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THE JOURNAL OF COMPARATIVE NEUROLOGY 404:114–126 (1999) Morphology of Feedback Neurons in the Mushroom Body of the Honeybee, Apis mellifera BERND GRÜNEWALD* Institut für Neurobiologie, Freie Universität Berlin, D-14195 Berlin, Germany ABSTRACT The anatomy of ␥-aminobutyric acid (GABA)-immunoreactive, recurrent feedback neurons in the mushroom body (MB) of the honeybee, Apis mellifera, was investigated by using intraneuropilar injections of cobalt ions and light microscopic techniques. Each MB contains approximately 110 GABA-immunoreactive neurons, and approximately 50% of them are feedback neurons, i.e., they connect the MB output regions—the ␣-lobe, -lobe, and pedunculus—with its input regions—the calyces. Their somata are located in the lateral protocerebral lobe, and their primary neurites project medially and bifurcate near the ␣-lobe. In the ␣-lobe feedback neurons form narrow banded, horizontal arborizations in the dorsal and median ␣-lobe; each cell innervates a certain ␣-lobe layer. The neurons form additional branches in the pedunculus and the -lobe. All calycal subcompartments—the lip, collar, and basal ring—are innervated by feedback neurons. However, individual feedback neurons innervate exclusively a certain subcompartment in both the median and lateral calyx. Due to the arrangement of intrinsic Kenyon cells, each calycal subcompartment is connected to its specific, corresponding layer in the ␣-lobe. Feedback neurons interconnect the ␣-lobe and the calyces in either a corresponding or a noncorresponding fashion. With respect to their branching pattern in the ␣-lobe, the basal ring and the collar neuropil receive input from feedback neurons innervating the corresponding dorsal and median ␣-lobe layers. By contrast, the lip region, which receives olfactory antennal input, is innervated by feedback neurons with arborizations in a noncorresponding dorsal ␣-lobe layer. J. Comp. Neurol. 404:114–126, 1999. r 1999 Wiley-Liss, Inc. Indexing terms: insects; neuroanatomy; protocerebrum; cobalt hexamine chloride; inhibitory feedback neurons The mushroom bodies (MB) are central neuropil structures in the protocerebrum of the insect brain. They are involved essentially in learning behavior and memory formation in insects (Apis: Menzel et al., 1974; Erber et al., 1980, 1987; Hammer and Menzel, 1995; Drosophila: Heisenberg et al., 1985; De Belle and Heisenberg, 1994; Connolly et al., 1996). Since their discovery (Dujardin, 1850), their anatomical organization, their connectivity within the insect protocerebrum, and their behavioral development have been described (Kenyon, 1896; Vowles, 1955; Goll, 1967; Steiger, 1967; Pearson, 1971; Schürmann, 1974; Strausfeld, 1976; Weiss, 1981; Mobbs, 1982, 1984; Rybak and Menzel, 1993; Withers et al., 1993; Fahrbach et al., 1995; for review, see Mobbs, 1985; Schürmann, 1987; Menzel et al., 1994). Detailed neuroanatomical studies of individual MB neurons or groups of neurons, however, are rare. This study describes the anatomy of ␥-aminobutyric acid (GABA)-immunoreactive feedback neurons within the MB neuropil of the honeybee, Apis r 1999 WILEY-LISS, INC. mellifera. This neuron group is particularly interesting, because it forms a major component of the insect MB and may be involved in olfactory learning (Erber et al., 1987; Hammer, 1997) and olfactory information processing (Laurent and Naraghi, 1994; Stopfer et al., 1997). Each MB of the worker honeybee consists of some 170,000 densely packed and parallel arranged, intrinsic Kenyon cells (Witthöft, 1967). Their dendritic arborizations build up the median and lateral calyces, which were identified ultrastructurally as the main input regions of the MB (Schürmann, 1974) and are subdivided into the Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: Pf 128/6 and SFB 515/C5. *Correspondence to: Bernd Grünewald, Institut für Neurobiologie, Freie Universität Berlin, Königin-Luise-Str. 28–30, D-14195 Berlin, Germany. E-mail: [email protected] Received 18 March 1998; Revised 25 August 1998; Accepted 28 August 1998 MB FEEDBACK NEURONS IN THE HONEYBEE main subcompartments: lip, collar, and basal ring. Kenyon cell axons form the two stalks of the pedunculus and branch halfway in the pedunculus to form the ␣-lobe and the -lobe, which are the main output regions of the MB (Schürmann, 1974). Each calycal subcompartment receives input from a certain sensory modality. The lip region receives olfactory input, the collar receives visual input, and the basal ring receives mixed chemosensory and visual input (Mobbs, 1982, 1984; Homberg, 1984; Gronenberg, 1986). The topographic organization of the MB indicates that the Kenyon cells maintain this modalityspecific input structure. Their radial arrangement in the concentric calycal subcompartments is transmitted into a stratified orientation in the ␣-lobe, and each calycal subcompartment can be allocated to a discrete band in the ␣-lobe, its