Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

Pain Facilitation and Activity-Dependent Plasticity in Pain Modulatory Circuitry: Role of BDNF-TrkB Signaling and NMDA Receptors

Pain modulatory circuitry in the brainstem exhibits considerable synaptic plasticity. The increased peripheral neuronal barrage after injury activates spinal projection neurons that then activate multiple chemical mediators including glutamatergic neurons at the brainstem level, leading to an increased synaptic strength and facilitatory output. It is not surprising that a well-established regulator of synaptic plasticity, brain-derived neurotrophic factor (BDNF), contributes to the mechanisms of descending pain facilitation. After tissue injury, BDNF and TrkB signaling in the brainstem circuitry is rapidly activated. Through the intracellular signaling cascade that involves phospholipase C, inositol trisphosphate, protein kinase C, and nonreceptor protein tyrosine kinases; N-methyl-D-aspartate (NMDA) receptors are phosphorylated, descending facilitatory drive is initiated, and behavioral hyperalgesia follows. The synaptic plasticity observed in the pain pathways shares much similarity with more extensively studied forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD), which typically express NMDA receptor dependency and regulation by trophic factors. However, LTP and LTD are experimental phenomena whose relationship to functional states of learning and memory has been difficult to prove. Although mechanisms of synaptic plasticity in pain pathways have typically not been related to LTP and LTD, pain pathways have an advantage as a model system for synaptic modifications as there are many well-established models of persistent pain with clear measures of the behavioral phenotype. Further studies will elucidate cellular and molecular mechanisms of pain sensitization and further our understanding of principles of central nervous system plasticity and responsiveness to environmental challenge.     

Presynaptic LTP and LTD of Excitatory and Inhibitory Synapses

Ubiquitous forms of long-term potentiation (LTP) and depression (LTD) are caused by enduring increases or decreases in neurotransmitter release. Such forms or presynaptic plasticity are equally observed at excitatory and inhibitory synapses and the list of locations expressing presynaptic LTP and LTD continues to grow. In addition to the mechanistically distinct forms of postsynaptic plasticity, presynaptic plasticity offers a powerful means to modify neural circuits. A wide range of induction mechanisms has been identified, some of which occur entirely in the presynaptic terminal, whereas others require retrograde signaling from the postsynaptic to presynaptic terminals. In spite of this diversity of induction mechanisms, some common induction rules can be identified across synapses. Although the precise molecular mechanism underlying long-term changes in transmitter release in most cases remains unclear, increasing evidence indicates that presynaptic LTP and LTD can occur in vivo and likely mediate some forms of learning.At several excitatory and inhibitory synapses, neuronal activity can trigger enduring increases or decreases in neurotransmitter release, thereby producing long-term potentiation (LTP) or long-term depression (LTD) of synaptic strength, respectively. In the last decade, many studies have revealed that these forms of plasticity are ubiquitously expressed in the mammalian brain, and accumulating evidence indicates that they may underlie behavioral adaptations occurring in vivo. These studies have also uncovered a wide range of induction mechanisms, which converge on the presynaptic terminal where an enduring modification in the neurotransmitter release process takes place. Interestingly, presynaptic forms of LTP/LTD can coexist with classical forms of postsynaptic plasticity. Such diversity expands the dynamic range and repertoire by which neurons modify their synaptic connections. This review discusses mechanistic aspects of presynaptic LTP and LTD at both excitatory and inhibitory synapses in the mammalian brain, with an emphasis on recent findings.     

Do stress and long-term potentiation share the same molecular mechanisms?

Stress is a biological, significant factor shown to influence hippocampal synaptic plasticity and cognitive functions. Although numerous studies have reported that stress produces a suppression in long-term potentiation (LTP; a putative synaptic mechanism underlying learning and memory), little is known about the mechanism by which this occurs. Because the effects of stress on LTP and its converse process, long-term depression (LTD), parallel the changes in synapticity that occur following the establishment of LTP with tetanic stimulation (i.e., occluding LTP and enhancing LTD induction), it has been proposed that stress affects subsequent hippocampal plasticity by sharing the same molecular machinery required to support LTP. This article summarizes recent findings from ours and other laboratories to assess this view and discusses relevant hypotheses in the study of stress-related modifications of synaptic plasticity.     

