Antinociceptive action of cannabidiol on thermal sensitivity and post-operative pain in male and female rats.

This study investigated the antinociceptive potential of cannabidiol (CBD) in male and female Wistar rats. The assessment and analysis included tail withdrawal to thermal stimulation (tail flick test) and mechanical allodynia induced by plantar incision injury (von Frey test). CBD reduced acute thermal sensitivity in uninjured animals and post-operative mechanical allodynia in males and females. In the tail flick test, CBD 30 mg/kg i.p. was required to induce antinociception in males. During the proestrus phase, females did not show a statistically significant antinociceptive response to CBD treatment despite a noticeable trend. In contrast, in a separate group of rats tested during the late diestrus phase, antinociception varied with CBD dosage and time. In the post-operative pain model, CBD at 3 mg/kg decreased mechanical allodynia in males. Similarly, this dose reduced allodynia in females during proestrus. However, in females during late diestrus, the lower dose of CBD (0.3 mg/kg) reduced mechanical allodynia, although the latency to onset of the effect was slower (90 min). The effectiveness of a 10-fold lower dose of CBD during the late diestrus stage in females suggests that ovarian hormones can influence the action of CBD. While CBD has potential for alleviating pain in humans, personalized dosing regimens may need to be developed to treat pain in women.

Sex and estrous cycle-linked differences in the effect of cannabidiol on panic-like responding in rats and mice.

Clinical and preclinical studies point towards anxiolytic actions of cannabidiol (CBD), but its effect in panic disorder has been less explored and few studies consider effects in females. We here compared the effect of CBD on the response of male and female rats and mice to a panicogenic challenge; exposure to low O2 (rats) or high CO2 (mice) paying attention in females to possible effects of estrous cycle phase. Male and female Sprague-Dawley rats and C57BL/6 J mice were exposed to 7% O2 for 5 min (rats) or 20% CO2 (mice) and escape behaviour, which has been associated with panic attacks, was quantified as undirected jumps towards the gas chamber's ceiling. The effect of pretreatment with CBD (1-10 mg kg-1 i.p. in rats or 10-60 mg kg-1 i.p. in mice) was tested. The results showed that low O2 (rats) or high CO2 (mice) evoked escape in both sexes. In female rats the response was estrous cycle-sensitive: females in late diestrus made significantly more jumps than females in proestrus. In female mice escape was not influenced by estrous cycle phase and CBD was panicolytic. In female rats CBD attenuated escape behaviour in late diestrus phase but not in proestrus. In male rats and mice CBD had no effect on escape behaviour. Therefore, CBD is panicolytic in female rats and mice but not in males. In rats the effect is estrous cycle-sensitive: rats were most responsive to CBD in late diestrus. In mice higher doses were required to elicit effects and estrous cycle had no effect.

Low doses of fluoxetine for the treatment of emotional premenstrual syndrome: a randomized double-blind, placebo-controlled, pilot study.

INTRODUCTION: The neuroactive metabolite of progesterone, allopregnanolone (ALLO), has been implicated in premenstrual syndrome (PMS) physiopathology and preclinical studies suggested that low doses of fluoxetine increase the ALLO brain concentration. OBJECTIVES: To assess which low dose of fluoxetine (2 mg/d, 5 mg/d or 10 mg/d), administered exclusively during the luteal phase of menstrual cycle, has a potential effect for preventing or mitigating emotional PMS symptoms. METHODS: In this randomized, double-blind, placebo-controlled pilot study, we followed 40 women (mean age = 29.7 +/- 7.4 years) with emotional PMS, during two menstrual cycles: cycle 1, without pharmacological intervention; and cycle 2, with pharmacological intervention. Participants took capsules, on average, seven days preceding the likely date of menses. We assessed the severity of PMS symptoms in both cycles using the Daily Record of Severity of Problems scale (DRSP). RESULTS: There was an increase in the DRSP scores during the late luteal phase of cycle 1, confirming the diagnosis of emotional PMS. Low doses of fluoxetine (5 mg/d: 33.5%; 10 mg/d: 48.4%) reduced DRSP total score in the day before menses (day-1) at cycle 2 compared with day-1 at cycle 1. Fluoxetine 10 mg/d had the most consistent decline in emotional PMS symptoms; 70% of the participants reported a reduction greater than 40% in the DRSP score. CONCLUSIONS: Low doses of fluoxetine, which may have no or few effect on the serotonergic system, but may interfere in the progesterone metabolization, seem to have some potential to mitigate emotional PMS symptoms. While the 10 mg/d of fluoxetine had the best performance on reducing emotional PMS symptoms, the 5 mg/d dose also seems to have some effect on emotional PMS symptoms. Further larger studies will help establish the lowest effective dose of flouxetine for PMS treatment.

