TBS-induced LTP and the NMDA- to AMPA-mediated EPSC ratio are reduced in adult <i>NesCreIrs2KO</i> mice.

<p><b>A</b>: In the presence of the GABA_A receptor antagonist SR95531 (6 µM), LTP was reliably induced in control mice (+/+; average EPSP slope change 60 min after induction: 151±6%, N = 4, n = 6) in response to a single theta-burst protocol (TBS). The level of LTP produced under the same conditions, 60 min following TBS, was significantly reduced in <i>NesCreIrs2KO</i> mice (−/−; average EPSP slope change 60 min after induction: 123±6%, N = 4, n = 5, p<0.005). Arrow indicates application of TBS. <b>B</b>: Sample EPSP traces (average of 4 consecutive sweeps) from individual experiments, taken prior to, and 60 min following TBS in control (+/+) and <i>NesCreIrs2KO</i> (−/−) mice. <b>C</b>: In the presence of SR95531 (6 µM), the ratio of NMDA-EPSC amplitude (holding potential: +40 mV) to AMPA-EPSC amplitude (holding potential: −70 mV) was significantly lower in <i>NesCreIrs2KO</i> (−/−; 0.32±0.04, N = 6, n = 11) compared to littermate controls (+/+; 0.44±0.04, N = 5, n = 12, p<0.05). <b>D</b>: EPSC traces (average of 10 consecutive sweeps) from whole-cell patch clamp experiments in <i>NesCreIrs2KO</i> (−/−) and control mice (+/+) illustrating the NMDA-EPSC (measured at a holding potential of +40 mV) and corresponding AMPA-EPSC (measured at a holding potential of −70 mV). NMDA-EPSC amplitude was measured 50 ms following stimulation to minimize contamination from the AMPA-mediated component. <b>E–I</b>: Representative immunoblot images of hippocampal subfield CA1 lysates from control (+/+) and <i>NesCreIrs2KO</i> mice (−/−) probed with antibodies against the total protein or specific phosphorylation sites of the AMPA receptor subunit GluR1 (<b>E–F</b>) and the NMDA subunits NR1 (<b>G</b>), NR2A (<b>H</b>), and NR2B (<b>I</b>). Quantifications of immunoblots showing the protein level or relative proportion of phosphorylated protein over the total are shown in the bar diagrams. <b>E–F</b>: The total level of the AMPA receptor subunit GluR1 was similar in control (100±8%) and <i>NesCreIrs2KO</i> mice (77±9%, n = 10, p = 0.063; <b>upper panels</b>). <b>E</b>: The proportion of GluR1 subunit phosphorylated at Ser845 (p-GluR1-S845) relative to total GluR1 protein was similar in control (100±4%, n = 5) and <i>NesCreIrs2KO</i> mice (93±8%, n = 5, p = 0.46; <b>middle and lower panels</b>). <b>F</b>: The proportion of GluR1 subunit phosphorylated at Ser831 (p-GluR1-S831) relative to total GluR1 protein was similar in control (Ser831: 100±13%; n = 5) and <i>NesCreIrs2KO</i> mice (Ser831: 103±18%, n = 5; p = 0.73; <b>middle and lower panels</b>). <b>G</b>: There was no change in the total level of the NMDA receptor subunit NR1 (control mice: 100±11%; <i>NesCreIrs2KO</i> mice: 93±8%, n = 5, p = 0.46; <b>upper panel</b>). <i>NesCreIrs2KO</i> mice displayed a reduced phosphorylation of NR1 at Ser897 (p-NR1; 45±9%, n = 6) relative to their control littermates (100±9%, n = 6, p<0.001; <b>middle and lower panels</b>). <b>H–I</b>: There were no changes in the total levels of the NMDA receptor subunits NR2A (<b>H</b>; control mice: 100±11%; <i>NesCreIrs2KO</i> mice: 89±11%, n = 7, p = 0.49), and NR2B (<b>I</b>; control mice: 100±17%; <i>NesCreIrs2KO</i> mice: 129±17%, n = 5, p = 0.55). Data are expressed as mean ± SEM. **p<0.01 (unpaired t-test).</p

Hypothalamic and brainstem <i>Nnat</i> expression after modified gastric bypass <i>versus</i> sham surgery.

