Postsynaptic signaling, plasticity, and pathophysiology at excitatory synapses in organotypic hippocampal slice cultures

Abstract

Fast excitatory transmission in the central nervous system occurs via the neurotransmitter glutamate. Understanding the properties of ionotropic glutamate receptors, their functional distribution, and the roles they play in signalling and plasticity will shed light on brain function critical for learning and memory. I combined hippocampal slice culturing techniques with optical photo-stimulation methods, fluorescence microscopy, and electrophysiological recording techniques to study the role of glutamate in signaling and plasticity at a cellular level with high resolution. I used photolysis of caged glutamate to selectively stimulate synaptic receptors at single synapses on the dendritic spines of CA1 pyramidal neurons in hippocampal slice cultures to study both short- and long-term forms of glutamate-mediated plasticity. I found that AMPA receptor subtypes were differentially expressed depending on whether the spine was located on an apical or basal dendrite, as shown by difference in postsynaptic paired pulse facilitation, rectification, and sensitivity to polyamines. AMPARs on apical spines, but not on basal spines, were blocked by endogenous and exogenous polyamines. Apical spines displayed postsynaptic facilitation and rectification. I demonstrated for the first time that long-term, NMDAR-dependent increases in the strength of AMPAR-mediated transmission known as long-term potentiation (LTP) could be induced solely by exogenous glutamate and occurred in a delayed, step-wise manner. UP was expressed at synaptic receptor locations on spine heads, but not at dendritic shaft receptors in the same cells. This potentiation could be reversed in an NMDAR-dependent manner. UP was accompanied by a decrease in the postsynaptic paired-pulse facilitation of the photolytic currents, indicating that a change in the subunit composition of the AMPARs underlying the response contributed to the potentiation. These results show that activation of postsynaptic glutamate receptors by glutamate is not only necessary, but sufficient, for the induction of NMDA receptor-dependent UP and reveal novel aspects of its expression. In addition, long-term changes in AMPA transmission strength were not accompanied by immediate structural alterations. I measured the spine head size before and after the induction of UP and found that spine head diameter was not changed by UP induction. Finally, to study chronic changes following traumatic brain injury, I performed synaptic stimulation to evoke pathophysiological dendritic responses in area CA1 one week after lesioning the Shaffer collalateral axon pathway. After deafferenting the excitatory inputs to the CA1 apical dendrites, I recorded pathological responses that resulted from postsynaptic hyperexcitability. I conclude that studying the function of the synaptic and extrasynaptic receptors is critical for understanding postsynaptic plasticity and pathophysiology.