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The Journal of Comparative Neurology 512:433– 452 (2009)

Tyramine as an Independent Transmitter and a Precursor of Octopamine in the Locust Central Nervous System: An Immunocytochemical Study ¨ GER NATALIA L. KONONENKO,* HEIKE WOLFENBERG, AND HANS-JOACHIM PFLU Freie Universita¨t Berlin, Institute of Biology, Neurobiology, D-14195 Berlin, Germany

ABSTRACT Octopamine and its precursor tyramine are biogenic amines that are found ubiquitously in insects, playing independent but opposite neuromodulatory roles in a wide spectrum of behaviors, ranging from locomotion and aggression to learning and memory. We used recently available antibodies to octopamine and tyramine to label the distribution of immunoreactive profiles in the brain and ventral nerve cord of the locust. In the brain and all ventral cord ganglia all known octopaminergic neurons were labeled with both the tyramine and octopamine antisera. In the brain the subesophageal ganglion and all fused abdominal ganglia we found somata that were only labeled by the tyramine antibody. Some prominent architectural features of the brain, like the protocerebral bridge, the central body, and associated neuro-

pils, also contain intensely labeled tyramine-immunoreactive fibers. In addition, tyraminergic fibers occur in all ganglia of the ventral cord. For known octopaminergic neurons of the thoracic ganglia, octopamine-immunoreactivity was confined to the cell body and to the varicosities or boutons, whereas fiber processes always expressed tyramine-immunoreactivity. The distribution of the tyramine and octopamine content within these neurons turned out to be dependent on how the animal was handled before fixation for immunocytochemistry. We conclude that tyramine is an independent transmitter in locusts, and that in octopaminergic neurons the ratio between octopamine and its precursor tyramine is highly dynamic. J. Comp. Neurol. 512:433– 452, 2009. © 2008 Wiley-Liss, Inc.

Indexing terms: insect; behavior; biogenic amines; neurotransmitter; neuromodulation; neuron compartmentalization

Octopamine is a biogenic amine first discovered in the salivary gland of Octopus (Erspamer and Boretti, 1951) which is widely distributed among invertebrate phyla. It has been shown to play a role in a diverse range of behaviors (Roeder, 1999; Pflu¨ger and Stevenson, 2005). Its functions range from mediating appetitive learning in Drosophila (Schwaerzel et al., 2003) and honey bees (Hammer, 1993; Hammer and Menzel, 1998) to modulating central motor networks (locust flight, Stevenson and Kutsch, 1988; crustacean stomatogastric ganglion, Flamm and Harris-Warrick, 1986a,b) and neuromuscular transmission (locust, Evans and O’Shea, 1977) as well as regulating muscular energy metabolism during motor behavior (locust, Mentel et al., 2003). It was previously thought that octopamine was solely responsible for mediating these behavioral effects. However, recent studies have shown that tyramine, the monoamine from which octopamine is synthesized, may play its own independent role as a transmitter in the nervous system. The evidence for this comes mainly from mutants like the tyramine-␤-hydroxylase (T␤H) mutant of Drosophila, which cannot synthesize octopamine and exhibits a severe locomotion phenotype (Saraswati et al., 2004; Fox et al., 2006; see also Hardie et al., 2007) as well as severe deficits in flight performance (Brembs et al., 2007), or from Caeno-

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rhabditis T␤H mutants which have severe phenotypes in egglaying, reversal behavior, and head movements in response to touch (Alkema et al., 2005). A number of studies have suggested that octopamine and tyramine cause opposite behavioral effects in honey bee (Fussnecker et al., 2006) and Drosophila larvae (Kutsukake et al., 2000), which was also supported by the results of rescue experiments on T␤Hmutants of Drosophila (Brembs et al., 2007). Tyraminergic neurons have been identified in the Drosophila brain (Nagaya et al., 2002) and tyraminergic fibers innervate the locust oviduct where tyramine acts on myogenic contractions (Donini

Additional Supporting Information may be found in the online version of this article. Grant sponsor: Alexander von Humboldt Foundation, Bonn, Germany (fellowship to N.L.K.); Grant sponsor: German Science Foundation, DFG; Grant number: GK 837 (Functional Insect Science, to H.J.P.). *Current address and correspondence to: Natalia L. Kononenko, Centre for the Biology of Memory, NTNU, Olav Kyrres gate 9, NO-7489 Trondheim, Norway. E-mail: [email protected] Received 19 September 2007; Revised 30 May 2008; Accepted 15 October 2008 DOI 10.1002/cne.21911 Published online in Wiley InterScience (www.interscience.wiley.com).

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and Lange, 2004). Additionally, specific tyramine receptors have been cloned (Drosophila, Saudou et al., 1990; honey bee, Blenau et al., 2000; Bombyx, Ohta et al., 2003; von NikischRosenegk et al., 1996; locust, VandenBroeck et al., 1995) and, correspondingly, specific uptake systems exist for octopamine (Roeder and Gewecke, 1990; Scavone et al., 1994) and, most likely, tyramine (Hiripi et al., 1994) with an octopamine transporter cloned in the cabbage looper, Trichoplusia ni (Malutan et al., 2002). Thus, it has become increasingly clear that these behavioral effects are not modulated by one monoamine, but rather are caused by the concerted action of two amines that may have opposing functions. This might even hold for mammals and pathologies such as Parkinson’s disease, in which both dopamine and serotonin may be involved (Scholtissen et al., 2006). In addition, both octopamine and tyramine occur in mammals in nanomolar quantities and are considered trace amines, although there is still considerable debate as to their functional roles (Berry, 2004; Zucchi et al., 2006; Lewin, 2006). Octopamine is synthesized in insects from tyramine, and as tyramine is now considered an independent transmitter, the question arises as to whether populations of exclusive tyraminergic neurons exist in insects. Also, within which compartments of an octopaminergic neuron can tyramine be detected, and is tyramine released in addition to octopamine by some neurons? It is very fortunate that the distribution of octopaminergic neurons in insects is well known and has been studied in both the brain and in the ganglia of the ventral nerve cord (locust, Konings et al., 1988; Braünig, 1991; Stevenson et al., 1992, 1994; cockroach, Eckert et al., 1992; Sinakevitch et al., 2005; tobacco sphinx moth, Dacks et al., 2005; honey bee, Kreissl et al., 1994; Sinakevitch et al., 2005; flies, Monastirioti et al., 1995; Sinakevitch and Strausfeld, 2006; for review, see Stevenson and Spo¨rhase-Eichmann, 1995; Burrows, 1996; Braünig and Pflu¨ger, 2001). Most octopaminergic neurons of the ventral cord ganglia belong to the class of dorsal or ventral unpaired median (DUM- or VUM-) neurons, which are efferent neurons and innervate muscles, glands, and sensory organs

(Hoyle, 1978; Watson, 1984; for review, see Braünig and Pflu¨ger, 2001). These neurons are activated (or inhibited) in parallel to various motor patterns (Burrows and Pflu¨ger, 1995; Baudoux et al., 1998; Duch and Pflu¨ger, 1999; Johnston et al., 1999; Mentel et al., 2008), and are known to exert important modulating effects on the peripheral target tissues. Consequently, they are the prime candidates for studying the release of closely related transmitters. The aim of this study is threefold: First, to describe the distribution of tyraminergic neuron populations within the locust brain and ventral cord ganglia; Second, to describe the distribution of tyramine and octopamine within a wellcharacterized class of dorsal unpaired median neurons (DUM neurons), and thus present findings on the formation of internal compartments. This will be achieved by double-labeling thoracic ganglia with antibodies specific against tyramine and octopamine; Third, to test the hypothesis that the relative levels of octopamine and its precursor tyramine, in wellknown and -characterized neurons, are dynamic and depend on the previous experience of the animal. Octopamine is known to be released when novel or arousing stimuli occur (Orchard and Lange, 1983; Adamo et al., 1995), for example, the delivery of a stressful stimulus. Does this affect the ratio of octopamine to its precursor tyramine?

MATERIALS AND METHODS Animals Brains and ventral cord ganglia of adult Schistocerca gregaria of either sex were examined; the animals were taken from our own crowded colony, maintained at a constant light regime (12:12/h light:dark) at ⬇30°C.

