The evidence for nitric oxide synthase immunopositivity in the monosynaptic Ia-motoneuron pathway of the dog

In this study, nitric oxide synthase immunohistochemistry supported by nicotinamide adenine dinucleotide phosphate diaphorase histochemistry was used to demonstrate the nitric oxide synthase immunoreactivity in the monosynaptic Ia-motoneuron pathway exemplified by structural components of the afferent limb of the soleus H-reflex in the dog. A noticeable number of medium-sized intensely nitric oxide synthase immunoreactive somata (1000-2000 microm(2) square area) and large intraganglionic nitric oxide synthase immunoreactive fibers, presumed to be Ia axons, was found in the L7 and S1 dorsal root ganglia. The existence of nitric oxide synthase immunoreactive fibers (6-8 microm in diameter, not counting the myelin sheath) was confirmed in L7 and S1 dorsal roots and in the medial bundle of both dorsal roots before entering the dorsal root entry zone. By virtue of the funicular organization of nitric oxide synthase immunoreactive fibers in the dorsal funiculus, the largest nitric oxide synthase immunoreactive fibers represent stem Ia axons located in the deep portion of the dorsal funiculus close to the dorsomedial margin of the dorsal horn. Upon entering the gray matter of L7 and S1 segments and passing through the medial half of the dorsal horn, tapered nitric oxide synthase immunoreactive collaterals of the stem Ia fibers pass through the deep layers of the dorsal horn and intermediate zone, and terminate in the group of homonymous motoneurons in L7 and S1 segments innervating the gastrocnemius-soleus muscles. Terminal fibers issued in the ventral horn intensely nitric oxide synthase immunoreactive terminals with long axis ranging from 0.7 to >or=15.1 microm presumed to be Ia bNOS-IR boutons. This finding is unique in that it focuses directly on nitric oxide synthase immunopositivity in the signalling transmitted by proprioceptive Ia fibers. Nitric oxide synthase immunoreactive boutons were found in the neuropil of Clarke's column of L4 segment, varying greatly in size from 0.7 to >or=15.1 microm in length x 0.7 to 4.8 microm wide. Subsequent to identification of the afferent nitric oxide synthase immunoreactive limb of the monosynaptic Ia-motoneuron pathway on control sections, intramuscular injections of the retrograde tracer Fluorogold into the gastrocnemius-soleus muscles, combined with nitric oxide synthase immunohistochemistry of L7 and S1 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive somata (1000-2000 microm(2) square area) in the dorsolateral part of both dorsal root ganglia, presumed to be proprioceptive Ia neurons. Concurrently, large nitric oxide synthase immunoreactive fibers were detected at the input and output side of both dorsal root ganglia. S1 and S2 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of S1 and S2 dorsal roots and in the dorsal funiculus of S1, S2 and lower lumbar segments. In addition, anterograde degeneration of large nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L7-S2 segments produces direct evidence that the afferent limb of the soleus H-reflex is nitric oxide synthase immunoreactive and presents new immunohistochemical characteristics of the monosynaptic Ia-motoneuron pathway, unseparably coupled with the performance of the stretch reflex.     

Crossed and uncrossed projections to cat sacrocaudal spinal cord: II. Axons from muscle spindle primary endings

The terminal fields of primary afferent fibers from tail muscle spindle primary endings were mapped within cat sacrocaudal spinal cord (S3-Ca7), using intra-axonal recording and horseradish peroxidase staining techniques. We sought to determine the ipsilateral and contralateral projection patterns and to relate these to the fibers' muscles of origin. Fifty-three group Ia fibers were successfully stained. Segmental collaterals originated from either the ascending or descending branch within the dorsal columns. Collaterals coursed rostromedially within the dorsal columns and traversed the medial aspect of the dorsal horn. Ipsilateral terminations were similar for all fibers. Within the ventral horn, boutons were consistently observed in the medial or central portions of lamina VII. In lamina VIII, a variable number of boutons was seen on fine branches emerging from larger fibers coursing ventrally. Clusters of terminals were plentiful in the regions of motoneurons, i.e., lamina IX and the nucleus commissuralis. Terminals were found in the adjacent white matter. In addition to ipsilateral terminations, some group Ia fibers (20 of 53) had collateral branches that crossed ventrally to the central canal, terminating within the midline ventral gray commissure and/or the contralateral ventral horn. Crossed projections always originated in medial (dorsal or ventral), but not lateral, muscles of the tail. These data suggest that ipsilateral projections of group Ia fibers make connections on sacrocaudal motoneurons, on neurons mediating segmental reflex functions and on neurons conveying ascending information. It is speculated that crossed and uncrossed connections between group Ia fibers from medial muscles and bilateral dendritic trees of motoneurons subserve synchronized co-contraction of synergistic muscles located on the two sides of the body, such as with dorsal or ventral flexion of the tail. Group Ia projections from lateral muscles, that are entirely ipsilateral, would be involved with lateral movements of the tail.     

