Three-photon fluorescence microscopy (3PM) has driven rapid progress in deep-tissue imaging beyond the depth limit of two-photon microscopy, with impacts in neuroscience, immunology, and cancer biology. Three-photon excitation places a premium on ultrashort pulses with high peak power in the 1300- and 1700-nm wavelength bands, which allow deepest imaging. The inefficiency and cost of current sources of these pulses present major barriers to the use of 3PM in biomedical research labs. Fiber sources of such pulses could potentially alleviate these problems, but the peak-power limitations of optical fibers have limited their use in 3PM. Here, we describe a fiber-based source of femtosecond pulses with multi-megawatt peak power. Femtosecond pulses at 1030 nm are launched into an antiresonant hollow-core fiber filled with argon. By varying only the gas pressure, pulses with hundreds of nanojoules of energy and sub-100 fs duration are obtained at wavelengths between 850 and 1700 nm. This approach is a new route to an efficient and potentially low-cost source for deep-tissue imaging. In particular, 960-nJ and 50-fs pulses are generated at 1300 nm with a conversion efficiency of 10%. The nearly 20-MW peak power is an order of magnitude higher than the previous best from a femtosecond solid-core fiber source at 1300 nm. As an example of the capabilities of the source, these pulses are used to image structure and neuronal activity in a mouse brain as deep as 1.1 mm below the dura.

We apply adaptive excitation to a fast two-photon microscope to image voltage activity in multiple neurons simultaneously at > 600 μm depth in awake mouse brains.

In her book, A Room of One’s Own, the famous author Virginia Woolf writes “One cannot think well, love well, sleep well if one has not dined well”. This is true. All animals need to forage for food and consume specific nutrients to maintain their physiological homeostasis, maximize their fitness and their reproduction. After decades of research in humans and many model organisms, we now know that our brain is one of the key players that control what, when, and how much we eat. In this review, we discuss the recent literature on neural control of food intake behaviors in mice and flies with the view that these two model organisms complement one another in efforts to uncover conserved principles brains use to regulate energy metabolism and food ingestion.

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.

Neurotrophin receptors regulate neuronal survival and network formation, as well as synaptic plasticity in the brain via interaction with their ligands. Here, we examined early changes in the expression of neurotrophin receptor genes Ntk1 (TrkA), Ntrk2 (TrkB), Ntrk3 (TrkC), Ngfr (p75NTR) and miRNAs that target theses gens in the mouse brain after induction of seizure activity by pentylenetetrazol. We found that expression of Ntrk3 and Ngfr was upregulated in the cortex and the hippocampus 1-3 hours after the seizures, while Ntrk2 expression increased after 3-6 hours in the anterior cortex and after 1 and 6 hours in the hippocampus. At the same time, the ratio of Bcl-2/Bax signaling proteins increased in the anterior and posterior cortex, but not in the hippocampus, suggesting the activation of anti-apoptotic signaling. Expression of miRNA-9 and miRNA-29a, which were predicted to target Ntrk3, was upregulated in the hippocampus 3 hours after pentylenetetrazol injection. Therefore, early cellular response to seizures in the brain includes induction of the Ntrk2, Ntrk3, Ngfr, miRNA-9, and miRNA-29a expression, as well as activation of Bcl-2 and Bax signaling pathways, which may characterize them as important mediators of neuronal adaptation and survival upon induction of the generalized brain activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure‐induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3 hr after pentylenetetrazole seizures. Such early activity‐induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription–translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate‐early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6 hr following seizures, urokinase and plasminogen activator inhibitor‐1 gene expression showed a delayed activation only at 3 days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity‐dependent neural tissue remodeling in pathological and normal conditions.

