Each sensory nerve arising from the geniculate ganglion expresses a unique fingerprint of neurotrophin and neurotrophin receptor genes

Neurons in the geniculate ganglion, like those in other sensory ganglia, are dependent on neurotrophins for survival. Most geniculate ganglion neurons innervate taste buds in two regions of the tongue and two regions of the palate; the rest are cutaneous nerves to the skin of the ear. We investigated the expression of four neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4, and five neurotrophin receptors, trkA, trkB, trkC, p75, and truncated trkB (Trn-B) in single sensory neurons of the adult rat geniculate ganglion associated with the five innervation fields. For fungiform papillae, a glass pipette containing biotinylated dextran was placed over the target papilla and the tracer was iontophoresed into the target papilla. For the other target fields, Fluoro-Gold was microinjected. After 3 days, geniculate ganglia were harvested, sectioned, and treated histochemically (for biotinylated dextran) or immunohistochemically (for Fluoro-Gold) to reveal the neurons containing the tracer. Single labeled neurons were harvested from the slides and subjected to RNA amplification and RT-PCR to reveal the neurotrophin or neurotrophin receptor genes that were expressed. Neurons projecting from the geniculate ganglion to each of the five target fields had a unique expression profile of neurotrophin and neurotrophic receptor genes. Several individual neurons expressed more than one neurotrophin receptor or more than one neurotrophin gene. Although BDNF is significantly expressed in taste buds, its primary high affinity receptor, trkB, was not prominently expressed in the neurons. The results are consistent with the interpretation that at least some, perhaps most, of the trophic influence on the sensory neurons is derived from the neuronal somata, and the trophic effect is paracrine or autocrine, rather than target derived. The BDNF in the taste bud may also act in a paracrine or autocrine manner on the trkB expressed in taste buds, as shown by others.     

Regenerating retinal axons of goldfish respond to a repellent guiding component on caudal tectal membranes of adult fish and embryonic chick.

On a substrate of rostral/caudal tectal membrane stripes of adult fish, regenerating temporal retinal axons avoid the caudal membranes. Thus they behave like embryonic chick axons on chick E9 membranes. The caudal membranes of adult fish contain a repellent component that, as has previously been shown in the chick, is inactivated by the enzyme PI-PLC. Fish axons respond not only to their own but also to the repellent component of embryonic chick membranes. Fish and more so chick E9 caudal membranes have an outgrowth reducing effect on fish axons that is also abolished by PI-PLC treatment and is weaker on chick E16 membranes. Thus adult fish tecta express a guiding component for retinal axons related to that in the embryonic chick.     

Redeployment of trigeminal motor axons during metamorphosis.

As a consequence of the degeneration and replacement of the jaw muscle fibers in the leopard frog, Rana pipiens, trigeminal motoneurons innervate different targets before and after metamorphosis. This investigation examined the morphological correlates of the reassignment of trigeminal motoneurons during the initial phases of myofiber turnover. Specifically, silver-cholinesterase histochemistry and electron microscopy were used to 1) identify the fate of motor axons within the neuromuscular junctions (NMJs) applied to degenerating larval myofibers and 2) to determine the origin(s) of the motor axons that innervate the postmetamorphic muscle fibers of the jaw. The results demonstrate that the NMJs are retained on larval myofibers throughout their degeneration and are readily identifiable on the residual larval basal laminae that remain after involution of the sarcoplasm. Light and electron microscopic observations provide evidence that both pre- and post-synaptic elements are present on the degenerating fibers. Furthermore, morphometric analyses indicate that the preponderance (86%) of motor axons supplying adult muscle fibers originates from the larval NMJs. This condition suggests that metamorphic redeployment of trigeminal motoneurons occurs through the resumption of growth at the axon terminal supplying larval muscle rather than through the proximal collateralization of these axons and resorption of larval terminals.     

Support of trigeminal sensory neurons by nonneuronal p75 neurotrophin receptors

The p75 neurotrophin receptor (p75NTR) binds all four mammalian neurotrophins, including neurotrophin-3 (NT-3) required for the development of select sensory neurons. This study demonstrated that many gustatory and somatosensory neurons of the tongue depend upon p75NTR. Each of thousands of filiform papillae at the front of the tongue as well as each somatosensory prominence at the back of the tongue has a small cluster of p75NTR-positive epithelial cells that is targeted by somatosensory innervation. This expression of p75NTR by epithelial target cells required NT-3 but not adult innervation. NT-3-secreting cells were adjacent to the p75NTR-positive target cells of each somatosensory organ, as demonstrated in NT-3(lacZneo) transgenic mice. In NT-3 null mutant mice, there were few lingual somatosensory neurons. In p75NTR null mutant mice, the lingual somatosensory axons were likewise absent or had deficient terminal arborizations. Cell culture indicated that substrate p75NTR can influence neuronal outgrowth. Specifically, dissociated trigeminal sensory neurons more than doubled their neurite lengths when grown on a lawn of p75NTR-overexpressing fibroblasts. This enhancement of neurite outgrowth by fibroblast p75NTR raises the possibility that epithelial target cell p75NTR may help to promote axonal arborization in vivo. The co-occurrence in p75NTR null mice of a 35% reduction in geniculate ganglion taste neurons and a shortfall of taste buds is consistent with the established role of gustatory innervation in prompting mammalian taste receptor cell differentiation.     

