Roger A. Nicoll

Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses

Marijuana affects brain function primarily by activating the G-protein-coupled cannabinoid receptor-1 (CB1), which is expressed throughout the brain at high levels. Two endogenous lipids, anandamide and 2-arachidonylglycerol (2-AG), have been identified as CB1 ligands. Depolarized hippocampal neurons rapidly release both anandamide and 2-AG in a Ca2+-dependent manner. In the hippocampus, CB1 is expressed mainly by GABA (γ-aminobutyric acid)-mediated inhibitory interneurons, where CB1 clusters on the axon terminal. A synthetic CB1 agonist depresses GABA release from hippocampal slices. These findings indicate that the function of endogenous cannabinoids released by depolarized hippocampal neurons might be to downregulate GABA release. Here we show that the transient suppression of GABA-mediated transmission that follows depolarization of hippocampal pyramidal neurons is mediated by retrograde signalling through release of endogenous cannabinoids. Signalling by the endocannabinoid system thus represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs.

Evidence for silent synapses: Implications for the expression of LTP

Recent work has suggested that some proportion of excitatory synapses on hippocampal CA1 pyramidal cells that express NMDA receptors (NMDARs) may not express functional AMPA receptors (AMPARs), thus making these synapses silent at the resting membrane potential. In agreement with this hypothesis, we demonstrate here that it is possible to stimulate synapses that yield no detectable excitatory postsynaptic currents (EPSCs) when the cell is held at -60 mV; yet at positive holding potentials (+30 to +60 mV), EPSCs can be elicited that are completely blocked by the NMDAR antagonist, D-APV. When these functionally silent synapses are subjected to an LTP induction protocol, EPSCs mediated by AMPARs appear and remain for the duration of the experiment. This conversion of silent synapses to functional synapses is blocked by D-APV. These results suggest that LTP may involve modification of AMPARs that, prior to LTP, were either not present in the postsynaptic membrane or electrophysiologically silent. This mechanism may account for several experimental results previously attributed to presynaptic changes in quantal content.

AMPA Receptor Trafficking at Excitatory Synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.

Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms

Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms.

Long-term potentiation in the CA1 region of the hippocampus is the most extensively studied model of activity-dependent synaptic plasticity in the mammalian brain. Its induction normally involves activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors, which are thought to control the occurrence of long-term potentiation at individual synapses. Recent work in the hippocampus indicates that NMDA receptor activation does not necessarily lead to induction of long-term potentiation but instead may elicit a repertoire of distinct forms of synaptic plasticity including short-term potentiation or long-term depression. Furthermore, mechanisms exist such that the induction of long-term potentiation can be inhibited by modest activation of NMDA receptors. Experimental results are beginning to clarify the mechanistic relationships between these different phenomena, although much remains unknown. Whatever their underlying mechanisms, these additional forms of NMDA-receptor-dependent synaptic plasticity confer increased flexibility to neural circuits involved in information processing and storage.

Role of AMPA Receptor Cycling in Synaptic Transmission and Plasticity

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

G Protein-Coupled Inwardly Rectifying K+ Channels (GIRKs) Mediate Postsynaptic but Not Presynaptic Transmitter Actions in Hippocampal Neurons

To study the role of G protein-coupled, inwardly rectifying K+ (GIRK) channels in mediating neurotransmitter actions in hippocampal neurons, we have examined slices from transgenic mice lacking the GIRK2 gene. The outward currents evoked by agonists for GABA(B) receptors, 5HT1A receptors, and adenosine A1 receptors were essentially absent in mutant mice, while the inward current evoked by muscarinic receptor activation was unaltered. In contrast, the presynaptic inhibitory action of a number of presynaptic receptors on excitatory and inhibitory terminals was unaltered in mutant mice. These included GABA(B), adenosine, muscarinic, metabotropic glutamate, and NPY receptors on excitatory synapses and GABA(B) and opioid receptors on inhibitory synapses. These findings suggest that a number of G protein-coupled receptors activate the same class of postsynaptic K+ channel, which contains GIRK2. In addition, the GIRK2 channels play no role in the inhibition mediated by presynaptic G protein-coupled receptors, suggesting that the same receptor can couple to different effector systems according to its subcellular location in the neuron.

Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number

Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). However, mechanisms involved in recruiting AMPARs to synapses are unknown. Here we use hippocampal slice cultures and biolistic gene transfections to study the targeting of AMPARs to synapses. We show that AMPARs are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 (postsynaptic density protein of 95 kDa) to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPARs to synapses without changing the number of surface AMPARs. At the same time, we show that stargazin overexpression drastically increases the number of extra-synaptic AMPARs, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs.

Increased Expression of α-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering after Endocytosis

The protein alpha-synuclein accumulates in the brain of patients with sporadic Parkinsons disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. alpha-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest overexpression of alpha-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of alpha-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology.

Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro.

1. The actions of serotonin (5‐HT) on pyramidal cells of the CA1 region of the rat hippocampus were characterized using intracellular recording in in vitro brain slices. 2. 5‐HT typically evokes a biphasic response consisting of a hyperpolarization which is followed by a longer‐lasting depolarization. These effects on membrane potential are accompanied by a decrease in the calcium‐activated after‐hyperpolarization (a.h.p). 3. Detailed analysis using 5‐HT antagonists and agonists indicates that the hyperpolarization is mediated by a 5‐HT1A receptor. Spiperone is the most effective antagonist of the response and the selective 5‐HT1A agonist, 8‐OHDPAT, behaves as a partial agonist at this receptor. In agreement with the distribution of 5‐HT1A binding sites, responses to 5‐HT were most prominent in the stratum radiatum. 4. The hyperpolarizing response is associated with a decrease in input resistance, is blocked by extracellular barium and intracellular caesium, is unaffected by the chloride gradient, and its reversal potential shifts with the extracellular concentration of potassium as predicted for a response mediated by a selective increase in potassium permeability. 5. The depolarizing response and reduction in the a.h.p. could be studied in isolation by blocking the hyperpolarizing response with either pertussis toxin or spiperone. The pharmacology of these responses did not correspond to that of any of the 5‐HT binding sites reported in C.N.S. tissue. Although the depolarization and blockade of the a.h.p. have the same time course it is unclear if they are mediated by the same or different receptors. 6. The depolarization most likely results from a decrease in resting potassium conductance. However, neither a blockade of the M current nor the a.h.p. current can account for the depolarization. 7. Blockade of phosphodiesterase activity by 3‐isobutyl‐1‐methylxanthine (IBMX) did not enhance the depressant action of 5‐HT on the a.h.p., making it unlikely that this action is mediated by cyclic AMP. 8. Blockade of the a.h.p. by 5‐HT reduces spike frequency adaptation and counteracts the inhibitory action of 5‐HT on 5‐HT1A receptors. This excitatory action outlasts the hyperpolarizing action. 9. In summary 5‐HT acts on at least two distinct receptors on hippocampal pyramidal cells, one coupled to the opening of potassium channels and a second coupled to a decrease in a resting potassium conductance and a decrease in the a.h.p.