John G. Hildebrand

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

Summary1.A subset of olfactory projection neurons in the brain of maleManduca sexta is described, and their role in sex pheromone information processing is examined.2.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 (Figs. 4, 7, 9, 13 and 15).3.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 (Fig. 1).4.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 (Fig. 2).5.Two broad physiological categories were found: (a) cells that were excited by stimulation of the ipsilateral antenna with pheromones (29 out of 41), and (b) cells that received a mixed input (inhibition and excitation) from pheromone pathways (12 out of 41).6.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 (Fig. 3).7.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 (Figs. 5, 6 and 8). Some of these neurons had dendritic arborizations restricted to only a portion of the MGC neuropil (Fig. 9), whereas most had arborizations throughout the MGC.8.Of the cells in the second category, 9 out of 12 were excited by bombykal, inhibited by C-l 5, and showed a mixed response to the natural pheromone blend (Figs. 11 and 12). For the other 3 out of 12 cells, the response polarity was reversed for the two chemically-identified odors (Fig. 14).9.Two additional neurons, which were not tested with olfactory stimuli, were tonically inhibited in response to electrical stimulation of the ipsilateral antennal nerve (Fig. 15).10.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 known 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.

Anatomy of antenno-cerebral pathways in the brain of the sphinx moth Manduca sexta

SummaryIn the moth Manduca sexta, the number and morphology of neuronal connections between the antennal lobes and the protocerebrum were examined. Cobalt injections revealed eight morphological types of neurons with somata adjacent to the AL neuropil that project in the inner, middle, and outer antenno-cerebral tracts to the protocerebrum. Neurons innervating the macroglomerular complex and many neurons with fibers in the inner antennocerebral tract have uniglomerular antennal-lobe arborizations. Most neurons in the middle and outer antenno-cerebral tracts, on the other hand, seem to innervate more than one glomerulus. Protocerebral areas receiving direct input from the antennal lobe include the calyces of the mushroom bodies, and circumscribed areas termed “olfactory foci” in the lateral horn of the protocerebrum and several other regions, especially areas in close proximity to the mushroom bodies. Fibers in the inner antenno-cerebral tract that innervate the male-specific macroglomerular complex have arborizations in the protocerebrum that are distinct from the projections of sexually non-specific neurons. Protocerebral neurons projecting into the antennal lobe are much less numerous than antennal-lobe output cells. Most of these protocerebral fibers enter the antennal lobe in small fiber tracts that are different from those described above. In the protocerebrum, these centrifugal cells arborize in olfactory foci and also in the inferior median protocerebrum and the lateral accessory lobes. The morphological diversity of connections between the antennal lobes and the protocerebrum, described here for the first time on a single-cell level, suggests a much greater physiological complexity of the olfactory system than has been assumed so far.

Odour-plume dynamics influence the brain's olfactory code

The neural computations used to represent olfactory information in the brain have long been investigated. Recent studies in the insect antennal lobe suggest that precise temporal and/or spatial patterns of activity underlie the recognition and discrimination of different odours, and that these patterns may be strengthened by associative learning. It remains unknown, however, whether these activity patterns persist when odour intensity varies rapidly and unpredictably, as often occurs in nature. Here we show that with naturally intermittent odour stimulation, spike patterns recorded from moth antennal-lobe output neurons varied predictably with the fine-scale temporal dynamics and intensity of the odour. These data support the hypothesis that olfactory circuits compensate for contextual variations in the stimulus pattern with high temporal precision. The timing of output neuron activity is constantly modulated to reflect ongoing changes in stimulus intensity and dynamics that occur on a millisecond timescale.

