Hierarchical order of coexisting pre- and postsynaptic forms of long-term potentiation at synapses in amygdala

Synaptic rules that may determine the interaction between coexisting forms of long-term potentiation (LTP) at glutamatergic central synapses remain unknown. Here, we show that two mechanistically distinct forms of LTP could be induced in thalamic input to the lateral nucleus of the amygdala (LA) with an identical presynaptic stimulation protocol, depending on the level of postsynaptic membrane polarization. One form of LTP, resulting from pairing of postsynaptic depolarization and low-frequency presynaptic stimulation, was both induced and expressed postsynaptically ("post-LTP"). The same stimulation in the absence of postsynaptic depolarization led to LTP, which was induced and expressed presynaptically ("pre-LTP"). The inducibility of coexisting pre- and postsynaptic forms of LTP at synapses in thalamic input followed a well-defined hierarchical order, such that pre-LTP was suppressed when post-LTP was induced. This interaction was mediated by activation of cannabinoid type 1 receptors by endogenous cannabinoids released in the lateral nucleus of the amygdala in response to activation of the type 1 metabotropic glutamate receptor. These results suggest a previously unknown mechanism by which the hierarchy of coexisting forms of long-term synaptic plasticity in the neural circuits of learned fear could be established, possibly reflecting the hierarchy of memories for the previously experienced fearful events according to their aversiveness level.     

Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.     

Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.     

Delivery of AMPA receptors to perisynaptic sites precedes the full expression of long-term potentiation

Trafficking of AMPA subtype glutamate receptors (AMPARs) from intracellular compartments to synapses is thought to be a major mechanism underlying the expression of long-term potentiation (LTP), a cellular substrate for learning and memory. However, it remains unclear whether the AMPAR trafficking that takes place during LTP is due to a targeted insertion of AMPARs directly into the synapse or delivery to extrasynaptic sites followed by translocation into the synapse. Here, we provide direct physiological evidence that LTP induced by a theta-burst pairing and tetanic stimulation protocols causes the rapid delivery of AMPARs to a perisynaptic site. Perisynaptic AMPARs do not normally detect synaptically released glutamate but can do so when the glial glutamate transporter EAAT1 is inhibited. AMPARs can be detected at this perisynaptic site before, but not after, the full expression of LTP. The appearance of perisynaptic AMPARs requires postsynaptic exocytosis, PKA signaling, and the C-terminal region of GluR1 subunit of AMPARs but not actin polymerization. Actin polymerization after LTP induction is required to retain AMPARs at the perisynaptic site after their appearance. Low-frequency stimulation given shortly after LTP induction leads to activity-dependent removal of perisynaptic AMPARs and suppresses the subsequent expression of LTP. These results demonstrate that AMPARs are rapidly trafficked to perisynaptic sites shortly after LTP induction and suggest that the delivery and maintenance of perisynaptic AMPARs may serve as a checkpoint in the expression of LTP.     

The neurotrophin receptor p75NTR modulates long-term depression and regulates the expression of AMPA receptor subunits in the hippocampus

Neurotrophins are involved in the modulation of synaptic transmission, including the induction of long-term potentiation (LTP) through the receptor TrkB. Because previous studies have revealed a bidirectional mode of neurotrophin action by virtue of signaling through either the neurotrophin receptor p75NTR or the Trk receptors, we tested the hypothesis that p75NTR is important for longterm depression (LTD) to occur. Although LTP was found to be unaffected in hippocampal slices of two different strains of mice carrying mutations of the p75NTR gene, hippocampal LTD was impaired in both p75NTR-deficient mouse strains. Furthermore, the expression levels of two (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, GluR2 and GluR3, but not GluR1 or GluR4, were found to be significantly altered in the hippocampus of p75NTR-deficient mice. These results implicate p75NTR in activity-dependent synaptic plasticity and extend the concept of functional antagonism of the neurotrophin signaling system.     

