Gary Lynch

Changes in Synaptic Morphology Accompany Actin Signaling during LTP

Stabilization of long-term potentiation (LTP) is commonly proposed to involve changes in synaptic morphology and reorganization of the spine cytoskeleton. Here we tested whether, as predicted from this hypothesis, induction of LTP by theta-burst stimulation activates an actin regulatory pathway and alters synapse morphology within the same dendritic spines. TBS increased severalfold the numbers of spines containing phosphorylated (p) p21-activated kinase (PAK) or its downstream target cofilin; the latter regulates actin filament assembly. The PAK/cofilin phosphoproteins were increased at 2 min but not 30 s post-TBS, peaked at 7 min, and then declined. Double immunostaining for the postsynaptic density protein PSD95 revealed that spines with high pPAK or pCofilin levels had larger synapses (+60–70%) with a more normal size frequency distribution than did neighboring spines. Based on these results and simulations of shape changes to synapse-like objects, we propose that theta stimulation markedly increases the probability that a spine will enter a state characterized by a large, ovoid synapse and that this morphology is important for expression and later stabilization of LTP.

Brain-Derived Neurotrophic Factor Rescues Synaptic Plasticity in a Mouse Model of Fragile X Syndrome

Mice lacking expression of the fragile X mental retardation 1 (Fmr1) gene have deficits in types of learning that are dependent on the hippocampus. Here, we report that long-term potentiation (LTP) elicited by threshold levels of theta burst afferent stimulation (TBS) is severely impaired in hippocampal field CA1 of young adult Fmr1 knock-out mice. The deficit was not associated with changes in postsynaptic responses to TBS, NMDA receptor activation, or levels of punctate glutamic acid decarboxylase-65/67 immunoreactivity. TBS-induced actin polymerization within dendritic spines was also normal. The LTP impairment was evident within 5 min of induction and, thus, may not be secondary to defects in activity-initiated protein synthesis. Protein levels for both brain-derived neurotrophic factor (BDNF), a neurotrophin that activates pathways involved in spine cytoskeletal reorganization, and its TrkB receptor were comparable between genotypes. BDNF infusion had no effect on baseline transmission or on postsynaptic responses to theta burst stimulation, but nonetheless fully restored LTP in slices from fragile X mice. These results indicate that the fragile X mutation produces a highly selective impairment to LTP, possibly at a step downstream of actin filament assembly, and suggest a means for overcoming this deficit. The possibility of a pharmacological therapy based on these results is discussed.

LTP consolidation: substrates, explanatory power, and functional significance.

Long-term potentiation (LTP) resembles memory in that it is initially unstable and then, over about 30 min, becomes increasingly resistant to disruption. Here we present an hypothesis to account for this initial consolidation effect and consider implications that follow from it. Anatomical studies indicate that LTP is accompanied by changes in spine morphology and therefore likely involves cytoskeletal changes. Accordingly, theta bursts initiate calpain-mediated proteolysis of the actin cross-linking protein spectrin and trigger actin polymerization in spine heads, two effects indicative of cytoskeletal reorganization. Polymerization occurs within 2 min, has the same threshold as LTP, is dependent on integrins, and becomes resistant to disruption over 30 min. We propose that the stabilization of the new cytoskeletal organization, and thus of a new spine morphology, underlies the initial phase of LTP consolidation. This hypothesis helps explain the diverse array of proteins and signaling cascades implicated in LTP, as well as the often-contradictory results about contributions of particular molecules. It also provides a novel explanation for why LTP is potently modulated by factors likely to be released during theta trains (e.g., BDNF). Finally, building on evidence that normal patterns of activity reverse LTP, we suggest that consolidation provides a delay that allows brain networks to sculpt newly formed memories.

Brain-Derived Neurotrophic Factor Restores Synaptic Plasticity in a Knock-In Mouse Model of Huntington's Disease

Asymptomatic Huntingtons disease (HD) patients exhibit memory and cognition deficits that generally worsen with age. Similarly, long-term potentiation (LTP), a form of synaptic plasticity involved in memory encoding, is impaired in HD mouse models well before motor disturbances occur. The reasons why LTP deteriorates are unknown. Here we show that LTP is impaired in hippocampal slices from presymptomatic HdhQ92 and HdhQ111 knock-in mice, describe two factors contributing to this deficit, and establish that potentiation can be rescued with brain-derived neurotrophic factor (BDNF). Baseline physiological measures were unaffected by the HD mutation, but LTP induction and, to a greater degree, consolidation were both defective. The facilitation of burst responses that normally occurs during a theta stimulation train was reduced in HD knock-in mice, as was theta-induced actin polymerization in dendritic spines. The decrease in actin polymerization and deficits in LTP stabilization were reversed by BDNF, concentrations of which were substantially reduced in hippocampus of both HdhQ92 and HdhQ111 mice. These results suggest that the HD mutation discretely disrupts processes needed to both induce and stabilize LTP, with the latter effect likely arising from reduced BDNF expression. That BDNF rescues LTP in HD knock-in mice suggests the possibility of treating cognitive deficits in asymptomatic HD gene carriers by upregulating production of the neurotrophin.

