Image_3_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.TIF

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

Table_2_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.docx

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

Data_Sheet_1_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.pdf

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

Table_1_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.docx

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

Image_1_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.TIFF

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

Image_2_A subpopulation of agouti-related peptide neurons exciting corticotropin-releasing hormone axon terminals in median eminence led to hypothalamic-pituitary-adrenal axis activation in response to food restriction.TIF

The excitatory action of gamma-aminobutyric-acid (GABA) in the median-eminence (ME) led to the steady-state release of corticotropin-releasing hormone (CRH) from CRH axon terminals, which modulates the hypothalamic-pituitary-adrenal (HPA) axis. However, in ME, the source of excitatory GABAergic input is unknown. We examined agouti-related peptide (AgRP) expressing neurons in the arcuate nucleus as a possible source for excitatory GABAergic input. Here, we show that a subpopulation of activated AgRP neurons directly project to the CRH axon terminals in ME elevates serum corticosterone levels in 60% food-restricted mice. This increase in serum corticosterone is not dependent on activation of CRH neuronal soma in the paraventricular nucleus. Furthermore, conditional deletion of Na+-K+-2Cl– cotransporter-1 (NKCC1), which promotes depolarizing GABA action, from the CRH axon terminals results in significantly lower corticosterone levels in response to food restriction. These findings highlight the important role of a subset of AgRP neurons in HPA axis modulation via NKCC1-dependent GABAergic excitation in ME.</p

MHCI molecules are expressed in the nucleus accumbens core in WT mice.

<p>Staining for MHCI with OX-18 antibody (upper-left), Neurogranin (upper-center) and the merged image (upper-right) are shown. Lower images represent negative controls without OX-18. Scale bar represents 50 µm.</p

Comparison of AMPA receptor densities.

<p>A) A raw electron micrograph image of SDS-FRL replica immunolabeled with pan-AMPA antibody (left), and an analyzed image (right). A representative replica from a wild type mouse treated with saline (WT-Sal) is shown. The blue area indicates intra-membrane particle clusters and the black dots indicate immunogold particles. B) Densities of pan-AMPA, GluR1, and GluR2 in WT-Sal mice and β2m mice treated with saline (β2m^−/−-Sal). The densities normalized to WT in each receptor were: pan-AMPA: β2m^−/−-Sal, 110.0±8.5%; GluR1: β2m^−/−-Sal, 92.9±7.8%; GluR2: β2m^−/−-Sal, 95.4±1.5%, n = 6 [for each receptor 6 replicas from three animals, 30 synapses per replica]. C) Comparison of pan-AMPA, GluR1 and GluR2 densities between WT-Sal mice and wild type mice treated with cocaine (WT-Coc). The densities normalized to saline group in each receptor were: pan-AMPA: WT-Coc, 119.4±8.3%; GluR1: WT–Coc, 135.3±8.9%; GluR2: WT–Coc, 105.6±9.6%, n = 6 [for each receptor six replicas from three animals, 30 synapses per replica]. D) Comparison of pan-AMPA, GluR1 and GluR2 densities between β2m^−/−-Sal mice and β2m^−/− mice treated with cocaine (β2m^−/−-Coc). The densities normalized to saline group in each receptor were: pan-AMPA: β2m^−/−-Coc, 130.9±10.0%; GluR1: β2m^−/−-Coc, 117.5±5.8%; GluR2: β2m^−/−-Coc, 119.7±6.2%, n = 6 [for each receptor six replicas from three animals, 30 synapses per replica]. E) Representative electron micrographs of the replicas from WT-Sal and WT-Coc mice. The blue area indicates intra-membrane particle clusters and the black dots indicate immunogold particles. F) Representative electron micrographs of the replicas from β2m^−/−-Sal and β2m^−/−-Coc mice. The red area indicates intra-membrane particle clusters and the black dots indicate immunogold particles. Scale bars represent 200 nm. *p<0.05.</p

Comparison of the fEPSP slopes.

