Biomedical subjects

Jack Waters

Publications and source records attributed to Jack Waters.

12 recordsLinked to original sources

PubMed2006

Postsynaptic excitability is necessary for strengthening of cortical sensory responses during experience-dependent development.

Sensory experience is necessary for normal cortical development. This has been shown by sensory deprivation and pharmacological perturbation of the cortex. Because these manipulations affect the cortical network as a whole, the role of postsynaptic cellular properties during experience-dependent development is unclear. Here we addressed the developmental role of somatodendritic excitability, which enables postsynaptic spike timing-dependent forms of plasticity, in rat somatosensory cortex. We used short interfering RNA (siRNA)-based knockdown of Na+ channels to suppress the somatodendritic excitability of small numbers of layer 2/3 pyramidal neurons in the barrel cortex, without altering the ascending sensory pathway. In vivo recordings from siRNA-expressing cells revealed that this manipulation interfered with the normal developmental strengthening of sensory responses. The sensory responsiveness of neighboring cortical neurons was unchanged, indicating that the cortical network was unchanged. We conclude that somatodendritic excitability of the postsynaptic neuron is needed for the regulation of synaptic strength in the developing sensory cortex.

Animals↗

PubMed2006

Background synaptic activity is sparse in neocortex.

Neurons are continually exposed to background synaptic activity in vivo. This is thought to influence neural information processing, but background levels of excitation and inhibition remain controversial. Here we show, using whole-cell recordings in anesthetized rats, that spontaneous depolarizations ("Up states") in neocortical pyramidal neurons are driven by sparse, mostly excitatory synaptic activity (less than five inputs per millisecond; approximately 10% inhibitory). The mean synaptic conductance change is small (<10 nS at the soma) and opposed by anomalous rectification, resulting in a net increase in input resistance during Up states. These conditions enhance the effectiveness of each synapse at depolarized potentials. Hence, neocortical networks are relatively quiet at rest, and the effect of synaptic background is weaker than previously thought.

Action Potentials↗

PubMed2005

Backpropagating action potentials in neurones: measurement, mechanisms and potential functions.

Here we review some properties and functions of backpropagating action potentials in the dendrites of mammalian CNS neurones. We focus on three main aspects: firstly the current techniques available for measuring backpropagating action potentials, secondly the morphological parameters and voltage gated ion channels that determine action potential backpropagation and thirdly the potential functions of backpropagating action potentials in real neuronal networks.

Action Potentials↗

PubMed2004

Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo.

It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from alpha-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopy directed whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

Animals↗

PubMed2004

Boosting of action potential backpropagation by neocortical network activity in vivo.

Action potentials backpropagate into the dendritic trees of pyramidal neurons, reporting output activity to the sites of synaptic input and provoking long-lasting changes in synaptic strength. It is unclear how this retrograde signal is modified by neural network activity. Using whole-cell recordings from somata, apical trunks, and dendritic tuft branches of layer 2/3 pyramidal neurons in vivo, we show that network-driven subthreshold membrane depolarizations ("up states") occur simultaneously throughout the apical dendritic tree. This spontaneous synaptic activity enhances action potential-evoked calcium influx into the distal apical dendrite by promoting action potential backpropagation. Hence, somatic feedback to the dendrites becomes stronger with increasing network activity.

Action Potentials↗

PubMed2004

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

Animals↗

PubMed2004

Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons.

Sindbis virus-based vectors have been successfully used for transient heterologous protein expression in neurons. Their main limitation arises from infection-associated cytotoxicity, attributed largely to a progressive shut down of host cell protein synthesis. Here we evaluated a modified Sindbis vector, based on a viral strain containing a point mutation in the second nonstructural protein, nsP2 P726S, described to delay inhibition of protein synthesis in BHK cells [Virology 228 (1997) 74], for heterologous expression in neurons in vitro and in vivo. First, we constructed an optimized helper vector, termed DH-BB(tRNA/TE12), for production of SINrep(nsP2S(726)) viral particles with low levels of helper RNA co-packaging and high neurospecificity of infection. Second, we determined that hippocampal primary neurons infected with SINrep(nsP2S(726)) virus expressing EGFP showed a delayed onset of viral induced cytotoxicity and higher levels of EGFP expression in comparison to cells infected with wild type SINrep5 EGFP-expressing virus. However, a strong decrease in protein synthesis still occurred by day 3 postinfection. The SINrep(nsP2S(726)) vector is thus well suited for rapid high level expression within this time window. As an experimental example, we demonstrate the applicability of this system for high-resolution two-photon imaging of dendritic spines in vivo.

Animals↗

PubMed2003

Mitochondria and release at hippocampal synapses.

