Immunohistochemical localization of alpha(1a)-adrenoreceptors in muscle spindles of rabbit masseter muscle.

The expression of alpha(1a)-adrenoreceptors (alpha(1a)-ARs) within the muscle spindles of rabbit masseter muscle was investigated. The alpha(1a)-ARs were detected by immunohistochemical fluorescent method and examined along the entire length of 109 cross serially sectioned spindles. The sympathetic fibers were visualized by the immunofluorescent labeling of the noradrenaline synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). In order to recognize the intrafusal muscle fiber types, antibodies for different myosin heavy chain isoforms (MyHCI) were used. TH and DBH immunolabeled nerve fibers have been observed within the capsule lamellar layers, in the periaxial fluid space and close to intrafusal muscle fibers. The alpha(1a)-ARs were detected on the smooth muscle cells of the blood vessels coursing in the muscle and in the capsule lamellar layers or within the periaxial fluid space of the spindles. Moreover, at the polar regions of a high percentage (88.1%) of muscle spindles a strong alpha(1a)-ARs immunoreactivity was present on the intrafusal muscle fibers. In double immunostained sections for alpha(1a)-ARs and MyHCI it was evidenced that both bag, and nuclear chain fibers express alpha(1a)-ARs. The receptors that we have detected by immunofluorescence may support a direct control by adrenergic fibers on muscle spindle.

Central distribution of nociceptive intradental afferent nerve fibers in the rat.

The central distribution of intradental afferent nerve fibers was investigated by combining electron microscopic observations with a selective method for inducing degeneration of the A delta- and C-type afferent fibers. Degenerating terminals were found on the proprioceptive mesencephalic trigeminal neurons and on dendrites in the neuropil of the trigeminal motor nucleus after application of capsaicin to the rat's lower incisor tooth pulp. The results give anatomical evidence of new sites of central projection of intradental A delta- and C-type fibers whereby the nociceptive information from the tooth pulp can affect jaw muscle activity.

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.

Central projections and entries of capsaicin-sensitive muscle afferents.

The entry pathway and central distribution of A delta and C muscle afferents within the central nervous system (CNS) were investigated by combining electron microscopy and electrophysiological analysis after intramuscular injection of capsaicin. The drug was injected into the rat lateral gastrocnemius (LG) and extraocular (EO) muscles. The compound action potentials of LG nerve and the evoked field potentials recorded in semilunar ganglion showed an immediate and permanent reduction in A delta and C components. The morphological data revealed degenerating unmyelinated axons and terminals in the inner sublamina II and in the border of laminae I-II of the dorsal horn at L4-L5 and C1-C2 (subnucleus caudalis trigemini) spinal cord segments. Most degenerating terminals were the central bouton (C) of type I and II synaptic glomeruli. Furthermore, degenerating peripheral axonal endings (V2) presynaptic to normal C were found. Since V2 were previously found degenerated after cutting the oculomotor nerve (ON) or L4 ventral root, we conclude that some A delta and C afferents from LG and EO muscles entering the CNS by ON or ventral roots make axoaxonic synapses on other primary afferents to promote an afferent control of sensory input.

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.

Collaterals of recurrent laryngeal nerve fibres innervate the thymus: a fluorescent tracer and HRP investigation of efferent vagal neurons in the rat brainstem.

The origin and course of efferent vagal fibers, which innervate the rat thymus, were investigated by a fluorescent retrograde double labeling method, using Fast blue (FB) and Diamidino yellow dihydrochloride (DY) as tracers. In the same animal, one tracer was injected into the cranial portion of the right lobe of the thymus and the other dye was deposited around the cut end of the right recurrent laryngeal nerve. The neuronal population giving origin to the recurrent nerve was mapped by using retrograde labeling with HRP applied to the central stump of the nerve. The HRP retrograde axonal transport showed that most efferent vagal fibers of the recurrent nerve have their perikarya in the nucleus retroambigualis (NRA), nucleus ambiguus (NA), and to a lesser extent in the nucleus retrofacialis (NRF). In fluorescent retrograde double labeling of thymus and recurrent laryngeal nerve both single and double labeled cells were found. The cells labeled by the injections into the thymus were colocalized with the neurons labeled by the tracer deposited in the recurrent laryngeal nerve to the NRA, NA, and NRF. Moreover along the rostrocaudal extent of the NRF and NA double labeled cells were present, showing that some of the thymic efferents are collaterals of the recurrent nerve fibers. Our experiments shown that some thymic vagal fibres originate from neurons of nucleus dorsalis nervi vagi (NDV) as demonstrated both by HRP and FB injected thymuses. The possible role of these efferents in thymic function is briefly discussed.