Patchy and laminar terminations of medial geniculate axons in monkey auditory cortex

The object of this study was to identify the terminal distributions of thalamocortical axons arising in chemically characterized subdivisions of the medial geriiculate complex. Large injections of wheat germ agglutinin-conjugated horseradish peroxidase or small injections of Phaseolus vulgaris leucoagglutinin were made in the medial geniculate complex of Macaca fuscata. The terminal distributions of labeled axons in the cortex were correlated with auditory cortical fields demonstrable by different intensities of immunoreactivity for parvalbumin. Fibers from the ventral nucleus terminated mainly in layer IV and deep portion of layer III (IIIB), with additional terminations in layers I-IIIA and in layer VI. In layers IIIB-IV, a major terminal plexus was formed by a small number of dense patches, 300–500 μm in diameter, surrounded by smaller satellite patches. The patches conformed to a similarly lobulated pattern of parvalbumin fiber immunoreactivity. Terminations of some individually labeled thalamocortical fibers were restricted to a single patch, whereas others innervated more than one patch by collateral branches. Fibers from the dorsal nuclei ending in areas of less dense parvalbumin immunoreactivity surrounding the primary auditory cortex formed much larger terminal patches centered largely in layer IIIB. Fibers from the magnocellular nucleus had relatively few terminal branches but innervated extremely wide areas by collaterals of single axons. Two types of axons arose from the magnocellular nucleus, one terminating preferentially in middle cortical layers and the other exclusively in layer I. These may arise respectively from parvalbumin- and calbindin-immunoreactive cell populations in the magrnocellular nucleus. © Wiley-Liss, Inc.     

Unmyelinated axons in the trigeminal motor root of human and cat

The fiber composition of the human and cat trigeminal “motor roots” were studied utilizing the electron microscope. Twelve to twenty percent of fibers in the human trigeminal motor root are unmyelinated whereas 9–15% are unmyelinated in the cat. The only previous examination of the fiber composition of the peripheral trigeminal motor nerve utilized the light microscope and indicated that less than 5% of fibers were unmyelinated in cat. No study of the fiber composition of the motor nerve root is available. The present results are similar to those recently obtained by others for spinal ventral roots. The function of unmyelinated fibers in the trigeminal “motor root” is unknown, however indirect evidence, both laboratory and clinical, suggests a potential sensory function for them. The findings question seriously the concept that the functional separation of the nervous system into motor and sensory systems has anatomical correlates in the spinal and cranial nerve roots. The results relate directly to our conceptualization of the nervous system and also to the design of methods for the treatment of intractable pain.     

Growing tips of embryonic cerebellar axons in vivo

Few studies have focused on the transformation of growth cones to mature synaptic arbors. To study these events in developing axons in vivo, we have labeled growing cerebellar axons with horseradish peroxidase (HRP) in postnatal stages [Mason and Gregory, 1984]. This report will provide the first data on embryonic cerebellar axons, and will ask whether growth cones differ in tracts and in target tissue and what features characterize axons that enter the cerebellum in fetal periods. During the earliest embryonic (E) periods examined (E16-19), axons in tracts have enlarged growth cones with lamellopodia and short filopodia that contain small and large vesicles. In contrast, axons within the cerebellar anlage from E16-postnatal day (P) 5 have fine calibers with a minimum of branching, and have small tapered growing tips. If synaptic contacts are made by such growing tips, there is little concomitant change in their shape. As target cells from layers and as their dendrites extend (P5-P7), growing tips and synaptic boutons differentiate according to the type of synaptic arrangement in which they engage. Enlarged, irregular expansions of growing tips correspond to synaptic contacts with multiple dendritic partners and are filled with large and small clear vesicles. Filopodia arising from such swellings, like the small undifferentiated growing tips of the type seen on embryonic axons, contain a mixture of vesicle types but make simple synapses with single profiles. Many axons make both kinds of synaptic structures, especially during the period when maturing axons give rise to long filopodia. Thus, growing tips have immature forms long after synaptogenesis begins, and use filopodial structures to elaborate synaptic arrangements. This analysis should elucidate the changes in growth cone form and cytology that reflect cell-cell interactions during synaptogenesis.     