Sex-dependent differences in the anxiolytic-like effect of cannabidiol in the elevated plus-maze.

RATIONALE: Cannabidiol (CBD), the major non-psychoactive constituent of cannabis, has therapeutic potential for the treatment of anxiety. Most preclinical studies investigate only acute effects of CBD and only in males, yet the drug is most likely to be used over a sustained period in clinical practice. OBJECTIVES: The objectives of this study were to investigate the anxiolytic-like effect of CBD in female rats compared to males and to determine whether the responsiveness of females was influenced by the stage of the estrous cycle. METHODS: We carried out experiments to compare the effect of CBD in male and female rats in the elevated plus maze (EPM) in response to acute and short-term (4 days) administration through a complete cycle in females. RESULTS: Male and female rats behaved in a similar manner in the EPM, but females in the late diestrus (LD) phase exhibited more anxiety-like behavior than at other stages, the difference reaching statistical significance compared to proestrus stages. CBD produced anxiolytic-like effects in both sexes, but female rats were responsive only in LD and 10-fold lower dose than males. After sub-chronic (4 days) treatment, responsiveness to CBD was maintained in females in LD, but females in proestrus remained unresponsive to CBD treatment. CONCLUSIONS: We suggest that there are sex differences in the anxiolytic-like effects of CBD in rats that reflect different underlying mechanisms: based on literature data, gonadal hormone status linked to GABAA receptor expression in females, and 5-HT1A receptor activation in males.

Neonatal maternal deprivation facilitates the expression of a panic-like escape behavior in adult rats.

A wealth of evidence associates disruptions of the parent-infant relationship (e.g. childhood parental loss or parental neglect) with the later appearance of panic disorder. In rodents, neonatal maternal separation and maternal deprivation (MD) are reported to increase the expression of anxiety-related defensive responses in adult animals. However, little is known about the long-term consequences of these early-life stressors in animal models of panic. We here investigated the effects of a single 24 h-episode of MD on post-natal day 11 (PND 11) in adult male Wistar rats submitted to two animal models that associate escape expression with panic attacks: the elevated T-maze and exposure to severe hypoxia (7% O2). We also investigated the involvement of serotonin (5-HT) in the observed changes. Although neonatal MD did not affect the behavioral responses measured in the elevated T-maze, it facilitated the expression of escape during hypoxia exposure, indicating a panicogenic-like effect. Pre-test administration of the 5-HT synthesis inhibitor, para-chlorophenylalanine (PCPA; 4 daily injections of 100 mg/kg) facilitated escape attempts in non-deprived animals during the hypoxia challenge, but did not interfere with the expression of this behavior in maternally-deprived rats. The levels of 5-HT1A receptors in key panic- and anxiety-associated areas, the dorsal periaqueductal gray and amygdala, respectively, were not different between previously deprived and non-deprived animals. Plasma corticosterone levels were significantly increased by hypoxia exposure, independently of the animals' previous stress condition or PCPA administration. Therefore, MD on PND 11 predisposes the adult animal to the panic-evoking effects of severe hypoxia, a stimulus also reported to induce panic attacks in humans. The lack of PCPA effect on the pro-escape consequence of MD may be indicative that 5-HT signaling is impaired in the stressed animal.

Panic-like responses of female Wistar rats confronted by Bothrops alternatus pit vipers, or exposure to acute hypoxia: Effect of oestrous cycle.