Ultrastructural evidence related to presynaptic inhibition of primary muscle afferents in Clarke's column of the cat

As part of an investigation on excitatory synaptic transmission in the mammalian CNS, we have examined ultrastructural details of the synaptic connection between primary afferent fibers and dorsal spinocerebellar tract (DSCT) neurons in Clarke's column of the cat spinal cord. Single primary muscle afferents (group Ia and Ib) and DSCT neurons were identified and stained intracellularly with HRP. The terminations of these afferent fibers were examined in serial sections under the EM. Five of 6 Ib boutons and 1 of 14 Ia boutons were contacted by small presynaptic boutons. An example was illustrated in which only 1 out of 7 boutons arising from the same Ia fiber and contacting the same postsynaptic DSCT neuron was contacted by a presynaptic bouton. It is likely that the presynaptic contacts are responsible for presynaptic inhibition of synaptic transmission between primary afferents and DSCT neurons. We have proposed that the observed differences in presynaptic contacts from bouton to bouton may be one of the causes of a nonuniformity in the probability of transmitter release between release sites at this connection.     

Projections and terminations of single respiratory axons in the cervical spinal cord of cat

Position, divergence, branching, and termination patterns of single, respiratory axons were studied in cat cervical spinal cord by injecting horseradish peroxidase (HRP) intra-axonally. We stained 12 axons which were characterized by their firing patterns and by electrical stimulation. Five axons discharged during inspiration (I); the remaining 7 discharged during expiration (E). No injected axon was evoked by stimulating ipsilateral phrenic nerve roots while 7 (4 I, 3 E) of 12 were excited at a short latency from stimulating at a medullary site (on the midline, 1-2 mm rostral to the obex, approximately 3 mm below the dorsal medullary surface) where many bulbospinal respiratory axons decussate. All injected stem axons were located in the ventral and ventrolateral funiculi, traversed in a rostrocaudal direction, and were stained for lengths ranging from 3.6 to 12.4 mm. Mean axonal diameter was 2.9 microns. In 6 axons (4 I, 2 E), 14 collaterals were stained: 1 on each E axon, 2 on one I axon, 3 each on 2 others and 4 on another I axon. Collaterals emerged perpendicularly from the descending stem axon and projected directly to the ventral horn. The average distance between neighboring collaterals was 1.0 mm (n = 7). Collaterals did not arborize until they were near or within the ventral horn. Both en passant and terminaux types of presynaptic boutons were found primarily within the rostrocaudal cylinder that defined the phrenic motor column. In addition, some boutons were located dorsomedial to the phrenic motor column. We conclude that I axons, presumably of medullary origin, have multiple collaterals which terminate primarily in the phrenic motor column. However, the same axon can have terminals in different regions of the ventral horn, which are known to contain dendrites of phrenic motoneurons.     

Primary afferent projections of the major splanchnic nerve to the spinal cord and gracile nucleus of the cat

