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## **Meet the editor**

Thomas Heinbockel, PhD, is currently a professor and director of Graduate Studies in the Department of Anatomy, Howard University College of Medicine, Washington, DC, USA. Dr. Heinbockel's laboratory engages in multidisciplinary research to elucidate organizational principles of neural systems in the brain, specifically the limbic and olfactory system. His research has been di-

rected at understanding brain mechanisms of information processing and their relation to neurological and neuropsychiatric disorders. Dr. Heinbockel studied biology at the Philipps-University, Marburg, Germany. His studies of the brain started during his MS thesis work at the Max Planck Institute for Behavioral Physiology, Starnberg/Seewiesen, Germany. Subsequently, he completed his PhD degree in Neuroscience at the University of Arizona, Tucson, Arizona, USA. After graduating, he was a research associate at the Institute of Physiology, Otto von Guericke University School of Medicine, Magdeburg, Germany. Prior to his arrival at Howard University, Dr. Heinbockel held joint research faculty appointments in the Department of Anatomy and Neurobiology and the Department of Physiology at the University of Maryland School of Medicine, Baltimore, Maryland, USA. He still maintains an adjunct appointment in these departments.

## Contents

#### **Section 3 Plasticity and Neurological Diseases 151**

Chapter 10 **Neuroplasticity in Bipolar Disorder: Insights from**

**Neuroimaging 193** Marlos Vasconcelos Rocha, Fabiana Nery, Amanda Galvão-de-Almeida, Lucas de Castro Quarantini and Ângela Miranda-Scippa

## Preface

**Section 3 Plasticity and Neurological Diseases 151**

**Processes 153**

**Epilepsy 167**

**Neuroimaging 193**

Torres

**VI** Contents

Chapter 8 **Plasticity of Dendritic Spines. Not Only for Cognitive**

Meghan A. Van Zandt and Janice R. Naegele

Chapter 10 **Neuroplasticity in Bipolar Disorder: Insights from**

Ignacio González-Burgos, Dulce A. Velázquez-Zamora, David González-Tapia, Nallely Vázquez-Hernández and Néstor I. Martínez-

Marlos Vasconcelos Rocha, Fabiana Nery, Amanda Galvão-de-Almeida, Lucas de Castro Quarantini and Ângela Miranda-Scippa

Chapter 9 **GABAergic Synapse Dysfunction and Repair in Temporal Lobe**

Synaptic plasticity is a topic that I first encountered in graduate school, and it has accompa‐ nied me on my research journey ever since. The topic fascinates me today as much as it did when I attended graduate courses, namely, that the brain and the nervous system are plas‐ tic. They can change and adapt to their environment, and it is this plasticity that allows us to learn and also to forget. Synaptic plasticity forms the basis of many brain functions and dys‐ functions and is the basis of our ability to think and remember. The study of synaptic plasti‐ city has flourished over the years with the advancement of technical breakthroughs and is a timely scientific endeavor today just like it was several decades ago. This book contributes to our understanding of synaptic plasticity at the molecular, biochemical, and cellular sys‐ tems and behavioral level and informs the reader about its clinical relevance.

The book contains ten chapters that fall into the following three broad sections: (1) "Mecha‐ nisms of Synaptic Plasticity," (2) "Neural Plasticity," and (3) "Plasticity and Neurological Diseases." The book presents comprehensive reviews in these different areas written by ex‐ perts in their respective fields. The mechanisms of synaptic plasticity and its relation to neu‐ rological diseases are featured prominently as a recurring theme throughout most chapters. This book will be a most valuable resource for neuroscientists and other scientists alike. In addition, it will contribute to the training of current and future neuroscientists who find the changing nervous system as fascinating as many generations before them.

In Section 1 "Mechanisms of Synaptic Plasticity," the first chapter, written by Thomas Hein‐ bockel, introduces to the topic of this book by discussing the cellular elements, mechanisms, and functions of synaptic plasticity (Chapter 1: "Introductory Chapter: Mechanisms and Function of Synaptic Plasticity"). The chapter starts with a strong historical component to outline where our terminology and perspective on brain plasticity have their origin, contin‐ ues with recent examples of synaptic plasticity in different brain systems, and concludes with new directions of research on synaptic plasticity.

In Chapter 2 "Post-Transcriptional Mechanisms of Neuronal Translational Control in Synap‐ tic Plasticity," Dylan J. Kiltschewskij and Murray J. Cairns address novel findings of multidi‐ mensional regulation of neuronal mRNA translation achieved by a number of posttranscriptional mechanisms and discuss how these mechanisms of transcriptomic regu‐ lation are linked together to achieve high-order spatiotemporal control of postsynaptic translation. The authors highlight mRNA distal trafficking via RNA-binding proteins, small noncoding microRNA, brain-enriched long noncoding RNA, and novel circular RNA in‐ volved in posttranscriptional regulation of gene expression through modulation of both mRNA and miRNA function.

Chapter 3 "Mitochondrial Regulators of Synaptic Plasticity in the Ischemic Brain" by Han-A Park and Elizabeth A. Jonas describes how an inadequate blood supply and lack of oxygen in the brain, that is, cerebral ischemic insults, regulate neuronal intracellular mechanisms and signaling pathways. The authors address how, after neuronal injury, synaptic plasticity is regulated at different stages such as rehabilitation and recovery and which regulators of synaptic plasticity are at play.

Chapter 4 "Molecular Mechanisms of Drug-Induced Plasticity" by R.J. Oliver and Nora I. Perrone-Bizzozero reviews the regulation of addiction-related genes. These genes impact synaptic plasticity and behavior and may provide new pharmacological treatment strategies that reverse aberrant drug-evoked forms of plasticity. Acute and addiction-related behavio‐ ral exposures to drugs of abuse can lead to changes in synaptic plasticity that are persistent and similar to the chronic relapse state of patients with substance abuse disorders and can mirror many synaptic features of other forms of learning and memory.

Chapter 5 by Masako Isokawa "The Ghrelin Receptor Regulates Dendritic Spines and the NMDA Receptor–Mediated Synaptic Transmission in the Hippocampus" details the in‐ volvement of ghrelin and its receptor in extrahypothalamic functions such as hippocampal learning and memory. The author explains the cellular and molecular mechanisms underly‐ ing ghrelin-regulated hippocampal synaptic transmission and plasticity and its interaction with the endogenous cannabinoid system. Ghrelin, the ghrelin receptor, and the endocanna‐ binoid system may be critical for successful acquisition of metabolic state-dependent learn‐ ing and adaptive appetitive behavior.

Section 2 "Neural Plasticity" comprises two chapters, starting with Chapter 6 written by Karolina A. Bearss and Joseph F.X. DeSouza "Plasticity in Damaged Multisensory Net‐ works" This chapter discusses functional and anatomical locations as well as how neural networks of unimodal senses intersect and interact with multimodal sensory processes. The authors review research on damage occurring within unimodal and multimodal sensory networks and on cross modal plasticity in multisensory areas following brain damage with potential benefits of plastic reorganization of the cortex.

Chapter 7 by Stefan M. Golaszewski "Synaptic Plasticity by Afferent Electrical Stimulation" discusses the effect of afferent electrical stimulation on synaptic transmission and synaptic plasticity in general terms and within the sensorimotor cortex. The author describes a spe‐ cial protocol using whole-hand afferent electrical stimulation. The author provides evidence for the induction of synaptic plasticity within the sensorimotor cortex regarding short-inter‐ val intracortical inhibition, intracortical facilitation, and motor cortex excitability verified with functional magnetic resonance imaging and transcranial magnetic stimulation techni‐ ques.

The third section (Section 3, "Plasticity and Neurological Diseases") comprises three chap‐ ters. Chapter 8 "Plasticity of Dendritic Spines. Not Only for Cognitive

Processes" written by Ignacio González-Burgos, Dulce A. Velázquez-Zamora, David Gonzá‐ lez-Tapia, Nallely Vázquez-Hernández, and Néstor I. Martínez-Torres, discusses experimen‐ tal evidence that dendritic spines can express plastic changes that are not directly related to cognition. Traditionally, plastic changes of dendritic spines have been related to specific functional effects in the postsynaptic neuron such as learning of new information or memo‐

ry storage. The authors provide a discussion of the possible functional meaning of spine changes unrelated to cognition.

Chapter 3 "Mitochondrial Regulators of Synaptic Plasticity in the Ischemic Brain" by Han-A Park and Elizabeth A. Jonas describes how an inadequate blood supply and lack of oxygen in the brain, that is, cerebral ischemic insults, regulate neuronal intracellular mechanisms and signaling pathways. The authors address how, after neuronal injury, synaptic plasticity is regulated at different stages such as rehabilitation and recovery and which regulators of

Chapter 4 "Molecular Mechanisms of Drug-Induced Plasticity" by R.J. Oliver and Nora I. Perrone-Bizzozero reviews the regulation of addiction-related genes. These genes impact synaptic plasticity and behavior and may provide new pharmacological treatment strategies that reverse aberrant drug-evoked forms of plasticity. Acute and addiction-related behavio‐ ral exposures to drugs of abuse can lead to changes in synaptic plasticity that are persistent and similar to the chronic relapse state of patients with substance abuse disorders and can

Chapter 5 by Masako Isokawa "The Ghrelin Receptor Regulates Dendritic Spines and the NMDA Receptor–Mediated Synaptic Transmission in the Hippocampus" details the in‐ volvement of ghrelin and its receptor in extrahypothalamic functions such as hippocampal learning and memory. The author explains the cellular and molecular mechanisms underly‐ ing ghrelin-regulated hippocampal synaptic transmission and plasticity and its interaction with the endogenous cannabinoid system. Ghrelin, the ghrelin receptor, and the endocanna‐ binoid system may be critical for successful acquisition of metabolic state-dependent learn‐

Section 2 "Neural Plasticity" comprises two chapters, starting with Chapter 6 written by Karolina A. Bearss and Joseph F.X. DeSouza "Plasticity in Damaged Multisensory Net‐ works" This chapter discusses functional and anatomical locations as well as how neural networks of unimodal senses intersect and interact with multimodal sensory processes. The authors review research on damage occurring within unimodal and multimodal sensory networks and on cross modal plasticity in multisensory areas following brain damage with

Chapter 7 by Stefan M. Golaszewski "Synaptic Plasticity by Afferent Electrical Stimulation" discusses the effect of afferent electrical stimulation on synaptic transmission and synaptic plasticity in general terms and within the sensorimotor cortex. The author describes a spe‐ cial protocol using whole-hand afferent electrical stimulation. The author provides evidence for the induction of synaptic plasticity within the sensorimotor cortex regarding short-inter‐ val intracortical inhibition, intracortical facilitation, and motor cortex excitability verified with functional magnetic resonance imaging and transcranial magnetic stimulation techni‐

The third section (Section 3, "Plasticity and Neurological Diseases") comprises three chap‐

Processes" written by Ignacio González-Burgos, Dulce A. Velázquez-Zamora, David Gonzá‐ lez-Tapia, Nallely Vázquez-Hernández, and Néstor I. Martínez-Torres, discusses experimen‐ tal evidence that dendritic spines can express plastic changes that are not directly related to cognition. Traditionally, plastic changes of dendritic spines have been related to specific functional effects in the postsynaptic neuron such as learning of new information or memo‐

ters. Chapter 8 "Plasticity of Dendritic Spines. Not Only for Cognitive

mirror many synaptic features of other forms of learning and memory.

synaptic plasticity are at play.

VIII Preface

ing and adaptive appetitive behavior.

ques.

potential benefits of plastic reorganization of the cortex.