Anxiety-related diseases are more than twice as common in women than in men, and in women, symptoms may be exacerbated during the late luteal phase of the menstrual cycle. Despite this, most research into the underlying mechanisms, which drives drug development, have been carried out using male animals. In an effort to redress this imbalance, we compared responses of male and female Wistar rats during exposure to two unconditioned threatening stimuli that evoke panic-related defensive behaviours: confrontation with a predator (Bothrops alternatus) and acute exposure to hypoxia (7% O2 ). Threatened by venomous snake, male and female rats initially displayed defensive attention, risk assessment, and cautious interaction with the snake, progressing to defensive immobility to overt escape. Both males and females displayed higher levels of risk assessment but less interaction with the predator. They also spent more time in the burrow, displaying inhibitory avoidance, and more time engaged in defensive attention, and non-oriented escape behaviour. In females, anxiety-like behaviour was most pronounced in the oestrous and proestrus phases whereas panic-like behaviour was more pronounced during the dioestrus phase, particularly during late dioestrus. Acute hypoxia evoked panic-like behaviour (undirected jumping) in both sexes, but in females, responsiveness in late dioestrus was significantly greater than at other stages of the cycle. The results reveal that females respond in a qualitatively similar manner to males during exposure to naturally occurring threatening stimuli, but the responses of females is oestrous cycle dependent with a significant exacerbation of panic-like behaviour in the late dioestrus phase.

Enhanced responsiveness to hypoxic panicogenic challenge in female rats in late diestrus is suppressed by short-term, low-dose fluoxetine: Involvement of the dorsal raphe nucleus and the dorsal periaqueductal gray.

BACKGROUND: Acute hypoxia, which is panicogenic in humans, also evokes panic-like behavior in male rats. Panic disorder is more common in women and susceptibility increases during the premenstrual phase of the cycle. AIMS: We here investigated for the first time the impact of hypoxia on the expression of panic-like escape behavior by female rats and its relationship with the estrous cycle. We also evaluated functional activation of the midbrain panic circuitry in response to this panicogenic stimulus and whether short-term, low-dose fluoxetine treatment inhibits the hyper-responsiveness of females in late diestrus. METHODS: Male and female Sprague Dawley rats were exposed to 7% O2. Females in late diestrus were also tested after short-term treatment with fluoxetine (1.75 or 10 mg/kg, i.p.). Brains were harvested and processed for c-Fos and tryptophan hydroxylase immunoreactivity in the periaqueductal gray matter (PAG) and dorsal raphe nucleus (DR). RESULTS: Acute hypoxia evoked escape in both sexes. Overall, females were more responsive than males and this is clearer in late diestrus phase. In both sexes, hypoxia induced functional activation (c-Fos expression) in non-serotonergic cells in the lateral wings of the DR and dorsomedial PAG, which was greater in late diestrus than proestrus (lowest behavioral response to hypoxia). Increased responding in late diestrus (behavioral and cellular levels) was prevented by 1.75, but not 10 mg/kg fluoxetine. DISCUSSION: The response of female rats to acute hypoxia models panic behavior in women. Low-dose fluoxetine administered in the premenstrual phase deserves further attention for management of panic disorders in women.

The Frequency-Dependence of Pre- and Postganglionic Nerve Stimulation of Pig and Rat Bladder.

