Representation of primary plant odorants in the antennal lobe of the moth Heliothis virescens using calcium imaging

The primary olfactory centre, the antennal lobe of Heliothis virescens moths, contains 62 glomeruli which process plant odour information and four male-specific glomeruli which form the macroglomerular complex, involved in processing information about pheromone and interspecific signals. Using calcium imaging, we recorded the spatio-temporal activity pattern of the glomeruli in the anterior antennal lobe during stimulation with odorants produced by plants or insects. Each odorant elicited specific excitatory responses in one or a few glomeruli: the major pheromone component did so exclusively in the large glomerulus of the macroglomerular complex and the plant odours exclusively in the ordinary glomeruli. Eight glomeruli, with corresponding plant odour responses and positions, were identified within each sex. Glomeruli responded specifically to linalool, beta-ocimene/beta-myrcene or germacrene D/alpha-farnesene. Responses to two essential plant oils covered the response areas of their major constituents, as well as activating additional glomeruli. Stronger activation in the AL due to increased odour concentration was expressed as increased response strength within the odorant-specific glomeruli as well as recruitment of less sensitive glomeruli.     

Differential interactions of sex pheromone and plant odour in the olfactory pathway of a male moth

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.     

Calcium responses to pheromones and plant odours in the antennal lobe of the male and female moth Heliothis virescens

In male moths, the primary olfactory integration centre, the antennal lobe, consists of two systems. The macroglomerular complex processes pheromone information, while the ordinary glomeruli process plant odour information. Females lack a macroglomerular complex. We measured the spatial representation of odours using in-vivo optical recording. We found that: (1) pheromone substances elicited activity exclusively in the MGC. No response was found in female antennal lobes. (2) Plant odours elicited combinatorial activity patterns in the ordinary glomeruli in both males and females. No response was found in the MGC of male moths. (3) A clean air puff often led to activity, in both males and females, suggesting that mechano-sensory information is also processed in the antennal lobe. (4) With an interstimulus interval of 5 or 10 s, strongly activated glomeruli were able to follow the temporal structure of the stimulus, while others lost their phase-locking. Some glomeruli showed "off" responses. These properties were odour dependent. This confirms and extends previous studies, showing the functional significance of the two subsystems for processing olfactory information. Pheromones are coded in a combinatorial manner within the macroglomerular complex, with each glomerulus corresponding to one information channel. Plant odours are coded in an across-glomeruli code in the ordinary glomeruli.     

Concurrent modulation of neuronal and behavioural olfactory responses to sex and host plant cues in a male moth

Mating has profound effects on animal physiology and behaviour, not only in females but also in males, which we show here for olfactory responses. In cotton leafworm moths, Spodoptera littoralis, odour-mediated attraction to sex pheromone and plant volatiles are modulated after mating, producing a behavioural response that matches the physiological condition of the male insect. Unmated males are attracted by upwind flight to sex pheromone released by calling females, as well as to volatiles of lilac flowers and green leaves of the host plant cotton, signalling adult food and mating sites, respectively. Mating temporarily abolishes male attraction to females and host plant odour, but does not diminish attraction to flowers. This behavioural modulation is correlated with a response modulation in the olfactory system, as shown by electro-physiological recordings from antennae and by functional imaging of the antennal lobe, using natural odours and synthetic compounds. An effect of mating on the olfactory responses to pheromone and cotton plant volatiles but not to lilac flowers indicates the presence of functionally independent neural circuits within the olfactory system. Our results indicate that these circuits interconnect and weigh perception of social and habitat odour signals to generate appropriate behavioural responses according to mating state.     

Glomerular interactions in olfactory processing channels of the antennal lobes

An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.     

Responses of protocerebral neurons in Manduca sexta to sex-pheromone mixtures

Male Manduca sexta moths are attracted to a mixture of two components of the female’s sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male’s antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.     