Chapter 9 "GABAergic Synapse Dysfunction and Repair in Temporal Lobe Epilepsy" by Me‐ ghan Van Zandt and Janice R. Naegele reviews recent progress in finding experimental ap‐ proaches to treat pharmacoresistant seizures as seen in medial temporal lobe epilepsy. One strategy and its molecular mechanisms described by the authors is to replace dysfunctional hippocampal GABAergic inhibitory interneurons through neural stem cell transplantation of GABAergic progenitor cells, an approach that has yielded synapse formation and seizure reductions and improved cognitive effects in a rodent model.

In Chapter 10 "Neuroplasticity in Bipolar Disorder: Insights from Neuroimaging " Marlos Rocha et al., are reviewing the major cortical and subcortical structures of the brain involved in bipolar disorder, a disease that is caused by a combination of genetic vulnerability and environmental stressors with abnormalities in neurotransmitter and neuroendocrine sys‐ tems and intracellular signaling pathways. The author describes findings of structural and functional neuroimaging in this disease and synthesizes impaired major cellular plasticity mechanisms and potential neuroplastic effects of mood stabilizers on structural and func‐ tional findings from the neuroimaging studies.

I am grateful to InTech Open Access Publisher for initiating this book project and for asking me to serve as its editor. I thank Iva Simcic at InTech for guiding me through the publication process and for moving the book ahead in a timely fashion. I would like to thank all contrib‐ utors of this book for taking the time to first write a chapter proposal, compose their chap‐ ter, and, lastly, make my requested revisions to them. Hopefully, all contributors will continue their neuroplasticity research with many intellectual challenges and exciting new directions. I would like to thank my wife Dr. Vonnie D.C. Shields, associate dean and pro‐ fessor, Towson University, Towson, MD, and our son Torben Heinbockel for the time that I was able to spend working on this book project during the past year. Finally, I am grateful to my parents Erich and Renate Heinbockel for their continuous support and interest in my work over many years.

**Thomas Heinbockel, PhD**

Professor and Director of Graduate Studies Department of Anatomy Howard University College of Medicine Washington, USA

**Mechanisms of Synaptic Plasticity**

## **Introductory Chapter: Mechanisms and Function of Synaptic Plasticity**

## Thomas Heinbockel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67891

## **1. Introduction**

Many everyday experiences such as reading a book like this one, classroom learning, drug taking, or stressful situations can result in changes of our brain at different levels. These changes can manifest themselves in altering both the structure and function of neural circuits. Neural circuits are built by neurons, which form points of contacts with each other, the synapses [1]. A given neuron can form thousands of synapses on its dendrites, cell body and axon, and through synaptic transmission, communicates information with other neurons in the nervous system. It is at the synapses that changes in brain function occur through modification of synaptic transmission termed synaptic plasticity (reviewed in [2]). Below, a description of synaptic plasticity is provided in terms of its historical context, mechanisms of its different forms, and directions of research on synaptic plasticity.

## **2. A brief history of synaptic plasticity**

The term plasticity has its origin in science more than 100 years ago and has been attributed to the famous Spanish scientist and founder of modern neuroscience Santiago Ramón y Cajal [3, 4]. His idea that the brain can store information by modifying synaptic connections was expressed in 1894 [5], even 3 years before Charles Sherrington introduced the term synapse for connections between neurons [6, 7]. Subsequently, Ramón y Cajal discovered that neurons are unique entities and synapses are the points of communication between them, the neuron doctrine [8]. It was also Ramón y Cajal who insisted that small spiny protrusions of dendrites, dendritic spines, were not an artifact but real and that they have a key role in mediating synaptic connectivity [9].

The idea and concept of synaptic plasticity gained prominence in the late 1940s with pioneering work by the Polish neurophysiologist Konorski [10] and the Canadian psychologist Hebb [11]. Konorski described plasticity as "permanent functional transformations," and Hebb attributed testable physiologic characteristics to synaptic plasticity [6]. Synaptic plasticity

means that the connections between nerve cells in the brain are not static but can undergo changes, they are plastic. Mammalian brains are remarkably plastic, which implies an ability to modify existing neural circuits and to alter future behavior, emotions, and responses to sensory input [12]. Synaptic plasticity refers to activity-dependent changes in the efficacy of synaptic communication and has been proposed to be critically involved in the remarkable capacity of the brain to translate transient experiences into apparently unlimited numbers of memories that can last for many years.

Even though the notion of synaptic plasticity dates back to the end of the nineteenth century, it took almost 80 years before experimental evidence was obtained to demonstrate that synapses are capable of long-lasting changes in synaptic strength [13]. Timothy Bliss and Terry Lomo experimentally induced an increase in the synaptic strength of neurons in the mammalian hippocampus as a result of electrical stimulation. Such an increase in postsynaptic responses is now called long-term potentiation (LTP). Further experimentation by Serena Dudek and Mark Bear [14] revealed the ability of synapses to change in two directions, namely to increase (LTP) or decrease (long-term depression, LTD) in strength, i.e., synapses undergo activity-driven bidirectional modification. Both LTP and LTD have been found in various brain regions, most prominently the hippocampus [2, 15], cerebellum [16], cerebral cortex [17–19], and the amygdala [20–25] where sensory input has been linked to motor output in fear conditioning paradigms.

## **3. Synaptic and neural plasticity**

Principally, synaptic plasticity refers to the strengthening or weakening of synaptic contacts as a result of increasing or decreasing activity levels of the neurons involved in a particular neural circuit. Synaptic plasticity implies direct regulation of pre- and/or postsynaptic neurons through alterations of the synaptic machinery. Examples include changes (a) of the number of neurotransmitter receptors in the postsynaptic membrane, (b) in the quantity of neurotransmitters released from the presynaptic neuron into a synapse, or (c) in receptor sensitivity to the released neurotransmitters [26–29]. Synaptic plasticity has been found at synapses that convey glutamate-mediated excitation and at other synapses that mediate GABAergic inhibition [2, 30]. Synaptic plasticity takes place at different time scales, from tens of milliseconds to life-long changes in synaptic transmission. Therefore, synaptic plasticity can be classified as either short-term or long-term. Short-term synaptic plasticity occurs at time periods from subsecond to minutes whereas long-term synaptic plasticity changes the efficacy of synapses for hours to years and is thought to form lasting memories that are stored in brain circuits.

The terms neuroplasticity, neural plasticity, or brain plasticity are used in a broader context to indicate changes that occur throughout a person's life either at the synapse or whole neurons or even entire brain regions. The basic premise is the same, namely that certain aspects of the brain or brain function can be changed throughout life [31]. This was not always understood to be the case. Previous studies of the brain suggested the existence of a critical period early in life during which the brain is amenable to changes of structure and function (plastic) and would remain unchangeable thereafter (static) (reviewed in [30, 32]). Likewise, synapses were considered as simple relay stations for information transfer from one neuron to another or from a neuron to a muscle cell. These relay stations were thought to be established during development and to remain in place throughout life with a relatively fixed synaptic strength of the connection. Neuroscience textbooks nowadays appreciate the extreme plasticity of most synapses such that they are able to change their strength as a result of either their own activity or through activity in another pathway [30].

### **4. Plasticity, memory, and learning**

means that the connections between nerve cells in the brain are not static but can undergo changes, they are plastic. Mammalian brains are remarkably plastic, which implies an ability to modify existing neural circuits and to alter future behavior, emotions, and responses to sensory input [12]. Synaptic plasticity refers to activity-dependent changes in the efficacy of synaptic communication and has been proposed to be critically involved in the remarkable capacity of the brain to translate transient experiences into apparently unlimited numbers of

Even though the notion of synaptic plasticity dates back to the end of the nineteenth century, it took almost 80 years before experimental evidence was obtained to demonstrate that synapses are capable of long-lasting changes in synaptic strength [13]. Timothy Bliss and Terry Lomo experimentally induced an increase in the synaptic strength of neurons in the mammalian hippocampus as a result of electrical stimulation. Such an increase in postsynaptic responses is now called long-term potentiation (LTP). Further experimentation by Serena Dudek and Mark Bear [14] revealed the ability of synapses to change in two directions, namely to increase (LTP) or decrease (long-term depression, LTD) in strength, i.e., synapses undergo activity-driven bidirectional modification. Both LTP and LTD have been found in various brain regions, most prominently the hippocampus [2, 15], cerebellum [16], cerebral cortex [17–19], and the amygdala [20–25] where sensory input has been linked to motor output in fear conditioning paradigms.

Principally, synaptic plasticity refers to the strengthening or weakening of synaptic contacts as a result of increasing or decreasing activity levels of the neurons involved in a particular neural circuit. Synaptic plasticity implies direct regulation of pre- and/or postsynaptic neurons through alterations of the synaptic machinery. Examples include changes (a) of the number of neurotransmitter receptors in the postsynaptic membrane, (b) in the quantity of neurotransmitters released from the presynaptic neuron into a synapse, or (c) in receptor sensitivity to the released neurotransmitters [26–29]. Synaptic plasticity has been found at synapses that convey glutamate-mediated excitation and at other synapses that mediate GABAergic inhibition [2, 30]. Synaptic plasticity takes place at different time scales, from tens of milliseconds to life-long changes in synaptic transmission. Therefore, synaptic plasticity can be classified as either short-term or long-term. Short-term synaptic plasticity occurs at time periods from subsecond to minutes whereas long-term synaptic plasticity changes the efficacy of synapses for hours to years and is thought to form lasting memories that are stored in brain circuits.

The terms neuroplasticity, neural plasticity, or brain plasticity are used in a broader context to indicate changes that occur throughout a person's life either at the synapse or whole neurons or even entire brain regions. The basic premise is the same, namely that certain aspects of the brain or brain function can be changed throughout life [31]. This was not always understood to be the case. Previous studies of the brain suggested the existence of a critical period early in life during which the brain is amenable to changes of structure and function (plastic) and would remain unchangeable thereafter (static) (reviewed in [30, 32]). Likewise, synapses were considered as simple relay stations for information transfer from one neuron to another or

memories that can last for many years.

4 Synaptic Plasticity

**3. Synaptic and neural plasticity**

Plasticity is now known to be an intrinsic property of the brain such that it is not limited by its own genome but can adapt to external stressors, physiological alterations, and a person's experiences. Plasticity manifests itself as dynamic shifts in the strength of preexisting connections across distributed neural networks and as modifications of the mapping between behavior and neural activity that take place in response to changes in afferent input or efferent demand [32]. Not only can existing connections undergo rapid changes, the establishment of new connections through dendritic growth and arborization can follow [33–36]. Synaptic and/ or neural plasticity is the mechanism for development and learning, but it is also the basis of much brain pathology as seen in various neurological disorders, and maladaptive synaptic plasticity may contribute to neuropsychiatric disorders [2].

While synaptic plasticity is a key concept in itself for brain function and dysfunction, it has become central to our understanding of the mechanisms of learning and memory. Synaptic plasticity is intimately related to learning and memory because memories are thought to be represented by neural networks that are connected at synapses. One critical concept in this regard is the Hebbian theory [11], which proposes an explanation for neuronal adaptation during the learning process and is considered a basic mechanism for synaptic plasticity. Hebb postulated that coincident activity of synaptically connected neurons leads to lasting changes in the efficacy of synaptic transmission. Experimental evidence supports this hypothesis by demonstrating that modifiable synapses exist in brain and form the basis for learning and memory. Under conditions when a presynaptic neuron repeatedly and persistently stimulates a postsynaptic neuron, i.e., when both neurons are active, synaptic connections are modifiable in their efficacy. Hebb's theory has been summarized in a more colloquial way by Siegrid Löwel's phrase: "Cells that fire together, wire together [37]." One important aspect of Hebb's theory relates to the exact timing of activity of the presynaptic neuron in relation to postsynaptic activity. The presynaptic cell needs to generate action potentials just before the postsynaptic cell and not at the same time, a concept known as spike-timing-dependent plasticity [38].

