1. Yoav Livneh yoav.livneh@mail.huji.ac.il02-6585163

2. neurogenesis and migration Refinement of synaptic connections outgrowth of axons and dendrites, and synaptogenesis Neuronal development (generally)

3. Adult neurogenesis (hippocampus) * also occurs in the adult olfactory bulb

4. Neuronal development (in the adult hippocampus) Ge et al., J Physiol, 2008

5. GABA in neuronal development Ben-Ari, Nature Reviews: Neuroscience, 2002

6. 1. Genesis of neurons (and migration). 2. Outgrowth of axons and dendrites, and synaptogenesis. 3. Refinement of synaptic connections.

7. Development of GABA (and glutamate) currents in adult-born hippocampal neurons tonic GABA currents evoked GABA currents evoked Glu. currents 3 dpi7 dpi14 dpi28 dpi DCX (doublecortin) - a marker for immature neurons Bicuculine – GABAa-R antagonist CNQX – AMPA-R antagonist

8. The big question – What is the role of these tonic excitatory GABA currents?

9. Exploration and manipulation of GABA currents in adult-born hippocampal neurons exc.inh. - GABA is excitatory from 3 to ~14 dpi. - Afterwards GABA currents mature to become inhibitory. - shRNA-mediated knockdown of NKCC1 (chloride transporter) eliminated the excitatory nature of GABA (high [Cl] in is not maintained). ~inh. Perforated patch – initially keeps the original intracellular content. Whole-cell patch – content is very rapidly replaced with the electrode’s content.

10. How will this manipulation affect adult-born neuron development?

11. Effects of eliminating excitatory GABA currents – part I (dendritic development) Excitatory GABA currents are essential (necessary) for proper dendritic development - Shorter total dendritic length. - Less branches. - Occupy a smaller volume of space.

12. Effects of enhancing excitatory GABA currents The opposite, complementary, trends were observed with administration of a GABA agonist (suggesting it might also be sufficient). (should be positively correlated) Pentobarbital – GABA agonist

13. Effects of eliminating excitatory GABA currents – part II (inhibitory synaptic integration) Excitatory GABA currents are essential for proper inhibitory synaptic integration evokedspontaneous - Less neurons with evoked inhibitory responses. - Lower evoked response amplitudes. - Lower spontaneous response frequencies (but similar amplitudes).

14. Effects of eliminating excitatory GABA currents – part II (excitatory synaptic integration) Excitatory GABA currents are essential for proper excitatory synaptic integration evoked spontaneous - Dramatically less neurons with evoked excitatory responses. - Lower evoked response amplitudes. - Lower spontaneous response frequencies (but similar amplitudes).

15. Effects of eliminating excitatory GABA currents from birth (KO mice) The same trends were observed with NKCC1 KO mice.

16. Conclusions and interpretations Conclusions: -At the initial stages of the development of adult-born neurons, GABA is excitatory due to high intracellular Cl concentration (mediated by the transporter NKCC1). - This excitatory action of GABA is essential (necessary and perhaps sufficient) for proper development of dendrites and synaptic connections. Interpretations: - New neurons initially “listen” to network activity, before they “talk” (what they “hear” might affect their integration). - Tonic excitatory GABA might act through low-threshold (-57 mV) T- type calcium channels (expressed in these neurons). Calcium then initiates changes in gene expression, cytoskeletal rearrangement, etc.

17. Questions?