Characterizing Purkinje Cell Responses and Cerebellar Influence on Fluid Licking in the Mouse

Rodents consume water by performing stereotypical, rhythmic licking movements which are believed to be driven by central pattern generating circuits located in the brainstem. Temporal aspects of rhythmic licking behavior have been shown to be represented in the olivo-cerebellar system in the form of population complex spike activity. These findings suggest that the olivo-cerebellar system is involved in the generating circuitry responsible for licking rhythm in rodents. However, the representation of licking in the simple spike activity of Purkinje cells and the consequences of loss of cerebellar function on licking behavior has not been quantified. I investigated the influence of the cerebellum on the maintenance of rhythm in murine fluid-licking. In one set of experiments, I characterized Purkinje cell activity in healthy mice during fluid licking. Use of a head-restrained preparation allowed recordings of well-isolated single units during repeated experimental sessions. Thus, a large number of neurons were tested for their relationship with behavior and detailed spatial maps of behavior related neuronal activity were generated as exemplified here with recordings from lick-related Purkinje cells in the cerebellum. The data show a multifaceted representation of licking behavior in the simple spike activity of a large population of Purkinje cells distributed across Crus I, Crus II, and lobus simplex of mouse cerebellar cortex. Lick related Purkinje cell simple spike activity was changed in a manner that was either rhythmic, in phase with the lick rhythm, or nonrhythmic with a decrease or increase in firing in relation to licks but not phasically. For rhythmically responsive units, signal modulation was marked by the introduction of a phasic variation in the frequency of spikes. A subpopulation of lick related Purkinje cells exhibited different activity patterns during short and long interlick intervals (ILIs). I examined the role of the cerebellum in fluid-licking by using several models of cerebellar ataxia with distinct causes. First, I observed fluid-licking in animals over several days to determine how the microstructure of the behavior may also be altered. The first model involved animals that underwent cerebellectomies. Surgical removal of the cerebellum resulted in significant slowing of the lick rhythm but did not affect the mouse’s ability to perform the gross licking movement. Thus, the cerebellum is involved in the modulation but not in the generation of the licking rhythm. Next, I observed changes to behavior in animals with a genetic cause to their ataxia, the Cerebellin1 (CBLN1) knockout and heterozygous mice (Morgan et al., 1988). The CBLN1 gene is a member of a family of proteins that have been found primarily in the Purkinje cell/parallel fiber synapse and is thought to stabilize the connection. Although removal of the gene does not alter the numbers of neurons or their spatial relations, the mutation results in moderate to severe ataxia. While these animals also varied significantly from their wild type counterparts in lick rate and microstructure, the changes were not all similar to the cerebellectomized model of ataxia. For example, cerebellectomized mice licked significantly slower with an average ILI of 135 ± 8 ms (mean ± S.D.) compared to 117 ± 7 ms whereas in cbln1 KO had a faster lick rate (110 ± 4 ) than wild type counterparts (121 ± 6 ), with all of these values significant with p \u3c 0.05. These observations show that the removal of the normal functions of the cerebellum can alter fluid-licking resulting in bidirectional rate changes. An alternative possibility is that there may be compensatory process. Lastly, I used a chemically-induced model of cerebellar ataxia by injecting the GABA agonist muscimol in the medial and lateral deep cerebellar nuclei. This transient cerebellar ataxia resulted in a similar slowing of the licking rhythm as in the cerebellectomized mice with the eventual recovery of the fluid-licking behavior to normal as the effect of muscimol wore off. My work to characterize the role of the cerebellum in the maintenance of fluid- licking rhythm and behavior microstructure has resulted in the development of experimental procedures for the recording of neuronal activity in awake and behaving mice. It is an important and necessary step towards neurophysiological investigation of normal and pathological mouse brain function. I have presented the first characterization of simple spike activity, the main cerebellar cortical output signal, during fluid-licking. Furthermore, my results show that the cerebellum is also involved in the control of fluid intake or homeostasis as the intervals between drinking events were abnormally long in mice with cerebellar ataxia. Electrophysiological recordings of individual Purkinje cells from the cerebellar cortex demonstrated variations in spike activity capable of influencing the rhythmicity of fluid licking. While licking still occurred with relative regularity in ataxic animals, the lick rates slowed significantly for mice with surgically induced ataxia and pharmalogically induced ataxia. For animals with a genetic origin to ataxia, lick rates increased. Regularity of licking remained evident despite the change in interlick interval duration. Any alteration of lick timing could ultimately affect the coordination of licking with other orofacial movements. Future investigations may benefit from this work by investigating if therapeutic interventions for cerebellar ataxias show a recovery of typical behavior or adapt the neurophysiological recordings to other behaviors in awake mice