It is now generally accepted that memories are stored as alterations in the strength of synaptic connections between neurons [30]. Alterations in synaptic efficacy have been traced for hours to months, and therefore, LTP is both the most widely studied and the most popular candidate cellular mechanism for storing information in neural circuits over long-time periods. Irrespective of the usefulness of LTP and LTD as examples of long-lasting synaptic plasticity, some authors have cautioned that it is not clear how LTP and LTD relate to memory, i.e., the causal link between LTP and memory has not been demonstrated convincingly (reviewed in Ref. [30]), especially for hippocampal LTP. Other forms of memory and plasticity have allowed linking cellular events and circuitry to behavior, e.g., classical conditioning in the invertebrate model *Aplysia*, eye-blink conditioning, and amygdala-dependent fear conditioning [30, 39, 40]. Particularly, cerebellar LTD and amygdalar LTP are considered to directly underlie memory-associated behavioral changes [41, 42].

### **5. Endocannabinoids as mediators of synaptic plasticity**

Over the past two decades, a new set of signaling molecules has been implicated in synaptic plasticity, namely, endogenously generated cannabinoids, the endocannabinoids (eCBs) [2, 43–54]. Two endocannabinoids, N-arachidonoylethanol-amide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) have been found to be the natural agonists of cannabinoid receptors in the brain, CB1R [46]. These signaling molecules are unusual neurotransmitters because they are not stored in synaptic vesicles in synaptic terminals. Instead, endocannabinoids are made on-demand from membrane lipids of activated neurons and are released nonsynaptically. Nevertheless, they have been shown to be involved in synaptic plasticity in many neural systems in both short-term and long-term plasticity, learning and memory such as extinction of aversive memories [52–56]. Endocannabinoids are known to play a role in synapse formation, neurogenesis, and a number of bodily functions such feeding [57, 58], anxiety, pain reception, and recovery after brain injury [59–62]. Endocannabinoids serve as intercellular messengers in the brain [46]. They act in a retrograde fashion at synapses and presynaptically regulate both glutamatergic and GABAergic synapses to alter releaseprobability in synaptic plasticity. Endocannabinoids mediate short-term synaptic plasticity through a form of neuronal communication known as DSI, Depolarization-induced Suppression of Inhibition (reviewed in [46, 53, 54]). During DSI, when a principal neuron is activated through experimental current injection or activation of metabotropic glutamate or acetylcholine receptors, the inhibitory input onto that principal neuron is transiently reduced or abolished. When a postsynaptic principal neuron experiences a brief increase in intracellular calcium concentration, it synthesizes and releases endocannabinoids that travel to the presynaptic neuron and bind to cannabinoid receptors triggering an intracellular messenger cascade. The result is a transient decline of incoming inhibitory signals in the form of GABA arriving from presynaptic neurons. During DSI, endocannabinoids travel from the postsynaptic cell to the presynaptic GABA-releasing one and through activation of CB1R turn off neurotransmitter release. Endocannabinoids, thereby, act as retrogradesignaling molecules. DSI works as a transient local effect because endocannabinoids are lipids that cannot diffuse widely in the extracellular watery space of neurons. DSI allows neurons to disconnect briefly from other neurons or alter the strength of synapses made onto them through relieve of their inhibition [46]. DSI is a regulatory process allowing neurons to control their own synaptic excitability in an activity-dependent manner. A corresponding form of short-term synaptic plasticity has been described in the cerebellum, DSE, Depolarization-induced Suppression of Excitation, which reduces synaptic excitation by suppressing presynaptic glutamate release [44].

In addition to serving a role in mediating short-term synaptic plasticity, endocannabinoids have been shown to be critical in several forms of long-term synaptic plasticity. In the hippocampus, endocannabinoids evoke long-term depression at inhibitory, but not excitatory, synapses [63]. Endocannabinoid-mediated LTD (eCB-LTD) was described in the cerebellum [64], in the glutamatergic synapses onto medium spiny neurons in the striatum [65, 66] and at synapses between layer V pyramidal neurons in the neocortex [67]. Here, eCB-LTD does not depend on postsynaptic activation of metabotropic glutamate receptors but requires coincident activation ofpresynaptic ionotropic glutamate (NMDA)receptors. eCB-LTD in both the dorsal and the ventral striatum with the nucleus accumbens requires postsynaptic activation of group I metabotropic glutamate receptors [2, 68–70]. Differences exist regarding a requirement for concomitant presynaptic activity [71], the known involvement of anandamide as the endocannabinoid [72] and the presence of postsynaptic D2 dopamine receptors [73, 74] in the dorsal striatum.

## **6. Developments and directions of synaptic plasticity research**

Synaptic plasticity has become an overriding theme of brain research in order to understand the nervous system in its function and dysfunction. Over the past several decades, researchers have attempted and succeeded in deciphering molecular and cellular synaptic changes that are the basis for behavior and disease [75–77]. However, even though our understanding of synaptic plasticity has grown tremendously, pivotal questions regarding plasticity and its function remain to this day, e.g., how do the different forms of synaptic plasticity compliment or interfere with each other [55, 78].

Technical advances in neuroscience research are also a major catalyst for progress in synaptic plasticity research. Most recently, among these advances are genetic, optical, and optogenetic methods that allow researchers to manipulate single cells or neural circuits with subcellular precision, at microsecond timescales or through longitudinal electrophysiological and optical recordings [79–89]. Novel experimental and conceptual approaches will pave the way to a more complete understanding of the functional consequences of synaptic plasticity and its implication for health and disease.

## **Acknowledgements**

in Ref. [30]), especially for hippocampal LTP. Other forms of memory and plasticity have allowed linking cellular events and circuitry to behavior, e.g., classical conditioning in the invertebrate model *Aplysia*, eye-blink conditioning, and amygdala-dependent fear conditioning [30, 39, 40]. Particularly, cerebellar LTD and amygdalar LTP are considered to directly

Over the past two decades, a new set of signaling molecules has been implicated in synaptic plasticity, namely, endogenously generated cannabinoids, the endocannabinoids (eCBs) [2, 43–54]. Two endocannabinoids, N-arachidonoylethanol-amide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) have been found to be the natural agonists of cannabinoid receptors in the brain, CB1R [46]. These signaling molecules are unusual neurotransmitters because they are not stored in synaptic vesicles in synaptic terminals. Instead, endocannabinoids are made on-demand from membrane lipids of activated neurons and are released nonsynaptically. Nevertheless, they have been shown to be involved in synaptic plasticity in many neural systems in both short-term and long-term plasticity, learning and memory such as extinction of aversive memories [52–56]. Endocannabinoids are known to play a role in synapse formation, neurogenesis, and a number of bodily functions such feeding [57, 58], anxiety, pain reception, and recovery after brain injury [59–62]. Endocannabinoids serve as intercellular messengers in the brain [46]. They act in a retrograde fashion at synapses and presynaptically regulate both glutamatergic and GABAergic synapses to alter releaseprobability in synaptic plasticity. Endocannabinoids mediate short-term synaptic plasticity through a form of neuronal communication known as DSI, Depolarization-induced Suppression of Inhibition (reviewed in [46, 53, 54]). During DSI, when a principal neuron is activated through experimental current injection or activation of metabotropic glutamate or acetylcholine receptors, the inhibitory input onto that principal neuron is transiently reduced or abolished. When a postsynaptic principal neuron experiences a brief increase in intracellular calcium concentration, it synthesizes and releases endocannabinoids that travel to the presynaptic neuron and bind to cannabinoid receptors triggering an intracellular messenger cascade. The result is a transient decline of incoming inhibitory signals in the form of GABA arriving from presynaptic neurons. During DSI, endocannabinoids travel from the postsynaptic cell to the presynaptic GABA-releasing one and through activation of CB1R turn off neurotransmitter release. Endocannabinoids, thereby, act as retrogradesignaling molecules. DSI works as a transient local effect because endocannabinoids are lipids that cannot diffuse widely in the extracellular watery space of neurons. DSI allows neurons to disconnect briefly from other neurons or alter the strength of synapses made onto them through relieve of their inhibition [46]. DSI is a regulatory process allowing neurons to control their own synaptic excitability in an activity-dependent manner. A corresponding form of short-term synaptic plasticity has been described in the cerebellum, DSE, Depolarization-induced Suppression of Excitation, which reduces synaptic excitation

underlie memory-associated behavioral changes [41, 42].

6 Synaptic Plasticity

by suppressing presynaptic glutamate release [44].

**5. Endocannabinoids as mediators of synaptic plasticity**

This work was supported in part by grants from the National Science Foundation (NSF IOS-1355034) and the Charles and Mary Latham Trust Fund.

## **Conflict of interest**

The author declares that there is no conflict of interests regarding the publication of this chapter.

## **Author details**

Thomas Heinbockel

Address all correspondence to<EMAIL_ADDRESS>
Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

## **References**

[15] Bear MF, Abraham WC. Long-term depression in hippocampus. Annu Rev Neurosci 1996; 19:437-462.

**Author details**

8 Synaptic Plasticity

Thomas Heinbockel

**References**

neuroplasticity

Address all correspondence to<EMAIL_ADDRESS>
Spring Harbor Laboratory Press, 2012, 397 p.

Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

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## **Post-Transcriptional Mechanisms of Neuronal Translational Control in Synaptic Plasticity**

Dylan Kiltschewskij and Murray J. Cairns

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67603

#### **Abstract**

The dynamic complexity of synaptic function is matched by extensive multidimen‐ sional regulation of neuronal mRNA translation which is achieved by a number of post‐ transcriptional mechanisms. The first key aspect of this regulatory capacity is mRNA distal trafficking through RNA‐binding proteins, which governs the transcriptomic composition of post‐synaptic compartments. Small non‐coding microRNA and asso‐ ciated machinery have the capacity to precisely coordinate neural gene networks in space and time by providing a flexible specificity dimension to translational regulation. This RNA‐guided subcellular fine‐tuning of protein synthesis is an exquisite mecha‐ nism used in neurons to exert control of synaptic properties. Emerging evidence also implicates brain‐enriched long non‐coding RNA and novel circular RNA in posttran‐ scriptional regulation of gene expression through the modulation of both mRNA and miRNA functions, thereby exemplifying the complex nature of neuronal translation. Herein, we review current knowledge of these regulatory systems and analyse how these mechanisms of transcriptomic regulation may be linked together to achieve high‐ order spatiotemporal control of post‐synaptic translation.

**Keywords:** translation, mRNA trafficking, messenger ribonucleoprotein, microRNA, P‐bodies, circular RNA

## **1. Introduction**

The early phases of long‐term potentiation (LTP) and long‐term depression (LTD) are char‐ acterised by post‐translational modification and trafficking of AMPA glutamate receptors, modifying reactivity of the post‐synaptic membrane following an appropriate inducing stim‐ ulus [1]. While this is an effective means of altering synaptic transmission in the short term, consolidation of these changes into late‐phase synaptic plasticity requires a substantially

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

deeper response, driven by novel transcription and translation of messenger RNA (mRNA) [2]. Translation is a particularly interesting aspect of this system as discrete foci of polyribo‐ somes have been observed in dendritic spines engaging in protein synthesis, indicating a role for localised distal translation in dendritic function which has since proven critical in synaptic plasticity, and indeed, learning and memory [2–5].

