The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

Axonal and transynaptic spread of prions.

UNLABELLED: Natural transmission of prion diseases depends upon the spread of prions from the nervous system to excretory or secretory tissues, but the mechanism of prion transport in axons and into peripheral tissue is unresolved. Here, we examined the temporal and spatial movement of prions from the brain stem along cranial nerves into skeletal muscle as a model of axonal transport and transynaptic spread. The disease-specific isoform of the prion protein, PrP(Sc), was observed in nerve fibers of the tongue approximately 2 weeks prior to PrP(Sc) deposition in skeletal muscle. Initially, PrP(Sc) deposits had a small punctate pattern on the edge of muscle cells that colocalized with synaptophysin, a marker for the neuromuscular junction (NMJ), in >50% of the cells. At later time points PrP(Sc) was widely distributed in muscle cells, but <10% of prion-infected cells exhibited PrP(Sc) deposition at the NMJ, suggesting additional prion replication and dissemination within muscle cells. In contrast to the NMJ, PrP(Sc) was not associated with synaptophysin in nerve fibers but was found to colocalize with LAMP-1 and cathepsin D during early stages of axonal spread. We propose that PrP(Sc)-bound endosomes can lead to membrane recycling in which PrP(Sc) is directed to the synapse, where it either moves across the NMJ into the postsynaptic muscle cell or induces PrP(Sc) formation on muscle cells across the NMJ. IMPORTANCE: Prion diseases are transmissible and fatal neurodegenerative diseases in which prion dissemination to excretory or secretory tissues is necessary for natural disease transmission. Despite the importance of this pathway, the cellular mechanism of prion transport in axons and into peripheral tissue is unresolved. This study demonstrates anterograde spread of prions within nerve fibers prior to infection of peripheral synapses (i.e., neuromuscular junction) and infection of peripheral tissues (i.e., muscle cells). Within nerve fibers prions were associated with the endosomal-lysosomal pathway prior to entry into muscle cells. Since early prion spread is anterograde and endosome-lysosomal movement within axons is primarily retrograde, these findings suggest that endosome-bound prions may have an alternate fate that directs prions to the peripheral synapse.

Bifurcation of axons from cranial sensory neurons is disabled in the absence of Npr2-induced cGMP signaling.

Axonal branching is a prerequisite for the establishment of complex neuronal circuits and their capacity for parallel information processing. Previously, we have identified a cGMP signaling pathway composed of the ligand C-type natriuretic peptide (CNP), its receptor, the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent kinase Iα (cGKIα) that regulates axon bifurcation of dorsal root ganglion (DRG) neurons in the spinal cord. Now we asked whether this cascade also controls axon bifurcation elsewhere in the nervous system. An Npr2-lacZ reporter mouse line was generated to clarify the pattern of the CNP receptor expression. It was found that during the period of axonal outgrowth, Npr2 and cGKIα were strongly labeled in neurons of all cranial sensory ganglia (gV, gVII, gVIII, gIX, and gX). In addition, strong complementary expression of CNP was detected in the hindbrain at the entry zones of sensory afferents. To analyze axon branching in individual Npr2-positive neurons, we generated a mouse mutant expressing a tamoxifen-inducible variant of Cre recombinase expressed under control of the Npr2-promoter (Npr2-CreER(T2)). After crossing this strain with conditional reporter mouse lines, we revealed that the complete absence of Npr2 activity indeed prohibited the bifurcation of cranial sensory axons in their entrance region. Consequently, axons only turned in either an ascending or descending direction, while collateral formation and growth of the peripheral arm was not affected. These findings indicate that in neurons of the cranial sensory ganglia, as in DRG neurons, cGMP signals are necessary for the execution of an axonal bifurcation program.

[Odontoblast-like cell phenotype differentiation of cranial neural crest stem cell in vivo].

OBJECTIVE: To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo. METHODS: Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry. RESULTS: With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells. CONCLUSION: By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

Differential distribution of calbindin D28k and parvalbumin among functionally distinctive sets of structures in the macaque brainstem.

