Expression of iaEPO and iaEPOR in the central nervous system of Hirudo medicinalis

Figure 2: Frontal sections through the midbrain of a mouse labelled with anti-EPOR serum (red fluorescence) and the nuclear marker DAPI (blue fluorescence). a-a``: Overview of a midbrain section with a distinct, homogeneous and cytoplasmic expression of EPOR-like immunoreactivity in several cells. b-d: Detailed view of EPOR-immunopositive cells with neuronal morphology.

Sections of mouse midbrains were also labelled with anti-EPOR serum and several cells with a distinct, homogeneous, cytoplasmic expression of EPOR immunoreactivity were detected (Figure 2). Detailed analysis (Figure 2b-d) revealed a typical neuronal morphology.

2.3.2 Expression of iaEPO and iaEPOR in the central nervous system of Hirudo medicinalis

The central nervous system of the leech H. medicinalis comprises 32 ganglia, 4 of which are fused to the head ganglion and 7 to a tail ganglion. In between, 21 segmental ganglia are individually iterated along paired connectives (Nicholls & Essen 1974). Horizontal sections through the head ganglion and the segmental ganglia were labelled with anti-EPO (Figure 3) and anti-EPOR serum (Figure 4).

For iaEPO immunostaining segmental ganglia of leeches were cut horizontally with the vibratome into slices of 40µm thickness and labelled with the anti-EPO serum. EPO-like immunoreactivity was detected in serveral neuronal cells throughout all segmental ganglia.

Figure 3a displays a section through a hemiganglion with two iaEPO-immunopositive cells (higher magnification in Figure 3a`). One of these cells shows a strong immunoreactivity in

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the soma and weak immunoreactivity in its primary neurite (Figure 3a`, asterisk). The other cell contains EPO-like immunoreactivity concentrated in the vicinity of the cell membrane of the soma and its projections (Figure 3a`, arrowhead). In addition, fine and extensively branching fibres in the central neuropil region of the ganglion are distinctly labelled. Other examples of cells from different segmental ganglia that contained iaEPO immunoreactivity are summarised in Figure 3b-d.

Figure 3: Horizontal sections through segmental ganglia of the leech Hirudo medicinalis labelled with anti-EPO serum. a, a`: Section through one hemiganglion with two iaEPO-immunopositive cells. Higher magnification in a`

reveals one cell with an intensive cytoplasmic staining (asterisk) and one cell with staining restricted along the outer cell membrane (arrowhead). b-d: Detailed view of other iaEPO-immunopositive cells.

For iaEPOR immunoreactivity, the head ganglion of leeches was embedded in paraffin wax and horizontally cut into slices of 8µm thickness (Figure 4). A pair of strongly iaEPOR-immunopositive cells was detected in the most anterior part of the head ganglion, the supraesophageal ganglion (Figure 4a, arrowheads, higher magnification in 4a`, a``). Other iaEPOR-immunopositive cells were detected in the more posterior part of the head ganglion, the subesophageal ganglion (Figure 4b, c). All cells that contained EPOR-like immuno-reactivity had a distinct, homogeneous cytoplasmic staining.

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Figure 4: Horizontal section through the head ganglion of the leech Hirudo medicinalis labelled with anti-EPOR serum. a-a``: White arrowheads mark two iaEPOR-immunopositive cell bodies that are displayed at a higher magnification in a` and a``. b, c: Further examples of neuronal cells in the subesophageal ganglion that express iaEPOR immunoreactivity.

SupraEG: supraesophageal ganglion, SubEG: subesophageal ganglion

2.3.3 Expression of iaEPO and iaEPOR in the central nervous system of Procambarus spec.

Brain sections of the crayfish Procambarus spec. were labelled with anti-EPO serum and anti-EPOR serum and the immunoreactivity was either visualised with a diaminobenzidine (DAB) reaction (EPO, Figure 5) or fluorophore-coupled streptavidin (EPOR, Figure 6).

EPO-like immunoreactivity was detected both in fibres invading neuropil regions and in cell bodies of different cell types. Figure 5a and 5a` show a frontal section through a hemibrain with iaEPO-immunoreactive fibres that project in-between the olfactory and accessory lobe to central regions of the protocerebrum. Furthermore, iaEPO immunoreactivity with different patterns of distribution was detected in a number of cell bodies (Figure 5b-d). Various iaEPO-immunopositive cells showed a distinct staining in close association with the cell membranes of somata and neurites (Figure 5b, arrowhead). In contrast, entirely labelled cells with small somata (7-11µm) seemed to enclose the cell bodies of larger unlabelled cells (Figure 5c, arrowheads). A third type of iaEPO-immunopositive cells contained homogeneous staining throughout its entire cytoplasm (Figure 5d, arrowhead).

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Figure 5: Frontal sections through the brain of Procambarus spec. labelled with anti-EPO serum (brown colour) and methylenblue. a, a`: Section through a hemibrain with iaEPO-expressing fibres in the deutocerebrum. The dashed line in a marks the region that is presented at higher magnification in a`. b: iaEPO-immunopositive cells with intensive staining associated with the membranes of somata and neurites (arrowhead). c: iaEPO-immunoreactive cells with small somata (arrowheads) enclose other cell bodies with labelled processes.

d: iaEPO-immunopositive cell with homogeneous cytoplasmic staining (arrowhead).

OL: olfactory lobe, AL: accessory lobe

Figure 6: Frontal sections through the brain of Procambarus spec. labelled with anti-EPOR serum (red fluorescence) and the nuclear marker DAPI (blue fluorescence). a-a``: Section through a hemibrain showing an iaEPOR-immunolabelled cell body with a homogeneous cytoplasmic staining that is presented at higher magnification in a`. An iaEPOR-immunopositive fibre in the protocerebrum is presented at higher magnification in a``. b, c: Other examples of cells that displayed iaEPOR immunoreactivity located in the tritocerebrum of the brain.

OL: olfactory lobe, AL: accessory lobe, CB: central body

For iaEPOR immunohistochemistry the brain sections of Procambarus spec. were labelled with anti-EPOR serum (red fluorescence) and the nuclear marker DAPI (blue fluorescence).

Figure 6a provides an overview of one section through a hemibrain with an iaEPOR-immunopositive cell located in a cluster of unlabelled cells lateral to the olfactory and accessory lobe (higher magnification in Figure 6a`). This cell has a homogeneous staining in

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the cytoplasm of the soma. Furthermore, iaEPOR expressing fibres project posteriorly to the central body (higher magnification in Figure 6a``). Other cells that contained homogeneous cytoplasmic staining of iaEPOR were detected in the tritocerebrum of the brain (Figure 6b, c).

2.3.4 Expression of iaEPO and iaEPOR in the central nervous system of