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Export Date: 15 September 2021 Correspondence Address: Fagerstedt, K.W.; Department of Pathology, Finland; email: klaus.wj.fagerstedt@helsinki.fi Mixed neuroendocrine-non-neuroendocrine neoplasms (MINEN) are rare tumors that consist of at least 30% of both neuroendocrine and non-neuroendocrine components. The data concerning the pathogenesis of MINEN suggest a monoclonal origin. We describe a spontaneously immortalized cell line derived from gastric MINEN called GNEN-1. Primary tumor consisted of components of high-grade neuroendocrine carcinoma and adenocarcinoma. The GNEN-1 cell line was initiated from metastatic tumor cells of peritoneal fluid and expresses a purely neuroendocrine phenotype. The GNEN-1 cell line grows as monolayers and has retained the neuroendocrine phenotype with positivity for chromogranin A in immunohistochemistry. Electron microscopy showed cytoplasmic dense core granules and axon hillocks. The karyotype revealed alterations typical of both adenocarcinoma and neuroendocrine carcinoma such as trisomy 7 and 8. GNEN-1 cells were also positive for stanniocalcin-1, a marker of poor prognosis in gastric carcinomas. Expression of several markers related to neuroendocrine tumors was found. There have been only a few studies on the pathogenesis of MINEN and management of the disease due to the rarity of this tumor type. Here we describe for the first time an immortalized cell line derived from mixed gastric NEN. The GNEN-1 line offers a tool for future research on gastric NEN. © 2021 The authors.

The position of the axon initial segment (AIS) is thought to play a critical role in neuronal excitability. In particular, empirical studies have found correlations between a distal shift in AIS position and a reduction of excitability. Yet, theoretical work has suggested that the neuron should become more excitable as the distance between soma and AIS is increased, because of increased electrical isolation. Specifically, resistive coupling theory predicts that the action potential (AP) threshold decreases with the logarithm of the axial resistance (Ra) between the middle of the AIS and the soma. However, no direct experimental evidence has been provided so far to support this theoretical prediction. We therefore examined how changes in Ra at the axon hillock impact the voltage threshold (Vth) of the somatic AP in L5 pyramidal neurons. Increasing Ra by mechanically pinching the axon between the soma and the AIS was found to lower the spike threshold by ~6 mV. Conversely, decreasing Ra by replacing a weakly mobile ion (gluconate) by a highly mobile ion (chloride) elevated the spike threshold. All Ra-dependent changes in spike threshold could be reproduced in a Hodgkin-Huxley compartmental model. We conclude that in L5 pyramidal neurons, excitability increases with axial resistance, and therefore with a distal shift of the AIS.

Over the past two decades, substantial amount of work has been conducted to characterize different odorant receptors, neuroanatomy and odorant response properties of the early olfactory system of Drosophila melanogaster. Yet many odorant receptors remain only partially characterized, and the odorant transduction process and the axon hillock spiking mechanism of the olfactory sensory neurons (OSNs) have yet to be fully determined. Identity and concentration, two key characteristics of the space of odorants, are encoded by the odorant transduction process. Detailed molecular models of the odorant transduction process are, however, scarce for fruit flies. To address these challenges we advance a comprehensive model of fruit fly OSNs as a cascade consisting of an odorant transduction process (OTP) and a biophysical spike generator (BSG). We model odorant identity and concentration using an odorant-receptor binding rate tensor, modulated by the odorant concentration profile, and an odorant-receptor dissociation rate tensor, and quantitatively describe the mechanics of the molecular ligand binding/dissociation of the OTP. We model the BSG as a Connor-Stevens point neuron. The resulting spatio-temporal encoding model of the Drosophila antenna provides a theoretical foundation for understanding the neural code of both odorant identity and odorant concentration and advances the state-of-the-art in a number of ways. First, it quantifies on the molecular level the spatio-temporal level of complexity of the transformation taking place in the antennae. The concentration-dependent spatio-temporal code at the output of the antenna circuits determines the level of complexity of olfactory processing in the downstream neuropils, such as odorant recognition and olfactory associative learning. Second, the model is biologically validated using multiple electrophysiological recordings. Third, the model demonstrates that the currently available data for odorant-receptor responses only enable the estimation of the affinity of the odorant-receptor pairs. The odorant-dissociation rate is only available for a few odorant-receptor pairs. Finally, our model calls for new experiments for massively identifying the odorant-receptor dissociation rates of relevance to flies., Author summary Identity and concentration, intrinsically embedded in the odorant space, are two key characteristics of olfactory coding that define the level of complexity of neural processing throughout the olfactory system in the fruit fly. In this paper we advance a theoretical foundation for understanding these two characteristics by quantifying mathematically the odorant space and devising a biophysical model of the olfactory sensory neurons (OSNs). To validate our modeling approach, we propose and apply an algorithm to estimate the affinity value and the dissociation rate, the two characteristics that define odorant identity, of multiple odorant-receptor pairs. We then evaluate our model with a multitude of odorant waveforms and demonstrate that the model output reproduces the temporal responses of OSNs obtained from in vivo electrophysiology recordings. Furthermore, we evaluate the model at the OSN population level and quantify on the molecular level the spatio-temporal level of complexity of the transformation taking place between the odorant space and the OSNs. The resulting concentration-dependent spatio-temporal code determines the level of complexity of the input space driving olfactory processing in the downstream neuropils. Lastly, our model demonstrates that the currently available data for OSN responses only enables estimation of affinity value. This calls for new experiments for massively identifying the odorant-receptor dissociation rates of relevance to flies.

