Acetylcholinesterase activity and type C synapses in the hypoglossal,facial and spinal-cord motor nuclei of rats

Summary Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptie cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple active zones. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.In honour of Prof. P. van Duijn     

Evolution of the brainstem orofacial motor system in primates: a comparative study of trigeminal, facial, and hypoglossal nuclei

The trigeminal motor (Vmo), facial (VII), and hypoglossal (XII) nuclei of the brainstem comprise the final common output for neural control of most orofacial muscles. Hence, these cranial motor nuclei are involved in the production of adaptive behaviors such as feeding, facial expression, and vocalization. We measured the volume and Grey Level Index (GLI) of Vmo, VII, and XII in 47 species of primates and examined these nuclei for scaling patterns and phylogenetic specializations. Allometric regression, using medulla volume as an independent variable, did not reveal a significant difference between strepsirrhines and haplorhines in the scaling of Vmo volume. In addition, correlation analysis using independent contrasts did not find a relationship between Vmo size or GLI and the percent of leaves in the diet. The scaling trajectory of VII volume, in contrast, differed significantly between suborders. Great ape and human VII volumes, furthermore, were significantly larger than predicted by the haplorhine regression. Enlargement of VII in these taxa may reflect increased differentiation of the facial muscles of expression and greater utilization of the visual channel in social communication. The independent contrasts of VII volume and GLI, however, were not correlated with social group size. To examine whether the human hypoglossal motor system is specialized to control the tongue for speech, we tested human XII volume and GLI for departures from nonhuman haplorhine prediction lines. Although human XII volumes were observed above the regression line, they did not exceed prediction intervals. Of note, orang-utan XII volumes had greater residuals than humans. Human XII GLI values also did not differ from allometric prediction. In sum, these findings indicate that the cranial orofacial motor nuclei evince a mosaic of phylogenetic specializations for innervation of the facial muscles of expression in the context of a generally conservative scaling relationship with respect to medulla size.     

Genioglossus muscle activity and serotonergic modulation of hypoglossal motor output in obese Zucker rats.

Obese Zucker rats have a narrower and more collapsible upper airway compared with lean controls, similar to obstructive sleep apnea (OSA) patients. Genioglossus (GG) muscle activity is augmented in awake OSA patients to compensate for airway narrowing, but the neural control of GG activity in obese Zucker rats has not been investigated to determine whether such neuromuscular compensation also occurs. This study tests the hypotheses that GG activity is augmented in obese Zucker rats compared with lean controls and that endogenous 5-hydroxytryptamine (5-HT) contributes to GG activation. Seven obese and seven lean Zucker rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and they were implanted with GG and diaphragm wires for respiratory muscle recordings. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid and the 5-HT receptor antagonist mianserin (100 microM). Compared with lean controls, respiratory rates were increased in obese rats across sleep-wake states (P=0.048) because of reduced expiratory durations (P=0.007); diaphragm activation was similar between lean and obese animals (P=0.632). Respiratory-related, tonic, and peak GG activities were also similar between obese and lean rats (P>0.139). There was no reduction in GG activity with mianserin at the hypoglossal motor nucleus, consistent with recent observations of a minimal contribution of endogenous 5-HT to GG activity. These results suggest that despite the upper airway narrowing in obese Zucker rats, these animals have a sufficiently stable airway such that pharyngeal muscle activity is normal across sleep-wake states.     

Craniofacial sequelae of lesions to facial and trigeminal motor nuclei in growing rats

Unilateral electrolytic lesions were made in the left-side facial motor nucleus (FMNu) of six Sprague-Dawley rats at 35 days of age in order to correlate craniofacial sequelae with changed motoneuron function. Experimental and control rats were killed at 22, 32, 42, and 52 days postoperatively to provide muscle weight, brain histology, and dry skull preparations for analyses. Dissection, muscle weight, motoneuron count, and osteometric data revealed that lesion-side facial and masticatory muscles were affected by the lesions. Paired t-tests indicated that significant differences existed between weights of experimental lesion- and nonlesion-side anterior digastric, temporalis, masseteric complex, and medial pterygoid muscles, numbers of facial and trigeminal motoneurons, and several skeletal dimensions of the skull. Basi-cranial dimensions of experimental animals were least affected by the lesion, whereas zygomatic arch, dorsal facial region, and mandibular condyle dimensions were most affected. Statistical analyses also detected significant differences between experimental and control groups for several skeletal dimensions of the skull. Data indicated that damage to the trigeminal motor nucleus (TMNu) was secondary to the primary lesion in the FMNu. Motoneurons within the facial and trigeminal neuromuscular complexes (FNC and TNC) play an important role in craniofacial growth and development.     