Splanchnic afferent projections to the spinal cord and gracile nucleus were labeled following the application of HRP to the central cut end of the major splanchnic nerve. Labeled afferent fibers were detected in the ipsilateral dorsal column, in Lissauer's tract (LT), in laminae 1, 5, 7, and 10, and in the dorsal gray commissure at T1-T13 levels of the spinal cord. Afferent projections were not identified in laminae 2-4. Collaterals from LT projected ventrally along the lateral and medial margins of the dorsal horn (called lateral and medial pathways, respectively). Afferents in the lateral pathway formed small bundles, spaced rostrocaudally at intervals of 300-1,000 microns, which passed medially at the base of the dorsal horn into laminae 5, 7, and 10 and to the contralateral spinal cord. Some afferents in the lateral pathway projected to the intermediolateral nucleus where labeled sympathetic preganglionic neurons were located. Afferents in the medial pathway entered the lateral aspect of the dorsal column and projected as a group near the midline rostrally to the medulla. The dorsal column pathway terminated in the ventral gracile nucleus in four or five clusters, each occupying a region ranging in size from 0.01-0.1 mm3 and separated in the rostrocaudal axis by distances of 400-800 microns. These clusters were concentrated in the middle and caudal portions of the nucleus below the obex. A comparison of the present results with those from earlier experiments on the central projections of afferent fibers from the heart, kidney, and pelvic organs demonstrates a consistent pattern of visceral afferent termination in the thoracolumbar and sacral segments of the spinal cord. This is not unexpected, since visceral afferent pathways to different organs perform similar functions, such as the transmission of nociceptive information and the initiation of autonomic reflexes.     

Morphology of single mesencephalic trigeminal neurons innervating masseter muscle of the cat

The morphology of functionally identified single axons of mesencephalic trigeminal neurons was studied in the cat by the method of intra-axonal injection of horseradish peroxidase (HRP). Each axon can be divided into united (U), peripheral (P) and central branches (C). The united axon (U) descends from its soma within the tract of the trigeminal mesencephalic nucleus to the dorsal aspect of the trigeminal motor nucleus (Vmo), where it splits into peripheral and descending central branches with a Y-shaped bifurcation. The peripheral axon (P) joins the motor root of the trigeminal nerve to exit the brainstem. The central axon (C) travels caudally within the juxtatrigeminal regions (or lateral reticular formation). All 3 branches issue axon collaterals that distribute terminal boutons within the dorsolateral subdivision of Vmo, supra- and intertrigeminal regions. Collaterals emanating from the central axon (C) except for its proximal segment travel ventrolaterally within the juxtatrigeminal regions, and send their terminal branches into the lateral boundaries adjacent to the spinal trigeminal nucleus. The trajectory of terminal branches distinguishes group Ia afferents from the possible group II afferents. The majority of terminal boutons are found to distribute in the supra- and intertrigeminal regions for group II afferent fibers and in the dorsolateral subdivision of Vmo for group Ia afferents.     

Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study

The distribution of two calcium-binding proteins, parvalbumin (PV) and calbindin-D 28K (CaBP), was studied by the peroxidase-anti-peroxidase immunohistochemical method at the light and electron microscopic level in the rat spinal cord and dorsal root ganglia. The possible coexistence of these two proteins was also investigated. PV-positive neurons were revealed in all layers of the spinal cord, except lamina I, which was devoid of labelling. Most of the PV-positive cells were found in the inner layer of lamina II, lamina III, internal basilar nucleus, central gray region, and at the dorsomedial and ventromedial aspects of the lateral motor column in the ventral horn. Neuronal processes intensely stained for PV sharply delineated inner lamina II. With the electron microscope most of them appeared to be dendrites, but vesicle containing profiles were also found in a smaller number. CaBP-positive neurons appeared to be dispersed all over the spinal gray matter. The great majority of them were found in laminae I, II, IV; the central gray region; the intermediolateral nucleus; and in the ventral horn just medial to the lateral motor column. Laminae I and II were densely packed with CaBP-positive punctate profiles that proved to be dendrites and axons in the electron microscope. A portion of labelled neurons in lamina IV and on the ventromedial aspect of the lateral motor column in the ventral horn disclosed both PV- and CaBP-immunoreactivity. All of the funiculi of the spinal white matter contained a large number of fibres immunopositive for both PV and CaBP. The highest density of CaBP-positive fibres was found in the dorsolateral funiculus, which was also densely packed with PV-positive fibres. PV-positive fibres were even more numerous in the dorsal part of the dorsal funiculus. The territory of the gracile funiculus in the brachial cord and that of the pyramidal tract in its whole extent were devoid of labelled fibres. In the thoracic cord, the dorsal nucleus of Clarke received a large number of PV-positive fibres. Dorsal root ganglia displayed both PV- and CaBP-immunopositivity. The cell diameter distribution histogram of PV-positive neurons disclosed two peaks--one at 35 microns and the other at 50 microns. CaBP-positive cells in the dorsal root ganglia corresponded to subgroups of small and large neurons with mean diameters of 25 microns and 45 microns, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology of single vestibulospinal collaterals in the upper cervical spinal cord of the cat. II. Collaterals originating from axons outside the ventral funiculi.

