Faculty Bibliography

BACKGROUND:transients in neuronal somata, dendrites, and synapses. NEW METHOD/UNASSIGNED: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: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)/UNASSIGNED:These transgenic lines allows calcium 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.

Mitochondrial calcium ([Ca²⁺]_mito) dynamics plays vital roles in regulating fundamental cellular and organellar functions including bioenergetics. However, neuronal [Ca²⁺]_mito dynamics in vivo and its regulation by brain activity are largely unknown. By performing two-photon Ca²⁺ imaging in the primary motor (M1) and visual cortexes (V1) of awake behaving mice, we find that discrete [Ca²⁺]_mito transients occur synchronously over somatic and dendritic mitochondrial network, and couple with cytosolic calcium ([Ca²⁺]_cyto) transients in a probabilistic, rather than deterministic manner. The amplitude, duration, and frequency of [Ca²⁺]_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, Ca²⁺/calmodulin kinase II is mechanistically involved in modulating the dynamic coupling process. Thus, activity-dependent dynamic [Ca²⁺]_mito-to-[Ca²⁺]_cyto coupling affords an important mechanism whereby [Ca²⁺]_mito decodes brain activity for the regulation of mitochondrial bioenergetics to meet fluctuating neuronal energy demands as well as for neuronal information processing.

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.

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.

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.

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, 2018.

Dendritic spines are the postsynaptic sites of most excitatory synapses in the mammalian brain. In vivo imaging of dendritic spines in the mouse cerebral cortex indicates that spines are highly plastic during development and become remarkably stable in adulthood. In my presentation, I will discuss how learning experiences and subsequent sleep regulate the plasticity and maintenance of dendritic spines. Because dendritic spines are the key elements for information acquisition and retention, understanding how they are regulated by sleep in the living brain provides important insights into the functions of sleep in learning and memory

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.