Investigation of the Functional Effects of Two Novel Ampakines in the CNS
The ionotropic glutamate AMPA ((R,S)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor mediates the majority of excitatory transmission in the CNS. AMPA receptors play a crucial role in both basal neurotransmission and synaptic plasticity events (such as long-term potentiation, LTP). Compounds that ‘potentiate’ AMPA receptor function (‘Ampakines’) are known to positively modulate glutamatergic AMPA receptor-gated currents, by slowing the deactivation and desensitisation rate of the receptors, in the presence of the endogenous agonist glutamate. Ampakines have been shown to facilitate LTP induction, improve cognition, and as such have potential in the treatment of conditions such as depression and psychoses (schizophrenia). The main aim of this thesis was to investigate the functional actions of two novel Ampakines, Org 26576 and Org 24448, in the mouse brain. The studies described in this thesis were designed to address this and are outlined as follows: 1. Characterisation and validation of an in vivo semi-quantitative model of [14C]-2-deoxyglucose autoradiography in the C57Bl/6J mouse The first study sought to develop and characterise a model of [14C]-2-deoxyglucose autoradiography, to allow measurement of regional alterations in local cerebral glucose use (LCGU) in the mouse CNS. Following intraperitoneal injection of [14C]-2-deoxyglucose in C57Bl/6J mice, the radiolabelled brains were sectioned and exposed to x-ray film. The resultant autoradiograms were semi-quantitatively analysed for relative optical densities in predetermined regions of interest. The baseline LCGU values in different brain regions were found to be consistent with previously published data. The model was also able to replicate the effects of a well-characterised compound, the NMDA receptor antagonist MK-801 (0.5 mg/kg), in respect to functional cerebral changes. Characteristic effects such as prominent increases in LCGU in the limbic system, and decreases in the somatosensory cortex were reproduced in the model. Thus the semi-quantitative [14C]-2-deoxyglucose model was reproducible and accurate and thus could be further used to investigate the effects of the novel Ampakines, Org 26576 and Org 24448, on cerebral function. 2. Investigation into the effects of acute administration of the novel Ampakines Org 26576 and Org 24448 on functional activity in the murine cerebrum Following the establishment of the methodology, regional alterations in LCGU in response to the Ampakines Org 26576 and Org 24448 were investigated using [14C]-2-deoxyglucose autoradiography. Both Org 26576 and Org 24448 produced regionally selective, dose-dependent increases in LCGU in the mouse cerebrum when administered acutely (~1 hr). The compounds displayed similar yet functionally distinct profiles of activation, the highest levels of activation occurred in areas of the limbic system (hippocampus), sensory systems, and various nuclei (raphe nucleus). Their effects were blocked by pre-administration of the potent selective AMPA receptor antagonist, NBQX (10 mg/kg), which itself had minimal effects on LCGU. These data provide an anatomical basis for the cerebral activation induced by these compounds, which are directly AMPA receptor mediated. Areas activated also closely correlated with brain regions implicated in various psychiatric conditions, and as such is suggestive of a potential therapeutic benefit of these compounds in conditions such as depression and schizophrenia. 3. Investigation into the effects of chronic administration of the novel Ampakines Org 26576 and Org 24448 on functional activity, neurogenesis and receptor/signalling alterations in the murine cerebrum Following the demonstration that acute administration of Org 26576 and Org 24448 displayed regionally selective and dose-dependent alterations in LCGU, the effect of chronic administration of the Ampakines Org 26576 and Org 24448 on regional functional alterations ([14C]-2-deoxyglucose autoradiography), neurogenesis (BrdU labelling), and proteins levels (GluR, MAPK, LynK and CREB) (Western blot analysis) were investigated. Chronic administration (7 and 28 days) of Org 26576 (1 mg/kg) and Org 24448 (10 mg/kg) induced functional cerebral increases in the mouse cerebrum particularly in areas of the mesocorticolimbic system, which were not only rapid in onset, with significant effects visible after 7 days administration; but importantly were also persistent and long lasting. Chronic administration of the compounds had no significant effect on the level of neurogenesis or on the levels AMPA receptor subunits (GluR1,2,3), and signalling pathways (MAPK/LynK-CREB pathway), implicated in AMPA/Ampakine signalling, in the murine hippocampus. These data show that the Ampakines Org 26576 and Org 24448 when administered chronically can potentiate complex neural networks intimately associated with disease states, the effects of which are maintained over prolonged periods. There was no evidence that this involved an effect on neurogenesis or the MAPK/LynK-CREB signalling pathway. 4. Modulation of AMPA receptor kinetics by Org 26576 and Org 24448 influences synaptic plasticity in the murine hippocampus The ability of Org 26576 and Org 24448 to modify baseline kinetic properties of AMPA receptors and a paradigm of synaptic plasticity, LTP, in the mouse hippocampus was investigated using electrophysiology. Both Org 26576 and Org 24448 produced dose-dependant increases in fEPSP amplitude without affecting the half-width of responses, in acute hippocampal slices. Concentrations of both compounds, equating to functionally active levels witnessed in vivo, potentiated a stable form of LTP; whilst higher EC50 concentrations prevented the maintenance of LTP. These results are suggestive that Org 26576 and Org 24448 are effective in boosting the neural correlate of cognition, LTP, and may have potential in treating cognitive deficits, for example those associated with depression, schizophrenia or Alzheimer’s disease. The data presented in this thesis illustrate that the novel Ampakines Org 26576 and Org 24448 centrally modulate brain regions and circuitry intimately associated with conditions such as depression and schizophrenia (psychoses), with effects that are rapid in onset and persistent over chronic periods of administration. Specifically targeting the glutamatergic system through the use of these compounds may provide an innovative approach to treat various conditions that may be partly due to a compromise of normal excitatory glutamatergic neurotransmission.