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Recognizing the affective states of social counterparts and responding appropriately fosters successful social interactions. However, little is known about how the affective states are expressed and perceived and how they influence social decisions. Here, we show that male and female mice emit distinct olfactory cues after experiencing distress. These cues activate distinct neural circuits in the piriform cortex (PiC) and evoke sexually dimorphic empathic behaviors in observers. Specifically, the PiC → PrL pathway is activated in female observers, inducing a social preference for the distressed counterpart. Conversely, the PiC → MeA pathway is activated in male observers, evoking excessive self-grooming behaviors. These pathways originate from non-overlapping PiC neuron populations with distinct gene expression signatures regulated by transcription factors and sex hormones. Our study unveils how internal states of social counterparts are processed through sexually dimorphic mechanisms at the molecular, cellular, and circuit levels and offers insights into the neural mechanisms underpinning sex differences in higher brain functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nerve growth factor (NGF) is critical for neuronal physiology during development and adulthood. Despite the well-recognized effect of NGF on neurons, less is known about whether NGF can actually affect other cell types in the central nervous system (CNS). In this work, we show that astrocytes are susceptible to changes in ambient levels of NGF. First, we observe that interfering with NGF signaling in vivo via the constitutive expression of an antiNGF antibody induces astrocytic atrophy. A similar asthenic phenotype is encountered in an uncleavable proNGF transgenic mouse model (TgproNGF#72), effectively increasing the brain proNGF levels. To examine whether this effect on astrocytes is cell-autonomous, we cultured wild-type primary astrocytes in the presence of antiNGF antibodies, uncovering that a short incubation period is sufficient to potently and rapidly trigger calcium oscillations. Acute induction of calcium oscillations by antiNGF antibodies is followed by progressive morphological changes similar to those observed in antiNGF AD11 mice. Conversely, incubation with mature NGF has no effect on either calcium activity nor on astrocytic morphology. At longer timescales, transcriptomic analysis revealed that NGF-deprived astrocytes acquire a proinflammatory profile. In particular, antiNGF-treated astrocytes show upregulation of neurotoxic transcripts and downregulation of neuroprotective mRNAs. Consistent with that data, culturing wild-type neurons in the presence of NGF-deprived astrocytes leads to neuronal cell death. Finally, we report that in both awake and anesthetized mice , astrocytes in layer I of the motor cortex respond with an increase in calcium activity to acute NGF inhibition using either NGF-neutralizing antibodies or a TrkA-Fc NGF scavenger. Moreover, in vivo calcium imaging in the cortex of the 5xFAD neurodegeneration mouse model shows an increased level of spontaneous calcium activity in astrocytes, which is significantly reduced after acute administration of NGF. In conclusion, we unveil a novel neurotoxic mechanism driven by astrocytes, triggered by their sensing and reacting to changes in the levels of ambient NGF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tiberi, Carucci, Testa, Rizzi, Pacifico, Borgonovo, Arisi, D’Onofrio, Brandi, Gan, Capsoni and Cattaneo.)

There is a demand for noninvasive methods to ameliorate disease. We investigated whether 40-Hz flickering light entrains gamma oscillations and suppresses amyloid-β in the brains of APP/PS1 and 5xFAD mouse models of Alzheimer's disease. We used multisite silicon probe recording in the visual cortex, entorhinal cortex or the hippocampus and found that 40-Hz flickering simulation did not engage native gamma oscillations in these regions. Additionally, spike responses in the hippocampus were weak, suggesting 40-Hz light does not effectively entrain deep structures. Mice avoided 40-Hz flickering light, associated with elevated cholinergic activity in the hippocampus. We found no reliable changes in plaque count or microglia morphology by either immunohistochemistry or in vivo two-photon imaging following 40-Hz stimulation, nor reduced levels of amyloid-β 40/42. Thus, visual flicker stimulation may not be a viable mechanism for modulating activity in deep structures., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Increased low frequency cortical oscillations are observed in people with neuropathic pain, but the cause of such elevated cortical oscillations and their impact on pain development remain unclear. By imaging neuronal activity in a spared nerve injury (SNI) mouse model of neuropathic pain, we show that neurons in dorsal root ganglia (DRG) and somatosensory cortex (S1) exhibit synchronized activity after peripheral nerve injury. Notably, synchronized activity of DRG neurons occurs within hours after injury and 1-2 days before increased cortical oscillations. This DRG synchrony is initiated by axotomized neurons and mediated by local purinergic signaling at the site of nerve injury. We further show that synchronized DRG activity after SNI is responsible for increasing low frequency cortical oscillations and synaptic remodeling in S1, as well as for inducing animals' pain-like behaviors. In naive mice, enhancing the synchrony, not the level, of DRG neuronal activity causes synaptic changes in S1 and pain-like behaviors similar to SNI mice. Taken together, these results reveal the critical role of synchronized DRG neuronal activity in increasing cortical plasticity and oscillations in a neuropathic pain model. These findings also suggest the potential importance of detection and suppression of elevated cortical oscillations in neuropathic pain states., (© 2023. The Author(s).)