Spatial representation of the glomerular map in the Drosophila protocerebrum

In the fruit fly, Drosophila, olfactory sensory neurons expressing a given receptor project to spatially invariant loci in the antennal lobe to create a topographic map of receptor activation. We have asked how the map in the antennal lobe is represented in higher sensory centers in the brain. Random labeling of individual projection neurons using the FLP-out technique reveals that projection neurons that innervate the same glomerulus exhibit strikingly similar axonal topography, whereas neurons from different glomeruli display very different patterns of projection in the protocerebrum. These results demonstrate that a topographic map of olfactory information is retained in higher brain centers, but the character of the map differs from that of the antennal lobe, affording an opportunity for integration of olfactory sensory input.     

A combined perceptual, physico-chemical, and imaging approach to 'odour-distances' suggests a categorizing function of the Drosophila antennal lobe

How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3-octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours.     

Dose-dependent response characteristics of antennal lobe neurons in the male moth Agrotis segetum (Lepidoptera: Noctuidae)

Responses of antennal lobe neurons to different amounts of the female-produced pheromone blend and to its individual components were investigated in Agrotis segetum males using intracellular recording methods. We identified three physiological types of antennal lobe neurons, categorized according to their response thresholds to single pheromone components and to the blend: generalist neurons, component-specific neurons and blend-specific neurons. Response and specificity of antennal lobe neurons were largely dose dependent. In most cases specific responses occurred only at low stimulus amounts, while increasing concentrations often resulted in an increase of the number of pheromone stimuli to which the neuron responded. Dose-response relationships often differed between different stimuli activating a neuron. Accepted: 24 May 1997     

Representation of a mixture of pheromone and host plant odor by antennal lobe projection neurons of the silkmoth Bombyx mori

Pheromone-source orientation behavior can be modified by coexisting plant volatiles. Some host plant volatiles enhance the pheromonal responses of olfactory receptor neurons and increase the sensitivity of orientation behavior in the Lepidoptera species. Although many electrophysiological studies have focused on the pheromonal response of olfactory interneurons, the response to the mixture of pheromone and plant odor is not yet known. Using the silkmoth, Bombyx mori, we investigated the physiology of interneurons in the antennal lobe (AL), the primary olfactory center in the insect brain, in response to a mixture of the primary pheromone component bombykol and cis-3-hexen-1-ol, a mulberry leaf volatile. Application of the mixture enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the AL. In contrast, the mixture of pheromone and cis-3-hexen-1-ol had little influence on the responses of projection neurons innervating the ordinary glomeruli whereas other plant odors dynamically modified the response. Together this suggests moths can process plant odor information under conditions of simultaneous exposure to sex pheromone.     

Antennal receptive fields of pheromone-responsive projection neurons in the antennal lobes of the male sphinx moth Manduca sexta

Stimulation of the antenna of the male moth, Manduca sexta, with a key component of the female's sex pheromone and a mimic of the second key component evokes responses in projection neurons in the sexually dimorphic macroglomerular complex of the antennal lobe. Using intracellular recording and staining techniques, we studied the antennal receptive fields of 149 such projection neurons. An antennal flagellum was stimulated in six regions along its proximo-distal axis with one or both of the pheromone-related compounds while activity was recorded in projection neurons. These neurons fell mainly into two groups, based on their responses to the two-component blend: neurons with broad receptive fields that were excited when any region of the flagellum was stimulated, and neurons selectively excited by stimulation of the proximal region of the flagellum. Projection neurons that were depolarized by stimulation of one antennal region were not inhibited by stimulation of other regions, suggesting absence of antennotopic center-surround organization. In most projection neurons, the receptive field was determined by afferent input evoked by only one of the two components. Different receptive-field properties of projection neurons may be related to the roles of these neurons in sensory control of the various phases of pheromone-modulated behavior of male moths. Accepted: 30 January 1998     