In a study of brainstem in the cynomolgus monkey, we found that the distribution of calbindin D28K (CB) and parvalbumin (PV) is nonoverlapping among functionally distinct sets of brainstem structures. Nuclei involved in representation and regulation of the organism's internal state contain CB, whereas those involved in the representation of the external environment and the representation or execution of externally directed actions contain only PV. Moreover, our findings indicate that different nuclei known as components of the ascending reticular activating system (ARAS) contain either CB or PV or both, suggesting that this system in primates operates with both CB and PV. In line with previously reported findings, we also found that unmyelinated pathways contain only CB, whereas myelinated pathways contain PV. Distribution of CB and PV in the macaque brainstem follows a pattern comparable to, but in some instances significantly different than, the pattern previously reported in the rat. We argue that the nonoverlapping distribution of CB and PV among different structures of the brainstem might reflect underlying differences in the physiological, anatomic, and perhaps phylogenetic properties of these structures. Considering our recent findings of selective vulnerability of brainstem structures to Alzheimer's disease, the present data suggest that the majority of macaque brainstem nuclei that contain CB are vulnerable to neurofibrillary tangles in humans. By contrast, only few nuclei that contain PV exhibit pathologic changes. Some of these nuclei are affected with a high number of neuritic plaques without ever developing neurofibrillary tangles.

Alpha-melanophore-stimulating hormone in the brain, cranial placode derivatives, and retina of Xenopus laevis during development in relation to background adaptation.

The amphibian Xenopus laevis can adapt the color of its skin to the light intensity of the background. A key peptide in this adaptation process is alpha-melanophore-stimulating hormone (alpha-MSH), which is derived from proopiomelanocortin (POMC) and released by the endocrine melanotrope cells in the pituitary pars intermedia. In this study, the presence of alpha-MSH in the brain, cranial placode derivatives, and retina of developing Xenopus laevis was investigated using immunocytochemistry, to test the hypothesis that POMC peptide-producing neurons and endocrine cells have a common embryonic origin and a common function, i.e., controlling each other's activities and/or being involved in the process of physiological adaptation. The presence of alpha-MSH-positive cells in the suprachiasmatic nucleus, ventral hypothalamic nucleus, epiphysis, and endocrine melanotrope and corticotrope cells, which are all involved in regulation of adaptation processes, has been detected from stage 37/38 onward. This is consistent with the presumed common origin of these cells, the anterior neural ridge (ANR) of the neural-plate-stage embryo. The olfactory epithelium and the otic and epibranchial ganglia also contain alpha-MSH, indicating that these placodal derivatives originate from a common placodal domain continuous with the ANR. Furthermore, we demonstrate the presence of alpha-MSH in a subpopulation of retinal ganglion cells (RGCs), which is possibly also derived from the ANR. Immunoreactivity for alpha-MSH in RGCs that are located in the dorsal part of the retina is dependent on the background light intensity, suggesting that these cells are involved in the regulation of background adaptation. Taken together, the results support the hypothesis that POMC peptide-producing cells have a common embryonic origin and are involved in adaptation processes.

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri.

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates.

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study.

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.

Innervation pattern of substance P- and calcitonin gene-related peptide-immunoreactive nerves of the cerebral arteries in the quail.

The pattern of cerebrovascular substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) innervation was investigated in the quail. SP- and CGRP-IR nerves were relatively a few in the rostral part of the anterior circulation, and very scanty or lacking in its caudal part and the whole of the posterior circulation. A significant finding was that the anterior circulation in the majority of individuals is furnished with a varying proportion of SP-IR nerves with or without CGRP immunoreactivity. There was a good correlation in the expression of CGRP immunoreactivity between SP-IR cells in the ophthalmic division of the trigeminal ganglion and SP-IR nerves supplying the major cerebral arteries. In the quail, SP- and CGRP-IR fiber bundles are usually present in the internal ethmoidal artery (IEA). From these and other findings, it is most probable that cerebral perivascular SP- and CGRP-IR nerves are mainly derived from the same categories of neurons in the primary sensory ganglion via the IEA. The close association of varicose SP-IR axons to the nerve cells in the pial arteries suggests that these intrinsic neurons may play some vasocontrolling roles through the modulatory effect of their pericellular SP-IR axons.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

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.

Unilateral absence of the terminal nerve and distribution of gonadotropin-releasing hormone immunoreactive neurons in the brain of the common mole-rat (Cryptomys, Rodentia).