Memories can be modified by new experience in a specific or generalized manner. Changes in synaptic connections are crucial for memory storage, but it remains unknown how synaptic changes associated with different memories are distributed within neuronal circuits and how such distributions affect specific or generalized modification by novel experience. Here we show that fear conditioning with two different auditory stimuli (CS) and footshocks (US) induces dendritic spine elimination mainly on different dendritic branches of layer 5 pyramidal neurons in the mouse motor cortex. Subsequent fear extinction causes CS-specific spine formation and extinction of freezing behavior. In contrast, spine elimination induced by fear conditioning with >2 different CS-USs often co-exists on the same dendritic branches. Fear extinction induces CS-nonspecific spine formation and generalized fear extinction. Moreover, activation of somatostatin-expressing interneurons increases the occurrence of spine elimination induced by different CS-USs on the same dendritic branches and facilitates the generalization of fear extinction. These findings suggest that specific or generalized modification of existing memories by new experience depends on whether synaptic changes induced by previous experiences are segregated or co-exist at the level of individual dendritic branches., (© 2023. The Author(s).)

Enzymes are critical for cellular functions, and malfunction of enzymes is closely related to many human diseases. Inhibition studies can help in deciphering the physiological roles of enzymes and guide conventional drug development programs. In particular, chemogenetic approaches enabling rapid and selective inhibition of enzymes in mammalian cells have unique advantages. Here, we describe the procedure for rapid and selective inhibition of a kinase in mammalian cells by bioorthogonal ligand tethering (iBOLT). Briefly, a non-canonical amino acid bearing a bioorthogonal group is genetically incorporated into the target kinase by genetic code expansion. The sensitized kinase can react with a conjugate containing a complementary biorthogonal group linked with a known inhibitory ligand. As a result, tethering of the conjugate to the target kinase allows selective inhibition of protein function. Here, we demonstrate this method by using cAMP-dependent protein kinase catalytic subunit alpha (PKA-Cα) as the model enzyme. The method should be applicable to other kinases, enabling their rapid and selective inhibition., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In vivo imaging has been widely used for investigating the structure and function of neurons typically located within ~ 800 μm below the cortical surface. Due to light scattering and absorption, it has been difficult to perform in-vivo imaging of neurons in deep cortical and subcortical regions of large animals with two-photon microscopy. Here, we combined a thin-wall quartz capillary with a GRIN lens attached to a prism for large-volume structural and calcium imaging of neurons located 2 mm below the surface of rabbit and monkey brains. The field of view was greatly expanded by rotating and changing the depth of the imaging probe inside a quartz capillary. Calcium imaging of layer 5/6 neurons in the rabbit motor cortex revealed differential activity of these neurons between quiet wakefulness and slow wave sleep. The method described here provides an important tool for studying the structure and function of neurons located deep in the brains of large animals., (© 2022. The Author(s).)

