Antibodies used in the study.

<p>Antibodies used in the study.</p

Schematic drawing shows the proposed mechanism for GABA_AR and NMDAR cooperation during early postnatal development.

<p>Our results that GABA_A and NMDA receptors are co-expressed in GABAergic synapses can provide the anatomical basis for a new model of the generation of SSAs during the postnatal period. The GABA_AR mediated current leads to postsynaptic depolarization in the GABAergic synapse that allows activation of NMDARs located in the same synapse. This homosynaptic receptor activation in GABAergic synapses causes strong local depolarization that leads to a subsequent heterosynaptic activation of NMDARs in otherwise silent (AMPA receptor-lacking) glutamatergic synapses. The schematic drawing illustrates the position of GABAAR and NMDAR at synapses, where their presence has already been proven by others or in our current study.</p

Analysis of the expression of NMDAR subunits in GABAergic and glutamatergic synapses at postnatal day 6–7. A

<p>, Percentage of synapses positive for different NMDAR subunits out of all identified glutamatergic (white columns) and GABAergic synapses (black columns). <b>B</b>, Linear density of labeling (gold particle/µm) for different NMDAR subunits in glutamatergic synapses (white columns), GABAergic synapses (black columns) and extrasynaptic membranes (grey columns), measured on 100 nm thick electron microscopic sections. The extrasynaptic density of labeling was 0.09, 0.03 and 0.02 immunogold particles/µm for GluN1, GluN2B and GluN2A subunits, respectively. <b>C</b>, Size (µm²) of glutamatergic (white column) and GABAergic (black column) synapses. Data shown in A, B and C were measured from preembedding experiments. <b>D</b>, Density of labeling (gold particle/µm) for GluN1 subunits in glutamatergic synapses (white column), GABAergic synapses (black column) and extrasynaptic (E.S.) membranes (grey column, 0.03 gold particles/µm), measured from quantitative post-embedding experiments.</p

Quantitative post-embedding immunogold labeling reveals NMDAR expression levels in GABAergic and glutamatergic synapses at postnatal day 6–7.

<p>The same synapses were reacted with antibodies against different epitopes on adjacent ultrathin sections using the mirror technique. 10 nm immunogold particles label presynaptic glutamatergic (vGluT1 positive) terminals in A1 and B1. Intensified immunogold particles label presynaptic GABAergic (GAD65/67 positive) terminals in C1 and D1. 10 nm immunogold particles (arrows) label GluN1 subunits in A2-3, B2, C2, D2. Note that the postembedding immunoreaction is on the surface of the sections, therefore, the position of gold particles can be on either side of the postsynaptic membrane, even if the labeled epitope is purely postsynaptic. Images from adjacent sections of the same synapses are displayed in A1-3, in B1-2, in C1-2 and in D1-2. Scale bar is 200 nm for all images.</p

NMDAR subunits are expressed postsynaptically in both GABAergic and glutamatergic synapses at postnatal day 6–7.

<p>Electron micrographs show combined immunogold-immunoperoxidase reactions from stratum radiatum of the hippocampal CA1 region. Synapses of vGluT1 positive presynaptic terminals (marked by dark reaction product in A, C, E1 and E2) contain postsynaptic GluN1 (black particles in A, arrows), GluN2B (black particles in C, arrows), and GluN2A subunits (black particles in E1 and E2, arrows). Synapses of GAD67-positive presynaptic terminals (marked by dark reaction product in B, D1, D2, F1, F2 and F3) contain postsynaptic GluN1 (black particles in B, arrows), GluN2B (black particles in D1 and D2, arrows), and GluN2A subunits (black particles on F1, F2 and F3, arrows). Serial images show the same synapse in D1 and D2; E1 and E2; F1, F2 and F3. Scale bar is 300 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