A paired terminal nerve with gonadotropin-releasing hormone-immunoreactive (GnRHir) neurons was found in five of six specimens of the Zambian common mole-rat (Cryptomys sp.). In these animals the distribution of GnRHir neurons in the CNS was approximately even on both sides. One adult female lacked a right terminal nerve, yet exhibited a comparable total number of GnRHir neurons, most of which were located on the left side of the brain, i. e., on that side where the terminal nerve was present. An additional population of GnRHir cells was detected in the area of the parafascicular and dorsomedial thalamic nuclei of three non-reproductive adult females, but not in young animals (one female, two males). The additional GnRHir cells, referred to as dark spot cells (DSCs) since their perikarya exhibit large or small strongly immunoreactive vacuoles, were present on both sides of the brain in equal numbers even in the specimen with unilateral absence of the terminal nerve. Obviously, the lack of one terminal nerve correlates with a drastic reduction in the number of ipsilateral genuine neurons but leaves the DSCs unaffected.

Coexistence of FMRFAMIDE-like and LHRH-like immunoreactivity in the terminal nerve and forebrain of the big brown bat, Eptesicus fuscus.

The coexistence of molluscan cardioexcitatory neuropeptide (FMRFAMIDE) and luteinizing hormone-releasing hormone (LHRH) was studied in the nervous system of the big brown bat, Eptesicus fuscus, with immunocytochemistry. Within mammals, this is the first report of the coexistence of these neuropeptides in the terminal nerve. In juvenile and adult bats, both neuropeptides are distributed identically throughout the terminal nerve (tn), and they coexist in many parts of the prosencephalon from the olfactory bulb as far caudally as the interpeduncular nucleus. Peripherally, on the basal surface of the forebrain, fibers and a few perikarya, which may belong to the tn, form a loose plexus. Within the brain wall, regions of maximal immunoreactivity (ir) are the habenula, medial preoptic area, arcuate nucleus, and the infundibulum. Whereas in most areas of the prosencephalon (e.g., stria terminalis and bed nuclei, amygdaloid complex) fibers show stronger immunoreactivity to FMRFAMIDE, labeling of fibers in the habenula and infundibulum is largely identical for both neuropeptides. The arcuate nucleus contains a large number of perikarya and is the major source of both FMRFAMIDE- and LHRH-ir within the forebrain. A number of fibers run along the ependyma of the ventricular system and seem to terminate here; this is particularly evident in the median eminence and infundibular stalk. In the big brown bat, there seems to exist a continuum of FMRFAMIDE- and LHRH-ir throughout the tn and those structures of the forebrain that are known to be engaged in the control of mating behavior, reproduction, and rhythmicity. Concerning the hypothalamo-hypophyseal-gonadal axis, the arcuate nucleus may serve as a central hub between the olfactory/terminal input and superior areas including the limbic system. In contrast to LHRH immunoreactivity, FMRFAMIDE-like ir extends throughout the brainstem and cervical spinal cord. This system may also be involved in the processing and modulation of autonomic input via the parabrachial and solitary nuclei, the rhombencephalic central gray, and its projection into the hypothalamus (paraventricular nucleus), thus facilitating feed-back of gonadotropic influences of the terminal nerve and prosencephalon.

Immunohistochemical demonstration of cytokeratin in human embryonic neurons arising from placodes.

Sensory neurons of the olfactory, trigeminal, facial, vestibulo-cochlear, glossopharyngeal and vagal nerves, and neurons migrating along the olfactory nerve to the brain have special anlagen, made up of placodes located in the epithelial layer. To investigate the characteristic phenotype of placode-derived neurons, immunohistochemical analysis of intermediate filaments was conducted on formalin-fixed human embryonic tissues. Neurons arising from placodes including luteinizing-hormone releasing hormone (LHRH) neurons migrating from the olfactory placode to the brain had immunoreactivity to antibodies specific to cytokeratin, AE1 and CAM5.2 during the embryonic stage. However, this immunoreactivity disappeared during the late embryonic to the post-embryonic stage and was not observed in the roots of these nerves in the post-natal stage. Immunoreactivity was detected in both the somata and processes, and the distribution differed from that described in rodent brain neurons. With this exception, no other human peripheral neurons, including spinal dorsal root ganglia, had immunoreactivity with anti-cytokeratin antibodies throughout the entire developmental stage. Although the cephalic neural crest also directly generates neurons to most of the cranial sensory ganglia, we could not find any evidence that it contributed to the genesis of cytokeratin-positive embryonic neurons. We concluded that cytokeratin is an intermediate filament common to human embryonic neurons of cephalic placodal origin and that this immunohistochemical marker may be useful in analyzing the developmental sequence of several congenital diseases involving the cranial nerves, such as Moebius syndrome and Goldenhar syndrome.