Learning induces the formation of new synapses in addition to changes of existing synapse strength. However, it remains unclear whether new synapses serve different functions from existing synapses. By performing two-photon structural and Ca 2+ imaging of postsynaptic dendritic spines in layer 2/3 pyramidal neurons, we show that new spine formation increases in the mouse motor cortex 8-24 h after motor training. New spines, not existing spine populations, are preferentially active when mice perform the learned task rather than a new task. New spine activity is also more synchronized with dendritic/somatic activity when the learned task, not a new task, is carried out. Furthermore, new spines are formed to increase the task specificity in a subset of neurons, and their survival is not affected when a new task is learned. These findings suggest that newly formed synapses preferentially increase the task specificity of neurons over existing synapses at the retention stage of motor learning., Competing Interests: Declaration of interests The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Including apolipoprotein E-ε4 (APOE-ε4) status and older age into consideration may increase the accuracy of plasma Aβ 42 /Aβ 40 detecting Aβ+ individuals, but the rationale behind this remains to be fully understood. Besides, both Aβ pathology and vascular diseases are related to neurodegeneration and cognitive decline, but it is still not fully understood how APOE-ε4 modulates these relationships. In this study, we examined 241 non-demented Alzheimer's Disease Neuroimaging Initiative participants to investigate the associations among age, white matter hyperintensities (WMH), hypertension, hyperlipidemia, body mass index (BMI), plasma Aβ 42 /Aβ 40 measured by liquid chromatography tandem mass spectrometry, and 18 F-florbetapir Aβ PET as well as their prediction of longitudinal adjusted hippocampal volume (aHCV) and cognition in APOE-ε4 carriers and non-carriers. We found older age predicted faster WMH increase (p = 0.024) and cortical Aβ accumulation (p = 0.043) in APOE-ε4 non-carriers only, whereas lower plasma Aβ 42 /Aβ 40 predicted faster cortical Aβ accumulation (p < 0.018) regardless of APOE-ε4 status. While larger WMH and underweight predicted (p < 0.05) faster decreases in aHCV and cognition in APOE-ε4 non-carriers, lower plasma Aβ 42 /Aβ 40 predicted (p < 0.031) faster decreases in aHCV and cognition in APOE-ε4 carriers. Higher Aβ PET also predicted faster rates of aHCV (p = 0.010) in APOE-ε4 carriers only, but was related to faster rates of cognitive decline (p < 0.022) regardless of APOE-ε4 status. These findings may provide novel insights into understanding different mechanisms underlie neurodegeneration and cognitive decline in non-demented elderly adults with and without APOE-ε4 allele, which may help the design of anti-Alzheimer's clinical trials., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests Agnel Sfeir is a co-founder, consultant, and shareholder in Repare Therapeutics. Gord Fishell is a co-founder, consultant, and shareholder in Regel Therapeutics. Alexander Schier, Michael Dustin, Wen-Biao Gan, Alexandra Joyner, Ruth Lehmann, David Ron, David Roth, William S. Talbot, Deborah Yelon, and Arturo Zychlinsky declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.

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., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: The authors have declared that no competing interests exist.

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. CX3CR1 CreER :R26 iDTR 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-β1, Arg1, IL-10, IL-4, and Ym1 as well as a significant decline of pro-inflammatory factors TNF-α, iNOS, and IL-1β 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.

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-HT 2B receptor, the main serotonin receptor expressed by microglia, might represent a valuable candidate. First, we observed that Htr2b -/- mice, knock-out for the 5-HT 2B 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-HT 2B 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-HT 2B receptors is necessary to prevent microglia overreactivity in adulthood., (© 2020 Wiley Periodicals LLC.)

Perturbed neuronal Ca 2+ homeostasis is implicated in Alzheimer's disease, which has primarily been demonstrated in mice with amyloid-β deposits but to a lesser and more variable extent in tauopathy models. In this study, we injected AAV to express Ca 2+ indicator in layer II/III motor cortex neurons and measured neuronal Ca 2+ 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 Ca 2+ dyshomeostasis was related to pathological tau protein. Under running conditions, we found abnormal neuronal Ca 2+ activity in tauopathy mice compared to age-matched wild-type mice with higher frequency of Ca 2+ transients, lower amplitude of peak Ca 2+ transients and lower total Ca 2+ activity in layer II/III motor cortex neurons. While at resting conditions, only Ca 2+ frequency was increased. Brain levels of soluble pathological tau correlated better than insoluble tau levels with the degree of Ca 2+ dysfunction in tauopathy mice. Furthermore, tau monoclonal antibody 4E6 partially rescued Ca 2+ 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 Ca 2+ dyshomeostasis is causally linked to pathological tau protein. These findings also reveal more pronounced neuronal Ca 2+ dysregulation in tauopathy mice than previously reported by two-photon imaging that can be partially corrected with an acute tau antibody treatment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In many parts of the nervous system, experience-dependent refinement of neuronal circuits predominantly involves synapse elimination. The role of sleep in this process remains unknown. We investigated the role of sleep in experience-dependent dendritic spine elimination of layer 5 pyramidal neurons in the visual (V1) and frontal association cortex (FrA) of 1-month-old mice. We found that monocular deprivation (MD) or auditory-cued fear conditioning (FC) caused rapid spine elimination in V1 or FrA, respectively. MD- or FC-induced spine elimination was significantly reduced after total sleep or REM sleep deprivation. Total sleep or REM sleep deprivation also prevented MD- and FC-induced reduction of neuronal activity in response to visual or conditioned auditory stimuli. Furthermore, dendritic calcium spikes increased substantially during REM sleep, and the blockade of these calcium spikes prevented MD- and FC-induced spine elimination. These findings reveal an important role of REM sleep in experience-dependent synapse elimination and neuronal activity reduction.

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.