Preparation of nervous tissue Brains and ganglia were dissected on ice in fixative solution (6.25% glutaraldehyde, 75% picric acid, 5% (glacial) acetic acid, 1% sodium metabisulfite (SMB)) and fixed for 3 hours. Then they were washed 2 ⴛ 5 minutes each in 1% SMB in

Abbreviations 1.OC ACa aL AL AMMC ant bL Ca CBU CBL DC DMT dors DUM lat la La LAL LH Lo lVAC med md Me Me,i, Me,o

First optical chiasm Anterior calyx ␣ lobe of the mushroom body Antennal lobe Antennal mechanosensory and motor center Anterior ␤ lobe of the mushroom body Calyx Upper division of the central body Lower division of the central body Deutocerebrum Dorsal median tract Dorsal Dorsal unpaired median Lateral Labial (neuromere of the SEG) Lamina Lateral accessory lobe Lateral horn of the protocerebrum Lobula Lateral VAC Median Mandibular (neuromere of the SEG) Medulla Inner and outer layer of the medulla

mPC mx mVAC MVT N n OA OA-l-ir P PB PC pPC Pi PM post SMP SEG TA TA-l-ir T␤H TC VAC vent VMT VUM

Median protocerebrum Maxillar (neuromere of the SEG) Median VAC Median ventral tract Noduli Nucleus Octopamine Octopamine-like-immunoreactivity Peduncle Protocerebral bridge Protocerebrum Posterior protocerebrum Pars intercerebralis Protocerebral medulla cells Posterior Superior median protocerebrum Subesophageal ganglion Tyramine Tyramine-like-immunoreactivity Tyramine-␤-hydroxylase Tritocerebrum Ventral association center Ventral Ventral median tract Ventral unpaired median

The Journal of Comparative Neurology TYRAMINE AND OCTOPAMINE IN LOCUST distilled water, dehydrated in an ascending ethanol series (50%, 70%, 90%, 100% for 10 minutes each), permeabilized in a 1:1 mixture of 100% ethanol and methylsalicylate for 5 minutes, and rehydrated in a descending ethanol series (100%, 90%, 70%, 50%, 10 minutes each), where rehydration in 70% and 50% ethanol was done at 37°C. Specimens were then washed 6 ⴛ 5 minutes each in warm (37°C) 0.1 M TrisHCl-buffer containing 0.45% SMB (Tris-HCl-SMB, pH 7.6) and subsequently treated with enzyme (0.1% hyaluronidase / 0.1% collagenase-dispase in 0.05 M Tris-buffer pH 7.6) for 30 minutes at 37°C.

Immunocytochemistry on thick vibratome sections After enzymatic treatment the ganglia were washed 1 ⴛ 15 minutes in 0.1 M Tris-HCl-SMB, pH 7.6, and subsequently embedded in a gelatin/albumin mixture (4.8% gelatin from porcine skin, Type A, G 2500, Sigma, St. Louis, MO; and 12% albumin from chicken egg white, Grade II, A 5253, Sigma, in demineralized water), oriented transversally, horizontally, or sagittally, and postfixed overnight in 10% paraformaldehyde in 0.1 M Tris-HCl-SMB, pH 7.6. Thick sections of the brains and thoracic ganglia (50 ␮m, 70 ␮m, or 120 ␮m) were cut with a vibrating blade microtome (VT 1000S, Leica, Wetzlar, Germany), washed in 0.1 M Tris-HCl-SMB, pH 7.6, for 15 minutes, and then for 10 minutes in 1% Na-borohydride (Sigma) in 0.1M Tris-HCl-SMB, pH 7.6. After this, sections were washed in 1% Triton X-100 in 0.1 M Tris-HCl-SMB, pH 7.6, and preincubated with 1% Triton X-100 in 0.1 M Tris-HCl containing 10% normal goat serum (NGS, ISN Biomedicals, Meckenheim, Germany) overnight at 4°C. Incubation with primary antibodies lasted for up to 120 hours at 7°C. A mix of anti-octopamine and antityramine antibodies was used to determine whether octopaminergic cell bodies also contain tyramine. The primary antioctopamine antibody (monoclonal anti-mouse, immunogen: octopamine-thyroglobulin-glutaraldehyde conjugate, Bioscience, Jena, Germany; see also Dacks et al., 2005) was diluted at 1:1,000 in the antisera diluent (1% Triton X-100, 0.05% sodium azide (NaN3) in 0.1 M Tris-HCl). The primary anti-tyramine antibody (polyclonal rabbit anti-p-tyramine, AB 124, lot 0606032868, immunogen: p-tyramine-glutaraldehydeN-alpha-acetyl-L-lysine-N-methylamide, Chemicon-Millipore, Bedford, MA) was diluted at 1:500 in the same antisera diluent. Next, sections were washed 6 ⴛ 20 minutes each in 1% Triton X-100 in 0.1 M Tris-HCl, and subsequently incubated in a mixture of Cy2, Cy3, or Cy5 conjugated secondary antibodies (goat anti-mouse conjugated to Cy3 or Cy5, 1:200, and goat anti-rabbit conjugated to Cy2, Dianova, Pinole, CA; 1:300) for 12 hours using standard immunofluorescence techniques.

Immunocytochemistry on wholemount preparations For immunocytochemistry on wholemount preparations ganglia were washed after enzyme treatment for 1 ⴛ 15 minutes in 0.1 M Tris-HCl-SMB, pH 7.6, and then for 10 minutes in 1% Na-borhydride in 0.1 M Tris-HCl-SMB, pH 7.6. After this, ganglia were washed in 3% Triton X-100 in 0.1 Tris-HCl-SMB, pH 7.6, and preincubated with 3% Triton X-100 in 0.1 M Tris-HCl containing 3% NGS (ISN Biomedicals) overnight at 4°C. Incubation with primary antibodies lasted for up to 120 hours at 7°C. The primary anti-octopamine antibody (monoclonal mouse anti-octopamine, Jena, Bioscience) was diluted

435 1:500 in the antisera diluent (1% Triton X-100, 0.05% sodium azide (NaN3), containing 1% NGS in 0.1 M Tris-HCl). The primary anti-tyramine antibody (polyclonal anti-rabbit, Chemicon, Temecula, CA) was diluted 1:500 in the same antisera diluent. Next, ganglia were washed 6 ⴛ 20 minutes each in 1% TX-100 in 0.1 M Tris-HCl, and subsequently incubated in a mixture of Cy2, Cy3, or Cy5 conjugated secondary antibodies (goat anti-mouse conjugated to Cy3 or Cy5, 1:200, and goat anti-rabbit conjugated to Cy2, Dianova, 1:300) for 12 hours using standard immunofluorescence techniques.

Experience-dependent tyramine/octopamine immunocytochemistry To study a presumed dynamic in the content of the biogenic amine transmitters tyramine and octopamine, we performed immunocytochemistry on differently handled locusts. The control group (total n ⴝ 6) was kept undisturbed and either cooled in the refrigerator (n ⴝ 3) or kept at the same temperatures as the stressed group between 3 and 12 hours (n ⴝ 3) before quick dissection. The handled group (stressed) (total n ⴝ 6) was exposed to a number of stressful stimuli including hot air currents, moving visual stimuli, or loud noises (for example, rattling of keys) for up to 15 minutes before quickly dissecting them (3 times ⴛ 2 animals treated, n ⴝ 6). Immediately after opening the cuticle cold fixative was added to the preparation (see above). In the control group no differences in staining that would indicate effects of the different temperatures were seen.

Determination of crossreactivity To establish the specificity of the tyramine antibody and its crossreactivity with octopamine in the tissue, the primary tyramine antiserum was omitted, or sections were immunostained with the primary antiserum that earlier had been subjected to a preadsorption treatment with 1) D,L-octopamine hydrochloride (Sigma) in a concentration of 0.1 mM, 1 mM, and 10 mM, and 2) tyramine hydrochloride (Sigma) in a concentration of 0.1 mM, 1 mM, and 10 mM, or 3) 1 mM dopamine-hydrochloride (Sigma). For preadsorption the TA antiserum, diluted at working dilution in 1% Triton X-100 in 0.1 M Tris-HCl, pH 7.6, was incubated with the preadsorption component on a shaking table overnight at 4°C. Immunoreactive labeling with the antibody against tyramine in the locust central nervous system (CNS) is specific and repeatable from one animal to the next, revealing somata, neurites, and processes (Fig. 2a– d). After preadsorption with tyramine hydrochloride (1 mM), specific labeling is abolished. However, preadsorption with D,L-octopamine hydrochloride (1 mM) as well as dopamine-hydrochloride (1 mM) resulted in a retained pattern of tyramine-immunoreactivity, although the staining intensity appeared slightly weaker. However, as this antibody was previously described to exhibit crossreactivity to dopamine in the leech (Crisp et al., 2002), some further tests using the ELISA technique (enzyme-linked immunosorbent assay, courtesy Uli Mu¨ller, Saarbru¨cken, Suppl. Fig. 1) and the dotblot technique (courtesy Wolfgang Blenau, Potsdam, Suppl. Fig. 2) were applied. The ELISA study revealed some crossreactivity to dopamine, which was, however, not confirmed in the dot-blot study. Therefore, a well-described antiserum against dopamine was used (Boer et al., 1984; Meek et al., 1989) on vibratome sections and compared with similar vibratome sections in which the tyramine-antibody was applied



(Suppl. Fig. 3). These comparisons confirmed that none of the dopaminergic neurons or profiles were labeled by the tyramine antibody. This is consistent with the published literature on the distribution of dopaminergic neurons and processes within the CNS (Scha¨fer and Rehder, 1989; Ho¨rner, 1999; Wendt and Homberg, 1992). This demonstrates that this antibody does not reveal any crossreactivity to dopamine, at least within the biological tissue of the locusts. The following descriptions are based on the analyses of 18 brains and 21 ventral nerve cord ganglia (6 subesophageal ganglia (SEG), 12 thoracic ganglia, 3 abdominal ganglia).

