TH-GAL4 expressing neurons in the Drosophila brain as visualized with TH-GAL4&3x0003E;UAS-mCD8::GFP. (A–C) Anterior view. (D–F) Posterior view. (A,D) Anti-GFP staining (green) showing cell bodies and processes of TH-GAL4 expressing cells. The brain was counterstained (magenta) with an anti-nc82 antibody. Scale bar, 50 μm. (B,E) The cell bodies were identified in the protocerebrum by thresholding images taken from the brains shown in panels (A) and (D). Dotted lines encircle groups of cells. (C,F) Schematic diagram of (B,E) indicating the name, location and typical number of TH-GAL4 expressing cell clusters in the protocerebrum (Tanaka et al., ). The protocerebral anterior medial (PAM) cluster resides near the anterior face of the brain in the anterior inferiormedial protocerebrum. The protocerebral anterior lateral (PAL) cluster is lateral to the dorsal portion of the vertical lobes in the middle superiorlateral protocerebrum (Figure A). The protocerebral posterior medial (PPM) clusters all reside on the posterior face of the brain in the posterior superiormedial protocerebrum (PPM1), the posterior inferiormedial protocerebrum (PPM2) and the superior posterior slope (PPM3, PPM4). Similarly, the protocerebral posterior lateral (PPL) clusters are also near the posterior face of the brain in the posterior inferiorlateral protocerebrum (PPL1), the posterior lateral protocerebum (PPL2c, PPL4, and PPL2ab), or near the boundary of the superior posterior slope and the posterior lateral protocerebrum (PPL3). The PPL5 neuron is located near the dorsal edge of the lateral horn. The PPD neuron is located at the dorsal surface of the brain, posterior to the tips of the vertical lobes.

Dopaminergic neurons as identified by anti-TH staining compared with those identified by TH-GAL4>UAS-mCD8::GFP expression. Medial is left in all panels except (A) and (E) which shows both hemispheres; dorsal is up in all panels. Panel (D) is an anterior view. All others are viewed from a posterior perspective. Scale bar in all panels: 20 μm. (A) Six clusters of neurons (green) were positive for anti-TH in the posterior protocerebrum. These correspond to the clusters PPM1, PPM2, PPM3, PPL1, PPL2c, and PPL2ab (Figure ). Blue, TOTO-3 staining cell nuclei in the brain. (B) In addition to the six clusters of cells identified in (A), TH-GAL4>UAS-mCD8::GFP expression was also observed in four groups of cells that are TH-negative, labeled as PPD, PPL5, and PPL3 in this panel and PPL4 in panel (H). Green, anti-TH immunostaining. Red, TH-GAL4>UAS-mCD8::GFP, identified with an anti-GFP antibody unless otherwise noted. Blue, TOTO-3. (C) The PPL3 cell is one large neuron lateral to the PPM3 cluster (partially shown) and ventromedial to the PPL1 group (partially shown). (D) High magnification view of the PAM cluster. PAM (Figure ) neurons positive for anti-TH (green) far outnumber those that express TH-GAL4>UAS-mCD8::GFP (magenta). All cells identified with TH-GAL4>UAS-mCD8::GFP were also anti-TH positive. (E) For PPM1, anti-TH stains two small cells, while TH-GAL4 expresses in additional large cells. (F) For PPL2c, anti-TH stains only two cells, while TH-GAL4 typically expresses in three additional cells. (G–I) Anti-TH immunostaining (G) and anti-GFP immunostaining (H) of the PPL1 group. (I) is an overlay of (G) and (H) with TOTO-3 staining. All anti-TH positive cells are also labeled by TH-GAL4. TH-GAL4 may express in one or two additional cells. Arrow: a cell that is strongly labeled by TH-GAL4 but only faintly by anti-TH. Arrowhead: a cell that is modestly labeled by TH-GAL4 but faintly by anti-TH. Open arrowhead: one of the small cells labeled by TH-GAL4 that are scattered in several areas on the surface of the brain. This cell is not counted as a PPL1 neuron. The PPL4 neuron is one large cell ventral to the PPL1 cluster. It is only labeled by TH-GAL4 ((H), also Figure ) but not anti-TH. (J–L) Anti-TH immunostaining (J) and anti-GFP immunostaining (K) of the PPL2ab group. (L) is overlay of (J) and (K) with TOTO-3 staining. The same set of cells is labeled by both anti-TH and TH-GAL4.

