives had slower latency (F(3,663) = 3.49, p = 0.016) and more impaired PPI than Biotype 2 and 3 relatives (F(3,663) = 2.77, p = 0.041). ConclusionStartle latency is prolonged in psychotic disorders that cross traditional diagnostic categories. These data suggest a genetic difference between biotypes that span across clinically defined diagnoses.

Effect of mild and chronic neonatal hypothyroidism on sensory information processing in a rodent model: A behavioral and electrophysiological study

Thyroid hormones are essential for neonatal brain development. It is well established that absence of thyroid hormones during critical periods of development can alter sensory functions such as visual and auditory processing, but there are few studies on rat somatosensory system development at mild, long-term, and irreversible neonatal hypothyroidism. Thus, the current study was conducted to investigate whether chronic thyroid hormone insufficiency would alter different cognitive aspects of tactile information processing and sensory motor filtering at behavioral or neuronal levels. Neonatal Wistar male rats were exposed to 0 and 6 ppm of propylthiouracil for 150 days. Behavioral tests including tactile discrimination tests and acoustic startle reflex test were performed. Using extracellular single unit recording technique, barrel cortex neurons’ excitatory and inhibitory responses to controlled displacement of whiskers were evaluated. Results indicated that percentage of correct choice in tactile learning and discrimination of a new texture decreased in hypothyroid group compared to the control group (P < 0.05). In addition, acoustic startle reflex of hypothyroid group significantly decreased compared to the control rats when the prepulse intensity was 71 dB (P < 0.05). Data obtained from electrophysiological tests showed that spontaneous activity and response magnitude of barrel cortex neurons decreased in hypothyroid group compared to the control group (P < 0.05). It is concluded that, thyroid hormones can regulate tactile and auditory sensory processing in male rats, and mild and long-term absence of these hormones can result in deficiency in natural functions of these sensory systems.

Artificial rearing affects the emotional state and reactivity of pigs post-weaning

AbstractArtificial rearing involves removing piglets from their mother at seven days of age and feeding them milk replacer until weaning. Early-life rearing conditions can influence piglets’ mental development, as reflected by their emotional state and reactivity. This study compared the post-weaning emotional state and reactivity of pigs which were either sow-reared or artificially reared pre-weaning. Behavioural tests (startle test, novel object test, human-animal relationship test and open door test) were conducted one week post-weaning (weaner 1, 34 [± 0.6] days old), one week after movement to weaner 2 (69 [±1.2] days old) and to finisher (100 [± 1.3] days old) stages. Qualitative Behavioural Assessments (QBA) were conducted on the same days in weaner 2 and finisher stages. QBA descriptors were computed by PCA and all other data were analysed using linear models. Artificially reared pigs were less fearful of human contact in weaner 1 (45.1 [± 8.43] vs 81.3 [± 7.89]%) and finisher (25.8 [± 5.19] vs 45.7 [± 6.00]%) stages; but there was no difference in the other tests. Artificially reared pigs had a higher QBA score (more positive) than sow-reared pigs in weaner 2 (54.49 [± 10.102] vs 17.88 [± 9.94]) but not in finisher (70.71 [± 8.860] vs 52.76 [± 9.735]) stage. In conclusion, artificially reared pigs appeared to have a more positive emotional state transiently post-weaning and a lower fearfulness towards humans, which are likely mediated by their pre-weaning conditions. These data emphasise the need to consider the entire life of the animals to fully evaluate the long-term impacts of a rearing system.

Eye Blink Rates and Eyelid Twitches as a Non-Invasive Measure of Stress in the Domestic Horse.

