Disruption of rat deep cerebellar perineuronal net alters eyeblink conditioning and neuronal electrophysiology.

The perineuronal net (PNN) is a specialized type of extracellular matrix found in the central nervous system. The PNN forms on fast spiking neurons during postnatal development but the ontogeny of PNN development has yet to be elucidated. By studying the development and prevalence of the PNN in the juvenile and adult rat brain, we may be able to understand the PNN's role in development and learning and memory. We show that the PNN is fully developed in the deep cerebellar nuclei (DCN) of rats by P18. By using enzymatic digestion of the PNN with chondroitinase ABC (ChABC), we are able to study how digestion of the PNN affects cerebellar-dependent eyeblink conditioning in vivo and perform electrophysiological recordings from DCN neurons in vitro. In vivo degradation of the PNN resulted in significant differences in eyeblink conditioning amplitude and area. Female animals in the vehicle group demonstrated higher levels of conditioning as well as significantly higher post-probe conditioned responses compared to males in that group, differences not present in the ChABC group. In vitro, we found that DCN neurons with a disrupted PNN following exposure to ChABC had altered membrane properties, fewer rebound spikes, and decreased intrinsic excitability. Together, this study further elucidates the role of the PNN in cerebellar learning in the DCN and is the first to demonstrate PNN degradation may erase sex differences in delay conditioning.

Changes in membrane properties of rat deep cerebellar nuclear projection neurons during acquisition of eyeblink conditioning.

Previous studies have shown changes in membrane properties of neurons in rat deep cerebellar nuclei (DCN) as a function of development, but due to technical difficulties in obtaining viable DCN slices from adult animals, it remains unclear whether there are learning-related alterations in the membrane properties of DCN neurons in adult rats. This study was designed to record from identified DCN cells in cerebellar slices from postnatal day 25-26 (P25-26) rats that had a relatively mature sensory nervous system and were able to acquire learning as a result of tone-shock eyeblink conditioning (EBC) and to document resulting changes in electrophysiological properties. After electromyographic electrode implantation at P21 and inoculation with a fluorescent pseudorabies virus (PRV-152) at P22-23, rats received either four sessions of paired delay EBC or unpaired stimulus presentations with a tone conditioned stimulus and a shock unconditioned stimulus or sat in the training chamber without stimulus presentations. Compared with rats given unpaired stimuli or no stimulus presentations, rats given paired EBC showed an increase in conditioned responses across sessions. Whole-cell recordings of both fluorescent and nonfluorescent DCN projection neurons showed that delay EBC induced significant changes in membrane properties of evoked DCN action potentials including a reduced after-hyperpolarization amplitude and shortened latency. Similar findings were obtained in hyperpolarization-induced rebound spikes of DCN neurons. In sum, delay EBC produced significant changes in the membrane properties of juvenile rat DCN projection neurons. These learning-specific changes in DCN excitability have not previously been reported in any species or task.

Virtual Reality Analgesia in Labor: The VRAIL Pilot Study-A Preliminary Randomized Controlled Trial Suggesting Benefit of Immersive Virtual Reality Analgesia in Unmedicated Laboring Women.

This pilot study investigated the use of virtual reality (VR) in laboring women. Twenty-seven women were observed for equivalent time during unmedicated contractions in the first stage of labor both with and without VR (order balanced and randomized). Numeric rating scale scores were collected after both study conditions. Significant decreases in sensory pain -1.5 (95% CI, -0.8 to -2.2), affective pain -2.5 (95% CI, -1.6 to -3.3), cognitive pain -3.1 (95% CI, -2.4 to -3.8), and anxiety -1.5 (95% CI, -0.8 to -2.3) were observed during VR. Results suggest that VR is a potentially effective technique for improving pain and anxiety during labor.

Sex differences in a rabbit eyeblink conditioning model of PTSD.

