Cyfip2 allelic variation in C57BL/6J and C57BL/6NJ mice alters free-choice ethanol drinking but not binge-like drinking or wheel-running activity.

BACKGROUND: Since the origin of the C57BL/6 (B6) mouse strain, several phenotypically and genetically distinct B6 substrains have emerged. For example, C57BL/6J mice (B6J) display greater voluntary ethanol consumption and locomotor response to psychostimulants and differences in nucleus accumbens synaptic physiology relative to C57BL/6N (B6N) mice. A non-synonymous serine to phenylalanine point mutation (S968F) in the cytoplasmic FMR1-interacting protein 2 (Cyfip2) gene underlies both the differential locomotor response to cocaine and the accumbal physiology exhibited by these substrains. We examined whether Cyfip2 allelic variation underlies B6 substrain differences in other reward-related phenotypes, such as ethanol intake and wheel-running activity. METHODS: We compared voluntary ethanol consumption, wheel-running, and binge-like ethanol drinking in male and female B6J and B6NJ mice. When substrain differences were observed, additional experiments were performed in two novel mouse models in which the B6N Cyfip2 mutation was either introduced (S968F) into the B6J background or corrected (F968S) via CRISPR/Cas9 technology. RESULTS: B6J consumed significantly more ethanol than B6NJ and allelic variation in Cyfip2 contributed substantially to this substrain difference. In contrast, B6NJ displayed significantly more daily wheel-running than B6J, with Cyfip2 allelic variation playing only a minor role in this substrain difference. Lastly, no substrain differences were observed in binge-like ethanol drinking. CONCLUSIONS: These results contribute to the characterization of behavior-genetic differences between B6 substrains, support previous work indicating that free-choice and binge-like ethanol drinking are dependent on partially distinct genetic networks, and identify a novel phenotypic difference between B6 substrains in wheel-running activity.

Affective Disruption During Forced Ethanol Abstinence in C57BL/6J and C57BL/6NJ Mice.

BACKGROUND: In alcohol-dependent individuals, acute alcohol withdrawal results in severe physiological disruption, including potentially lethal central nervous system hyperexcitability. Although benzodiazepines successfully mitigate such symptoms, this treatment does not significantly reduce recidivism rates in postdependent individuals. Instead, persistent affective disturbances that often emerge weeks to months after initial detoxification appear to play a significant role in relapse risk; however, it remains unclear whether genetic predispositions contribute to their emergence, severity, and/or duration. Interestingly, significant genotypic and phenotypic differences have been observed among distinct C57BL/6 (B6) substrains, and, in particular, C57BL/6J (B6J) mice have been found to reliably exhibit higher voluntary ethanol (EtOH) intake and EtOH preference compared to several C57BL/6N (B6N)-derived substrains. To date, however, B6 substrains have not been directly compared on measures of acute withdrawal severity or affective-behavioral disruption during extended abstinence. METHODS: Male and female B6J and B6NJ mice were exposed to either a 7-day chronic intermittent EtOH vapor (CIE) protocol or to ordinary room air in inhalation chambers. Subsequently, blood EtOH concentrations and handling-induced convulsions were evaluated during acute withdrawal, and mice were then tested weekly for affective behavior on the sucrose preference test, light-dark box test, and forced swim test throughout 4 weeks of (forced) abstinence. RESULTS: Despite documented differences in voluntary EtOH intake between these substrains, we found little evidence for substrain differences in either acute withdrawal or long-term abstinence between B6J and B6NJ mice. CONCLUSIONS: In B6J and B6NJ mice, both the acute and long-term sequelae of EtOH withdrawal are dependent on largely nonoverlapping gene networks relative to those underlying voluntary EtOH drinking.

A Surgical Mouse Model for Advancing Laryngeal Nerve Regeneration Strategies.