Background: The genetically encoded calcium (Ca 2+ ) sensor GCaMP6 has been widely used for imaging Ca 2+ transients in neuronal somata, dendrites, and synapses., New Method: Here we describe five new transgenic mouse lines expressing GCaMP6F (fast) or GCaMP6S (slow) in the central and peripheral nervous system under the control of theThy1.2 promoter., Results: These transgenic lines exhibit stable and layer-specific expression of GCaMP6 in multiple brain regions. They have several unique features compared to existing Thy1.2-GCaMP6 mice, including sparse expression of GCaMP6 in layer V pyramidal neurons of the cerebral cortex, motor neurons in the spinal cord, as well as sensory neurons in dorsal root ganglia (DRG). We further demonstrate that these mouse lines allow for robust detection of Ca 2+ transients in neuronal somata and apical dendrites in the cerebral cortex of both anesthetized and awake behaving mice, as well as in DRG neurons., Comparison With Existing Method(s): These transgenic lines allows Ca 2+ imaging of dendrites and somas of pyramidal neurons in specific cortical layers that is difficult to achieve with existing methods., Conclusions: These GCaMP6 transgenic lines thus provide useful tools for functional analysis of neuronal circuits in both central and peripheral nervous systems., Competing Interests: Declarations of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mitochondrial calcium ([Ca 2+ ] mito ) dynamics plays vital roles in regulating fundamental cellular and organellar functions including bioenergetics. However, neuronal [Ca 2+ ] mito dynamics in vivo and its regulation by brain activity are largely unknown. By performing two-photon Ca 2+ imaging in the primary motor (M1) and visual cortexes (V1) of awake behaving mice, we find that discrete [Ca 2+ ] mito transients occur synchronously over somatic and dendritic mitochondrial network, and couple with cytosolic calcium ([Ca 2+ ] cyto ) transients in a probabilistic, rather than deterministic manner. The amplitude, duration, and frequency of [Ca 2+ ] cyto transients constitute important determinants of the coupling, and the coupling fidelity is greatly increased during treadmill running (in M1 neurons) and visual stimulation (in V1 neurons). Moreover, Ca 2+ /calmodulin kinase II is mechanistically involved in modulating the dynamic coupling process. Thus, activity-dependent dynamic [Ca 2+ ] mito -to-[Ca 2+ ] cyto coupling affords an important mechanism whereby [Ca 2+ ] mito decodes brain activity for the regulation of mitochondrial bioenergetics to meet fluctuating neuronal energy demands as well as for neuronal information processing.

The dorsal root ganglia (DRG) contain the somas of first-order sensory neurons critical for somatosensation. Due to technical difficulties, DRG neuronal activity in awake behaving animals remains unknown. Here, we develop a method for imaging DRG at cellular and subcellular resolution over weeks in awake mice. The method involves the installation of an intervertebral fusion mount to reduce spinal movement, and the implantation of a vertebral glass window without interfering animals' motor and sensory functions. In vivo two-photon calcium imaging shows that DRG neuronal activity is higher in awake than anesthetized animals. Immediately after plantar formalin injection, DRG neuronal activity increases substantially and this activity upsurge correlates with animals' phasic pain behavior. Repeated imaging of DRG over 5 weeks after formalin injection reveals persistent neuronal hyperactivity associated with ongoing pain. The method described here provides an important means for in vivo studies of DRG functions in sensory perception and disorders.

The activities of neuronal populations exhibit temporal sequences that are thought to mediate spatial navigation, cognitive processing, and motor actions. The mechanisms underlying the generation and maintenance of sequential neuronal activity remain unclear. We found that layer 2 and/or 3 pyramidal neurons (PNs) showed sequential activation in the mouse primary motor cortex during motor skill learning. Concomitantly, the activity of somatostatin (SST)-expressing interneurons increased and decreased in a task-specific manner. Activating SST interneurons during motor training, either directly or via inhibiting vasoactive-intestinal-peptide-expressing interneurons, prevented learning-induced sequential activities of PNs and behavioral improvement. Conversely, inactivating SST interneurons during the learning of a new motor task reversed sequential activities and behavioral improvement that occurred during a previous task. Furthermore, the control of SST interneurons over sequential activation of PNs required CaMKII-dependent synaptic plasticity. These findings indicate that SST interneurons enable and maintain synaptic plasticity-dependent sequential activation of PNs during motor skill learning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple brain regions including the amygdala and prefrontal cortex are crucial for modulating fear conditioning and extinction. The primary motor cortex is known to participate in the planning, control, and execution of voluntary movements. Whether and how the primary motor cortex is involved in modulating freezing responses related to fear conditioning and extinction remains unclear. Here we show that inactivation of the mouse primary motor cortex impairs both the acquisition and extinction of freezing responses induced by auditory-cued fear conditioning. Fear conditioning significantly increases the elimination of dendritic spines on apical dendrites of layer 5 pyramidal neurons in the motor cortex. These eliminated spines are further apart from each other than expected from random distribution along dendrites. On the other hand, fear extinction causes the formation of new spines that are located near the site of spines eliminated previously after fear conditioning. We further show that fear conditioning decreases and fear extinction increases somatic activities of layer 5 pyramidal neurons in the motor cortex respectively. Taken together, these findings indicate fear conditioning and extinction induce opposing changes in synaptic connections and somatic activities of layer 5 pyramidal neurons in the primary motor cortex, a cortical region important for the acquisition and extinction of auditory-cued conditioned freezing responses.