Recent studies have shown that vestibulospinal axons reach the upper cervical spinal cord of the cat via several different funicular routes. The purpose of this study was to describe the projections of those axons travelling outside the well-recognized pathways in the ventral funiculi. These axons are located in the dorsal columns, dorsolateral funiculi, and lateral funiculi. Collaterals of these axons were stained following extracellular injections of Phaseolus vulgaris leucoagglutinin in the medial and descending vestibular nuclei. The trajectories of individual collaterals were reconstructed from serial histological sections. Collaterals arising from axons in the same funiculus usually had the same characteristic appearance. Axons in the lateral funiculi, ipsilateral or contralateral to their cells of origin, gave rise to collaterals that had a simple structure and usually followed a horizontal trajectory across laminae VII and VIII. The boutons of these collaterals were distributed throughout the mediolateral extent of laminae VI and VII and the dorsal half of lamina VIII. In contrast, axons in the dorsolateral funiculi, ipsilateral or contralateral to their cells of origin, terminated primarily in laminae IV and V. Many collaterals of these axons projected either rostrally or caudally and had a narrow mediolateral distribution. The combined distribution of boutons from collaterals originating from axons in the dorsal columns included the dorsal horn and intermediate zone. Although these collaterals were less common and formed a heterogeneous group, they were easily distinguished from collaterals originating from axons travelling in other funiculi. These results indicate that vestibulospinal axons travelling outside the ventral funiculi comprise several distinct systems. Each system travels by a different funicular route and is distinguished by differences in collateral morphology and termination zones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trajectory of group Ia afferent fibers stained with horseradish peroxidase in the lumbosacral spinal cord of the cat: three dimensional reconstructions from serial sections.

A reconstruction was made of the intramedullary trajectory of 23 physiologically identified Ia afferents from cat hind limb muscles (medial gastrocnemius, soleus, plantaris, flexor digitorum-hallucis longus, and hamstring). The afferents were stained by intra-axonally injected HRP. The axons of these afferents were traced over distances of 5.8 mm to 15.7 mm rostrocaudally. In the dorsal funiculus fibers from all the muscles showed a similar course and similarly bifurcated into an ascending and a descending branch. The mean diameters of stem axons, ascending branches, and descending branches were 6.6 micrometer, 5.8 micrometer, and 3.0 micrometer, respectively. Within the analyzed lengths of the spinal cord five to eleven collaterals were given off from the two branches. The distances between adjacent collaterals of the ascending and descending branches averaged 1200 micrometer and 790 micrometer, respectively. The collaterals as a rule passed through the medial half of the dorsal horn before they entered the deeper parts of the gray matter. The terminal distribution areas common to all Ia collaterals were: (1) the medial half of the base of the dorsal horn, mainly lamina VI: (2) lamina VII; and (3) lamina IX. The numbers of terminals were largest in lamina IX and smallest in lamina VII. The density of terminals in lamina IX was highest in the homonymous motor cell column. The terminal distribution areas of adjacent collaterals showed no overlap in the sagittal plane. Terminal branches carried one bouton terminal and up to six boutons en passage with an average of 1.8 terminals per terminal branch. Apparent axosomatic and axodendritic contacts were seen on small-sized and medium-sized neurons in laminae V-VI, medium-sized neurons in lamina VII, and large neurons in lamina IX. One motoneurons was contacted by an average of 3.3 terminals. In addition to the common features, Ia collaterals of various muscles of origin showed some differences in their trajectories in the ventral horn, and in their terminations in the gray matter.     

Arginine-vasopressin enhances the responses of lateral septal neurons in the rat to excitatory amino acids and fimbria-fornix stimuli

Ia synapses in laminae VI and IX of the cat's spinal cord were examined in the electron microscope following iontophoretic injection of horseradish peroxidase (HRP) into single, identified, Ia afferent fibers from gastrocnemius muscles. Ia boutons contacting motoneuron dendrites in lamina IX contained spherical synaptic vesicles and generally contracted only one postsynaptic profile. The Ia boutons were often postsynaptic to smaller P-type axonal terminal. Consequently Ia boutons may be classified as S-boutons with axo-axonic contacts.     

