Localisation of recurrent laryngeal nerve motoneurons in the sheep by means of retrograde fluorescent labelling.

The purpose of this investigation was to determine the central distribution of the efferent neurons of the recurrent laryngeal nerve (RLN) in the sheep by the use of the retrograde transport of the fluorescent tracer Fast Blue. The distribution of the RLN neurons was also compared with that of the neurons simultaneously labelled by injection of another tracer, Diamidino Yellow dihydrochloride, into the cervical trunk of the vagus nerve (CTV). Injections of the tracer into the CTV resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve, in the nucleus ambiguus, in the nucleus retroambigualis and in the reticular formation surrounding the nucleus ambiguus. Following injections of the tracer into the RLN, labelling of neurons was seen over a wide area of the ipsilateral nucleus ambiguus and in the nucleus retroambigualis. Species differences in the distribution of the efferent component of the RLN are discussed, in particular ruminants compared to nonruminants.

The mesencephalic trigeminal nucleus of the duck: development and apoptosis.

The normal development of the mesencephalic trigeminal nucleus (MesV) of the white Peking duck (Anas platyrhynchos) was studied from the 9th day of incubation until hatching and during adulthood. In the early days of embryonic development, neurons are present in the posterior commissure and in the mesenchymal tissue outside the leptomeninges in addition to those in the tectal commissure (TC) and in the optic tectum. Following the internucleosomal cleavage of DNA, a massive loss of neurons in the MesV starts in the 11-day embryo and continues until the 15th day of incubation. On the 16th day, the nucleus consists of a numerically larger medial division located in the TC and a smaller lateral division within the stratum griseum periventriculare as is found in the adult animal. The programmed cell death occurring in the MesV is discussed herein and correlated with the analogous apoptotic phenomena observed in the trigeminal motor nucleus.

Selective deafferentation of hand cutaneous territory is followed by changes in fibre type distribution of a forearm muscle in the horse.

Based on previous observations that capsaicin can selectively damage group III and IV afferents and induce muscle fibre transformation, we hypothesized that eliminating, by means of capsaicin, the group III and IV afferents of a peripheral territory it could lead to a fibre transformation in a muscle involved in the flexor reflexes of the same peripheral territory. Therefore, capsaicin was injected into the palmar nerves of the forelimb of the horse to investigate if eliminating group III and IV afferents from the hand of the horse a muscle fibre transition would occur in the flexor carpi radialis (FCR) muscle, which is involved in the flexor reflexes of the finger itself. 120 days after capsaicin injection, type I slow fibres increased and type IIA fast fibres decreased. We presume that the long lasting deafferentation of the ergo-nociceptive fibres causes a plastic remodelling in the central nervous system and indirectly influences the motoneuron excitability via short or long loop-pathways enhancing their tonic discharge.

Nerve fiber composition of the intracranial portion of the oculomotor, trochlear, and abducens nerves in the sheep.

In the present investigation, the fiber content and the diameter spectra of the intracranial portion of the three oculomotor nerves (oculomotor, trochlear, and abducens nerves) were analysed in sheep by light and electron microscopy. It was determined that up to 14.98% of fibers in the oculomotor nerve, 17.01% in the trochlear nerve, and 11.87% in the abducens nerve were unmyelinated. The myelinated fibers showed a bimodal distribution in their size spectrum in all three nerves, with a majority of large myelinated axons, but a considerable proportion of small myelinated fibers, as well. The sensory function of the unmyelinated fibers present in the three oculomotor nerves is discussed also on the basis of our previous morphofunctional investigations.

Cerebellar afferents from neurons in the extraocular motor nuclei: a fluorescent retrograde double-labeling study in the sheep.

The fluorescent retrograde double labeling technique has been used to identify within the extraocular motor nuclei of the sheep the neurons projecting to the cerebellum and to provide evidence whether they are motor neurons sending collaterals to the cerebellum or a separate population of neurons. The study was performed on eight sheep. The fluorescent tracers used were Fast Blue and the diamidino yellow dihydrochloride. In one and the same animal a fluorescent tracer was injected into the extraocular muscles (EOMs) and the other into bilateral points of the vermal folia II-V and paramedian lobule, or into the vermal folia VI, VIIA and VIIB, or into the underlying fastigial nuclei. Within the oculomotor, trochlear, and abducens nuclei, almost all of the motor neurons were labeled by the tracer injected into the EOMs and only a few cells were fluorescent for the tracer infiltrated into the cerebellum. These latter labelings were present bilaterally, and their number and distribution did not show apparent differences after injecting the paramedian lobule and the vermal folia or the fastigial nucleus. Along the rostrocaudal extent of the oculomotor and trochlear nuclei, the neurons projecting to the cerebellum were intermingled with the motor neurons located in the nuclear area facing the medial longitudinal fasciculus. In the abducens nucleus they were restricted to the caudal pole of the nucleus, which is located ventrolaterally to the genu of the facial nerve. Double-labeled neurons were never found. The absence of double-labeled cells, in spite of the efficiency of the tracer infiltration into the EOMs and into the cerebellum, demonstrates that the cerebellar projections from the extraocular motor nuclei are not collaterals of the motor neurons, but axons of a separate population of neurons.