Agrotis segetum雄蛾触角叶神经元对性信息素时间编码的机制分析

应用压力注射,在Agrotis segetum雄蛾触角叶（AL）中33个对性信息素有反应的MGC神经元上探计了对性信息素反应模式的形成机制,压力注射100mmol/L GABA进入AL神经网引起神经元一个慢的超极化电位,并有一个长时程的放电抑制相,与用性信息素刺激诱导的神经元分应很相似,但GABA并不影响神经元对性信息素刺激的去极化反应,低Cl^-溶液可减弱AL神经元对性信息素刺激的超极化反应,甚至使超极化相逆转为兴奋反应,抑制相消失。压力注射Bicuculline使神经元放电频率增加。压力注射Bicuculline的同时给予性信息素刺激,可使性信息素刺激所致的神经元放电增加进一步加强;Bicuculline可使性信息素刺激引起的神经元超极化幅度变小,放电抑制时间变短,甚至其抑制相完全被逆转为正常放电,无超极化反应和抑制相存在,结果表明,AL神经元对性信息系反应的超极化相与GABA受体有关。     

Central nervous processing of behaviourally relevant odours in solitary and gregarious fifth instar locusts, Schistocerca gregaria

Physiological and morphological characteristics of antennal lobe neurons of solitary and gregarious fifth-instar nymphs of the desert locust, Schistocerca gregaria, were studied using intracellular recording and staining techniques. Physiological characteristics of antennal lobe neurons of both locust phases responding to stage-dependent aggregation pheromones, egg-laying attractants, a putative sex pheromone and plant-associated volatiles are described. Antennal lobe neurons showed excitatory, inhibitory, combined excitatory and inhibitory and delayed responses. In addition, one neuron␣showing an initial inhibition followed by an excitation and inhibition response was found. Pheromone-specific-, plant-specific- and pheromone-plant-generalist neurons were found in both locust phases. Antennal lobe neurons displayed stage- and phase-dependent differences in the processing of aggregation pheromone component input. Nymphal antennal lobe neurons showed stage-dependent response characteristics highly correlated with the preferential behavioural attraction to the nymphal aggregation pheromone. Phase-dependent differences were found in the response spectra and the sensitivity of the same neuron types. Neurons of solitary locusts responded significantly more frequently to some of the tested components than neurons of gregarious locusts. Furthermore, antennal lobe neurons of solitary locusts showed a higher sensitivity to most of the tested compounds. Accepted: 4 July 1998     

Male-specific,sex pheromone-selective projection neurons in the antennal lobes of the mothManduca sexta

A subset of olfactory projection neurons in the brain of male Manduca sexta is described, and their role in sex pheromone information processing is examined. These neurons have extensive arborizations in the macroglomerular complex (MGC), a distinctive and sexually dimorphic area of neuropil in the antennal lobe (AL), to which the axons of two known classes of antennal pheromone receptors project. Each projection neuron sends an axon from the AL into the protocerebrum. Forty-one projection neurons were characterized according to their responses to electrical stimulation of the antennal nerve as well as olfactory stimulation of antennal receptors. All neurons exhibited strong selectivity for female sex pheromones. Other behaviorally relevant odors, such as plant volatiles, had no obvious effect on the activity of these neurons. Two broad physiological categories were found: cells that were excited by stimulation of the ipsilateral antenna with pheromones (29 out of 41), and cells that received a mixed input (inhibition and excitation) from pheromone pathways (12 out of 41). Of the cells in the first category, 13 out of 29 were equally excited in response to stimulation of the antenna with either the principal natural pheromone (bombykal) or a mimic of a second unidentified pheromone ('C-15') and were similarly excited by the natural pheromone blend. The remaining 16 out of 29 cells responded selectively, and in some cases, in a dose-dependent manner, to stimulation of the antenna with bombykal or C-15, but not both. Some of these neurons had dendritic arborizations restricted to only a portion of the MGC neuropil, whereas most had arborizations throughout the MGC. Of the cells in the second category, 9 out of 12 were excited by bombykal, inhibited by C-15, and showed a mixed response to the natural pheromone blend. For the other 3 out of 12 cells, the response polarity was reversed for the two chemically-identified odors. Two additional neurons, which were not tested with olfactory stimuli, were tonically inhibited in response to electrical stimulation of the ipsilateral antennal nerve. These observations suggest that some of the male-specific projection neurons may signal general pheromone-triggered arousal, whereas a smaller number can actively integrate inputs from the two know receptor classes (Bal- and C-15-selective) and may operate as 'mixture detectors' at this level of the olfactory subsystem that processes information about sex pheromones.     