Iatrogenic recurrent laryngeal nerve (RLN) injury is a morbid complication of anterior neck surgical procedures. Existing treatments are predominantly symptomatic, ranging from behavioral therapy to a variety of surgical approaches. Though laryngeal reinnervation strategies often provide muscle tone to the paralyzed vocal fold (VF), which may improve outcomes, there is no clinical intervention that reliably restores true physiologic VF movement. Moreover, existing interventions neglect the full cascade of molecular events that affect the entire neuromuscular pathway after RLN injury, including the intrinsic laryngeal muscles, synaptic connections within the central nervous system, and laryngeal nerve anastomoses. Systematic investigations of this pathway are essential to develop better RLN regenerative strategies. Our aim was to develop a translational mouse model for this purpose, which will permit longitudinal investigations of the pathophysiology of iatrogenic RLN injury and potential therapeutic interventions. C57BL/6J mice were divided into four surgical transection groups (unilateral RLN, n = 10; bilateral RLN, n = 2; unilateral SLN, n = 10; bilateral SLN, n = 10) and a sham surgical group (n = 10). Miniaturized transoral laryngoscopy was used to assess VF mobility over time, and swallowing was assessed using serial videofluoroscopy. Histological assays were conducted 3 months post-surgery for anatomical investigation of the larynx and laryngeal nerves. Eight additional mice underwent unilateral RLN crush injury, half of which received intraoperative vagal nerve stimulation (iVNS). These 8 mice underwent weekly transoral laryngoscopy to investigate VF recovery patterns. Unilateral RLN injury resulted in chronic VF immobility but only acute dysphagia. Bilateral RLN injury caused intraoperative asphyxiation and death. VF mobility was unaffected by SLN transection (unilateral or bilateral), and dysphagia (transient) was evident only after bilateral SLN transection. The sham surgery group retained normal VF mobility and swallow function. Mice that underwent RLN crush injury and iVNS treatment demonstrated accelerated and improved VF recovery. We successfully developed a mouse model of iatrogenic RLN injury with impaired VF mobility and swallowing function that can serve as a clinically relevant platform to develop translational neuroregenerative strategies for RLN injury.

Recurrent laryngeal nerve transection in mice results in translational upper airway dysfunction.

The recurrent laryngeal nerve (RLN) is responsible for normal vocal-fold (VF) movement, and is at risk for iatrogenic injury during anterior neck surgical procedures in human patients. Injury, resulting in VF paralysis, may contribute to subsequent swallowing, voice, and respiratory dysfunction. Unfortunately, treatment for RLN injury does little to restore physiologic function of the VFs. Thus, we sought to create a mouse model with translational functional outcomes to further investigate RLN regeneration and potential therapeutic interventions. To do so, we performed ventral neck surgery in 21 C57BL/6J male mice, divided into two groups: Unilateral RLN Transection (n = 11) and Sham Injury (n = 10). Mice underwent behavioral assays to determine upper airway function at multiple time points prior to and following surgery. Transoral endoscopy, videofluoroscopy, ultrasonic vocalizations, and whole-body plethysmography were used to assess VF motion, swallow function, vocal function, and respiratory function, respectively. Affected outcome metrics, such as VF motion correlation, intervocalization interval, and peak inspiratory flow were identified to increase the translational potential of this model. Additionally, immunohistochemistry was used to investigate neuronal cell death in the nucleus ambiguus. Results revealed that RLN transection created ipsilateral VF paralysis that did not recover by 13 weeks postsurgery. Furthermore, there was evidence of significant vocal and respiratory dysfunction in the RLN transection group, but not the sham injury group. No significant differences in swallow function or neuronal cell death were found between the two groups. In conclusion, our mouse model of RLN injury provides several novel functional outcome measures to increase the translational potential of findings in preclinical animal studies. We will use this model and behavioral assays to assess various treatment options in future studies.

Mice Lacking Brain-Derived Serotonin Have Altered Swallowing Function.