Cerebellar and spinal projections of the coeruleus complex in the duck: a fluorescent retrograde double-labeling study.

The double fluorescent retrograde tracing technique was used to identify, within the coeruleus complex (Co complex) of the duck, the nerve cells projecting to the cerebellar cortex and to the spinal cord. This technique was also used to investigate the possibility that the cerebellar and spinal projections of the Co complex are collaterals of the same axons. In the same animal, nuclear Diamidino yellow dihydrochloride (DY) fluorescent tracer was placed into the cerebellar cortex of folia V-VII, and cytoplasmic fluorescent Fast blue (FB) dye was injected into C3-C4 spinal cord segments. FB labeled multipolar somata and DY fluorescent nuclei were intermingled within the dorsal caudal region of the locus coeruleus (LCo) and within the dorsal division of the nucleus subcoeruleus (dSCo). Moreover, in the LCo, a low proportion of double-labeled neurons (about 3-4% of labelings) was evidenced among single-labeled neurons. In the ventral division of the nucleus subcoeruleus (vSCo), occasional DY labeled nuclei were found, whereas FB-labeled cells were frequently present. The present findings reveal the location of the coeruleocerebellar and coeruleospinal projecting neurons within the Co complex of the duck. They are intermingled in the caudal portion of the LCo and along the rostrocaudal extent of the subjacent dSco. The LCo and the dSCo are the major source of the projections to the folia V-VII, whereas the vSCo contributes very slightly to the innervation of the cerebellar injected areas. Moreover, the double-labeling study demonstrates that in the duck a low percentage of neurons within the ventrolateral portion of the caudal region of the LCo projects both to the cerebellar cortex of folia V-VII and to C3-C4 spinal cord segments via collaterals. Therefore, these neurons simultaneously influence the cerebellar cortex and spinal cord. The possibility that the projections studied are noradrenergic and that they play a role in feeding is discussed.

Partial transformation from fast to slow muscle fibers induced by deafferentation of capsaicin-sensitive muscle afferents.

Mechanical and histochemical characteristics of the lateral gastrocnemius (LG) muscle of the rat were examined 21 days after capsaicin injection into the LG muscle. The capsaicin caused a decrease in generation rate of twitch and tetanic tension and an increase in fatigue resistance of LG muscle. The histochemical muscle fiber profile evaluated by myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide tetrazolium reductase methods showed an increase of type I and IIC fibers and a decrease of the type IIB in whole muscle, and a decrease of the IIA, IIX fibers in the red part accompanied by their increase in the white part. Therefore the capsaicin treatment, which selectively eliminated fibers belonging to the III and IV groups of muscle afferents, induced muscle fiber transformation from fast contracting fatiguing fibers to slowly contracting nonfatiguing ones.

Mesencephalic trigeminal nucleus neurons supplying the jaw closing muscles have no spinal projection: a fluorescent double-labeling study in birds and mammals.

BACKGROUND: The present study deals with the possibility that the mesencephalic trigeminal nucleus (MeV) neurons that innervate the muscle spindles of the jaw closing muscles may also have collaterals projecting to the cervical spinal cord. At the same time, we reexamine the morphology of these cells and their location within the MeV. METHODS: The fluorescent retrograde tracers Fast Blue (FB) and Diamidino Yellow dihydrochloride (DY) were injected into the jaw closing muscles and C2-C3 spinal cord segments, respectively, of duck, rat, and rabbit in one series of experiments. In a second series of animals, the targets of the tracers were reversed. RESULTS: Retrogradely double-labeled cells (FB+DY) were not found in the MeV. On the contrary, the tracer injected into the muscles retrogradely labeled only large unipolar MeV cells, whereas the tracer injected into C2-C3 spinal cord segments labeled only small multipolar cells which were intermingled with the MeV somata of muscle spindle afferents. CONCLUSIONS: These findings exclude the possibility of spinal projections via collaterals of MeV cells supplying muscle spindles of jaw closing muscles in duck, rat, and rabbit. Moreover, the retrograde double-labeling technique evidences two cellular populations within the MeV of the duck, rat, and rabbit: large unipolar neurons which are the cell bodies of primary afferents from jaw closing muscles and small multipolar cells projecting to the upper cervical spinal cord.

In situ detection of apoptosis in regressing corpus luteum of pregnant sow: evidence of an early presence of DNA fragmentation.

