The data reported here stem from three sets of triple labeling experiments i.e. combinations of markers (see material and methods):

1: synapsin + actin + nuclei;

2: RFamide + synapsin + nuclei;

3: glutamine synthetase + serotonin + nuclei.

In the figures, we use color-coded abbreviations to identify the markers:

SYN: synapsin immunoreactivity

RF: RFamide-like immunoreactivity

GS: glutamine synthetase-like immunoreactivity

5HT: serotonin-immunolocalization

ACT: phalloidin histochemistry to label actin

NUC: nuclear counter stain with bisbenzimide

The experiments provided a consistent picture of the gross brain anatomy so that we were able to compile a schematic drawing of the C. clypeatus brain (Fig. 2). We will first give an overview about the labeling pattern with these three marker sets (Figs. 3, 4, 5) and then will describe specific brain structures in more detail, from the protocerebrum across the deuto- and tritocerebrum to end with the eyestalk neuropils. We determined the sex of the specimens that were studied according to their pleopod morphology, but did not encounter any sex-specific differences of their brains. Most of the brain structures (except for example the central body) are bilaterally paired. For simplicity we will describe only one brain hemisphere (mostly the right side) in the understanding that mirror symmetrical structures are present in the contralateral hemisphere.

Figure 2 A: Idealized schematic drawing of the C. clypeatus brain (dorsal view) compiled from ca. 4–5 successive sections (80 μm) of several animals at a mid horizontal level. B: Schematic cartoons of ventral to dorsal sections of the main olfactory lobe (ON) and the side olfactory lobe (xON) to show the localization of the median (mF) and posterior foramina (pF) and the patches of non-columnar neuropil (dotted areas labeled with letters A-F). Arrows labeled 9 and 10 show the input of olfactory interneurons. The arrow labeled OGT shows the exit of the olfactory globular tract. Abbreviations: 6, 9, 10 cell clusters 6, 9, 10, A1Nv nerve of antenna 1, A2Nv nerve of Antenna 2, AcN acessory lobe/neuropil, AMPN anterior medial protocerebral neuropil, AnN antenna 2 neuropil, CA cerebral artery, Cap cap neuropil of the hemiellipsoid body, CB central body, CEC circumesophageal connectives, CO1, CO2 core neuropils 1 and 2 of the hemiellipsoid body, ICh inner optic chiasm, IL1, IL2 intermediate layers 1 and 2 of the hemiellipsoid body, La Lamina (lamina ganglionaris), LAN lateral antenna 1 neuropil, Lo Lobula (medulla interna), LoP Lobula "plate", LPI lateral protocerebral interneurons, MAN median antenna 1 neuropil, Me Medulla (medulla externa), mF median foramen, MT Medulla terminalis, OCh outer optic chiasm, OGT olfactory globular tract, OGTN olfactory globular tract neuropil, OGTNa accessory olfactory globular tract neuropil, ON olfactory lobe/neuropil, PB protocerebral bridge, pF posterior foramen, PMPN posterior medial protocerebral neuropil, PT protocerebral tract, VC ventral neuropil column, X chiasm of the olfactory globular tract. Full size image

Figure 3 Low power views of a ventral to dorsal section series featuring anti-synapsin immunohistochemistry (SYN; green) with actin (ACT; red) and nuclear (NUC; blue) counter stains; conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning. In the first two most ventral slices A and B, the left hemisphere is shown). Numbers 9 and 10 identify cell clusters. Letters A to F identify the non-columnar olfactory neuropils. Other abbreviations: AcN accessory lobe/neuropil, AMPN anterior medial protocerebral neuropil, AnN antenna 2 neuropil, LAN lateral antenna 1 neuropil, mF median foramen, OGT olfactory globular tract, ON olfactory lobe/neuropil, PC protocerebrum, pF posterior foramen, PMPN posterior medial protocerebral neuropil, xON side olfactory lobe/neuropil, VC ventral neuropil column. Full size image

Figure 4 Low power views of a dorsal to ventral series of vibratome sections triple labeled for synapsin immunoreactivity (SYN; red), RFamide-like immunoreactivity (RF; green), and the nuclear marker (NUC; blue); conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning. Note that section E is from another animal and inserted here because it provides a better view of the central body (CB). Section E is not perfectly horizontal so that the medial foramina of the olfactory lobes are not visible here. The arrows in C to F point to the cerebral artery that pierces the brain. Numbers 6, 9, 10 identify cell clusters. Letters A to F identify the non-columnar olfactory neuropils. Other abbreviations: AcN accessory lobe/neuropil, AMPN anterior medial protocerebral neuropil, AnN antenna 2 neuropil, LAN lateral antenna 1 neuropil, mF median foramen, OGT olfactory globular tract, ON olfactory lobe/neuropil, PC protocerebrum, pF posterior foramen, PMPN posterior medial protocerebral neuropil, xON side olfactory lobe/neuropil, VC ventral neuropil column. Full size image

Figure 5 Low power views of horizontal vibratome sections triple labeled for serotonin immunoreactivity (5HT; green), glutamine synthetase-like immunoreactivity (GS; red), and the nuclear marker (NUC; blue); conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning. A is more ventral than B1 and B2. B1 and B2 show different labels in the same section. Strong glutamine synthetase-like immunoreactivity is present in all brain neuropils except the olfactory lobes. The inset in B1 shows a higher magnification of the boxed region in B2. Putative ensheathing glia cells are arranged around the periphery of the lateral antenna 1 neuropil. Number 9 identifies cell cluster (9) that houses local olfactory interneurons that are serotonergic and innervate the olfactory lobes Other abbreviations: AMPN anterior medial protocerebral neuropil, AnN antenna 2 neuropil, LAN lateral antenna 1 neuropil, ON olfactory lobe/neuropil, PC protocerebrum, PMPN posterior medial protocerebral neuropil. Full size image