A neuron sprouts an axon, and its branches to innervate many target neurons that are divergent in their functions. In order to efficiently regulate the diversified cells, the axon branches should differentiate functionally to be compatible with their target neurons, i.e., a function compatibility between presynaptic and postsynaptic partners. We have examined this hypothesis by using electrophysiological method in the cerebellum, in which the main axon of Purkinje cell projected to deep nucleus cells and the recurrent axons innervated the adjacent Purkinje cells. The fidelity of spike propagation is superior in the recurrent branches than the main axon. The capabilities of encoding spikes and processing GABAergic inputs are advanced in Purkinje cells versus deep nucleus cells. The functional differences among Purkinje's axonal branches and their postsynaptic neurons are preset by the variable dynamics of their voltage-gated sodium channels. In addition, activity strengths between presynaptic and postsynaptic partners are proportionally correlated, i.e., active axonal branches innervate active target neurons, or vice versa. The physiological impact of the functional compatibility is to make the neurons in their circuits to be activated appropriately. In conclusion, each cerebellar Purkinje cell sprouts the differentiated axon branches to be compatible with the diversified target cells in their functions, in order to construct the homeostatic and efficient units for their coordinated activity in neural circuits.

The electrical connectivity in the inferior olive (IO) nucleus plays an important role in generating well-timed spiking activity. Here we combined electrophysiological and computational approaches to assess the functional organization of mice IO nucleus. Spontaneous fast and slow subthreshold events were commonly encountered during in vitro recordings. We show that the fast events represent a regenerative response in unique excitable spine-like structures in the axon hillock, whereas the slow events reflect the electrical connectivity between neurons (‘spikelets’). Recordings from cell pairs revealed the synchronized occurrence of distinct groups of spikelets; their rate and distribution enabled an accurate estimation of the number of connected cells and is suggestive of a clustered organization. This study thus provides a new perspective on the functional and structural organization of the olivary nucleus, insights into two different subthreshold non-synaptic events, and a novel experimental and theoretical approach to the study of electrically-coupled networks.

Traumatic injury often results in axonal severance, initiating obligatory Wallerian degeneration of distal segments, whereas proximal segments often survive. Calcium ion (Ca2+ ) influx at severed proximal axonal ends activates pathways that can induce apoptosis. However, this same Ca2+ -influx also activates multiple parallel pathways that seal the plasmalemma by inducing accumulation and fusion of vesicles at the lesion site that reduce Ca2+ -influx and enhance survival. We examined whether various inhibitors of Ca2+ /calmodulin-dependent protein kinases (CaMKs), and/or dimethyl sulfoxide (DMSO), a common solvent for biologically active substances, affected the ability of a hippocampal-derived neuronal cell line (B104 cells) to seal membrane damage following axotomy. Axolemmal sealing frequencies were assessed at different transection distances from the axon hillock and at various times after Ca2+ -influx (PC times) by observing whether transected cells took-up fluorescent dyes. Inhibition of CaMKII by tatCN21 and KN-93, but not inhibition of CaMKI and CaMKIV by STO-609, affected axonal sealing frequencies. That is, CaMKII is a component of previously reported parallel pathways that induce membrane sealing, whereas CaMKI and CaMKIV are not involved. The effects of these CaMKII inhibitors on plasmalemmal sealing depended on their mechanism of inhibition, transection distance, and PC time. DMSO at low concentrations (90 µM-28 mM or 0.00064%-0.2% v/v) significantly increased membrane-sealing frequencies at most PC times and transection distances, possibly by permeabilizing the plasmalemma to Ca2+ . Inhibition of CaMKII, DMSO, PC time, and the transection distance significantly affect plasmalemmal sealing that is critical to somal survival in traumatic lesions.

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.

In cortical pyramidal neurons the presynaptic terminals controlling transmitter release are located along unmyelinated axon collaterals, far from the original action potential (AP) initiation site, the axon initial segment (AIS). Once initiated, APs will need to reliably propagate over long distances and regions of geometrical inhomogeneity like branch points (BPs) to rapidly depolarize the presynaptic terminals and confer temporally precise synaptic transmission. While axon pathologies such as demyelinating diseases are well established to impede the fidelity of AP propagation along internodes, to which extent myelin loss affects propagation along BPs and axon collaterals is not well understood. Here, using the cuprizone demyelination model, we performed optical voltage-sensitive dye (VSD) imaging from control and demyelinated layer 5 pyramidal neuron axons. In the main axon, we find that myelin loss switches the modality of AP propagation from rapid saltation towards a slow continuous wave. The duration of single AP waveforms at BPs or nodes was, however, only slightly briefer. In contrast, by using two-photon microscopy-guided loose-seal patch recordings from axon collaterals we revealed a presynaptic AP broadening in combination with a reduced velocity and frequency-dependent failure. Finally, internodal myelin loss was also associated with de novo sprouting of axon collaterals starting from the primary (demyelinated) axon. Thus, the loss of oligodendrocytes and myelin sheaths bears functional consequences beyond the main axon, impeding the temporal fidelity of presynaptic APs and affecting the functional and structural organization of synaptic connectivity within the neocortex.

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth. Galectin-4 sorts to axon plasma membrane segments by binding to sulfatide-containing axonal carriers and, being bivalent, it organizes the transport of L1, and likely other glycoproteins, by attaching them to the same carriers specifically through their LacNAc termini. We propose that this mechanism would underlie L1 functional clustered organization on the axon membrane, required for proper axon growth., This study was funded by i) EC Marie Curie RTN (contract no. 2005-019561), ii) EU 7th Framework Program (under grant agreement no. 26060, and iii) Carlos III Health Institute and Castilla-La-Mancha Health Service (SESCAM) EMER program (EMER07/026).

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100-150 microns wide. Electron-microscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cell's own dendritic field in an area 100-200 microns in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization. The axon formed specialized, 10-50 microns long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments. It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells...

