N-Cofilin Can Compensate for the Loss of ADF in Excitatory Synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content

Spatiotemporal Properties of the Action Potential Propagation in the Mouse Visual Cortical Slice Analyzed by Calcium Imaging

The calcium ion (Ca2+) is an important messenger for signal transduction, and the intracellular Ca2+ concentration ([Ca2+]i) changes in response to an excitation of the cell. To reveal the spatiotemporal properties of the propagation of an excitatory signal with action potentials in the primary visual cortical circuit, we conducted a Ca2+ imaging study on slices of the mouse visual cortex. Electrical stimulation of layer 4 evoked [Ca2+]i transients around the stimulus electrode. Subsequently, the high [Ca2+]i region mainly propagated perpendicular to the cortical layer (vertical propagation), with horizontal propagation being restricted. When the excitatory synaptic transmission was blocked, only weak and concentric [Ca2+]i transients were observed. When the action potential was blocked, the [Ca2+]i transients disappeared almost completely. These results suggested that the action potential contributed to the induction of the [Ca2+]i transients, and that excitatory synaptic connections were involved in the propagation of the high [Ca2+]i region in the primary visual cortical circuit. To elucidate the involvement of inhibitory synaptic connections in signal propagation in the primary visual cortex, the GABAA receptor inhibitor bicuculline was applied. In this case, the evoked signal propagated from layer 4 to the entire field of view, and the prolonged [Ca2+]i transients were observed compared with the control condition. Our results suggest that excitatory neurons are widely connected to each other over the entire primary visual cortex with recurrent synapses, and inhibitory neurons play a fundamental role in the organization of functional sub-networks by restricting the propagation of excitation signals

Subdivisions of the Auditory Midbrain (N. Mesencephalicus Lateralis, pars dorsalis) in Zebra Finches Using Calcium-Binding Protein Immunocytochemistry

The midbrain nucleus mesencephalicus lateralis pars dorsalis (MLd) is thought to be the avian homologue of the central nucleus of the mammalian inferior colliculus. As such, it is a major relay in the ascending auditory pathway of all birds and in songbirds mediates the auditory feedback necessary for the learning and maintenance of song. To clarify the organization of MLd, we applied three calcium binding protein antibodies to tissue sections from the brains of adult male and female zebra finches. The staining patterns resulting from the application of parvalbumin, calbindin and calretinin antibodies differed from each other and in different parts of the nucleus. Parvalbumin-like immunoreactivity was distributed throughout the whole nucleus, as defined by the totality of the terminations of brainstem auditory afferents; in other words parvalbumin-like immunoreactivity defines the boundaries of MLd. Staining patterns of parvalbumin, calbindin and calretinin defined two regions of MLd: inner (MLd.I) and outer (MLd.O). MLd.O largely surrounds MLd.I and is distinct from the surrounding intercollicular nucleus. Unlike the case in some non-songbirds, however, the two MLd regions do not correspond to the terminal zones of the projections of the brainstem auditory nuclei angularis and laminaris, which have been found to overlap substantially throughout the nucleus in zebra finches

Multidimensional Characterization and Differentiation of Neurons in the Anteroventral Cochlear Nucleus

Multiple parallel auditory pathways ascend from the cochlear nucleus. It is generally accepted that the origin of these pathways are distinct groups of neurons differing in their anatomical and physiological properties. In extracellular in vivo recordings these neurons are typically classified on the basis of their peri-stimulus time histogram. In the present study we reconsider the question of classification of neurons in the anteroventral cochlear nucleus (AVCN) by taking a wider range of response properties into account. The study aims at a better understanding of the AVCN's functional organization and its significance as the source of different ascending auditory pathways. The analyses were based on 223 neurons recorded in the AVCN of the Mongolian gerbil. The range of analysed parameters encompassed spontaneous activity, frequency coding, sound level coding, as well as temporal coding. In order to categorize the unit sample without any presumptions as to the relevance of certain response parameters, hierarchical cluster analysis and additional principal component analysis were employed which both allow a classification on the basis of a multitude of parameters simultaneously. Even with the presently considered wider range of parameters, high number of neurons and more advanced analytical methods, no clear boundaries emerged which would separate the neurons based on their physiology. At the current resolution of the analysis, we therefore conclude that the AVCN units more likely constitute a multi-dimensional continuum with different physiological characteristics manifested at different poles. However, more complex stimuli could be useful to uncover physiological differences in future studies