The ultrastructure of group Ia afferent fiber synapses in the lumbosacral spinal cord of the cat

Ia synapses in laminae VI and IX of the cat's spinal cord were examined in the electron microscope following iontophoretic injection of horseradish peroxidase (HRP) into single, identified, Ia afferent fibers from gastrocnemius muscles. Ia boutons contacting motoneuron dendrites in lamina IX contained spherical synaptic vesicles and generally contracted only one postsynaptic profile. The Ia boutons were often postsynaptic to smaller P-type axonal terminal. Consequently Ia boutons may be classified as S-boutons with axo-axonic contacts.     

Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoid-HRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP).

The central projections of the rat sciatic, saphenous, median, and ulnar nerves were labeled by injecting each nerve with 0.05 mg B-HRP, or 0.5 mg WGA-HRP, or a mixture of both. The B-HRP labeled large dorsal root ganglion cells (30-50 microns) and, correspondingly, 98% of axons labeled in a rootlet were meyelinated; although all sizes of myelinated axons were labeled, a greater proportion fell in the large ranges (2-6.5 microns axon diameter) than in the small ranges (0.5-2 microns). Primary afferents labeled with B-HRP were distributed in laminae I, III, IV, and V of the dorsal horn and extended into the intermediate grey and the ventral horn; Clarke's column and the respective dorsal column nuclei were also densely labeled. Motoneurons of the nerve were densely labeled by B-HRP, including extensive regions of their dendritic trees. In contrast, WGA-HRP labeled small dorsal root ganglion cells (15-25 microns) and in the dorsal rootlets, 84% of the labeled axons were nonmyelinated; the small population of labeled myelinated afferents mainly fell within the smaller ranges (0.5-2.0 microns). Terminal fields of WGA-HRP labeled afferents were restricted to the superficial dorsal horn (laminae I-III), and to limited regions in the dorsal column nuclei. Sciatic nerve projections traced by labeling with B-HRP alone or in combination with WGA-HRP were more extensive than previously described when using either native HRP or WGA-HRP. Afferents to the dorsal horn extended from L1-S1, to Clarke's nucleus from T8-L1, to the ventral horn from L2-L5, and extended throughout the medial and dorsal region of the gracilie nucleus. Motoneurons were found from L4-L6. Using the same tracers, saphenous projections extended in the superficial dorsal horn from caudal L1 to rostral L4, in the deep dorsal horn to mid L4 and along the length of the central part of the gracilie nucleus. The median nerve projected to the internal basilar nucleus from C1-C6, the dorsal horn from C3-T2, Clarke's nucleus from T1-T6, the external cuneate nucleus, and a large central area throughout the length of the cuneate nucleus. Motoneurons were located in dorsolateral and ventrolateral nuclear groups from C4 through C8. The ulnar nerve projections were less extensive but also included the internal basilar nucleus from C1-C6, the medial region of the dorsal horn from C4-T1, Clarke's nucleus from T1-T6, the external cuneate nucleus, and the medial part of the cuneate nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology of single primary spindle afferents of the intercostal muscles in the cat

A reconstruction was made of the trajectory of primary spindle afferents from the intercostal muscles in the spinal cord of the cat. Intraaxonal recordings were performed from the primary spindle afferents that were identified by their response to lung inflation and stimulus threshold to activate the action potentials. The afferents were stained by using intraaxonal injection of horseradish peroxidase (HRP). Results were obtained mainly from internal intercostal Ia fibers, which entered the spinal cord and bifurcated into ascending and descending branches. The ascending branches could be traced up to 10.7 mm, and the descending branches could be traced up to 7.3 mm. The ascending branches extended to the next segment. Collaterals ranging from one to six were given off from these branches. The distances between adjacent collaterals ranged from 0.9 mm to 4.7 mm. Each collateral had similar morphological characteristics. The collaterals entered the dorsal horn and ran toward lamina IX through the medial half of the gray matter. Fine branches and boutons were given off in laminae V, VII, VIII, and IX. The aggregations of these branches were found in lamina VII, mainly in the region of Clarke's column and in the ventral and ventrolateral regions thereof and in lamina IX, mainly in the nucleus lateromedialis. Most terminals did not contact the somata of target neurons in all laminae in which terminals were found. However, a few terminals were found to contact large neurons in lamina IX. In addition to these aggregates, there were some terminals scattered throughout the ventral horn. Thus, it was concluded that single intercostal Ia afferents project to the region of Clarke's column, to the intercostal motor nucleus, and to the intermediate regions. J. Comp. Neurol. 398:459–472, 1998. © 1998 Wiley-Liss, Inc.     

