Our movements, sensory perceptions of what happen around us and the generation of an answer to them depends on a complex network of neurons in our central and peripheral nervous system. Santiago Ramon y Cajal was the first to postulate the existence of the neuron as a basic unit of brain function. The name ‘neuron’ was proposed by the German Anatomist Heinrich W. Waldeyer. Ramón y Cajal proposed in his law of dynamic polarization that nerve impulse transmission always occurs from the protoplasmic branches and cell body to the axon or functional expansion, and that every neuron has, therefore, a receiving device, the soma and extensions protoplasmic (dendrites), an emission apparatus, the axon, and a dispensing apparatus, the nerve terminal arborization¹. With this idea, Ramón y Cajal opened two issues that are now important research fields. First, how is the morphology/structure of a neuron acquired and second, what are the cellular and molecular mechanisms that enable the transmission of nerve impulses from one neuron to another. Even more, this complex neuronal network is also modulated by other type of brain cells, glial cells (astrocytes, microglia, oligodendrocytes and Schwann cells). Glial cells support neuronal survival and modulate nerve impulse transmission between neurons. Even if we know the existence of neurons and different types of glial cells since the end of the 19th century, we do not yet fully understand the mechanisms that regulate their development and participation in brain physiological and pathological states.

Due to the growing complexity of the study in human brains, cellular and animal models are useful tools to study these diseases and to find therapeutic targets. Brain diseases represent a high direct and indirect economic cost for states, but also an emotional cost for patients and their families. Psychiatric diseases that affect neuronal connectivity and excitability represent between 1 and 3% of the population, depending on disease type and geographical location. It is calculated that around 1% of the population suffers a kind of schizophrenia, while the number is around 3% for epileptic seizures, and higher for depression. The exact cause of the appearance of some of the psychiatric diseases is not known, but a neurodevelopmental hypothesis has arisen proposing that they are generated early in development due viral infections or genetic modifications. On another hand, the cause of most of the neurodegeneratives diseases is unknown, which renders it difficult to find a cure for them.

The neuron is the cell type with a higher morphological and functional complexity. The acquisition of this complexity begins with the specification of one of the initial neurites as the axon and its elongation. Thus, the neuron acquires its morphological polarity, differentiating a somatodendritic domain (postsynaptic) and an axonal domain (presynaptic). The specification of an axon during neuronal polarization is an essential stage for the correct transfer of synaptic information in the nervous system. This idea was designed by Santiago Ramón y Cajal about 120 years ago (cf. figure 1).

Over the past 30 years, numerous studies have explored the molecular and cellular mechanisms regulating the neuronal polarity. The intracellular mechanisms that regulate axon specification and further growth are still poorly understood. Moreover, Interactions of glial cells (oligodendrocytes or Schwann cells) and neurons have been shown to be critical not only for axon development, but also for efficient and rapid (saltatory) propagation of action potentials along the axon through the myelin sheath enwrapping the axon (cf. Figure 2, p. 22). Despite the known data on neuronal morphogenesis in situ, the understanding of the regulatory intracellular mechanisms involved in neuritogenesis and axon elongation requires experimental systems where neurons can be manipulated during differentiation. The generation of a standardized culture of hippocampal neurons, with defined stages, allowed advancing in the study of the molecular mechanisms responsible for the formation and elongation of the axon (figure 2). This model has confirmed Cajal´s idea of neuronal polarization and reproduces functional and morphological changes observed in neurons in situ. It has extensively contributed to the research and understanding of neuronal functions².

At present, we know with greater accuracy how neuronal polarity and axon formation are established. In this phenomenon, at least two intracellular signaling pathways are involved, the PI3-kinase (Phosphoinositide 3-kinase) pathway and a less known one that involves the LKB1 protein (tumor suppressor kinase Lkb1). These pathways regulate the dynamics of actin and microtubule cytoskeleton, which plays an essential role in axon growth, axon membrane proteins transport and the establishment of axonal subdomains that will allow conduction of nerve impulses. This impulse is generated in response to the signals received by the dendritic spines, dendrites and soma, is integrated into the AIS, generating an action potential, which is transmitted along the axon and maintained by nodes of Ranvier until it reaches the presynaptic terminals that liberate neurotransmitters. For this machine to work properly, each domain of the neuron (somatodendritic, AIS, nodes of Ranvier and presynaptic terminals) must fulfill its specific function and therefore needs its own specific set of functional proteins (neurotransmitter receptors, ion channels and proteins capable of anchoring them at a particular precise position). Therefore, deregulation of cytoskeletal signaling, the absence or lack of protein function or an inaccurate localization of these proteins can generate a dysfunction of the neuronal network, causing mental or neurodegenerative diseases. A better knowledge of axon structure and function will allow a better understanding of neuronal plasticity and excitability at the level of the axon, and a more comprehensive search of strategies to eliminate or to bypass the defects that generate diseases affecting the nervous system.

