Central projections of neurons in the lower unit complex of the anterior optic tubercle (AOTU-LUC).

<p>(A-E) Neurobiotin (NB) injection into the AOTU-LUC reveals central projection areas. Cartoon in A illustrates injection site. (A-C) Direct volume rendering from projections in the contralateral AOTU-LUC shows three morphologically different types of TuTu1 neuron (TuTu1a, TuTu1b, TuTu1c). (A’-C’) Maximum intensity projections of six (A’) or three (B’, C’) adjacent slices from preparations shown in A-C, combined with anti-synapsin labeling (syn-ir, grey). (A’) TuTu1a neurons connecting only the dorsal areas of the AOTU-LUC. (B’, C’) Two similar types of TuTu1 neuron with ramifications in the ventral and median areas of the AOTU-LUC. These neurons had dense (green arrows) and sparse (white arrows) ramification areas. While TuTu1b neurons ramified densely within their dorsal, and sparsely within their ventral branching areas (B’), the opposite was true for TuTu1c neurons (C’). (D, E) Projections of TuLAL1 neurons from the AOTU-LUC to the medial and lateral bulb. (D) Overview (direct volume rendering) shows course of the axons which run within the AOTU-LAL tract. The tract separates into two fascicles that innervate the lateral or the medial bulb (LBU, MBU), respectively. The cell bodies of TuLAL1 neurons were located medially of the AOTU (asterisk). Also stained are axons of TuTu1 neurons that run in the intertubercle tract (ITT). (E) Direct volume rendering of large synaptic terminals of TuLAL1 neurons within the median and the lateral bulbs. (E’) Maximum intensity projection of three adjacent slices of preparation shown in E combined with anti-synapsin labeling (grey) illustrates the projection areas of TuLAL1 neurons with respect to the central complex. AL, antennal lobe; CBU, central body upper division; CBL, central body lower division; UU, upper unit of AOTU. All views in frontal plane. Scale bars: 30 μm in A-C; 100 μm in D; 50 μm in E.</p

Transmedulla neurons: Ramifications within the medulla.

<p>Tracer injection into the lower unit of the anterior optic tubercle labels transmedulla neurons within a thin layer of the dorsal half of the medulla (ME) which extend into the dorsal rim area of the medulla (MEDRA). (A) Direct volume rendering of dextran Texas Red labeling superimposed on synapsin-ir slice. The neurons have their cell bodies at the distal face of the medulla (asterisk), branch in the MEDRA, run through a thin layer within the medulla and enter the 2nd optic chiasm. (B) Transmedulla neurons, labeled through Dex-TR injection into the AOTU-LUC, lie in the same layer (arrows) as a narrow band of synapsin-ir (cyan). (C) Higher magnification/resolution shows that synapsin-ir punctae are next to, but not identical to swellings of the transmedulla neurons, suggesting synaptic input onto the latter. (D-F) Direct volume rendering of two neurobiotin-injected sibling transmedulla neurons. (D) Extracellular iontophoretic injection of Neurobiotin into the AOTU-LUC labeled with streptavidin-Cy3 shows two sibling transmedulla. From an extensive meshwork of branches within the MEDRA, a single, unbranched neurite runs in dorsoventral direction through the medulla. (E, F) Higher magnification/resolution images of MEDRA (E) and a more ventral part of the neurite running dorsoventrally through the medulla (F). Arrows indicate small processes both medially and laterally of the neurite. LA, lamina; LO, Lobula. All views in frontal plane. Scale bars: 100 μm in A, B, and D; 20 μm in C, E, and F.</p

Absence of PDF-, 5HT, and GABA-ir in the lower unit complex of the anterior optic tubercle (AOTU-LUC).

<p>Synapsin-immunoreactivity (syn-ir, magenta) and immunoreactivity to antisera against PDH (PDH-ir), 5HT (5HT-ir) and GABA (GABA-ir), shown in green, in the anterior optic tubercle. (A) The entire AOTU is devoid of PDH-ir. (B) 5HT-ir was found in the upper unit of the anterior optic tubercle (UU), but not in the AOTU-LUC. (C) GABA-ir, green was found in the AOTU-UU, but not in the AOTU-LUC. AL, antennal lobe. All views in frontal plane. All scale bars: 30 μm.</p

Spatial relationship between transmedulla neurons and GABA-ir fibers.

<p>(A) Confocal image of GABA-ir neurons (green) and transmedulla neurons stained by dextran Texas Red injection into the AOTU-LUC (magenta). GABA-ir is found throughout the medulla, but is more concentrated in some layers than in others. (B, C) Higher magnification/resolution images of the areas indicated in A show close proximity, but no colocalization of the two stainings. (B) GABA-ir is also present within the dorsal rim area of the medulla (MEDRA). (C) Varicose GABA-ir is in close proximity to the transmedulla neurons. ME, medulla. All views in frontal plane. Scale bars: 100 μm in A; 30 μm in B, C.</p

Transmedulla neurons: Ramifications within the lower unit complex of the anterior optic tubercle (AOTU-LUC).

