RESUMO
Multiphoton microscopy has enabled us to image cellular dynamics in vivo. However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 0.65-1.04 µm. Here we develop a femtosecond fiber laser system that produces â¼150 fs pulses at 1.8 µm. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 µm using a Raman shift fiber. The 1.8 µm pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is â¼1 µJ, sufficient for in vivo imaging. We successfully observe TurboFP635-expressing cortical neurons at a depth of 0.7 mm from the brain surface by three-photon excitation and Clover-expressing astrocytes at a depth of 0.15 mm by four-photon excitation.
RESUMO
Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 µm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.
RESUMO
Noxious stimuli on the colorectum cause colorectal contractions through activation of descending monoaminergic pathways projecting from the supraspinal defecation center to the spinal defecation center. Since it is known that substance P is involved in the response to peripheral noxious stimuli in the spinal cord, we investigated the effects of intrathecally administered substance P at L6-S1 levels on colorectal motility in rats that were anesthetized with α-chloralose and ketamine. Intrathecally administered substance P enhanced colorectal motility, even after transection of the thoracic spinal cord at the T4 level. Severing the pelvic nerves, but not the colonic nerves, abolished substance P enhanced colorectal motility. In the spinal cord at L6-S1 levels, expression of mRNA coding neurokinin (NK) 1-3 receptors was detected by RT-PCR. Immunohistological experiments revealed that preganglionic neurons of the pelvic nerves express NK1 receptors, whereas expression of NK2 receptors was not found. In addition, substance P-containing fibers densely innervated around the preganglionic neurons expressing NK1 receptors. An intrathecally administered NK1 receptor antagonist (spantide) attenuated capsaicin-induced colorectal contractions. These results suggest that the colokinetic action of substance P is mediated by the NK1 receptor in the spinal defecation center. Our findings indicate that substance P may function as a neurotransmitter in the spinal defecation center.NEW & NOTEWORTHY We found that intrathecally administered substance P enhanced colorectal motility in anesthetized rats. Neurokinin (NK) 1 receptors, but not NK2 receptors, were detected in preganglionic neurons of the pelvic nerves. Blockade of NK1 receptors in the spinal cord attenuated the enhanced colorectal motility in response to intracolonic noxious stimuli. The findings indicate that substance P may function as a neurotransmitter in the spinal reflex pathway controlling defecation.
Assuntos
Neoplasias Colorretais , Defecação , Animais , Defecação/fisiologia , Motilidade Gastrointestinal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1 , Medula Espinal/fisiologia , Substância P/farmacologiaRESUMO
Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of long-term potentiation (LTP), known as metaplasticity. However, the metaplastic regulation of structural LTP (sLTP) remains unclear. We investigate glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAA receptor antagonists. We find that the neuronal excitation decreases the glutamate uncaging-evoked Ca2+ influx mediated by GluN2B-containing NMDA receptors and suppresses sLTP induction. In addition, single-spine optogenetic stimulation using paCaMKII indicates the suppression of CaMKII signaling. While the inhibition of Ca2+ influx is protein synthesis independent, the paCaMKII-induced sLTP suppression depends on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., dual inhibition of Ca2+ influx and CaMKII signaling). This dual inhibition mechanism may contribute to robust neuronal protection in excitable environments.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Região CA1 Hipocampal/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Espinhas Dendríticas/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de GABA-A/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Through the decades, 2-photon fluorescence microscopy has allowed visualization of microstructures, such as synapses, with high spatial resolution in deep brain tissue. However, signal transduction, such as protein activity and protein-protein interaction in neurons in tissues and in vivo, has remained elusive because of the technical difficulty of observing biochemical reactions at the level of subcellular resolution in light-scattering tissues. Recently, 2-photon fluorescence microscopy combined with fluorescence lifetime imaging microscopy (2pFLIM) has enabled visualization of various protein activities and protein-protein interactions at submicrometer resolution in tissue with a reasonable temporal resolution. Thus far, 2pFLIM has been extensively applied for imaging kinase and small GTPase activation in dendritic spines of hippocampal neurons in slice cultures. However, it has been recently applied to various subcellular structures, such as axon terminals and nuclei, and has increased our understanding of spatially organized molecular dynamics. One of the future directions of 2pFLIM utilization is to combine various optogenetic tools for manipulating protein activity. This combination allows the activation of specific proteins with light and visualization of its readout as the activation of downstream molecules. Here, we have introduced the recent application of 2pFLIM for neurons and present the utilization of a new optogenetic tool in combination with 2pFLIM.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica , Neurônios , Hipocampo , Microscopia de Fluorescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/metabolismo , Transdução de SinaisRESUMO
Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Potenciação de Longa Duração/fisiologia , Optogenética/métodos , Sinapses/metabolismo , Animais , Eletrofisiologia , Feminino , Células HeLa , Hipocampo/metabolismo , Hipocampo/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinapses/fisiologiaRESUMO
The central nervous system is involved in regulation of defaecation. It is generally considered that supraspinal regions control the spinal defaecation centre. However, signal transmission from supraspinal regions to the spinal defaecation centre is still unclear. In this study, we investigated the regulatory role of an anorexigenic neuropeptide, α-MSH, in the spinal defaecation centre in rats. Intrathecal administration of α-MSH to the L6-S1 spinal cord enhanced colorectal motility. The prokinetic effect of α-MSH was abolished by severing the pelvic nerves. In contrast, severing the colonic nerves or thoracic cord transection at the T4 level had no impact on the effect of α-MSH. RT-PCR analysis revealed MC1R mRNA and MC4R mRNA expression in the L6-S1 spinal cord. Intrathecally administered MC1R agonists, BMS470539 and SHU9119, mimicked the α-MSH effect, but a MC4R agonist, THIQ, had no effect. These results demonstrate that α-MSH binds to MC1R in the spinal defaecation centre and activates pelvic nerves, leading to enhancement of colorectal motility. This is, to our knowledge, the first report showing the functional role of α-MSH in the spinal cord. In conclusion, our findings suggest that α-MSH is a candidate for a neurotransmitter from supraspinal regions to the spinal defaecation centre.