Image acquisition All vibratome sections and wholemount preparations were imaged with a laser-scanning confocal microscope (Leica TCS SP2, Leica, Germany). Series composed of 10 – 45 optical sections (z-increment 8 ␮m) were scanned at a resolution of 1024 ⴛ 1024 in 8-bit depth by using HC PL 10ⴛ/0.40 imm or HC PL 20ⴛ/0.70 imm/corr oil apochromat objectives. Two different laser lines (argon/krypton laser (488 nm) and Helium/ Neon laser (633 nm)) were used to acquire image stacks of double-labeled preparations. For high-resolution 3D analysis small sampling areas on the dendrites and cell bodies were scanned in all channels with the high-resolution immersion objective lens (HC ⴛ PL 63ⴛ, immersion oil, NA 1.32, zoom 2.4, pinhole 1.00 arbitrary units (Airy disk 100%), z-increment 300 nm (d ⴝ 1.22 ␭ / (NAobjⴙNAcdn), approximately 80 images at 1024 ⴛ 1024 pixels, 8-bit sampling, sequential frameby-frame mode). Postacquisition, all image files were processed with Amira 4.1.2 software (TGS, Mercury Computer Systems, Merignac, France). Images were median filtered to reduce photomultiplier tube noise. Only minor adjustments of contrast and gamma of the images were made to make dim features visible in the figures presented. For colocalization analysis of two fluorochromes, recorded in two separate channels, the “correlation plot” module (Amira 4.1.2 ) was applied. This module computes a 2D correlation histogram of two uniform scalar fields (e.g., image stacks) and accordingly detects regions of correlated intensity in two images of the same object. The result of correlation analysis is applied for an image segmentation where the regions of high correlation are represented in a third color (e.g., magenta). Finally, snapshots were arranged into figures with Corel Draw 12 (Ottawa, Canada) and Adobe Photoshop 8 (CS2) (San Jose, CA).

Terminology Due to the fact that, in addition to octopamine (OA), all octopaminergic single cells or cell clusters also possess immunoreactivity to tyramine (TA), they are referred to here as clusters OA1-4/TA. Single cell bodies, and their cluster showing immunoreactivity only to tyramine antibody, are called soma/cluster TA1-10. A statement like “5 to 10 pairs of cell bodies” means that on each side of the brain 5 to 10 single cell bodies exist. The terminology for brain and ganglionic structures is based on the nomenclature of Tyrer and Gregory (1982), Pflu¨ger et al. (1988), and Homberg (1994). Sectional planes and definitions of brain structures are shown in Figure 1. In brief, the brain is assumed to be aligned to the ventral nerve cord and, therefore, all axes of the locust nervous system are defined according to the body axis.

RESULTS Tyramine- and octopamine-like-immunoreactive neurons in the locust brain In the locust brain we found that tyramine-likeimmunoreactivity (TA-l-ir) is expressed by six bilaterally symmetrical sets of neurons (sets TA2,3,6 –9) together with four pairs of TA-l-ir single cells (TA1,4,5,10) which together total 70 – 80 neurons. In addition to these, we identified four TA-l-ir neuron clusters that also expressed octopamine-likeimmunoreactivity (clusters OA1-4/TA) and consisted of over 40 neurons. All labeled OA-l-ir cells expressed TA-l-ir, and they correspond to the groups of octopaminergic cells previously described in insects (Figs. 1, 2, 4; locust: Stevenson and Spo¨rhase-Eichmann, 1995; cockroach: Sinakevitch et al., 2005). In Table 1 the numbers of neurons in OA- and TAimmunoreactive clusters are summarized and compared with those from other studies on locusts and cockroaches.

TA-l-ir neurons (TA1-10 clusters) TA1 cells. One pair of cells is located on the most dorsal surface of the locust midbrain and is situated laterally and posterior to the antennal lobe region from which they send a neurite into the tritocerebral neuropil (Figs. 1a, 2a). TA2 cluster. The small cells of the TA2 cell cluster, with soma sizes of about 12 ␮m in diameter, are located above and anterior to the protocerebral bridge and are comprised of ⬇5–9 pairs of cell bodies as well as one well-defined cluster consisting of 7 pairs of small neurons (see Fig. 1a and arrowhead in 2a). From our results it is clear that these cells densely supply the protocerebral bridge (Figs. 3a, 4d, 5e). The neurons of this TA2 cluster most likely correspond to tangential and columnar neurons, which were described in Homberg (1994) and found to supply the protocerebral bridge, the central body complex, as well as the lateral accessory lobe. TA3 cluster. This is located on either side of the pars intercerebralis (Figs. 1a, 2b,c, 3e,f). In all our stainings these small cells with soma sizes of ⬇12 ␮m only expressed TA-l-ir rather than OA-l-ir even under different handling conditions. OA-l-ir cells in a similar location were described for cockroaches by Sinakevitch et al. (2005, G1 cluster) and for the locust brain by Stevenson and Spo¨rhase-Eichmann (1995, C7 cluster) (see also Table 1). These cells (number >10) project to the supramedian protocerebrum (see arrow in Fig. 2c and Fig. 3f). According to Stevenson and Spo¨rhase-Eichmann (1995), the other cells of this cluster project into the corpora cardiaca, TA4 cluster. This consists of two single cells on each side of the brain and lies underneath the lateral calyx (Figs. 1a, 2c). In this study the axons of these cells were followed to their branching within the lateral horn of the protocerebrum. TA5 cluster. A single pair of cells was stained with the antibody against tyramine at the anterior margin of the lobula (Fig. 1b). The TA5 neurite can be traced as it runs toward the outer layer of the medulla. TA6 cluster. This cluster is comprised of numerous (>10) small neurons (20 –25 ␮m in soma diameter) located posteriorly and ventrally at the margin of the lobula and projecting to the inner layer of the medulla as well as to the outer surface of the lobula (Fig. 1b). TA7 cluster. This group of small neurons (20 ␮m in soma diameter) consists of 4 –5 pairs of cell bodies and is located posterior to the lateral horn of the protocerebrum (Figs. 1a,

The Journal of Comparative Neurology TYRAMINE AND OCTOPAMINE IN LOCUST

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Figure 1. Diagram showing locations of TA-l-ir (TA1-10) and OA/TA-l-ir (OA1-4/TA) cells and their clusters in the locust midbrain (a) and optic lobe (b). Filled circles represent TA-l-ir cell bodies; open circles indicate OA/TA-l-ir neurons. The insert shows the sectional planes and section definitions used in the present study. For abbreviations, see list. Scale bar ⴝ 200 ␮m.

2b). The projection patterns of the primary neurites of these cells suggest that they project within the lateral horn of the protocerebrum rather than innervate the optic lobes. TA8 cluster. Three pairs of small neurons were located laterodorsal to the antennal lobes. This cluster lies underneath the OA3/TA cluster (see below) (Figs. 1a, 2c, 3a). TA9 cluster. Two pairs of cells are located at the ventral midline of the protocerebrum (Figs. 1a, 2c) and lie above the upper anterior margin of the antennal lobes. TA10 cells. A single pair of cells with a large soma diameter of ⬇60 ␮m, located within the medioventral group of neurons and previously described from ethyl gallate stains of the brain by Williams (1975), was found to be immunopositive against tyramine (Figs. 1a, 2c,d, 4d), but immunonegative against octopamine (Fig. 4d). The neurite of this cell can be followed posteriorly to the tritocerebrum and may continue through the circumesophageal connective. Other members of this medioventral group of cells (Williams 1975) form the

OA1/TA cluster, and thus exhibit both TA-l-ir and OA-l-ir, which was first described by Konings et al. (1988).

Neurons expressing both OA-l-ir and TA-l-ir (OA1-4/TA) OA1/TA cluster. Four pairs of relatively large (60 ␮m and 40 ␮m soma diameter) and three pairs of small cell bodies (20 ␮m soma diameter) were labeled medioventrally in the deutocerebrum (Figs. 1a, 2b, 3a). All these neurons showed both anti-OA and anti-TA immunoreactivity and, therefore, were named the OA1/TA cluster. This medioventral group of cells was first described by Williams (1975) and Stern et al. (1995). Stern (1999) distinguished several subtypes within this group: 1) PM4a and PM4b cells (with a soma diameter ⬇60 ␮m), and 2) PM5 cells having a smaller soma diameter ⬇40 ␮m. From the results of the present study it is clear that the axons of the large four cells form a bundle and extend posteriorly and send processes to the antennal mechanosensory and motor center



Figure 2. Distribution of TA-l-ir within the midbrain of the locust. The tyramine antibody reveals single neurons or clusters of immunostained neurons and their fibers (a,b), as well as densely labeled neuropiles (b– d). Insert in (a) shows schematic side view of the locust midbrain indicating horizontal planes of selected vibratome sections, 70 ␮m thick (from (a), most dorsal section, to (d), most ventral section) (for detailed explanation, see text). For abbreviations, see list. Scale bar ⴝ 200 ␮m (applies to a– d). TABLE 1. Comparison of OA-l-ir ad TA-l-ir Cell Classes Found in the Brain of the Cockroach (1) and the Locust (2, 3) Class (2)

Class (3)

Number (1)

