From Wiring Together to Firing Together: The Marvelous Neuron
Neuron: Structure/Function, Cellular/Molecular
Neuron - a late nineteenth century Greek term, refers to highly specialized "nerve cells" that conduct electrical impulses (Action potential). This innate propensity to generate and conduct electrical potentials is a unique hallmark of all "excitable cells" of which the neurons are the most specialized. A neuron exhibits a highly complex repertoire of specialized membranous structures, embedded Ion channels, second messengers, genetic and epigenetic elements and unique complements of various proteins such as the receptors. A "synapse," which is the functional building block of all communicating neurons, refers to the juxtaposed point of contact between two excitable cells. As the nerve impulse invades the "presynaptic terminal," it elicits the release of chemical messenger/s - the neurotransmitter into the synaptic cleft. The diffused chemical neurotransmitter substance, such as dopamine, Serotonin or a proteineous peptide (substance P for example) then binds to its respective receptor located on the "postsynaptic" side of the terminal and invokes an electrical response. Synapses are analogous to electrical bulbs that light up when electric current traveling through the nerve cables (Cable theory) is switched on by the neuron. Thus, synapses serve as the functional unit of all neuronal connectivity upon which hinge its marvelous attributes - ranging from the control of simple reflexes (Reflexes) to complex motor patterns, learning and memory, cognition, emotions etc. Perturbations - emanating from either genetic, cellular and molecular malfunction or an injury - disrupt lines of communications between neurons thus rendering the nervous system dysfunctional. Therefore, central to our comprehension of all brain functions and its repair lie an in-depth understanding of the cellular and molecular elements that make up the neuronal architecture.
From Wiring Together to Firing Together: The Marvelous Neuron
The astonishing structural and functional traits of the human brain have eluded many intriguing minds for centuries - and yet our understanding of even the very basic neuronal elements, such as the synapse, remains pedestrian. Notwithstanding tremendous efforts by the neuroscience community over the decades, the sheer numbers of brain cells (tens of billions) and the intricate nature of their connectivity continue to offer formidable challenges. While tools are being developed to visualize and record the activities of functionally active neurons embedded deep within the brain, an alternative paradigm is to understand how the nervous system is put together during development in the first instance. A developmental approach to understanding nervous system function and dysfunction is aimed at drawing up the road maps that were originally used to orchestrate the neuronal connectivity patterns. Once a blue print of all such essential, cellular and molecular components used to lay down the original neuronal maps are "de-coded," one might be in a much better position to recapitulate these steps in an adult brain to help "rewire" its damaged connectivity.
A variety of animal model systems are being used to define elements that foster neuronal proliferation, migration and differentiation - steps that are central to the normal wiring of the brain. The steps that enable a neuron to get to its final and well-defined destination in the nervous system are highly complex and rely upon a variety of intrinsic cell-cell signaling and extrinsic factors. Having arrived at its final destination, a neuron begins to develop its axonal and dendritic architecture, which is highly ordered and equipped with navigational tools that would enable these newly born processes to reach out to select groups of target cells that are often located at some distance. Such "search and select" tasks are assigned by neurons to highly specialized structures, termed growth cones located at the tip of an extending neurite (axon or dendrite). Every growth cone, fueled by specific chemotropic molecules and Growth Factors, follows a precise roadmap, rarely deviating from its defined trajectory that is designed for it to seek out its specific target/s. Growth cones are assisted in their navigational tasks by a variety of cell-cell interacting and diffusible molecules comprising the extracellular milieu. A number of molecules, such as netrin, slit etc. and their interacting receptors are eloquently described and discussed in detail by Spencer et al. (axonal pathfinding and network assembly).