Considering the importance of dendritic translation in strengthening alterations to the post‐ synaptic compartment, the role of mRNA in connecting neuronal transcription and distal translation is crucial and understandably subject to significant modulation. To this extent, neurons exploit a variety of post‐transcriptional regulatory mechanisms to ensure an array of over 2500 unique, dendritically localised mRNAs are only translated when appropriate [5]. At present, it is clear that a variety of mRNA‐binding proteins (RBPs) act to selectively distribute mRNAs to individual dendritic spines and may additionally act as enhancers or inhibitors of translation. Non‐coding RNA, specifically microRNA (miRNA), provides a potent and spe‐ cific dimension to translational control, which themselves may be regulated by other novel, brain‐enriched, non‐coding RNA.

Despite extensive investigation of neuronal mechanisms of translation control, we still lack a clear understanding of how these systems work both individually and synergistically to regu‐ late the localisation and appropriate timing of translation. This chapter therefore explores recent advances in the field of neuronal post‐transcriptional translational regulation, with particular focus on miRNA and how these small non‐coding RNAs interact with a variety of intermediates to exert precise control over neuronal protein synthesis.

## **2. Intracellular trafficking supplies dendrites with mRNA**

#### **2.1. Cis‐acting factors**

Neuronal subcellular compartments such as dendritic spines rely on distal trafficking of mRNA from the soma to provide a stockpile of transcripts for protein synthesis. Considering the functional similarity and close proximity of these compartments, precise localisation of mRNA is critical for modulation of the dendritic transcriptome, which influences transla‐ tion in response to stimuli such as synaptic transmission. This raises a key question; how can thousands of unique mRNAs successfully localise in functionally similar subcellular neuronal compartments?

Part of the answer lies within the mRNA itself as its 3′ UTR is encoded with *cis*‐acting sequences known as localisation elements (LE), which signal the desired site of transcript localisation. LEs were initially observed in chicken embryonic myoblasts whereby isoform‐specific 3′ UTR sequences were shown to direct the accumulation of α‐actin and β‐actin in peripheral and perinuclear regions, respectively [6]. Dendrite‐specific LEs have since been identified in key mediators of synaptic plasticity, including AMPA receptors, MAP2, αCaMKII and BDNF [7–10], indicating LEs play a critical role in the post‐synaptic localisation of specific mRNAs. The AMPA receptor is a particularly interesting example as "flip" and "flop" splice variants have been shown to respectively localise in dendrites and the soma [7], revealing alternative splicing may serve to edit LE expression and subcellular localisation through removal of undesirable LEs in specific transcript splice variants. Furthermore, MAP2 long and short 3′ UTR variants generated by alternative polyadenylation have been shown to, respectively, tar‐ get the dendrites and soma [9], implicating polyadenylation as another possible mechanism through which LE expression may be altered.

Following the discovery of LEs, a subset of studies has focussed on identifying a mechanis‐ tic basis through which these sequences facilitate selective mRNA trafficking. Considering the role of proteins in physically transporting mRNA (covered next), a distinct possibility is "digital" LEs form secondary structures to facilitate "analogue" recognition by RNA‐binding proteins (RBP) [11]. This proposed mechanism of LE function is strongly supported by the identification of 3′ UTR stem‐loop LEs required for the transport of K10, ASH1 and Oskar mRNAs in non‐neuronal models [12–14]. In the context of synaptic function, a 5′ UTR stem‐ loop motif has recently been shown to direct sensorin mRNA to the synapse in *Aplysia*, both demonstrating the role of stem‐loops in moderating synaptic mRNA localisation and reveal‐ ing that LEs may not be purely confined to the 3′ UTR [15].

#### **2.2. Trans‐acting factors**

deeper response, driven by novel transcription and translation of messenger RNA (mRNA) [2]. Translation is a particularly interesting aspect of this system as discrete foci of polyribo‐ somes have been observed in dendritic spines engaging in protein synthesis, indicating a role for localised distal translation in dendritic function which has since proven critical in synaptic

Considering the importance of dendritic translation in strengthening alterations to the post‐ synaptic compartment, the role of mRNA in connecting neuronal transcription and distal translation is crucial and understandably subject to significant modulation. To this extent, neurons exploit a variety of post‐transcriptional regulatory mechanisms to ensure an array of over 2500 unique, dendritically localised mRNAs are only translated when appropriate [5]. At present, it is clear that a variety of mRNA‐binding proteins (RBPs) act to selectively distribute mRNAs to individual dendritic spines and may additionally act as enhancers or inhibitors of translation. Non‐coding RNA, specifically microRNA (miRNA), provides a potent and spe‐ cific dimension to translational control, which themselves may be regulated by other novel,

Despite extensive investigation of neuronal mechanisms of translation control, we still lack a clear understanding of how these systems work both individually and synergistically to regu‐ late the localisation and appropriate timing of translation. This chapter therefore explores recent advances in the field of neuronal post‐transcriptional translational regulation, with particular focus on miRNA and how these small non‐coding RNAs interact with a variety of

Neuronal subcellular compartments such as dendritic spines rely on distal trafficking of mRNA from the soma to provide a stockpile of transcripts for protein synthesis. Considering the functional similarity and close proximity of these compartments, precise localisation of mRNA is critical for modulation of the dendritic transcriptome, which influences transla‐ tion in response to stimuli such as synaptic transmission. This raises a key question; how can thousands of unique mRNAs successfully localise in functionally similar subcellular neuronal

Part of the answer lies within the mRNA itself as its 3′ UTR is encoded with *cis*‐acting sequences known as localisation elements (LE), which signal the desired site of transcript localisation. LEs were initially observed in chicken embryonic myoblasts whereby isoform‐specific 3′ UTR sequences were shown to direct the accumulation of α‐actin and β‐actin in peripheral and perinuclear regions, respectively [6]. Dendrite‐specific LEs have since been identified in key mediators of synaptic plasticity, including AMPA receptors, MAP2, αCaMKII and BDNF [7–10], indicating LEs play a critical role in the post‐synaptic localisation of specific mRNAs. The AMPA receptor is a particularly interesting example as "flip" and "flop" splice variants have been shown to respectively localise in dendrites and the soma [7], revealing alternative

intermediates to exert precise control over neuronal protein synthesis.

**2. Intracellular trafficking supplies dendrites with mRNA**

plasticity, and indeed, learning and memory [2–5].

brain‐enriched, non‐coding RNA.

16 Synaptic Plasticity

**2.1. Cis‐acting factors**

compartments?

While encoding mRNAs with LEs constitutes an elegant mechanism by which transcripts may be directed to subcellular compartments, neurons require an efficient physical mecha‐ nism of mRNA distribution to ensure all dendrites and their spines are adequately supplied with transcripts. Such a system is provided by a specialised group of RBPs, the neuronal microtubule network and associated motor proteins, all of which act in concert to facilitate mRNA trafficking from the soma to far distal regions of the neuron.

Current evidence heavily implicates neuronal messenger ribonucleoprotein (mRNP) gran‐ ules as the primary vehicles of neuronal mRNA trafficking as observed through their trans‐ location in neurites and mRNA/protein enrichment [16, 17]. These granules are composed of aggregated mRNA‐RBP complexes, which are hypothesised to form upon *cis‐trans* interac‐ tion between RBPs and target mRNA LEs (**Figure 1**), conferring specificity to this system while additionally providing a means of translationally repressing mRNAs during transport [18, 19]. At present, the neuronally enriched fragile X mental retardation protein (FMRP) and Staufen family proteins are the chief candidate RBPs involved in neuronal mRNA trafficking as evidenced by their enrichment within mRNP granules while complexed with a variety of plasticity‐relevant mRNAs including CaMKIIα, MAP1b, β‐actin and poly(A)‐binding protein (PABP) [20–25]. An important aspect of these studies is that neuronal RBPs clearly target a variety of unique mRNAs, therefore suggesting RBPs recognise a number of different LEs, or alternatively, LEs for the same subcellular compartment share similar structural proper‐ ties. Regardless, the key advantage of this system is the need for relatively few unique RBPs relative to the number of mRNAs requiring transport. In contrast to this observation, zip‐ code‐binding protein 1 (ZBP1) specifically engages in activity‐dependent trafficking of β‐actin mRNA to the axon and dendrites through recognition of a 54nt LE within the 3′ UTR [26, 27]. While other binding partners may exist, it is possible that ZBP1 acts as a highly specific medi‐ ator of β‐actin expression due to the high demand for this protein in neuronal cytoskeletal remodelling [28].

**Figure 1.** Assembly of neuronal messenger ribonucleoprotein granules. (1) During transcription, mRNAs are inherently encoded with cis‐acting localisation elements in the 3′ UTR. (2) These sequences form secondary structures such as stem‐ loops, facilitating recognition by RNA‐binding proteins. (3) Aggregation of multiple mRNA‐RBP complexes results in the formation of neuronal mRNP granules, which transport mRNAs to distal locations in a translationally repressed state.

The overall significance of RBPs in neuronal mRNA trafficking is further exemplified by their hypothesised role as adaptors, linking transcripts to motor proteins for distal transport via microtubules. Supporting this view, mRNP granule‐associated FMRP has been shown to directly interact with KIF3c and KIF5 kinesin motor protein subunits [22, 29], while similarly, ZPB1 directly binds the tail domain of KIF11 while complexed with β‐actin mRNA [30]. The role of microtubules in this process has been further elucidated by the observed microtubule‐ dependent translocation of Staufen granules to the dendrites in response to synaptic activity as seen through GFP‐fusion experiments [31]. It is interesting to note that Dynein is another candidate motor protein involved in mRNA microtubular trafficking; however, its role is cur‐ rently only described in *Drosophila* embryonic extracts [32].

#### **2.3. "Synaptic Tag and Capture" and "Sushi Belt" theories**

Evidence pertaining the activity‐dependent migration of neuronal mRNP granules to the dendrites implies that these granules bear functional significance in the regulation of activity‐ dependent translation [31, 33]. However, despite our extensive knowledge of neuronal mRNA trafficking, it is still unclear how specific neuronal mRNP granules are successfully delivered to individual dendritic spines following stimuli such as synaptic transmission. A commonly accepted model regarding this question was initially proposed in 1997 by Frey and Morris, who presented the "Synaptic Tag and Capture" theory in the context of LTP [34, 35] which has since received significant attention, and indeed, refinement [36–38]. At the core of this theory, strong synaptic stimuli capable of inducing late‐phase LTP "tags" specific dendritic spines, signalling for the uptake of plasticity‐related proteins and mRNAs from dendritic shafts and the soma [34]. While evidence obtained since the original proposal of this model has sup‐ ported synaptic tag and capture, the identity of the tag, whether a single molecule or state of the synapse, has yet to be elucidated [38].

More recently, Doyle and Kiebler [18] have proposed an alternate "Sushi Belt" theory of den‐ dritic mRNA trafficking with stronger emphasis on neuronal mRNP granules. This theory suggests that mRNPs constitutively circulate dendritic shafts through microtubules and motor proteins, analogous to a sushi conveyor belt. Application of a potentiating stimulus to any particular spine "tags" it, resulting in recruitment of mRNP granules to that spine through the action of dynamic microtubules when novel translation is required. Importantly, this theory accounts for both basal and activity‐dependent mRNP transport in addition to anterograde and retrograde mRNP translocation, which has previously been observed [16]. Considering current knowledge of mRNA dendritic transport, however, it remains a distinct possibility that facets from both of these models may likely underlie the true mechanism behind both basal and activity‐dependent neuronal mRNA trafficking.

