Neuroligin 2 is localized postsynaptically at cholinergic synapses in the neocortex.

<p>Images demonstrate double immunohistochemical reactions for ChAT (dark, homogenous DAB precipitate) combined with NLGN2 (black intensified gold particles) in somatosensory (S1) and prefrontal cortices (PFC). Serial sections of the same synapses are shown in B_1–2, D_1–2 and E_1–2. In both areas, ChAT-positive boutons (b_1–5) form type II synaptic contacts on dendrites (d, A, C, D_1–2) and spines (s, B_1–2, E_1–2) that express NLGN2 at the postsynaptic membranes (open arrowheads label synaptic edges). The innervated spines also received a type I synapse from a ChAT-negative terminal (B_1–2, black arrowheads). In C the postsynaptic dendrite of bouton b₃ receive an additional, type I synaptic input (black arrowheads) from an unlabeled terminal. These type I synapses in B_1–2 and C do not contain NLGN2. In contrast, another ChAT-negative, putative GABAergic bouton (b_neg) establishes a type II, NLGN2-positive synapse (black arrowheads) with a dendrite in D_1–2. Scale bar is 200 nm for all images.</p

Neuroligin 2 is present postsynaptically at both GABAergic and cholinergic synapses in the hippocampus.

<p>Electron micrographs from combined immunogold/immunoperoxidase experiments for NLGN2 (immunogold: black particles) and ChAT (DAB: dark, homogenous reaction product) reveal the presence of NLGN2 at ChAT-negative and ChAT-positive type II synapses in the CA1 area. Arrowheads indicate synapse-edges. A, A pyramidal cell body receives a synapse from a ChAT-negative bouton (b_neg) that expresses NLGN2 postsynaptically in a WT mouse. B, C, In contrast, the same type of immunostaining in a NLGN2-KO mice shows no NLGN2-immunoreactive synapses, demonstrating the specificity of the antibody. A GABAergic terminal (b_neg) from str. pyramidale, lacking gold particles at the postsynaptic site is shown (B). An example of a synapse of a ChAT-positive bouton (b₁) on a dendrite (d) in str. radiatum that is immunonegative for NLGN2 in KO mouse (C). D–I: NLGN2 immunogold labeling is present at the postsynaptic site of synapses established by ChAT-positive axon terminals (b_2–5) on dendrites (d) and spines (s) in str. radiatum (D–G) and oriens (H, I) of WT mice. Serial images show the same synapse in D₁ and D₂; E₁ and E₂; F₁ and F₂; G₁ and G₂. E_1–2 demonstrates that some of the presynaptic profiles were small-diameter, intervaricose-like segments of ChAT-positive axons (b₃). In F_1–2 and H, the postsynaptic targets of boutons b₄ and b₆ are putative pyramidal dendrites (Pd) the latter of which is identified by the presence of spines (s). I, Occasionally, we found ChAT-positive presynaptic elements that formed synapses with two postsynaptic targets. Here, bouton b7 forms a synapse with a dendrite and a spine, which receives a type I synapse (black arrowheads). Note, that in many cases, synaptic junctions of ChAT-positive terminals are atypical (E, F, H, I). Scale bar is 200 nm for all images.</p

Cholinergic projection neurons of the basal forebrain and neostriatal cholinergic interneurons express NLGN2 in their inputs synapses.

<p>Images from combined immunogold/immunoperoxidase experiments show that dendrites of cholinergic cells (dChAT, dark, homogenous DAB precipitate) express NLGN2 (intensified gold particles) at postsynaptic membranes of type II synapses (open arrowheads) in the medial septum (A: MS), vertical- and horizontal diagonal band of Broca (B: VDB; C, D: HDB), substantia innominata/ventral pallidum (E: SI/VP) and caudate putamen (F: CPu). In B and C two unlabeled dendrites (d_neg) also express NLGN2 in their type II synapses (black arrowheads). Scale bar is 200 nm for all images.</p

MRR stimulation selectively activates brain areas involved in emotional control.

<p>Optic stimulation selectively increased the expression of the activity marker c-Fos in two sub-regions of the medial prefrontal cortex (<b>A</b>), the whole periaqueductal gray (<b>B</b>), and the paraventricular nucleus of the hypothalamus (<b>C</b>). PrL and the amygdala was activated by cage transfer, but not stimulation (<b>D</b>); the hippocampus showed no responses (<b>E</b>). Panel <b>F</b> is a 3D illustration of the Multiple Regression analysis presented in the text. <i>BLA</i>: basolateral amygdala; <i>CA1</i>: CA1 region of the hippocampus; <i>CeA</i>: central amygdala; <i>Cg1</i>: anterior cingulate cortex; <i>DG</i>: dentate girus of the hippocampus; <i>dl-</i>: dorsolateral; <i>dm-</i>: dorsomedial; <i>IL</i>: infralimbic cortex, <i>l-</i>: lateral; <i>MeA</i>: medial amygdala; <i>PAG</i>: periaqueductal gray; <i>PrL</i>: prelimbic cortex; <i>PVN</i>: paraventricular nucleus of the hypothalamus; <i>vl-</i>: ventrolateral. * p<0.05 significant difference from home-cage controls; # p<0.05 significant difference from “no ChR2”.</p

The acute effects of MRR- and shock-conditioning are differentiated by freezing.

