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Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration.

Netrin-1 is a bifunctional secreted protein that directs axon extension in various groups of developing axonal tracts. The transmembrane DCC (deleted in colorectal cancer) receptor is described as netrin-1 receptor and is involved in the attractive effects of netrin-1. In this study, we examined the spatio-temporal expression patterns of both netrin-1 and DCC in the rat olfactory system at different stages of development and during axonal regeneration following unilateral bulbectomy. High DCC expression was detected on the pioneer olfactory axons as they are extending toward the telencephalon. This expression was transient since from embryonic day 16 onwards, DCC was no longer detected along the olfactory nerve path. From embryonic day 14 until birth, DCC was also expressed within the mesenchyme surrounding the olfactory epithelium. During the same period, netrin-1 protein was detected along the trajectory of olfactory axons up to the olfactory bulb and its expression pattern in the nasal mesenchyme largely overlapped that of DCC. Moreover, netrin-1 continued to be present during the two first post-natal weeks, and a weak protein expression still persisted in the dorso-medial region of the olfactory epithelium in adult rats. While unilateral bulbectomy induced a transient up-regulation of netrin-1 in the lamina propria, particularly in the dorso-medial region of the neuroepithelium, no DCC expression was detected on the regenerating olfactory axons. In the developing olfactory bulb, the extension of mitral cell axons was associated with DCC presence while netrin-1 was absent along this axonal path. DCC was also highly expressed in the newly formed glomeruli after birth, and a weak DCC expression was still detected in the glomerular layer in adult rats. Taken together, these data support the notion that netrin-1, via DCC expressed on axons, may play a role in promoting outgrowth and/or guidance of pioneering olfactory axons toward the olfactory bulb primordium. Moreover, association of netrin-1 with mesenchymal DCC may provide a permissive environment to the growth of both pioneer and later-growing axons. The maintenance of netrin-1 expression in the nasal mesenchyme of adult rats as well as its regional up-regulation following unilateral bulbectomy infer that netrin-1, even in the absence of DCC, may be involved in the process of axonal growth of newly differentiated olfactory receptor neurons probably through the use of other receptors.

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Calibre and Microtubule Content of the Non-Medullated and Myelinated Domains of Optic Nerve Axons of Rats.

Calibres and microtubule contents of the non-medullated and myelinated domains of optic nerve axons of adult rats were studied with the electron microscope. The cross-sectional areas of the non-medullated domain was 0.25 microm2, and that of the myelinated domain 0.40 microm2, that is, greater by 59%. The increase in size was uneven across the axonal population; it was marked in fine and medium sized axons, and modest in the largest axons. The number of microtubules increased with axonal size; the density, however, decreased from 85 mirotubules/microm2 in 0.1 microm2 axons to about 20 in 1.2 microm2 axons. In axons of equal cross sectional area, the microtubular density of the myelinated and non-medullated domains was the same. Microtubular density values of optic axons resemble those of dorsal roots more than those of peripheral nerve axons of equal calibre. The facts that optic axons increase in size and gain microtubules behind the eyeball while the microtubular packing decreases suggest a local regulation of the axonal cytoskeleton.

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Differential physiology and morphology of phasic and tonic motor axons in a crayfish limb extensor muscle

The main leg extensor muscle of the crayfish Procambarus clarkii is innervated by two excitatory motor axons (phasic and tonic) and by the common inhibitory axon. The axons and their terminals were investigated using vital fluorescent dyes, antibodies and electron microscopy. Correlative physiological observations were made using intracellular microelectrodes and focal 'macro-patch' electrodes. The excitatory axons innervate the muscle fibres more extensively than does the inhibitory axon. Striking morphological and physiological differences between the two excitatory axons were discerned. The tonic motor axon contains many mitochondria and has varicose junctional terminals; the phasic axon has fewer mitochondria and thin terminals. In freely moving animals, the tonic axon is very active, whereas the phasic axon is inactive but fires in brief bursts during walking. Throughout the muscle, tonic excitatory postsynaptic potentials (EPSPs) are very small at low frequencies and facilitate greatly at higher frequencies. The EPSPs of the phasic axon are larger at low frequencies and depress with maintained stimulation. At identified tonic terminals, quantal content at 1­10 Hz is very low, whereas at identified phasic terminals, quantal content is 50- to 200-fold greater. The results indicate that transmitter release is regulated differently at the synapses of the two axons.

