Intracellular blockade of inhibitory synaptic responses in visual cortical layer IV neurons

1. Inhibitory postsynaptic potentials (IPSPs) evoked by stimulation at the white-matter/layer VI border were recorded intracellularly from visual cortical layer IV neurons maintained in vitro. These IPSPs, typically not apparent at resting membrane potentials, were measured at membrane potentials 15-25 mV depolarized from resting levels. The effects of two chloride channel blockers on these IPSPs were investigated. 2. 4,4'-Dinitro-stilbene-2,2'-disulfonic acid (DNDS) was found to inhibit IPSPs as it diffused into the postsynaptic cell from an intracellular micropipette, leaving only the presumed pure excitatory postsynaptic potential (EPSP) component of the evoked compound PSP. Input resistance, resting membrane potential, spike accommodation, and EPSPs at resting membrane potentials were not significantly affected. 3. A novel chloride channel blocker 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]a rene (TS-TM-calix[4]arene) was found to potently inhibit IPSPs recorded at depolarized membrane potentials. The TS-TM-calix[4]arene, similar to DNDS, did not affect input resistance, resting membrane potential, spike accommodation, and EPSPs at resting membrane potentials. 4. To confirm that DNDS and TS-TM-calix[4]arene were indeed blocking IPSPs, similar experiments were performed on monosynaptic IPSPs evoked by stimulation of layer V in the presence of 2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both DNDS and TS-TM-calix[4]arene were effective in blocking monosynaptic IPSPs. 5. Consistent with the notion that DNDS and TS-TM-calix[4]arene block IPSPs by inhibiting gamma-aminobutyric acid-A (GABAA) receptor channels, the decrease in input resistance caused by extracellular application of muscimol was also significantly inhibited by intracellular use of these compounds. 6. These data suggest that DNDS and TS-TM-calix[4]arene applied intracellularly may be useful for the study of the function of GABAA-mediated synaptic inhibition of cortical neurons. Because only neurons impaled by the recording electrodes are influenced by the drugs, this method offers an advantage over extracellular application of GABAA blockers in that entire networks of neurons are not influenced.