Axon outgrowth requires plasma membrane expansion, which results from post-Golgi vesicular transport and fusion. However, the molecular mechanisms regulating post-Golgi vesicular trafficking for membrane expansion and axon outgrowth remain unclear. Here, we show that Rab33a expression became upregulated during axon outgrowth of cultured rat hippocampal neurons. Rab33a was preferentially localized to the Golgi apparatus and to synaptophysin-positive vesicles that are transported along the growing axon. Previous studies showed that synaptophysin is localized to post-Golgi vesicles transported by fast axonal transport in developing neurons. Reduction of Rab33a expression by RNAi (RNA interference) inhibited the anterograde transport of synaptophysin-positive vesicles, leading to their decrease in axonal tips. Furthermore, this treatment reduced membrane fusion of synaptophysin-positive vesicles at the growth cones and inhibited axon outgrowth. Overexpression of Rab33a, on the other hand, induced excessive accumulation of synaptophysin-positive vesicles and concurrent formation of surplus axons. These data suggest that Rab33a participates in axon outgrowth by mediating anterograde axonal transport of synaptophysin-positive vesicles and their concomitant fusion at the growth cones.

Many neuronal cytosolic and nuclear proteins are post-translationally modified by the reversible addition of O-linked N-acetylglucosamine (O-GlcNAc) on serines and threonines. The cellular functions of O-GlcNAc modifications in neuronal development are not known. We report that O-GlcNAc-modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O-GlcNAc levels on proteins through specific inhibition of O-GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O-GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O-GlcNAcase. Western analysis revealed that O-GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O-GlcNAc modification-specific influences in neuronal development in primary culture, and indicate specific roles for O-GlcNAc in the regulation of axon morphology. © 2008 Wiley Periodicals, Inc. Develop Neurobiol 69: 162–173, 2009

SummaryAxon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

Correct function of neuronal networks is enabled by a delicate interplay among neurons communicating with each other. One of the keys is the communication at chemical synapses where neurotransmitters like glutamate, GABA, and glycine enable signal transfer over the synaptic cleft. Thereby, the neurotransmitters are released from the presynapse and bind as ligands to specific receptors at the postsynaptic side to allow for modulation of the postsynaptic membrane potentials. The postsynaptic electrical signal, which is highly modulated by voltage-gated ion channels, spreads over the dendritic tree and is thus integrated to allow for generation of action potentials at the axon hillock. This concert of receptors and voltage-gated ion channels depends on correct function of all its components. Misfunction of receptors and/or voltage-gated potassium channels (VGKC) leads to diverse adverse effects in patients. Such malfunctions can be the result of inherited genetic alterations or pharmacological side effects by drugs. Recently, autoantibodies targeting receptor or channel complexes like NMDAR, AMPAR, GABA-receptors, glycine receptors, LGI1 or CASPR2 (previously termed as VGKC-complex antibodies) have been discovered. The presence of specific autoantibodies against these targets associates with severe forms of antibody-mediated encephalitis. Understanding the molecular details of autoantibody actions on receptor and VGKC complexes is highly desirable and may open the path to develop specific therapies to treat humoral autoimmune encephalitis. Here, we summarize the current knowledge and discuss technical approaches to fill the gap of knowledge. These techniques include electrophysiology, biochemical approaches for epitope mapping, and in silico modeling to simulate molecular interactions between autoantibody and its molecular target.

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of Kv7 potassium channels and voltage-gated sodium (Nav) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these Kv7 channels and the functional impact of colocalization with Nav channels remain poorly understood. Here, we quantitatively examined Kv7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. Kv7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ∼12 (proximal) to 150 pS μm−2 (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by Kv7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (∼15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic Kv7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal Kv7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains Kv7.2/7.3 channels were found to increase Nav channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, Kv7 clustering near axonal Nav channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.

The F-wave is a result of the backfire of α-motoneurons following an antidromic inva‐ sion of propagated impulses across the axon hillock (Kimura, 1974). Its occurrence reflects excitability changes in the spinal motor neurons, as reported in patients with spasticity (Odusote & Eisen, 1979) and in healthy subjects with isometric contraction (Suzuki, Fujiwara & Takeda, 1993). In our previous study that investigated the nervous system of hemiplegic patients, excitability of spinal neural function was evaluated using F-wave data of patients with cerebrovascular disease (CVD) (Suzuki, Fujiwara & Takeda, 1993). We also reported that the persistence and amplitude ratio of F/M in patients with CVD were affected by the grade of muscle tonus, tendon reflex, or voluntary movement. Persistence reportedly depends on the number of neuromuscular units activated, while the ampli‐ tude ratio of F/M depends on their excitability (Eisen and Odusote, 1979). Therefore, we concluded that F-wave measurement was an effective neurological test for evaluating muscle tonus and voluntary movements.

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3a/b and b-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or b-catenin knockdown with shRNAs decreased the levels of phosphorylated-b-catenin, ankyrinG, and voltagegated sodium channels at the AIS, both ‘‘in vitro’’ and ‘‘in vivo’’, therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and b-catenin at the AIS., This work was supported by grants SAF2009-12249-C02-02 and SAF2009-12249- C02-01 from the Ministerio de Ciencia e Innovacion (Spain), by the Centro de Investigacio´ n Biome´ dica en Red de Enfermedades Neurodegenerativas (CIBERNED, Spain), INSERM and Agence National de la Recherche (EXCION, EPISOM). Mo´nica Tapia is supported by a CSIC-JAE fellowship.

Summary Background GABAergic interneurons regulate the balance and dynamics of neural circuits, in part, by elaborating their strategically placed axon branches that innervate specific cellular and subcellular targets. However, the molecular mechanisms that regulate target-directed GABAergic axon branching are not well understood. Results Here we show that the secreted axon guidance molecule, SEMA3A, expressed locally by Purkinje cells, regulates cerebellar basket cell axon branching through its cognate receptor Neuropilin-1 (NRP1). SEMA3A was specifically localized and enriched in the Purkinje cell layer (PCL). In sema3A −/− and nrp1 sema−/sema− mice lacking SEMA3A-binding domains, basket axon branching in PCL was reduced. We demonstrate that SEMA3A-induced axon branching was dependent on local recruitment of soluble guanylyl cyclase (sGC) to the plasma membrane of basket cells, and sGC subcellular trafficking was regulated by the Src kinase FYN. In fyn -deficient mice, basket axon terminal branching was reduced in PCL, but not in the molecular layer. Conclusions These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.

