Natural Spike Trains Trigger Short- and Long-Lasting Dynamics at Hippocampal Mossy Fiber Synapses in Rodents

Synapses exhibit strikingly different forms of plasticity over a wide range of time scales, from milliseconds to hours. Studies on synaptic plasticity typically use constant-frequency stimulation to activate synapses, whereas in vivo activity of neurons is irregular.Using extracellular and whole-cell electrophysiological recordings, we have here studied the synaptic responses at hippocampal mossy fiber synapses in vitro to stimulus patterns obtained from in vivo recordings of place cell firing of dentate gyrus granule cells in behaving rodents. We find that synaptic strength is strongly modulated on short- and long-lasting time scales during the presentation of the natural stimulus trains.We conclude that dynamic short- and long-term synaptic plasticity at the hippocampal mossy fiber synapse plays a prominent role in normal synaptic function

Excitatory microcircuits within superficial layers of the medial entorhinal cortex

The distinctive firing pattern of grid cells in the medial entorhinal cortex (MEC) supports its role in the representation of space. It is widely believed that the hexagonal firing field of grid cells emerges from neural dynamics that depends on the local microcircuitry. However, local networks within the MEC are still not sufficiently characterized. Here, applying up to eight simultaneous whole-cell recordings in acute brain slices, we demonstrate the existence of unitary excitatory connections between principal neurons in the superficial layers of the MEC. In particular, we find prevalent feed-forward excitation from pyramidal neurons in layer III and layer II onto stellate cells in layer II, which might contribute to the generation or the inheritance of grid-cell patterns

Cellular and molecular aspects of synaptic transmission and plasticity of the hippocampal mossy fiber synapse