PURPOSE: The urinary bladder generates phasic contractions via action potentials generated in pre- and then postganglionic neurons. Whilst the frequency-dependence of postganglionic neurons to generate contractions has been quantified, the dynamic range of preganglionic neurons is less clear and if intramural ganglia exert frequency-dependent modulation of transmission between pre- and postganglionic neurons. The phosphodiesterase type 5 inhibitor sildenafil reduces neurotransmitter release from postganglionic fibres to detrusor smooth muscle and an additional question was if there was also a preganglionic action. This study aimed to compare the frequency range of bladder contractile activation by pre- and postganglionic stimulation in pig and rat bladders and if sildenafil exerted additional preganglionic actions. METHODS: An arterially-perfused ex vivo pig bladder preparation was used for preganglionic (pelvic nerve) and mixed pre-and postganglionic (direct bladder wall) stimulation at 36°C and postganglionic mediated contractions achieved by field-stimulation of in vitro isolated detrusor strips. With rats, pelvic nerve stimulation was carried out in vivo and postganglionic stimulation also with isolated detrusor strips. RESULTS: All contractions were abolished by 2% lignocaine indicating they are nerve-mediated. Stimulation targets were verified with hexamethonium that completely abolished pelvic nerve responses by had no effect on detrusor strips; responses to mixed bladder wall stimulation were partially reduced. The frequency-dependence of contractile activation was similar whether by pre- or postganglionic stimulation in both pigs and rats. Sildenafil reduced contractions to preganglionic stimulation significantly more than to postganglionic stimulation. Mixed pre- and postganglionic stimulation were reduced by an intermediate extent. CONCLUSION: Intramural ganglia offer no frequency-dependent modulation under the experimental conditions used here and the sildenafil data are consistent with multiple sites of action underlying generation of bladder contractions. A translational aspect of these findings is discussed in terms of setting stimulation parameters for neuromodulation protocols.

Effect of Estrous Cycle on Behavior of Females in Rodent Tests of Anxiety.

Anxiety disorders are more prevalent in women than in men. In women the menstrual cycle introduces another variable; indeed, some conditions e.g., premenstrual syndrome, are menstrual cycle specific. Animal models of fear and anxiety, which form the basis for research into drug treatments, have been developed almost exclusively, using males. There remains a paucity of work using females and the available literature presents a confusing picture. One confound is the estrous cycle in females, which some authors consider, but many do not. Importantly, there are no accepted standardized criteria for defining cycle phase, which is important given the rapidly changing hormonal profile during the 4-day cycle of rodents. Moreover, since many behavioral tests that involve a learning component or that consider extinction of a previously acquired association require several days to complete; the outcome may depend on the phase of the cycle on the days of training as well as on test days. In this article we consider responsiveness of females compared to males in a number of commonly used behavioral tests of anxiety and fear that were developed in male rodents. We conclude that females perform in a qualitatively similar manner to males in most tests although there may be sex and strain differences in sensitivity. Tests based on unconditioned threatening stimuli are significantly influenced by estrous cycle phase with animals displaying increased responsiveness in the late diestrus phase of the cycle (similar to the premenstrual phase in women). Tests that utilize conditioned fear paradigms, which involve a learning component appear to be less impacted by the estrous cycle although sex and cycle-related differences in responding can still be detected. Ethologically-relevant tests appear to have more translational value in females. However, even when sex differences in behavior are not detected, the same outward behavioral response may be mediated by different brain mechanisms. In order to progress basic research in the field of female psychiatry and psychopharmacology, there is a pressing need to validate and standardize experimental protocols for using female animal models of anxiety-related states.

Acute and chronic cardiorespiratory consequences of focal intrahippocampal administration of seizure-inducing agents. Implications for SUDEP.

The risk factors for SUDEP are undoubtedly heterogenous but the main factor is the frequency of generalized tonic-clonic seizures with apnoea and/or cardiac abnormalities likely precipitating the lethal event. By its very nature modelling SUDEP experimentally is challenging, yet insights into the nature of the lethal event and precipitating factors are vital in order to understand and prevent fatalities. Acute animal models, which induce status epilepticus (SE), can be used to help understand pathophysiological processes during and following seizures, which sometimes lead to death. The most commonly used method to induce seizures and status epilepticus is systemic administration of an ictogenic agent. Microinjection of such agents into restricted regions within the brain induces a more localised epileptic focus and circumvents the risk of direct actions on cardiorespiratory control centres. Both approaches have revealed substantial cardiovascular and respiratory consequences, including death as a result of apnoea, which may be of central origin, obstructive due to laryngospasm or, at least in genetically modified mice, a result of spreading depolarisation to medullary respiratory control centres. SUDEP is by definition a result of epilepsy, which in turn is diagnosed on the basis of two or more unprovoked seizures. The incidence of tonic-clonic seizures is the main risk factor, raising the possibility that repeated seizures cause cumulative pathological and/or pathophysiological changes that contribute to the risk of SUDEP. Chronic experimental models, which induce repeated seizures that in some cases lead to death, do show progressive development of pathophysiological changes in the myocardium, e.g. prolongation of QT the interval of the ECG or, over longer periods, ventricular hypertrophy. However, the currently available evidence indicates that seizure-related deaths are primarily due to apnoeas, but cardiac factors, particularly cumulative cardiac pathophysiologies due to repeated seizures, are potential contributing factors.