Overview over the C. clypeatusbrain

Fig. 3 shows a ventral to dorsal section series featuring anti-synapsin immunohistochemistry (green) with actin (red) and nuclear (blue) counter stains. In the two most ventral slices (Fig. 3A, B; the left hemisphere is shown), tangential sections of the olfactory neuropil or olfactory lobe (ON) can be seen. In Crustacea the ON receives afferent chemosensory input from olfactory receptor neurons on the paired first antennae. The olfactory neuropil is composed of numerous column-like structures with strong synapsin-immunoreactivity (SYNir), the "olfactory" glomeruli. Despite their columnar shape we will refer to these neuropil elements as glomeruli since this term is well introduced in the literature. In cross sections (Fig. 3A), these structures appear as round profiles whereas in the following sections it becomes apparent that the glomeruli are arranged parallel to each other around the periphery of the lobe. The centre of the lobe is devoid of SYNir, yet actin labeling shows that this core is filled with bundles of fibrous material (Fig. 3C, D). Histochemical labeling of cell nuclei reveals a densely packed cluster with hundreds if not thousands of neuronal somata to be associated with the olfactory lobe. This cluster most probably corresponds to cluster (10), which is known to house olfactory projection neurons in other malacostracan Crustacea [52]. In C. clypeatus, cluster (10) is located medially and posteriorly to the ON in the most ventral aspect but also extends more dorsally, where it wraps around the posterior part of the ON (Fig. 3C). In subsequent sections, a side lobe of the olfactory neuropil (xON) becomes visible that in the more ventral sections seems to be separate from the main ON (Fig. 3B). Proceeding further dorsally, however, it becomes apparent that this side lobe is connected to the main ON (Fig. 3C–F). Medial to the ON, SYNir reveals a horizontal column of loose, unstructured neuropil that extends in an anterior-posterior direction, the ventral neuropil column (VC; Fig. 3D). Further dorsally (Fig. 3E–H), two compact, medially situated neuropils become visible displaying strong SYNir: the lateral antenna 1 neuropil (LAN), and the antenna 2 neuropil (AnN). At this level, in the protocerebrum (PC), unstructured immunolabelled neuropil is visible. Between the protocerebrum and the anterior part of the ON, a second compact cell cluster with densely packed nuclei is visible. This is most likely cell cluster (9) that houses local olfactory interneurons [52]. This cell cluster extends through at least five 80 μm sections and once again houses hundreds or thousands of neurons (Fig. 3F–J). In other sections, nuclear labeling shows that the brain is surrounded by a thick layer of cell nuclei, but we could not differentiate which of these belong to the perineurium and which may be neurons. The accessory lobe (AcN) is an assemblage of small SYNir glomeruli and is located medially to the olfactory lobe close to the point where the olfactory globular tract (OGT) emerges from the latter (Fig. 3H). The ON clearly is the dominating structure of the C. clypeatus brain and dorso-ventrally stretches through the entire section series. In the most dorsal section, once again cross sections of the radially arranged olfactory glomeruli are visible (Fig. 3J).

Fig. 4 shows ventral to dorsal section series of another specimen that was processed for anti-synapsin immunohistochemistry (red), RFamide-like immunohistochemistry (green) and a nuclear counter stain (blue). This series reveals a few additional structures compared to Fig. 3 but the general arrangement and size of the main neuropils is similar in this and several other specimens that we examined. The orange color of the olfactory lobes indicates that in the olfactory glomeruli, SYNir and RFamide-like immunoreactivity (RFir) are mostly co-localized (Fig. 4A, B). In the middle of the brain, however, where the glomeruli are sectioned longitudinally, it becomes clear that the cap region of the glomeruli shows only SYNir (red) but not RFir (green). In the ventral neuropil column, RFir fibers are embedded and RFir somata are located between this column and the ONs (Fig. 4B, C). The protocerebrum is filled with a loose network of RFir fibers. In this section series, the subdivision of the protocerebral neuropil in an anterior and a posterior component, that is so typical of decapod crustaceans [52], is visible. These are the anterior (AMPN) and posterior medial protocerebral neuropils (PMPN; Fig. 4D–F). The central body (CB) is a transverse, unpaired protocerebral neuropil that extends across the midline, is embedded between the two aforementioned protocerebral compartments, and displays strong RFir (Fig. 4E). A thick, paired fiber bundle, the olfactory globular tract, leaves the olfactory lobes in a medial direction and surprisingly seems to display both RFir and SYNir (Fig. 4F). The left and right portions of this tract touch each other at the midline, slightly above the central body, where they form a characteristic chiasm. The two bundles then separate again to veer antero-laterally and exit the medial brain by joining the protocerebral tract to target the lateral protocerebrum in the eyestalks (see below). Cell cluster (6) is situated anteriorly between the two arms of the protocerebral tract. The accessory lobe, being situated close to the origin of the olfactory globular tract displays both RFir and SYNir. Medially, a block of diffuse neuropil, the median antenna 1 neuropil (MAN) is embedded between the two arms of the olfactory globular tract (Fig. 4F). In the most dorsal section (Fig. 4G) it becomes apparent that many cell somata in cluster (9) display strong RFir.

Fig. 5 shows two horizontal sections of another specimen that was processed for anti-serotonin immunohistochemistry (green), glutamine synthetase-like immunohistochemistry (red) and a nuclear counter stain (blue). Cell somata with strong glutamine synthetase-like immunoreactivity (GSir) surround all brain neuropils with the exception of the ONs. Within the ON, there is a very faint and diffuse labeling. We were unable to decide if it represents a specific signal or just unspecific background labeling. A tissue layer displaying weak GSir, presumably the perineurium [53], surrounds the entire brain (Fig. 5B). Within the cell clusters known to comprise neuronal cell bodies such as cluster (9), typically very few or no GSir somata were present (Fig. 5B1, 6A). Those neuropils surrounded by GSir cells also display strong immunolabelling in the neuropil core (Fig. 5A, B1, 6A). At higher magnification, the GSir cells at the periphery of the neuropil can be seen to extend processes into the neuropil (Fig. 5B1 inset). These cells are typically bi- or tripolar (Fig. 6B, C). Comparing the labeling pattern observed here to other studies on crustacean glia cells [54–56] suggests that GSir in C. clypeatus is strongly localized in a certain type of glia cells, the ensheathing glia [53] but not in neurons. Serotonin-immunoreactivity (5HTir) is widespread throughout the C. clypeatus brain. The protocerebrum, the lateral antenna 1 neuropil, as well as the antenna 2 neuropil display strong 5HTir (Fig. 5B2, 6A). A population of serotonergic olfactory interneurons with somata within cell cluster (9) gives rise to a strong innervation of the ON.

