Terminal Arborizations Research Articles

Linking cell surface receptors to microtubules: tubulin folding cofactor D mediates Dscam functions during neuronal morphogenesis.

Formation of functional neural networks requires the coordination of cell surface receptors and downstream signaling cascades, which eventually leads to dynamic remodeling of the cytoskeleton. Although a number of guidance receptors affecting actin cytoskeleton remodeling have been identified, it is relatively unknown how microtubule dynamics are regulated by guidance receptors. We used Drosophila olfactory projection neurons to study the molecular mechanisms of neuronal morphogenesis. Dendrites of each projection neuron target a single glomerulus of ∼50 glomeruli in the antennal lobe, and the axons show stereotypical pattern of terminal arborization. In the course of genetic analysis of the dachsous mutant allele (ds(UAO71)), we identified a mutation in the tubulin folding cofactor D gene (TBCD) as a background mutation. TBCD is one of five tubulin-folding cofactors required for the formation of α- and β-tubulin heterodimers. Single-cell clones of projection neurons homozygous for the TBCD mutation displayed disruption of microtubules, resulting in ectopic arborization of dendrites, and axon degeneration. Interestingly, overexpression of TBCD also resulted in microtubule disruption and ectopic dendrite arborization, suggesting that an optimum level of TBCD is crucial for in vivo neuronal morphogenesis. We further found that TBCD physically interacts with the intracellular domain of Down syndrome cell adhesion molecule (Dscam), which is important for neural development and has been implicated in Down syndrome. Genetic analyses revealed that TBCD cooperates with Dscam in vivo. Our study may offer new insights into the molecular mechanism underlying the altered neural networks in cognitive disabilities of Down syndrome.

The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations.

Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. We previously reported that axon terminal arborization in Drosophila larval neuromuscular junctions (NMJs) is enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs) remains unaltered despite the increase in synaptic bouton numbers. Here we determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29°C). We found that synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (<15°C to >30°C). However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs), which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut) encoding adenylyl cyclase (AC) exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends our previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g., slowpoke (slo). In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Shaker (Sh) IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size.

Reorganization of spinal neural circuitry and functional recovery after spinal cord injury

Reorganization of spinal neural circuitry and functional recovery after spinal cord injury

Nicotinic potentiation of frog retinotectal transmission in tectum layer F by α3β2, α4β2, α2β4, α6β2, or α7 acetylcholine receptor subtypes

ObjectiveThe aim of the study was to explore the effect of semi-specific antagonists and agonists of the nicotinic acetylcholine receptors on the paired-pulse facilitation and nicotinic tonic and phasic potentiation of the frog retinotectal synaptic transmission. Materials and methodsThe experiments were performed in vivo on adult frogs, Rana temporaria. An individual retina ganglion cell (or its retinotectal fiber) was stimulated by current pulses delivered through multichannel stimulating electrode positioned on the retina. Responses to a discharge of a single retinal ganglion cell were recorded in the tectum by an extracellular carbon-fiber microelectrode positioned in the terminal arborization of the retinotectal fiber in the tectum layer F. The effect of the antagonists and agonists of the nicotinic acetylcholine receptors on the tectal responses has been tested. ResultsWe found that the antagonists, MLA and DHβE, and agonists, RJR-2403 and choline, of the nicotinic acetylcholine receptors of the α3β2, α4β2, α2β4, α6β2 or α7 subtypes have had no effect on the phasic and tonic potentiation of the retinotectal transmission. The paired-pulse facilitation of the retinotectal transmission was not appreciably affected by the antagonists, but the choline, agonist of the α7 subtype receptor, has significantly decreased the paired-pulse facilitation. ConclusionsThe tonic and phasic potentiation of the retinotectal transmission in the tectum layer F were not mediated by the receptors of α3β2, α4β2, α2β4, α6β2 or α7 subtype. The results suggest that presynaptic nicotinic acetylcholine receptors of the frog optic fibers are different from those of the mammalian optic fibers.

The roles of tubulin-folding cofactors in neuronal morphogenesis and disease.

Microtubules play important roles in neuronal morphogenesis, including cellular polarization, neurite growth, and branching. A microtubule is a polymer of α- and β-tubulin heterodimers that are formed by a multistep process assisted by at least five tubulin-folding cofactors (TBCA–E) (Lopez-Fanarraga et al., 2001). Newly synthesized α- and β-tubulins associate with the cytosolic chaperonin complex (CCT), and then the quasi-native tubulins interact with five tubulin-folding cofactors. The α- and β-tubulins first interact with TBCB and TBCA, which are further transferred to TBCE and TBCD, respectively. The α- and β-tubulins, TBCC, TBCD, and TBCE form a supercomplex, and guanosine triphosphate (GTP) hydrolysis occurs within the supercomplex, releasing a tubulin heterodimer. In addition to their functions in tubulin folding and tubulin heterodimer formation, excess amounts of TBCD or TBCE may interact with tubulin heterodimers, leading to their degradation. Thus, tubulin-folding cofactors may play a role in both the synthesis and degradation of tubulin heterodimers.

