Guilherme Neves

Synaptic plasticity, memory and the hippocampus: a neural network approach to causality

Two facts about the hippocampus have been common currency among neuroscientists for several decades. First, lesions of the hippocampus in humans prevent the acquisition of new episodic memories; second, activity-dependent synaptic plasticity is a prominent feature of hippocampal synapses. Given this background, the hypothesis that hippocampus-dependent memory is mediated, at least in part, by hippocampal synaptic plasticity has seemed as cogent in theory as it has been difficult to prove in practice. Here we argue that the recent development of transgenic molecular devices will encourage a shift from mechanistic investigations of synaptic plasticity in single neurons towards an analysis of how networks of neurons encode and represent memory, and we suggest ways in which this might be achieved. In the process, the hypothesis that synaptic plasticity is necessary and sufficient for information storage in the brain may finally be validated.

Stochastic yet biased expression of multiple Dscam splice variants by individual cells

The Drosophila melanogaster gene Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. We analyzed the Dscam mRNA isoforms expressed by different cell types and individual cells. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR documented that individual cells express several different Dscam isoforms and allowed an estimation of the diversity that is present. For example, we estimate that each R3/R4 photoreceptor cell expresses 14–50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of its cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere.

Analysis of Dscam Diversity in Regulating Axon Guidance in Drosophila Mushroom Bodies

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.

The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells

1 The kinetics of exocytosis and endocytosis were studied in the giant synaptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane surface area, and fluorescence measurements of exocytosis using the membrane dye FM1‐43. 2 Three phases of exocytosis occurred during maintained depolarization to 0 mV. The first component was complete within about 10 ms and involved a pool of 1200–1800 vesicles (with a total membrane area equivalent to about 1.6% of the surface of the terminal). The second component of exocytosis involved the release of about 4400 vesicles over 1 s. The third component of exocytosis was stimulated continuously at a rate of about 1000 vesicles s−1. 3 After short depolarizations (< 200 ms), neither the FM1‐43 signal nor the capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca2+ channels. The fall in capacitance could therefore be used to monitor endocytosis independently of exocytosis. The capacitance measured after brief stimuli began to fall immediately, recovering to the pre‐stimulus baseline with a rate constant of 0.8 s−1. 4 The amount of exocytosis measured using the capacitance and FM1‐43 techniques was similar during the first 200 ms of depolarization, suggesting that the most rapidly released vesicles could be detected by either method. 5 After a few seconds of continuous stimulation, the net increase in membrane surface area reached a plateau at about 5%, even though continuous exocytosis occurred at a rate of 0.9% s−1. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0.2 s−1. The average rate of endocytosis during maintained depolarization was therefore considerably slower than the rate observed after a brief stimulus. 6 After longer depolarizations (> 500 ms), both the capacitance and FM1‐43 signals continued to rise for periods of seconds after closure of Ca2+ channels. The continuation of exocytosis was correlated with a persistent increase in [Ca2+]i in the synaptic terminal, as indicated by the activation of a Ca2+‐dependent conductance and measurements of [Ca2+]i using the fluorescent indicator furaptra. 7 The delayed fall in membrane capacitance after longer depolarizations occurred along a double exponential time course indicating the existence of two endocytic processes: fast endocytosis, with a rate constant of 0.8 s−1, and slow endocytosis, with a rate constant of 0.1 s−1. 8 Increasing the duration of depolarization caused an increase in the fraction of membrane recovered by slow endocytosis. After a 100 ms stimulus, all the membrane was recycled by fast endocytosis, but after a 5 s depolarization, about 50% of the membrane was recycled by slow endocytosis. 9 These results demonstrate the existence of fast and slow endocytic mechanisms at a synapse and support the idea that prolonged stimulation leads to an increase in the amount of membrane retrieved by the slower route. The rise in cytoplasmic Ca2+ that occurred during longer depolarizations was correlated with stimulation of continuous exocytosis and inhibition of fast endocytosis. The results also confirm that transient and continuous components of exocytosis coexist in the synaptic terminal of depolarizing bipolar cells.

Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells

To investigate the regulation of endocytosis by Ca2+, we have made capacitance measurements in the synaptic terminal of depolarizing bipolar cells from the retina of goldfish. After a brief depolarization, all of the excess membrane was retrieved rapidly (τ ≈1 s). But when the rise in free [Ca2+] was reduced by the introduction of Ca2+ buffers [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) or EGTA], a large fraction of the membrane was retrieved by a second, slower mechanism (τ ≥ 10 s). The block of fast endocytosis by EGTA could be overcome by increasing the amplitude of the Ca2+ current, demonstrating that Ca2+ influx was the trigger for fast endocytosis. These manipulations of the Ca2+ signal altered the relative proportions of fast and slow endocytosis but did not modulate the rate constants of these processes. A brief stimulus that triggered fast endocytosis did not generate a significant rise in the spatially averaged [Ca2+], indicating that Ca2+ regulated endocytosis through an action close to the active zone. The slow mode of retrieval occurred at the resting [Ca2+]. These results demonstrate that Ca2+ influx couples fast endocytosis and exocytosis at this synapse.

Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells.

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

The actions of barium and strontium on exocytosis and endocytosis in the synaptic terminal of goldfish bipolar cells.

1 We investigated the properties of Ca2+‐sensitive steps in the cycling of synaptic vesicles by comparing the actions of Ca2+, Ba2+ and Sr2+ in the synaptic terminal of depolarizing bipolar cells isolated from the retina of goldfish. FM1‐43 fluorescence and capacitance measurements demonstrated that exocytosis, endocytosis and vesicle mobilization were maintained when external Ca2+ was replaced by either Ba2+ or Sr2+. 2 The rapidly releasable pool of vesicles (RRP) was equivalent to 1.5 % of the membrane surface area when measured in the presence of 2.5 mm Ca2+, but only 0.4 % in 2.5 mm Sr2+. The relative sizes of the RRP in Ca2+, Sr2+ and Ba2+ were 1.0, 0.28 and 0.1, respectively. We conclude that a smaller proportion of docked vesicles are available for fast exocytosis triggered by the influx of Sr2+ or Ba2+ compared to Ca2+. 3 The slow phase of exocytosis was not altered when Ca2+ was replaced by Ba2+, but it was accelerated 1.6‐fold in Sr2+. The peak concentrations of Ca2+, Sr2+ and Ba2+ (measured using Mag‐fura‐5) were ≈4, ≈14 and ≈60 μm, respectively. The order of efficiency for the stimulation of slow exocytosis was Ca2+≈ Sr2+ > Ba2+. 4 Exocytosis was prolonged after the influx of Sr2+ and Ba2+. Sr2+ was cleared from the synaptic terminal with the same time constant as Ca2+ (1.3 s), but Ba2+ was cleared 10‐100 times more slowly. Although Ba2+ stimulates the slow release of a large number of vesicles, it did so less efficiently than Ca2+ or Sr2+. 5 The recovery of the membrane capacitance was equally rapid in Sr2+ and Ca2+, demonstrating that the fast mode of endocytosis could be triggered by either cation.

The LIM Homeodomain Protein Lhx6 Regulates Maturation of Interneurons and Network Excitability in the Mammalian Cortex

Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst+ interneurons, but relatively minor and localized changes in Pva+ interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst+ interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

Transcription factor LIM homeobox 7 (Lhx7) maintains subtype identity of cholinergic interneurons in the mammalian striatum

The generation and maintenance of a plethora of neuronal subtypes is essential for normal brain function. Nevertheless, little is known about the molecular mechanisms that maintain the defining characteristics of neurons following their initial postmitotic specification. Using conditional gene ablation in mice, we demonstrate here that the homeodomain protein LIM homeobox (Lhx)7 is essential for maintaining the morphological and molecular characteristics of cholinergic interneurons of the striatum. Lhx7-depleted cholinergic interneurons extinguish expression of several subtype-specific markers, including choline acetyl transferase and Isl1, and are respecified into Lhx6-expressing mature GABAergic interneurons. Additional expression studies support a model where Lhx7 controls the choice between cholinergic or GABAergic identity by gating a cross inhibitory regulation between Isl1 and Lhx6. By demonstrating that the switch between alternative striatal interneuron fates depends on persistent activity of a single transcription factor, we provide evidence that the intrinsic plasticity of mammalian forebrain neuronal subtypes is maintained after the initial specification and lineage commitment and possibly throughout life.

The variable transmembrane domain of Drosophila N-cadherin regulates adhesive activity

ABSTRACT Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.