Hongbo Jia

Dendritic organization of sensory input to cortical neurons in vivo

In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.

Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo two-photon calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.

Calcium imaging in the living brain: prospects for molecular medicine.

Calcium imaging has revolutionized the approaches for functional analyses in the living brain of animal experimental models. Changes in intracellular calcium concentration are strictly linked to the electrical activity in neurons and produce signals that are effectively detected by optical methods. Distinctive features of fluorescence-based calcium imaging are its high temporal resolution in the millisecond range and its high spatial resolution in the micrometer range. Recent progress includes the development of fluorometric calcium sensors, new approaches for targeted labeling with these sensors and the implementation of powerful imaging techniques, especially two-photon microscopy. An important and rapidly evolving field of current research is the use of calcium imaging for the analysis of in vivo mouse models for various brain diseases, such as Alzheimers disease, stroke and epilepsy.

Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator.

Significance We introduce a two-photon imaging method with improved depth penetration for the recording of neuronal activity with single-cell resolution in the intact brain of living animals. This method relies on the use of the fluorometric Ca2+-sensitive dye Cal-590, which is effectively excited by infrared light (1,050 nm). By combining population Ca2+ imaging and electrical recordings in vivo, we demonstrate that neuronal activity can be monitored in all six layers of the mouse cortex. In combination with spectrally different Ca2+ indicators, Cal-590 can be used for the simultaneous imaging of neuronal activity in distinct neuronal populations. In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd = 561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to −900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.

LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5–6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscopes robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

Linear integration of spine Ca2+ signals in layer 4 cortical neurons in vivo

Significance In the mammalian brain, sensory information reaches the cortex through thalamic axons, which provide a strong drive to layer 4 (L4). The thalamic inputs are characterized by their synchronous mode of activation and their high efficacy. Here, by using two-photon imaging in vivo, we analyze sensory-stimulation–evoked calcium transients on the level of individual excitatory synapses located on dendritic spines in L4 spiny stellate cells. We demonstrate that the rapid transfer of peripheral signals to the cortical network is associated with the activation of synapses that are widely dispersed throughout the entire dendritic tree. The dendritic integration of the synaptic responses is linear and does not involve cooperativity between individual synapses. Sensory information reaches the cortex through synchronously active thalamic axons, which provide a strong drive to layer 4 (L4) cortical neurons. Because of technical limitations, the dendritic signaling processes underlying the rapid and efficient activation of L4 neurons in vivo remained unknown. Here we introduce an approach that allows the direct monitoring of single dendritic spine Ca2+ signals in L4 spiny stellate cells of the vibrissal mouse cortex in vivo. Our results demonstrate that activation of N-methyl-D-aspartate (NMDA) receptors is required for sensory-evoked action potential (AP) generation in these neurons. By analyzing NMDA receptor-mediated Ca2+ signaling, we identify whisker stimulation-evoked large responses in a subset of dendritic spines. These sensory-stimulation–activated spines, representing predominantly thalamo-cortical input sites, were denser at proximal dendritic regions. The amplitude of sensory-evoked spine Ca2+ signals was independent of the activity of neighboring spines, without evidence for cooperativity. Furthermore, we found that spine Ca2+ signals evoked by back-propagating APs sum linearly with sensory-evoked synaptic Ca2+ signals. Thus, our results identify in sensory information-receiving L4 cortical neurons a linear mode of dendritic integration that underlies the rapid and reliable transfer of peripheral signals to the cortical network.

Primary Auditory Cortex is Required for Anticipatory Motor Response

The ability of the brain to predict future events based on the pattern of recent sensory experience is critical for guiding animals behavior. Neocortical circuits for ongoing processing of sensory stimuli are extensively studied, but their contributions to the anticipation of upcoming sensory stimuli remain less understood. We, therefore, used in vivo cellular imaging and fiber photometry to record mouse primary auditory cortex to elucidate its role in processing anticipated stimulation. We found neuronal ensembles in layers 2/3, 4, and 5 which were activated in relationship to anticipated sound events following rhythmic stimulation. These neuronal activities correlated with the occurrence of anticipatory motor responses in an auditory learning task. Optogenetic manipulation experiments revealed an essential role of such neuronal activities in producing the anticipatory behavior. These results strongly suggest that the neural circuits of primary sensory cortex are critical for coding predictive information and transforming it into anticipatory motor behavior.

The Catalytic Effect of Modified Bentonite on the Pyrolysis of Oily Sludge

To raise the recovery of oil during pyrolysis, reactions were performed on oily sludge using catalysts such as a molecular sieve and an Fe-containing molecular sieve catalyst in a tubular furnace reactor. The catalysts were prepared through hydrothermal method and characterized by various characterization methods. The results of the sample for the test have shown that more time could significantly increase the oil yield. The rate of oil recovery was increased with the increase of the catalytic in the initial phase and decreased at the back of the stage. In addition, higher temperature contributed to higher oil production, and at the same time the optimal ratio of molecular sieve catalytic to sludge was found to be 0.035. Research has revealed that the content of oil recovery rate could reach up to 85.52%. The effects of catalytic on the recovery improve rate of oil in oily sludge samples presented the following decreasing order of the molecular sieve and the Fe-containing molecular sieve.

Targeted Patching and Dendritic Ca 2+ Imaging in Nonhuman Primate Brain in vivo

Nonhuman primates provide an important model not only for understanding human brain but also for translational research in neurological and psychiatric disorders. However, many high-resolution techniques for recording neural activity in vivo that were initially established for rodents have not been yet applied to the nonhuman primate brain. Here, we introduce a combination of two-photon targeted patching and dendritic Ca2+ imaging to the neocortex of adult common marmoset, an invaluable primate model for neuroscience research. Using targeted patching, we show both spontaneous and sensory-evoked intracellular dynamics of visually identified neurons in the marmoset cortex. Using two-photon Ca2+ imaging and intracellular pharmacological manipulation, we report both action-potential-associated global and synaptically-evoked NMDA (N-methyl-D-aspartate) receptor-mediated local Ca2+ signals in dendrites and spines of the superficial-layer cortical neurons. Therefore, we demonstrate the presence of synaptic Ca2+ signals in neuronal dendrites in living nonhuman primates. This work represents a proof-of-principle for exploring the primate brain functions in vivo by monitoring neural activity and morphology at a subcellular resolution.