Brain-derived neurotrophic factor and TrkB modulate visual experience-dependent refinement of neuronal pathways in retina.

Sensory experience refines neuronal structure and functionality. The visual system has proved to be a productive model system to study this plasticity. In the neonatal retina, the dendritic arbors of a large proportion of ganglion cells are diffuse in the inner plexiform layer. With maturation, many of these arbors become monolaminated. Visual deprivation suppresses this remodeling. Little is known of the molecular mechanisms controlling maturational and experience-dependent refinement. Here, we tested the hypothesis that brain-derived neurotrophic factor (BDNF), which is known to regulate dendritic branching and synaptic function in the brain, modulates the developmental and visual experience-dependent refinement of retinal ganglion cells. We used a transgenic mouse line, in which a small number of ganglion cells were labeled with yellow fluorescence protein, to delineate their dendritic structure in vivo. We found that transgenic overexpression of BDNF accelerated the laminar refinement of ganglion cell dendrites, whereas decreased TrkB expression or retina-specific deletion of TrkB, the cognate receptor for BDNF, retarded it. BDNF-TrkB signaling regulated the maturational formation of new branches in ON but not the bilaminated ON-OFF ganglion cells. Furthermore, BDNF overexpression overrides the requirement for visual inputs to stimulate laminar refinement and dendritic branching of ganglion cells. These experiments reveal a previously unrecognized action of BDNF and TrkB in controlling cell-specific, experience-dependent remodeling of neuronal structures in the visual system.

Using a Time-Driven Activity-Based Costing Model To Determine the Actual Cost of Services Provided by a Transgenic Core.

Laboratory animal programs and core laboratories often set service rates based on cost estimates. However, actual costs may be unknown, and service rates may not reflect the actual cost of services. Accurately evaluating the actual costs of services can be challenging and time-consuming. We used a time-driven activity-based costing (ABC) model to determine the cost of services provided by a resource laboratory at our institution. The time-driven approach is a more efficient approach to calculating costs than using a traditional ABC model. We calculated only 2 parameters: the time required to perform an activity and the unit cost of the activity based on employee cost. This method allowed us to rapidly and accurately calculate the actual cost of services provided, including microinjection of a DNA construct, microinjection of embryonic stem cells, embryo transfer, and in vitro fertilization. We successfully implemented a time-driven ABC model to evaluate the cost of these services and the capacity of labor used to deliver them. We determined how actual costs compared with current service rates. In addition, we determined that the labor supplied to conduct all services (10,645 min/wk) exceeded the practical labor capacity (8400 min/wk), indicating that the laboratory team was highly efficient and that additional labor capacity was needed to prevent overloading of the current team. Importantly, this time-driven ABC approach allowed us to establish a baseline model that can easily be updated to reflect operational changes or changes in labor costs. We demonstrated that a time-driven ABC model is a powerful management tool that can be applied to other core facilities as well as to entire animal programs, providing valuable information that can be used to set rates based on the actual cost of services and to improve operating efficiency.

Increased host neuronal survival and motor function in BMT Parkinsonian mice: involvement of immunosuppression.

We examined the potential of bone marrow transplantation (BMT) to rescue dopaminergic neurons in a mouse model of Parkinson's disease (PD). A BMT from mice transgenic for green fluorescent protein (GFP(+)) given either before or after administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) led to the accumulation of transplanted adult GFP(+) bone-marrow-derived cells (BMDC) in the substantia nigra, where dopaminergic neurodegeneration occurs in PD. Post-BMT, mice exposed to MPTP had substantially greater numbers of endogenous tyrosine hydroxylase-positive neuronal cell bodies in the substantia nigra and increased dopamine transporter-positive projections into the striatum compared to controls. Moreover, motor function was restored to normal within 1 month post-MPTP in BMT-treated mice assayed by a rotarod behavioral test. The effect of BMT on PD was indirect, as no evidence of BMDC fusion with or transdifferentiation into dopaminergic neurons was observed. BMDC activated by BMT or associated factors could play a trophic role in rescuing damaged cells. Alternatively, the beneficial effects of BMT are due to immunosuppression reflected by a reduction in the proportion of T-cells and a reduction of T-cell proliferation in BMT mice. These findings highlight that when immunosuppression is required for transplantation studies, the amelioration of symptoms may not be due to the transplant itself. Further, they suggest that the immune system plays a role in the development of characteristics typical of PD.

