Harvey Grill Lab

Representative immunofluorescent analysis showing the extent of AAV-shCtrl (A) and AAV-LepR (B) spread, with EGFP-expressing neurons being identified in nuclei that contain the LepR: medial nucleus tractus solitarius (mNTS) and area postrema (AP). EGFP-expressing neurons are also found in non-LepR-expressing nuclei: dorsal motor nucleus of the vagus (DMV), dorsolateral NTS (SolDL) and solitary commissural (SolC). cc = central canal. (C) Representative qPCR reveals significant suppression in LepR mRNA in mNTS/AP micropunched tissue for AAV-LepR treated rats compared to AAV-shCtrl rats. * = P< 0.05. (D) Daily Kcal intake for LepRKD and shCtrl rats pre- and post-viral delivery while maintained on chow or high fat (HF) diet (60% Kcal from fat). * = P< 0.05 for bracketed weekly averages.

Figures taken from Hayes, Skibicka, Leichner, Guarnieri, DiLeone, Bence, Grill (Cell Metabolism 2010)

Cumulative body weight of chow-maintained LepRKD and shCtrl rats pre- and post-mNTS/AP directed AAV delivery. * = P< 0.05 for bracketed weekly averages. Inset graph shows inguinal and total WAT mass for LepRKD and shCtrl rats. * = P<0.05.

Figures taken from Hayes, Skibicka, Leichner, Guarnieri, DiLeone, Bence, Grill (Cell Metabolism 2010)

(A) Cumulative 15% sucrose intake following IP injection of CCK (3μg/kg) or vehicle (0.9% saline) for LepRKD and shCtrl rats pre- and 4 weeks-post mNTS-directed AAV delivery. * = P< 0.05. (B) LepRKD rats showed increased pAMPKα2 levels in mNTS/AP micropunched tissue compared to shCtrl rats under ad libitum fed conditions. Representative immunoblots for total AMPK and pAMPKα2 are shown. Relative pAMPKα2 = the ratio of pAMPKα2 to total AMPK. * = P< 0.05. (C) Cumulative chow intake for LepRKD and shCtrl rats following 4th icv delivery of compound C (15μg) or vehicle (DMSO). * = P< 0.05.

Figures taken from Hayes, Skibicka, Leichner, Guarnieri, DiLeone, Bence, Grill (Cell Metabolism 2010)

Suppression of Intake by Hindbrain GLP-1R Activation Is Meditated by Reduced AMPK Signaling(A) Decreased phosphorylation of AMPKα2 in caudal DVC tissue lysates following fourth i.c.v. Ex-4 (0.3 μg) administration. Representative immunoblots for pAMPKα2 and total AMPKα are shown. *p < 0.05 from aCSF. Cumulative chow intake (B) and 24 hr BW change (C) following fourth i.c.v. delivery of the AMPK activity promoter AICAR (300 μg) or Ex-4 (0.1 μg) alone or in combination, counterbalanced with vehicle/vehicle (aCSF/aCSF). Chow intake recorded via automated feedometers that continuously record food intake to the nearest ± 0.1 g/m/24 hr. Data are mean ± SEM. *p < 0.05 from vehicle/vehicle. #p = 0.065 from vehicle/Ex-4.

Figures taken from Hayes, Leichner, Zhao, Lee, Chowansky, Zimmer,De Jonghe, Kanoski, Grill, Bence (Cell Metabolism 2011)

Intracellular Signaling Pathways in NTS GLP-1R-Expressing Neurons Mediating Food Intake Suppression by Hindbrain GLP-1R LigandsGastric vagal afferent signaling increases endogenous NTS-derived GLP-1, which activates endemic NTS GLP-1R-expressing neurons (Hayes et al., 2009a) to engage cAMP-dependent increase in PKA activity. Increased PKA activity concomitantly increases phosphorylation of p44/42-MAPK and decreases phosphorylation of AMPK. The combined increase in PKA and p44/42-MAPK activity together with decreased AMPK activity is hypothesized to increase CREB-mediated nuclear transcription and protein synthesis. mTOR, mammalian target of rapamycin; CAMKK, calmodulin-dependent protein kinase kinase; CAMKII, calcium/calmodulin-dependent protein kinase II; VGCC, voltage gated calcium channel; MEK, mitogen-activated protein kinase kinase.

