M6a expression in neurons of the hippocampal formation and the mPFC; quantitative in situ hybridization with emulsion autoradiography.

<p>Upper panel: Numbers of silver grains per neuron reveal reduced M6a mRNA expression after stress in dentate gyrus granule neurons and in CA3 pyramidal neurons, but enhanced M6a mRNA expression in neurons of the prelimbic and infralimbic cortex. Lower panel: Examples of sections from the dentate gyrus (left) and the infralimbic cortex (right) showing silver grains over cells that were counter stained with methyl-green (cyan). Significant differences between groups as determined by Student's <i>t</i>-test: *, p<0.05, **, p<0.01.</p

Autoradiograms showing M6a expression in the hippocampal formation (A) and the prefrontal cortex (B) as revealed by in situ hybridization.

<p>Abbreviations: ACx, anterior cingulated cortex; CA1, hippocampal region CA1; CA3, hippocampal region CA3; CA4, hippocampal region CA4; DG, dentate gyrus; gcl, granule cell layer; IL, infralimbic cortex; PL, prelimbic cortex; pyr, pyramidal cell layer.</p

Immunofluorescence showing M6a immunoreactivity in the stratum lucidum (str.luc.) of the hippocampus (A, C) and in the infralimbic cortex (B, D) as revealed by confocal microscopy.

<p>A: M6a (red) and synaptophysin immunoreactivity (green) in the stratum lucidum. Note that the giant mossy fiber terminals (mft) which are strongly synaptophysin positive surround the unlabeled dendrites (d) of the CA3 pyramidal neurons. B: M6a (red) and synaptophysin immunoreactivity (green) in the infralimbic cortex; axons (ax) are M6a immunopositive, dendrites (d) are unlabeled. Note that there is occasional colocalization (yellow) of M6a and synaptophysin in the axonal terminals that surround the unlabeled soma of the pyramidal neuron (pyr). C: M6a (red) and MAP-2 immunoreactivity (green) in the stratum lucidum. Note that the dendrites (d) of CA3 pyramidal neurons which are strongly MAP-2 positive are not labelled by the M6a antibody. D: M6a (red) and MAP-2 immunoreactivity (green) in the infralimbic cortex. Note that there is no colocalization of M6a and MAP-2 in the axons/axonal terminals (ax) that surround the MAP-2 immunopositive soma of the pyramidal neurons (pyr).</p

Quantitative real-time PCR showing M6a expression in the hippocampus (upper pannel) and prefrontal cortex (lower pannel).

<p>Analysis with primers recognizing the 3′-UTR of M6a, which is common to all isoforms of M6a, revealed a significant stress-induced downregulation in the hippocampus. Subsequent analysis with isoform-specific primers showed that M6a isoform Ib, but not Ia, is regulated by stress. In the prefrontal cortex, both isoforms show a tendency towards upregulation but fail to reach significance. Data are expressed as percentage of the mean control±SEM (standard error of the mean), n = 9/group. Significant differences between groups as determined by Student's <i>t</i>-test: *, p<0.05, **, p<0.01. UTR, untranslated region.</p

Effects of chronic restraint stress on body weight gain and adrenal weight.

<p>Left: Animals exposed to chronic restraint stress exhibited reduced body weight gain coinciding with the onset of restraint; data are from 11 controls and 10 stressed animals. Right: Adrenal weight of stressed rats is significantly increased compared to controls (10 animals/group). Data are expressed as mean±SEM (standard error of the mean). Significant differences between groups as determined by Student's <i>t</i>-test: *, p<0.05.</p

Immunocytochemical detection of M6a expression in the hippocampus.

<p>(A) shows no immunoreactivity in the granule cell layer (gcl) whereas the hilus (h) is strongly stained. A laminated pattern of immunoreactivity is detected in the molecular layer (ml) of the dentate gyrus, in stratum radiatum (rad) and stratum oriens (or) of region CA1. B (enlarged area from the box in A), mossy fibers terminating in the stratum lucidum (str.luc.) are strongly labeled by the M6a antibody whereas pyramidal neurons (pyr) are not stained.</p

Primer used for quantitative RT-PCR.

<p>Primer used for quantitative RT-PCR.</p

Alignment of marmoset (Cj), human (Hs), chimpanzee (Pt), and macaque (Mm) CDNF amino acid sequences by CLUSTAL O (version 1.2.0) software.

<p>Identical amino acids are marked with an asterisk, physicochemically highly similar with a colon, and similar with a dot. Predicted signal sequences (SignalP 4.1 Server <a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>) of marmoset, chimpanzee and macaque CDNF (residues 1–26; underlined) are equal in length to the verified signal peptide of human CDNF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149776#pone.0149776.ref001" target="_blank">1</a>]. Eight conserved cysteine residues with identical spacing of the mature CDNF are indicated in yellow. N-linked glycosylation site of human CDNF at position 57N [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149776#pone.0149776.ref013" target="_blank">13</a>] and O-linked glycosylation site at position 181T [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149776#pone.0149776.ref052" target="_blank">52</a>] is indicated in green. The C-terminal ER-retention motif KAEL/KTEL is boxed. Alpha-helical regions of CDNF are indicated above the sequences according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149776#pone.0149776.ref007" target="_blank">7</a>] (helices α1-α5 and turn of 3₁₀ helix) and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149776#pone.0149776.ref009" target="_blank">9</a>] (helices α6-α8). The division between N-terminal saposin-like domain and C-terminal SAP-domain is indicated by a vertical dashed line.</p

Amino acid identity (%) of CDNF proteins between human and selected nonhuman primates calculated by EMBOSS Needle software.

<p>Signal peptide sequences have been omitted.</p

Time course of experiments.

<p>In the tolerability approach (upper scheme) high concentrations of CDNF (10 or 15μg per day) were intrastriatally delivered for 28 days via osmotic minipumps followed by subsequent histopathological assessment. Efficacy of CDNF and GDNF was tested in the 6-OHDA model (lower scheme). Treatment effects were evaluated <i>in vivo</i> by SPECT imaging and post mortem IHC.</p