Simple SummaryEye blink rate has been used as an indicator of stress in humans and, due to its non-invasive nature, could be useful to measure stress in horses. Horses exhibit both full and half blinks as well as eyelid twitches. We exposed 33 horses to stressful situations such as separation from herdmates, denied access to feed and sudden introduction of a novel object, and determined that full and half eye blinks decrease in these situations. Feed restriction was the most stressful for the horse as indicated by increased heart rate, restless behaviour and high head position. The decrease in eye blink rate during feed restriction was paralleled with an increase in eyelid twitches. There was no increase in eyelid twitches or heart rate with the other treatments indicating that the horses did not find these overly stressful, but they did focus their attention more during these situations. Observation of eye blinks and eyelid twitches can provide important information on the stress level of horses with a decrease in eye blinks and an increase in eyelid twitches in stressful environments.Physiological changes provide indices of stress responses, however, behavioural measures may be easier to determine. Spontaneous eye blink rate has potential as a non-invasive indicator of stress. Eyelid movements, along with heart rate (HR) and behaviour, from 33 horses were evaluated over four treatments: (1) control—horse in its normal paddock environment; (2) feed restriction—feed was withheld at regular feeding time; (3) separation—horse was removed from visual contact with their paddock mates; and (4) startle test—a ball was suddenly thrown on the ground in front of the horse. HR data was collected every five s throughout each three min test. Eyelid movements and behaviours were retrospectively determined from video recordings. A generalized linear mixed model (GLIMMIX) procedure with Sidak’s multiple comparisons of least squares means demonstrated that both full blinks (16 ± 12b vs. 15 ± 15b vs. 13 ± 11b vs. 26 ± 20a full blinks/3 min ± SEM; a,b differ p < 0.006) and half blinks (34 ± 15ab vs. 27 ± 14bc vs. 25 ± 13c vs. 42 ± 22a half blinks/3 min ± SEM; a,b,c differ p < 0.0001) decreased during feed restriction, separation and the startle test compared to the control, respectively. Eyelid twitches occurred more frequently in feed restriction (p < 0.0001) along with an increased HR (p < 0.0001). This study demonstrates that spontaneous blink rate decreases while eyelid twitches increase when the horse experiences a stressful situation.

19 Safety in numbers: Social isolation increases behavioral responses of cattle during startle tests

Abstract Social isolation confounds cattle fear tests. Fear tests often require individuals to be isolated from the herd, possibly eliciting other emotions (i.e. panic and grief) outside of fear. To test this, steer (n = 32) behavior during an individual startle test (IST) and a group startle test (GST) were video recorded. During IST, one steer was placed in a solid sided pen and, after 60 seconds, two umbrellas opened simultaneously. Behavior was video recorded for an additional four minutes. For GST, the same procedures were followed, except 4 familiar steers were in the pen simultaneously. Within each test, steers were evaluated in a random order. IST retesting (IST_2) occurred five weeks after the first IST (IST_1). The GST took place one week after IST_2 testing completed. Video recordings of IST_1, IST_2, and GST were decoded for frequency and duration of behavior (steps, standing bouts, time spent standing, eliminations). Relationships among behavior, test, and minute of test were analyzed using Generalized Linear Mixed Model (PROC MIXED). Minute and test affected number of steps (P &lt; 0.0001) and time spent standing (P &lt; 0.0001). During GST, steers performed fewer steps than in the IST_1 (P &lt; 0.0001) and IST_2 (P = 0.0003). There were no differences in step count between IST_1 and IST_2. Steers spent more time standing during GST than IST_1 (P &lt; 0.0001) and IST_2 (P = 0.004). No differences in time-spent standing were seen between IST_1 and IST_2. Number of standing bouts were impacted by test (P = 0.0008). Steers stood more during GST than in IST_1 (P = 0.0007) and IST_2 (PP = 0.03) with no differences observed between IST_1 and IST_2. The IST is repeatable, yet the strength of the behavioral response is reduced during GST. This is the first quantifiable representation of the impact of social isolation on the fear response in beef cattle.