Endocytosis and recycling of AMPA receptors lacking GluR2/3

Excitatory synapses in the mammalian brain contain two types of ligand-gated ion channels: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). AMPARs are responsible for generating excitatory synaptic responses, whereas NMDAR activation triggers long-lasting changes in these responses by modulating the trafficking of AMPARs toward and away from synapses. AMPARs are tetramers composed of four subunits (GluR1-GluR4), which current models suggest govern distinct AMPAR trafficking behavior during synaptic plasticity. Here, we address the roles of GluR2 and GluR3 in controlling the recycling- and activity-dependent endocytosis of AMPARs by using cultured hippocampal neurons prepared from knockout (KO) mice lacking these subunits. We find that synapses and dendritic spines form normally in cells lacking GluR2/3 and that upon NMDAR activation, GluR2/3-lacking AMPARs are endocytosed in a manner indistinguishable from GluR2-containing AMPARs in wild-type (WT) neurons. AMPARs lacking GluR2/3 also recycle to the plasma membrane identically to WT AMPARs. However, because of their permeability to calcium, GluR2-lacking but not WT AMPARs exhibited robust internalization throughout the dendritic tree in response to AMPA application. Dendritic endocytosis of AMPARs also was observed in GABAergic neurons, which express a high proportion of GluR2-lacking AMPARs. These results demonstrate that GluR2 and GluR3 are not required for activity-dependent endocytosis of AMPARs and suggest that the most important property of GluR2 in the context of AMPAR trafficking may be its influence on calcium permeability.     

TARP modulation of synaptic AMPA receptor trafficking and gating depends on multiple intracellular domains

Previous work has established stargazin and its related family of transmembrane AMPA receptor regulatory proteins (TARPs) as auxiliary subunits of AMPA receptors (AMPARs) that control synaptic strength both by targeting AMPARs to synapses through an intracellular PDZ-binding motif and by modulating their gating through an extracellular domain. However, TARPs γ-2 and γ-8 differentially regulate the synaptic targeting of AMPARs, despite having identical PDZ-binding motifs. Here, we investigate the structural elements that contribute to this functional difference between TARP subtypes by using domain transplantation and truncation. We identify a component of synaptic AMPAR trafficking that is independent of the TARP C-terminal PDZ-binding motif, and we establish previously uncharacterized roles for the TARP intracellular N terminus, loop, and C terminus in modulating both the trafficking and gating of synaptic AMPARs.     

Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Dynamic changes in synaptic strength are thought to be critical for higher brain function such as learning and memory. Alterations in synaptic strength can result from modulation of AMPA receptor (AMPAR) function and trafficking to synaptic sites. The phosphorylation state of AMPAR subunits is one mechanism by which cells regulate receptor function and trafficking. Receptor phosphorylation is in turn regulated by extracellular signals; these include neuronal activity, neuropeptides, and neuromodulators such as dopamine and norepinephrine (NE). Although numerous studies have reported that the neuropeptide pituitary adenylate cyclase activating polypeptide 38 (PACAP38) alters hippocampal CA1 synaptic strength and GluA1 synaptic localization, its effect on AMPAR phosphorylation state has not been explored. We determined that PACAP38 stimulation of hippocampal cultures increased phosphorylation of S845, and decreased phosphorylation of T840 on the GluA1 AMPAR subunit. Increases in GluA1 S845 phosphorylation primarily occurred via PAC1 and VPAC2 receptor activation, whereas a reduction in GluA1 T840 phosphorylation was largely driven by PAC1 receptor activation and to a lesser extent by VPAC1 and VPAC2 receptor activation. GluA1 S845 phosphorylation could be blocked by a PKA inhibitor, and GluA1 T840 dephosphorylation could be blocked by a protein phosphatase 1/2A (PP1/PP2A) inhibitor and was partly blocked by a NMDA receptor (NMDAR) antagonist. These results demonstrate that the neuropeptide PACAP38 inversely regulates the phosphorylation of two distinct sites on GluA1 and may play an important role modulating AMPAR function and synaptic plasticity in the brain.AMPA-type glutamate receptors (AMPARs) are a tetrameric assembly composed of the GluA1, 2, 3, or 4 subunits. Within the adult hippocampus, receptors consist of primarily GluA1/2 and GluA2/3 complexes (1). Because AMPARs conduct the majority of excitatory transmission in the brain, modulation of AMPAR synaptic transmission is a powerful tool by which the cell can regulate synaptic strength and cell firing. Furthermore, it is hypothesized that complex behaviors such as learning, memory, and drug addiction involve alterations in synaptic strength (2, 3).The cell can regulate synaptic strength through changes in AMPAR conductance, trafficking, and tethering at synaptic sites. Such changes can be achieved through alterations in AMPAR expression, binding partners, and posttranslational modifications (4). A number of GluA1 and GluA2 phosphorylation sites have been proposed to play a role in AMPAR trafficking and synaptic plasticity. GluA1 S845 and T840 are two phosphorylation sites particularly relevant to this study. GluA1 S845 is phosphorylated by PKA and cGMP-dependent protein kinase II (5, 6). Its phosphorylation levels are regulated by NMDA receptors (NMDARs) (7), β-adrenergic receptors (8, 9), and muscarinic cholinergic receptors (9), and during homeostatic scaling (10), long-term depression (LTD) (11), and emotionally stressful conditions (8). Likewise, GluA1 S845 phospho-mutants show GluA1 trafficking and LTD deficits (12–14). In contrast, the GluA1 T840 site is less well characterized. PKC, calcium/calmodulin-dependent protein kinase II, protein phosphatase 1/2A (PP1/PP2A), and NMDAR activity have been reported to regulate GluA1 T840 phosphorylation (15–17). GluA1 T840 phosphorylation has also been found to enhance channel conductance (18).PACAP38 (pituitary adenylate cyclase activating polypeptide 38) is a neuropeptide that has been shown to regulate hippocampal CA1 synaptic strength (19–22). PACAP38 can bind to and activate three different G protein coupled receptors, the PAC1, VPAC1, and VPAC2 receptors, which can lead to elevated cyclic AMP and Ca²⁺ levels, and activation of phospholipase C and phospholipase D (23). In the hippocampus, PACAP38 stimulation has been shown to alter synaptic strength (19–22) and AMPAR excitatory postsynaptic currents (EPSCs) (24) and to reduce GluA1 synaptic localization (25). PACAP knockout mice are impaired in contextual fear conditioning and novel object recognition (26), and PAC1 receptor knockouts exhibit impaired contextual fear conditioning (27). Given the ability of PACAP38 to regulate basal synaptic transmission and AMPAR EPSCs (24), we hypothesized that PACAP38 stimulation could alter AMPAR phosphorylation levels.We found that PACAP38 stimulation led to increased GluA1 S845 phosphorylation and decreased GluA1 T840 phosphorylation. We also demonstrated that unique signaling pathways were used to drive these phosphorylation changes. Although activation of the PAC1 and VPAC2 receptor elicited a robust increase in GluA1 S845 phosphorylation, only PAC1 receptor activity could elicit a robust decrease in GluA1 T840 phosphorylation. In addition, a PKA inhibitor blocked the increase in S845 phosphorylation, while a PP1/PP2A inhibitor blocked the decrease in T840 phosphorylation and a NMDAR antagonist partially blocked the decrease in T840 phosphorylation.     

Relevance of synaptic tagging and capture to the persistence of long-term potentiation and everyday spatial memory

Memory for inconsequential events fades, unless these happen before or after other novel or surprising events. However, our understanding of the neurobiological mechanisms of novelty-enhanced memory persistence is mainly restricted to aversive or fear-associated memories. We now outline an "everyday appetitive" behavioral model to examine whether and how unrelated novelty facilitates the persistence of spatial memory coupled to parallel electrophysiological studies of the persistence of long-term potentiation (LTP). Across successive days, rats were given one trial per day to find food in different places and later had to recall that day's location. This task is both hippocampus and NMDA receptor dependent. First, encoding with low reward induced place memory that decayed over 24 h; in parallel, weak tetanization of CA1 synapses in brain slices induced early-LTP fading to baseline. Second, novelty exploration scheduled 30 min after this weak encoding resulted in persistent place memory; similarly, strong tetanization--analogous to novelty--both induced late-LTP and rescued early- into late-LTP on an independent but convergent pathway. Third, hippocampal dopamine D1/D5 receptor blockade or protein synthesis inhibition within 15 min of exploration prevented persistent place memory and blocked late-LTP. Fourth, symmetrically, when spatial memory was encoded using strong reward, this memory persisted for 24 h unless encoding occurred under hippocampal D1/D5 receptor blockade. Novelty exploration before this encoding rescued the drug-induced memory impairment. Parallel effects were observed in LTP. These findings can be explained by the synaptic tagging and capture hypothesis.     