Mutation of Npr2 leads to blurred tonotopic organization of central auditory circuits in mice.

Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.

Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

Neurotrophic factors for spinal cord repair: Which, where, how and when to apply, and for what period of time?

A variety of neurotrophic factors have been used in attempts to improve morphological and behavioural outcomes after experimental spinal cord injury (SCI). Here we review many of these factors, their cellular targets, and their therapeutic impact on spinal cord repair in different, primarily rodent, models of SCI. A majority of studies report favourable outcomes but results are by no means consistent, thus a major aim of this review is to consider how best to apply neurotrophic factors after SCI to optimize their therapeutic potential. In addition to which factors are chosen, many variables need be considered when delivering trophic support, including where and when to apply a given factor or factors, how such factors are administered, at what dose, and for how long. Overall, the majority of studies have applied neurotrophic support in or close to the spinal cord lesion site, in the acute or sub-acute phase (0–14 days post-injury). Far fewer chronic SCI studies have been undertaken. In addition, comparatively fewer studies have administered neurotrophic factors directly to the cell bodies of injured neurons; yet in other instructive rodent models of CNS injury, for example optic nerve crush or transection, therapies are targeted directly at the injured neurons themselves, the retinal ganglion cells. The mode of delivery of neurotrophic factors is also an important variable, whether delivered by acute injection of recombinant proteins, sub-acute or chronic delivery using osmotic minipumps, cell-mediated delivery, delivery using polymer release vehicles or supporting bridges of some sort, or the use of gene therapy to modify neurons, glial cells or precursor/stem cells. Neurotrophic factors are often used in combination with cell or tissue grafts and/or other pharmacotherapeutic agents. Finally, the dose and time-course of delivery of trophic support should ideally be tailored to suit specific biological requirements, whether they relate to neuronal survival, axonal sparing/sprouting, or the long-distance regeneration of axons ending in a different mode of growth associated with terminal arborization and renewed synaptogenesis.This article is part of a Special Issue entitled SI: Spinal cord injury.

Long-term seizure suppression and optogenetic analyses of synaptic connectivity in epileptic mice with hippocampal grafts of GABAergic interneurons.

Studies in rodent epilepsy models suggest that GABAergic interneuron progenitor grafts can reduce hyperexcitability and seizures in temporal lobe epilepsy (TLE). Although integration of the transplanted cells has been proposed as the underlying mechanism for these disease-modifying effects, prior studies have not explicitly examined cell types and synaptic mechanisms for long-term seizure suppression. To address this gap, we transplanted medial ganglionic eminence (MGE) cells from embryonic day 13.5 VGAT-Venus or VGAT-ChR2-EYFP transgenic embryos into the dentate gyrus (DG) of adult mice 2 weeks after induction of TLE with pilocarpine. Beginning 3-4 weeks after status epilepticus, we conducted continuous video-electroencephalographic recording until 90-100 d. TLE mice with bilateral MGE cell grafts in the DG had significantly fewer and milder electrographic seizures, compared with TLE controls. Immunohistochemical studies showed that the transplants contained multiple neuropeptide or calcium-binding protein-expressing interneuron types and these cells established dense terminal arborizations onto the somas, apical dendrites, and axon initial segments of dentate granule cells (GCs). A majority of the synaptic terminals formed by the transplanted cells were apposed to large postsynaptic clusters of gephyrin, indicative of mature inhibitory synaptic complexes. Functionality of these new inhibitory synapses was demonstrated by optogenetically activating VGAT-ChR2-EYFP-expressing transplanted neurons, which generated robust hyperpolarizations in GCs. These findings suggest that fetal GABAergic interneuron grafts may suppress pharmacoresistant seizures by enhancing synaptic inhibition in DG neural circuits.

A fourth generation of neuroanatomical tracing techniques: exploiting the offspring of genetic engineering.