Dopaminergic innervation of the mushroom bodies. Scale bar in all panels: 20 μm. (A–C) Innervation of the lobes as viewed from the anterior. (A) TH-GAL4 driven GFP expression in the vertical lobes, the junction (arrow), and the heel (double arrows). Note the sharp vertical boundary of green fluorescence just medial to the junction in the horizontal lobes, giving the impression that dopaminergic innvervation excludes much of the horizontal lobes. Magenta, anti-FasII. Arrowhead, a PAM neuron. Double arrowhead, PAL neurons. Medial is to the left; lateral to the right. (B) TH-GAL4 driven UAS-syt.eGFP, showing dense synaptic puncta (green) within several zones of innervation of the vertical lobes, including the junction (J), the lower stalk (LS), the upper stalk (US), the tip of the α′ lobe (α′) and the tip of the α lobe (α). Magenta, anti-nc82. (C) Robust anti-TH immunostaining of the medial tips of the horizontal lobes (arrow). Medial is to the left; lateral to the right. Arrowhead, PAM neurons. Double arrowhead, PAL neurons. (D) Dorsal view of dopaminergic innervation of the distal peduncle. Green, TH-GAL4 driving UAS-syt.eGFP (green) showing dense puncta in the distal peduncle (arrow), heel (arrowhead) and the central complex (fan-shaped body, FB; ellipsoid body, EB). Magenta, anti-nc82. Posterior (P) is up; anterior (A) is down. (E) Anti-TH signal (green) was observed in the distal peduncle, outlined by c305a-GAL4>UAS-mCD8::GFP (magenta). Anterior view. Medial is to the left; lateral to the right. (F) TH-GAL4>UAS-mCD8::GFP expression (green) in the calyx from a posterior perspective. Magenta, anti-nc82. Cell bodies of TH-GAL4 positive neurons (PPL1 and PPM2) in the posterior cortex can be seen. (G) TH-GAL4 driven UAS-syt.eGFP expression (green) in the calyx from a posterior viewpoint. Arrowhead, protocerebral bridge. Open arrowhead, PPL1 neurons. Magenta, anti-nc82. (H) Anti-TH signal (green) in the calyx. The calyx is shown as the void space surrounded by the densely packed mushroom body cell bodies (magenta, TOTO-3 nuclear stain). Arrow head, protocerebral bridge. Open arrowhead, PPL1 neurons.

