Axonal Segments Research Articles

Cell type‐specific and activity‐dependent dynamics of action potential‐evoked Ca2+ signals in dendrites of hippocampal inhibitory interneurons

In most central neurons, action potentials (APs), generated in the initial axon segment, propagate back into dendrites and trigger considerable Ca(2+) entry via activation of voltage-sensitive calcium channels (VSCCs). Despite the similarity in its underlying mechanisms, however, AP-evoked dendritic Ca(2+) signalling often demonstrates a cell type-specific profile that is determined by the neuron dendritic properties. Using two-photon Ca(2+) imaging in combination with patch-clamp whole-cell recordings,we found that in distinct types of hippocampal inhibitory interneurons Ca(2+) transients evoked by backpropagating APs not only were shaped by the interneuron-specific properties of dendritic Ca(2+) handling but also involved specific Ca(2+) mechanisms that were regulated dynamically by distinct activity patterns. In dendrites of regularly spiking basket cells, AP-evoked Ca(2+) rises were of large amplitude and fast kinetics; however, they decreased with membrane hyperpolarization or following high-frequency firing episodes. In contrast, AP-evoked Ca(2+) elevations in dendrites of Schaffer collateral-associated cells exhibited significantly smaller amplitude and slower kinetics, but increased with membrane hyperpolarization. These cell type-specific properties of AP-evoked dendritic Ca(2+) signalling were determined by distinct endogenous buffer capacities of the interneurons examined and by specific types of VSCCs recruited by APs during different patterns of activity. Furthermore, AP-evoked Ca(2+) transients summated efficiently during theta-like bursting and were associated with the induction of long-term potentiation at inhibitory synapses onto both types of interneurons. Therefore, the cell type-specific profile of AP-evoked dendritic Ca(2+) signalling is shaped in an activity-dependent manner, such that the same pattern of hippocampal activity can be differentially translated into dendritic Ca(2+) signals in different cell types. However, Cell type-specific differences in Ca(2+) signals can be 'smoothed out' by changes in neuronal activity, providing a means for common, cell-type-independent forms of synaptic plasticity.

Diffuse Traumatic Axonal Injury in the Mouse Induces Atrophy, c-Jun Activation, and Axonal Outgrowth in the Axotomized Neuronal Population

Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI) and is associated with much of its morbidity. Little is known regarding the long-term retrograde neuronal consequences of TAI and/or the potential that TAI could lead to anterograde axonal reorganization and repair. To investigate the repertoire of anterograde and retrograde responses triggered by TIA, Thy1-YFP-H mice were subjected to mild central fluid percussion injury and killed at various times between 15 min and 28 d post-injury. Based upon confocal assessment of the endogenous neuronal fluorescence, such injury was found to result in diffuse TAI throughout layer V of the neocortex within yellow fluorescent protein (YFP)-positive axons. When these fluorescent approaches were coupled with various quantitative and immunohistochemical approaches, we found that this TAI did not result in neuronal death over the 28 d period assessed. Rather, it elicited neuronal atrophy. Within these same axotomized neuronal populations, TAI was also found to induce an early and sustained activation of the transcription factors c-Jun and ATF-3 (activating transcription factor 3), known regulators of axon regeneration. Parallel ultrastructural studies confirmed that these reactive changes are consistent with atrophy in the absence of neuronal death. Concurrent with those events ongoing in the neuronal cell bodies, their downstream axonal segments revealed, as early as 1 d post-injury, morphological changes consistent with reactive sprouting that was accompanied by significant axonal elongation over time. Collectively, these TAI-linked events are consistent with sustained neuronal recovery, an activation of a regenerative genetic program, and subsequent axonal reorganization suggestive of some form of regenerative response.

Rescue of neurons from undergoing hallmark tau-induced Alzheimer's disease cell pathologies by the antimitotic drug paclitaxel

Through the use of live confocal imaging, electron microscopy, and the novel cell biological platform of cultured Aplysia neurons we show that unfolding of the hallmark cell pathologies induced by mutant-human-tau (mt-human-tau) expression is rescued by 10 nM paclitaxel. At this concentration paclitaxel prevents mt-human-tau-induced swelling of axonal segments, translocation of tau and microtubules (MT) to submembrane domains, reduction in the number of MTs along the axon, reversal of the MT polar orientation, impaired organelle transport, accumulation of macro-autophagosomes and lysosomes, compromised neurite morphology and degeneration. Unexpectedly, higher paclitaxel concentrations (100 nM) do not prevent these events from occurring and in fact facilitate them. We conclude that antimitotic MT-stabilizing reagents have the potential to serve as drugs to prevent or slow down the unfolding of tauopathies.