Number (2)

Number2 (3)

Location (2,3)

G3a,b — G5b Not named1

C1 C2 C5 C8

OA1/TA OA2/TA OA3/TA OA4/TA

13 pairs

6–8 pairs 1 pair 1 pair Numerous

7 pairs (20–60) 1 pair (50–60) 4 pairs (30–60) Numerous, > 20 (12–18)

G6a G4a G1 G0 — Not named1 — — G2a

— — C7 — — C6 — —

TA1 TA2 TA3 TA4 TA5 TA6 TA7 TA8 TA9

— — 14 pairs

— — - variable — — — — — —

1 pair (20–30) 12–16 pairs (12–20) Numerous, > 10 (12–20) 2 pairs (15–20) 1 pair (30) Numerous, > 10 (20–25) 4–5 pairs (15–20) 3 pairs (15–20) 2 pairs (15–20)

—

—

TA10

—

—

1 pair (60)

Ventro-posterior, medial to AL Median, DC-TC border Dorsal, lateral to AL Outer face of Me (around first optical chiasm) Dorsal, laterally posterior to AL Anterior-dorsal to PB Pars intercerebralis Median, anterior to LH Anterior to Lo Posterior to Lo Median, posterior to LH Ventral, lateral to AL Above the upper inner margin of AL Ventroposterior, anterior to OA/TA1 cluster

Class (1)

Numerous 11 pairs 6 pairs — 14 pairs —

References: (1) Sinakevitch et al., 2005, (2) Stevenson, Spo¨rhase-Eichman, 1995, (3) present study. 1 These cell clusters are present in the article, but were not named by the author. 2 The approximate sizes of cell bodies are shown in parentheses in ␮m. Note: Classification by Stevenson, Spo¨rhase-Eichman (1995) and by Sinakevitch et al. (2005) is based on the use of a polyclonal OA antibody only, whereas the classification in the present study is based on the use of both monoclonal anti-OA and polyclonal anti-TA antibodies. For abbreviations, see list.

(AMMC, Fig. 2b), and then turn dorsolaterally to pass over the dorsal surface of the protocerebrum (see Fig. 2a). The axons from the two largest neurons (PM4a,b) extend straight to the ipsilateral optic lobe, where they arborize in the lobula and medulla. The axons from the two intermediately sized neurons deviate from the axon bundle and then bifurcate. One branch

rejoins the axon bundle projecting into the ipsilateral optic lobe, while the other one projects across the midline of the brain to join the axon bundle entering the contralateral optic lobe (see dashed white line circles in Fig. 2a). These axons along with those of the OA3/TA-cluster (see below) run within the posterior optic tracts (see Strausfeld, 1974). The axons


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Figure 3. Transverse (a– d) and horizontal (e– g) sections through the midbrain of the locust, showing details of TA-l-ir in major neuropilar regions. (a) TA-l-ir fibers within the protocerebral bridge (PB) originating from cells of the TA2 cluster (marked with asterisk). Note the absence of immunoreactive processes within the peduncle (P) and ␣-Lobe (aL) of the mushroom bodies. (b) Section through the noduli (N) of the central complex (see also (e) for a horizontal view). Strong TA-l-ir is present within the median protocerebrum (mPC) and the lower division of the central body (CBL) (marked by a white bracket). The arrow points to the chiasm of TA-l-ir fibers underneath the central complex (see also ci and cii). ␤-Lobes (bL) of mushroom bodies show no TA-l-ir. (ci) Section through the CBL, showing that TA-l-ir processes in the lateral accessory lobe (LAL) are formed by fibers originating from two ventral tracts (marked by arrowheads). Neurites of these tracts run either ipsi- or contralaterally (forming a chiasm, marked by the arrow, ci and cii) to innervate posterior protocerebrum (pPC), or they continue anteriorly to innervate supra median protocerebrum (SMP), shown in (cii) in a horizontal section. (d) Strong tyraminergic innervation within the posterior region of the LAL, originating from fibers of the ventral fiber tracts (marked by arrowheads). (e,f) The distribution of TA-l-ir in central body neuropils and the pars intercerebralis (Pi). The paired noduli of the central complex (N, marked by double arrowheads) are extensively supplied by bundles of tyraminergic fibers coming from the neurons of the OA/TA3 cluster (see Fig. 2a). TA3 cell cluster belongs to the pars intercerebralis (Pi) (see Fig. 3e). The white bracket in (f) marks the CBL, where fine TA-l-ir processes were revealed, and corresponds to the location of the bracket in (b). (g) Horizontal section through antennal lobe (AL) showing dense innervation by TA-l-ir fibers. For abbreviations, see list. Scale bars ⴝ 50 ␮m.

that belong to the three pairs of smaller cells can be followed toward the regions of the central body and lateral accessory lobes (see solid white line circle in Fig. 2b). We did not observe any descending axons to the subesophageal ganglion from neurons of this cluster as described by Konings et al. (1988). OA2/TA cells. A single pair of large cell bodies (⬇60 ␮m soma diameter) situated lateroposteriorly in the tritocerebrum also expressed both OA-l-ir and TA-l-ir (Figs. 1a, 4b,c). We observed extension of the OA2/TA axons posteriorly toward the tritocerebral commissure, but the final branching pattern could not be resolved in our stains.

OA3/TA cluster. This cluster is a group of four pairs of cells with large soma diameter (⬇40 – 60 ␮m) located laterally and anteriorly in relation to the antennal lobe region (Figs. 1a, 2a). The cells of this OA3/TA cluster became immunopositive for octopamine only in the group of animals that were stressed (handled) before preparation, whereas in the nonstressed (control) group, these cells only showed TA-l-ir (see Fig. 10a). Neurites of two cells of the OA3/TA group run posteriorly to the tritocerebrum and descend further to the subesophageal ganglion (see solid white line circle in Fig. 2a). Another two cells run anteriorly to join the neurites of the OA1/TA cluster



Figure 4. Correlation between TA-l-ir (green) and OA-l-ir (magenta) within the midbrain of the locust. (a) Horizontal section through the upper central body complex (CBU) showing distribution of TA-l-ir (ai) and OA-l-ir (aii). Double-labeled OA1/TA somata are white (aiii). Posterior part of lateral accessory lobe (LAL) is extensively labeled by TA-l-ir (two arrows in ai). Fine octopaminergic innervation of the anterior part of the CBU is marked by a white asterisk in (aii). Innervation of the peduncle (P) is missing. (b) Cells of OA1/TA and OA2/TA cluster in the deutocerebrum (DC). Tyramine and octopamine are colocalized within the cell bodies of these clusters (white). Note strong arborization of both TA-l-ir and OA-l-ir fibers within the antennal mechanosensory and motor center (AMMC). (c) Transverse section through the DC region marked in (b) with a dashed rectangle. Cell bodies of OA1/TA cluster are labeled from 1 to 7. Two relatively big somata (60 ␮m) are labeled 1 and 2 and correspond to PM5 cells; two cells with a somata diameter of about 40 ␮m are labeled 3 and 4 and correspond to the PM4a,b cells. Smaller cells of this cluster are labeled from 5 to 7. (d) Horizontal section through the central body complex. Noduli (N) within the lower division of the central body (CBL) are labeled with tyramine antibody, as well as the PB and a posterior region of LAL. The TA10 cell is labeled only with antibody against tyramine, while the cells of OA1/TA cluster show both TA-l-ir and OA-l-ir. For abbreviations, see list. Scale bars ⴝ 200 ␮m in a,d; 100 ␮m in b,c.

(see dashed white line circles in Fig. 2a) and to project laterally within the posterior optic tract (see double arrowheads in Fig. 2a). Additionally, the neurites of these cells supply a variety of neuropils in the lateral and medial protocerebrum and in the deutocerebrum posterior to the antennal lobes and above the central complex. OA4/TA cluster. Numerous (>20) small cells of this cluster (medulla amacrines, see Homberg, 1994) with soma diameters of about 12 ␮m, show both TA-l-ir and OA-l-ir, and have their somata around the first optical chiasm. Their fibers enter the outer layer of the medulla and thus innervate this optic neuropil (Fig. 1b, see also Fig. 5c).