We have developed a rabbit model of posttraumatic stress disorder (PTSD) which recapitulates several core features of PTSD, particularly hyperarousal and conditioned responding to trauma-associated cues. The work conducted with this model has all been done in male rabbits and, given sex differences in PTSD prevalence, it is important to expand our animal model of PTSD to include female rabbits to determine if they develop core features of PTSD, and if those core features can be treated. This is particularly important because, contrary to human studies, nearly all animal studies have found that males are consistently more vulnerable to various forms of acute and chronic stress than females. Using eyeblink conditioning in which we paired tone with a brief periorbital shock, we found that although both male and female rabbits acquired identical levels of conditioning, females showed more hyperarousal after conditioning but seemed to respond somewhat better to treatment.

Grouping subjects based on conditioning criteria reveals differences in acquisition rates and in strength of conditioning-specific reflex modification.

Averaging behavioral data such as the nictitating membrane response (NMR) across subjects can conceal important individual and group differences. Analyses were conducted of NMR data from rabbits that were grouped based on the point during NMR conditioning when subjects produced 8 conditioned responses (CR) in a set of 10 trials. This resulted in five groups (Early Day 1, Late Day 1, Early Day 2, Late Day 2, Early Day 3) in which group differences in CR acquisition rates were found. Percent (%) CRs were not found to increase monotonically and between-session differences in % CR were found. Conditioning-specific reflex modification (CRM) of the NMR is a type of enhanced reflexive responding of the NMR that is detected when the unconditioned stimulus (US) is presented in the absence of the conditioned stimulus (CS) following paired classical conditioning. CRM occurred in some subjects in all five groups. Subjects from both the group that was fastest and the group that was slowest to reach the learning criterion had unconditioned response (UR) topographies following NMR conditioning that strongly resembled the CR-UR response sequence elicited during NMR conditioning. This finding was most pronounced when the US duration used to assess CRM was equivalent to that used during NMR conditioning, further evidence to support the hypothesis that CRM is a CR that has generalized from the CS to the US. While grouping data based on conditioning criteria did not facilitate identifying individuals more predisposed to exhibiting CRM, strong CRM only occurred in the groups that reached the conditioning criterion the fastest.

Effects of systemic glutamatergic manipulations on conditioned eyeblink responses and hyperarousal in a rabbit model of post-traumatic stress disorder.

Glutamatergic dysfunction is implicated in many neuropsychiatric conditions, including post-traumatic stress disorder (PTSD). Glutamate antagonists have shown some utility in treating PTSD symptoms, whereas glutamate agonists may facilitate cognitive behavioral therapy outcomes. We have developed an animal model of PTSD, based on conditioning of the rabbit's eyeblink response, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). The optimal treatment to reduce both CRs and CRM is unpaired extinction. The goals of the study were to examine whether treatment with the N-methyl-D-aspartate glutamate receptor antagonist ketamine could reduce CRs and CRM, and whether the N-methyl-D-aspartate agonist D-cycloserine combined with unpaired extinction treatment could enhance the extinction of both. Administration of a single dose of subanesthetic ketamine had no significant immediate or delayed effect on CRs or CRM. Combining D-cycloserine with a single day of unpaired extinction facilitated extinction of CRs in the short term while having no impact on CRM. These results caution that treatments may improve one aspect of the PTSD symptomology while having no significant effects on other symptoms, stressing the importance of a multiple-treatment approach to PTSD and of animal models that address multiple symptoms.

Applying to subspecialty fellowship: clarifying the confusion and conflicts!

Of graduating obstetrics and gynecology residents, 40% apply for fellowship training and this percentage is likely to increase. The fellowship interview process creates a substantial financial burden on candidates as well as significant challenges in scheduling the multiple interviews for residents, residency programs, and fellowship programs. Coverage with relatively short lead time is needed for some resident rotations, multiple residents may request time off during overlapping time periods, and applicants may not be able to interview based on conflicting interview dates or the inability to find coverage from other residents for their clinical responsibilities. To address these issues, we propose that each subspecialty fellowship within obstetrics and gynecology be allocated a specified and limited time period to schedule their interviews with minimal overlap between subspecialties. Furthermore, programs in close geographic areas should attempt to coordinate their interview dates. This will allow residents to plan their residency rotation schedules far in advance to minimize the impact on rotations that are less amenable to time away from their associated clinical duties, and decrease the numbers of residents needing time off for interviews during any one time period. In addition, a series of formal discussions should take place between subspecialties related to these issues as well as within subspecialties to facilitate coordination.

