4 Discussion
4.2 An in vivo / in vitro approach to study laminar connectivity of functionally characterized L2/3
4.2 An in vivo / in vitro approach to study laminar connectivity of
differences to the published in vivo / in vitro approaches. The novel approach described here does not rely on time-consuming photo activation of individual, functionally characterized neurons, and instead uses a constantly present structural marker. This is a main advantage over the pa-GFP approaches that require pre-selection of target neurons, and as an additional step, their individual photo-activation during the in vivo experiment. Compared to the method introduced by Mrsic-Flogel and colleagues, a constantly present structural marker removes the limitation where the in vitro experiment has to be carried out within hours after in vivo imaging, before the neurons lose the OGB-1 label. Furthermore, the main limitation of both published in vivo / in vitro approaches by using a synthetic calcium indicator is that this restricts the study of neuronal activity to a single time point in vivo. The use of a GECI as performed here overcomes this fundamental limitation and provides the potential for repeated, chronic in vivo investigation, expanding the combined in vivo / in vitro analysis for studying cell-type specific neuronal plasticity, learning-related changes and alterations during the course of disease.
4.2.2 Applications of the in vivo / in vitro approach
The described in vivo / in vitro approach is applicable for in vivo imaging of neuronal activity in mouse cortex with 2PLSM, and therefore allows studying neuronal activity underlying behavior (Keller et al. 2012, Petreanu et al. 2012, Li et al. 2015) and assessing alterations in neuronal activity in mouse models of disease (Liebscher et al. 2016, Hamm et al. 2017). The developed in vivo / in vitro approach is particularly suited to monitor neuronal activity repeatedly over long periods of time, for example during learning, experience-dependent plasticity or disease progression or remission, to investigate corresponding changes in neuronal activity and subsequently the underlying alterations at the neuronal circuit and cellular level.
Apart from mice, this approach can be adapted to any mammalian model organisms where cortical in vivo 2-photon calcium imaging has been established, including organisms such as rats (Scott et al. , Ohki et al. 2005), ferrets (Smith et al. 2015), cats (Ohki et al. 2006) and non-human primates (Heider et al. 2010, Li et al. 2017).
For relating the recorded neuronal activity in vivo to the underlying neuronal substrate at the level of neuronal circuits, neurons and synapses, separate recordings are usually performed on the same cell type in vivo and in brain slices (Barnes et al. , Keck et al. 2013, Kuhlman et al. 2013). The established
approach allows performing such recordings on the very same neurons, thus allowing direct correlation of in vivo activity with the underlying cellular and synaptic properties as well as the neuronal connectivity.
The in vivo part of the approach can be adapted to miniature 1- or 2-photon microscopy for investigations in freely behaving animals (Ziv et al. 2013, Resendez et al. 2016, Zong et al. 2017) beyond the head-fixed anesthetized (Mittmann et al. 2011, Rose et al. 2016) or awake (Pakan et al. 2016, Rose et al. 2016) condition. Likewise, the in vitro part can be extended to any method applied to acute brain slices, such as multiple patch recordings, to assess local connectivity between functionally characterized neurons (Hofer et al. 2011, Ko et al. 2011), pathway-specific circuit and synapse mapping using optogenetics (Petreanu et al. 2009), and in addition cell type characterization by single-cell PCR (Cadwell et al. 2016) or by immunohistochemistry for assessing neuronal markers
4.2.3 Limitation of the in vivo / in vitro approach
While the in vivo / in vitro approach allows efficient matching of cells between the in vivo and in vitro preparations, there are some limitations with regard to specific scientific questions. For studies in early development, the use of viral injections to deliver GECIs and fluorescent markers is not suitable, as viral transduction takes 2-3 weeks. A possible solution for this is to use transgenic animals constitutively expressing a calcium indicator in neurons (e.g. Tg(tetO-GCaMP6s)2Niell, Wekselblatt et al. (2016)) or alternativelys to perform in utero viral injections (Itah et al. 2004) or DNA electroporation (Potter et al.
2003).
As in previous in vivo calcium imaging approaches, the brain area of interest needs to be made accessible optically for 2-photon calcium imaging in order to be able to study the same cells in vivo and in vitro. While the long-term cranial window method has been used in young animals (P3, Portera-Cailliau et al. (2005)), the brain slice preparation puts an upper limit on the age that can be effectively studied.
The older the animal, the more difficult the brain slice preparation will be for obtaining high quality acute tissue slices. Therefore, the described approach has only been used for brain slice preparations from animals up to P100.
Another critical point is that denser labelling and a larger area of expression in the brain, beyond the area of interest, decreases throughput, as identification of the correct brain slice followed by the unambiguous identification of cells characterized in vivo takes more time. The density of expression has to be adapted to the individual experimental needs in order to achieve the appropriate balance between
imaging a sufficient number of cells in vivo, and the time it takes to successfully re-identify cells in vitro.
Critically, the time taken for cell re-finding in vitro must be minimized in order to maximize the number of cells that can be recorded before the brain slices degrade.
4.2.4 Conclusion and outlook
The in vivo / in vitro approach developed in this thesis closes the gap between chronic in vivo imaging of neuronal activity, and investigating neuronal circuit connectivity and cellular as well as synaptic physiology with high resolution methods in vitro. The described approach is reliable and time-efficient, yielding a high-throughput. Moreover, it can be adopted to various animal models, brain areas and allows studying individual cell types embedded in neuronal circuits.
In future experiments, the in vivo / in vitro approach has enormous potential for studying plasticity-related changes of individual cells, and the underlying circuit rewiring at high resolution.