Alex H. Babayan

Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosines effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTPs vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.

The Neuron-specific Chromatin Regulatory Subunit BAF53b is Necessary for Synaptic Plasticity and Memory

Recent exome sequencing studies have implicated polymorphic Brg1-Associated Factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Postmitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, which suggests a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appeared to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. Our results provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders.

Physiological Activation of Synaptic Rac>PAK (p-21 Activated Kinase) Signaling Is Defective in a Mouse Model of Fragile X Syndrome

The abnormal spine morphology found in fragile X syndrome (FXS) is suggestive of an error in the signaling cascades that organize the actin cytoskeleton. We report here that physiological activation of the small GTPase Rac1 and its effector p-21 activated kinase (PAK), two enzymes critically involved in actin management and functional synaptic plasticity, is impaired at hippocampal synapses in the Fmr1-knock-out (KO) mouse model of FXS. Theta burst afferent stimulation (TBS) caused a marked increase in the number of synapses associated with phosphorylated PAK in adult hippocampal slices from wild-type, but not Fmr1-KO, mice. Stimulation-induced activation of synaptic Rac1 was also absent in the mutants. The polymerization of spine actin that occurs immediately after theta stimulation appeared normal in mutant slices but the newly formed polymers did not properly stabilize, as evidenced by a prolonged vulnerability to a toxin (latrunculin) that disrupts dynamic actin filaments. Latrunculin also reversed long-term potentiation when applied at 10 min post-TBS, a time point at which the potentiation effect is resistant to interference in wild-type slices. We propose that a Rac>PAK signaling pathway needed for rapid stabilization of activity-induced actin filaments, and thus for normal spine morphology and lasting synaptic changes, is defective in FXS.

Synaptic evidence for the efficacy of spaced learning

The superiority of spaced vs. massed training is a fundamental feature of learning. Here, we describe unanticipated timing rules for the production of long-term potentiation (LTP) in adult rat hippocampal slices that can account for one temporal segment of the spaced trials phenomenon. Successive bouts of naturalistic theta burst stimulation of field CA1 afferents markedly enhanced previously saturated LTP if spaced apart by 1 h or longer, but were without effect when shorter intervals were used. Analyses of F-actin-enriched spines to identify potentiated synapses indicated that the added LTP obtained with delayed theta trains involved recruitment of synapses that were “missed” by the first stimulation bout. Single spine glutamate-uncaging experiments confirmed that less than half of the spines in adult hippocampus are primed to undergo plasticity under baseline conditions, suggesting that intrinsic variability among individual synapses imposes a repetitive presentation requirement for maximizing the percentage of potentiated connections. We propose that a combination of local diffusion from initially modified spines coupled with much later membrane insertion events dictate that the repetitions be widely spaced. Thus, the synaptic mechanisms described here provide a neurobiological explanation for one component of a poorly understood, ubiquitous aspect of learning.

Impairment of synaptic plasticity by the stress mediator CRH involves selective destruction of thin dendritic spines via RhoA signaling

Stress is ubiquitous in modern life and exerts profound effects on cognitive and emotional functions. Thus, whereas acute stress enhances memory, longer episodes exert negative effects through as yet unresolved mechanisms. We report a novel, hippocampus-intrinsic mechanism for the selective memory defects that are provoked by stress. CRH (corticotropin-releasing hormone), a peptide released from hippocampal neurons during stress, depressed synaptic transmission, blocked activity-induced polymerization of spine actin and impaired synaptic plasticity in adult hippocampal slices. Live, multiphoton imaging demonstrated a selective vulnerability of thin dendritic spines to this stress hormone, resulting in depletion of small, potentiation-ready excitatory synapses. The underlying molecular mechanisms required activation and signaling of the actin-regulating small GTPase, RhoA. These results implicate the selective loss of dendritic spine sub-populations as a novel structural and functional foundation for the clinically important effects of stress on cognitive and emotional processes.

Estrogen promotes learning-related plasticity by modifying the synaptic cytoskeleton.

Estrogens acute, facilitatory effects on glutamatergic transmission and long-term potentiation (LTP) provide a potential explanation for the steroids considerable influence on behavior. Recent work has identified mechanisms underlying these synaptic actions. Brief infusion of 17ß-estradiol (E2) into adult male rat hippocampal slices triggers actin polymerization within dendritic spines via a signaling cascade beginning with the GTPase RhoA and ending with inactivation of the filament-severing protein cofilin. Blocking this sequence, or actin polymerization itself, eliminates E2s effects on synaptic physiology. Notably, the theta burst stimulation used to induce LTP activates the same signaling pathway as E2 plus events that stabilize the reorganization of the sub-synaptic cytoskeleton. These observations suggest that E2 elicits a partial form of LTP, resulting in an increase of fast excitatory postsynaptic potentials (EPSPs) and a reduction in the threshold for lasting synaptic changes. While E2s effects on the cytoskeleton could be direct, results described here indicate that the hormone activates synaptic tropomyosin-related kinase B (TrkB) receptors for brain-derived neurotrophic factor (BDNF), a releasable neurotrophin that stimulates the RhoA to cofilin pathway. It is therefore possible that E2 acts via transactivation of neighboring receptors to modify the composition and structure of excitatory contacts. Finally, there is the question of whether a loss of acute synaptic actions contributes to the memory problems associated with estrogen depletion. Initial tests found that ovariectomy in middle-aged rats disrupts RhoA signaling, actin polymerization, and LTP consolidation. Acute applications of E2 reversed these defects, a result consistent with the idea that disturbances to actin management are one cause of behavioral effects that emerge with reductions in steroid levels.

