Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Nkx6-1 controls the identity and fate of red nucleus and oculomotor neurons in the mouse midbrain.

Little is known about the cues controlling the generation of motoneuron populations in the mammalian ventral midbrain. We show that Otx2 provides the crucial anterior-posterior positional information for the generation of red nucleus neurons in the murine midbrain. Moreover, the homeodomain transcription factor Nkx6-1 controls the proper development of the red nucleus and of the oculomotor and trochlear nucleus neurons. Nkx6-1 is expressed in ventral midbrain progenitors and acts as a fate determinant of the Brn3a(+) (also known as Pou4f1) red nucleus neurons. These progenitors are partially dorsalized in the absence of Nkx6-1, and a fraction of their postmitotic offspring adopts an alternative cell fate, as revealed by the activation of Dbx1 and Otx2 in these cells. Nkx6-1 is also expressed in postmitotic Isl1(+) oculomotor and trochlear neurons. Similar to hindbrain visceral (branchio-) motoneurons, Nkx6-1 controls the proper migration and axon outgrowth of these neurons by regulating the expression of at least three axon guidance/neuronal migration molecules. Based on these findings, we provide additional evidence that the developmental mechanism of the oculomotor and trochlear neurons exhibits more similarity with that of special visceral motoneurons than with that controlling the generation of somatic motoneurons located in the murine caudal hindbrain and spinal cord.

Neural organization of the pathways from the superior colliculus to trochlear motoneurons.

The neural organization of the pathways from the superior colliculus (SC) to trochlear motoneurons was analyzed in anesthetized cats using intracellular recording and transneuronal labeling techniques. Stimulation of the ipsilateral or contralateral SC evoked excitation and inhibition in trochlear motoneurons with latencies of 1.1-2.3 and 1.1-3.8 ms, respectively, suggesting that the earliest components of excitation and inhibition were disynaptic. A midline section between the two SCs revealed that ipsi- and contralateral SC stimulation evoked disynaptic excitation and inhibition in trochlear motoneurons, respectively. Premotor neurons labeled transneuronally after application of wheat germ agglutinin-conjugated horseradish peroxidase into the trochlear nerve were mainly distributed ipsilaterally in the Forel's field H (FFH) and bilaterally in the interstitial nucleus of Cajal (INC). Consequently, we investigated these two likely intermediaries between the SC and trochlear nucleus electrophysiologically. Stimulation of the FFH evoked ipsilateral mono- and disynaptic excitation and contralateral disynaptic inhibition in trochlear motoneurons. Preconditioning stimulation of the ipsilateral SC facilitated FFH-evoked monosynaptic excitation. Stimulation of the INC evoked ipsilateral monosynaptic excitation and inhibition, and contralateral monosynaptic inhibition in trochlear motoneurons. Preconditioning stimulation of the contralateral SC facilitated contralateral INC-evoked monosynaptic inhibition. These results revealed a reciprocal input pattern from the SCs to vertical ocular motoneurons in the saccadic system; trochlear motoneurons received disynaptic excitation from the ipsilateral SC via ipsilateral FFH neurons and disynaptic inhibition from the contralateral SC via contralateral INC neurons. These inhibitory INC neurons were considered to be a counterpart of inhibitory burst neurons in the horizontal saccadic system.

The effects of unilateral eighth nerve block on fictive VOR in the turtle.

Multiunit activity during horizontal sinusoidal motion was recorded from pairs of oculomotor, trochlear, or abducens nerves of an in vitro turtle brainstem preparation that received inputs from intact semicircular canals. Responses of left oculomotor, right trochlear and right abducens nerves were approximately aligned with leftward head velocity, and that of the respective contralateral nerves were in-phase with rightward velocity. We examined the effect of sectioning or injecting lidocaine (1-2 microL of 0.5%) into the right vestibular nerve. Nerve block caused a striking phase shift in the evoked response of right oculomotor and left trochlear nerves, in which (rightward) control responses were replaced by a smaller-amplitude response to leftward table motion. Such "phase-reversed" responses were poorly defined in abducens nerve recordings. Frequency analysis demonstrated that this activity was advanced in phase relative to post-block responses of the respective contralateral nerves, which were in turn phase-advanced relative to pre-block controls. Phase differences were largest (approximately 10 degrees) at low frequencies (approximately 0.1 Hz) and statistically absent at 1 Hz. The phase-reversed responses were further investigated by eliminating individual canal input from the left labyrinth following right nVIII block, which indicated that the activation of the vertical canal afferents is the source of this activity.