Along axon structure the axon initial segment (AIS) is the most important axonal subdomain. Several evidences support this idea. First, integrity of AIS is necessary to confer the axonal identity, and its disruption gives axon some dendritic properties, as the appearance of dendritic spines³. Second, the AIS is the place where neuronal action potentials are generated due to the high concentration of voltage gated ion channels. The structure of the AIS shows a unique composition from its cytoskeleton to its plasmatic membrane and function. The scaffold holding the AIS is composed by microtubules, with special characteristics in their organization and composition, and by a dense actin microfilaments cystokeleton. This cytoskeleton scaffold controls proteins traffic towards the axon and serves as a barrier to define axonal protein composition, a function that is complementary with the protein diffusion barrier properties of AIS plasmatic membrane. This membrane is characterized by a high density concentration of adhesion molecules, glycoproteins and ion channels. It is this high concentration of ion channels what allows the generation of action potentials. Three main types of ion channels are densely concentrated at the AIS, voltage gated sodium channels (Na_v), necessary to launch the action potential, voltage gated potassium channels (K_v), which control the action potential, and voltage gated calcium channels (Ca_v), that mediate calcium influx in response to membrane depolarization, shaping the action potential and controlling patterns of repetitive firing. These channels are concentrated and distributed all along the AIS or show a proximal or distal distribution at the AIS depending on their different subtypes. Concentration at the AIS is due to an adaptor or scaffold protein called ankyrinG that interacts and retains sodium and potassium channels through the AIS motif⁴. Other adaptor proteins such as bIV-spectrin contribute to the stabilization of the AIS, and recently different kinases have been described to play an important role in AIS regulation. The combination of the different ion channels, their differential distribution and their activation at different membrane potentials allows the neuron to control neuronal excitability and the generation of different replies depending on input signals. Moreover, the ion channels composition is different in different types of neurons.

One important question has arisen during the last 5 years: how is modulated the expression of ion channels at the axon and AIS membrane, the nodes of Ranvier, or the presynaptic terminals. The answer to this question is necessary to understand how neuronal excitability is deregulated not only in different types of psychiatric diseases, but also in some neurodegenerative diseases or after brain trauma, such as brain vascular accidents.

Altered activity or expression of ion channels at the axon generates brain and neuromuscular diseases. For example, Angelman syndrome is a neuro-genetic brain disease characterized by intellectual, developmental, motor and behavioral disorders, epilepsy or autism. This syndrome is due to a deletion in the UBE3A gene and at present there is no cure. A mouse model of this syndrome has recently contributed to the understanding of some neuronal alterations. In fact, voltage gated sodium channels and the scaffolding protein ankyrinG show a significantly higher expression at the AIS of hippocampal neurons⁵. Another disease that affects neuronal excitability is the Benign familial neonatal epilepsy caused by mutations in Kv7 potassium channels and characterized by neuronal hyperexcitability. This hyperexcitability is also observed when Kv1 potassium channels are inactivated, as shown by Dominique Debanne’s laboratory (INSERM UNIS 1072, Unité de Neurobiologie des canaux Ioniques et de la Synapse, Marseille⁶). Even more, brain vascular accidents cause a disruption of the AIS which compromise neuronal function or viability. One of the ways to find a therapeutic cure to the pathologies should be go through the comprehension of the mechanisms deregulated in these alterations and how to compensate them.

In this sense, my laboratory and other research groups are working to understand how the AIS is formed and regulated. Our research group has described the role of GSK3 (Glycogen synthase kinase) and beta-catenin in the regulation of ankyrinG and voltage gated ion channels at the AIS. Both are necessary to maintain physiological neuronal excitability⁷, and their inhibition or suppression reduces the number of AIS sodium channels and neuronal excitability. Interestingly, GSK3 is a kinase involved in neurodegenerative and psychiatric diseases and in the target of lithium, a treatment commonly used in psychiatric disorders. In fact, modifications in GSK3 and ankyrinG expression levels have been described in schizophrenic patients ‘postmortem’ brains (cf. reference 7). Further work is necessary to understand how GSK3 is regulated at the AIS and how this can influence the appearance of psychiatric or neurodegenerative diseases, such as Alzheimer´s disease. Furthermore, other kinases such as CK2 (casein kinase 2), cdk5 (cyclin dependent kinase 5) and probably phosphatases do play an important role in the regulation of the AIS. Another important research line that is being developed it is the role of the AIS plasticity/movement along the axon which regulates the response generated by the axon. Briefly, AIS can elongate or modify its distance to the soma generating adapted response. It is expected that this type of modifications also need also regulation of the microtubules and of the actin cytoskeleton that supports the AIS. In this sense, our work has described that AIS microtubules show differential characteristics compared to the rest of the axon, and this is due to the absence of HDAC6 (tubulin deacetylase) expression at the AIS8. Other scientific works sugget that AIS’s length and position depends on scaffolding proteins at the axon that determines the end of the AIS⁹. It seems that calcium channels may also play a role in this plasticity, and it would be necessary to better understand the role of calcium in the diseases and physiology of AIS. Some information arising involves a calcium dependent protease– calpain–in the cleavage of ankyrinG and sodium channels, but also in AIS integrity. Calcium entry in neurons is mediated by different membrane receptors and ion channels. The best known are the ionotropic glutamate receptors (i.e. AMPA, NMDA receptors) and voltage-gated calcium channels, but calcium can enter neurons by other regulated mechanisms, such as store-operated intracellular calcium entry (SOCE), TRP channels (transient receptor potential channels), and others. Our group has been working during the last years on a type of purinergic receptors, P2X, that modulate calcium entry in axons and are activated by extracellular ATP, liberated by neurons, glial cells and cell death in brain injury. One of these receptors, P2X7 modulates in a negative way axonal growth, and the inhibition of its activity promotes axonal elongation¹⁰. P2X7 antagonists are at present considered as therapeutical agents to promote axonal regeneration after spinal cord damage. The role of this ATP-operated calcium channel remains mostly unknown in neurons and may have important implications in the regulation of axonal structure and function. The questions arising from these studies are what proteins regulate calcium balance, how is calcium balance modulated, where does it take place, and which specific functions does calcium regulation exert on the AIS. At least one of these questions can be answered, the cisternal organelle in AIS shares multiple characteristics with the calcium store compartments in dendritic spines, and probably acts in a similar way, modulating intracellular calcium concentration in the AIS, and serving as a buffering compartment¹¹. Our working group presently tries to answer some of these questions in order to understand neuronal excitability regulation in AIS. A plethora of questions remains unanswered and these answers are essential to understanding and fighting brain diseases. Let me also add that all private and governmental contributions to this effort are necessary and will, in the future, return to society as therapies for diseases that we do not understand at present. Just think of how past efforts in biological and medical studies have contributed to our present welfare.