corresponding zone (Mobbs, 1982; Rybak, 1994). Therefore, it was concluded that olfactory information is processed mainly in the ventral parts of the ␣-lobe that correspond to the lip region, that visual information is processed mainly in the median ␣-lobe corresponding to the collar region, and that basal ring information is represented in the dorsal layers of the ␣-lobe. Output neurons typically arborize in the ␣-lobe, in the pedunculus, or in the -lobe and connect the MB with other brain areas (Mobbs, 1984; Schürmann, 1987; Rybak and Menzel, 1993). They respond to a large variety of stimuli, and individual neurons often respond to different sensory modalities (Erber, 1978; Homberg and Erber, 1979; Gronenberg, 1987; Mauelshagen, 1993). Similar response patterns were observed in MB output neurons of other species, like crickets and cockroaches (Schildberger, 1981, 1983, 1984; Li and Strausfeld, 1997). This indicates that, at least at the MB output level, the separation between sensory modalities is abolished. Rather, output neurons form large dendritic arborizations and, thus, would be capable of integrating information of different sensory modalities (Mauelshagen, 1993; Rybak and Menzel, 1993). Therefore, the MB has been regarded as a sensory integration center (Erber et al., 1987). Feedback connections are major components in the insect MB (ants: Goll, 1967; crickets: Schürmann, 1973; Schildberger, 1983; flies: Strausfeld, 1976; honeybees: Mobbs, 1982; Bicker et al., 1985; locusts: Leitch and Laurent, 1996; moths: Homberg et al., 1987; orthopterans: Weiss, 1978). In the honeybee MB, they connect the ␣-lobe, the -lobe, and the pedunculus with the ipsilateral median and lateral calyx. The feedback neurons form spines in the lobes and in the pedunculus and form bleb-like structures in the calyces (Mobbs, 1982; Gronenberg, 1987; Rybak and Menzel, 1993), indicating postsynaptic terminals in the lobes and presynaptic endings in the calyces for these neurons. Feedback neurons, thus, would transmit information from the output of the MB back to its input regions. Because they are immunoreactive to GABA (Bicker et al., 1985), feedback neurons probably provide an extrinsic inhibitory feedback loop of the MB. It is still unclear whether feedback neurons interconnect corresponding or noncorresponding zones of the MB. This is interesting, because, if there is information transfer between noncorresponding MB zones through feedback neurons, then this may explain several aspects of the multimodal response behavior of MB output neurons. Therefore, this study investigates the anatomy of feedback neurons in the honeybee MB with special attention to the innervated areas in the ␣-lobe and the calyces. A preliminary account 115 Fig. 1. Schematic diagram of the mushroom body (MB) within the honeybee brain. Only the right brain hemisphere is represented. The extrinsic feedback loop is indicated, and arrows represent the putative flow of information. The corresponding MB zones—lip and ventral ␣-lobe, collar and median ␣-lobe, basal ring and dorsal ␣-lobe—are each indicated by the same gray shading. Arrows indicate calycal regions that presumably are innervated by feedback neurons. ␣, ␣-Lobe;  -lobe; MC, median calyx; LC, lateral calyx; Pe, pedunculus; Li, lip; Co, collar; Br, basal ring; AL, antennal lobe; LO, lobula; ME, medulla; CB, central body; OC, lateral ocellus. Dorsal (d) and lateral (l) directions are indicated. Scale bar ⫽ 100 µm. of this work has appeared in abstract form (Grünewald, 1995). MATERIALS AND METHODS Animals and preparation Worker honeybees, Apis mellifera carnica, were caught between 9:00 a.m. and 10:00 a.m. at the hive entrance with a perspex pyramid hold at a distance of 50–100 cm from the hive entrance. Thus, mostly departing foragers were caught. The age of each bee was estimated by checking external criteria (e.g., hairs on the thorax) and internal morphologic features (e.g., color and number of head glands). Young bees were excluded from further analyses. After immobilization by cooling, the bees were fixed in small metal tubes by a stripe of sticky wax between head and thorax. After recovering, animals were fed with a drop of sucrose solution (25% concentration). To allow for stable and reproducible positioning of the injection pipette, strong movements of the brain were minimized by using the head preparation developed by Mauelshagen (1993). For this, bees were decapitated, the isolated heads were mounted on a perfusion chamber that was supplied continuously Fig. 2. Arborizations of feedback neurons in the ␣-lobe and the pedunculus of the mushroom body. A: Group of several cobalt-stained neurons. Stained profiles are restricted to the dorsal (d) and median ␣-lobe (m). No staining is observed in the ventral portion (v). Arrow indicates the ␣-exit. B: The ␣-lobe is subdivided into six horizontal bands. Dorsal bands 1 and 2 comprise axons of Kenyon cells that form dendrites in the basal ring neuropil of the