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Terminal arbors of individual, physiologically identified geniculocortical axons in the tree shrew's striate cortex.

We studied the terminal patterns of single, physiologically identified geniculocortical axons in the striate cortex of the tree shrew by using intracellular recording and labeling methods. Axons were classified by their response to the onset (ON-center) or offset (OFF-center) of a light stimulus presented to the ipsilateral or contralateral eye. Then, we attempted to penetrate each axon for labeling with horseradish peroxidase. We recovered 23 axons and studied 16 of these in detail. Light microscopic reconstructions of these axons revealed several distinct terminal patterns within cortical layer IV. ON-center axons had terminal arbors that ended mainly in the upper part of layer IV (IVa), while OFF-center axons ended in the lower part of layer IV (IVb). Within layers IVa and IVb, axons driven by the ipsilateral eye and those driven by the contralateral eye had overlapping distributions. However, their terminal arbors differed in size, in shape, and in the number of boutons. Compared with contralateral eye arbors, ipsilateral eye axons were on average three times larger in lateral extent (925 microns vs. 325 microns), spread over four times the surface area (0.13 mm2 vs. 0.03 mm2), and supported one and one-half times as many terminal boutons (1,647 vs. 1,086). The ipsilateral eye axons had more boutons at the edges of layer IV (i.e., the upper part of layer IVa and the lower part of layer IVb), while those from the contralateral eye axons were more evenly distributed. These results show that each functional class of geniculocortical fiber has a different laminar and areal arrangement of boutons and we consider the significance of these differences for visual cortical function.

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Carbon disulfide axonopathy. Another experimental model characterized by acceleration of neurofilament transport and distinct changes of axonal size.

The role of axonal transport in the development of structural changes of axons can be examined using experimental models. Two different compounds, 2,5-hexanedione (2,5-HD) and carbon disulfide (CS2), cause axonopathies characterized by the formation of neurofilaments (NF) containing enlargements in preterminal regions of central and peripheral axons. These axonopathies are excellent experimental models of the giant axonal neuropathies, a group of acquired and inherited human diseases of the central and peripheral nervous system. We previously reported that following administration of 2,5-HD, transport of NF is accelerated while number of NF and cross-sectional area are decreased in regions of the axon proximal to the enlargements. We proposed that acceleration of NF transport leads to a 'longitudinal' redistribution of NF which are decreased proximally and increased distally where they form the NF containing axonal enlargements. We have now carried out morphometric, transport and immunocytochemical studies in primary visual axons of rats exposed to CS2. NF-containing axonal enlargements were observed in optic tract and superior colliculus and they increased in number in a proximodistal direction. There was no detectable axonal degeneration and the cross-sectional area of axons proximal to the enlargements was decreased. Transport of NF was markedly accelerated. Immunostaining showed that all 3 NF subunits and phosphorylated epitopes of the 200-kDa NF subunit were present in the NF-containing axonal enlargements. All these findings were similar to those previously observed in the 2,5-HD axonopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

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Inhibition of proteolysis blocks anterograde-retrograde conversion of axonally transported vesicles.

To test the hypothesis that proteolysis is required for anterograde-retrograde (A-R) conversion of membranous organelles at axon tips, a new experimental paradigm was developed. By cutting the sciatic nerves of rats, a concentrated population of axon tips was produced, and proteases in the axon tips were locally inhibited by immersing the cut end of the nerve into a solution containing protease inhibitors (E-64 or leupeptin). Membranous organelles were pulse-labeled with [3H]leucine at the nerve cell body, and the amount of retrogradely transported radiolabeled vesicles from the axon tips was quantified with a proximal collection ligature. The results show that protease inhibition decreased the amount of radioactivity that was transported retrogradely from the axon tips and correspondingly increased the amount that remained in the tips. Ultrastructural analyses showed that the protease-inhibited axon tips were greatly distended by 40-80 nm membranous tubules. By contrast, the control axon tips had relatively few of these membranous tubules. These results show that protease inhibition at the axon tip blocks the removal of membranous elements from the axon tips by retrograde transport. We propose that proteolysis is an A-R converting mechanism which is critically required at the axon tip for the conversion of 40-80 nm membranous tubules into retrograde organelles. Apparently, the 40-80 nm membranous tubules are normally transient intermediates in the A-R conversion pathway, and they rapidly accumulate in the axon tip if the mechanisms that convert them into retrograde organelles are blocked. These 40-80 nm tubules also accumulate in certain pathologies and in the aging process.(ABSTRACT TRUNCATED AT 250 WORDS)