When CA3 pyramidal cell axons are stimulated repetitively at low frequencies (10-20 Hz) the fiber volley detected in CA1 stratum radiatum is unchanged indicating faithful propagation. However at higher stimulation frequencies (50-100 Hz), the fiber volley is briefly potentiated with shorter latency, but quickly becomes depressed with longer latency. Antidromic action potentials in response to high frequency stimulation become reduced in amplitude, are wider and are prone to failure. To determine the mechanisms responsible for these experimental observations of frequency dependent action potential failure we turned to computational models. We constructed a computational model of a CA3 pyramidal cell with detailed axon morphology taken from the http://Neuromorpho.org database [1]. Synaptic boutons were added to axon collaterals and diameters from the axon hillock to the initial segment to the early proximal axon were modified. Ion channels modeled include Nav1.2, Nav1.6, Kv and Km with kinetics and densities taken from Hu et al. (2009) [2]. The model included a mechanism to track extracellular potassium and intracellular sodium accumulation and the effect this has on the Nernst potentials. The size of the extracellular space for potassium accumulation was varied among simulations. Results were obtained for both low (20 Hz) and high (100 Hz) stimulation frequencies. When stimulation frequency was 20 Hz, there were no propagation failures orthodromically or antidromically. When stimulation frequency was 100 Hz and the space for extracellular potassium accumulation was large, occasional action potential failures were observed in the antidromic direction as the action potential approached the diameter expansions beginning at the proximal axon. However no failures were observed in the orthodromic direction, even at diameter expansions at synaptic boutons. When the space for extracellular potassium accumulation was small, action potential failures became more frequent in the antidromic direction with action potentials first becoming wider and smaller and then dropping out for a period; this again occurred on the approach to the axon diameter expansion. In the orthodromic direction, action potential failures were observed at branch points. Interestingly, the action potential latency of successive action potentials in the train increased gradually until failure occurred. Sodium channel inactivation was found to increase gradually in cases where failure was eventually observed. Our results suggest that ion accumulation, morphology and sodium inactivation kinetics interact to cause frequency dependent action potential failures in CA3 axons. At this time we cannot rule out additional contributions from synaptic transmission at boutons.

The shape of action potentials invading presynaptic terminals, which can vary significantly from spike waveforms recorded at the soma, may critically influence the probability of synaptic neurotransmitter release. Revealing the conductances that determine spike shape in presynaptic boutons is important for understanding how changes in the electrochemical context in which a spike is generated, such as subthreshold depolarization spreading from the soma, can modulate synaptic strength. Utilizing recent improvements in the signal-to-noise ratio of voltage-sensitive dye imaging in mouse brain slices, we demonstrate that intracortical axon collaterals anden passantpresynaptic terminals of layer 5 pyramidal cells exhibit a high density of Kv1 subunit-containing ion channels, which generate a slowly inactivating K+current critically important for spike repolarization in these compartments. Blockade of the current by low doses of 4-aminopyridine or α-dendrotoxin dramatically slows the falling phase of action potentials in axon collaterals and presynaptic boutons. Furthermore, subthreshold depolarization of the soma broadened action potentials in collaterals bearing presynaptic boutons, an effect abolished by blocking Kv1 channels with α-dendrotoxin. These results indicate that action potential-induced synaptic transmission may operate through a mix of analog–digital transmission owing to the properties of Kv1 channels in axon collaterals and presynaptic boutons.

Background The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier? Results We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS. Conclusions α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

The development of morphological neuronal polarity starts by the formation and elongation of an axon. At the same timeó the axon initial segment (AIS) is generated and creates a diffusion barrier which differentiate axon and somatodendriticó compartment. Different structural and functional proteins that contribute to the generation of neuronal action potential areóconcentrated at the axon initial segment. While axonal elongation is controlled by signalling pathways that regulateócytoskeleton through microtubule associated proteins and tubulin modifications, the microtubule cytoskeleton under theóAIS is mostly unknown. Thus, understanding which proteins modify tubulin, where in the neuron and at whichódevelopmental stage is crucial to understanding how morphological and functional neuronal polarity is achieved. In thisóstudy performed in mice and using a well established model of murine cultured hippocampal neurons, we report that theótubulin deacetylase HDAC6 is localized at the distal region of the axon, and its inhibition with TSA or tubacin slows downóaxonal growth. Suppression of HDAC6 expression with HDAC6 shRNAs or expression of a non-active mutant of HDAC6 alsoóreduces axonal length. Furthermore, HDAC6 inhibition or suppression avoids the concentration of ankyrinG and sodiumóchannels at the axon initial segment (AIS). Moreover, treatment of mouse cultured hippocampal neurons with detergents toóeliminate the soluble pool of microtubules identified a pool of detergent resistant acetylated microtubules at the AIS, notópresent at the rest of the axon. Inhibition or suppression of HDAC6 increases acetylation all along the axon and disrupts theóspecificity of AIS cytoskeleton, modifying the axonal distal gradient localization of KIF5C to a somatodendritic and axonalólocalization. In conclusion, our results reveal a new role of HDAC6 tubulin deacetylase as a regulator of microtubuleócharacteristics in the axon distal region where axonal elongation takes place, and allowing the development of acetylatedómicrotubules microdomains where HDAC6 is not concentrated, such as the axon initial segment. © 2010 Tapia et al.

