Songbirds, Steroids, and Adult Neurogenesis. Brad Walters Biological Sciences Lehigh University September, 2008

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1 Songbirds, Steroids, and Adult Neurogenesis Brad Walters Biological Sciences Lehigh University September, 2008

2 Songbirds, steroids, and neurogenesis

3 Some tissues regenerate well

4 Others, not so good The absence of evidence is not evidence of absence!!!

5 Visualizing Neurogenesis: nucleotides (e.g. thymidine) get incorporated into DNA strands during replication Altman, 1962 [ 3 H] Thymidine incorporates into dividing cells!

6 Some songbird brains change dramatically across seasons Brenowitz et al, 1991 Margoliash, 2005 Nottebohm, 1981

7 Nucleus Axon Hillock Do the changes in volume result from Neurogenesis? Dendrites Axon A neuron stained with [ 3 H]thymidine, counterstained with methylene blue. Bar = 50µm Goldman and Nottebohm, 1983 Electron micrograph of a new neuron in HVC. N = nucleus, A = axonal hillock, D = dendrite,

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9 But are they really neurons? Voltage (mv) Time (msec) Paton and Nottebohm, 1984

10 No But seriously Are they really neurons? The retrograde tracer, Fluorogold was injected into RA, a nucleus known to be innervated by axons projecting from HVC. Kirn and Nottebohm, 1991

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12 Spring Seasonal neurogenesis in songbirds suggests the involvement of steroid hormones. Fall Kirn and Nottebohm, 1993 Smith et al. 1997

13 Testosterone increases neurogenesis Bottjer and Dignan, 1988

14 However, Testosterone can be metabolized in the brain 5-α Reductase Aromatase Testosterone Dihydrotestosterone (DHT) 17β-Estradiol (Estrogen)

15 So which steroid is it? C T DHT E 2 DHT+E 2 C T DHT E 2 DHT+E 2 Tramontin et al. 2003

16 Estrogen increases neurogenesis in adult songbirds Average # of HVC Neurons/Section 18 Average # of New HVC Neurons/Section HVC Neurons * N = 7 N = 8 [3H] Positive HVC Neurons * N = 7 N = No E E Replaced No E E Replaced 1 2 adapted from Hidalgo et al. 1995

17 Estrogens enhance mammalian neurogenesis. Tanapat et al. 1999

18 Proliferation Migration Integration Perpetuation Differentiation Adapted from Gage, 2003

19 Which process or processes are being affected by Estrogen provision? Functional Neurogenesis is comprised of 1. Proliferation 2. Migration 3. Differentiation 4. Perpetuation (Survival) 5. Integration

20 Does E affect neural stem cell Two main questions: proliferation? Do estrogens increase the number of cells entering the cell cycle at any given point in time? Do estrogens increase the rate at which cells proceed through the cell cycle?

21 Does E affect neuronal migration? E acts on cells involved in the migration process. E influences migration away from VZ. Williams et al., 1999

22 Does E affect Differentiation? Does E affect cell fate directly? Does E affect factors that affect cell fate? Fold Change of mrna expression 200 BMP Brànnvall et al Fadrozole Saline Modified from Walters and Saldanha, 2008

23 Perpetuation (Survival) * * FAD FAD+E2 Saldanha et al. 2005

24 With regard to adult neurogenesis, estrogen affects Proliferation: tentative yes Migration: tentative yes Differentiation: maybe Survival: yes Integration:??? This is the next big question!!!

25 OTHER FACTORS KNOWN TO INFLUENCE NEUROGENESIS INCLUDE: HORMONES Glucocorticoids (-) NEUROTRANSMITTERS Serotonin (+) Testosterone (+) Norepinephrine (+) Progesterone (+) Glutamate (-) BEHAVIOR Exercise (+) Enriched Environment (+) Gage, 2003

26 Summary Brain Injury and Neurodegenerative diseases can be devastating to the adult brain. However, neurogenesis in the CNS does occur throughout life. Thymidine analogs are a powerful, but limited, tool for measuring this process. Songbirds are an indispensable model organism in this field. Steroid hormones affect neurogenesis. Estrogen increases neurogenesis most likely by increasing survival as well as via proliferation (probably), and possibly by affecting cell fate and/or migration. There are many factors, often working together, that affect neurogenesis. Clinical applications will depend heavily on the site specific migration and integration of new neurons.

27 Acknowledgements PI Colin Saldanha Collaborators Ryan Wynne Work funded by: NIH (NINDS)