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Emma Wade
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1 SUPPLEMENTARY INFORMATION doi: /nature09554 Supplementary Figure 1: Optical Tracing with New Photoactivatable GFP Variants Reveals Enhanced Labeling of Neuronal Processes We qualitatively compare the fluorescent signal after photoconversion of photoactivatable GFPs in single DA1 PNs. An identical photoactivation protocol was applied to photoconvert the DA1 PN cell body in male flies in which frugal4 directs expression of PA-GFP, C3PA-GFP or SPA-GFP. Photoconversion of the new photoactivatable GFPs yields increased fluorescent signal in DA1 axon terminals in the lateral horn. Similar results were obtained using different insertions of the photoactivatable GFP transgenes, suggesting that the differences in fluorescent intensity we observe in the DA1 axonal processes are likely related to the intrinsic properties of the fluorophores and not due to differences in fluorophore expression levels. Scale bars, 10 μm. Dorsal (D) and medial (M) axes are indicated. w w w. n a t u r e. c o m / NATURE 1

RESEARCH SUPPLEMENTARY INFORMATION b c Supplementary Figure 2: Dimorphic Lateral Horn Neurons Project to the Lateral Triangle and SMP Tract a, The direct photoactivation of the male-specific DC1

2 RESEARCH SUPPLEMENTARY INFORMATION b c Supplementary Figure 2: Dimorphic Lateral Horn Neurons Project to the Lateral Triangle and SMP Tract a, The direct photoactivation of the male-specific DC1 neurons reveals processes that interdigitate with the axons of DA1 PNs in the lateral horn (LH). DC1 neurons also extend processes into the lateral triangle (arrow) and the SMP tract (arrowhead). b, The male-specific DC2 cluster, labeled through direct photoactivation, innervates the lateral triangle (arrow). c, Direct photoactivation of the LC1 cluster in males (left) reveals rich projections into the lateral horn where they interdigitate with the DA1 axonal arbor. In males, LC1 processes also extend into the lateral triangle (arrow) and the SMP tract (arrowhead). In females (right), direct photoactivation of the LC1 cluster reveals a smaller number of neurons than is observed in the male. LC1 neurons in the female are also anatomically dimorphic and exhibit more restricted processes that do not project contralaterally. d, The LC2 cluster labeled by direct photoactivation is not overtly dimorphic and exhibits more restricyed projections in both males (left) and females (right). In all images, neurons express either C3PA-GFP or SPA-GFP directed by frugal4. The DA1 PNs were labeled by photoactivation of the DA1 glomerulus. Schematics illustrate the approximate location of photoactivated neurons and their projections within the brain. The basal fluorescence originating from the cell bodies and projections of nearby neurons has been masked for clarity. Scale bar, 20 μm. Dorsal (D) and medial (M) axes are indicated. 2 WWW. NATURE. COM / NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3: Fru+ Clusters Are the Major Recipients of DA1 Input Photoactivation of DA1 axon terminals in flies in which photoactivatable GFP expression

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3: Fru+ Clusters Are the Major Recipients of DA1 Input Photoactivation of DA1 axon terminals in flies in which photoactivatable GFP expression is restricted to Fru+ neurons (left) or is ubiquitous (right) reveals a similar constellation of labeled neuronal clusters. C3PA-GFP expression was driven in Fru+ neurons by fru GAL4 or panneuronally by elav-gal4. In both examples, the DA1 glomerulus was first photoactivated to reveal DA1 axonal projections in the lateral horn. Photoactivation of DA1 axon terminals when C3PA-GFP is ubiquitously expressed labels the Fru+ lateral horn clusters identified in this study as well as a small number of additional Fru- neurons. Projections from photoactivated lateral horn neurons into the SMP tract are labeled with an arrowhead. Note that photoactivation of the DA1 glomerulus in flies that express C3PA-GFP ubiquitously labels both DA1 PNs and a population of Fru- interneurons whose cell bodies surround the antennal lobe. Scale bar, 20 μm. Dorsal (D) and medial (M) axes are indicated. 3

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4: LC1 Neurons Produce the Inhibitory Neurotransmitter GABA Immunostaining of a male fly brain in which fru GAL4 directs expression of

4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4: LC1 Neurons Produce the Inhibitory Neurotransmitter GABA Immunostaining of a male fly brain in which fru GAL4 directs expression of CD8-GFP (green) using an antibody recognizing the neurotransmitter GABA (red). LC1 neurons form part of a larger cluster of GABAergic neurons in the lateral protocerebrum 4

SUPPLEMENTARY INFORMATION RESEARCH a b Supplementary Figure 5: The DC1 Cluster is Composed of Multiple Anatomic and Functional Classes of Neurons a, Photoactivation of a single DC1 cell body labels

