Inhibitory interactions among olfactory glomeruli do not necessarily reflect spatial proximity

Abstract

Inhibitory interactions shape the activity of output neurons in primary olfactory centers and promote contrast enhancement of odor representations. Patterns of interglomerular connectivity, however, are largely unknown. To test whether the proximity of glomeruli to one another is correlated with interglomerular inhibitory interactions, we used intracellular recording and staining methods to record the responses of projection (output) neurons (PNs) associated with glomeruli of known olfactory tuning in the primary olfactory center of the moth Manduca sexta. We focused on Toroid I, a glomerulus in the male-specific macroglomerular complex (MGC) specialized to one of the two key components of the conspecific females' sex pheromone, and the adjacent, sexually isomorphic glomerulus 35, which is highly sensitive to Z-3-hexenyl acetate (Z3-6:OAc). We used the two odorants to activate these reference glomeruli and tested the effects of olfactory activation in other glomeruli. We found that Toroid-I PNs were not inhibited by input to G35, whereas G35 PNs were inhibited by input to Toroid-I PNs. We also recorded the responses of PNs arborizing in other sexually isomorphic glomeruli to stimulation with the sex pheromone and Z3-6:OAc. We found that inhibitory responses were not related to proximity to the MGC and G35: both distant and adjacent PNs were inhibited by stimulation with the sex pheromone, some others were affected by only one odorant, and yet others by neither. Similar results were obtained in female PNs recorded in proximity to female-specific glomeruli. Our findings indicate that inhibitory interactions among glomeruli are widespread and independent of their spatial proximity.

FIG. 1.

Morphological features and typical odor responses of projection neurons (PNs) arborizing in 3 neighboring glomeruli in the antennal lobe (AL) of male Manduca sexta. The Cumulus (A) and Toroid I (B) are the 2 main glomeruli of the male-specific macroglomerular complex (MGC); the adjacent glomerulus 35 (G35, C) is a sexually isomorphic glomerulus. MGC PNs and G35 PNs have their cell bodies, respectively, in the medial group of AL neuronal cell bodies (MC in A and B) and in the anterior group (AC in C). Electrophysiological traces obtained from a Cumulus PN (D), a Toroid-I PN (E), and a G35 PN. All PN classes gave excitatory responses to stimulation with their respective key odor inputs, the sex-pheromone components E10,E12,Z14-16:Al (here substituted by the stable chemical mimic, E11,Z13-15:Al or “C15”) and E10,Z12-16:Al (bombykal), and the plant volatile Z-3-hexenyl acetate (Z3-6:OAc). Cumulus PNs and Toroid-I PNs, respectively, were inhibited by stimulation with bombykal and C15 (D and E, deflections below the resting membrane potential indicated by ---), i.e., each by excitatory input to the other MGC glomerulus. These PNs were not inhibited by Z3-6:OAc (D and E, 3rd row), the compound that activates PNs in the adjacent G35. Stimulation with C15 or bombykal inhibited G35 PNs (F, 1st and 2nd rows). The solvents used to dilute the pheromone components and Z3-6:OAc (cyclohexane and mineral oil, respectively—solvent controls) elicited no response (not shown in this figure). —, the activation of the device controlling the stimulus delivery system and the duration (200 ms) of the stimulus. Concentrations or amount of compounds are indicated between parentheses below each record. Calibration: 10 mV, 500 ms. D, dorsal; L, lateral. Scale bars: 150 μm.

FIG. 2.

Quantification of the responses of Toroid-I PNs (left, means ± SE, n = 11 PNs) to stimulation with bombykal (bomb), C15, Z3-6:OAc, and the 2 respective solvent controls (cyclohexane in the case of pheromone components, mineral oil in the case of Z3-6:OAc). A: the net number of spikes in a 1-s period after the onset of antennal stimulation. B: the amplitude of the odor-evoked deflection (negative values indicate hyperpolarization). PNs were stimulated with 20 ng of bombykal (except 1 PN was stimulated with 2 ng), and with 20, 200, or 500 ng of C15 loaded on filter paper. Stimulus duration was 200 ms (except 500 ms in 3 PNs). Asterisks indicate significant differences between C15 and the respective control (Wilcoxon matched pairs tests; *: P < 0.05, ***: P < 0.005). In neither case was the response to Z3-6:OAc statistically different from the response evoked by mineral oil (Wilcoxon matched pairs tests: P > 0.05). C: time course of the responses of Toroid-I PNs. The net number of spikes (means ± SE, n = 8 PNs) during a 1-s period after the odor had reached the antenna was broken down into 20 50-ms bins. First row: net response to stimulation with the C15 (•) and the cyclohexane control; 2nd row: net response to stimulation with Z3-6:OAc 10⁻² vol/vol (•) and the mineral oil control. This panel includes only those PNs that were stimulated with 200-ms pulses. *, statistical differences between the response to the odorant and the respective control for each time bin (Wilcoxon matched pair tests, P < 0.05). Note that the response to C15 was statistically different from that to the control in 11 consecutive time bins. The response to Z3-6:OAc was different from the response to the control in only 1 of the 20 time bins, which is the false positive rate expected under a P = 0.05 criterion. These results show that Toroid-I PNs do not receive inhibitory input from the adjacent G35 glomerulus or any other Z3-6:OAc-activated glomerulus.

FIG. 3.