Melatonin receptors in red deer fetuses (Cervus elaphus).

Red deer (Cervus elaphus) exhibit highly seasonal rhythms in physiology and behaviour. The influence of photoperiod on the timing of these changes begins in utero where the fetus receives photoperiodic information via the diurnal pattern of maternal melatonin secretion. The potential sensitivity of deer fetuses to melatonin was ascertained by mapping specific receptors and characterizing them using 2-[125I]iodomelatonin and quantitative autoradiography in vitro. Specific binding occurred from day 31 of gestation onwards (term = 233 days) over the spinal nerves and respiratory system. At later stages of gestation binding occurred over the brain, particularly the brainstem, the pituitary gland, thyroid gland, gastrointestinal tract including the pancreas, metanephros, cochlea of the ear, spinal cord, and spinal and cranial nerves. Binding was abolished in the presence of 10(-7) mol melatonin l-1 and diminished in the presence of 10(-4) mol GTP gamma S l-1 (guanosine-5-O-(3-thiotriphosphate)), confirming that binding represented functional G-protein-coupled melatonin receptors. Characterization studies, carried out on fetal lung, revealed that binding was time-dependent, reaching equilibrium at about 3 h at room temperature (22 degrees C), and saturable with a dissociation constant (Kd) of 104 pmol l-1. This study demonstrates the presence of G-protein-coupled melatonin receptors over a wide range of tissues in red deer fetuses from early in gestation, indicating that in addition to its role in the communication of photoperiodic information to the fetus in this species, melatonin may be involved in fetal growth and development.

Immunohistochemical and Ultrastructural characterization of the terminal nerve ganglion cells of the ayu, Plecoglossus altivelis (Salmoniformes, Teleostei).

BACKGROUND: Little is known on the cytological properties of the terminal nerve ganglion (TNG) cells in teleosts (Demski, 1993. Acta Anat., 148:81-95). MATERIALS AND METHODS: To characterize the TNG cells of a salmonoid fish, Plecoglossus altivelis, we adopted immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The majority of the TNG cells formed a compact mass halfway between the olfactory sac and the olfactory bulb, whereas a few cells were scattered in the ventromedial region of the olfactory bulb. The cell had a voluminous perikaryon that was positive to antisera against gonadotropin-releasing hormone (GnRH), molluscan cardioexcitatory tetrapeptide (FM-RFamide), and neuropeptide Y (NPY). Immunostaining of consecutive sections with each antiserum showed the coexistence of these antigens in the same cells and their processes. Most of the processes originating from the cells projected centrally to the basal forebrain, including the optic nerve. With TEM, the cells revealed a peptidergic nature, i.e., the presence of abundant granular endoplasmic reticula and well-developed Golgi bodies in association with vesicles that were 70-100 nm in diameter. Occasionally, the cells adjoined one another directly without the intervention of glial sheets. Synaptic contacts were frequent in the proximal region of the processes, where thin lateral processes of the cells and axon terminals of unknown origin were intermingled with each other. Terminal buttons being engulfed by the soma were commonly seen. CONCLUSIONS: The TNG cells of the salmonoid fish share many cytological characteristics with the cells of the nucleus olfactoretinalis of advanced teleosts such as acanthopterygians.

Comparison between two HNK-1-related antibodies immunoreactivity (HNK-1-anti-leu 7 and anti-HNK-1/N-CAM) during rat cephalogenesis.

The fixation sites of two antibodies, HNK-1-anti-leu 7 and anti- HNK-1/N-CAM, were studied during visceral cephalogenesis in the rat. The labelling patterns of both antibodies were different. Anti-leu 7 staining was restricted to trunk neural crest cells, the peripheral nervous system, myotomes and premuscular cells, the eye rudiment and the olfactory vesicle. On the other hand, anti- HNK-1/N-CAM extended to the otocyst, several epithelia (including tooth germs) and precartilages. Anti-HNK-1/N-CAM staining of somites and cranial nerve ganglia was more precocious than anti-leu 7 labelling. We conclude that both antibodies, despite several common sites labelling, display quite different immunological properties, and that the expression "HNK-1" positive has no absolute significance.

