Situación fiscal

In-vitro research refers to studies conducted using some components of the living organism, but outside the organism and in a controlled environment. The capa- bility of in-vitro neuronal culture has been fundamental in the advancement of understanding of brain functions. In vivo on the other hand refers to studies per- formed on whole living organisms. The landmark work by [29,30] demonstrated -

for the first time - the successful growth of nerve fibres of embryonic tissue outside the body. His studies not only answered fundamental questions about the growth and development of nerve cells to nerve fibres, but transformed biological science to a new direction in development studies in neuroscience.

Over the past century, a diverse plethora of techniques have been developed for in-vitro neuronal culture. Figure 2.4 shows the emergence timeline over the last 100+ years of different neuronal culture techniques. A detailed review of different techniques is reported in [31].

Fundamentally, there are two main approaches for an in-vitro neuronal culture - Acute or Organotypic Slice [32] and Dissociated culture. The dissociated culture method has been a preferred method to study individual neurons or group of neurons for decades due to the ease of dissociating neurons and maintaining the culture. Even though all the experiments for the work in this thesis utilise the dissociated culture technique, a brief overview of slice culture is presented for completeness.

Figure 2.4: Emergence of cell culture methods. The timeline shows introduction of new neuronal culture methods over the past century. [31]

2.3.1

Brain slice culture

Slice culture utilises thinly sliced (100 − 500µm thick) sections of the brain in a controlled in-vivo like environment. The complex neural circuitry and the spatial distribution of the neurons established during the development stage are rela- tively undisturbed along with tissue functions in a tissue slice under favourable in vitro conditions [33]. The main advantage of such brain slices is the partial preservation of functional and structural properties of the original intact brain.

Such brain slices are used for a diverse range of studies - from intrinsic devel- opmental studies, network dynamics to synaptic plasticity. In many cases, brain slices are the preferred choice for many neuroscientists due to the structural and functional integrity of the slice culture. With the development of Micro(also referred as Multi) Electrode Arrays(MEAs), brain slices may be coupled with MEAs to record electrophysiological activities [34,35]. It is straightforward to record data from slices when coupled with MEAs, in comparison to the dissoci- ated cultured networks which can take many weeks for the culture to mature to start measurements. An example of a rat brain (hippocampus) slice coupled with CMOS based MEAs is shown in Figure 2.5.

Figure 2.5: Rat hippocampal slice recorded by high density Multi Electrode Ar- rays(4096 electrodes). Screenshot from [36]

Although the main advantage of the slice culture is its partial morphological and functional integrity, the same feature becomes undesirable when the study focuses on the dynamical evolution of the neuronal network from an unstruc- tured initial condition under some external stimulation. Slice cultures are not ideal in studies that focus on inducing specific network response or how the net- work connectivity evolves with time when started from a random unstructured distribution of neuronal cells. Dissociated cultures on the other hand provides the desired flexibility and control to study a plethora of research topics.

2.3.2

Dissociated culture

Demonstration of growth and maturation of tissues not just inside the organism (in-vivo) but also outside of the body (in-vitro) has opened new and innovative opportunities for neuroscientists [37] to study nervous system and neuronal dy- namics that underlie the foundation of higher level behaviours such as cognition, memory, learning and more.

Among neuroscientists, the dissociated neuronal culture is a common method to study neurobiology due to the relative ease with which the brain tissues can be dissociated to isolate neurons, maintain and measure physiological phenomenon. Such dissociated cultures can be kept healthy for a long period (up to months) making them an ideal experimental setup to study a variety of brain functions. Dissociated neuronal cultures grown on planar MEAs provide non invasive ex- tracellular electrophysiological recordings (further discussed in Section 2.4) of the neurons at different time points under different desired pharmacological or electrical stimulation conditions.

Neurons grown in dissociated cultures form seemingly random synaptic inter- connections without maintaining the in-vivo like network. However, neurons tend to retain the morphological and physiological properties that correspond closely to the in-vivo properties [38]. Hence, such dissociated cultures can be seen as a reasonably simplified biological model of areas of the brain. Such a model is an appealing experimental setup to study a wide range of brain functions due to the fact that despite its simplicity, such a simple model exhibits rich neuronal dynamics [39].

Cultured neurons provide a “close to ideal“ substrate to study brain functions since the cultured neurons can be grown healthily for months. This allows for a controlled experimental setup to start a biological network without any pre- configured interconnections, and investigate and alter these connections to study overall network dynamics in response to different stages in network change or controlled external perturbations. Neuronal mechanisms such as long-term po- tentiation (LTP) or long-term depression (LTD) are particularly interesting to study because these mechanisms are synaptically analogous to learning. Neu- ronal culture models to study network dynamics will be discussed further in the

next chapter.