Benjamin Odermatt

Distinct Types of Astroglial Cells in the Hippocampus Differ in Gap Junction Coupling

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA‐type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter‐controlled EGFP expression and patch‐clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing β‐Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP‐EGFP‐positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes.

A genetically encoded reporter of synaptic activity in vivo.

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.

Selective Permeability of Different Connexin Channels to the Second Messenger Cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte-astrocyte gap junctions

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO‐1 and ZONAB. Here, we investigated the localization of multi‐PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47‐knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild‐type mice. These MUPP1‐positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte–astrocyte (O/A) gap junctions, but were not found at astrocyte–astrocyte gap junctions. In Cx47‐knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO‐1‐positive puncta remained. In Cx32‐knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47‐knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47‐dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47‐knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.

Encoding of Luminance and Contrast by Linear and Nonlinear Synapses in the Retina

Summary Understanding how neural circuits transmit information is technically challenging because the neural code is contained in the activity of large numbers of neurons and synapses. Here, we use genetically encoded reporters to image synaptic transmission across a population of sensory neurons—bipolar cells in the retina of live zebrafish. We demonstrate that the luminance sensitivities of these synapses varies over 104 with a log-normal distribution. About half the synapses made by ON and OFF cells alter their polarity of transmission as a function of luminance to generate a triphasic tuning curve with distinct maxima and minima. These nonlinear synapses signal temporal contrast with greater sensitivity than linear ones. Triphasic tuning curves increase the dynamic range over which bipolar cells signal light and improve the efficiency with which luminance information is transmitted. The most efficient synapses signaled luminance using just 1 synaptic vesicle per second per distinguishable gray level.

Computational processing of optical measurements of neuronal and synaptic activity in networks

Imaging of optical reporters of neural activity across large populations of neurones is a widely used approach for investigating the function of neural circuits in slices and in vivo. Major challenges in analysing such experiments include the automatic identification of neurones and synapses, extraction of dynamic signals, and assessing the temporal and spatial relationships between active units in relation to the gross structure of the circuit. We have developed an integrated set of software tools, named SARFIA, by which these aspects of dynamic imaging experiments can be analysed semi-automatically. Key features are image-based detection of structures of interest using the Laplace operator, determining the positions of units in a layered network, clustering algorithms to classify units with similar functional responses, and a database to store, exchange and analyse results across experiments. We demonstrate the use of these tools to analyse synaptic activity in the retina of live zebrafish by multi-photon imaging of SyGCaMP2, a genetically encoded synaptically localised calcium reporter. By simultaneously recording activity across tens of bipolar cell terminals distributed throughout the IPL we made a functional map of the ON and OFF signalling channels and found that these were only partially separated. The automated detection of signals across many neurones in the retina allowed the reliable detection of small populations of neurones generating “ectopic” signals in the “ON” and “OFF” sublaminae. This software should be generally applicable for the analysis of dynamic imaging experiments across hundreds of responding units.

Synaptic mechanisms of adaptation and sensitization in the retina

Sensory systems continually adjust the way stimuli are processed. What are the circuit mechanisms underlying this plasticity? We investigated how synapses in the retina of zebrafish adjust to changes in the temporal contrast of a visual stimulus by imaging activity in vivo. Following an increase in contrast, bipolar cell synapses with strong initial responses depressed, whereas synapses with weak initial responses facilitated. Depression and facilitation predominated in different strata of the inner retina, where bipolar cell output was anticorrelated with the activity of amacrine cell synapses providing inhibitory feedback. Pharmacological block of GABAergic feedback converted facilitating bipolar cell synapses into depressing ones. These results indicate that depression intrinsic to bipolar cell synapses causes adaptation of the ganglion cell response to contrast, whereas depression in amacrine cell synapses causes sensitization. Distinct microcircuits segregating to different layers of the retina can cause simultaneous increases or decreases in the gain of neural responses.

Clathrin-mediated endocytosis : the physiological mechanism of vesicle retrieval at hippocampal synapses

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin‐mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called ‘kiss‐and‐run’, which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of ‘kiss‐and‐run’ in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis (τ= 15 s) operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C‐terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin‐mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.

Imaging phluorin-based probes at hippocampal synapses.

Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles.

Comment on "The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles".

Zhang et al. (Research Articles, 13 March 2009, p. 1448) reported that synaptic vesicles usually release neurotransmitter through a kiss-and-run mechanism occurring within 1 second but that full collapse of the vesicles becomes more prevalent with repeated stimuli. We report that the kinetics of vesicle retrieval do not change during a stimulus train, with endocytosis occurring in 10 to 15 seconds.