Influence of aging and behavioral experience on expression of GluN1 splice variants of the NMDA receptor in prefrontal cortex of mice brain

As the aging population continues to grow the world over, age related complications become more and more apparent among the elderly population. One such complication is age associated memory impairment, which makes the elderly more dependent on caregivers early on. NMDA receptors in the brain are important for memory formation, consolidation and retrieval. Expression of NMDA receptors declines with age, which is associated with declines in memory observed during aging. Age-related changes in the protein and mRNA expression of some of the splice forms of the GluN1 (GluN1, NR1) subunit of the NMDA receptor have been seen in mice and rats. The present study was designed to determine whether individual splice forms of the GluN1 subunit of the NMDA receptor within prefrontal / frontal cortical regions contribute to memory deficits during aging and whether experience in learning tasks can influence the expression of the splice forms. mRNA expression of 4 splice forms GluN1[subscript X11], GluN1[subscript X10], GluN1[subscript 0XX] and GluN1[subscript 1XX] (GluN1-1, GluN1-3, GluN1-a and GluN1-b, respectively) and mRNA for all known splice forms (GluN1-pan) were examined by in situ hybridization. mRNA for the C-terminal splice forms, GluN1[subscript X11] (GluN1-1; +C1 and +C2 cassettes) and GluN1[subscript X10] (GluN1-3; +C1 and +C2'), showed significant declines during aging in several brain regions even though overall GluN1-pan mRNA expression was not significantly affected by aging. This work provides evidence that these splice forms are more influenced by aging than the subunit as a whole. There was an increase in the expression of GluN1[subscript 0XX] (GluN1-a; -N1 cassette) splice form in the behaviorally-experienced old mice relative to the younger groups. Old mice with the highest levels of mRNA expression for the GluN1[subscript 0XX] (GluN1-a) splice form in orbital cortex showed the best performances in spatial working and reference memory tasks, but the poorest performances in a cued, associative learning task. These results suggest that the GluN1[subscript 0XX] subunit splice variant may be important for spatial memory performance in the old animals. Protein expression of GluN1 subunits containing C-terminal cassettes C2 or C2' were observed to decline with increasing age, regardless of experience. In middle-age animals, higher expressions of the GluN1 subunit and C2' cassette proteins were associated with good reference memory on initial search. In the aged animals, higher protein expression of GluN1 subunits containing C1 cassettes and the whole population of GluN1 subunits were found to be associated with better performance in the final phase of probe trials but this appeared to be due to perseveration or delays in applying an accurate search. These results provide support for the theory that there is heterogeneity in the effect of aging on the expression of the GluN1 subunits containing different splice cassettes. It also suggests that the GluN1 subunit might be most important for good reference memory during middle age. The next study was undertaken to determine if the GluN1[subscript 0XX] splice form is required for good performance in reference memory tasks in young mice. Mice were injected with 5µl of either siRNA specific to GluN1[subscript 0XX], control siRNA or vehicle alone into ventro-lateral orbital regions of both sides of the brain using a stereotaxic apparatus. A fourth group of mice did not receive any injections. Five days post-injection, mice were tested for their performance in a spatial reference memory task for 4 days using the Morris water maze. There was a 10 -19% reduction in GluN1[subscript 0XX] splice variant expression for mice after siRNA treatment in ventro-lateral orbital regions of the brain. Decline in performance in the first half of reference memory were observed in the mice receiving siRNA specific for GluN1[subscript 0XX] splice form, as compared to the mice injected with control siRNA and/or vehicle. These results suggest an important role of the GluN1[subscript 0XX] splice variant in orbital regions for spatial reference memory acquisition and/or consolidation in the early stages of memory training. These results suggest that there is a complex interaction between GluN1 splice form expression and performance of memory tasks during aging. Future studies designed to differentiate between involvement of splice variants in particular stages of memory formation would be helpful

Multifunctional Drug Carriers with Programmable Properties.

In recent decades, drug-loaded carrier systems have become a viable option to replace or augment current, unsuccessful therapies that address a myriad of disease conditions. In this area, a successful therapy needs to address the following parameters: specificity, long circulation, biocompatibility/biodegradability, desired pharmacokinetics of single & multiple therapeutics, carrier-cell interactions, and potential imaging capabilities. While various formulations have tackled some of these characteristics, a concrete methodology to design multifunctional particles addressing all parameters is currently lacking. Herein, the method of electrohydrodynamic (EHD) co-jetting and downstream processing are used to design multifunctional particles with tunable characteristics that could be used to address all desired parameters. Specifically, each of the chapters address (i) the development of carriers with selective and orthogonal surface modifications using functional polymers incorporated into individual compartments for the addition of stealth, targeting, or therapeutic capabilities; (ii) the development of carriers with programmable physical properties such as shape, size, porosity, roughness, and charge that can determine the fate of a particle in the body, and the testing of nanoparticles in animal models to determine their biodistribution; (iii) the development of particles with complex degradation and release kinetics of therapeutics using rapidly degrading or stimuli-responsive (pH, light, & oxidative stress) polymers; (iv) the design of multifunctional particles loaded with multiple therapeutics for cochlear delivery and their testing to determine the therapeutics’ release kinetics, the particles’ persistence & biodistribution in the cochlea, and the general biocompatibility of the system. Accordingly, this dissertation suggests the potential establishment of a toolbox that can be used for the design of particles with programmable properties for individual applications in the effective delivery of therapeutics.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111636/1/rahmani_1.pd

Assessment of Noise Effects on Sensitive Animal Communities

There is a statutory requirement to protect certain animals and to assess the environmental effects of new developments on wildlife. However, there is no formal guidance on how such assessments should be undertaken. This research has developed an assessment process specific to animals, which enables informed judgement as to the likely short or long-term impacts. Published animal responses have been analysed to identify particular trends and response thresholds, and a standard procedure for assessing noise effects on animals has been developed. The procedure assigns significance criteria (no effect, slight, moderate and severe) that take account of the physiological and behavioural responses exhibited following exposure to noise. The significance rating determines whether mitigation is required. Particular combinations of noise, animals and habitat that are especially sensitive to environmental noise are identified as off-road vehicles, helicopters, very quiet habitats, and animals having special hearing characteristics. An assessment threshold is proposed based on key factors such as the noise level, source distance, and other site-specific circumstances. If LAmax noise levels are greater than 80 dB or the separation between the animals and the noise source is less than 1,000m, an assessment is recommended. For fish and marine mammals, if the Received Level (RL) is greater than 140 dB re: 1μPa rms an assessment is recommended. Slight responses may still arise below these thresholds but moderate or severe responses would not be expected. Circumstances most likely to affect animal responses are a rapid onset of noise, and the presence of helicopters, sonic booms, low flying aircraft, artillery/rockets, blasting/explosions, fireworks, motorboats or float planes. The assessment methodology is tested on two animal species (black grouse and golden plover) using data from a planning application for military development, and retrospectively to mammals at a wildlife park where low-flying jets had caused moderate/severe responses