Silver lampreys (Ichthyomyzon unicuspis) lack a gonadotropin-releasing hormone- and FMRFamide-immunoreactive terminal nerve.

The terminal nerve is a ganglionated cranial nerve with peripheral processes that enter the nasal cavity and centrally directed processes that enter the forebrain. Members of all classes of gnathostomes have been found to possess a terminal nerve, some components of which demonstrate immunoreactivity to the peptides Phe-Met-Arg-Phe-NH2 (FMRFamide) and gonadotropin-releasing hormone (GnRH). To explore the possibility that lampreys possess a terminal nerve, we examined the distribution of these peptides in the silver lamprey, Ichthyomyzon unicuspis, by using antisera to FMRFamide and to four forms of GnRH. We found cells with FMRFamide-like immunoreactivity in the preoptic area and the isthmal gray region of the mesencephalon, and found labeled fibers throughout the preoptic-infundibular region. Occasional labeled fibers were scattered through many regions of the brain, including the optic nerve and olfactory bulb; however, unlike species that possess a terminal nerve, lampreys have no immunoreactive cells or fibers in the olfactory nerve or nasal epithelia. In addition, we observed GnRH-immunoreactive cell bodies in the preoptic area of all animals and in the ventral hypothalamus of one individual. Most of the labeled fibers extended ventrally to the hypothalamus, with other fibers extending throughout the striatum and hypothalamic-neurohypophyseal region. A few fibers in other regions, including the optic nerve, were also labeled; we detected no immunoreactivity in the olfactory bulb, olfactory nerve, or nasal epithelia. The use of different GnRH antisera resulted in remarkably similar patterns of labeling of both cells and fibers. In summary, we did not observe either GnRH or FMRFamide-like immunoreactivity in the olfactory regions that represent the typical path of terminal nerve fibers, nor were we able to locate a terminal nerve ganglion. We conclude that lampreys may lack a terminal nerve, and that the previously described fiber bundle extending from the nasal sac to the ventral forebrain may constitute an extra-bulbar olfactory pathway.

Brainstem motoneuron pools that are selectively resistant in amyotrophic lateral sclerosis are preferentially enriched in parvalbumin: evidence from monkey brainstem for a calcium-mediated mechanism in sporadic ALS.

Some brainstem motoneuron groups appear more resistant to the process of neurodegeneration in ALS (for example, oculomotor, trochlear, and abducens nuclei) than others (for example, trigeminal, facial, ambiguus, and hypoglossal nuclei). The possibility that the differential presence of the calcium-chelating protein parvalbumin might underlie this difference in vulnerability was examined immunohistochemically as a way to determine whether a calcium-mediated mechanism might be involved in ALS. In normal monkey brainstem, we found that the abundance of parvalbumin-containing neurons in the oculomotor, trochlear, and abducens nuclei was approximately 90% of the abundance of choline acetyltransferase (CHAT)-containing motoneurons. In contrast, the abundance of parvalbumin-containing neurons in the other brainstem motor nuclei innervating skeletal muscle (trigeminal, facial, ambiguus, and hypoglossal) was only about 30-60% of the abundance of CHAT-containing motoneurons. Since some of these motoneuron pools contain nonmotoneuron internuclear neurons that might be parvalbumin-containing, we also carried out double-label studies to specifically determine the percentage of cholinergic motoneurons that contained parvalbumin in each of these motoneuron pools. We found that 85-100% of the oculomotor, trochlear, and abducens motoneurons were parvalbumin-containing. In contrast, only 20-30% of the trigeminal, facial, ambiguus, and hypoglossal motoneurons were parvalbumin-containing. These results raise the possibility that motoneuron death in sporadic ALS is related to some defect that promotes cytosolic calcium accumulation in motoneurons. This excess calcium entry may promote cell death via an excitotoxic pathway. Motoneurons rich in parvalbumin may resist the deleterious effects of this putative calcium gating defect because they are better able to sequester the excess calcium.