Pubertal Neurogenesis

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Pubertal Neurogenesis

• As an animal goes through puberty, what are its
  effects on neurogenesis in the adolescent brain?

By Carly Christensen
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Overview

• Part I What is Puberty?
• Looking at the neural basis of puberty and
  adolescence
• Pubertal hormones and behavioral maturation
• Part II Puberty and Neurogenesis
• What is neurogenesis?
• The areas of the brain that were examined
• Dentate Gyrus of the Hippocampus, and Forebrain
  Subventricular Zone
• Hypothalamus
• Part III Unanswered Questions and Future
  Direction
• What about the other structures involved in
  reproduction?
• What happens when neurogenesis is ablated?

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Part I
What is Puberty?
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What is Puberty and Adolescence?
According to Cheryl Sisk and Douglas Foster in
The Neural Basis of Puberty and Adolescence

• Puberty and adolescence mark the changing of a
  child into an adult.
• Puberty refers to the activation of the
  hypothalamic-pituitary-gonadal axis that
  cumulates in gonadal maturation.
• Adolescence is the maturation of adult social and
  cognitive behaviors.
• The transition from puberty to adulthood involves
  both gonadal and behavioral maturation.

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Adolescent Maturation of Reproductive Behavior
Steroid hormones remodel and activate neural
circuits during adolescent brain development,
which leads to the development of sexual salience
of sensory stimuli, sexual motivation, and
expression of copulatory behaviors in certain
social contexts.
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• Transient activation of the HPG axis during late
  prenatal/ early postnatal life causes an increase
  in gonadal steroids and in turn sexual
  differentiation. GnRH secretion decreases after
  HPG activation, and hormone pulse slows
  throughout the prepubertal period. After this
  period of quiescence, puberty begins when GnRH
  secretion gradually increases and levels off to
  stimulate gonadotropin and steroid hormone
  secretion, resulting in complete gonadal
  maturation and reproductive behavior.
• Possible triggers that induce re-emergence of
  GnRH secretion at puberty are melatonin, body
  fat, and leptin.
• Copulatory behavior can still be activated by a
  dose of testosterone in castrated males during
  adolescence, but not in prepubertal males.
• Remodeling of the brain during adolescence
  includes increased myelination and decreased gray
  matter volume in cortical areas, synaptic
  elaboration and pruning in striatum and PFC, cell
  death in primary visual cortex, and changes in
  connectivity in the amygdala and PFC.
• These rearrangements are thought to be linked to
  making decisions, planning, drug sensitivity, and
  reward seeking behavior.

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GnRH neurons are key to gonadal and behavioral
maturation

• GnRH is a decapeptide produced by specialized
  neurons that secrete pulses of hormone from nerve
  terminals in the median eminence of the basal
  hypothalamus.
• The brain initiates activation of the GnRH system
  at puberty onset, leading to an increase in
  steroid hormone production.
• Steroid hormones in turn modulate GnRH secretion
  in the brain, and organize and activate neural
  circuits mediating reproductive behavior during
  adolescence.

The pubertal increase in GnRH neuronal activity
and gonadotropin secretion is timed by a
developmental clock and fine-tuned by integration
of permissive signals
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Pubertal Hormones Organize the Brain

• In an article by Schulz and Sisk et al., evidence
  that adolescent brain development is a
  prerequisite for the activation of reproductive
  behavior was reported in the Syrian Hamster.
• It was also found that pubertal gonadal hormones
  shape adolescent brain development through their
  organization of neural circuits.

Perinatal hormone secretions sexually
differentiate behavioral neural circuits and
pubertal hormone secretions improve and finish
these processes during adolescence to allow for
the display of sex-typical social behaviors in
adulthood
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Adolescent Exposure to Testicular Hormones

• Figure 3 shows that testicular hormone
  deprivation during puberty causes deficits in
  masculine reproductive behavior.
• Males deprived of testicular hormones during
  puberty (No-T_at_P) exhibited diminished mounting
  and T_at_P (with testicular hormones) displayed
  significantly more mounts.
• Figure 5 shows that adolescent testicular
  hormones defeminize lordosis behavior.
• Lordosis a posture assumed by some female
  mammals during mating, in which the back arches
  downward
• Males exposed to testicular hormones during
  pubertal development display longer lordosis
  latencies than males deprived of pubertal
  testicular hormones .

