Cerebral spinal fluid GABA concentrations are high in day 5C7 postpartum female rats when dams are allowed to physically interact with pups [58] and this may also be true for the production and release of endogenous benzodiazepines or other positive modulators of the GABAA receptor [59C61]. male rats was also included. We found that [3H]muscimol binding did not differ among groups in any site but that diestrous virgin females had greater [3H]flunitrazepam binding in the CA1 and dentate gyrus of the hippocampus compared to mid-pregnant females and males. Notably, postpartum and diestrous virgin females did not significantly differ in binding of either ligand in any site examined. This is the first study to evaluate the densities of GABAA and benzodiazepine binding sites simultaneously across three female reproductive says and sex with a focus on brain sites influencing anxiety-related behaviors. The results suggest that changes other GABAA receptor characteristics, such as subunit composition or increased presynaptic GABA release during interactions with offspring, must instead play a greater role in the postpartum suppression of stress in laboratory rats. = 8), were housed singly beginning 7 days after mating and sacrificed on day 10 (1 day) of pregnancy. The other mated females were allowed to eventually give birth (= 8), and members of this future postpartum group were individually housed 5C6 days before parturition. Their litters were culled to contain 8 pups (4 females and 4 males) within 48 hr after parturition and the dams were sacrificed on day 7 (1 day) postpartum. Subjects in the virgin female group (= 8) were singly housed after 70 days of age, vaginally smeared daily and the cytology examined under a microscope to determine stage of the estrous cycle, and rats were sacrificed on a day of diestrus. A group of singly housed, sexually naive adult males was also included (= 8). Matings were scheduled so that some subjects from each group were usually sacrificed concurrently. Sections from two of the males and two of the pregnant females were inadvertently omitted from the [3H]flunitrazepam autoradiography, resulting in these groups having a sample size of six for the [3H]flunitrazepam analysis. 2.2 Tissue collection Subjects were rendered unconscious with CO2 and rapidly decapitated using a guillotine. Brains were quickly removed, placed on dry ice, and stored at ?80C until Pyrimethamine further processing. Brains were later coronally sectioned with a cryostat into six series of 15-m sections thaw-mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and stored at ?80C until further processing. 2.3 Autoradiography Two series of slides were thawed at room temperature and the sections were fixed in 1% paraformaldehyde for 2 min. They were then preincubated twice in 50 mM Tris-HCl buffer (pH 7.4) at 4C for 15 min each. One series of sections was then incubated in 50 mM Tris-HCl buffer made up of 10 nM [3H]muscimol (20C40 Ci/mmol; PerkinElmer, Waltham, MA) [32,33], and the other series was incubated in 50 mM Tris-HCl buffer made up Pyrimethamine of 2 nM [3H]flunitrazepam (70C87 Ci/mmol; Perkin-Elmer, Waltham, MA) [34C36], for 1 hr at 4C. Nonspecific binding was determined by incubating slides of adjacent sections in comparable solutions of [3H]muscimol or [3H]flunitrazepam that contained 100 M Rabbit Polyclonal to GNRHR GABA or 10 M flunitrazepam (Sigma-Aldrich, St. Louis, MO), respectively; nonspecific binding was found to be negligible. After incubation, sections were washed twice in 4C 50 mM Tris-HCl buffer for 30 sec each, dipped in 4C dH2O, then laid out to dry overnight at 4C. The following day, slides were moved to autoradiography cassettes (Fisher Scientific, Pittsburgh, PA) each made up of a set of tritium microscale standards (3C110 nCi/mg and 0.1C16 nCi/mg; Perkin-Elmer, Waltham, MA) and all were exposed to Hyperfilm MP film at room heat (Amersham; Perkin-Elmer, Waltham, MA). All groups had slides included in each cassette. Sections incubated with [3H]muscimol were uncovered for 14C28 weeks, while those incubated with [3H]flunitrazepam were uncovered for 14 weeks. Durations of exposure were based on test films developed at various time points. Film was developed and fixed using a Kodak X-OMAT 1000A Processor (Kodak Co., Rochester, NY). Films were analyzed by placing them on a light box (0.35 Amps, 60 Hz; Knox Manufacturing