A systematic review of the development and application of home cage monitoring in laboratory mice and rats

Funding Information: Open Access funding enabled and organized by Projekt DEAL. VV is supported by Jane and Aatos Erkko Foundation. Funding Information: The Vienna BioCenter Core Facilities (VBCF) Preclinical Phenotyping Facility acknowledges funding from the Austrian Federal Ministry of Education, Science & Research; and the City of Vienna. Funding Information: This article is based upon work from COST Action “Improving biomedical research by automated behaviour monitoring in the animal home-cage” (TEATIME; CA20135; cost-teatime.org) supported by COST (European Cooperation in Science and Technology). Funding Information: PK, PM, AJ, BL, CTR, LL, and KH were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC 2002/1 “Science of Intelligence” —project number 390523135. Funding Information: This article is based upon work from COST Action “Improving biomedical research by automated behaviour monitoring in the animal home-cage” (TEATIME; CA20135; cost-teatime.org) supported by COST (European Cooperation in Science and Technology). Publisher Copyright: © 2023, The Author(s).Background: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. Results: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011–2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4–12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. Conclusions: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.publishersversionpublishe

Monitoring System for Laboratory Mice Transportation: A Novel Concept for the Measurement of Physiological and Environmental Parameters

Laboratory mice are used in biomedical research as “models” for studying human disease. These mice may be subject to significant levels of stress during transportation that can cause alterations that could negatively affect the results of the performed investigation. Here, we present the design and realization of a prototypical transportation container for laboratory mice, which may contribute to improved laboratory animal welfare. This prototype incorporates electric potential integrated circuit (EPIC) sensors, which have been shown to allow the recording of physiological parameters (heart rate and breathing rate) and other sensors for recording environmental parameters during mouse transportation. This allows for the estimation of the stress levels suffered by mice. First experimental results for capturing physiological and environmental parameters are shown and discussed

Behavioral verification of associative learning in whisker-related fear conditioning in mice

Fear-conditioning is one of the most widely used paradigms in attempts to unravel the processes and mechanisms underlying learning and plasticity. In most Pavlovian conditioning paradigms an auditory stimulus is used as the conditioned stimulus (CS), but conditioning to a tactile CS can also be accomplished. The whisker-to-barrel tactile system in mice offers a convenient way to investigate the brain pathways and mechanisms of learning and plasticity of the cerebral cortex. To support the claim that an animal learns during conditioning sessions and that the resulting plastic changes are associative in nature, objective measures of behavior are necessary. Multiple types of conditioned responses can develop depending on the training situation, CS and unconditioned stimulus (UCS) characteristics. These include physiological responses such as salivation, heart rate, and galvanic skin reaction, and also behavioral responses such as startle reflex potentiation or suppression of an ongoing behavior. When studying learning with the whisker system in behaving mice, stimulation of individual whiskers in a well-controlled manner may require animal restraint, which has the disadvantage of limiting the observation of potential behavioral responses. Stimulation of whiskers in a neck-restraining apparatus evokes head movements. When whisker stimulation (CS) is paired with an aversive UCS during conditioning, the number of head movements decrease in the course of the training. This reaction, called minifreezing, resembles the frequently used behavioral measure known as the freezing response. However, this is only applicable for freely moving animals. This article will review experimental evidence confirming that minifreezing is a relevant index of association formation between the neutral CS and aversive UCS

Refinements to rodent head fixation and fluid/food control for neuroscience

The use of head fixation in mice is increasingly common in research, its use having initially been restricted to the field of sensory neuroscience. Head restraint has often been combined with fluid control, rather than food restriction, to motivate behaviour, but this too is now in use for both restrained and non-restrained animals. Despite this, there is little guidance on how best to employ these techniques to optimise both scientific outcomes and animal welfare. This article summarises current practices and provides recommendations to improve animal wellbeing and data quality, based on a survey of the community, literature reviews, and the expert opinion and practical experience of an international working group convened by the UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Topics covered include head fixation surgery and post-operative care, habituation to restraint, and the use of fluid/food control to motivate performance. We also discuss some recent developments that may offer alternative ways to collect data from large numbers of behavioural trials without the need for restraint. The aim is to provide support for researchers at all levels, animal care staff, and ethics committees to refine procedures and practices in line with the refinement principle of the 3Rs