Otolithic and extraocular muscle proprioceptive influences on the spatial organization of the vestibulo- and cervico-ocular quick phases.

The cervico-ocular reflex (COR) was studied alone or in combination with the vestibulo-ocular reflex (VOR) in the rabbit. Step stimulations of the body with respect to the fixed head induced small slow compensatory responses followed by large compensatory quick phases (QP). These responses remained aligned with the horizon at different head pitch angles. The QP reorientation in space was due to the gravity influence on the otolithic receptors. The vestibular induced QPs exhibit a similar pattern. Because of this reorientation, the reduction of the amplitude of the vestibular induced QPs, due to the addition of the COR, was maintained even at different static head positions. The electrolytic lesion of the ophthalmic branch of the trigeminal nerve deeply affected the space orientation of the COR. In particular, the cervically induced compensatory QPs of the eye ipsilateral to the lesion showed a remarkable variability of their trajectories and they lost space reorientation. These findings suggest that the coordinate system controlling the QPs is influenced by signals originating from both head position in space and eye position in the orbit.

The morphology and location of atrial specific granules and the demonstration of atrial natriuretic factor in porcine, lapine and bovine heart by immunoelectronmicroscopy.

The atrial specific granules (ASGs) were studied in samples collected from the right and left auricles of conventionally slaughtered cows (10), pigs (16) and rabbits (8). In addition, the presence of atrial natriuretic factor (ANF) was detected by immunocytochemistry. Mature ASGs, characterized by the presence of highly osmiophilic and electron-dense material surrounded by a membrane, were present in all atrial myoendocrine cells and their diameters ranged from 100 to 470 nm in pigs, from 100 to 235 nm in cattle, and from 125 to 275 nm in rabbits. Immunoelectronmicroscopical studies revealed the presence of ANF in the ASGs of pigs and cattle, whereas anti-ANF polyclonal serum failed to detect any significative reaction in lapine ASGs. The ultrastructural features of the ASGs of pigs, cattle and rabbits described may be useful in comparing the morphological picture of several cardiac endocrine pathological conditions.

Somatotopic organization of second order neurons of the eye muscle proprioception.

Unit activity was recorded extracellularly from lamb's nucleus principalis and pars oralis of trigeminal nuclear complex following moderate manual stretching of individual extraocular muscles. The oral portion of the spinal trigeminal nucleus and the main sensory nucleus have been investigated by systematic exploration of the second-order neurons of the eye muscle proprioception. Such responses were somatotopically organized. In particular, each single extraocular muscle was represented along the main dorso-ventral axis in this manner: superior oblique and superior rectus in a dorsal layer; inferior rectus and inferior oblique in an intermediate layer, while the medial rectus and the lateral rectus were represented more ventrally. In a few experiments this representation was not observed, due to intermingling of the units. The topographic organization of eye muscle proprioception in the trigeminal nuclear complex described above closely corresponds to that reported by previous authors in the Gasser ganglion.

A somatotopic and functional organization of the masticatory proprioceptors in the mesencephalic trigeminal nucleus of the frog.

A stratified organization of the first-order neurons of the masticatory proprioception has been observed in the frog mesencephalic trigeminal nucleus (MeNV). Extracellular records of unit activity have shown that in this nucleus neurons responding either to jaw raising muscles or to jaw lowering muscles reveal a somatotopy. A consistent topography pattern is evident by exploring in dorsoventral direction the layers 4 and 6 in the optic tectum. The jaw closing muscles are represented in the dorsal part of the MeNV, whereas the jaw opening ones are distributed more ventrally. Since MeNV neurons represents the afferent limb of the masticatory reflex, the motor effects elicited by electrical stimulation of these cells have been tested.

A scanning electron microscopic study of the mesencephalic trigeminal nucleus in duck and rabbit.

The mesencephalic trigeminal nucleus (MTN) cells of both young and adult ducks as well as of rabbits were investigated by scanning electron microscope. The rabbit showed only ovoid unipolar cells, while the duck also presented polyhedral cells. Few of these latter revealed processes originating from their surface and were recognized as multipolar cells. Some differences between the MTN cell surface of young and adult ducks were noticed. Synaptic bulbs were observed on the MTN cells in both duck and rabbit.

Afferent fibers and sensory ganglion cells within the oculomotor nerve in some mammals and man. I. Anatomical investigations.