115 Safety in numbers: Social isolation increases behavioral responses of cattle during startle tests

Abstract Social isolation confounds cattle fear tests. Fear tests often require individuals to be isolated from the herd, possibly eliciting other emotions (i.e. panic and grief) outside of fear. To test this, steer (n = 32) behavior during an individual startle test (IST) and a group startle test (GST) were video recorded. During IST, one steer was placed in a solid sided pen and, after 60 seconds, two umbrellas opened simultaneously. Behavior was video recorded for an additional four minutes. For GST, the same procedures were followed, except 4 familiar steers were in the pen simultaneously. Within each test, steers were evaluated in a random order. IST retesting (IST_2) occurred five weeks after the first IST (IST_1). The GST took place one week after IST_2 testing completed. Video recordings of IST_1, IST_2, and GST were decoded for frequency and duration of behavior (steps, standing bouts, time spent standing, eliminations). Relationships among behavior, test, and minute of test were analyzed using Generalized Linear Mixed Model (PROC MIXED). Minute and test affected number of steps (P &lt; 0.0001) and time spent standing (P &lt; 0.0001). During GST, steers performed fewer steps than in the IST_1 (P &lt; 0.0001) and IST_2 (P = 0.0003). There were no differences in step count between IST_1 and IST_2. Steers spent more time standing during GST than IST_1 (P &lt; 0.0001) and IST_2 (P = 0.004). No differences in time-spent standing were seen between IST_1 and IST_2. Number of standing bouts were impacted by test (P = 0.0008). Steers stood more during GST than in IST_1 (P = 0.0007) and IST_2 (PP = 0.03) with no differences observed between IST_1 and IST_2. The IST is repeatable, yet the strength of the behavioral response is reduced during GST. This is the first quantifiable representation of the impact of social isolation on the fear response in beef cattle.

Effect of dexmedetomidine on long-term sensorimotor gating system after sevoflurane anaesthesia in neonatal rats

Objective To evaluate the effect of dexmedetomidine on the long-term sensorimotor gating system after sevoflurane anaesthesia in neonatal rats. Methods One hundred forty-four clean-grade healthy male Sprague-Dawley rats, aged 4-6 days, weighing 8-15 g, were divided into 4 groups (n=36 each) using a random number table method: control group (group C), sevoflurane group (group S), dexmedetomidine plus sevoflurane group (group D+ S), and dexmedetomidine plus α2 receptor antagonist atipamezole plus sevoflurane group (group D+ A+ S). In group S, anesthesia was induced with 6% sevoflurane for 3 min and maintained with 2.1% sevoflurane, and the anesthesia time was 6 h in total.Dexmedetomidine 25 μg/kg was intraperitoneally injected in group D. In group D+ A+ S, dexmedetomidine 25 μg/kg and atipamezole 250 μg/kg were intraperitoneally injected, and the other treatments were similar to those previously described in group S. Twelve rats in each group were randomly selected after anesthesia and sacrificed, and blood samples were collected for determination of serum corticosterone concentrations by enzyme-linked immunosorbent assay.Twenty-four rats were randomly selected in each group, and prepulse inhibition (PPI) of startle test was performed at 70 days after birth.PPI rate (PP3%, PP6%, PP12%) was calculated.The serum corticosterone concentration was measured by restraint stress test on 80 days after birth. Results There was no significant difference in PP6% or PP12% among the four groups (P>0.05). Compared with group C, PP3% was significantly decreased, and the serum corticosterone concentration was increased after the end of anesthesia and during restraint stress test at 80 days after birth in S and D+ A+ S groups (P 0.05). Compared with group S, PP3% was significantly increased, and the serum corticosterone concentration was decreased after the end of anesthesia and during restraint stress test at 80 days after birth in group D (P 0.05). Compared with group D, PP3% was significantly decreased, and the serum corticosterone concentration was increased after the end of anesthesia and during restraint stress test at 80 days after birth in group D+ A+ S (P<0.05). Conclusion Dexmedetomidine can alleviate the damage to long-term sensorimotor gating system after sevoflurane anesthesia in neonatal rats, and the mechanism may be related to activating central α2 receptors and improving hypothalamic-pituitary-adrenal axis hyperfunction. Key words: Dexmedetomidine; Anesthetics, inhalation; Infant, newborn; Adolescent; Sensorimotor gating system

Effect of electrical stimulation at auricular points combined with sound masking on the expression of CREB, BDNF and TrkB in the auditory cortex of tinnitus rats