Aerospace Medicine and Biology: A continuing bibliography, supplement 216

One hundred twenty reports, articles, and other documents introduced into the NASA scientific and technical information system in January 1981 are listed. Topics include: sanitary problems; pharmacology; toxicology; safety and survival; life support systems; exobiology; and personnel factors

Development of a Carbon Nanotube-Based Micro-CT and its Applications in Preclinical Research

Due to the dependence of researchers on mouse models for the study of human disease, diagnostic tools available in the clinic must be modified for use on these much smaller subjects. In addition to high spatial resolution, cardiac and lung imaging of mice presents extreme temporal challenges, and physiological gating methods must be developed in order to image these organs without motion blur. Commercially available micro-CT imaging devices are equipped with conventional thermionic x-ray sources and have a limited temporal response and are not ideal for in vivo small animal studies. Recent development of a field-emission x-ray source with carbon nanotube (CNT) cathode in our lab presented the opportunity to create a micro-CT device well-suited for in vivo lung and cardiac imaging of murine models for human disease. The goal of this thesis work was to present such a device, to develop and refine protocols which allow high resolution in vivo imaging of free-breathing mice, and to demonstrate the use of this new imaging tool for the study many different disease models. In Chapter 1, I provide background information about x-rays, CT imaging, and small animal micro-CT. In Chapter 2, CNT-based x-ray sources are explained, and details of a micro-focus x-ray tube specialized for micro-CT imaging are presented. In Chapter 3, the first and second generation CNT micro-CT devices are characterized, and successful respiratory- and cardiac-gated live animal imaging on normal, wild-type mice is achieved. In Chapter 4, respiratory-gated imaging of mouse disease models is demonstrated, limitations to the method are discussed, and a new contactless respiration sensor is presented which addresses many of these limitations. In Chapter 5, cardiac-gated imaging of disease models is demonstrated, including studies of aortic calcification, left ventricular hypertrophy, and myocardial infarction. In Chapter 6, several methods for image and system improvement are explored, and radiation therapy-related micro-CT imaging is present. Finally, in Chapter 7 I discuss future directions for this research and for the CNT micro-CT.Doctor of Philosoph

Etude expérimentale des dynamiques temporelles du comportement normal et pathologique chez le rat et la souris

155 p.Modern neuroscience highlights the need for designing sophisticated behavioral readout of internal cognitive states. From a thorough analysis of classical behavioral test, my results supports the hypothesis that sensory ypersensitivity might be the cause of other behavioural deficits, and confirm the potassium channel BKCa as a potentially relevant molecular target for the development of drug medication against Fragile X Syndrome/Autism Spectrum Disorders. I have also used an innovative device, based on pressure sensors that can non-invasively detect the slightest animal movement with unprecedented sensitivity and time resolution, during spontaneous behaviour. Analysing this signal with sophisticated computational tools, I could demonstrate the outstanding potential of this methodology for behavioural phenotyping in general, and more specifically for the investigation of pain, fear or locomotion in normal mice and models of neurodevelopmental and neurodegenerative disorders

Aerospace medicine and biology: A continuing bibliography with indexes, supplement 130, July 1974

This special bibliography lists 291 reports, articles, and other documents introduced into the NASA scientific and technical information system in June 1974

Centrifuge facility conceptual system study. Volume 2: Facility systems and study summary

The Centrifuge Facility is a major element of the biological research facility for the implementation of NASA's Life Science Research Program on Space Station Freedom using nonhuman species (small primates, rodents, plants, insects, cell tissues, etc.). The Centrifuge Facility consists of a variable gravity Centrifuge to provide artificial gravity up to 2 earth G's' a Holding System to maintain specimens at microgravity levels, a Glovebox, and a Service Unit for servicing specimen chambers. The following subject areas are covered: (1) Holding System; (2) Centrifuge System; (3) Glovebox System; (4) Service System; and (5) system study summary