Intracellular distribution and axonal transport of PKD in neurons

Event Abstract Back to Event Intracellular distribution and axonal transport of PKD in neurons T. Szórádi1*, F. Klacsmann1, F. Vajda1, K. Tárnok1 and K. Schlett1 1 Eötvös Loránd University, Department of Physiology and Neurobiology, Hungary Protein Kinase D (PKD) is a serine/threonine kinase, known to regulate dendritic maintenance and secretory transport in neurons. PKD-regulated axonal events, however, are not identified yet. In this study we quantitatively examined the intracellular distribution of EGFP-tagged wild type, point or deletion mutants of PKD in transfected primary hippocampal neurons. While the majority of the investigated mutants were evenly distributed, dominant-negative kinase-inactive form of PKD1 or a construct lacking the kinase domain were found to be excluded from the axons and restricted to the somatodendritic compartment. Similar uneven intracellular localization was observed when PKD1 mutants lacking the PH (Pleckstrin homology) domain or amino acids 80-146 were used. These findings suggested that beside kinase activity, the N terminal region also regulates PKD’s axonal presence. In order to identify the potential transport differences responsible for the altered intracellular distribution of the mutant PKD proteins, live cell recordings were made from transfected axonal segments. In several cases, vesicle-like transportation of EGFP-tagged PKD mutants was observed. Detailed analysis of the dynamic properties (e.g. cumulative distance, straight distance, mean velocity) of the fluorescent vesicle-like structures is in progress and will help to clarify the regulation of PKD’s intracellular distribution. This work will help to elucidate the potential role of PKD in axons, as well. Keywords: Molecular and cellular neurobiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Molecular and cellular neurobiology Citation: Szórádi T, Klacsmann F, Vajda F, Tárnok K and Schlett K (2011). Intracellular distribution and axonal transport of PKD in neurons. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00049 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. T. Szórádi, Eötvös Loránd University, Department of Physiology and Neurobiology, Budapest, Hungary, szoradi.tamas@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers T. Szórádi F. Klacsmann F. Vajda K. Tárnok K. Schlett Google T. Szórádi F. Klacsmann F. Vajda K. Tárnok K. Schlett Google Scholar T. Szórádi F. Klacsmann F. Vajda K. Tárnok K. Schlett PubMed T. Szórádi F. Klacsmann F. Vajda K. Tárnok K. Schlett Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The spike initiation in neurons with low input resistance

Event Abstract Back to Event The spike initiation in neurons with low input resistance Simon Lehnert1* and Christian Leibold1, 2 1 Ludwig-Maximilians-Universität München, Department of Biology II, Germany 2 Bernstein Center for Computational Neuroscience Munich, Germany Spike initiation is generally assumed to occur in the axon's initial segment (AIS). In neurons with very low input resistance, however, this may no longer hold true, because the soma constitutes a huge current sink. As an example, we consider the principal cells of the medial superior olive (MSO). These neurons are very fast coincidence detectors in the auditory brainstem that encode low frequency azimuthal sound localization by interaural time differences. Their enormously low time constant of only a few hundred microseconds yields from a very low input resistance of about 5MΩ at rest [1], which arises from the expression of low-voltage-activated potassium channels and hyperpolarization-activated unspecific cation channels. Since these cells receive a huge amount of slowly-decaying inhibition [1], it is likely that their input resistance in vivo is even much smaller than at rest. Hence the question arises: how are these cells able to elicit action potentials (AP) at all? By using a multi-compartemental model of an MSO cell, we found that the spike initiation segment (SIS) in the model is still the AIS. The reason for this is the electrotonic independence of the AIS from the leaky soma. The input resistance of the axonal segments is considerably higher than that of the soma and hence an input current is able to evoke an AP in the AIS, while an AP in the soma is hardly distinguishable from a subthreshold response. We conclude, that the excitability of the axon enables the cell to convey information downstream, although the soma itself is not excitable. Thus, neurons with low input resistance cannot be realistically modeled using only a single compartment.