calyces. Median band 3 corresponds to the collar region, and ventral bands 4–6 correspond to the lip region of the ipsilateral calyx (according to Mobbs, 1982). C: Some feedback neurons form fine, narrow, horizontal arborizations in the dorsal band 2 of the ␣-lobe (arrows). D–F: Serial sections of a group of three feedback neurons photographed at 60 µm (D), 150 µm (E), and 250 µm (F) below the anterior surface of the brain. These neurons innervate median band 3 of the ␣-lobe. Entrance point of feedback neurites into the ␣-lobe is indicated (arrow in D). Profiles that branch radially around the ␣-lobe are also stained (star in D). Arrows in E and F indicate the stained neurite bundle that runs within the protocerebrocalycal tract toward the calyces. Weakly stained profiles in the ventral ␣-lobes (stars within the ␣-lobe in C and D) belong to nonfeedback neurons. Frontal sections. Dorsal (d), median (m), and lateral (l) directions are indicated by arrows in this and all following figures. For other abbreviations, see Figure 1. Scale bars ⫽ 50 µm. MB FEEDBACK NEURONS IN THE HONEYBEE 117 with aerated saline [135 mM NaCl, 5 mM KCl, 10 mM MgCl2, 1.6 mM CaCl2, and 80 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.25]. After stabilizing the brain by carefully dissecting the esophagus with its connecting muscles, the anterior surface of the brain was exposed around the injection site. Injection pipettes (borosilicate glass; 1.0 mm outer diameter, 0.58 mm inner diameter) were pulled with a horizontal micropipette puller (P87; Sutter Instruments, Novato, CA). Pipette tips were filled with an aqueous 4% solution of cobalt-hexaminechloride (Sigma, St. Louis, MO), and the tissue resistance of pipettes ranged between 20 M⍀ and 100 M⍀. Capillaries were positioned in the mediolateral ␣-lobe, which was easy to recognize visually. They were lowered vertically into the brain, and the actual depth of the pipette was monitored by using a potentiometer connected to the fine adjustment of the micromanipulator (Leitz, Wetzlar, Germany). Due to the small tip diameter of pipettes, neural activity could be recorded extracellularly (10 ⫻ preamplification). Thus, the site at which the bundle of feedback neurons entered the ␣-lobe at a depth of 80–120 µm depth was identified, and the injection pipette could be positioned precisely. Cobalt histology and microscopy Cobalt ions were injected iontophoretically for 10–30 minutes with 10–20 nA depolarizing current pulses (200msec duration; 1–3 Hz) applied with a stimulator (SD9; Grass Instrument Co., Quincy, MA) that was connected to an intracellular preamplifier (Simmonds, Cambridge, United Kingdom). Thus, small amounts of cobalt ions were injected into the ␣-lobe to stain only a few cells and to reduce background staining around the injection site. After diffusion of cobalt ions (1–3 hours), brains were excised and placed in phosphate-buffered saline (PBS), pH 7.4, followed by precipitation of cobalt with aqueous 5% ammonium sulfide solution (4 minutes). Subsequently, specimens were fixed in Carnoy’s solution (45 minutes) and block intensified (Bacon and Altman, 1977; Rybak and Menzel, 1993). After dehydration in graded ethanol, the specimens were embedded with propylene oxide in Durcupan (Fluka, Deisenhofen, Germany), and frontal and sagittal sections were cut (20–50 µm, Leitz microtome). Serial sections were reconstructed by using a camera lucida attachment and photographed (Agfapan black-andwhite, 25 ASA film; Gevaert NV, Montsel, Belgium) at a Wild-Polyvar microscope (Leica, Bensheim, Germany). For this study, 28 specimens were evaluated. Each consisted of 1–20 marked neurons; thus, more than 200 neurons were stained and analyzed. According to their branching patterns, the feedback neurons could be classified into different classes. However, estimates on the total numbers of neurons per class were not determined, because such estimates are unreliable and can be misleading. This is due to the fact that, depending on the pipette position, one group of neurons may incorporate more cobalt ions than another or one group may be stained more homogeneously than the other. In addition, in those specimens in which many feedback neurons were stained, the different groups could not be distinguished unambiguously. RESULTS General morphology The feedback neurons belong to a group of cells termed A3 neurons by Rybak and Menzel (1993). By counting Fig. 3. Camera lucida drawing of ␣-lobe projections of feedback neurons. A: Two neurons with tree-like arborizations in dorsal ␣-lobe band 1. B: Group of three neurons that innervate band 3 and form additional branches dorsally around the ␣-lobe. Scale bar ⫽ 100 µm. stained somata and fibers in some specimens (n ⫽ 3), it was estimated that they formed a subpopulation of about 50–60 fibers in the ventral somata cluster of the A3 neurons (A3-v, according to Rybak and Menzel, 1993) out of a total of approximately 110 GABA-immunoreactive MB neurons, which is