Ultrastructural localization of phospholipid synthesis in the rat trigeminal nerve during myelination.

A method for the ultrastructural localization of acyltransferase enzymes involved in phospholipid metabolism has been applied to the developing rat trigeminal nerve. Determination of acyltransferase levels in the nerve indicated that a peak of activity occurs at the 8th day after birth with gradual declines of activity up to 15 days. Morphological surveys and determinations of cholesterol levels suggested that heavy myelin formation occurs in the nerve during this latter period. Fixed nerves incubated in a medium for localization of acyltransferases indicated deposition of reaction product associated with Golgi cisternae, intracellular smooth vesicles, and the plasma membrane of the Schwann cell in the incipient stages of myelin formation. Golgi-derived vesicles appeared to move toward the Schwann cell surface and fuse with the plasma membrane. Activity continued to be detectable in the plasma membrane of the internal mesaxon as long as cytoplasm was evident and mature myelin membrane was not yet formed. Cells in which myelin formation appeared advanced showed little or no enzyme marker. Consistent with cytochemical observations were biochemical determinations of acyltransferases which showed high levels of the enzymes in microsomes, while no activity could be detected in the myelin fraction. Acyltransferase reaction product was also observed in the Golgi apparatus of ganglion cell bodies, axoplasmic smooth vesicles, and the axolemma. Localization of acyltransferase enzymes in Schwann cells, ganglion cell bodies, and axons during development of the nerve is discussed in relation to membrane biogenesis in the nervous system.

Differences in the distribution of catecholamine varicosities in cat and rat reticular formation.

The distribution of catecholamine varicosities within the brainstem reticular formation of the immature cat was determined by means of the formaldehyde-induced fluorescence technique. A continuous pattern of intense, green, medium-sized varicosities exists at nearly all brainstem levels. At most of these levels the varicosities appear within the boundaries of reticular formation nuclei. However, in rostral mesencephalon, some of the varicosities of the pattern lie in proximity to perikarya of the red nucleus. In addition, numerous varicosities in caudal medulla appear to extend from the pattern into nonreticular formation nuclei. A comparable pattern of reticular formation fluorescence is absent in the rat and this finding is believed to represent a true interspecies difference.

Cerebral arterial innervation by nerve fibers containing calcitonin gene-related peptide (CGRP): I. Distribution and origin of CGRP perivascular innervation in the rat.

The origin, density and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in cerebral perivascular nerves and the trigeminal ganglion of rats were examined in this study. CGRP immunoreactive axons were abundant on the walls of the rostral circulation of the major cerebral arteries in the circle of Willis. The fibers form a grid- or meshwork of longitudinal and circumferential axons studded with numerous varicose swellings. The density of CGRP fibers was particularly high at the bifurcation of major arteries. A few CGRP fibers cross the midline to innervate arteries on the contralateral side of the arterial tree. The arteries of the caudal circulation were sparsely innervated by CGRP fibers. In the trigeminal ganglion, about 30% of the ganglion cells had CGRP immunoreactivity. The cell size of most (75%) of CGRP neurons was less than 30 micron in diameter. There was no significant difference in staining density between small and large CGRP neurons. Unilateral transection of the maxillary and mandibular divisions of the trigeminal nerve caused a substantial decrease of CGRP immunoreactivity in the ipsilateral dorsal two-thirds of the trigeminal nucleus and cervical spinal cord but did not noticeably change the diameter of the vascular lumen or the densities of CGRP fibers in the walls of the cerebral arteries. In contrast, unilateral transection that included the ophthalmic division eliminated CGRP fibers on the ipsilateral cerebral arteries and eliminated CGRP immunoreactivity throughout the trigeminal nucleus in the brainstem and rostral cervical cord. In addition, these lesions caused a significant reduction in the diameter of the denervated arteries. The present study demonstrates that CGRP, a putative neurotransmitter/neuromodulator, is especially abundant in the rostral cerebral circulation and is derived from the ipsilateral ophthalmic division of the trigeminal nerve. In addition, the loss of CGRP perivascular nerves is associated with a reduction of the arterial lumen. This suggests that CGRP is a strong candidate as a nerve-derived trophic factor at trigeminal terminals and provides additional evidence that CGRP is a component in the trigeminovascular system influencing vascular diameter.