The extraocular motor nuclei: organization and functional neuroanatomy.

The organization of the motoneuron subgroups in the brainstem controlling each extraocular eye muscle is highly stable through the vertebrate species. The subgroups are topographically organized in the oculomotor nucleus (III) and are usually considered to form the final common pathway for eye muscle control. Eye muscles contain a unique type of slow non-twitch, fatigue-resistant muscle fiber, the multiply innervated muscle fibers (MIFs). The recent identification the MIF motoneurons shows that they too have topographic organization, but very different from the classical singly innervated muscle fiber (SIF) motoneurons. The MIF motoneurons lie around the periphery of the oculomotor nucleus (III), trochlear nucleus (IV), and abducens nucleus (VI), slightly separated from the SIF subgroups. The location of four different types of neurons in VI are described and illustrated: (1) SIF motoneurons, (2) MIF motoneurons, (3) internuclear neurons, and (4) the paramedian tract neurons which project to the flocculus. Afferents to the motoneurons arise from the vestibular nuclei, the oculomotor and abducens internuclear neurons, the mesencephalic and pontine burst neurons, the interstitial nucleus of Cajal, nucleus prepositus hypoglossi, the supraoculomotor area and the central mesencephalic reticular formation and the pretectum. The MIF and SIF motoneurons have different histochemical properties and different afferent inputs. The hypothesis that SIFs participate in moving the eye and MIFs determine the alignment seems possible but is not compatible with the concept of a final common pathway.

Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans.

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

Intracranial portion of the trochlear nerve and dorsal oblique muscle composition in dog: a structural and ultrastructural study.

In the present investigation the right intracranial portion of the trochlear nerves and dorsal oblique muscle of the right ocular globe were removed from six adult dogs and analyzed by light and electron microscopy. Unmyelinated fibers were observed in the analyzed nerves. The number, diameter, area, and density of myelinated fibers were determined, as were corresponding axon area and diameter and myelin sheath thickness. Frequency histograms of myelin sheath thickness and fiber size show a bimodal distribution with a similar proportion of large and small fibers. Muscle samples were taken from the central portion of the muscle belly, subsequently frozen, cut, and stained with m-ATPase at pH 4.6. Fibers were classified as Type 1 or Type 2 according to their reaction to the m-ATPase and detailed morphologic and morphometric studies were made. The muscles showed two clearly distinct layers, a central layer and a peripheral layer, chiefly composed of Type 2 fibers. The fibers in the central layer were larger in size than those in the peripheral layer.

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys.

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.

Vestibuloocular reflex of the adult flatfish. III. A species-specific reciprocal pattern of excitation and inhibition.

