Viewing Animal Migration Through a Social Lens

Evidence of social learning is growing across the animal kingdom. Researchers have long hypothesized that social interactions play a key role in many animal migrations, but strong empirical support is scarce except in a few unique systems and species. In this review, we aim to catalyze advances in the study of social migrations by synthesizing research across disciplines and providing a framework for understanding when, how, and why social influences shape the decisions animals make during migration. Integrating research across the fields of social learning and migration ecology will advance our understanding of the complex behavioral phenomena of animal migration and help to inform conservation of animal migrations in a changing world

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS

Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons

Synaptic mAb7a-specific gephyrin clusters are reduced upon CDK5 knockdown whereas VIAAT puncta were not reduced in perisomatic dendritic segments.

<p>Hippocampal neurons (div14) were immunolabeled with anti-GFP antibodies to detect infected neurons (upper panel), with phosphospecific anti-gephyrin mAb7a antibody (middle panel), and with anti-VIAAT antibody (lower panel). (A) Non-infected cells; (A') CDK5-kd2 knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of the number of VIAAT puncta, mAb7a puncta and overlapping mAb7a/VIAAT puncta (synaptic mAb7a clusters). Mean ± SE. Each value was calculated from analysis of 15 cells from n = 3 independent cultures, ANOVA with post-hoc test: *** P< 0.001, * P<0.05.</p

S270 phosphorylation is different at different synapses.

<p>Relative fluorescence intensities of double labelling experiments using the pan-gephyrin antibody Ab-175 and the phospho-specific antibody mAb7a were measured in focal planes of confocal microscopy images. (A) Focal plane showing dendritic gephyrin clusters detected with Ab-175 or (B) with the phospho-specific mAb7a antibody. The scale bar represents 10 µm. (A′, B′, E) Upon treatment with CDK5 inhibitor SU9516 (5 µM) for six hours the phosphorylation of gephyrin is significantly reduced. (A′′) After replacement of medium with SU9516 by conditioned medium without inhibitor, cells were fixed and stained after a “recovery” time of two hours. Note increasing phosphorylation as indicated by mAb7a staining seen in B′′ compared to B′. The same analysis in C, C′, C′′ and D′, D′′, D′′ for “somatic” gephyrin clusters, revealing decrease and increase of gephyrin phosphorylation upon SU9516 treatment and “recovery”, respectively (D′′ compared to D′). (E) Quantification of mAb7a-specific phosphorylation comparing the fluorescence intensities of single clusters stained with mAb7a and Ab175 (ratios). <b>c.d</b>.: control dendritic clusters; <b>c.s</b>.: control somatic clusters; i.d.: CDK5 inhibitor treatment for 6 h, dentritic clusters h, dentritic clusters; i.s.: CDK5 inhibitor treatment for 6 h, somatic clusters h, somatic clusters; <b>1d</b>: 1 hour recovery from inhibition, dendritic clusters hour recovery from inhibition, dendritic clusters; <b>1s</b>: 1 hour recovery from inhibition, somatic clusters hour recovery from inhibition, somatic clusters; <b>2d</b>: 2 hour recovery from inhibition, dendritic clusters hour recovery from inhibition, dendritic clusters; <b>2s</b>: 2 hour recovery from inhibition, somatic clusters. Quantification was done with 660 clusters (total) from nine cells from three (n = 3) independent cultures. Mean ± SE. ANOVA with post-hoc test: ** P<0.01.</p

Gephyrin is dephosphorylated at Ser270 by PP1/PP2A phosphatase.

