I investigate the use of vertical cavity surface emitting lasers (VCSELs) for portable optical neural imaging. In particular, I attempt to use one laser source to produce the different illumination needed for three techniques, imaging flow, oxygenation, and fluorescence. Our group's goal is to combine these techniques using a single source in a simple imaging format, which can be modified for portable use in awake animals. We exploit tuning properties of VCSELs to implement two distinct operation schemes with different illumination characteristics. Single mode operation provides high speckle contrasts, used to map flow speeds in laser speckle contrast imaging (LSCI). Sweep operation provides low noise, allowing us to image small signal changes with intrinsic optical signal imaging (IOSI), which relate to blood oxygenation. Finally, we use our VCSEL devices for on-chip integrated fluorescence sensing. Future work aims to develop a fully portable format, using CMOS detectors and integrated circuit control.

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

Anatomical connectivity and single neuron coding suggest a dissociation of information representation within the lateral and medial entorhinal cortex, a brain region with widespread connections to cortical areas. We aimed to expand this idea by examining differential contribution of these two sub-regions to the retrieval of non-spatial and spatial memory. Inactivation of lateral, but not medial regions severely impaired the retrieval of recently and remotely acquired non-spatial memory while spatial memory remained intact. To link functioning of the lateral entorhinal cortex with the known roles of the hippocampus and medial prefrontal cortex for memory retrieval, communication with these two regions was detected as synchronized oscillations in local field potentials. We found that stronger communication between the lateral entorhinal and prefrontal cortex during stimulus-free periods correlated with better memory performance. The lateral entorhinal cortex therefore may serve as a gateway of memory-related information between the medial prefrontal and other cortical regions.

Auditory sensitivity has often been measured by identifying neural threshold in real-time (online) which can introduce bias in the audiograms that are produced. We tested this by recording auditory nerve activity of the notodontid moth Nadata gibbosa elicited by bat-like ultrasound and analysing the response offline. We compared this audiogram with a published online audiogram showing that the bias introduced can result in a difference in the audiogram shape. In the second part of our study we compared offline audiograms using spike number as threshold with others that used spike period and stimulus/spike latency, variables that have been suggested as providing behaviourally functional criteria. These comparisons reveal that functional audiograms are more flatly tuned than simple spike audiograms. The shapes of behavioural audiograms are discussed in the context of the selection pressure that maintains their shape, bat predation.

I investigate the use of vertical cavity surface emitting lasers (VCSELs) for portable optical neural imaging. In particular, I attempt to use one laser source to produce the different illumination needed for three techniques, imaging flow, oxygenation, and fluorescence. Our group's goal is to combine these techniques using a single source in a simple imaging format, which can be modified for portable use in awake animals. We exploit tuning properties of VCSELs to implement two distinct operation schemes with different illumination characteristics. Single mode operation provides high speckle contrasts, used to map flow speeds in laser speckle contrast imaging (LSCI). Sweep operation provides low noise, allowing us to image small signal changes with intrinsic optical signal imaging (IOSI), which relate to blood oxygenation. Finally, we use our VCSEL devices for on-chip integrated fluorescence sensing. Future work aims to develop a fully portable format, using CMOS detectors and integrated circuit control.

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

The molecular mechanisms that regulate survival of embryonic neural precursors are still relatively ill-defined. Here, we have asked whether the p53 family member p63 plays any role during this developmental window, focusing upon the embryonic cerebral cortex. We show that genetic knockdown of p63 either in culture or in the embryonic telencephalon causes apoptosis of cortical precursors and newly-born cortical neurons, and that this can be rescued by expression of ΔNp63, but not TAp63 isoforms. This cortical precursor apoptosis is the consequence of deregulated p53 activity, since both basal precursor apoptosis and that induced by loss of p63 are rescued by coincident genetic silencing of p53. Finally, we demonstrate that the third p53 family member, ΔNp73, does not regulate survival of cortical precursor cells, but that it collaborates with ΔNp63 to ensure the survival of newly-born cortical neurons. Thus, the balance of ΔNp63 versus p53 determines the life versus death of embryonic cortical precursors. To assess if p63 plays a similar role in the adult brain, we examined mice haploinsufficient in p63 (p63+/-); we observed that aging, but not young, mice show deficits in short term memory and vertical exploratory behaviour. These results establish a foundation upon which the role of p63 in aging can be further characterized. Given the importance of p73 in aging and in preventing neurodegeneration, we aimed to create a conditional p73 knock out mouse (floxed p73). This mouse will allow for lineage-specific characterization of p73 function and will circumvent the reduced life span displayed by 4 mice lacking p73 (p73-/-). The work presented in this thesis has advanced our knowledge on the role of the p53 family members (p53, p63, and p73) in regulating survival and death in the nervous system. This knowledge will be important to understand neuropathology and to design appropriate therapeutic interventions.