Directional specificity and patterning of sensory axons in trigeminal ganglion-whisker pad cocultures

In the rodent trigeminal pathway, trigeminal axons invade the developing whisker pad from a caudal to rostral direction. We investigated directional specificity of embryonic day (E) 15 rat trigeminal axons within this peripheral target field using explant cocultures. E15 trigeminal axons readily grow into the same age whisker pad explants and form follicle-related patterns along a caudal to rostral direction. They also can grow into this target from its lateral aspects. In contrast, they are unable to invade the whisker pad from the rostral (nasal) pole. We did not find any correlation between the distribution of extracellular matrix molecules and trigeminal axon growth preferences. We also examined age-related changes in trigeminal axon responsiveness to directional cues. E19 trigeminal axons readily grew into E15 whisker pad explants from either the caudal or the rostral pole. These results suggest the presence of growth permissive and repulsive cues that guide sensory axons in the whisker pad. Furthermore, trigeminal axons lose their responsiveness to growth inhibitory cues at later stages of development.     

Spinal and medullary dorsal cell axons in the trigeminal nerve in lampreys

The ophthalmic branch of the trigeminal nerve was labeled with horseradish peroxidase (HRP) in 3 species of adult lampreys. In each species, some medullary and spinal dorsal cells were retrogradely labeled by HRP. Approximately 30% of spinal dorsal cells were labeled ipsilateral to the injection; an occasional contralateral spinal dorsal cell was also labeled. Intracellular recordings confirmed the anatomical findings.     

Distribution and synaptic organization of dopaminergic axons in the lateral geniculate nucleus of the rat

In the present study, immunocytochemistry with an antiserum against dopamine (DA) revealed hitherto unknown terminal fields of DA axons in the lateral geniculate nucleus (LGN) of the rat. The innervation of all subdivisions of the LGN is achieved by a common set of afferent fibers that branch to form terminal fields of uneven density. The ventral lateral geniculate nucleus (LGv) receives slightly more DA axons than the dorsal lateral geniculate nucleus (LGd), whereas within the latter, DA afferents innervate the lateral part of the nucleus slightly more densely. Labeled axon terminals and varicosities, examined in single and serial ultrathin sections, were found in the extraglomerular neuropil in the LGd and in the neuropil of the LGv characterized by relatively simple synaptic relationships. They formed predominantly asymmetrical synaptic contacts with dendritic profiles. Occasionally, the postsynaptic elements were found to be presynaptic dendrites of presumptive interneurons. Some of the possible roles of this newly demonstrated DA afferent system in the physiology of the LGN and in the pathophysiology of diseases associated with impairment of dopaminergic activity are discussed.     

Ultrastructure and immunohistochemistry of the trigeminal peripheral myelinated axons in patients with neuralgia

Objective

Detailed ultrastructural and immunohistochemical examination of the trigeminal axons surrounded by the peripheral type of the myelin could add new information about the extent of the trigeminal nerve lesion in neuralgia.

Patients, materials and methods

The examination comprised, firstly, the 10 trigeminal nerve roots (TNRs) in which the neurovascular contact was found in 20% of the cases, and the 2 additional control TNRs. Secondly, the biopsy specimens were taken from 6 patients with trigeminal neuralgia and 2 patients with trigeminal neuropathy following a partial TNR rhizotomy. The specimens were examined under the electron microscope (EM) and/or using the immunohistochemical (IHC) methods.

Results

In addition to the central zone of demyelination, the EM examination of the TNR also revealed alterations of the peripheral myelin, i.e. deformation, thickening, demyelination and remyelination, as well as changes of the peripheral axons, that is, atrophy or hypertrophy, neurofilaments increase, loss of the myelin and sprouting occasionally. Some Schwann cells were also damaged. The IHC examination usually showed a moderate immune reaction against neuron-specific enolase (NSE) and protein gene product 9.5 (PGP9.5), but sporadically weaker reaction against the S-100 protein, synaptophysin (SY), neurofilament protein (NFP) and glial fibrillary acidic protein (GFAP). The substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity was weak at some sites, but strong at some other places.

Conclusions

The pathological changes affect not only the central nerve fibers of the TNR, but also some of the peripheral axons, their myelin sheath and Schwann cells. These are signs of the retrograde ultrastructural and biochemical alterations, which could participate in the pathophysiological mechanism underlying the trigeminal neuralgia.     