The intricate sensorimotor neural circuits that control swallowing are heavily reliant on serotonin (5-hydroxytryptamine [5-HT]); however, the impact of 5-HT deficiency on swallow function remains largely unexplored. We investigated this using mice deficient in tryptophan-hydroxylase-2 (TPH2), the enzyme catalyzing the rate-limiting step in 5-HT synthesis. Videofluoroscopy was utilized to characterize the swallowing function of TPH2 knockout (TPH2-/-) mice as compared with littermate controls (TPH2+/+). Results showed that 5-HT deficiency altered all 3 stages of swallowing. As compared with controls, TPH2-/- mice had significantly slower lick and swallow rates and faster esophageal transit times. Future studies with this model are necessary to determine if 5-HT replacement may rescue abnormal swallowing function. If so, supplemental 5-HT therapy may have vast applications for a large population of patients with a variety of neurologic disorders resulting in life-diminishing dysphagia, particularly amyotrophic lateral sclerosis and Parkinson's disease, for which 5-HT deficiency is implicated in the disease pathogenesis.

Affective Behavior in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice during Long-Term Alcohol Abstinence.

BACKGROUND: While the acute alcohol withdrawal syndrome has been well characterized both in human clinical studies and in experimental animals, much less is known regarding long-term affective disturbances that can sometimes persist during protracted abstinence. Nevertheless, since relapse often occurs long after acute detoxification and may be predicted by persistent affective disruption, a better understanding of the long-term behavioral consequences of prior alcohol dependence may lead to improved strategies for relapse prevention. METHODS: Male and female Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice from the second selection replicate (WSP-2, WSR-2) were exposed to a 10-day chronic-intermittent ethanol vapor protocol (CIE) or plain air and then tested repeatedly on the sucrose preference test (SPT), marble burying test (MBT), and the light-dark box test (LDT) over 7 weeks of (forced) abstinence. RESULTS: While WSP and WSR mice differed significantly on tests of anxiety-like behavior (LDT, MBT), we found little evidence for long-term affective disruption following CIE in either line. The major exception was in the LDT, in that WSP but not WSR mice displayed longer latencies to enter the light compartment following CIE relative to air-controls. CONCLUSIONS: Selective breeding for acute withdrawal severity has resulted in differences in anxiety-like behavior between WSP and WSR mice. In contrast, however, genes contributing to the severity of acute withdrawal convulsions appear to have little overlap with those predisposing to affective disruption during long-term abstinence.

A Thermogenic-Like Brown Adipose Tissue Phenotype Is Dispensable for Enhanced Glucose Tolerance in Female Mice.

The prevailing dogma is that thermogenic brown adipose tissue (BAT) contributes to improvements in glucose homeostasis in obesogenic animal models, though much of the evidence supporting this premise is from thermostressed rodents. Determination of whether modulation of the BAT morphology/function drives changes in glucoregulation at thermoneutrality requires further investigation. We used loss- and gain-of-function approaches including genetic manipulation of the lipolytic enzyme Pnpla2, change in environmental temperature, and lifestyle interventions to comprehensively test the premise that a thermogenic-like BAT phenotype is coupled with enhanced glucose tolerance in female mice. In contrast to this hypothesis, we found that 1) compared to mice living at thermoneutrality, enhanced activation of BAT and its thermogenic phenotype via chronic mild cold stress does not improve glucose tolerance in obese mice, 2) silencing of the Pnpla2 in interscapular BAT causes a brown-to-white phenotypic shift accompanied with inflammation but does not disrupt glucose tolerance in lean mice, and 3) exercise and low-fat diet improve glucose tolerance in obese mice but these effects do not track with a thermogenic BAT phenotype. Collectively, these findings indicate that a thermogenic-like BAT phenotype is not linked to heightened glucose tolerance in female mice.

Automated Quantification of Vocal Fold Motion in a Recurrent Laryngeal Nerve Injury Mouse Model.

