Nitric oxide-containing neurons in long-term grafts in a rat model of Parkinson's disease.

The role that nitric oxide may play in modulating graft function in long-term fetal ventral mesencephalic grafts in an animal model of Parkinson's disease was investigated. Mature grafts harvested from the entire fetal ventral mesencephalon possessed a large number of neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase-containing neurons throughout the graft intermingled with dopaminergic neurons. The morphological and neurochemical characteristics of these NADPH-diaphorase neurons resembled those in centers adjacent to the substantia nigra of adult brain but not that of the striatum. Pretreatment with the nNOS blocker, 7-nitroindazole, resulted in contralateral rotations following methamphetamine challenge in long-term grafted animals that previously showed normalized rotational behavior. In contrast, mature grafts derived from fetal ventral mesencephalon without the midline areas possessed only a few nNOS-containing neurons within the grafts, and a similar methamphetamine challenge following 7-nitroindazole pretreatment in long-term grafted rats that previously showed normalized rotational behavior resulted in random movements. Our results indicate that nitric oxide-containing neurons inadvertently included during grafting may affect graft function, and excluding the midline areas of the ventral mesencephalon during tissue harvesting may minimize this effect.

Role of the mesolimbic cholinergic projection to the septum in the production of 22 kHz alarm calls in rats.

The role of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) to septum in the production of 22 kHz ultrasonic vocalization was studied in adult rats, using behavioral-pharmacological and anatomical tracing methods. Direct application of carbachol, a muscarinic agonist, into the lateral septal region induced species-typical 22 kHz alarm calls. The septum receives cholinergic input from LDT, thus, activation with glutamate of predominantly cholinergic neurons of the LDT induced comparable 22 kHz alarm calls in the same animals. This glutamate-induced response from LDT was significantly reduced when the lateral septum was pretreated with scopolamine, a cholinergic antagonist. To investigate the localization of the cell groups projecting to septum, the fluorescent retrograde tracer, fluorogold, was pressure injected into the lateral septum and sections from these brains were also immunostained against choline acetyltransferase (ChAT) to visualize cholinergic cell bodies. Several ChAT-fluorogold double-labeled cells within the boundaries of the LDT were found, while other fluorogold-labeled regions did not contain double-labeled cells. These results provide both direct and indirect evidence that at least a part of the mesolimbic ascending cholinergic projection from LDT to septum is involved in the initiation of the 22 kHz vocalization. It is concluded that the septum is an integral part of the medial cholinoceptive vocalization strip and the 22 kHz alarm vocalization is triggered from septum by the cholinergic input from the LDT.

Subpallidal outputs to the nucleus accumbens and the ventral tegmental area: anatomical and electrophysiological studies.

The goal of this study was to investigate the functional organization of the subpallidal-->accumbens direct and indirect feedback loops by both anatomical and electrophysiological methods. The results of the dextran-conjugated rhodamine injections into the subpallidal area has shown three distinct projections: (1) a substantial pathway from the subpallidal area to the ventral tegmental area, (2) a more diffuse rostral projection from the subpallidal area to the core area of the nucleus accumbens, and (3) a sparse pathway projecting rostrodorsally from the subpallidal area toward the thalamic regions. Electrical or chemical stimulation of the subpallidal region, which was studied by the axonal tracer, evoked inhibitory responses in the majority (60 and 80%, respectively) of the accumbens and ventral tegmental area neurons in a standard extracellular recording study. Less than 1/3 of the accumbens or ventral tegmental area cells showed an increase in the mean firing rate. The majority (77.5%) of all responded neurons had a latency of less than 10 ms. Furthermore, injection of glutamate into the subpallidal area not only altered the firing pattern of the accumbens neurons, but also attenuated their excitatory responses elicited by the electrical stimulation of the ventral subiculum. Our results indicate that the subpallidal area plays a predominantly inhibitory role in the ventral tegmental area-accumbens-subpallidal circuitry, presumably by its GABAergic projections, and may also modulate subicular input into the nucleus accumbens.