Mitochondria are present in some, but not all presynaptic terminals in the hippocampus. Mitochondria are capable of sequestering and storing large amounts of calcium, but it is unclear whether they influence release probability at these synapses. Using FM dye imaging techniques and confocal microscopy, we have examined the relationship between mitochondrial presence/absence and presynaptic vesicle release from rat hippocampal neurones in primary dissociated culture at room temperature. Following staining with the mitochondrial dye mitotracker green, we were able to resolve putative individual mitochondria associated with neuronal processes. The majority of mitochondria were positionally stable, although some exhibited periods of rapid motility (up to 0.4 microm/s) interspersed with periods of immobility. Co-staining with mitotracker green and the synaptic vesicle dye FM 4-64 indicated that 180 of 506 (36%) synapses were devoid of mitochondria. A comparison of vesicular release in response to stimulation at 1 Hz and at 10 Hz revealed no differences in release properties between synapses with and without mitochondria.

Animals↗

PubMed2003

Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo.

Pyramidal neurons in layer 2/3 of the neocortex are central to cortical circuitry, but the intrinsic properties of their dendrites are poorly understood. Here we study layer 2/3 apical dendrites in parallel experiments in acute brain slices and in anesthetized rats using whole-cell recordings and Ca2+ imaging. We find that backpropagation of action potentials into the dendritic arbor is actively supported by Na+ channels both in vitro and in vivo. Single action potentials evoke substantial Ca2+ influx in the apical trunk but little or none in the dendritic tuft. Supralinear Ca2+ influx is produced in the tuft, however, when an action potential is paired with synaptic input. This dendritic supralinearity enables layer 2/3 neurons to integrate ascending sensory input from layer 4 and associative input to layer 1.

Action Potentials↗

PubMed2002

Ca2+ imaging in the mammalian brain in vivo.

Changes in intracellular free calcium ion concentration ([Ca(2+)](i)) have been visualized over more than two decades using fluorescent dyes and optical microscopy. So far, however, most imaging studies have been performed on isolated cells or brain tissue. Here, we review approaches to measure cellular [Ca(2+)](i) changes in vivo, i.e. within the intact brain of a living animal. In particular we describe the application of two-photon microscopy to the mammalian central nervous system, which has recently enabled studies of Ca(2+) dynamics in individual dendrites in anaesthetized rats. New developments in microscopy and labeling techniques are creating further opportunities to study Ca(2+) dynamics in vivo and are likely to make measurements of spatio-temporal [Ca(2+)](i) distributions feasible even in awake, behaving mammals.

Animals↗

PubMed2002

Vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses.

Hippocampal synapses display a range of release probabilities. This is partially the result of scaling of release probability with the total number of releasable vesicles at each synapse. We have compared synaptic release and vesicle pool sizes across a large number of hippocampal synapses using FM 1-43 and confocal fluorescence microscopy. We found that the relationship between the number of recycling vesicles at a synapse and its release probability is dependent on firing frequency. During firing at 10 Hz, the release probability of each synapse is closely related to the number of recycling vesicles that it contains. In contrast, during firing at 1 Hz, different synapses turn over their recycling vesicle pools at different rates leading to an indirect relationship between recycling vesicle pool size and release probability. Hence two synapses may release vesicles at markedly different rates during low frequency firing, even if they contain similar numbers of vesicles. Both further kinetic analyses and manipulation of the number of vesicles in the readily releasable pool using phorbol ester treatment suggested that this imprecise scaling observed during firing at 1 Hz resulted from synapse-to-synapse differences in the proportion of recycling vesicles partitioned into the readily releasable pool. Hence differential partitioning between vesicle pools affects presynaptic weighting in a frequency-dependent manner. Since hippocampal single unit firing rates shift between 1 Hz and 10 Hz regimes with behavioural state, differential partitioning may be a mechanism for encoding information in hippocampal circuits.

Action Potentials↗

PubMed2002

Stability and plasticity of developing synapses in hippocampal neuronal cultures.

To explore mechanisms governing the formation, stability, and elimination of synapses during neuronal development, we used FM 1-43 fluorescence imaging to track vesicle turnover at >7000 individually identified developing synapses between embryonic rat hippocampal neurons in culture. The majority of presynaptic boutons were stable in efficacy and position over a period of 1.5 hr. Activity, evoked by burst-patterned field stimulation, decreased presynaptic function across the population of boutons, an effect that required NMDA receptor activation. Decreased FM 1-43 staining correlated with low synapsin-I and synaptophysin immunoreactivities, suggesting that decreased presynaptic function was commensurate with synaptic disassembly. These observations provide new information on the stability of developing presynaptic function and suggest that NMDA receptor activation may regulate the stability of developing synapses.

Animals↗