Intravital imaging of hair follicle regeneration in the mouse.

Hair follicles are mammalian skin organs that periodically and stereotypically regenerate from a small pool of stem cells. Hence, hair follicles are a widely studied model for stem cell biology and regeneration. This protocol describes the use of two-photon laser-scanning microscopy (TPLSM) to study hair regeneration within a living, uninjured mouse. TPLSM provides advantages over conventional approaches, including enabling time-resolved imaging of single hair follicle stem cells. Thus, it is possible to capture behaviors including apoptosis, proliferation and migration, and to revisit the same cells for in vivo lineage tracing. In addition, a wide range of fluorescent reporter mouse lines facilitates TPLSM in the skin. This protocol also describes TPLSM laser ablation, which can spatiotemporally manipulate specific cellular populations of the hair follicle or microenvironment to test their regenerative contributions. The preparation time is variable depending on the goals of the experiment, but it generally takes 30-60 min. Imaging time is dependent on the goals of the experiment. Together, these components of TPLSM can be used to develop a comprehensive understanding of hair regeneration during homeostasis and injury.

Chronic cellular imaging of entire cortical columns in awake mice using microprisms.

Two-photon imaging of cortical neurons in vivo has provided unique insights into the structure, function, and plasticity of cortical networks, but this method does not currently allow simultaneous imaging of neurons in the superficial and deepest cortical layers. Here, we describe a simple modification that enables simultaneous, long-term imaging of all cortical layers. Using a chronically implanted glass microprism in barrel cortex, we could image the same fluorescently labeled deep-layer pyramidal neurons across their entire somatodendritic axis for several months. We could also image visually evoked and endogenous calcium activity in hundreds of cell bodies or long-range axon terminals, across all six layers in visual cortex of awake mice. Electrophysiology and calcium imaging of evoked and endogenous activity near the prism face were consistent across days and comparable with previous observations. These experiments extend the reach of in vivo two-photon imaging to chronic, simultaneous monitoring of entire cortical columns.

Selective functional interactions between excitatory and inhibitory cortical neurons and differential contribution to persistent activity of the slow oscillation.

The neocortex depends upon a relative balance of recurrent excitation and inhibition for its operation. During spontaneous Up states, cortical pyramidal cells receive proportional barrages of excitatory and inhibitory synaptic potentials. Many of these synaptic potentials arise from the activity of nearby neurons, although the identity of these cells is relatively unknown, especially for those underlying the generation of inhibitory synaptic events. To address these fundamental questions, we developed an in vitro submerged slice preparation of the mouse entorhinal cortex that generates robust and regular spontaneous recurrent network activity in the form of the slow oscillation. By performing whole-cell recordings from multiple cell types identified with green fluorescent protein expression and electrophysiological and/or morphological properties, we show that distinct functional subpopulations of neurons exist in the entorhinal cortex, with large variations in contribution to the generation of balanced excitation and inhibition during the slow oscillation. The most active neurons during the slow oscillation are excitatory pyramidal and inhibitory fast spiking interneurons, receiving robust barrages of both excitatory and inhibitory synaptic potentials. Weak action potential activity was observed in stellate excitatory neurons and somatostatin-containing interneurons. In contrast, interneurons containing neuropeptide Y, vasoactive intestinal peptide, or the 5-hydroxytryptamine (serotonin) 3a receptor, were silent. Our data demonstrate remarkable functional specificity in the interactions between different excitatory and inhibitory cortical neuronal subtypes, and suggest that it is the large recurrent interaction between pyramidal neurons and fast spiking interneurons that is responsible for the generation of persistent activity that characterizes the depolarized states of the cortex.

The columnar and laminar organization of inhibitory connections to neocortical excitatory cells.

The cytoarchitectonic similarities of different neocortical regions have given rise to the idea of 'canonical' connectivity between excitatory neurons of different layers within a column. It is unclear whether similarly general organizational principles also exist for inhibitory neocortical circuits. Here we delineate and compare local inhibitory-to-excitatory wiring patterns in all principal layers of primary motor (M1), somatosensory (S1) and visual (V1) cortex, using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derived largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B received extensive translaminar inhibition. These neurons were prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex and absent in M1. Although inhibitory connection patterns were stereotypical, the abundance of individual motifs varied between regions and cells, potentially reflecting functional specializations.

A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis.

Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.

A mechanism intrinsic to the vesicle fusion machinery determines fast and slow transmitter release at a large CNS synapse.