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Influence of temperature on slow flow in populations of regenerating axons with different elongation velocities.

The slow intra-axonal transport mechanisms may be subdivided into 4 elementary processes: (a) protein synthesis; (b) release of the slow material from the cell body; (c) transport of the molecules along the axon; and (d) deposition of the material from the moving phase into the immobile axonal constituents. These 4 components of the slow flow mechanisms are differently affected by temperature and react differently to axonal regeneration. They have been studied at temperatures ranging from 10 to 35 degrees C, in 3 different populations of regenerating C-fibers of the garfish olfactory nerve. These 3 classes of axons have very different elongation properties as determined by elongation rates ranging from a minimum of 0.3 mm/day at 14 degrees C to a maximum of 12.4 mm/day at 35 degrees C. From this study several properties of the slow intra-axonal transport mechanism can be underlined: (a) in regenerating nerves the rate of slow flow is multiplied by a factor of 3.3 as compared to the rate characteristic of an intact nerve. A similar increase was measured in every regenerating phase regardless of the specific elongation velocity of the growing axons. This accelerated rate of slow flow has been measured previously in degenerating axons detached from their perikarya: therefore the rate increase appears to be triggered by the injury rather than the needs of the neuron; (b) protein synthesis represents one of the main steps controlling axonal elongation. Neurons with the most active level of protein synthesis grow with the highest elongation velocity; (c) the somal releasing times decrease exponentially with temperature but are identical to the values measured in intact neurons; (d) finally the height of the slow flow peak decreases exponentially as a function of the distance, as seen previously in intact nerves, but a significantly larger amount of radioactivity reaches the end of the nerve. It can be concluded that slow flow is intimately linked with the regeneration process. The rate of slow flow and more specifically of Scb represents the upper limit that can be reached by the elongation velocities of regenerating fibers when all the growth conditions are maximal. When the regenerating conditions are not at their peak, the axonal elongation proceeds at velocities which can be very significantly slower than the rate of slow flow and might be determined by the level of protein synthesis in the perikarya. Axonal elongation, however, can never proceed at a rate faster than slow flow.

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Lesion-induced establishment of the crossed corticorubral projections in kittens is associated with axonal proliferation and topographic refinement.

The aberrant crossed corticorubral projection of the cat, which is very weak compared to the uncrossed one at about 1 month postnatal, becomes pronounced following unilateral lesions of the sensorimotor cortex. In order to determine whether or not terminal proliferation of pre-existing axons underlie this enlargement, the morphological changes of the crossed axons were examined, using the anterograde tracer Phaseolus vulgar- is leukoagglutinin (PHA-L). The crossed corticorubral axons in normal kittens were mostly simple in morphology with infrequent branching and did not often exhibit growth-cone-like axonal endings at 1 month postnatal. Two to 5 days after unilateral lesions of the sensorimotor cortex placed at this age, the axons were as simple as those in normal animals but ended in growth cones more frequently. Seven to 10 days post-lesion, the axons often bore side-branches which ended in growth cones. Two to 3 weeks post-lesion axons with sprays of finger-like fine sprouts occurred throughout the projection zone. There was no clear topography for the crossed projection in normal animals, but at 1-2 weeks post-lesion the axons started to show a certain amount of localization in the regions of the red nucleus which corresponded to the densely innervated region on the ipsilateral side. The topography of the crossed projections roughly mirrors that of the ipsilateral projection at about 1 month post-lesion. Thus, the lesions of the sensorimotor cortex induce substantial growth and proliferation of the crossed corticorubral axons. The post-lesion changes in axonal morphology and topographic refinement are reminiscent of developmental events. It is likely that the lesions permit the crossed axons, which normally fail to develop, to develop like the uncrossed ones.

