Properties of glutamatergic synaptic transmission recorded from striatal MSNs.

<p>No significant changes in PPR (<b>A</b>), average sEPSC frequency and amplitude (<b>B</b>), and NMDA/AMPA ratio (<b>C</b>) were observed between LacZ and CNTF slices. Traces: <b>A:</b> representative paired pulse-evoked EPSCs (normalized on the first black EPSC); <b>B:</b> examples of sEPSCs recordings; <b>C:</b> representative example of EPSCs recorded from a striatal MSNs (LacZ) showing where the AMPA and NMDA receptor-mediated component of the EPSC was measured (gray dots); note that after 50 ms (upper gray dot) the AMPA component is negligible, as shown by the trace in the presence of AP-5 (40 µM). HP in <b>A</b> and <b>B</b> was −60 mV.</p

The neuroprotective effect of CNTF is mediated by GTs.

<p><b>A:</b> γ-DGG has a significantly higher inhibitory effect in CNTF slices compared to LacZ: the histogram represents the ratio between EPSC amplitude in the presence of 0.5 mM γ-DGG and in control conditions (*p<0.05, Mann-Whitney test). Traces show representative EPSCs (HP = -60 mV). <b>B:</b> in the presence of the GTs inhibitor TBOA, there is little and similar FP recovery from QA excitotoxicity in both groups. <b>C:</b> summary of FP amplitude data at t = 45 min in the absence and in the presence of 30 µM TBOA (*p<0.05, Mann-Whitney test).</p

CNTF partially prevents QA-induced FP reduction in corticostriatal slices.

<p><b>A:</b> time-course of the effect of QA on striatal FP amplitude in LacZ vs. CNTF slices. Note how FP loss in CNTF is attenuated and the recovery improved compared to LacZ. The inset shows that the input/output ratio (volley/FP) is similar in both groups. <b>B:</b> FP amplitude at different times in the two groups: FP recovery after QA washout is significantly improved in CNTF compared to LacZ rats (*p<0.05, **p<0.01, Mann-Whitney test).</p

CNTF activates astrocytes and protects striatal neurons against QA excitotoxicity <i>in vivo</i>.

<p><b>A:</b> CNTF activates astrocytes that re-express vimentin (red) and overexpress GFAP around GFP-positive MSNs neurons of the striatum. <b>B:</b> rats from Vehicle (Veh.), LacZ and CNTF groups were injected with 80 nmol QA and the lesion (*) volume was assessed 15 days later on NeuN-immunostained sections. CNTF significantly decreased lesion size (^§p<0.001 vs. Veh. and LacZ groups, ANOVA and Scheffé's test).</p

Absence of LAMP5 does not alter brain structure nor spine density in the OB.

<p>(A) Cresyl violet staining of forebrain and OB sections shows no obvious structural changes in the brain in the absence of LAMP5. (B) Dendrites of control (<i>LAMP5 flox/+</i>) and mutant (<i>LAMP5 flox/flox</i>) newly generated granule neurons observed in the EPL of the OB 28 days after their electroporation (28dpe) with GFP and Cre recombinase expression plasmids. (C) Quantification of spine density on the dendrites of control (CTL) and KO newly integrated neurons. Dendrite morphology and spine density are unchanged in <i>LAMP5</i> KO. Dendrites n = 26 (flox/+) and 27 (flox/flox). Statistics: Wilcoxon test p-value = 0.9645. Scale bars: A, 1mm (0.5 mm for the OB); B, 20 μm (left), 5 μm (right). ST: Striatum; Th: Thalamus.</p

<i>LAMP5</i> knockout mice show normal VGAT distribution.

<p>(A, B) Immunostaining of LAMP5 and VGAT on GP (A) and OB (B) histological sections show no change in tissue and subcellular distribution in the absence of LAMP5. (C) Signal intensity of VGAT staining in the GP and in the OB, assessed by ImageJ analysis, is also unchanged. Mean intensity was calculated over 3 photomicrographs. Scale bar: A, 100 μm (left), 5 μm (right); C, 50 μm (left), 5 μm (right).</p

LAMP5 is specifically expressed in GABAergic synapses.