Automated kymograph analysis for profiling axonal transport of secretory granules

This paper describes an automated method to profile the velocity patterns of small organelles (BDNF granules) being transported along a selected section of axon of a cultured neuron imaged by time-lapse fluorescence microscopy. Instead of directly detecting the granules as in conventional tracking, the proposed method starts by generating a two-dimensional spatio-temporal map (kymograph) of the granule traffic along an axon segment. Temporal sharpening during the kymograph creation helps to highlight granule movements while suppressing clutter due to stationary granules. A voting algorithm defined over orientation distribution functions is used to refine the locations and velocities of the granules. The refined kymograph is analyzed using an algorithm inspired from the minimum set cover framework to generate multiple motion trajectories of granule transport paths. The proposed method is computationally efficient, robust to significant levels of noise and clutter, and can be used to capture and quantify trends in transport patterns quickly and accurately. When evaluated on a collection of image sequences, the proposed method was found to detect granule movement events with 94% recall rate and 82% precision compared to a time-consuming manual analysis. Further, we present a study to evaluate the efficacy of velocity profiling by analyzing the impact of oxidative stress on granule transport in which the fully automated analysis correctly reproduced the biological conclusion generated by manual analysis.

Differential Subcellular Recruitment of Monoacylglycerol Lipase Generates Spatial Specificity of 2-Arachidonoyl Glycerol Signaling during Axonal Pathfinding

Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB(1) cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGLα/β) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGLα and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGLα undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB(1)R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.

The specific origin of the simple and complex spikes in Purkinje neurons

The specific origin of the simple and complex spikes in Purkinje neurons

APC/CFzr/Cdh1-dependent regulation of cell adhesion controls glial migration in the Drosophila PNS

Interactions between neurons and glia are a key feature during the assembly of the nervous system. During development, glial cells often follow extending axons, implying that axonal outgrowth and glial migration are precisely coordinated. We found that the anaphase-promoting complex/cyclosome (APC/C) co-activator fizzy-related/Cdh1 (Fzr/Cdh1) is involved in the non-autonomous control of peripheral glial migration in postmitotic Drosophila neurons. APC/C(Fzr/Cdh1) is a cell-cycle regulator that targets proteins that are required for G1 arrest for ubiquitination and subsequent degradation. We found that Fzr/Cdh1 function is mediated by the immunoglobulin superfamily cell adhesion molecule Fasciclin2 (Fas2). In motor neurons Fzr/Cdh1 is crucial for the establishment of a graded axonal distribution of Fas2. Axonal Fas2 interacts homophilically with a glial isoform of Fas2. Glial migration is initiated along axonal segments that have low levels of Fas2 but stalls in axonal domains with high levels of Fas2 on their surfaces. This represents a simple mechanism by which a subcellular gradient of adhesiveness can coordinate glial migration with axonal growth.

Transcriptional profiling of the injured sciatic nerve of mice carrying the Wld(S) mutant gene: Identification of genes involved in neuroprotection, neuroinflammation, and nerve regeneration

Wallerian degeneration (WD) involves the fragmentation of axonal segments disconnected from their cell bodies, segmentation of the myelin sheath, and removal of debris by Schwann cells and immune cells. The removal and downregulation of myelin-associated inhibitors of axonal regeneration and synthesis of growth factors by these two cell types are critical responses to successful nerve repair. Here, we analyzed the transcriptome of the sciatic nerve of mice carrying the Wallerian degeneration slow (Wld S) mutant gene, a gene that confers axonal protection in the distal stump after injury, therefore causing significant delays in WD, neuroinflammation, and axonal regeneration. Of the thousands of genes analyzed by microarray, 719 transcripts were differentially expressed between Wld S and wild-type (wt) mice. Notably, the Nmnat1, a transcript contained within the sequence of the Wld S gene, was upregulated by five to eightfold in the sciatic nerve of naive Wld S mice compared with wt. The injured sciatic nerve of wt could be further distinguished from the one of Wld S mice by the preferential upregulation of genes involved in axonal processes and plasticity (Chl1, Epha5, Gadd45b, Jun, Nav2, Nptx1, Nrcam, Ntm, Sema4f), inflammation and immunity (Arg1, Lgals3, Megf10, Panx1), growth factors/cytokines and their receptors (Clcf1, Fgf5, Gdnf, Gfrα1, Il7r, Lif, Ngfr/p75 NTR, Shh), and cell adhesion and extracellular matrix (Adam8, Gpc1, Mmp9, Tnc). These results will help understand how the nervous and immune systems interact to modulate nerve repair, and identify the molecules that drive these responses.