Luteolysis has been shown to be correlated with apoptosis in rats, sheep, and cows. In pigs, apoptosis has already been demonstrated as regards atretic follicles. The present study has been conducted to evaluate whether apoptosis occurs during corpora lutea regression in the pregnant pig and to investigate the temporal relationship between apoptosis and functional luteolysis. The apoptotic process has been studied through the research of oligonucleosome fragmentation by means of classical electrophoresis methods and by in situ detection on histological luteal sections. The latter method allows the identification of apoptosis and the localization of apoptotic cells. Pregnant sows were cloprostenol (PGF2 alpha analog) treated and ovariectomized 0, 6, 12, 24, 48, and 72 hr after treatment. Corpora lutea were utilized for progesterone and DNA extraction and in situ evaluation of apoptosis. Clear evidence of apoptosis was seen earlier with the in situ technique (6 hr for stromal tissue, 12 hr for luteal cells) than with the classical method (24 hr). Apoptosis was, however, apparent after plasma and tissue progesterone had reached basal levels. In conclusion, these results are consistent with the hypothesis that apoptosis occurs during luteolysis in pigs. Moreover, the data obtained with the in situ technique made it possible to identify signs of structural regression in stromal tissue first than in parenchymal cells. A two-stage activation of apoptosis has been discussed to explain structural changes that occur during luteolysis after cloprostenol treatment in swine corpora lutea.

Motoneuron organisation of the muscles of the spinal accessory complex of the sheep investigated with the fluorescent retrograde tracer technique.

Retrograde transport of the fluorescent tracers Diamidino Yellow dihydrochloride and Fast Blue was used to determine the location of the spinal nucleus of the accessory nerve in the sheep. We also considered whether in this species the sternocephalic, brachiocephalic, omotransversarius and trapezius muscles, i.e. the muscles of the spinal accessory complex, are supplied by more than one population of motoneurons. The spinal accessory nucleus extends as a single column of neurons from C1 to C7 spinal cord segments and occupies a lateral position within the ventral horn. The most rostral portion of this column is located dorsolaterally, whereas the remaining portion from C2 to C7 occupies a ventrolateral position. At C1 and C4 levels the nucleus also possesses some cells with a medial location. All the muscles of the spinal accessory complex receive their motor innervation both from the spinal accessory nucleus and from motoneurons forming the cervical spinal nerves. A double motor innervation of these muscles is thus present in the sheep.

Localization of motoneurons innervating the extraocular muscles of the sheep by retrograde fluorescent tracers.

Retrograde transport of the fluorescent tracers Fast blue, Evans blue, Diamidino yellow dihydrochloride, and Propidium iodide was used to determine the location of the motoneurons innervating the extraocular muscles of the sheep. An extensive superposition among the motor pools of the oculomotor nucleus (ON) has been observed. In the rostral third of the ON, a considerable merging occurs between obliquus ventralis and rectus medialis motoneurons and also between rectus ventralis and rectus medialis motoneurons. In the middle third of the ON, rectus dorsalis and levator palpebrae superioris motoneurons are intermingled with each other, and also with obliquus ventralis motoneurons dorsally and with rectus medialis motoneurons ventrally. The rostral portion of the trochlear nucleus overlaps with the caudal pole of the ON. The motoneurons innervating the obliquus dorsalis muscle are mainly contralateral with few ipsilateral exceptions. The retractor bulbi muscle receive the innervation by both the abducens and accessory abducens nuclei.

High incidence of multiple-bag fiber muscle spindles in the articularis humeri muscle of the horse.

The articularis humeri (AH) muscle of the horse is a small muscle composed of histochemically identified type I and IIA extrafusal fibers and a large number of muscle spindles. A total of 150 complete spindles with both spindle poles available were examined in serial transverse sections. On the basis of myosin ATPase-staining reactions after alkaline and acid preincubations, four types of intrafusal fibers, namely, bag1, bag2, "mixed" bag, and chain fibers, were identified. A high proportion of the spindle population (62.6%) consisted of multiple-bag spindles containing three or more (up to six) bag fibers. Also one-bag-fiber spindles were observed. The one-bag-fiber spindles containing a bag2 fiber could be traced into tandem linkages. "Mixed" bag intrafusal fibers, differing in their ATPase staining profile at the two poles, were found in spindles containing also at least one bag1 and one bag2 fiber. An unusually long extracapsular tract (up to 5,500 microns) of the bag intrafusal fibers was observed.

Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry.