## **3. microRNAs specifically and potently regulate post‐synaptic translation**

MicroRNAs (miRNAs) are a class of short, ∼22nt non‐coding RNAs that function as target‐ specific guides in the degradation and translational repression of mRNA. Since their initial discovery in 1993 [39, 40], the role of miRNA in providing combinatorial specificity to trans‐ lational regulation has been highlighted through the discovery of more than 3700 unique miRNA participating in over 366,000 experimentally supported miRNA‐target interactions [41]. Profiling studies have further identified the CNS as a source of significant miRNA enrich‐ ment whereby a subset of miRNA and associated factors regulate synaptic plasticity and are subject to activity‐dependent turnover, indicating a key role in the maintenance of synaptic function [42]. Consequently, dysregulation of miRNA function is thought to contribute to the pathogenesis of complex neurological diseases, in particular, neuropsychiatric conditions characterised by impairments in learning and memory [43].

#### **3.1. miRNA biogenesis and target interaction**

The overall significance of RBPs in neuronal mRNA trafficking is further exemplified by their hypothesised role as adaptors, linking transcripts to motor proteins for distal transport via microtubules. Supporting this view, mRNP granule‐associated FMRP has been shown to directly interact with KIF3c and KIF5 kinesin motor protein subunits [22, 29], while similarly, ZPB1 directly binds the tail domain of KIF11 while complexed with β‐actin mRNA [30]. The role of microtubules in this process has been further elucidated by the observed microtubule‐ dependent translocation of Staufen granules to the dendrites in response to synaptic activity as seen through GFP‐fusion experiments [31]. It is interesting to note that Dynein is another candidate motor protein involved in mRNA microtubular trafficking; however, its role is cur‐

**Figure 1.** Assembly of neuronal messenger ribonucleoprotein granules. (1) During transcription, mRNAs are inherently encoded with cis‐acting localisation elements in the 3′ UTR. (2) These sequences form secondary structures such as stem‐ loops, facilitating recognition by RNA‐binding proteins. (3) Aggregation of multiple mRNA‐RBP complexes results in the formation of neuronal mRNP granules, which transport mRNAs to distal locations in a translationally repressed state.

Evidence pertaining the activity‐dependent migration of neuronal mRNP granules to the dendrites implies that these granules bear functional significance in the regulation of activity‐ dependent translation [31, 33]. However, despite our extensive knowledge of neuronal mRNA trafficking, it is still unclear how specific neuronal mRNP granules are successfully delivered

rently only described in *Drosophila* embryonic extracts [32].

18 Synaptic Plasticity

**2.3. "Synaptic Tag and Capture" and "Sushi Belt" theories**

miRNAs are derived from the transcription and processing of miRNA genes (**Figure 2**) located in a diverse array of genomic locations, including coding, non‐coding and inter‐ genic regions [44, 45]. Transcription of miRNA genes by RNA polymerase II yields primary miRNA (pri‐miR), which house one (monocistronic) or multiple (polycistronic) miRNA spe‐ cies and are post‐transcriptionally supplemented with 5′ methylguanosine cap and 3′ poly(A)

**Figure 2.** miRNA biogenesis. (1) Transcription of miRNA genes yields pri‐miRNA, which adopt a hairpin secondary structure and contain 5′ cap and 3′ poly(A) tail similarly to mRNA. (2) These structures are cleaved from the pri‐miRNA by Drosha and cofactor DGCR8, yielding a ∼70nt pre‐miRNA which is (3) transported to the cytosol through exportin‐5. (4) Further processing by dicer cleaves the pre‐miRNA hairpin loop, leaving a miRNA duplex which is (5) loaded into one of four human argonaute proteins. (6) The RISC is considered mature following selection of a guide strand and subsequent ejection of the passenger strand.

tail structures [46–48]. Complementary base pairing between the miRNA sequence and its antisense counterpart forms a secondary hairpin structure which is liberated from the pri‐ mary transcript via cleavage, mediated by the nuclear microprocessor Drosha and co‐factor DGCR8 [49]. The resultant ∼70nt miRNA precursor (pre‐miR) is exported to the cytoplasm through the action of Exportin‐5 in a Ran‐GTP‐dependent manner [50, 51] wherein the cyto‐ plasmic RNase III Dicer cleaves the hairpin loop structure from this pre‐miR [52, 53]. This produces a ∼22nt double‐stranded miRNA duplex which associates with one of four ortho‐ logues of the Argonaute (AGO) protein family, thereby forming an immature RNA‐induced silencing complex (RISC) [54]. Maturation of this complex is achieved through positive selec‐ tion of a 5′ uridine containing "guide" [55] strand from the miRNA duplex and subsequent ejection of the antisense "passenger" strand from the RISC [56–58]. Incomplete Watson‐Crick base complementarity between the miRNA seed region (nucleotides 2–8) and miRNA rec‐ ognition elements encoded in the mRNA 3′ UTR (and occasionally, 5′ UTR and CDS) subse‐ quently acts as a targeting mechanism for the RISC, facilitating translational repression and degradation of specific mRNAs [59–62]. This redundancy in miRNA‐mRNA interaction is a particularly critical characteristic of miRNA function as it enables a single miRNA to regu‐ late a many unique transcripts, eliminating the need for excessively large numbers of very specific miRNA.

#### **3.2. miRNA regulation of gene expression**

#### *3.2.1. 5′ to 3′ exonucleic decay*

The initial stages of cap‐dependent mRNA translation are primed by interaction between 5′ bound eukaryotic initiation factors (eIF) and 3′ poly(A)‐binding proteins (PABP), which both circularise mRNA for efficient translation and promote assembly of a translation initiation complex in the 5′ UTR [63]. The mRNA 5′ methylguanosine cap and 3′ poly(A) tail are there‐ fore considered as two critical *cis‐*acting factors in translation initiation and are thus ideal targets for translational repression and degradation by miRNA and the RISC [64].

An extensive body of research has since revealed that the RISC induces activation of the 5′ to 3′ mRNA decay pathway to facilitate degradation of target mRNAs. The first step of this pathway is characterised by recruitment of the PAN2‐PAN3 and CCR4‐NOT deadenyl‐ ation complexes to target mRNA, which trigger sequential cleavage of the 3′ poly(A) tail [65, 66]. Co‐immunoprecipitation and structural analysis suggest that the RISC indirectly recruits these deadenylation factors through the action of GW182 proteins, which bind both AGO family members in addition to the PAN3 and NOT1/9 deadenylase subunits [67–70]. Erosion of the poly(A) tail subsequently stimulates the removal of the 5′ methyl‐ guanosine cap via DCP2 and associated decapping factors, recruited to the target mRNA via CCR4‐NOT [71]. The exposed mRNA is then subject to 5′ to 3′ digestion by XRN1 exo‐ nuclease [71].

#### *3.2.2. Inhibition of active translation*

tail structures [46–48]. Complementary base pairing between the miRNA sequence and its antisense counterpart forms a secondary hairpin structure which is liberated from the pri‐ mary transcript via cleavage, mediated by the nuclear microprocessor Drosha and co‐factor DGCR8 [49]. The resultant ∼70nt miRNA precursor (pre‐miR) is exported to the cytoplasm through the action of Exportin‐5 in a Ran‐GTP‐dependent manner [50, 51] wherein the cyto‐ plasmic RNase III Dicer cleaves the hairpin loop structure from this pre‐miR [52, 53]. This produces a ∼22nt double‐stranded miRNA duplex which associates with one of four ortho‐ logues of the Argonaute (AGO) protein family, thereby forming an immature RNA‐induced silencing complex (RISC) [54]. Maturation of this complex is achieved through positive selec‐ tion of a 5′ uridine containing "guide" [55] strand from the miRNA duplex and subsequent ejection of the antisense "passenger" strand from the RISC [56–58]. Incomplete Watson‐Crick base complementarity between the miRNA seed region (nucleotides 2–8) and miRNA rec‐ ognition elements encoded in the mRNA 3′ UTR (and occasionally, 5′ UTR and CDS) subse‐ quently acts as a targeting mechanism for the RISC, facilitating translational repression and degradation of specific mRNAs [59–62]. This redundancy in miRNA‐mRNA interaction is a particularly critical characteristic of miRNA function as it enables a single miRNA to regu‐ late a many unique transcripts, eliminating the need for excessively large numbers of very

**Figure 2.** miRNA biogenesis. (1) Transcription of miRNA genes yields pri‐miRNA, which adopt a hairpin secondary structure and contain 5′ cap and 3′ poly(A) tail similarly to mRNA. (2) These structures are cleaved from the pri‐miRNA by Drosha and cofactor DGCR8, yielding a ∼70nt pre‐miRNA which is (3) transported to the cytosol through exportin‐5. (4) Further processing by dicer cleaves the pre‐miRNA hairpin loop, leaving a miRNA duplex which is (5) loaded into one of four human argonaute proteins. (6) The RISC is considered mature following selection of a guide strand and

specific miRNA.

subsequent ejection of the passenger strand.

20 Synaptic Plasticity

RISC‐mediated degradation of mRNA has long been recognised as the canonical mecha‐ nism through which miRNAs specifically modulate the transcriptome and therefore transla‐ tion. Emerging evidence suggests the RISC may also function to repress translation through a number of mechanisms (**Figure 3**), foremost of which is inhibition of active translation. This form of translational repression arose as a potential facet of RISC function following the initial discovery of miRNA in association with polysomes [72]; however, no evidence was uncovered regarding a link between miRNA binding and arrest of active translation. A similar discovery has also been observed for the dendrite‐abundant translational repres‐ sor FMRP, which has been shown to form translationally active complexes with ribosomes in mouse cortical preparations [73]. Further studies have identified CDS‐bound phosphor‐ ylated FMRP is associated with stalled ribosomes, leading to the current hypothesis that FMRP halts active protein synthesis through binding actively translating ribosomes [74–76]. This holds significance for neuronal miRNA function since FMRP has been shown to inter‐ act with AGO1 [77], AGO2 [78], MOV10 (RISC RNA helicase) [79] and specific microRNAs [80], raising the possibility that miRNA and the RISC may act as a targeting mechanism for FMRP. Considering these interactions together in conjunction with the dendritic enrich‐ ment of FMRP, miRNA‐ and FMRP‐mediated stalling of actively translating ribosomes could constitute an exciting dimension to miRNA function particularly relevant to synaptic translation.

**Figure 3.** Mechanisms of miRNA‐mediated translational repression. The RISC acquires target mRNAs through complementary base pairing, triggering one of a number of repressive pathways. (1) Transcript degradation is stimulated through RISC recruitment of deadenylase complexes to cleave the mRNA poly(A) tail, followed by decapping and exonuclease decay. (2) Cleavage of the poly(A) tail reduces the capacity for target mRNA to act as a substrate for protein synthesis, leading to translational repression likely reversible by cytoplasmic poly(A) polymerases. (3) The RISC may additionally stimulate dissociation of eIFs from the mRNA 5′ cap, or alternatively, (4) recruit FMRP to halt active synthesis through ribosome stalling.