Mapping the Acoustic Soundscape off Vancouver Island Using the NEPTUNE Canada Ocean Observatory.

NEPTUNE Canada is a cabled ocean observatory system containing five nodes located in the northeast Pacific Ocean. Using passive acoustic data recorded at two nodes (Folger Passage Deep and Barkley Canyon Axis) between June 2010 and May 2011, we sought to quantify the levels of vessel traffic and the occurrence of biological sounds to determine the potential impact of anthropogenic sound in masking acoustic communication. The results from a comparison of the relative amplitude and occurrence of low-frequency biotic sounds to broadband sounds resulting from vessel traffic are presented. Additional contributions to the marine soundscape from self-generated instrument noise are discussed.

Dietary cholesterol degrades rabbit long term memory for discrimination learning but facilitates acquisition of discrimination reversal.

We have shown previously that feeding dietary cholesterol before learning can improve acquisition whereas feeding cholesterol after learning can degrade long term memory. To examine these different findings within a single paradigm, we fed groups of rabbits 2% cholesterol or normal chow with or without 0.12 ppm copper added to the drinking water following two-tone discrimination learning of the nictitating membrane response in which a 8-kHz tone (conditioned stimulus, CS+) was followed by air puff and a 1-kHz tone (CS-) was not. After eight weeks on the diet, we assessed the rabbits' conditioned responding during testing and retraining. We then reversed the two-tone discrimination and assessed responding to the 1-kHz tone CS+ and the 8-kHz CS-. During testing, rabbits given cholesterol without copper had lower levels of responding to CS+ than rabbits in the other groups suggesting they did not retain the discrimination as well. However, during a brief discrimination retraining session, their response levels to the CS+ returned to the level of the other groups, demonstrating a return of the memory of the original discrimination. At the end of discrimination reversal, these same rabbits exhibited superior discrimination indexed by lower response levels to CS- but similar levels to CS+, suggesting they were better able to acquire the new relationship between the two tones by inhibiting CS- responses. These results add to our previous data by showing cholesterol diet-induced degradation of an old memory and facilitation of a new memory can both be demonstrated within a discrimination reversal paradigm. Given discrimination reversal is a hippocampally-dependent form of learning, the data support the role of cholesterol in modifying hippocampal function as we have shown previously with in vitro brain slice recordings.

Subacute fluoxetine enhances conditioned responding and conditioning-specific reflex modification of the rabbit nictitating membrane response: implications for drug treatment with selective serotonin reuptake inhibitors.

Extensive research on the rabbit nictitating membrane response (NMR) has shown that the NMR reflex can become exaggerated following classical fear conditioning. This learning-related change is referred to as conditioning-specific reflex modification (CRM) and is observed in the absence of the conditioned stimulus. The aim of the current study was to examine the sensitivity of the CRM paradigm to serotonergic manipulation with fluoxetine, a commonly prescribed selective serotonin reuptake inhibitor for anxiety disorders. To assess the effect of fluoxetine on exaggerated reflexive responding indicative of CRM and on conditioned cued fear, rabbits underwent delay NMR conditioning (pairings of tone and periorbital shock) and were tested for CRM, followed by 5 days of daily fluoxetine (0.03, 0.3, or 3.0 mg/kg) or saline injections. CRM was reassessed 1 day and 1 week later, followed by a retention test of conditioned responses (CRs) to the tone. Fluoxetine (3.0 mg/kg) enhanced CRM and retention of conditioned responses, a week after treatment ceased, and this is in agreement with the reports on increased anxiety-like behaviors in other animal models and humans. The CRM paradigm, therefore, may provide important insight into the mechanisms underlying the paradoxical selective serotonin reuptake inhibitor effects.

Eyeblink tract tracing with two strains of herpes simplex virus 1.

BACKGROUND: Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. NEW METHOD: We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. RESULTS AND COMPARISON WITH EXISTING METHODS: Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. CONCLUSIONS: Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Neurovascular changes measured by time-of-flight MR angiography in cholesterol-fed rabbits with cortical amyloid beta-peptide accumulation.