The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigated by the use of fluorescent tracers in double-labeling experiments. For this purpose Diamidino yellow (DY) injection into the oculomotor nerve was combined with Fast blue (FB) injection either into the extraocular muscles (EOMs), or the cornea, or the superior eyelid. Double-labeled DY + FB cells were found in the ophthalmic region of the trigeminal ganglion in addition to single-labeled DY or FB cells. The DY and DY + FB-labeled trigeminal cells were analysed immunocytochemically for their content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and cholecystokinin-8 (CCK-8)-like. All single-labeled DY cells showed SP-, CGRP- or CCK-8-like immunoreactivity. Double-labeled DY + FB neurons innervating the EOMs were immunoreactive for each of the three peptides, whereas double-labeled neurons supplying the cornea were only CGRP-like positive. The findings suggest that, in the sheep, trigeminal neurons which send their process centrally through the oculomotor nerve supply the EOMs, the cornea, and the superior eyelid and contain neuropeptides which are usually associated with pain sensation.

Analysis of the sternotrachealis muscle fibers in some Anseriformes: histochemistry and sex differences.

Histochemical characteristics and sizes of the fibers of the sternotrachealis (ST) muscle have been investigated in some Anseriformes (mallard, Pekin duck, Muscovy duck, and goose) of both sexes. A sexual dimorphism has been shown in the muscle of the species examined. In the mallard and Pekin duck, the male ST muscle shows type IIIA fibers in addition to the type I, IIA, and IIB fibers observed also in the female. In the Muscovy duck, the male muscle has only type I and IIA fibers, whereas the female muscle presents type I fibers and both types IIA and IIB fibers. Moreover, the mean frequencies for each fiber type were significantly different between males and females. In the goose, both male and female muscles present only type I and IIA fibers. In all the species examined, the mean areas of each fiber type are significantly different between male and female, being always larger in the male muscles. The anatomical sexual dimorphism observed in the ST muscle is discussed in relation to function.

Histochemical profile of articularis humeri muscle in the horse.

In this study the histochemical fiber type analysis of the articularis humeri muscle of the horse was performed. This muscle is composed of type I and type IIA fibers. A large number of spindles has been observed and the presence of these receptors could help to understand the role played by this muscle.

An immunohistochemical approach to the intrafusal fibers of extraocular muscle spindles in sheep, cow, and pig.

Intrafusal muscle fibers of the extraocular muscles (EOMs) of the sheep, cow, and pig were studied histochemically and immunohistochemically. In sheep and cow spindles, three intrafusal fiber types, namely the bag1, bag2, and chain fibers, were identified by a combination of standard histochemical methods and immunohistochemical staining with antibodies selective for slow-tonic (antitonic ALD) and slow twitch (anti-I BA-D5) myosin. The bag1 and bag2 fibers appeared immunologically different on the basis of their differential reactivity with the two antisera. Anti-tonic ALD preferentially stained the bag1 fibers, whereas anti-I BA-D5 labeled the bag2 fibers. Chain fibers did not react with either antisera. In the pig EOM spindles, in general, one bag and some chain intrafusal fibers were identified. The bag fiber was labeled by anti-tonic ALD, but it did not react with the anti-I BA-D5. These findings point to the existence in pig EOM spindles of only one bag fiber antigenically similar to the bag1 fiber of the other species examined.

Simultaneous cell death in the trigeminal ganglion and in ganglion neurons present in the oculomotor nerve of the bovine fetus.

A well-developed ganglion and scattered ganglion cells are present in the intracranial portion of the oculomotor nerve during the first half of fetal life in the ox. In the second half of fetal life a dramatic reduction of the ganglion cells associated with the oculomotor nerve occurs because of spontaneous cell death. Concomitantly, the same phenomenon of cell death is found in the trigeminal ganglion, especially in its rostromedial portion. Free degenerating perikarya can be found in the cavernous sinus.

Different intrafusal fiber composition of spindles in sheep and pig extraocular muscles.

Histochemical profiles of intrafusal fibers have been examined in muscle spindles of extraocular muscles of sheep and pig. Results show that in the sheep the intrafusal content presents, in addition to chain fibers, at least one bag1 and one bag2 fiber, whereas in the pig almost all the spindles are one-bag-fiber [corrected] spindles.

The fine structure of the area postrema of the sheep.

The ultrastructural features of the area postrema (AP) were investigated in the suckling lamb, weaned lamb and adult sheep. No morphological differences were observed between lambs and sheep. Unciliated ependymal cells, linked by zonulae adherentes-type junctions and gap junctions, cover the AP ventricular surface. Clusters of pyriform neurons, glial cells, and axons are present in the parenchyma. The blood vessels are surrounded by wide perivascular spaces, which present an inner and outer basal lamina. The capillaries are of the fenestrated type. Perivascular glial cells rest on the outer basal lamina of the perivascular space and form a continuous ensheathment with their cell bodies or with flattened interdigitating processes. Along adjacent perivascular glial processes gap junctions are present. From our ultrastructural observations it appears that the overall cellular morphology of AP of the sheep does not differ substantially from that of monogastric mammals.