Firing of SST interneurons during 4-AP induced LFPs.

<p>Representative recordings of 4-AP-induced LFPs (A1,B1) and SST interneuron firing (A2,B2) during epileptiform activity. 4-AP provoked two classes of LFPs, detectable in CA3 (gray) and DG (black) hippocampal regions via extracellular pMEA recording (A1,B1): LFP_R-CA3 and LFP_P-CA3/DG (indicated with *). Juxtacellular patch-clamp recordings (A2,B2) from two visually identified SST- interneurons demonstrate increased AP firing of SST-interneurons in correspondence to distinct LFPs. Sections of the traces in <b>A</b> are shown at an expanded time scale in panel <b>B</b>.</p

4-AP-induced LFPs provoke AP firing from SST-interneurons.

<p>(<b>A</b>) Representative images of GCaMP3 signal in a visual field containing several SST interneurons before (left) and during (right) LFP_P-CA3/DG. (B) Simultaneous optical (B1) and juxtacellular (B3) recordings of SST activity during hippocampal LFPs (B2). B1: Fluorescent Ca²⁺ signals (ΔF/F) recorded from the ROIs indicated in (<b>A</b>). B2: Distinct LFPs recorded from pMEA electrodes located in the DG (black) and the CA3 (grey) regions.B3: Simultaneously recorded juxtacellular trace from a visually identified SST interneuron. (<b>C</b>) Same recording configuration as in (<b>B</b>) in the presence of the iGlurR antagonists, CPP and NBQX. Sections of traces in B3 and C3 (dotted line) are expanded below.</p

Distribution of somatostatin-positive interneurons in the hilus of SST-Ires-Cre mice.

<p>Representative fluorescence micrographs (Z-projection of confocal stacks) illustrating the expression of td-Tomato (<b>A</b>) and GCaMP3 (<b>B</b>) in the hippocampal hilus of SST-Ires-Cre reporter mice (postnatal days 18 and 14, respectively). GCaMP3 imaging was performed in the presence of 40 mM KCl to achieve maximal activation of the Ca²⁺ sensor.</p

Voltage-clamp recordings of LFP-induced currents in SST- interneurons.

<p>(A) Extracellular pMEA recording of LFPs (A1) together with concurrent optical imaging (A2) and juxtacellular (A3) or voltage-clamp (A4) recording (Vh = −50 mV, holding current 45 pA) from two SST- interneurons. The inward currents observed were significantly longer and larger in correspondence to the LFP_P-CA3/DG than to the LFP_R-CA3. Perfusion with NBQX/CPP (<b>B</b>) abolished the excitatory current revealing the occurrence of sustained IPSCs in association to the CPP/NBQX LFP. Note the increase in spontaneous IPSCs during the time between the LFPs occurrence. (<b>C</b>) Voltage-clamp recording (Vh = −52 mV, holding current 37 pA) from another SST- interneuron shown at different time scales illustrating the occurrence of inward EPSCs and less frequent outward IPSCs. Perfusion with NBQX/CPP (<b>D</b>) in the same neurons abolishes all EPSCs and reveals the occurrence of IPSCs.</p

LFPs evoke spontaneous EPSPs with a large synchronous depolarization in SST- interneurons.

<p>GCaMP3 fluorescence (orange) recorded simultaneously with extracellular LFPs (A1) during juxtacellular (A2, red) and intracellular current-clamp (A3, blue) recording from two SST- interneurons. The depolarization and spike burst duration were longer in correspondence to the LFP_P-CA3/DG than to the LFP_R-CA3 (A). Perfusion with CPP/NBQX (<b>B</b>) induced sustained spiking of the SST- interneuron with juxtacellular recording, but not for the SST-interneuron recorded in current clamp mode (resting membrane potential = −67 mV). Note the biphasic Ca²⁺ transient in cell #1 during CPP/NBQX application.</p

Soluble ICAM-5 affects an increase in GluA1 surface staining along dendrites in particular.

