A Preliminary Study Of Optogenetics In Classical Eyeblink Conditioning

The history of classical conditioning can goes back to one hundred years ago, and one of the most simple is eyeblink conditioning. It is also the most widely used model of associative learning and memory. Eyeblink conditioning is divided into two models:delay & trace. In general, the peripheral stimulus (such as visual or auditory stimulus) serve as the conditioned stimulus to establish a model of classical eyeblink conditioning; At the same time, we found some reports indicated that using electricity to stimulate local brain regions (or nuclei) of peripheral audio-visual signal upload pathway (relay) also can do it. Existing data suggest that the prefrontal cortex is involved in the establishment or expression of eyeblink conditioning (delay pattern)in some cases, it is a challenge to the traditional conclusion:The prefrontal cortex is not involved in the delay pattern. Thus, we are also very interested in it. why does the delay pattern requires the prefrontal cortex in some special cases? Because the glutamatergic neurons are wide distribution in the prefrontal cortex, so we chose the glutamatergic neurons as the target. In particular,we want to explore the neural mechanism that glutamatergic neurons are involved in delay eyeblink conditioning.Optogenetics is a revolutionary technology. Optical channel sensitive genes are packaged to the related viruses by using the genetic engineering technique, and then,the channel proteins are transfected into the specific neurons, realizing the expression of channel proteins in the cell membrane of the specific neurons and the millisecond regulation of the certain cells or neurons on the basis of the different characteristics of various photosensitive proteins(such as Chr2).Because of the unique advantages of optogenetics, we can use it to prove the hypothesis of delay requiring glutamatergic neurons in the prefrontal cortex in some cases. Through retrieval in the pubmed, we don’t found the report of serving light activing certain neurons in the brain as CS to establish the EBC model. Thus, we realized the specific manipulation of glutamatergic neurons in the medial prefrontal cortex by optogenetics, and serve light activing the neurons as CS to verify the validity of using this way to establish the EBC model and study the intrinsic neural mechanismsMaterials and methods:We microinjected AAV2/8-CaMKⅡα-ChR2-mCherry into the rat medial prefrontal cortex by using stereotaxie apparatus, after 3-4 weeks, use fluorescence microscopy and confocal laser scanning microscopy to determine whether the neuron membrane surface have the expression of red fluorescent protein and the position is in mPFC, the specificity of expression of photosensitive gene in glutamatergic neurons. We observe electrophysiological characteristics of the glutamatergic neurons expressed with AAV2/8-CaMKIIa-ChR2-mCherry in light stimulations of different frequencies to understand the physiological function of the ChR2 channel, laying a solid foundation for further behavior studies. On the other hand, we complete the behavior experiment by manipulating ChR2 channels expressed in glutamatergic neurons in free activities animal mPFC:we microinjected virus AAV2/8-CamK Ⅱα-ChR2-mCherry into the right side of rat mPFC, after 4 weeks, establish optical-neural interfaces through operation and obligate the hole for injecting drugs. We serve the blue light (470 nm,10 mW/mm2, 30 ms wave width,20 Hz mPFC) activing glutamatergic neurons as CS and electricity stimulating the orbicularis oculi muscles as US to observe whether it can establish delay-EBC; After establishing it, we inject the Short-term deactivation drug muscimol (GABA-A receptor agonist, chloride ion channel opening lead to large chloride ions into cells and make cells inactivate), into the position of lighting in mPFC by the micro injection technology, observing the effect to delay-EBC.Results:(1) Virus is injected into mPFC,94.6 ± 0.9%glutamate with CaMKⅡα expression have ChR2-mCherry protein expression,and 98.87+ 0.3% neurons with ChR2-mCherry protein expression are glutamatergic neurons, indicated that In regulation of CamK Ⅱα promoter, adeno- associated virus vector can make photosensitive gene express in glutamatergic neurons, specifically. Glutamate neurons with ChR2-mCherry protein can active to the blue light(470nm,20 mW/mm2, wave width:5 ms,5-80 Hz)stably. Compared to High frequencies, low frequencies (5,10 and 20Hz) have better activations. We can establish eyeblink conditioning by serving the blue light (470 nm,10 mW/mm2,30 ms wave width,20 Hz mPFC) activing glutamatergic neurons as CS,the effect of injecting the short-term deactivation drug muscimol to delay-EBC conditioned reaction is obvious.Conclusion:Successful, we constructed the optogenetics platform for classical eyeblink conditioning. Furthermore, we found that serving specific activation of glutamate neurons in mPFC as CS can establish the delay-EBC, proving the rationality of our hypothesis. Therefore,its significance is not only in the establishment of technical platform,but also in the contribution of further researching the mechanism of the associative learning and memory in free animal.