Figure 3
Figure 5
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Figure 6

• Adolescent exposure to testicular hormones
  organizes male agonistic (combative) behavior.
• In Figure 6, adolescent testicular hormones
  increase aggressive and decrease submissive
  behaviors in adulthood.
• Males exposed to testicular hormones during
  puberty (T_at_P) displayed more attacks and fewer
  escape dashes than males deprived (No-T_at_P)
  regardless of testosterone treatment.
• _______________________________________
• Adolescent exposure to ovarian hormones
  defeminizes female reproductive behavior.
• Figure 7 represents the finding that pubertal
  ovarian hormones defeminize lordosis behavior.
• Females exposed to ovarian hormones during
  puberty (O_at_P) display longer lordosis latencies
  than females deprived of pubertal ovarian
  hormones (No-O_at_P).

Figure 7
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Conclusions

• Gonadal steroid hormones are important at both
  stages of behavioral development because of their
  ability to influence cell survival, cell
  phenotype, synaptic organization, and neural
  circuitry.
• During the adolescent period of development, both
  ovarian and testicular secretions have organizing
  actions, which cause the adult behaviors of
  females and males to relate.
• Support for adolescence being a sensitive period
  
• The fact that steroid hormones bring out
  different behavioral responses before and after
  adolescence.
• The effects on behavior resulting from the
  absence of hormones during adolescence are not
  reversed by hormone replacement in adulthood.

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Part II
Puberty and Neurogenesis
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Defining characteristics of Adult Stem Cells

• Proliferate
• Multipotential
• Last life-time
• (extensive self-renewal)

Taken from Dr. Kippins slides
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Neurogenesis

• The generation of new neurons after birth has
  been identified in various structures of the
  adult brain including the forebrain
  subventricular zone (SVZ), hippocampal dentate
  gyrus (DG), and paraventricular nucleus (PVN) of
  the hypothalamus.
• These areas were examined in the various papers
  that will be discussed today.

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The effect of Maturation on Neurogenesis

• According to He and Crews et al. adolescence is
  marked by risk-taking, exploration, novelty
  seeking, social interaction, activity, and play
  along with changes in hormones and growth
  factors.
• It has been shown that overproduction of axons
  and synapses occur during early puberty and rapid
  pruning follows later in adolescence, while
  neurogenesis in the SVZ and DG continues
  throughout life in humans and rodents.
• Jun He and Fulton Crews set out to determine if
  neurogenesis changes during the transition from
  adolescence to adulthood.

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Subjects, Materials, Methods

• IGF-1 is a growth factor during postnatal
  development known to enhance neurogenesis in
  developing mice.
• Transgenic mice that over-express IGF-1 show
  overgrowth of the brain causing higher brain
  weight compared to wildtype controls.
• Transgenic mice that over-express IGF binding
  protein-1 (IGFBP-1) causes a reduction in
  neurogenesis resulting in brain growth
  retardation (activation of IGFBP-1 inhibits IGF-1
  bioactivity).
• BrdU (exogenous mitotic marker bromodeoxyuridine)
  and DCX (endogenous neuronal marker doublecortin)
  were used to measure the level of neurogenesis in
  this study.
• BrdU Injections were given once a day 300
  mg/kg/day for 2 days. Animals were killed 24h
  after last injection.

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Increased Proliferation in Adolescent DG

• Figure 1 represents the number of BrdU cells in
  the hippocampus of adult and adolescent brains.
• The number of BrdU cell/ DG section is
  significantly higher in adolescent hippocampus
  than adults in all genetic background of strains
  (IGF-1, IGFBP-1, and WT).
• Figure 2 represents BrdU cells in DG of
  hippocampus.
• AC) Adolescent hippocampus
• BD) Adult hippocampus.

Figure 1
Figure 2
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Increased Differentiation of Neuroprogenitors in
Adolescent Hippocampus

• Figure 3 represents the expression of DCX
  (doublecortin) in the hippocampus of adolescent
  and adult brains.
• The DCX immunoreactivity is significantly higher
  in adolescent hippocampus than those of the
  adults in all strands of mice.
• Figure 4 represents DCX expression in the
  hippocampus of adolescent and adult brains.
  Adolescent mice were 30 days old, while adult
  mice were 120 days old.