Tyramine- and octopamine-like-immunoreactivity within main brain neuropils Central complex. Figure 3 shows details of TA-l-ir in the main neuropils associated with the central complex of the

locust brain. The central complex consists of the protocerebral bridge at the dorsal–posterior margin of the median protocerebrum and the central body (see Homberg, 1994). The dorsal part of the protocerebral bridge receives extensive innervation by TA-l-ir fibers (Fig. 3a, see also Fig. 5e), originating from small size somata above and behind the bridge (see asterisk in Fig. 3a). Cell bodies of these neurons probably belong to the TA2 cluster. In Figure 5a,b, where both the OA and TA antibodies were applied simultaneously to reveal the colocalization between these two neurotransmitters, TA-l-ir within the protocerebral bridge is revealed by fibers and their varicosities or “boutons” (Fig. 5a), whereas OA-l-ir is only expressed by numerous varicosities which in horizontal sections are aligned perpendicular to tyraminergic fibers (Fig. 5bi and bii). Since not all tyraminergic boutons were doublelabeled by the octopamine antibody (see arrows in Fig. 5biii), we conclude that this part of the central complex also re-


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Figure 5. Details of TA-l-ir (green) and OA-l-ir (magenta) within the protocerebral bridge (a,b,e), optic (c,d) and antennal lobes (f,g). (a) Horizontal section through the PB showing TA-l-ir and OA-l-ir. The dashed square refers to the area shown in high-magnification 3ⴛ 0.3 ␮m optical sections in (b) (bi, TA-l-ir, bii, OA-l-ir, biii, composite). See double-labeled (white in biii) and exclusive tyraminergic varicosities (arrows in biii). (c) Horizontal section through the medulla (Me) showing tyraminergic cell bodies and the distribution of TA-l-ir and OA-l-ir in the outer, Me(o), and inner, Me(i), layer of the medulla, indicated by white brackets. (d) Amira 3D-reconstruction of the dashed square region in (c), representing a 3-␮m optical section. Note different color code. OA-l-ir is yellow (di), TA-l-ir is blue (dii). The “Correlation plot” analysis (diii) (see Materials and Methods) shows that all OA-l-ir processes colocalized with TA-l-ir ones (magenta labels). Note the pure tyraminergic profiles (blue, marked with the dashed circle) (ei) Dense TA-l-ir and OA-l-ir in the midbrain, including PB (horizontal section). The TA-l-ir fiber within the anterior optic tract is labeled with a double arrowhead (see also Fig. 2a). (eii) One octopaminergic fiber innervates the anterior calyx (ACa) of the mushroom body (arrows in ei and eii correspond to the same location). (f) A 70-␮m thick optical sections of the AL neuropil (double-labeled parts are white) showing OA-l-ir and TA-l-ir. Arrowheads point to pure tyraminergic varicosities on a fiber. (g) Single optic section (0.3 ␮m thick) showing labeling of a large varicosity in AL with TA-antibody (gi) and OA-antibody (gii), and the composite (giii), marked by a white circle in (f). Scale bars ⴝ 20 ␮m in a,c; 5 ␮m in b; 10 ␮m in d; 200 ␮m in ei; 150 ␮m in eii; 15 ␮m in f; 0.5 ␮m in g.

ceives innervation from purely tyraminergic neurons. The lower division of the central body (Fig. 3b,f) and the protocerebral bridge (Fig. 3a) show the highest density of TA-l-ir, whereas in most of the upper division of the central body it is absent (Fig. 3b,e). However, the anterior part of the upper division showed positive immunoreactivity to the antibody against OA (see Fig. 4a,d). The observed TA-l-ir in the lower division of the central body may come from TA-l-ir fibers forming a chiasm underneath the central complex (see arrow in Fig. 3b). Noduli, or so-called “ventral tubercles” (Hertweck 1931), lying ventral to the upper division of the central body and caudal to its lower division, receive extensive innervation

by bundles of tyraminergic fibers coming from the neurons of the OA3/TA cluster (Fig. 3e, see also Fig. 2a,b). Neuropils, closely associated with the central complex such as the supramedian protocerebrum (Fig. 3cii,e,f, see also Fig. 2d) and the lateral accessory lobe (Fig. 3d, see also Fig. 2b,c), possess rich TA-l-ir, coming from fibers of the two ventral tracts (marked with arrowheads in Fig. 3ci and d). Fibers of these tracts run either ipsi- or contralaterally, thus forming a chiasm (marked by an arrow in Figs. 3b and 3cii), and upward to innervate the posterior protocerebrum, or they continue anteriorly to innervate the supramedian protocerebrum. Unpaired neurons of the subesophageal ganglion (Braünig, 1991) most

The Journal of Comparative Neurology 442 likely also contribute processes to these ventral tracts, and therefore to the mentioned neuropils. Optic lobes. Extensive TA-l-ir (Fig. 5c,d) and OA-l-ir (Fig. 5d) processes arborize in the lobula and medulla (see also Fig. 1b). In the locust at least four cells from the OA1/TA cluster (PM4a,b and PM5 cells; see Stern et al., 1995, Stern 1999) send neurites within the ipsi- and contralateral posterior optic tracts to the optic lobes. Another cluster of numerous neurons expressing both OA- and TA-l-ir is located at the outer layer of the medulla. Additionally, we discovered two pure TA-l-ir clusters (TA5 and TA6), whose perikarya are lying at the border between the lobula and medulla. All these mentioned neurons may contribute to the observed distribution of OA/TA-l-ir in the optic lobes. The fibers branching within a distal part of the lobula may come from PM4a,b or PM 5 cells, whereas numerous varicose processes in the proximal part may be the result of arborizations of the cells from the TA5 and TA6 clusters. The inner as well as the outer layers of the medulla receive innervation from TA-l-ir fibers, whereas OA-l-ir processes are found mainly within the outer medulla layer (Fig. 5c). The OA4/TA cluster, as well as the cells from the OA1/TA cluster, may contribute to the OA-l-ir in the outer layer of the medulla. Figure 5diii shows the Amira 4.1 “correlation plot” analysis of the outer layer of the medulla. All OA-l-ir processes (Fig. 5di, yellow label) within a selected region of the medulla are indeed colocalized with TA-l-ir (Fig. 5diii, overlapping regions, magenta label). Exclusive TA-l-ir innervation of the medulla was found within the third layer of the outer medulla, and can be clearly seen in the frontal view of the plot diagram (marked with the dashed circle in Fig. 5diii). Antennal lobes. Numerous TA-l-ir fibers invade the antennal lobes (Fig. 3g), where extensive branching can be observed between glomeruli. Again, only TA-l-ir fibers within the antennal lobe neuropil (Fig. 5f, arrowheads) are stained, whereas varicosities are always double-labeled. Figure 5g shows a high-magnification image of one antennal lobe varicosity (marked by a white circle in Fig. 5f), which shows regional segregation of tyramine and octopamine staining. Octopamine (Fig. 5gii) seems to be restricted to more terminal varicose structures (“boutons”), whereas tyramine (Fig. 5gi) is more evenly distributed. It appears that not the whole varicosity is double-labeled, as in the composite still some green areas remain (Fig. 5giii). This may be an indication that tyramine may be present and more evenly distributed in the cytoplasm, whereas octopamine is more concentrated and, perhaps, associated with vesicles. Mushroom bodies. The peduncle, ␣- and ␤-lobe exhibited no TA-l-ir, and the calyces reveal only little. In contrast, the calyces do exhibit OA-l-ir, which is shown in Figure 5eii. The fiber marked by an arrow expresses OA-l-ir. This fiber may originate from unpaired octopaminergic neurons of the subesophageal ganglion, which have processes that innervate the calyces (Braünig, 1991).

Tyramine- and octopamine-immunoreactivity within the ventral nerve cord Subesophageal ganglion. Figure 6 shows the distribution of OA-l-ir (magenta) and TA-l-ir (green) within the subesophageal ganglion. The total number (n ⴝ 24) of localities and relative sizes of cell bodies correspond to those previously identified by octopamine immunocytochemistry using a polyclonal antiserum (Braünig, 1991; Stevenson and Spo¨rhase-Eichmann, 1995) with

N.L. KONONENKO ET AL. the exception of two pairs of TA-l-ir neurons. In addition to all the known dorsal and ventral unpaired median neurons, a pair of ventral OA-l-ir neurons was also labeled. In a projection view from the side of a whole subesophageal ganglion (Fig. 6a) the double-labeled cell bodies can be seen on the dorsal and ventral surface as well as the distribution of immunoreactivity within central parts of the neuropils. Note the different distribution of immunoreactivity within the neuropils (Fig. 6a,g). TA-l-ir (green) labels the anterior part of the neuropil (mandibular neuromere, md in Fig. 6g), whereas parts with predominantly OA-l-ir (magenta) are confined to the neuropil of the maxillar (mx) neuromere (Fig. 6a,g). Parts of the maxillar and the posterior neuropil of the labial (la in Fig. 6gii) neuromere are double-labeled (white parts in Fig. 6a). The neuropil of the labial neuromere predominantly expresses TA-l-ir (green). A confocal scan of 176 ␮m from the dorsal surface (“b” indicated in Fig. 6a) reveals that all dorsal midline neurons as well as some posterior neuron somata are double-labeled (white, Fig. 6b). Figure 6c,d shows the maximal intensity projections of two image stacks each (z-increment 8 ␮m) taken from the areas indicated in Figure 6a. In both panels double-labeled neuron somata (white) showing TA-l-ir and OAl-ir can be seen, but there is also a pair of exclusive TA-l-ir neurons (arrowheads) which reveal only TA-l-ir in the mandibular (Fig. 6c) and maxillar neuromeres (Fig. 6g). Higher magnifications of the densely labeled neuropils again show TA-l-ir fibers and more varicose profiles revealing OA-l-ir (Fig. 6e). What appears double-labeled in white in Figure 6e is an artifact resulting from color addition of differently labeled profiles in maximal intensity projection of multiple image stacks, which was verified by viewing three 0.3-␮m-thick optical sections (Fig. 6f). This clearly revealed the TA-l-ir fibers (green) and the OA-l-ir more varicose profiles (magenta). Thoracic-abdominal nervous system. In the thoracic ganglia all neurons that were previously described to express OA (Stevenson et al., 1992; see also Stevenson and Spo¨rhaseEichmann, 1995) also show TA-l-ir, and thus were doublelabeled. This is demonstrated in Figure 7a,b, which shows the cluster of posterior (DUM) neuron somata in the mesothoracic ganglion double-labeled (white, Fig. 7ai) including the paired ventral cells marked by asterisks in the insert (Fig. 7aii). Both the somata and primary neurites are double-labeled, as well as varicose profiles in the neuropil, whereas many fibers in the neuropil express only TA-l-ir. This is underlined by highermagnification projection views in Figure 7d (area indicated in the dashed box of Fig. 7c) and which show the TA-l-ir in fibers and varicose profiles (Fig. 7di), the OA-l-ir of varicose profiles alone (Fig. 7dii), and the composite (white, Fig. 7diii). This is shown at even higher magnification in Figure 7e. In the metathoracic ganglion, which is fused with the first three abdominal neuromeres (A1 to 3 in Fig. 7fi) pure TA-l-ir cells can be seen in the third abdominal neuromere (double arrowheads in Fig. 7fi). These cells are not revealed in Figure 7fii, which shows the somata of OA-l-ir neurons. The paired ventral cells (asterisks in Fig. 7fi) are again double-labeled (Fig. 7fiii) both by TA-l-ir and OA-l-ir. As all somata of the well-known DUM neurons of thoracic ganglia are double-labeled by the tyramine and octopamine antibody, we examined the soma region more closely (Fig. 8). A projection view in Figure 8a clearly shows labeling of the DUM cell body region by both TA-l-ir and OA-l-ir. The dashed line area in Figure 8a indicates higher magnifications of the