Anatomy of glutamic acid decarboxylase immunoreactive neurons and axons in the rat medial geniculate body

This is a study of the form, density, and distribution of glutamic acid decarboxylase (GAD) immunoreactive neurons and puncta (axon terminals) in the adult rat medial geniculate complex. GAD-positive elements were stained by either the peroxidase-antiperoxidase or avidin-biotin procedures. Thalamic architectonic subdivisions were defined independently in Golgi, Nissl, plastic-embedded semi-thin, and fiber-stained preparations, and from investigations of medial geniculate connectivity. GAD-positive neurons represent only approximately 1% of medial geniculate neurons. They occur in the three major medial geniculate subdivisions (ventral, dorsal, and medial). There is variability between subdivisions in the form and number of such neurons, and among the puncta. In the ventral division, immunopositive somata may have sparsely branched dendrites as long as 300-400 microns and capped with varicose expansions or bouton-like sprays of appendages. These closely appose the somata or primary dendrites of other cells; the axons of these GAD-positive neurons are also immunostained. In the dorsal division there are fewer GAD-positive neurons and their structure is different. Their dendrites are rarely immunoreactive for more than 100-150 microns; nor can their immunostained axons be traced very far. In the medial division the number of GAD-positive neurons, considering the relatively small size of this division, was high. These neurons rarely have immunostained dendrites, and more than one type of neuron is immunoreactive. The average somatic diameter of GAD-positive neurons is about 60% of that of non-immunostained cells in semi-thin material; however, the range of somatic area and the dendritic variability of these neurons suggest that cells representing more than one population are immunopositive and include all but the largest neurons. The puncta also show regional differences. Small (0.5-2 microns in diameter), medium (2-3 microns), or large (greater than 3 microns) puncta occur. In the ventral division, the predominantly medium-sized puncta are about four times as numerous on a unit/area basis than in the dorsal division, where they are far smaller and more delicate; medial division puncta are as numerous as those in the ventral division, but are much larger and coarser, and may form perisomatic arrangements. Controls were devoid of specific immunostaining.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tenascin-R as a repellent guidance molecule for newly growing and regenerating optic axons in adult zebrafish

In adult fish, in contrast to mammals, new optic axons are continuously added to the optic projection, and optic axons regrow after injury. Thus, pathfinding of optic axons during development, adult growth, and adult regeneration may rely on the same guidance cues. We have shown that tenascin-R, a component of the extracellular matrix, borders the optic pathway in developing zebrafish and acts as a repellent guidance molecule for optic axons. Here we analyze tenascin-R expression patterns along the unlesioned and lesioned optic pathway of adult zebrafish and test the influence of tenascin-R on growing optic axons of adult fish in vitro. Within intraretinal fascicles of optic axons and in the optic nerve, newly added optic axons grow in a tenascin-R immunonegative pathway, which is bordered by tenascin-R immunoreactivity. In the brain, tenascin-R expression domains in the ventral diencephalon, in non-retinorecipient pretectal nuclei and in some tectal layers closely border the optic pathway in unlesioned animals and during axon regrowth. We mimicked these boundary situations with a sharp substrate border of tenascin-R in vitro. Optic axons emanating from adult retinal explants were repelled by tenascin-R substrate borders. This is consistent with a function of tenascin-R as a repellent guidance molecule in boundaries for adult optic axons. Thus, tenascin-R may guide newly added and regenerating optic axons by a contact-repellent mechanism in the optic pathway of adult fish.     

Powering of bulk transport (varicosities) and differential sensitivities of directional transport in growing axons

Goldfish retinal ganglion cell (RGC) axons, regenerating in vitro, have varicosities, intervening phase-dense inclusions (IPDIs) and particles that are mobile. Varicosities contain an aggregate complex of cytomembranes embedded in a cytoskeletal matrix, and, when they saltate, they represent a form of bulk transport. While movement of varicosities is normally infrequent, the incidence of movement can be greatly increased by alkalinization with NH4Cl. However, alkalinization also lowers the phase density of varicosities to reveal that motile hyperdense particles appear to be responsible for powering the translocation of varicosities and IPDIs. Other effects of alkalinization include a selective arrest of all anterograde movements and approximately a 10-fold reduction in the rate of retrograde mobility of particles and IPDIs. In mildly permeabilized axons, 20 microM orthovanadate selectively arrests retrogradely directed particle movements, while 100 microM arrests both antero- and retrograde transport. In addition to demonstrating in RGC axons that antero- and retrograde mechanisms exhibit differential pharmacological and pH sensitivities, the observations indicate that a heterogenous bulk mass can be translocated in growing axons by a passive 'piggyback' mechanism.     

