Coincident Pre- and Postsynaptic Activation Induces Dendritic Filopodia via Neurotrypsin-Dependent Agrin Cleavage

SummaryThe synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS

Neuron to Astrocyte Communication via Cannabinoid Receptors Is Necessary for Sustained Epileptiform Activity in Rat Hippocampus

Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1) receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus

Modulation of hippocampal network oscillation by PICK1-dependent cell surface expression of mGlu3 receptors

International audienceMetabotropic glutamate receptor type 3 (mGlu3) controls the sleep/wake architecture which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptors expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in wild-type male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during REM sleep, NREM sleep and wake states after intra-ventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia

Calsyntenin-1 regulates targeting of dendritic NMDA receptors and dendritic spine maturation in CA1 hippocampal pyramidal cells during postnatal development

Calsyntenin-1 is a transmembrane cargo-docking protein important for kinesin-1-mediated fast transport of membrane-bound organelles that exhibits peak expression levels at postnatal day 7. However, its neuronal function during postnatal development remains unknown. We generated a knock-out mouse to characterize calsyntenin-1 function in juvenile mice. In the absence of calsyntenin-1, synaptic transmission was depressed. To address the mechanism, evoked EPSPs were analyzed revealing a greater proportion of synaptic GluN2B subunit-containing receptors typical for less mature synapses. This imbalance was due to a disruption in calsyntenin-1-mediated dendritic transport of NMDA receptor subunits. As a consequence of increased expression of GluN2B subunits, NMDA receptor-dependent LTP was enhanced at Schaffer collateral-CA1 pyramidal cell synapses. Interestingly, these defects were accompanied by a decrease in dendritic arborization and increased proportions of immature filopodia-like dendritic protrusions at the expense of thin-type dendritic spines in CA1 pyramidal cells. Thus, these results highlight a key role for calsyntenin-1 in the transport of NMDA receptors to synaptic targets, which is necessary for the maturation of neuronal circuits during early development

Selective silencing of individual dendritic branches by an mGlu2-activated potassium conductance in dentate gyrus granule cells

Group II metabotropic glutamate receptors (mGlu-IIs) modulate hippocampal information processing through several presynaptic actions. We describe a novel postsynaptic inhibitory mechanism mediated by the mGlu2 subtype that activates an inwardly rectifying potassium conductance in the dendrites of DG granule cells of rats and mice. Data from glutamate-uncaging experiments and simulations indicate that mGlu2-activated potassium conductance uniformly reduces the peak amplitude of synaptic inputs arriving in the distal two-thirds of dendrites, with only minor effects on proximal inputs. This unique shunting profile is consistent with a peak expression of the mGlu2-activated conductance at the transition between the proximal and middle third of the dendrites. Further simulations under various physiologically relevant conditions showed that when a shunting conductance was activated in the proximal third of a single dendrite, it effectively modulated input to this specific branch while leaving inputs in neighboring dendrites relatively unaffected. Therefore, the restricted expression of the mGlu2-activated potassium conductance in the proximal third of DG granule cell dendrites represents an optimal localization for achieving the opposing biophysical requirements for uniform yet selective modulation of individual dendritic branches

The duration but not the initiation of ED is reduced following disruption of neuroglial interactions.

<p>A, Compounds reported to disrupt neuroglial and astrocyte coupling do not modify the initiation of ED in response to 4-AP. The probability of ED initiation was not changed either by antagonists for gap junctions or by a solution that inhibits neuroglial interactions consisting of MRS 2179 (4 µM) to block P2Y1 receptors and MPEP (10 µM) to block mGluR5. The probability of ED initiation was not changed either by a cocktail containing antagonists for P2Y1R, mGluR5 and CB1R (AM251, 4 µM) or CB1R antagonist alone. But ED initiation is prevented when synaptic transmission is blocked with TTX (1 µM), or reduced when ionotropic glutamate receptors are blocked with NBQX (50 µM). B, Application of antagonists for P2Y1 and mGlu5 receptors increases the frequency of ED induced by 4-AP, while MRS alone has not effect. C, Mean ED frequency 4–6 and 8–10 minutes after initiation. D, E, Addition of AM251 to the MRS 2179 and MPEP solution significantly reduces ED frequency.</p

CB1 receptors expressed by astrocytes contribute significantly to maintenance of ED in CA1.