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Figure 6. OA-l-ir (magenta) and TA-l-ir (green) cell groups of a locust subesophageal ganglion. (a) A subesophageal ganglion (whole-mount) in a side view showing the distribution of OA-l-ir and TA-l-ir within dorsal or ventral cell bodies and within distinct areas of the neuropil. The brackets (marked with the letters (b– d)) indicate the thickness of the optical slices through the wholemount ganglion and refer to the confocal image stacks, which are shown in (b– d). (b) All somata are double-labeled (white) by TA-l-ir (green) and OA-l-ir (magenta). (c) An image stack of more ventral sections (indicated in (a), white brackets), and reveals two double-labeled somata (white), and two smaller paired cell bodies within the maxillar neuropil, which are only labeled by the antibody against TA. Also note in (c) the densely labeled fibers by TA-l-ir. (d) Again, two paired small cell bodies are shown that are only labeled by TA-l-ir. (e) The densely packed fibers labeled by TA-l-ir of the maxillar neuropil are revealed (the area indicated by the white asterisk in (a) corresponds to 36 optical sections, 0.3 mm each). (f) A higher magnification of the area indicated by the white asterisk in (e) reveals fibers labeled by the antibody against TA and terminal varicosities labeled by OA-antibody (corresponds to three optical sections, 0.3 ␮m each). (g) Side view of the subesophageal neuropils shows clear differences in the distribution of TA-l-ir (gi) and OA-l-ir (gii). For abbreviations, see list. Scale bars ⴝ 50 ␮m in b– d,g; 20 ␮m in e; 5 ␮m in f.

DUM neuron soma shown in Figure 8bi– biii. The cytoplasm of the soma itself is labeled by either TA-l-ir (Fig. 8bi), or even more densely by OA-l-ir (Fig. 8bii). In this single section only a very small number of profiles are double-labeled (see white profiles in the merged Fig. 8biii), and in many places profiles exclusively labeled by OA-l-ir (magenta) exist, whereas profiles labeled by TA-l-ir (green) are exclusively almost nonex-

istent. Profiles labeled by TA-l-ir appeared more clustered, which may, perhaps, point to their association with the Golgi complex. Vesicles stained with the OA antibody are smaller in diameter than those stained by the TA-antibody, and their distribution appears more scattered. In addition, we noticed some finger-like elongations from many DUM-neuron somata (Fig. 8ci– ciii), which were always free of TA-l-ir but exhibited

Figure 7. The distribution of OA-l-ir (magenta) and TA-l-ir (green) in a locust thoracic ganglia. (a) Horizontal (70 ␮m thick) vibratome section showing the projection view of the composite between OA-l-ir and TA-l-ir. Note the white-stained DUM-cell somata as well as the double-labeled paired ventral cells (asterisks) in insert (aii). (b) In a stack of confocal images (five optical sections, each 8 ␮m thick) the DUM-cell cluster, as well as the primary neurite, are marked by the OA-antibody (bi) as well as by the TA-antibody (bii); see white labels in composite in (biii). Note that mostly varicose profiles are labeled by both antibodies (see white dots in composite), whereas many fibers are only labeled by TA-l-ir (green), see also an intensely labeled longitudinal fiber in (a) (arrowhead). (c) A composite of a projection view of an image stack of 5 ⴛ 8 ␮m optical sections shows parts of the neuropil with double-labeled profiles (white) and fibers showing predominantly TA-l-ir (green). The dashed square refers to a neuropilar area shown in higher magnification in (d), TA-l-ir in green (di) shows predominantly labeled fibers, and in OA-l-ir in magenta (dii) shows predominantly varicose profiles. Note that varicose profiles are double-labeled (white in diii), whereas fibers express mostly TA-l-ir. The dashed square in (diii) refers to an even higher magnified part of the neuropil shown in (e) (3 ⴛ 0.3 ␮m optical sections) in which varicose profiles (i.e., fiber terminals) are double-labeled (white, eiii). Note that fibers labeled by TA-l-ir (ei and eiii) possess varicosities that are not double-labeled. (f) Cell bodies of the abdominal neuromeres of a locust metathoracic ganglion (A1 to A3) express TA-l-ir (fi, green) and OA-l-ir (fii, magenta). The composite in (fiii) reveals that paired cells exist that are only labeled by TA-l-ir (double arrowheads in fi). In (fi) paired ventral cells in A1 and A2 ganglia are labeled with asterisks. For abbreviations, see list. Scale bars ⴝ 100 ␮m in a; 50 ␮m in b,c,f; 10 ␮m in d; 1 ␮m in e.



OA-l-ir (see asterisk in Fig. 8cii and ciii). Such elongations from the soma had been noticed before, for example in Figure 8d in an intracellularly stained DUM3,4,5-neuron (courtesy of Dr. E.

Heidel; see also Kononenko and Pflu¨ger, 2007). In addition, in all our specimens the intensity of immunostaining within the DUM-neuron cluster was not uniform, but variable (e.g., Fig.

Figure 7


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Figure 8. DUM cell bodies of a mesothoracic ganglion labeled with antibodies against TA (green) and OA (magenta). The dashed line square in (a) represents a magnified part of a DUM cell body (n, nucleus) shown in (b). (bi) Vesicles stained with the antibody against TA (double arrows in bi). (bii) Vesicles, stained with the OA-antibody. (biii) A composite of panels (bi) and (bii). Note that exclusive octopaminergic vesicles exist within the cytoplasm (dashed line circle). The section corresponds to a single 0.3-␮m-thick optical section. (c) A 70-␮m-thick vibratome section shows several mesothoracic DUM cell bodies double-labeled by TA-l-ir (ci) and OA-l-ir (cii) (see merged picture in ciii). Note that “finger-likeelongations” from the cell body (see asterisk in cii and ciii) only reveal OA-l-ir. (d) Such elongations are not artifacts, as they are also observed in individually stained DUM-neurons (see asterisk); the arrow refers to the primary neurite that originates from the soma. For abbreviations, see list. Scale bars ⴝ 5 ␮m in a; 1 ␮m in b; 15 ␮m in c.

8c). However, except for the stressed (handled) group of animals, we always found double-labeled octopaminergic neurons. Figure 9 shows the distribution of TA-l-ir (rows i) and OA-l-ir (rows ii) in the seventh abdominal ganglion (Fig. 9d, dorsal view, and Fig. 9e, ventral view) and the terminal abdominal ganglion (Fig. 9a, dorsal view, and Fig. 9b, ventral view), which form the ganglia associated with the genital organs. The wellknown dorsal and ventral unpaired neurons of these ganglia are all double-labeled, except two pairs of very small cell ventral bodies within the ninth abdominal neuromere (arrows in Fig. 9bi) of the terminal ganglion, which only show TA-l-ir. In projection views of whole ganglia viewed laterally from the side, the difference in labeling between TA-l-ir (Fig. 9ci, terminal ganglion, and Fig. 9fi, seventh abdominal ganglion) and OA-l-ir (Fig. 9cii and Fig. 9fii) can be clearly seen. Distinct parts of the median and ventral neuropil are densely labeled by TA-l-ir, most of which may actually result from descending TA-l-ir fiber(s) (see double arrowheads in Figs. 9ai, ci, ei, and fi).