The first three generations of neuroanatomical tract-tracing methods include, respectively, techniques exploiting degeneration, retrograde cellular transport and anterograde cellular transport. This paper reviews the most recent development in third-generation tracing, i.e., neurochemical fingerprinting based on BDA tracing, and continues with an emerging tracing technique called here 'selective fluorescent protein expression' that in our view belongs to an entirely new 'fourth-generation' class. Tracing techniques in this class lean on gene expression technology designed to 'label' projections exclusively originating from neurons expressing a very specific molecular phenotype. Genetically engineered mice that express cre-recombinase in a neurochemically specific neuronal population receive into a brain locus of interest an injection of an adeno-associated virus (AAV) carrying a double-floxed promoter-eYFP DNA sequence. After transfection this sequence is expressed only in neurons metabolizing recombinase protein. These particular neurons promptly start manufacturing the fluorescent protein which then accumulates and labels to full detail all the neuronal processes, including fibers and terminal arborizations. All other neurons remain optically 'dark'. The AAV is not replicated by the neurons, prohibiting intracerebral spread of 'infection'. The essence is that the fiber projections of discrete subpopulations of neurochemically specific neurons can be traced in full detail. One condition is that the transgenic mouse strain is recombinase-perfect. We illustrate selective fluorescent protein expression in parvalbumin-cre (PV-cre) mice and choline acetyltransferase-cre (ChAT-cre) mice. In addition we compare this novel tracing technique with observations in brains of native PV mice and ChAT-GFP mice. We include a note on tracing techniques using viruses.

Afferent-hair cell connectivity as a possible source of spike train irregularity in turtle vestibular bouton afferents

Vestibular afferents have been categorized as “regular” or “irregular” depending on the variability of their spike trains [1,2]. It is thought that the difference in regularity among afferents results from differences in the types of voltage-dependent conductances present in the cell. For example afferents may differ in their complements of potassium A, D, and M currents or in currents that produce a small vs. prominent afterhyperpolarization (AHP) [3-5]. Because bouton afferents from turtle utricle vary widely in morphology depending on their location in the utricle, we sought to determine if afferent morphology might also play a role in spike train irregularity. Although we found that morphology itself is not a factor in determining spike train variability, we did find that spike train variability can be affected by afferent/hair cell connectivity. Morphological reconstructions of bouton afferents from the lateral extrastriolar (LES), striola, juxtastriola and medial extrastriolar (MES) regions of turtle utricle were done with Neurolucida. Models based on these reconstructions were constructed for 16 afferents. Voltage-dependent conductance distributions in different regions (axon, soma, terminal arbor, varicosities, nodes, myelin) were identical in each cell model. Given the locations of synapses in the terminal arbor and the density of hair cells in each region, we were able to estimate the number of synapses formed between individual hair cells and an afferent. In the LES, striola and juxtastriola most synapses on an afferent were formed with unique hair cells, whereas in the MES single hair cells more often made multiple synapses with an afferent. To determine the effect of this difference in connectivity we modeled Poisson release from hair cells to synapses in the terminal arbor to generate spike trains. The coefficient of variation (cv) as a function of interspike interval (ISI) was calculated to estimate spike train variability with different average release rates. We found that cv as a function of ISI was nearly identical for LES, striola and juxtastriola afferents, but was the same for MES afferents only if connectivity in the MES was assumed to be one afferent synapse per hair cell. However, cv was much larger when the MES afferents were modeled with multiple synapses between an afferent and a hair cell, as calculated from synaptic location and hair cell density. To see if we could make an LES afferent more irregular, we modified connectivity to have more multiple synapses between individual hair cells and the afferent. As predicted, cv increased. These results suggest that afferent-hair cell connectivity may be a source of spike train irregularity. Given the large number of multiple contacts between a calyx and its presynaptic hair cell, we expect that connectivity contributes to spike train variability in calyx-bearing (calyx and dimorph) afferents.

Mitotoxicity in distal symmetrical sensory peripheral neuropathies.

Chronic distal symmetrical sensory peripheral neuropathy is a common neurological complication of cancer chemotherapy, HIV treatment and diabetes. Although aetiology-specific differences in presentation are evident, the clinical signs and symptoms of these neuropathies are clearly similar. Data from animal models of neuropathic pain suggest that the similarities have a common cause: mitochondrial dysfunction in primary afferent sensory neurons. Mitochondrial dysfunction is caused by mitotoxic effects of cancer chemotherapeutic drugs of several chemical classes, HIV-associated viral proteins, and nucleoside reverse transcriptase inhibitor treatment, as well as the (possibly both direct and indirect) effects of excess glucose. The mitochondrial injury results in a chronic neuronal energy deficit, which gives rise to spontaneous nerve impulses and a compartmental neuronal degeneration that is first apparent in the terminal receptor arbor--that is, intraepidermal nerve fibres--of cutaneous afferent neurons. Preliminary data suggest that drugs that prevent mitochondrial injury or improve mitochondrial function could be useful in the treatment of these conditions.

