In vivo STED microscopy: A roadmap to nanoscale imaging in the living mouse.

Superresolution microscopy techniques are now widely used, but their application in living animals remains a challenging task. The first superresolution imaging in a live vertebrate was demonstrated with STED microscopy in the visual cortex of an anaesthetized mouse. Here, we explain the requirements for a simple but robust in vivo STED microscope as well as the surgical preparation of the cranial window and the mounting of the mouse in detail. We have developed a mounting stage with a heating plate to keep the mouse body temperature stable and that can be adjusted to the optical axis of the microscope. We have optimised the design to avoid inducing thermal drift, which is critical for nanoscale imaging. STED microscopy with a resolution of 60 nm requires special cranial window preparation to avoid motion artefacts. We have implemented a drain tube to reduce the fluid between the glass window and the surface of the brain, which has been identified as the main cause for the motion artefacts. Together, these advances in the preparation allow the use of a simple intraperitoneal anaesthesia and make the previously used venous infusion and artificial respiration obsolete.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.

Nanoscopy of filamentous actin in cortical dendrites of a living mouse.

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 µm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43-70 nm resolution reveal dynamical changes in spine morphology.

In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice.

The vesicular glutamate transporter VGLUT1 loads synaptic vesicles with the neurotransmitter glutamate and thereby determines glutamate release at many synapses in the mammalian brain. Due to its function and selective localization, VGLUT1 is one of the most specific markers for glutamatergic synaptic vesicles. It has been used widely to identify glutamatergic synapses, and its expression levels are tightly correlated with changes in quantal size, modulations of synaptic plasticity, and corresponding behaviors. We generated a fluorescent VGLUT1(Venus) knock-in mouse for the analysis of VGLUT1 and glutamatergic synaptic vesicle trafficking. The mutation does not affect glutamatergic synapse function, and thus the new mouse model represents a universal tool for the analysis of glutamatergic transmitter systems in the forebrain. Previous studies demonstrated synaptic vesicle exchange between terminals in vitro. Using the VGLUT1(Venus) knock-in, we show that synaptic vesicles are dynamically shared among boutons in the cortex of mice in vivo. We provide a detailed analysis of synaptic vesicle sharing in vitro, and show that network homeostasis leads to dynamic scaling of synaptic VGLUT1 levels.

Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy.

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex. In general, the spine head geometry and PSD95 assemblies were highly dynamic, their changes depended linearly on their original size but correlated only mildly. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more uniformly. The topography of the PSD95 nanoorganization was more dynamic after environmental enrichment; changes in size were smaller but more correlated than in mice housed in standard cages. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.

Fatigability of spinal reflex transmission in a mouse model (SOD1(G93A) ) of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons. To analyze the progressive motor deficits during the course of this disease, we investigated fatigability and ability of recovery of spinal motor neurons by testing monosynaptic reflex transmission with increasing stimulus frequencies in the lumbar spinal cord of the SOD1(G93A) mouse model for ALS in a comparison with wild-type (WT) mice. Monosynaptic reflexes in WT and SOD1(G93A) mice without behavioral deficits showed no difference with respect to their resistance to increasing stimulus frequencies. During the progression of motor deficits in SOD1(G93A) mice, the vulnerability of monosynaptic reflexes to higher frequencies increased, the required time for reflex recovery was extended, and recovery was often incomplete. Fatigability and demand for recovery of spinal motor neurons in SOD1(G93A) mice rose with increasing motor deficits. This supports the assumption that impairment of the energy supply may contribute to the pathogenesis of ALS.

Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins.

Despite the tremendous success of super-resolution microscopy, multi-color in vivo applications are still rare. Here we present live-cell multi-label STED microscopy in vivo and in vitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations in vivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2. Combining the positive and negative switching RSFPs Padron and Dronpa-M159T enables dual-label STED microscopy. All labels are recorded quasi-simultaneously by parallelized on- and off-switching of the RSFPs within the fast-scanning axis. Depletion is performed by a single STED beam so that all channels automatically co-align. Such an addition of a second or third marker merely requires a switching laser, minimizing setup complexity. Our technique enhances in vivo STED microscopy, making it a powerful tool for studying multiple synaptic nano-organizations or the tripartite synapse in vivo.

