Optogenetics:

Optogenetics is a technique that combines the use of non-specific cationic channels known as channelrhodopsins, and their activation through illumination by lasers. It can be used to activate specific cells in specific cell populations, generating electrical excitability in these cells, which can then activate downstream targets. It is rapidly emerging as an essential technique in furthering the field of neuroscience, especially as pharmaceutical companies such as GSK and Novartis move away from funding drugs, and instead look to generate a comprehensive guide of neural circuitry.

From the outside, the brain may appear as a pink mass. There is nothing to distinguish it from any other organ of the body, bar its characteristic sulci and gyri, and its pale pink shine. Yet beneath those glistening pia which keep it all together, liesthe most complex machine that has ever existed to our knowledge. The brain, simplistically, can be imagined as a computer. Made up of components that lie in parallel, they act like a circuit, a continuous network. In the brain, these components are the neurons; and there are around 86 billion of them (). Each neuron can receive around 10,000 inputs, and output onto 10,000 more.

An activated neuron may transmit its signal onto another neuron, which in turn becomes activated, and so on. Suddenly the scale at which the brain works became both apparent and incomprehensible. The task is further complicated by the fact that there is not just one type of neuron. In fact, there are thousands of different types, all with specific roles and functions that are carried out in order

to keep ourselves cognitively able. Examples of common neurons include pyramidal neurons, purkinje cells, granule cells, motor neurons, tripolar neurons, basket cells, stellate cells, and the list goes on and on. All of these neurons are also divided into sub-types, each one being subtly different due to their age and location within the central nervous system. Their differences lie in the expression of proteins and behaviours/functions.

I feel that the scale of the task presented to neuroscientists may be slowly becoming apparent. But how does optogenetics fit in?

So far, we havent been able to activate a small number of cells in a specific region of the brain. Genetic modifications has enabled us to alter the expression of certain receptors on cell types, resulting in monitored behavioural and physiological changes. This is has led to a vast amount of discoveries,creating an exponential development in neuroscience research. But we have merely been modulating cells; pure activation and silencing has been impossible simply using genetic tools. What optogenetics brings to us is a whole new way of approaching neuroscience. No more are researchers bound to pure genetic manipulation in order to deduce the roles of neurons and their circuits. Using optogenetics we can activate or silence specific neurons in specific cell populations, in fully alive, unencumbered, freely moving animals, and gather large amounts of data.

How does optogenetics work?

There is a very simple organism on our planet, an alga, known as Chlamydomonas reinhardtii. This alga relies on light for photosynthesis for energy. It is able to move towards light due to presence of an eye spot. This eye spot contains the non-specific cationic receptor channel known as channelrhodopsin (ChR). Activation by photons leads to opening of the channel, which allows an influx of cations across the cell membrane. This then leads to depolarisation of the membrane, generating an electrical charge along the cell. The cell becomes excitable, and this in turn causes the Chlamydomonas to move towards the source of light.

In optogenetics, the DNA that codes for the ChR is taken and inserted into the genome of certain cells in the brain. This is done through stereotaxic injection of adeno-associated viruses, containing the ChR DNA, into the relevant brain area of an animal model. Stereotaxic injections can target brain areas anywhere from 1 mm to 0.5 mm in size, and occasionally even smaller (however these have varying degrees of success). Using a genetic tool known as Cre, we can express ChR in a Cre-dependent manner. This means that ChRs are expressed not only in a certain area, but in certain neurons in a certain area, that contain the Cre

sequence in their DNA. The Cre sequence can be added to specific neurons using genetic manipulation techniques. For example, we can express ChRs in D1 (dopamine receptor 1) expressing cells in the prefrontal cortex, or pyramidal neurons in the hippocampus. The neurons in the targeted cell areas now express ChRs on their surface. A laser emitting device is then inserted into the brain, and can be turned on at specific times and events. The neurons expressing ChRs are activated by the presence of the laser and can therefore easily be traced.

An example of a use of optogenetics to research behavioural changes, and their relevant nuclei in the brain, can be seen in an experiment conducted by Karl Deisseroth and Josh Johansen. Activation of neurons in the lateral nucleus of the amygdala using optogenetics allowed for implantation of false fear memories in rats. Normally, during fear conditioning (a traditionally Pavlovian model) a tone is played followed by a footshock. The animals quickly learn to associate the two, and upon hearing a tone they freeze an instinctual response to the presence of a threat (in this case the footshock). Using optogenetics, the tone was substituted with direct activation of the lateral nucleus of the amygdala. The lateral nucleus was activated using the laser at the same time as the electric foot shock. After several trials, activation of the lateral nucleus alone was enough to elicit the instinctual freezing behaviour seen in rats that underwent normal fear conditioning using a tone. This study proved that the tone activates neurons in the lateral nucleus of the amygdala, and is responsible for mediating fear responses. This sort of work has potentially massive implications in mental health fields, especially post-traumatic stress disorder (PTSD) and anxiety disorders.

Furthermore, work by Jerry Silver at the Case Western Reserve University in Cleveland, Ohio, showed that selective expression of ChR in specific neurons in the cervical spinal cord following spinal cord injury brought about recovery of breathing and diaphragm activity. These neurons had previously lost their presynaptic activation due to the disruption of the spinal cord. But expression of the ChRs allowed for an alternative site leading to activation.

More recently, halorhodopsin has also been discovered, and activation of these receptors leads to the silencing of cells.

The medical potentials offered by treatments stemming from optogenetics are obviously massive. Not only are medical advances apparent, but optogenetics also makes the generation of a comprehensive guide of neural circuitry more realistic; a great step towards increasing our knowledge. The experiments mentioned above are only two examples of a whole multitude of papers being released. Both of these potentials have not gone amiss. Headed by Barack Obama, a new initiative known as Brain Research through Advancing Innovative Neurotechnologies (BRAIN) has been implemented. With $100m being approved from the US budget for the first year of operation, there should without doubt be yet more advances in the field of neuroscience. The US government is not alone in taking notice. Large pharmaceutical companies have begun to withdraw funding for drug research into mental health disorders, and instead are looking to projects that map neural circuitry, with the hope of generating more specific diagnoses, and therefore more specific treatments. There are

currently no drugs on the market that can claim to a cure a neural disorder, and those that treat them are rarely without side-effects. Therefore the move away seems obvious and unavoidable. It is becoming increasingly apparent that mental disorders are not just a result of a chemical imbalance, although they do play a role. It appears that connections between different populations, and the strength and/or weaknesses of these connections are heavily involved in neural diseases.

For now, using optogenetics techniques in humans still faces a multitude of problems. However, the potential cannot be denied, and the increasing research into, and use of, optogenetics, surely is a step in the right direction. For now, their use in animal models is becoming increasingly prolific and successful, and we should not have to hope, but expect, great advances in the not too distant future.