An understanding of the logic of odor perception requires a functional analysis of odor-evoked patterns of activity in neural assemblies in the brain. We have developed a sensitive imaging system in the Drosophila brain that couples two-photon microscopy with the specific expression of the calcium-sensitive fluorescent protein, G-CaMP. At natural odor concentration, each odor elicits a distinct and sparse spatial pattern of activity in the antennal lobe that is conserved in different flies. Patterns of glomerular activity are similar upon imaging of sensory and projection neurons, suggesting the faithful transmission of sensory input to higher brain centers. Finally, we demonstrate that the response pattern of a given glomerulus is a function of the specificity of a single odorant receptor. The development of this imaging system affords an opportunity to monitor activity in defined neurons throughout the fly brain with high sensitivity and excellent spatial resolution.

Courtship is an innate sexually dimorphic behaviour that can be observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate this behaviour are developmentally programmed. In Drosophila, courtship involves a complex yet stereotyped array of dimorphic behaviours that are regulated by Fru(M), a male-specific isoform of the fruitless gene. Fru(M) is expressed in about 2,000 neurons in the fly brain, including three subpopulations of olfactory sensory neurons and projection neurons (PNs). One set of Fru(+) olfactory neurons expresses the odorant receptor Or67d and responds to the male-specific pheromone cis-vaccenyl acetate (cVA). These neurons converge on the DA1 glomerulus in the antennal lobe. In males, activation of Or67d(+) neurons by cVA inhibits courtship of other males, whereas in females their activation promotes receptivity to other males. These observations pose the question of how a single pheromone acting through the same set of sensory neurons can elicit different behaviours in male and female flies. Anatomical or functional dimorphisms in this neural circuit might be responsible for the dimorphic behaviour. We therefore developed a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoactivatable green fluorescent protein. Here we show, using this technique, that the projections from the DA1 glomerulus to the protocerebrum are sexually dimorphic. We observe a male-specific axonal arbor in the lateral horn whose elaboration requires the expression of the transcription factor Fru(M) in DA1 projection neurons and other Fru(+) cells. The observation that cVA activates a sexually dimorphic circuit in the protocerebrum suggests a mechanism by which a single pheromone can elicit different behaviours in males and in females.

1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives ipsilateral axonal projections from superior and inferior noses. This network exhibits an approximately 0.7-Hz intrinsic oscillation in its local field potential (LFP). 2. Intracellular recordings show that the lobe contains at least two classes of neurons with activity phase locked to the oscillation. Neurons in one class produce periodic bursts of spikes, followed by a period of hyperpolarization and subsequently a depolarizing afterpotential. There is a small but significant chance for a second burst to occur during the depolarizing afterpotential; this leads to a double event in the LFP. Bursting neurons constitute approximately 10% of the neurons in the lobe. 3. Neurons in the other class fire infrequently and do not produce periodic bursts of action potentials. However, they receive strong, periodic inhibitory input during every event in the LFP. These nonbursting cells constitute the major fraction of neurons in the lobe. There is a clear correlation between the periodic burst of action potentials in the bursting neurons and the hyperpolarization seen in nonbursting neurons. 4. Optical techniques are used to image the spatially averaged transmembrane potentials in preparations stained with voltage-sensitive dyes. The results of simultaneous optical and electrical measurements show that the major part of the optical signal can be interpreted as a superposition of the intracellular signals arising from the bursting and nonbursting neurons. 5. Successive images of the entire PC lobe show waves of electrical activity that span the width of the lobe and travel its full length along a longitudinal axis. The direction of propagation in the unperturbed lobe is always from the distal to the proximal end. The wavelength varies between preparations but is on the order of the length of the preparation. 6. One-dimensional images along the longitudinal axis of the lobe are used to construct a space-time map of the optical activity, from which we calculate the absolute contribution of bursting and nonbursting neurons to the optical signal. The contribution of the intracellular signals from the two cell types appears to vary systematically across the lobe; bursting cells dominate at middle and proximal locations, and nonbursting cells dominate at distal locations. 7. The direction and form of the waves can be perturbed either by microsurgical manipulation of the preparation or by chemical modulation of its synaptic and neuronal properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigating how information propagates between layers in the olfactory system is an important step toward understanding the olfactory code. Each glomerular output projection neuron (PN) receives two sources of input: the olfactory receptor neurons (ORNs) of the same glomerulus and interneurons that innervate many glomeruli. We therefore asked how these inputs interact to produce PN output. We used receptor gene mutations to silence all of the ORNs innervating a specific glomerulus and recorded PN activity with two-photon calcium imaging and electrophysiology. We found evidence for balanced excitatory and inhibitory synaptic inputs but saw little or no response in the absence of direct ORN input. We next asked whether any transformation of activity occurs at successive layers of the antennal lobe. We found a strong link between PN firing and dendritic calcium elevation, the latter of which is tightly correlated with calcium activity in ORN axons, supporting the idea of glomerular propagation of olfactory information. Finally, we showed that odors are represented by a sparse population of PNs. Together, these results are consistent with the idea that direct receptor input provides the main excitatory drive to PNs, whereas interneurons modulate PN output. Balanced excitatory and inhibitory interneuron input may provide a mechanism to adjust PN sensitivity.antennal lobe ͉ gain modulation ͉ olfaction ͉ smell ͉ sparse code S ensory systems have the difficult task of encoding the identity and intensity of behaviorally relevant stimuli in the environment. The question of how these stimuli are encoded and propagated from the periphery to higher brain centers is central to systems neuroscience. The stereotypic organization of the Drosophila olfactory system and the identification of the odorant receptor genes make the fly an attractive model system in which to study the successive processing of sensory information.Drosophila olfactory receptor neurons (ORNs) in the antennae detect odors and relay neural activity to the antennal lobe in the brain. An adult fly expresses Ϸ50 odorant receptor genes (1-6). In the antennal lobe, axons of ORNs expressing the same receptor gene project with precision to spatially invariant glomeruli (4-7). Most second-order projection neurons (PNs) send dendrites to individual glomeruli in the antennal lobe and send axons to the mushroom body and the lateral horn of the protocerebrum (8-11).Imaging experiments in the insect antennal lobe (12-14) reveal a spatial map of glomerular activity. It has been hypothesized that each glomerulus is a functional unit and that the pattern of glomerular activity encodes the quality of odors (15). However, the Drosophila antennal lobe contains many GABAergic inhibitory interneurons (10,16,17), as well as a group of recently identified cholinergic excitatory interneurons (18). These two classes of interneurons apparently have opposing effects on PNs, and the extent to which they contribute to PN output is unknown. There are two general models for the function...

The temporal dynamics of electrical activity in an olfactory organ, the procerebral lobe of the terrestrial mollusc Limax maximus, is studied. The lobe exhibits intrinsic oscillations in its field potential. Intracellular recordings show that the lobe contains two classes of neurons, both with activity phase-locked to the oscillation. Neurons in one class produce periodic bursts of spikes while those in the other class rre infrequently but receive strong, periodic inhibition whose onset coincides with the burst. The large-scale activity of these neurons is imaged in preparations stained with voltagesensitive dyes. We observe waves of electrical activity that span the width of the lobe and travel its full length along a longitudinal axis. Simultaneous optical and intracellular recordings show that the form of the wave reflects the electrical activity of both dasses of neurons. The application of natural odor stimuli causes the electrical activity along the lobe to transiently switch from the state with propagating waves to one with spatially uniform oscillations. The behavioral and computational relevance of this change in global timing is discussed.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

SUMMARY The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.