<p>(A) Hippocampal neurons were immunolabeled for gephyrin with antibody mAb7a, detecting gephyrin phosphorylation at S270. Neurons were fixed and immunolabeled at div14. (A) Control cells, non-treated; (B) cultures treated with aminopurvalanol A (5 µM) for 6 hours hours; (C) cultures treated with aminopurvalanol A (5 µM) and okadaic acid (250 nM nM) for 6 hours hours; (D) cultures treated with okadaic acid (250 nM nM) for 6 hours. Note a bright mAb7a immunostaining in D, revealing that gephyrin which is present in the soma seems to be dephosphorylated under physiological conditions. The scale bar represents 30 hours. Note a bright mAb7a immunostaining in D, revealing that gephyrin which is present in the soma seems to be dephosphorylated under physiological conditions. The scale bar represents 30 µm. (A'–D') Enlargement of selected dendritic areas shown in A–D. (E) Quantification of the number of gephyrin (mAb7a) puncta above a certain threshold. Quantification was done with 15 cells from 3 independent cultures. Mean ± SE. ANOVA with post-hoc test: *** P<0.001. (F) Quantification of the fluorescence intensity (average gray value) of those puncta above a certain threshold measured in E. Quantification was done with 15 cells from 3 independent cultures. Mean ± SE. ANOVA with post-hoc test: * P<0.05.</p

Gephyrin and GABA_A receptor γ2 puncta are reduced upon CDK5/2/1 inhibition.

<p>(A) Hippocampal neurons were double-immunolabeled for gephyrin with antibody mAb7a (red, upper panel) and with anti-VIAAT antibody (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days; (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed gephyrin puncta (yellow, merge) under A, compared to A' and A''. The scale bar represents 15 µm. (B) Hippocampal neurons were double-immunolabeled for GABA_A receptors with anti-γ2-subunit antibody (red, upper panel) and with anti-VIAAT antibodies (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days. (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed γ2-subunit puncta (yellow, merge) under B, compared to B' and B''. (C) Quantification of the number of gephyrin (mAb7a) puncta. Quantification was done with about 10 cells from 4 independent cultures. Mean ± S.E.; ANOVA with post-hoc test: * P<0.05. (D) Quantification of the number of GABA_A receptors with anti-γ2-subunit antibody. Mean ± SE. Quantification was done with 49 cells from n = 4 independent cultures (control), 34 cells from n = 3 independent cultures (2 days) and 18 cells from n = 3 independent cultures (3 days). ANOVA with post-hoc test: ** P<0.01.</p

CDK5 knockdown correlates with reduced numbers of GABA_A receptor clusters containing γ2-subunits.

<p>Hippocampal neurons (div14) were immunolabeled with anti GFP antibodies to detect infected neurons (upper panel), with phosphospecific anti gephyrin mAb7a antibody (middle panel), and with anti-GABA_A receptors γ2-subunit of (lower panel). (A) Non-infected cells; (A') CDK5-kd2 knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of the number of GABA_A receptor γ2 puncta on three proximal dendritic segments of 30 µm. 20 or 18 cells from n = 3 independent cultures of control or mismatch neurons and 25 cells (69 dendrites) from n = 4 independent cultures of kd2-infected neurons, mean ± SE. ANOVA with post-hoc test, *** P<0.001. (C) Quantification of the relative fluorescence intensities of GABA_A receptor γ2 puncta. Mean ± SE. Each value was calculated from data from the same cell numbers as in B from n = 3 independent cultures. ANOVA with post-hoc test: *** P<0.001, ** P<0.01.</p

CDK5 knockdown virus infection results in reduced CDK5 expression and reduced numbers of phospho-gephyrin clusters in cultured hippocampal neurons.

<p>Hippocampal neurons (div14) were stained with anti-GFP antibodies to detect infected neurons (upper panel), with anti-CDK5 antibody to quantify CDK5 expression levels (middle panel) and with the phosphospecific anti-gephyrin mAb7a antibody (lower panel). (A) Non-infected cells; (A') CDK5-knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of CDK5 fluorescence intensities of neurons infected with three different CDK5 knockdown viruses (kd1, kd2 and kd3). CDK5 knockdown cells were compared to non-infected neurons. n = 3, mean ± SD (C) Quantification of mAb7a cluster numbers of hippocampal neurons infected with three different CDK5 knockdown viruses (kd1, kd2, kd3) compared to non-infected and control shRNA (mismatch). 30 cells from n = 4 independent cultures (for each of non-infected, mismatch, kd2, kd1) or 30 cells from n = 3 independent cultures (kd3), mean ± SE, ANOVA with post-hoc test, ***P<0.001.</p