Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord. I. Methods for preparing implants from dissociated cells

Our goal was to devise methods of implanting defined populations of the cellular constituents of peripheral nerve into regions of spinal cord injury. This objective derived from the knowledge that the cellular environment of peripheral nerve is known to be supportive of axon regeneration from both central and peripheral neurons. Two of the constituents of the peripheral nerve environment known to influence axonal growth are the Schwann cell and extracellular matrix (particularly basal lamina), both of which can be obtained in culture. We describe here large-scale methods of establishing purified populations of rat sensory neurons to which purified populations of Schwann cells were added. These essentially monolayer preparations were then scrolled and cut into lengths of proper shape and size to provide implants for sites of spinal cord injury in newborn rats. We also describe methods enabling the addition of leptomeningeal components to the implants; this addition contributes a proliferating population of vascular endothelial cells (identified by immunostaining) to the otherwise vasculature-free neuron/Schwann cell implant. Light and electron microscopic observations were made to characterize the implants. When the implant was ready for use, it contained Schwann cells that were differentiated, i.e., had begun to ensheathe axons and form basal lamina. The use of a medium containing human plasma to foster endothelial cell growth led to increased neurite fasciculation and Schwann cell migratory activity in the outgrowth, particularly when the neurons and Schwann cells were cultured on leptomeninges. The second paper in this series reports the deportment of these implants and their influence on corticospinal tract growth after placement into regions of dorsal column injury in neonatal rats (Kuhlengel et al., J. Comp. Neurol 293:74-91, 1990).     

Regeneration of primary sensory axons into the adult rat spinal cord via a peripheral nerve graft bridging the lumbar dorsal roots to the dorsal column

This study investigated the feasibility of using a peripheral nerve autograft (NAG) to promote and guide regeneration of sensory axons from the caudal lumbar dorsal roots to the rostral dorsal column following a lower thoracic cordotomy in adult rats. After a left hemicordotomy at the T13 vertebra level and ipsilateral L3 and L4 rhizotomies, a peripheral NAG (peroneal nerve) was connected to the distal roots stumps, then implanted into the left dorsal column 10 mm rostral to hemicordotomy site (n = 12). After surgery, all animals of the experimental group experienced complete anesthesia in their left hindlimb. Three months later, a slight response to nociceptive stimulation reappeared in L3 and/or L4 dermatomes in 6 of the 12 experimental animals. None of these animals exhibited self-mutilation. Nine months after surgery, we performed retrograde tracing studies by injecting horseradish peroxidase (HRP) into the left dorsal column 30 mm rostral to the NAG implantation site. In eight animals, we found HRP-stained neurons in the left L3 and/or L4 dorsal root ganglia (DRG). The mean number of HRP-stained neurons per DRG was 71 +/- 92 (range 2-259). In control groups, no HRP-stained neurons were found in L3 or L4 DRG. Histological analysis of the NAG showed evidence of axonal regeneration in all 8 animals with positive retrograde labeling of DRG neurons. However, we did not find a statistical correlation between the number of HRP-stained neurons and the degree of sensory recovery. This study demonstrates that an NAG joining dorsal roots to the dorsal column, thus shunting the original CNS-PNS junction, can support regeneration of central axons from DRG primary sensory neurons into the dorsal column over distances of at least 30 mm despite the inhibitory influence of the CNS white matter.     

Functional reconnections established by central respiratory neurons regenerating axons into a nerve graft bridging the respiratory centers to the cervical spinal cord

The present work investigated, in adult rats, the long-term functional properties and terminal reconnections of central respiratory neurons regenerating axons within a peripheral nerve autograft bridging two separated central structures. A nerve graft was first inserted into the left medulla oblongata, in which the respiratory centers are located. Three months later, a C3 left hemisection was performed, and the distal tip of the graft was implanted into the C4 left spinal cord at the level of the phrenic nucleus, a natural central inspiratory target. Six to eight months after medullary implantation, the animals (n = 12) were electrophysiologically investigated to test 1) the phrenic target reinnervation by analyzing the phrenic responses elicited by bridge electrical stimulation and 2) the bridge innervation by unitary recordings of the spontaneous activity of regenerated axons within the nerve bridge. In the control group (n = 6), the medullary site of implantation corresponded to the dorsolateral medulla, a region known to be an unsuitable site for inducing respiratory axonal regrowth after nerve grafting. Stimulation of the nerve bridge never elicited phrenic nerve response, and no respiratory units were found within the nerve bridge. In the experimental group (n = 6), the proximal tip of the nerve bridge was implanted within the ventrolateral medulla at the level of the respiratory centers. Electrical stimulation of the nerve bridge induced phrenic nerve responses that reflected a postsynaptic activation of the phrenic target. Subsequent unitary recordings from teased fibers within the bridge revealed the presence of regenerated inspiratory fibers exhibiting discharge patterns typical of medullary inspiratory neurons, which normally make synaptic contacts with the inspiratory phrenic target. These results indicate that, when provided with an appropriate denervated target, central respiratory neurons with regenerated axons along a nerve bridge can remain functional for a long period and can make precise and specific functional reconnections with central homotypic target neurons.     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.     