Heterogeneity of release probability p between vesicles in the readily releasable pool (RRP) is expected to strongly influence the kinetics of depression at synapses, but the underlying mechanism(s) are not well understood. To test whether differences in the intrinsic Ca2+ sensitivity of vesicle fusion might cause heterogeneity of p, we made presynaptic Ca2+-uncaging measurements at the calyx of Held and analyzed the time course of transmitter release by EPSC deconvolution. Ca2+ uncaging, which produced spatially homogeneous elevations of [Ca2+]i, evoked a fast and a slow component of release over a wide range of [Ca2+]i, showing that mechanism(s) intrinsic to the vesicle fusion machinery cause fast and slow transmitter release. Surprisingly, the number of vesicles released in the fast component increased with Ca2+-uncaging stimuli of larger amplitudes, a finding that was most obvious below approximately 10 microM [Ca2+]i and that we call "submaximal release" of fast-releasable vesicles. During trains of action potential-like presynaptic depolarizations, submaximal release was also observed as an increase in the cumulative fast release at enhanced release probabilities. A model that assumes two separate subpools of RRP vesicles with different intrinsic Ca2+ sensitivities predicted the observed Ca2+ dependencies of fast and slow transmitter release but could not fully account for submaximal release. Thus, fast and slow transmitter release in response to prolonged [Ca2+]i elevations is caused by intrinsic differences between RRP vesicles, and an "a posteriori" reduction of the Ca2+ sensitivity of vesicle fusion after the onset of the stimulus might cause submaximal release of fast-releasable vesicles and contribute to short-term synaptic depression.

The synaptic vesicle protein CSP alpha prevents presynaptic degeneration.

Cysteine string protein alpha (CSPalpha)--an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40 chaperones--is thought to regulate Ca2+ channels and/or synaptic vesicle exocytosis. We now show that, in young mice, deletion of CSPalpha does not impair survival and causes no significant changes in presynaptic Ca2+ currents or synaptic vesicle exocytosis as measured in the Calyx of Held synapse. At 2-4 weeks of age, however, CSPalpha-deficient mice develop a progressive, fatal sensorimotor disorder. The neuromuscular junctions and Calyx synapses of CSPalpha-deficient mice exhibit increasing neurodegenerative changes, synaptic transmission becomes severely impaired, and the mutant mice die at approximately 2 months of age. Our data suggest that CSPalpha is not essential for the normal operation of Ca2+ channels or exocytosis but acts as a presynaptic chaperone that maintains continued synaptic function, raising the possibility that enhanced CSPalpha function could attenuate neurodegenerative diseases.

Presynaptic capacitance measurements and Ca2+ uncaging reveal submillisecond exocytosis kinetics and characterize the Ca2+ sensitivity of vesicle pool depletion at a fast CNS synapse.

The intracellular Ca2+ sensitivity of synaptic vesicle fusion is an important determinant of transmitter release probability, but it is unknown for most CNS synapses. We combined whole-cell membrane capacitance measurements and Ca2+ uncaging at the large calyx of Held nerve terminals to determine the Ca2+ sensitivity of synaptic vesicle fusion at a glutamatergic CNS synapse, independent of recording EPSCs. Capacitance increases measured 30-50 msec after elevating the intracellular Ca2+ concentration ([Ca2+]i) by Ca2+ uncaging were half-maximal at approximately 5 microm [Ca2+]i. At 10 microm [Ca2+]i, capacitance increases reached maximal values (256 +/- 125 fF; mean +/- SD), indicating the depletion of an average pool of approximately 4000 readily releasable vesicles. Vesicle pool depletion was confirmed in cross-depletion experiments, in which capacitance responses were measured after Ca2+ uncaging, or after combined stimuli of prolonged presynaptic depolarizations and Ca2+ uncaging. To analyze the Ca2+-dependent rates of vesicle pool depletion, the capacitance rise after Ca2+ uncaging was fitted with single- or double-exponential functions. The fast time constants of double-exponential fits, and the time constants of single-exponential fits were 2-3 msec at 10-15 microm [Ca2+]i and reached submillisecond values at 30 microm [Ca2+]i. These results suggest that three to five readily releasable vesicles can fuse within <1 msec at each active zone of a calyx of Held, given that [Ca2+]i rises sufficiently high. Submillisecond kinetics of exocytosis are reached at significantly lower [Ca2+]i than at ribbon-type sensory synapses previously investigated by capacitance measurements.