Pamela M. England

Photoinactivation of Native AMPA Receptors Reveals Their Real-Time Trafficking

AMPA receptors mediate the majority of the fast excitatory transmission in the central nervous system. Much evidence suggests that the fast trafficking of AMPA receptors into and out of the postsynaptic membrane underlies changes in synaptic strength thought to be necessary for higher cognitive functions such as learning and memory. Despite the abundance of research conducted in this area, a direct, real-time functional assay that measures the trafficking of native AMPA receptors has been lacking. Toward this aim, we use a photoreactive, irreversible antagonist of AMPA receptors, ANQX, to rapidly silence surface AMPA receptors and investigate directly the trafficking of native AMPA receptors in real time. We find that the most dynamic movement of AMPA receptors occurs by lateral movement across the surface of neurons. Fast cycling of surface AMPA receptors with receptors from internal stores does occur but exclusively at extrasynaptic somatic sites. The cycling of synaptic AMPA receptors only occurs on a much longer timescale with complete exchange requiring at least 16 hr. This cycling is not dependent on protein synthesis or action potential driven network activity. These data suggest a revised model of AMPA receptor trafficking wherein a large internal store of AMPA receptors exchanges rapidly with extrasynaptic somatic AMPA receptors, and these newly inserted AMPA receptors then travel laterally along dendrites to reside stably at synapses.

Mechanism and Time Course of Cocaine-Induced Long-Term Potentiation in the Ventral Tegmental Area

Synaptic plasticity in the ventral tegmental area (VTA) has been implicated in the acquisition of a drug-dependent state. Even a single exposure to cocaine in naive animals is sufficient to trigger sustained changes on VTA glutamatergic synapses that resemble activity-dependent long-term potentiation (LTP) in other brain regions. However, an insight into its time course and mechanisms of action is limited. Here, we show that cocaine acts locally within the VTA to induce an LTP-like enhancement of AMPA receptor-mediated transmission that is not detectable minutes after drug exposure but is fully expressed within 3 h. This cocaine-induced LTP appears to be mediated via dopamine D5 receptor activation of NMDA receptors and to require protein synthesis. Increased levels of high-conductance GluR1-containing AMPA receptors at synapses are evident at 3 h after cocaine exposure. Furthermore, our data suggest that cocaine-induced LTP might share the same molecular substrates for expression with activity-dependent LTP induced in the VTA by a spike-timing-dependent (STD) protocol, because we observed that STD LTP is significantly reduced or not inducible in VTA neurons previously exposed to cocaine in vivo or in vitro.

A Chemical-Genetic Approach to Studying Neurotrophin Signaling

Trk tyrosine kinases are receptors for members of the neurotrophin family and are crucial for growth and survival of specific populations of neurons. Yet, the functions of neurotrophin-Trk signaling in postnatal development as well as maintenance and plasticity of the adult nervous system are less clear. We report here the generation of mice harboring Trk knockin alleles that allow for pharmacological control of Trk kinase activity. Nanomolar concentrations of either 1NMPP1 or 1NaPP1, derivatives of the general kinase inhibitor PP1, inhibit NGF and BDNF signaling in TrkA(F592A) and TrkB(F616A) neurons, respectively, while no such Trk inhibition is observed in wild-type neurons. Moreover, oral administration of 1NMPP1 leads to specific inhibition of TrkA(F592A), TrkB(F616A), and TrkC(F167A) signaling in vivo. Thus, Trk knockin mice provide valuable tools for selective, rapid, and reversible inhibition of neurotrophin signaling in vitro and in vivo.

TrkB kinase is required for recovery, but not loss, of cortical responses following monocular deprivation

Changes in visual cortical responses that are induced by monocular visual deprivation are a widely studied example of competitive, experience-dependent neural plasticity. It has been thought that the deprived-eye pathway will fail to compete against the open-eye pathway for limited amounts of brain-derived neurotrophic factor, which acts on TrkB and is needed to sustain effective synaptic connections. We tested this model by using a chemical-genetic approach in mice to inhibit TrkB kinase activity rapidly and specifically during the induction of cortical plasticity in vivo. Contrary to the model, TrkB kinase activity was not required for any of the effects of monocular deprivation. When the deprived eye was re-opened during the critical period, cortical responses to it recovered. This recovery was blocked by TrkB inhibition. These findings suggest a more conventional trophic role for TrkB signaling in the enhancement of responses or growth of new connections, rather than a role in competition.

TrkB signaling is required for both the induction and maintenance of tissue and nerve injury-induced persistent pain

Activation of primary afferent nociceptors produces acute, short-lived pain, and tissue or nerve injury induces long-term enhancement of nociceptive processing, manifested as hypersensitivity to thermal and mechanical stimulation. Here we used a chemical–genetic and pharmacological approach to study the contribution of the receptor tyrosine kinase, type 2 (TrkB) to the generation and maintenance of injury-induced persistent pain. We performed the studies in wild-type mice and transgenic (TrkBF616A) mice that express mutant but fully functional TrkB receptors. By injecting a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 (1NM-PP1), it is possible to produce highly selective inhibition of TrkB autophosphorylation in adult mice, without interfering with the activity of other protein kinases. We report that oral administration of 1NM-PP1, at doses that blocked phosphorylation of TrkB in the spinal cord, had no effect in behavioral tests of acute heat, mechanical, or chemical pain sensitivity. However, the same pretreatment with 1NM-PP1 prevented the development of tissue- or nerve injury-induced heat and mechanical hypersensitivity. Established hypersensitivity was transiently reversed by intraperitoneal injection of 1NM-PP1. Although interfering with TrkB signaling altered neither acute capsaicin nor formalin-induced pain behavior, the prolonged mechanical hypersensitivity produced by these chemical injuries was prevented by 1NM-PP1 inhibition of TrkB signaling. We conclude that TrkB signaling is not only an important contributor to the induction of heat and mechanical hypersensitivity produced by tissue or nerve injury but also to the persistence of the pain.