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity.

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption and the storage of long-term information, spines need to be plastic and stable at the same time. Here, we advanced in vivo STED nanoscopy to superresolve distinct features of spines (head size and neck length/width) in mouse neocortex for up to 1 month. While LTP-dependent changes predict highly correlated modifications of spine geometry, we find both, uncorrelated and correlated dynamics, indicating multiple independent drivers of spine remodeling. The magnitude of this remodeling suggests substantial fluctuations in synaptic strength. Despite this high degree of volatility, all spine features exhibit persistent components that are maintained over long periods of time. Furthermore, chronic nanoscopy uncovers structural alterations in the cortex of a mouse model of neurodegeneration. Thus, at the nanoscale, stable dendritic spines exhibit a delicate balance of stability and volatility.

NO mediates microglial response to acute spinal cord injury under ATP control in vivo.

To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two-photon laser-scanning microscopy (2P-LSM), we identified the messenger nitric oxide (NO) as a modulator of injury-activated microglia. Local tissue damages evoked by high-power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO-donor spermine NONOate (SPNO) or the NO-dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High-tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.

Long-lasting post-mortem activity of spinal microglia in situ in mice.

As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion-directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser-induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post-mortem, in the progressively degrading nervous tissue. For this purpose, we used time-lapse two-photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5-10 hr post-mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post- mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue.

In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex.

The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex. Adult knock-in mice expressing eGFP fused to the endogenous PSD95 protein were imaged at time points from 1 min to 6 h. Superresolved large assemblies of PSD95 show different sub-structures; most large assemblies were ring-like, some horse-shoe or figure-8 shaped, and shapes were continuous or made up of nanoclusters. The sub-structure appeared stable during the shorter (minute) time points, but after 1 h, more than 50% of the large assemblies showed a change in sub-structure. Overall, these data showed a sub-morphology of large PSD95 assemblies which undergo changes within the 6 hours of observation in the anaesthetised mouse.

Acute muscle inflammation enhances the monosynaptic reflexes and c-fos expression in the feline spinal cord.

The aim of this research was to study the changes of the motor reflex activity (monosynaptic reflex (MSR) of the flexor and extensor muscles) and Fos immunoreactivity in lumbo-sacral spinal cord after acute induced myositis of m. gastrocnemius-soleus (GS). The experiments were carried out on ischaemic decerebrated, spinalized in C1 cats. After infiltration of the GS muscle with carrageenan (2%) MSRs of flexors and extensors showed a significant increase in amplitude +127+/-24.5% and +155+/-28.5%, respectively, p<0.05. The exposed effect was initiated within 30 min and achieved a maximum 2.8h after the intramuscular injections of carrageenan. After analysis of dynamics of the MSRs, animals were perfused and c-fos expression in the spinal segments L6-S1 was evaluated. In comparison to sham-operated animals, the number of Fos-immunoreactive (Fos-ir) cells was noticeably increased in the lumbar cord of cats with carrageenan-induced myositis. The labeled cells were concentrated in the ipsilateral laminae I/II, neck of the dorsal horn (V/VI) and intermediate zone (VII), however, clear predominance of their concentration was found in the deep laminae. The effect of muscle inflammation was also expressed as a significant decline in the number of NADPH-d-reactive cells (p<0.05) in ipsilateral laminae I/II of L6/L7. The results show that the input from acutely inflamed muscles may induce an increase of the reflex responsiveness of flexors and extensors which is not mediated via the gamma-spindle-loop and which coincides with a significant increase in c-fos expression in the deep laminae of the lumbar spinal cord.