The present research shows that sensory ganglion cells are located within the oculomotor nerve of monkeys and man. Furthermore, afferent fibers have been found in the IIIrd nerve of all the animals examined (lamb, pig, cat, dog and monkey). These fibers have their perikarya prevalently in the semilunar ganglion. Their pathway could be studied after section of either the trigeminal ophthalmic branch or of the intracranial portion of the IIIrd nerve. Following these operations, degenerating fibers were found entering the brain stem through the oculomotor nerve. In the brain stem, they were traced through the pons and the medulla and were seen to end in the spinal cord, within the subnucleus gelatinosus of the nucleus caudalis trigemini. Their degenerating endings found in the neuropil of the SG Rolandi, represented peripheral axonal endings of the glomeruli, rather than central axonal endings, as was the case after trigeminal rhizotomy. On the basis of these different degenerating patterns, the conclusion can be reached that the perikarya of the afferent fibers located in the semilunar ganglion represent, in reality, a ganglion of the IIIrd nerve.

Localization and somatotopy of sensory cells innervating the extraocular muscles of lamb, pig and cat. Histochemical and electrophysiological investigation.

The localization of sensory cells innervating the extraocular muscles (EOMs) was studied in the lamb, pig and cat in which horseradish peroxidase (HRP) was injected into each EOM. Electrophysiological techniques were also used to search for EOM stretch sensitive units in the semilunar ganglion. In lamb and pig labeling was observed in the semilunar ganglion only, while in cat labeled neurons were present in both the semilunar ganglion and mesencephalic trigeminal nucleus. In the semilunar ganglion of all these species a clear somatotopic organization of EOM afferents was observed. The histochemical somatotopic pattern of EOM afferents in the semilunar ganglion of lamb and pig was substantially in agreement with the electrophysiological arrangement. The responses recorded to EOM stretch in the semilunar ganglion of the pig were characterized by a low threshold and a slow adaptation as previously found in the lamb; on the contrary, in the semilunar ganglion of the cat only a few units were found, which showed high stretch threshold and quick adaptation.

Influence of oculomotor nerve afferents on central endings of primary trigeminal fibers.

Painful fibers running in the third nerve and originating from the ophthalmic trigeminal area send their central projections at level of substantia gelatinosa of nucleus caudalis trigemini. The central endings of these fibers form axoaxonic synapses with trigeminal fibers entering the brain stem through the trigeminal root. The effect of electrical stimulation of the third nerve central stump on the central endings of trigeminal afferent fibers consists in an increased excitability, possibly resulting in a presynaptic inhibition. This inhibitory influence is due to both direct and indirect connections of the third nerve afferent fibers with the trigeminal ones.

On the nature of the afferent fibers of oculomotor nerve.

The oculogyric nerves contain afferent fibers originating from the ophthalmic territory, the somata of which are located in the ipsilateral semilunar ganglion. These primary sensory neurons project to the Subnucleus Gelatinosus of the Nucleus Caudalis Trigemini, where they make presynaptic contact with the central endings of the primary trigeminal afferents running in the fifth cranial nerve. After complete section of the trigeminal root, the antidromic volleys elicited in the trunk of the third cranial nerve by stimulating SG of NCT consisted of two waves belonging to the A delta and C groups. The area of both components of the antidromic volleys decreased both after bradykinin and hystamine injection into the corresponding cutaneous region and after thermic stimulation of the ipsilateral trigeminal ophthalmic territory. The reduction of such potentials can be explained in terms of collision between the antidromic volleys and those elicited orthodromically by chemical and thermic stimulation. Also, capsaicin applied on the nerve induced an immediate increase, followed by a long lasting decrease, of orthodromic evoked response area. These findings bring further support to the nociceptive nature of the afferent fibers running into the oculomotor nerve.

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.

Reflex and reticular modulation of first-order proprioceptive neurons of the mesencephalic trigeminal nucleus.

Several investigations have shown that the vagus nerve and the reticular formation can affect the reflex responses of the masticatory muscles. The present research has been devoted to analyze the mechanism of such modulations of the masseteric reflex in the lamb. Extracellular records of the electrical activity of the mesencephalic trigeminal nucleus (MTN) was carried out in immobilized lambs by means of tungsten microelectrodes. Units were found which responded to lowering the jaw and to stretching the masseter muscle: they were identified as the first-order neurons of the masticatory proprioception on the basis of their electrophysiological properties. Single-shock or repetitive electrical stimulations of the cervical vagus nerve and of the bulbo-pontine reticular formation could affect the unitary discharge of the MTN: different patterns of activation and inhibition of the MTN units were seen; however, the activation was the most prominent effect. The responses did not depend upon the circulatory effects of the vagal stimulation. Thus the conclusion can be reached that the vagus and the reticular substance can modulate the masseteric reflex at level of the perikarya of the afferent pathway. Such a statement is supported also by the presence of synaptic boutons on the soma of the MTN neurons.