Substance P-like immunoreactive fibers in the trigeminal sensory nuclei of the pit viper, Trimeresurus flavoviridis.

Substance P-like immunoreactive nerve fibres were located in the trigeminal sensory system of the infrared-sensitive snake, Trimeresurus flavoviridis, using the immunohistochemical method. There are two trigeminal sensory systems in the medulla of this animal: the descending nucleus and the lateral descending nucleus. The descending nucleus is equivalent to the trigeminal spinal nucleus in other vertebrates, and the lateral descending nucleus is a special trigeminal sensory nucleus belonging to the infrared sensory system. In the present study we determined that the lateral descending nucleus is completely ensheathed by large numbers of substance P-like immunoreactive fibers. The distribution of these fibers seems to be similar to that of the thin vagal unmyelinated fibers, rather than to that of the thick trigeminal myelinated fibers. More substance P-like immunoreactive nerve fibers were observed in the lateral descending tract than in the descending tract. Almost no dense substance P-like immunoreactive fibers were found in these tracts rostral to the lateral descending nucleus or rostral to the subnucleus caudalis of the descending nucleus. The substance P-like immunoreactive fibers in the lateral descending tract extended to those of Lissauer's tract of the spinal cord, and the substance P-like immunoreactive fibers surrounding the Lissauer's tract were similar in appearance to those of the lateral descending nucleus. This nucleus seems to have developed from the elements existing in Lissauer's tract, and also to have a similar modulating function. The primary nucleus of the infrared sensory system is the most substance P-like immunoreactive nucleus in the trigeminal sensory system of this animal. Even in the trigeminal sensory system, substance P-like immunoreactive fibers seem not to be related solely to the nociceptive sensation.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study.

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

Pia arachnoid contains substance P originating from trigeminal neurons.

Immunoreactive substance P is present in measurable amounts in pia arachnoid from rat, cat, dog and calf. Levels of substance P in this tissue are comparable to those found in peripheral structures receiving innervation from dorsal root or trigeminal ganglia. Separation and measurement of bovine pia-arachnoid extract by reverse phase high performance liquid chromatography and radioimmunoassay reveals a single peak of activity with a retention time identical to that of substance P. Unilateral lesions of the trigeminal ganglia decrease substance P levels within cat pia arachnoid and accompanying blood vessels ipsilaterally by greater than 50%. These data indicate that most of the substance P surrounding pial blood vessels resides within afferent nerve fibres from trigeminal ganglion cells.

Nerve fibers surrounding intracranial and extracranial vessels from human and other species contain dynorphin-like immunoreactivity.

Dynorphin B(20-32) was visualized by immunohistochemistry in guinea-pig and rat perivascular nerve fibers and was measured by radioimmunoassay within the walls of feline, canine, bovine and human cephalic and systemic arteries and veins. Canine vessels contained the highest levels. When human blood vessels or trigeminal ganglia were subjected to reverse-phase high-performance liquid chromatography, dynorphin immunoreactivity exhibited a retention time identical to that of synthetic dynorphin B. No differences in dynorphin-like immunoreactivity were measurable between feline systemic arteries and veins, or between cephalic and systemic vessels. The highest amounts were present in leptomeninges devoid of large pial arteries. Relatively high levels were also measured in feline and human trigeminal ganglia and feline superior cervical and sphenopalatine ganglia, three sources of projecting perivascular axons. Levels did not diminish, however, in ipsilateral feline cephalic vessels following either unilateral trigeminal or superior cervical ganglionectomies. Hence, dynorphin-containing fibers may project from parasympathetic cell bodies or perhaps from intrinsic brain sources. Previously published reports indicate that the kappa agonist dynorphin does not modify vessel tone when added in vitro but does inhibit release of neurotransmitters from afferent and sympathetic axons via prejunctional receptors. These observations suggest a pharmacological role for dynorphin on sensory and autonomic functions of the vasculature.

