Dopamine neurons of the brain marked by the expression of TH (blue) that also express a protein called Calbindin (red) appear pink.

Optogenetics: Controlling the Brain with Light

By Shabana Khan

The neuroscience field has been revolutionised by a powerful technique known as, optogenetics, originally invented to tackle some of the most crucial questions on the most important and complex organ — the Brain.

The word ‘optogenetics’ as it suggests is a term given to a technique now used by neuroscientists that combines both — the control of ‘genes’ using ‘light’ to understand how nerve cells, also known as ‘neurons’, work together in groups within different areas of the brain to mediate a particular behavioural function. Using this technique, by flashing blue light, a gene can be switched ‘on’ and ‘off’, the properties of the neurons that express this gene can be manipulated causing them to activate or inactivate resulting in unravelling the function that they are important for.

Traditionally, neurons have been manipulated using electrical, pharmacological and genetic approaches to understand the underlying function for which they are important. However, these types of manipulations have lacked either cell-type specificity (spatial) or precision of neuronal signalling timing (temporal).

Why is spatial and temporal specificity important in studying neuronal function?

Spatial: In the brain, there are approximately a 100 billion neurons, which are extremely heterogeneous. This means that there are a multitude of cell types with a range of unique and different functions. Cell-type can be characterised in a number of different ways. What makes a particular type or ‘subset’ of neuron different from another in the brain can be the combination of genes that it expresses, proteins that it has — these include transcription factors, receptors and signalling factors, hormones or a particular neurochemical termed neurotransmitter. For example, all the neurons in the brain that use dopamine as their neurotransmitter can be identified by their expression of a protein enzyme called tyrosine hydroxylase (TH). Yet all the dopamine-producing neurons in the brain do not have the same function. Several functions are attributed to dopamine neurons including regulation of movement, motivation to eat and addiction behaviour such as gambling. This is because within the group of TH-expressing dopamine neurons, there are subgroups of neurons with different expression profiles of genes, receptors, hormones and transcription factors. In order to identify and understand the function of each subtype of neuron, it is important to be sub-type, sub-group, cell-type specific i.e. only those dopamine neurons that control your motivation to eat.

Temporal: The timing of gene expression in a particular subgroup of neurons is crucial in understanding both the function of that gene and the function of the neurons themselves. The expression of sets of genes, transcription factors and signalling proteins are not always uniform or constant in a group of neurons. In fact, their expression can be turned ‘on’ or ‘off’ rapidly at any given time during the development of a neuron or even in it’s day to day functional mature state. Some genes are only transiently expressed, some during only the early and/or late-development stages whereas others are expressed throughout the age of a neuron since it is born to adulthood. Even the levels of gene expression can change at different times during a neuron’s maturation. So timing is crucial in understanding what the function and behavioural outcome of a particular cell-specific cluster of neurons is.

How does optogenetics technology fulfil both spatial and temporal specificity, and allow us to study neuron function?

Optogenetics works by making use of light-sensitive ion channels called opsins. Several opsins are found naturally in photoreceptor cells of the retina in the eyes of animals and contain a pigment called retinal. Channelrhodopsin is a specific type of retinal opsin light-sensitive ion channel that opens in response to blue light. Expressing these channelrhodopsins in a genetically-defined manner, so a specific subgroup of neurons marked by a particular gene they express also express the light-sensitive ion channels, these neurons can then be manipulated using optical light to activate them. Optical recordings can then measure the activation of the neurons using optogenetic sensors for calcium (e.g. GCaMP) or neurotransmitters (e.g. GluSnERs) while the animals carry out very specific behaviours. In this way, the expression of channelrhodopsins in groups of neurons, essentially allow light to control the electrical excitability, calcium influx and other cellular processes. This process allows for the manipulation of groups of neurons in both a spatial (gene-specific expression of the channelrhodopsins) and a temporal (turning these gene-specific neurons ‘on’ and ‘off’ using light) specific manner.

Experimentally, how does optogenetics work?

To conduct an optogenetics experiment in the lab, the scientist would:

  1. Assemble a genetic construct that contains a genetic ‘promoter’ that drives the expression of the gene that is added to the contsruct downstream. This downstream gene defines the specific group of neurons but also has an encoded opsin (e.g. channelrhodopsin) protein which is light-sensitive.
  2. This construct is then inserted into a virus.
  3. The virus is then injected into the brain of test animals used in the experiment. The light-sensitive channelrhodopsins will be expressed in only the specific group of neurons that express the particular gene in which the channelrhodopsin was originally encoded into.
  4. A fibre optic cable and a recording electrode are inserted into the brain of the same animal.
  5. Laser light is used to open the channelrhodopsin ion channels in those neurons allowing for excitation or activation of those neurons, which is measured by the recording electrode.
The six steps to optogenetics — Image source: http://optogenetics.weebly.com/why--how.html

Applications of optogenetics I: What behaviours have been discovered using optogenetics?

Optogenetic approaches to understanding brain function have unravelled several underlying behaviours and attributed them to the specific neurons and their circuits in the brain.

Homeostatic functions — These include identification of neurons and brain circuits responsible for movement regulation (Kravitz et al., 2010), hunger, thirst, energy balance (Oka et al., 2015; Jennings et al., 2015), respiration (Abbott et al., 2011), arousal, sleep and circadian rhythm (Anaclet et al., 2014; Jones et al., 2015).

Social behaviours — These include motivation, reward, cognition and memory, which have also been extensively studied using optogenetic approaches. The underlying brain circuits and groups of neurons that control and modulate social behaviours such as reward mechanisms, fear and anxiety have been identified.

