Effect of high sucrose in Rab3a KO cells.

<p><i>A</i>, representative traces of spontaneous postsynaptic currents in melanotrophs from WT and Rab3a-ablated pituitary glands before and after sucrose (500 mM) administration. Cells were voltage-clamped at the membrane potential of -80 mV. Increased postsynaptic activity during 500 mM sucrose intervention implied an intact innervation from hypothalamic neurons. <i>B</i>, spontaneous postsynaptic current (SPC) frequency. Numbers on bars indicate number of tested cells. ^∗<i>P</i><0.05 versus control (without sucrose).</p

Constitutive and KCl-triggered release in Rab3a KO melanotrophs.

<p>Specimens from blood plasma and pituitary slices or pituitary tissue culture medium were subjected to the competitive radioimmunoassay. <i>A</i>, plasma concentration of α-MSH (N= 5 mice per group). <i>B</i>, constitutive release of α-MSH was measured from the pituitary tissue culture medium after 24 hours. Pituitary slices were then homogenized and <b><i>C</i></b>, the cellular content of α-MSH was determined. <i>D</i>, KCl-stimulation (70 mM) provoked strong depolarisation of pituitary slices and triggered full secretory release of α-MSH from cellular stores. <i>E</i>, constitutive release of β-endorphin after 24 hours incubation at 37°C. Samples (pituitary slices) were collected and <b><i>F</i></b>, the cellular content of β-endorphin was quantified. ^∗<i>P</i><0.05 versus WT. </p

Alpha-MSH and pro-opiomelanocortin in Rab3a KO melanotrophs.

<p>Representative confocal microscopy micrographs from immunocytochemistry of γ-MSH/ pro-opiomelanocortin (POMC) (top) and α-MSH (bottom) in WT and Rab3a KO pituitary slices. Arrow indicates the intermediate lobe (PI). AP=anterior part, PP=posterior part. Rab3a KO melanotrophs contained POMC, but lacked α-MSH positive signal. </p

Initial high Ca²⁺ sensitive phase in Rab3a KO melanotrophs.

<p><i>A</i>-<i>C</i>, slow photo-release of caged Ca²⁺ triggered a multiphasic ΔC_m. The marked area is magnified in panel C. <i>D</i>, [Ca²⁺]_i threshold. <i>E</i>, cumulative ΔC_m. <i>F</i>, time derivative of the ΔC_m presented in B as a function of [Ca²⁺]_i during the first 2 s of the slow photo-release of caged Ca²⁺. Note that high Ca²⁺ sensitive phase (vesicle pool) was greatly reduced in Rab3a KO cells (arrow), and that Ca²⁺ triggered ΔC_m at significantly higher [Ca²⁺]_i in Rab3a KO melanotrophs compared to WT cells (inset); the inset shows normalized C_m rate fitted by the Hill function and is displayed as a function of [Ca²⁺]_i. <i>G</i>, half effective [Ca²⁺]_i (EC₅₀). <i>H</i>, amplitude of high Ca²⁺ sensitive pool (HCSP) quantified at 1.5 µM [Ca²⁺]_i . Numbers on bars indicate the number of tested cells. ^∗<i>P</i><0.05 versus WT.</p

Rescue experiments in Rab3a KO melanotrophs.

<p><i>A</i>, Semliki forest virus transduction of a WT-Rab3a plasmid restored α-MSH in Rab3a KO pituitary slice. <i>B</i>, Representative cumulative ΔC_m traces show depolarisation-induced secretory response in WT cells (black circles), Rab3a KO cells (grey triangles), Rab3a-deficient melanotrophs infected with Semliki forest virus harbouring a GTP-ase deficient Rab3a mutant (Rab3aQ81L, white triangles) or WT-Rab3a mutant (Rab3aKO+WT-Rab3a, grey circles). A repetitive stimulation of 75 pulses (40 ms stimulation, 10 Hz from -80 mV to 10 mV) evoked Ca²⁺ -dependent exocytosis. The marked area is magnified in panel C. <i>C</i>, The linear component was attenuated in Rab3aQ81L and Rab3a KO cells, but restored in Rab3a KO melanotrophs overexpressing a WT-Rab3a mutant. <i>D</i>, cumulative ΔC_m. <i>E</i>, average slope of the linear component. <i>F</i>, High voltage-gated Ca²⁺ current density. Numbers on bars indicate the number of tested cells.∗P<0.05 versus WT. </p

Depolarisation-induced secretory response in Rab3a KO melanotrophs.

<p><b><i>A</i>_<i>1</i></b>, Representative ΔC_m traces show Ca²⁺-dependent exocytosis triggered by a train of 50 depolarisation pulses (40 ms duration, at 10 Hz) in WT and Rab3a KO melanotrophs. Grey area confines the linear component (first second of depolarisation-<b><i>A</i>_<i>2</i></b>) from the subsequent threshold component. <i>B</i>, cumulative ΔC_m after 50 depolarisation pulses. <i>C</i>, summary of average slope of the linear component (grey shaded area-panel A₁) and threshold component. <i>D</i>, normalized I-V plot. Voltage-activated Ca²⁺ currents evoked by 300 ms voltage ramps (from -80 mV to 60 mV) were normalized to resting membrane cell capacitance. <i>E</i>, High voltage-activated (HVA) Ca²⁺ current density. Numbers on bars indicate the number of tested cells. ^∗<i>P</i><0.05 versus WT.</p

Effect of cAMP on Ca²⁺-dependent exocytosis in Rab3a KO melanotrophs.