Terminal axonal arborization and synaptic bouton formation critically rely on abp1 and the arp2/3 complex.

Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the Drosophila nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. Abp1 knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied abp1 knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.

In vivo measures of nigrostriatal neuronal response to unilateral MPTP treatment

A single unilateral intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into non-human primates causes injury to the nigrostriatal pathway including nigral cell bodies, axons and striatal terminal fields. In this model, motor parkinsonism correlates well with the loss of nigral dopaminergic cell bodies but only correlates with in vitro measures of nigrostriatal terminal fields when nigral cell loss does not exceed 50%. The goals of this study are to determine the relationship of motor parkinsonism with the degree of injury to nigrostriatal axons, as reflected by in vitro fiber length density measures, and compare in vivo with in vitro measures of striatal terminal fields. We determined axon integrity by measuring fiber length density with tyrosine hydroxylase (TH) immunohistology and dopamine transporter (DAT) density with DAT immunohistology. We then calculated the terminal arbor size and compared these measures with previously published data of quantified in vivo positron emission tomography (PET) measures of presynaptic dopaminergic neurons, autoradiographic measures of DAT and vesicular monoamine transporter type 2 (VMAT2), striatal dopamine, nigral cell counts, and parkinsonian motor ratings in the same animals. Our data demonstrate that in vivo and in vitro measures of striatal terminal fields correlate with each other regardless of the method of measurement. PET-based in vivo striatal measures accurately reflect in vitro measures of DAT and VMAT2. Terminal arbor size and other terminal field measures correlate with nigral TH immunoreactive (TH-ir) cell counts only when nigral TH-ir cell loss does not exceed 50%. Fiber length density was the only striatal measure that linearly correlated with motor ratings (Spearman: r=−0.81, p<0.001, n=16).

The therapeutic effects of human adipose-derived stem cells in a rat cervical spinal cord injury model.

Spinal cord injury triggers a cascade of degenerative changes leading to cell death and cavitation. Severed axons fail to regenerate across the scar tissue and are only capable of limited sprouting. In this study, we investigated the effects of adult human adipose-derived stem cells (ASC) on axonal regeneration following transplantation into the injured rat cervical spinal cord. ASC did not induce activation of astrocytes in culture and supported neurite outgrowth from adult rat sensory dorsal root ganglia neurons. After transplantation into the lateral funiculus 1 mm rostral and caudal to the cervical C3-C4 hemisection, ASC continued to express brain-derived neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor-2 for 3 weeks but only in animals treated with cyclosporine A. Transplanted ASC stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone with some terminal arborizations reaching the caudal spinal cord. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure of the lesion scar with numerous astrocytic processes extended into the middle of the trauma zone in a chain-like pattern and in close association with regenerating axons. The density of the astrocytic network was also significantly decreased. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the activity of OX42-positive microglial cells was markedly reduced. However, ASC did not support recovery of forelimb function. The results suggest that transplanted ASC can modify the structure of the glial scar and stimulate axonal sprouting.

Branch management: mechanisms of axon branching in the developing vertebrate CNS

The remarkable ability of a single axon to extend multiple branches and form terminal arbors enables vertebrate neurons to integrate information from divergent regions of the nervous system. Axons select appropriate pathways during development, but it is the branches that extend interstitially from the axon shaft and arborize at specific targets that are responsible for virtually all of the synaptic connectivity in the vertebrate CNS. How do axons form branches at specific target regions? Recent studies have identified molecular cues that activate intracellular signalling pathways in axons and mediate dynamic reorganization of the cytoskeleton to promote the formation of axon branches.

Inhibition of Dendritic L-Type Calcium Current by Memantine in Frog Tectum

The aim of the study was to explore the effects of memantine on responses elicited in the frog tectum by the bursts of spikes of moderate strength of a single retina ganglion cell and to gain an insight about the effect of memantine on the L-type Ca(2+) current. The experiments were performed in vivo on adult frogs (Rana temporaria). An individual retina ganglion cell (or its retinotectal fiber) was stimulated by current pulses delivered through a multichannel stimulating electrode positioned on the retina. Responses to the discharge of a single retinal ganglion cell were recorded in the tectum by an extracellular carbon-fiber microelectrode positioned in the terminal arborization of the retinotectal fiber in the tectum layer F. The solution of memantine (1-amino-3,5-dimethyladamantane) hydrochloride (30 or 45 μM) was applied onto the surface of the tectum by perfusion at a rate of 0.4 mL/min. Memantine (30-45 μM) largely inhibited the L-type Ca(2+) channel-mediated slow negative wave and late discharges seen in the tectum responses without any effect on fast synaptic retinotectal transmission. Our results suggest that the neuroprotective effect of memantine could arise not only through the inhibition of the NMDA receptor current but also through the suppression of the L-type Ca(2+) current.