<p>Figure 4 shows data from live cell surface staining for GluA1 in control and ICAM-5 treated hippocampal neurons at 14 and 21 DIV. Indicated cultures were treated with 2.5 µg/ml soluble ICAM-5 and surface staining performed 1 hour later. Representative images are shown in A and C, while quantitative data is shown in B and D. The mean and standard error for percent control values were 100 +/- 8.8, n=21 for the DIV 14 control group; 187.5 +/- 16.4, n=16 for the DIV 14 ICAM-5 group; 100 +/- 5.8, n=25 for the DIV 21 control group; and 146.3 +/- 7.9, n=25 for the DIV 21 ICAM-5 group. Differences in GluA1 staining between control and ICAM-5 treated cultures are significant at <i>p</i>< 0.01 (*) at both 14 and 21 DIV.</p

The ICAM-5 ectodomain stimulates an increase in surface levels of the glutamate receptor subunit GluA1.

<p>Rat hippocampal neurons were unstimulated (control) or stimulated for 60 min. with 1 µg/ml of the ICAM-5 ectodomain (R & D Systems). Surface proteins were then biotinylated, and biotinylated proteins pulled down to be analyzed by Western blot. As can be appreciated, ICAM-5 was associated with an increase in surface GluA1 (A). Blots from separate experiments are shown. Densitometric analysis showing the fold increase in GluA1 band intensity in ICAM-5 versus control treated cultures in shown in (B). The mean and standard error for the fold increase from 6 replicate experiments is shown, and the difference between control and ICAM-5 groups is significant at <i>p</i> < 0.1 (*<i>p</i>=0.05). A representative blot for GluA2 in surface protein preparations is shown in (C), and densitometric analysis showing the fold change in GluA2 band intensity from 3 replicate experiments follows in (D). The mean and standard error for the fold change from 3 replicate experiments is shown, and the difference between control and ICAM-5 groups is not significant (<i>p</i>= 0.6).</p

Schematic representation of MMP-dependent ICAM-5 signaling at the synapse.

<p>In figure 6 we show a hypothetical model in which MMPs are rapidly released from preformed peri-synaptic stores to cleave ICAM-5 (green and lavender) at a membrane proximal site. The released N terminal fragment can bind unengaged integrins (red ovals) to stimulate intracellular signaling cascades leading to increased phosphorylation and membrane insertion of GluA1 subunits. Following ectodomain shedding, the C terminal fragment of ICAM-5 could undergo additional processing followed by internalization and degradation. It is worth noting that following MMP or ADAM mediated shedding, select CAMs are further processed by intramembranous proteolysis. ICDs thus generated may be degraded or, in some cases, influence gene transcription.</p

The ICAM-5 ectodomain affects an increase in mini excitatory post synaptic current (mEPSC) frequency.

<p>Stimulation of rat hippocampal neurons with 1 µg/ml ICAM-5 ectodomain (60 min. pretreatment) is associated with an increase in mEPSC frequency. In these experiments, 1,468 events from 11 control cells and 2353 events from 16 ICAM-5 stimulated cells were evaluated using standard techniques [74]. Representative tracings are shown in (A) while the average mEPSC frequency is shown in (B) and amplitude in (C). The difference between mEPSC frequency in control and ICAM treated neurons was significant (*<i>p</i> < 0.05, Student’s <i>t</i> test).</p

Phosphorylation of GluA1 at serine-845 is increased by soluble ICAM-5.

<p>Rat hippocampal neurons were unstimulated (control) or treated for 60 min. with 1 µg/ml of soluble ICAM-5 and lysates tested by Western blot for phospho-serine 845 GluA1. A representative blot is shown in (A) while densitometric analysis of blots from 5 experiments using distinct cultures is shown in (B). The mean and standard error for the fold increase is shown, and the fold increase is significant at <i>p</i> < 0.1 (*<i>p</i>= 0.06).</p