The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking) shows a continuous relationship between frequency and stimulation current (f-Istim) and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking) shows a discontinuous f-Istim relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model). In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane., Author Summary All activity of the brain is manifested in electrical oscillatory patterns, shaped by the firing dynamics of the many neurons forming the brain networks. The underlying mechanisms of the firing pattern in the single neurons are still not fully understood. The distribution and identity of different channel types have been suggested as critical factors. We have suggested that the density of channels in the membrane is a fundamental complementary mechanism. In a hippocampal soma membrane model study we have shown that altering the ion channel densities can cause the membrane to switch between two qualitatively different firing patterns. Here we extend the analysis to two axon membranes. Unexpectedly, both show that channel density alterations do not cause switches between different firing behaviours. We believe that this is an important property of axon membranes, explaining their limited flexibility.

The ability of neurons to form a single axon and multiple dendrites underlies the directional flow of information transfer in the central nervous system. Dendrites and axons are molecularly and functionally distinct domains. Dendrites integrate synaptic inputs, triggering the generation of action potentials at the level of the soma. Action potentials then propagate along the axon, which makes presynaptic contacts onto target cells. This article reviews what is known about the cellular and molecular mechanisms underlying the ability of neurons to initiate and extend a single axon during development. Remarkably, neurons can polarize to form a single axon, multiple dendrites, and later establish functional synaptic contacts in reductionist in vitro conditions. This approach became, and remains, the dominant model to study axon initiation and growth and has yielded the identification of many molecules that regulate axon formation in vitro ( Dotti et al. 1988). At present, only a few of the genes identified using in vitro approaches have been shown to be required for axon initiation and outgrowth in vivo. In vitro, axon initiation and elongation are largely intrinsic properties of neurons that are established in the absence of relevant extracellular cues. However, the importance of extracellular cues to axon initiation and outgrowth in vivo is emerging as a major theme in neural development ( Barnes and Polleux 2009). In this article, we focus our attention on the extracellular cues and signaling pathways required in vivo for axon initiation and axon extension.

Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca2+-signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca2+/calmodulin kinase kinase (CaMKK) and its downstream target Ca2+/calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the γ-isoform of CaMKI as shRNA to CaMKIγ, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKIγ, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKIγ as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKIγ and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKIγ, which subsequently promote axon formation.

The distal end of the axon initial segment (AIS) is the preferred site for action potential initiation in cortical pyramidal neurons because of its high Na(+) channel density. However, it is not clear why action potentials are not initiated at the proximal AIS, which has a similarly high Na(+) channel density. We found that low-threshold Na(v)1.6 and high-threshold Na(v)1.2 channels preferentially accumulate at the distal and proximal AIS, respectively, and have distinct functions in action potential initiation and backpropagation. Patch-clamp recording from the axon cut end of pyramidal neurons in the rat prefrontal cortex revealed a high density of Na(+) current and a progressive reduction in the half-activation voltage (up to 14 mV) with increasing distance from the soma at the AIS. Further modeling studies and simultaneous somatic and axonal recordings showed that distal Na(v)1.6 promotes action potential initiation, whereas proximal Na(v)1.2 promotes its backpropagation to the soma.

In most neurons, Na+channels in the axon are complemented by others localized in the soma and dendrites to ensure spike back-propagation. However, cerebellar granule cells are neurons with simplified architecture in which the dendrites are short and unbranched and a single thin ascending axon travels toward the molecular layer before bifurcating into parallel fibers. Here we show that in cerebellar granule cells, Na+channels are enriched in the axon, especially in the hillock, but almost absent from soma and dendrites. The impact of this channel distribution on neuronal electroresponsiveness was investigated by multi-compartmental modeling. Numerical simulations indicated that granule cells have a compact electrotonic structure allowing excitatory postsynaptic potentials to diffuse with little attenuation from dendrites to axon. The spike arose almost simultaneously along the whole axonal ascending branch and invaded the hillock the activation of which promoted spike back-propagation with marginal delay (

Axonal action potentials initiate the cycle of inter-neuronal, synaptic communication that is key to our understanding of nervous system functioning. The field has accumulated vast knowledge of the signature action potential waveform, firing patterns, and underlying channel properties of many cell types, but in most cases this information comes from somatic intracellular/whole-cell recordings, which necessarily measure a mixture of the currents compartmentalized in the soma, dendrites, and axon. Because the axon in many neuron types appears to be the site of lowest threshold for action potential initiation, the channel constellation in the axon is of particular interest. However, the axon is more experimentally inaccessible than the soma or dendrites. Recent studies have developed and applied single-fiber extracellular recording, direct intracellular recording, and optical recording techniques from axons toward understanding the behavior of the axonal action potential. We are starting to understand better how specific channels and other cellular properties shape action potential threshold, waveform, and timing: key elements contributing to downstream transmitter release. From this increased scrutiny emerges a theme of axons with more computational power than in traditional conceptualizations.

Transient receptor potential channels (TRPC) are plasma membrane, nonselective cationic channels and have been proposed as candidates involved in the regulation of cellular Ca2+ influx [D.E. Clapham, L.W. Runnels, C., Strubing, The TRP ion channel family, Nat. Rev. Neurosci. 2 (2001) 387-396; A. Martorana, C. Giampa, Z. DeMarch, M.T. Viscomi, S. Patassini, G. Sancesario, G. Bernardi, F.R. Fusco, Distribution of TRPC1 receptors in dendrites of rat substantia nigra: a confocal and electron microscopy study, Eur. J. Neurosci. 24 (2006) 732-738]. Studies on regional localization patterns of TRPCs are necessary to provide helpful guidelines for correlating current types with particular channels. In this study, we examined the distribution of one particular member of TRPC superfamily, namely, TRPC6, in the substantia nigra of normal rat brain. Single and double label immunohistochemistry were employed to perform both light and confocal microscopy observations. Our single label studies showed that, in the substantia nigra, TRPC6 labeled the perikarya with a diffuse and intense immunoreaction product distributed throughout cell cytoplasm whereas only a light immunostaining was observed in the cell nuclei. No labeling of axon or terminals was observed, although TRPC6 was evenly distributed in the neuropil. Our dual label studies showed a TRPC6 immunoreactivity pattern that was localized into the proximal dendrites and axon hillock of the large dopaminergic neurons identified by TH immunoreaction. Furthermore, our double label immunofluorescence study for TRPC6 and mGluR1 showed a complete co-localization of the two markers in the substantia nigra. Moreover, TRPC6 did not co-localize with synaptophysin. Thus, our study shows the postsynaptic localization of TRPC6 and its association with mGluR1 in the midbrain dopamine neurons.