Unified quantitative model of AMPA receptor trafficking at synapses

Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.     

Stabilization of Ca2+-permeable AMPA receptors at perisynaptic sites by GluR1-S845 phosphorylation

AMPA receptor (AMPAR) channel properties and function are regulated by its subunit composition and phosphorylation. Certain types of neural activity can recruit Ca²⁺-permeable (CP) AMPARs, such as GluR1 homomers, to synapses likely via lateral diffusion from extrasynaptic sites. Here we show that GluR1-S845 phosphorylation can alter the subunit composition of perisynaptic AMPARs by providing stability to GluR1 homomers. Using mice specifically lacking phosphorylation of the GluR1-S845 site (GluR1-S845A mutants), we demonstrate that this site is necessary for maintaining CP-AMPARs. Specifically, in the GluR1-S845A mutants, CP-AMPARs were absent from perisynaptic locations mainly due to lysosomal degradation. This regulation was mimicked by acute desphosphorylation of the GluR1-S845 site in wild-type mice by NMDA application. Furthermore, long-term depression (LTD) was associated with a reduction in perisynaptic CP-AMPAR levels. Our findings suggest that GluR1-S845 is necessary for maintaining CP-AMPARs on the surface, especially at perisynaptic sites, and suggest that the regulation of these receptors is involved in synaptic plasticity.     

Spontaneous transmitter release recruits postsynaptic mechanisms of long-term and intermediate-term facilitation in Aplysia

Whereas short-term (minutes) facilitation at Aplysia sensory–motor neuron synapses is presynaptic, long-term (days) facilitation involves synaptic growth, which requires both presynaptic and postsynaptic mechanisms. How are the postsynaptic mechanisms recruited, and when does that process begin? We have been investigating the possible role of spontaneous transmitter release from the presynaptic neuron. In the previous paper, we found that spontaneous release is critical for the induction of long-term facilitation, and this process begins during an intermediate-term stage of facilitation that is the first stage to involve postsynaptic as well as presynaptic mechanisms. We now report that increased spontaneous release during the short-term stage acts as an orthograde signal to recruit postsynaptic mechanisms of intermediate-term facilitation including increased IP3, Ca²⁺, and membrane insertion and recruitment of clusters of AMPA-like receptors, which may be first steps in synaptic growth during long-term facilitation. These results suggest that the different stages of facilitation involve a cascade of pre- and postsynaptic mechanisms, which is initiated by spontaneous release and may culminate in synaptic growth.     

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine–glutamate interplay.Hippocampal dopaminergic neuromodulation participates in several cognitive functions including novelty detection and long-term memory storage (1, 2). As a consequence, impairments in hippocampal neuromodulatory transmission affect synaptic plasticity at glutamatergic synapses, prevent learning and memory formation, and have been proposed to be a cellular substrate for neurodevelopmental psychiatric disorders such as schizophrenia (3). In the hippocampus and cortex, pyramidal neurons express mostly dopamine D1 and D5 receptors along their dendritic tree (4–6). Their recruitment affects the trafficking and surface expression of glutamate NMDA receptors (NMDARs), two processes that are essential for excitatory neurotransmission and synaptic plasticity. Indeed, activating dopamine D1 receptors (D1Rs) promotes the surface expression and function of NMDAR and thereby favors the long-term potentiation of excitatory glutamate synapses (7–10). Reciprocally, the activation of NMDAR modulates D1R surface expression and signaling (11). The bidirectional dialogue between dopamine and glutamate NMDAR-associated signaling thus involves changes in membrane receptor content and trafficking.Although this functional interaction is usually considered as relying on intracellular protein kinase signaling cascades (7, 10, 12), physical interactions between D1R and NMDAR at the plasma membrane were recently reported to stabilize laterally diffusing surface D1R in spines, modulate D1R- and NMDAR-mediated signaling, and influence working memory (13–16). Thus, direct interactions between these receptors could contribute to the regulation of their surface distributions and play a major role in the dopamine–glutamate interplay (15, 17). In particular, because the regulation of NMDAR synaptic content involves surface diffusion processes in and out of synaptic and extrasynaptic compartments (18), the possibility emerges that dopamine might modulate NMDAR-dependent synaptic transmission by tuning NMDAR lateral dynamics through D1R–NMDAR physical interactions. To address this question and investigate the role of the D1R/NMDAR surface crosstalk in synaptic physiology, we here assessed the surface distribution and trafficking of D1 and NMDA receptors in rat hippocampal neurons using a combination of high-resolution single-nanoparticle tracking, bulk imaging, and electrophysiology.     