Influence of estrous cycle stage on acquisition and expression of fear conditioning in female rats.

It is unclear whether all animal models of anxiety-like states developed using males are appropriate for use in females. In females, tests involving a learning component might be influenced not only by estrous cycle stage on the test day but also by the stage during the conditioning process. We used two tests - conditioned freezing (CF) and fear potentiated startle (FPS) to compare responsiveness of male rats and females conditioned and/or tested in proestrus (P) or late diestrus (LD). For CF all rats displayed a similar freezing response regardless of sex or estrous cycle stage. In terms of FPS, males and females conditioned in P and tested in P or LD, and females conditioned in LD and tested in LD all showed potentiated startle. The response waned during the test session in males and in females conditioned in P, but not in those conditioned in LD. In contrast, FPS was not apparent in the first half of the test session in females conditioned in LD and tested in P but developed in the second half. We suggest that fear learning during P and LD is robust but may be initially be obscured in rats tested in P because of generalization to the CS due to high estrogen. Estrous cycle stage is an important consideration which must be taken into account in designing behavioural tests in females.

Sacral nerve stimulation for refractory OAB and idiopathic urinary retention: Can phenotyping improve the outcome for patients: ICI-RS 2019?

AIMS: Sacral nerve stimulation (SNS) is widely used to treat refractory idiopathic overactive bladder (OAB) and idiopathic urinary retention. However, clinical outcomes are variable and understanding predictive factors for success or side-effects would enable personalization of therapy and optimization of outcomes. At the International Consultation on Incontinence-Research Society meeting 2019, a Think Tank was convened to discuss how advances in the basic science study of SNS may be translatable into clinical practice to improve outcomes of patients undergoing SNS treatment. METHODS: We conducted a literature review and expert consensus meeting focusing on current methods of phenotyping patients and specifically, how advances in basic science research of the mechanism of action of SNS can be translated into clinical practice to improve patient selection for therapy. RESULTS: The terms "Idiopathic OAB" and "idiopathic urinary retention" encompass several underlying pathophysiological phenotypes. Commonly, phenotyping is based on clinical and urodynamic factors. Animal studies have demonstrated that high-frequency stimulation can produce rapid onset, reversible conduction block in peripheral nerves. Altering stimulation parameters may potentially enable personalization of therapy depending upon the clinical indication in the future. Similarly, advances in conditional and closed-loop stimulation may offer greater efficacy for certain patients. Phenotyping based on psychological comorbidity requires further study to potentially optimize patient selection for therapy. CONCLUSIONS: Idiopathic OAB and idiopathic urinary retention are heterogenous conditions with multiple potential underlying phenotypes. Tailoring stimulation parameters to the needs of each individual according to phenotype could optimize outcomes. Assessing psychological comorbidity may improve patient selection. Areas for further research are proposed.

Cardiac effects of repeated focal seizures in rats induced by intrahippocampal tetanus toxin: Bradyarrhythmias, tachycardias, and prolonged interictal QT interval.

OBJECTIVE: To determine electrical changes in the heart in a chronic, nonstatus model of epilepsy. METHODS: Electrocorticography (ECoG) and electrocardiography (ECG) of nine animals (five made epileptic by intrahippocampal injection of tetanus neurotoxin (TeNT) and four controls), are monitored continuously by radiotelemetry for up to 7 weeks. RESULTS: Epileptic animals develop a median of 168 seizures, with postictal tachycardias reaching a mean of 487 beats/min and lasting a mean of 661 seconds. Ictal changes in heart rate include tachycardia and in the case of convulsive seizures, bradyarrhythmias resembling Mobitz type 1 second-degree atrioventricular block; notably the P-R interval increased before block. Postictally, the amplitude of T wave increases. Interictally, QT dependence on RR is modest and conventional QT corrections prove ineffective. Interictal QT intervals, measured at a heart rate of 400 bpm, increased from 65 to 75 ms, an increase dependent on seizure incidence over the preceding 10-14 days. SIGNIFICANCE: Repeated seizures induce a sustained tachycardia and increase in QT interval of the ECG and evoke arrhythmias including periods of atrioventricular block during Racine type 4 and 5 seizures. These changes in cardiac function may predispose to development in fatal arrhythmias and sudden death in humans with epilepsy.