<p>(A) Immunofluorescence labeling for LAMP5 and GAD65 proteins demonstrates co-labelling in GABAergic axon terminals in the GP (arrowheads). (B) LAMP5 immunoreactivity never overlaps with vGLUT1 (arrowheads), thus it is absent from glutamatergic synapses. (C, C') In the OB, LAMP5 is localized on the synapses (arrowheads) of newly generated granule neurons, labeled with GFP by in vivo brain electroporation. (D) Electron microscopy immunogold labeling validates synaptic localization of LAMP5. Electron dense gold particles are always associated with symmetric synaptic densities (arrow) typical of GABAergic synapses formed by granule neurons onto mitral cell dendrites. (E) Like in the GP, glutamatergic synapses in the OB are devoid of LAMP5 staining (arrowheads). Scale bars: 5 μm in A,B,C,E, F; 0,1 μm in D.</p

Short-term plasticity of the striatopallidal synapse is altered in <i>LAMP5</i> deficient mice.

<p>(A) Top: Representative traces of mIPSCs from WT and KO mice. Bottom: Cumulative inter-event interval (left) and amplitude (right) distributions of mIPSCs obtained in WT and KO mice (n = 400 events per cell). The frequency of mIPSCs is significantly increased in <i>LAMP5</i> KO compared to WT mice (<i>p</i> < 0.001; WT amplitude n = 3 mice, n = 4 slices, n = 7 cells / KO amplitude n = 2 mice, n = 4 slices, n = 5 cells; WT frequency n = 3 mice, n = 4 slices, n = 7 cells / KO frequency n = 2 mice, n = 4 slices, n = 6 cells). (B) Parasagittal section of the mouse brain showing the recording and stimulation sites. Evoked striatopallidal GABAergic IPSCs are blocked by the GABA_A receptor antagonist, picrotoxin (ptx). St: striatum, GP: globus pallidus. (C) Mean PPR values from WT and KO mice at different interstimulus intervals. Sample traces of PPR are shown above the graph (traces were scaled to first IPSCs). * p < 0.05, ** p < 0.01 vs. WT mice. (D) Synaptic depression during repeated stimulation (10 pulses at 20 and 50 Hz) in WT mice was replaced by facilitation in KO mice. Representative traces to 20 and 50 Hz trains are shown in WT and KO mice. * p < 0.05, ** p < 0.01 vs. WT mice. Error bars represent SEM.</p

Differential expression of <i>LAMP5</i> mRNA and protein in the brain.

<p>(A,C) In situ hybridization and (B,D) immunohistochemistry for LAMP5 on sagittal brain sections (A,B) and on coronal olfactory bulb (OB) sections (C,D). Strongest expression of <i>LAMP5</i> mRNA is found in the neocortex (CX), piriform cortex (Pir), hippocampus (Hp), striatum (ST) and the granular cell layer (GCL) of the OB. LAMP5 protein is strongly present in the Globus Pallidus/Ventral Pallidum complex (GP/VP), the Substantia Nigra pars reticulata (SNr) and the entopeduncular nucleus (EP), that are the main output structures of the striatal GABAergic projection neurons, and in the external plexiform layer (EPL), in which granule cells positioned in the granule cell layer (GCL) form GABAergic synapses. (E-F) Schematic representation of <i>LAMP5</i> mRNA (light grey) and protein (dark grey) expression in the rodent forebrain (E) and OB (F). (G) qRT-PCR analysis of <i>LAMP5</i> expression in different brain tissue samples. Coherent with immunohistochemical stainings, strongest expression of <i>LAMP5</i> mRNA is detected in the cortex, striatum and the OB. (H) Western blotting and its quantification (I) demonstrates that LAMP5 protein is strongly expressed in GP and SNr while striatal tissue (ST) and cortex show only weak signal. Th: thalamus; Scale bar: 1 mm in A,B; 0.5 mm in C,D.</p

<i>LAMP5</i> deficient mice validate mAb 34.2 anti-LAMP5 antibody specificity.

<p>(A) Targeting strategy to generate a <i>LAMP5</i> deficient mouse line. The targeting vector was designed to remove exons 3–5 after CRE-induced recombination, leading to a null allele. <i>LAMP5</i> null animals are viable and therefore used for most analyses. (B) Western blot of KO (-/-), heterozygote (+/-) and wild type (+/+) mouse brains demonstrates absence of protein in homozygous KOs. (C) Immunohistochemistry with mAb 34.2 anti-LAMP5 antibody on <i>LAMP5</i>^<i>+/+</i> and <i>LAMP5</i>^<i>-/-</i> tissue sections of GP and OB validates the absence of LAMP5 protein in KOs. Cortex was always negative. Scale bars: C, 200 μm for GP and OB; 100 μm for cortex.</p