Lights, Genes, and Action! Understanding the Functions of Neural Circuits
By Jung Soo Lee ’12, thurj Staff
Throughout the bodies of most animals, specialized cells called neurons form intricate networks that serve as highways for information transmission and processing. These numerous junctions (also known as synapses) allow neurons to relay information throughout the entire body. Take the nematode Caenorhabditis elegans (C. elegans), for example. In the nervous system of this miniscule roundworm of approximately one millimeter in length, over 7000 synapses form connections between only 302 neurons.
While previous studies have elucidated the details of the physical connections among these neurons, little is yet known about the functional nature of these synaptic links. However, scientists at the FAS Center for Systems Biology and Harvard Medical School have recently been able to devise a novel optical method that allows a deeper examination of neural circuit function.
Zengcai Guo, a researcher in the lab of Professor Sharad Ramanathan (an assistant professor of Molecular and Cellular Biology and Applied Physics) describes this new approach as “a powerful new tool for analyzing small neural circuits [that] allows scientists to directly measure how neurons talk to each other.”
While the physical map of the nervous system of C. elegans is well characterized, Guo relates that “To my surprise, it’s still difficult to understand how the nervous system [regulates] animal behaviors even with the ‘wiring diagram’ of C. elegans mapped out. One crucial piece of information missing is that we typically don’t know whether a synaptic connection between neurons is excitatory or inhibitory.”
Although C. elegans is a popular model organism, neurological studies of the roundworm have proven to be rather challenging – its neurons are relatively small and a tough cuticle covers the length of its entire body. As a result, only one neuron could be studied at a time, undermining previous efforts to study the activities of neural circuits. In late 2009, however, Zengcai Guo, Professor Anne C. Hart of the Harvard Medical School, and Professor Ramanathan of Harvard University published a finding in the journal Nature Methods that offers a glimpse into a technique that could revolutionize future studies of synaptic function. This new approach uses an optical method to excite specific neurons while simultaneously measuring the activity of other subsets of neurons.
By combining various genetic techniques with an intricate system involving lasers and mirror arrays, these researchers were able to observe the interactions among distinct groups of neurons. One neural system scientists examined using this method was composed of synaptic connections between ASH sensory neurons and AVA and AVD command interneurons. ASH neurons react to various types of harmful mechanical and chemical stimuli, causing the worm to initiate an avoidance response through the AVA and AVD command interneurons, which are required for backward movement. Previous studies had demonstrated that ASH neurons form synaptic connections with the AVA and AVD command interneurons and that the activation of ASH neurons causes the simultaneous activation of AVA and AVD command interneurons.
First, the researchers activated channelrhodopsin-2 (CHR2) expressing ASH sensory neurons. Originally found in green algae, ChR2 is an ion channel that can be activated by specific wavelengths of light. Upon activation of these channels in ASH neurons, the optical system allowed the scientists to simultaneously measure neuronal activity in AVA and AVD command interneurons through the detection of calcium activity by the expression of GCaMP, which increases fluorescence in the presence of calcium.
The simultaneous activation of specific neurons and measurement of activity in other groups of neurons were accomplished through the use of a sophisticated system of mirrors and light sources. In this process, the initial activation of ChR2 is executed by a light source that hits a digital light processing mirror array. Each of these miniature mirrors can be precisely manipulated in order to accurately orient the light source on specific locations on the C. elegans. Meanwhile, a system involving a laser, spinning disc, and camera would measure calcium activity in other neurons through the detection of GCaMP fluorescence.
The researchers first confirmed that light activation of ASH sensory neurons results in the classic C. elegans avoidance response. Then they recorded GCaMP fluorescence levels in AVA and AVD command neurons, and successfully showed that the intensity of GCaMP fluorescence in these interneurons increased after activation of the ASH neurons.
The development of this method was not without challenges. One major setback was finding a way to overcome the limitations of the overlapping excitation spectra of both ChR2 and GCaMP. “GCaMP is a genetically encoded calcium indicator. Like fluorescent proteins, it does not emit light [by itself],” says Guo. “However, GCaMP emits green light when exposed to blue light. ChR2 is an opsin protein which functions as light sensitive channels. When exposed to blue light, ChR2 molecules undergo conformational changes and allow ions to flux into cells expressing ChR2.” However, Guo explained, because both ChR2 and GCaMP respond to blue light, their excitation spectra are not always distinct. Thus, researchers had to devise a method to effectively separate neural activation using ChR2 and calcium activity measurement through GCaMP. In order to do this, Ramanathan and colleagues opted to use a high-power light source to activate neurons and a lower-intensity laser to measure GCaMP fluorescence.
The researchers were also faced with the difficulty of precisely activating only neurons of interest, leading to the development of the mirror array that permits the accurate aiming of light at a specific group of neurons.
The successful development of this method is a major breakthrough that will allow scientists to better understand how our neurons build connections and form networks. “The idea of combining light sensitive channels and genetically encoded calcium sensors to develop an all optical method is quite general,” describes Guo. “We expect this method can be widely used in C. elegans and zebrafish larvae (as it’s also transparent) to study small neural circuits. It might also be adapted to study local neural circuits in high reorder animals such as mice.” Even though the many synapses in C. elegans have previously been mapped, this method will allow scientists to understand exactly how those connections actually function in order to relay information throughout the entire organism to evoke certain behaviors and responses. These types of research and methods will prove to be crucial and invaluable as scientists strive to apply fundamental findings at the molecular and cellular level to the greater understanding of organism function as a whole.
Optical interrogation of neural circuits in Caenorhabditis elegans. Zengcai V Guo, Anne C Hart, and Sharad Ramanathan. Nature Methods, December 2009, Vol. 6 No. 12