Basic science under threat: Lessons from the Skirball Institute.

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises.

Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.

Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1(CreER) mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1(CreER) to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia showed deficits in multiple learning tasks and a significant reduction in motor-learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal tropomyosin-related kinase receptor B phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal that microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through BDNF signaling.

Clear optically matched panoramic access channel technique (COMPACT) for large-volume deep brain imaging.

To understand neural circuit mechanisms underlying behavior, it is crucial to observe the dynamics of neuronal structure and function in different regions of the brain. Since current noninvasive imaging technologies allow cellular-resolution imaging of neurons only within ~1 mm below the cortical surface, the majority of mouse brain tissue remains inaccessible. While miniature optical imaging probes allow access to deep brain regions, cellular-resolution imaging is typically restricted to a small tissue volume. To increase the tissue access volume, we developed a clear optically matched panoramic access channel technique (COMPACT). With probe dimensions comparable to those of common gradient-index lenses, COMPACT enables a two to three orders of magnitude greater tissue access volume. We demonstrated the capabilities of COMPACT by multiregional calcium imaging in mice during sleep. We believe that large-volume in vivo imaging with COMPACT will be valuable to a variety of deep tissue imaging applications.

BDNF produced by cerebral microglia promotes cortical plasticity and pain hypersensitivity after peripheral nerve injury.

Peripheral nerve injury-induced mechanical allodynia is often accompanied by abnormalities in the higher cortical regions, yet the mechanisms underlying such maladaptive cortical plasticity remain unclear. Here, we show that in male mice, structural and functional changes in the primary somatosensory cortex (S1) caused by peripheral nerve injury require neuron-microglial signaling within the local circuit. Following peripheral nerve injury, microglia in the S1 maintain ramified morphology and normal density but up-regulate the mRNA expression of brain-derived neurotrophic factor (BDNF). Using in vivo two-photon imaging and Cx3cr1CreER;Bdnfflox mice, we show that conditional knockout of BDNF from microglia prevents nerve injury-induced synaptic remodeling and pyramidal neuron hyperactivity in the S1, as well as pain hypersensitivity in mice. Importantly, S1-targeted removal of microglial BDNF largely recapitulates the beneficial effects of systemic BDNF depletion on cortical plasticity and allodynia. Together, these findings reveal a pivotal role of cerebral microglial BDNF in somatosensory cortical plasticity and pain hypersensitivity.

Sleep promotes the formation of dendritic filopodia and spines near learning-inactive existing spines.

Changes in synaptic connections are believed to underlie long-term memory storage. Previous studies have suggested that sleep is important for synapse formation after learning, but how sleep is involved in the process of synapse formation remains unclear. To address this question, we used transcranial two-photon microscopy to investigate the effect of postlearning sleep on the location of newly formed dendritic filopodia and spines of layer 5 pyramidal neurons in the primary motor cortex of adolescent mice. We found that newly formed filopodia and spines were partially clustered with existing spines along individual dendritic segments 24 h after motor training. Notably, posttraining sleep was critical for promoting the formation of dendritic filopodia and spines clustered with existing spines within 8 h. A fraction of these filopodia was converted into new spines and contributed to clustered spine formation 24 h after motor training. This sleep-dependent spine formation via filopodia was different from retraining-induced new spine formation, which emerged from dendritic shafts without prior presence of filopodia. Furthermore, sleep-dependent new filopodia and spines tended to be formed away from existing spines that were active at the time of motor training. Taken together, these findings reveal a role of postlearning sleep in regulating the number and location of new synapses via promoting filopodial formation.

The serotonin 2B receptor is required in neonatal microglia to limit neuroinflammation and sickness behavior in adulthood.