Neural Representations of Airflow in Drosophila Mushroom Body

The Drosophila mushroom body (MB) is a higher olfactory center where olfactory and other sensory information are thought to be associated. However, how MB neurons of Drosophila respond to sensory stimuli other than odor is not known. Here, we characterized the responses of MB neurons to a change in airflow, a stimulus associated with odor perception. In vivo calcium imaging from MB neurons revealed surprisingly strong and dynamic responses to an airflow stimulus. This response was dependent on the movement of the 3^rd antennal segment, suggesting that Johnston''s organ may be detecting the airflow. The calyx, the input region of the MB, responded homogeneously to airflow on. However, in the output lobes of the MB, different types of MB neurons responded with different patterns of activity to airflow on and off. Furthermore, detailed spatial analysis of the responses revealed that even within a lobe that is composed of a single type of MB neuron, there are subdivisions that respond differently to airflow on and off. These subdivisions within a single lobe were organized in a stereotypic manner across flies. For the first time, we show that changes in airflow affect MB neurons significantly and these effects are spatially organized into divisions smaller than previously defined MB neuron types.     

Mixture interactions in moth olfactory physiology: examining the effects of odorant mixture, concentration, distal stimulation, and antennal nerve transection on sensillar responses

The insect olfactory system is challenged to decipher valid signals from among an assortment of chemical cues present in the airborne environment. In the moth, Heliothis virescens, males rely upon detection and discrimination of a unique blend of components in the female sex pheromone to locate mates. The effect of variable odor mixtures was used to examine physiological responses from neurons within sensilla on the moth antenna sensitive to female sex pheromone components. Increasing concentrations of heliothine sex pheromone components applied in concert with the cognate stimulus for each neuronal type resulted in mixture suppression of activity, except for one odorant combination where mixture enhancement was apparent. Olfactory receptor neuron (ORN) responses were compared between moths with intact and transected antennal nerves to determine whether specific instances of suppression might be influenced by central mechanisms. Type A sensilla showed little variation in response between transected and intact preparations; however, recordings from type B sensilla with transected antennal nerves exhibited reduced mixture suppression. Testing by parallel stimulation of distal antennal segments while recording and stimulating proximal segments dismissed the possibility of interneuronal or ephaptic effects upon sensillar responses. The results indicate that increasing concentrations of "noncognate" odorants in an odor mixture or antennal nerve transection can produce variation in the intensity and temporal dynamics of physiological recordings from H. virescens ORNs.     

Spatial organization of antennal olfactory sensory neurons in the female Spodoptera littoralis moth: differences in sensitivity and temporal characteristics

Single-cell recordings from olfactory sensory neurons (OSNs), housed in sensilla located at the base and at the tip of the antenna, showed selective responses to plant odors and female sex pheromone in this polyphagous moth. A spatial variation existed in sensitivity: OSNs present on the more proximal segment (P) were more sensitive than those on the more distal segment (D). OSNs of the 2 locations also differed in temporal characteristics: OSNs on P had shorter latency and displayed more phasic responses, whereas those on D had more tonic responses, especially at low stimulus concentrations. The 196 OSNs responding to our 35 monomolecular stimuli in the screening were housed in 32 functional sensillum types: 27 in basiconic, 3 in long-trichoid, 2 in coeloconic, and 3 in auricillic sensilla. The OSNs in basiconic, coeloconic, and auricillic sensilla responded to plant-associated odorants, whereas OSNs in long-trichoid sensilla responded to female-produced sex pheromone components. Short-trichoid sensilla showed spontaneous activity, but no responses to any odorant tested. OSN specificity to plant stimuli ranged from highly specific to broadly tuned, but it did not differ clearly from females in more specialized moths. OSN response diversity is discussed in terms of olfactory coding, behavior, and ecological specialization.     