Botulinum toxin A for refractory OAB and idiopathic urinary retention: Can phenotyping improve outcome for patients: ICI-RS 2019?

AIMS: Botulinum toxin A (BTX-A) is a well-established treatment for refractory idiopathic overactive bladder (OAB). It has also been used with short-term success in treating idiopathic urinary retention. However, efficacy and complication rates are variable and predicting those likely to benefit most from treatment would enable personalization of therapy and optimization of outcomes. At the International Consultation on Incontinence-Research Society (ICI-RS) meeting in 2019 a Think Tank addressed the question of how we can improve the way we phenotype patients undergoing BTX-A treatment. METHODS: The Think Tank conducted a literature review and expert consensus meeting focussing on how advances in basic science research of the mechanism of action of BTX-A, as well as assessment of psychological comorbidity, can be translated into clinical practice to improve patient selection for therapy. RESULTS: Idiopathic OAB and idiopathic urinary retention are heterogenous conditions encompassing several phenotypes with multiple potential pathophysiological mechanisms. Animal models have demonstrated a central nervous system mechanism of action of intravesically injected BTX-A and this has been confirmed in human functional MRI studies, but whether this tool can be used to predict outcome from treatment remains to be determined. Phenotyping based on psychological comorbidity using validated screening tools should be studied as a way to potentially optimize patient selection for therapy. CONCLUSIONS: Advances in basic science research into the mechanism of action of BTX-A have improved our understanding of the pathophysiology of OAB and may lead to novel ways to phenotype patients. Psychological assessment is another way in which phenotyping may be improved. Areas for further research are proposed.

Brainstem activity, apnea, and death during seizures induced by intrahippocampal kainic acid in anaesthetized rats.

OBJECTIVE: To investigate how prolonged seizure activity affects cardiorespiratory function and activity of pre-Bötzinger complex, leading to sudden death. METHODS: Urethane-anesthetized female Long-Evans rats were implanted with nasal thermocouple; venous and arterial cannulae; and electrodes for electrocardiography (ECG) and hippocampal, cortical, and brainstem recording. Kainic acid injection into the ventral hippocampus induced status epilepticus. RESULTS: Seizures caused hypertension, tachycardia, and tachypnea punctuated by recurrent transient apneas. Salivation increased considerably: in 11 of 12 rats, liquid with alkaline pH consistent with saliva was expelled from the mouth. Most transient apneas were obstructive: nasal airflow ceased, while, in 83%, efforts to breathe persisted as continued rhythmic activity of respiratory pre-Bötzinger neurons, inspiratory electromyography (EMG), and excursions of the chest wall and abdomen. Blood pressure oscillated in time with respiratory efforts. This pattern also occurred in a minority of cases (16%) of incomplete apnea, but not in rare cases (1%) of transient central apneas. During transient obstructive apneas, the frequency of all inspiratory efforts decreased abruptly by ~30%, suggesting a resetting of the central respiratory rhythm generator. Twenty-two of thirty-one rats died, due either to obstructive apnea (12) or central apnea following progressive slowing of respiration (10). Most rats dying of central apnea had experienced several transient obstructive apneas. Negative DC field potential shifts of the brainstem followed the final breath, consistent with previous reports on spreading depolarization in mouse models. Timing suggests that the DC shift is a consequence rather than cause of respiratory collapse. Cardiac activity continued for tens of seconds. SIGNIFICANCE: Seizure activity in forebrain induces pronounced autonomic activation and disrupts activity in medullary respiratory centers, resulting in death from either obstructive or central apnea. These results directly inform mechanisms of death in status epilepticus, and indirectly provide clues to mechanisms of sudden unexpected death in epilepsy (SUDEP).