Dopaminergic innervation of the horizontal lobes of the mushroom bodies. (A–D) Anti-TH immunostaining in the horizontal lobes. Maxiumum projection images of one hemisphere of frontal sections near the γ, β′ or β lobe. Medial is to the left; lateral to the right. Scale bar in all panels: 20 μm. (A1) Anti-TH immunostaining (green) of a brain from NP1131-GAL4>UAS-mCD8::GFP flies. (A2) Anti-GFP immunostaining (magenta) of the same brain. (A3) Merge of (A1) and (A2). Note the discontinuous signal (double arrowhead) between the junction (J), and the medial segments of the γ lobe. This discontinuity in signal is also apparent in panel (A1). (B1) Anti-TH immunostaining (green) of a brain from c739-GAL4>UAS-mCD8::GFP flies. (B2) Anti-GFP immunostaining (magenta) of the same brain. (B3) Merge of (B1) and (B2). Rather widespread innervation of the β lobe by dopaminergic processes was observed, with a strong horizontal bar of signal across the dorsal aspect of the β lobe. (C1) Anti-TH immunostaining (green) of a brain from c305a-GAL4>UAS-mCD8::GFP flies. (C2) Anti-GFP immunostaining (magenta) of the same brain. (C3) Merge of (C1) and (C2). Note the robust innervation of the medial tip of the β′ lobe by dopaminergic processes. The open arrowheads point to the fibers connecting the ipsi- and contralateral tips of the medial lobes. (D) Anti-TH (green) and anti-GFP immunostaining (magenta) of a brain from OK107-GAL4>UAS-mCD8::GFP flies. (E) Dorsal view of a sample containing three flip-out PAM neurons (arrows) showing that PAM neurons innervate the horizontal lobes. These neurons extend processes posteriorly to the horizontal lobes (arrowheads) and to more posterior areas of the brain. The open arrowhead points to the fibers connecting the ipsi- and contralateral tips of the horizontal lobes. Green, TH-GAL4 flip-out GFP. Magenta, anti-nc82. Posterior (P) is up; anterior (A) is down. (F) Frontal section of a sample containing three flip-out PAM neurons. Only the processes at the tips of the horizontal lobes are shown in this image. The open arrowheads point to the fibers connecting the ipsi- and contralateral tips of the horizontal lobes. Green, TH-GAL4 flip-out GFP. Magenta, anti-FasII. (G) Ventral-anterior view of a flip-out PAM neuron that innervates the tips of the β lobes (arrows). The arrowhead points to the processes that extend to more posterior areas of the brain. Green, TH-GAL4 flip-out GFP. Magenta, anti-FasII. (H) Anterior view of a flip-out PAM neuron (double arrowhead) that innervates the tips of the γ lobes (arrows). Green, TH-GAL4 flip-out GFP. Magenta, anti-nc82.

PPL1 neurons innervate distinct areas of the mushroom body lobes. Green, anti-GFP from TH-GAL4 flip-out clones. Magenta, anti-FasII immunostaining in panel (D), anti-nc82 immunostaining in all others. Grayscale, GFP signal with background removed along with other unrelated structures. Scale bar in all panels: 20 μm. (A) One type of PPL1 neuron innervates the tip of the α lobe. Posterior view. Medial is to the left; lateral to the right. The cell body is located lateral to the calyx (C). (B1,B2) Dorsal view of another PPL1 neuron. Posterior (P) is up; anterior (A) is down. (B1) Cell body is located lateral to the calyx. The primary process runs along the anterior margin of the calyx rather than entering it. There was no indication at this level of resolution that the calyx is innervated by PPL1 neurons. (B2) The tips of the α lobes (α), but not the α′ lobes (arrowheads), are innervated bilaterally by the PPL1 neuron cell body shown in (B1). (C) The entire track of the neuron in panels (B1) and (B2) viewed from a dorsal perspective. Arrow points to the fiber providing innervation to the contralateral α lobe tip. There is considerable branching of the neuron in the dorsal ipsilateral protocerebrum prior to contacting the ipsilateral α lobe. (D) One type of PPL1 neuron bilaterally innervates the tip of α′ lobe. Robust innervation of the tip of the α′ lobe is observable in this PPL1 clone. The arrow points to the fiber connecting the two hemispheres. Only the processes innervating the α′ lobe tip are from the PPL1 neuron clone (above dotted line); other labeled processes are from PPM3 clones in this brain that innervate the fan-shaped body (below dotted line). The cell body for the PPL1 neuron is not visible in this image stack that represents an anterior region of the brain. (E) One type of PPL1 neuron bilaterally innervates the upper stalk of the vertical lobes. The upper stalk is the columnar region of the vertical lobes that is ventral to the tips of the α and α′ lobes (arrowheads). The arrow points to the fiber connecting the two hemispheres. All of the fluorescent processes in the right hemisphere from this viewpoint as well as the fiber link is from the clone bilaterally innervating the upper stalks of the vertical lobes. The fluorescent processes in the left hemisphere ventromedial to the left upper stalk is from another clone in the brain. (F) One type of PPL1 neuron