Growth-associated protein 43 immunoreactivity in the superficial dorsal horn of the rat spinal cord is localized in atrophic C-fiber,and not in sprouted A-fiber,central terminals after peripheral nerve injury

Peripheral nerve injury induces the up-regulation in dorsal root ganglion cells of growth-associated protein 43 (GAP-43) and its transport to the superficial laminae of the dorsal horn of the spinal cord, where it is located primarily in unmyelinated axons and growth-cone like structures. Peripheral nerve injury also induces the central terminals of axotomized myelinated axons to sprout and form novel synaptic contacts in lamina II of the dorsal horn. To investigate whether the sprouting of A-fiber central terminals into lamina II is the consequence of GAP-43 incorporation into their terminal membranes, we have used an ultrastructural analysis with double labelling to identify the localization of GAP-43 immunoreactivity. Transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to identify C-fiber terminals. Transganglionic transport of the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label A-fiber sciatic nerve central terminals in combination with GAP-43 immunocytochemistry. GAP-43 was found to colocalize only with WGA-HRP- and not with B-HRP-labelled synapses or axons. In addition, many single-labelled GAP-43 synapses were observed. Many of the WGA-HRP-labelled terminals that were characterized by degenerative changes were GAP-43 immunoreactive. Our results indicate that peripheral nerve injury induces novel synapse formation of A fibers in lamina II but that up-regulated levels of GAP-43 are present mainly in other axon projections to the superficial dorsal horn. J. Comp. Neurol. 386:111–118, 1997. © 1997 Wiley-Liss, Inc.     

p75NTR expression in rat urinary bladder sensory neurons and spinal cord with cyclophosphamide-induced cystitis

A role for nerve growth factor (NGF) in contributing to increased voiding frequency and altered sensation from the urinary bladder has been suggested. Previous studies have examined the expression and regulation of tyrosine kinase receptors (Trks) in micturition reflexes with urinary bladder inflammation. The present studies examine the expression and regulation of another receptor known to bind NGF, p75(NTR), after various durations of bladder inflammation induced by cyclophosphamide (CYP). CYP-induced cystitis increased (P < or = 0.001) p75(NTR) expression in the superficial lateral and medial dorsal horn in L1-L2 and L6-S1 spinal segments. The number of p75(NTR)-immunoreactive (-IR) cells in the lumbosacral dorsal root ganglia (DRG) also increased (P < or = 0.05) with CYP-induced cystitis (acute, intermediate, and chronic). Quantitative, real-time polymerase chain reaction also demonstrated significant increases (P < or = 0.01) in p75(NTR) mRNA in DRG with intermediate and chronic CYP-induced cystitis. Retrograde dye-tracing techniques with Fastblue were used to identify presumptive bladder afferent cells in the lumbosacral DRG. In bladder afferent cells in DRG, p75(NTR)-IR was also increased (P < or = 0.01) with cystitis. In addition to increases in p75(NTR)-IR in DRG cell bodies, increases (P < or = 0.001) in pericellular (encircling DRG cells) p75(NTR)-IR in DRG also increased. Confocal analyses demonstrated that pericellular p75(NTR)-IR was not colocalized with the glial marker, glial fibrillary acidic protein (GFAP). These studies demonstrate that p75(NTR) expression in micturition reflexes is present constitutively and modified by bladder inflammation. The functional significance of p75(NTR) expression in micturition reflexes remains to be determined.     

Development of substance P-immunoreactive neurons in cranial sensory ganglia of the rat

Substance P-like immunoreactivity has been observed in fetal and adult cranial sensory ganglia. It first appears at day 16 of gestation in sensory neurons of trigeminal, superior-jugular, petrous and nodose ganglia, as well as in the autonomic myenteric plexus, and at day 17 in cervical dorsal root ganglion cells. Substance P immunoreactivity can be visualized much earlier (day 12) in the central nervous system. The ganglionic immunoreactivity subsequently increases during fetal life but drops at birth. The reactive material is first diffuse, then slowly becomes granular, and is mostly concentrated in coarse perinuclear inclusions in adult sensory neurons. Most substance P-positive neurons in trigeminal and superior-jugular ganglia are small, but medium-sized and large positive neurons are also observed in the trigeminal, petrous and nodose ganglia.Our observations give a precise picture of the development of substance P immunoreactivity in sensory neurons and are in general agreement with previous reports on some fetal and adult rat sensory ganglia. They indicate that in the rat, maturation of peripheral substance P-containing sensory neurons is slower than that of central substance P neurons or equivalent sensory neurons in other species. The examination of fetal material allows the observation of numerous immunoreactive sensory neurons which cannot be visualized after birth. We hypothesize a possible different embryonic origin (neural crest or placodal) for small nociceptive and larger substance P-containing neurons in rat cranial sensory ganglia.     

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segements lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly. Since S14 and S28 but not S11.5 kdalton are transported via the dorsal root, the dorsal root section would be predicted to produce a relatively greater decrease in S14 and S28 than in S11.5 kdalton. Therefore, failure to find a selective loss of S14 and S28 suggests that dorsal rhizotomy affects dorsal horn IRS content not only by removing afferent input but possibly also by modifyinh the processing of IRS by the remaining somatostatinergic neurons.     