Inherited photoreceptor degenerations (IPDs) are the most common monogenic cause of blindness in humans. To discover genes that may influence the risk of death in IPDs, microarray studies were used, and ET-2 was identified as the most differentially expressed transcript. ET-2 mRNA was 32-fold (p< 0.004), 70-fold (p< 0.009) and 72-fold (p<0.0009) increased in the Rds+/- , Tg(RHO P347S) and Rd1-/- mouse models of IPD, respectively, and the ET-2 peptide was minimally three-fold upregulated in Rds+/- retinas. The increased ET-2 transcript was detected solely in the photoreceptors (PRs) of Rds+/- and Tg(RHO P347S) retinas, but not in wild-type (wt) retinas by in situ hybridization. To determine the biological role of ET-2 in IPDs, mouse IPD models were crossed to ET-2-null mice. At age 40 and 15 days, ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/- retinas showed, respectively, a 41% (n=6;p<0.003) and 49% rescue of PR degeneration (n=5;p<0.007). Unexpectedly, however, the PRs in ET-2-/- Rd1-/-retinal explants were not rescued (n=6;p>0.05), suggesting that the rescue observed in vivo might be due to extraocular mechanisms. Additionally, the expression of ET-2 mRNA from an rAAV5-CBA-ET-2 vector in ET-2-/-; Rd1-/- retinas did not restore PR degeneration (n=6;p>0.05). A survey of the extraocular phenotypes of ET-2 null mice showed them to be hypoxic owing to aberrant lung development, with a loss of normal alveolarization of the lung. Erythropoietin (EPO) levels were 11-fold elevated in the serum of ET-2 null mice (n=7;p<0.05) and retinal vascular endothelial growth factor (VEGF) was increased 4-fold (n=4;p<0.05). To examine the role of hypoxia in PR degeneration and to exclude increased EPO levels as the sole factor accounting for the rescue of mutant PRs in ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/- mice in vivo, the effect of hypoxia on PR death in Rd1-/- retinal explants was examined. Rd1-/- explants cultured in 6% O2 from PN10 to PN17 showed a 32% rescue of PR death (n=5;p<0.05). Although ET-2 may mediate PR death through a direct role in mutant PRs, the PR rescue observed in ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/-retinas may also result from systemic hypoxia due to poor lung function in ET-2-/- animals.

The vestibular system provides us with sensory information that is essential for maintaining balance and stability. When sensory input is lost due to unilateral vestibular damage (UVD), our ability to maintain stable gaze and posture becomes compromised. Over time, vestibular function is partially restored through a process known as vestibular compensation, which is associated with the rebalancing of activity in the vestibular nuclear complex (VNC) of the brainstem. However, the physiological mechanisms associated with vestibular compensation remain elusive. We addressed several different experimental objectives pertaining to plasticity and sensory adaptation associated with vestibular compensation. First, we demonstrated that systemic manipulation of γ-amino-butyric acid type B (GABAB) receptors altered the course of vestibular behavioural recovery within the first several hours after UVD. Second, we showed that immunohistochemical labeling of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR4 was elevated in the VNC on the intact compared to lesioned side acutely following UVD. Third, we produced preliminary data suggesting that excitatory responses to vestibular nerve stimulation may be acutely potentiated by UVD on the intact side. Finally, we established that rapid sensory adaptation may increase the dynamic ranges of vestibular neurons and perhaps improve limited vestibular reflex function in the long term. Acutely following UVD, potentiation of vestibular nerve synapses appear to be associated with an increase in GluR4 subunit expression in the contralesional VNC. Also, such potentiation could be enhanced by acute modifications in pre-synaptic GABAB receptor activation. In the long term, and independent of these plastic changes, sensory adaptation may enable the vestibular system to overcome the persistent limitations imposed by UVD.

While the hippocampus may play an essential role in the expression of memories soon after training, over time these memories are thought to become increasingly dependent on coordinated activity in a broad network of cortical and subcortical brain regions. However, the distributed nature of this representation has made it challenging to define the neural elements of the memory trace, and lesion and electrophysiological approaches provide only a narrow window into what is appreciated to be a much more global network. Here global mapping approaches are used to identify networks of brain regions that are activated and co-activated following recall of recent and remote contextual fear memory in mice. Analysis of Fos expression across 84 brain regions allowed for the description of brain-wide activity and interactivity of brain regions associated with memory expression. Activity analysis revealed that remote memory engages a broad collection of cortical and subcortical regions in comparison to recent memory expression. Interactivity analyses revealed that functional connectivity associated with fear memories depends on memory age and is altered in mutant mice that exhibit premature forgetting. In-depth functional connectivity analysis of remote long-term fear memory indicates that memory recall engages a network that has a distinct thalamic-hippocampal-cortical signature. This network is concurrently integrated and segregated and therefore has small-world properties as well as a resilient core of highly inter-connected regions. Centrality measures identify a collection of regions that may play a critical role in the function of the network including expected regions such as the anterior cingulate cortex and prelimbic cortex as well as novel regions including the reuniens thalamic nucleus. Post conditioning lesions of the reuniens lead to mild deficits in contextual fear memory expression providing support for the idea that identified hub regions may play a critical role in the function of the network. These results identify and describe functional activity and interactivity of brain regions underlying recent and remote fear memory expression and provide strong evidence for reorganization and distribution of the functional organization of memories over time.