Neuronal encoding of environmental information has long been in the focus of neuroscience. Neurophysiological studies show that when animals become familiar with space or objects, neuronal activity changes due to these factors. However, despite a considerable amount of experimental data, the specific neural bases of space and objects encoding, and the nature of their disturbance in aging or pathology remains an open question. In this work, we test the hypothesis that neurons in different regions of the brain, hippocampus and retrosplenial cortex (RSC) encode spatial and object information respectively, and that natural aging impairs these two forms of memory differently. To test this hypothesis, we developed an approach combining optical recording of neuronal activity and assessment of animal behavior during novel object recognition (NOR) and object place recognition (OPR) tasks, as well as cognitive enhancement in young and old animals. First, we examined what forms of memory are affected in old mice, and whether it is possible to restore the age‐impaired memory pharmacologically. We trained young (2–3 months) and old (18–21 months) mice in NOR and OPR tasks and then tested short‐term 120 min memory and long‐term 24 h memory. We showed that both young and old mice demonstrate short‐term NOR and OPR memories, and that P2 cognitive enhancer that belongs to a new class of sAPP‐mimetics did not potentiate this memory. On the other hand, both old and young mice failed to form NOR and OPR long‐term memories. Interestingly, administration of P2 produced long‐term memory manifestation only in the old mice.Next, we recorded the object‐type and object‐place related calcium activity in the RSC and hippocampus CA1 of young mice using genetically encoded YtnC sensor and fiber‐optic photometry (Fig. 1, A). We found an increase in the number of calcium events in the RSC when animals examined new position of the familiar object. We also discovered decrease in the RSC activity during exploration of a novel object.Using multiphoton microscopy and NVista minimicroscopy we registered GCaMP6 calcium activity of identified RSC (Fig. 1, B) and CA1 (Fig. 1, C) neurons in young mice during NOR and OPR tasks performed in the Mobile HomeCage. Different classes of neurons were identified – those demonstrating an increase and decrease of activity during place and objects exploration, as well as cells that were active during specific behavioral acts.Thus, we showed that both object‐type and object‐place memory was specifically expressed in the activity of RSC and CA1 neurons. Both object‐type and object‐place long‐term memories were missing in the old mice but were brought to the surface by administration sAPP‐mimetic cognitive enhancer. In the future, we will test how memory‐related brain activity changes in NOR and OPR tasks in old animals, and whether these changes in neuronal activity could be compensated pharmacologically.Support or Funding InformationSupported by RSF 20‐15‐00283 and RFBR 17‐00‐00215, 19‐315‐80020, 17‐29‐07083, 18‐32‐20212Neuronal calcium activity in novel object recognition and object place recognition tasks. A: fiber‐optic photometry of YtnC calcium signal from retrosplenial cortex (RSC) and hippocampus CA1; red line – baseline. B: two‐photon microscopy of identified GCaMP6S neurons in the retrosplenial cortex; asterisks show calcium events – spikes. Ñ: NVista minimicroscopy of GCaMP6S neurons in hippocampus CA1; arrows show calcium spikes.Figure 1

The parietal cortex in rodents has an integrative function and participates in sensory and spatial processing, movement planning and decision-making. However, much less is known about its functions in associative memory processing. Here using Fos immunohistochemical mapping of neuronal activity and two-photon imaging in Fos-eGFP mice we show an involvement of anterior part of the parietal cortex (PtA) in the formation and retrieval of recent fear memory in mice. Using ex vivo c-fos imaging we demonstrate the specific activation of the PtA during recent memory retrieval. In vivo two-photon c-fos imaging confirms these results as well as establishes the activation of the PtA neurons during fear memory formation. Additionally, we describe a design of Fos-Cre-GCaMP transgenic mice to investigate long-term changes of calcium dynamics in neurons captured with Fos-TRAP technique during fear conditioning training.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

In this study, we describe use of Cre-mediated recombination to obtain a permanent genetic labeling of the brain neuronal networks activated during a new experience in animals. This method utilizes bitransgenic Fos-Cre-eGFP mice in which a green fluorescent protein is expressed upon tamoxifen-induced Cre-recombination only in the cells where immediate early gene c-fos expression takes place due to the new experience. We used the classical fear conditioning model to show that ex vivo microscopy of the eGFP protein in Fos-Cre-eGFP mice enables mapping of the neurons of the various brain regions that undergo Cre-recombination during acquisition of a new experience. We exposed the animals to the new environment in brief sessions and demonstrated that double immunohistochemical staining enables a characterization of the types of neocortical and hippocampal neurons that undergo experience-dependent Cre-recombination. Notably, Fos-Cre-eGFP labeled cells appeared to belong to excitatory pyramidal neurons rather than to various types of inhibitory neurons. We also showed that a combination of genetic Cre-eGFP labeling with immunohistochemical staining of the endogenous c-Fos protein allows one to identify and compare the neuronal populations that are activated during two different episodes of new experiences in the same animal. This new approach can be used in a wide spectrum of tasks that require imaging and a comparative analysis of cognitive neuronal networks.