Figure 6 A-C: Higher magnifications of the protocerebrum and median deutocerebrum. Horizontal vibratome sections double labeled for serotonin immunoreactivity (5HT; green) and glutamine synthetase-like immunoreactivity (GS; red); confocal laser scan microscopy. The boxed area in A is shown in a higher magnification in C. The neuropils surrounded by GSir cells display strong immunolabelling in the neuropil core. The bi- or tripolar GSir cells, presumably ensheathing glia cells, extend processes into the neuropil. The arrow in A identifies a large serotonergic neuron. Many more serotonergic somata are located in cell cluster (9). D1, D2: Double labeled sections showing synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green); confocal laser scan microscopy. In D2, only the green channel is visualized at a slightly higher magnification to show the central body (CB). The arrowheads in D1 identify peptidergic cell somata. E, F: The protocerebral bridge (PB) displays synapsin immunoreactivity (E) and serotonin immunoreactivity (F; confocal laser scan microscopy). The arrowheads identify cell somata within cell cluster (6). The X labels a synaptic region in the chiasm of the olfactory globular tract. Abbreviations: 9 cell cluster (9), AcN accessory lobe/neuropil, AMPN anterior medial protocerebral neuropil, AnN antenna 2 neuropil, LAN lateral antenna 1 neuropil, MAN median antenna 1 neuropil, OGT olfactory globular tract, ON olfactory lobe/neuropil, PC protocerebrum, PMPN posterior medial protocerebral neuropil, PT protocerebral tract. Full size image

Protocerebrum

The protocerebrum can be subdivided into an anterior and a posterior component, the anterior (AMPN) and posterior medial protocerebral neuropils (PMPN; Fig. 6D1, 7A1, 7B1). This subdivision is most obvious in the middle of the section series (Fig. 4D–F), at the level of the olfactory globular tract chiasm (see below), whereas more ventrally (Fig. 4C) and more dorsally (Fig. 4G, 5B1, B2, 6A) such a distinction is not possible. In the protocerebral tract that links the anterior median protocerebral neuropil to the lateral protocerebrum, RFir fibers are present (Fig. 6D1). Anteriorly, the protocerebral neuropil is adjoined by the cell cluster (6) in which neuronal somata with both RFir (arrowheads in Fig. 6D1, E, 7A1) and 5HTir (arrowheads in Fig. 6F) are located. At the interface between the anterior (AMPN) and posterior medial protocerebral neuropils, a transverse, unpaired neuropil extends across the midline, the central body (CB; Fig. 6D1, D2). The central body is innervated by a dense plexus of fine RFir (Fig. 6D2) and 5HTir fibers (data not shown). Several thick RFir commissural fiber bundles accompany the central body posteriorly (Fig. 6D1, D2) and dorsally (Fig. 7A1). Behind these commissural fibers, the cerebral artery pierces the brain in a dorso-ventral direction (arrowheads in Fig. 4C–F, asterisk in Fig. 6D1, D2). The protocerebral bridge is located anteriorly and slightly dorsal to the central body at the level of the olfactory globular tract chiasm (Fig. 6E, F). Its bilaterally symmetrical neuropil compartments adjoin each other at the midline and show positive SYNir and 5HTir but not any RFir.

Figure 7 A1, A2: A horizontal section double labeled for synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green) to show the course of the olfactory globular tract (OGT); confocal laser scan microscopy. The X labels the chiasm of the olfactory globular tract. Arrowheads identify peptidergic somata in the anteriorly located cell cluster (6). Arrows point towards a bundle of neurites from cluster (9) olfactory interneurons that penetrate into the olfactory lobe. The cerebral artery is labeled with an asterisk. The boxed area in A2 is shown in a higher magnification in Fig. 2. B1, B2, C: Triple labeled section showing actin histochemistry (ACT; red), Synapsin immunoreactivity (SYN; green), and localization of nuclei (NUC; blue) to show the course of the olfactory globular tract (OGT); conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning. The X labels the chiasm of the olfactory globular tract. A2 shows the red channel only, and B2 and C the red and green channel. The inset in B2 shows the green and the blue channel. Abbreviations: 6 cell cluster (6), AcN accessory lobe/neuropil, AMPN anterior medial protocerebral neuropil, LAN lateral antenna 1 neuropil, MAN median antenna 1 neuropil, OGT olfactory globular tract, ON olfactory lobe/neuropil, PMPN posterior medial protocerebral neuropil. Full size image

The olfactory globular tract

The olfactory globular tract links the olfactory and accessory neuropils to the lateral protocerebrum. In Decapoda, this tract is formed by the axons of the projection neurons with their somata in cell cluster (10) and this tract represents the major output pathway of the olfactory system (reviews [52, 57, 58]). In the brain of C. clypeatus, actin labeling revealed the course of this tract (Fig. 7B1, C, 13A). After emerging from the olfactory lobes it courses antero-medially to meet its contralateral counterpart in a chiasm slightly dorsal to the central body (the chiasm is identified by the X in Fig. 6E, 7A–C). Its two arms then separate again to proceed antero-laterally to leave the brain via the protocerebral tract and to target the lateral protocerebrum (see below). Surprisingly, synapsin labeling provided evidence for synaptic material to be associated with two regions of the olfactory globular tract, namely a region within the chiasm (Fig. 6E), and a long section between the olfactory lobe and the chiasm (Fig. 3H, 7A2, B2, C; see also Fig. 13A). This section of the olfactory globular tract also displays strong RFir (Fig. 7A1). At a higher magnification it becomes clear that the regions of SYNir and RFir in this stretch of the olfactory globular tract almost completely overlap (Fig. 8) suggesting the presence of synapses with RFamide-like neuropeptides to be associated with the tract. This synaptic region may correspond to the olfactory globular tract neuropil as found in other Decapoda [52]. The olfactory globular tract is laterally accompanied by a small, spherical neuropil, the accessory olfactory globular tract neuropil (OGTNa) that displays SYNir but not RFir (Fig. 8, 13A).