In addition to various growth-permissive molecules described above, a growth cone's navigational ability is also empowered by a number of well-defined growth repulsive factors that are either membrane-bound or diffused along its path, en route towards targets. These growth-repulsive molecules such as the Samaphorins, NI35 etc. will, on the one hand, deter growth cone's entry into the wrong territory, and on the other hand, they serve to prevent wiring among functionally "unrelated" neurons. An intriguing aspect of these growth-suppressive or -repulsive molecules is their continued presence in the adult brain - which incidentally offers formidable challenge to brain repair after trauma and injury. Numerous studies in which the activities of these growth-inhibitory molecules were neutralized have uncovered an innate regenerative capacity of the adult neurons - thus underscoring their therapeutic importance vis-à-vis functional recovery from stroke and injury. Metz and Faraji have defined some of these growth inhibitory molecules in nervous system development and regeneration and have identified their underlying mechanisms. These authors have also offered several therapeutic strategies that might involve perturbation of these growth-inhibitory molecules to ensure functional regeneration and recovery after nerve injury or neuronal degeneration.
In the vicinity of its target tissue, a growth cone slows its advance and makes physical contacts with potential target cells. Cell-cell interactions via a variety of membrane-bound molecules such as neuroligans and neuregulin etc. trigger inductive changes not only in the presynaptic cells but also its postsynaptic partner. On the presynaptic side, the growth cone undergoes dramatic structural changes that begin with the retraction of filopidia while lamellopodia transform into a club shaped structure. Transmitter vesicles and other related synaptic proteins descend into the bulbous ending, which comes to rest at the juxtaposed postsynaptic site. In addition, Ca2+ channels (Calcium channels - an overview) and other elements of the synaptic machinery specifically cluster presynaptically. At the postsynaptic site, neurotransmitter receptors and their respective second messenger molecules cluster - concomitant with the postsynaptic density (PSD). Initially, neurons make myriads of synaptic contacts, which are subsequently refined through activity-dependent mechanisms. The molecular machinery mediating cell-cell contact coupled with the activity-dependent mechanisms are central to establishing a precise balance between inhibitory and excitatory synapses and their respective partners. Interplay between various molecules mediating cell-cell interactions and the underlying mechanisms have been described by Arstikaeitis and El-Husseini (synapse formation: neuromuscular junction vs. central nervous system) and Colicos (activity-dependent synaptic plasticity). While El-Husseini's lab takes advantage of powerful molecular techniques to unravel various elements of the synaptogenic program, Colicos lab uses novel photoconductive stimulation techniques to decipher how activity-dependent mechanisms either strengthen or weaken certain synapses. Several recent studies from these and other labs have shed significant light on to the mechanisms by which neurons recognize their potential targets and establish synaptic connectivity. Because some developmental aspects of synapse formation are also recapitulated in the adult brain during synaptic plasticity that underlies learning and memory, many investigators are taking advantage of activity- or plasticity-related changes in the adult brain to understand how synapses may form and subsequently refine during development. The plasticity-related induction of new synapses or the awakening of the silent Synapses has thus provided greater insight into mechanisms that regulate synapse formation during development (activity-dependent synaptic plasticity) This area of research is not only important for our understanding of the mechanisms underlying nervous system development but also synaptic plasticity that forms the basis for learning and memory in the intact animals.
Due in large measure to the complex nature of the neuronal connectivity in the adult brain where cell-cell interactions are often difficult to study at the level of single pre- and postsynaptic neurons, a number of labs have opted to explore various model system approaches to define mechanisms underlying synapse formation. For instance, the neuromuscular junction (NMJ) and various invertebrate models have been extensively used to define both the cellular and molecular mechanisms underlying target cell selection, specific synapse formation and synaptic refinement. As a result of these studies as highlighted by Feng in the chapter synaptic transmission: model systems we now know a great deal about various steps that determine the specificity of synapse formation both at the NMJ and between central neurons. Molecules such as Agrin that are synthesized and secreted by Motoneuron (motor neurons) have been shown to bring about specific inductive changes required for the assembly of the postsynaptic machinery at the NMJ. Similarly, postsynaptic cells have been shown to induce clustering of Ca2+ channels and other elements of the synaptic machinery at the presynaptic terminal. Newly formed synapses have since been shown to undergo activity-dependent refinement and consolidation.