Actin-associated neurabin-protein phosphatase-1 complex regulates hippocampal plasticity

Protein phosphatase-1 (PP1) has been implicated in the control of long-term potentiation (LTP) and depression (LTD) in rat hippocampal CA1 neurons. PP1 catalytic subunits associate with multiple postsynaptic regulatory subunits, but the PP1 complexes that control hippocampal LTP and LTD in the rat hippocampus remain unidentified. The neuron-specific actin-binding protein, neurabin-I, is enriched in dendritic spines, and tethers PP1 to actin-rich postsynaptic density to regulate morphology and maturation of spines. The present studies utilized Sindbis virus-mediated expression of wild-type and mutant neurabin-I polypeptides in organotypic cultures of rat hippocampal slices to investigate their role in synaptic plasticity. While wild-type neurabin-I elicited no change in basal synaptic transmission, it enhanced LTD and inhibited LTP in CA1 pyramidal neurons. By comparison, mutant neurabins, specifically those unable to bind PP1 or F-actin, decreased basal synaptic transmission, attenuated LTD and increased LTP in slice cultures. Biochemical and cell biological analyses suggested that, by mislocalizing synaptic PP1, the mutant neurabins impaired the functions of endogenous neurabin-PP1 complexes and modulated LTP and LTD. Together, these studies provided the first biochemical and physiological evidence that a postsynaptic actin-bound neurabin-I-PP1 complex regulates synaptic transmission and bidirectional changes in hippocampal plasticity.     

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.     

Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses

Activity-induced modification of neuronal connections is essential for the development of the nervous system and may also underlie learning and memory functions of mature brain. Previous studies have shown an increase in dendritic spine density and/or enlargement of spines after the induction of long-term potentiation (LTP). Using two-photon time-lapse imaging of dendritic spines in acute hippocampal slices from neonatal rats, we found that the induction of long-term depression (LTD) by low-frequency stimulation is accompanied by a marked shrinkage of spines, which can be reversed by subsequent high-frequency stimulation that induces LTP. The spine shrinkage requires activation of NMDA receptors and calcineurin, similar to that for LTD. However, spine shrinkage is mediated by cofilin, but not by protein phosphatase 1 (PP1), which is essential for LTD, suggesting that different downstream pathways are involved in spine shrinkage and LTD. This activity-induced spine shrinkage may contribute to activity-dependent elimination of synaptic connections.     

Aquaporin-4 water channels and synaptic plasticity in the hippocampus

Aquaporin-4 (AQP4) is the major water channel expressed in the central nervous system (CNS) and is primarily expressed in glial cells. Many studies have shown that AQP4 regulates the response of the CNS to insults or injury, but far less is known about the potential for AQP4 to influence synaptic plasticity or behavior. Recent studies have examined long-term potentiation (LTP), long-term depression (LTD), and behavior in AQP4 knockout (KO) and wild-type mice to gain more insight into its potential role. The results showed a selective effect of AQP4 deletion on LTP of the Schaffer collateral pathway in hippocampus using an LTP induction protocol that simulates pyramidal cell firing during theta oscillations (theta-burst stimulation; TBS). However, LTP produced by a different induction protocol was unaffected. There was also a defect in LTD after low frequency stimulation (LFS) in AQP4 KO mice. Interestingly, some slices from AQP4 KO mice exhibited LTD after TBS instead of LTP, or LTP following LFS instead of LTD. These data suggest that AQP4 and astrocytes influence the polarity of long-term synaptic plasticity (potentiation or depression). These potentially powerful roles expand the influence of AQP4 and astrocytes beyond the original suggestions related to regulation of extracellular potassium and water balance. Remarkably, AQP4 KO mice did not show deficits in basal transmission, suggesting specificity for long-term synaptic plasticity. The mechanism appears to be related to neurotrophins and specifically brain-derived neurotrophic factor (BDNF) because pharmacological blockade of neurotrophin trk receptors or scavenging ligands such as BDNF restored plasticity. The in vitro studies predicted effects in vivo of AQP4 deletion because AQP4 KO mice performed worse using a task that requires memory for the location of objects (object placement). However, performance on other hippocampal-dependent tasks was spared. The results suggest an unanticipated and selective role of AQP4 in synaptic plasticity and spatial memory, and underscore the growing appreciation of the role of glial cells in functions typically attributed to neurons. Implications for epilepsy are discussed because of the previous evidence that AQP4 influences seizures, and the role of synaptic plasticity in epileptogenesis.     

Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala

Stimulated exocytosis and endocytosis of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors (AMPARs) have been proposed as primary mechanisms for the expression of hippocampal CA1 long-term potentiation (LTP) and long-term depression (LTD), respectively. LTP and LTD, the two most well characterized forms of synaptic plasticity, are thought to be important for learning and memory in behaving animals. Both LTP and LTD can also be induced in the lateral amygdala (LA), a critical structure involved in fear conditioning. However, the role of AMPAR trafficking in the expression of either LTP or LTD in this structure remains unclear. In this study, we show that NMDA receptor-dependent LTP and LTD can be reliably induced at the synapses of the auditory thalamic inputs to the LA in brain slices. The expression of LTP was prevented by post-synaptic blockade of vesicle-mediated exocytosis with application of a light chain of Clostridium tetanus neurotoxin and was associated with increased cell-surface AMPAR expression. In contrast, the expression of LTD was prevented by post-synaptic application of a glutamate receptor 2-derived interference peptide, which specifically blocks the stimulated clathrin-dependent endocytosis of AMPARs, and was correlated with a reduction in plasma membrane-surface expression of AMPARs. These results strongly suggest that regulated trafficking of post-synaptic AMPARs is also involved in the expression of LTP and LTD in the LA.     

On the Mechanism of Synaptic Depression Induced by CaMKIIN,an Endogenous Inhibitor of CaMKII

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca²⁺ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.     

Cholinergic neurotransmission is essential for perirhinal cortical plasticity and recognition memory

We establish the importance of cholinergic neurotransmission to both recognition memory and plasticity within the perirhinal cortex of the temporal lobe. The muscarinic receptor antagonist scopolamine impaired the preferential exploration of novel over familiar objects, disrupted the normal reduced activation of perirhinal neurones to familiar compared to novel pictures, and blocked production of long-term depression (LTD) but not long-term potentiation (LTP) of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for the involvement of cholinergic mechanisms in synaptic plastic processes within perirhinal cortex that are necessary for recognition memory.     

The AAA+ ATPase Thorase regulates AMPA receptor-dependent synaptic plasticity and behavior

The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase Thorase, which regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP, and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory.     

Synaptic Long-Term Potentiation and Depression in the Rat Medial Vestibular Nuclei Depend on Neural Activation of Estrogenic and Androgenic Signals

Estrogenic and androgenic steroids can be synthesised in the brain and rapidly modulate synaptic transmission and plasticity through direct interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used whole cell patch clamp recordings in brainstem slices of male rats to explore the influence of ER and AR activation and local synthesis of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the long-term synaptic changes induced in the neurons of the medial vestibular nucleus (MVN). Long-term depression (LTD) and long-term potentiation (LTP) caused by different patterns of high frequency stimulation (HFS) of the primary vestibular afferents were assayed under the blockade of ARs and ERs or in the presence of inhibitors for enzymes synthesizing DHT (5α-reductase) and E2 (P450-aromatase) from testosterone (T). We found that LTD is mediated by interaction of locally produced androgens with ARs and LTP by interaction of locally synthesized E2 with ERs. In fact, the AR block with flutamide prevented LTD while did not affect LTP, and the blockade of ERs with ICI 182,780 abolished LTP without influencing LTD. Moreover, the block of P450-aromatase with letrozole not only prevented the LTP induction, but inverted LTP into LTD. This LTD is likely due to the local activation of androgens, since it was abolished under blockade of ARs. Conversely, LTD was still induced in the presence of finasteride the inhibitor of 5α-reductase demonstrating that T is able to activate ARs and induce LTD even when DHT is not synthesized. This study demonstrates a key and opposite role of sex neurosteroids in the long-term synaptic changes of the MVN with a specific role of T-DHT for LTD and of E2 for LTP. Moreover, it suggests that different stimulation patterns can lead to LTD or LTP by specifically activating the enzymes involved in the synthesis of androgenic or estrogenic neurosteroids.     