Figure 3
Figure 4
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Increased Proliferation in SVZ of Adolescents

• Figure 5 shows enhanced BrdU-immunoreactivity in
  the forebrain SVZ of the adolescents compared to
  the adults.
• BrdU-labeling is significantly higher in the SVZ
  of the adolescents than in the adults.
• On the right, adolescents are the 30 day olds and
  adults are the 120 day olds.

Figure 5
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Conclusions Further Implications

• Both forebrain and hippocampal neurogenesis is
  significantly reduced during brain maturation
  from adolescence to adults.
• Their results suggest that the influence of IGF-1
  on neurogenesis most likely occurs before the
  animals reach adolescence, since the IGF-1
  transgenic mice didnt contribute to the decrease
  in neurogenesis.
• The high level of neurogenesis in adolescent
  brain found in this study could be due to the
  high level of neuroplasticity during adolescence.

• The significant decline of neurogenesis may
  indicate a critical window of opportunity where
  the neuronal circuitry is still modifiable for
  further adaptation.
• Any manipulation during this critical period
  could lead to more damage in adulthood (i.e.
  alcohol-induced reduction of neurogenesis).
• Therefore, the development of the adolescent
  brain is a period of susceptibility and prospect.

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Leptin Promotes Adult Neurogenesis in the
Hippocampus

• Leptin is an adipose-derived hormone encoded by
  the obese (ob) gene that is linked to various
  physiological processes within the hippocampus.
• Leptin is known for its role in the control of
  food intake and body weight, which is believed to
  be mediated by interaction with LepRb in the
  hippocampus.
• It has also been found that leptin facilitates
  spatial learning and memory and produces
  anti-depressant effects.
• Adult neurogenesis has been thought to mediate
  hippocampal-dependent learning and therapeutic
  actions of anti-depressants.
• In the study by Garza et al., the impact of
  leptin on cell proliferation, differentiation,
  and survival in the DG of adult mice was
  examined.

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Effect of Leptin on Cell Proliferation

• Figure 1 shows the effects of leptin
  administration on cell proliferation in the DG of
  adult mice
• BrdU was used to label cell proliferation
• Mice were injected i.p. with leptin (1mg/kg) or
  vehicle twice daily for 1,5, or 14 days followed
  by BrdU labeling
• A) Acute treatment (1d)
• B) Short-term treatment (5d)
• C) Chronic treatment (14d)
• D E) show BrdU-labeled cells in adult DG of
  mice treated for 14 days
• D1 E1) show BrdU-labeled cells in PV thalamus
  treated for 14 days
• F) shows high magnification of proliferating
  cells

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Effect of Leptin on Cell Differentiation

• Figure 2 shows the effect of leptin
  administration on cell differentiation.
• Mice were injected i.p. with leptin (1mg/kg) or
  vehicle twice daily for 14 days followed by BrdU
  labeling and were perfused 28 days later.
• A) A significantly higher number of BrdU cells
  remained in the leptin-treated group compared to
  the control group 28d after BrdU
• B) The effect of leptin on the of BrdU-labeled
  cells double-labeled for NeuN or GFAP (higher for
  leptin-treated groups, not significant)
• C) Co-localization of BrdU with NeuN under
  confocal microscope
• D) Confocal microscope images show
  co-localization of BrdU with GFAP

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The Effect of Leptin on Proliferation

• Figure 4 shows the expression of the long form
  leptin receptor (LepRb) in adult hippocampal
  stem/progenitor cells
• B) cells stained with Nestin (green) are for
  leptin receptor (red)
• Figure 5 shows the effects of leptin treatment on
  proliferation of adult hippocampal
  stem/progenitor cells
• Cells were treated with various concentrations of
  leptin (1-30nM) for 48h and labeled with BrdU
  (10microM) in the last 4h of incubation
• A) The number of BrdU-labeled cells was increased
  by leptin treatment at concentrations of 1nM and
  3nM compared to the control
• B) Microscopic images represent BrdU-labeled
  adult hippocampal progenitor cells

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Effect of Leptin on Differentiation of Cultured
Cells