ATP is released into the rabbit eye by antidromic stimulation of the trigeminal nerve.

Antidromic stimulation of the trigeminal nerve produces an irritative response in the rabbit eye characterized by ipsilateral miosis, hyperemia, elevated intraocular pressure, and a disruption of the blood-aqueous barrier. The latter is a bilateral effect. The mediator or mediators involved in this response of the eye are unknown. Increased ATP levels in aqueous humor could be found after trigeminal stimulation. Treatment of rabbits with dipyridamole further increased ATP levels in aqueous humor after stimulation, confirming the findings of Holton that stimulation of sensory nerves causes a release of ATP. Intravitreal injections of ATP could not reproduce the ocular irritative response; however, an iridial hyperemia of long latency and an increase in aqueous humor protein levels were produced. The mechanism of this part of the reaction requires further study.

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system.

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations. The lack of any rise in the brainstem CGRP content raises the possibility that this process spares central terminations. In contrast, the level of neuropeptide Y, a peptide mainly contained in sympathetic terminals, fell to 35% of control values in the iris and pial arteries whilst the trigeminal ganglion and brainstem concentrations remained unchanged. The possible relevance of these observations to the clinical syndrome of postsympathectomy pain (sympathalgia) is discussed. There are similarities between the delayed onset of the human pain state and the delayed rise in sensory peptides after sympathectomy.

Insulin receptors in the peripheral nervous system: a structural and functional analysis.

Although the brain is known to contain specific insulin receptors, there is no information on whether these receptors are also present in the peripheral nervous system (PNS). The present studies sought to provide this information by characterizing insulin binding in bovine autonomic (superior cervical) and sensory (trigeminal) ganglia. It was found that both ganglia contain specific, high-affinity receptors for insulin. Like insulin receptors in other tissue, these receptors could be solubilized and purified on wheat germ agarose columns and were found to have tyrosine-specific kinase activity. SDS-PAGE and autoradiography revealed that the apparent molecular weight (Mr) of the PNS insulin receptor was approximately 133 kDa which is similar to the Mr of hepatic receptors, but is approximately 10 kDa larger than the insulin receptor found in the brain. Because the vasculature of autonomic and sensory ganglia is fenestrated, it is possible that PNS insulin receptors are exposed to blood-borne insulin.

A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin-like immunoreactivity with retrogradely labeled trigeminal ganglion cells innervating the eye.

To evaluate the intraganglionic organization of ocular sensory neurons in the guinea pig, we studied the retrograde axonal transport from the eye to the trigeminal ganglion of cholera toxin B subunit and then applied immunohistochemistry for substance P, calcitonin gene-related peptide and cholecystokinin. Retrogradely labeled cells were observed only in the anteromedial portion of the ipsilateral ganglion. We observed no somatotopical organization to trigeminal neurons containing any of these three peptides, either for cells projecting to the eye or for the ganglion as a whole. The relative proportion of neurons immunoreactive for each of these three peptides was similar among the population of neurons retrogradely labeled with cholera toxin B and among the population of neurons without direct projections to the eye.

Comparison of the distribution of Na+,K+-ATPase and myelin-associated glycoprotein (MAG) in the optic nerve, spinal cord and trigeminal ganglion of shiverer (shi/shi) and control (+/+) mice.