To explore the effect of electrical stimulation at auricular points (EAS) combined with sound masking on the expression of cAMP-response element binding protein (CREB), brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) in the auditory cortex of tinnitus rats. A total of 27 adult male SD rats were randomly divided into a control group, a model group and an EAS group. The rats in the model group and the EAS group were intervened with intraperitoneal injection of sodium salicylate to induce tinnitus model, while the rats in the control group were intervened with injection of 0.9% NaCl solution. After the model was successfully established, the rats in the EAS group were treated with electrical stimulation at "Shenmen" (TF4) and "Yidan" (CO11), combined with sound masking; the treatment was given once a day for 15 days. The gap prepulse inhibition of acoustic startle (GPIAS) and prepulse inhibition (PPI) testing were performed using the acoustic startle reflex starter package for rats. The expression of BDNF, TrkB, CREB and p-CREB in the auditory cortex of each group were measured with Western Blot analysis. ① Compared with the control group, the GPIAS values in 12 kHz, 16 kHz, 20 kHz and 28 kHz were significantly decreased in the model group (all P<0.05); compared with the model group, GPIAS values in 12 kHz, 16 kHz, 20 kHz and 28 kHz were significantly increased in the EAS group (all P<0.05). ② Compared with the control group, the expression of BDNF and p-CREB in the model group was significantly increased (P<0.01), and the expression of TrkB in the model group was significantly increased (P<0.05); the differences of expression of BDNF, TrkB, CREB and p-CREB between the model group and the EAS group had no statistics significance (all P>0.05). EAS could improve the GPIAS values of high-frequency background sound in tinnitus rats, which may be related with the upregulation of the BDNF/TrkB/CREB signaling pathway in the auditory cortex, leading to the reversion of the maladaptive plasticity.

Intrauterine infection affects early growth and neurobehavioral development in neonatal rats

Objective:To explore the effects of intrauterine infection on early growth and neurobehavioral development in neonatal rats. Methods:Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats with gestation of 15 d to establish the intrauterine infection model, and the effect on the delivery of pregnant rats was observed. The neonatal rat brain tissue was stained with Hematoxylin-Eosin and the cerebral white matter damage was assessed. Immunohistochemical staining and Western blot analysis were performed to evaluate the expression of glial fibrillary acidic protein (GFAP), 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and neurofilament (NF) in pup brains. Birth weight and early growth development indices were monitored,and neurobehavioral tests were performed to access the change of neurobehavioral development in neonatal rats. Results:The white blood cell count increased significantly in the uterus and placenta of the pregnant rats after intrauterine E. coli infection and no significant impact was observed on the delivery of pregnant rats. Weak staining and focal rarefaction of cerebral white matter from rats at P7 in intrauterine infection group were observed. The expression of GFAP markedly increased (P<0.05) in infection group, while the level of CNPase and NF in pup brains at P7 significantly decreased (P<0.05 or P<0.01). Compared with control group, the neonatal rats in infection group had lower birth weight and slower weight gain during the suckling period (P<0.05 or P<0.01), and the completion times of ear opening, eye opening, surface righting, negative geotaxis, acoustic startle and swimming test in infection group were significantly delayed (P<0.05 or P<0.01). Conclussion:Intrauterine infection in pregnant rats can induce cerebral white matter damage and retardation of early growth and neurobehavioral development in neonatal rats.

Effects of a novel blend of citrus botanical oils on equine behavior during a startle test and while trailering

Effects of a novel blend of citrus botanical oils on equine behavior during a startle test and while trailering

Increased metabolic activity and hysteretic enhanced GABAA receptor binding in a rat model of salicylate-induced tinnitus.

Tinnitus is relevant to neural hyperactivity in the central nervous system (CNS). Normal quantity and functioning of the γ-aminobutyric acid (GABA) receptor are crucial for maintaining the balance between excitation and inhibition in the brain. In this study, we applied a rat model of tinnitus via long-term salicylate administration. The combination of the gap pre-pulse inhibition of acoustic startle (GPIAS) and pre-pulse inhibition (PPI) tests were used to detect tinnitus-like behavior, and rats receiving 7 or 14 consecutive days of salicylate administration showed evidence of tinnitus. After positron emission tomography (PET) scan, we found that the metabolic activity was increased after salicylate treatment followed by enhanced GABAA receptor binding with cessation of salicylate administration in the auditory cortex (AC), medial prefrontal cortex (mPFC), hippocampus (HP), cingulate cortex (CiC) and insular (InC). The inferior colliculus (IC) showed an elevated metabolic activity with no change in the GABAA receptor binding. All the alterations returned to baseline several days after cessation of salicylate treatment despite a mismatch between the time-course of them. By contrast, we found alterations in neither the metabolic activity nor the GABAA receptor binding in the amygdala (AMY) and cerebellum (CRB). These findings indicate that enhanced neural activity in the auditory and limbic system may contribute to the development of tinnitus, while the hysteretic increase of GABAA receptor binding in specific areas of the CNS may be a compensation for hyperactivity, which may be involved in tinnitus relieving.