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., Competing Interests: The authors declare no conflict of interest.

The cyclic nucleotide cGMP is an intracellular second messenger with important roles in neuronal functions and animals' behaviors. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze the second messengers cGMP and cAMP. Inhibition of phosphodiesterase 9 (PDE9), a main isoform of PDEs hydrolyzing cGMP, has been shown to improve learning and memory as well as cognitive function in rodents. However, the role of PDE9 in regulating neuronal structure and function in vivo remains unclear. Here we used in vivo two-photon microscopy to investigate the effect of a selective PDE9 inhibitor PF-04449613 on the activity and plasticity of dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex. We found that administration of PF-04449613 increased calcium activity of dendrites and dendritic spines of layer V pyramidal neurons in mice under resting and running conditions. Chronic treatment of PF-04449613 over weeks increased dendritic spine formation and elimination under basal conditions. Furthermore, PF-04449613 treatment over 1-7 days increased the formation and survival of new spines as well as performance improvement after rotarod motor training. Taken together, our studies suggest that elevating the level of cGMP with the PDE9 inhibitor PF-04449613 increases synaptic calcium activity and learning-dependent synaptic plasticity, thereby contributing to performance improvement after learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018., (© 2018 Wiley Periodicals, Inc.)

The anterior cingulate cortex (ACC) is thought to be important for acute pain perception as well as the development of chronic pain after peripheral nerve injury. Nevertheless, how ACC neurons respond to sensory stimulation under chronic pain states is not well understood. Here, we used an in vivo two-photon imaging technique to monitor the activity of individual neurons in the ACC of awake, head restrained mice. Calcium imaging in the dorsal ACC revealed robust somatic activity in layer 5 (L5) pyramidal neurons in response to peripheral noxious stimuli, and the degree of evoked activity was correlated with the intensity of noxious stimulation. Furthermore, the activation of ACC neurons occurred bilaterally upon noxious stimulation to either contralateral or ipsilateral hind paws. Notably, with nerve injury-induced neuropathic pain in one limb, L5 pyramidal neurons in both sides of the ACC showed enhanced activity in the absence or presence of pain stimuli. These results reveal hyperactivity of L5 pyramidal neurons in the bilateral ACC during the development of neuropathic pain.

Background: Alzheimer's disease (AD) is characterized by amyloid deposition, tangle formation as well as synapse loss. Synaptic abnormalities occur early in the pathogenesis of AD. Identifying early synaptic abnormalities and their underlying mechanisms is likely important for the prevention and treatment of AD., Methods: We performed in vivo two-photon calcium imaging to examine the activities of somas, dendrites and dendritic spines of layer 2/3 pyramidal neurons in the primary motor cortex in the APPswe/PS1dE9 mouse model of AD and age-matched wild type control mice. We also performed calcium imaging to determine the effect of Aβ oligomers on dendritic calcium activity. In addition, structural and functional two-photon imaging were used to examine the link between abnormal dendritic calcium activity and changes in dendritic spine size in the AD mouse model., Results: We found that somatic calcium activities of layer 2/3 neurons were significantly lower in the primary motor cortex of 3-month-old APPswe/PS1dE9 mice than in wild type mice during quiet resting, but not during running on a treadmill. Notably, a significantly larger fraction of apical dendrites of layer 2/3 pyramidal neurons showed calcium transients with abnormally long duration and high peak amplitudes during treadmill running in AD mice. Administration of Aβ oligomers into the brain of wild type mice also induced abnormal dendritic calcium transients during running. Furthermore, we found that the activity and size of dendritic spines were significantly reduced on dendritic branches with abnormally prolonged dendritic calcium transients in AD mice., Conclusion: Our findings show that abnormal dendritic calcium transients and synaptic depotentiation occur before amyloid plaque formation in the motor cortex of the APPswe/PS1dE9 mouse model of AD. Dendritic calcium transients with abnormally long durations and high amplitudes could be induced by soluble Aβ oligomers and contribute to synaptic deficits in the early pathogenesis of AD.