Physiological responses and central nervous projections of antennal olfactory receptor neurons in the adult desert locust, Schistocerca gregaria (Orthoptera: Acrididae)

Olfactory receptor neurons present in two morphological sensillum types on the male Schistocerca gregaria antenna were for the first time investigated physiologically when stimulated with behaviourally relevant odours. Neurons present in trichoid/basiconic sensilla showed clear excitatory responses to compounds present in the male-produced aggregation pheromone and also to a plant produced compound. Sensilla could be categorised physiologically according to the responses of their receptor neurons to the tested stimuli. Also receptor neurons present in sensilla coeloconica responded to aggregation pheromone components, but always in an inhibitory fashion. These neurons could, however, be excited by a plant produced compound and by some acids present in the nymphal odour. The antennal lobe of the male S. gregaria was observed to contain about 1000 very small glomerular structures. Single receptor neurons were stained from the antenna to the antennal lobe using a cobalt lysine technique. These stainings revealed a multi glomerular axonal branching pattern of antennal receptor neurons.Abbreviations AN antennal nerve - AL antennal lobe - RN receptor neuron     

Transplant Antennae and Host Brain Interact to Shape Odor Perceptual Space in Male Moths

Behavioral responses to odors rely first upon their accurate detection by peripheral sensory organs followed by subsequent processing within the brain’s olfactory system and higher centers. These processes allow the animal to form a unified impression of the odor environment and recognize combinations of odorants as single entities. To investigate how interactions between peripheral and central olfactory pathways shape odor perception, we transplanted antennal imaginal discs between larval males of two species of moth Heliothis virescens and Heliothis subflexa that utilize distinct pheromone blends. During metamorphic development olfactory receptor neurons originating from transplanted discs formed connections with host brain neurons within olfactory glomeruli of the adult antennal lobe. The normal antennal receptor repertoire exhibited by males of each species reflects the differences in the pheromone blends that these species employ. Behavioral assays of adult transplant males revealed high response levels to two odor blends that were dissimilar from those that attract normal males of either species. Neurophysiological analyses of peripheral receptor neurons and central olfactory neurons revealed that these behavioral responses were a result of: 1. the specificity of H. virescens donor olfactory receptor neurons for odorants unique to the donor pheromone blend and, 2. central odor recognition by the H. subflexa host brain, which typically requires peripheral receptor input across 3 distinct odor channels in order to elicit behavioral responses.     

Aim‐then‐shoot anemotaxis involved in the hopping approach of potato tuberworm moth Phthorimaea operculella toward a sex pheromone source

Male moths locate conspecific females by pheromone‐induced upwind flight maintained by detecting a visual flow, termed optomotor anemotaxis. Their behavioural pattern is characterized by an upwind surge in response to a pheromone stimulus and crosswind casting after odour loss, which is considered to be reset and restarted on receipt of another pheromone pulse. However, pheromone‐stimulated males of the potato tuberworm moth Phthorimaea operculella exhibit a series of short and straight intermittent flights, or hops, when moving upwind. It is unclear whether they navigate by employing the same behavioural pattern and wind detection mechanism as that used by flying moths. To analyze odour‐modulated anemotaxis in male potato tuberworm moths, a flat wind tunnel is constructed to give regular odour stimuli to an insect regardless of its location. Moths are subjected to pheromone pulses of different frequencies to test whether they show a behavioural pattern that is reset and restarted by a pheromone pulse. Moths on the ground are also subjected to crosswind shear to examine their detection of wind direction. Path analyses reveal that males surge upwind when they receive a pheromone pulse and exhibit casting by successive hops when they lose odour. This behavioural pattern appears to be similar to that of flying moths. When the direction of the airflow is switched orthogonally, males adjust their course angle accordingly when they are on the ground. It is suggested that, instead of optomotor anemotaxis, this ‘aim‐then‐shoot’ system aids the detection of wind direction, possibly by mechanosensory means.