Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe

Abstract

Drosophila olfactory receptor neurons project to the antennal lobe, the insect analog of the mammalian olfactory bulb. GABAergic synaptic inhibition is thought to play a critical role in olfactory processing in the antennal lobe and olfactory bulb. However, the properties of GABAergic neurons and the cellular effects of GABA have not been described in Drosophila, an important model organism for olfaction research. We have used whole-cell patch-clamp recording, pharmacology, immunohistochemistry, and genetic markers to investigate how GABAergic inhibition affects olfactory processing in the Drosophila antennal lobe. We show that many axonless local neurons (LNs) in the adult antennal lobe are GABAergic. GABA hyperpolarizes antennal lobe projection neurons (PNs) via two distinct conductances, blocked by a GABAA- and GABAB-type antagonist, respectively. Whereas GABAA receptors shape PN odor responses during the early phase of odor responses, GABAB receptors mediate odor-evoked inhibition on longer time scales. The patterns of odor-evoked GABAB-mediated inhibition differ across glomeruli and across odors. Finally, we show that LNs display broad but diverse morphologies and odor preferences, suggesting a cellular basis for odor- and glomerulus-dependent patterns of inhibition. Together, these results are consistent with a model in which odors elicit stimulus-specific spatial patterns of GABA release, and as a result, GABAergic inhibition increases the degree of difference between the neural representations of different odors.

Figure 1.

Many antennal lobe LNs are GABAergic, but most PNs are not. A, Projection of a 2 μm confocal Z-stack through the center of both antennal lobes. GABA (magenta) colocalizes with CD8GFP (green) driven by GH298-Gal4, which targets a subpopulation of LNs. A minority of GFP-positive cells are GABA negative (arrow). The box outlined with a dotted line is enlarged below to show each channel individually. In all images, dorsal is up and ventral is down.B, GABA does not colocalize with CD8GFP driven by GH146-Gal4, which labels a subpopulation of PNs. All PNs are GABA negative, with the exception of approximately six ventral PNs (arrow). Scale bars, 20 μm.

Figure 2.

GABA hyperpolarizes antennal lobe neurons via both GABA_A and GABA_B receptors. A, A 1 s microiontophoretic pulse of GABA at the soma suppresses spontaneous spiking of an LN recorded in cell-attached mode (left). GABA has a similar effect after rupturing the patch (right). B1, GABA was microiontophoresed periodically into the neuropil, and the amplitude of the GABA-evoked hyperpolarization was monitored over time in whole-cell recordings. The graph plots mean ± SEM hyperpolarization amplitude as a percentage of control. Picrotoxin blocked the GABA response in LNs but suppressed less than one-half of the GABA response in PNs. B2, Representative traces show GABA response before and after addition of picrotoxin in an LN (left) and a PN (right). The arrows mark GABA pulses. C1, CGP54626 blocks the picrotoxin-resistant GABA response in PNs. C2, Representative traces show GABA response in a PN before and after addition of picrotoxin and after addition of CGP54626. C3, Subtracted traces show the picrotoxin-sensitive component and CGP54626-sensitive component from traces in C2. D1, CGP54626 does not affect the GABA response in LNs. D2, Representative traces show the GABA response in an LN before and after addition of CGP54626. Error bars represent SEM.

Figure 3.

GABA_B receptors contribute to long inhibitory epochs during PN odor responses. A, Rasters show spikes in a PN during odor stimulation (gray bar; 1 s). Each row represents a different trial. After adding picrotoxin, the number of spikes elicited in the first few hundred milliseconds after stimulus onset is increased, but the late inhibitory epoch is unaffected. B, CGP54626 blocks the late inhibitory epoch in a PN odor response. C, Group data averaged over all cell-odor combinations (mean ± SEM) show that both picrotoxin and CGP54626 increase PN spike rate during the first 500 ms of the odor response, although the effect of picrotoxin is stronger. D, CGP54626 also increases the odor-evoked PN spike rate later in the response, 1.5-2.5 s after stimulus onset, when picrotoxin has no effect. Error bars represent SEM.

Figure 4.

GABA_B receptors mediate odor-dependent, glomerulus-dependent synaptic inhibition. A-F, Rasters show spikes in individual PNs before (top) and after (bottom) CGP54626 was added. In many cells (A, B), GABA_B blockade removed an inhibitory epoch from the odor response. In other cells (C, D), there was little effect. Although CGP54626 did not change the response to pentyl acetate in glomerulus DM5 (C), it did affect the response to methyl salicylate (E). C and E are from the same cell. A and F contrast the effect of CGP54626 on different odor responses in glomerulus VM3. A and F are from the same cell. G, The coefficient of variation of the mean PN peristimulus-time histogram shows two peaks (solid line). CGP54626 (dashed line) selectively decreases the second peak, indicating a decrease in the tendency of odor responses to diverge over time. Each CV value is plotted on the x-axis in the center of the 200 ms bin over which it was calculated.

Figure 5.

Morphological heterogeneity among multiglomerular LNs. A, Projection of a 15 μm confocal Z-stack through the center of both antennal lobes showing the morphology of a representative LN filled with biocytin. A primary neurite enters the antennal lobes from the lateral side and arborizes throughout many glomeruli. B, An example of three different LNs that innervate anterolateral glomeruli to varying degrees. Each image is a projection of a 15 μm stack through the most anterior portion of the right antennal lobe. Scale bars: A, B, 20 μm. C, The spatial pattern of innervation density varies across different LNs. Density[as a percentage of the maximum (% of max) density for that LN] is plotted for four densely innervated glomeruli (VA2, DM1, DM6, V) and four glomeruli that are more sparsely innervated (VA1d, VA1lm, DA1, DL3).

Figure 6.

Heterogeneity in odor responses among LNs. A, Rasters compare the responses of two different LNs (same as cells LN7 and LN8 in B) with three odors (gray bar; 500 ms). The first of these responds more weakly to hexanal than to linalool or methyl salicylate. The second cell responds equally strongly to all three odors, but latency of the methyl salicylate response is shortest. B, Odor preferences vary across LNs. The number of odor-evoked spikes [as a percentage of the maximum (% of max) response for each cell] is plotted for each odor.