Responses and morphological features of PNs in sexually isomorphic glomeruli. A–C, G, and H: PNs in glomeruli adjacent to or 1–2 glomeruli away from the MGC and G35; D–F, I, and J: PNs in glomeruli distant to the MGC. Records shown in A, B, D, and E were obtained, respectively, from the neurons shown in G–J. PNs in both adjacent (A and G) and distant (D and I) glomeruli were inhibited by stimulation with either odorant. PNs in both adjacent (B and H) and distant (F and J) glomeruli were inhibited only by Z3-6:OAc. C shows an example of a PN that was inhibited only by the sex pheromone, and E and J show an example of a PN that was not inhibited by either key component of the sex pheromone. PNs were stimulated with the odor compounds and concentrations indicated. The respective control stimuli (cyclohexane and mineral oil) elicited no response (not shown in this figure). The resting potential is indicated (···). These results show that not all PNs, regardless of their position, receive inhibitory input from the MGC or G35 (or any other glomeruli activated by Z3-6:OAc). Calibration bars: 10 mV in all records. Scale bars = 200 μm. ···, the outline of the MGC glomeruli. The cell body of the PN shown in I (not visible in this section) was in the anterior group of neuronal cell bodies.

FIG. 4.

Quantification of the responses of PNs in glomeruli adjacent to or 1–2 glomeruli away from the MGC or distant to the MGC and G35 to stimulation with the sex-pheromone blend (A and B) and Z3-6:OAc (C and D). A and C: the net number of spikes in a 1-s period postantennal stimulation. B–D: the amplitude of the odor-evoked change in membrane potential. ○, the average response (control-subtracted for clarity) of each PN to illustrate the response variability. •, the average response across PNs. The net number of spikes and the change in membrane potential evoked by the pheromone blend were statistically different from those evoked by the cyclohexane solvent-control stimulus in PNs in glomeruli both nearby and distant to the MGC (A and B, *, Wilcoxon matched pairs tests, n = 11 and 14, P < 0.05). The responses evoked by Z3-6:OAc were statistically different from that to the mineral-oil control stimulus only in PNs in glomeruli distant from the MGC and G35 (C and D, *; Wilcoxon matched pairs tests; G35 PNs were not included in this analysis because Z3-6:OAc evokes a strong excitatory response in these PNs). PNs were stimulated with pheromone blend (200 or 500 ng; duration: 200 or 500 ms), the cyclohexane control (200 or 500 ms), Z3-6:OAc (1:100 vol/vol in most cases; G35 PNs were stimulated with 1:1,000 or 1:10,000 vol/vol; duration = 200 ms), and the mineral oil control (200 ms; see methods for an explanation of the different concentration/stimulus duration used). These results indicate that many PNs in sexually isomorphic glomeruli receive inhibitory input from the MGC regardless of their position, and that PNs in distant glomeruli receive inhibitory input from G35 and/or other Z3-6:OAc activated glomeruli.

FIG. 5.

Responses of PNs in sexually isomorphic glomeruli of female ALs near the female-specific lateral large female glomerulus (latLFG) and G35 to stimulation with linalool and Z3-6:OAc. These odorants respectively strongly stimulated PNs in the latLFG and G35 and to a less extent, PNs in other glomeruli (Reisenman et al. 2004, 2005; Roche King et al. 2000). A: morphology of a PN with arborizations restricted to a glomerulus near the latLFG (···) and G35 (not visible in this section; image obtained after sectioning). This PN had its cell body in the lateral group of neuronal cell bodies (LC). Scale bar: 200 μm. B: electrophysiological responses of this PN to stimulation with the mineral oil control, Z3-6:OAc, and [±]linalool. Note that both Z3-6:OAc and [±]linalool elicited hyperpolarization in this PN (deflections below the resting potential, ···). Calibration bars: 5 mV in all panels. C: deflection evoked by stimulation with [±]linalool and Z3-6:OAc in PNs (n = 9) with dendritic arborizations in the neighborhood of the latLFG and G35. ○ the average response (control-subtracted for clarity) of each PN to illustrate the response variability across neurons (only 6 PNs could be tested with Z3-6:OAc). •, the average response across PNs. Concentration of odorants was 10⁻³ or 10⁻² vol/vol (duration = 200 ms). The response to [±]linalool, but not to Z3-6:OAc, was statistically different from the response to the control (, Wilcoxon matched pairs test, P < 0.05). These results show that the latLFG and/or any other linalool activated glomeruli cause inhibition in PNs in nearby glomeruli.

FIG. 6.

Examples of morphological types of local interneurons (LNs) in the ALs of female M. sexta. LNs are confined to the ALs. A: this type of LN exhibits a symmetrical arborization pattern in which the dendrites branch radially from the major neurite in the central, coarse neuropil and ramify widely in the glomeruli. B: this type of LN is distinguished by the marked asymmetry in the branching pattern of their neurites into the glomeruli and also ramifies widely in the glomeruli. C and D: this type of LN shows an asymmetric pattern, but the arborizations are limited to a smaller number of glomeruli. D: confocal microscopic image obtained from the neuron shown in C after embedding in plastic and sectioning. This neuron had 2 main neurites, one connecting a large number of glomeruli (C and D, →), which includes the latLFG (D, ···), and the other connecting fewer glomeruli (C, ▵). Scale bars: 100 μm. This figure illustrates the different types of LNs that mediate interglomerular interactions. The different morphologies provide a neuronal substrate for proposed global and glomerulus-specific inhibitory networks (Silbering and Galizia 2007).