SummaryAxon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

A mature neuron is typically polarized both structurally and functionally, with a single long axon and several dendrites. Neuronal polarity is essential for unidirectional signal flow from somata or dendrites to axons. The initial event in establishing a polarized neuron is the specification of a single axon. Early in neuronal development, one immature neurite becomes differentiated from other neurites to form an axon. Although studies in the past two decades have yielded a catalog of structural, molecular, and functional differences between axons and dendrites, we are only now beginning to understand the molecular mechanisms involved in the establishment of neuronal polarity.In the last few years, neuronal polarity-regulating molecules have been revealed. There are two major signaling cascades in neuronal polarization. Several groups, including ours, reported that the phosphatidylinositol 3-kinase (PI3-kinase)/Akt/glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein-2 pathway is important for axon specification and elongation. Recent studies have revealed that the positive feedback loop composed of Rho family small GTPases and the Par3/Par6/atypical protein kinase C complex plays a role in the initial events of neuronal polarization downstream of PI3-kinase. Here, we discuss the roles of signaling molecules for axon specification.

Background Messenger RNA (mRNA) comprises three major parts: a 5'-UTR (UnTranslated Region), a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g.) neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs) found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region) in the 3'-UTR. Methods We compared the cis-acting signal of Tau with mRNAs in the axonal regions of neurons using the ClustalW program for alignment of sequences and the Mfold program for analysis of secondary structures. Results We found that at least 3 out of 12 mRNA analyzed (GRP75, cofilin and synuclein) have a sequence similar to the cis-acting signal of Tau in the 3'-UTR. This could indicate that these messengers are localized specifically in the axon. The Mfold program showed that these mRNAs have a similar "bubble" structure in the putative sequence signal. Conclusion Hence, we suggest that a U-rich sequence in the 3'-UTR region of the mRNA could act as a signal for its localization in the axon in neuronal cells. Sequences homologous to the DTE sequence of BC1 mRNA could direct the messenger to the dendrites. Messengers with homologues of both types of sequence, e.g. β-actin, might be located in both dendrites and axon.

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence time-lapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth.

Voltage-gated Na+ channels play a major role in the generation and propagation of action potentials in central neurones. Their properties and distribution over the excitable membrane are essential factors that determine the shape of the individual action potential as well as the pattern of complex firing behaviour. It has been shown that most Na+ channels are located in the axon hillock and axon initial segment, whereas their density in the soma is considerably lower (Catterall, 1981; Boudier et al. 1985; Wollner & Catterall, 1986; Angelides et al. 1988; Safronov et al. 1997). Increased density of Na+ channels in the axonal membrane lowers the threshold at which action potentials occur, making the axon initial segment the favourable site for action potential initiation (Coombs et al. 1957a,b; Stuart & Sakmann, 1994; Safronov et al. 1997). A number of studies have indicated that the process of neuronal maturation is accompanied by an increase in total Na+ conductance (Spitzer, 1979; O'Dowd et al. 1988; Huguenard et al. 1988; McCobb et al. 1990). It has also been shown that complex forms of electrophysiological activity appear with the growth and morphological differentiation of neurites possessing active ionic conductances (Gruol & Franklin, 1987). In addition, molecular biology studies have revealed a striking change in the expression of different Na+ channel mRNAs in the rat brain occurring within the first month of postnatal development (Beckh et al. 1989). Unfortunately, still very little is known about the time course and spatial pattern of Na+ channel expression in the intact neurone during the period when most developmental changes in membrane excitability occur. Such a problem, however, can be solved by applying the ‘entire soma isolation’ (ESI) method, which was recently developed for studying the distribution of Na+ and K+ channels in intact neurones (Safronov et al. 1997; Wolff et al. 1998). The ESI method has allowed an estimation of the partial contributions of somatic and axonal channels to the total Na+ current of a whole neurone. It was shown that only 1/7 of all Na+ channels in a spinal dorsal horn neurone are present in the soma, whereas the majority are located in the axon initial segment. In this study the ESI method was further employed to examine the changes in proportions of somatic and axonal Na+ currents in dorsal horn neurones during postnatal development. Our previous ESI experiments performed using 2- to 9-day-old rats (in most cases 3- to 6-day-old rats) have also revealed that the soma of dorsal horn neurones (with no axon) is not able to generate action potentials, showing only passive and local responses (Safronov et al. 1997; Wolff et al. 1998). The concept of a non-excitable soma, however, does not account for changes in neuronal excitability in the early stages of postnatal development. For example, the possibility cannot be excluded that at birth the neurones with poorly developed axons and dendrites generate simple somatic spikes. On the other hand, a possible increase in the density of Na+ channels in the somatic membrane during cell maturation might finally enable the soma to generate spikes. Therefore, further investigation of somatic excitability at different stages of postnatal development is required. In this paper, ESI experiments were performed on spinal neurones to study the expression of Na+ channels and changes in soma excitability during the first postnatal weeks (days 0-39). It was found that the total Na+ current of a neurone progressively increases due to expression of new Na+ channels in the axonal but not the somatic membrane. The isolated somata of neurones did not generate action potentials at any stage of development studied (days 0, 3-7, 15, 21, 32 and 39). Intact dorsal horn neurones from birth to maturation therefore appear to generate action potentials of axonal origin.