Somatostatin-like neurons are expressed in fetal neocortical homografts in adult rat spinal cord

Fetal central nervous system homografts to adult spinal cord are considered a potential aid for recovery of function after paraplegia. This study utilizes somatostatin (SOM) immunohistochemistry to study the organization of an embryonic day 14 (E14) neocortical homograft into the spinal cord of an adult host over 6 postoperative months. Although the E14 homograft does not contain SOM-positive cells, SOM-reactive neurons are expressed by 30 days postimplantation and are still present in 6-month-old homografts. SOM-immunoreactive neurons are bitufted or multipolar and have dendrites that are confined to the graft. The homograft contains SOM-immunoreactive axons entering and/or exiting from lamina II in the host dorsal horn and SOM-positive homografted neurons send axons into the host ventral columns. These data show that the SOM peptide neocortical phenotype is preserved in homografts to spinal cord but there is anatomical host-homograft integration.     

Characterization of nerve growth factor binding to embryonic rat spinal cord neurons

The binding of iodinated beta-nerve growth factor, [125I]-NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 x 10(-11) M and 4 x 10(-9) M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]-NGF binding to each metrizamide fraction showed that the motoneuron-enriched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.     

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injur y

The purpose of this work was to investigate whether, by intranasal administration, the nerve growth factor bypasses the blood-brain barrier and turns over the spinal cord neurons and if such therapeutic approach could be of value in the treatment of spinal cord injury. Adult Sprague-Dawley rats with intact and injured spinal cord received daily intranasal nerve growth factor administration in both nostrils for 1 day or for 3 consecutive weeks. We found an in-creased content of nerve growth factor and enhanced expression of nerve growth factor receptor in the spinal cord 24 hours after a single intranasal administration of nerve growth factor in healthy rats, while daily treatment for 3 weeks in a model of spinal cord injury improved the deifcits in locomotor behaviour and increased spinal content of both nerve growth factor and nerve growth factor receptors. These outcomes suggest that the intranasal nerve growth factor bypasses blood-brain barrier and affects spinal cord neurons in spinal cord injury. They also suggest exploiting the possible therapeutic role of intranasally delivered nerve growth factor for the neuroprotection of damaged spinal nerve cells.     

Anterograde transport of horseradish-peroxidase conjugated isolectin B4 from Griffonia simplicifolia I in spinal primary sensory neurons of the rat

Anterograde transport of the isolectin B4 from Griffonia simplicifolia I (B4) conjugated to horseradish peroxidase (HRP) was investigated in rat somatic and visceral primary sensory neurons at different spinal levels. Injection of B4-HRP into the L5 dorsal root ganglion (DRG) resulted in labelling in the sural nerve, but not in the gastrocnemius nerves. Free nerve endings and lanceolate-like nerve endings were labelled in the lateral hindpaw skin. Labelled fibres were also observed in the greater splanchnic nerve following B4-HRP injection into the T10–11 DRGs. Electron microscopic examination of the labelled nerves showed that B4-HRP labelled exclusively unmyelinated axons. In the spinal cord, labelling was observed in the superficial dorsal horn, and additionally, although much more sparse, in the medial and lateral collateral projections following injections into the T10–11 DRGs. The results suggest that B4-HRP should be a suitable anterograde tracer of unmyelinated cutaneous and splanchnic but not muscle primary afferent fibres.     

Immunohistochemical detection of glutamate in rat vagal sensory neurons

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

Development of commissural neurons in the embryonic rat spinal cord.

Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.     

Ascending myelinated axons of primary sensory neurons of the sciatic nerve in the posterior column of the rat spinal cord

The present study was undertaken to obtain morphologic data about the posterior column of the spinal cord to characterize ascending myelinated axons of primary sensory neurons of the sciatic nerve. By applying doxorubicin to the right sciatic nerve in eight male Wistar rats, selective degeneration of centrally directed axons of these neurons in the posterior column was produced. Epon-embedded transverse sections of the posterior column at spinal cord segments C1, C3, C8, T6, L3 and L5 showed a circumscribed area (R) that contained a cluster of degenerated myelinated fibers. To characterize area R, its size and distances between various defined points on transverse sections of the posterior column were measured and compared at several spinal segments. The location of area R was illustrated in representative rats. The posterior intermediate septum corresponded to the lateral border of area R at C8 and T6. To characterize the putatively degenerating and degenerated myelinated fibers, area L in the left posterior column, corresponding to area R, was defined, and subsequently the number and size distribution of normal-appearing myelinated fibers in areas R and L were evaluated at C3, T6 and L3 in four rats. After comparative evaluation of these data, it was concluded that large myelinated fibers degenerated preferentially in area R. The number of putatively degenerating and degenerated myelinated fibers in area R at segments C3 and T6 was estimated to be 38.6% and 50.1%, respectively, of that at segment L3. Received: 25 August 1995 / Revised, accepted: 25 January 1996     

Pathfinding and growth termination of primary trigeminal sensory afferents in the embryonic rat hindbrain

Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets.