Activity of estrogen receptor ß expressing neurons in the medial amygdala regulates preference toward receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference toward the odor of sexually receptive (RF) over nonreceptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERß) expressed in the medial amygdala (MeA) in male preference toward RF. To further delineate the role played by ERß in the MeA in the neuronal network regulating male preference, we developed a new ERß-iCre mouse line using the CRISPR-Cas9 system. Fiber photometry Ca2+ imaging revealed that ERß-expressing neurons in the postero-dorsal part of the MeA (MeApd-ERß+ neurons) were more active during social investigation toward RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERß+ neuronal activity abolished a preference to RF in "RF vs. XF," but not "RF vs. IM," tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERß+ neurons. Fiber photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERß+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERß+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs. XF) but not sex-based preference (i.e., RF vs. IM) in male mice.

Limb-clasping, cognitive deficit and increased vulnerability to kainic acid-induced seizures in neuronal glycosylphosphatidylinositol deficiency mouse models.

Posttranslational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, >150 human GPI-anchored proteins have been discovered and ~30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from inherited glycosylphosphatidylinositol deficiency (IGD) with intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko) or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus.

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme CaMKII plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on runaway synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

LOV2-based photoactivatable CaMKII and its application to single synapses: Local Optogenetics.

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.

In vivo three- and four-photon fluorescence microscopy using a 1.8 µm femtosecond fiber laser system.

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 ∼150 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 ∼1 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.

Imaging single CaMKII holoenzymes at work by high-speed atomic force microscopy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic plasticity. It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.

Imaging intracellular protein interactions/activity in neurons using 2-photon fluorescence lifetime imaging microscopy.

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.

Chronic neuronal excitation leads to dual metaplasticity in the signaling for structural long-term potentiation.

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.