<p>A) <i>Nnat</i>-β showed a significant reduction in the hypothalamus (**P = 0.003) after modified gastric bypass (n = 8) compared to sham surgery (n = 7) whilst <i>Nnat</i>-α did not reduce significantly, consistent with a bypass-specific effect on <i>Nnat</i>-β expression; B) expression of <i>Nnat</i>-α and <i>Nnat</i>-β did not differ in the brainstem after modified gastric bypass (n = 8) compared to sham surgery (n = 7); <i>key – GBP = modified gastric bypass surgery, Sham = sham surgery, AU = arbitrary units where Nnat expression was standardised using an endogenous reference gene (ubiquitin (Ubc) for hypothalamus, hypoxanthine guanine phosphoriboribosyl transferase (Hprt) for brainstem).</i></p

Metaplasticity cannot be induced in juvenile <i>NesCreIrs2KO</i> mice.

<p><b>A</b>: A priming stimulus (10 Hz; small arrow) applied 20–30 min prior to TBS (large arrow) significantly inhibited subsequent LTP induction in juvenile control mice (+/+) for at least 50 min (average EPSP slope change: 10 Hz primed: 104±4%, N = 7, n = 7), when compared to un-primed LTP recorded 50 min following induction (average EPSP slope change: Control: 158±9%, N = 7, n = 7, p<0.0005). <b>B</b>: In juvenile <i>NesCreIrs2KO</i> mice (−/−), the same priming stimulus (10 Hz; small arrow) did not affect TBS-induced LTP (average EPSP slope change: 10 Hz primed; 135±5%, N = 7, n = 10), compared with the un-primed LTP measured 50 min post-TBS (average EPSP slope change: Control; 126±11%, N = 5, n = 7; p = 0.47). Insets in <b>A</b> and <b>B</b> show representative EPSP traces (average of 4 consecutive sweeps) taken prior to and 50 min following TBS, from individual experiments in control (<b>A</b>) and <i>NesCreIrs2KO</i> (<b>B</b>) slices to which 10 Hz priming stimuli had previously been applied. Scale bars: 0.4 mV; 10 ms. <b>C</b>: Enhancing NMDA receptor activity by lowering the concentration of extracellular Mg²⁺ to 1 mM and increasing the Ca²⁺ concentration to 3 mM, caused an attenuation of TBS-induced LTP in juvenile control mice (+/+) (average EPSP slope change: Low Mg²⁺/High Ca²⁺: 135±3%, N = 5, n = 6) compared with LTP recorded for 60 min under standard ionic conditions (average EPSP slope change: Control: 160±9%, N = 7, n = 7, p<0.05). <b>D</b>: The same ionic conditions to enhance NMDA receptor activity did not significantly alter the level of LTP obtained in <i>NesCreIrs2KO</i> mice (−/−) (average EPSP slope change: Low Mg²⁺/High Ca²⁺: 127±4, N = 4, n = 5) compared with that recorded in control conditions 60 min following induction (average EPSP slope change: Control: 128±11%, N = 5, n = 7). <b>E</b>: Metaplasticity induced in control mice (+/+) by altering extracellular divalent cation concentrations to enhance NMDA receptor activity yielded an LTP of similar magnitude to that observed in <i>NesCreIrs2KO</i> mice (−/−) under the same conditions. <b>F</b>: Representative EPSP traces (average of 4 consecutive sweeps) from individual experiments performed in low Mg²⁺/high Ca²⁺ conditions, taken prior to application of TBS and following 60 min of LTP in both control (+/+) and <i>NesCreIrs2KO</i> (−/−) mice.</p

Changes in body-weight, leptin and gut hormones after surgery.

<p>GBP = modified gastric bypass surgery; data presented as Mean (Standard Error of Mean) or P value (<i>t</i> test).</p

Isoform-specific <i>Nnat</i> expression in the hypothalamus after surgery in relation to weight-loss and circulating leptin.