Figure 8 Higher magnification of the section shown in Fig. 6A2, synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green); confocal laser scan microscopy. Peptidergic olfactory interneurons the somata of which are located in cell cluster (9) project their neurites through the median foramen (mF) into the olfactory lobe. In the medial antenna 1 neuropil, the lateral antenna 1 neuropil, and the non-columnar olfactory neuropils F, large, round RFamidergic profiles (putative peptidergic release sites) are embedded whereas in the posterior median protocerebral neuropil (PMPN) finer profiles are present. The glomeruli in the accessory neuropil (AcN) show both, synapsin- and RFamide-like immunoreactivity. Within the olfactory globular tract (OGT), a synaptic neuropil region is embedded that also shows RFamide-like immunoreactivity. The accessory olfactory globular tract neuropil (OGTNa) that is associated with the olfactory globular tract, shows synapsin immunoreactivity but not any peptidergic labeling. Full size image

Deutocerebrum: the olfactory neuropils

Two large clusters of olfactory interneurons are associated with the olfactory neuropils, cell cluster (10) with projection neuron somata, and cluster (9) that houses local olfactory interneurons (Fig. 3, 4). None of the projection neurons in cluster (10) displays 5HTir or RFir. Yet, in cluster (9) large populations of local interneurons are present that display strong 5HTir (Fig. 5B2, 9A, C) or RFir (Fig. 6D1, D2, 7A1, A2, 8) and the neurites of which extend into the core of the ON. We did not analyze if some cluster (9) neurons co-localize serotonin and RFamide. Between the ON and the lateral antenna 1 neuropil, the soma of at least one large serotonergic neuron is located (arrowheads in Fig. 6A, 9B, C) but the axonal projection could not be traced. Bundles of fine neurites of the cluster (9) interneurons enter the ONs from the medial side in a thick bundle that then branches out into finer bundles (Fig. 8, 9, 11B, C1, C2), in which fibers approach the proximal part of the olfactory glomeruli. In specimens processed for 5HTir, a large anterior (aB) and posterior bundle (pB) of cluster (9) neurites can be distinguished, where fibers spread out towards the bases of glomeruli (arrows in Fig. 9C).

Figure 9 A-C: Serotonergic innervation of the olfactory lobes. Double labeled sections showing glutamine synthetase-like immunoreactivity (GS; red) and serotonin immunoreactivity (5HT; green); confocal laser scan microscopy. A large population of serotonergic local interneurons in cell cluster (9) sends neurites into the core of the olfactory lobe (ON) by passing the median foramen (mF). This foramen is flanked by the non-columnar olfactory neuropils B and F the latter of which is connected by a neuropil bridge to neuropil E. The thick neurite bundles from cluster (9) interneurons, after entering the olfactory lobe, split into a large anterior (aB) and posterior bundle (pB). These bundles then branch out into finer bundles the fibers in which approach the proximal part of the olfactory glomeruli (arrows in C). Between the olfactory neuropil (ON) and the lateral antenna 1 neuropil (LAN), the soma of at least one large serotonergic neuron is located (arrowheads in B, C) but the axonal projection could not be traced. The dotted line in C encircles a blood vessel (BV). Full size image

As noted above, the ON is composed of numerous glomeruli that display strong SYNir and are arranged parallel to each other around the periphery of the lobe (Fig. 10, 11). Examining single optical sections from a z stack gives an idea of the dense packing of these glomeruli (Fig. 10A1, A2). They are elongate, cylindrical structures and their distal part is slightly larger than the proximal part. The glomeruli have a length of around 150 μm. A 3D reconstruction (Fig. 10B) confirmed that, as seen in tangential sections (Fig. 3A, 11A), the cross-sectional profile of these structures is more or less round. The diameter of the glomeruli in cross sections is around 20 μm. The periphery of the ONs is entirely packed with glomeruli with the exception of two spared spaces, the foramina, where fiber bundles enter or exit the lobes. These are the median foramen (mF; Fig. 3E, F, 4D, 8, 9A, C, 11B), through which the neurites from cluster (9) interneurons pass into the lobe and through which the olfactory globular tracts exits is, and the posterior foramen (pF), through which the axons of cluster (10) neurons enter the lobe (Fig. 3C, D, 4C; see also Fig. 1B). Double labeling for SYNir and RFir revealed a regionalization of the glomeruli. Whereas SYNir is present throughout the entire glomeruli, the cap region is devoid of RFir (terminology according to [59]). However, RFir is present in the base region and is particularly strong in the subcap region (Fig. 11B, C1, C2, D, E).

Figure 10 Immunolocalization of synapsin in the olfactory lobes shows that synaptic neuropil is confined to the olfactory glomeruli and the non-columnar olfactory neuropil (letters B, D, F). A1, A2: two optical sections (Apotome structured illumination technique) from different levels of one vibratome section. The non-columnar olfactory neuropils B and F are merged here and line the dorsal part of the median foramen. The non-columnar olfactory neuropil D is embedded within the olfactory glomeruli. The boxed area in A2 is shown in a higher magnification in C. This optical section (Apotome structured illumination technique) shows that there is not any overlap between the synaptic regions of both types of olfactory neuropils (columnar versus non-columnar). D shows a surface reconstructions obtained from a z-series of confocal images that were directly loaded into Amira and processed for semiautomatic segmentation using Amira's "wrap" module. Full size image

Figure 11 Horizontal vibratome sections to show the localization of the non-columnar olfactory neuropils and the regionalization of the olfactory glomeruli. Double labeling (C1-E) for synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green), or triple labeling for these two substances plus the nuclear marker (NUC; A, B); conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning (A, B) and confocal laser scan microscopy (C1-E). A: superficial, tangential section of the right olfactory lobe (compare inset and Fig. 3D) to show the localization of non-columnar olfactory neuropil A. Cell cluster (10) is also visible. B: Overview over the arrangement of non-columnar olfactory neuropils C-F and the medial foramen (mF). The inset shows a low power view of this section (compare Fig. 3D). C1, C2: these two images show the non-columnar olfactory neuropils in B at a higher magnification. The images are from a stack of 30 optical sections covering 22 μm in the z direction. Image C1 is a projection of 6 sections covering z = 9.1 μm to z = 12.9 μm. Image C2 is a projection of 6 sections covering z = 18.2 μm to z = 21.2 μm. The non-columnar olfactory neuropil C is merged with D. Both are closely associated with the proximal bases of olfactory glomeruli and in some places even seem to overlap with the glomeruli (Fig. 9A1, 10B-C). D: single optical section (0.7 μm) from this stack at z = 1.5 μm showing the non-columnar olfactory neuropil C. It is not possible to decide if neuropil C does in fact connect with the bases of the glomeruli. E: Single optical section (0.44 μm) from another specimen. The olfactory glomeruli are subdivided into a cap region (Cp) that shows only synapsin immunoreactivity (red) and a subcap (Sc) and base (Ba) region in which synapsin immunoreactivity overlaps with RFamide-like immunoreactivity (yellow/orange color). The boxed area is shown in a higher magnification in Fig. 11. Full size image