generally consistent with earlier observations (Bicker et al., 1985; Rybak and Menzel, 1993). The somata of the feedback neurons are located in a cluster ventrally in the anterior lateral protocerebral lobe in a depth of about 80 µm below the anterior brain surface, close to the border of the lobula (Figs. 1, 7, 9). The primary neurite projects dorsally and medially and bifurcates at the dorsolateral margin of the ␣-lobe. One branch loops ventrally and enters the ␣-lobe laterally at a depth of 80–120 µm (␣-exit). This branch gives rise to stratified, horizontal arborizations within the ␣-lobe perpendicular to the Kenyon cell axons and sends several collaterals posteriorly. These run parallel to the Kenyon cells within the ␣-lobe, bifurcate at the posterior border of 118 B. GRÜNEWALD Fig. 4. Arborizations of feedback neurons in the -lobe, pedunculus, and basal ring of the calyces. A,B: Some neurons form processes in the -lobe that are located at the -lobe margins. Two different specimens. C: Most neurons form loose networks of fine profiles throughout the whole -lobe extension. In the distal pedunculus, cobalt staining of feedback neurons is limited to the finger-like structures that are formed by those Kenyon cell profiles that build up the ␣-lobe. The stained pedunculus areas correspond to the median and dorsal ␣-lobe layers. D: Camera lucida drawing of pedunculus and -lobe innervations of a single, cobalt-stained neuron (reconstruction of the projections of the same neuron in the lip region of the calyces is shown in Fig. 5D). Arborizations in the proximal median pedunculus (arrowhead) probably were stained incompletely. E: Group of feedback neurons that innervates a dorsal portion of the basal ring region (arrows). Stained profiles also are seen in the proximal pedunculus (arrowheads). Frontal sections. For abbreviations, see Figure 1. Scale bars ⫽ 50 µm in A–C and E, 100 µm in D. the ␣-lobe (about 200 µm deep), and innervate the -lobe and the pedunculus. The other branch runs outside the MB within the protocerebral-calycal tract (p.c.t.) in a dorsal and posterior direction, bifurcates between the median and lateral calyx, and sends collaterals into the inner ring tracts (i.r.t.) that run between the peduncular stalk and the calyx (Figs. 5, 6). Each feedback neuron innervates both the median and the lateral calyx (see, e.g., Fig. 4E). From the i.r.t., several fine branches project distally into the calycal subcompartment, which is innervated homogeneously (Figs. 5D, 6D). In addition to the branches within the MBs, the feedback neurons form small-diameter branches with few and fine, ring-like arborizations around the dorsal ␣-lobe. Some neurons also possess tree-like arborizations at the bifurcation site between the calyces (Figs. 7, 9). The projection areas of the feedback neurons are restricted to the ipsilateral brain hemisphere: The neurons do not project to the contralateral brain site. Projection areas in the MB compartments ␣ -Lobe. The ␣-lobe is organized in several horizontal layers from dorsal to ventral (bands 1–6; Mobbs, 1982), with each layer comprising the axons of Kenyon cells that MB FEEDBACK NEURONS IN THE HONEYBEE 119 Fig. 5. Cobalt staining of feedback neurons in the calyces. A,B: Two groups of neurons were stained that innervate either the collar (stars) or the basal ring (arrowheads). Photomicrographs of two 50-µm frontal sections of the same specimen (B is 100 µm farther posterior than A). The arrow in A indicates the inner ring tract. The collar is innervated densely by stained profiles throughout its whole proximodistal extension (B). The lip regions are free of any cobalt-stained profiles. C: Another stained neuron that arborizes in the collar region of the calyx. Innervation of the proximal pedunculus by feedback neurons (arrowheads) reaches up to the calyces. Arrow indicates the inner ring tract. D: Camera lucida drawing of the innervation of the collar neuropil by two cobalt-stained feedback neurons with similar morphology. From the inner ring tract (arrowhead), feedback neurons send several collaterals distally that innervate the whole collar neuropil. The arrow indicates the neurite that runs within the protocerebrocalycal tract. For abbreviations, see Figure 1. Scale bars ⫽ 50 µm in A–C, 100 µm in D. form dendrites in a certain calycal subcompartment (Figs. 1, 2B). The feedback neurons arborize exclusively in the dorsal and median bands of the ␣-lobe, namely, dorsal bands 1 and 2 and median band 3 (Fig. 2A). In the anterior-to-posterior ␣-lobe extension, they innervate an area that extends from about 30 µm to about 100 µm below the anterior surface of the ␣-lobe. According to their arborization pattern in the ␣-lobe, three different types of neurons can be distinguished: first, loosely stratified neurons that form tree-like and less dense arborization patterns in band 1 or 3 (Figs. 2D, 3); second, narrowly stratified neurons that form dense bands with a small anterior-to-posterior