Figures taken from Hayes, Leichner, Zhao, Lee, Chowansky, Zimmer,De Jonghe, Kanoski, Grill, Bence (Cell Metabolism 2011)

A–D, Effect of PBN PGE2 administration (0.1 μl) on TC (A), HR (B), SPA (C), and 24-h food intake (D); E, photomicrograph with a representative injection in the PBN. Line graphs represent across-rat average parameter measurements throughout the recording period. The bracketed time period on the line graph x-axis indicates the periods used in the histograms below. The histograms below provide 1.5-h postinjection averages + sem for each parameter at each dose. **, P < 0.005; ***, P < 0.0005. LPB, Lateral parabrachial nucleus; LPBC, lateral parabrachial nucleus, central part; LPBE, lateral parabrachial nucleus, external part; KF, Killiker-Fuse nucleus; MPB, medial parabrachial nucleus.

Figures taken from Skibicka, Alhadeff, Leichner, Grill (Endocrinology 2011)

A–D, Effect of PVH PGE2 administration (0.1 μl) on TC (A), HR (B), SPA (C), and 24-h food intake (D); E, photomicrograph with a representative injection in the PVH. Line graphs represent across-rat average parameter measurements throughout the recording period. The bracketed time period on the line graph x-axis indicates the periods used in the histograms below. The histograms below provide 1.5 h postinjection averages + sem for each parameter at each dose. *, P < 0.05; **, P < 0.005. PaDC, Paraventricular hypothalamic nucleus, dorsal cap; PaLM, paraventricular hypothalamic nucleus, lateral magnocellular part; PaMP, paraventricular hypothalamic nucleus, medial parvicellular part; PaV, paraventricular hypothalamic nucleus, ventral part.

Figures taken from Skibicka, Alhadeff, Leichner, Grill (Endocrinology 2011)

Food intake. Leptin administration to the ventral and dorsal hippocampus reduced food intake in rats. Chow intake is shown at 1, 3, 6, and 24 h following bilateral intra-hippocampal leptin injections at dark onset in ad libitum fed rats (*p<0.05).

Figures taken from Kanoski, Hayes, Greenwald, Fortin,Gianessi, Gilbert, Grill (Neuropsychopharmacology 2011)

Spatial and non-spatial memory consolidation. Ventral but not dorsal hippocampal leptin delivery (0.4 μg) after training suppressed memory consolidation for the spatial location of food relative to vehicle treatment (a). Leptin did not influence the consolidation of an appetitive non-spatial response task (b) (*p<0.05).

Figures taken from Kanoski, Hayes, Greenwald, Fortin,Gianessi, Gilbert, Grill (Neuropsychopharmacology 2011)

Cumulative chow intake at 1 h and 3 h (A), at 6 h and 24 h (B), and δ body weight at 24 h (C) after ICV (vehicle or exendin-(9–39), 100 μg) and ip (vehicle, liraglutide, 10 μg/kg; or exendin-4, 3.0 μg/kg) drug coadministration (* denotes significant difference compared with ICV vehicle/ip vehicle treatment; † denotes significant difference compared with ICV vehicle/ip liraglutide treatment; ‡ denotes significant difference compared with ICV vehicle/ip exendin-4 treatment). P < 0.05 = significant difference; data are mean ± sem.

Figures taken from Kanoski,Fortin, Arnold, Grill, Hayes (Endocrinology 2011)

(A) Compared to rats fed ad libitum, 24h food deprivation increased pAMPK levels in NTS-enriched tissue (DVC). These data indicate that pAMPK levels in DVC and liver tissues are similarly responsive to energy status, with food deprivation increasing pAMPK in both the liver (control) and NTS by approximately 25%. (B) Elevated pAMPK levels in NTS-enriched tissue of 48h food deprived rats is reduced following a 2h reefed. Representative immunoblots for total AMPK and pAMPK are shown.

Figures taken from Hayes, Skibicka, Bence and Grill (Endocrinology 2009)

(A) Compound C (an AMPK inhibitor; 1.0μg/100nl) delivered to the caudal mNTS, at the AP level, significantly suppressed food intake at 3h, 6h and 24h, as well as 24h body weight gain compared to intakes and body weight following vehicle injections. (B) By contrast, when compound C was delivered to the rostral mNTS (at the 4th ventricle level) neither food intake nor 24h body weight gain was affected. * = P<0.05 from respective vehicle intakes and body weights. Photographs of representative histological sections are provided to show intraparenchymal injections of pontamine sky blue in the (C) caudal mNTS (at the level of the AP) and (D) rostral mNTS (at the level of the 4th ventricle). AP, area postrema; CC, central canal; DMV, dorsal motor nucleus of the vagus; mNTS, medial nucleus of the solitary tract.