In der vorliegenden kumulativen Habilitationsschrift wurden verschiedene Aspekte der synaptischen Kurzzeit- und Langzeitdynamik an unterschiedlichen synaptischen Verbindungen des Hipppocampus untersucht. Der Schwerpunkt lag dabei auf der Moosfasersynapse. An der Moosfasersynapse konnte zunächst demonstriert werden, dass Adenosin eine tonische Hemmung von spannungsabhängigen Kalziumkanälen vermittelt. Es konnte die Modulation der P/Q- und N-Typ Kanäle pharmakologisch eingegrenzt werden. Über diesen Mechanismus wird auf extrinsische Weise die Freisetzungswahrscheinlichkeit an dieser Synapse auf einem niedrigen Niveau gehalten, was wiederum zu den Voraussetzungen für die besonders stark ausgeprägte Fazilitierungsdynamik gehört. Im Vergleich hierzu erfolgt die Einflussnahme von Munc13-2 auf die Freisetzungswahrscheinlichkeit auf intrinsischem Wege. Dieses Protein konnte als Bestandteil des Freisetzungsapparates als besonders bedeutsam für die Moosfasersynapse nachgewiesen werden. Der Verlust von Munc13-2 in einer Deletionsmutante führte zu eine deutlich verringerten Freisetzungswahrscheinlichkeit und als Konsequenz zu einer stark erhöhten Kurzzeitfazilitierung an den Moosfasern. Die Langzeitplastizität hingegen war durch den Verlust von Munc 13-2 nicht beeinträchtigt. Im Gegensatz dazu waren die Kernparameter der synaptischen Transmission an drei weiteren hippokampalen Synapsen, nämlich Schaffer-Kollateral-, Assoziational-Komissural-, sowie inhibitorischen Synapsen auf CA3 Pyramidenzellen durch den Verlust von Munc13-2 unbeeinträchtigt. Eine weitere bedeutsame Einflussgrösse an den Moosfaserterminalen stellen ionotrope glutamaterge Autorezeptoren vom Kainattyp dar. Diesbezüglich konnte in der vorliegenden Arbeit demonstriert werden, dass die GluK6 Untereinheit entscheidender Bestandteil der Zusammensetzung des präsynaptischen Kainatrezeptors ist und nicht wie vielfach postuliert die GluK5 Untereinheit. Die Aktivierung der GluK6 enthaltenden KAR trägt sowohl zur Kurzzeitdynamik an der Moosfasersynapse bei als auch zum Setzen der Induktionsschwelle für die Langzeitplastizität. Eine Involvierung von intrazellulären Kalziumspeichern in der durch KAR initiierten Signalkaskade konnte hier experimentell nicht nachgewiesen werden. Die präsynaptischen ionotropen Kainatrezeptoren sind darüber hinaus auch für die Vermittelung einer präsynaptischen Form der Assoziativität an Moosfasersynapsen bedeutsam. Die Existenz dieses wichtigen Charakteristikums der Langzeitpotenzierung war für die Moosfasersynapse lange Zeit fraglich geblieben. Die Rolle der KAR für die Kooperativität zwischen Moosfasern als auch für die Assoziativität zwischen AC- und Moosfasersynapsen konnte hier durch den Einsatz entsprechender Stimulationsprotokolle sowie mittels pharmakologischer Werkzeuge erstmalig demonstriert werden. Eine andere ungeklärte Frage betraf die Quelle des für die Induktion der Moosfaser-LTP notwendigen präsynaptischen Kalziumioneninflux. In diesem Zusammenhang gelang es den R-Typ Kalziumkanal als wesentlich zu identifizieren. Ebenso wie im Falle des präsynaptischen Kainatrezeptors besteht auch für den R-Typ Kalziumkanal allerdings keine absolute Notwendigkeit für die Auslösung der LTP. Induktionsprotokolle höherer Intensität sind in der Lage andere Quellen für den Kalziuminflux (P/Q- und N-Typ Kanäle) zu rekrutieren. Die Induktionsschwelle wird allerdings durch die R-Typ Kanäle herabgesetzt. Interessanterweise scheint die basale synaptische Transmission an Moosfasern weitgehend unabhängig von R-Typ Kanälen zu sein. Ein weiterer Gegenstand der vorgelegten Untersuchungen betraf die mögliche Funktion der Proteine 4.1N und 4.1G im Zuge der postsynaptischen Expression von Langzeit-Potenzierung an Schaffer-Kollateral-Synapsen. Es konnte keine Beteiligung dieser beiden Paraloge am Transport von AMPAR-Untereinheiten in die postsynaptische und extrasynaptische Membran nachgewiesen werden, obgleich Vorbefunde dies nahegelegt hatten. Vielmehr scheint eine ausgeprägte funktionelle Redundanz der 4.1 Paraloge vorzuliegen, so dass erst das Fehlen aller zerebral exprimierten Paraloge zu einem elektrophysiologisch detektierbaren Phänotyp führt.In this cumulative thesis varius aspects of synaptic short-term as well as long-term plasticity were studied at different synaptic connections of the hippocampus of rodents. The main focus of the work was directed towards the mossy fiber synapse. At the mossy fiber synapse it could be demonstrated that adenosine leads to a tonic inhibition of voltage dependent calcium channels. A modulation of both P/Q- and N-type channels could be dissected by pharmacological means. Through this mechanism the release probability at this synapse is extrinsically maintained at a low level which in turn is a prerequisite for the synapse´s pronounced short-term dynamics. In comparison Munc13-2 influences the release probability in an intrinsic fashion. This active zone protein was found to be of special importance at the mossy fiber synapse. Loss of Munc13-2 in deletion mutants resulted in a severe reduction of the release probability and consequently to a strongly enhanced short-term facilitation at mossy fibers. Long-term plasticity was unaffected by the deletion of Munc13-2. In contrast the basic parameters of synaptic transmission at three other hippocampal synapses, namely Schaffer collateral, associational-commissural as well as inhibitory synapses onto CA3 pyramidal neurons, were unimpaired by the loss of Munc13-2. Another important factor at mossy fiber terminals are kainate autoreceptors which belong to the group of ionotropic glutamate receptors. In this context, it could be demonstrated in the present study that the GluK6 subunit constitutes a crucial component of the presynaptic kainate receptors, whereas the GluK5 subunit seems not relevant. Activation of GluK6 containing kainate receptors substantially contributes to the short-term dynamics at mossy fiber synapses as well as to setting the induction threshold for long-term plasticity. An involvement of intracellular calcium stores in the kainate receptor triggered signaling cascade could not be expermientally detected in this work. Moreover presynaptic ionotropic kainate receptors participate in mediating a presynaptic form of associativity at mossy fiber synapses. The existence of this important characteristic of long-term potentiation was questioned for a long time for mossy fiber synapses. Another important question concerned the source of the presynaptic calcium which is necessary for the induction of mossy fiber long-term potentiation. In this regard we were able to identify the R-type voltage dependent calcium channel as fundamental. As for the presynaptic kainate receptor there is also no absolute requirement for the R-type calcium channel for induction of long-term potentiation. Higher intensity induction protocolls are capable of recruiting other calcium sources for calcium influx (namely P/Q-type and N-type channels). But he induction threshold is substantially lowered by R-type channels. Interestingly basal synaptic transmission at mossy fiber synapses seems independent of R-type channels. Another topic of the investigations presented here regarded the possible role of proteins 4.1N and 4.1G in the postsynaptic expression of long-term potentiation at Schaffer collateral synapses. We were unable to detect any participation of either paralog in the transport of AMPA receptor subunits into the postsynaptic or extrasynaptic membrane, despite published results hinting at this possibility. It rather seems that there exists a strong functional redundancy of the 4.1 paralogs. For this reason only the loss of all cerebrally expressed paralogs may lead to an electrophysiologically detectable phenotype

Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the Bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System in Escherichia coli▿

The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (EIIs). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and EI of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the EIIs of E. coli. In contrast, activation of the E. coli BglG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 16, 17, 20, 24, 27, 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive EIIs

Place field specific spiking activity of dentate gyrus granule cells triggers long-term potentiation of mossy fiber synaptic responses <i>in vitro</i>.

<p>(<b>A</b>) Presentation of spike train 1 (indicated by grey area) led to potentiation of mossy fiber fEPSP amplitudes in this examplary recording. Constant stimulation frequency before and after delivery of spike train was 0.05 Hz. Application of DCGIV at the end of experiment blocked mossy fiber synaptic transmission. Upper traces show averages of 10 sweeps under control condition and 30 min after presentation of spike train 1. (<b>B</b>) Summary of n = 5 such experiments. Presentation of spike train led to reliable long-term potentiation of fEPSP amplitudes to ∼130% of control values 25 min after spike train 1. (<b>C</b>) Time-resolved plot of another place field specific spike episode (spike train 2, up) and continuous recording of mossy fiber fEPSP response to single presentation of this spike train (lower part). Stimulus artifacts are cut for visual clarity. Please note different timescale compared to spike train 1. (<b>D</b>) Examplary mossy fiber synaptic fEPSP recording, where a single presentation of spike train 2 (grey bar, not drawn to scale) leads to long-term potentiation of fEPSP responses. Arrow points to frequency facilitation paradigm (switch of stimulation frequency from 0.05 Hz to 1 Hz for 20 stimuli). Application of DCGIV (1 µM) at the end of experiment blocked mossy fiber fEPSPs. Upper traces show averages of 10 sweeps each under control condition and 25 minutes after presentation of spike train. Constant stimulation frequency was 0.05 Hz. (<b>E</b>) Repetitive presentation of spike train 2 (5x with 30 s pauses inbetween) resulted in pronounced long-term potentiation of mossy fiber fEPSP amplitudes in this examplary experiment. Upper traces show averages of 10 sweeps each under control condition and 25 minutes after repetitive presentation of spike train. (<b>F</b>) Summary of n = 6 experiments with single presentation of spike train (open circles) and n = 7 experiments with repetitive presentation (filled circles). Both paradigms led to significant long-term potentiation of response amplitudes to ∼150% and ∼230% of control values, respectively. Data shows mean ± sem. Upper dashed lines in subpanels indicate basal response amplitudes to constant stimulation at 0.05 Hz.</p

Natural spike train induced long-term potentiation is strongly reduced in the presence of elevated cAMP levels.

