Jess - Hormone Monster

Study Objective & Hypothesis

• Investigate how gonadal steroid hormones (testosterone, estradiol) affect perineuronal net (PNN) development in the medial prefrontal cortex (mPFC) of male and female mice.

• Examine the age-dependent and sex-specific sensitivity of PNNs to hormone exposure during juvenile, adolescent, or adult periods.

• PNN density is influenced by both age and gonadal hormone exposure.

• The age at which an animal receives gonadal steroid hormones will influence PNN density in the mPFC

  • Animals exposed to hormones during adolescence would show increased PNN density compared to juvenile or adult exposure.

Background

• PNNs: Extracellular structures surrounding parvalbumin (PV) interneurons; involved in stabilizing synaptic connections and regulating plasticity

  • Parvalbumin (PV) interneurons: inhibitory GABAergic cells that play a crucial role in modulating excitatory signals within neural circuits, impacting overall brain function and development.

• PNNs increase during adolescence and may signal the closure of sensitive periods in brain development.

  • Net development crystalizes learning & neural circuits

  • Disruption or variation in PNN development is linked to mental health disorders (e.g., schizophrenia, bipolar disorder).

• Abnormalities in pubertal timing (early onset in girls, late onset in boys) increase the risk of developing a mental health disorder (Conley & Rudolph, 2009; Richburg, 2021).

  • The brain is more susceptible to experience-dependent modifications such as pruning and myelination (Scott et al., 1974; Johnson et al., 2009; Selemon, 2013)

• The medial prefrontal cortex (mPFC) undergoes significant development during adolescence and contributes to changes in emotional regulation and decision making

Methods

Subjects:

• 72 C57BL/6 male and female mice.

  • (n = 4 per group per sex)

  • Sex (male, female), Age (juvenile, adolescent, adult), Treatment (GDX + blank, GDX + T/E), sham = baseline comparison

Hormonal Manipulation:

• Gonadectomy (GDX) or sham surgery before puberty (PD 22)

• Hormone capsule: 17β-estradiol for females, testosterone for males, or blank silastic capsules implanted between the shoulder blades at one of these ages:

  • Juvenile (PD 23)

  • Adolescent (PD 40)

  • Adult (PD 63)

Brain Collection

• Brains collected 1 week after silastic capsule implantation

  • PD 30, 47, or 70

• Animals were euthanized by cardiac perfusion

  • Blood samples were collected, but they were insufficient for radioimmunoassay

• 24 hr fixed, 48 hr in 30% sucrose, flash frozen before 40 µm sectioning

Fluorescent Immunohistochemistry

• WFA - PNNs - 488 streptavidin

• ?? - Cyanine5

• DAPI

• PNN density (mm²): PNN # / sampled cross-sectional area mm²

Regions analyzed:

• PL/A32 (Prelimbic Cortex)

  • Primate Broadmann’s Area (BA) 32

    • Located in the anterior cingulate cortex, balances emotion and reason, maintaining emotional equilibrium by facilitating communication between cognitive and emotional brain regions

• IL/A25 (Infralimbic Cortex)

  • Primate BA25

    • Located in the subgenual cingulate cortex (ventromedial prefrontal cortex), implicated in the regulation of autonomic and endocrine functions and maybe processing emotional information

Results

PL/A32 Region

• Females:

  • No main effect of E2 treatment or interaction between age and E2 treatment

  • Significant main effect of age

    • PNN density increased from juvenile (lowest density) to adolescent/adult (highest density).

  • Juvenile estradiol treatment showed decreased PNN density compared to blank juveniles.

    • No effect in adolescents or adults.

  • Sham females: PNN density is based on age

• Males:

  • Significant main effect of age

    • PNN density of juvenile males was significantly lower than adolescents and adults

  • Significant main effect of testosterone

    • testosterone-treated males showed a higher PNN density than blank-treated males

  • Trending (p = 0.09) interaction between age and testosterone treatment

    • Adult males treated with testosterone showed a higher PNN density than adult males treated with the blank

  • Sham males: no age-related PNN changes.

IL/A25 Region

• Females:

  • No hormone effects

    • No significant main effect of age or hormone treatment, nor an interaction

  • Sham females: gradual increase in PNN density across development.

    • Juveniles show a lower PNN density than adolescents shams, who show a marginally lower PNN density than adults

• Males:

  • No effect of age or testosterone in GDX animals (blank or T).

  • Sham males: no age-related changes.

Discussion

• Age- and region-specific hormone sensitivity:

  • PL/A32 is sensitive to hormone exposure; IL/A25 is not.

• Sex-specific differences:

  • Juvenile female PNNs are sensitive to E2, but not in adol. or adult

  • Adult male PNNs are sensitive to T, starting in adolescence

  • A decrease in PNNs in juvenile females may be related to an increase in synaptic pruning that occurs in early adolescence

    • Reduced PNNs allows for space for pruning

• Suggests sensitive periods for PNN development that are influenced by hormone timing:

  • Females: sensitive period in juvenile phase.

  • Males: sensitive period in adulthood.

• Adolescence may mark the beginning of a prolonged sensitive period, not a discrete one.

• PNNs may themselves have a sensitive period to sex steroid hormones

  • PNN development is particularly sensitive to the presence of hormones at a specific time. Blank animals did not behave the same as the hormone-treated animals