The morphology of corticofugal axons to the dorsal lateral geniculate nucleus in the cat

The structural features of corticogeniculate axons were studied in adult cats after labeling them with horseradish peroxidase (HRP). Injections of HRP into the optic radiations near the dorsal lateral geniculate nucleus result in Golgi-like filling of both geniculate relay neurons and corticogeniculate axons. In the present material at least two main types of axons could be defined. The most common type is called the type I axon because it so closely resembles the type I axons described by Guillery ('66, '67) in Golgi preparations. These fine axons have smooth surfaces and consistent fiber diameter. Most terminal swellings are at the ends of short collateral branches and these swellings form asymmetric synaptic contacts onto small and medium-sized dendrites. Type I axons typically innervate more than one lamina as well as interlaminar zones and they clearly arise from the cerebral cortex. The second type of axon is called the beaded axon because of its numerous swellings, en passant. These swellings frequently are larger than those on type I axons and they differ from previously described corticogeniculate axon terminals in their ultrastructural features. That is, their synaptic contacts appear symmetrical and they form axosomatic contacts. Because of these differences, the possibility that beaded axons are of subcortical origin, particularly from the perigeniculate nucleus, is discussed. When type I axons and geniculate relay neurons are filled in the same region of the nucleus it is possible to identify probable sites of synaptic contact by using the light microscope. Such analyses indicate that corticogeniculate axons synapse directly onto relay cells, primarily on peripheral dendritic branches. Further, it appears that single axons contact many geniculate neurons and that single neurons are contacted by many axons.     

Synaptic connections between corticogeniculate axons and interneurons in the dorsal lateral geniculate nucleus of the cat

Anatomical evidence is provided for direct synaptic connections by axons from visual cortex with interneurons in lamina A of the cat's dorsal lateral geniculate nucleus. Corticogeniculate axon terminals were labeled selectively with 3H-proline and identified by means of electron microscopic autoradiography. Interneurons in the lateral geniculate nucleus were stained with antibodies that had been raised against gamma aminobutyric acid (GABA). We found that corticogeniculate terminals synapsed with dendrites stained positively for GABA about three times as often as with unstained dendrites. Of the corticogeniculate terminals that contacted GABA-positive dendrites, 97% made synaptic connections with dendritic shafts. Only 3% synapsed with F profiles, the vesicle-filled dendritic appendages characteristic of lateral geniculate interneurons. These results suggest that the corticogeniculate pathway in the cat is directed primarily at interneurons and is organized synaptically to influence the integrated output of these cells, rather than the local interactions in which their dendritic specializations participate.     

Quantitative effects of NGF on the growth of embryonic frog axons

Sparse, dissociated cultures of embryonic Xenopus CNS neurons were grown with and without NGF. Under both conditions the same number of neurons survived and extended neurites, and under both conditions the neurites moved at approximately the same overall rates and with the same degree of straightness. On the other hand, neurons in the NGF-supplemented cultures had more neurites and these neurites branched 64% more often. Detailed measurements showed that the axons elongated 44% faster in NGF and that this increase could be ascribed to a selective increase in the stepping rate of axonal elongation. These observations raise the possibility that NGF may selectively modulate the rate of movement of the core cytoskeleton of the axon.     

Target specific differentiation of peripheral trigeminal axons in rat-chick chimeric explant cocultures.

Avian and rodent trigeminal ganglion (TG) neurons share common features in their neurotrophin requirements and axonal projections between the sensory periphery and the brainstem. In rodents, the whisker pad (WP) is a major peripheral target of the infraorbital (IO) nerve component of the TG. The chick IO nerve is much smaller and innervates the maxillary process (MP). In the embryonic WP, IO axons course in fascicles from a caudal to rostral direction and form terminal plexuses around follicles. In the chick, IO axons travel as a thin bundle to the MP and branch out with no specific patterning. We cocultured E15 rat TG with E5-6 chick MP or chick TG with rat WP explants to examine target influences on trigeminal axon growth patterns as visualized with DiI labeling or neurofilament immunohistochemistry. Chick TG axons showed robust growth into WP explants, and the ganglion increased in size. Thick bundles of axons traveled between rows of follicles and formed a distinct pattern as they developed terminal arbors around individual follicles. In contrast, rat TG axon growth was sparse in chick MP explants and the ganglion size reduced over time. Furthermore, rat TG axons did not show any patterning in the chick MP. Similar target-specific growth patterns were observed when TG explants were given a choice between chick MP and rat WP explants. Collectively these results indicate that both the chick and rat TG cells respond to similar target-specific peripheral cues in the establishment of innervation density and patterning in peripheral orofacial targets.