| BackgroundBefore the 1990s, it was widely thought that neurons in the adult human brain did not regenerate. Consequently, it was thought that if neurons died during your adult life for any reason (e.g., oxidative stress, stroke, neurodegenerative disease, head trauma, normal aging), they would never be replaced. However, in the 1960s, Altman and colleagues challenged this idea, reporting that new neurons are continuously and spontaneously born in at least two regions of the adult rat brain:the subgranular zone of the hippocampus with cells migrating to the granule layer of the dentate gyrus and the subventricular zone with cells migrating to the olfactory bulb. Due to the longstanding dogma that postnatal neurogenesis is non-existent in mammals, it took more than three decades before the Altman and Das (1965) discovery was broadly accepted.Multi-potent stem cells reside in the subgranular zone of the dentate gyrus and the subventricular zone. These stem cells divide asymmetrically producing one daughter progenitor cell and one stem cell. The progenitor cell can then divide asymmetrically producing daughter cells that differentiate into either astrocytes or neurons and one progenitor cell retaining the capacity to divide multiple times. Studying the growth, survival and differentiation of these new cells often involves exogenous administration of the thymidine analog, bromodeoxyuridine (BrdU) that labels dividing cells. Neurogenesis in adults is dynamically regulated by a number of intrinsic as well as extrinsic factors. Endogenous extrinsic factors in the local microenvironment, often referred to as the "neurogenic niche" or "stem cell niche", include neural precursor cells, surrounding mature cells, cell-to-cell interactions, cilia, secreted factors, and neurotransmitters. Microenvironments of the SVZ and SGZ, but not other brain regions, are thought to have specific factors that are permissive for the differentiation and integration of new neurons, as evidenced by a pivotal study showing that adult hippocampal astrocytes promote neuronal differentiation of adult-derived hippocampal progenitor cells in vitro.Acetylcholine (ACh), as an important neurotransmitter, was the first discovered neurotransmitter. It is involved in many significant functions, such as the regulation of learning, memory, emotion, sleeping and so on. Although the distribution of cholinergic neurons is just in some certain regions, their axons dominate almost the entire brain. For example, there are rare cholinergic neurons in the hippocampus, but it is suffused with cholinergic nerve fibers from the basal forebrain. Many studies have revealed that GABAergic neurons, glutamate neurons and dopaminergic neurons play an important role on the regulation of newborn neurons in the dentate gyrus of the hippocampus, however, little has been reported about the cholinergic neurons and neurogenesis.ObjectiveThe purpose of the present study consists of three main parts:First, we specificly inhibited or activated cholinergic neurons by means of transgenic or optogenetic methods, and then observed the relationship between cholinergic neurons and neurogenesis in the dentate gyrus.Second, exploring the regulatory mechanism of cholinergic neurons on newborn neurons.Third, building the HD 3D image of cholinergic neurons in the whole mouse brain.MethodsThe effect of inhibited cholinergic neurons on the neurogenesis in the dentate gyrus:In order to specifically inhibit the cholinergic neurons of mouse brain, we created the Kir2.1 transgenic mouse (loxp-stop-loxp-Kir2.1-tdTomato). Then we crossed the Kir2.1 mouse with ChAT-Cre mouse to get the generations (ChAT+/Kir2.1+,ChAT+/Kir2.1-). Next, according to the different modes of administration, we divided the two types of transgenic offspring mice into four groups: ChAT+/Kir2.1+/Tamoxifen, ChAT+/Kir2.1+/Oil, ChAT+/Kir2.1-/Tamoxifen ChAT+/Kir2.1-/Oil. After the full expression of Kir2.1, those mice were intraperitoneally injected with Brdu and counted the number of it in the Id,4d,7d, 14d,28d, and 42d. Last, making the statistical analysis of the number