Simultaneous recordings from the soma and apical dendrite of layer V neocortical pyramidal cells of young rats show that, for any location of current input, an evoked action potential (AP) always starts at the axon and then propagates actively, but decrementally, backward into the dendrites. This back-propagating AP is supported by a low density (-gNa = approximately 4 mS/cm2) of rapidly inactivating voltage-dependent Na+ channels in the soma and the apical dendrite. Investigation of detailed, biophysically constrained, models of reconstructed pyramidal cells shows the following. (i) The initiation of the AP first in the axon cannot be explained solely by morphological considerations; the axon must be more excitable than the soma and dendrites. (ii) The minimal Na+ channel density in the axon that fully accounts for the experimental results is about 20-times that of the soma. If -gNa in the axon hillock and initial segment is the same as in the soma [as recently suggested by Colbert and Johnston [Colbert, C. M. & Johnston, D. (1995) Soc. Neurosci. Abstr. 21, 684.2]], then -gNa in the more distal axonal regions is required to be about 40-times that of the soma. (iii) A backward propagating AP in weakly excitable dendrites can be modulated in a graded manner by background synaptic activity. The functional role of weakly excitable dendrites and a more excitable axon for forward synaptic integration and for backward, global, communication between the axon and the dendrites is discussed.

The spontaneous activity of first-order neurons (neuroreceptors of the mucosa) and second-order neurons (mitral cells of the bulb) was recorded extracellularly in the frog olfactory system. To assess the influence of peripheral inputs upon mitral cells, the bulb was either normally connected or partially deafferented. Our first set of findings concern the firing behavior. We found that most neurons generated interspike intervals (ISIs) that were stationary in mean and variance, and were not serially correlated at first and second order. Individual spikes in mitral cells and bursts of spikes in neuroreceptors were found to be generated by a Poisson process. Stochastic modeling suggests that the Poissonian behavior depends on the mean value of the membrane potential at the axon hillock. In these models, the mean potential in mitral cells would be far below the firing threshold and in neuroreceptors it would fluctuate at random between two states, one close to resting potential (between bursts) and the other close to the firing threshold with occasional crossings (within bursts). Secondly, partially deafferented mitral cells had significantly higher activity and lower variance than mitral cells receiving normal afferent input. This effect gives evidence that peripheral inputs influence mitral cells at rest not only through direct excitation but also through indirect inhibition exerted by local neurons. Thus, the unstimulated state of the olfactory bulb would not be qualitatively different from its stimulated state in the sense that both states involve the same types of synaptic interactions. Consequently, understanding the synaptic relationships that take place in the bulb network can benefit from studies of its spontaneous activity.

Understanding the mechanisms and dynamics of extracellular neural stimulation is very important for the development of electro-neural interfaces in general, and for the design of stimulation waveforms and electrode configurations for neural prosthetic implants, in particular. The most common type of neural stimulation is extracellular, and yet its mechanisms and dynamics have scarcely been explored and described. Its strength-duration relationship is often assumed to be similar to the classical intracellular dependence, with a slope asymptotically approaching 1/τ at pulse durations τ shorter than chronaxy. The current study explores the basic mechanisms of extracellular stimulation and derives its strength-duration curve in a wide range of stimulus durations for various waveforms and cell shapes. We used two different models of active membrane properties: the Hodgkin-Huxley model of the squid giant axon [1], and a six-channel salamander retinal ganglion cell model [2]. Three cell geometries were analyzed: an idealized planar cell with two uniformly polarized flat surfaces, and more realistic spherical and cylindrical shapes corresponding to the soma and unmyelinated axon or axon hillock. The strength-duration relationship was found to differ significantly from classical intracellular models. For the Hodgkin-Huxley model at pulse durations between 4 μs and 5 ms stimulation is dominated by sodium channels, and has a slope of approximately –0.72 in log-log coordinates. At shorter durations it is dominated by the potassium channels, and has a much lower slope of about –0.13. With pulses shorter than cell polarization time (typically about 0.1-1 μs), it is dominated by polarization dynamics, and asymptotically approaches the classical –1 slope. For retinal ganglion cells we demonstrate that extracellular stimulation can have not only lower but also upper thresholds, and may be impossible below certain pulse durations. For both cell models we have found that in some stimulation regimes the stimulus can hyperpolarize cells, suppressing rather than stimulating spiking behavior. Thresholds for burst stimuli can be either higher or lower than that of a single pulse, depending on pulse duration. The modeled thresholds were found to be comparable to published experimental data obtained with rabbit retinal ganglion cells. These results provide a biophysical basis for understanding stimulation dynamics, and guidance for optimizing the efficacy and safety of extracellular neural stimulation.

Early electrophysiological studies in the mammalian hippocampus reported that orthodromic depolarization of pyramidal cells evoked action potential discharge (presumed Na+ dependent) both at the axon hillock and at one or more sites in the dendritic arborization (Cragg and Hamlyn, 1955; Andersen, 1959, 1960; Spencer and Kandel, 1961; Andersen and Lomo, 1966). Although tetrodotoxin (TTX)-sensitive spikes have been recorded at the dendritic level (Wong et al., 1979; Benardo et al., 1982; Miyakawa and Kato, 1986; Turner et al., 1989), the site for initiation of these potentials has not yet been determined. In this study, we examine the site for initiation of Na+ spike discharge over the cell axis of rat hippocampal CA1 pyramidal neurons. Intrasomatic and intradendritic recordings were obtained from pyramidal neurons of hippocampal slices maintained in vitro. Spike discharge was evoked by alvear (antidromic) stimulation or orthodromically by stimulation of afferent inputs in stratum oriens (SO) or stratum radiatum (SR). Antidromic and orthodromic spikes were greatest in amplitude in somatic recordings and declined over the apical dendritic axis, while spike half-width was shortest at the cell body and increased with distance from stratum pyramidale. Measurements of orthodromic spike threshold revealed that the only location at which spikes discharged at a consistent membrane potential at threshold intensity (voltage threshold) was the cell body region. Finally, at threshold intensity, SR-evoked intradendritic spikes were blocked by local application of TTX in stratum pyramidale, while spike blockade at suprathreshold intensity required the diffusion of TTX into the apical dendritic region. These results indicate that, for threshold intensities of stimulation, antidromic and orthodromic spike discharge in CA1 pyramidal cells is initiated in the region of the cell body layer, subsequently conducting over the apical dendrites in a retrograde fashion. In contrast, SR-evoked orthodromic spike discharge exhibits an intensity-dependent shift in the site of origin up to 200 microns within the apical dendritic arborization.