LTP inhibits LTD in the hippocampus via regulation of GSK3beta

Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. Here we show that GSK3beta mediates an interaction between two major forms of synaptic plasticity in the brain, N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3beta inhibitors block the induction of LTD. Furthermore, the activity of GSK3beta is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3beta activity. This regulation of GSK3beta during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. We conclude that the regulation of GSK3beta activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information.     

Complexity of calcium signaling in synaptic spines

Long-term potentiation and long-term depression are thought to be cellular mechanisms contributing to learning and memory. Although the physiological phenomena have been well characterized, little consensus of their underlying molecular mechanisms has emerged. One reason for this may be the under-appreciated complexity of the signaling pathways that can arise if key signaling molecules are discretely localized within the synapse. Recent findings suggest an unanticipated degree of structural organization at the synapse, and improved methods in cellular imaging of living tissue have provided much-needed information about the intracellular dynamics of Ca(2+), thought to be critical for both LTP and LTD. In this review, we briefly summarize some of these developments, and show that a more complete understanding of cellular signaling depends on the successful integration of traditional biochemistry and molecular biology with the spatial and temporal details of synaptic ultrastructure. Biophysically realistic computer simulations can have an important role in bridging these disciplines.     

Synaptic plasticity in the mesolimbic dopamine system

Long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical mechanisms that contribute to the neural circuit modifications that mediate all forms of experience-dependent plasticity. It has, however, been difficult to demonstrate directly that experience causes long-lasting changes in synaptic strength and that these mediate changes in behaviour. To address these potential functional roles of LTP and LTD, we have taken advantage of the powerful in vivo effects of drugs of abuse that exert their behavioural effects in large part by acting in the nucleus accumbens (NAc) and ventral tegmental area (VTA); the two major components of the mesolimbic dopamine system. Our studies suggest that in vivo drugs of abuse such as cocaine cause long-lasting changes at excitatory synapses in the NAc and VTA owing to activation of the mechanisms that underlie LTP and LTD in these structures. Thus, administration of drugs of abuse provides a distinctive model for further investigating the mechanisms and functions of synaptic plasticity in brain regions that play important roles in the control of motivated behaviour, and one with considerable practical implications.     

Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.     

Molecular constraints on synaptic tagging and maintenance of long-term potentiation: a predictive model

Protein synthesis-dependent, late long-term potentiation (LTP) and depression (LTD) at glutamatergic hippocampal synapses are well characterized examples of long-term synaptic plasticity. Persistent increased activity of protein kinase M ζ (PKMζ) is thought essential for maintaining LTP. Additional spatial and temporal features that govern LTP and LTD induction are embodied in the synaptic tagging and capture (STC) and cross capture hypotheses. Only synapses that have been "tagged" by a stimulus sufficient for LTP and learning can "capture" PKMζ. A model was developed to simulate the dynamics of key molecules required for LTP and LTD. The model concisely represents relationships between tagging, capture, LTD, and LTP maintenance. The model successfully simulated LTP maintained by persistent synaptic PKMζ, STC, LTD, and cross capture, and makes testable predictions concerning the dynamics of PKMζ. The maintenance of LTP, and consequently of at least some forms of long-term memory, is predicted to require continual positive feedback in which PKMζ enhances its own synthesis only at potentiated synapses. This feedback underlies bistability in the activity of PKMζ. Second, cross capture requires the induction of LTD to induce dendritic PKMζ synthesis, although this may require tagging of a nearby synapse for LTP. The model also simulates the effects of PKMζ inhibition, and makes additional predictions for the dynamics of CaM kinases. Experiments testing the above predictions would significantly advance the understanding of memory maintenance.