• Figure 6 shows the effects of leptin treatment on
  differentiation of cultured adult hippocampal
  stem/progenitor cells
• Cells that were treated with leptin (1nM) for 48h
  and labeled with BrdU (10microM) in the last 4h
  of incubation were allowed to differentiate for 8
  days before fixation
• A) Microscopic images show that BrdU-labeled
  cells differentiated into neuronal (TuJ1 in red)
  or glial (GFAP in green) cells
• B) The of BrdU-labeled cells that were TuJ1 or
  GFAP was not altered by leptin treatment

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Significance

• Leptin was demonstrated to promote adult
  hippocampal neurogenesis both in vitro and in
  vivo.
• Leptin-stimulated neurogenesis resulted from
  increased cell proliferation, as leptin showed no
  significant effect on cell differentiation and
  survival.
• Cell proliferation in the DG was increased by
  chronic, not short-term or acute, administration
  of leptin.
• Leptin is known to suppress appetite, increase
  energy expenditure, and reduce body weight gain.
  Since dietary restriction and physical activity
  have been shown to increase hippocampal
  neurogenesis, leptins effects on neurogenesis
  may be induced by negative energy balance
  following chronic administration.
• LEPTIN INCREASES THE PRODUCTION OF NEW NEURONS IN
  THE ADULT DENTATE GYRUS.

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Neurogenesis in the PVN of the Pig Hypothalamus

• In the article by Raymond et al., there is
  evidence suggesting that neurogenesis occurs in
  the adult hypothalamus, including centers
  containing oxytocin and vasopressin producing
  neurons.
• The pig hypothalamus contains nuclei that release
  the hormones oxytocin (OT) and vasopressin (VP),
  which include the supraoptic nucleus (SON),
  vasopressin and oxytocin-containing nucleus
  (VON), and PVN.
• Oxytocin plays a role in stimulating prolactin
  release from the anterior pituitary gland and
  regulates ingestive behaviors.
• Vasopressin is an anti-diuretic that has been
  suggested to play a role in reproductive and
  sexual behavior.
• In a study by Rankin et al., it was hypothesized
  that there is existence of proliferating neurons
  in the VON, and the occurrence of neuronal
  proliferation is greater in adolescent pigs than
  in mature pigs.
• The VON is located bilateral to the third
  ventricle in the anterior of the pigs
  hypothalamus and contains VP and OT neurons.
• It increases in size, volume, and neuron number
  during puberty (16-30 weeks) and continues to
  grow into adulthood.

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Visualizing Methods

• OT immunoreactivity was identified by the
  presence of dark blue-black precipitate in the
  cytoplasm of cells. These cells were identified
  as neurons because of their morphology and
  antigenic reaction to the functional OT marker.
• PCNA immunoreactivity was identified by the
  presence of a brown granular precipitate in the
  nuclei of neurons and glia.
• VP immunoreactivity was identified by the
  presence of red granular precipitate in the
  cytoplasm of neurons.
• Figure 1 represents transverse sections through
  the pig hypothalamus showing the rostrocaudal
  progression of the PVN.

Figure 1
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Neurogenesis of oxytocin-containing neurons in
the PVN

• Oxytocin-containing neurons were observed in the
  SON and PVN.
• Within the PVN, OT-containing neurons were
  observed at the dorsal end of the PVMM
  subnucleus.
• PCNA cells occurred most frequently in the PVLM
  subnucleus of the PVN and were more numerous than
  OT-containing neurons.
• Figure 2 is a nissl-stained photomicrograph
  showing the subnuclei of the pig PVN.
• Figure 3 is a photomicrograph of a Nissl-stained
  section of the PVN. There is a high density of
  large spindle-shaped neurons in the PVLM and
  lower density of the medium-sized neurons in the
  PVMM. The PVMP contains a low density of small
  neurons.

Figure 2
Figure 3
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• Neurons double-labeled for OT and PCNA were found
  in the SON and PVN of all pigs.
• They were identified as neurons based on their
  size and morphology, thought to be neurons
  producing a neurotransmitter such as vasopressin.
• In the tissue stained for VP and OT, the density
  of VP-containing neurons was highest in the PVLM
  subnucleus.
• There was a significantly higher number of OT
  PCNA labeled neurons in the PVN on lactating sows
  and adult gilts compared to puberty gilts.