Among various proteins that are selectively targeted at both the pre- and postsynaptic sites are the ion channels. For instance, Ca2+ (Calcium channels - and overview), Na+ (Sodium channels) and K+ channels (Neuronal potassium channels) are specifically targeted at select synaptic sites, and this targeting is essential not only for normal synapse formation but also the synaptic transmission. In the chapter ion channels from development to disease Pham et al. demonstrate how various ion channel sub-types are selectively gated at various synaptic and extrasynaptic sites to serve their well-defined roles in a wide variety of cell types. Perturbation or mutations to various ion channels sub-types either in non-excitable or excitable cells may result in pathologies, such as the neonatal diabetes and epilepsy, respectively.
In addition to ion channel targeting to specific synaptic sites, the function of various other synapse-specific and Ca2+-dependent proteins are also highly regulated. Intricate interplays between myriads of synaptic vesicle-associated proteins have been an area of intense investigation recently. A combination of biochemical, molecular, imaging and electrophysiological approaches have served to identify how synaptic vesicles might be targeted, primed, docked, released and recycled at the synaptic sites. As outlined by Coorssen in the chapter synaptic proteins and regulated exocytosis newly synthesized synaptic vesicles leave the cell body by a series of well-defined pathways. These vesicles are then specifically targeted to select synaptic sites where they get tethered, docked and primed for release. An action potential-induced Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) (Calcium channels - and overview) is a critical step, which triggers fusion and exocytosis. Following their release at the synapse, the synaptic vesicles undergo endocytosis and are recycled for subsequent re-release. Although the spatio-temporal patterns of the synaptic vesicle behavior have been well characterized, this area of research, however, continues to enjoy its fair share of controversies.
The opening of the VGCCs invokes Ca2+ entry into the cytosol. This Ca2+ is then rapidly taken up by the fast endogenous Ca2± buffers, the mitochondria as well as the SERCA - sarco-endoplasmic reticulum Ca2+-ATPase pumps (Ion transport). These three steps thus exert a critical regulatory control over the magnitude of the rapid, Ca2+-mediated signaling. In addition to these fast acting steps, the Ca2+ homeostasis is also maintained by slower endogenous Ca2+ buffers, such as the mitochondria, the SERCA pumps, the plasma membrane plasma membrane NCX - Na+-Ca2+ exchanger and the PMCA - plasma membrane Ca2+-ATPase pumps (Ion transport) - all of which curtail subsequent Ca2+ signaling. The role/s of these various Ca2+-regulatory steps are not only cell type-specific, but they also vary within a cell from its somal to extrasomal compartments. Recent advances in various imaging and molecular techniques are enabling a greater understanding of the mechanisms by which various regulatory steps maintain Ca2+ homeostasis and these are described by Amy Tse et al. (influence of Ca2+ homeostasis on neurosecretion).
In contrast to classical transmitters such a dopamine, serotonin and Acetylcholine, much less is known about the secretary machinery that regulates the release of dense-cored vesicles containing neuro-hormones or peptides. Fred Tse (non-synaptic release) and colleagues have developed reliable carbon fiber amperometry approaches to define the kinetics of transmitters (such as catecholamines) release at the resolution of single granule cells. The Tse lab and others have also demonstrated the involvement of the SNARE complex in the release machinery to provide direct evidence that kinetics of release probability is highly variable from cell to cell and relies, in many important ways, on Ca2+ sensitivity of the system. Because the release of polypeptides and peptidergic neurochemical substances occurs at a relatively slower time scale, a great deal is now known about the cellular and molecular mechanisms underlying their mode of release. A variety of peptide messengers have now been shown to regulate important neuronal programs in a number of species.
In their chapter neuropeptides in energy balance Chee and Colmers describe how neuropeptides modulate hypothalamic circuitry to regulate energy balance. Their work underscores the importance of peptides such as melacocortin, corticotrophin-releasing hormones (CRH) and CRH-like peptide and neuropeptide Y, agouti-related peptide (AgRP), melanin-concentrating hormone (MCH), orexin etc. in regulating food intake and body metabolism. This is an impressive list of candidate molecules that appear to be specifically released to regulate energy balance in various animal models. Deciphering their precise roles is the focus of many laboratories and the studies are deemed important for obesity research.
