学位論文内容の要旨（Summary of dissertation）

博士の専攻分野の名称
博士（医 学）（Degree conferred: Doctor of Philosophy）

氏 名：リトル ケリス アリシア
（Name of recipient: Lyttle Kerise Alecia ）学位論文題名（Title of dissertation）
Postnatal stress exposure infers depressive-like behavior and morphological changes in rats
（ラット幼若期ストレスはうつ様行動と形態学的変化を引き起こす）
Background and Objectives
The early postnatal period is characterized by increased sensitivity to adverse exposures.
Studies have shown that exposure to early adverse experiences confer increased susceptibility to thedevelopment of depressive disorders. The modulatory effects of the early environment on adultpsychiatry are thought to be mediated through enduring morphological changes in executive and limbic
brain systems. The raphe nuclei serotonergic system is implicated in the regulation of emotional
response. The executive brain system represented by the medial prefrontal cortex (mPFC) is of particularinterest due to its role in the control of higher cognitive and executive functions including the regulationof emotional behavior. Notably, altered serotonergic parameters and prefrontal cortex abnormalities havebeen observed in depression. Although clinical studies have implied the negative effects of early life
stress on brain development, causality and the detailed morphogenetic changes has not been clearly
elucidated. The raphe nuclei serotonergic system is undergoing final arborisation while the prefrontal
cortex is still undergoing significant maturation in the third postnatal week (Spear, 2000), during which
rat pups are subjected to stress exposure. In particular, layer II/III of the prefrontal cortex is the lastcortical layer to mature (Clancy et al., 2001) and is the site of processing of long-range inputs fromsubcortical limbic structures and may thus be susceptible to postnatal stress exposure. In the present
study, I used an established model of early postnatal stress to investigate the presentation of depressivelike behavior and the neural mechanisms involved by focusing on raphe nuclei tryptophan hydroxylase 2(TPH2) and glutamic acid decarboxylase (GAD) protein levels and mPFC layer II/III pyramidal spine
densities using a rodent model. Additionally, I investigated the pharmacotherapeutic value of theselective serotonin reuptake inhibitor (SSRI), fluvoxamine on the behavioral deficits and alteredneuronal morphology observed in rats subjected to 3 week footshock stress. Because it is well known
that repeated treatment with SSRIs causes neurotrophic effects (Duman and Monteggia, 2006;
Licznerski and Duman, 2013), I hypothesized that SSRI treatment could recover juvenile stress-induced
behavioral and morphological impairments.
Methods
Male rats used for experiments were the offspring of timed-pregnant Wistar/ST rats. Pups wereweaned at postnatal day 21 and group-housed (4 – 5 per cage). Food and water were provided ad libitum.
At the post-adolescent stage behavioral characterization of locomotor activity and depressive-likebehavior was carried out. The open field test was used for locomotor activity assessment in a squarechamber (90 cm long × 90 cm wide × 40 cm high). Locomotor activity was assessed by measuring total
distance travelled and the total number of crossings (crossings of the lines made by the division of thechamber into 9 cm × 9 cm squares) during a 15 min interval. LimeLight 2 software (Actimetrics USA)was used to score behavioral parameters.
Characterization of depressive-like behavior was assessed by the rat 2 day forced swim test withanalysis of day 2 behavioral data. Forced swim test analysis was done by assessment of the occurring
behavioral parameter at the end of each 5 sec interval. Behavioral parameters assessed included: (1)immobility, swimming and climbing. Increased counts of immobile behavior is accepted as a depressivelike phenotype (Cryan et al., 2005).
Assessment of raphe nuclei TPH2 and GAD67 levels was performed by western blot. Dorsaland median raphe sections were assessed separately.
The Golgi-Cox staining technique was used for neuronal reconstruction of mPFC infralimbic
and prelimbic cortex layer II/III pyramidal neuron using the FD Rapid GolgiStain™ Kit (FDNeurotechnologies, Inc.). Brains were extracted fresh and rinsed in purified water Millipore™. Brains
were immediately transferred to the Golgi-Cox solution and stored in the dark for at least 2 weeks.
Brains were further processed in Solution C for a minimum 48 hr at 4 °C. 100 µm mPFC coronal
sections were used for photomicrographic analysis of pyramidal neuron spine densities.
Statistical data analysis was performed using IBM® SPSS® Statistics 21. Most parameters wereanalysed separately using a two-factor ANOVA with stress (no-FS or 3wFS) and the drug (vehicle orfluvoxamine) as between-subject factors. In cases in which there was a significant stress × druginteraction, it was followed by a one-factor ANOVA. P < 0.05 was considered statistically significant.
Results
Post-adolescent behavioral and morphological studies identified the presentation of increased
depressive-like behaviors, elevated dorsal raphe TPH2 levels and reduced spine densities of layer II/IIIpyramidal neuron in the infralimbic cortex, but not in the prelimbic cortex of rats exposed to juvenile
stress. Repeated fluvoxamine treatment recovered the increased depressive-like behavior and reduced
spine densities observed in rats exposed to footshock stress.
Discussion
Juvenile stress exposure induced increased depressive-like behavior congruent with otherstudies which have demonstrated that adverse early-life stress results in behavioral deficits during
adulthood. Investigation of raphe nuclei TPH2 identified a significant increase in dorsal raphe TPH2levels following juvenile stress exposure which is consistent with previous studies of elevated dorsal
raphe TPH2 following stress exposure. No significant changes in dorsal raphe GAD67 nor median raphe
TPH2 and GAD67 levels may relate to the predominant role of dorsal raphe serotonergic mechanisms inregulation of stress-induced behavioral impairments.
Reduced apical and basal spine densities were observed in IL layer II/III pyramidal neurons ofjuvenile stressed rats. Layer II/III pyramidal neurons arborize throughout mPFC layers I and II/IIIenabling the sampling of diverse inputs from other brain regions (Spruston, 2008) including subcorticallimbic structures involved in emotional arousal and it is expected that reduced spine densities would
infer deficiencies in emotional regulation. It is likely juvenile stress-induced increased corticosterone
levels mediate spine atrophy because Liu and Aghajanian (2008) demonstrated that glucocorticoid
receptor antagonist reversed stress-induced reduction of spine density in the mPFC though theyexamined layer 5 in adult rats. 14 day oral administration of 10 mg/kg fluvoxamine recovered bothincreased forced swim test immobility and reduced spine densities associated with juvenile stress. It is
likely that repeated fluvoxamine administration activates neurotrophic pathways responsible forbehavioral and dendritic recovery.
Conclusion
The results demonstrate cortical and serotonergic sensitivities to stress exposures during thejuvenile stage which mediate behavioral and morphological impairments, and provide a clue to findtherapeutics for early life stress-induced emotional dysfunctions.