Thomas A. Christensen

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.

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.

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.

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.

GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth, Manduca sexta

Summary1.Responses of neurons in the antennal lobe (AL) of the mothManduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials (Fig. 2A). Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons (Fig. 2B).2.The IPSP in projection neurons reverses below the resting potential (Fig. 3), is sensitive to changes in external (Fig. 4) and internal (Fig. 5) chloride concentration, and thus is apparently mediated by an increase in chloride conductance.3.The IPSP is reversibly blocked by 100 μM picrotoxin (Fig. 6) or bicuculline (Fig. 7).4.Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity (Fig. 8). GABA responses are associated with an increase in neuronal input conductance (Fig. 9) and a reversal potential below the resting potential (Fig. 11).5.Application of GABA blocks inhibitory synaptic inputs (Fig. 12 A) and reduces or blocks excitatory inputs (Fig. 12B). EPSPs can be protected from depression by application of GABA (Fig. 12B).6.Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth (Fig. 13).

Characterization and Coding of Behaviorally Significant Odor Mixtures

For animals to execute odor-driven behaviors, the olfactory system must process complex odor signals and maintain stimulus identity in the face of constantly changing odor intensities [1-5]. Surprisingly, how the olfactory system maintains identity of complex odors is unclear [6-10]. We took advantage of the plant-pollinator relationship between the Sacred Datura (Datura wrightii) and the moth Manduca sexta[11, 12] to determine how olfactory networks in this insects brain represent odor mixtures. We combined gas chromatography and neural-ensemble recording in the moths antennal lobe to examine population codes for the floral mixture and its fractionated components. Although the floral scent of D. wrightii comprises at least 60 compounds, only nine of those elicited robust neural responses. Behavioral experiments confirmed that these nine odorants mediate flower-foraging behaviors, but only as a mixture. Moreover, the mixture evoked equivalent foraging behaviors over a 1000-fold range in dilution, suggesting a singular percept across this concentration range. Furthermore, neural-ensemble recordings in the moths antennal lobe revealed that reliable encoding of the floral mixture is organized through synchronized activity distributed across a population of glomerular coding units, and this timing mechanism may bind the features of a complex stimulus into a coherent odor percept.

Combinatorial odor discrimination in the brain: Attractive and antagonist odor blends are represented in distinct combinations of uniquely identifiable glomeruli

The rules governing the central discrimination of odors are complex and poorly understood, but a growing body of evidence supports the hypothesis that olfactory glomeruli may represent functionally distinct coding modules in the brain. Testing this hypothesis requires that both the functional characteristics and the spatial position of the glomerulus under study be uniquely identifiable. To address these questions, we examined a specialized array of glomeruli (the macroglomerular complex; MGC) in the antennal lobe of male moths that receives input from olfactory receptor cells tuned specifically to female‐released odorants that either promote upwind flight (conspecific sex pheromones) or inhibit it (interspecific antagonists). By using a three‐dimensional reconstruction method based on high‐resolution laser‐scanning confocal microscopy, we generated precise spatial maps of the MGC glomeruli in two related noctuid species with similar pheromone chemistry, Heliothis virescens and Helicoverpa zea. To determine the breadth of tuning of individual MGC glomeruli in processing information about these social signals, we used intracellular recording and staining methods to examine the responses of projection (output) neurons that innervate MGC glomeruli and that each project an axon to higher integrative centers. In both species, a close correspondence was found between the odor specificity of the projection neurons and the glomerulus (or glomeruli) supplied by them. The binary blend of pheromone components for each species was represented by neural activity in only two distinct glomeruli in both H. virescensand H. zea. Odorants that antagonize upwind flight when they are added to the respective pheromonal blendsevoked excitatory activity in output neurons restricted to a third glomerulus in the MGCs of both species. In summary, these results suggest that the selective activation of different combinations of functionally distinct MGC glomeruli is a general means for discriminating these specific attractant and antagonist chemical signals in the brain. J. Comp. Neurol. 400:35–56, 1998.

Chemical communication in heliothine moths

Abstract1.Projection patterns of olfactory receptor neurons, specifically tuned to the two principal components of the female H. virescens sex pheromone blend, and to a third pheromone-like compound of possible antagonistic significance, were examined using a combined electrophysiological and morphological technique.2.The macroglomerular complex consists of four major glomerular subdivisions.3.In the sensillum type containing a receptor neuron detecting the main pheromone component, Z11-16: AL, two cells were present. When the sensillum was stimulated with Z11-16:AL, a single axon, stained by a method that selectively stains active neurons, was seen projecting into the large a glomerulus. The b glomerulus was innervated by a second neuron in a few double stainings.4.In a second sensillum type, one receptor neuron tuned to the second major pheromone component, Z9-14:AL, was found. In these sensilla, one or two receptor neurons of unknown specificity were also observed. When the sensillum was stimulated with Z9-14: AL, a single neuron projecting into glomerulus a or two neurons projecting into glomerulus a were most often observed.5.In the third sensillum type, one neuron specifically tuned to Z11-16:AC projected to glomerulus c, and a second cell of unknown specificity projected to the same area.6.The axonal arborizations of different physiological receptor neuron types involved in the detection of the pheromone blend do not display a clearcut morphological separation into different glomeruli in the MGC. A separation between neurons detecting attracting and repelling odours was, however, present.

Multitasking in the Olfactory System: Context-Dependent Responses to Odors Reveal Dual GABA-Regulated Coding Mechanisms in Single Olfactory Projection Neurons

Studies of olfaction have focused mainly on neural processing of information about the chemistry of odors, but olfactory stimuli have other properties that also affect central responses and thus influence behavior. In moths, continuous and intermittent stimulation with the same odor evokes two distinct flight behaviors, but the neural basis of this differential response is unknown. Here we show that certain projection neurons (PNs) in the primary olfactory center in the brain give context-dependent responses to a specific odor blend, and these responses are shaped in several ways by a bicuculline-sensitive GABA receptor. Pharmacological dissection of PN responses reveals that bicuculline blocks GABAA-type receptors/chloride channels in PNs, and that these receptors play a critical role in shaping the responses of these glomerular output neurons. The firing patterns of PNs are not odor-specific but are strongly modulated by the temporal pattern of the odor stimulus. Brief repetitive odor pulses evoke fast inhibitory potentials, followed by discrete bursts of action potentials that are phase-locked to the pulses. In contrast, the response to a single prolonged stimulus with the same odor is a series of slow oscillations underlying irregular firing. Bicuculline disrupts the timing of both types of responses, suggesting that GABAA-like receptors underlie both coding mechanisms. These results suggest that glomerular output neurons could use more than one coding scheme to represent a single olfactory stimulus. Moreover, these context-dependent odor responses encode information about both the chemical composition and the temporal pattern of the odor signal. Together with behavioral evidence, these findings suggest that context-dependent odor responses evoke different perceptions in the brain that provide the animal with important information about the spatiotemporal variations that occur in natural odor plumes.

Multi-unit recordings reveal context-dependent modulation of synchrony in odor-specific neural ensembles.

We used neural ensemble recording to examine odor-evoked ensemble patterns in the moth antennal (olfactory) lobe. Different odors are thought to evoke unique spatiotemporal patterns of glomerular activity, but little is known about the population dynamics underlying formation of these patterns. Using a silicon multielectrode array, we observed dynamic network interactions within and between glomeruli. Whereas brief odor pulses repeatedly triggered activity in the same coding ensemble, the temporal pattern of synchronous activity superimposed on the ensemble was neither oscillatory nor odor specific. Rather, synchrony strongly depended on contextual variables such as odor intensity and intermittency. Also, because of emergent inhibitory circuit interactions, odor blends evoked temporal ensemble patterns that could not be predicted from the responses to the individual odorants. Thus even at this early stage of information processing, the timing of odor-evoked neural representations is modulated by key stimulus factors unrelated to the molecular identity of the odor.