AMPA receptors and synaptic plasticity: a chemist's perspective

The ability of the mammalian brain to undergo experience-based adaptations is among its most important and fascinating properties. Such plasticity is reflected in the capacity of neuronal activity to continuously modify the neural circuitry that underlies thought, feeling and behavior. The locus of this plasticity occurs at the level of synapses, the specialized junctions where one neuron receives chemical signals from another. Synaptic connections become stronger or weaker in response to specific patterns of activity. This activity drives regulated changes in the neurotransmitter released by presynaptic neurons and in the receptors localized on postsynaptic neurons. Detailed studies of these receptors have advanced our understanding of synaptic plasticity. However, many key questions remain unresolved, and over the past decade innovative chemical approaches have emerged to tackle them. Here we review these chemical tools and their application to unraveling the molecular basis of synaptic plasticity.

6-Azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione (ANQX) Forms an Irreversible Bond To the Active Site of the GluR2 AMPA Receptor†

AMPA receptors mediate fast excitatory synaptic transmission and are essential for synaptic plasticity. ANQX, a photoreactive AMPA receptor antagonist, is an important biological probe used to irreversibly inactivate AMPA receptors. Here, using X-ray crystallography and mass spectroscopy, we report that ANQX forms two major products in the presence of the GluR2 AMPAR ligand-binding core (S1S2J). Upon photostimulation, ANQX reacts intramolecularly to form FQX or intermolecularly to form a covalent adduct with Glu705.

Developing a complete pharmacology for AMPA receptors: a perspective on subtype-selective ligands.

AMPA receptors are a family of ligand-gated ion channels that play central roles in rapid neural signaling and in regulation of synaptic strength. Additionally, these receptors are implicated in a number of major psychiatric and neurological diseases. A comprehensive understanding of the roles that AMPA receptors play in the mammalian nervous system has been hampered by the dearth of ligands available to select between individual AMPA receptors subtypes. Here we provide a perspective on opportunities for developing a complete pharmacology for AMPA receptors.

Critical role of trkB receptors in reactive axonal sprouting and hyperexcitability after axonal injury

Traumatic brain injury (TBI) causes many long-term neurological complications. Some of these conditions, such as posttraumatic epilepsy, are characterized by increased excitability that typically arises after a latent period lasting from months to years, suggesting that slow injury-induced processes are critical. We tested the hypothesis that trkB activation promotes delayed injury-induced hyperexcitability in part by promoting reactive axonal sprouting. We modeled penetrative TBI with transection of the Schaffer collateral pathway in knock-in mice having an introduced mutation in the trkB receptor (trkB(F616A)) that renders it susceptible to inhibition by the novel small molecule 1NMPP1. We observed that trkB activation was increased in area CA3 1 day after injury and that expression of a marker of axonal growth, GAP43, was increased 7 days after lesion. Extracellular field potentials in stratum pyramidale of area CA3 in acute slices from sham-operated and lesioned mice were normal in control saline. Abnormal bursts of population spikes were observed under conditions that were mildly proconvulsive but only in slices taken from mice lesioned 7-21 days earlier and not in slices from control mice. trkB activation, GAP43 upregulation, and hyperexcitability were diminished by systemic administration of 1NMPP1 for 7 days after the lesion. Synaptic transmission from area CA3 to area CA1 recovered 7 days after lesion in untreated mice but not in mice treated with 1NMPP1. We conclude that trkB receptor activation and reactive axonal sprouting are critical factors in injury-induced hyperexcitability and may contribute to the neurological complications of TBI.

Rapid Photoinactivation of Native AMPA Receptors on Live Cells Using ANQX

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, a major subtype of ionotropic glutamate receptors, mediate the majority of fast excitatory synaptic transmission in the mammalian brain. The constitutive and regulated trafficking of AMPA receptors into and out of excitatory synapses ensures rapid responses to synaptically released glutamate and provides a mechanism for synaptic plasticity. To permit the direct, quantitative, real-time measurement of native AMPA receptor trafficking in live neurons, we designed and utilized a membrane-impermeable, photoreactive AMPA receptor antagonist to rapidly and irreversibly inactivate surface receptors with ultraviolet (UV) light. The photoreactive antagonist, 6-azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione (ANQX), is an aryl azide that, when irradiated with UV light, becomes a highly reactive nitrene that can covalently cross-link to and thus irreversibly antagonize bound AMPA receptors. Thus, ANQX provides a means of rapidly silencing surface-exposed AMPA receptors. Combined with a functional AMPA receptor assay, such as continuous recording of AMPA receptor–mediated ionic currents, ANQX provides a means of directly monitoring native AMPA receptor trafficking in real time.