Ongoing neurogenesis in the adult brain is a fundamental process of neural plasticity. The songbird is an established model for studying neuroprotection, neurogenesis, and neuronal turnover due to dramatic plastic changes in morphology and function in the nuclei that control avian song production. HVC, a region of the brain that times the production of song, doubles in size at the beginning of the breeding season, largely as a result of an increase in new neuron incorporation. In the nonbreeding season the song control circuit rapidly regresses in size. Within 7 days HVC regresses to non-breeding condition volume, and neuron number decreases by around 25% (> 68,000 neurons) via neuronal apoptosis. We tested the hypothesis that regression of HVC via neuronal apoptosis upon transition into nonbreeding conditions is tightly linked to proliferation of neural stem cells (NSC) in the nearby ventricular zone and changes in singing behavior. We also asked whether new neurons recently incorporated into the song control system were retained or lost through transition into non-breeding conditions. We rapidly transitioned birds from breeding to nonbreeding conditions and measured neuronal death and survival, NSC proliferation, and song behavior over a time-course of 28 days. We found that some but not all neurons incorporated into HVC during the previous breeding season persist at least 28 days into the nonbreeding season, suggesting that both new and mature neurons must undergo apoptosis during transition into nonbreeding conditions. Interestingly, we found that proliferation in the VZ was tightly linked to the amount of cell death occurring within HVC. We also quantified song degradation following rapid transition into nonbreeding conditions and correlated the observed changes in behavior to the cellular changes occurring within HVC. These findings demonstrate a relationship between cell death and neural stem cell proliferation.

 Vesicular Glutamate Transporters (vGluts) are proteins that package glutamate into vesicles prior to synaptic transmission. Three isoforms have been identified in mammals (vGlut-1-3), with 1 and 2 showing a mostly complementary pattern of expression, and 3 co-localizing in a small number of cells with both 1 and 2. Knockout mice for the vGlut1 gene die shortly after weaning, indicating a crucial role for vGlut1 in development. Intriguingly, avian genomes contain no vGlut-1 orthologs, despite the fact that lizards, a common ancestor of both mammals and birds, have orthologs of both vGlut-1 and -2. The fact that birds function without vGlut1 suggests there may be an increased functional role for vGlut2 in the avian brain. To further demonstrate the presence and potential role of vGlut2 in avian pallial circuitry, we used immunocytochemical methods to study the brain distribution of vGlut2 protein in zebra finch. To confirm the specificity of antibodies to vglut2, we first performed co-localization of anterograde tracer with vGlut-2 in known glutamatergic terminals in RA that originated from pallial song nucleus HVC. Electron microscopy further confirmed that labeling was restricted to synaptic terminals containing vesicles, and in situ hybridization was used to confirm the presence of mRNA in somata. We found that a large number of vGlut2-positive cells are present throughout the zebra finch pallium, and also found evidence for vGlut-2 positive synapses in three different song nuclei (nucleus HVC, Area X, and robust nucleus of the arcopallium), which were imaged and analyzed for size, and the potential identity of post-synaptic elements. Our results are consistent with the notion that avian vGlut2 has largely taken over vGlut1 functions. We believe our study contributes important facts to the long disputed evolutionary relationship between the avian pallium and the mammalian cortex.

Cervical spinal injury can result in a loss of function in the hands and arms. Restoration of hand and arm function is nearly 5-fold more important to patients than restoration of other functions. A major difficulty in restoring function lies in overcoming the formation of perineuronal nets (PNNs) and the accumulation of chondroitin sulfate proteoglycans (CSPGs), which contribute to the scar around the lesion site. The disruption of CSPGs in both the scar and PNNs is possible with Chondroitinase ABC (ChABC), and more chronically, through the use of a novel intracellular sigma peptide (ISP). The inhibition of CSPGs allows for axonal regeneration, which may be guided through the use of intraspinal microstimulation (ISMS). Preliminary results shows that the combination of these approaches act synergistically, leading to much greater recovery than through ChABC and ISMS alone. Functional recovery is assessed through a series of behavioral tests, including a trained forelimb reaching task and the Irvine, Beatties & Bresnahan test (IBB). These behavioral tasks are designed to measure attributes that are important for upper limb rehabiliation, such as spasticity, ability to grasp, movement in the wrist and digits, and plantar contact. If successful, this treatment may be used to rewire spinal circuits around a lesion and restore function below the site of the lesion.

Research in our lab has demonstrated that stimulation of the spinal cord may enhance rehabilitation and restoration of forelimb function after spinal cord injury. Building on previous experiments, the goal of this project is to see how the combination of Chondroitinase (a treatment of enzymes) and spinal stimulation could promote the recovery of forelimb function in rats with chronic spinal cord injury. We trained animals on a skilled forelimb reaching task and determined their dominant hand. Once trained to proficiency, animals received a contusion injury to the lateral aspect of the 4th and 5th cervical segments resulting in paresis of their dominant forelimb. Animals were then randomly assigned to groups receiving stimulation, Chase treatment, or the combination of the two treatments. We are conducting a battery of forelimb functional tests, including skilled forelimb reaching, each week for the duration of the study in order to record their progress and observe their improvement using the injured forelimb. It is the anticipated result that the addition of Chonroitinase during rehabilitation will result in an increase of the formation of synapses in circuits bypassing the lesion, which may be investigated using neuroanatomical tracing techniques. If successful, our results can be utilized in human medicine to improve the healing of a spinal cord-related injury and promote rehabilitation.

 Spinal cord injury accounts for 29% of paralysis patients in the US, and the recovery of hand and arm function is the highest treatment priority in individuals with tetraplegia.  Induced pluripotent stem (iPS) cells can be directed toward specific cell fates, such as functioning neurons or astrocytes, making them an exciting new tool in regenerative medicine for those that suffer from spinal cord injury paralysis.  We investigated the effect of neurally-fated iPS cells on recovery of forelimb function after a cervical spinal cord contusion injury in a rat model.  Neurally-fated iPS cells were injected above and below the lesion in an attempt to replace cells lost to injury.  We transplanted cells four weeks after injury to mimic a chronic spinal cord injury model.  Forelimb function was quantified before and after the injury for 15 weeks using three forelimb behavior tasks.  Tasks included a forelimb reaching task (FRT), a cylinder-rearing task, and a cereal-eating task (IBB).  Results from these tasks will be analyzed to quantify any improvement in the experimental animals that received iPS cells versus the controls.  If treatment provides a significant difference in recovery rate, then the introduction of iPS cells in damaged spinal cords could provide a possible regenerative treatment for paralysis patients.