Integrin Dynamics Produce a Delayed Stage of Long-Term Potentiation and Memory Consolidation

Memory consolidation theory posits that newly acquired information passes through a series of stabilization steps before being firmly encoded. We report here that in rat and mouse, hippocampus cell adhesion receptors belonging to the β1-integrin family exhibit dynamic properties in adult synapses and that these contribute importantly to a previously unidentified stage of consolidation. Quantitative dual immunofluorescence microscopy showed that induction of long-term potentiation (LTP) by theta burst stimulation (TBS) activates β1 integrins, and integrin-signaling kinases, at spine synapses in adult hippocampal slices. Neutralizing antisera selective for β1 integrins blocked these effects. TBS-induced integrin activation was brief (<7 min) and followed by an ∼45 min period during which the adhesion receptors did not respond to a second application of TBS. Brefeldin A, which blocks integrin trafficking to the plasma membrane, prevented the delayed recovery of integrin responses to TBS. β1 integrin-neutralizing antisera erased LTP when applied during, but not after, the return of integrin responsivity. Similarly, infusions of anti-β1 into rostral mouse hippocampus blocked formation of long-term, object location memory when started 20 min after learning but not 40 min later. The finding that β1 integrin neutralization was effective in the same time window for slice and behavioral experiments strongly suggests that integrin recovery triggers a temporally discrete, previously undetected second stage of consolidation for both LTP and memory.

LTP Induction Translocates Cortactin at Distant Synapses in Wild-Type But Not Fmr1 Knock-Out Mice

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactins movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactins interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter proteins use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

Synaptic Abnormalities in the Infralimbic Cortex of a Model of Congenital Depression

Multiple lines of evidence suggest that disturbances in excitatory transmission contribute to depression. Whether these defects involve the number, size, or composition of glutamatergic contacts is unclear. This study used recently introduced procedures for fluorescence deconvolution tomography in a well-studied rat model of congenital depression to characterize excitatory synapses in layer I of infralimbic cortex, a region involved in mood disorders, and of primary somatosensory cortex. Three groups were studied: (1) rats bred for learned helplessness (cLH); (2) rats resistant to learned helplessness (cNLH); and (3) control Sprague Dawley rats. In fields within infralimbic cortex, cLH rats had the same numerical density of synapses, immunolabeled for either the postsynaptic density (PSD) marker PSD95 or the presynaptic protein synaptophysin, as controls. However, PSD95 immunolabeling intensities were substantially lower in cLH rats, as were numerical densities of synapse-sized clusters of the AMPA receptor subunit GluA1. Similar but less pronounced differences (comparable numerical densities but reduced immunolabeling intensity for PSD95) were found in the somatosensory cortex. In contrast, non-helpless rats had 25% more PSDs than either cLH or control rats without any increase in synaptophysin-labeled terminal frequency. Compared with controls, both cLH and cNLH rats had fewer GABAergic contacts. These results indicate that congenital tendencies that increase or decrease depression-like behavior differentially affect excitatory synapses.

Rapid Effects of Oestrogen on Synaptic Plasticity: Interactions with Actin and Its Signalling Proteins

Oestrogen rapidly enhances fast excitatory postsynaptic potentials, facilitates long‐term potentiation (LTP) and increases spine numbers. Each effect likely contributes to the influence of the steroid on cognition and memory. In the present review, we first describe a model for the substrates of LTP that includes an outline of the synaptic events occurring during induction, expression and consolidation. Briefly, critical signalling pathways involving the small GTPases RhoA and Rac/Cdc42 are activated by theta burst‐induced calcium influx and initiate actin filament assembly via phosphorylation (inactivation) of cofilin. Reorganisation of the actin cytoskeleton changes spine and synapse morphology, resulting in increased concentrations of AMPA receptors at stimulated contacts. We then use the synaptic model to develop a specific hypothesis about how oestrogen affects both baseline transmission and plasticity. Brief infusions of 17β‐oestradiol (E2) reversibly stimulate the RhoA, cofilin phosphorylation and actin polymerisation cascade of the LTP machinery; blocking this eliminates the effects of the steroid on transmission. We accordingly propose that E2 induces a weak form of LTP and thereby increases synaptic responses, a hypothesis that also accounts for how it markedly enhances theta burst induced potentiation. Although the effects of E2 on the cytoskeleton could be a result of the direct activation of small GTPases by oestrogen receptors on the synaptic membrane, the hormone also activates tropomyosin‐related kinase B receptors for brain‐derived neurotrophic factor, a neurotrophin that engages the RhoA‐cofilin sequence and promotes LTP. The latter observations raise the possibility that E2 produces its effects on synaptic physiology via transactivation of neighbouring receptors that have prominent roles in the management of spine actin, synaptic physiology and plasticity.