Topography and organization of cranial nerve nuclei in the sand lizard, Lacerta agilis

Cobaltic-lysine complex compound was used to label cranial nerves of the ventrolateral (branchiomotor) and dorsomedial (somatomotor) nuclear columns in the sand lizard, Lacerta agilis. The dendritic arborizations and axonal trajectories of neurons of the respective nuclei were reconstructed from serial sections. A fairly uniform neuronal morphology was found in the nuclei of the ventrolateral column: a spindle-shaped perikaryon gave rise to dorsomedial and ventrolateral dendritic trees, the latter arborizing in a characteristic broomlike manner within a narrow region in the lateral white matter. Axons of all neurons converged upon the medial longitudinal fasciculus and after making a hairpin turn formed the corresponding motor roots. A group of small neurons constituted a separate subnucleus within the V motor nucleus. The VII and IX nuclei were fused into a single nuclear complex. The nucleus ambiguus was found dorsal to the XII nucleus and lateral to the dorsal vagal nucleus. The latter nucleus extended rostrally to the caudal pole of the VI nucleus, and its neurons sent axons to the VII, IX, and X nerves. The term "dorsal visceromotor column" designates the extended dorsal vagal nucleus. A number of small polygonal neurons lying scattered in the lateral part of the medulla were labeled via the VII, IX, and X nerves. This loose aggregate of labeled neurons was termed the "lateral visceromotor area." On the basis of nuclear topography and cellular morphology, the existence of a bulbar XI nucleus was excluded. Three different types of neurons could be distinguished in the dorsomedial nuclear column. Neurons with oval or spherical perikarya and radially oriented dendrites constituted the nuclei innervating external eye muscles. Except for the IV nucleus, axons followed a ventral trajectory. The accessory VI nucleus was composed of a second type of neuron with elongated soma and dorsoventral dendrite orientation; the dorsally directed axon turned ventrally at the VI nucleus. The XII nucleus contains a third type of neuron with strongly decussating dendrites. The distinct differences in the neuronal morphology did not support the classical assumption that all of the nuclei of the dorsomedial motor column supply muscles derived from somitic mesoderm. Sensory fibers of the trigeminal nerve formed the familiar spinal tract, which partially decussated in the medullospinal transition zone and could be followed as far as the lumbar segments on the ipsilateral side of the spinal cord. Neurons of the mesencephalic root of the trigeminal nerve were localized in the optic tectum; their descending fibers joined the medial aspect of the spinal tract.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decussations of the descending paraventricular pathways to the brainstem and spinal cord autonomic centers.

Decussations of descending fibers of the hypothalamic paraventricular nucleus (PVN) were investigated by using Phaseolus vulgaris-leucoagglutinin (PHA-L) in intact and brainstem-operated rats. Fibers descend ipsilaterally along the brainstem and spinal cord and decussate at four levels: 1) Supramamillary decussations (SM). PVN fibers reach this area through the lateral hypothalamus and along the third ventricle in the dorsal hypothalamus. In the posterior hypothalamus some fibers crossover in the SM and terminate in the supramamillary region bilaterally. 2) Pontine tegmentum. PVN fibers run in the lateral part of the tegmentum arching to the basis of the pons. Some fibers crossover under the fourth ventricle. The locus ceruleus and the Barrington's nucleus receive bilateral innervation with ipsilateral dominance. 3) Commissural part of the nucleus of the solitary tract (NTS). The major crossover of PVN fibers is found here. The decussated fibers form a dense network here, and loop rostralward to innervate the entire NTS. A midsagittal knife-cut through the NTS eliminated paraventricular-fibers on the contralateral side. Synaptic contacts between PHA-L-labeled boutons and tyrozine hydroxilase-positive neurons were verified in the NTS. The caudal ventrolateral medulla also receives bilateral innervation. 4) Lamina X of the thoracic spinal cord. Paraventricular fibers enter the lateral funiculus ipsilaterally and innervate the intermediolateral cell column (IML). Some fibers cross the midline ventral and dorsal to the central canal running to the contralateral IML, at the level of the decussation. Our results demonstrated that paraventricular projections form a continuous descending pathway on their side of origin, and provide crossover fibers which may terminate segmentally without forming long tracts after crossover.     

