Odorant-evoked nitric oxide signals in the antennal lobe of Manduca sexta

Abstract

The gaseous signaling molecule nitric oxide (NO) can affect the activities of neurons and neural networks in many different systems. The strong expression of NO synthase (NOS) in the primary synaptic neuropil (the antennal lobe in insects and the olfactory bulb in vertebrates) of the olfactory system of most organisms, and the unique spheroidal geometry of olfactory glomeruli in those neuropils, have led to suggestions that NO signaling is important for processing olfactory information. No direct evidence exists, however, that NO signals are produced in olfactory glomeruli. We investigated the production of NO in the antennal lobe of the moth, Manduca sexta, by using immunocytochemistry and real-time optical imaging with a NO-sensitive fluorescent marker, diaminofluorescein diacetate. We confirmed that NOS was expressed in the axons of olfactory receptor neurons projecting to all glomeruli. Soluble guanylyl cyclase, the best characterized target of NO, was found in a subset of postsynaptic antennal lobe neurons that included projection neurons, a small number of GABA-immunoreactive neurons, and a serotonin-immunoreactive neuron. We found that odorant stimulation evoked NO signals that were reproducible and spatially focused. Different odorants evoked spatially distinct patterns of NO production. Increased concentrations of pheromone and plant odorants caused increases in peak signal intensity. Increased concentrations of plant odorants also evoked a dramatic increase in signal area. The results of these experiments show clearly that odorant stimulation can evoke NO production in the olfactory system. The NO signals produced are likely to play an important role in processing olfactory information.

Figure 1.

Immunocytochemical labeling of NOS protein in the ALs and central brain. Strong NOS labeling is in most or all of the ORN axons in both ALs, which are shown in serial sections from anterior (A) to posterior (E). The mushroom body lobes (F, dashed outlines), central complex (G, dashed outline), mushroom body calyces (H, dashed outlines), and other parts of the central brain do not express NOS at such high levels.

Figure 3.

Immunocytochemical labeling of NOS and MsGCα1 in the AL. A, All glomeruli (G, dashed outlines) including the MGC, contain NOS-positive ORN axons. B, Large subsets of intrinsic AL neurons in the medial and lateral clusters (MC and LC, respectively), and a subset of antennal mechanosensory (AM) axons are MsGCα1 positive. C, The expression patterns for NOS and MsGCα1 are closely apposed in glomeruli. D, A single optical section of an image of a glomerulus shows that NOS and MsGCα1 are not coexpressed in glomeruli. D, Dorsal; L, lateral.

Figure 2.

MsGCα1 antiserum recognizes MsGCα1 protein in the adult brain of M. sexta by Western blot analysis. The predominant band of 78 kDa recognized by the antiserum matches the predicted size for MsGCα1 of 78.4 kDa.

Figure 4.

Characterizing MsGCα1-positive neurons with other immunocytochemical markers. A, A large subset of neurons in the lateral cluster (LC), but not the medial cluster (MC), are GABA immunoreactive. B, Both clusters contain MsGCα1-positive neurons. C, A small subset of the GABA-immunoreactive neurons also contains MsGCα1 (yellow cell bodies). D, Each AL houses one 5-HT-immunoreactive neuron. E, F, The serotonin-immunoreactive neuron expresses MsGCα1 highly. D, Dorsal; L, lateral.

Figure 5.

Typical NO signals for the odorants tested. The images show the relative change (dF/F) in activity after stimulation. Each image is an average of four frames at the peak of the signal. Each image is false-color coded and scaled to its entire intensity range. The solid white outlines are an estimate of the AL border. The dashed white lines mark the center of the image. The dotted white outlines are an estimate of the visible MGC area. A, NO signals in a single male AL for 10 μg of (+/-)-linalool. B, A graph of the temporal dynamics of the peak of the NO signal for A is shown relative to control. The stimulus pulses are indicated by red bars. NO signals in the same AL for geraniol (C), 1-octanol (D), PAA (E), and 1 μg of EEZ (F). G, H, In a different male AL, the NO signal for 10 μg of (+/-)-linalool is reduced by C-PTIO and l-NAME. These pharmacological antagonists of NO signaling significantly reduced the average peak dF/F in three animals tested (1.11 ± 0.21% before treatment, n = 6 stimulations; 0.88 ± 0.23% during treatment, n = 6 stimulations; p < 0.001). Note that this level of reduction (∼21%) is similar to that reported in the literature for these antagonists of NO signaling (Pittner et al., 2003). D, Dorsal; L, lateral.

Figure 6.

Peak magnitudes and temporal dynamics of dF/F. A, Average peak magnitude of odorant-evoked NO signals, which were calculated from the most activated glomerulus of each response. B, Temporal dynamics of the averaged signals for each plant odorant (10 μg) tested. C, Temporal dynamics of the averaged signals for 1 μg of pheromone. Red bars indicate stimulus pulses. Paraffin oil, n = 20 stimulations in seven animals; geraniol, n = 11 responses in seven animals; (+/-)-linalool, n = 14 responses in six animals; 1-octanol, n = 8 responses in five animals; PAA, n = 13 responses in seven animals; cyclohexane, n = 8 stimulations in three animals; pheromone, n = 8 responses in three animals. All odorant-evoked signals were significantly different from controls (p < 0.0001 for all).

Figure 7.

Concentration-dependent NO signals for (+/-)-linalool (A-D) and pheromone mixture (E-H). The images represent every other frame of a recording. Each group of images begins at the onset of the stimulus and ends at or after the absolute peak of the signal. For simplicity, the onset of the stimulus is defined as time 0 in these examples. All images are false-color coded. The luminance, contrast, and gamma settings were kept the same within each odorant series. The white outlines are an estimate of the AL border. A-D, Responses to (+/-)-linalool. A, The NO signal evoked by 10 μg of (+/-)-linalool. B, The NO signal for 100 μg was stronger and involved a larger area of the AL. C, The NO signal for 1 mg was the strongest and involved several neighboring glomeruli. Note that the signal has faded by the final frame. Because diaminofluoresceins are integrative dyes, the decrease in signal is likely attributable to photo-bleaching. D, The peaks of the signals are grouped according to concentration. The concentration groups were compared in one-way ANOVA (p < 0.0001; n = 6 animals). E-H, Responses to pheromone mixture. E, The NO signal evoked by 10 ng of the pheromone mixture. The focus on this group of images was shifted slightly in relation to the other two groups. F, The NO signal for 100 ng was stronger. G, The NO signal for 1 μg was the strongest, yet neighboring glomeruli did not appear to be activated. H, The peaks of the signals are grouped according to concentration. The concentration groups were compared in one-way ANOVA (p < 0.0001; n = 3 animals). D, Dorsal; L, lateral.