Neural circuit refers to the channel in the brain which is formed by the interconnection of neurons to transmit certain information. The simplest neural circuit is knee jerk reflex circuit: after hammering below the knee, sensory information is generated from the muscle spindle and enters the spinal cord through sensory neurons; Then the movement information is transmitted from the motor neurons to the spinal cord and then to the muscles, which controls the contraction of the quadriceps femoris and the relaxation of the biceps. The neural circuit of the central nervous system is often much more complicated than knee jerk reflex, which not only involves multiple brain regions, but also has a complex connection structure.
No matter how complex reflection is, it is also composed of three parts: input (sensory information), intermediate processing and output (behavior). Simple as knee jerk reflex circuit, its input is knocking near the knee and its output is kicking behavior caused by muscle contraction; The complex courtship behavior of flies is manifested in specific environment, time and the existence of female fruit flies. The output is a series of behaviors of pursuing female fruit flies, such as following, singing and smelling. Input and output are often easy to control and observe, and the nervous system is like a black box. We only know its input and output, but we know nothing about its working mechanism.
In order to find out the truth of neural circuits, biology has accumulated a lot of methods to solve the following questions: What are the components of a neural circuit? What is the function of each component? How are these functions realized?
To analyze the components of neural circuits and their functions, that is, to distinguish the parts related to a specific function from all 1000 billion neurons in the brain. This requires us to correspond some characteristics of neurons with some functions of the nervous system. What are the characteristics of neurons? Morphology is not enough to distinguish different circuits (although sometimes appearance features are useful, such as dark dopaminergic neurons in substantia nigra of midbrain). Generally speaking, the highest resolution has two characteristics: ① the temporal and spatial characteristics of gene expression, which is the fundamental reason for the differences between any cells in an individual; ② Temporal and spatial characteristics of action potential release and synaptic transmission are the basis for the nervous system to function. For the characteristics of gene expression, we can use molecular biology/genetics to detect; We have electrophysiological methods to detect the release of action potential.
Next, how do we relate features to specific functions of the nervous system? In order to explain it more vividly, we take the courtship behavior of fruit flies as an example. One way of thinking is as follows: ① Observing the brain of Drosophila during courtship and seeing which neurons are active in turn, we can simply think that these neurons are related to courtship. The problem of this idea is how to record the activity of each neuron in the brain with high temporal and spatial resolution in living fruit flies-this is obviously unrealistic, but we can use various technologies to approach this goal, which will be described later. Another idea is to inhibit or enhance the function of some neurons, and then observe how the courtship behavior of fruit flies is affected-whether it is enhanced (like Teddy in estrus) or weakened (dismissing sexy female fruit flies). This method does not need real-time recording, but only needs to observe the behavior. Its problem lies in how to inhibit or enhance the function of specific parts of neurons.
Let's talk about the second way of thinking. When we unplug the network card, the computer can't surf the Internet, so we think that the function of the network card is to connect the network. Similarly, the brain can be studied in the same way. In the mid-20th century, a patient named H.M. was surgically removed from the hippocampus, and it was difficult to form a new (declarative) memory, which was very helpful for the scientific community to understand the memory-related circuits. However, the resolution of the operation is limited, and only a part of brain tissue with similar space can be removed. Modern biology can not only change the activity of neurons permanently (from birth to death) by molecular biology/genetics methods, but also operate neurons in real time (from a few seconds to several hours) by chemical control, temperature control and light control.
Gene manipulation is the main method to permanently change the activity of neurons, that is, to reduce the expression of some endogenous genes and enhance the expression of some endogenous (or exogenous) genes. The inhibition of gene expression can be screened by forward genetics or reverse genetics, such as source recombination-mediated knockout and RNAi-mediated knockout. Exogenous genes can be increased by transgene or knockout, while over-expression of endogenous genes can increase the enhancer/promoter regulatory elements upstream of the target gene.
The operation of neurons sometimes needs to be carried out in a specific time and space segment. Double expression systems, such as Cre/LoxP and Gal4/UAS, provide modular and tissue-specific genetic manipulation means, thus improving the spatial resolution of the operation. Virus injection, optogenetics, chemical genetics and other methods provide a means to temporarily change the gene expression characteristics in specific areas, thus improving the time resolution of surgery.
Let's talk about the first idea first. How to observe the activity of neurons in vivo? What is the difference between active neurons and inactive neurons? Active neurons have dense action potentials, a large number of ion channels are open, and there are transmembrane ion currents. Electrophysiological technique recorded the former, and calcium imaging technique (GECI) (the most commonly used GCaMP) recorded the latter. GEVI records the changes of membrane potential, and the time resolution is higher than GECI, but the fluorescence intensity is insufficient. Another problem of the above imaging technology is that the thick brain tissue in the living body is a great challenge to the microscope. At present, the most advanced two-photon technology can only penetrate the tissue of about 500mm and maintain the resolution. In addition, fMRI records the accelerated blood flow near active neurons. Although its spatial resolution is very low, as a non-invasive method, it is of great significance in human brain research.
Through the above two ways of thinking, we often get such a result: "xxx neurons in region A and region B may be related to X behavior, and region C also has some influence on this behavior ...". But how are these areas connected? Where is the boundary of this area? What kinds of neurons are there in each region? The above two experiments are difficult to completely answer these questions. At this time, it is necessary to use anatomical methods to observe the brain statically.
How to mark a specific cell to distinguish it from the surrounding cells? Dyeing is a common method in biology. When GFP is transferred into mice, some specific cell types can be labeled. Marcm (mosaic analysis with repressible cell marker) technology can inactivate the fluorescent pigments of most cells in a cluster of cells of the same species, leaving only a small part with fluorescence, so as to observe the morphology of a single cell more clearly. Photoconversion/switchable green fluorescent protein can mark neurons with light-induced specificity; Campari (calcium-modulated optically movable ratio measurement integrator) can specifically label active neurons, which provides a tool for dynamic recording in vivo.
Besides observing the morphology of individual cells, we also want to know the connections between neurons. GRASP technology and functional maps can test whether there is a connection between two neurons, while transmembrane tracers based on chemicals or viruses can find out the upstream or downstream neurons of a neuron.
The above are the molecular biology methods commonly used in the study of neural circuits.
Network neuroscience. ? Nat neuroscience? 20,? 353–364(20 17)doi: 10. 1038/nn . 4502