Localization of neurons innervating masticatory muscle spindle and periodontal receptors in the mesencephalic trigeminal nucleus and their reflex actions.

The mesencephalic trigeminal nucleus was studied in anaesthetized and curarized rabbits by recording the unitary activity through extracellular microelectrodes and identifying the constituent cell types. Two types of units were found, namely primary afferents supplying jaw raising muscle spindles and periodontal or gingival mechanoreceptors. These two groups of neurons exhibited a rostrocaudal somatotopy: the former occupied the entire rostral portion of the nucleus (A7-P2.3; trochlear decussation being taken as an arbitrary 0 level), the latter was located caudally (P3-P4.5) while the somata of both types of afferent fibres were present between P2.2 and P3. No evidence was found for representation of both tendon organs of jaw muscles and joint receptors. Among the units innervating muscle spindles, secondary afferents were largely more numerous than the primary ones. Among periodontal and gingival mechanoreceptor afferents, incisors were the most widely represented, followed by interalveolar gingiva and molars; the axonal conduction velocity ranged between 9 and 40 m/sec and between 8 and 16 m/sec for ipsilaterally and contralaterally projecting neurons, respectively. The motor responses obtained by electrical stimulation of discrete areas of the MTN confirmed the presence of a high degree of segregation between the two different populations of neurons. In fact, jaw raising movements are obtained when stimulating the area within A7 and P2 containing the somata of spindle afferent neurons, while only jaw opening movements are elicited by stimulation of the caudal levels of the nucleus. These data also show that the periodontal neurons whose somata are located in the MTN participate in the jaw opening reflex, just as the more numerous periodontal mechanoreceptors whose somata are located in the Gasser ganglion. Soma-somatic and soma-axon hillock gap junctions were found among the neurons of the MTN, particularly in the caudal third of the nucleus.

Epileptogenic stimulation of the cortical masticatory area and the mesencephalic trigeminal nucleus.

1. The effects of epileptogenic stimulation of the cerebral masticatory area on the somata of the first-order neurons of masticatory proprioception localized in the mesencephalic trigeminal nucleus (MTN) were studied in curarized, lightly anaesthetized rabbits. 2. Low-frequency stimulations inducing modest cortical after-discharges fired the silent MTN units with latencies of 0.3-0.8 sec, and induced alternate excitatory and inhibitory effects on the active units. On some occasions the tonic afferent discharge was changed into rhythmic bursts, which occurred at the constant frequency of 2.5-3.5/sec for the entire range of stimulation frequencies used, i.e. from 0.3 to 10/sec. 3. High-frequency cortical stimulations eliciting true epileptic seizures induced a transient increase in discharge of the MTN units, followed by a prolonged inhibition. 4. All these effects were attributed to activation of reticular pathways acting on the some of the recorded proprioceptive neurons. Such a modulation of the proprioceptive input elicited by the epileptic masticatory cortex may contribute to the motor effects and to the changes of the masseteric reflex which occur during convulsive seizures.

Afferent fibers and sensory ganglion cells within the oculomotor nerve in some mammals and man. II. Electrophysiological investigations.

The main aim of the present study was to localize with electrophysiological techniques the central projections and terminations of the aberrant trigeminal fibres contained in the oculomotor nerve of the lamb. After severing a trigeminal root, single-shock electrical stimulation of the trigeminal axons present in the central stump of the ipsilateral oculomotor nerve evoked field potentials in the area of, i) the subnucleus gelatinosus of the nucleus caudalis trigemini at the level of C1-C2; ii) the main sensory trigeminal nucleus; iii) the descending trigeminal nucleus and tract; iv) the adjacent reticular formation. Units whose discharge rate was influenced by such a stimulation were also found in the same territories. These regions actually exhibited degenerations after cutting an oculomotor nerve. We conclude, therefore, that the trigeminal fibres which leave the Vth nerve at the level of the cavernous sinus and enter the brain stem through the IIIrd nerve, end in the same structures which receive the terminations of the afferent fibres entering the brain stem through the sensory trigeminal root.