Short-term, low-dose fluoxetine prevents oestrous cycle-linked increase in anxiety-like behaviour in female rats.

BACKGROUND AND AIMS: We sought a robust behavioural test that evoked increased anxiety-like behaviour during the late dioestrus phase of the oestrous cycle (similar to the premenstrual period in women) and tested whether this could be prevented by acute low-dose fluoxetine (FLX). METHODS: Female Wistar rats in different stages of their cycle were exposed to four different tests of anxiety-like behaviour. RESULTS: No oestrous cycle differences were detected in fear potentiated startle or conditioned freezing to an aversive context. In a light switch-off test where rats move from one compartment of a shuttle-box to the other to turn off an aversive light, females displayed enhanced responding in late dioestrus. During isolation restraint stress females in late dioestrus emitted three times more 22 kHz ultrasound vocalisations (USV) than at other cycle stages. Using the USV test, short-term administration of low-dose FLX (1.75 mg kg-1, i.p.) designed to blunt the sharp fall in brain allopregnanolone concentration during late dioestrus but without affecting 5-HT systems, prevented the increase in isolation stress-evoked USVs. CONCLUSIONS: The light switch-off and isolation restraint-induced USV tests evoke unconditioned adverse emotional responses that are ethologically relevant and sensitive to oestrous cycle stage. The USV test fulfils many criteria required of a model for premenstrual syndrome in women. Using the USV test, short-term administration of FLX to increase brain allopregnanolone concentration without affecting 5-HT systems prevented the increased USV responding in late dioestrus. Short-term low-dose FLX treatment may have potential to alleviate development of adverse premenstrual symptoms in women.

Suppression of Urinary Voiding "on Demand" by High-Frequency Stimulation of the S1 Sacral Nerve Root in Anesthetized Rats.

OBJECTIVES: High-frequency (kHz) stimulation of preganglionic pelvic nerve afferents can inhibit voiding in both anesthetized and conscious rats. The afferents travel via the S1 sacral nerve root, which is easier to access than the distal pelvic nerve fibers within the abdominal cavity. We therefore investigated whether voiding could be inhibited by high-frequency stimulation at S1 and how this compared to distal pelvic nerve stimulation. METHODS: Urethane-anesthetized rats were instrumented to record bladder pressure and abdominal wall electromyogram and to stimulate the distal preganglionic pelvic nerve bundle and S1 sacral root. Saline was infused continuously into the bladder to evoke repeated voiding. Stimulation was initiated within 1-2 sec of the onset of the steep rise in bladder pressure signaling an imminent void. RESULTS: In six rats, stimulation of the distal pelvic nerve bundle (1-3 kHz sinusoidal waveform 1 mA, 60 sec) supressed the occurrence of an imminent void. Voiding resumed within 70 ± 13.0 sec (mean ± SEM) of stopping stimulation. Stimulation (using the same parameters) of the S1 root at the level of the sacral foramen suppressed voiding for the entire stimulation period in three rats and deferred voiding for 35-56 sec (mean 44.0 ± 3.2 sec) in the remaining three. Stimulation at either site when the bladder was approximately half full, as estimated from previous intervoid intervals, had no effect on voiding. CONCLUSIONS: This preliminary study provides proof-of-concept for the sacral root as an accessible target for high-frequency stimulation that may be developed as an "on demand" neuromodulation paradigm to suppress unwanted urinary voids. CONFLICT OF INTEREST: The authors reported no conflict of interest.

The endogenous opioid system modulates defensive behavior evoked by Crotalus durissus terrificus: Panicolytic-like effect of intracollicular non-selective opioid receptors blockade.