10ai. The cells of the OA2/TA cluster, in contrast, reveal both TA-l-ir and OA-l-ir. When the animals are exposed to “stressing” stimuli the OA3/TA-cluster now reveals both OA-l-ir (Fig. 10bi) and TA-l-ir (Fig. 10bii, see also composite in Fig. 10biii). Figure 10c shows the characteristic DUM neuron cluster on the dorsal surface of a metathoracic ganglion (maximal intensity projection of 10 image stacks (z-increment 8 ␮m) from a control animal that was not handled (nonstressed, Fig. 10c) or an animal that was handled and exposed to stressing stimuli (Fig. 10d). In contrast to the control animals, in which all metathoracic DUM neurons are double-labeled by both TA-l-ir and OA-l-ir, this is different in the handled animals. Large DUM-somata are now intensely labeled by OA-l-ir but no longer exhibit TA-l-ir. However, some anterior, smaller diameter DUM-neuron somata still reveal TA-l-ir, and unlike in the control animals, now the secondary neurites are intensely labeled by TA-l-ir.

Previous handling (“stressing”) of the animal affects the octopamine-like-immunoreactivity of neurons

DISCUSSION Do neurons exist that exclusively express tyramine-like-immunoreactivity?

Figure 10a shows the neurons of the OA3/TA cluster close to the antennal lobes of a control animal which was not handled (“stressed”) and which only revealed TA-l-ir (Fig. 10aii and aiii, composite). Note the absence of OA-l-ir in this cluster, whose location is marked by a dashed line circle in Figure

The first step in the tyramine synthesis pathway is its formation from tyrosine by the action of tyrosine decarboxylase. Tyramine itself is the substrate of the enzyme T␤H, which converts it to octopamine (see Roeder, 2005). Consequently, this enzyme should be present in all neurons that synthesize



Figure 9. TA-l-ir (i) and OA-l-ir (ii) neurons and their central arborizations in wholemount preparations of a female terminal (a– c) and seventh (d–f) abdominal ganglia. (a,d) Ten optical sections (8 ␮m each) representing the dorsal surface of the ganglia. (b,e) Six optical sections (8 ␮m each) representing the ventral surface of the ganglia. (c,f) Side views of wholemount ganglia (xz maximal intensity projection, Amira 4.1.2). Arrows in (bi) point to paired cell bodies of tyraminergic neurons within the ventral part of terminal ganglion. Double arrows in (ai), (ci), (ei), and (fi) indicate one tyraminergic fiber running within VMT. Dashed circles mark the neuropilar areas of the ganglia, where tyraminergic nerve fibers make extensive arborizations. Scale bars ⴝ 100 ␮m in a,b,d,e; 50 ␮m in c,f.

octopamine, and thus all octopaminergic neurons must also contain tyramine, and this is indeed what we find in our study. All tyraminergic neurons identified in the present study fall into two main categories: 1) neurons showing both TA-l-ir and OA-l-ir and changing their content depending on previous handling, and 2) neurons that under the behavioral conditions tested by us (“stressed/unstressed”) express only TA-l-ir, but not OA-l-ir. This allows for speculation that the first group of

neurons possesses T␤H, while the second one is lacking this enzyme. Such clusters as TA5, TA8, and TA10 in the brain as well as two pairs of TA-l-ir ventral neurons in the subesophageal ganglion were identified in regions that we never found to contain octopamine or dopamine or any other catecholamines and, thus, appear to be indeed exclusively tyraminergic. All remaining TA-l-ir neurons and their clusters correspond to the previously identified OA-l-ir neurons either in cockroaches or


447

Figure 10. Differences in the handling of locusts by either keeping them unmanipulated (undisturbed, control animals, a,c) or exposing them to stressing stimuli (b,d), cause differential labeling of brain octopaminergic neurons (a,b) as well as of metathoracic DUM neurons (c,d) by OA (magenta) or TA antibodies (green). Third panel (iii) is a composite (white) between the OA-antibody (i) and the TA-antibody (ii) labeling. (a,b) The OA3/TA cluster within the deutocerebrum became immunopositive for octopamine only in the group of stressed animals (dashed line circle in bi), whereas in the control group these cells show only TA-l-ir (dashed line circle in (ai) shows no labeled cell bodies). Note the similar branching pattern of OA3/TA cluster in control and stressed animals (in (aiii) and (biii) marked by arrow) as well as the bundle of axons from OA1/TA cluster (in (biii) marked by white line circle) and neurites of other tyraminergic neurons (in (biii) marked by dashed line circle). (c,d) In the stressed group the TA-l-ir within the cell body of large DUM-neurons, mainly those innervating muscles associated with the legs, disappears completely, whereas in the control animals all DUM somata are double-labeled. However, in the stressed group the secondary neurites of DUM-neurons are intensely expressing TA-l-ir, whereas in the control group the secondary neurites are marked much less by both OA-l-ir and TA-l-ir. For abbreviations, see list. Scale bars ⴝ 100 ␮m in a,c,d; 150 ␮m in b.

The Journal of Comparative Neurology 448 in locusts (see Table 1) and, therefore, may synthesize octopamine under very specific behavioral conditions that exclude locomotor behaviors. However, in our study only one population of TA-l-ir neurons (cluster OA3/TA) exhibited OA-l-ir after a particular handling of the animal. On the other hand, throughout the whole CNS we indeed observed varicosities and “boutons” that were not double-labeled but only exhibited TA-l-ir. There are two possibilities: i) either the axons of OA/TA-l-ir neurons contain different types of varicosities, ones that are double-labeled, containing both monoamines, and others that only contain tyramine, or ii) these tyraminergic varicosities may belong to exclusive TA-l-ir neurons, which do not express the enzyme T␤H. When tracing the varicosities in a few optical sections, we always found the pure TA-l-ir fibers not matching the distribution of double-labeled varicosities, suggesting that the second possibility of exclusive TA-l-ir profiles may indeed be more likely. However, a definite final answer about neurons exclusively expressing TA-l-ir can only be given if the distribution of the enzyme T␤H will be additionally known, and if the nervous system is studied immediately after very specific behaviors such as, for example, courtship, egg-laying, or aggressive encounters.

Distribution of octopamine- and tyramine-like immunoreactivity Brain and subesophageal ganglion. Lehman et al. (2006) determined the cellular localization of T␤H in the honey bee brain (cerebral ganglion) by means of in situ hybridization. They identified only three groups of neurons expressing T␤H, although five clusters of OA-l-ir neurons were reported by Kreissl et al. (1994) and seven clusters divided into 15 subclusters by Sinakevitch et al. (2005). The three clusters of neurons identified by Lehman et al. (2006) correspond to our anti-octopamine staining in the locust brain (OA1/TA, OA2/TA, and OA3/TA, clusters). In the cockroach brain nine clusters of OA-l-ir neurons (including two clusters located within the optic lobes that remained unnamed) were described by Sinakevitch et al. (2005). If compared with our results on the locust brain, only three of these cell groups correspond to the OA-l-ir neurons, whereas the other six cell groups correspond to the TA1, TA2, TA3, TA4, TA6, and TA9 cell clusters (see also Table 1). Compared to the findings described in Stevenson and Spo¨rhase-Eichmann (1995) for the locust brain, they described six clusters of OA-l-ir neurons, four of which have similar locations as our OA1-4/TA clusters and two of which correspond to our TA3 and TA7 cluster (see also Table 1). The correlation between the location of OA-l-ir clusters described in the brains of honey bees, cockroaches, and locusts and some TA-l-ir clusters, identified in the present study, may perhaps be explained by differences in handling or by different physiological conditions of the animals that quickly shift the ratio between tyramine and octopamine. We have shown the existence of pure tyraminergic processes within the medulla, the protocerebral bridge, and the antennal lobes, as well as within all neuropils of the subesophageal ganglion. TA-l-ir has also been detected in nonoctopaminergic regions of the Drosophila (Nagaya et al., 2002) and Caenorhabditis elegans (Alkema et al., 2005) nervous systems. We suggest that pure TA-l-ir processes may belong to neurons that do not express T␤H (presumably clusters TA5, TA8, and TA10 and two pairs of TA-l-ir ventral neurons in the subesophageal ganglion) and therefore cannot synthesize oc-

N.L. KONONENKO ET AL. topamine. A precedent for the synthesis of independent neurotransmitters through the differential expression of a decarboxylase and a ␤-hydroxylase in neurons exists in vertebrates: dopaminergic neurons in substantia nigra express dopamine-decarboxylase, but not dopamine-␤hydroxylase, while noradrenergic neurons in locus coeruleus possessing dopamine-␤-hydroxylase show both dopaminergic and noradrenergic immunoreactivity (Hartman et al., 1972; Lamouroux et al., 1987; Mercer et al., 1991; Chatelin et al., 2001, Kononenko, unpubl. obs.). All these observations can be taken as a strong indication that tyramine is indeed not simply a biosynthetic precursor of octopamine, but is an independent neurotransmitter within the brain and subesophageal ganglion of the locust and released from distinct populations of TA-l-ir cells. Thoracic and abdominal ganglia. In control animals that did not receive stressing stimuli before dissection (i.e., taken from the cage and then cooled in the refrigerator or left undisturbed at similar temperatures as used for stressing the animals), the previously well-described populations of thoracic octopaminergic neurons, 1) the dorsal and ventral unpaired median (DUM or VUM) neurons, and 2) the paired ventral cells (Stevenson et al., 1992, 1994) were always stained by both antibodies in our study. Theoretically, these neurons could release both tyramine and octopamine. Evidence for such a corelease of noradrenaline (norepinephrine) and dopamine from noradrenergic neurons exists in the cerebral cortex (Devoto et al., 2001). As tyramine and octopamine seem to have opposite effects on the muscular system (Nagaya et al., 2002) a differential release of both tyramine and octopamine from DUM neuron terminals could increase the modulatory capacity immensely. Indeed, preliminary experiments on a central pattern generator network in the moth, Manduca sexta, suggest differential effects of octopamine and tyramine on component neurons (Vierk, Duch, and Pflu¨ger, in prep.).

Compartmentalization of TA- and OA-l-ir in known populations of neurons In thoracic ganglia much is known about the dendritic structure and projection patterns of OA-l-ir neurons (Hoyle, 1978; Watson, 1984; Kononenko and Pflu¨ger, 2007) and, thus, conclusions about the distribution of the two monoamines can be made from the staining patterns. In undisturbed animals the somata of DUM/VUM neurons, like the somata of the paired ventral cells, are always double-labeled, but this is different for the neurites. The secondary neurites only exhibit TA-l-ir, whereas the primary neurites may still exhibit some immunoreactivity to both octopamine and tyramine. In contrast to this, many terminal varicosities or “boutons” were again doublelabeled, thus expressing immunoreactivity to both monoamines. This may suggest that in an individual octopaminergic neuron a regional compartmentalization exists with respect to the synthesis and storage of octopamine, the principal transmitter released, and tyramine, its precursor. Whether in addition to release from varicosities of terminal fibers there exists a somatic release of these amines into a volume space surrounding all somata of the lineage of the unpaired median neuroblast (Campbell et al., 1995) cannot be decided from our experiments, but octopamine itself affects firing of octopaminergic DUM-neurons (Achenbach et al., 1987; see also Wicher et al., 2001), and the axon terminals of the DUMETi-neuron, the DUM-neuron of the locust extensor tibiae muscle, contain

The Journal of Comparative Neurology TYRAMINE AND OCTOPAMINE IN LOCUST autoreceptors that may limit the amount of octopamine released (Howell and Evans, 1998). The other compartments where octopamine is synthesized from tyramine are the varicosities or “boutons” of terminal fibers. In C. elegans, Alkema et al. (2005) observed punctuate staining with an antibody produced against C. elegans T␤H, where mainly synaptic regions of the cells (RIC interneurons) were stained, whereas weaker staining was observed in neuronal processes and cell bodies. By contrast, T␤H staining was predominantly located in an identified cell body of C. elegans mutants defective in the unc-104 gene, which encodes a neuron-specific kinesin, required for the anterograde transport of synaptic vesicles. In our experiments, like in many previous immunocytochemical studies (Konings et al., 1988; Eckert et al., 1992; Stevenson et al., 1992; Kreissl et al., 1994; Dacks et al., 2005; Sinakevitch et al., 2005), anti-octopamine staining often looks somewhat “blebby,” and high-resolution confocal images indicate that indeed octopamine could be synthesized directly in varicosities. This would suggest that in locusts T␤H, like the closely related vertebrate dopamine-␤hydroxylase, is associated with the membranes of synaptic vesicles (Nelson and Molinoff, 1976; Potter and Axelrod, 1963; Hartman and Udenfriend, 1972; Olschowka et al., 1981) allowing synthesis of octopamine only within the vesicles. Future immunocytochemical studies of these neurons should try to reveal the distribution of T␤H and to match it with the distribution of tyramine and octopamine presented in this study. In addition, the subcellular distribution of tyramine and octopamine within the soma and the terminal fiber structures can only be conclusively studied by means of electron microscopy and applying immunogold-staining methods.

Handling such as exposing animals to “stressing” stimuli affects the octopamine:tyramine ratio in neurons Immunocytochemistry reveals the presence of an antigen, provided it is contained in sufficient quantity at a particular moment of an animal’s life history. As octopaminergic neurons are easily activated by stimuli of arousal or stress (Hoyle and Dagan, 1978; Field et al., 2008) such a stimulation preceding the fixation of the nervous system may alter the staining pattern. Indeed, keeping the locusts undisturbed (control animals) and, thus, preventing most of their motor activity, always produced staining by both antibodies. Therefore, under these conditions a balance or certain ratio between tyramine and octopamine exists. In contrast, “stressing” the animals by visual and acoustic stimuli so that they would walk and jump (but not fly) in their cage and, in addition, exposing them to hot air stimuli resulted in a marked shift toward staining with only the octopamine antibody in the soma, and an absence of staining by the tyramine antibody. This suggests that octopamine can be synthesized from tyramine very rapidly, and, thus, the ratio between tyramine and octopamine is quickly changed. As DUM neurons are involved in different motor behaviors and their soma sizes correspond to different types of these neurons (Watson, 1984; Gras et al., 1990; Ramirez and Pearson, 1991a,b; Burrows and Pflu¨ger, 1995; Baudoux et al., 1998; Duch and Pflu¨ger, 1999; Duch et al., 1999; Johnston et al., 1999; Heidel and Pflu¨ger, 2006; Mentel et al., 2008), these may indeed affect the immunocytochemical staining intensity by the two antibodies. Variability in OA-l-ir staining intensity was known from labeling the same types of neurons

449 with previously used polyclonal octopamine antibodies in different insects. This suggests that both the anti-tyramine and anti-octopamine antibodies detect different amounts of these monoamines within the cell bodies, and that this is dependent on the animal’s earlier handling or behavioral history. Results of our experiments on animals that underwent a different handling support this. From vertebrate models it is known that expression of tyrosine hydroxylase and dopamine-␤-hydroxylase is coregulated and depends on various experimental situations. For instance, reserpine (Ciaranello et al., 1975), nerve growth factor (Acheson et al., 1984), as well as neural stimulation (Axelrod, 1972) specifically increase the activity of these two enzymes by increasing their synthesis. Weiner and Rabadjija (1968), using an isolated guinea pig hypogastric nerve–vas deferens preparation, have shown that electrical nerve stimulation in vitro is associated with an accelerated formation of noradrenaline from tyrosine. Our findings also suggest that synthesis of these two biogenic amines within DUM neurons is highly dynamic and dependent on how the animal was handled and, perhaps, on its whole previous behavioral state.

Specificity of the antibodies In our study we used a monoclonal mouse octopamine antibody whose specificity had previously been extensively tested on the Manduca sexta CNS (Dacks et al., 2005). In insects a 21% crossreactivity to tyramine was discovered, whereas all other crossreactivities, for example, to adrenaline (epinephrine) and noradrenaline (norepinephrine), do not play a role due to a lack of these transmitters in insects (Maxwell et al., 1978; see also Dacks et al., 2005). In addition, we used a polyclonal rabbit tyramine antibody which should stain all known OA-l-ir neurons in the brain and in all ventral cord ganglia, since tyramine is the immediate precursor from which octopamine is synthesized (see Roeder, 2005). This was indeed the case, with not a single exception. Interestingly, we found cells which, despite the above-mentioned crossreactivity of the octopamine-antibody used, were only labeled by the tyramine-antibody, and even after the bouquet of stressing stimuli were not found to label with the octopamine antibody. This could either mean that within these neurons the octopamine content was so low that it was below the threshold of detection by this antibody, or that they only contain tyramine. We favor the latter explanation, as the staining pattern did not change if the animals were treated in the same way and several TA-l-ir cell bodies were identified in regions that we never found to contain octopamine or dopamine. Of course, the possibility remains that octopamine may be synthesized in these neurons depending on some very specific behaviors that were not tried by us. Extensive specificity tests with the tyramine antibody (see supplementary material) could not completely resolve all questions about crossreactivity, in particular, as crossreactivity to dopamine was reported in the leech (Crisp et al., 2002). Therefore, we performed stainings on vibratome sections of similar brain areas with a known dopamine antibody and compared these to the stainings with the tyramine antibody. In no case did we find that there was any overlap in the staining pattern between the two antibodies (see Suppl. Fig. 3). In addition, preadsorption of the tyramine antibody with dopamine still revealed staining of TA-l-ir neurons. Therefore, we are convinced that, at least in the locust, the two antibodies



(anti-TA and anti-OA) can be successfully used and label the appropriate neuronal processes

ACKNOWLEDGEMENTS We thank Dipl. Biol. Bettina Stocker, Dr. Ju¨rgen Rybak, and Mary Wurm (all Berlin), as well as two anonymous reviewers for valuable discussions and critically reading the article. We thank Drs. James Ainge and Rosamund Langston (both Trondheim, Norway) for correcting the English version of the article. We also thank Prof. Dr. Uli Mu¨ller, Saarbru¨cken, and Dr. Wolfgang Blenau (Potsdam) for their enormous help with characterizing the tyramine antibody, and Dr. Paul A. Stevenson (Leipzig) for his generous gift of the dopamine antibody.

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