extension (Fig. 2C) mostly in the narrow dorsal band 2; and third, neurons that form only a few, short, sparsely arborized branches in the median ␣-lobe. These neurons arborize rather extensively in the pedunculus (Figs. 4D, 10). Any individual feedback neuron arborizes in only one particular ␣-lobe band and innervates this band in its complete median-to-lateral extension. Feedback neurons do not innervate ventral bands 4–6 of the ␣-lobe. Pedunculus. Kenyon cell axons in the pedunculus are arranged in a horseshoe-like pattern with concentric rings that correspond to the calycal subcompartments (Howse, 1974). Most feedback neurons form finger-like, dense, and richly arborized projections in the pedunculus in addition to their ␣-lobe arborizations (Figs. 2E,F, 4C, 5C). The innervated pedunculus area reaches from anterior, just 120 B. GRÜNEWALD Fig. 6. Innervation pattern of feedback neurons in the calyces. A–C: Cobalt-stained group of fibers that arborize either in the lip or in the basal ring region (arrowheads). The collar does not contain stained profiles. The arrows indicate the inner ring tract. Serial 50-µm-thick sections were photographed at three planes (B was cut 150 µm posterior to A, and C was cut 200 µm posterior to A). D: Camera lucida drawing of a single neuron that arborizes in the lip and forms bleb-like terminals. It is the same neuron for which the pedunculus and -lobe projections are shown in Figure 4D. The arrowhead indicates the protocerebrocalycal tract, and the arrow indicates the inner ring tract. For abbreviations, see Figure 1. Scale bars ⫽ 50 µm in A–C, 100 µm in D. beneath the ␣-lobe (distal pedunculus), to posterior, at the border to the calyces (proximal pedunculus). In the distal pedunculus, the neurons form short, fine arborizations that are organized in layers (Figs. 2F, 4C) with a mediolateral extension that is limited to the finger-like structures of those Kenyon cell axon bundles that project frontally to form the ␣-lobe. In the pedunculus, the feedback neurons innervate those layers that correspond to the median and dorsal ␣-lobe bands. Thus, feedback neurons may collect information along their course in the MB from a certain Kenyon cell subpopulation. The dorsal pedunculus is divided into a lateral and a median stalk connecting the lobes with the lateral and median calyx. Individual feedback neurons innervate both stalks up to the very dorsoposterior end (Fig. 10), closely adjacent to the calyces.  -Lobe. The -lobe lies perpendicular to the ␣-lobe, is inclined toward the median, and is directly adjacent to the contralateral -lobe. The -lobe is innervated by two different types of feedback neurons: first, neurons that form diffuse, loosely, and less ramified arborizations throughout the whole -lobe, forming a loosely woven network of profiles within the -lobe (Figs. 4C, 7, 9); and second, neurons that innervate the -lobe by a few collaterals, whose rather dense ramifications are restricted to the lateral and median margins of the -lobe (Figs. 4A,B,D, 10). Calyces. The cup-shaped calyces comprise the dendritic terminals of the Kenyon cells, which form the radially arranged subcompartments of the lip, collar, and basal ring. The feedback neurons innervate all subcompartments (Figs. 4E, 5, 6); however, the branching area covered by an individual cell is restricted to one certain subcompartment. Because the feedback neurons innervate both ipsilateral calyces symmetrically, a given subcompartment in the median calyx and its corresponding subcompartment in the lateral calyx are innervated by the same neuron group. MB FEEDBACK NEURONS IN THE HONEYBEE Fig. 7. Camera lucida drawing of three FN2 neurons connecting dorsal ␣-lobe band 1 with the lip region of the calyces. The -lobe is innervated diffusely. These neurons form additional arborizations around the ␣-lobe (star) and beneath the calyces (arrowhead). Arborizations in the pedunculus are omitted. Oe, esophagus. For other abbreviations, see Figure 1. Scale bar ⫽ 100 µm. The collaterals that run within the i.r.t. send several fine branches at regularly distances into the distal parts of the calycal subcompartment (see, e.g., Figs. 5A,C,D, 6D, 8). Due to its asymmetry in size and shape, the lateral calyx is innervated by approximately 20–30 branches, and the median calyx is innervated by 10–15 branches. Each branch ramifies into a fine dendritic tree of about 50–80 µm in diameter. The areas covered by the dendritic trees of adjacent branches of an individual neuron overlap partially (Figs. 5D, 10). Thus, the complete calycal subcompartment is innervated homogeneously by feedback neuron projections. In addition to their arborizations within the MB, some feedback neurons posses fine processes either dorsally around the ␣-lobe (Figs. 2D, 3B, 7, 9), at the bifurcation of the neurons ventrally beneath the calyces (Fig. 7), or at their neurite close to its entrance into the lateral ␣-lobe (not shown, but previously described by Rybak