Figure 4- Photomicrograph from the PVN of a
tissue section stained immunohistochemically for
OT, proliferating cell nuclear antigen (PCNA) and
counterstained with hematoxylin. The unlabeled
neuron (small arrowhead), glial cells (double
arrow), and neurons for OT (large arrow), PCNA
(large arrowhead) and OT PCNA (small arrow).
Figure 5- Comparison of mean OT, OTPCNA, PCNA,
unlabeled neurons and total counts in PVN of
puberty gilts, adult gilts, and lactating sows
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Postnatal Neurogenesis in the VP and
OT-containing nucleus of the HypothalamusImmunohi
stochemistry

• Figure 1 represents photomicrographs of PCNA-VP
  stains in two hypothalamic nuclei of the pig
• Granular blue- VP immunohistochemistry
• Brown- PCNA immunohistochemistry
• A) VON of 249-week-old dry sow
• Densely packed
• B) VON of 23-week-old gilt (young female)
• Smaller and less dense than older sow
• C) Double-labeled (VP-PCNA) neuron in VON of
  adolescent gilt (short arrow)
• Long arrow- VP without PCNA
• D) SON of 23-week-old gilt (mostly VP neurons,
  less PCNA)
• E) Double labeled (PCNA-VP) neuron in SON of an
  adolescent gilt

Figure 1
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Quantitative Analysis

• Figure 2 shows the comparison of mean VON volume
  between adolescent gilts and mature dry sows
• VON volume is significantly larger in mature dry
  sows than in adolescent gilts
• Figure 3 shows the comparison of the mean VP-PCNA
  neurons counts in VON of adolescent gilts and
  mature dry sows
• VP counts are significantly greater in mature
  sows, while PCNA-VP counts are significantly
  greater in adolescent gilts
• PCNA-VP cells indicate the generation of a new
  neuron

Figure 2
Figure 3
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Discussion

• The first study showed that the PVN of the adult
  female pig contains proliferating neurons (PCNA),
  OT-containing neurons and newly generated
  OT-containing neurons.
• Because the proportion of double-labeled cells
  was significantly higher in lactating sows than
  in puberty gilts, but there was no overall
  significant difference in the number of neurons
  in the PVN, it can be considered that
  neurogenesis may occur to replace neurons that
  have been lost or damaged.
• The replacement of these old neurons with new,
  short-lived neurons may allow for constant
  upgrade of brain circuits.
• As pigs experience puberty and repeated estrous
  cycles as they age, the prepubertal neurons may
  be replaced with those needed for brain function
  by a sexually mature adult mammal.
• Age may be an influencing factor in neurogenesis
  of adult pigs. The up-regulation of OT-containing
  neurons may be correlated with age and driven by
  sexual maturation.
• The decrease in proportion of double-labeled
  (PCNA-VP) neurons with age suggests a slowing of
  the recruitment of neurons in the old dry sow.

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Importance?

• The significant volume increase of the VON
  between adolescence and maturity, increase in
  number of neurons, and recently proliferated
  neurosecretory cells suggests recruitment of new
  neurons in the hypothalamus.
• The identification of recently mitotic VP neuron
  suggests that postnatal neurogenesis is used by
  the VON to increase in size from adolescence to
  maturity. It also shows that neurogenesis in the
  hypothalamus is occurring into adulthood.
• The number and proportion of double-labeled
  (PCNA-VP) cells was significantly higher in
  adolescent pigs, while the number of PCNA-OT
  cells was greater in lactating sows and adult
  gilts.
• Since the hypothalamus is important to
  reproductive regulation, neurogenesis could be
  related to the reproductive requirements of the
  animal.

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What Have We Learned?

