Local field potentials during sharp wave/ripples in <i>GluA4^HC−/−</i> mice.

<p>(A) Representative examples of SWRs recorded during a rest trial in a control and a <i>GluA4^HC−/−</i> mouse. Top trace: raw signal. Bottom trace: band-pass filtered (125–250 Hz) signal. (B) Mean length of SWR epochs and frequency of SWR occurrence in control and <i>GluA4^HC−/−</i> mice. (C) Mean time-frequency representation of power centered on the peak power of each SWR epoch. (D) Mean power spectrum of SWRs in control and <i>GluA4^HC−/−</i> mice. (E) Mean peak ripple frequency and mean peak power during SWRs in control and <i>GluA4^HC−/−</i> mice. (F) Mean waveform of ripples centered on the peak power of each SWR epoch and aligned on the positive-to-negative zero-crossing. (G) Mean ripple amplitude in control and <i>GluA4^HC−/−</i> mice. *: <i>p</i><0.05.</p

Theta oscillations in <i>GluA4^HC−/−</i> mice.

<p> (A) Representative examples of theta oscillations recorded during exploratory trials in control and <i>GluA4^HC−/−</i> mice. Top trace: raw signal. Bottom trace: band-pass filtered (5–14 Hz) signal. (B) Mean power spectra in the theta frequency range when mice ran at different speed. The peak power and peak frequency was similar in the control and <i>GluA4^HC−/−</i> mice. (C) Polar plot of the preferred theta phase and theta vector length of pyramidal cells in control and <i>GluA4^HC−/−</i> mice. Each dot represents a neuron. Phase 0 is the positive-to-negative zero-crossing of the theta oscillation. The theta vector length of each cells is equal to the distance between the dot and the center of the plot. The short lines in the right-top corner indicate the mean preferred phase of the recorded neurons. (D) Mean theta vector length for all pyramidal cells in control and <i>GluA4^HC−/−</i> mice. (E) Distribution of preferred theta phase for pyramidal cells in control and <i>GluA4^HC−/−</i> mice. (F) Mean firing probability at different theta phases for pyramidal cells. (G–J) Same as C–F but for interneurons. Abbreviations: Int., interneurons; Pyr., pyramidal cells. **: <i>p</i><0.005.</p

GluA4 deletion affects the recruitment of interneurons and spatial working memory.

<p>(A) Representative averaged excitatory postsynaptic currents (EPSCs) from a fast-spiking interneuron in control and <i>GluA4^HC−/−</i> mice. Arrows indicate the time points at which the AMPA and NMDA components were measured. Traces in control and <i>GluA4^HC−/−</i> mice were normalized to the size of the EPSC measured at +70 mV. (B) AMPA/NMDA ratio in fast-spiking cells and pyramidal cells of control and <i>GluA4^HC−/−</i> mice. (C) Percentage of correct choices during the acquisition of an appetitive hippocampus-dependent spatial reference memory task on an elevated Y-maze in control and <i>GluA4^HC−/−</i> mice. The dotted line represents chance levels. (D) Percentage of correct choices during hippocampus-dependent rewarded alternation task on the elevated T-maze for control and <i>GluA4^HC−/−</i> mice. (E) Reference memory errors per block (maximum of 16 errors) for control and <i>GluA4^HC−/−</i> mice during reference memory acquisition of the radial arm maze task (doors prevented working memory errors). (F) Number of working memory (top) and reference memory (bottom) errors during simultaneous assessment of working and reference memory on the radial arm maze. Abbreviations: FS, fast-spiking interneurons; Pyr., pyramidal cells. **: <i>p</i><0.01, ***: <i>p</i><10<i>⁻</i>¹¹.</p

GluA4 ablation in the dorsal hippocampus.

<p>(A) Cre-mediated recombination of the <i>ROSA26</i> reporter gene after AAV-Cre injection into the dorsal hippocampus. Coronal sections at two anteroposterior levels of the hippocampus (left two panels) and a third section at higher magnification of the CA1 hippocampal region (right panel) showing pan-neuronal X-gal staining. Scale bar: 150 µm. (B) Representative Western blot from the dorsal hippocampus in control and <i>GluA4^HC−/−</i> mice. (C) Quantification of GluA4 expression level in the dorsal hippocampus from Western blot analysis. Data are expressed as percentage of control levels (mean ± SEM). (D) Representative Western blot of the ventral hippocampus in control and <i>GluA4^HC−/−</i> mice. (E) Quantification of GluA4 expression level in the ventral hippocampus from Western blot analysis. Abbreviations: so, stratum oriens; sr, stratum radiatum. ***: <i>p</i><10⁻¹⁰.</p

Spatial firing during trials in the open field and the zigzag maze.

<p>(A) Representative examples of firing rate maps in the open field from 10 simultaneously recorded pyramidal cells in a control and a <i>GluA4^HC−/−</i> mouse. Numbers above each map indicate the peak firing rate in Hz. (B) Spatial information score in the open field for pyramidal cells in control and <i>GluA4^HC−/−</i> mice (mean ± SEM). (C) Sparsity score in the open field for pyramidal cells in control and <i>GluA4^HC−/−</i> mice. (D) Peak firing rate of firing fields in the open field. (E) Mean place field size in the open field. (F) Stability of place firing rate maps across two trials in the open field. Representative recording of one cell during two trials in the open field (1 hr inter-trial interval, left panel). Stability of place firing rate maps with different spatial information scores (right panel). (G) Representative examples of firing rate maps in the zigzag maze from 4 pyramidal cells recorded in a control and a <i>GluA4^HC−/−</i> mouse. South- and northbound runs (indicated by arrows) are plotted separately (top and bottom rows). Numbers above each map indicate the peak firing rate in Hz. (H) Spatial information score in the zigzag maze for pyramidal cells in the zigzag maze. (I) Sparsity score in the zigzag maze for pyramidal cells. (J) Peak firing rate in the 2-dimensional firing rate maps in the zigzag maze for pyramidal cells of control and <i>GluA4^HC−/−</i> mice. (K) Mean size of the firing fields detected in 1-dimensional firing rate maps of the zigzag maze. *: <i>p</i><0.05, ***: <i>p</i><10<i>⁻</i>⁷.</p

Cell activity during sharp wave/ripples in <i>GluA4^HC−/−</i> mice.

<p>(A) Firing rate of pyramidal cells during SWRs. Time 0 represents the peak power of SWRs. The inset shows the mean firing rate during SWRs. (B) Same as A but for interneurons. (C) Proportion of SWRs in which a pyramidal fire from 0 to 5 spikes. Pyramidal cells in <i>GluA4^HC−/−</i> mice were more likely than that of control mice to fire between 1 to 5 spikes during a SWR. (D) Polar plot of the preferred ripple phase and ripple vector length of pyramidal cells (left) and interneurons (right) in control and <i>GluA4^HC−/−</i> mice. Each dot represents a neuron. Phase 0 is the positive-to-negative zero-crossing of the ripple. The ripple vector length of each cells is equal to the distance between the dot and the the center of the plot. The short lines in the right-top corner indicate the mean preferred phase of the recorded neurons. (E) Mean firing probability at different ripple phases for pyramidal cells (left) and interneurons (right) in control and <i>GluA4^HC−/−</i> mice. Abbreviations: Int., interneurons; Pyr., pyramidal cells. **: <i>p</i><0.01, ***: <i>p</i><10<i>⁻</i>⁵.</p

Data_Sheet_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.PDF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Image_2_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Image_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p