<p>A, Two-photon fluorescence calcium imaging of CA1 cells during 4-AP perfusion of the slice. Fluorescence signals from neurons and astrocytes during 4-AP in the presence or absence of the CB1 receptor antagonist AM251 (4 µM). The plotted points represent the averages of the responses for each individual cell. B, Blocking CB1 receptors significantly reduced integrated area of calcium induced fluorescence measured 1–4 min after ED initiation. This inhibition was markedly particularly important in astrocytes (p<0.001 Mann-Whitney U test) C, Similarly, average time course show that ED in CA1 pyramidal cells recorded in current-clamp mode is reduced in the presence of the CB1 antagonist AM251. D, Representative traces for data shown in C. E, Mean ED frequency at two time points after initiation of ED for CA1 pyramidal cells show that the reduction induced by AM251 is highly significant. F, In the presence of GABA_A and GABA_B receptor antagonists to block interneuron signaling, blocking CB1 receptors still results in a significant reduction in ED but much less effective in reducing ED frequency. G, When astrocyte signaling is inhibited by BAPTA injection (40 mM) under conditions where GABAergic transmission is blocked, the blockade of CB1 receptors no longer reduces 4-AP-induced ED.</p

Astrocytes participate in the maintenance of epileptiform activity in CA1.

<p>A, A field recording in the CA1 area and a patch-clamp recording from a CA1 pyramidal cell show a typical initial depolarization event followed by epileptiform discharge (ED) in response to continuous application of 4-AP (100 µM). The insets show examples of individual EDs taken from the left traces at an expanded time base. B, Averaged time course of ED frequency following the initial depolarization as measured with patch-clamp recording (n = 23). C, Two-photon fluorescence imaging in hippocampal organotypic slice cultures during ED showing co-activation of neuronal and glial networks. Top right shows an overlaid image with neurons (green) loaded with OGB-1 and astrocytes (red) with sulforhodamine 101. Traces show the calcium rise both in neurons and astrocytes during 4-AP-induced epileptiform activity. Below are images of cellular calcium levels before (left) and during (right) epileptiform activity. Scale bar = 30 µm. D, Time course of fluorescence changes in CA1 pyramidal cells and in astrocytes during 4-AP superperfusion (n = 164 neurons and 146 astrocytes in 8 slice cultures). The plotted points represent averages of the mean cellular responses for each slice. E, Chelating calcium with intracellular BAPTA in astrocytes decreases the frequency of ED recorded in a CA1 pyramidal cell. Top: Images show a patch-clamp pipette approaching the CA1 stratum radiatum (left), an astrocyte patched with the pipette containing BAPTA (40 mM) and the fluorescent dye Alexa 488 (middle), and the diffusion of the fluorescent marker throughout a small network of astrocytes following depolarization (1 nA injection) to open gap-junctions (right). Bottom left: representative current clamp recordings from a CA1 pyramidal cell during ED when a neighboring astrocyte was injected with control intracellular solution (black trace) or with BAPTA (gray trace). Sample activity at expanded time base is shown at right. Bottom Right: Mean ED frequency at two time points after initiation of ED for CA1 pyramidal cells with a neighboring astrocyte filled with control intracellular solution versus a 40 mM BAPTA intracellular solution (n = 4). F, current-clamp traces recorded from single CA1 pyramidal cells in 4 different slice cultures showing ED activity in control or after incubation for three hours with two antagonists of connexins, carbenoxolone (100 µM) and mefloquine (25 µM).</p