Anxiety and exploratory behavior in the African striped mouse, Rhabdomys, taxa are partially modified by the physical rearing environment.

The environment may modulate genetic influences on behavioral expression. We investigated whether the physical rearing environment modulates anxiety and exploratory behavior in four populations, representing three species, of the striped mouse Rhabdomys. One population originated from an arid, open habitat and the others from grassy, covered habitats, and two species occurred in sympatry. We raised captive individuals of all populations in treatments that simulated cover or no cover for two generations and investigated the behavior of resulting adults in an open-field, light-dark and startle response tests. We expected that, when raised without cover, the arid population would be less anxious and more exploratory than grassland populations, but found the opposite in the open-field test only. We also expected that all individuals would be anxious and less exploratory when raised under cover, which was the case for anxiety in a light-dark test, but individuals from the no cover treatment were more anxious in the open-field test. Only one population × treatment interaction was detected in which the arid population was least exploratory. Therefore, the physical rearing environment had less of an influence than phylogeny on the development of anxiety and exploration in Rhabdomys.

Activation of somatodendritic 5-HT1A autoreceptors reduces the acquisition and expression of cued fear in the rat fear-potentiated startle test

RationaleFear conditioning is an important factor in the etiology of anxiety disorders. Previous studies have demonstrated a role for serotonin (5-HT)1A receptors in fear conditioning. However, the relative contribution of somatodendritic 5-HT1A autoreceptors and post-synaptic 5-HT1A heteroreceptors in fear conditioning is still unclear.ObjectiveTo determine the role of pre- and post-synaptic 5-HT1A receptors in the acquisition and expression of cued and contextual conditioned fear.MethodsWe studied the acute effects of four 5-HT1A receptor ligands in the fear-potentiated startle test. Male Wistar rats were injected with the 5-HT1A receptors biased agonists F13714 (0–0.16 mg/kg, IP), which preferentially activates somatodendritic 5-HT1A autoreceptors, or F15599 (0–0.16 mg/kg, IP), which preferentially activates cortical post-synaptic 5-HT1A heteroreceptors, with the prototypical 5-HT1A receptor agonist R(+)8-OH-DPAT (0–0.3 mg/kg, SC) or the 5-HT1A receptor antagonist WAY100,635 (0–1.0 mg/kg, SC).ResultsF13714 (0.16 mg/kg) and R(+)-8-OH-DPAT (0.03 mg/kg) injected before training reduced cued fear acquisition. Pre-treatment with F15599 or WAY100,635 had no effect on fear learning. In the fear-potentiated startle test, F13714 (0.04–0.16 mg/kg) and R(+)-8-OH-DPAT (0.1–0.3 mg/kg) reduced the expression of cued and contextual fear, whereas F15599 had no effect. WAY100,635 (0.03–1.0 mg/kg) reduced the overall startle response.ConclusionsThe current findings indicate that activation of somatodendritic 5-HT1A autoreceptors reduces cued fear learning, whereas 5-HT1A receptors seem not involved in contextual fear learning. Moreover, activation of somatodendritic 5-HT1A autoreceptors may reduce cued and contextual fear expression, whereas we found no evidence for the involvement of cortical 5-HT1A heteroreceptors in the expression of conditioned fear.

The Role of Cholinergic Midbrain Neurons in Startle and Prepulse Inhibition.