G protein-activated inwardly rectifying potassium channels mediate depotentiation of long-term potentiation

Excitatory synapses in the brain undergo activity-dependent changes in the strength of synaptic transmission. Such synaptic plasticity as exemplified by long-term potentiation (LTP) is considered a cellular correlate of learning and memory. The presence of G protein-activated inwardly rectifying K⁺ (GIRK) channels near excitatory synapses on dendritic spines suggests their possible involvement in synaptic plasticity. However, whether activity-dependent regulation of GIRK channels affects excitatory synaptic plasticity is unknown. In a companion article we have reported activity-dependent regulation of GIRK channel density in cultured hippocampal neurons that requires activity of NMDA receptors (NMDAR) and protein phosphatase-1 (PP1) and takes place within 15 min. In this study, we performed whole-cell recordings of cultured hippocampal neurons and found that NMDAR activation increases basal GIRK current and GIRK channel activation mediated by adenosine A₁ receptors, but not GABA_B receptors. Given the similar involvement of NMDARs, adenosine A₁ receptors, and PP1 in depotentiation of LTP caused by low-frequency stimulation that immediately follows LTP-inducing high-frequency stimulation, we wondered whether NMDAR-induced increase in GIRK channel surface density and current may contribute to the molecular mechanisms underlying this specific depotentiation. Remarkably, GIRK2 null mutation or GIRK channel blockade abolishes depotentiation of LTP, demonstrating that GIRK channels are critical for depotentiation, one form of excitatory synaptic plasticity.     

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity–dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor–mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.     

Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats

Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that “long thin” but not “mushroom” spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.Alcohol addiction is a major public health problem in the Western world. In the United States alone, about 15% of adults have an alcohol-related disorder at some point in their life, and alcohol abuse costs the economy over $220 billion per y in medical care and productivity loss (1). A general consensus has emerged on drug addiction as a substance-induced, aberrant form of neural plasticity (2, 3). The nucleus accumbens (Nacc) plays a central role in the neural circuits that are responsible for goal-directed behaviors (4, 5) and in addictive states. Its activity is heavily modulated by glutamate- (GLU) and dopamine- (DA) containing projections that originate in cortical and limbic regions and converge on a common postsynaptic target: the medium spiny neuron (MSN). Furthermore, DA modulates GLU inputs to Nacc neurons (6, 7), both by directly influencing synaptic transmission and by modulating voltage-dependent conductances (8). Accordingly, interactions between DA and GLU are involved in drug-induced locomotor stimulation and addiction (9, 10) and may represent useful potential therapeutic targets (11, 12). In the distal portion of the dendrites of MSNs a significant subpopulation of spines shows a particular synaptic architecture, called the “striatal microcircuit” or “synaptic triad” (13, 14), which is characterized by a double, discrete, and reciprocal interaction between DA and GLU afferents: The former establishes synaptic contact on the spine neck, whereas the latter reaches the head (13). This classical, widely accepted picture has been integrated with the coexistence of DA and GLU on the same neurons (15), but because this phenomenon appears to regress with growth in vitro (16) and its role is unclear at present (17), in the present study GLU and DA will be considered as originating from cortex and ventral tegmental area (VTA), respectively.At present, little information is available concerning the effects produced by ethanol withdrawal in dependent rats (18), although a selective increase in the density of mushroom-type spines following chronic intermittent ethanol intake has recently been reported on the basal dendrites of layer V neurons of the rodent prefrontal cortex (19). In addition, reduced expression of tyrosine hydroxylase (TH) has been demonstrated in the ventral striatum of rats maintained on a chronic ethanol-containing diet (20), and a decrease of neurofilament protein immunoreactivity in the VTA (21) has been reported. Thus, in the present work, we sought to investigate possible alterations produced by ethanol withdrawal on mesocorticolimbic transmission by exploring critical elements whose presence is strictly correlated with DAergic and GLUergic function, respectively: TH- and dopamine transporter (DAT)-positive fibers and postsynaptic density 95 (PSD-95). Spine density, morphology, and morphometry of MSNs in the Nacc shell were also investigated to obtain structural insights into pre- and postsynaptic elements of the triad simultaneously. Although considered impossible until recently (22), we have developed a new method (23) that allows visualizing the finest morphological details of spinous neurons (Golgi-Cox staining) together with the immunofluorescent neuronal elements under study. By exploiting this novel approach, we are able to visualize (in the same slice) spine morphology, TH- and DAT-positive fibers, and PSD-95–positive elements to gather information on DA and GLU transmission. Notably, because recent work suggests (24, 25) a potential relationship between spine shape, synaptic function, and morphological rearrangements of the spines as forms of developmental or experience-dependent plasticity (26), we performed patch-clamp experiments in Nacc shell slices obtained from ethanol-withdrawn rats to evaluate whether long-term depression (LTD) formation and its underlying synaptic currents are modified by experimental conditions.     