and Menzel, 1993). Different classes of feedback neurons The feedback neurons were classified with respect to their branching areas in the ␣-lobe layers and in the calycal subcompartments. According to the initial question of whether the feedback neurons interconnect corresponding or noncorresponding output and input regions, four 121 different classes of feedback neurons (FN1–FN4) can be distinguished (Table 1). FN1 neurons connect dorsal band 1 of the ␣-lobe with the basal ring of the calyces. In the ␣-lobe, their arborizations are less dense, and additional branches are send into the pedunculus. This group of feedback neurons, which has been described previously (Rybak and Menzel, 1993), interconnects corresponding MB zones. FN2 neurons connect dorsal band 1 of the ␣-lobe with the lip region of the calyces (Fig. 7), and their tree-like arborizations in the ␣-lobe loosely innervate the whole extension of band 1 (Fig. 3A). Some of these neurons form fine processes dorsally around the ␣-lobe and small dendritic trees at the bifurcation point of the main branch between the calyces. FN2 neurons interconnect noncorresponding MB zones. FN3 neurons project into dorsomedian band 2 of the ␣-lobe and into the basal ring of the calyces (Figs. 2C, 4E, 8). They form condensed, narrow, stratified arborizations in the ␣-lobe and also send collaterals into the pedunculus. FN4 neurons connect median band 3 of the ␣-lobe with the collar of the calyces (Figs. 2D,E, 5C, 9). They densely innervate this ␣-lobe layer, project toward the pedunculus, and innervate the -lobe by diffusely arborized processes. FN3 and FN4 neurons connect corresponding MB zones. The results indicate that the basal ring receives inhibitory input from its corresponding ␣-lobe bands 1 and 2 and that the collar receives input from corresponding band 3. By contrast, the lip is innervated by feedback neurons (FN2 neurons) of the noncorresponding ␣-lobe band 1, whose corresponding calycal subcompartment would be the basal ring. In addition to these four feedback neuron classes, another type of feedback neuron exists that connects the pedunculus with the calyces but does not form banded arborizations in the ␣-lobe. These neurons densely innervate the pedunculus from its distal part at the origin of the ␣-lobe to its proximal part at the calyces. In the median ␣-lobe, these neurons form only few, short, and sparsely arborized branches (Figs. 4D, 10). This group, which consists of several different types of neurons connecting the pedunculus with the lip and collar region of the calyces, was not characterized further. DISCUSSION Technical considerations Extracellular, intraneuropilar injections of cobalt ions were employed successfully to reveal the neuronal connectivity of the MB (Strausfeld and Hausen, 1977; Mobbs, 1984; Rybak and Menzel, 1993). The original technique employed (e.g., see Rybak and Menzel, 1993) to study the neural connectivity of the MB of the honeybee was modified in this study to stain fewer neurons, to achieve maximum resolution of fine arborizations, and to inject cobalt solution directly into the bundle of feedback neurons. Cobalt ions were injected iontophoretically, instead of pressure injection, by using pipettes with very small tip diameters. This reduced the pool of injected cobalt ions and probably injured fewer cells, which resulted in staining fewer neurons that were distinguishable even in the area around the injection site. Therefore, reconstructions could be performed at the level of small cell groups with arborizations that could be identified unambiguously. Spontaneous neuronal discharge activity could be recorded extracellularly because of the small tip diameters of the pipettes and the use of an attached intracellular amplifier. Thus, pipette 122 B. GRÜNEWALD Fig. 8. Anatomical reconstruction of two FN3 neurons that connect the narrow band 2 of the dorsal ␣-lobe with the basal ring of the calyces. Pedunculus arborizations are omitted. Innervation of the -lobe was stained very weakly in this specimen and was not reconstructed. For abbreviations, see Figure 1. Scale bar ⫽ 100 µm. positioning was very precise, and the feedback neuron bundle was identified at its entrance into the ␣-lobe, which increased the probability of specifically and exclusively staining feedback neurons. The use of the head preparation strongly reduced movements of the whole brain by eliminating rhythmic hemolymph pumping and muscle contractions. This allowed for stable positioning of the injection pipette for an extended period of time. Organization of the MB Sensory input into the honeybee MB calyces is organized in a modality-specific way. The lip region is innervated by projection neurons from the ipsilateral antennal lobe that transmit chemosensory information into the MB (Mobbs, 1982; Homberg, 1984). The collar is innervated by fiber tracts from the ipsilateral lobula and medulla that transmit visual information into the MB (Mobbs, 1982; Gronenberg, 