Layer-specific changes of KCC2 and NKCC1 in the mouse dentate gyrus after entorhinal denervation

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl− concentration and cell volume of neurons and/or glia. The Cl− extruder KCC2 is expressed at higher levels than the Cl− transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl− concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis

Die Rollen der Autismus-Kandidatengene Neuroligin-3, Neuroligin-4 und Neurobeachin in der synaptischen Plastizität und der Exzitations-Inhibitions-Balance im Gyrus Dentatus

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals. In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.Autismus-Spektrum-Störung (ASS) ist eine häufig vorkommende neurologische Entwicklungsstörung mit einem mannigfaltigen klinischen Erscheinungsbild. Obwohl schon viele genetische Risikofaktoren identifiziert und in Mausmodellen untersucht worden sind, sind die neurophysiologischen Mechanismen, die zu einer Ausbildung eines autistischen Phänotyps führen, immer noch unklar. Basierend auf der hohen Komorbiditätsrate von ASS und Epilepsie wurde die Hypothese aufgestellt, dass das Gleichgewicht zwischen der Exzitation und Inhibition in neuronalen Netzwerken der betroffenen Personen gestört sein könnte. In dieser Dissertation wurde die Aktivität von Neuronen im Netzwerk und an der Synapse in drei genetisch veränderten Mausmodellen, die charakteristische Verhaltensauffälligkeiten der autistischen Störung aufzeigen, untersucht: im Neurobeachin (Nbea) haploinsuffizienten, im Neuroligin-3 (Nlgn3) Knock-out (KO), und im Nlgn4 KO Mausmodell. Diese Proteine sind alle an der Synapsenbildung und/oder -funktion im zentralen Nervensystem beteiligt. Daher wurde vermutet, dass die Reduktion oder Deletion dieser Proteine die Balance zwischen exzitatorischer und inhibitorischer synaptischer Übertragung in einzelnen Neuronen sowie in neuronalen Netzwerken ändern könnte. Extrazelluläre Ableitungen im hippocampalen Gyrus dentatus von anästhesierten Mäusen haben gezeigt, dass das Exzitations-Inhibitions(E-I)-Gleichgewicht in Nbea haploinsuffizienten sowie Nlgn4 KO-Mäusen reduziert war, aber in Nlgn3 KO-Mäusen trotz einer Verminderung der exzitatorischen synaptischen Übertragung unverändert war. Unerwarteterweise war die intrinsische Erregbarkeit der Körnerzellen in allen drei Mausmodellen verändert. Diese Ergebnisse weisen darauf hin, dass eine homöostatische Erhöhung der intrinsischen Erregbarkeit die Reduktion der exzitatorischen synaptischen Transmission in Nlgn3 KO-Mäusen kompensieren kann, wohingegen die verminderte intrinsische Erregbarkeit in Nbea haploinsuffizienten sowie Nlgn4 KO-Mäusen das E-I-Ungleichgewicht verursacht. Insgesamt zeigen diese Befunde, dass der Einfluss von genetischen Faktoren auf das E-I-Gleichgewicht ein potenzieller Mechanismus ist, der zur Ausbildung von ASS führen könnte

Synaptic Plasticity and Excitation-Inhibition Balance in the Dentate Gyrus: Insights from In Vivo Recordings in Neuroligin-1, Neuroligin-2, and Collybistin Knockouts

The hippocampal dentate gyrus plays a role in spatial learning and memory and is thought to encode differences between similar environments. The integrity of excitatory and inhibitory transmission and a fine balance between them is essential for efficient processing of information. Therefore, identification and functional characterization of crucial molecular players at excitatory and inhibitory inputs is critical for understanding the dentate gyrus function. In this minireview, we discuss recent studies unraveling molecular mechanisms of excitatory/inhibitory synaptic transmission, long-term synaptic plasticity, and dentate granule cell excitability in the hippocampus of live animals. We focus on the role of three major postsynaptic proteins localized at excitatory (neuroligin-1) and inhibitory synapses (neuroligin-2 and collybistin). In vivo recordings of field potentials have the advantage of characterizing the effects of the loss of these proteins on the input-output function of granule cells embedded in a network with intact connectivity. The lack of neuroligin-1 leads to deficient synaptic plasticity and reduced excitation but normal granule cell output, suggesting unaltered excitation-inhibition ratio. In contrast, the lack of neuroligin-2 and collybistin reduces inhibition resulting in a shift towards excitation of the dentate circuitry

Overview of reconstructed morphologies.

<p>Overview of reconstructed morphologies.</p

Branch diameters in reconstructed dendritic trees as a function of SO.