<p><i>A</i>-<i>B</i>, representative traces of total membrane capacitance changes (ΣΔC_m) recorded during a train of 200 depolarisation pulses (40 ms duration, 10 Hz, from -80 mV to 10 mV) in WT and Rab3a KO cells treated without and with 200 μM cAMP in the pipette solution. The marked area is enlarged in panel B. Note that cAMP did not affect the linear component in Rab3a KO cells (grey area, enlarged inset). Note also a rapid increase in the ΣΔC_m as part of the subsequent threshold component (arrow) in Rab3a KO melanotrophs treated with 200 μM cAMP. <i>C</i>, average slope of the linear component. <i>D</i>, High voltage-activated Ca²⁺ current density. Numbers on bars indicate number of tested cells. ^∗<i>P</i><0.05 versus WT control (ctrl). ^#<i>P</i><0.05 versus cAMP-treated WT cells.</p

The experience-enabled enhancement of the optomotor reflex of the open eye after monocular deprivation (MD) was compromised in both PT-lesioned PSD-95 WT and PSD-95 KO mice.

<p>In contrast, enhancements of spatial vision were present in nonlesioned PSD-95 WT and PSD-95 KO mice. (A, B) Spatial frequency threshold of the optomotor response of the open eye in cycles per degree (cyc/deg) plotted against days after MD. After 7 days of MD, nonlesioned PSD-95 KO mice (A) as well as sham-treated control mice (B; data from Greifzu et al., 2011) showed a significant increase in the spatial frequency threshold of the optomotor reflex of the open eye. This experience-enabled increase was abolished by a PT in S1 (A, B). (C-F) Contrast sensitivity thresholds of the optomotor reflex of the open eye at 6 different spatial frequencies before (day 0) and 7 days after MD. For both nonlesioned PSD-95 KO (C) and PSD-95 WT mice (D), there was an increase in contrast sensitivity after 7 days of MD. After PT, this experience-enabled increase was absent in both groups (E, F).</p

Location of the photothrombotically induced cortical stroke lesion in a PSD-95 KO mouse in S1 (PT, red dashed line).

<p>(A) Top view of a representative mouse brain illustrating the lesion location in S1, on average 1 mm anterior to the anterior border of the primary visual cortex (V1, blue dashed line). (B) Nissl-stained frontal section through the lesion (same animal as in A). (C) Higher magnification composite image of the superficial vascular pattern of the brain and the superimposed optically recorded retinotopic map of the binocular part of V1 of a PSD-95 KO mouse in which the PT-lesion (L) was very close to V1; nevertheless, OD-plasticity was present in this animal (average ODI = 0.00). Scale bar all, 1 mm.</p

In adult PSD-95 KO mice, ocular dominance plasticity was preserved after a PT-stroke in S1.

<p>Optically recorded activity maps of the contralateral (contra) and ipsilateral (ipsi) eye in the binocular region of mouse primary visual cortex (V1) in PT-lesioned PSD-95 WT (A) and PSD-95 KO mice (B) after monocular deprivation (MD), and their quantification (C, D). Open and closed eyes indicated by a white or black circle. (A, B) Grayscale coded response magnitude maps of the contra- and ipsilateral eye (including average V1-activation of the illustrated example) and the histogram of OD-scores including the average OD-index (top row), color-coded polar maps of retinotopy and 2-dimensional OD-maps after MD (lower row) are illustrated. (A) In PSD-95 WT littermates, activity patches evoked by stimulation of the contralateral eye were darker than those of the ipsilateral eye, the average ODI was positive, and warm colors prevailed in the OD-maps, indicating contralateral dominance. In contrast, in PSD-95 KO mice, the contra- and ipsilateral eye activated V1 about equally strong, colder colors appeared in the OD-map, and the histogram of OD-scores shifted to the left (B). Scale bar: 1 mm. (C) Optically imaged OD-indices in PT-lesioned PSD-95 WT and PSD-95 KO mice (light red and red triangles). For comparison, ODIs of control (sham-treated) and PT-lesioned Bl6/J mice are illustrated (white and grey squares). Symbols represent ODI values of individuals, means are marked by horizontal lines. (D) V1-activation elicited by stimulation of the contralateral (C) or ipsilateral (I) eye in PSD-95 WT and PSD-95 KO mice after PT and MD. Note that V1-activtation after stimulation of the contra- and ipsilateral eye was not different in PSD-95 KO mice, indicating preserved juvenile-like OD-plasticity in V1 despite the S1-lesion.</p