References

Ramón y Cajal S., ‘Comunicación acerca de la significación fisiológica de las expansiones protoplásmicas y nerviosas de la sustancia gris’, in Primer Congreso Médico-Farmaceútico regional, Valencia, 1891. Revista de Ciencias Médicas de Barcelona, 17, 671-679, 715-723 (1891); Ramón y Cajal S., ‘Leyes de la morfología y el dinamismo de las células nerviosas’. Revista Trimestral Micrográfica, 2, 1-28 (1897).

Dotti, C. G., Sullivan, C. A. and Banker, G. A. (1988), ‘The establishment of polarity by hippocampal neurons in culture’ in Journal of Neuroscience 8, 1454-68; Goslin, K. and Banker, G. (1989). Experimental observations on the development of polarity by hippocampal neurons in culture, in J Cell Biol 108, 1507-16; Kaech, S. and Banker, G. (2006), Culturing hippocampal neurons, Nat Protoc 1, 2406-15.

Schafer DP, Jha S, Liu F, Akella T, McCullough LD, Rasband MN,  ‘Disruption of the AIS cytoskeleton is a new mechanism for neuronal injury’ in Journal of Neuroscience 29(42):13242-54 (2009).

Garrido JJ, Giraud P, Carlier E, Fernandes F, Moussif A, Fache MP, Debanne D, Dargent B., ‘A targeting motif involved in sodium channel clustering at the axonal initial segment’ in Science 300(5628):2091-4 (2003).

Kaphzan H, Buffington SA, Jung JI, Rasband MN, Klann E., ‘Alterations in intrinsic membrane properties and the AIS in a mouse model of Angelman syndrome’ in Journal of Neuroscience 31(48):17637-48 (2011).

Cudmore RH, Fronzaroli -Molinieres L, Giraud P, Debanne D., ‘Spike-time precision and network synchrony are controlled by the homeostatic regulation of the D-type potassium current’ in Journal of Neuroscience 30(38):12885-95 (2010).

Tapia M, Del Puerto A, Puime A, Sánchez-Ponce D, Fronzaroli-Molinieres L, Pallas-Bazarra N, Carlier E, Giraud P, Debanne D, Wandosell F, Garrido JJ., ‘GSK3 and β-catenin determines functional expression of sodium channels at the AIS’, in Cell Mol Life Sci 70 (1):105-20 (2013).

Tapia M, Wandosell F, Garrido JJ., ‘Impaired function of HDAC6 slows down axonal growth and interferes with AIS development’, in PLoS One, 2010 Sep 23;5(9):e12908.

Galiano MR, Jha S, Ho TS, Zhang C, Ogawa Y, Chang KJ, Stankewich MC, Mohler PJ, Rasband MN., ‘A distal axonal cytoskeleton forms an intra-axonal boundary that controls AIS assembly’ in Cell. 2012 May 25; 149(5):1125-39.

del Puerto A, Díaz-Hernández JI, Tapia M, Gomez-Villafuertes R, Benitez MJ, Zhang J, Miras-Portugal MT, Wandosell F, Díaz-Hernández M, Garrido JJ. ‘Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation’ in Journal of Cell Sci. 1;125:176-88 (2012).

Sánchez-Ponce D, DeFelipe J, Garrido JJ, Muñoz A., ‘In vitro maturation of the cisternal organelle in the hippocampal neuron’s AIS’ in Mol Cell Neurosci 48(1):104-16 (2011).