Apart from the glomeruli, a second type of neuropil in the ONs, the non-columnar olfactory neuropil (ncON), displays strong SYNir, 5HTir and RFir. Six patches of this diffusely structured neuropil can be reproducibly identified in different specimens and will be denoted with capital letters A-F in the following (see summary diagram Fig. 1B). Patch A of the ncON (Fig. 3B, 4B, 11A) is seen in ventrally located tangential sections of the ON in a medio-posterior position to the main ON. It stretches further dorsally towards the point where the extra olfactory lobe joins the main lobe (Fig. 4B). Patch B is located anteriorly to the median foramen (Fig. 3G, 8, 9A, C). In sections dorsal to the median foramen, it merges with patch F that is located posterior to this foramen. The merged patches B and F thus line the dorsal part of the median foramen (Fig. 3H, 5B2, 8, 9A, C, 10A1, A2, 11, 13A). Hence, it appears that the thick bundles of cluster (9) interneurons on passing into the olfactory lobe are surrounded by ncON. Patch C of the ncON is closely linked to patch D by a broad neuropil bridge. Both patches are located antero-laterally in the ONs and are flanked by the columnar neuropil (Fig. 3C–H, 4C, D, 7B2, 9C, 10A1, A2, C, 11B–E). Patch E of the ncON is associated with the posterior foramen from where it stretches further dorsally, there being embedded within columnar neuropil (Fig. 3D, E, 4D, 11B, C1, C1, 9C). Patch E is linked to patch F by a neuropil bridge at the level of those sections that show the median foramen (Fig. 9F). Patches C-E are located very close to the proximal bases of olfactory glomeruli and in some places even seem to merge with the glomeruli (Fig. 10A1, 11B–C). However, analyses of single optical sections (Fig. 10C, 11D) could not unequivocally answer the question if fiber connections between the ncON and the glomeruli exist. Double labeling of the ncON showed numerous round RFir swellings to be embedded within the SYNir neuropil (Fig. 11B–E). However, close inspection of single confocal sections failed to show a co-localization of both labels (Fig. 12) suggesting that, if RFamide-like peptides are released into the ncON, synapsins are not associated with these release sites (at least as far as detectable with our methods).

Figure 12 Higher magnification of the non-columnar olfactory neuropil (merged patches C/D) shown in Fig. 10E. Double labeling for synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green); confocal laser scan microscopy, single optical section (0.44 μm). Numerous round RFamide-like immunoreactive swellings of ca 10 μm diameter, putative non-synaptic peptide release sites, are embedded within the synapsin immunoreactive neuropil, but both labels are not co-localized. Full size image

Deutocerebrum: other neuropils

The median antenna 1 neuropil (MAN) extends across the brain posterior to the protocerebrum behind the cerebral artery and is on both sides flanked by the arms of the olfactory globular tract (Fig. 6D1, D2, 7A1, A2, 8). Anteriorly, it seems to be continuous with the protocerebral neuropils. It displays both, strong SYNir and Rfir, the latter being distributed in rather coarse profiles (Fig. 6D2, 8). The lateral antenna 1 neuropil (LAN) in decapod crustaceans is known to receive afferents from the mechanoreceptors of the antenna 1 (Sandeman et al. 1992, 1993). In C. clypeatus, it caudally adjoins the median antenna 1 neuropil (Fig. 3F–H, 4D–F, 5A, B, 6A, 7A1, 7, 9A–C, 13A) and in some sections seems to be connected to this neuropil (Fig. 3F, 4F, 5A, 7A2). Similar to the non-columnar olfactory neuropil, large, round RFir profiles are embedded in the SYNir neuropil of this structure (Fig. 8).

The accessory neuropil (AcN) or accessory lobe is another conspicuous feature of the C. clypeatus deutocerebrum. It is located medially to the ON, close to where the olfactory globular tract exits the latter (Fig. 3G, H, 4E, F, 6D1, D2, 7, 8, 13A). The accessory lobe is composed of about 50–80 small, spherical glomeruli, all of which have a diameter of around 10–15 μm (Fig. 13B–D). In specimens processed for SYNir, the synaptic areas of the glomeruli appear well separated from each other and the glomeruli do not show any recognizable substructures (Fig. 13C–E). Actin labeling reveals a bundle of fibers extending anteriorly from the accessory lobe towards the olfactory globular tract (Fig. 13B). However, we were not able to determine if this fiber bundle connects to the latter tract. Fig. 13F shows a specimen processed for GSir. The arrow identifies the soma of a glia cell, the processes of which branch and associat with several glomeruli in the accessory lobe.

Figure 13 Details of the accessory lobe (AcN); confocal laser scan microscopy. A: Double labeling for synapsin immunoreactivity (SYN; green) and actin (ACT; red). The accessory lobe is located close to the exit point of the olfactory globular tract (OGT) from the olfactory lobe. Other abbreviations: F patch F of the non-columnar olfactory neuropil, LAN lateral antenna 1 neuropil, OGTNa accessory olfactory globular tract neuropil. B: Histochemical localization of actin (ACT) shows two fiber bundles (arrows) that extend from the accessory lobe towards the olfactory globular tract (OGT) but seem to pass underneath it. Color coded three dimensional visualization of a confocal stack, use red-green glasses to view. C: Color coded three dimensional visualization of a confocal stack (use red-green glasses to view) of a synapsin labeled specimen shows that the accessory lobes is composed of an array of evenly spaced spherical glomeruli. D: a surface reconstruction of four glomeruli obtained from a z-series of confocal images (synapsin immunoreactivity) that were directly loaded into Amira and processed for semiautomatic segmentation using Amira's "wrap" module. E: single optical section of the glomeruli (synapsin labeling) to show the regular structure of the synaptic neuropil within the glomeruli. F: Double labeling for serotonin immunoreactivity (5HT; green) and glutamine synthetase-like immunoreactivity (GS; red). A single glutamine synthetase positive cell (arrow) just adjacent to the lateral antenna 1 neuropil (LAN) extends branches towards and penetrates into several glomeruli of the accessory lobe. Full size image

Tritocerebrum

The antenna 2 neuropil (AnN) receives afferent input from the second antenna but also provides motor innervation for the muscles that move this antenna [52]. It is located posteriorly to the lateral antenna 1 neuropil and is visible in the middle of the section stacks (Fig. 4E, F, 5D, 6B1, B2, 7A, 10C, 14). With neither SYNir nor RFir could we recognize any compartmentalization or substructures in this neuropil (Fig. 14). Yet, once again we observed large and very prominent RFir swellings in the neuropil suggesting the presence of peptide release sites (Fig. 14 inset).