While it is generally believed that most proteins such as the neuropeptides destined for various extrasomal sites (axons, dendrites and synapses), are synthesized at the soma and then selectively transported to these regions, this dogma has however, been recently challenged. Specifically, several recent studies have provided ample convincing evidence that the extrasomal compartments are able to synthesize a host of synapse- and plasticity-specific proteins de novo. Support for this notion stems from earlier studies where a host of mRNA species were identified in dendrites and axons where they were selectively targeted to specific synaptic sites following an activity-dependent mechanism. Subsequent studies using a number of molecular and radio-labeling techniques demonstrated that the targeted mRNA was indeed able to translate specific protein locally. Furthermore, injection of foreign mRNA into the extrasomal compartments was also shown not only to result in the production of encoded proteins but also that these proteins were functional. The impact of this research, which is highlighted by van Minnen in extrasomal protein synthesis in neurons are far-reaching and perhaps will be one of the most exciting areas of neuroscience in the years to come.
Once the developmental program has established a complete repertoire of synaptic connectivity, the neuronal networks are put to work through myriad modes of neuronal communication. These range from excitatory to inhibitory to mixed excitatory/inhibitory connections. While the synaptic transmission in general is predominantly chemical, the role of electrically coupled networks cannot be underestimated. Specifically, in addition to conventional chemical synapses, many neurons may also connect to each other through gap junctions where the membranes of two neurons become contiguous. Current in one cell may pass unabated to another without the need for a synaptic delay. While such gap junctions are predominant during development, their presence in the adult nervous system is only beginning to be realized in most vertebrates. In invertebrates, however, electrically coupled networks are quite common where they are often recruited to trigger fast escape responses that are critical for their survival and thus cannot afford the synaptic delays which are the hallmark of most chemical synapses. The precise nature of both structural and functional attributes of gap junction/tight junction or electrically coupled cells is wonderfully described by Wildering in the chapter on electrical synapses. Blocking gap junctions during early development has been shown to perturb nervous system development; their precise functions in the adult mammalian brain are, however, yet to be fully understood. It is nevertheless generally agreed that one of the hallmarks of gap junctions is to synchronize pattern activity either during a patterned motor program or pathological discharges such as epilepsy.
One of the most fascinating aspects of the neuronal uniqueness is the ability of a network of central pattern-generating neurons to exhibit rhythmical activity in the absence of the peripheral feedback. These networks of neurons, often termed central pattern generators (CPG), control a variety of rhythmical behaviors such as locomotion, respiration, feeding, mastication etc. Because CPG neurons can generate fictive, patterned activity underlying a rhythmical behavior, even in an isolated preparation, a great deal is known about intrinsic membrane properties that generate a well-organized motor output. In some instance, neurons are known to possess pacemaker potentials, which can generate endogenous bursting patterns; however, the rhythmogenesis is always a network phenomenon in both vertebrates and invertebrates. In a serious of chapters written by Bell (peripheral feedback and rhythm generation), Straub (central pattern generator) and Whelan (neurotransmitters and pattern generation) we learn a great deal about various intrinsic membrane properties (pacemaker potential, endogenous bursters, conditional bursters, etc) and synaptic interactions (excitatory/inhibitory, half-center model, reciprocal inhibition, postinhibitory rebound excitation, ramp generators, recurrent inhibition etc.) underlying patterned motor activity. Even though the CPG neurons have been known to generate patterned activity in the absence of any peripheral feedback, Bell (peripheral feedback and rhythm generation) demonstrates how peripheral feedback could be critical for the initiation, modulation and termination of the patterned activity. He specifically focuses on the role of hypoxia-sensitive chemosensory drive from the carotid body chemoreceptors, and how it affects the patterned respiratory discharges. Bell then discusses how these networks of rhythm-generating neurons are similar in both vertebrate and invertebrate animals - assuring us that the fundamental building blocks of CPG neurons are likely conserved throughout the animal kingdom. While Straub (central pattern generator) illuminates various membrane and network properties that are the hallmark of pattern generation, Whelan (neurotransmitters and pattern generation) examines structural, functional and transmitter (serotonin, dopamine etc) organization of the CPG underlying locomotor behavior in mammals. Whereas in some invertebrate models, command neurons are thought to be sufficient and necessary to trigger a patterned discharge, it is generally believed that the rhythm generation is a function of polymorphic nature of the network. In this configuration, the network exhibits a highly dynamic repertoire of activity patterns thus allowing greater flexibility within the network. Neuronal networks are thus known not to be hardwired, rather they exhibit great flexibility - allowing a subset of neurons to switch between inter-related networks. A similar re-organization of the network behavior is observed following trauma and injury whereby uninjured neurons either take on additional assignments or switch their roles from one to another.