Severe peripheral infections induce an adaptive sickness behavior and an innate immune reaction in various organs including the brain. On the long term, persistent alteration of microglia, the brain innate immune cells, is associated with an increased risk of psychiatric disorders. It is thus critical to identify genes and mechanisms controlling the intensity and duration of the neuroinflammation induced by peripheral immune challenges. We tested the hypothesis that the 5-HT2B receptor, the main serotonin receptor expressed by microglia, might represent a valuable candidate. First, we observed that Htr2b-/- mice, knock-out for the 5-HT2B receptor gene, developed, when exposed to a peripheral lipopolysaccharide (LPS) challenge, a stronger weight loss compared to wild-type mice; in addition, comparison of inflammatory markers in brain, 4 and 24 hr after LPS injection, showed that Htr2b deficiency leads to a prolonged neuroinflammation. Second, to assess the specific contribution of the microglial 5-HT2B receptor, we investigated the response to LPS of conditional knock-out mice invalidated for Htr2b in microglia only. We found that deletion of Htr2b in microglia since birth is sufficient to cause enhanced weight loss and increased neuroinflammatory response upon LPS injection at adult stage. In contrast, mice deleted for microglial Htr2b in adulthood responded normally to LPS, revealing a neonatal developmental effect. These results highlight the role of microglia in the response to a peripheral immune challenge and suggest the existence of a developmental, neonatal period, during which instruction of microglia through 5-HT2B receptors is necessary to prevent microglia overreactivity in adulthood.

Increased neuronal activity in motor cortex reveals prominent calcium dyshomeostasis in tauopathy mice.

Perturbed neuronal Ca2+ homeostasis is implicated in Alzheimer's disease, which has primarily been demonstrated in mice with amyloid-ß deposits but to a lesser and more variable extent in tauopathy models. In this study, we injected AAV to express Ca2+ indicator in layer II/III motor cortex neurons and measured neuronal Ca2+ activity by two photon imaging in awake transgenic JNPL3 tauopathy and wild-type mice. Various biochemical measurements were conducted in postmortem mouse brains for mechanistic insight and a group of animals received two intravenous injections of a tau monoclonal antibody spaced by four days to test whether the Ca2+ dyshomeostasis was related to pathological tau protein. Under running conditions, we found abnormal neuronal Ca2+ activity in tauopathy mice compared to age-matched wild-type mice with higher frequency of Ca2+ transients, lower amplitude of peak Ca2+ transients and lower total Ca2+ activity in layer II/III motor cortex neurons. While at resting conditions, only Ca2+ frequency was increased. Brain levels of soluble pathological tau correlated better than insoluble tau levels with the degree of Ca2+ dysfunction in tauopathy mice. Furthermore, tau monoclonal antibody 4E6 partially rescued Ca2+ activity abnormalities in tauopathy mice after two intravenous injections and decreased soluble pathological tau protein within the brain. This correlation and antibody effects strongly suggest that the neuronal Ca2+ dyshomeostasis is causally linked to pathological tau protein. These findings also reveal more pronounced neuronal Ca2+ dysregulation in tauopathy mice than previously reported by two-photon imaging that can be partially corrected with an acute tau antibody treatment.

Specific depletion of resident microglia in the early stage of stroke reduces cerebral ischemic damage.

BACKGROUND: Ischemia can induce rapid activation of microglia in the brain. As key immunocompetent cells, reactive microglia play an important role in pathological development of ischemic stroke. However, the role of activated microglia during the development of ischemia remains controversial. Thus, we aimed to investigate the function of reactive microglia in the early stage of ischemic stroke. METHODS: A Rose Bengal photothrombosis model was applied to induce targeted ischemic stroke in mice. CX3CR1CreER:R26iDTR mice were used to specifically deplete resident microglia through intragastric administration of tamoxifen (Ta) and intraperitoneal injection of diphtheria toxin (DT). At day 3 after ischemic stroke, behavioral tests were performed. After that, mouse brains were collected for further histological analysis and detection of mRNA expression of inflammatory factors. RESULTS: The results showed that specific depletion of microglia resulted in a significant decrease in ischemic infarct volume and improved performance in motor ability 3 days after stroke. Microglial depletion caused a remarkable reduction in the densities of degenerating neurons and inducible nitric oxide synthase positive (iNOS+) cells. Importantly, depleting microglia induced a significant increase in the mRNA expression level of anti-inflammatory factors TGF-ß1, Arg1, IL-10, IL-4, and Ym1 as well as a significant decline of pro-inflammatory factors TNF-α, iNOS, and IL-1ß 3 days after stroke. CONCLUSIONS: These results suggest that activated microglia is an important modulator of the brain's inflammatory response in stroke, contributing to neurological deficit and infarct expansion. Modulation of the inflammatory response through the elimination of microglia at a precise time point may be a promising therapeutic approach for the treatment of cerebral ischemia.

Branch-specific dendritic Ca(2+) spikes cause persistent synaptic plasticity.