innervates the lower stalk and junction. View from the anterior. Arrowhead points to processes in the anterior inferior medial protocerebrum from the same PPL1 neuron that innervates the junction and lower stalk. (G) Dorsal view of another PPL1 neuron that bilaterally innervates the junction and lower stalk. The PPL1 neuron ramifies broadly throughout the anterior inferior medial protocerebrum en route to the junction and lower stalk. Arrow points to the fiber providing innervation to the contralateral junction and lower stalk. (H) One type of PPL1 neuron bilaterally innervates the heel and distal peduncle. In this projection image from an anterior perspective, the heel obscures the distal peduncle. Arrowhead, processes from the PPL1 neuron innervating the heel, exhibiting broad ramification in the superior medial protocerebrum prior to contacting the ipsilateral heel/distal peduncle. (I) Dorsal view at two levels of another PPL1 neuron innervating the heel. Panel (I1) shows the neurons at the dorsal/ventral level of the tips of the vertical lobes and panel (I2) shows the neuron at the level of the heel. Posterior (P) is up; anterior (A) is down. Panel (I1) shows that the more dorsal processes of this neuron ramify around the anterior surface of the vertical lobes before innervating the heel (double arrowhead in (I2)) and the distal peduncle (arrow in (I2)). (J) Entire track of the neuron in panel (I). The neuron extends a process that branches extensively around the ipsilateral vertical lobe in the anterior protocerebrum, prior to innervating the ipsilateral heel and the distal peduncle. Arrow points to the fiber providing innervation to the contralateral heel and peduncle. (K) Cell body of the PPL1 neuron shown in panel (F). The arrow points to the primary neurite that projects to the mushroom bodies. The arrowhead points to short processes. (L) One type of PPL1 neuron innervates the anterior superior and inferior medial protocerebrum. (L1) Image from a dorsal perspective taken at a level near the tips of the vertical lobes showing broad innervation of the anterior superior medial protocerebrum. (L2) An image from a deeper plane showing the PPL1 neuron branches that surround the horizontal lobes (HL) in the anterior inferior medial protocerebrum. This is more obvious in the right hemisphere as viewed because of the tilt of the specimen. (M) Depiction of the neuron shown in panel (L). The arrow points to the fiber providing for bilateral innervation of the anterior superior and inferior medial protocerebrum. (N) One type of PPL1 neuron extends a process (double arrowhead) to a region posterior to the tips of the vertical lobes (open arrowhead). One secondary branch (arrow) innervates the superior arch (S). Another branch (arrowhead) extends ventrally to a region medial to the vertical lobes and posterior to the horizontal lobes. (O) Dorsal view of another PPL1 neuron that has the same innervation pattern as the one shown in panel (N). Arrow head points to the cell body lateral to the calyx. There is dense innervation of the region posterior to the vertical lobes (V). Only the most dorsal portion of the brain is shown here.

Two types of PPL2ab neuron innervate the calyx of the mushroom body. In contrast to the PPL1 neurons that bilaterally innervate the mushroom body lobes, the innervation of each calyx is unilateral. Scale bar in all panels: 20 μm. (A) Posterior view of one type of PPL2ab neuron. The arrow points to the cell body. The primary neurite from this neuron extends dorsally. One branch innervates the calyx (C). A second branch innervates the adjacent posterior inferior lateral protocerebrum and the ventral portion of the lateral horn (LH). A third branch projects medially and innervates the middle inferior medial protocerebrum. Medial is to the left, lateral to the right. (B) Projection image of the neuron in panel (A) from a lateral point of view. Anterior is to the right, posterior to the left. (C) Posterior view of aother type of PPL2ab neuron (arrow points to the cell body). The primary neurite from this neuron extends dorsally and ramifies to innervate the calyx, the entire lateral horn and the middle superior protocerebrum. Medial is to the left, lateral to the right. (D) Projection image of the neuron in panel (C) from a lateral point of view. Anterior is to the right, posterior to the left. (E) Posterior view of a PPL2ab neuron that does not project to the calyx. This neuron extends a dorso-medial branch that travels along the posterior optical track (POT) and ramifies near the dorsal end of esophagus in both hemispheres, a lateral branch that innervates the lobula (lo), and dorsal processes that innervates broad areas from the posterior lateral protocerebrum, the superior posterior slope, and the middle inferior protocerebrum. (F) Posterior view of another PPL2ab neuron that does not project to the calyx. It innervates the posterior inferior lateral protocerebrum and the posterior superior lateral protocerebrum.