Structure and development of antennae in a moth, Manduca sexta

Abstract The antenna of the moth, Manduca sexta , comprises two small basal segments and a long (2 cm) flagellum, which is divided into nearly 80 annuli. The annuli bear cuticular scales and small sensory organs, sensilla. A trachea, a blood vessel, and two nerve trunks run through the lumen of the antenna and into the head. Sensilla are arranged in an orderly pattern that is repeated on each flagellar annulus. Each flagellum bears about 10 5 sensilla, which contain about 2.5 × 10 5 primary sensory neurons. Clumps of undifferentiated cells (imaginal disks), present in the larva, form pupal antennae during the larval-pupal molt. During the subsequent metamorphic development of the adult, cell divisions, changes in cell shape, and cellular differentiation transform pupal into adult antennae. Sensilla and scales arise and differentiate in the antenna during metamorphosis; regions in which sensilla and scales will arise can be recognized before overt differentiation occurs. All of the flagellar annuli develop synchronously. The dense innervation and neuronal simplicity of antennal flagella, as well as their synchronous development at a late and accessible stage in the animals life cycle, suit them for studies of neuronal differentiation.

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons).Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.

Olfactory systems: Common design, uncommon origins?

In both vertebrates and invertebrates, odorant molecules reach the dendrites of olfactory receptor cells through an aqueous medium, which reflects the evolutionary origin of these systems in a marine environment. Important recent advances, however, have demonstrated striking interphyletic differences between the structure of vertebrate and invertebrate olfactory receptor proteins, as well as the organization of the genes encoding them. While these disparities support independent origins for odor-processing systems in craniates and protostomes (and even between the nasal and vomeronasal systems of craniates), olfactory neuropils share close neuroanatomical and physiological characters. Whereas there is a case to be made for homology among members of the two great protostome clades (the ecdysozoans and lophotrochozoans), the position of the craniates remains ambiguous.

Organization and Synaptic Ultrastructure of Glomeruli in the Antennal Lobes of the Moth Manduca sexta: A Study Using Thin Sections and Freeze-Fracture

The antennal lobe of the brain of Manduca sexta comprises a central area of coarse neuropil surrounded by dense, spheroidal glomeruli, where all synaptic interactions between antennal-nerve axons and the second-order neurons of the lobe occur. Neuronal interactions in the glomeruli are complex, involving several types of neuritic profiles and mediated by synapses with a one-to-many ratio of pre- to postsynaptic elements. Presynaptic profiles in the glomeruli have been categorized into three types, containing round clear vesicles, large numbers of large dense-cored vesicles, and pleiomorphic clear vesicles, respectively. Preliminary studies of horseradish peroxidase-filled axons and neurons indicate that antennal-nerve axons form synapses without large numbers of dense-cored vesicles and that antennal-lobe neurons not only receive synapses but also may synapse onto other elements in the antennal lobe. A typical synaptic contact involves multiple postsynaptic elements apposed in pairs to an individual presynaptic element. The presynaptic element contains a bar-shaped membrane-associated density, which follows a shallow groove in the membrane and is flanked by synaptic vesicles. Postsynaptic elements are lined by membrane-associated densities in the region opposite to the synaptic bar, and may be observed to participate in serial synapses. Freeze-fracture replicas of the glomerular neuropil contain many membrane specializations that are thought to be presynaptic, some of which resemble those of vertebrate excitatory synapses. At these apparently presynaptic regions, large particles cluster in the P face of the membrane and are often surrounded by plasmalemmal deformations presumably representing sites of exo- or endocytosis. The shape of the predominant type of presynaptic membrane specialization (a plaque) does not match the shape of the presynaptic membrane-associated density (a bar); this raises the possibility that vesicle release occurs at isolated ‘active zones’ along the presynaptic bar. Postsynaptic sites are represented by clusters of large particles in the E face of the postsynaptic membrane.

Physiology and morphology of projection neurons in the antennal lobe of the male mothManduca sexta

