A key feature of the mammalian brain is its capacity to adapt in response to experience, in part by remodeling of synaptic connections between neurons. Excitatory synapse rearrangements have been monitored in vivo by observation of dendritic spine dynamics, but lack of a vital marker for inhibitory synapses has precluded their observation. Here, we simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbor of pyramidal neurons in the adult mammalian cortex using large volume high-resolution dual color two-photon microscopy. We find that inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience. Further, we find inhibitory synapse and dendritic spine remodeling to be spatially clustered, and that clustering is influenced by sensory input. Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.

While inhibition has been implicated in mediating plasticity in the adult brain, the mechanism remains unclear. Here we present a structural mechanism for the role of inhibition in experience-dependent plasticity. Using chronic in vivo two-photon microscopy in the mouse neocortex we show that experience drives structural remodeling of superficial layer 2/3 interneurons in an input- and circuit-specific manner, with up to 16% of branch tips remodeling. Visual deprivation initially induces dendritic branch retractions accompanied by loss of inhibitory inputs onto neighboring pyramidal cells. The resulting decrease in inhibitory tone, also achievable pharmacologically by the antidepressant fluoxetine, provides a permissive environment for further structural adaptation, including addition of new synapse bearing branch tips. Our findings suggest that therapeutic approaches that reduce inhibition, when combined with an instructive stimulus, could facilitate restructuring of mature circuits impaired by damage or disease, improving function and perhaps enhancing cognitive abilities.

Summary Older concepts of a hard-wired adult brain have been overturned in recent years by in vivo imaging studies revealing synaptic remodeling, now thought to mediate rearrangements in microcircuit connectivity. Using three-color labeling and spectrally resolved two-photon microscopy, we monitor in parallel the daily structural dynamics (assembly or removal) of excitatory and inhibitory postsynaptic sites on the same neurons in mouse visual cortex in vivo. We find that dynamic inhibitory synapses often disappear and reappear again in the same location. The starkest contrast between excitatory and inhibitory synapse dynamics is on dually innervated spines, where inhibitory synapses frequently recur while excitatory synapses are stable. Monocular deprivation, a model of sensory input-dependent plasticity, shortens inhibitory synapse lifetimes and lengthens intervals to recurrence, resulting in a new dynamic state with reduced inhibitory synaptic presence. Reversible structural dynamics indicates a fundamentally new role for inhibitory synaptic remodeling – flexible, input-specific modulation of stable excitatory connections.

We became aware from a reader that the data graphed in Figure 2E resembled the data graphed in 2F, although the legend indicated otherwise. We investigated and found that panel 2E was indeed a placeholder that was prepared with the data from 2F. Unfortunately, in one of the revisions, we inserted the placeholder rather than the correct final panel. All of the data and statistics we report in the manuscript text and legend are based on the true figure, so these remain correct as published. We are grateful to the reader who brought this unfortunate mistake to our attention and apologize for any confusion caused to our colleagues in the community. The correct panel E is shown below.

The aim of the study was to evaluate the effect of sex and age on the thickness of the retinal layer in normal eyes using spectral-domain optical coherence tomography (SD-OCT).Fifty healthy subjects between the ages of 20 and 80 had their retinal layers measured using SD-OCT at Seoul St. Mary's Hospital. Mean thickness and volume were measured for 9 retinal layers in the fovea, the pericentral ring, and the peripheral ring. The differences of sex- and age-related thickness and volume in each retinal layer were analyzed.The retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL) were thinnest in the fovea area, whereas the outer nuclear layer (ONL), photoreceptor layer (PHL), and retinal pigment epithelium (RPE) were thickest at similar locations. Mean thickness of the RNFL, GCL, IPL, and OPL was significantly greater in men than women. However, mean thickness of the ONL was greater in women than in men. When compared between patients < 30 years and > 60 years of age, the thickness and volume of peripheral RNFL, GCL, and pericentral and peripheral IPL were significantly larger in the younger group than the older group. Conversely, the thickness and volume of foveal INL and IR were larger in the older group than in the younger group.The thickness and volume of the retinal layer in normal eyes significantly vary depending on age and sex. These results should be considered when evaluating layer analysis in retinal disease.

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

Abstract. The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude. 291-295 (1996). 6. K. König, P. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, "Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress," J. Microsc. 183(Pt 3), 197-204 (1996). 7. J. R. Lakowicz, "Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching," Scanning Microsc. Suppl. 10, 213-224 (1996 high-resolution measurements reveal a lack of correlation," Nat. Med. 3(2), 177-182 (1997). 14. I. P. Torres Filho, M. Leunig, F. Yuan, M. Intaglietta, and R. K. Jain, "Noninvasive measurement of microvascular and interstitial oxygen profiles in a human tumor in SCID mice," Proc. Natl. Acad. Sci. U.S.A. French, "Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source," Opt. Lett. 32(23), 3408-3410 (2007). 32.