1986). The basal ring receives mixed sensory input from various parts of the brain (Mobbs, 1982). This inputspecific organization is maintained throughout the MB (Fig. 11) and within the layers of the pedunculus and the ␣-lobe, where information is processed onto MB extrinsic output (Mobbs, 1982; Rybak and Menzel, 1993). Individual output neurons often respond to a large variety of stimuli MB FEEDBACK NEURONS IN THE HONEYBEE 123 Fig. 9. Three FN4 neurons arborize in median ␣-lobe band 3 and connect this layer with its corresponding collar neuropil of the calyces. This type of feedback neuron was stained frequently during this study. The -lobe is innervated diffusely by these neurons. Peduncular arborization are not shown. This neuron type forms additional fine and less arborized, ring-like branches dorsally around the ␣-lobe (arrow). Arrowhead indicates the ␣-exit point. For abbreviations, see Figure 1. Scale bar ⫽ 100 µm. of different modalities in the honeybee MB (Erber, 1978; Homberg and Erber, 1979; Mauelshagen, 1993) and in crickets (Schildberger, 1981, 1983, 1984) and cockroaches (Li and Strausfeld, 1997). This indicates that information from different sensory sources is integrated either at the level of the MB extrinsic neurons or farther upstream within the MB, at the level of the Kenyon cells. The finding that feedback neurons of the honeybee MB connect noncorresponding MB zones, like the dorsal ␣-lobe with the lip region of the calyx, indicates that there is information transfer between noncorresponding zones of the MB. Therefore, the feedback neurons may mediate one suggested function of the MB as a sensory integration center (Erber et al., 1987; Menzel et al., 1994). of different sensory stimuli, including olfactory and gustatory stimuli (Erber, 1978; Homberg and Erber, 1979; Gronenberg, 1987; Grünewald, 1995). Individual neurons often respond to stimuli of different sensory modalities. In the calyces, feedback neurons probably form output synapses onto Kenyon cells. Feedback neurons possess bleb-like structures at their calycal terminals, indicative of presynaptic endings (Gronenberg, 1987; Rybak and Menzel, 1993). Cultured Kenyon cells of honeybees express GABA-mediated chloride currents (Rosenboom et al., 1994). In the MB of Calliphora, an antibody against a GABAA receptor subunit stains Kenyon cells (Brotz et al., 1997). Thus, GABA release from feedback neurons may induce hyperpolarizing currents in postsynaptic Kenyon cells. Furthermore, intracellular recordings from Kenyon cells of Schistocerca MB reveal inhibitory postsynaptic potentials during odor responses that probably are evoked by activity in MB feedback neurons (Laurent and Naraghi, 1994), and GABA-immunoreactive processes were found presynaptic to Kenyon cells in the calyces (Leitch and Laurent, 1996). However, two observations suggest a more complex pattern of innervation. First, Leitch and Laurent Feedback neurons of the MB Feedback neurons form extensive branching areas in the lobes and in the pedunculus, where they collect information from large numbers of Kenyon cells. Hence, their morphology suggests that feedback neurons show rather broad response spectra. Indeed, intracellular recordings have revealed that the neurons respond to a large variety 124 B. GRÜNEWALD Fig. 10. Group of two cobalt-stained neurons that form extensive projections within the pedunculus but that innervate the ␣-lobe very sparsely (arrowheads). These neurons innervate the pedunculus densely and form band-like arborizations at the margins of the -lobe. They innervate the collar region of the calyces. For abbreviations, see Figure 1. Scale bar ⫽ 100 µm. TABLE 1. Classification of Feedback Neurons1 subcompartments. However, the subcompartments are innervated differentially: The collar and basal ring are innervated by feedback neurons from the corresponding median (FN4) and dorsal (FN1 and FN3) ␣-lobe layers, and the lip region is innervated from a noncorresponding dorsal ␣-lobe layer (FN2) that corresponds to the basal ring. This finding suggests that processing of olfactory information within the MB may be different than for example, visual information processing. Feedback neurons do not arborize within ventral ␣-lobe bands 4–6, which would be the corresponding layers of the lip region and the region where antennal information is processed (Mobbs, 1982; Rybak, 1994; Joerges and Faber, personal communication). Therefore, the question arose: Where do the feedback neurons receive olfactory input if not from Kenyon cell axons within the ventral ␣-lobe? Several explanations may be considered. First, although the lip is the main input area for olfactory input, Mobbs (1982) described input from the antennal lobe into the basal ring. Antennal olfactory information, therefore, also may be present in the dorsal ␣-lobe bands. There, feedback neurons may receive olfactory input from intrinsic Kenyon cells of the basal ring. Second, Kenyon cells from various calycal subcompartments may converge onto single ␣-lobe bands (Rybak and Menzel, 1993; Rybak, 1994), indicating that, within an individual layer in the pedunculus or the ␣-lobe, different sensory modalities are processed together (Fig. 11). Band 1 2 3 ␣-Lobe position Dorsal Mediodorsal Median Calyx Basal ring Collar Lip FN12 — — FN42 FN23 — — FN32 — 1According to their ␣-lobe-to-calyx innervation, feedback neurons form corresponding and noncorresponding connections. 