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A research in Zhejiang University may promote the autism treatment

Edit:0916032 Date:2018-03-08

The film "Ocean Paradise" strikes a deep chord in all of our hearts and arouses the public's attention to autism. Autism is a developmental disorder characterized by troubles with social interaction and communication, and by restricted and repetitive behavior. To date, there is no effective treatment for this disease, and the effectiveness of clinical practice training and rehabilitation methods is undetermined.

On March 1st, the top journal of neuroscience, Neuron, published a study on social behaviors of an autistic mouse model entitled "Gamma Oscillation Dysfunction in mPFC Leads to Social Deficits in Neuroligin 3 R451C Knockin Mice", whose work was done by the team led by Professor Jianhong Luo from Zhejiang University.
The results of this study suggest that abnormal brain oscillations of a specific pattern in the medial prefrontal cortex (mPFC) causes social disorders in mice with autism, which can be restored during adulthood by manipulating specific types of neurons (PV-positive interneurons) in the mPFC. This finding may also apply to other models of autism and suggest the feasibility of treating adult patients with autism. This discovery brings new ideas to Autism treatment.
This four-and-half-a-year research was conducted by co-first authors Dr. Wei Cao and Dr. Shen Lin and co-corresponding authors Professor Jianhong Luo and associate Professor Junyu Xu of Zhejiang University School of Medicine.

Schematic diagram of autistic mice before and after treatment:

The image, in the style of Chinese paper-cut, shows two states of autistic mice before and after optogenetic therapy. On the left, the mice can’t communicate successfully and feel lost due to the abnormal oscillations during social interaction. However, when the mice are treated with patterned optogenetic stimulation, the happy chat and dance scene is shown on the right. The notes represent the oscillations in the mPFC, and the notes on the right emphasis on theta-gamma rhythm.

The study found that autistic phenotype of NL3 R451C KI mouse was derived from decreased excitability of PV interneurons. 
The online "Mendelian Genetics" database shows that changes in nearly 30 core genes can cause autism whose functions are mostly associated with synaptic function. Synapses, as the basic structure of neuron communication, help to build a neural network of connecting neurons.
Prof. Jianhong Luo explained: "Neuroligin (NL) is a cell-adhesion molecule that helps to "stick" the synaptic structure together and allows the normal information transmission between neurons. Mutations in this gene may lead to autism, so the study started with an autistic mouse model of the NL3 gene mutation. "
●How to measure the social behavior of mice?
Social interaction refers to the social intercourse between people and is a way that people use to convey information, exchange ideas, to achieve a certain purpose in all social activities. Mouse is a gregarious animal whose social behavior is conducted by mutual sniffing, chasing and so on.
The team of Prof. Jianhong Luo’s used three-chamber sociability and social novelty test to measure the number of sniffing and the cumulative duration in per unit time, and then evaluated the social disorder in autistic mice.

In the third phase, KI mice did not show a preference for new stranger 2, namely, social novelty defects.

●Experiment 1: The medial prefrontal cortex was found to be related to social behavior.
Specific behaviors are dominated by specific neuronal activity in the brain, leaving specific markers within the cell after neuronal activity, allowing scientists to track these markers and establish their association with specific behaviors.
The group assumed that when mice are socializing, neurons in one part of the brain issue social commands. By looking back at molecular traces and comparing the differences between normal mice and autistic mice, the team found abnormalities in the medial prefrontal cortex in the autistic mice.
Left: Experimental design of secial behavior and molecular markers detection. Right: Molecular markers of mPFC regions of WT and KI mice in a social novelty group.

●Experiment 2: The social deficit of KI mice was found to be associated with abnormalities of PV ihterneurons in mPFC.
Following the identification of the associated brain regions, the next step is to locate the smaller structural units, finding the root that causes of the abnormalities at the cellular level and exploring the effectiveness of genetic variation on cellular function.
The mPFC tissue has two main types of neurons: the excitatoty neurons and inhibitory interneurons, connected to form a functional network. The team found that there was a problem within PV positive interneurons. PV interneurons is representatives of inhibitory interneurons that represent a subpopulation of high-metabolizing and high-electrical activity, which are more widely distributed and are the cellular basis of low-frequency gamma-oscillation.
"It took a long time to find and confirm that the defect comes from the PV interneurons. The autonomic neurons in the autistic mice have reduced excitability and are not easily discharged, and the research group has spent." Prof. Jianhong Luo said, "It is very time-consuming to find out exactly the exact component with problem in the integrated circuit system, and it is necessary to do a variety of exquisite electrophysiological experiments to confirm a certain kind of neuronal dysfunction.”
The action potential number of PV interneurons (fast-spiking) that responsed to the same depolarizing current was decreased, i.e. the excitability decreased.