<p>A–B) <i>Nnat</i>-α expression in the hypothalamus did not correlate with either change in body-weight or with circulating leptin after surgery; C–D) by contrast <i>Nnat</i>-β expression showed positive correlation with change in body-weight and weak positive correlation with circulating leptin after surgery; <i>key – Nnat-α expression shown with open squares, Nnat-β with filled circles, AU = arbitrary units where Nnat expression was standardised using ubiquitin (Ubc) as a reference gene</i>.</p

Change in body weight and leptin after chronic dietary switches.

<p>Control = standard dietary chow maintained, Control-HF = standard dietary chow switch to high-fat diet, Control-CR = standard dietary chow switch to caloric restriction; HF-Control = high-fat diet switch to standard dietary chow, HF = high-fat diet maintained, HF-CR = high-fat diet switch to caloric restriction; data presented as P value (post-hoc ANOVA).</p

LTP and phosphorylation of Akt and GSK-3β are reduced in juvenile <i>NesCreIrs2KO</i> mice.

<p><b>A</b>: LTP induced by a single TBS in slices with intact inhibitory synaptic transmission was significantly reduced in juvenile <i>NesCreIrs2KO</i> mice (−/−; average EPSP slope change: 128±11%, N = 5, n = 7) compared to values obtained from littermate controls (+/+; average EPSP slope change: 160±9%, N = 7, n = 7, p<0.05), 60 min following induction. <b>B</b>: Representative EPSP traces (average of 4 consecutive sweeps) from individual experiments taken immediately prior to TBS, and following 60 min of LTP in both control (+/+) and <i>NesCreIrs2KO</i> (−/−) mice. <b>C</b>: Immunoblot analysis from CA1 lysates showing that neither the total level of p38MAPK nor the amount of phosphorylated p38MAPK (Thr180/Tyr182) are altered by brain-deletion of IRS-2 in <i>NesCreIrs2KO</i> (−/−; 102±8%) mice compared with control littermates (+/+; 100±14%, n = 5–7; p = 0.91). The bar diagram on the right shows a summary of the data obtained from 5–7 mice per experimental group. <b>D</b>: Immunoblot analysis from CA1 specific lysates showing that neither the total level of p42/44 MAPK (ERK) nor the amount of ERK phosphorylated at Thr202 and Tyr204 are altered by brain-deletion of IRS-2 in <i>NesCreIrs2KO</i> mice. For p42 MAPK: +/+ 100±6.5%; −/− 94.8±5.5%; n = 5; p = 0.56. For p44 MAPK: +/+ 100±6.4%; −/− 101.5±4.7%; n = 5; p = 0.86. <b>E</b>: Immunoblot analysis from CA1 lysates showing reduced Akt/protein kinase B phosphorylation levels at Thr308 and Ser473 in <i>NesCreIrs2KO</i> mice (−/−; Ser473: 74±8.%; Thr308: 61±9%, n = 7) compared to controls (+/+; Ser473: 100±8%; Thr308: 100±9%, n = 7; p = 0.04 for Ser473, p = 0.01 for Thr308). The bar diagram on the right shows a summary of the data obtained from 7 mice per experimental group. <b>F</b>: Immunoblot analysis from CA1 lysates showing reduced phoshosphorylation of GSK-3β at Ser9 in <i>NesCreIrs2KO</i> mice (−/−; 48±10%, n = 5) compared to controls (+/+; 100±10%, n = 5; p = 0.007). The bar diagram on the right shows a summary of the data obtained from 5 mice per experimental group. Data are expressed as mean ± SEM. *p<0.05; **p<0.01 (unpaired t-test).</p

Expression of <i>Nnat</i> isoforms in response to fasting versus <i>ad-libitum</i> feeding.

<p>A) Hypothalamic <i>Nnat</i>-α and -β showed a non-significant reduction in response to overnight fasting (n = 10) when compared to <i>ad-libitum</i> fed counterparts (n = 10); B) but a significant reduction after 24-h fasting (n = 10) compared to feeding (n = 11), equivalent for both isoforms (**<i>Nnat</i>-α P = 0.005, **<i>Nnat</i>-β P = 0.007); C–D) <i>Nnat</i> isoforms did not show a consistent or significant change after overnight fasting in the stomach or duodenum (n = 10 fasted, n = 10 <i>ad-libitum</i> fed in both tissues); <i>key – AU = arbitrary units where Nnat expression was standardised using ubiquitin (Ubc) as a reference gene</i>.</p