Pavel Osten

Evoked Axonal Oxytocin Release in the Central Amygdala Attenuates Fear Response

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

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 α-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 microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

Novel Anchorage of GluR2/3 to the Postsynaptic Density by the AMPA Receptor–Binding Protein ABP

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

The AMPA Receptor GluR2 C Terminus Can Mediate a Reversible, ATP-Dependent Interaction with NSF and α- and β-SNAPs

In this study, we demonstrate specific interaction of the GluR2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide–sensitive fusion protein (NSF) and α- and β-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2–NSF–SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2–NSF–SNAP complex is similar to that of the t-SNARE syntaxin–NSF–SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

PICK1 Targets Activated Protein Kinase Cα to AMPA Receptor Clusters in Spines of Hippocampal Neurons and Reduces Surface Levels of the AMPA-Type Glutamate Receptor Subunit 2

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Cα (PKCα) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1–PKCα complexes is strongly induced by TPA, and PICK1–PKCα complexes are cotargeted with PICK1–GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCα to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane.

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Serial two-photon tomography for automated ex vivo mouse brain imaging.

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

Stargazin Reduces Desensitization and Slows Deactivation of the AMPA-Type Glutamate Receptors

The AMPA-type glutamate receptors mediate the majority of the fast excitatory synaptic transmission and critically contribute to synaptic plasticity in the brain, hence the existence of numerous trafficking proteins dedicated to regulation of their synaptic delivery and turnover. Stargazin (also termed γ2) is a member of a recently identified protein family termed transmembrane AMPA receptor regulatory proteins (TARPs). TARPs physically associate with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. Here, we report that next to its trafficking roles, stargazin may also act as a positive allosteric modulator of AMPA receptor ion channel function. Coexpression of stargazin with AMPA receptor subunits, either in Xenopus oocytes or in human embryonic kidney 293 cells, significantly reduced receptor desensitization in response to glutamate. Receptor deactivation rates were also slowed, and the recovery from desensitization was accelerated. Structurally, based on the data showing a tight correlation between desensitization and the stability of the AMPA receptor intradimer interface, we propose that binding of stargazin may stabilize the receptor conformation. Functionally, our data suggest that AMPA receptors complexed with stargazin (and possibly also with other TARPs) at the postsynaptic membrane are significantly more responsive to synaptically released glutamate compared with AMPA receptors lacking stargazin/TARP interaction. The putative existence of such two states of synaptic AMPA receptors, with and without stargazin/TARP binding, may provide a novel mechanism for regulation of excitatory synaptic strength during development and/or in synaptic plasticity in the adult brain.

Rap2-JNK Removes Synaptic AMPA Receptors during Depotentiation

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.

Protein synthesis-dependent formation of protein kinase Mzeta in long- term potentiation

The maintenance of long-term potentiation (LTP) in the CA1 region of the hippocampus has been reported to require both a persistent increase in phosphorylation and the synthesis of new proteins. The increased activity of protein kinase C (PKC) during the maintenance phase of LTP may result from the formation of PKMzeta, the constitutively active fragment of a specific PKC isozyme. To define the relationship among PKMzeta, long-term EPSP responses, and the requirement for new protein synthesis, we examined the regulation of PKMzeta after sub-threshold stimulation that produced short-term potentiation (STP) and after suprathreshold stimulation by single and multiple tetanic trains that produced LTP. We found that, although no persistent increase in PKMzeta followed STP, the degree of long-term EPSP potentiation was linearly correlated with the increase of PKMzeta. The increase was first observed 10 min after a tetanus that induced LTP and lasted for at least 2 hr, in parallel with the persistence of EPSP enhancement. Both the maintenance of LTP and the long-term increase in PKMzeta++ were blocked by the protein synthesis inhibitors anisomycin and cycloheximide. These results suggest that PKMzeta is a component of a protein synthesis-dependent mechanism for persistent phosphorylation in LTP.