The Hippocampus as a Model for Studying Anatomical Plasticity in the Adult Brain

The suggestion that intact axons might grow new branches (“sprout”) in response to damage of their neighbors appears to have been made at various times throughout the century-long argument that revolved around the issue of peripheral nerve regeneration. One of the earliest references was made by Haighton, who in 1795 reported a series of “physiological” studies on regeneration (or “reproduction”) which included controls for “a difficulty which naturally presents itself here, and this is, the possibility of the stomach and vocal organs having received an additional supply of nervous energy from another source” (p. 198). Exner (1885), according to Edds (1953), provided a clear description of sprouting at the neuromuscular junction, and Kennedy in his 1897 review listed several authors who mentioned the need to control for growth by undamaged nerves into deafferented sites in evaluating studies on regeneration.

MYOSIN IIB REGULATES ACTIN DYNAMICS DURING SYNAPTIC PLASTICITY AND MEMORY FORMATION

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

The substrates of memory: defects, treatments, and enhancement.

Recent work has added strong support to the long-standing hypothesis that the stabilization of both long-term potentiation and memory requires rapid reorganization of the spine actin cytoskeleton. This development has led to new insights into the origins of cognitive disorders, and raised the possibility that a diverse array of memory problems, including those associated with diabetes, reflect disturbances to various components of the same mechanism. In accord with this argument, impairments to long-term potentiation in mouse models of Huntingtons disease and in middle-aged rats have both been linked to problems with modulatory factors that control actin polymerization in spine heads. Complementary to the common mechanism hypothesis is the idea of a single treatment for addressing seemingly unrelated memory diseases. First tests of the point were positive: Brain-Derived Neurotrophic Factor (BDNF), a potent activator of actin signaling cascades in adult spines, rescued potentiation in Huntingtons disease mutant mice, middle-aged rats, and a mouse model of Fragile-X syndrome. A similar reversal of impairments to long-term potentiation was obtained in middle-aged rats by up-regulating BDNF production with brief exposures to ampakines, a class of drugs that positively modulate AMPA-type glutamate receptors. Work now in progress will test if chronic elevation of BDNF enhances memory in normal animals.

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.

Reactive Synaptogenesis in the Adult Nervous System

In 1885 Exner suggested that the recovery of muscular contraction observed after partial transection of a motor nerve, but prior to regeneration of the damaged fibers, resulted from collateral growth of intact fibers and reinnervation of the muscle. Subsequently Edds (1950), Hoffman (1950), and others (Weddell et al., 1946; Hones et al., 1945; Weiss and Edds, 1946) demonstrated conclusively that the transection of few motor fibers could in fact result in axon collateral sprouting by the remaining undamaged fibers. This phenomenon was extended to connections between neurons when Murray and Thompson (1957) provided direct anatomical evidence for axon collateral sprouting in the partially denervated sympathetic ganglion and Liu and Chambers (1958) reported evidence for axon sprouting in the spinal cord. Over the last 20 years there has been an explosive growth of research on axon sprouting in the central nervous system. It is now clear that the phenomenon exists and can be highly selective in terms of which fibers sprout, which neurons are reinnervated, and at what ages it can be demonstrated.

Reduced hypothermic effects of cholinomimetic agents following chronic anticholinesterase treatment.

Abstract Tolerance to diisopropylfluorophosphate (DFP), an irreversible anticholinesterase agent which produces hypothermia following an acute injection, developed rapidly for temperature regulation. The third injection of a chronic regimen of DFP treatment produced little or no hypothermia, in rats. The hypothermie effects of systemically administered pilocaprine, a tertiary cholinomimetic agent, were reduced or eliminated following chronic administration of DFP. Similarly, the hypothermie effects of intrahypothalamically administered carbachol were also reduced following chronic administration of DFP. These results support other recent findings of a reduced sensitivity to cholinomimetics following chronic anticholinesterase treatment and suggest that the muscarinic cholinergic receptors in the anterior hypothalamus may alter their sensitivity to acetylcholine. This alteration in the sensitivity of the cholinergic receptors may be one mechanism underlying the development of tolerance to DFP.