Distal to proximal development of peripheral nerves requires the expression of neurofilament heavy

At the initiation of radial growth, neurofilaments are likely to consist primarily of neurofilament light and medium as neurofilament heavy expression is developmentally delayed. To better understand the role of neurofilament heavy in structuring axons, axonal diameter and neurofilament organization were measured in proximal and distal segments of the sciatic nerve and along the entire length of the phrenic nerve. Deletion of neurofilament heavy reduced axonal diameters and neurofilament number in proximal nerve segments. However, neurofilament spacing was greater in proximal versus distal phrenic nerve segments. Taken together, these results suggest that loss of neurofilament heavy reduces radial growth in proximal axonal segments by reducing the accumulation of neurofilaments. As neurofilament heavy expression is developmentally delayed, these results suggest that without neurofilament heavy, the neurofilament network is established in a distal to proximal gradient perhaps to allow distal axonal segments to develop prior to proximal segments.

Trajectory and terminal distribution of single centrifugal axons from olfactory cortical areas in the rat olfactory bulb

The olfactory bulb receives a large number of centrifugal fibers whose functions remain unclear. To gain insight into the function of the bulbar centrifugal system, the morphology of individual centrifugal axons from olfactory cortical areas was examined in detail. An anterograde tracer, Phaseolus vulgaris leucoagglutinin, was injected into rat olfactory cortical areas, including the pars lateralis of the anterior olfactory nucleus (lAON) and the anterior part of the piriform cortex (aPC). Reconstruction from serial sections revealed that the extrabulbar segments of centrifugal axons from the lAON and those from the aPC had distinct trajectories: the former tended to innervate the pars externa of the AON before entering the olfactory bulb, while the latter had extrabulbar collaterals that extended to a variety of targets. In contrast to the extrabulbar segments, no clear differences were found between the intrabulbar segments of axons from the lAON and from the aPC. The intrabulbar segments of centrifugal axons were mainly found in the granule cell layer but a few axons extended into the external plexiform and glomerular layer. Approximately 40% of centrifugal axons innervated both the medial and lateral aspects of the olfactory bulb. The number of boutons found on single intrabulbar segments was typically less than 1000. Boutons tended to aggregate and form complex terminal tufts with short axonal branches. Terminal tufts, no more than 10 in single axons from ipsilateral cortical areas, were localized to the granule cell layer with varying intervals; some tufts formed patchy clusters and others were scattered over areas that extended for a few millimeters. The patchy, widespread distribution of terminals suggests that the centrifugal axons are able to couple the activity of specific subsets of bulbar neurons even when the subsets are spatially separated.

Distribution of perineuronal nets in the human superior olivary complex

Perineuronal nets (PNNs) are specialized assemblies of chondroitin sulfate proteoglycans (CSPGs) in the central nervous system that form a lattice-like covering over the cell body, primary dendrites and initial axon segment of select neuronal populations. PNNs appear to play significant roles in development of the central nervous system, neuronal protection, synaptic plasticity and local ion homeostasis. In seven human brainstems (average age = 81 years), we have utilized Wisteria floribunda (WFA) histochemistry and immunocytochemistry for CSPG to map the distribution of PNNs within the nuclei of the human superior olivary complex (SOC). Within the SOC, the majority of net-bearing neurons are situated in the most medially situated nuclei, especially the superior paraolivary nucleus and medial nucleus of the trapezoid body. Net-bearing neurons are consistently found in the ventral nucleus of the trapezoid body and posterior periolivary nucleus, but to a lesser extent in the lateral nucleus of the trapezoid body. Finally, perineuronal nets are typically absent from the lateral and medial superior olives.