<p>(<b>A</b>) Application of the adenylate cyclase activator forskolin (50 µM) enhances synaptic transmission in this exemplary experiment and strongly reduces long-term potentiation induced by repetitive (5 x) delivery of spike train 2. Traces on top are averages of five consecutive sweeps taken at the time point indicated by the numbers in the graph. Triangle denotes frequency facilitation paradigm for 20 pulses with 1 Hz, arrow indicates time point of spike train 2 application, second horizontal bar represents application of DCGIV (1 µM) at the end of experiment. (<b>B</b>) Summary plot displaying the drastically reduced potentiation for n = 4 such experiments (closed circles). Values are normalized to the amplitude in forskolin before train delivery. For comparison, the potentiation elicited by spike train 2 in the absence of drugs (open circles, same dataset as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009961#pone-0009961-g002" target="_blank">Figure 2F</a>) is overlayed. In the presence of forskolin the potentiation was reduced to 143.4±11.5% (p<0.001, compared to control).</p

Species-specific differences in synaptic transmission and plasticity

Synaptic transmission and plasticity in the hippocampus are integral factors in learning and memory. While there has been intense investigation of these critical mechanisms in the brain of rodents, we lack a broader understanding of the generality of these processes across species. We investigated one of the smallest animals with conserved hippocampal macroanatomy—the Etruscan shrew, and found that while synaptic properties and plasticity in CA1 Schaffer collateral synapses were similar to mice, CA3 mossy fiber synapses showed striking differences in synaptic plasticity between shrews and mice. Shrew mossy fibers have lower long term plasticity compared to mice. Short term plasticity and the expression of a key protein involved in it, synaptotagmin 7 were also markedly lower at the mossy fibers in shrews than in mice. We also observed similar lower expression of synaptotagmin 7 in the mossy fibers of bats that are evolutionarily closer to shrews than mice. Species specific differences in synaptic plasticity and the key molecules regulating it, highlight the evolutionary divergence of neuronal circuit functions

Mossy fiber synaptic LTP - induced by place field specific spiking activity of dentate gyrus granule cells - is presynaptically expressed.

<p>(<b>A</b>) Examplary whole-cell recording of mossy fiber synaptic responses in CA3 pyramidal cell. Repetitive presentation of spike train 2 (grey bars, compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009961#pone-0009961-g002" target="_blank">Figure 2</a>) induces long-term potentiation of EPSC amplitudes. Upper traces show averages of 10 sweeps each under control condition and 20 min after presentation of spike train. Constant stimulation frequency outside of spike train 2 was 0.1 Hz. CA3 pyramidal cell was held in voltage-clamp condition at -60 mV, also during presentation of spike train. Upper dashed line indicates basal response amplitudes to constant stimulation at 0.1 Hz. (<b>B</b>) Summary of n = 5 whole-cell experiments where repetitive presentation of spike train 2 induces long-term potentiation of mossy fiber EPSC amplitudes. Potentiation to ∼220% of control values was visible 30 min after spike train. Data was binned to 0.5 min time points and depicts mean ± sem. (<b>C</b>) CV² analysis of data from experiments in A. The change in the squared coefficient of variation in control versus LTP condition shows a linear dependence on the change in the mean response amplitude. (<b>D</b>) The mean rate of failures of synaptic transmission is decreased after expression of LTP. Upper traces show 50 individual sweeps (grey) and mean sweeps (black) in control and LTP condition of an exemplary whole-cell recording. Note the large incidence of synaptic failures under control conditions.</p

Natural spike trains induce mossy fiber LTP indepedent of NMDAR and mGluR activation.

<p>(<b>A</b>) Exemplary mossy fiber fEPSP recording under blockage of NMDA- and mGlu-receptors. Repetitive presentation of spike train 2 still induced significant long-term potentiation. Arrow points to frequency facilitation paradigm. Upper traces are averages of 10 sweeps each under control condition and 25 min after presentation of spike trains. Constant stimulation frequency was 0.05 Hz. (<b>B</b>) Summary of n = 6 such experiments and experiments under control conditions, respectively. Repetitive presentation of spike train 2 resulted in potentiation of response amplitudes to ∼220% of control values 30 min after presentation of spike trains. Data was binned to 1 min time points and depicts mean ± sem. Upper dashed lines in subpanels indicate basal response amplitudes to constant stimulation at 0.05 Hz.</p