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.

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.

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 cortex (V1), using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derive largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B receive extensive translaminar inhibition. These neurons are prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex, and absent in M1. Although inhibitory connection patterns are stereotypical, the abundance of individual motifs varies between regions and cells, potentially reflecting functional specializations.

Summary 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 deeplayer 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.

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 Ca

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.

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.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008