Comparison of motor endplate and central synapses. An ultrastructural study]

The fine structural characteristics of motor endplate (paradigm of a cholinergic synapse) and central synapses are briefly summarized giving major emphasis on recent observations in freeze fractured material. Special attention is paid to the concepts of "Active Zones" (presynaptic membrane complex) and "Specific Sites" (postsynaptic membrane complex). Comparison and functional interpretations are made specifically with respect to secretion and receptor mechanisms that are so characteristic of chemically transmitting junctions.     

Quantitative studies on the motor root of the mouse facial nerve. An electron-microscopic study.

Fiber count analysis was performed with the electron microscope on the motor root of the facial nerve in six mice. On an average, 3,433 (84.9%) of the total nerve fibers (4,046) were myelinated and 592 (14.6%) unmyelinated. The motor root consisted mostly of large myelinated fibers (large fiber group), but a part of the root consisted of small myelinated and unmyelinated fibers (small fiber group). The nerve fibers of the small fiber group appear to correspond with those of the intermediate nerve, and to pass through the motor root and enter the intermediate nerve near the geniculate ganglion.     

Superior laryngeal and hypoglossal afferents tonically influence upper airway motor excitability in anesthetized rats.

Upper airway (UA) muscle activity is stimulated by changes in UA transmural pressure and by asphyxia. These responses are reduced by muscle relaxation. We hypothesized that this is due to a change in afferent feedback in the ansa hypoglossi and/or superior laryngeal nerve (SLN). We examined 1) the glossopharyngeal motor responses to UA transmural pressure and asphyxia and 2) how these responses were changed by muscle relaxation in animals where one or both of these afferent pathways had been sectioned bilaterally. Experiments were performed in 24 anesthetized, thoracotomized, artificially ventilated rats. Baseline glossopharyngeal activity and its response to UA transmural pressure and asphyxia were moderately reduced after bilateral section of the ansa hypoglossi (P < 0.05). Conversely, bilateral SLN section increased baseline glossopharyngeal activity, augmented the response to asphyxia, and abolished the response to UA transmural pressure. Muscle relaxation reduced resting glossopharyngeal activity and the response to asphyxia (P < 0.001). This occurred whether or not the ansa hypoglossi, the SLN, or both afferent pathways had been interrupted. We conclude that ansa hypoglossi afferents tonically excite and SLN afferents tonically inhibit UA motor activity. Muscle relaxation depressed UA motor activity after section of the ansa hypoglossi and SLN. This suggests that some or all of the response to muscle relaxation is mediated by alterations in the activity of afferent fibers other than those in the ansa hypoglossi or SLN.     

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.     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

Developmental study of cytochrome oxidase activity in the brain stem respiratory nuclei of postnatal rats.

We utilized cytochrome oxidase (CO) as a marker of neuronal functional activity to examine metabolic changes in brain stem respiratory nuclei of rats from newborn to 21 day of age. The pre-B?tzinger complex (PBC), upper airway motoneurons of nucleus ambiguus (NA(UAM)), ventrolateral nucleus of solitary tract (NTS(VL)), and medial and lateral parabrachial nuclei (PB(M) and PB(L), respectively) were examined at postnatal days (P) 0, 1, 2, 3, 4, 5, 7, 14, and 21. CO histochemistry was performed, and the intensity of CO reaction product was quantitatively analyzed by optical densitometry. In addition, CO histochemistry was combined with neurokinin-1 receptor (NK1R) immunogold-silver staining to doubly label neurons of PBC in P14 animals. The results showed that levels of CO activity generally increased with age in all of the nuclei examined. However, a significant decrease was found in NA(UAM) at P3 (P < 0.01), and a distinct plateau of CO activity was noted at P3 in PBC and at P3 and P4 in NTS(VL), PB(M), and PB(L). Of the neurons examined in PBC, 83% were doubly labeled with CO and NK1R. Of these, CO activity was high in 33.9%, moderate in 27.3%, and light in 38.8% of neurons, suggesting different energy demands in these metabolic groups that may be related to their physiological or synaptic properties. The transient decrease or plateau in CO activity at P3 and P4 implies a period of synaptic adjustment or reorganization during development, when there may be decreased excitatory synaptic drive or increased inhibitory synaptic drive, or both, in these brain stem respiratory nuclei. The adjustment, in turn, may render the system less responsive to respiratory insults. This may bear some relevance to our understanding of pathological events during postnatal development, such as occurs in sudden infant death syndrome.     

Dendro-dendritic and reciprocal synapses in the primate motor cortex.

Dendro-dendritic synapses have been observed infrequently in the deep layers of the motor cortex. The presynaptic dendrites are of a varicose type and themselves receive a considerable density of synapses both of the asymmetric and symmetrical type. The ultrastructure of the dendro-dendritic synapse itself shows the typical arrangement of presynaptic and postsynaptic membrane densities, often with presynaptic dense projections, and the membrane specialization is of the symmetrical type. There is the usual cleft containing electron-dense material between the presynaptic and postsynaptic profiles. The synaptic vesicles occur in a small cluster confined to a region close to the presynaptic membrane specialization; some of the vesicles are flattened and were shown by tilt analysis to be of the discoid type. Two examples were found of reciprocal dendro-dendritic synapses, both components being of the symmetrical type. A single axon terminal may make a synapse on to both dendrites involved in a dendro-dendritic synapse.     