The brain has an extraordinary capacity for memory storage, but how it stores new information without disrupting previously acquired memories remains unknown. Here we show that different motor learning tasks induce dendritic Ca(2+) spikes on different apical tuft branches of individual layer V pyramidal neurons in the mouse motor cortex. These task-related, branch-specific Ca(2+) spikes cause long-lasting potentiation of postsynaptic dendritic spines active at the time of spike generation. When somatostatin-expressing interneurons are inactivated, different motor tasks frequently induce Ca(2+) spikes on the same branches. On those branches, spines potentiated during one task are depotentiated when they are active seconds before Ca(2+) spikes induced by another task. Concomitantly, increased neuronal activity and performance improvement after learning one task are disrupted when another task is learned. These findings indicate that dendritic-branch-specific generation of Ca(2+) spikes is crucial for establishing long-lasting synaptic plasticity, thereby facilitating information storage associated with different learning experiences.

Fear extinction reverses dendritic spine formation induced by fear conditioning in the mouse auditory cortex.

Fear conditioning-induced behavioral responses can be extinguished after fear extinction. While fear extinction is generally thought to be a form of new learning, several lines of evidence suggest that neuronal changes associated with fear conditioning could be reversed after fear extinction. To better understand how fear conditioning and extinction modify synaptic circuits, we examined changes of postsynaptic dendritic spines of layer V pyramidal neurons in the mouse auditory cortex over time using transcranial two-photon microscopy. We found that auditory-cued fear conditioning induced the formation of new dendritic spines within 2 days. The survived new spines induced by fear conditioning with one auditory cue were clustered within dendritic branch segments and spatially segregated from new spines induced by fear conditioning with a different auditory cue. Importantly, fear extinction preferentially caused the elimination of newly formed spines induced by fear conditioning in an auditory cue-specific manner. Furthermore, after fear extinction, fear reconditioning induced reformation of new dendritic spines in close proximity to the sites of new spine formation induced by previous fear conditioning. These results show that fear conditioning, extinction, and reconditioning induce cue- and location-specific dendritic spine remodeling in the auditory cortex. They also suggest that changes of synaptic connections induced by fear conditioning are reversed after fear extinction.

Long-range remote focusing by image-plane aberration correction.

Laser scanning plays an important role in a broad range of applications. Toward 3D aberration-free scanning, a remote focusing technique has been developed for high-speed imaging applications. However, the implementation of remote focusing often suffers from a limited axial scan range as a result of unknown aberration. Through simple analysis, we show that the sample-to-image path length conservation is crucially important to the remote focusing performance. To enhance the axial scan range, we propose and demonstrate an image-plane aberration correction method. Using a static correction, we can effectively improve the focus quality over a large defocusing range. Experimentally, we achieved ∼three times greater defocusing range than that of conventional methods. This technique can broadly benefit the implementations of high-speed large-volume 3D imaging.

Contrast gain through simple illumination control for wide-field fluorescence imaging of scattering samples.

Wide field fluorescence microscopy is the most commonly employed fluorescence imaging modality. However, a major drawback of wide field imaging is the very limited imaging depth in scattering samples. By experimentally varying the control of illumination, we found that the optimized illumination profile can lead to large contrast improvement for imaging at a depth beyond four scattering path lengths. At such imaging depth, we found that the achieved image signal-to-noise ratio can rival that of confocal measurement. As the employed illumination control is very simple, the method can be broadly applied to a wide variety of wide field fluorescence imaging systems.

Pupil plane actuated remote focusing for rapid focal depth control.

Laser scanning is widely employed in imaging and material processing. Common laser scanners are often fast for 2D transverse scanning. Rapid focal depth control is highly desired in many applications. Although remote focusing has been developed to achieve fast focal depth control, the implementation is limited by the laser damage to the actuator near laser focus. Here, we present a new method named pupil plane actuated remote focusing, which enables sub-millisecond response time while avoiding laser damage. We demonstrate its application by implementing a dual-plane two-photon laser scanning fluorescence microscope for in vivo recording of calcium transient of neurons in mouse neocortex.

Jitter suppression for resonant galvo based high-throughput laser scanning systems.