PURPOSE: To test the hypothesis that narrowing of cranial blood vessels in cholesterol-fed rabbits is a function of the duration of the high cholesterol diet. Such neurovascular changes, caused by elevated serum cholesterol, are linked to stroke and Alzheimer's disease risk. MATERIALS AND METHODS: Four groups of New Zealand White rabbits were studied. Six were fed a normal diet, 19 were fed a 2% cholesterol diet with 0.12 ppm copper in the drinking water for 8 weeks, 10 weeks, or 12 weeks. Time-of-flight (TOF) MR angiography (MRA) at 3 Tesla was used to measure arterial diameters in 11 vessels. Previously published data for amyloid beta-peptide (Abeta) accumulation in the brains measured postmortem were correlated to vessel diameters. Ventricular volumes of rabbits were measured on group-averaged data. RESULTS: Several vessel diameters decreased with cholesterol diet duration. The posterior communicating arteries showed the largest significant effect. Abeta accumulation was inversely correlated with arterial diameter. Ventricular volumes between the normal diet and 12 weeks cholesterol-fed groups were not significantly different. CONCLUSION: Reduction in vessel diameter of medium-sized vessels but not large vessels was measured in these hypercholesterolemic rabbits. The vessel diameter narrowing and cortical Abeta deposition occurred before measurable ventricular enlargement.

Timing and Outcomes of an Indication-Only Use of Intravenous Cannulation During Spontaneous Labor.

INTRODUCTION: In the United States, most women presenting in spontaneous labor undergo intravenous (IV) cannulation on admission to hospital labor and birth units. There is limited evidence for this routine practice in pregnant women at low risk for adverse outcomes during labor or birth. METHODS: A retrospective, exploratory, descriptive study of an indication-only practice of IV cannulation on admission for women presenting in spontaneous labor and cared for by a nurse-midwife service was performed. Descriptive data included the timing of IV cannula placement (admission, during labor or postpartum period, or not at all) and indications for placement. Maternal outcomes of interest were estimated blood loss, postpartum hemorrhage rates, and management; neonatal outcome was 5-minute Apgar scores. RESULTS: Records for 1069 women cared for by nurse-midwives who presented in spontaneous labor were reviewed. In this cohort, 445 (41.6%) had IV access established on admission, 325 (30.4%) had an IV cannula placed during labor or postpartum, and 299 (28%) never had IV access during their hospital stay. For the 325 women with IV cannulas placed after admission, 25 (7.7%) were placed urgently for excessive postpartum bleeding. Further analysis of the subset of women who had a postpartum hemorrhage after vaginal birth (defined as >500 mL estimated blood loss) indicated that urgent IV cannulation was not associated with a lower mean postpartum hemoglobin or hematocrit or an increase in blood transfusion rate when compared with women who had an IV cannula placed earlier in their labor course. DISCUSSION: Indication-only IV cannulation for women experiencing an uncomplicated labor and birth is a reasonable practice in settings where IV access can be established urgently if needed.

Conditioning-specific reflex modification of the rabbit's nictitating membrane response and heart rate: behavioral rules, neural substrates, and potential applications to posttraumatic stress disorder.

Interest in classical conditioning is usually focused on anticipatory responses to a stimulus associated with a significant event, and it is assumed that responses to the event itself are reflexive, involuntary, and relatively invariant. However, there is compelling evidence that both the rabbit nictitating membrane response (NMR) and heart rate response (HR), well-known reflexive reactions to aversive events, can change quite dramatically as a function of learning when measured in the absence of the conditioned stimulus. In the case of NMR conditioning, a simple blink is transformed into a larger and more complex response. For HR conditioning, reflexive heart rate acceleration can actually change to heart rate deceleration. In both cases, the reflex comes to resemble the conditioned response and follows some of the same behavioral laws. This change in response to the aversive event itself or weaker forms of that event is called conditioning-specific reflex modification (CRM). CRM may force us to reevaluate the behavioral and neural consequences of classical conditioning and may have important consequences for the treatment of conditions such as posttraumatic stress disorder.

Delayed unpaired extinction as a treatment for hyperarousal of the rabbit nictitating membrane response and its implications for treating PTSD.