Glycine at hypoglossal motor nucleus: genioglossus activity, CO(2) responses, and the additive effects of GABA.

There is evidence for glycine and GABA(A)-receptor-mediated inhibition of hypoglossal motoneurons in vitro. However, comparable studies have not been performed in vivo, and the interactions of such mechanisms with integrative reflex respiratory control have also not been determined. This study tests the hypotheses that glycine at the hypoglossal motor nucleus (HMN) will suppress genioglossus (GG) muscle activity, even in the presence of hypercapnic respiratory stimulation, and the effects of glycine will be blocked by strychnine. We also determined whether coapplication of glycine and muscimol (GABA(A)- receptor agonist) to the HMN is additive in suppressing GG activity. Twenty-four urethane-anesthetized, tracheotomized, and vagotomized rats were studied. Diaphragm and GG activities, the electroencephalogram, and blood pressure were recorded. Microdialysis probes were implanted into the HMN for delivery of artificial cerebrospinal fluid (control), glycine (0.0001-10 mM), or muscimol (0.1 microM). Increasing glycine at the HMN produced graded suppression of GG activity (P < 0.001), although the GG still responded to stimulation with 7% inspired CO(2) (P = 0.002). Strychnine (0.1 mM) reversed the glycine-mediated suppression of GG activity, whereas combined glycine and muscimol were additive in GG muscle suppression. It remains to be determined whether the recruitment of such glycine and GABA mechanisms explains the periods of major GG suppression in behaviors such as rapid eye movement sleep.     

An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m² and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).     

Acetylcholinesterase activity of motor and sensory nerve fibers in the spinal nerve roots of the rat

Summary The histochemical and cytochemical distribution of acetylcholinesterase activity in the anterior and posterior spinal nerve roots and ganglia of the rat was demonstrated by the Karnovsky method using acetyl and butyrylthiocholine as substrates and eserine and DFP as inhibitors. Light and electron microscopic examination of transverse frozen sections enabled the simultaneous visualization of end product in relationship to the various fiber components of each nerve root. While the enzymatic activity of the anterior roots was consistantly observed in the large extrafusal and small intrafusal motor fibers a relatively greater amount of precipitate occurred in aggregates of myelinated and unmyelinated fibers believed to represent preganglionic sympathetic nerves. In contrast, no significant enzymatic activity could be demonstrated in the myelinated nerve fibers of the posterior root. In the sensory sytem, the limited enzymatic precipitate was largely restricted to the unmyelinated afferent fibers and to their small cell bodies in the dorsal root ganglia. The ultrastructural distribution of enzymatic activity was located in the granular endoplasmic reticulum and perinuclear spaces of the ganglion cells. Within peripheral nerves this end product occurred between the apposing axonal and Schwann cell membranes and along the membranous aspect of occasional axoplasmic vesicles of both myelinated and unmyelinated nerve fibers.This study was supported by grants NB 04161-04 and NB 04161-05 of the National Institute of Neurological Diseases and Blindness. — The author would like to thank MissMaria C. la Valle for her skillful technical assistance.     

Acetylcholinesterase and butyrylcholinesterase activity in the atria of the heart of adult albino rats

In experiments on adult albino rats the authors used the substances BW 284 C51 (1.5-bis(allyldimethylammoniumphenyl)-pentane-3-one-dibromide) as a specific inhibitor of acetylcholinesterase (AChE) and ethopropazine (10-(2-diethylaminopropyl) phenothiazine hydrochloride) as a specific inhibitor of butyrylcholinesterase (BuChE) to determine the two enzyme activities in atrial homogenates and to investigate changes after AChE or BuChE inhibition of the negative chronotropic effect of acetylcholine (ACh) on atria incubated in vitro. AChE accounted for only 12% and BuChE for 88% of the total ability of atrial homogenates to hydrolyse acetylcholine. The concentration of exogenous ACh needed to reduce the spontaneous frequency of contractions of the isolated right atrium by 30, 60, or 90/min fell by 78%, 79% and 84% respectively after BW 284 C51 inhibition of AChE and by 95%, 94% and 94% after simultaneous inhibition of AChE and BuChE. The significance of AChE in control of the negative chronotropic effect of ACh is thus evidently significantly greater than would correspond to the percentual proportion of AChE in cholinesterase activities in the atria of the rat heart. In can be assumed that AChE is functionally associated with parasympathetic innervation of the heart and that it is probably present in a high concentration in the primary pacemaker region.