<p>A) Comparison between WT mice and β2m^−/− mice after high frequency stimulation (HFS) at 100 Hz. HFS induced LTP in both groups (WT: 125.8±4.5%, n = 9/N = 5, T = 1, p<0.01; β2m^−/−: 141.8±5.7%, n = 11/N = 6, T = 0, p<0.001; Signed rank test), whereas LTP was enhanced in the β2m^−/− group (t(18) = 2.107, p<0.05; Student’s t-test). B) Comparison between WT and β2m^−/− mice after low frequency stimulation (LFS) at 10 Hz. LFS induced LTD in WT mice (79.2±5.9%, n = 9/N = 6, T = 1, p<0.05), whereas it was ineffective in β2m^−/− mice (103.2±7.3%, n = 9/N = 6, T = 18, p = 0.65). At 45–50 min after LFS, the fEPSP slope in β2m^−/− mice was significantly higher than in WT mice (t(16) = 2.548, p<0.05). C) Comparison between WT and β2m^−/− mice after 1 Hz stimulation. Neither LTP nor LTD was induced by this stimulation (WT: 103.4±7.1%, n = 7/N = 6, T = 9, p = 0.47; β2m^−/−: 100.7±5.7%, n = 7/N = 6, T = 12, p = 0.81). There was no significant difference in the fEPSP slope at 45–50 min after 1 Hz/15 min stimulation between genotypes (t(12) = 0.302, p = 0.768). D) Comparison of paired pulse ratios (PPRs). (Left) PPRs in WT and β2m^−/− mice at 30, 50 and 100 ms inter stimulus interval (ISI). There was no significant difference in the PPRs of WT (ISI 30 ms: 117.9±2.4%; ISI 50 ms: 124.5±2.6%; ISI100 ms: 111.7±1.0%) (n = 9/N = 5) and β2m^−/− (ISI 30 ms: 116.8±1.9%; ISI 50 ms: 123.2±2.3%; ISI 100 ms: 111.5±0.8%) (n = 9/N = 5) mice, in all tested ISIs (ISI 30 ms: t(16) = 0.349, p = 0.731; ISI 50 ms: t(16) = 0.355, p = 0.727; ISI 100 ms: t(16) = 0.164, p = 0.872). (Right) Representative traces of fEPSPs at 50 ms inter stimulus interval (blue: WT; red: β2m^−/−). Vertical scale bars represent 100 µV, and horizontal scale bars represent 10 ms. *p<0.05. n = slices/N = animals.</p

Behavioral sensitization elicited by repeated cocaine exposure.

<p>After three days of saline injections, WT and β2m^−/− mice were divided into groups that received daily injections of saline or cocaine (20 mg/kg) for 7 days. These groups were composed of WT mice treated with saline (WT-Sal) (n = 6) or cocaine (WT-Coc) (n = 6) and β2m^−/− mice treated with saline (β2m^−/−-Sal) (n = 6) or cocaine (β2m^−/−-Coc) (n = 6). Mice of both genotypes showed increased locomotor activity following cocaine but not saline injections. Analysis was conducted over a 8-day period from day 3 to day 10 in WT-Coc and β2m^−/−-Coc groups. Two-way ANOVA with repeated measures revealed significant interactions of day × genotype (F(7, 70) = 2.892, p<0.05), a main effect of day (F(7, 70) = 42.285, P<0.001) and genotype (F(1, 10) = 5.027, p<0.05). Bonferroni post-hoc test revealed significant differences at day 7, day 9 and day 10. Furthermore, the β2m^−/−-Coc group displayed a larger response to a challenge dose of cocaine after withdrawal (Day 24) than the WT-Coc group. Two-way ANOVA revealed a main effect of genotype (F(1, 20) = 19.674, p<0.001) and treatment (F(1, 20) = 121,488, p<0.001), and a significant interaction of genotype × treatment (F(1, 20) = 13.138, p<0.01). A significant difference was observed between genotypes treated with cocaine (p<0.001, Bonferroni post-hoc test). *p<0.05 β2m^−/−-Coc versus WT-Coc; ***p<0.001 β2m^−/−-Coc versus WT-Coc.</p