BACKGROUND: There is a controversy regarding the key role played by opioid peptide neurotransmission in the modulation of panic-attack-related responses. AIMS: Using a prey versus rattlesnakes paradigm, the present work investigated the involvement of the endogenous opioid peptide-mediated system of the inferior colliculus in the modulation of panic attack-related responses. METHODS: Wistar rats were pretreated with intracollicular administration of either physiological saline or naloxone at different concentrations and confronted with rattlesnakes ( Crotalus durissus terrificus). The prey versus rattlesnake confrontations were performed in a polygonal arena for snakes. The defensive behaviors displayed by prey (defensive attention, defensive immobility, escape response, flat back approach and startle) were recorded twice: firstly, over a period of 15 min the presence of the predator and a re-exposure was performed 24 h after the confrontation, when animals were exposed to the experimental enclosure without the rattlesnake. RESULTS: The intramesencephalic non-specific blockade of opioid receptors with microinjections of naloxone at higher doses decreased both anxiety- (defensive attention and flat back approach) and panic attack-like (defensive immobility and escape) behaviors, evoked in the presence of rattlesnakes and increased non-defensive responses. During the exposure to the experimental context, there was a decrease in duration of defensive attention. CONCLUSIONS: These findings suggest a panicolytic-like effect of endogenous opioid receptors antagonism in the inferior colliculus on innate (panic attack) and conditioned (anticipatory anxiety) fear in rats threatened by rattlesnakes.

Acid reflux induced laryngospasm as a potential mechanism of sudden death in epilepsy.

OBJECTIVE: Recent research suggests that obstructive laryngospasm and consequent respiratory arrest may be a mechanism in sudden unexpected death in epilepsy. We sought to test a new hypothesis that this laryngospasm is caused by seizures driving reflux of stomach acid into the larynx, rather than spontaneous pathological activity in the recurrent laryngeal nerve. APPROACH: We used an acute kainic acid model under urethane anesthesia to observe seizure activity in Long-Evans rats. We measured the pH in the esophagus and respiratory activity. In a subset of experiments, we blocked acid movement up the esophagus with a balloon catheter. MAIN RESULTS: In all cases of sudden death, terminal apnea was preceded by a large pH drop from 7 to 2 in the esophagus. In several animals we observed acidic fluid exiting the mouth, sometimes in large quantities. In animals where acid movement was blocked, sudden deaths did not occur. No acid was detected in controls. SIGNIFICANCE: The results suggest that acid movement up the esophagus is a trigger for sudden death in KA induced seizures. The fact that blocking acid also eliminates sudden death implies causation. These results may provide insight to the mechanism of SUDEP in humans.

Suppression of Urinary Voiding by Conditional High Frequency Stimulation of the Pelvic Nerve in Conscious Rats.

Female Wistar rats were instrumented to record bladder pressure and to stimulate the left pelvic nerve. Repeated voids were induced by continuous infusion of saline into the bladder (11.2 ml/h) via a T-piece in the line to the bladder catheter. In each animal tested (n = 6) high frequency pelvic nerve stimulation (1-3 kHz, 1-2 mA sinusoidal waveform for 60 s) applied within 2 s of the onset of a sharp rise in bladder pressure signaling an imminent void was able to inhibit micturition. Voiding was modulated in three ways: (1) Suppression of voiding (four rats, n = 13 trials). No fluid output or a very small volume of fluid expelled (<15% of the volume expected based on the mean of the previous 2 or 3 voids). Voiding suppressed for the entirety of the stimulation period (60 s) and resumed within 37 s of stopping stimulation. (2) Void deferred (four rats, n = 6 trials). The imminent void was suppressed (no fluid expelled) but a void occurred later in the stimulation period (12-44 s, mean 24.5 ± 5.2 s after the onset of the stimulation). (3) Reduction in voided volume (five rats, n = 20 trials). Voiding took place but the volume of fluid voided was 15-80% (range 21.8-77.8%, mean 45.3 ± 3.6%) of the volume expected from the mean of the preceding two or three voids. Spontaneous voiding resumed within 5 min of stopping stimulation. Stimulation during the filling phase in between voids had no effect. The experiments demonstrate that conditional high frequency stimulation of the pelvic nerve started at the onset of an imminent void can inhibit voiding. The effect was rapidly reversible and was not accompanied by any adverse behavioral side effects.