The axon of the pyramidal neuron in the cerebral cortex arises either directly from the perikaryon or as a branch from a basal dendrite. When it arises from the perikaryon, an axon hillock is present. The hillock is a region in which there is a transition between the cytological features of the perikaryon and those of the initial segment of the axon. Thus, in the hillock there is a diminution in the number of ribosomes and a beginning of the fasciculation of microtubules that characterize the initial segment. Not all of the microtubules entering the hillock from the perikaryon continue into the initial segment. Distally, the axon hillock ends where the dense undercoating of the plasma membrane of the initial segment commences. Dense material also appears in the extracellular space surrounding the initial segment. The initial segment of the pyramidal cell axon contains a cisternal organelle consisting of stacks of flattened cisternae alternating with plates of dense granular material. These cisternal organelles resemble the spine apparatuses that occur in the dendritic spines of this same neuron. Axo-axonal synapses are formed between the initial segment and surrounding axon terminals. The axon terminals contain clear synaptic vesicles and, at the synaptic junctions, both synaptic complexes and puncta adhaerentia are present.

In fifty-seven resting nerve fibers of the squid Sepioteuthis sepioidea impaled from outside to inside, three potential difference (PD) levels were recorded. In twelve nerve fibers the position of the tip of the micropipette, when it was recording one of the PD levels, was labeled with carmine, and the minute spot of dye was localized histologically. The first PD level of -10 to -26 mv, was located in the endoneurium cells; the second level, formed by a single PD of -33 to -46 mv, was located in the Schwann cell; and the third level, formed by a single PD of -50 to -65 mv, was located in the axon. In sixteen nerve fibers the independence among these PD levels was demonstrated as follows. One micropipette was inserted inside the axon for recording its PD; a second one was inserted inside one endoneurium cell or inside one Schwann cell for recording the PD; and a third micropipette was inserted inside the axon to supply current to depolarize the axon. When the axon was depolarized no significant change was observed in the PD of either the endoneurium cell or the Schwann cell. In all nerve fibers action potentials were registered from the axon only. This identifies the axolemma (axon plasma membrane) as the excitable membrane.

SummaryAction potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 μm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.

SummaryDeveloping neurons can change axonal and dendritic fate upon axonal lesion [1–6], but it is unclear whether neurons retain such plasticity when they are synaptically interconnected [7, 8]. To address whether polarity is reversible in mature neurons, we cut the axon of GFP-labeled hippocampal neurons in dissociated and organotypic cultures and found that a new axon arose from a mature dendrite. The regenerative response correlated with the length of the remaining stump: proximal axotomies (35 μm) induced axon regrowth, similar to what is seen in young neurons [3]. Searching for a putative landmark in the distal axon that could determine axon identity, we focused on the stability of microtubules, which regulate initial neuronal polarization during early development [9]. We found that functionally polarized neurons contain a distinctively high proportion of stable microtubules in the distal axon. Moreover, pharmacological stabilization of microtubules was sufficient to induce the formation of multiple axons out of differentiated dendrites. Our data argue that mature neurons integrated in functional networks remain flexible in their polarity and that mechanisms acting during initial axon selection can be reactivated to induce axon growth out of functionally mature dendrites.

Ca2+ release from the endoplasmic reticulum (ER) contributes to Ca2+ transients in frog sympathetic ganglion neurons. Here we use video-rate confocal fluo-4 fluorescence imaging to show that single action potentials reproducibly trigger rapidly rising Ca2+ transients at 1–3 local hot spots within the peripheral ER-rich layer in intact neurons in fresh ganglia and in the majority (74%) of cultured neurons. Hot spots were located near the nucleus or the axon hillock region. Other regions exhibited either slower and smaller signals or no response. Ca2+ signals spread into the cell at constant velocity across the ER in nonnuclear regions, indicating active propagation, but spread with a (time)1/2 dependence within the nucleus, consistent with diffusion. 26% of cultured cells exhibited uniform Ca2+ signals around the periphery, but hot spots were produced by loading the cytosol with EGTA or by bathing such cells in low-Ca2+ Ringer's solution. Peripheral hot spots for Ca2+ release within the perinuclear and axon hillock regions provide a mechanism for preferential initiation of nuclear and axonal Ca2+ signals by single action potentials in sympathetic ganglion neurons.

SummarySemaphorin 3A (Sema3A) is a secreted factor known to guide axon/dendrite growth and neuronal migration. We found that it also acts as a polarizing factor for axon/dendrite development in cultured hippocampal neurons. Exposure of the undifferentiated neurite to localized Sema3A suppressed its differentiation into axon and promoted dendrite formation, resulting in axon formation away from the Sema3A source, and bath application of Sema3A to polarized neurons promoted dendrite growth but suppressed axon growth. Fluorescence resonance energy transfer (FRET) imaging showed that Sema3A elevated the cGMP but reduced cAMP and protein kinase A (PKA) activity, and its axon suppression is attributed to the downregulation of PKA-dependent phosphorylation of axon determinants LKB1 and GSK-3β. Downregulating Sema3A signaling in rat embryonic cortical progenitors via in utero electroporation of siRNAs against the Sema3A receptor neuropilin-1 also resulted in polarization defects in vivo. Thus, Sema3A regulates the earliest step of neuronal morphogenesis by polarizing axon/dendrite formation.