#### *3.2.3. Poly(A) tail erosion and cytoplasmic polyadenylation*

While RISC induction of mRNA decay constitutes an effective mechanism through which translation may be fine‐tuned for cellular needs, in the neuronal context, this mechanism relies heavily on both transcriptional activity and mRNA trafficking to maintain transcrip‐ tomic supply at dendrites. Consequently, a system by which the translational competency of mRNAs could be modulated during periods of quiescence and in response to plasticity‐ inducing stimuli would significantly relieve pressure applied by RISC degradation of transcripts. A major candidate mechanism through which this may be achieved is modula‐ tion of mRNA poly(A) tail length, since this structure and associated PABPs facilitate 5′–3′ interactions required for translation initiation [63]. The manipulation of reporter mRNA in HeLa cultures [81] and cell‐free systems [82] has indeed revealed let‐7 translational arrest of reporter luciferase depends on the expression of a poly(A) tail, emphasising the role of the poly(A) tail as a conduit through which translational competency may be altered by miRNA. A requirement for such a system to exist, however, is the functional decou‐ pling of poly(A) tail erosion and mRNA decay. Recent studies illustrate that this occurs in embryonic culture systems such that deadenylated targets of miRNA, including miR‐35‐42, miR‐51‐56 and miR‐155, have been shown to accumulate in a stable manner [83, 84], sug‐ gesting miRNA‐mediated poly(A) tail cleavage does not necessarily instigate mRNA decay. This potential dimension to miRNA function becomes particularly interesting in the neu‐ ral context, as cytoplasmic polyadenylation has the potential to reinstate the translational activity of poly(A) deficient mRNAs. Poly(A) polymerase GLD‐2 is a promising contender for this function due to its neuronal enrichment, cytoplasmic localisation and participation in translational enhancement via poly(A) tail extension [85, 86]. In addition to these features, GLD‐2 function increases the abundance and stability of poorly translated mRNAs and is also required for the induction of LTP, suggesting that cytoplasmic polyadenylation could play a significant role in synaptic plasticity [85, 87, 88]. Regardless, the role of GLD‐2 and deadenylase counterpart PARN (poly(A)‐specific ribonuclease) in bidirectional regulation of poly(A) tail length and neuronal translation as part of the cytoplasmic polyadenylation complex illustrates the significance of cytoplasmic polyadenylation in neuronal translational control [85].

#### *3.2.4. Inhibition of translation initiation*

Further emerging evidence implicates miRNA and the RISC in direct inhibition of translation initiation through interaction with 5′ cap‐bound eIFs responsible for mediating ribosomal assembly in the mRNA 5′ UTR [63]. At present, AGO1‐RISC has been shown to indirectly stimulate disassociation of eIF4E via GW182, and eIF4A through a mechanism independent of GW182, inhibiting the assembly of an eIF4F translation initiation complex [89, 90]. While it is clear that this interaction acts to prevent translation of target mRNAs, there is uncertainty regarding whether this is another mechanism of translationally arresting mRNA or simply a precursor to mRNA degradation. The recent discovery of an RNA‐independent association between CCR4‐NOT subunit NOT1 and eIF4A2 RNA helicase involved in RISC degradation of target mRNA [91] suggests the latter is likely but does not rule out the possibility of eIF interference as a means of transcript repression.

#### **3.3. microRNAs in synaptic function**

*3.2.3. Poly(A) tail erosion and cytoplasmic polyadenylation*

synthesis through ribosome stalling.

22 Synaptic Plasticity

While RISC induction of mRNA decay constitutes an effective mechanism through which translation may be fine‐tuned for cellular needs, in the neuronal context, this mechanism relies heavily on both transcriptional activity and mRNA trafficking to maintain transcrip‐ tomic supply at dendrites. Consequently, a system by which the translational competency of mRNAs could be modulated during periods of quiescence and in response to plasticity‐ inducing stimuli would significantly relieve pressure applied by RISC degradation of transcripts. A major candidate mechanism through which this may be achieved is modula‐ tion of mRNA poly(A) tail length, since this structure and associated PABPs facilitate 5′–3′ interactions required for translation initiation [63]. The manipulation of reporter mRNA in HeLa cultures [81] and cell‐free systems [82] has indeed revealed let‐7 translational arrest of reporter luciferase depends on the expression of a poly(A) tail, emphasising the role of the poly(A) tail as a conduit through which translational competency may be altered by miRNA. A requirement for such a system to exist, however, is the functional decou‐ pling of poly(A) tail erosion and mRNA decay. Recent studies illustrate that this occurs in embryonic culture systems such that deadenylated targets of miRNA, including miR‐35‐42, miR‐51‐56 and miR‐155, have been shown to accumulate in a stable manner [83, 84], sug‐ gesting miRNA‐mediated poly(A) tail cleavage does not necessarily instigate mRNA decay.

**Figure 3.** Mechanisms of miRNA‐mediated translational repression. The RISC acquires target mRNAs through complementary base pairing, triggering one of a number of repressive pathways. (1) Transcript degradation is stimulated through RISC recruitment of deadenylase complexes to cleave the mRNA poly(A) tail, followed by decapping and exonuclease decay. (2) Cleavage of the poly(A) tail reduces the capacity for target mRNA to act as a substrate for protein synthesis, leading to translational repression likely reversible by cytoplasmic poly(A) polymerases. (3) The RISC may additionally stimulate dissociation of eIFs from the mRNA 5′ cap, or alternatively, (4) recruit FMRP to halt active

> Considering the array of confirmed and emerging miRNA functions, miRNA degradation and/ or repression of transcripts constitutes an effective mechanism for fine‐tuning highly dynamic translation at the post‐synaptic compartment. While miRNAs are thought to be largely sta‐ ble mRNA species, new evidence suggests that miRNAs are subject to activity‐dependent synthesis and rapid turnover in dendritic spines [42]. Dendritic synthesis of miRNA became particularly apparent following the discovery of pre‐miRs [92] and their inactive process‐ ing enzyme Dicer at the post‐synaptic density (PSD) whereby activity‐dependent stimulation of Calpain I was sufficient to induce Dicer function [93]. These data were supported by the detection of mature miRNA species enriched in mouse forebrain synaptoneurosomes [92]. Activity‐dependent degradation of miRNA has also recently been observed in mouse retinal neurons; however, the exact mechanism of degradation remains elusive [94]. Interestingly, exosome release may act as a means of post‐synaptic miRNA clearance, as MAP1B‐enriched neuronal exosomes released in response to depolarisation have recently been shown to con‐ tain miRNA (miR‐638, miR‐149\*, miR‐4281 and let‐7e) normally downregulated by depolari‐ sation in the dendritic compartment [95].

Supporting the critical role of miRNA in synaptic function, microarray analysis and deep‐ sequencing have revealed that subsets of miRNA are subject to differential expression up to 24‐h post‐induction of hippocampal synaptic plasticity [96, 97]. These data have since been extended through the development of a conditional *Dicer 1* knockout in the adult mouse fore‐ brain shown to result in enhanced LTP, learning and memory [98]. Reflecting on these dis‐ coveries, it is perhaps not surprising that a number of neuron‐enriched miRNA have been described in terms of the regulation of synaptic function.

Arguably the most studied miRNA in the context of synaptic function, miR‐132 is widely regarded for its role in the regulation of synaptic plasticity in brain regions such as the visual cortex [99] and hippocampus [100]. Following neuronal activity, miR‐132 is subject to a rapid increase in CREB‐mediated (cAMP response element‐binding protein) transcription, resulting in stimulation of dendritic growth and branching through suppression of p250GAP (p250 GTPase activating protein) [101, 102]. These modifications to dendritic morphology are characterised by expression of stubby, mushroom‐like dendritic spines which act to potentiate synaptic transmis‐ sion [80]. miR‐132 expression additionally directs the upregulation of NR2A, NR2B and GluR1 glutamate receptor subunits following BDNF stimulation [103], revealing this miRNA may direct synaptic plasticity through guiding both morphological and physiological change. A particularly interesting aspect of miR‐132 function, however, is its relationship with FMRP, which provides novel insight regarding potential interplay between miRNA and FMRP homologues. Specifically, miR‐132 effects on dendritic morphology are abolished following FMRP knockdown, uncovering a functional relationship between these molecules likely to be responsible for miR‐132‐associated regulation of plasticity‐relevant transcripts [80]. A similar relationship has been observed for miR‐125b whereby FMRP deficits inhibit miR‐125b negative regulation of both dendritic spine morphology and NR2A [80], further supporting the role of FMRP in neuronal miRNA function.

Aside from miR‐132, several recent studies have implicated a variety of other neuron‐enriched or specific miRNAs in the modification of synaptic function. miR‐134 is a key example which modulates dendritic spine volume through the regulation of *Limk1* expression, resulting in dendritic spine volume deficits when miR‐134 is overexpressed [104, 105]. This function of miR‐134 in the negative regulation of dendritic spine morphology is reflected in the impair‐ ment of mouse contextual fear learning following miR‐134 overexpression in the CA1 hip‐ pocampus; however, post‐transcriptional miR‐134 regulation of the CREB transcription factor appears to significantly contribute to this deficiency [106]. Another miRNA involved in the inhibition of synaptic plasticity is miR‐137 which decreases dendritic complexity and length through downregulation of mind bomb‐1 ubiquitin ligase while additionally inhibiting pre‐ synaptic vesicle release [107, 108]. miR‐137 also directly influences AMPA receptor expression through the regulation of GluA1, leading to synaptic silencing and deficits in hippocampal learning when overexpressed in mice [109].

Considering these examples alone, it is evident that miRNAs modulate a variety of synaptic characteristics through post‐transcriptional regulation of a number of unique mRNA spe‐ cies, emphasising the importance of miRNA in coordinating a number of gene expression networks to fine‐tune synaptic plasticity. Moreover, miRNA species appear to provide the basis through which the induction of plastic alterations to the synapse may be bidirectionally regulated. This not only implicates specific miRNA species such as miR‐132 in the enhance‐ ment of synaptic plasticity, but also additionally implies miRNA including miR‐134 and miR‐137 act as a breaking mechanism to prevent excessive potentiation or depression of indi‐ vidual synapses. Despite this extensive understanding of miRNA function, many are yet to be fully characterised in relation to synaptic and indeed neuronal function.

Supporting the critical role of miRNA in synaptic function, microarray analysis and deep‐ sequencing have revealed that subsets of miRNA are subject to differential expression up to 24‐h post‐induction of hippocampal synaptic plasticity [96, 97]. These data have since been extended through the development of a conditional *Dicer 1* knockout in the adult mouse fore‐ brain shown to result in enhanced LTP, learning and memory [98]. Reflecting on these dis‐ coveries, it is perhaps not surprising that a number of neuron‐enriched miRNA have been

Arguably the most studied miRNA in the context of synaptic function, miR‐132 is widely regarded for its role in the regulation of synaptic plasticity in brain regions such as the visual cortex [99] and hippocampus [100]. Following neuronal activity, miR‐132 is subject to a rapid increase in CREB‐mediated (cAMP response element‐binding protein) transcription, resulting in stimulation of dendritic growth and branching through suppression of p250GAP (p250 GTPase activating protein) [101, 102]. These modifications to dendritic morphology are characterised by expression of stubby, mushroom‐like dendritic spines which act to potentiate synaptic transmis‐ sion [80]. miR‐132 expression additionally directs the upregulation of NR2A, NR2B and GluR1 glutamate receptor subunits following BDNF stimulation [103], revealing this miRNA may direct synaptic plasticity through guiding both morphological and physiological change. A particularly interesting aspect of miR‐132 function, however, is its relationship with FMRP, which provides novel insight regarding potential interplay between miRNA and FMRP homologues. Specifically, miR‐132 effects on dendritic morphology are abolished following FMRP knockdown, uncovering a functional relationship between these molecules likely to be responsible for miR‐132‐associated regulation of plasticity‐relevant transcripts [80]. A similar relationship has been observed for miR‐125b whereby FMRP deficits inhibit miR‐125b negative regulation of both dendritic spine morphology and NR2A [80], further supporting the role of FMRP in neuronal miRNA function. Aside from miR‐132, several recent studies have implicated a variety of other neuron‐enriched or specific miRNAs in the modification of synaptic function. miR‐134 is a key example which modulates dendritic spine volume through the regulation of *Limk1* expression, resulting in dendritic spine volume deficits when miR‐134 is overexpressed [104, 105]. This function of miR‐134 in the negative regulation of dendritic spine morphology is reflected in the impair‐ ment of mouse contextual fear learning following miR‐134 overexpression in the CA1 hip‐ pocampus; however, post‐transcriptional miR‐134 regulation of the CREB transcription factor appears to significantly contribute to this deficiency [106]. Another miRNA involved in the inhibition of synaptic plasticity is miR‐137 which decreases dendritic complexity and length through downregulation of mind bomb‐1 ubiquitin ligase while additionally inhibiting pre‐ synaptic vesicle release [107, 108]. miR‐137 also directly influences AMPA receptor expression through the regulation of GluA1, leading to synaptic silencing and deficits in hippocampal

Considering these examples alone, it is evident that miRNAs modulate a variety of synaptic characteristics through post‐transcriptional regulation of a number of unique mRNA spe‐ cies, emphasising the importance of miRNA in coordinating a number of gene expression networks to fine‐tune synaptic plasticity. Moreover, miRNA species appear to provide the basis through which the induction of plastic alterations to the synapse may be bidirectionally

described in terms of the regulation of synaptic function.

