Correlation of AMPA receptor subunit composition with synaptic input in the mammalian cochlear nuclei

Abstract

The composition of AMPA receptors in patches excised from somata and dendrites of six cell types in the mammalian cochlear nuclei was probed and compared electrophysiologically and pharmacologically with the rapid application of glutamate. Cells excited predominantly by auditory nerve fibers had AMPA receptors with exceptionally rapid gating (submillisecond deactivation and desensitization time constants). The nonlinear current-voltage relationship in the presence of spermine showed that few of these receptors had GluR2 subunits, and the insensitivity of desensitization to cyclothiazide indicated that they contained mostly flop splice variants. At synapses made by parallel fibers, AMPA receptors were slowly gating (time constants of deactivation and desensitization >1 msec) and contained higher levels of GluR2 and flip isoforms. However, receptors at auditory nerve synapses on cells that also receive parallel fiber input, the fusiform cells, had intermediate properties with respect to kinetics and contained GluR2 and flip isoforms. Given the diverse biophysical properties, patterns of innervation, patterns of electrical activity, and targets of each cell type in vivo, these data indicate that the kinetics and permeation properties of AMPA receptors are linked to factors associated with synaptic connectivity.

Fig. 1.

A, AMPA receptors in auditory nerve targets have faster rates of deactivation and desensitization than receptors in parallel fiber targets. Outside-out patches were exposed to either ≤1 or 100 msec pulses of 10 mml-glutamate. Short exposures led to the opening of the channels followed by deactivation after removal of the glutamate (light traces). The channels sometimes started to desensitize during the 1 msec exposure. Single exponential fits to the deactivation of these channels were 0.43, 0.36, 0.37, 0.36, and 0.46 msec for bushy, octopus, T stellate, tuberculoventral cells, and the fusiform basal dendrite, respectively. The time constant of deactivation for the fusiform apical dendrite and the cartwheel cell were 1.17 and 1.99 msec. Time constants for deactivation were significantly faster in auditory nerve targets than in the parallel fiber targets (p < 0.01; ttest). During long exposures to glutamate (100 msec), channels opened briefly and then desensitized in the continued presence of glutamate (dark traces). Single exponential fits to the desensitization of these channels were 0.97, 0.91, 0.90, and 0.82 msec for bushy, octopus, T stellate, and tuberculoventral cells, respectively. Desensitization of receptors from the basal dendrite of a fusiform cell was fit by two exponentials, 1.70 (58%) and 6.36 msec (42%). Receptors in the fusiform apical dendrite and the cartwheel cell had time constants of desensitization of 5.62 and 4.80 msec.B, The time constant of decay of the fastest mEPSCs matched the time constant of deactivation. Left,Response in a patch from an octopus cell to a 1 msec pulse of glutamate. Right, Ensemble average of 127 of the fastest mEPSCs from the same cell before excision of patch. Single exponential fits are superimposed on the current traces.

Fig. 2.

Cyclothiazide slows desensitization in both auditory nerve targets and parallel fiber targets. Left,AMPA receptors in an octopus and cartwheel cell desensitize during a 100 msec exposure to 10 mm glutamate.Middle, Glutamate-evoked currents in the presence of 200 μm cyclothiazide show a dramatic slowing of desensitization, but not a complete block in both cell types, and there was little potentiation of the current peak. Cyclothiazide was less effective in blocking desensitization in the octopus cell.Right, Currents evoked with a 3 sec exposure to 10 mm glutamate and 200 μm cyclothiazide desensitized to >90% in the octopus cell and 82% in the cartwheel cell.

Fig. 3.

AMPA receptors in auditory nerve and parallel fiber targets contain subunits in the flop isoform. A,Glutamate-evoked currents in the presence of 200 μmcyclothiazide desensitized in all cell types but with a slower time course than under control conditions. B, Currents evoked by glutamate in the presence of 200 μm cyclothiazide desensitized by >90% in receptors from cells with only auditory nerve fiber input. Currents in the fusiform basal dendrites, apical dendrites, and in the cartwheel cell desensitized to ∼75%.C, Pooled data from at least five patches for each cell type show that AMPA receptors in auditory nerve targets desensitized to a similar extent in cyclothiazide (dark bars) as in the absence (light bars). In contrast, AMPA receptors in fusiform basal and apical dendrites and cartwheel cells desensitized significantly less in the presence of cyclothiazide compared with control (p < 0.01; t test). In addition, in the presence of cyclothiazide, AMPA receptors in fusiform and cartwheel cells desensitized significantly less than those in cells with only auditory nerve input (p< 0.01; t test). Error bars indicate SD.

Fig. 4.

AMPA receptors in auditory nerve targets recover from desensitization faster than in parallel fiber targets. Recovery from desensitization (I_test/I_condition) of the AMPA receptors was plotted as a function of interstimulus interval. A single exponential described the time course of recovery for all cell types after a delay of 5 msec in bushy, octopus, T stellate, tuberculoventral, and fusiform cells and 25 msec in cartwheel cells. Each point represents the average value from at least five different patches from each cell type with the SD (error bars).

Fig. 5.

In the presence of intracellular polyamines, current–voltage relationships are inwardly rectifying in cells with mainly auditory nerve input and linear in fusiform and cartwheel cells. Patches were exposed to glutamate for 100 msec at a range of holding potentials from +80 to −100 mV in 20 mV steps. Normalized peak currents obtained from patches with 100 μm spermine in the pipette were plotted as a function of the membrane potential. The current–voltage plots exhibited inward rectification at positive membrane potentials in cells with mainly auditory nerve input, whereas those in fusiform and cartwheel cells were linear throughout the entire voltage range. Each point represents the normalized mean with the SD shown as the error bars.

Fig. 6.

AMPA receptors in cells with mainly auditory nerve input have larger single-channel currents than cells with parallel fiber input. A,Left, 50 consecutive currents evoked by 100 msec exposures to glutamate in a patch from an octopus cell at −65 mV. The mean current is superimposed (light gray). Right, The variance of the current data on the left. B, Plots of the variance versus the mean current for at least 30 consecutive currents for single patches from all neurons. The parabolic fits are superimposed on the data.

Fig. 7.

The properties of receptors in fusiform dendrite patches are the same as synaptic receptors. A,Left, Four consecutive sweeps of spontaneous mEPSCs recorded in whole-cell mode at −65 mV. Middle, mEPSCs evoked in response to a 60 msec spritz of sucrose saline on an apical dendrite. Right, mEPSCs evoked in response to a 60 msec spritz of sucrose saline on a basal dendrite. The data are shown before (top) and after (bottom) application of PhTX. B, Sucrose-evoked mEPSCs have faster kinetics in basal dendrites than in apical dendrites. Left,Histograms of the single exponential decay time constants fit to individual events from the cells illustrated above.Right, Ensemble average currents from 75 events for each dendrite with single exponential fits superimposed.

Fig. 8.

PhTX blocks spontaneously occurring and sucrose-evoked mEPSCs in a T stellate cell. Left, Four consecutive current sweeps of spontaneous currents before (top) and after (bottom) PhTX at −65 mV.Right, Sucrose applications for 60 msec to the cell body before and after PhTX at −65 mV.