In contrast to their central counterparts, most peripheral neurons are able to regenerate their axonal projections after injury (Regeneration). Although this regeneration appears to re-capitulate developmental patterns of growth, the reinnervation is often incomplete, mismatched and often accompanied with neuropathic pain. Tremendous efforts are therefore being made to improve the outcome of peripheral injuries by either manipulating the extracellular environment or the surgical interventions. Zochodne (axon degeneration and regeneration of peripheral neurons) provides a very comprehensive account for cellular and molecular changes that occur immediately after a peripheral injury (neurapraxia, axonotmesis, neurotemesis and how this signal is conveyed to the cell body to activate the "regenerative program." It is generally believed that an immediate injury response triggers a massive Ca2+ influx, which in turn activates a cascade of events that lead to the microtubular disorganization and neurofilament dissolution. Subsequent SC activation then results in microphage invasion, an upregulation of cytokines and chemokines - including IL-1β, IL-6, IFN-γ, TNF-α, MCP-1 (monocyte chemoattractant protein-1) and MIP-1α (macrophage inflammatory protein 1α) followed by a complete breakdown of myelin. In the presence of appropriate trophic factors, nitric oxide, and various substrate adhesion molecules, a neuron then triggers its regenerative program, which begins with the initiation of new neurites and the re-establishment of synaptic connectivity. In contrast with the above described crush injuries, nerve transsections often result in Wallerian degeneration (neurotmesis or Sunderland Type V injury), which involves breakdown of axons and myelin distal to the injury site. It is interesting to note that the regeneration re-activates many but not all elements of the developmental program and as a consequence the functional recovery after nerve injury is often incomplete. Several novel approaches are being developed to enhance the clinical outcomes of nerve injury and are described in detail by Midha (peripheral nerve regeneration and nerve repair). Specifically, Midha provides extensive overview vis-à-vis the pros and cons of nerve grafts, electrical stimulation paradigm and nerve conduits that are being used clinically. This chapter also provides a detailed account of various bio-engineering approaches that are being developed to create nerve conduits that may, in the future, play "active" rather than passive roles in promoting nerve regeneration. This approach most certainly holds tremendous potential and is being perused extensively by Zochodne and Midha labs.
A neuron is considered as the functional unit of the nervous system, whereas a synapse serves as a gatekeeper of all neuronal communication. Over the past 50 years our understanding of both the structural and functional attributes of neurons and synapses has been enhanced tremendously. Specifically, a great deal is now known about the intrinsic membrane properties that contribute to neuronal excitability and shape its unique characteristics. Every unique neuronal trait in turn, makes specific contributions to synaptic properties of the network in which it is embedded. Neurochemical, electrochemical and/or electro-electrical properties empower a network to generate rhythmical patterns, which in turn control important behaviors - ranging from simple reflexes to complex motor patterns and learning and memory. These connectivity patterns are orchestrated early during development and are constantly re-organized and reconfigured throughout life. Perturbation to either the intrinsic membrane or synaptic properties renders the nervous system dysfunctional thus resulting in the permanent loss of neuronal function. Restoration of this connectivity is perhaps one of the greatest challenges facing the neuroscientists - an area that requires extensive efforts not only by the basic scientists, clinical investigators but also the bio-medical engineers and nano-engineers. A multidisciplinary approach is likely to yield bionic hybrids, which can then be interfaced with neurons to resort lost brain function. For instance, bio-compatible and neuron-friendly chips that can be interfaced with networks of brain cells will not only enhance our understanding of brain function but also regain the lost nervous system function. Although challenging - this appears to be the most promising avenue towards regeneration and functional repair of the injured nervous system.