Immunohistochemical localization of calretinin in the dorsal root ganglion and spinal cord of the rat

Calretinin (CR). a recently identified calcium-binding protein, is present in nervous tissue, including sensory pathways, where it may play an important role in regulation of cellular activity. Using immunocytochemistry, we examined the cellular localization of CR in dorsal root ganglia (DRG) and spinal cord of normal rats and after multiple unilateral dorsal root ganglionectomies. In DRG, CR-immunoreactive cell bodies and axons were a small subpopulation (10%) of medium- to large-sized neurons. In the spinal cord, CR-like immunoreactivity (LI) in neurons and fibers was found in all laminae except motoneurons. Dense fiber networks were also found in Clarke's column. The densest staining of both cell bodies and fibers was in the superficial laminae, especially lamina II, and in the lateral spinal and lateral cervical nuclei. CR-immunoreactive fibers were also observed in the fasciculi cuneatus and gracilis. Fasciculus gracilis exhibited the greatest number of labeled axons at the lumbosacral levels, but few labeled axons were found at the rostral thoracic and cervical levels. In contrast, the corticospinal tract at the base of the dorsal column was devoid of CR-immunoreactive fibers. Unilateral multiple lumbar ganglionectomies resulted in a loss of CR-LI in the dorsal columns ipsilateral to the surgery. In the spinal gray matter ipsilateral to the ganglionectomies, CR-LI was reduced in Clarke's column and slightly enhanced in the medial third of lamina II. Our observations demonstrate a unique distribution pattern of CR-LI compared to other calcium-binding proteins in the spinal cord, and suggest a role for CR in nociceptive and proprioceptive pathways.     

Neural and anatomic characteristics of peripheral afferent fibers in the milk ejection reflex

Conduction velocities were measured and certain morphologic characteristics were examined of the abdominal mammary nerve in two- to ten-day postpartum rats. This nerve enters the spinal cord at the spinal segmental level T-12. Overall conduction velocity was (Mean +/- S.D.) 18.9 +/- 2.25 m/sec with a major peak at 9.7 +/- 0.72 m/sec. The distribution of conduction velocities in the nerve was similar to that of a typical spinal nerve. Nerve fiber diameters measured between about 1 and 25 microns with peaks at 4.9, 10.5, and 18.9 microns. Injection into the peripheral nerve of fluorescent dye, Lucifer yellow CH (LY), or wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) after ventral root rhizotomy permitted study of the distribution of primary afferents in the spinal cord. The terminal field of these fibers centered around the dorsal cap of Clarke's column and the lateral spinal nucleus, bilaterally. The distribution of WGA-HRP was more restricted than that of LY. A large number of LY-staining fibers were also found ipsilaterally in the medial portion of the intermediomedial column. A smaller amount of LY-staining was present contralaterally in the area of the spinothalamic tract. It is concluded that afferent impulses resulting from mammary stimulation in the milk ejection reflex are probably carried in a mixed spinal nerve whose primary afferent field lies mainly in ipsilateral spinal structures, although there is some evidence for crossing fibers. The data suggest that considerable opportunity exists for interaction with major sensory afferent fiber systems as well as with autonomic fibers. Hence, the spinal path of afferent information relevant for the milk ejection reflex may well be diffuse and it may involve several sensory modalities.     

Terminals of group la primary afferent fibres in Clarke''s column are enriched with l-glutamate-like immunoreactivity

Group Ia muscle spindle afferent fibres form giant terminals in Clarke's column which can be identified by morphological criteria. Postembedding Immunogold reactions were performed on tissue from the column using antiserum which recognized fixed L-glutamate in tissue. Giant terminals were heavily labelled with gold particles and quantitative analysis revealed that they contained significantly higher concentrations of L-glutamate in comparison with adjacent structures. L-Glutamate-enrichment of giant boutons is further evidence supporting the idea that this amino acid is a neurotransmitter at Ia synapses.     