Odor and shock response properties of dopaminergic processes in four subareas of the mushroom body lobes. (A) General response properties shown from one TH-GAL4>UAS-G-CaMP fly. Left, basal fluorescence. Middle, response to 0.1% methylcyclohexnol (MCH) presented to the antennae of the fly. Right, Response to 0.01 mA electric shock presented to the legs. All three images are averages of five successive frames either before (basal) or at the peak responses (MCH and shock). The tips of α lobe and α′ lobe were imaged simultaneously. MCH elicits a robust response in the PPL1 neuron processes that innervate the α′ tip and a much more modest response in those that innervate the tip. Electric shock elicited a robust response in the processes that innervate the α tip and a much more modest response in those that innervate the α′ tip. PPL1 neurons therefore exhibit differential response properties to these stimuli. (B) Group data. (Left panel) At the same current level, the response to electric shock was significantly higher in lower stalk/junction (n = 12) and α tip (n = 6) than in the α′ tip (n = 6) or the upper stalk (n = 12). The response increased in the lower stalk/junction with increasing current (n = 12 at each current level). (Middle panel) Response to MCH was significantly higher in the tip of α′ (n = 31) than in the tip of α (n = 31), the upper stalk (n = 45), or the lower stalk/junction (n = 36). It was also significantly higher in the lower stalk/junction than in the α lobe tip. (Right panel) Responses in the upper stalk increased with increased odor concentration (n = 7 for each point). Data points represent mean ± SEM. *Indicates significant difference (p ≤ 0.0002). Each animal was subjected to only one stimulus at one dose to avoid complications due to prior exposure.

Odor response properties of the dopaminergic neurons innervating the lobes after pairing odor with electric shock. (A) Training paradigms including naïve, forward, backward, CS-only, and US-only. For each fly, odor was presented for 3 s at 5 min prior to conditioning and the calcium responses were monitored. The odor was presented again at 10 min after conditioning and the calcium responses were again monitored. Flies were trained between the Pre and Post presentations of odors and the percentage ratio of the Post/Pre response determined and plotted in panels (B,C). (B) Responses in the upper stalk and lower stalk/junction 10 min after conditioning with 0.3% octanol (OCT) and 0.01 mA electric shock. n = 10–21 for each group. Data represents mean ± SEM. *Indicates significant difference (p < 0.0001). Forward, backward, and CS-only conditioning each reduced the subsequent calcium response to odor in the upper stalk region. There were no significant differences observed due to conditioning in the lower stalk/junction region, although there was a trend for an increased response with forward and US-only conditioning. (C) Responses in the upper stalk, lower stalk/junction, α lobe tip, and α′ lobe tip after conditioning with 0.1% methylcyclohexanol (MCH) and 0.01 mA electric shock. n = 6–12 for each group. Data represents mean ± SEM. *Indicates significant difference (p < 0.005). The response properties due to conditioning with MCH in the upper stalk and lower stalk/junction were similar to those observed with OCT, with decreased responses in the upper stalk after forward and CS-only conditioning, and no significant differences in the lower stalk/junction. The α and α′ lobe tips responded to conditioning in ways that were similar to the upper stalk responses, with reduced responses to forward, backward, or CS-only conditioning that were either significant or were trends that failed to reach significance.