Na+,K+ ATPase and myelin-associated glycoprotein (MAG) were studied by immunocytochemistry on paraffin sections of the spinal cord, optic nerve and trigeminal ganglion of adult control (+/+) and CNS myelin-deficient shiverer (shi/shi) mice. Immunostaining for Na+, K+-ATPase outlined the periphery of nerve fibers in the spinal cord white matter, optic nerve and trigeminal ganglion of +/+ and shi/shi mice. Immunostaining for Na+,K+-ATPase appeared somewhat denser in the optic nerve and spinal cord lateral funiculi of shi/shi than in +/+ mice. In addition, immunostaining for Na+,K+-ATPase was demonstrated at the plasmalemma of presumed satellite cells situated at the periphery of ganglion cell bodies in the trigeminal ganglion of both species of mice. Immunostaining for MAG was localized along the periphery of nerve fibers in the spinal cord funiculi (with little immunostaining within gray horns), optic nerve and trigeminal ganglion of both +/+ and shi/shi mice. The major differences between shi/shi and +/+ mice were that the number of MAG-immunostained nerve fibers was greatly reduced in the spinal cord funiculi and the density of immunostaining was slightly increased in the optic nerve of shi/shi mice. The numbers of MAG-immunostained nerve fibers in trigeminal ganglion were similar in both species. Also, the cytoplasm of some oligodendrocyte-like cells was found densely immunostained for MAG in the spinal cord and optic nerve of shi/shi mice, but not of +/+ mice. This light microscopic study provides evidence that the defective shiverer gene leads to a decrease in MAG deposition and to aggregations of MAG-like material within perikarya of oligodendrocyte-like cells in regions of the CNS.

Mitogenic effect of myelinated vs unmyelinated garfish nerve extracts.

Mitogenic activities in crude extracts of unmyelinated olfactory nerves and myelinated trigeminal nerves of the garfish Lepisosteus osseus were compared. Extracts of each nerve type were added in a range of protein concentrations to serum-starved, subconfluent cultures of BALB/c 3T3 cells. At low protein concentrations (50-250 micrograms/ml) myelinated nerve extracts produced more [3H]thymidine incorporation in the cultured cells than unmyelinated nerve extracts, while at higher concentrations (500-1000 micrograms/ml), the latter caused as much DNA synthesis as the myelinated nerve extracts, surpassing them at the highest concentrations tested. The results suggest that both axonal and myelin components contribute to the growth-promoting activity in nerve tissue.

Selective localization of alpha-fetoprotein and serum albumin within the sensory ganglia cells of developing chicken.

The presence of alpha-fetoprotein (AFP) and serum albumin (SA) has been described within developing brain cells of mammals and chicken. In order to avoid the influence of the choroid plexuses in the transport, via the cerebrospinal fluid, of these proteins to brain cells, we studied the presence of AFP and SA within chicken sensory ganglion cells during ontogeny using the indirect immunoperoxidase technique. Our results show that before day 9 of egg incubation, both AFP and SA can be detected within the trigeminal and spinal ganglia cells. Henceforth, the labeling for AFP starts decreasing in the spinal ganglia but not in the trigeminal ganglion. The labeling for SA remains constant in both structures. Differences between AFP and SA staining in the spinal ganglia are maximal on day 11 of incubation, when AFP is no longer detected. It is concluded that at this time, the uptake of AFP by spinal ganglia cells is switched-off while SA is still taken up. As the blood concentration of both proteins is similar at this time, the finding reported here gives support to the advanced hypothesis suggesting the presence of specific receptors for AFP and/or SA in embryonic neural cells.

Trigeminal origin of beta-preprotachykinin products in feline pial blood vessels.

A specific and sensitive radioimmunoassay measured the presence of immunoreactive neurokinin A within feline and human pial arteries. Immunoreactivity exhibited a retention time identical to that of synthetic peptide when acid extracts from feline and human blood vessels were subjected to reverse-phase high-performance liquid chromatography. As shown previously for substance P, levels of immunoreactive neurokinin A decreased significantly in the vessels from the ipsilateral rostral circle of Willis 19-24 days following unilateral trigeminal ganglionectomy. Hence, trigeminal projections to cerebral blood vessels contain both products of beta-preprotachykinin mRNA.