Aging↗

Inhibition of sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along direct but not transcytotic axonal pathways.

Glycosphingolipids are constituents of lipid rafts which might function in sorting apical and axonal cargoes in the trans-Golgi network. In fact, two GPI-linked proteins, Thy1 and PrPC, require lipid raft lipids for sorting to the axon. It was previously shown that inhibition of glycosphingolipid synthesis by FumonisinB1 (FB1) impairs axon outgrowth but not axon specification, leading to the hypothesis that formation of axonally-targeted vesicles is coupled to sphingolipid synthesis. Since the axonal cell adhesion molecule L1/NgCAM can partition into membrane rafts biochemically, we asked whether correct targeting to the axon is FB1-sensitive, similarly to GPI-linked proteins. We previously showed that cultured hippocampal neurons use more than one trafficking pathway to the axon: a transcytotic pathway and a direct pathway. We show here that reducing raft lipid levels does not disrupt axonal targeting of L1/NgCAM along either pathway. Unexpectedly, FB1 selectively slowed the kinetics of surface expression of a truncated NgCAM using the direct pathway, but not of NgCAM using the transcytotic pathway. Therefore, the formation and/or transport of a subset of axonally-targeted vesicles are coupled to sphingolipid synthesis. Our results yield a mechanism for the axon outgrowth defect observed in FB1.

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Direct evidence for coherent low velocity axonal transport of mitochondria.

Axonal growth depends on axonal transport. We report the first global analysis of mitochondrial transport during axonal growth and pauses. In the proximal axon, we found that docked mitochondria attached to the cytoskeletal framework that were stationary relative to the substrate and fast axonal transport fully accounted for mitochondrial transport. In the distal axon, we found both fast mitochondrial transport and a coherent slow transport of the mitochondria docked to the axonal framework (low velocity transport [LVT]). LVT was distinct from previously described transport processes; it was coupled with stretching of the axonal framework and, surprisingly, was independent of growth cone advance. Fast mitochondrial transport decreased and LVT increased in a proximodistal gradient along the axon, but together they generated a constant mitochondrial flux. These findings suggest that the viscoelastic stretching/creep of axons caused by tension exerted by the growth cone, with or without advance, is seen as LVT that is followed by compensatory intercalated addition of new mitochondria by fast axonal transport.

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Molecular mechanisms of axonal damage in inflammatory central nervous system diseases.

PURPOSE OF REVIEW: Axonal dysfunction and damage is an early pathological sign of autoimmune central nervous system disease, viral and bacterial infections, and brain trauma. Axonal injury has attracted considerable interest during the past few years because the degree of axonal damage appears to determine long-term clinical outcome. RECENT FINDINGS: Advanced magnetic resonance spectroscopic imaging techniques have suggested that axonal loss and dysfunction is responsible for the persistent neurological deficits that occur in patients with multiple sclerosis. Histopathological methods have shown that axonal damage is defined primarily by dysfunction of axonal transport, and finally by complete transection and degeneration of axons. Recent studies have demonstrated that the extent of axonal damage in the primary demyelinating lesion of multiple sclerosis patients is associated with the number of activated microglia/macrophages and cytotoxic CD8+ T lymphocytes. In addition, diffuse axonal dysfunction independent of demyelination develops in normal appearing white matter, possibly due to indirect effects of inflammation. SUMMARY: The fact that axonal damage in response to overt inflammatory reactions may occur gradually, leaving a window for therapeutical intervention, has important clinical implications. Determination of the exact molecular mechanism might help in finding new therapies for inflammatory axonal damage.

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Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

5' Untranslated Regions↗

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.

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Multi-segmental innervation of single pontine reticulospinal axons in the cervico-thoracic region of the cat: anterograde PHA-L tracing study.