Effects of Breeding Configuration on Maternal and Weanling Behavior in Laboratory Mice.

Although numerous studies have evaluated the effect of housing density on the wellbeing of laboratory mice, little is known about the effect of breeding configuration on mouse behavior. The 8th edition of the Guide for the Care and Use of Laboratory Animals lists the recommended minimal floor area per animal for a female mouse and her litter as 51 in.2 We sought to determine the effects of pair, trio, and harem breeding configurations on the maternal and weanling behavior of C57BL/6J (B6) and 129S6/SvEvTac (129) mice on the basis of nest scores and performance in pup retrieval tests, open-field test (OFT), elevated plus maze, and tail suspension test; we concurrently evaluated cage microenvironment, reproductive indices, and anatomic and clinical pathology. Harem breeding configurations enhanced B6 maternal behaviors as evidenced by significantly shorter pup retrieval times. Trio- and harem-raised B6 weanlings showed increased exploratory behaviors, as evidenced by greater time spent in the center of the OFT, when compared with pair-raised B6 mice. Conversely, breeding configuration did not alter pup retrieval times for 129 mice, and on the day of weaning trio- and harem-raised 129 mice demonstrated increased anxiety-like behavior, as evidenced by greater time spent in the periphery of the OFT, when compared with pair-raised counterparts. Behavioral differences were not noted on subsequent days for either strain. Trio- and harem-raised B6 and 129 weanling mice had significantly higher weaning weights than weanlings raised in a pair breeding configuration. Trio and harem breeding in a standard 67-in.2 shoebox cage did not detrimentally affect the evaluated welfare parameters in either C57BL/6J or 129S6/SvEvTac mice.

The Behavioral Effects of Single Housing and Environmental Enrichment on Adult Zebrafish (Danio rerio).

Environmental enrichment provides laboratory-housed species the opportunity to express natural behavior and exert control over their home environment, thereby minimizing stress. We sought to determine whether providing an artificial plant in the holding tank as enrichment influenced anxiety-like behaviors and place-preference choice in adult zebrafish. Fish were housed singly or in social groups of 5 for 3 wk in 1 of 4 experimental housing environments: single-housed enriched (n = 30), single-housed barren (n = 30), group-housed enriched (n = 30), and group-housed barren (n = 30). On week 4, individual fish were selected randomly from each of the experimental housing environments and tested by using novel-tank, light-dark, and place-preference tests. Housing fish singly in a barren environment increased anxiety-like behaviors in the novel-tank and light-dark behavioral tests. Single-housed zebrafish in barren tanks as well as zebrafish group-housed with conspecifics, both with and without plant enrichment, spent more time associating with conspecifics than with the artificial plant enrichment device during the place-preference test. Single-housed fish maintained in enriched tanks displayed no preference between a compartment with conspecifics or an artificial plant. Our results suggest the addition of an artificial plant as enrichment may benefit single-housed zebrafish when social housing is not possible.

Use of microisolator caging in a risk-based mouse import and quarantine program: a retrospective study.

The expanding use of genetically engineered mice has placed increasing demands upon research facilities to import mutant strains from noncommercial sources. Conventional quarantine strategies use relatively large amounts of space per animal and/or require specialized equipment (e.g., cubicles, isolators, and ventilated racks). We have retrospectively assessed a quarantine program instituted 4 years ago that used a small amount of space and minimized the need for special equipment. Shipments presumed, in light of health reports, to be free of agents excluded from our colonies were housed in static microisolator caging in a shared quarantine room. Rather than functioning as an "all-in-all-out" area, the room continually received new shipments, which were released intermittently as multi-shipment groups after testing was performed. Noninvasive testing of the imported mice was combined with nonsurvival sampling of sentinels that had been exposed to shipped animals via direct contact and/or exposure to soiled bedding. During the 4-year period examined, the vast majority of shipments presumed to be free of excluded agents showed no evidence of contamination when screened. When active infection was detected in the shared room, the procedures in place proved sufficient to prevent cross-contamination of other shipments. The use of sentinel animals to detect the shedding of infectious agents during quarantine was found to be an effective strategy that minimized the potential impact of invasive testing on quarantined animals. Although the program we describe is not appropriate for all situations, this type of approach may be considered by institutions wishing to explore alternatives to conventional quarantine strategies during periods of restricted space availability.

Use of molecular methods for genetic monitoring of an institutional mouse breeding colony.

It is important to perform genetic quality control on inbred mouse strains to minimize variability of genetic backgrounds. We used sensitive molecular methods to examine the genetic integrity of inbred mouse substrains maintained at an academic institution. Our goal, in part, was to compare the different molecular genetic monitoring methods to determine which were most sensitive, efficient, and beneficial in our genetic monitoring program. We examined the sensitivity and efficiency of simple sequence length polymorphism (SSLP) analysis of microsatellites and restriction fragment length polymorphism (RFLP) analysis of minisatellites with commercial, human, and synthetic mouse minisatellite probes. Although no polymorphisms were detected with the microsatellite analysis, certain minisatellite probes detected a small degree of polymorphism between our mouse substrains and the commercially available strains used as controls. Minisatellite probes also detected intra-substrain variation within our colonies; this variation probably represents mutations in highly unstable loci rather than genetic variation. Our analysis indicated that the genetic integrity of in-house C57BL/Ka, BALB/cKa, and C3H/Km inbred substrains had remained intact over 35 generations. Genetic monitoring by RFLP minisatellite analysis was more sensitive and efficient in detecting substrain differences than was SSLP microsatellite analysis. On the basis of these results, we established a strategy for future analysis of the in-house breeding colony.

Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Gavaging adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 µl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

Cage change influences serum corticosterone and anxiety-like behaviors in the mouse.

Environmental variables and husbandry practices can influence physiology and alter behavior in mice. Our study evaluated the effects of cage change on serum corticosterone levels and anxiety-like behaviors in C57BL/6 male mice. We examined the effects of 3 different methods of performing cage transfer and of transferring mice to a clean or a dirty familiar cage microenvironment. The 3 different handling methods were forceps transfer, gentle transfer with gloved hands, and a passive transfer technique that did not involve active handling. Active handling methods and transfer to both clean and dirty cage microenvironments significantly increased serum corticosterone 15 min after cage change; however, at 60 min after cage change, levels were comparable to those of unmanipulated mice. Although the effects were transient, cage change altered anxiety-like behaviors in the open field when behavioral testing was performed on the same day. These results demonstrate that the timing of cage change can influence behavioral results, an effect that is an important consideration for rodent behavioral studies.

A comprehensive laboratory animal facility pandemic response plan.

The potential of a severe influenza pandemic necessitates the development of an organized, rational plan for continued laboratory animal facility operation without compromise of the welfare of animals. A comprehensive laboratory animal program pandemic response plan was integrated into a university-wide plan. Preparation involved input from all levels of organizational hierarchy including the IACUC. Many contingencies and operational scenarios were considered based on the severity and duration of the influenza pandemic. Trigger points for systematic action steps were based on the World Health Organization's phase alert criteria. One extreme scenario requires hibernation of research operations and maintenance of reduced numbers of laboratory animal colonies for a period of up to 6 mo. This plan includes active recruitment and cross-training of volunteers for essential personnel positions, protective measures for employee and family health, logistical arrangements for delivery and storage of food and bedding, the removal of waste, and the potential for euthanasia. Strategies such as encouraging and subsidizing cryopreservation of unique strains were undertaken to protect valuable research assets and intellectual property. Elements of this plan were put into practice after escalation of the pandemic alerts due to influenza A (H1N1) in April 2009.

Construction noise decreases reproductive efficiency in mice.

Excessive noise is well known to impair rodent health. To better understand the effect of construction noise and to establish effective noise limits during a planned expansion of our vivarium, we analyzed the effects of construction noise on mouse gestation and neonatal growth. Our hypothesis was that high levels of construction noise would reduce the number of live births and retard neonatal growth. Female Swiss Webster mice were individually implanted with 15 B6CBAF1/J embryos and then exposed to 70- and 90-dBA concrete saw cutting noise samples at defined time points during gestation. In addition, groups of mice with litters were exposed to noise at 70, 80, or 90 dBA for 1 h daily during the first week after parturition. Litter size, birth weight, incidence of stillborn pups, and rate of neonatal weight gain were analyzed. Noise decreased reproductive efficiency by decreasing live birth rates and increasing the number of stillborn pups.