Effects of self- and cross-reinnervation on the numbers of motoneurons regenerating to forearm muscles in young and adult rats.

The present study was carried out to compare the ability of motoneurons to regenerate to functionally appropriate and inappropriate muscles, following axotomy at different stages of postnatal development. Five-, 10-, 21-day-old and adult rats of both sexes were used. In one group, the right median and radial nerves were cut and reunited. In a second group, the cut nerves were cross reunited and, in a third group the nerves were merely exposed. Following survival periods of up to one year, the extent of motoneuron regeneration through the repaired nerves was determined by injecting the retrogradely transported tracers horseradish peroxidase (HRP) and Fast Blue into the flexor and extensor muscles of the right forearm. The results were expressed in terms of the difference between the number of labelled motoneurons on the experimental side of the spinal cord and the number on the control side, the latter having been labelled by injection of HRP and Fast Blue into the muscles of the left forearm. Comparisons were then made between the groups with respect to the age at which axotomy occurred, and the target of regeneration. The results showed that when axotomy was performed in 5- and 10-day-old rats, significantly fewer motoneurons were labelled, irrespective of whether or not the target was functionally appropriate, than when axotomy was performed in adulthood. The difference was most likely due to a lower survival rate of motoneurons following axotomy in neonates. No difference was found, however, between the numbers of labelled median and radial nerve motoneurons following self- versus cross-reinnervation in any age group. This suggests that, in both adult and neonatal rats, motoneurons which survive axotomy are able to regenerate equally well to functionally appropriate or inappropriate muscles.

Afferent organization of the lateral reticular nucleus in the rat: an anterograde tracing study.

The organization of the afferent projections to the lateral reticular nucleus of the rat was investigated following placement of horseradish peroxidase-conjugated wheatgerm agglutinin into the red nucleus, fastigial nucleus, various levels of the spinal cord or the sensorimotor area of the cerebral cortex. The pattern of distribution of anterogradely labelled profiles visualized with tetramethylbenzidine revealed that the caudal three-fourths of the lateral reticular nucleus received a large, topographically organized projection from the entire length of the contralateral spinal cord. The lateral part of the rostral half of the lateral reticular nucleus received a small projection from the contralateral red nucleus, the dorsal part of the middle third of the nucleus received a diffuse projection from the contralateral fastigial nucleus, and the extreme rostromedial part of the nucleus received a sparse projection from the contralateral cerebral cortex. The dorsal part of the middle third of the lateral reticular nucleus also received a small projection from the ipsilateral cervical spinal cord. The distribution of afferent fibres from different levels of the spinal cord, red nucleus, and fastigial nucleus overlapped substantially in the middle third of the lateral reticular nucleus, whereas the cerebral cortical receiving area was separate. These data suggest that the middle third of the lateral reticular nucleus integrates spinal and supraspinal impulses to the cerebellum, while the rostral part of the nucleus is involved in a separate cerebral cortico-cerebellar pathway.

Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures.

The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P-immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P-containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase-immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase-immunoreactive dendrites. These findings reveal that two types of transmitter-specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase.

Efferent connections of the A1 noradrenergic cell group: a DBH immunohistochemical and PHA-L anterograde tracing study.

