Median raphe region stimulation alone generates remote, but not recent fear memory traces

The median raphe region (MRR) is believed to control the fear circuitry indirectly, by influencing the encoding and retrieval of fear memories by amygdala, hippocampus and prefrontal cortex. Here we show that in addition to this established role, MRR stimulation may alone elicit the emergence of remote but not recent fear memories. We substituted electric shocks with optic stimulation of MRR in C57BL/6N male mice in an optogenetic conditioning paradigm and found that stimulations produced agitation, but not fear, during the conditioning trial. Contextual fear, reflected by freezing was not present the next day, but appeared after a 7 days incubation. The optogenetic silencing of MRR during electric shocks ameliorated conditioned fear also seven, but not one day after conditioning. The optogenetic stimulation patterns (50Hz theta burst and 20Hz) used in our tests elicited serotonin release in vitro and lead to activation primarily in the periaqueductal gray examined by c-Fos immunohistochemistry. Earlier studies demonstrated that fear can be induced acutely by stimulation of several subcortical centers, which, however, do not generate persistent fear memories. Here we show that the MRR also elicits fear, but this develops slowly over time, likely by plastic changes induced by the area and its connections. These findings assign a specific role to the MRR in fear learning. Particularly, we suggest that this area is responsible for the durable sensitization of fear circuits towards aversive contexts, and by this, it contributes to the persistence of fear memories. This suggests the existence a bottom-up control of fear circuits by the MRR, which complements the top-down control exerted by the medial prefrontal cortex

Neuroligin 2 is expressed in synapses established by cholinergic cells in the mouse brain.

Neuroligin 2 is a postsynaptic protein that plays a critical role in the maturation and proper function of GABAergic synapses. Previous studies demonstrated that deletion of neuroligin 2 impaired GABAergic synaptic transmission, whereas its overexpression caused increased inhibition, which suggest that its presence strongly influences synaptic function. Interestingly, the overexpressing transgenic mouse line showed increased anxiety-like behavior and other behavioral phenotypes, not easily explained by an otherwise strengthened GABAergic transmission. This suggested that other, non-GABAergic synapses may also express neuroligin 2. Here, we tested the presence of neuroligin 2 at synapses established by cholinergic neurons in the mouse brain using serial electron microscopic sections double labeled for neuroligin 2 and choline acetyltransferase. We found that besides GABAergic synapses, neuroligin 2 is also present in the postsynaptic membrane of cholinergic synapses in all investigated brain areas (including dorsal hippocampus, somatosensory and medial prefrontal cortices, caudate putamen, basolateral amygdala, centrolateral thalamic nucleus, medial septum, vertical- and horizontal limbs of the diagonal band of Broca, substantia innominata and ventral pallidum). In the hippocampus, the density of neuroligin 2 labeling was similar in GABAergic and cholinergic synapses. Moreover, several cholinergic contact sites that were strongly labeled with neuroligin 2 did not resemble typical synapses, suggesting that cholinergic axons form more synaptic connections than it was recognized previously. We showed that cholinergic cells themselves also express neuroligin 2 in a subset of their input synapses. These data indicate that mutations in human neuroligin 2 gene and genetic manipulations of neuroligin 2 levels in rodents will potentially cause alterations in the cholinergic system as well, which may also have a profound effect on the functional properties of brain circuits and behavior

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

New Silver-Gold Intensification Method of Diaminobenzidine for Double-Labeling Immunoelectron Microscopy

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3′-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level

Amyloid β induces interneuron-specific changes in the hippocampus of APPNL-F mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and amyloid-beta (Aβ) depositions generated by the proteolysis of amyloid precursor protein (APP) in the brain. In APPNL-F mice, APP gene was humanized and contains two familial AD mutations, and APP-unlike other mouse models of AD-is driven by the endogenous mouse APP promoter. Similar to people without apparent cognitive dysfunction but with heavy Aβ plaque load, we found no significant decline in the working memory of adult APPNL-F mice, but these mice showed decline in the expression of normal anxiety. Using immunohistochemistry and 3D block-face scanning electron microscopy, we found no changes in GABAA receptor positivity and size of somatic and dendritic synapses of hippocampal interneurons. We did not find alterations in the level of expression of perineuronal nets around parvalbumin (PV) interneurons or in the density of PV- or somatostatin-positive hippocampal interneurons. However, in contrast to other investigated cell types, PV interneuron axons were occasionally mildly dystrophic around Aβ plaques, and the synapses of PV-positive axon initial segment (AIS)-targeting interneurons were significantly enlarged. Our results suggest that PV interneurons are highly resistant to amyloidosis in APPNL-F mice and amyloid-induced increase in hippocampal pyramidal cell excitability may be compensated by PV-positive AIS-targeting cells. Mechanisms that make PV neurons more resilient could therefore be exploited in the treatment of AD for mitigating Aβ-related inflammatory effects on neurons

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