Neuropathic pain involves long-lasting modifications of pain pathways that result in abnormal cortical activity. How cortical circuits are altered and contribute to the intense sensation associated with allodynia is unclear. Here we report a persistent elevation of layer V pyramidal neuron activity in the somatosensory cortex of a mouse model of neuropathic pain. This enhanced pyramidal neuron activity was caused in part by increases of synaptic activity and NMDA-receptor-dependent calcium spikes in apical tuft dendrites. Furthermore, local inhibitory interneuron networks shifted their activity in favor of pyramidal neuron hyperactivity: somatostatin-expressing and parvalbumin-expressing inhibitory neurons reduced their activity, whereas vasoactive intestinal polypeptide-expressing interneurons increased their activity. Pharmacogenetic activation of somatostatin-expressing cells reduced pyramidal neuron hyperactivity and reversed mechanical allodynia. These findings reveal cortical circuit changes that arise during the development of neuropathic pain and identify the activation of specific cortical interneurons as therapeutic targets for chronic pain treatment.

It is well established that visual deprivation has a profound impact on the responsiveness of neurons in the developing visual cortex. The effect of visual deprivation on synaptic connectivity remains unclear. Using transcranial two-photon microscopy, we examined the effect of visual deprivation and subsequent recovery on dendritic spine remodeling of layer 5 pyramidal neurons in the mouse primary visual cortex. We found that monocular deprivation (MD), but not binocular deprivation (BD), increased dendritic spine elimination over 3 days in the binocular region of 4-week-old adolescent mice. This MD-induced dendritic spine elimination persisted during subsequent 2-4 days of binocular recovery. Furthermore, we found that average dendritic spine sizes were decreased and increased following 3-day MD and BD, respectively. These spine size changes induced by MD or BD tended to be reversed during subsequent binocular recovery. Taken together, these findings reveal differential effects of MD and BD on synaptic connectivity of layer 5 pyramidal neurons and underscore the persistent impact of MD on synapse loss in the developing visual cortex.

Background: Microglia are known as resident immune cells in the brain. β-amyloid (Aβ) plaques in the brain of Alzheimer's disease (AD) are surrounded by microglia, but whether and how microglia affect the formation and maintenance of plaques remains controversial., Methods: We depleted microglia by injecting diphtheria toxin (DT) in CX 3 CR1 CreER/+ :R26 DTR/+ (CX 3 CR1-iDTR) mice crossed with APPswe/PSEN1dE9 (APP/PS1) mice. Intravital time-lapse imaging was performed to examine changes in the number and size of Congo Red-labeled amyloid plaques over 1-2 weeks. We also examined spine density and shaft diameter of dendrites passing through plaques in a PSAPP mouse model of AD (PS1 M146L line 6.2 × Tg2576) crossed with Thy1 YFP H-line mice., Results: We found that DT administration to CX 3 CR1-iDTR mice efficiently ablated microglia within one week and that microglia repopulated in the second week after DT administration. Microglia depletion didn't affect the number of amyloid plaques, but led to ~13% increase in the size of Aβ plaques within one week. Moreover, microglia repopulation was associated with the stabilization of plaque size during the second week. In addition, we found dendritic spine loss and shaft atrophy in the distal parts of dendrites passing through plaques., Conclusion: Our results demonstrate the important role of microglia in limiting the growth of Aβ plaques and plaque-associated disruption of neuronal connection.

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., Competing Interests: The authors declare no conflict of interest.

The functions and underlying mechanisms of rapid eye movement (REM) sleep remain unclear. Here we show that REM sleep prunes newly formed postsynaptic dendritic spines of layer 5 pyramidal neurons in the mouse motor cortex during development and motor learning. This REM sleep-dependent elimination of new spines facilitates subsequent spine formation during development and when a new motor task is learned, indicating a role for REM sleep in pruning to balance the number of new spines formed over time. Moreover, REM sleep also strengthens and maintains newly formed spines, which are critical for neuronal circuit development and behavioral improvement after learning. We further show that dendritic calcium spikes arising during REM sleep are important for pruning and strengthening new spines. Together, these findings indicate that REM sleep has multifaceted functions in brain development, learning and memory consolidation by selectively eliminating and maintaining newly formed synapses via dendritic calcium spike-dependent mechanisms.

Microglia and peripheral monocytes contribute to hypersensitivity in rodent models of neuropathic pain. However, the precise respective function of microglia and peripheral monocytes has not been investigated in these models. To address this question, here we combined transgenic mice and pharmacological tools to specifically and temporally control the depletion of microglia and monocytes in a mouse model of spinal nerve transection (SNT). We found that although microglia and monocytes are required during the initiation of mechanical allodynia or thermal hyperalgesia, these cells may not be as important for the maintenance of hypersensitivity. Moreover, we demonstrated that either resident microglia or peripheral monocytes are sufficient in gating neuropathic pain after SNT. We propose that resident microglia and peripheral monocytes act synergistically to initiate hypersensitivity and promote the transition from acute to chronic pain after peripheral nerve injury.