<p><b>A</b>. <i>Left</i>: Average normalised diameters (between minimum and maximum values) for nodes of different SO in reconstructed dendritic trees and their standard deviations (shaded areas); <i>right</i>: same, but with SO values normalised between 0 and 1 (standard deviations omitted for clarity). <b>B</b>. Diameter values as a function of topological subtree size for all branch points in the trees, divided in panels for each cell type. Grey line corresponds to the average diameter for branch points of the same subtree size and is shown until the number of data points available for mean calculation was less than 0.1% of the data points for topological subtree size 2. Different colours denote data from different cell types (see legend at top of figure).</p

Neuroligin-3 regulates excitatory synaptic transmission and EPSP-spike coupling in the dentate gyrus in vivo

Neuroligin-3 (Nlgn3), a neuronal adhesion protein implicated in autism spectrum disorder (ASD), is expressed at excitatory and inhibitory postsynapses and hence may regulate neuronal excitation/inhibition balance. To test this hypothesis, we recorded field excitatory postsynaptic potentials (fEPSPs) in the dentate gyrus of Nlgn3 knockout (KO) and wild-type mice. Synaptic transmission evoked by perforant path stimulation was reduced in KO mice, but coupling of the fEPSP to the population spike was increased, suggesting a compensatory change in granule cell excitability. These findings closely resemble those in neuroligin-1 (Nlgn1) KO mice and could be partially explained by the reduction in Nlgn1 levels we observed in hippocampal synaptosomes from Nlgn3 KO mice. However, unlike Nlgn1, Nlgn3 is not necessary for long-term potentiation. We conclude that while Nlgn1 and Nlgn3 have distinct functions, both are required for intact synaptic transmission in the mouse dentate gyrus. Our results indicate that interactions between neuroligins may play an important role in regulating synaptic transmission and that ASD-related neuroligin mutations may also affect the synaptic availability of other neuroligins

SO-sorted topological measures in binary trees.

<p><b>A</b>. Sample binary trees taken from all possible 9,694,845 unsorted and 10,905 sorted trees of degree 16. Coloured dots denote node SO (black– 1, green– 2, red– 3, blue– 4, cyan– 5). <b>B</b>. Depiction of all possible distributions of segment numbers with SO in binary trees of degree 16; distributions for different trees are coloured differently. Bold green and bold magenta lines show distributions for the so-called “herringbone” tree (green box in A) and for the complete binary tree (magenta box in A), respectively. <b>C</b>. All possible distributions of branch numbers with SO in binary trees of degree 16. Bold green and bold magenta lines show distribution for the “herringbone” tree and for the complete binary tree, respectively. Bold grey line shows distribution for a tree that follows the trend of 4^1−<i>k</i> for SO <i>k</i> from dashed lines in E and H. <b>D</b>. Averages (lines with markers) and standard deviations (shaded areas) of normalised segment number distributions with SO for all possible sorted and unsorted binary trees of degree 16 divided into trees of segment Strahler number (SN) 2, 3, and 4. Dashed line indicates the distribution obtained from a complete binary tree (see main text). <b>E</b>. Similar to D but with normalised branch number distributions. Dashed line here indicates the asymptotic power relation obtained for large random binary trees (see main text). <b>F</b>. Sketch illustrating the Galton-Watson (GW) type random branching process. Terminal nodes either stop growing or branch out with probabilities <i>P</i>_<i>st</i> = 0.5 (red) and <i>P</i>_<i>br</i> = 0.5 (blue), respectively. <b>G, H</b>. Similar to D and E but for GW random binary trees divided into groups of SN 2–6. <b>I</b>. Branch bifurcation ratio between subsequent orders for the same GW random binary trees as in G and H. Magenta line indicates lower bound (see main text) and grey line denotes asymptotic bifurcation ratio for large random binary trees (see main text). <b>J</b>. Average (lines with markers) and standard deviations (shaded areas) of normalised topological subtree sizes for all topological nodes as a function of SO in GW random binary trees divided into SN 3–6. Magenta dashed line indicates relation obtained for a complete binary tree (CBT) of node SN 6 (see main text). Grey dashed lines represent mirror images (4^{<i>k</i>−<i>SN</i>}, for SO <i>k</i> and SN 3–6) of the power relation in E (see main text). Note that in segment distributions per SO such as in D and G, nearly half of the segments are terminal segments (i.e. SO 1) in all cases, a property of binary trees.</p

Linear regression fits for overall bifurcation ratios of reconstructed neuronal morphologies (see main text).

<p>Linear regression fits for overall bifurcation ratios of reconstructed neuronal morphologies (see main text).</p

SO-sorted topological measures in minimum spanning trees (MSTs).

<p><b>A</b>. Example synthetic MST model dendrites (2D circular morphology) generated with 500 target points and differing balancing factors (<i>bf</i>). Colours and legend to the right indicate <i>bf</i> in the remaining panels. <b>B—E</b>. Similar plots to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005615#pcbi.1005615.g002" target="_blank">Fig 2G–2J</a>. For B, C, and E, each line represents the mean of 614–970 (SN 3), 398–940 (SN 4), 625–967 (SN 5), and 248–964 (SN 6) model trees per <i>bf</i>. For better clarity, standard deviations are not shown.</p