Calcitonin gene-related peptide-immunoreactive nerve fibers in mandibular periosteum of rat: evidence for primary afferent origin.

Peptidergic neurons may play a role in the local regulation of bone mineralization. The neuropeptide vasoactive intestinal peptide (VIP) increases bone resorption in vitro, while calcitonin gene-related peptide (CGRP) has been shown to inhibit bone resorption in vitro. We have previously reported that sympathetic nerves with VIP-immunoreactivity innervate bone and periosteum. In the present study we sought to determine if CGRP fibers, like VIP fibers, exist in periosteum and what their origin might be. In whole-mount preparations of mandibular periosteum from rat, CGRP- and VIP-immunoreactive (IR) nerve fibers were present as networks within the periosteum. In preparations using two-color immunofluorescence, most CGRP-IR fibers were also immunoreactive for substance P (SP). In rats in which the subperiosteal space subjacent to the mandibular molars was injected with Fast blue or Fluoro-gold, retrogradely labeled cells were seen in ipsilateral trigeminal ganglia, superior cervical ganglia, and nodose ganglia. Individual cells labeled with both CGRP immunoreactivity and retrograde tracer were seen only in the mandibular portion of the trigeminal ganglion. These data suggest that CGRP-IR nerve fibers in periosteum may be of primary afferent origin. Given the reported effects of CGRP on bone mineralization, the present results suggest that primary afferent nerves containing CGRP and SP, as well as sympathetic nerves containing VIP, may play a role in focal bone remodeling.

DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves.

Twelve calves infected with bovine herpesvirus type 1 (BHV-1) were killed when in a latent state of infection. Latency was verified 30 days after virus inoculation of the calves by seroconversion, absence of virus shedding, and in 2 calves, by recrudescence of the infection after they were treated with dexamethasone. By in situ hybridization techniques and autoradiography, DNA of BHV-1 was detected in 13 of 23 trigeminal ganglia of latently infected calves. Viral DNA was restricted to the nucleus of nerve cells. Single neurons harboring BHV-1 DNA were observed in 4.9% of the sections (n = 325) of the trigeminal ganglia. The results obtained correspond to those known from herpes simplex virus infections in mice. The implications for the virus-host relationship are discussed.

[Biochemical and immunocytochemical study of the axonal domain of mature and immature microtubules]. / Etude biochimique et immunocytochimique du domaine axonal des microtubules matures et immatures.

The axonal domain of microtubules was analyzed using biochemistry and immunocytochemistry. High resolution isofocusing was used to study the cold-labile and cold-stable fractions of various samples: thalamus, optic nerve, sciatic nerve, brachial plexus and trigeminal nerve. The cold-labile fraction from the thalamus, i.e. from a central nervous system cell population contains 20 well-resolved isotypes as the axonal fraction from the central nervous system. The cold-stable fraction from the peripheral nervous system contains only 18 isotypes. All the cold-stable fractions are characterized by an important relative quantity of isotypes 7-9, with specific patterns tissue dependent. The cold-labile fractions are specific in both the central and peripheral nervous system. Immunocytochemistry using anti-tubulin and anti-MAP2 specific antibodies was used in PEG semi-thin sections of the embryonic tectal plate at stage E10. In the axonal profiles, cold-stable fragments of microtubules were observed. The perikaryon of the young neurons, both migrating and post-migratory, contains MAP2 as the cold-labile fractions of microtubules of the axonal process. These results suggest that; tubulin is involved in the cold-stability of the axonal domain, but this role seems tissue dependent; MAP2 is a marker of neuronal differentiation; in the growing axonal process, cold-stable fragments are present and the cold-labile pool of microtubules contain a transiently expressed protein, MAP2.