Laser scanning has been widely used in material processing and optical imaging. Among the established scanners, resonant galvo scanners offer high scanning throughput and 100% duty cycle and have been employed in various laser scanning microscopes. However, the common applications of resonant galvo often suffer from position jitters which could introduce substantial measurement artifacts. In this work, we systematically quantify the impact of position sensor, data acquisition system and air turbulence and provide a simple solution to achieve jitter free high-throughput measurement.

Line scanning mechanical streak camera for phosphorescence lifetime imaging.

Phosphorescence lifetime measurement holds great importance in life sciences and material sciences. Due to the long lifetime of phosphorescence emission, conventional approaches based on point scanning time-domain recording suffer from long recording time and low signal-to-noise ratio (SNR). To overcome these difficulties, we developed a line scanning mechanical streak camera for parallel and high SNR imaging. This design offers three key advantages. First, hundreds to thousands of pixels can be recorded simultaneously at high throughput. Second, hundreds of excitation can be accumulated on a single camera frame and read out at once with high quantum efficiency (QE) and low read noise. Third, the system is very simple, only requiring a camera and a scanner. Using a confocal line scanning configuration, we imaged samples of various lifetime ranging from tens of nanoseconds to hundreds of microseconds, which demonstrated the versatility and advantages of this method.

Efficient Position Decoding Methods Based on Fluorescence Calcium Imaging in the Mouse Hippocampus.

Large-scale fluorescence calcium imaging methods have become widely adopted for studies of long-term hippocampal and cortical neuronal dynamics. Pyramidal neurons of the rodent hippocampus show spatial tuning in freely foraging or head-fixed navigation tasks. Development of efficient neural decoding methods for reconstructing the animal's position in real or virtual environments can provide a fast readout of spatial representations in closed-loop neuroscience experiments. Here, we develop an efficient strategy to extract features from fluorescence calcium imaging traces and further decode the animal's position. We validate our spike inference-free decoding methods in multiple in vivo calcium imaging recordings of the mouse hippocampus based on both supervised and unsupervised decoding analyses. We systematically investigate the decoding performance of our proposed methods with respect to the number of neurons, imaging frame rate, and signal-to-noise ratio. Our proposed supervised decoding analysis is ultrafast and robust, and thereby appealing for real-time position decoding applications based on calcium imaging.

Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Large-scale femtosecond holography for near simultaneous optogenetic neural modulation.

For better understanding of brain functions, optogenetic neural modulation has been widely employed in neural science research. For deep tissue in vivo applications, large-scale two-photon based near simultaneous 3D laser excitation is needed. Although 3D holographic laser excitation is nowadays common practice, the inherent short coherence length of the commonly used femtosecond pulses fundamentally restricts the achievable field-of-view. Here we report a technique for near simultaneous large-scale femtosecond holographic 3D excitation. Specifically, we achieved two-photon excitation over 1.3 mm field-of-view within 1.3 milliseconds, which is sufficiently fast even for spike timing recording. The method is scalable and compatible with the commonly used two-photon sources and imaging systems in neuroscience research.

Opposite effects of fear conditioning and extinction on dendritic spine remodelling.

It is generally believed that fear extinction is a form of new learning that inhibits rather than erases previously acquired fear memories. Although this view has gained much support from behavioural and electrophysiological studies, the hypothesis that extinction causes the partial erasure of fear memories remains viable. Using transcranial two-photon microscopy, we investigated how neural circuits are modified by fear learning and extinction by examining the formation and elimination of postsynaptic dendritic spines of layer-V pyramidal neurons in the mouse frontal association cortex. Here we show that fear conditioning by pairing an auditory cue with a footshock increases the rate of spine elimination. By contrast, fear extinction by repeated presentation of the same auditory cue without a footshock increases the rate of spine formation. The degrees of spine remodelling induced by fear conditioning and extinction strongly correlate with the expression and extinction of conditioned fear responses, respectively. Notably, spine elimination and formation induced by fear conditioning and extinction occur on the same dendritic branches in a cue- and location-specific manner: cue-specific extinction causes formation of dendritic spines within a distance of two micrometres from spines that were eliminated after fear conditioning. Furthermore, reconditioning preferentially induces elimination of dendritic spines that were formed after extinction. Thus, within vastly complex neuronal networks, fear conditioning, extinction and reconditioning lead to opposing changes at the level of individual synapses. These findings also suggest that fear memory traces are partially erased after extinction.

Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina.

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.