Summary1.We have used intracellular recording and staining, followed by reconstruction from serial sections, to characterize the responses and structure of projection neurons (PNs) that link the antennal lobe (AL) to other regions of the brain of the male sphinx mothManduca sexta.2.Dendritic arborizations of the AL PNs were usually restricted either to ordinary glomeruli or to the male-specific macroglomerular complex (MGC) within the AL neuropil. Dendritic fields in the MGC appeared to belong to distinct partitions within the MGC (Figs. 2, 3). PNs innervating the ordinary glomeruli had arborizations in a single glomerulus (uniglomerular) (Figs. 6, 7, 9, 11, 12A) or in more than one ordinary glomerulus of one AL (multiglomerular) (Figs. 12B, C, 14, 15), or in one case, in single glomeruli in both ALs (bilateral-uniglomerular) (Fig. 16). One PN innervated the MGC and many or all ordinary glomeruli of the AL (Fig. 13).3.PNs with dendritic arborizations in the ordinary glomeruli and PNs associated with the MGC typically projected both to the calyces of the ipsilateral mushroom body and to the lateral protocerebrum, but some differences in the patterns of termination in those regions have been noted for the two classes of PNs (Figs. 2, 3, 6, 7, 9, 16). One PN conspicuously lacked branches in the calyces but did project to the lateral protocerebrum (Fig. 14). The PN innervating the MGC and many ordinary glomeruli projected to the calyces of the ipsilateral mushroom body and the superior protocerebrum (Fig. 13).4.Crude sex-pheromone extracts excited all neurons with arborizations in the MGC, although some were inhibited by other odors (Figs. 3, 4). One P(MGC) was excited by crude sex-pheromone extract and by a mimic of one component of the pheromone blend but was inhibited by another component of the blend (Fig. 5).5.PNs with dendritic arborizations in ordinary glomeruli were excited (Figs. 7, 8, 10) or inhibited (Figs. 9, 11) by certain non-pheromonal odors. Some of these PNs also responded to mechanosensory stimulation of the antennae (Figs. 10, 11, 15, 16).6.The PN with dendritic arborizations in the MGC and many ordinary glomeruli was excited by crude sex-pheromone extracts and non-pheromonal odors and also responded to mechanosensory stimulation of the antenna (Fig. 13).

Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta

SummaryWe have prepared and characterized specific rabbit antisera against γ-aminobutyric acid (GABA) coupled covalently to bovine serum albumin and keyhole-limpet hemocyanin. Using these antisera in immunocytochemical staining procedures, we have probed the antennal lobes and their afferent and efferent fiber tracts in the sphinx moth Manduca sexta for GABA-like immunoreactivity in order to map putatively GABAergic central neurons in the central antennal-sensory pathway. About 30% of the neuronal somata in the large lateral group of cell bodies in the antennal lobe are GABA-immunoreactive; cells in the medial and anterior groups of antennal-lobe cells did not exhibit GABA-like immunoreactivity. GABA-immunoreactive neurites had arborizations in all of the glomeruli in the antennal lobe. Double-labeling experiments involving tandem intracellular staining with Lucifer Yellow and immunocytochemical staining for GABA-like immunoreactivity demonstrated that at least some of the GABA-immunoreactive cells in the antennal lobe are amacrine local interneurons. Several fiber tracts that carry axons of antennal-lobe projection neurons exhibited GABA-immunoreactive fibers. Among the possibly GABA-containing projection neurons are several cells, with somata in the lateral group of the antennal lobe, that send their axons directly to the lateral protocerebmm.

Origin and morphogenesis of sensory neurons in an insect antenna.

Abstract Each antennal flagellum of the moth, Manduca sexta, contains about 2.5 × 105 primary sensory neurons. The neurons are components of small sensory organs (sensilla) and send axons through antennal nerves to the brain. The neurons, sensilla, and nerves differentiate as the antenna develops, during the 18 days of metamorphosis from pupa to adult. Neurons arise from divisions of epidermal cells between 25 and 60 hr after pupal ecdysis and elaborate axons and dendrites soon thereafter. Neurons have the bipolar form, ciliated dendrite, and glial sheath characteristic of the adult within a few days of their birth. The axons grow along small pupal nerves to form the adult antennal nerves, and the dendrites grow beyond the apical margin of the epidermis, where they are enveloped by a growing process of the sensillas trichogen cell. Cuticle secreted by the trichogen cell forms the seta or sensory hair of the sensillum. Later, the neuronal somata migrate from the basal to the apical margin of the epidermis. Finally, the cytoplasm withdraws from the seta, leaving the dendrites imprisoned in a cylinder of cuticle. All of the neurons in the flagellum differentiate nearly synchronously, facilitating correlation of morphogenetic results presented here with biochemical and electrophysiological analyses of the developing neurons.