Spinocerebellar projections from the upper lumbar segments in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase

The projection fields of the dorsal spinocerebellar tract (DSCT) arising from Clarke's column, marginal neurons of Clarke's column, and lamina V neurons in the upper lumbar segments were studied by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in the cat. To label only these neuron groups with uncrossed ascending axons, the spinal cord was lesioned rostral and contralateral to the WGA-HRP injections. Following injections of WGA-HRP into the L1-L4 segments, labeled terminals were seen in sublobules Ia-VIc and VIIb-VIIIb, the simple lobule, the paramedian lobule, and the dorsal paraflocculus. About 70-80% and 20-30% of the total number of labeled terminals were in the anterior and the posterior lobe, respectively; the projections were predominantly ipsilateral to the cells of origin (about 87% or more labeled terminals of the total number in each of sublobules IIb-Va). The labeled terminals were abundant in sublobule IIb (6-11%), lobule III (12-27%), and sublobules IVa (14-17%) and IVb (14-21%). In the mediolateral extent of the lobules in the anterior lobe, the labeled terminals were most numerous between 1.1 and 3.0 mm lateral to the midline (45-75% of the total number of labeled terminals on the ipsilateral side). In the posterior lobe labeled terminals were numerous in sublobule VIIIb (13.6%) and sublobule C of the paramedian lobule (15-19%). The projection fields in the horizontal plane of the lobules were reconstructed from a series of cross sections through each lobule. In the anterior lobe the labeled terminals were distributed in eight major areas. In sublobules IIb-III, areas 1-3 were located within 1.0 mm of the midline in zone A of Voogd; areas 4-6, between 1.0 and 2.5 mm lateral to the midline in zones B-C1; and areas 7 and 8, lateral to 3.0 mm from the midline in zones C2 and C3. Areas 1-6 extended apicobasally in the middle part of the lobules. In sublobule VIIIb projections were confined to three longitudinal areas whereas in the paramedian lobule the projection areas were less distinct. The projection pattern of the lumbar DSCT was different from that of the thoracic DSCT reported previously. In the anterior lobe the thoracic DSCT projects to five areas in the medial (zone A) and the lateral part (zone B) of the vermis and to four areas in the intermediate region of the hemisphere (zones C1-C3).     

The topographic organization of associational fibers of the olfactory system in the rat, including centrifugal fibers to the olfactory bulb

This study analyzed the topographic organization of the associational fibers within the olfactory cortex of the rat, by using the autoradiographic method. Small injections of 3H-leucine were placed in all of the subdivisions of the olfactory cortex, to label selectively the fibers arising in each area. Intracortical fibers were identified from all of the olfactory cortical areas except the olfactory tubercle and were classified into two major systems (the layer Ib system and the layer II-deep Ib system) on the basis of their laminar pattern of termination (see Luskin and Price, '83). The layer Ib fiber system arises in the anterior olfactory nucleus, piriform cortex, and lateral entorhinal area, and is broadly organized in relation to the lateral olfactory tract. Cortical areas deep to or near the lateral olfactory tract are preferentially interconnected with areas near the tract, while parts of the cortex lateral and caudal to the lateral olfactory tract are most heavily interconnected with areas lateral, caudal, and medial to the tract. Commissural projections from the anterior olfactory nucleus and the anterior piriform cortex match some (but not all) components of the ipsilateral layer Ib fiber system. The layer II-deep Ib fiber system arises in three small areas--the ventral tenia tecta, the dorsal peduncular cortex, and the periamygdaloid cortex. The fibers from the ventral tenia tecta terminate in layer II of the anterior olfactory nucleus and are topographically organized. The fibers from the dorsal peduncular cortex and the periamygdaloid cortex are more widely distributed, especially in the lateral and caudal parts of the cortex. Two other intracortical projections do not fit into either of these fiber systems. The nucleus of the lateral olfactory tract projects bilaterally to the islands of Calleja and the medial edge of the anterior piriform cortex. The anterior cortical nucleus projects to many parts of the olfactory cortex, but the fibers end in both superficial and deep parts of layer I (layer Ia and Ib). There are projections from several of the olfactory cortical areas to the cortical areas surrounding the olfactory cortex. Virtually all of the olfactory areas also project to the ventral and dorsal endopiriform nuclei deep to the piriform cortex and/or to the polymorph zone deep to the olfactory tubercle. In addition, projections have been demonstrated to the deep amygdaloid nuclei, especially from the more ventromedial and caudal parts of the olfactory cortex.