In juvenile flatfish the vestibuloocular reflex (VOR) circuitry that underlies compensatory eye movements adapts to a 90 degrees relative displacement of vestibular and oculomotor reference frames during metamorphosis. VOR pathways are rearranged to allow horizontal canal-activated second-order vestibular neurons in adult flatfish to control extraocular motoneurons innervating vertical eye muscles. This study describes the anatomy and physiology of identified flatfish-specific excitatory and inhibitory vestibular pathways. In antidromically identified oculomotor and trochlear motoneurons, excitatory postsynaptic potentials (EPSPs) were elicited after electrical stimulation of the horizontal canal nerve expected to provide excitatory input. Electrotonic depolarizations (0.8-0.9 ms) preceded small amplitude (<0.5 mV) chemical EPSPs at 1.2-1.6 ms with much larger EPSPs (>1 mV) recorded around 2.5 ms. Stimulation of the opposite horizontal canal nerve produced inhibitory postsynaptic potentials (IPSPs) at a disynaptic latency of 1.6-1.8 ms that were depolarizing at membrane resting potentials around -60 mV. Injection of chloride ions increased IPSP amplitude, and current-clamp analysis showed the IPSP equilibrium potential to be near the membrane resting potential. Repeated electrical stimulation of either the excitatory or inhibitory horizontal canal vestibular nerve greatly increased the amplitude of the respective synaptic responses. These observations suggest that the large terminal arborizations of each VOR neuron imposes an electrotonic load requiring multiple action potentials to maximize synaptic efficacy. GABA antibodies labeled axons in the medial longitudinal fasciculus (MLF) some of which were hypothesized to originate from horizontal canal-activated inhibitory vestibular neurons. GABAergic terminal arborizations were distributed largely on the somata and proximal dendrites of oculomotor and trochlear motoneurons. These findings suggest that the species-specific horizontal canal inhibitory pathway exhibits similar electrophysiological and synaptic transmitter profiles as the anterior and posterior canal inhibitory projections to oculomotor and trochlear motoneurons. Electron microscopy showed axosomatic and axodendritic synaptic endings containing spheroidal synaptic vesicles to establish chemical excitatory synaptic contacts characterized by asymmetrical pre/postsynaptic membrane specializations as well as gap junctional contacts consistent with electrotonic coupling. Another type of axosomatic synaptic ending contained pleiomorphic synaptic vesicles forming chemical, presumed inhibitory, synaptic contacts on motoneurons that never included gap junctions. Altogether these data provide electrophysiological, immunohistochemical, and ultrastructural evidence for reciprocal excitatory/inhibitory organization of the novel vestibulooculomotor projections in adult flatfish. The appearance of unique second-order vestibular neurons linking the horizontal canal to vertical oculomotor neurons suggests that reciprocal excitation and inhibition are a fundamental, developmentally linked trait of compensatory eye movement circuits in vertebrates.

Differential expression of Group I metabotropic glutamate receptors in motoneurons at low and high risk for degeneration in ALS.

Glutamate excitotoxicity has been suggested to play a role in amyotrophic lateral sclerosis (ALS), yet it remains unclear why some groups of motoneurons (MNs) are more vulnerable to degeneration than others. Our aim was to compare, in normal adult rats, the expression of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) in MNs normally affected in ALS (XII and spinal MNs) with those which are spared (III and IV MNs). RT-PCR analysis of tissue punches taken from III and XII motor nuclei revealed mRNA for both 'a' and 'b' splice variants of the mGluR1 and mGluR5 receptor subtypes, with expression of the 'a' variant dominant for both receptor subtypes in III and XII nuclei. Immunolabeling for mGluR1a protein was strong in vulnerable (XII and spinal) but negligible in the resistant (III and IV) MNs. Immunoreactivity for mGluR5 was not detected in the cell bodies or proximal dendrites of any MN pool examined. Greater expression of mGluR1a receptor protein within vulnerable MN pools may predispose these neurons to neurodegeneration as seen in ALS.

Morphological identification of nitric oxide sources and targets in the cat oculomotor system.

Nitric oxide (NO) production by specific neurons in the prepositus hypoglossi (PH) nucleus is necessary for the correct performance of eye movements in alert cats. In an attempt to characterize the morphological substrate of this NO function, the distribution of nitrergic neurons and NO-responding neurons has been investigated in different brainstem structures related to eye movements. Nitrergic neurons were stained by either immunohistochemistry for NO synthase I or histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The NO targets were identified by cyclic guanosine monophosphate (cGMP) immunohistochemistry in animals treated with a NO donor immediately before fixation of the brain. Connectivity between cells of the NO-cGMP pathway was analyzed by injections of the retrograde tracers horseradish peroxidase or fast blue in different structures. The motor nuclei commanding extraocular muscles did not contain elements of the NO-cGMP pathway, except for some scattered nitrergic neurons in the most caudal part of the abducens nucleus. The PH nucleus contained the largest number of nitrergic cell bodies and a rich neuropil, distributed in two groups in medial and lateral positions in the caudal part, and one central group in the rostral part of the nucleus. An abundant cGMP positive neuropil was the only NO-sensitive element in the PH nucleus, where no cGMP-producing neuronal cell bodies were observed. The opposite disposition was found in the marginal zone between the PH and the medial vestibular nuclei, with a large number of NO-sensitive cGMP-producing neurons and almost no nitrergic cells. Both nitrergic and NO-sensitive cell bodies were found in the medial and inferior vestibular nuclei and in the superior colliculus, whereas the lateral geniculate nucleus contained nitrergic neuropil and a large number of NO-sensitive cell bodies. Some of the cGMP-positive neurons in the marginal zone and medial vestibular nucleus projected to the PH nucleus, predominantly to the ipsilateral side. These morphological findings may help to explain the mechanism of action of NO in the oculomotor system.

GABA-immunoreactive internuclear neurons in the ocular motor system of lampreys.

The presence of internuclear neurons in the abducens and oculomotor nuclei of lampreys [González, M.J., Pombal, M.A., Rodicio, M.C. and Anadón, R., Internuclear neurons of the ocular motor system of the larval sea lamprey, J. Comp. Neurol. 401 (1998) 1-15] indicates that coordination of eye movements by internuclear neurons appeared early during the evolution of vertebrates. In order to investigate the possible involvement of inhibitory neurotransmitters in internuclear circuits, the distribution of gamma-aminobutyric acid (GABA) in the extraocular motor nuclei of the lamprey was studied using immunocytochemical techniques. Small GABA-immunoreactive (GABAir) neurons were observed in the three ocular motor nuclei. Numerous GABAir neurons were observed in the group of internuclear neurons of the dorsal rectus oculomotor subnucleus. A second group of GABAir neurons was observed among and below the trochlear motoneurons. Two further groups of GABAir interneurons, periventricular and lateral, were located in the abducens nucleus among the cells of the caudal rectus and the ventral rectus motor subnuclei, respectively. In addition to the presence of GABAir neurons, in all the ocular motor nuclei the motoneurons were contacted by numerous GABAir boutons. Taken together, these results suggest that GABA is involved as a neurotransmitter in internuclear pathways of the ocular motor system of lampreys.

Localization of motoneurons innervating the extraocular muscles in the chameleon (Chamaeleo chameleon).

The topography and localization of motoneurons innervating the six extraocular muscles in the chameleon (Chamaeleo chameleon) was studied following HRP injection in each of these individual muscles. Four muscles were innervated ipsilaterally: medial rectus, inferior rectus, inferior oblique and lateral rectus. The medial rectus muscle was innervated by the dorsomedial part of the oculomotor nucleus. The innervation to the inferior rectus muscle arose from the lateral part of the intermediate oculomotor subnucleus, which extended to the lateral part of the dorsal subdivision. The lateral rectus muscle was innervated by the abducens nucleus, which was composed by two subgroups of labeled cells, respectively observed in the principal and accessory abducens subnuclei, whereas efferents to the inferior oblique muscle originated from both the ventral and intermediate oculomotor subnuclei. The contralateral pattern consisted of motoneurons innervating the superior rectus and the superior oblique that were located respectively in the caudal portion of the ventral oculomotor nucleus and in the trochlear nucleus. These results confirmed data reported in most vertebrate species, and were discussed from a comparative and functional point of view.

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.

Further observations on the presence of ganglion cells in the oculomotor nerves of mammals and fish: number, origin and probable functions.

The oculomotor nerves (3rd, 4th and 6th) of some species of fish and mammals have been studied to establish the presence, number, true topography and probable functional role of the ganglion cells located along the trunk. The finding of typical pseudo-unipolar ganglion cells is always unpredictable and extremely variable, from an inter- and intra-specific point of view, in members of the two zoological classes studied.

The oculomotor and trochlear nuclei in the one humped camel (Camelus dromedarius).

