Final Report Summary - CP-AMPAR TRAFFICKING (Molecular mechanisms regulating the trafficking of calcium-permeable and -impermeable AMPA receptors in synaptic plasticity)
In the brain, cells communicate through specialised contact points called synapses. A presynaptic neuron releases a chemical transmitter that binds to receptors located on a postsynaptic cell. In mammalian brain, the main excitatory neurotransmitter is glutamate, and fast excitatory transmission is mediated mostly by activation of AMPA type glutamate receptors. These receptors are protein complexes embedded in the cell outer membrane. Glutamate binding causes the complex to open and allows cations to flow through the membrane. There are 2 types of AMPARs; the most common (calcium impermeable, CI- AMPARs), allows flux of monovalent cations. The other, the calcium permeable AMPARs (CP-AMPARs) also allows calcium to pass. When open, both subtypes pass a depolarising current that can trigger release of a neurotransmitter packet by the cell onto its target.
Messages are thus relayed from one neuron to another by a sequence of chemical and electrical signals. In addition to generating electric currents, open CP-AMPARs let calcium into the cell. This feature is of particular interest since the entry of calcium through CP-AMPARs is linked to neuronal development, long-term changes in synapse strength or plasticity (for information storage about past synaptic activity: 'memory'), pain perception, and cell death after ischemia. Despite the importance of calcium entry in both physiological and pathological aspects of a neurons life, the molecular mechanisms underlying CP-AMPAR delivery to synapses remain unclear.
We, thus, focused our attention on this question and, in particular, explored the potential role of recently discovered AMPA auxiliary subunits, TARPs, in this process. Cerebellar stellate cells (SCs) are an ideal neuron for addressing this issue as they contain both types of AMPARs, the relative expression of each being tightly regulated and compartmentalised at the neuronal surface. Because the prototypical TARP, Stargazin (Stg), is highly expressed in the cerebellum, and Stargazer mice (stg-/-, which naturally lack the protein) display severe cerebellar defects (poor motor control), we used stg-/- SC to investigate CP-AMPAR trafficking from the TARP perspective.
By combining high-resolution electrophysiology (recording of electric activity) in cerebellar slices, and fluorescence-based protein imaging in SCs maintained in culture, we aimed to get a clearer picture of CP- vs CI- AMPARs distribution in relation to TARPs expression. We, thus, compared AMPAR currents in normal vs stg-/- SCs. We recorded postsynaptic currents evoked by minimal glutamate release from intact presynaptic terminals. We then extended our study to extrasynaptic receptors; currents were evoked by rapidly applying glutamate onto patches of membrane excised from cell soma.
While AMPARs are clustered at synapses, they are present at a lower density in the entire membrane. These extrasynaptic receptors are exposed to glutamate spillover during synchronised activity, and activated by high glutamate brain levels in some pathologies and trauma. Finally, they constitute a reserve pool that can be added to synapses during plasticity events that alter transmission. It was thus crucial to extract the maximum data about both synaptic and extrasynaptic AMPARs from the recorded currents.
A careful analysis of channel properties, as well as the use of subtype specific drugs, allowed an estimation of the proportion of CP- vs CI-AMPAR in each compartment, and showed this was affected by the lack of Stg. This, together with images obtained in culture, suggested that other TARPs are present in SC and could act in concert with Stg to control CP-/CI-AMPARs ratio at synapse. Some of our conclusions, which are still confidential, will be of great interest to the wide scientific community working on AMPARs. A manuscript presenting these will be soon submitted for review as a full length scientific paper.
Messages are thus relayed from one neuron to another by a sequence of chemical and electrical signals. In addition to generating electric currents, open CP-AMPARs let calcium into the cell. This feature is of particular interest since the entry of calcium through CP-AMPARs is linked to neuronal development, long-term changes in synapse strength or plasticity (for information storage about past synaptic activity: 'memory'), pain perception, and cell death after ischemia. Despite the importance of calcium entry in both physiological and pathological aspects of a neurons life, the molecular mechanisms underlying CP-AMPAR delivery to synapses remain unclear.
We, thus, focused our attention on this question and, in particular, explored the potential role of recently discovered AMPA auxiliary subunits, TARPs, in this process. Cerebellar stellate cells (SCs) are an ideal neuron for addressing this issue as they contain both types of AMPARs, the relative expression of each being tightly regulated and compartmentalised at the neuronal surface. Because the prototypical TARP, Stargazin (Stg), is highly expressed in the cerebellum, and Stargazer mice (stg-/-, which naturally lack the protein) display severe cerebellar defects (poor motor control), we used stg-/- SC to investigate CP-AMPAR trafficking from the TARP perspective.
By combining high-resolution electrophysiology (recording of electric activity) in cerebellar slices, and fluorescence-based protein imaging in SCs maintained in culture, we aimed to get a clearer picture of CP- vs CI- AMPARs distribution in relation to TARPs expression. We, thus, compared AMPAR currents in normal vs stg-/- SCs. We recorded postsynaptic currents evoked by minimal glutamate release from intact presynaptic terminals. We then extended our study to extrasynaptic receptors; currents were evoked by rapidly applying glutamate onto patches of membrane excised from cell soma.
While AMPARs are clustered at synapses, they are present at a lower density in the entire membrane. These extrasynaptic receptors are exposed to glutamate spillover during synchronised activity, and activated by high glutamate brain levels in some pathologies and trauma. Finally, they constitute a reserve pool that can be added to synapses during plasticity events that alter transmission. It was thus crucial to extract the maximum data about both synaptic and extrasynaptic AMPARs from the recorded currents.
A careful analysis of channel properties, as well as the use of subtype specific drugs, allowed an estimation of the proportion of CP- vs CI-AMPAR in each compartment, and showed this was affected by the lack of Stg. This, together with images obtained in culture, suggested that other TARPs are present in SC and could act in concert with Stg to control CP-/CI-AMPARs ratio at synapse. Some of our conclusions, which are still confidential, will be of great interest to the wide scientific community working on AMPARs. A manuscript presenting these will be soon submitted for review as a full length scientific paper.