of Brdu among every group in each time point.Observing the reverse effect of newborn neurons after the cholinergic neurons in the basal forebrain were activated:We mated the ChAT+/Kir2.1+ mice with ChR2 mice to get the offspring mice (ChAT+/Kir2.1+/ChR2+, ChAT+/Kir2.1+/ChR2-). Fibers were embedded in the basal forebrain after the Tamoxifen induce. Then, these mice were intraperitoneally injected with Brdu, and the mice were divided into four groups:ChAT+/Kir2.1+/ChR2+/light, ChAT+/Kir2.1+/ChR2+/no light, ChAT+/Kir2.1+/ChR2-/light, ChAT+/Kir2.1+/ChR2-/no light. After the light treatment from 14d to 27d, their Brdu number on 28d and 42d were counted and the statistical analysis was made.Observe the change of newborn neurons number after the AChRs of newborn neurons in dentate gyrus were blocked:Based on the mice model above, these mice were injected retrovirus-(M1)shRNA before the experiment of light stimulation. Then, we studied the number of Brdu on 28d and 42d just as above. Study the expression of AChRs on the newborn neurons:We injected retrovirus (RV-GFP) into the dentate gyrus of some 1-month-old C57BL/6 mice. Then, we detected the AChRs of newborn neurons on 4d,7d,14d,21d, 28d and 42d by means of electrophysiology in the use of cholinergic receptor inhibitors. At the same time, the immunohistochemistry were also used to detect the AChRs on newborn neurons.The HD 3D reconstruction of cholinergic neurons in a whole mouse brain:We cut the brain of a 3-month-old mouse into sagittal serial sections (16?m). Then all these brain sections were stained by means of ChAT-antibody and were scanned by a large-scan microscope. These large images were aligned by the Photoshop software, and then they were reconstructed into a HD 3D image by the Imaris software. Moreover, the regions we interested were also intercepted to make the HD 3D images.ResultsAt the first four time points (1d,4d,7d and 14d), there were no difference about the number of Brdu among all groups, but the number of Brdu in the guoup of ChAT+/Kir2.1+/Tamoxifen was decreased significantly compared to the other three groups at the last two time points (28d and 42d). After we had given the treatment of light, the number of Brdu in the guoup of ChAT+/Kir2.1+/Tamoxifen became to recovery at the last two time points.In order to further investigate which class (or classes) of AChRs was involved in such regulation, we observed that the newborn neurons at 4d and 7d had no response to antagonists of M-type and N-type. At the 14d,21d and 28d, they had evident response to the antagonists of M-type and M1-type, instead of the antagonists of M2-M5-type and N-type. Then, when the M1 of newborn neurons were blocked, the tendency of recovery disappeared.Moreover, we build the HD 3D image of cholinergic neurons in a whole mouse brain. The distribution of every group of cholinergic neurons was clear in the 3D image, and the number of cholinergic neurons in specific regions was as follows: 11,395 ± 571 cholinergic neurons in the Cpu; 12,117 ± 613 cells in the BFB and 4457 ± 197 cholinergic neurons in the regions of MS and nucleus of the vertical limb and horizontal limb of the diagonal band (V/HDB); 1252±55 cells in the pedunculopontine nucleus (Ch5) and the laterodorsal tegmental nucleus of the rostal brainstem (Ch6); and 2212 ± 103 cells in the facial nucleus of the brainstem and 307 ± 25 cells in the dorsal motor nucleus of the brainstem. Furthermore, the number of all the cholinergic neurons in the central nervous system, except the cortex, was less than 40,000 in the 3-month-old male C57BL/6 mouse, but the cholinergic fibers were found to be widely distributed in nearly all the regions of the brain, which suggests that each of the cholinergic neuron is greatly significant because it dominates a wide area.DiscussionCholinergic neurons in the basal forebrain might regulate the survival but not the genesis and differentiation of newborn neurons in the dentate gyrus by M1 receptor, which perhaps provide an effective avenue for the treatment of memory deficits. |
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