1. Volume 138, Issue 7, Pages 2418-2425 (June 2010)
Riluzole Normalizes Early-Life Stress-Induced Visceral Hypersensitivity in Rats: Role of Spinal Glutamate Reuptake Mechanisms 
Romain–Daniel Gosselin, Richard M. O'Connor, Monica Tramullas, Marcela Julio–Pieper, Timothy G. Dinan, John F. Cryan 
Gastroenterology 
Volume 138, Issue 7, Pages (June 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
Riluzole reverses stress-induced visceral hypersensitivity. (A) Maternally separated (MS) rats and nonseparated (NS) controls were treated with vehicle or riluzole (5 mg/kg, intraperitoneally), and visceral sensitivity to colorectal distension (10 minutes at 40 mm Hg) was determined as the number of abdominal contractions. An increase in abdominal contractions in response to distension is observed in separated animals. Riluzole counteracts the visceral hypersensitivity observed in separated rats (F(3,28) = 4.058; Newman–Keuls test, *P < .05) with no detectable effect in nonseparated animals (n = 9 for vehicle NS and MS, n = 8 and 6 for riluzole NS and riluzole MS, respectively). (B) Riluzole has no effect on acute nociception in the Hot Plate, Randall–Sellito, or Hargreaves tests (n = 8 per group). (C) Rotarod testing following administration of various doses of riluzole shows no impairment of motor coordination after treatment with 2.5, 5, or 10 mg/kg riluzole.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Effect of riluzole on [3H]D-aspartate transport in vitro. (A) Riluzole dose-dependently increases [3H]D-aspartate reuptake in cultured astrocytes with a significant effect for 1 μM riluzole (F(8,27) = 29.8; Dunnett's test, *P < .05 vs no riluzole treatment; n = 4 wells). (B) Western blots showing that primary cultures of spinal astrocytes expressing the astrocytic marker glial fibrillary acidic protein (GFAP) also express the glutamate transporters excitatory amino-acid transporter (EAAT)-1 and EAAT-2.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Maternal separation is associated with a reduction in excitatory amino-acid transporter (EAAT)-1 in spinal astrocytes. (A) Western-blot analysis of spinal EAAT and γ-amino butyric acid transporter (GAT) in separated rats. A significant decrease in spinal EAAT-1 is detected in comparison to nonseparated rats (**P < .01, Student's t test, n = 5), with no change in EAAT-2 or GAT transporters. (B) Immunofluorescence analysis showing a drop in EAAT-1 immunoreactivity in the dorsal spinal cord of separated rats (*P < .05, Student's t test, n = 5). Scale bar: 200 μm. (C) Double immunofluorescence showing the colocalization (yellow, right images) of glial fibrillary acidic protein (GFAP) (red, left images) with EAAT-1 (green, middle images) in the spinal cord of nonseparated rats (upper panels) or maternally separated rats (lower panels). Scale bar: 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Maternal separation is not associated with alterations in the gross spinal astrocytic phenotype. (A) Microphotograph showing glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal spinal cord of nonseparated (left panel) and maternally separated rats (right panel). Scale bar: 200 μm. (B) No change in spinal GFAP immunoreactivity was noticeable in pixel density, cell density per mm2, or in the mean length of astrocytic processes following maternal separation (n = 5 rats). (C) Western blot confirming that maternal separation had no effect on spinal GFAP expression. All Western blot results were expressed as a ratio over actin and normalized to nonseparated rats (n = 5 rats).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
Inhibition of spinal glutamate transport produces visceral hypersensitivity. (A) Intrathecal injection of glutamate transporter blocker DL-threo-β-benzyloxyaspartate (TBOA) results in a visceral hypersensitivity to colorectal distension (F(2,30) = 6.7; Dunnett's test, *P < .05, **P < .01, vs vehicle treatment; n = 10 for vehicle and 1.2 ng TBOA, n = 13 for 12 ng TBOA). (B) TBOA blocks [3H]D-aspartate reuptake in rat primary spinal astrocytes (***P < .0001, Student's t test, n = 4 wells).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions