Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR.     

Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals

It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.     

Autoradiographic detection of kinin receptors in the human medulla of control,hypertensive, and diabetic donors

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B, and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4-1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.     

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: A histological,fast Dil and immunocytochemical study in the hypoglossal nucleus

We have previously reported on our investigation of motoneuron cell death (MCD) in the rat nucleus ambiguus (NA). This article focuses on the other major upper respiratory tract motor nucleus: the hypoglossal. The hypoglossal nucleus (XII) contains motoneurons to the tongue and, as such, plays a critical role in defining patterns of respiration, deglutition, and vocalization. Motoneuron counts were made in XII in a developmental series of rats. In addition, the neural tracer fast DiI was used to ensure that all hypoglossal motoneurons had migrated into the nucleus at the time cell death was assessed. Furthermore, an antibody to γ-aminobutyric acid (GABA) was used to determine the potential effect of inadvertently counting large interneurons on motoneuron counts. Cell death in XII was shown to occur entirely prenatally with a loss of 35% of cells between embryonic day 16 (E16) and birth. Fast DiI tracings of the prenatal hypoglossal nerve indicated that all motoneurons were present in a well-defined nucleus by E15. Immunocytochemical staining for GABA demonstrated considerably fewer interneurons than motoneurons in XII. These findings in XII, in comparison with those previously reported for NA, demonstrate differences in the timing and amount of cell death between upper respiratory tract motor nuclei. These differences establish periods during which one nucleus may be preferentially insulted by environmental or teratogenic factors. Preferential insults may underlie some of the upper respiratory tract incoordination pathologies seen in the newborn such as the sudden infant death syndrome (SIDS). © 1995 John Wiley & Sons, Inc.     

A5 cells are silenced when REM sleep-like signs are elicited by pontine carbachol.

The A5 noradrenergic neurons are considered important for cardiorespiratory regulation. We hypothesized that A5 cells are silenced during rapid eye movement (REM) sleep, thereby contributing to cardiorespiratory changes and suppression of hypoglossal (XII) motoneuronal activity. We used an anesthetized, paralyzed, and artificially ventilated rat in which pontine microinjections of carbachol trigger signs of REM sleep, including hippocampal theta rhythm, motor suppression, and silencing of locus coeruleus neurons. All 16 putative noradrenergic A5 cells recorded were strongly suppressed when the REM sleep-like episodes were elicited and also after intravenous clonidine. Antidromic mapping showed that none of six neurons tested projected to the XII nucleus, whereas three of five projected to the nucleus of the solitary tract and two of four to the rostral ventrolateral medulla. Bilateral microinjections of clonidine into the A5 regions did not alter XII nerve activity. These data suggest that A5 neurons are silenced during natural REM sleep. This will lead to decreased norepinephrine release and may alter synaptic transmission in the nucleus of the solitary tract and rostral ventrolateral medulla without, however, a detectable impact on XII motoneurons.     

Androgen receptors in cranial nerve motor nuclei of male and female rats

This study compared AR proteins in four cranial nerve motor nuclei among male and female rats that were intact, gonadectomized, or gonadectomized and given TP by immunohistochemistry. AR-immunoreactive (ir) neurons were found, in descending order of abundance, in the nucleus ambiguus, hypoglossal nucleus, and the facial and trigeminal motor nuclei of both males and females of intact and gonadectomized plus TP rats. Virtually every neuron of the nucleus ambiguus was AR-ir. In contrast, AR-ir neurons were either restricted to a specific area of the hypoglossal nucleus, or randomly distributed in the facial and trigeminal motor nuclei. The predominant AR-ir site shifted from cell nuclei to the cytoplasm, depending upon the presence or absence of ligand. Sex differences in the amount and staining intensity of AR-ir neurons were discernable in all four motor nuclei of intact rats, and these differences were maintained in gonadectomized plus TP rats, with the exception of the nucleus ambiguus. The immunostaining results were complemented by results from AR binding studies. Cytosolic AR binding values for the hypoglossal and facial motor nuclei of females were only approximately 50% of those of males despite the absence of a sex difference in neuron number. These results indicate that intrinsic sex differences in AR levels and androgenic regulation of AR exist in cranial nerve motor nuclei, and that there are differences in the abundance and distribution pattern of AR responsive neurons in cranial nerve motor nuclei. These results are consistent with the idea that sex differences in AR could account for sex differences observed in nerve regeneration and neuron loss following cranial nerve injury.     

Changes in localized arrangement into the hypoglossal nucleus after the neurectomy of unilateral hypoglossal nerve (medial branch) in cats

We studied changes in orofacial behavior and the arrangement of bilateral hypoglossal nuclei after the neurectomy of the medial branch of the unilateral hypoglossal nerve in cats. After recovery from surgery in a head holder, the animals were acclimated to take and chew fish paste (1.8 g) from a spoon and lick milk from a wetted paintbrush. Next we performed a neurectomy in the unilateral hypoglossal nerve after training. We firstly recorded behavior during the taking of fish paste and licking of milk, and then performed a neurectomy in the unilateral hypoglossal nerve. After nerve cutting, the cats’ tongue deviated toward the cut side when they licked food, and bilateral activities of EMGs in the genioglossus muscles became stable in about 1 month. After that, we injected two kinds of fluorescent dye (10% Evans blue, EB, and 3% Fast blue, FB) into the bilateral genioglossus muscles using syringes (0.15 ml in each), respectively. Although each injection of FB and EB into the bilateral genioglossus muscles in normal cats revealed cells positively stained with each dye in the hypoglossal nuclei of each injection site, in cats 1 month after nerve cutting, fluorescent dye was only observed in positive cells in the hypoglossal nucleus of the intact side and the dye injected into the neurectomy side showed a mixture into positive cells of the intact side. The findings suggest that muscles in the neurectomy side may be compensated by regeneration of the peripheral nerves on the intact side.     

Immunohistochemical mapping of adrenomedullin in the human medulla oblongata

We studied by immunocytochemistry the expression of adrenomedullin (AM) in the human medulla oblongata, sampled from 13 adult subjects (mean age: 38 years), whose medical history was negative for neurological and neurovascular pathologies. Immunoreactive neurons were found in the medulla oblongata with statistically significant differences among the various nuclei (one-way ANOVA, P < 0.001). The hypoglossal nucleus showed higher AM expression than that of the spinal tract of the trigeminal nerve (P < 0.05), solitary tract nucleus (P < 0.05), nucleus intercalatus (P < 0.05), and area postrema (P < 0.05). The arcuate nucleus and inferior olivary nuclear complex showed lower AM expression than the hypoglossal nucleus (P < 0.001), vestibular nuclei (P < 0.01), cuneate and gracile nuclei (P < 0.05), lateral column of the reticular formation (P < 0.05), and nucleus ambiguous (P < 0.05). Furthermore the nuclei were grouped with reference to their function, into somatic sensitive nuclei, somatic motor nuclei, visceral nuclei, reticular formation, and nuclei involved in cerebellar functions. The ANOVA revealed statistically significant differences (P < 0.001) in mean AM scores among the different groups. Nuclei involved in cerebellar function showed the lowest mean AM score (P < 0.05). The difference in AM score between somatic motor nuclei and visceral nuclei was also statistically significant (P < 0.05). Widespread AM immunoreactivity in the nuclei of the medulla oblongata may account for the role of the peptide in neuronal function and regulation of regional blood flow. Differences in the expression of AM in the nuclei studied indicate the different involvement of AM in neurotransmission and neuromodulation.     

Bilateral brainstem connections of the rat supratrigeminal region

Efferent and afferent connections of the supratrigeminal region were studied in the rat using iontophoretically delivered horseradish peroxidase and Phaseolus vulgaris leuco-agglutinin. Projections of supratrigeminal efferents were found to the contralateral supratrigeminal region, to the ipsi- and contralateral trigeminal motor nuclei and the medullary reticular formation, and to the ipsilateral facial and hypoglossal motor nuclei. Neurons projecting to the supratrigeminal region were located in the contralateral supratrigeminal nucleus, in the ipsilateral mesencephalic trigeminal nucleus and bilaterally in the medullary reticular formation. This organization is discussed with respect to bilateral oral motor control mechanisms.     

Patterning of skeletal muscle

Recent studies challenge the view that signals provided by motor neurons are required to activate subsynaptic nuclei and induce postsynaptic specializations in developing skeletal muscle. New findings show that acetylcholine receptor genes are expressed and that acetylcholine receptor clusters form preferentially in the prospective synaptic region of muscle independently of motor innervation. These results indicate that developing myotubes are patterned by mechanisms intrinsic to developing muscles and raise the possibility that patterning of muscles may influence the growth pattern of motor axons and the sites where synapses form.     

Lifelong Expression of Apolipoprotein D in the Human Brainstem: Correlation with Reduced Age-Related Neurodegeneration

The lipocalin apolipoprotein D (Apo D) is upregulated in peripheral nerves following injury and in regions of the central nervous system, such as the cerebral cortex, hippocampus, and cerebellum, during aging and progression of certain neurological diseases. In contrast, few studies have examined Apo D expression in the brainstem, a region necessary for survival and generally less prone to age-related degeneration. We measured Apo D expression in whole human brainstem lysates by slot-blot and at higher spatial resolution by quantitative immunohistochemistry in eleven brainstem nuclei (the 4 nuclei of the vestibular nuclear complex, inferior olive, hypoglossal nucleus, oculomotor nucleus, facial motor nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and Roller`s nucleus). In contrast to cortex, hippocampus, and cerebellum, apolipoprotein D was highly expressed in brainstem tissue from subjects (N = 26, 32−96 years of age) with no history of neurological disease, and expression showed little variation with age. Expression was significantly stronger in somatomotor nuclei (hypoglossal, oculomotor, facial) than visceromotor or sensory nuclei. Both neurons and glia expressed Apo D, particularly neurons with larger somata and glia in the periphery of these brainstem centers. Immunostaining was strongest in the neuronal perinuclear region and absent in the nucleus. We propose that strong brainstem expression of Apo D throughout adult life contributes to resistance against neurodegenerative disease and age-related degeneration, possibly by preventing oxidative stress and ensuing lipid peroxidation.     

Cortical and subcortical control of tongue movement in humans: a functional neuroimaging study using fMRI.

We have used voluntary tongue contraction to test whether we can image activation of the hypoglossal nuclei within the human brain stem by using functional magnetic resonance imaging (fMRI). Functional images of the whole brain were acquired in eight subjects by using T2-weighted echo planar imaging (blood oxygen level development) every 6.2 s. Sequences of images were acquired during 12 periods of 31-s "isometric" rhythmic tongue contraction alternated with 12 periods of 31-s tongue relaxation. Noise arising from cardiac- and respiratory-related movement was removed either by filtration (high pass; cutoff 120 s) or by inclusion in the statistical analysis as confounding effects of no interest. For the group, tongue contraction was associated with significant signal increases (P < 0.05 corrected for multiple comparisons) in the sensorimotor cortex, supplementary motor area, operculum, insula, thalamus, and cerebellum. For the group and for six of eight individuals, significant signal increases were also seen within the medulla (P < 0.001, predefined region of interest with no correction for multiple comparisons); this signal is most likely to reflect neuronal activation associated with the hypoglossal motor nuclei. The data demonstrate that fMRI can be used to detect, simultaneously, the cerebral and brain stem control of tongue movement.     

CORRELATED EVOLUTION OF GENOME SIZE AND CELL VOLUME IN DIATOMS (BACILLARIOPHYCEAE)1

A correlation between genome size and cell volume has been observed across diverse assemblages of eukaryotes. We examined this relationship in diatoms (Bacillariophyceae), a phylum in which cell volume is of critical ecological and biogeochemical importance. In addition to testing whether there is a predictive relationship across extant species, we tested whether evolutionary divergences in genome size were correlated with evolutionary divergences in cell size (using independent contrasts). We estimated total DNA content for 16 diatom species using a flow cytometer and estimated cell volumes using critical dimensions with scaling equations. Our independent contrast analyses indicated a significant correlated evolution between genome size and cell volume. We then explored the evolutionary and ecological implications of this evolutionary relationship. Diatom cell volume is an important component of the global carbon cycle; therefore, understanding the mechanisms that drive diatom genome evolution has both evolutionary and ecological importance.     

Neuronal expression of peripherin,a type III intermediate filament protein,in the mouse hindbrain

Peripherin is a 57 kDa Type III intermediate filament protein associated with neurite extension, neuropathies such as amyotrophic lateral sclerosis, and cranial nerve and dorsal root projections. However, knowledge of peripherin expression in the CNS is limited. We have used immunoperoxidase histochemistry to characterise peripherin expression in the mouse hindbrain, including the inferior colliculus, pons, medulla and cerebellum. Peripherin immunolabelling was observed in the nerve fibres and nuclei that are associated with all cranial nerves [(CN) V–XII] in the hindbrain. Peripherin expression was prominent in the cell bodies and axons of the mesenchephalic trigeminal nucleus and the pars compacta region of nucleus ambiguus, and in the fibres that comprise the solitary tract, the descending spinal trigeminal tract and the trigeminal and facial nerves. A small proportion of peripherin positive fibres in CN VIII likely arise from cochlear type II spiral ganglion neurons. Peripherin positive fibres were also observed in the inferior cerebellar peduncle and folia in the intermediate zone of the cerebellum. Antibody specificity was confirmed by absence of labelling in hindbrain tissue from peripherin knockout mice. This study shows that in the adult mouse hindbrain, peripherin is expressed in discrete neuronal subpopulations that have sensory, motor and autonomic functions.     

The anatomy and motor nerve distribution of the eye muscles in the crayfish

Summary The gross anatomy of the muscles in the crayfish compound eye and the distribution of brain oculomotor neurons were studied by a variety of anatomical and physiological techniques. There are 11 major muscles in each eye. These vary considerably in size and influence upon eye movements and in their source of motor innervation. Muscles that cause defensive eyestalk withdrawal are controlled by axons of a giant motor neuron cluster. Muscles that move the eyecup in vertical planes are innervated by cells of an anterior motor cluster, as well as by cells in the medulla terminalis. Muscles which move the eyecup horizontally are supplied by neurons of the lateral motor cluster. The separation of the oculomotor system into different neuronal groups that supply different sets of muscles thus reflects functional specializations of the component divisions.I am grateful to Mr. Gene Lorton for his technical assistance with some phases of this work. I also thank Sharon Greene for executing the illustration in Figure 1 and Susan Suarez for illustrating Figure 2. This work was supported by USPHS research grant NS04989.     

Inhibition of carbonic anhydrase isoforms I,II, IV,VII and XII with carboxylates and sulfonamides incorporating phthalimide/phthalic anhydride scaffolds

We report a panel of carboxylates and sulfonamides incorporating phthalic anhydride and phthalimide moieties in their structure and their interaction with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). They were synthesized from substituted anthranilic acids and trimellitic anhydride chloride, followed by reaction with primary amines and were tested for the inhibition of five physiologically relevant CA isoforms, the human (h) hCA I, II, IV, VII and XII, some of which are involved in serious pathologies (CA II, IV and XII in glaucoma; CA VII in epilepsy; CA XII in some solid tumors). The carboxylic acids were generally poor inhibitors of isoforms hCA I, II and IV but were highly effective, low nanomolar inhibitors of hCA VII and XII. The sulfonamides inhibited all isoforms significantly, and some of them were sub-nanomolar hCA VII inhibitors, although their isoform selectivity was lower compared to the carboxylates. This study proves that carboxylic acids incorporating a phthalic anhydride/phthalimide based scaffold may lead to isoform-selective inhibitors by applying the tail approach, mostly used up until now for obtaining sulfonamide, sulfamide and sulfamate CA inhibitors.     

Effect of chronic intermittent hypoxia on noradrenergic activation of hypoglossal motoneurons

In obstructive sleep apnea patients, elevated activity of the lingual muscles during wakefulness protects the upper airway against occlusions. A possibly related form of respiratory neuroplasticity is present in rats exposed to acute and chronic intermittent hypoxia (CIH). Since rats exposed to CIH have increased density of noradrenergic terminals and increased α(1)-adrenoceptor immunoreactivity in the hypoglossal (XII) nucleus, we investigated whether these anatomic indexes of increased noradrenergic innervation translate to increased sensitivity of XII motoneurons to noradrenergic activation. Adult male Sprague-Dawley rats were subjected to CIH for 35 days, with O(2) level varying between 24% and 7% with 180-s period for 10 h/day. They were then anesthetized, vagotomized, paralyzed, and artificially ventilated. The dorsal medulla was exposed, and phenylephrine (2 mM, 10 nl) and then the α(1)-adrenoceptor antagonist prazosin (0.2 mM, 3 × 40 nl) were microinjected into the XII nucleus while XII nerve activity (XIIa) was recorded. The area under integrated XIIa was measured before and at different times after microinjections. The excitatory effect of phenylephrine on XII motoneurons was similar in sham- and CIH-treated rats. In contrast, spontaneous XIIa was more profoundly reduced following prazosin injections in CIH- than sham-treated rats [to 21 ± 7% (SE) vs. 40 ± 8% of baseline, P < 0.05] without significant changes in central respiratory rate, arterial blood pressure, or heart rate. Thus, consistent with increased neuroanatomic measures of noradrenergic innervation of XII motoneurons following exposure to CIH, prazosin injections revealed a stronger endogenous noradrenergic excitatory drive to XII motoneurons in CIH- than sham-treated anesthetized rats.     

Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.