Literature on the organization of the oculomotor and trochlear nuclei of large animals is scanty. There were no reports on the organization of the oculomotor and trochlear nuclei of the camel, hence this study. Nine brains were used for the study. The brainstems were double-embedded in celloidin and paraffin and were cut serially at 24 microns and stained with toluidine blue. Light microscopic studies of the nuclei showed that the principal oculomotor nuclei were not subdivided and were composed of large multipolar nerve cell bodies that had a mean length of 30 +/- 5 microns. The nucleus was 2.4 mm long, 0.7 mm wide and 1.1 mm high. The Edinger-Westphal nucleus was small and was made up of elongated oval cell bodies that had a mean length of 33 +/- 5 microns and a mean diameter of 10 +/- 2 microns. The trochlear nucleus was located caudal to the oculomotor nucleus from which it was separated by a gap. The nerve cell bodies of the trochlear nuclei were similar to those of the oculomotor nuclei. The cell bodies had a mean length of 20 +/- 2.5 microns and a mean width of 18 +/- 3 microns. The caudal central nucleus was indistinct. It was concluded, that the oculomotor and trochlear nuclei of the camel are similar in their general organization to those of other animals but differences exist in the development and organization of the component parts.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy.

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Axon-glial relations during regeneration of axons in the adult rat anterior medullary velum.

The anterior medullary velum (AMV) of adult Wistar rats was lesioned in the midsagittal plane, transecting all decussating axons including those of the central projection of the IVth nerve. At selected times up to 200 days after transection, the degenerative and regenerative responses of axons and glia were analyzed using transmission and scanning electron microscopy and immunohistochemistry. In particular, both the capacity of oligodendrocytes to remyelinate regenerated fibers and the stability of the CNS/PNS junctional zone of the IVth nerve rootlet were documented. Transected central AMV axons exhibited four patterns of fiber regeneration in which fibers grew: rostrocaudally in the reactive paralesion neuropil (Group 1); randomly within the AMV (Group 2); into the ipsilateral IVth nerve rootlet, after turning at the lesion edge and growing recurrently through the old degenerated contralateral central trochlear nerve trajectory (Group 3); and ectopically through paralesion tears in the ependyma onto the surface of the IVth ventricle (Group 4). Group 1-3 axons regenerated unperturbed through degenerating central myelin, reactive astrocytes, oligodendrocytes, microglia, and large accumulations of hematogenous macrophages. Only Group 3 axons survived long term in significant numbers, and all became myelinated by oligodendrocytes, ultimately establishing thin sheaths with relatively normal nodal gaps and intersegmental myelin sheath lengths. Schwann cells at the CNS/PNS junction of the IVth nerve rootlet did not invade the CNS, but astrocyte processes grew across the junction into the PNS portion of the IVth nerve. The basal lamina of the junctional glia limitans remained stable throughout the experimental period.

Comparative microanatomy of the lateral wall of the 'cavernous sinus' in humans and the olive baboon.

Despite many studies of the 'cavernous sinus' lateral wall, the anatomy of this area remains controversial. We performed a comparative microanatomical and histoarchitectural study in 14 humans and in 10 nonhuman primates (Papio cynocephalus anubis). Venous channels and cranial nerves were embedded in the 'interperiosteodural space'. The dura propria of the lateral wall could be removed without entering the venous compartment. The oculomotor and trochlear nerves were accompanied by an arachnoidal and dural sheath. The oculomotor nerve sheath stopped under the anterior clinoid process in baboons. The trigeminal ganglion was covered posteriorly with an arachnoid membrane and adhered firmly to the dura propria on lateral and anterior sections. The three branches of the trigeminal nerve had no arachnoid covering, except for arachnoid granulations in humans. In baboons, the oculomotor and trochlear nerves were thicker than in humans, while the ophthalmic nerve was thinner. The abducens nerve belonged to the lateral wall of the sinus in baboons and had no arachnoidal sheath except in the first millimeters of Dorello's canal. After leaving their arachnoidal and dural sheath, the intracavernous cranial nerves acquired a typical peripheral sheath. The venous channels in both species were true dural sinuses. Willis cords and adipose tissue were identified.