Figure 14 Details of the antenna 2 neuropil (AnN). Double labeling for synapsin immunoreactivity (SYN; red, A1) and RFamide-like immunoreactivity (RF; green, A1, A2). Note that in addition to numerous small peptidergic profiles which are embedded within the synaptic neuropil, several large peptidergic profiles are present that may represent non-synaptic release sites (inset). Full size image

Eyestalk neuropils: the lateral protocerebrum – medulla terminalis and hemiellipsoid body

Apart from the medial portion of the brain in the head capsule, another substantial part of the brain is located within the eyestalks. Fig. 15A, B and 15D feature images of the median brain and the eyestalk neuropils reproduced at the same scale to make the point that specifically one of the eyestalk neuropils, the hemiellipsoid body, almost matches the size of the olfactory lobes (ON). The medial protocerebrum is connected to the eyestalk neuropils via the protocerebral tract (PT; Fig. 15B), which includes the olfactory globular tract that ascends from the deutocerebrum. The eyestalks contain the lateral protocerebrum which is composed of the medulla terminalis (MT) and the hemiellipsoid body (HE). Furthermore, the four optic neuropils lamina (La), medulla (Me), lobula (Lo) and a small proximal lobula neuropil (LoP) are enclosed in the eyestalks (Fig. 15A–D, 18A). Because the spatial arrangement of the eyestalk neuropils is rather complicated, Fig. 15 presents low power views of four different specimens to demonstrate these relationships and also to provide an idea of the level of structural variation between individuals.

Figure 15 The eyestalk neuropils as shown in vibratome sections that were double labelled (E, F) for synapsin immunoreactivity (SYN; red) and RFamide-like immunoreactivity (RF; green), or triple labeling for these two substances plus the nuclear marker (NUC; A-E2); conventional fluorescence combined with the Apotome structured illumination technique for optical sectioning (A-E2) and confocal laser scan microscopy (E, F). A, B and D are rendered at the same scale to compare the size of a median hemi brain (A; see also Fig. 3D) with that of the eyestalk neuropils. The eyestalks contain the lateral protocerebrum which is composed of the medulla terminalis (MT) and the hemiellipsoid body (HE). Furthermore, the four optic neuropils lamina (La), medulla (Me), lobula (Lo) and lobula plate are enclosed in the eyestalks. The low power views presented in B-E2 show four different eyestalks to show the individual variation between specimens. The hemiellipsoid body (HE; encircled by a dotted line in B) is a large spherical neuropil that is associated with a compact, laterally situated cluster of densely packed neurons, the lateral protocerebral interneurons (LPI). With the synapsin label, several neuropil compartments are visible within the hemiellipsoid body, the peripheral cap neuropil (Cap) which is separated by the unlabelled intermediate layer 1 (IL 1) from the strongly synapsin immunoreactive core neuropil 1 (Co1). A second unlabeled intermediate layer (IL 2) separates core neuropil 1 from the more proximally located core neuropil 2 (Co2). Asterisks in B-E2 and F mark the point where the two intermediate layers meet. The boxed area in C1 is shown at a higher magnification in E. The demarcation between the medulla terminalis (MT) and the hemiellipsoid body is difficult to draw. The opposed arrows in B-E tentatively mark the border between these two structures. From the medulla terminalis, strongly labeled RFamide-like immunoreactive fibers invade the core neuropils 1 and 2 (Co1, Co2) of the hemielliposid body where they terminate in a circumscribed field with very fine varicosities (E, F). Intermediate layers IL1 and IL2 are unlabeled. The arrows in F identify patches of neuropil that seem to extend across the intermediate layer 1. Full size image

This paragraph will focus on the neuropils that constitute the lateral protocerebrum, the closely associated hemiellipsoid body (enclosed by dots in Fig. 15B) and the medulla terminalis (MT). The lobula ontogenetically derives from the medulla terminalis and hence is part of the lateral protocerebrum [60]. Nevertheless, in the present account it will be described together with the other two visual neuropils. The demarcation between the medulla terminalis and the hemiellipsoid body is difficult to draw and we will here use the RFir as a somewhat arbitrary landmark. The opposed arrows in Fig. 15B–E mark the border between the medulla terminalis which is filled by a loose network of peptidergic fibers and the hemiellipsoid body that displays strong SYNir. A cluster of RFir cell bodies flanks the medulla terminalis laterally and probably gives rise to at least some of the RFir innervation of this neuropil (inset in Fig. 16A2). In low power views, strongly RFir fibers in the protocerebral tract (Fig. 15B) seem to spread out and give rise to an intense peptidergic innervation of the medulla terminalis. At a higher magnification, the protocerebral tract carries only few distinct RFir fibers (Fig. 6D1, arrows in Fig. 16A2). Yet, even higher magnifications of the protocerebral tract close to the medulla terminalis (see frame in Fig. 16A2) reveals an extensive network of very fine RFir fibers within this tract (Fig. 16E). We could not determine if this network represents efferent fibers from the median brain to the lateral protocerebrum or if they originate from the local RFir neurons associated with the medulla terminalis.

Figure 16 Details of the hemielliposid body and the medulla terminalis; triple labeling for synapsin immunoreactivity (SYN; red), RFamide-like immunoreactivity (RF; green), plus the nuclear marker (NUC) shown in conventional fluorescence combined with the Apotome structured illumination technique (A1, B) and confocal laser scan microscopy (A2, C-E). A1: a massif cluster of lateral protocerebral interneurons (LPI) is associated with the hemiellipsoid body (HE). The dotted line demarks the field of strong innervation with peptidergic neurites. The medulla terminalis (MT) is filled with a loose meshwork of peptidergic fibers. The boxed area is shown at a higher magnification in B. A2: same specimen as in A1 but visualized with confocal microscopy and showing only the RFamide channel. Arrows identify single, peptidergic fibers within the protocerebral tract (PT). Peptidergic interneurons are associated with the medulla terminalis (inset). The boxed areas d and e are shown at a higher magnification in D and E. B: Already at moderate magnification, the core neuropil (Co1) displays a layered appearance. The dotted line demarks the field of strong innervation with peptidergic neurites. The cluster of lateral protocerebral interneurons is also visible (LPI) as are single cell nuclei (presumably endothelial cells of blood vessels) within the core neuropil. C: The medulla terminalis (MT) is filled with a loose meshwork of peptidergic fibers. D: Higher magnification of the boxed area in A2. Both the cap and core 1 neuropils are filled with numerous very small RFir profiles some of which are arranged in a string (see insets) suggesting an intense peptidergic innervation of these neuropils. E: higher magnification of the boxed area in A2 showing a meshwork of very fine peptidergic fibers within the olfactory globular tract close to its entrance into the medulla terminalis/hemiellipsoid body complex. Full size image

The hemiellipsoid body is a large (ca. 300 μm in diameter), spherical neuropil that is associated with a compact, laterally situated cluster of densely packed neurons, the lateral protocerebral interneurons (LPI; Fig. 15A–E, 16A1, B, 17A–C). With the synapsin label, several neuropil compartments are visible within the hemiellipsoid body. The peripheral cap neuropil is separated by the unlabelled intermediate layer 1 (IL 1) from the strongly SYNir core neuropil 1 (Co1). A second unlabeled intermediate layer (IL 2) separates core neuropil 1 from the more proximally located core neuropil 2 (Co2; Figs. 15, 16, 17). We suggest that the unlabelled intermediate layers 1 and 2, which have a common origin (asterisks in Fig. 15B, C1, D, E1, F, 17B), may be the sites where the fibers of the lateral protocerebral interneurons penetrate into the hemiellipsoid body. From the medulla terminalis, strongly labeled RFir fibers invade the core neuropils 1 and 2 of the hemielliposid body, where they terminate in a circumscribed field with very fine varicosities (Fig. 15E). This field is labeled with a dotted line in figures 16A1, B, 17D4). Apart from this strongly labeled field, high power views of the cap and those regions of the core neuropils that in low power views did not seem to display RFir, nevertheless demonstrate a weak but distinct peptidergic innervation. Fig. 16D demonstrates that both the cap and core 1 neuropils are filled with numerous very small RFir profiles, some of which are arranged in a string, suggesting an intense peptidergic innervation of these neuropils. This string-like appearance is even more apparent in tangential sections (Fig. 17; see below). Already at low magnification, in preparations with SYNir, the core neuropils one and two have a layered appearance (Fig. 15E1, 16A1). Higher power views reveal that not only the core but also the cap neuropils are clearly organized into parallel layers, or lamellae (Fig. 16B, 17D2–D4). Fig. 17B shows a transverse section of the hemiellipsoid body, the position of which is indicated in Fig. 17A. This section nicely demonstrates the arrangement of the intermediate layers 1 and 2 (the dotted lines delineates a damaged region of the tissue). Furthermore, the extensive cluster of lateral protocerebral interneurons is seen to stretch around both sides of the hemiellipsoid body. Fig. 17D1–D4 shows a series of tangential optical sections through the hemiellipsoid body, the positions of which are indicated in Fig. 17A. Section D1 is the most superficial and shows the cap region as well as the intermediate layer 1. Section D2 is slightly deeper and touches the upper part of core neuropil 1. The insets in sections D1–D3 demonstrate the string-like arrangement of tiny RFir profiles in the cap neuropil, and the inset in D4 shows similar profiles in the core neuropil 1. In sections D3 and D4 strands of lightly SYNir material (arrows in D3) seem to span across the intermediate layer 1 thus connecting the core neuropil 1 and the cap neuropil (see also arrows in Fig. 15F). This hemiellipsoid body sector, which is strongly invaded by RFir fibers, is identified by a dotted line in section D4. Sections with the nuclear counter stain reveal the presence of cell nuclei that are interspersed in the core 1 neuropil (Fig. 16B, 17C). These nuclei presumably belong to endothelial cells of blood vessels. In addition the interface between intermediate layer 1 and core neuropil 1 is lined with cell nuclei (arrows in Fig. 17C).

Figure 17 High power views of the hemiellipsoid body to reveal the lamellar organization of the cap and core neuropils. Triple labeling for synapsin immunoreactivity (SYN; red), RFamide-like immunoreactivity (RF; green), plus the nuclear marker (NUC) shown in conventional fluorescence combined with the Apotome structured illumination technique (A-C) and confocal laser scan microscopy (D1–D4). A: the position of sections D and D1–D4 is indicated here. The cluster of lateral protocerebral interneurons (LPI) is identified. B: transverse section of the hemiellipsoid body demonstrating the arrangement of the cap (Cap), core 1 (Co1) and core 2 (Co2) neuropils as well as the intermediate layers 1 and 2 (IL1, IL2; the dotted lines delineates a damaged region of the tissue). The extensive cluster of lateral protocerebral interneurons (LPI) is seen to stretch around both sides of the hemiellipsoid body. C: A tangential section with the nuclear counter stain reveals the presence of cell nuclei that are interspersed in the core 1 neuropil, presumably belonging to endothelial cells of blood vessels. In addition, the interface between intermediate layer 1 and core neuropil 1 is lined with cell nuclei (arrows). The extensive cluster of lateral protocerebral interneurons (LPI) stretches around the hemiellipsoid body. D1–D4: a series of tangential optical sections through the hemiellipsoid body (the positions are indicated in A). Note the lamellar organization of the cap and core neuropil. The entire image stack is composed of 29 optical sections of 0.76 μm thickness covering z = 21.2 μm. The four single images are projections of 3 optical sections covering z = 0 – 1.5 μm (D1; the most superficial section), z = 3.8 – 5.3 μm (D2), z = 9.1 – 10.6 μm (D3), and z = 18.2 – 19.7 μm (D4). The Cap (Cap) and core 1 (Co1) neuropils are visible as well as the intermediate layer 1 (IL1). The insets in sections D1–D3 demonstrate the string-like arrangement of tiny RFir profiles in the cap neuropil which are arranged parallel to the lamellae, and the inset in D4 shows similar profiles in the core neuropil 1. In sections D3 and D4, strands of lightly synapsin immunoreactive material seem to span across the intermediate layer 1 (arrows in D3). The dotted line in D4 identifies that sector of the core 1 neuropil that is strongly invaded by RFir fibers. Full size image