Treatment for PTSD (Post-traumatic stress disorder) is rarely available immediately after trauma and often delayed for weeks or months after an event. In a rabbit eyeblink conditioning model of PTSD, we have previously shown that presentations of a tone conditioned stimulus (CS) and shock unconditioned stimulus (US) in an explicitly unpaired manner known as unpaired extinction is effective in reducing CS responding and US hyperarousal even if shock intensity is reduced eight-fold and elicits only minimal responding. Here we determined if delayed delivery of unpaired extinction would still be effective in extinguishing hyperarousal. Rabbits were tested for sensitivity to shock before CS-US pairings and after six days of unpaired extinction presented a day, a week or a month after CS-US pairings. Hyperarousal was extinguished a day and a week after conditioning but not after a month suggesting a significant delay in "treatment" can make hyperarousal persist. We next assessed if this persistence of hyperarousal was associative by comparing rabbits given CS-US pairings to those given explicitly unpaired CS and US presentations, measuring hyperarousal a day and a month later, followed by unpaired extinction and hyperarousal assessment. After four weeks, there was an increase in responding for all rabbits but only rabbits receiving CS-US pairings showed a significant increase in associatively-mediated hyperarousal. Importantly, both paired and unpaired groups showed increased levels of responding after unpaired extinction suggesting treatment delayed for too long may no longer be effective and could cause generalized hyperarousal.

Propranolol produces short-term facilitation of extinction in a rabbit model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a learning-based anxiety disorder with significant public health challenges due to difficulties in treating the complex, multiple symptomology. We have developed an animal model of PTSD, based on Pavlovian eyeblink conditioning in rabbits, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). We have found previously that unpaired extinction is ideal for reducing both CRs and CRM simultaneously and shows sensitivity to systemic serotonergic and glutamatergic manipulations. The following study aimed to extend our work to examine the role of the noradrenergic system, dysregulation of which is strongly implicated as part of the neurobiology of PTSD and which may also play a role in the balance shift from fear reconsolidation to extinction during treatment. The goal of the following two studies was to examine whether the ß-adrenergic receptor antagonist propranolol combined with either a full or brief course of unpaired extinction treatment could enhance extinction of CRs and/or CRM. Results showed a within-session facilitation of propranolol on extinction of CRs, particularly during the first extinction session, and a short-term enhancement of extinction of CRM when extinction treatment was brief. However, neither benefit translated to long-term extinction retention for the majority of subjects. Findings suggest that propranolol may provide the most therapeutic benefit in situations of high arousal early in treatment, which may be more important for future patient compliance rather than long-term treatment outcomes.

Cholesterol enhances classical conditioning of the rabbit heart rate response.

The cholesterol-fed rabbit is a model of atherosclerosis and has been proposed as an animal model of Alzheimer's disease. Feeding rabbits cholesterol has been shown to increase the number of beta amyloid immunoreactive neurons in the cortex. Addition of copper to the drinking water of cholesterol-fed rabbits can increase this number still further and may lead to plaque-like structures. Classical conditioning of the nictitating membrane response in cholesterol-fed rabbits is retarded in the presence of these plaque-like structures but may be facilitated in their absence. In a factorial design, rabbits fed 2% cholesterol or a normal diet (0% cholesterol) for 8 weeks with or without copper added to the drinking water were given trace classical conditioning using a tone and periorbital electrodermal stimulation to study the effects of cholesterol and copper on classical conditioning of heart rate and the nictitating membrane response. Cholesterol-fed rabbits showed significant facilitation of heart rate conditioning and conditioning-specific modification of heart rate relative to normal diet controls. Consistent with previous research, cholesterol had minimal effects on classical conditioning of the nictitating membrane response when periorbital electrodermal stimulation was used as the unconditioned stimulus. Immunohistochemical analysis showed a significant increase in the number of beta amyloid positive neurons in the cortex, hippocampus and amygdala of the cholesterol-fed rabbits. Supplementation of drinking water with copper increased the number of beta amyloid positive neurons in the cortex of cholesterol-fed rabbits but did not produce plaque-like structures or have a significant effect on heart rate conditioning. The data provide additional support for our finding that, in the absence of plaques, dietary cholesterol may facilitate learning and memory.