One of the two major cholinergic centers of the mammalian brain is located in the midbrain, i.e., the pedunculopontine tegmentum (PPTg) and the adjacent laterodorsal tegmentum. These cholinergic neurons have been shown to be important for e.g., arousal, reward associations, and sleep. They also have been suggested to mediate sensorimotor gating, measured as prepulse inhibition of startle (PPI). PPI disruptions are a hallmark of schizophrenia and are observed in various other psychiatric disorders, where they are associated with, and often predictive of, other cognitive symptoms. PPI has been proposed to be mediated by a short midbrain circuitry including inhibitory cholinergic projections from PPTg to the startle pathway. Although the data indicating the involvement of the PPTg is very robust, some more recent evidence challenges that there is a cholinergic contribution to PPI. We here use transient optogenetic activation of specifically the cholinergic PPTg neurons in male and female rats to address their role in startle modulation in general, and in PPI specifically. Although we could confirm the crucial role of PPTg cholinergic neurons in associative reward learning, validating our experimental approach, we found that activation of cholinergic PPTg neurons did not inhibit startle responses. In contrast, activation of cholinergic PPTg neurons enhanced startle, which is in accordance with their general role in arousal and indicate a potential involvement in sensitization of startle. We conclude that noncholinergic PPTg neurons mediate PPI in contrast to the longstanding hypothetical view that PPI is mediated by cholinergic PPTg neurons.SIGNIFICANCE STATEMENT Activation of cholinergic neurons in the midbrain has been assumed to mediate prepulse inhibition of startle (PPI), a common measure of sensorimotor gating that is disrupted in schizophrenia and other psychiatric disorders. We here revisit this long-standing hypothesis using optogenetic activation of these specific neurons combined with startle testing in rats. In contrast to the hypothetical role of these neurons in startle modulation, we show that their activation leads to an increase of baseline startle and to prepulse facilitation. This supports recent data by others that have started to cast some doubt on the cholinergic hypothesis of PPI, and calls for a revision of the theoretical construct of PPI mechanisms.

Chronic exposure to cannabinoids before an emotional trauma may have negative effects on emotional function

Chronic direct activation of cannabinoid CB1 receptors (CB1r) may lead to downregulation of CB1r which may in turn result in a depression-like phenotype in certain individuals.We examined the effects of chronic cannabinoid receptor activation before exposure to an emotional traumatic event on CB1r expression in the basolateral amygdala (BLA) and CA1 and on protracted anxiety- and depression-like behaviors. We used exposure to severe shock and situational reminders (SRs) in an inhibitory apparatus as a model for emotional trauma.Chronic treatment with the CB1/2 receptor agonist WIN55,212-2 (1.2 mg/kg, i.p.) before shock exposure had differential effects on depression- and anxiety-like behavioral measures depending on withdrawal periods. In the 24 hrs withdrawal condition, WIN55,212–2 enhanced fear retrieval and impaired extinction, increased anhedonia and despair, but had a therapeutic effect in the startle test. In the 10 days withdrawal condition, WIN55,212–2 enhanced fear retrieval and impaired extinction without preventing the shock/SR-induced negative effects on anhedonia or startle response, but had a therapeutic effect in the despair test.Chronic treatment with WIN55,212–2 was found to down regulate CB1r protein levels in the BLA in the 10 days withdrawal condition, and to upregulate CB1r protein levels in the 24 hrs condition. In the CA1, rats chronically injected with vehicle or WIN55,212–2 demonstrated downregulation of CB1r protein levels.Chronic exposure to cannabinoids prior to an emotional trauma may have deleterious effects on emotional function suggesting that direct CB1/2 receptor activation may not be an optimal way to manipulate the endocannabinoid system in stressful individuals.

The contribution of contextual fear in the anxiolytic effect of chlordiazepoxide in the fear-potentiated startle test

