The epaulette shark, Hemiscyllium ocellatum is a tropical reef shark that can live in an environment with cyclic periods of low oxygen concentration, suggesting that it has a well-developed capacity for anaerobic metabolism and/or neuroprotective strategies. Previous investigations of hypoxia-tolerant teleosts and reptiles have focused on species that inhabit cold environments. The work comprising this thesis was focused on a tropical reef shark in order to determine the range of strategies for hypoxia survival that are used at higher environmental temperatures. The aims of this thesis were to: 1) Quantify the anaerobic response of the epaulette shark to hypoxia. 2) Determine whether homeostasis of the excitatory neurotransmitter glutamate is maintained after chronic exposure to hypoxia. 3) Examine the balance of excitatory and inhibitory neurotransmitters (glutamate and GABA respectively) in the hypoxia sensitive cerebellum. 4) Quantify changes in GABAA receptor binding in response to hypoxia.
Firstly, the extent to which the epaulette shark could tolerate hypoxia was tested by using an experimental regimen that reflected the cyclic and prolonged characteristics of its hypoxic environment. The treatment also used a more severe dose of hypoxia to increase the chance of eliciting neuroprotective physiological responses. It was determined that the epaulette shark could survive repeated exposure to extended periods of extreme hypoxia (0.39mg O2/L). This finding provided the basis for using the epaulette as an experimental model to examine neuroprotective strategies during hypoxia.
Since glutamate excitotoxicity, which leads to neuronal death, is an end point in the hypoxic cascade, this thesis also examined the regulation of glutamate homeostasis during hypoxia in the epaulette shark hindbrain and cerebellum, using glutamate immunohistochemistry. Glutamate-like immunoreactivity did not increase in either of these brain regions. In addition, a decrease in glutamate-like immunoreactivity was found in descending hindbrain tracts. These data reveal that the epaulette shark, like cold water hypoxia-tolerant vertebrates, is able to forestall, an excitotoxic build up of glutamate during hypoxia, and that the regulation of glutamate homeostasis is one of its key neuroprotective strategies.
The potential neuroprotective role of the GABAergic neurotransmitter system was also investigated in response to hypoxic exposure. Not only was an increased level of GABA-like immunoreactivity identified in cerebellar neurons, axons and dendrites, but also, the GABAA receptors differed from those of other vertebrates and exhibited altered binding characteristics in response hypoxia. This means that the two major excitatory and inhibitory neurotransmitter systems in the epaulette shark respond to hypoxia in a manner that protects the brain from glutamate mediated excitotoxicity and the consequent neuronal damage.
While examining benzodiazepine binding to GABAA receptors in the epaulette shark cerebellum, binding characteristics that are not shared with any other vertebrate group were observed. This unique feature, along with data regarding the surface anatomy, cytoarchitecture, and immunocytochemistry of the epaulette shark hindbrain and cerebellum has been reported for the first time in this thesis. These findings, combined with previous literature regarding hypoxia/anoxia tolerance in other vertebrates, have led us to conclude that the epaulette shark possesses a highly developed tolerance to hypoxia. Also, neuroprotective strategies are employed by the epaulette shark that are common with those used by other hypoxia tolerant animals, to delay the release of excitotoxic concentrations of glutamate during hypoxia. Furthermore the epaulette employs other unique neuroprotective strategies that are not held in common with previous studied vertebrates. This thesis has established the epaulette shark as a valuable experimental model to use in examining neuroprotective response to hypoxia.