Sensory neuron signaling to the brain: properties of transmitter release from olfactory nerve terminals

Abstract

Olfactory receptor neurons (ORNs) convey sensory information directly to the CNS via conventional glutamatergic synaptic contacts in olfactory bulb glomeruli. To better understand the process by which information contained in the odorant-evoked firing of ORNs is transmitted to the brain, we examined the properties of glutamate release from olfactory nerve (ON) terminals in slices of the rat olfactory bulb. We show that marked paired pulse depression is the same in simultaneously recorded periglomerular and tufted neurons, and that this form of short-term plasticity is attributable to a reduction of glutamate release from ON terminals. We used the progressive blockade of NMDA receptor (NMDAR) EPSCs by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine hydrogen maleate] and stationary fluctuation analysis of AMPA receptor (AMPAR) EPSCs to determine the probability of release (P(r)) of ON terminals; both approaches indicated that P(r) is unusually high (>/=0.8). The low-affinity glutamate receptor antagonists gamma-d-glutamylglycine and l-amino-5-phosphonovaleric acid blocked ON-evoked AMPAR- and NMDAR-mediated EPSCs, respectively, to the same extent under conditions of low and high P(r), suggesting that multivesicular release is not a feature of ON terminals. Although release from most synapses exhibits a highly nonlinear dependence on extracellular Ca(2+), we find that the relationship between glutamate release and extracellular Ca(2+) at ON terminals is nearly linear. Our results suggest that ON terminals have specialized features that may contribute to the reliable transmission of sensory information from nose to brain.

Figure 1.

ON terminals show strong presynaptic paired pulse depression. A₁, The average response of a simultaneously recorded PG and tufted cell to 5, 10, 25, and 50 msec paired pulse stimulation. A₂, Average PPR (EPSC₂ /EPSC₁) over a range of interstimulus intervals (n = 7 pairs). B₁, ON-evoked EPSC (V_h = –70 mV) before (Con) and after (CTZ) application of cyclothiazide (100 μm). B₂, Average effect of CTZ on PPR (left axis) and EPSC₁ amplitude (right axis; n = 5). C₁, AMPAR- and NMDAR-mediated paired pulse depression in a representative PG neuron. C₂, Average AMPAR- and NMDAR-mediated PPR are not significantly different (n = 7 cells; p > 0.2). D₁, Fifteen consecutive AMPAR-mediated responses in a PG neuron evoked by paired pulse stimulation of the ON layer. D₂, CV² ratio (EPSC₁ CV²/EPSC₂ CV²) versus PPR (25–100 msec ISI) in 11 experiments.

Figure 2.

Progressive blockade of ON-evoked NMDAR EPSCs by MK-801 indicates P_r from ON terminals is high. A₁, NMDAR EPSC amplitude versus episode number for a typical experiment. After establishing a stable ON-evoked NMDAR-mediated response (open circles), stimulation was halted, and MK-801 was introduced. In the presence of MK-801, the peak NMDAR EPSC amplitude (gray circles) declined rapidly when stimulation was resumed. A₂, Average response to ON stimulation before (control) and average of the first five responses in MK-801. A₃, NMDAR-mediated currents evoked by brief (15 msec) pressure application of glutamate (Glu; 100 μm) and ON stimulation in normal aCSF (2.5 mm Ca²⁺; gray circles) or low Ca²⁺ (1.25 mm; open circles) are inhibited progressively in the presence of MK-801 with a monoexponental time course (τ_glu = 0.89 episodes; τ_{EPSC 2.5} = 2.12 episodes; τ_{EPSC 1.25} = 4.7 episodes). Inset, ON-evoked NMDAR-mediated currents under control conditions and in MK-801. White dots represent fits to the data generated by the kinetic model (see Materials and Methods).

Figure 3.

Nonstationary fluctuation analysis of ON-evoked AMPAR-mediated EPSCs indicates P_r from ON terminals is high. A₁, Ten consecutive ON-evoked EPSCs under control conditions and in the presence of two different concentrations of Cd²⁺. A₂, Variance–mean plot for the representative experiment in A₁. The estimated P_r in this experiment is 0.96. B, The predicted (small closed circle) and measured (small open circle) quantal size for each experiment. The average predicted (large filled circle) and measured (large open circle) quantal sizes were not significantly different (p = 0.66; left axis). P_r for each of the six individual experiments (small open circles) and the average estimated for the population (large open circle) are plotted on the right axis.

Figure 4.

ON-evoked EPSCs are equally sensitive to low-affinity glutamate receptor antagonists under conditions of high or low P_r. A, γ-DGG (2 mm) blocks AMPAR-mediated EPSCs to the same extent, whether P_r is high (2.5 mm Ca²⁺) or low (0.5 mm Ca²⁺). Traces from two representative experiments are shown above; the average fractional block of EPSC₁ and EPSC₂ by γ-DGG when P_r is high (n = 12) and low (n = 10) shown below was indistinguishable (p > 0.6). B, l-APV (1 mm) inhibits NMDAR-mediated EPSCs evoked by ON stimulation to the same degree when P_r is high and low (n = 5; p > 0.5).

Figure 5.

ON transmitter release exhibits unusual Ca²⁺ cooperativity. A₁, ON-evoked glomerular fEPSPs from a representative experiment in normal (2.5 mm) and reduced Ca²⁺. A₂, Normalized amplitude of ON-evoked glomerular fEPSPs (filled circles) and AMPAR-mediated EPSCs (open circles) as a function of extracellular Ca²⁺. Each point represents the average of three to six experiments. Predicted relationships for data with an identical K_0.5 (1.19) and cooperativity (n) values of 2 and 4 are shown as dashed lines. B₁, LOT-evoked fEPSPs in piriform cortex from a representative experiment in normal (2.5) and reduced Ca²⁺ aCSF. B₂, Normalized amplitude of LOT-evoked fEPSPs; predicted relationships for n = 2 and 4 are shown as in A₂, with K_0.5 = 1.28. All experiments in this figure were performed at room temperature.