Conduction Block in PMP22 Deficiency

Patients with PMP22 deficiency present with focal sensory and motor deficits when peripheral nerves are stressed by mechanical force. It has been hypothesized that these focal deficits are due to mechanically induced conduction block (CB). To test this hypothesis, we induced 60-70% CB (defined by electrophysiological criteria) by nerve compression in an authentic mouse model of hereditary neuropathy with liability to pressure palsies (HNPP) with an inactivation of one of the two pmp22 alleles (pmp22(+/-)). Induction time for the CB was significantly shorter in pmp22(+/-) mice than that in pmp22(+/+) mice. This shortened induction was also found in myelin-associated glycoprotein knock-out mice, but not in the mice with deficiency of myelin protein zero, a major structural protein of compact myelin. Pmp22(+/-) nerves showed intact tomacula with no segmental demyelination in both noncompressed and compressed conditions, normal molecular architecture, and normal concentration of voltage-gated sodium channels by [(3)H]-saxitoxin binding assay. However, focal constrictions were observed in the axonal segments enclosed by tomacula, a pathological hallmark of HNPP. The constricted axons increase axial resistance to action potential propagation, which may hasten the induction of CB in Pmp22 deficiency. Together, these results demonstrate that a function of Pmp22 is to protect the nerve from mechanical injury.

Persistent reflection underlies ectopic activity in multiple sclerosis: a numerical study

Ectopic activity in multiple sclerosis (MS) patients has been traditionally attributed to hyperexcitability of the demyelinated axon segments. Here, we propose that the same outcome may be the result of persistent reflection--the continuous reactivation of the axonal nodes that limit a demyelinated internodal segment. Using computer simulations, we studied the patterns of impulse propagation for a wide range of electrophysiological conditions. In uniformly myelinated fibers, increasing the temperature enabled successful propagation with no blocks in more severe conditions of demyelination. Secondary activations that were originated at the paranodes were formed for temperatures lower than T = 305 K, and at the condition of high sodium channel excitability. Non-sustained and persistent reflections appeared in the case of focally demyelinated fibers, and only within a narrow range of parameters of high temperature and membrane excitability. Persistent reflection reached steady state in ionic currents within 4 ms, and was characterized with a very high activation frequency of 1.504 (+/- 0.039 kHz. We conclude that persistent reflection is a possible mechanism for ectopic activity in MS patients, being more prominent in higher temperatures and severe axonal demyelination. Eliminating these symptoms may be addressed by cooling the body or by applying pharmacological agents to alter excitability properties.

Mechanisms and Distribution of Ion Channels in Retinal Ganglion Cells: Using Temperature as an Independent Variable

Trains of action potentials of rat and cat retinal ganglion cells (RGCs) were recorded intracellularly across a temperature range of 7-37 degrees C. Phase plots of the experimental impulse trains were precision fit using multicompartment simulations of anatomically reconstructed rat and cat RGCs. Action potential excitation was simulated with a "Five-channel model" [Na, K(delayed rectifier), Ca, K(A), and K(Ca-activated) channels] and the nonspace-clamped condition of the whole cell recording was exploited to determine the channels' distribution on the dendrites, soma, and proximal axon. At each temperature, optimal phase-plot fits for RGCs occurred with the same unique channel distribution. The "waveform" of the electrotonic current was found to be temperature dependent, which reflected the shape changes in the experimental action potentials and confirmed the channel distributions. The distributions are cell-type specific and adequate for soma and dendritic excitation with a safety margin. The highest Na-channel density was found on an axonal segment some 50-130 microm distal to the soma, as determined from the temperature-dependent "initial segment-somadendritic (IS-SD) break." The voltage dependence of the gating rate constants remains invariant between 7 and 23 degrees C and between 30 and 37 degrees C, but undergoes a transition between 23 and 30 degrees C. Both gating-kinetic and ion-permeability Q10s remain virtually constant between 23 and 37 degrees C (kinetic Q10s = 1.9-1.95; permeability Q10s = 1.49-1.64). The Q10s systematically increase for T <23 degrees C (kinetic Q10 = 8 at T = 8 degrees C). The Na channels were consistently "sleepy" (non-Arrhenius) for T <8 degrees C, with a loss of spiking for T <7 degrees C.

