Characterization of the accessory olfactory pathway in the noctuid moth – from the labial pit organ to protocerebral centers

Insects detect external fluctuations in atmospheric carbon dioxide (CO2) for a variety of different reasons. Herbivorous species, like lepidopterans, can sense small variations in external CO2 for the purpose of finding a suitable host plant both in relation to feeding and egg laying. Lepidopteran species detect CO2 via a specialized organ located on the peripheral segment of the labial palps, the labial palp pit organ (LPO). Based on tracing of LPO sensory neurons targeting one distinct antennal-lobe (AL) glomerulus in the sphinx moth, the projections originating from the LPO was described as “an accessory olfactory pathway in Lepidoptera” already in the 1980 ties. Carbon dioxide is a ubiquitous gas abundant in Earth’s atmosphere and the concentration of this gas is currently increasing at an unprecedented rate. It is not known how the general rise in CO2 will influence organisms that use this gas as an environmental cue. Fluctuations in external CO2 may affect their behaviors in different ways, dependent on the species, ecological niche, and context. It is therefore important to acquire knowledge on the processing of atmospheric CO2 input and the neural circuits involved in this system. The noctuid moth, Helicoverpa armigera, is a suitable model to unravel neural principles typifying the CO2 pathway. Both physiological and morphological characterizations of the CO2 sensory neurons have been performed previously in this species. In this PhD project, the main aim was to obtain new knowledge about the CO2 pathway in moths, from the periphery to higher brain centers. The first part of the work was accomplished by carrying out focused mass staining experiments labeling the sensory neurons in the labial pit organ (LPO) specifically. The previous studies, which included staining of sensory neurons located both inside and outside the pit organ, reported three main target areas, including the LPO glomerulus (LPOG) in both ALs, the gnathal ganglion, and the ventral nerve cord. By performing selective labeling of the LPO sensory neurons inside the pit and the neurons located outside the pit, we could demonstrate that the LPO sensory neurons terminate in the LPOG exclusively while the additional sensory neurons target the two other regions, the gnathal ganglion and the ventral nerve cord. We also utilized the single-neuron labeling technique resulting in morphological identification of three main types of LPO sensory neurons; in addition to finding a bilateral and unilateral type, as expected, we also discovered a contralateral type. It is worth noticing that the CO2 information, being based on one signal molecule and targeting one distinct AL glomerulus, is conveyed along sensory projections forming such a complex and non-uniform pattern. The second part of this PhD study describes how CO2 information is relayed by second-order projection neurons (PNs) originating from the LPOG and terminating in higher brain centers. We investigated the morphologies of individual LPOG-PNs by means of the single-neuron labeling technique. Here, we discovered that the LPOG PNs differ significantly from the typical uniglomerular PNs originating in the ordinary glomeruli. The CO2 information is relayed to wide areas of the protocerebrum, but via other antennal lobe tracts (ALT) than the classical medial ALT. Many of the individually stained LPOG-PNs followed the relatively thin fiber bundle classified as the transverse ALT. Another unique property of the LPOG-PNs was that they often bypassed the calyces before terminating in the lateral horn (LH). Interestingly, we found that their terminal projections in the LH overlapped with projections of PNs connected with the ordinary glomeruli, possibly indicating that this is a site for integrating inputs from the plant odors and CO2. Since we found overlapping terminals of LPOG PNs and OG PNs in the LH, suggesting a crosstalk between these two sub-systems, we asked ourselves whether there might be a putative interaction between the CO2 and pheromone subsystem at the lower synaptic level, i.e., the AL. Having access to the behaviorally relevant stimuli connected with the male-specific glomeruli in this species, we were able carry out calcium-imaging experiments to test whether CO2 might affect pheromone-elicited responses. By staining the uniglomerular medial-tract PNs with a calcium-sensitive dye we could measure the odor-evoked response in the macroglomerular complex (MGC) during stimulation with the pheromone alone and in combination with CO2. The results showed decreased pheromone responses when the CO2 was added to the pheromone stimuli. When we resected the LPO, the suppression effect was eliminated for the lower concentrations of CO2, indicating the involvement of inhibitory local interneurons innervating both the LPOG and the MGC. The maintenance of the suppression for the high-concentration CO2, indicated an alternative input channel. Additional experiments including measurement of summated potentials from the antenna, indicated that high concentrations of CO2 may suppress the response of the antennal sensory neurons directly. Altogether, the results presented here expand our knowledge on how CO2 information is transmitted from the periphery to the higher brain areas