5 SUPPLEMENTARY INFORMATION RESEARCH a b Supplementary Figure 5: The DC1 Cluster is Composed of Multiple Anatomic and Functional Classes of Neurons a, Photoactivation of a single DC1 cell body labels individual projection patterns within the protocerebrum. Examination of the photoactivated processes from single DC1 neurons reveals that the cluster is composed of several anatomic classes of neurons characterized by distinct branch patterns within the protocerebrum. The approximate location of the DA1 axonal arbor (determined by subsequent photoactivation of the DA1 glomerulus) is indicated with a white arrow. One class of DC1 neurons enters the lateral horn ventrally and gives rise to dense terminations that intermingle with ventral DA1 axon branches (left.) A second class of DC1 neurons exhibits more limited overlap with DA1 neurons and also directs processes more ventrally out of the lateral horn (middle.) A third class of DC1 neurons extends ventrally and bypasses the lateral horn entirely (right.) Note that these images focus on the DC1 projections within the protocerebrum and do not show the full extent of contralateral projections into the SMP tract. b, Different anatomic classes of DC1 neurons represent different functional classes. We performed optical recordings of neurons in the DC1 cluster expressing GCaMP3 in response to stimulation of the DA1 glomerulus. Neurons responsive or non-responsive to DA1 stimulation were identified and subsequently labeled by electroporation with Texas Red dextran to examine the disposition of the DC1 processes in the lateral horn. We observe that a responsive DC1 neuron (arrowhead, left panel) belongs to the anatomic class that sends rich projections into the lateral horn (indicated with an arrow). A non-responsive DC1 neuron (arrowhead, right panel) belongs to the anatomic class that bypasses the lateral horn (arrow) and extends processes into more ventral non-olfactory brain regions. Scale bars, 20 μm. Medial (M) and dorsal (D) axes are indicated. 5

RESEARCH SUPPLEMENTARY INFORMATION DC1 lobula plate DC1 SOG Supplementary Figure 6: Neuronal Processes Interdigitate with Non-Olfactory Sensory Inputs DC1 dendrites interdigitate with DA1 PNs within

In initial experiments, we photoactivated the ventral DC1 dendrites to identify other classes of neurons that may form presynaptic connections with DC1 neurons.

6 RESEARCH SUPPLEMENTARY INFORMATION DC1 lobula plate DC1 SOG Supplementary Figure 6: Neuronal Processes Interdigitate with Non-Olfactory Sensory Inputs DC1 dendrites interdigitate with DA1 PNs within the lateral horn (LH) but also extend ventral branches into non-olfactory areas of the protocerebrum. In initial experiments, we photoactivated the ventral DC1 dendrites to identify other classes of neurons that may form presynaptic connections with DC1 neurons. Photoactivation of the DC1 ventral dendrites in flies that express C3PA-GFP or SPA-GFP directed by fru GAL4 identifies a rich collection of neurons (data not shown) including a population that innervates the lobula plate (a) and a population that innervates the SOG (b). a, Direct photoactivation of the population of lobula plate neurons (left) followed by direct photoactivation of the DC1 cluster (right), reveals that the processes of these visual projection neurons interdigitate with DC1 ventral dendrites. b, Direct photoactivation of an ascending neuron whose cell body resides in the ventral nerve cord (not shown) reveals projections in the brain that terminate within the SOG and the ventral protocerebrum. Subsequent direct photoactivation of the DC1 cluster reveals the overlap of ventral DC1 dendrites with the presumptive axons from this gustatory projection neuron. The background fluorescence originating from the cell bodies and projections of nearby non-photoactivated neurons has been masked for clarity. Scale bars, 20 μm. Dorsal (D) and medial (M) axes are indicated. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 7: An Odorant Mix that Fails to Activate DC1 Neurons is a Potent Stimulus for non-da1 PNs PNs were targeted for loose patch recordings in an intact fly preparation and the antenna was stimulated with a cocktail of odorants. The odorant mix was a 1:1000 dilution of each of 10 odorants (Benzaldehyde, Cyclohexanol, Geranyl acetate, 2-Heptanone, 1-Hexanol, Ethyl 3-hydroxybuterate, Isoamyl acetate, 3-Octanone, Methyl salicylate, Proprionic acid.) Raster plot (left) of the responses of 4 DA1 PNs (red) and 21 non-da1 PNs that innervate other glomeruli (black) elicited by a 1-sec application of the odorant mix reveals that the vast majority of PNs are activated by this olfactory stimulus. Peristimulus time histogram (average ± SEM, right) for DA1 PNs (red) or non-da1 PNs (black) indicates that although the odorant mix fails to activate DA1 PNs, it elicits high-frequency responses in PNs that innervate other glomeruli. PNs that innervate glomeruli other than DA 1 were identified as those that express GH146-Gal4 but reside in the cluster of PNs that lies dorsal to the antennal lobe. 7

Stereotyped connectivity and computations in higher order olfactory neurons Mehmet Fisek and Rachel I. Wilson

Stereotyped connectivity and computations in higher order olfactory neurons Mehmet Fisek and Rachel I. Wilson Supplementary Tale 1: Genotypes The genotypes used y figure are as follows: Figure 1a Figure