This study evaluated the extent to which a reduction in contextual fear contributes to the anxiolytic effect of benzodiazepines in the fear-potentiated startle response. To this end, chlordiazepoxide, an anxiolytic often used as positive control in preclinical drug studies, and zolpidem, known to have sedative properties and to be devoid of anxiolytic effects, were tested in two contexts: the same context as training had taken place and an alternative context. In addition, the level of muscle relaxation was assessed in a grip strength test. Chlordiazepoxide (2.5–10 mg/kg) decreased the fear-potentiated startle response, confirming its anxiolytic activity. In addition, it dose-dependently decreased the overall startle response in the same, but not the alternative context, and did not affect grip strength, indicating that chlordiazepoxide inhibits contextual fear in the absence of non-specific drug effects. Zolpidem (1.0–10 mg/kg) reduced the overall startle response in both contexts equally and decreased grip strength, indicating that its effects on fear-potentiated startle are due to non-specific drug effects, and not anxiolytic effects. The present findings show that chlordiazepoxide reduces contextual conditioned fear in the absence of non-specific drug effects. In addition, they show that training and testing rats in different contexts makes it possible to distinguish between cued, contextual and non-specific drug effects. As exaggerated contextual fear conditioning contributes to the fear generalization processes implicated in pathological anxiety, focus in screening of anxiolytic effects could be directed more towards the suppression of contextual fear and, therefore, this approach would be a valuable addition to standard preclinical screening.

Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

Protein S-acylation is a widespread post-translational modification that regulates the trafficking and function of a diverse array of proteins. This modification is catalysed by a family of twenty-three zDHHC enzymes that exhibit both specific and overlapping substrate interactions. Mutations in the gene encoding zDHHC9 cause mild-to-moderate intellectual disability, seizures, speech and language impairment, hypoplasia of the corpus callosum and reduced volume of sub-cortical structures. In this study, we have undertaken behavioural phenotyping, magnetic resonance imaging (MRI) and isolation of S-acylated proteins to investigate the effect of disruption of the Zdhhc9 gene in mice in a C57BL/6 genetic background. Zdhhc9 mutant male mice exhibit a range of abnormalities compared with their wild-type littermates: altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety; deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory; and a 36% reduction in corpus callosum volume revealed by MRI. Surprisingly, membrane association and S-acylation of H-Ras was not disrupted in either whole brain or hippocampus of Zdhhc9 mutant mice, suggesting that other substrates of this enzyme are linked to the observed changes. Overall, this study highlights a key role for zDHHC9 in brain development and behaviour, and supports the utility of the Zdhhc9 mutant mouse line to investigate molecular and cellular changes linked to intellectual disability and other deficits in the human population.

Sensorimotor Gating in Depressed and Euthymic Patients with Bipolar Disorder: Analysis on Prepulse Inhibition of Acoustic Startle Response Stratified by Gender and State.

Prepulse inhibition (PPI) of the acoustic startle reflex is an operational measure of sensorimotor gating. The findings on PPI deficits in bipolar disorder (BD) are inconsistent among studies due to various confounding factors such as gender. This study aimed to assess sensorimotor gating deficits in patients with BD stratified by gender and state (depressed/euthymic), and to explore related clinical variables. Subjects were 106 non-manic BD patients (26 BD I and 80 BD II; 63 with depression and 43 euthymic) and 232 age-, gender-, and ethnicity-matched (Japanese) healthy controls. Depression severity was assessed using the Hamilton Depression Rating Scale-21. The electromyographic activity of the orbicularis oculi muscle was measured by a computerized startle reflex test unit. Startle magnitude, habituation, and PPI were compared among the three clinical groups: depressed BD, euthymic BD, and healthy controls. In a second analysis, patients were divided into four groups using the quartile PPI levels of controls of each gender, and a ratio of the low-PPI group (<1st quartile of controls) was compared. Effects of psychosis and medication status were examined by the Mann-Whitney U test. Clinical correlates such as medication dosage and depression severity with startle measurements were examined by Spearman's correlation. Male patients with depression, but not euthymic male patients, showed significantly lower PPI at a prepulse of 86 dB and 120 ms lead interval than did male controls. More than half of the male patients with depression showed low-PPI. In contrast, PPI in female patients did not differ from that in female controls in either the depressed or euthymic state. Female patients with active psychosis showed significantly lower PPI than those without psychosis. Female patients on typical antipsychotics had significantly lower PPI, than those without such medication. PPI showed a significant positive correlation with lamotrigine dosage in male patients and lithium dosage in female patients. These findings suggest that sensorimotor gating is impaired in male BD patients with depression. However, we obtained no evidence for such abnormalities in female BD patients except for those with current psychosis. The observed associations between medication and startle measurements warrant further investigation.