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., (Copyright © 2016 the authors 0270-6474/16/362827-16$15.00/0.)

Many lines of evidence indicate that postsynaptic dendritic spines are plastic during development and largely stable in adulthood. It remains unclear to what degree presynaptic axonal terminals undergo changes in the developing and mature cortex. In this study, we examined the formation and elimination of fluorescently-labeled axonal boutons in the living mouse barrel cortex with transcranial two-photon microscopy. We found that the turnover of axonal boutons was significantly higher in 3-week-old young mice than in adult mice (older than 3 months). There was a slight but significant net loss of axonal boutons in mice from 1 to 2 months of age. In both young and adult barrel cortex, axonal boutons existed for at least 1 week were less likely to be eliminated than those recently-formed boutons. In adulthood, 80% of axonal boutons persisted over 12 months and enriched sensory experience caused a slight but not significant increase in the turnover of axonal boutons over 2-4 weeks. Thus, similar to postsynaptic dendritic spines, presynaptic axonal boutons show remarkable stability after development ends. This long-term stability of synaptic connections is likely important for reliable sensory processing in the mature somatosensory cortex., (© 2015 Wiley Periodicals, Inc.)

Previous studies have shown that sensory and motor experiences play an important role in the remodeling of dendritic spines of layer 5 (L5) pyramidal neurons in the cortex. In this study, we examined the effects of sensory deprivation and motor learning on dendritic spine remodeling of layer 2/3 (L2/3) pyramidal neurons in the barrel and motor cortices. Similar to L5 pyramidal neurons, spines on apical dendrites of L2/3 pyramidal neurons are plastic during development and largely stable in adulthood. Sensory deprivation via whisker trimming reduces the elimination rate of existing spines without significant effect on the rate of spine formation in the developing barrel cortex. Furthermore, we show that motor training increases the formation and elimination of dendritic spines in the primary motor cortex. Unlike L5 pyramidal neurons, however, there is no significant difference in the rate of spine formation between sibling dendritic branches of L2/3 pyramidal neurons. Our studies indicate that sensory and motor learning experiences have important impact on dendritic spine remodeling in L2/3 pyramidal neurons. They also suggest that the rules governing experience-dependent spine remodeling are largely similar, but not identical, between L2/3 and L5 pyramidal neurons., (© 2015 Wiley Periodicals, Inc.)

Retinitis pigmentosa, caused predominantly by mutations in photoreceptor genes, currently lacks comprehensive treatment. We discover that retinal microglia contribute non-cell autonomously to rod photoreceptor degeneration by primary phagocytosis of living rods. Using rd10 mice, we found that the initiation of rod degeneration is accompanied by early infiltration of microglia, upregulation of phagocytic molecules in microglia, and presentation of "eat-me" signals on mutated rods. On live-cell imaging, infiltrating microglia interact dynamically with photoreceptors via motile processes and engage in rapid phagocytic engulfment of non-apoptotic rods. Microglial contribution to rod demise is evidenced by morphological and functional amelioration of photoreceptor degeneration following genetic ablation of retinal microglia. Molecular inhibition of microglial phagocytosis using the vitronectin receptor antagonist cRGD also improved morphological and functional parameters of degeneration. Our findings highlight primary microglial phagocytosis as a contributing mechanism underlying cell death in retinitis pigmentosa and implicate microglia as a potential cellular target for therapy., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Severe spinal cord injury (SCI) can cause neurological dysfunction and paralysis. However, the early dynamic changes of neurons and their surrounding environment after SCI are poorly understood. Although methylprednisolone (MP) is currently the standard therapeutic agent for treating SCI, its efficacy remains controversial. The purpose of this project was to investigate the early dynamic changes and MP's efficacy on axonal damage, blood flow, and calcium influx into axons in a mouse SCI model. YFP H-line and Thy1-GCaMP transgenic mice were used in this study. Two-photon microscopy was used for imaging of axonal dieback, blood flow, and calcium influx post-injury. We found that MP treatment attenuated progressive damage of axons, increased blood flow, and reduced calcium influx post-injury. Furthermore, microglia/macrophages accumulated in the lesion site after SCI and expressed the proinflammatory mediators iNOS, MCP-1 and IL-1β. MP treatment markedly inhibited the accumulation of microglia/macrophages and reduced the expression of the proinflammatory mediators. MP treatment also improved the recovery of behavioral function post-injury. These findings suggest that MP exerts a neuroprotective effect on SCI treatment by attenuating progressive damage of axons, increasing blood flow, reducing calcium influx, and inhibiting the accumulation of microglia/macrophages after SCI.

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.