To characterize the fine morphology of individual reticulospinal axons at multiple spinal segments, localized injections of the anterograde neural tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), were made into the nucleus reticularis pontis oralis (NRPo) of the cat. Following survival periods of 6-8 weeks, labelled axons, between 1 and 8 microns in diameter, were found throughout the cervical and upper thoracic segments. Thick axons (diameter > or = 3 microns) were found to descend beyond the upper thoracic spinal cord, while most thin axons (diameter < 3 microns) ended in the upper cervical cord. From serial transverse sections (50 microns) of segments C3 to T2, in four cats, the trajectories of 23 single, thick reticulospinal axons were traced in continuity over distances of between 21.8 and 59.4 mm, corresponding to 3 and 8 segments, respectively. Most axons gave off at least one, and as many as four collaterals per segment, some preferentially in the cervical enlargement. The remainder gave off collaterals at most but not all segments. Detailed reconstruction of the collateralization and arborization in the spinal gray matter showed two major termination types, one where terminals remained ipsilateral to the stem axon, the other where additional collaterals extended across the midline from the ipsilateral gray matter to terminate in the contralateral gray matter. Axons tended to have collaterals of one type or the other, irrespective of the rostrocaudal level. Both ipsilateral and bilateral projections terminated mainly in laminae VII or VIII although the branching patterns varied from axon to axon. Individual stem axons, in general, showed similar termination patterns at each level.

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Development of axonal arbors of layer 6 pyramidal neurons in ferret primary visual cortex.

We followed the development of axonal arbors of layer 6 pyramidal neurons in ferret striate cortex to determine whether early developing axon collaterals are formed specifically in the correct target layers from the outset or achieve their adult specificity by the elimination of initially exuberant projections. These neurons were chosen for study because they are amongst the first to be generated in the developing ferret's striate cortex, and, in mature animals, these cells have axonal arbors that are highly specific for layer 4 and to a lesser extent layers 2/3 but have few collateral branches in layer 5. The axonal arbors of individual layer 6 pyramidal neurons were reconstructed following labeling in living slices prepared from the striate cortex of ferrets aged 13-35 days postnatal (P13-35). At the earliest ages (P13-15), axonal arbors consisted of a simple axon extending from the base of the cell body into the subplate or white matter and usually forming a few collateral branches but never ascending into layer 5. By P19-20, about one-half of the cells had extended axon collaterals into layer 5 or higher, and these already appeared to branch preferentially in layer 4. All of the cells from older animals had substantial axonal arbors in layers 2-4. By P26-28, there were approximately ten times as many axonal branches in layer 4 as in layer 5. Between P26-28 and P35, there was no significant change in the number of branches in layer 5, but the numbers of both branches and of axon collateral terminations in layer 4 approximately doubled. Thus, the extent of axonal arborization in layer 4 increases dramatically between P13 and P35, and growth is highly specific for correct target layers, with few branches formed in layer 5.

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Convergence and branching patterns of round, type 2 corticopulvinar axons.

Corticopulvinar connections consist of at least two morphologically distinct subpopulations. In one subgroup (E, type 1), axons have an "elongated" terminal field and thin, spinous terminations; in the other (R, type 2), axons have a small, round arbor and large, beaded terminations. Previous work (Rockland, 1996) indicates that E-type axons from several occipitotemporal areas branch extensively within and sometimes between pulvinar subdivisions, but that R-type axons tend to have spatially delimited arbors. The present report is a further investigation of R-type axons from areas V1 and MT and was initiated to test the generality of the previous findings. There are four main results: 1) By serial section reconstruction of anterogradely labeled axons, 10 of 25 axons originating in area V1 had two or three spatially separate arbors (8 and 2 axons, respectively). Sixteen axons analyzed from area MT, however, all had single arbors, although the arbors were often formed by the convergence of widely separate branches. 2) Multiple (at least 2-5) R-type corticopulvinar axons, from V1 or from MT, can converge in a single focus. 3) R-type axons originating from both areas V1 and MT can branch to other structures; namely, the superior colliculus, the pretectal area, and/or the reticular nucleus of the thalamus. 4) Finally, corticopulvinar terminations from area V1 are predominantly R-type, whereas those from MT are more predominantly E-type. These results thus provide additional evidence of the special relationship of area V1 to the pulvinar. They also emphasize that the idea of corticopulvinocortical "feedback loops," although convenient as a shorthand nomenclature, does not adequately convey the full complexity of the system.

