Tissue-Tissue Interaction-Triggered Calcium Elevation Is Required for Cell Polarization during Xenopus Gastrulation

The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates mechanical forces through cell-cell contact that initiates coordinated cell polarization through a transient increase in intracellular calcium

Modality-Specific Impairment of Hippocampal CA1 Neurons of Alzheimer’s Disease Model Mice

Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, while spatial representation is significantly impaired in the App knock-in mice. This is due to the overall reduction of active place cells but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in hippocampus.SIGNIFICANCE STATEMENT:Spatiotemporal memory impairments are common at the early stage of Alzheimer's disease patients. We demonstrate the different impairment patterns of place and time cells in the dorsal hippocampus of head-fixed App knock-in mouse by in vivo two-photon calcium imaging over months under the virtual reality spatiotemporal tasks. These results highlight that place cells were preferentially and gradually damaged nearby Aβ aggregates, while time cells were less vulnerable. We further show these impairments were due to neuronal hyperactivity that occurs near the Aβ deposition. We suggest the differential and gradual impairment in two major modalities of episodic memory under Aβ pathology

Orchestrated ensemble activities constitute a hippocampal memory engram

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory

The Properties of Specific Binding Site of 1251-Radioiodinated Myotoxin a, a Novel Ca Releasing Agent, in Skeletal Muscle Sarcoplasmic Reticulum1

ABSTRACT ABBREVIATIONS: MYTX, myotoxin a; MBED, 9-methyl-7-bromoeudistomin D; AMP-PCP, adenosine 5'-(j3,-y-methylene)triphosphate; MOPS, 3-(N-morpholino) propanesulfonic acid; SR, sarcoplasmic reticulum; HSR, the heavy fraction of fragmented SR; V, peak change in millivolts. 93

Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans

Abstract Background Tyramine, known as a “trace amine” in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans (C. elegans), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior. Results Using calcium imaging in freely moving C. elegans, we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns. Conclusion We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans

Additional file 1: of Role of tyramine in calcium dynamics of GABAergic neurons and escape behavior in Caenorhabditis elegans

Figure S1. ICaST system for optogenetic control and simultaneous calcium imaging in freely moving animals. The light paths are indicated by colored arrows. Details are described in the Materials and Methods and a previous report [27]. Figure S2. R-CaMP2 imaging in RME. (A) Representative images of a transgenic animal expressing both R-CaMP2 and EGFP in RME neurons in forward (top panels) and backward (bottom panels) movements. Transmitted-light images (TD), raw fluorescent images of R-CaMP2 and EGFP, fluorescent merged images, and pseudocolor ratio images (R-CaMP2/EGFP) are shown. (B) Fluorescent intensity ratio values (R=R-CaMP2/EGFP) of RME in a freely moving animal are plotted as a function of time. (C) Quantitative analysis of mean fluorescent ratio changes of RME during forward (gray) and backward (red) locomotion. The mean value of R during forward locomotion was normalized as 100%. Figure S3. tbh-1 mutants exhibit normal calcium responses in RME during backward locomotion. A representative calcium trace of RME in tbh-1(ok1193) mutants. Figure S4. tph-1 and cat-2 mutants exhibit normal calcium responses in RME during backward locomotion. (A) Biosynthetic pathways of serotonin and dopamine. Genes encoding synthetic enzymes are shown under the arrows. (B, C) Calcium dynamics of RME in tph-1 (mg280) (B) and cat-2 (jq6) (C) mutants during spontaneous locomotion. Table S1. C. elegans strains used in this study. Table S2. Transgenic lines generated in this study. Table S3. Mutations and primers for genotyping. Table S4. Primers for molecular biology. (PDF 359 kb

Electrophysiological properties of hippocampal neurons expressing R-CaMP1.07.

<p><b>A</b>, Average input resistance (left), membrane capacitance (middle), or resting potential (right) did not significantly differ between control and R-CaMP1.07 groups. Error bars, s.e.m. (<i>n</i> = 7 cells each). <i>P</i>>0.05, Student’s <i>t</i>-test. <b>B</b>, Representative spontaneous EPSCs recorded from a control and R-CaMP1.07-expressing cell at a holding potential of −70 mV (left). Cumulative probability of amplitudes of EPSCs is shown in the middle panel (Control, <i>n</i> = 11062 events; R-CaMP1.07, <i>n</i> = 10265 events). <i>P</i>>0.05, Kolmogorov-Smirnov test. Frequency of EPSCs is shown in the right panel. Error bars, s.e.m. (<i>n</i> = 7 cells each). <i>P</i>>0.05, Student’s <i>t</i>-test.</p

Characterization of R-CaMPs <i>in vitro</i> and in HeLa cells.

<p><b>A</b>, Schematic structures of R-CaMPs. Mutations are indicated with respect to R-GECO1. RSET and M13 are a tag that encodes hexahistidine and a target peptide for a Ca²⁺-bound CaM derived from MLCK, respectively. The amino-acid numbers of mApple and CaM are indicated in parentheses. <b>B</b>, Ca²⁺ affinity (<i>K</i>_d) and dynamic range (<i>F</i>_max/<i>F</i>_min). Error bars, s.d. (<i>n</i> = 3 each). <b>C</b>, Ca²⁺ titration curve. Curves were fit according to the Hill equation. The <i>K</i>_d is shown in <b>B</b>. Error bars, s.d. (<i>n</i> = 3 each). <b>D</b>, Normalized fluorescence and absorbance (inset) spectra of R-CaMP1.07 in 1 µM Ca²⁺ or 1 mM EGTA. <b>E</b>, Fluorescence images of HeLa cells expressing red fluorescent GECIs. <b>F</b>, Mean Δ<i>F</i>/<i>F</i> responses to the application of 100 µM ATP in HeLa cells. Error bars, s.d. (<i>n</i> = 163 cells for R-GECO1, 182 cells for R-CaMP1.01 and 166 cells for R-CaMP1.07). <b>G</b>, Baseline fluorescence and peak responses (Δ<i>F</i>/<i>F</i>) to the application of 100 µM ATP in HeLa cells. Error bars, s.d.</p

Simultaneous monitoring and manipulation of neuronal activity by co-expression of R-CaMP1.07 and ChR2.

<p><b>A</b>, A projection image of a CA3 pyramidal cell expressing R-CaMP1.07 and ChR2. The cell was whole-cell recorded to measure the number of APs. <b>B</b>, Imaging of ChR2-triggered-APs by changes in the R-CaMP1.07 fluorescence (top). APs were evoked by a pulse of 470-nm light with a duration of 300 to 3000 ms (blue region indicates time of photostimulation). Putative Ca²⁺ increases during the photostimulation are represented by broken lines (Bottom). The number of APs was recorded using the current-clamp mode. <b>C</b>, Δ<i>F</i>/<i>F</i> amplitude of the R-CaMP1.07 responses as a function of the number of APs induced by photostimulation. The peak Δ<i>F</i>/<i>F</i> amplitudes were calculated from the first frames after termination of the photostimulation. Individual data are plotted as black dots and their averages are shown in red. Error bars, s.e.m. (<i>n</i> = 4 cells).</p