Purpose: The aim of the present study was to explore the use of two-photon microscopy for investigating the therapeutic time window of methylprednisolone (MP) treatment after spinal cord injury (SCI)., Methods: Twenty-four YFP H-line mice were subjected to hemisection SCI and then divided into four groups. Group 1 received MP at 30 min post-injury; group 2 received MP at 8 h post-injury; group 3 received MP at 24 h post-injury; and group 4 received saline at 30 min post-injury. Post-injury axonal dieback was imaged in vivo using two-photon microscopy. After all imaging sessions, histological examination of the surviving neurons and microglial/macrophage accumulation was performed., Results: Two-photon imaging revealed the degree of progressive axon damage after SCI. Group 1 exhibited a shorter axonal dieback distance and slower axonal dieback speed than groups 2, 3, and 4 (p < 0.01). MAP-2 staining revealed greater neuronal survival in group 1 than in groups 2, 3, and 4 (p < 0.05). F4/80 staining revealed greater microglial/macrophage density in groups 2, 3, and 4 than in group 1 (p< 0.05)., Conclusions: MP therapy may help attenuate progressive axon damage, reduce neuronal death, and inhibit microglial/macrophage accumulation, especially when initiated shortly after SCI.

How sleep helps learning and memory remains unknown. We report in mouse motor cortex that sleep after motor learning promotes the formation of postsynaptic dendritic spines on a subset of branches of individual layer V pyramidal neurons. New spines are formed on different sets of dendritic branches in response to different learning tasks and are protected from being eliminated when multiple tasks are learned. Neurons activated during learning of a motor task are reactivated during subsequent non-rapid eye movement sleep, and disrupting this neuronal reactivation prevents branch-specific spine formation. These findings indicate that sleep has a key role in promoting learning-dependent synapse formation and maintenance on selected dendritic branches, which contribute to memory storage., (Copyright © 2014, American Association for the Advancement of Science.)

Study Design: Basic imaging experiment., Objective: To explore the use of 2-photon-excited fluorescence (2PEF) microscopy to investigate the therapeutic effect of methylprednisolone (MP) in mice with spinal cord injury (SCI)., Summary of Background Data: MP can alleviate secondary SCI through its anti-inflammatory effect; however, how MP regulates axonal dynamics in a compression SCI model is not well characterized. We used 2PEF microscopy to trace axonal dynamics in vivo during MP therapy., Methods: Two types of transgenic mice (weighing 23-25 g) including YFP-H line (n = 18) and CX3CR1-GFP (n = 18) were used for experimental procedure. Each type of mouse was randomly divided into 3 groups, and the sample size of every subgroup was 6. The sham groups including YFP-H line group (n = 6) and CX3CR1-GFP group (n = 6) received laminectomy only (group 1). SCI groups received saline treatment (group 2) and SCI groups received MP treatment (group 3). Hind limb motor function was evaluated using the Basso Mouse Scale. 2PEF microscopy was used to image in vivo axonal dynamics at baseline and at 0.5 hours, 24 hours, 48 hours, and 72 hours postinjury. Histology was employed to examine pathological changes and microglial/macrophage proliferation after all imaging sessions., Results: Group 1 exhibited no significant differences in hind limb motor function before versus after surgery. The Basso Mouse Scale scores were significantly lower in groups 2 and 3 than in group 1 (P < 0.05). Degree of recovery was higher in group 3 than in group 2 at 7 days postinjury (P < 0.05). The axons in group 1 remained intact at all time points. The survival rate of axons in groups 2 and 3 progressively decreased at 48 hours postinjury; at 72 hours postinjury, the axon survival rate was higher in group 3 than group 2 (P < 0.05). Histology revealed that group 3 presented milder damage in injured spinal cord than group 2. Microglial/macrophage proliferation was lower in group 3 than in group 2 (P < 0.05)., Conclusion: 2PEF microscopy is useful for detecting early changes, indicating axonal disruption in compression SCI. MP therapy may help alleviate axonal progressive damage and reduce the proliferation of microglia/macrophages in acute SCI., Level of Evidence: N/A.

Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. We showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D₂R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D₂R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics.

Appropriate distribution and supply of mitochondria to critical neuronal sites are thought to be necessary for the normal maintenance of neuronal architecture and activity, including synaptic plasticity and function. Imaging of neurons in vitro has provided understanding of the basic mechanisms of mitochondrial transport and the regulation of mitochondrial dynamics. However, in vivo imaging studies of neurons are preferable to in vitro approaches because of the advantage of being performed in their natural environment. Here, we present useful protocols to image and study axonal transport of mitochondria in vivo, in the peripheral nerves of mice. Imaging in motor and sensory axons of living mice allows researchers to analyze mitochondrial dynamics in two distinct neuronal populations that are often affected in peripheral neuropathies.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)