Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ABCB1 gene by PCR and isothermal amplification

Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within similar to 1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54;heterozygous for the mutation, n = 37;homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ABCB1 genotyping with less equipment than existing methods

Daily torpor: When heart and brain go cold - Nonlinear cardiac dynamics in the seasonal heterothermic Djungarian hamster

Djungarian hamsters (Phodopus sungorus) acclimated to short photoperiod display episodes of spontaneous daily torpor with metabolic rate depressed by ∼70%, body temperature (

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Behavioral and autonomic dynamics contextual fear conditioning in mice

Aversive conditioning to contextual stimulation was performed in mice implanted with ECG transmitters to investigate heart rate (HR) and behavioral responses during contextual retention. The dynamics of HR were analyzed by advanced nonlinear techniques to uncover central neuroautonomic outflow inferred from its sympathetic (SNS) and parasympathetic (PNS) projection onto the sinus node of the heart. Mice experienced a single foot shock (US, unconditioned stimulus) either immediately (USi) or late (USl) after placement in the conditioning context. Contextual memory was tested 24 h after training by reexposure to the conditioning context for 32 min. Only mice that experienced the USl exhibited a pronounced and sustained behavioral suppression (immobility) indicative of conditioned contextual fear. In contrast, HR was initially close to its maximal physiological limit (∼800 bpm) in all groups, and recovery towards baseline levels was sluggish, the most pronounced delay observed in the USl group. The results demonstrate that behavioral immobility was associated with maximum activation of autonomic system output in response to contextual reexposure. However, advanced complexity analysis of the variability of HR revealed uniform or stereotyped dynamical properties that were interpreted to reflect a generalized state of anticipatory emotional arousal experienced during reexposure to contextual stimuli. It is concluded that the dynamics of HR is a highly sensitive index of the autonomic nervous system response and emotional state elicited by sensory stimulation of an unfamiliar environment. © 2004 Elsevier B.V. All rights reserved

Heart rate dynamics and behavioral responses during acute emotional challenge in corticotropin-releasing factor receptor 1-deficient and corticotropin-releasing factor overexpressing mice

The role of corticotropin-releasing factor in autonomic regulation of heart rate, heart rate variability and behavior responses was investigated in two genetic mouse models: corticotropin-releasing factor receptor 1-deficient mice, and corticotropin-releasing factor-transgenic mice overexpressing corticotropin-releasing factor. Heart rate was recorded by radio-telemetry during novelty exposure and auditory fear conditioning. Locomotor activity and freezing served as behavioral indices. Locomotor activity and heart rate were invariably increased in response to novelty exposure in both corticotropin-releasing factor receptor 1-deficient mice and littermate wild-type controls. The heart rate responses during retention of conditioned auditory fear and the exponential relationship between heart rate and heart rate variability were unaffected by genotype. Moreover, conditioned fear responses inferred from multiple behavioral measures including freezing did not differ between corticotropin-releasing factor receptor 1-deficient and corticotropin-releasing factor receptor 1 wild-type control mice. Corticotropin-releasing factor-transgenic mice exhibited markedly reduced locomotor activity during novelty exposure when compared with littermate wild-type controls. Baseline and novelty-driven heart rate was slightly elevated in corticotropin-releasing factor-transgenic mice, whereas the novelty-induced increase of heart rate was not different between genotypes. In contrast, corticotropin-releasing factor-transgenic mice did not display a heart rate response indicative of conditioned auditory fear. It is concluded that corticotropin-releasing factor receptor 1-deficiency does not affect heart rate adjustment and behavioral responses to acute fearful stimuli. The resiliency of behavioral and cardiovascular patterns elevation argues against the involvement of corticotropin-releasing factor receptor 1 in acute emotional regulation on these two functional levels despite an absent corticosterone elevation in corticotropin-releasing factor receptor 1-deficient mice. It is hypothesized that the lack of a conditioned heart rate response in corticotropin-releasing factor-transgenic mice is attributable to an impairment of cognitive function. The results are compared with those of corticotropin-releasing factor receptor 2-deficient mice, and the role of the corticotropin-releasing factor system in cardiovascular regulation is discussed. © 2005 Published by Elsevier Ltd on behalf of IBRO

Blockade of 5-HT 1B receptors facilitates contextual aversive learning in mice by disinhibition of cholinergic and glutamatergic neurotransmission

Serotonergic (5-HT) neurotransmission plays a role in learning and memory processes, but the physiological role of various receptor subtypes is not well characterised. Among these, 5-H