Study of the role of adult neurogenesis in the olfactory bulb by optogenetic neuromodulation.

It is established that neurogenesis persists in the adult brain of all mammals including humans. Interestingly, this phenomenon is restricted to two specific brain areas consisting of the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus of the hippocampus (HC). The existence of adult neurogenesis throughout adult life created the hope for brain repair by cell replacement of the cells lost during neurodegenerative diseases. After 5 decades of research, it is clear that these stem cell sources will not meet the requirements for therapeutic purposes. However, it still provides an appropriate model system to gain knowledge on the unique skill of these stem cells to migrate, maturate and functionally integrate in a complex neuronal network in order to use engineered stem cell sources to their full potential. The process of adult neurogenesis is strictly controlled in the rodent brain. Only half of the SVZ-derived newborn neurons that arrive daily in the olfactory bulb (OB) are able to survive and functionally integrate while the other half undergoes apoptosis during the so-called critical period. Recent studies have shown that these newborn neurons ‘first listen before they talk’ as they receive synaptic input before they form their own functional synapses. The first extra-bulbar brain region contacting these newborn neurons is the olfactory cortex (OC) comprising two brain areas, the anterior olfactory nucleus (AON) and the piriform cortex (PC). In this thesis manuscript, we sought to investigate the specific role of this top-down connection of the AON in newborn neuronal survival.

First, we aimed to visualize and specifically target the connection of the AON pyramidal neurons to the newborn neurons in the OB using a transsynaptic tracer, the Wheat Germ Agglutin (WGA). Expression of a fusion of WGA and Cre recombinase in Cre-dependent YFP mice permitted us to differentially label the pre and post-synaptic partners of neurons in different brain regions. However, upon AAV-mediated expression of the WGA-Cre fusion protein in the AON pyramidal neurons, we could not observe transsynaptic labeling in the OB. Further evaluation of this transsynaptic tracing throughout the rodent brain revealed that the labeling efficiency is highly dependent on the brain region hampering its widespread use. 

Alternatively, we relied on the mouse CamKII promoter to specifically modulate the glutamatergic AON pyramidal neurons. First, we aimed to modulate the AON by repeated acute stimulation via the light-sensitive ion channel channelrhodopsin(L132C/T159C). However, upon insertion of the optical fiber, detrimental effects on the amount of newborn neurons in the OB were observed hampering the interpretation of the results. To circumvent the invasive optical fiber and simultaneously increase the target area, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that can be activated by a biologically inert compound Clozapine-N-Oxide (CNO). Upon expression of the stimulating hM3D DREADD, we confirmed an increased firing rate upon CNO administration that appeared to decrease upon multiple administrations. Nevertheless, chronic stimulation of the AON, during the critical period of the BrdU-labeled cohort of newborn neurons, resulted in a decreased BrdU+ density in the OB. The fact that this decrease was more pronounced upon bilateral stimulation of the AON and no cross-connectivity was observed between the anterior PC and the OB, prompted us to state that there is a direct contribution of the AON. Unexpectedly, we found that the BrdU+ density was also decreased upon bilateral hM4D mediated inhibition. The similar phenotype to olfactory deprivation suggests that, besides the top-down pathway from the cortex to the OB, also the bottom-up pathway from the OB to the AON and the PC was affected. Taken together, our results are in line with the postulated two stage model in which newborn neuron survival is controlled by the top-down connectivity and is based on the experience-dependent bottom-up stream of information.

Finally, we validated a new neural interface developed by Imec to enable the simultaneous light application and electrophysiological recording in a small ventral brain area such as the AON. We confirmed that this probe is able to stimulate AON neurons expressing channelrhodopsin(L132C/T159C) and record the neuronal response with high quality. Further, the small cross-sectional area and the flexibility of the probe shank cause a minimal probe tract reducing possible insertional artifacts.

In general, this thesis manuscript touches upon the complex signaling cascade involved in the integration of newborn neurons in an existing network. We found that the AON is a direct mediator of the integration but further research is necessary to fully understand this process. Further, it is clear that ongoing technological improvement is necessary to be able to specifically alter neuronal networks without compromising its integrity.