Co., Wooddale, IL) and relevant images captured using a Zeiss microscope and digital camera (Roper Scientific Photometrics, Tucsan, AZ). Brain areas were analyzed bilaterally (Physique 1) using.The optical density totals were averaged across the number of sections analyzed to standardize density values across brain sites. but that diestrous virgin females had greater [3H]flunitrazepam binding in the CA1 and dentate gyrus of the hippocampus compared to mid-pregnant females and males. Notably, postpartum and diestrous virgin females did not significantly differ in binding of either ligand in any site examined. This is the first study to evaluate the densities of GABAA and benzodiazepine binding sites simultaneously across three female reproductive says and sex with a focus on brain sites influencing anxiety-related behaviors. The results suggest that changes other GABAA receptor characteristics, such as subunit composition or increased presynaptic GABA release during interactions with offspring, must instead play a greater role in the postpartum suppression of stress in laboratory rats. = 8), were housed singly beginning 7 days after mating and sacrificed on day 10 (1 day) of pregnancy. The other mated females were allowed to eventually give birth (= 8), and members of this future postpartum group were individually housed 5C6 days before parturition. Their litters were culled to contain 8 pups (4 females and 4 males) within 48 hr after parturition and the dams were sacrificed on day 7 (1 day) postpartum. Subjects in the virgin female group (= 8) were singly housed after 70 days of age, vaginally smeared daily and the cytology examined under a microscope to determine stage of the estrous cycle, and rats were sacrificed on a day of diestrus. A group of singly housed, sexually naive adult males was also included (= 8). Matings were scheduled so that some subjects from each group were usually sacrificed concurrently. Sections from two of the males and two of the pregnant females were inadvertently omitted from the [3H]flunitrazepam autoradiography, resulting in these groups having a sample size of six for Pyrimethamine the [3H]flunitrazepam analysis. 2.2 Tissue collection Subjects were rendered unconscious with CO2 and rapidly decapitated using a guillotine. Brains were quickly removed, placed on dry ice, and stored at ?80C until further processing. Brains were later coronally sectioned with a cryostat into six series of 15-m sections thaw-mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and stored at ?80C until further processing. 2.3 Autoradiography Two series of slides were thawed at room temperature and the sections were fixed in 1% paraformaldehyde for 2 min. They were then preincubated twice in 50 mM Tris-HCl buffer (pH 7.4) at 4C for 15 min each. One series of sections was then incubated in 50 mM Tris-HCl buffer made up of 10 nM [3H]muscimol (20C40 Ci/mmol; PerkinElmer, Waltham, MA) [32,33], and the other series was incubated in 50 mM Tris-HCl buffer made up of 2 nM [3H]flunitrazepam (70C87 Ci/mmol; Perkin-Elmer, Waltham, MA) [34C36], for 1 hr at 4C. Nonspecific binding was determined by incubating slides of adjacent sections in comparable solutions of [3H]muscimol or [3H]flunitrazepam that contained 100 M GABA or 10 M flunitrazepam (Sigma-Aldrich, St. Louis, MO), respectively; nonspecific binding was found to be negligible. After incubation, sections were washed twice in 4C 50 mM Tris-HCl buffer for 30 sec each, dipped in 4C dH2O, then laid out to dry overnight at 4C. The following day, slides were moved to autoradiography cassettes (Fisher Scientific, Pittsburgh, PA) each made up of a set of tritium microscale standards (3C110 nCi/mg and 0.1C16 nCi/mg; Perkin-Elmer, Waltham, MA) and all were exposed to Hyperfilm MP film at room temperature (Amersham; Perkin-Elmer, Waltham, MA). All groups had slides included in each cassette. Sections incubated with [3H]muscimol were exposed for 14C28 weeks, while those incubated with [3H]flunitrazepam were exposed for 14 weeks. Durations of exposure were based on test films developed at various time points. Film was developed and fixed using a Kodak X-OMAT 1000A Processor (Kodak Co., Rochester, NY). Films were analyzed by placing them on a light box (0.35 Amps, 60 Hz; Knox Manufacturing Co., Wooddale, IL) and relevant images captured using a Zeiss microscope and digital camera (Roper.