In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

NADPH-diaphorase reactivity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord of cats submitted to acute muscle inflammation induced by injection of carrageenan.

The NADPH-diaphorase activity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord were studied in cats with acute unilateral myositis following injection of carrageenan into the m.m. gastrocnemius-soleus. In carrageenan-injected cats maximum in the mean number of intensely stained NADPH-diaphorase reactive (NADPH-dr) neurons was found in lamina VII (+100%) and VIII (+33%) of the contralateral ventral horn of the L6/L7 segments as compared with control animals. The maximumal level of Fos-immunoreactivity was registered in the same laminae with ipsilateral predominance (39.3±4.6 and 7.6±0.9 cells), in comparison with the contralateral side (13.6±0.8 and 5.5±0.6 cells, respectively; P<0.05). We also visualized low-intensely stained and double labelled (Fos immunoreactive+low-intensely stained NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. We visualized the double labelled (Fos-ir+NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. A significant difference in the mean number of RLCs was recorded between the ipsi- and contralateral sides in the lamina VII (13.6±2.5 vs. 4.9±0.7 cells, respectively). We suppose that activation of inhibitory RLCs in ipsilateral lamina VII could be directed on attenuation of activation of motoneurons during muscle pain development. Our study showed that a significant contralateral increase in the number of NADPH-dr cells is accompanied by an ipsilateral increase in c-Fos expression in lamina VII. These data may suggest that NADPH-dr neurons of the contralateral ventral horn through commissural connections also involved in the maintenance of the neuronal activity associated with acute muscle inflammation. It is also hypothesized, that during acute myositis, plastic changes in the ventral horn activate the processes of disinhibition due to an increase in the number of NADPH-d-reactive neurons in the spinal gray matter.

Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism.

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons.

Contractile characteristics of gastrocnemius-soleus muscle in the SOD1G93A ALS mouse model.

OBJECTIVES: In the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), a selective degeneration of fast-fatigable motor units and consequently an early decline of contractile force in individual fast-twitch muscles have been observed in the preclinical stage. However, most human muscles include fast and slow motor units. Gastrocnemius-soleus group (GS) contains such a mixture of units. METHODS: We have investigated changes in the mechanical properties of GS at different SOD1G93A stages in mice. For this purpose, the tibial nerve was repetitively stimulated with rectangular pulses and the force of GS twitches was recorded using a strain gauge fixed to the Achilles tendon. RESULTS: Isometric and tetanic force were attenuated but not before the first clinical signs developed. However, already at preclinical stages, single twitches showed a slower decay compared to control. Consequently, fusion of GS twitches occurred at lower stimulus rates. Furthermore, already preclinically, the temporal course of successive twitch amplitudes changed during repetitive stimulation at increasing rates. The peak amplitudes as well as the potentiation following decay (fatigue) were lower in preclinical mice than in control. DISCUSSION: The time-lapse analysis of the contractile pattern as well as of the twitch configuration of the mixed muscle GS have revealed distinctive differences between wild-type controls and preclinical SOD1G93A mice. It would be of interest to know whether these preclinical changes are also detectable in ALS patients.

Only minor spinal motor reflex effects from feline group IV muscle nociceptors.

The contribution of group III and IV muscle nociceptors activated by injection of KCl or bradykinin into the muscle artery (i.a.) of the gastrocnemius-soleus muscle to spinal motor reflex pathways was investigated in high spinal cats. Group I-III fibres were completely blocked by TTX, leaving group IV-fibre conduction intact. Thus, effects from i.a. KCl or bradykinin injection persisting after TTX were attributed to TTX resistant group IV fibres while the contribution of group III fibres was approximately defined by the difference between those effects and the control effects before TTX. Confirming former findings the chemical activation of group III and IV muscle afferents induced distinct reflex facilitation of the flexor posterior biceps semitendinosus and inhibition of the extensor quadriceps. After the block of all myelinated fibres by TTX the same stimuli induced only minor reflex effects mediated by the persistently conducting TTX resistant group IV afferents. It is concluded that the main functional meaning of group IV muscle afferents, which respond preferentially with a higher threshold to mechanical stimuli, is probably less related to reflex motor control than that of group III afferents.