<p>(A) Confocal image of synapsin-ir (syn-ir, magenta) and f-actin labeling (phalloidin, green) of the anterior optic tubercle. The AOTU-LUC has been previously divided into the lateral unit (lat. U) and the ventrolateral unit (vlat. U) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143244#pone.0143244.ref055" target="_blank">55</a>]. Synapsin/phalloidin labeling reveals that these two units are further structured into a complicated assembly of multiple small subcompartments. B-D) Direct volume rendering of AOTU-LUC projections from transmedulla neurons labeled through dextran Texas Red (Dex-TR) injection into the MEDRA. In each sample a different combination of focal projection areas is stained. Cartoon in (B) illustrates injection site. B’-D’) Single confocal sections (B’, C’) and maximum intensity projection of 10 adjacent slices (D’) of the neurons shown in B-D, combined with anti-synapsin labeling (grey). Projections from MEDRA neurons are exclusively found in the AOTU-LUC. AOT, anterior optic tract; UU, upper unit of the anterior optic tubercle. All views in frontal plane. All scale bars: 30 μm.</p

Spatial relationship between transmedulla neurons and 5HT-ir fibers.

<p>(A) Confocal image of 5HT-immunoreactive (5HT-ir) neurons (green) and transmedulla neurons labelled by dextran biotin/streptavidin Texas Red (magenta). Both stainings are restricted to the dorsal and medial parts of the medulla. (B, C) Higher magnification/resolution images of the areas indicated in A show close proximity, but no colocalization of the two stainings. (B) The dorsal rim area of the medulla (MEDRA) is devoid of 5HT-ir. (C) Transmedulla neurons overlap with the band of 5HT-ir, but the latter is wider and extends more medially. ME, medulla. All views in frontal plane. Scale bars: 100 μm in A; 30 μm in B, C.</p

Summary of the main findings.

<p>Frontal schematic diagram of the honeybee brain illustrates the main neuropils and the sky compass pathway. The dorsal rim area of the eye (DRA) is connected to the dorsal rim area of the medulla (MEDRA) through long visual fibers. Transmedulla neurons project from the MEDRA to the lower unit complex of the anterior optic tubercle (LUC). TuLAL1 neurons project from the LUC around the vertical lobe of the mushroom body (VL) to the median and lateral bulb (MBU, LBU). Three Types of TuTu1 neurons project to the contralateral LUC. Stippled grey lines in medulla (ME) indicate hypothetical unpolarized light input pathways. Also shown is immunoreactivity in the medulla to antisera against pigment dispersing factor (PDF) and 5HT (5HT). AL, antennal lobe; CBL, lower division of the central body; CBU, upper division of the central body; LA, lamina; LCA and MCA, lateral and medial calyx of the mushroom body; LO, lobula; PED, pedunculus. Scale bar: 200 μm.</p

Primary antibodies, dilutions and fixatives.

<p>GA, glutaraldehyde; GSA, glutathione-S-transferase; KLH, keyhole limpet hemocyanin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; PA, picric acid; PFA, paraformaldehyde.</p><p>Primary antibodies, dilutions and fixatives.</p

CRHR1-WT and CRHR1-STAVA localize throughout the neuronal plasma membrane including the excitatory post synapse.

<p>Primary hippocampal neurons at DIV 14–20 were transduced with CRHR1-WT and CRHR1-STAVA using AAV8. (A) The spatial pattern of CRHR1-WT and CRHR1-STAVA expression visualized by immunostaining of the GFP tag was confirmed using an anti-CRHR1 antibody. (B) Microtubule-associated protein 2 (MAP2) staining revealed the dendritic presence of the receptor. (C) Co-staining with the axon initial segment marker ankyrin G demonstrated CRHR1-WT and CRHR1-STAVA localization within axons. (D) The presynaptic marker synapsin indicated that CRHR1-WT and CRHR1-STAVA are present in the adjacent post synapse but not in the presynaptic axon terminal. (E) CRHR1-WT and CRHR1-STAVA did not co-localize with the inhibitory postsynaptic marker gephyrin but (F) they co-localized with the MAGUK and excitatory postsynaptic marker PSD95 in spines. (G) CRHR1-WT and mutant co-localized with the candidate interaction partner SAP97.</p

CRHR1 and candidate MAGUKs are co-expressed in the adult mouse brain.

<p>(A–F) Expression of mRNA of CRHR1, PSD95, SAP97, SAP102, PSD93, and MAGI2 was assessed by single <i>in situ</i> hybridization on consecutive coronal brain sections of wild-type mice. For comparison, representative photomicrographs of autoradiographs are shown in a false color display. CRHR1 expression is displayed in green, and PSD95, SAP97, SAP102, PSD93, and MAGI2 expression are displayed in red. The yellow signal reveals brain structures of overlapping expression of CRHR1 with respective MAGUKs. (G–K) Double <i>in situ</i> hybridization revealed co-expression of CRHR1 and interacting partners at the cellular level. Depicted are representative photomicrographs of coronal brain sections of CRHR1-GFP reporter mice. Blue boxes depict individual neurons shown below as magnifications (left: single MAGUK positive neuron, right: MAGUK and CRHR1 double positive neuron). Arrows indicate single positive cells stained by silver grains for CRHR1, and red arrowheads indicate cells expressing the candidate MAGUK only. White arrowheads indicate double-positive cells co-expressing CRHR1 and its respective interaction partners. CA: cornu ammonis, RTN: reticular thalamic nucleus.</p