The eyestalk neuropils: lamina, medulla, lobula (optic neuropils)

In decapod crustaceans, the visual input from the compound eyes is processed in three columnar optic neuropils, the lamina (lamina ganglionaris according to the older terminology), the medulla (medulla externa) and the lobula (medulla interna) all of which are enclosed in the eyestalk (compare Fig. 15, 18). Recent studies provide evidence for a fourth neuropil in crabs associated with the lobula, the lobula plate [61]. All neuropils can be identified by SYNir in C. clypeatus (Fig. 18A) but without any additional markers that would allow the visualization of the fiber composition it is not possible at the moment to decide if the additional proximal lobula neuropil in C. clypeatus corresponds to the lobula plate of brachyuran crabs. As in Drosophila, the labeling in the lamina is much weaker with the SYNORF antibody than in the other neuropils (Fig. 18A, B). SYNir identifies the plexiform layer of the lamina. In the projection of a confocal image stack the geometrical layout of the lamina is visible reflecting the arrangement of optic cartridges (Fig. 18B). Furthermore, a regular pattern of small RFir profiles is present in the plexiform layer of the lamina (Fig. 18C). The optic neuropils are surrounded by a layer of visual interneurons that we did not chart in any detail (inset in Fig. 18A). Many cell bodies are also present between the lamina and the medulla (Fig. 18C, D, 19), and the medulla and lobula (Fig. 19). The arrangement of cell nuclei between the lamina and the medulla, in a kind of negative image, reveals the course of fiber bundles that connect the lamina and the medulla (arrows in Fig. 18C, 19D1, D2).

Figure 18 The optic neuropils. A: overview (photomontage, confocal laser scan microscopy), vibratome section showing synapsin immunoreactivity (SYN; green in the inset), plus the nuclear marker (NUC; blue in the inset only; Apotome structured illumination technique). The three retinotopic neuropils from distal to proximal are the lamina (La), medulla (Me), Lobula (Lo) which is associated with an additional proximal lobula neuropil (LoP). The inset shows that the optic neuropils are surrounded by a cortex of neuronal somata. The boxed area is shown in E in a higher magnification. B: same specimen as A; maximum projection of several confocal sections to show the plexiform layer of the lamina. C, D: Triple labeling for synapsin immunoreactivity (SYN; red), RFamide-like immunoreactivity (RF; green), plus the nuclear marker (NUC) shown in conventional fluorescence combined with the Apotome structured illumination technique. In these tangential sections of the medulla, a regular arrangement of the labeled profiles signifies the ordered, retinotopic organization of this neuropil. E: higher magnification of the boxed area in A. In this image, contrast and brightness levels were artificially elevated, so that unspecific background staining reveals the presence of the inner optic chiasm, a cross-over of the fibers that connect the medulla and the lobula (enclosed between the arrows). Full size image

Figure 19 A, B: lobula (see A) and medulla (Me) and lobula (Lo; B1, B2); triple labeling for synapsin immunoreactivity (SYN; red), RFamide-like immunoreactivity (RF; green), plus the nuclear marker (NUC) shown in conventional fluorescence combined with the Apotome structured illumination technique. In the medulla, RFir is localized in three distinct parallel layers. In the lobula (see A), fourteen different layers can be recognized with this technique. Arrowheads label a conspicuous weakly labelled layer. Double arrows identify proximally located, large RFir profiles associated with the lobula. Arrows identify RFir somata of visual interneuons. C1–C3: lobula (Lo) and medulla (Me); triple labeling for serotonin immunoreactivity (5HT; green), glutamine synthetase-like immunoreactivity (RF; red), plus the nuclear marker (NUC; blue) shown in conventional fluorescence combined with the Apotome structured illumination technique. Arrowheads label a conspicuous weakly labelled layer (compare B2). D1–D3: lamina (Lo) and medulla (Me); triple labeling for serotonin immunoreactivity (5HT; green), glutamine synthetase-like immunoreactivity (RF; red), plus the nuclear marker (NUC; blue) shown in conventional fluorescence combined with the Apotome structured illumination technique. Arrows in D1 label the course of fiber bundles that link the lamina and the medulla. Arrows in D2 identify serotonergic somata located between the lamina and the medulla. Double arrows in D2 label serotonergic neurons associated laterally with the medulla. Full size image

In synapsin labeled preparations, it becomes clear that the medulla and lobula are composed of several parallel layers (Fig. 15, 18a). Darker, irregularly arranged areas in the medulla and lobula neuropil presumable show the course of blood vessels (Fig. 18a). A small but distinct additional neuropil is proximally associated with the lobula (LoP; Fig. 18A and inset). In tangential sections of medulla labelled for SYNir and RFir, a regular arrangement of the labeled profiles signifies the ordered, retinotopic organization of the medulla (Fig. 18D). In cross sections of the medulla, RFir is clearly localized in three distinct parallel layers (Fig. 19B1, B2). This neuropil is also strongly innervated by a cluster of serotonergic visual neurons located at the side of it (double arrow in Fig. 19D2). Additionally, serotonergic somata are located in the cell group between the lamina and the medulla (arrows in Fig. 19D2). Within the medulla neuropil, 5HTir is also arranged in parallel layers although less distinct than RFir (Fig. 19C2, C3). Cell somata with strong GSir, presumably ensheathing glia cells, surround the medulla laterally and distally and give rise to a strong glutamine synthetase signal within the neuropil (Fig. 19C1, D1). In an image with SYNir, in which the contrast and brightness levels were artificially elevated, unspecific background staining reveals the presence of the inner optic chiasm, a cross-over of the fibers that connect the medulla and the lobula (enclosed between the arrows in Fig. 18E). GSir is also strong in the third optic neuropil, the lobula. Within the neuropil, GSir shows the layered appearance of the lobula (Fig. 19C1, C3) that is also apparent with RFir (Fig. 19A, B1, B2) and 5HTir (Fig. 19C2). At least fourteen layers could be identified with RFir and SYNir but we did not analyze the layering in more detail (Fig. 19A). With all three markers, one conspicuous layer in the lobula is devoid of labeling (arrowheads in Fig. 19A, B1, B2, C1–3). A population of weakly labelled RFir cell somata is associated proximally with the lobula (single arrows in Fig. 19B1). Strongly RFir profiles line the most proximal neuropil layer of the lobula (double arrows in Fig. 19A, B1).