Different susceptibility of facilitatory and inhibitory spinal pathways to ischemia in the cat.

The sensitivity of different excitatory and inhibitory segmental reflex pathways to ischemia was investigated by monosynaptic reflex testing in the spinal cat. Spinal cord ischemia was established by aortal snare occlusion of 1-10 min duration. Excitatory and inhibitory spinal pathways showed statistically significant different susceptibility to ischemic impact. In the period of decreasing responses after the onset of ischemia the transmission through oligo- or polysynaptic, facilitatory or inhibitory pathways was found to be depressed earlier than that of monosynaptic pathways. The period from the end of ischemia until the beginning of recovery of reflex effects was significantly longer for inhibitory effects, compared to the monosynaptic reflexes alone.The results indicated that interneurones of excitatory segmental pathways may be less sensitive to ischemia than motoneurones, and motoneurones seem to be less sensitive to ischemia than interneurones of inhibitory pathways. In high spinal animals, with a relatively high level of extensor inhibition, the enhanced excitability of inhibitory interneurones to GS motoneurones may be responsible for their sensitivity to ischemia, due to an increased rate of O(2) consumption and exhaustion of high-energy phosphate resources.

Dysregulated expression of neuregulin-1 by cortical pyramidal neurons disrupts synaptic plasticity.

Neuregulin-1 (NRG1) gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions. Loss of NRG1 from cortical projection neurons resulted in increased inhibitory neurotransmission, reduced synaptic plasticity, and hypoactivity. Neuronal overexpression of cysteine-rich domain (CRD)-NRG1, the major brain isoform, caused unbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, abnormal spine growth, altered steady-state levels of synaptic plasticity-related proteins, and impaired sensorimotor gating. We conclude that an "optimal" level of NRG1 signaling balances excitatory and inhibitory neurotransmission in the cortex. Our data provide a potential pathomechanism for impaired synaptic plasticity and suggest that human NRG1 risk haplotypes exert a gain-of-function effect.

Changes in tetrodotoxin-resistant C-fibre activity during fatiguing isometric contractions in the rat.

It is by now well established that tetrodotoxin-resistant (TTX-R) afferent fibres from muscle in the rat exhibit a multisensitive profile, including nociception. TTX-R afferent fibres play an important role in motor control, via spinal and supraspinal loops, but their activation and function during muscle exercise and fatigue are still unknown. Therefore, the specific effect of isometric fatiguing muscle contraction on the responsiveness of TTX-R C-fibres has been investigated in this study. To quantify the TTX-R afferent input we recorded the cord dorsum potential (CDP), which is the result of the electrical fields set up within the spinal cord by the depolarisation of the interneurons located in the dorsal horn, activated by an incoming volley of TTX-R muscle afferents. The changes in TTX-R CDP size before, during and after fatiguing electrical stimulation of the gastrocnemius-soleus (GS) muscle have been taken as a measure of TTX-R C-unit activation. At the end of the fatiguing protocol, following an exponential drop in force, TTX-R CDP area decreased in the majority of trials (9/14) to 0.75 ± 0.03% (mean ± SEM) of the pre-fatigue value. Recovery to the control size of the TTX-R CDP was incomplete after 10 min. Furthermore, fatiguing trials could sensitise a fraction of the TTX-R C-fibres responding to muscle pinch. The results suggest a long-lasting activation of the TTX-R muscle afferents after fatiguing stimulation. The role of this behaviour in chronic muscle fatigue in connection with pain development is discussed. Accumulation of metabolites released into the interstitium during fatiguing stimulation might be one of the reasons underlying the C-fibres' long-lasting activation.