●Experiment 3: decreased excitability of PV neurons resulted in the abnormality of low frequency gamma oscillation.
Previous studies have shown that electroencephalography (EEG) is a macroeconomic form of local neuronal discharge that is closely related to the performance of specific functions in a given brain region, which in turn triggers social behavior. Normal mice in the social process, mPFC the energy of low frequency gamma oscillations obviously became high, and the main neuron discharge time is obviously matched. Obviously, this is a mechanism by which rhythm brain electricity triggers the encoding of social information, called "social rhythm."
The team analyzed the mPFC activity of mice by in-vivo recording and found that the amplitude of low-frequency gamma oscillations significantly decreased, and the loss of the phase coupling when coupled, and the main neuron discharge time was unmatched, all suggest abnormalities in the integration and processing of information in the medial prefrontal cortex of autistic mice. Pro. Jianhong Luo explained: "It is as if the command that issued a social order of their pace of work has been disrupted, and the order is also chaotic."
"The generation of low-frequency gamma oscillations often prompts local synchronized PV neurons to discharge synchronously," said Prof. Jianhong. "The research team concluded that when the PV neurons were abnormal, the discharge of the neurons in the mice was disturbed and the gamma band oscillation is impaired, leading to abnormal social behavior in mice."

The study on autistic mice likely mirrors what happened in human autism 
Optogenetics generally refers to the technology that combines optical and genetic approaches to precisely control the activity of specific neurons. By controlling the wavelength, time, frequency and other parameters of light, scientists can control the activation and deactivation of light-sensitive channel proteins on cells so as to effectively activate or inhibit neuronal activity, and precisely control neurons to perform activities at specific frequencies.

●Experiment 4: Optogenetics validates specific brain regions and specific types of neurons
After found PV neurons paly a key role in the disease of autistic mouse model of NL3 gene mutation, the group stimulated the neurons in the medial prefrontal cortex by optogenetics, according to the low-frequency gamma / theta oscillation pattern, and the mice were effectively rescued, as if "social rhythm" was opened using the "information key." The team further validated the social behavior of mice in the prefrontal cortex and PV neurons by using optogenetics. It is worth mentioning that it is a unique discovery that only using the right combination of frequencies to stimulate PV cells can effectively restore social function!
The prefrontal cortex was injected with virus expressing light-sensitive channels in PV interneurons and embedded in optical fiber. After recovery, the mice was tested in three-chamber social experiment with different stimulating patterns.

The research team said that according to the aforementioned discovery, it is possible to treat autism by precisely regulating the activity of specific neurons, and because the experimental model mice are already mature, the method can still have a therapeutic effect on adult autistic animals. The team believes that if there is a similar mechanism of human autism, we can use physical or medical means to improve the function of human prefrontal PV neurons to reconstruct the low frequency oscillation of the prefrontal cortex to achieve the purpose of improving the social skills of patients with autism. Social deficits, as a central symptom, the improvement of the defects may also have positive implications for the intellectual and verbal development of children with autism.
An anonymous review expert said the novelty of the study is based on the found that a particular cortical region is associated with a specific behavior and that the authors found that the gamma oscillations in the region and the PV interneurons are abnormal and thus provide behavioral, Links between changes in the network and autism-related mutations.

●Why does the research resluts of autism mice can be suitable for human autism?
This is because the basis of the study was to integrate the neuronal-linked gene mutations identified in autism patients into a mouse model by genetic engineering techniques, which also show behavior similar to autism and exist Social barriers.
In short, the basic message of genetic defects in mice comes from people with autism. Like humans, mice also have the NL3 gene, which has similar functions in neurodevelopment, and this is the corollary of the scientific basis.
Prof.Jianhong Luo said scientists studying animal models of disease seem to use a mirror to reflect the pathogenesis of human disease. But because of evolutionary differences, whether the conclusions of high-level function of the mouse model brain is applicable to humans also need more research to verify.
The study is mainly supported by major program key projects of "the foundation of the neural circuits of emotion and memory ", innovative team projects and surface projects of the National Natural Science Foundation of China.