Effects of Actin Filaments on NGF Retrograde Transport

Actin filament is an essential component of the cell cytoskeletal system under the physiological conditions. In addition to their roles in supporting cell shape, actin filaments act as molecular tracks for myosin motors that are involved in the movement of organelles such as mitochondria in the axon. However, how actin filaments regulate axonal transport processes are yet to be fully elucidated. Nerve growth factor binds and activates its receptor located at axon terminus, which intriguers the complex to be endocytosed sorted into signaling endosomes. NGF-containing endosomes are retrogradely transported from the axon terminus to the cell body. In this study, we investigated the effects of actin filiments on axonal transport by tracking the transport of single NGF modified with the quantum dot using microfluidic device. Embryonic DRG neurons were cultured in the microfluidic nerve cell chambers made by PDMS. The microfluidic chamber allows us to apply latrunculin B, an actin de-polymerization inhibitor, exclusively to the middle segment of axon. This treatment would not affect signaling processes in the cell body or the endocytosis process that happens at the axon termini. We monitored the retrograde transport of Qdot-NGF in actin-depleted axons using TIRF microscopy. We found that NGF axonal transport continues in axons that are depleted of actin filiments, confirming previous reports that NGF transport is a microtubule-based process. However, we found that the average speed of axonal transport slowed down in Latrunculin B treated axons. Detailed analysis of why actin depolymerization affects axonal transport is still in progress.

Chandelier and Interfascicular Neurons in the Adult Mouse Piriform Cortex

The structure of two neuron types native to the adult mouse piriform cortex (PC) is described. The first cell, termed an interfascicular neuron (IFN), lies between the axon fascicles of layer I. The IFN axon divides dichotomously and daughter fibrils run horizontally in the domain of layer Ia. The frequent apposition of the IFN axon to distal dendrites of the underlying pyramidal cells suggests an en passage synaptic interaction with them. A second neuron observed in layer II, or less frequently in layer III, matched in most respects the structure of the chandelier cell (CC) described elsewhere in the neo- and archi-cortex. In the PC, chandelier cells (PC-CC) display the following peculiarities. First, the PC-CC axonal field distributes in the neuropil of layers II and III and candlesticks are in close apposition to the initial axonal segment of the pyramidal cell, although somatic interactions cannot be rule out. Second, the PC-CC ascending dendrites pierce layer I, receiving short collaterals and boutons en passage from the olfactory axons therein. The possible role of IFN's and PC-CC and their interactions with the adjacent cells is discussed in the broad context of the cellular organization of the PC.

Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection

We describe the integrated use of pulsed laser microbeam irradiation and microfluidic cell culture methods to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, lambda = 532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 mum and 50 mum in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interferes with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a micropatterned cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.

Confocal analysis of cholinergic and dopaminergic inputs onto pyramidal cells in the prefrontal cortex of rodents

Cholinergic and dopaminergic projections to the rat medial prefrontal cortex (mPFC) are both involved in cognitive functions including attention. These neuronal systems modulate mPFC neuronal activity mainly through diffuse transmission. In order to better understand the anatomical level of influence of these systems, confocal microscopy with triple-fluorescent immunolabeling was used in three subregions of the mPFC of rats and Drd1a-tdTomato/Drd2-EGFP transgenic mice. The zone of interaction was defined as a reciprocal microproximity between dopaminergic and cholinergic axonal segments as well as pyramidal neurons. The density of varicosities, along these segments was considered as a possible activity-dependant morphological feature. The percentage of cholinergic and dopaminergic fibers in microproximity ranged from 12 to 40% depending on the layer and mPFC subregion. The cholinergic system appeared to have more influence on dopaminergic fibers since a larger proportion of the dopaminergic fibers were within microproximity to cholinergic fibers. The density of both cholinergic and dopaminergic varicosities was significantly elevated within microproximities. The main results indicate that the cholinergic and dopaminergic systems converge on pyramidal cells in mPFC particularly in the layer V. In transgenic mice 93% of the pyramidal cells expressed the transgenic marker for Drd2 expression, but only 22% expressed the maker for Drd1ar expression. Data presented here suggest that the modulation of mPFC by dopaminergic fibers would be mostly inhibitory and localized at the output level whereas the cholinergic modulation would be exerted at the input and output level both through direct interaction with pyramidal cells and dopaminergic fibers.