• The development of the adolescent brain is a
  period of plasticity.
• Both forebrain and hippocampal neurogenesis is
  significantly reduced during brain maturation
  from puberty to adulthood.
• Neurogenesis has been shown in the hypothalamus,
  hippocampus, and forebrain SVZ of the adolescent
  brain. The decrease in neurogenesis during brain
  maturation from adolescence to adulthood may be
  due to rewiring and strengthening of synapses as
  the brain matures.
• Leptin was demonstrated to promote adult
  hippocampal neurogenesis (DG), resulting from
  cell proliferation.
• Neurogenesis in the hypothalamus could be related
  to the reproductive requirements of the animal.
• Significantly increased numbers of proliferating
  OT-neurons were found in the lactating and adult
  sow, while there were greater amounts of
  proliferating VP-neurons in the adolescent gilt.
  Studies in several species have suggested various
  factors that can stimulate neurogenesis in
  different regions of the adult mammalian brain.
  These include the environment, photoperiod,
  growth factors, and hormones.

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Part III
There is Still Ambiguity
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Unanswered Questions

• There have only been a handful of studies on the
  topic of pubertal neurogenesis.
• What about the other areas of the brain that are
  affected by puberty?
• Are they affected by neurogenesis?
• What about the Olfactory Bulb? It is a major
  location of neurogenesis as well as puberty,
  since smell is an important aspect of rodents
  every day lives.
• In a study by Schreibman et al., a structural and
  functional link between olfactory and
  reproductive systems in platyfish was found. It
  was demonstrated by the connection of receptors
  in the nasal epithelium to a center in the brain
  that has a role in the reproductive system. There
  is large morphological increase in the nasal
  epithelium during sexual maturation.
• This needs to be studied further and related to
  neurogenesis.
• The two studies about the hypothalamus only used
  pigs. What about other species?
• What happens if neurogenesis is ablated? How
  would it affect puberty?

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The Sexual (Subcortex) Brain

• The medial preoptic area (MPOA) receives direct
  and indirect input from brain areas that are
  important for the assimilation of sexually
  relevant information.
• Olfactory stimulation is received by the
  olfactory bulbs (OB), the OB project to the
  medial amygdala (MeA), which relays information
  to the bed nucleus of stria terminalis (BST) and
  the MPOA.
• Additionally, the MPOA and MeA receive
  somatosensory input (from genitals) via the
  central tegmental field (CTF).
• In turn, the MPOA projects to the ventral
  tegmental area (VTA) and the brain stem (BS)
  which project to nucleus accumbens (NAc).
• Lateral hypothalamus involved in inhibiting mPOA
  and NAc.

Taken from Dr. Kippins slides
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Where to go in the Future

• Look at the effects of ablation of neurogenesis
  on puberty.
• Using GCV-activated GFAP-TK or mitotic toxins
• Study circuits in the brain related to puberty,
  and how they are affected by neurogenesis.
• Look at other regions in the brain besides the
  hypothalamus, hippocampus, and SVZ.
• Research on humans?

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References

• Garza, JC, Guo, M, Zhang, W, Lu, XY (2008).
  Leptin promotes adult hippocampal neurogenesis in
  vivo and in vitro. The Journal of biological
  chemistry,
• He , J., Crews, FT. (2007). Neurogenesis
  decreases during brain maturation from
  adolescence to adulthood. Pharmacology,
  biochemistry, and behavior. 86(2), 327-33.
• Rankin, SL, Partlow, GD, McCurdy, RD, Giles, ED,
  Fisher, KR (2003). Postnatal neurogenesis in
  the vasopressin and oxytocin-containing nucleus
  of the pig hypothalamus. Brain research. 971(2),
  189-96.
• Raymond, AD, Kucherepa , NN, Fisher, KR, Halina,
  WG, Partlow, GD (2006). Neurogenesis of
  oxytocin-containing neurons in the
  paraventricular nucleus (PVN) of the female pig
  in 3 reproductive states puberty gilts, adult
  gilts and lactating sows. Brain research.
  1102(1), 44-51.
• Schreibman, MP, Margolis-Kazan , H,
  Halpern-Sebold , L, O'Neill , PA, Silverman, RC
  (1984). Structural and functional links between
  olfactory and reproductive systems
  puberty-related changes in olfactory epithelium.
  Brain research. 302(1), 180-3.
• Schulz, KM, Sisk, CL (2006). Pubertal hormones,
  the adolescent brain, and the maturation of
  social behaviors Lessons from the Syrian
  hamster. Molecular and cellular endocrinology.
  254-255, 120-6.
• Sisk, CL, Foster, DL (2004). The neural basis
  of puberty and adolescence. Nature neuroscience.
  7(10), 1040-7.