Khodakhah, Kamran; (1993) Postsynaptic actions of excitatory amino acids investigated with flash photolysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Flash photolysis and whole cell patch clamp technique were combined with microspectrofluorimetry of calcium to study the role of inositol trisphosphate as a second messenger, and the role of L-glutamate as a neurotransmitter. Inactive photolabile analogous of inositol trisphosphate, caged InsP3, and fluorescent calcium indicators, Fluo-3 or Furaptra, was introduced into single cells via the whole-cell patch pipette. Astrocytes and neurones from primary cultures of cerebellar granule cells, hippocampal pyramidal cells, striatal neurones, and dorsal root ganglion neurones, and Purkinje cells from acutely prepared thin slices of the cerebellum were voltage clamped (holding potential -70mV). A 1ms pulse of near UV (300-350nm, 100mJ) was used to photolytically release a known concentration of InsP3 in the cytosol, and the changes in the intracellular free calcium concentration, [Ca2+], and conductance monitored. The results may be summarised as follows: (1) Photolytic release of InsP3 (100nM-30μM) in cultured neurones did not alter [Ca2+] or the membrane conductance. In glial cells from the same preparations, however, a rise in [Ca2+] was observed following intracellular release of InsP3 (0.2-5μM). The concentrations of InsP3 required to mobilise calcium in astrocytes is similar to that found in peripheral tissue. (2) Photolytic release of InsP3 (9-76μM) in voltage clamped Purkinje neurones in acutely prepared thick slices of the cerebellum resulted in a large increase in [Ca2+], often reaching 30μM when calibrated with Furaptra, and at the same time an outward current (200-900pA) in the presence or absence of external calcium. The outward current was the result of an increase of the membrane conductance to potassium ions, and under current clamp conditions hyperpolarised the cell to approximately -85mV. The responses occurred with a latency that decreased from a few hundred ms at low (9μM) to <15ms at high (76μM) InsP3 concentration. The time to peak also decreased from 900ms to <20ms over this concentration range, indicating increased calcium efflux from stores at high InsP3 concentration. The concentrations of InsP3 required to mobilise calcium in Purkinje neurones is higher than that required in astrocytes and peripheral tissue. The kinetics of calcium release is also faster than that in astrocytes and peripheral tissue, indicating an adaptation of InsP3 signalling in Purkinje cells. Photolabile analogues of L-glutamate would be useful to produce controlled pulses of L-glutamate to study synaptic transmission in the central nervous system. Several photolabile analogues of L-glutamate were characterised using whole cell patch clamp recording with granule cells and dorsal root ganglion neurones in culture. Five photolabile analogues of glutamate were dismissed due to low quantum yield, instability, or having activity at glutamate receptor sites. One photolabile analogue of glutamate, N(1,(2-nitrophenyl)ethoxycarbonyl)-L-glutamate, satisfied most of the criteria required for a caged neurotransmitter. Compared with L-glutamate, ImM caged glutamate in its un-photolysed form was >10000X less potent at the NMDA, and >50X at the non-NMDA receptors. When irradiated with a 1ms pulse of near UV light (300-350nm, 100mJ) it cleaved to release L-glutamate, 2-nitrosoacetophenone, and carbon dioxide with a quantum yield of 50%. Neither caged glutamate (1mM) nor its photolysis by-products had any potency as an antagonist at the excitatory amino acid sites. The rate of photolysis was found to have a half time of about 50ms at pH7, too slow to allow for studies of rate of receptor activation. The rate of photolysis, however, is pH dependent increasing 10 fold with one pH unit drop. The ability of the squid giant synapse to withstand acidic conditions was taken advantage of, and L-glutamate was photolytically released from caged glutamate equilibrated with the synapse at pH5.5. This abrupt release of L-glutamate elicited action potentials in the 3rd order fibre of the giant synapse providing good evidence of the role of L-glutamate as a neurotransmitter at this synapse.

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