Learning and memory — Learning behaviour has been extensively studied using optogenetics in specific neuronal groups within different anatomical brain areas including neurons of the amygdala (Johansen et al., 2010), midbrain and striatum (Brown et al., 2012). The ‘engram’ cells of the hippocampal region of the brain were identified to be key players of memory states using optogenenetics (Redondo et al., 2014).

Neuron development — Optogenetics applications have allowed various stages of embryonic and adult neuron development such as formation of synapses (the contact between two neurons), the wiring of their axons, micro-circuitry of axons to synapses within brain regions and long-term development to be understood in greater depth.

Applications of optogenetics II: How can optogenetics help the treatment of disease?

Alzheimer’s Disease & Memory Retrieval — Alzheimer’s disease (AD) patients suffer from a myriad of cognitive impairments and episodic memory loss. Episodic memory is attributed to the engram cells located in a brain region called the hippocampus. Until recently, it was unknown whether the memory loss observed in AD patients was due to the inability to store and encode those memories in the brain or as a result of the failure to retrieve those memories. A recent study published in the journal Nature, Roy et al., conducted optical activation of these hippocampal neurons directly in transgenic mouse models of early AD, which resulted in memory retrieval in these mice (Roy et al., 2016). The results of the optogenetic study revealed that AD mouse models exhibited normal storage of the memories and did not lose them but the problem lay in memory retrieval, which was rescued in these experiments. These findings provide hope towards recovering memory loss in AD patients in the future.

Epilepsy & Seizure Termination — Optogenetics methods have investigated the neuronal activity patterns that underlie seizure initiation, propagation and termination in animal models of epilepsy and have aided the identification of specific focal sites in the brain that can be targeted to immediately terminate an epileptic seizure. Interestingly, these seizure termination sites in the brain are different to the seizure initiation sites (Paz et al., 2013). Treatment of epileptic seizures may become possible with advanced optogenetic applications by silencing neurons that initiate and propagate seizures.

Parkinsonian Movement Disorders — The neurons involved in the regulation of movement and those that become affected in movement disorders such as Parkinson’s Disease (PD) have also been investigated using optogenetic methods. Two pathways are generally known to be involved in movement regulation — the direct and indirect pathways of the striatum. Optogenetics techniques have clearly demonstrated the role of the direct pathway in movement ‘initiation’ and the indirect pathway in movement ‘suppression’ in both healthy and PD states (Kravitz et al., 2010). These studies have allowed for in-depth understanding of movement regulation.

References

Abbott SB, Stornetta RL, Coates MB, Guyenet PG. Phox2b-expressing neurons of the parafacial region regulate breathing rate, inspiration, and expiration in conscious rats. J Neurosci. 2011 Nov 9;31(45):16410-22.
Anaclet C, Ferrari L, Arrigoni E, Bass CE, Saper CB, Lu J, Fuller PM. The GABAergic parafacial zone is a medullary slow wave sleep-promoting center. Nat Neurosci. 2014 Sep;17(9):1217-24.
Brown MT, Tan KR, O'Connor EC, Nikonenko I, Muller D, Lüscher C. Ventral tegmental area GABA projections pause accumbal cholinergic interneurons to enhance associative learning. Nature. 2012 Dec 20;492(7429):452-6.
Jennings JH, Ung RL, Resendez SL, Stamatakis AM, Taylor JG, Huang J, Veleta K,Kantak PA, Aita M, Shilling-Scrivo K, Ramakrishnan C, Deisseroth K, Otte S,Stuber GD. Visualizing hypothalamic network dynamics for appetitive and consummatory behaviors. Cell. 2015 Jan 29;160(3):516-27.
Johansen JP, Hamanaka H, Monfils MH, Behnia R, Deisseroth K, Blair HT, LeDoux JE. Optical activation of lateral amygdala pyramidal cells instructs associative fear learning. Proc Natl Acad Sci U S A. 2010 Jul 13;107(28):12692-7.
Jones JR, Tackenberg MC, McMahon DG. Manipulating circadian clock neuron firing rate resets molecular circadian rhythms and behavior. Nat Neurosci. 2015 Mar;18(3):373-5.
Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature. 2010 Jul 29;466(7306):622-6.
Oka Y, Ye M, Zuker CS. Thirst driving and suppressing signals encoded by distinct neural populations in the brain. Nature. 2015 Apr 16;520(7547):349-52.
Paz JT, Davidson TJ, Frechette ES, Delord B, Parada I, Peng K, Deisseroth K,Huguenard JR. Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury. Nat Neurosci. 2013 Jan;16(1):64-70.
Redondo RL, Kim J, Arons AL, Ramirez S, Liu X, Tonegawa S. Bidirectional switch of the valence associated with a hippocampal contextual memory engram. Nature. 2014 Sep 18;513(7518):426-30.
Roy DS, Arons A, Mitchell TI, Pignatelli M, Ryan TJ, Tonegawa S. Memory retrieval by activating engram cells in mouse models of early Alzheimer's disease. Nature. 2016 Mar 24;531(7595):508-12.

Shabana Khan is a final year Neuroscience Cell & Developmental Biology PhD Student working at The Francis Crick Institute formerly known as National Institute for Medical Research. Her PhD focuses on understanding the development of midbrain dopamine neurons in the brain and is funded by a scholarship award for four years from the Medical Research Council (MRC). Aside from being a Scientist in the lab, she can be described as a SciArt-ist, Coffee Addict, Swimmer and a Librocubicularist.

One clap, two clap, three clap, forty?

By clapping more or less, you can signal to us which stories really stand out.