2Corresponding connections. 3Noncorresponding connections. (1996) described presynaptic GABA terminals in the lobes and the pedunculi of locust MB. Second, Brotz et al. (1997) found antibody staining against a GABAA receptor also in the axons of Calliphora Kenyon cells. Therefore, Kenyon cells may receive inhibitory input not only in their main input areas, the calyces, but also at their axonal projections. In the calyces, the feedback neurons synapse probably onto large numbers of Kenyon cells, because the dendritic field of a Kenyon cell covers an area about 30 µm in diameter (Mobbs, 1982), whereas any individual feedback neuron homogeneously innervates a complete calycal subcompartment. Thus, great numbers of Kenyon cells may be under the inhibitory control of a single feedback neuron. The whole MB input region receives inhibitory feedback, because feedback neurons innervate all calycal MB FEEDBACK NEURONS IN THE HONEYBEE Fig. 11. Simplified schematic representation of the internal wiring of the honeybee MB. Arrows indicate the flow of information. Inputs into the calycal subcompartments are separated into sensory modalities. This modality-specific organization generally is maintained by the intrinsic Kenyon cells (K1–K5; Mobbs, 1982). In the ␣-lobe, Kenyon cells synapse onto output neurons and feedback neurons (the pedunculus is not shown). Feedback neurons (FN1–FN4) recurrently convey this information back to the calyces. In addition to the noncorresponding information transfer by feedback neurons of the FN2 group (see text), a subgroup of Kenyon cells of the collar region terminates in the ventral ␣-lobe (Rybak, 1994), indicating noncorresponding connections. Some individual Kenyon cells also have dendritic fields in neighboring calycal subcompartments (hatched area between collar and lip). These Kenyon cells may transmit olfactory information into the median ␣-lobe (Rybak and Menzel, 1993). The synaptic organization of the MB, however, may be more complex than indicated in the diagram. dors, Dorsal; vent, ventral; med, medial. For other abbreviations, see Figure 1. Third, the -lobe is a region where feedback neurons might receive olfactory input. At least some output neurons of the -lobe respond in an excitatory manner to olfactory stimuli (Homberg, 1984). Fourth, feedback neurons may get synaptic input outside the MB, because they form branches in the surrounding protocerebrum (Figs. 2D, 7, 9; Rybak and Menzel, 1993). MB output neurons that connect the ventral ␣-lobe with the protocerebrum have overlapping areas with feedback neurons and may provide additional synaptic input (Rybak and Menzel, 1993). Recently, Li and Strausfeld (1997) suggested that certain extrinsic neurons in the MB of cockroaches receive significant input outside the MB. Possible functions of recurrent inhibition in the MB Inhibitory feedback connections are essential neuronal components in a variety of systems. In the insect MB, they 125 may be involved in olfactory information processing. In both the locust (Laurent and Naraghi, 1994) and the honeybee (Stopfer et al., 1997), membrane potentials of Kenyon cells oscillate in response to odor stimulation. This odor-evoked oscillatory activity is generated within the antennal lobe and may be used for olfactory coding (Laurent, 1996). In the MB of the locust, oscillatory activity of Kenyon cells contains an inhibitory component that probably is mediated by inhibitory feedback neurons (Laurent and Naraghi, 1994). Inhibitory feedback loops are a basic feature of brain structures involved in learning-dependent plasticity, like the cerebellum (Thompson and Krupa, 1994) and the hippocampus (Bliss and Collingridge, 1993). During induction of hippocampal long-term potentiation, for example, GABAergic inhibitory input is reduced, resulting in enhanced postsynaptic depolarization (Bliss and Collingridge, 1993). The MB plays a key role in olfactory memory formation in insects (for reviews, see Hammer and Menzel, 1995; Davis, 1996; Menzel and Müller, 1996). Inhibitory feedback neurons may be necessary for the learningdependent response modulations of MB neurons, like the PE1 neuron, which is an individual, identified MB output neuron of the honeybee (Mauelshagen, 1993). The PE1 neuron undergoes a decrement in odor response frequency after associative olfactory learning. Inhibitory feedback neurons may regulate Kenyon cell activity and, thus, may modulate synaptic input to the PE1 neuron. 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