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Projection and innervation patterns of individual thalamic reticular axons in the thalamus of the adult rat: a three-dimensional, graphic, and morphometric analysis.

The gamma-aminobutyric acid-ergic thalamic reticular nucleus (Rt), which carries matching topographical maps of both the thalamus and cortex and in which constituent cells can synaptically communicate between each other, is the major extrinsic source of thalamic inhibitions and disinhibitions. Whether all the Rt axonal projections into the thalamus are similarly organized and have common projection and innervation patterns are questions of great interest to further our knowledge of the functioning of the Rt. The present study provides architectural and morphometric data of individual, anterogradely labeled axonal arbors that arose from distinct parts of the Rt. One hundred twenty-seven Rt neurons from all regions of Rt were marked juxtacellularly with biocytin or Neurobiotin in urethane-anesthetized adult rats. Eighteen two-dimensional and 14 three-dimensional reconstructions of single tracer-filled Rt neurons were made from serial, frontal, horizontal, or sagittal sections. Both the somatodendritic and axonal fields of tracer-filled Rt cells were mapped in three dimensions and illustrated to provide a complementary stereotaxic reference for future studies. Most marked units projected to a single nucleus of the anterior, dorsal, intralaminar, posterior, or ventral thalamus. Axons emerging from cells in distinct sectors of the Rt projected to distinct nuclei. Within a sector, neurons with separate dendritic fields innervated separate regions either in a single nucleus or into different but functionally related thalamic nuclei. Neurons with an overlap of their dendritic fields gave rise either to overlapping axonal arborizations or, more rarely, to distinct axonal arbors within two different thalamic nuclei implicated in the same function. In rare instances, an Rt axon could project within these two nuclei. Thalamic reticular axons commonly displayed a single well-circumscribed arbor containing a total of about 4,000 +/- 1,000 boutons. Every arbor was composed of a dense central core, which encompassed a thalamic volume of 5-63 x 10(6) microm3 and was made up of patches of maximal innervation density (10 +/- 4 boutons/tissue cube of 25 microm each side), surrounded by a sparse component. The metric relationships between the Rt axonal arbors and the dendrites of their target thalamocortical neurons were determined. Both the size and maximal innervation density of the axonal patches were found to fit in with the somatodendritic architecture of the target cells. The Rt axonal projections of adult rats are thus characterized by their (1) well-focused terminal field with a patchy distribution of boutons and (2) parallel organization with a certain degree of divergence. The role of the Rt-mediated thalamic inhibition and disinhibition may be to contrast significant with nonrelevant ongoing thalamocortical information.

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Synaptic connections of DB3 diffuse bipolar cell axons in macaque retina.

In primate retinas, the dendrites of DB3 diffuse bipolar cells are known to receive inputs from cones. The goal of this study was to describe the synaptic connections of DB3 bipolar cell axons in the inner plexiform layer. DB3 bipolar cells in midperipheral retina were labeled with antibodies to calbindin, and their axons were analyzed in serial, ultrathin sections by electron microscopy. Synapses were found almost exclusively at the axonal varicosities of DB3 axon terminals. There were 2.14 synaptic ribbons per varicosity. There were 33 varicosities per DB3 cell, giving an average of 71 ribbons per axon terminal. Because there were 1.5 postsynaptic ganglion cell dendrites per DB3 axonal varicosity, we estimate that there is at least 1 synapse per varicosity onto a parasol ganglion cell dendrite. There were 3.4 input synapses from amacrine cells per axonal varicosity. Among these were feedback synapses to the DB3 bipolar cell axon varicosities, which were made by 47% of the postsynaptic amacrine cell processes. Some of the feedback synapses could be from amacrine cells immunoreactive for cholecystokinin precursor or choline acetyltransferase, because both types of amacrine cells costratify with parasol cells and are known to be presynaptic to bipolar cells. AII amacrine cells were both presynaptic and postsynaptic to DB3 axons, a finding consistent with the large rod input to parasol ganglion cells reported in physiological experiments. DB3 bipolar cell axons also made frequent contacts with neighboring DB3 axons, and gap junctions were always found at these sites.

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