24 Synaptic Plasticity

learning when overexpressed in mice [109].

## **4. P‐bodies and mRNP granules: linking mRNA transport, translation and repression**

miRNA regulation of gene expression is sequestered within subcellular granules known as processing bodies (P‐bodies), proposed to form through aggregation of translationally arrested mRNA and mRNA degradation machinery [110]. These granules were initially described as cytoplasmic "speckles" termed GW‐bodies, observed as aggregates of GW182 complexed with mRNA [111]. P‐bodies have since been characterised as foci of mRNA degra‐ dation due to their enrichment of translationally repressive RBPs such as Argonaute proteins, DCP1 & DCP2 decapping enzymes, Pat1 & edc3 decapping factors and XRN1 exonuclease [110]. Considering these granules are both enriched at the post‐synaptic density and actively migrate to post‐synaptic compartments following neuronal activity [112–114], P‐bodies are currently heavily implicated in post‐synaptic translational regulation.

Neuronal mRNP granules similarly form by the aggregation of messenger RNPs to provide a vehicle for mRNA distal transport while maintaining mRNAs in a translationally arrested state [18]. Interestingly, *Drosophila* Staufen and FMRP mRNP granules are enriched with homologues of mRNA decay machinery including DCP1, XRN1 and AGO2 [24], demonstrat‐ ing these granules bear similarity to P‐bodies. In conjunction with this observation, an excit‐ ing interaction between these granules has recently been reported whereby 50% of dendritic P‐bodies have been shown to dock with neuronal mRNPs [113], raising the prospect that these granules exchange their contents.

Drawing from these observations, a fascinating scenario is the potential for neuronal RNP granules to unload their mRNA and mRNA degradation cargo to P‐bodies for repression until translation is appropriate, thereby acting as a means of mRNA storage with the potential for deg‐ radation if specific transcripts are not required. In support of this possibility, mapping of mRNA localisation during yeast glucose starvation and tetracycline transcriptional suppression indicates mRNA can associate with polysomes from P‐bodies [115], revealing mRNA may be subjected to translation following sequestration in P‐bodies. Adding to this evidence, cationic amino acid transporter–1 mRNA has recently been observed undergoing release from miR‐122 repression and subsequently associating with polysomes from P‐bodies during cellular stress [116], fur‐ ther implicating miRNA in repressing translation without necessarily instigating mRNA decay. While the potential underlying mechanisms driving mRNA de‐repression in P‐bodies remains unclear, *Drosophila* GLD‐2 poly(A) polymerase has been observed to co‐localise with FMR1 in neuronal mRNP granules [87] and may therefore be delivered to P‐bodies, lending to the pos‐ sibility of cytoplasmic poly(A) tail lengthening in de‐repressing mRNA.

In addition to their mRNA, P‐bodies themselves respond to cellular stimuli as synaptic input induces as much as a 60% decrease in dendritic P‐body abundance [113], while artificial stim‐ ulation of cap‐dependent translation decreases P‐body biogenesis in PNS sensory neurons [117]. Overall, these observations imply that these repressive granules could dissociate in a pro‐translational environment to release a collection of mRNAs for novel protein synthesis. A subset of dendritic P‐body‐like granules containing miRNAs repressed mRNAs and lacking XRN1, which respond to synaptic activation may represent a subcategory of P‐bodies specifi‐ cally designed for such a purpose [112]. Stress granules, which contain translationally stalled mRNAs, translational enhancers (such as eIFs and PABPs) and interact with P‐bodies, are another key candidate for mRNA storage; however, their functional relevance is limited to periods of exposure to stressful cellular stimuli [118].

## **5. Emerging non‐coding RNAs in post‐transcriptional regulation of synaptic translation**

#### **5.1. Long non‐coding RNA**

Long non‐coding RNA (lncRNA) is a broad category of >200nt non‐coding transcripts charac‐ terised by widespread genomic distribution and involvement in a variety of regulatory func‐ tions [119]. Like miRNA, lncRNAs inhabit a range of genomic regions including intergenic, telomeric and gene regulatory sequences [119]. Protein‐coding genes have also been identified as a source of lncRNA expression wherein these RNA may reside in antisense, overlapping, intronic or bidirectional configurations relative to protein‐coding sequences [120]. From these genes, lncRNAs are transcribed by RNA polymerase II following which many are subject to posttranscriptional modifications including alternative splicing, 5′ capping and polyadenyl‐ ation [121]. Despite this similarity to mRNA, recent studies have revealed the genomic origin of lncRNA dictates the functional role of individual lncRNA species, which may serve as reg‐ ulators of translation, transcription and epigenetic modifications, or act as miRNA precursors or sponges [120, 122]. Translational regulation is a particularly interesting aspect of lncRNA function due to the range of emerging post‐transcriptional mechanisms through which this may be achieved. This holds significance in the context of neuronal function as the adult brain has recently been identified as a source of substantial lncRNA enrichment, reflected in the discovery of 849 (of 1328 examined) lncRNAs subject to specific patterns of expression in mice [123]. Accordingly, a number of lncRNAs have exhibited roles in the regulation of syn‐ aptic translation and synaptic function, adding further complexity to the nature of neuronal translation.

The rodent lncRNA *Bc1* and its primate orthologue *BC200* are prime examples of neuronal lncRNAs, which modulate post‐synaptic translation of plasticity‐associated mRNA, including Arc, CaMKII and MAP1B [124]. Current studies indicate that *Bc1/BC200* instigates translational repression at somatodendritic compartments through inhibition of eIF4A and eIF4B activity at the mRNA 5′ cap, thereby suppressing translation initiation [125]. In conjunction with this observation, *BC200* alone has been shown to contain *cis‐*acting adenine‐rich elements which potentially facilitate interaction with PABPs [126], indicating this lncRNA may additionally interfere with 5′ to 3′ mRNA interactions required for translation initiation. FMRP may further contribute to the multidimensional nature of *Bc1/BC200* lncRNA function as oligonucleotide masking of an mRNA recognition motif within the *Bc1* lncRNA primary sequence inhibits FMRP interaction with MAP1B mRNA [124]. This suggests that *Bcl* lncRNA may potentially act as a guide in FMRP association with plasticity‐associated mRNA analogous to miRNA and the RISC; however, the exact functional mechanism of these relationships is yet to be elucidated.

In addition to *Bc1/BC200*, other emerging lncRNAs have been implicated in synaptic regula‐ tion through multiple post‐transcriptional mechanisms. lncRNA Malat1 is one such example, which modulates synaptic density through positive regulation of neuroligin 1 and SynCAM1 mRNA expression and alternative splicing of CaMKIIB [127]. Interestingly, this occurs within nuclear speckles, known for their role as sites of mRNA storage and processing, revealing this lncRNA modulates synaptic properties from the nucleus via a post‐transcriptional mecha‐ nism [127]. lncRNA Tsx has additionally been shown to enhance short‐term hippocampal memory in mice through an elusive mechanism, suggesting a role for this lncRNA in the regulation of synaptic plasticity [128].

Considering these examples together, it is evident that current understanding of the exact mechanisms through which various lncRNAs post‐transcriptionally modulate synaptic prop‐ erties is limited. Despite this gap in the literature, lncRNAs appear to bidirectionally regulate post‐synaptic translation, suggesting lncRNAs may act to fine‐tune protein synthesis rather than strongly influence gene expression networks. Supporting this theory, mRNAs with roles in synaptogenesis including *CamkIIa* and *Dag1* have been observed in complex with their anti‐ sense lncRNA counterparts in synaptoneurosomes of the adult mouse forebrain [129]. This further raises the possibility that lncRNAs could block translation via binding target mRNAs while additionally inhibiting miRNA‐mediated mRNA decay through masking miRNA rec‐ ognition elements. This distinct prospect further underscores the complex and multidimen‐ sional nature of post‐transcriptional lncRNA function in the regulation of synaptic properties and emphasises the need for extensive characterisation of individual lncRNA species.

#### **5.2. Circular RNA**

In addition to their mRNA, P‐bodies themselves respond to cellular stimuli as synaptic input induces as much as a 60% decrease in dendritic P‐body abundance [113], while artificial stim‐ ulation of cap‐dependent translation decreases P‐body biogenesis in PNS sensory neurons [117]. Overall, these observations imply that these repressive granules could dissociate in a pro‐translational environment to release a collection of mRNAs for novel protein synthesis. A subset of dendritic P‐body‐like granules containing miRNAs repressed mRNAs and lacking XRN1, which respond to synaptic activation may represent a subcategory of P‐bodies specifi‐ cally designed for such a purpose [112]. Stress granules, which contain translationally stalled mRNAs, translational enhancers (such as eIFs and PABPs) and interact with P‐bodies, are another key candidate for mRNA storage; however, their functional relevance is limited to

**5. Emerging non‐coding RNAs in post‐transcriptional regulation of**

Long non‐coding RNA (lncRNA) is a broad category of >200nt non‐coding transcripts charac‐ terised by widespread genomic distribution and involvement in a variety of regulatory func‐ tions [119]. Like miRNA, lncRNAs inhabit a range of genomic regions including intergenic, telomeric and gene regulatory sequences [119]. Protein‐coding genes have also been identified as a source of lncRNA expression wherein these RNA may reside in antisense, overlapping, intronic or bidirectional configurations relative to protein‐coding sequences [120]. From these genes, lncRNAs are transcribed by RNA polymerase II following which many are subject to posttranscriptional modifications including alternative splicing, 5′ capping and polyadenyl‐ ation [121]. Despite this similarity to mRNA, recent studies have revealed the genomic origin of lncRNA dictates the functional role of individual lncRNA species, which may serve as reg‐ ulators of translation, transcription and epigenetic modifications, or act as miRNA precursors or sponges [120, 122]. Translational regulation is a particularly interesting aspect of lncRNA function due to the range of emerging post‐transcriptional mechanisms through which this may be achieved. This holds significance in the context of neuronal function as the adult brain has recently been identified as a source of substantial lncRNA enrichment, reflected in the discovery of 849 (of 1328 examined) lncRNAs subject to specific patterns of expression in mice [123]. Accordingly, a number of lncRNAs have exhibited roles in the regulation of syn‐ aptic translation and synaptic function, adding further complexity to the nature of neuronal

The rodent lncRNA *Bc1* and its primate orthologue *BC200* are prime examples of neuronal lncRNAs, which modulate post‐synaptic translation of plasticity‐associated mRNA, including Arc, CaMKII and MAP1B [124]. Current studies indicate that *Bc1/BC200* instigates translational repression at somatodendritic compartments through inhibition of eIF4A and eIF4B activity at the mRNA 5′ cap, thereby suppressing translation initiation [125]. In conjunction with this observation, *BC200* alone has been shown to contain *cis‐*acting adenine‐rich elements which

periods of exposure to stressful cellular stimuli [118].