<p><b>(A</b>) Photomicrographs illustrating the location of the tip of the optic fibers and the distribution of GFP-labeled ChR2 expression. For stimulation patterns see the right-hand side of the figure. (<b>B</b>) and (<b>C</b>) MRR stimulation decreased exploration and increased ambulation only when it targeted the dorso-central region of the MRR ("central stimulation"). Partial stimulations (that reached ventral, lateral, anterior or posterior aspects of the MRR) were ineffective. (<b>D</b>) The rhythmic delivery of 50Hz theta bursts induced a corresponding rhythm of behavioral changes as indicated here by <i>ON-OFF responses</i>. Actual behavioral rhythms were similar to which is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181264#pone.0181264.g001" target="_blank">Fig 1D</a> and were presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181264#pone.0181264.s001" target="_blank">S1 Fig</a>. Note that behavior scoring was time-structured in a similar fashion in all groups to allow their comparison. (<b>E</b>) Central, but not partial MRR stimulations elicited “runs”, which were behaviorally similar to those observed in shocked mice. (<b>F</b>) Freezing was readily elicited by shock administration, but not by MRR-conditioning. <i>Aq</i>: aqueductus cerebri; <i>DRN</i>: dorsal raphe nucleus; <i>MRR</i>: median raphe region; <i>LineX</i>: line crossings; <i>Of</i>: optic fiber; <i>ON-OFF responses</i>: average changes in behavior elicited by the onset of stimulation <i>(ON responses)</i> and those elicited by their halting <i>(OFF responses)</i>; <i>stimulation/shock runs</i>: episodes of rapid ambulation without exploration. * p<0.01 significant difference from stimulation controls (either light-stimulated without ChR2 expression, or ChR2 expression without stimulation); # p<0.01 significant <i>ON-OFF</i> differences; ‡ p<0.01 significant difference from shock controls.</p

Channelrhodopsin (ChR2)-mediated optic stimulation robustly activated the MRR and altered behavior.

<p><b>(A</b>) Effects of <i>in vitro</i> stimulation on ^3H5-HT release from coronal brain slices including the MRR, which demonstrates the responsiveness of the area to photostimulation. The time-resolution of the curves is 1 min and covers the 10 min stimulation periods indicated by color. (<b>B</b>) The location of optic fibers in the <i>in-vivo</i> experiments, which are presented in panels C-E. All mice showed robust ChR2 expression in the MRR. Red and blue lines show iso-intensity lines of light penetration at 10% and 1% of release intensity, respectively (based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181264#pone.0181264.ref018" target="_blank">18</a>]). (<b>C</b>) MRR stimulation increased ambulation in freely moving animals. Mice were connected to the stimulation equipment by optic fibers, were transferred into a novel cage to mimic the conditions of the subsequent MRR-conditioning study and were stimulated at 50Hz theta-burst frequency by blue light (“stimulated (central)”). Controls were either stimulated in the absence of ChR2 expression (“no ChR2”), or ChR2 expression was induced, but light was not administered (“no light”). (<b>D</b>) Rhythmic decrease in exploration was induced by intermittent (rhythmic) stimulation of the MRR. (<b>E</b>) Rhythmic changes illustrated as <i>ON-OFF responses</i>, i.e. changes in behavior elicited by the onset of stimulations <i>(ON responses)</i> and those elicited by their halting <i>(OFF responses)</i>. <i>DRN</i>: dorsal raphe; <i>MRN</i>: median raphe; <i>MRR</i>: median raphe region; <i>LineX</i>: line crossings; <i>pMR</i>: paramedian raphe; <i>RtTg</i>: reticulotegmental nucleus of the pons. * p<0.01 significant difference from “no ChR2” and from “no light”; # p<0.01 significant <i>ON-OFF</i> difference.</p

Halorhodopsin-mediated silencing of the MRR ameliorates acute and remote, but not recent effects.

<p>Mice were submitted to electric shock conditioning either in a regular way or while their MRR was silenced by halorhodopsin illumination by yellow light. Halorhodopsin was expressed in all mice by a viral vector that carried the NpHR gene, and all mice were connected to optic fibers. (<b>A)</b> and (<b>B</b>) The acute effects of electric shocks were partially ameliorated by MRR silencing during the conditioning trial. (<b>C</b>) Effects were not secondary to alterations in pain perception, which was studied in the hot-plate and expressed as the temperature that consistently elicited paw licking. (<b>D</b>) Halorhodopsin silencing did not affect recent conditioned fear 24h after shock-conditioning, but markedly ameliorated remote freezing 7 days later. Note that freezing was statistically similar in MRR-silenced and non-shocked groups. * p<0.05 significant difference from non-shocked; # p<0.05 significant difference from shocked.</p