Figures taken from Hayes, Skibicka, Bence and Grill (Endocrinology 2009)

(A) Increasing hindbrain AMPK activity ( AICAR, an AMPK stimulator; 4th icv300μg) reversed the suppression of cumulative food intake at 2h and 4h following 4th icv leptin (5μg) administration. (B) In ad libitum fed rats, 4th icv administration of leptin (5μg) suppressed pAMPK levels in NTS-enriched tissue (DVC) 2h after 4th icv administration compared to control injections. This suppression in pAMPK levels in the DVC was reversed by 4th icv administration of AICAR (300μg), at a dose that was without effect on its own. (C) Conversely, no alterations in pAMPK levels were observed in hypothalamic lysates by 4th icv administration of leptin, AICAR, or their combination, thus indicating a hindbrain site of action for the 4th icv administered compounds. Representative immunoblots for total AMPK and pAMPK are shown. * = P<0.05 from vehicle intakes and respective vehicle tissue.

Figure taken from Hayes, Skibicka, Bence and Grill (Endocrinology 2009)

Effect of caudal brainstem melanocortin receptor blockade (1 μl, SHU 9119, 4th v. delivery) on caudal brainstem leptin receptor stimulation (1 μl, 4th v. delivery) on (a) core temperature, (b) heart rate and (c) spontaneous activity in chow fed rats. Line graphs represent across-rat average parameter measurements through the 8 h recording period. The bracketed time period on the line graph x-axis indicates the periods used in the histograms. The histograms provide 6 h post-injection averages + SEM for each parameter at each dose.

Figure taken from Skibicka and Grill, (Endocrinology 2009)

Effect of fourth ventricle MTII (MC3/4R agonist) injection in CD rats on TIBAT (A), TC (B), HR (C), and spontaneous activity (D) in CD rats. Line graphs represent across-rat average parameter measurements through the recording period. The bracketed time period on the line graph x-axis indicates the periods used in the histograms. Histograms represent 6 or 5 h (TIBAT) means + SEM. *, P 0.05; **, P 0.005; ***, P 0.0005. BPM, Beats per minute.

Figure taken from Skibicka and Grill(Endocrinology 2009)

Effect of medullary raphe MC4-R stimulation with 10 pmol MTII in CD rats on TIBAT. Line graph represents across-rat average parameter measurements through the 8-h recording period. The bracketed time period on the line graph x-axis indicates the periods used in the histograms. Histogram represents 6-h means + SEM. *, P 0.05.

Figure taken from Skibicka and Grill(Endocrinology 2009)

Reconstruction of injection sites based on microscopical analysis of dye injection at the same volume (100 nl) as the melanocortin agonist. Microscopical analysis revealed that three of four rats had placements within the medullary raphe. The dye injection placement for each animal is represented by solid line ovals. Dotted line circles represent negative placements. Placements shown were between -10.52 and -11.30 mm from bregma.

Figure taken from Skibicka and Grill(Endocrinology 2009)

A significant proportion of leptin-responsive cells respond to gastric distension. A representative merged microphotograph of double IHC (P-STAT3 and c-Fos) from gastric distension combined with leptin-treated rats is shown on the left. On the right are shown high magnifications (top, P-STAT3 green fluorescence IHC; middle, c-Fos red fluorescence IHC; bottom, merged microphotograph from the double IHC) of the area marked on the left. Examples of double-labeled cells are shown in yellow. cc, Central canal. Scale bars, 200 µm.

Figure taken from Huo, Maeng, Bjorbaek and Grill (Endocrinology 2007)

Leptin delivered to the hindbrain is sufficient to potentiate the intake-suppressive effects of an otherwise ineffective volume of gastric distension. Cumulative chow intake was not significantly affected by either 4-ml gastric distension or icv leptin treatment (5 µg/2 µl) alone at 30, 60, or 90 min. However, when combined, gastric distension and leptin significantly suppressed cumulative intakes at 60 and 90 min compared with vehicle/sham-distension intakes. *, P< 0.05.

Figure taken from Huo, Maeng, Bjorbaek and Grill (Endocrinology 2007)

Intraoral glucose (10%) intake (infused at 0.8 ml/min) for CD and control rats did not differ as a function of the neurological condition of the rat. Ex-4, a GLP-1R agonist; intraperitoneal at 1.2 and 3.0 µg/kg suppressed intake significantly in both control and CD rats, compared with respective vehicle intakes. The suppression of intake by peripheral Ex-4 did not differ as a function of the neurological condition of the rat (CD vs. control). *, P < 0.05 from respective vehicle.