Developmental regulation of protein interacting with C kinase 1 (PICK1) function in hippocampal synaptic plasticity and learning

AMPA-type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the mammalian central nervous system. Modulation of AMPAR trafficking supports several forms of synaptic plasticity thought to underlie learning and memory. Protein interacting with C kinase 1 (PICK1) is an AMPAR-binding protein shown to regulate both AMPAR trafficking and synaptic plasticity at many distinct synapses. However, studies examining the requirement for PICK1 in maintaining basal synaptic transmission and regulating synaptic plasticity at hippocampal Schaffer collateral-cornu ammonis 1 (SC-CA1) synapses have produced conflicting results. In addition, the effect of PICK1 manipulation on learning and memory has not been investigated. In the present study we analyzed the effect of genetic deletion of PICK1 on basal synaptic transmission and synaptic plasticity at hippocampal Schaffer collateral-CA1 synapses in adult and juvenile mice. Surprisingly, we find that loss of PICK1 has no significant effect on synaptic plasticity in juvenile mice but impairs some forms of long-term potentiation and multiple distinct forms of long-term depression in adult mice. Moreover, inhibitory avoidance learning is impaired only in adult KO mice. These results suggest that PICK1 is selectively required for hippocampal synaptic plasticity and learning in adult rodents.     

Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development

The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.     

Presynaptic m1 muscarinic receptors are necessary for mGluR long-term depression in the hippocampus

To investigate the role of M1 muscarininc acetylcholine receptors (m1 receptors) in metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), we produced mouse lines in which deletion of the m1 gene is restricted to the forebrain (FB–m1KO) or hippocampal CA3 pyramidal neurons (CA3–m1KO). Stimulation in FB–m1KO hippocampal slices resulted in excitatory postsynaptic potentials and long-term synaptic plasticity (long-term potentiation and LTD) similar to controls. The mice were deficient in (S)-3,5-dihydroxyphenylglycine hydrate (DHPG)-induced mGluR LTD, which correlated with a presynaptic increase in the release of neurotransmitters. Protein kinase C (PKC) activity, which is downstream from both mGluRs and m1 receptors, was reduced in CA3 but not in CA1. The presynaptic requirement of m1 receptors was confirmed by the lack of DHPG-induced mGluR LTD in the CA1 of slices from CA3–m1KO mice. mGluR LTD was rescued by stimulating PKC activity pharmacologically in CA3–m1KO mice. These data confirm a role for PKC activation in presynaptic induction of mGluR LTD and distinguish between the roles of mGluRs and m1 receptors.