RNA‐binding protein Vg1RBP regulates terminal arbor formation but not long‐range axon navigation in the developing visual system

Local synthesis of β-actin is required for attractive turning responses to guidance cues of growth cones in vitro but its functional role in axon guidance in vivo is poorly understood. The transport and translation of β-actin mRNA is regulated by the RNA-binding protein, Vg1RBP (zipcode-binding protein-1). To examine whether Vg1RBP plays a role in axon navigation in vivo, we disrupted Vg1RBP function in embryonic Xenopus laevis retinal ganglion cells by expressing a dominant-negative Vg1RBP and by antisense morpholino knockdown. We found that attractive turning to a netrin-1 gradient in vitro was abolished in Vg1RBP-deficient axons but, surprisingly, the long-range navigation from the retina to the optic tectum was unaffected. Within the tectum, however, the branching and complexity of axon terminals were significantly reduced. High-resolution time-lapse imaging of axon terminals in vivo revealed that Vg1RBP-GFP-positive granules accumulate locally in the axon shaft immediately preceding the emergence a filopodial-like protrusion. Comparative analysis of branch dynamics showed that Vg1RBP-deficient axons extend far fewer filopodial-like protrusions than control axons and indicate that Vg1RBP promotes filopodial formation, an essential step in branch initiation. Our findings show that Vg1RBP is required for terminal arborization but not long-range axon navigation and suggest that Vg1RBP-regulated mRNA translation promotes synaptic complexity.

Architecture of vagal motor units controlling striated muscle of esophagus: Peripheral elements patterning peristalsis?

Little is known about the architecture of the vagal motor units that control esophageal striated muscle, in spite of the fact that these units are necessary, and responsible, for peristalsis. The present experiment was designed to characterize the motor neuron projection fields and terminal arbors forming esophageal motor units. Nucleus ambiguus compact formation neurons of the rat were labeled by bilateral intracranial injections of the anterograde tracer dextran biotin. After tracer transport, thoracic and abdominal esophagi were removed and prepared as whole mounts of muscle wall without mucosa or submucosa. Labeled terminal arbors of individual vagal motor neurons (n=78) in the esophageal wall were inventoried, digitized and analyzed morphometrically. The size of individual vagal motor units innervating striated muscle, throughout thoracic and abdominal esophagus, averaged 52 endplates per motor neuron, a value indicative of fine motor control. A majority (77%) of the motor terminal arbors also issued one or more collateral branches that contacted neurons, including nitric oxide synthase-positive neurons, of local myenteric ganglia. Individual motor neuron terminal arbors co-innervated, or supplied endplates in tandem to, both longitudinal and circular muscle fibers in roughly similar proportions (i.e., two endplates to longitudinal for every three endplates to circular fibers). Both the observation that vagal motor unit collaterals project to myenteric ganglia and the fact that individual motor units co-innervate longitudinal and circular muscle layers are consistent with the hypothesis that elements contributing to peristaltic programming inhere, or are "hardwired," in the peripheral architecture of esophageal motor units.

Morphology of the olivocerebellar projection of the chick: An axonal reconstruction study

The projection pattern of the olivocerebellar (OC) axons, which terminate mainly as climbing fibers (CFs) in the cerebellar cortex, tightly reflects the compartmental and developmental organization of the cerebellum as revealed by mapping and reconstruction studies in the rat. The avian cerebellum is well lobulated and longitudinally compartmentalized like the mammalian cerebellum. However, the projection pattern of the OC axons has not been studied in detail for most areas of the avian cerebellum. In the present study, we reconstructed labeled chick OC axons resulting from a small focal injection of biotinylated dextran amine into the inferior olive to investigate their morphological characteristics, and to determine their relationship to the general morphology of the chick cerebellum. Labeled CFs were distributed basically in a single longitudinally elongated narrow band-shaped area in lobules I-VIII, but in multiple, transversely widened, band-shaped areas in lobules IX-X. Three of the four reconstructed OC axons terminated in a single longitudinally band-shaped area in lobules IXa-c, whereas the other one terminated in multiple mediolaterally separated areas in lobule IXc, which is part of the flocculus. Single OC axons branched into 14 CFs on average. Two CFs occasionally merged to form a single terminal arbor. Axons also had thin, non-CF collaterals that projected either to a cerebellar nucleus or to the cortex. The results indicate that the morphological characteristics of OC axons, including branching and termination, are basically conserved between the chick and the rat.