Figures taken from Hayes, Skibicka and Grill (Endocrinology 2008)

For control and CD rats, ip administration of Ex-4 (1.2 and 2.4 µg/kg) significantly suppressed 5 min gastric emptying of 0.9% saline, compared with vehicle in similar fashions. The gastric-emptying rates for the vehicle condition was not statistically different between control and CD rats, indicating that forebrain processing and forebrain-caudal brainstem communication is not necessary for the control of basal gastric emptying. *, P < 0.05 from respective vehicle.

Figures taken from Hayes, Skibicka and Grill (Endocrinology 2008)

Core temperature (Tc) in control and CD rats before and after injection of fourth icv Ex-4 (0.3 µg), ip Ex-4 (3.0 µg/kg), or vehicle. The histograms represent 6.5-h averages of Tc and show significant hypothermia after both fourth icv and ip Ex-4 administration for control and CD rats. *, P < 0.05 from respective vehicle Tc.

Figures taken from Hayes, Skibicka and Grill (Endocrinology 2008)

Exendin 9-39, a GLP-1R antagonist, applied to the mNTS at a ventricle subthreshold dose (1.0µg/100nl) resulted in a significant increase in food intake compared to intakes following vehicle injections in overnight food deprived rats. * = P<0.05 from respective vehicle intakes

Figures taken from Hayes, Bradley and Grill (Endocrinology 2009)

Gastric distension (9ml balloon distension for 15 min prior to food access) suppressed food intake compared to intake following sham distension in overnight food deprived rats. Hindbrain-directed blockade of GLP-1R (4th icv Exendin 9-39; 10µg) significantly attenuated the suppression of food intake by gastric distension. * = P<0.05

Figures taken from Hayes, Bradley and Grill (Endocrinology 2009)

Leptin induces P-STAT3 in 100% GLP-1-positive cells in the NTS of mice but not rats. Male Sprague Dawley rats or C57BL/6 mice were injected with leptin ip for 45 min. Series of coronal brain sections were subjected to combined P-STAT3 DAB and GLP-1 fluorescence double IHC. A and B, Shown are three representative merged photomicrographs from double IHC analyses of rats and mice, respectively. All sections are ordered in a rostral to caudal manner (from top to bottom). C, Shown are high magnifications (top, P-STAT3 DAB IHC; middle, GLP-1 green fluorescence IHC; bottom, merged photomicrograph from the double IHC) of the area marked in bottom of B. cc, Central canal; Cu, cuneate nucleus; Gr, gracile nucleus. Scale bars (A and B), 200 µm; (C), 50 µm.

Figure taken from Huo, Gamber, Grill, Bjorbaek (Endocrinology 2008)

Left: successive 5-min, group average core temperature (Tc) values of chronic decerebrate (CD; E) and pair-fed intact (■) rats during the 2.5-h pre-cold exposure room temperature baseline period, the 6-h cold exposure, and the post-cold period. SEs are denoted with light gray vertical lines. Right: group average temperature for 2-h intervals (one interval in the pre-cold exposure room temperature baseline period, three successive intervals during cold exposure period, and one post-cold return to room temperature interval). A: 4°C condition; B: 8°C condition; C: 12°C condition. Different lowercase letters above the histograms on right indicate instances of statistically significant differences (e.g., b denotes significant differences from a). Common letters indicate no significant difference (e.g., bc is not different from b but differs from a).

Figures taken from Nautiyal, Dailey, Brito, Brito, Harris, Bartness and Grill (AJP - Regu 2008)

Left: successive 30-s, group average heart rate (HR, beats/min) values of CD (E) and pair-fed intact (■) rats during the 2.5-h pre-cold exposure room temperature baseline period, the 6-h cold exposure, and the post-cold period. SEs are denoted with light gray vertical lines. Right: group average HR for 2-h intervals (one interval in the pre-cold exposure room temperature baseline period, three successive intervals during cold exposure period, and one post-cold return to room temperature interval). A: 4°C condition; B: 8°C condition; C: 12°C condition. Different lowercase letters above the histograms on right indicate instances of statistically significant differences (b denotes significant differences). Common letters indicate no significant difference.

Figures taken from Nautiyal, Dailey, Brito, Brito, Harris, Bartness and Grill (AJP - Regu 2008)