**synaptic translation**

26 Synaptic Plasticity

**5.1. Long non‐coding RNA**

translation.

Circular RNAs (circRNAs), as their name implies, are a class of circular long non‐coding RNAs produced by exonic or intronic splicing which lack 5' cap and 3' poly(A) tail structures and exhibit exonuclease‐resistance [130]. Unlike miRNA and RBPs which generally serve to regulate translation through direct modulation of mRNA characteristics, emerging evidence implicates circRNA as miRNA sponges, serving as a regulatory mechanism by which miRNA translational regulation may be dampened. A well‐characterised example is the brain‐enriched circRNA ciRS‐7 (also known as CDR1as) which contains as many as 74 conventional seed‐ binding sites for miR‐7 that allow this circRNA to function as a more potent miRNA sponge than traditional anti‐miRs used for miRNA knockdown [131, 132]. While this circRNA dem‐ onstrates highly specific miRNA sponging, it is unclear whether this is a unique example or common among other species. For comparison, circHIPK3 and circRNA mlb, respectively, knockdown 9 and 2 unique miRNA [133, 134].

circHIPK3 is a particularly interesting example as overexpression of this species triggers a reduction of midbrain size in developing zebrafish [133], indicating circRNA may exert sub‐ stantial influence over neuronal structure and function. Considering this, the function of this RNA class bears significant implications in the neuronal context since circRNAs exhibit sub‐ stantial brain enrichment and accumulate during nervous system development and with age [135, 136]. circRNA may also act to fine‐tune plasticity‐associated translation as evidenced through their subcellular enrichment within dendritic compartments of cortical pyramidal neurons and interneurons, and differential expression of at least 42 distinct species during bicuculline‐induced synaptic plasticity [134]. Together, these studies therefore implicate cir‐ cRNA in both neuronal development and plasticity and provide an exciting added dimen‐ sion to neuronal translational control. Further characterisation of individual circRNA will no doubt be integral in both determining the status of miRNA sponging as a primary circRNA function and the functional significance of these RNA in neuronal structure and function.

## **6. Towards a unified model of neuronal post‐transcriptional regulation of translation**

Since the initial discovery of extrasomatic neuronal translation, it has become evident that a number of regulatory systems fine‐tune neuronal protein synthesis in an exquisitely coordi‐ nated manner to control structural and physiological characteristics associated with synaptic plasticity. Since these regulatory systems are continually exhibiting varying degrees of overlap, deciphering the mechanisms through which these systems both function in isolation and inter‐ act is of critical importance in revealing the intricate details of neuronal post‐transcriptional regulation of translation.

A key example is the interplay between neuronal transport mRNP granules and P‐bodies at the post‐synaptic compartment [113]. The possibility of mRNP granules delivering cargo to P‐bod‐ ies is an enticing prospect further exemplified by the potential for P‐bodies to isolate and store repressed mRNAs for translation under suitable conditions [115]. What remains to be consid‐ ered is the potential for P‐bodies to export cargo to neuronal mRNP granules, perhaps acting as a means of mRNA recycling especially interesting in the context of the proposed "Sushi Belt" theory of neuronal mRNA transport [18]. As P‐bodies, neuronal mRNP granules, polysomes and stress granules display multiple degrees of functional interaction, an overlying mechanism of mRNA transport, storage, repression and translation is likely at play to exert considerable influence over the available pool of translatable mRNA at the post‐synaptic compartment.

With regard to miRNA function in the neuronal context, an increasing body of evidence underscores the fact that miRNAs are not simply mediators of mRNA decay. For neurons in particular, the existence of reversible systems of translational repression would present an energetically favourable mechanism of translational control capable of reducing total reliance on novel transcription and extra‐somatic mRNA trafficking to dendrites. Indeed, the role of cytoplasmic poly(A) polymerases such as GLD‐2 in enhancing activity‐dependent transla‐ tion [85] shows potential for cytoplasmic poly(A) tail modulation as a reversible mechanism of neuronal translational control, warranting investigation of the theoretical decoupling of miRNA repression and degradation of mRNA in the neuronal context. Furthermore, uncover‐ ing the functional relevance of FMRP in mediating miRNA ribosome stalling will likely reveal a neuron‐specific aspect to miRNA function which may prove critical in characterising the role of FMRP in the biogenesis of neuronal disease. Further miRNA studies in conjunction with high‐throughput ribosome profiling [137], poly(A) tail sequencing [138] and co‐immu‐ noprecipitation sequencing will prove critical in revealing the existence and functional signifi‐ cance of these repressive systems in coordinating post‐synaptic neuronal translation.

From the presented examples miR‐132, miR‐134 and miR‐137, plasticity‐relevant miRNA spe‐ cies clearly regulate dynamic morphological and physiological changes at the post‐synaptic compartment through interaction with a variety of targets. However, it is important to note that these miRNA constitute well‐described examples subject to intensive research. Additional miRNAs, such as miR‐9, miR‐22, miR‐124 and miR‐188, among others [139], have all displayed an association with synaptic plasticity and therefore stress the need for further characterisation of specific miRNA and how together they exquisitely coordinate gene expression networks in regulation of synaptic plasticity. This holds true for lncRNAs, which appear to regulate syn‐ aptic plasticity via an assortment of posttranscriptional mechanisms [119], stressing the need for further investigation of individual lncRNA species and the mechanisms through which they act. Considering the potential role of circRNA in regulating miRNA [131–134] and their post‐synaptic enrichment, emphasis should no doubt be additionally placed on characterising these non‐coding RNA in the context of neuronal translational control. Expression of circRNA is likely to partially account for the distinct complexity of neuronal translation and could act as novel therapeutic target or biomarker in neurological disease.

Continual advancement in high‐throughput technology and analysis will substantially fur‐ ther our ability to characterise and integrate these regulatory systems into a unified model of neuronal posttranscriptional regulation of neuronal translation. Increasing our knowledge of this aspect of the central dogma of molecular biology in the neuronal context will subse‐ quently facilitate characterisation of the molecular events underlying synaptic plasticity, fuel‐ ling our understanding of learning and memory.

## **Author details**

circHIPK3 is a particularly interesting example as overexpression of this species triggers a reduction of midbrain size in developing zebrafish [133], indicating circRNA may exert sub‐ stantial influence over neuronal structure and function. Considering this, the function of this RNA class bears significant implications in the neuronal context since circRNAs exhibit sub‐ stantial brain enrichment and accumulate during nervous system development and with age [135, 136]. circRNA may also act to fine‐tune plasticity‐associated translation as evidenced through their subcellular enrichment within dendritic compartments of cortical pyramidal neurons and interneurons, and differential expression of at least 42 distinct species during bicuculline‐induced synaptic plasticity [134]. Together, these studies therefore implicate cir‐ cRNA in both neuronal development and plasticity and provide an exciting added dimen‐ sion to neuronal translational control. Further characterisation of individual circRNA will no doubt be integral in both determining the status of miRNA sponging as a primary circRNA function and the functional significance of these RNA in neuronal structure and function.

**6. Towards a unified model of neuronal post‐transcriptional regulation of**

Since the initial discovery of extrasomatic neuronal translation, it has become evident that a number of regulatory systems fine‐tune neuronal protein synthesis in an exquisitely coordi‐ nated manner to control structural and physiological characteristics associated with synaptic plasticity. Since these regulatory systems are continually exhibiting varying degrees of overlap, deciphering the mechanisms through which these systems both function in isolation and inter‐ act is of critical importance in revealing the intricate details of neuronal post‐transcriptional

A key example is the interplay between neuronal transport mRNP granules and P‐bodies at the post‐synaptic compartment [113]. The possibility of mRNP granules delivering cargo to P‐bod‐ ies is an enticing prospect further exemplified by the potential for P‐bodies to isolate and store repressed mRNAs for translation under suitable conditions [115]. What remains to be consid‐ ered is the potential for P‐bodies to export cargo to neuronal mRNP granules, perhaps acting as a means of mRNA recycling especially interesting in the context of the proposed "Sushi Belt" theory of neuronal mRNA transport [18]. As P‐bodies, neuronal mRNP granules, polysomes and stress granules display multiple degrees of functional interaction, an overlying mechanism of mRNA transport, storage, repression and translation is likely at play to exert considerable influence over the available pool of translatable mRNA at the post‐synaptic compartment.

With regard to miRNA function in the neuronal context, an increasing body of evidence underscores the fact that miRNAs are not simply mediators of mRNA decay. For neurons in particular, the existence of reversible systems of translational repression would present an energetically favourable mechanism of translational control capable of reducing total reliance on novel transcription and extra‐somatic mRNA trafficking to dendrites. Indeed, the role of cytoplasmic poly(A) polymerases such as GLD‐2 in enhancing activity‐dependent transla‐ tion [85] shows potential for cytoplasmic poly(A) tail modulation as a reversible mechanism

**translation**

28 Synaptic Plasticity

regulation of translation.

Dylan Kiltschewskij1,2 and Murray J. Cairns1,2,3\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, Callaghan, Australia

2 Priority Research Centre for Brain and Mental Health Research, Hunter Medical Research Institute, New Lambton, Australia

3 Schizophrenia Research Institute, Sydney, Australia

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## **Mitochondrial Regulators of Synaptic Plasticity in the Ischemic Brain**

Han-A Park and Elizabeth A. Jonas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67126

#### **Abstract**

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Synaptic plasticity is a process by which neurons adapt or alter the strength of information transfer, and it is known to play a role in memory formation, learning, and recovery after injury. In this chapter, we describe how ischemic insults alter neuronal intracellular mechanisms and signaling pathways, and we discuss how, after neuronal injury, synaptic plasticity is regulated prior to and during death or rehabilitation and recovery. In addition, recently described regulators of synaptic plasticity will be introduced.

**Keywords:** ischemia, mitochondrial metabolism, neuroprotection, Bcl-xL, ATP synthase

## **1. Cellular mechanisms after cerebral ischemia**

Cerebral ischemia occurs as a result of a lack of, or insufficiency of, blood supply to the brain, which results in the failure to meet neuronal metabolic demands. Thrombotic or embolic stroke (focal), and cardiac arrest or cardiac surgery (global) are common causes of cerebral ischemia. The loss of oxygen and glucose flow to the brain eventually leads to neuronal energy deficits. These energy deficits result in the failure of adenosine triphosphate (ATP) dependent ion pumps expressed on the neuronal plasma membrane, permitting an unregulated surge of ion influx into the neuronal cytoplasm [1–3]. Calcium influx induces the release of neurotransmitters from presynaptic neurons and activation of postsynaptic glutamate receptors such as N-methyl-d-aspartate (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [4–6]. Uncontrolled opening of postsynaptic receptors provokes failure of intracellular ion homeostasis, resulting in excessive postsynaptic entrance of calcium or sodium through NMDA- or AMPA-regulated channels; this initiates signaling pathways.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.