OBJECTIVES/HYPOTHESIS: The goal of this study was to objectively examine vocal fold (VF) motion dynamics after iatrogenic recurrent laryngeal nerve (RLN) injury in a mouse surgical model. Furthermore, we sought to identify a method of inducing injury with a consistent recovery pattern from which we can begin to evaluate spontaneous recovery and test therapeutic interventions. STUDY DESIGN: Animal model. METHODS: The right RLN in C57BL/6J mice was crushed for 30 seconds using an aneurysm clip with 1.3-N closing force. Transoral laryngoscopy enabled visualization of VF movement prior to surgery, immediately post-crush, and at two endpoints: 3 days (n = 5) and 2 weeks (n = 5). VF motion was quantified with our custom motion-analysis software. At each endpoint, RLN samples were collected for transmission electron microscopy for correlation with VF motion dynamics. RESULTS: Our VF tracking software permitted automated quantification of several measures of VF dynamics, such as range and frequency of motion. By 2 weeks post-injury, the frequency of VF movement on the right (injured) side equaled the left, yet range of motion only partially recovered. These objective outcome measures enabled detection of VF dysfunction that persisted at 2 weeks post-crush. Transmission electron microscopy images revealed RLN degeneration 3 days post-crush and partial regeneration at 2 weeks, consistent with functional results obtained with automated VF tracking. CONCLUSIONS: Our motion-analysis software provides novel objective, quantitative, and repeatable metrics to detect and describe subtle VF dysfunction in mice that corresponds with underlying RLN degeneration and recovery. Adaptation of our tracking software for use with human patients is underway. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E247-E254, 2019.

Effects of Intraoperative Vagal Nerve Stimulation on the Gastrointestinal Microbiome in a Mouse Model of Amyotrophic Lateral Sclerosis.

The gastrointestinal microbiota (GM) plays a fundamental role in health and disease and contributes to the bidirectional signaling between the gastrointestinal system and brain. The direct line of communication between these organ systems is through the vagus nerve. Therefore, vagal nerve stimulation (VNS), a commonly used technique for multiple disorders, has potential to modulate the enteric microbiota, enabling investigation and possibly treatment of numerous neurologic disorders in which the microbiota has been linked with disease. Here we investigate the effect of VNS in a mouse model of amyotrophic lateral sclerosis (ALS). B6SJL-Tg(SOD1*G93A)dl1Gur (SOD1dl) and wildtype mice underwent ventral neck surgery to access the vagus nerve. During surgery, the experimental group received 1 h of VNS, whereas the sham group underwent 1 h of sham treatment. The third (control) group did not undergo any surgical manipulation. Fecal samples were collected before surgery and at 8 d after the initial collection. Microbial DNA was sequenced to determine the GM profiles at both time points. GM profiles did not differ between genotypes at either the initial or end point. In addition, VNS did not alter GM populations, according to the parameters chosen in this study, indicating that this short intraoperative treatment is safe and has no lasting effects on the GM. Future studies are warranted to determine whether different stimulation parameters or chronic use of VNS affect GM profiles.

High-dose rapamycin blocks mossy fiber sprouting but not seizures in a mouse model of temporal lobe epilepsy.

PURPOSE: The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and to measure the effect on seizure frequency. METHODS: Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/day) was administered. Starting 1 month after status epilepticus, mice were monitored by video 9 h per day, every day, for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomic analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections. KEY FINDINGS: Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naive controls. Seizure frequency was similar in vehicle-treated and rapamycin-treated mice. SIGNIFICANCE: These findings suggest that mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have antiseizure or antiepileptogenic effects in this model.

Factors affecting outcomes of pilocarpine treatment in a mouse model of temporal lobe epilepsy.

Pilocarpine-treated mice are an increasingly used model of temporal lobe epilepsy. However, outcomes of treatment can be disappointing, because many mice die or fail to develop status epilepticus. To improve animal welfare and outcomes of future experiments we analyzed results of previous pilocarpine treatments to identify factors that correlate with development of status epilepticus and survival. All treatments were performed by one investigator with mice of the FVB background strain. Results from 2413 mice were evaluated for effects of sex, age, body weight, and latency between administration of atropine methyl bromide and pilocarpine. All parameters correlated with effects on outcomes. Best results were obtained from male mice, 6-7 weeks old, and 21-25 g, with pilocarpine administered 18-30 min after atropine methyl bromide. In that group only 23% failed to develop status epilepticus, and 64% developed status epilepticus and survived. Those results are substantially better than that of the total sample in which 31% failed to develop status epilepticus and only 34% developed status epilepticus and survived.