Immunohistochemical localization of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) was employed to reveal the anatomical organization of the A1 noradrenergic cell group in the caudal ventrolateral medulla oblongata of the rat. Subsequently, the supraspinal efferent axonal projections of A1 were investigated with a view to elucidating the anatomical substrates underlying its postulated function in central fluid and cardiovascular homeostasis. Within the caudal medulla, DBH-positive/PNMT-negative (noradrenergic) neurons were observed extending bilaterally through the ventrolateral medullary reticular formation from upper cervical spinal cord levels to the level of the area postrema. At the rostral pole of A1, its neurons intermingled with PNMT-immunoreactive perikarya of the more rostrally situated C1 adrenergic cell group. Discrete injections of the anterogradely transported plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into A1 resulted in terminal labeling in a number of presumptive efferent target sites including the nucleus of the solitary tract, rostral ventrolateral medulla, dorsal parabrachial nucleus, Kolliker-Fuse nucleus, central grey, dorsomedial nucleus of the hypothalamus, perifornical region, zona incerta, lateral hypothalamus, paraventricular nucleus of the hypothalamus, supraoptic nucleus, bed nucleus of the stria terminalis, and organum vasculosum of the lamina terminalis. Tissue sections adjacent to those reacted for PHA-L were processed immunohistochemically for DBH to determine if anterogradely labeled terminals were localized in regions that demonstrated appropriate immunoreactivity. The majority of regions in which PHA-L terminal labeling was present also exhibited moderate to intense DBH activity. These experiments provide neuroanatomical evidence for direct efferent pathways from the A1 noradrenergic cell group to a number of supraspinal sites that have been reliably implicated in the neural circuitry underlying the central regulation of fluid and cardiovascular homeostasis. Furthermore, the results suggest a selective anatomical interrelation between A1 and sites in the basal forebrain and hypothalamus in which vasopressinergic neurons have been previously demonstrated. It is postulated that the noradrenergic A1 projections observed in this investigation represent the morphological substrate through which A1 exerts a significant influence on cardiovascular regulatory mechanisms.

The arrangement of forearm motoneurons in young and adult rats and the possibility of naturally occurring motoneuron death.

The normal number and arrangement of motoneurons contributing fibres, via the median and radial nerves, to the forearm muscles of the rat were determined at different stages of postnatal development. Horseradish peroxidase was applied directly to the proximal cut ends of the nerves in 5, 10, 21 days old and adult rats. At each stage of development, the motoneuronal pool for each nerve was confined to the ipsilateral, dorsolateral area of Lamina IX. In general, median nerve motoneurons were found dorsal and medial to those of the radial nerve and were located between the rostral parts of the C6-T1 segments of the spinal cord. The radial nerve motoneuronal pool extended between the caudal end of the C4 and rostral tip of the T1 segment. There was considerable overlap between the two populations, especially along the dorsoventral axis. Counts of labelled motoneurons revealed that significantly fewer were present in adult compared to 5 days old rats (P less than 0.05 for each nerve). Approximately 50% of the motoneurons were lost from each nerve over this period. Although results from other studies suggest that part of the apparent loss may have been due to deficiencies in the HRP tracing technique, the possibility of motoneuronal death cannot be entirely excluded in this study.

Simultaneous demonstration of choline acetyltransferase and glutamic acid decarboxylase immunoreactivity in the rat interpeduncular nucleus.

Double antigen immunohistochemistry was employed to simultaneously examine the distribution of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity in the rat interpeduncular nucleus (IPN) at the subnuclear and ultrastructural level. ChAT-immunoreactive axons of the fasciculus retroflexus (FR) innervated specific subnuclear divisions of the IPN that possessed GAD-immunoreactive somata and a high density of GAD-immunoreactive axons and terminals. At the ultrastructural level, each of the cholinoceptive subnuclei possessed a characteristic axodendritic synaptic contact. These morphologically distinct synapses were composed of terminals of ChAT-positive FR axons forming asymmetric contacts with dendritic profiles of GAD-positive neurons. An array of symmetric axodendritic contacts with GAD immunoreactivity located pre- and/or postsynaptically was also present in the cholinoceptive subnuclear divisions. The present study provides direct evidence for synaptic interactions between ChAT-immunoreactive FR axons and dendritic processes of GAD-immunoreactive neurons in the rat IPN. Also, GAD-positive terminals arising from possible intrinsic projections contact dendritic profiles of GAD-immunoreactive neurons in receipt of ChAT-positive FR terminals. These results reveal that putative cholinergic afferent inputs and GABAergic intranuclear projections simultaneously innervate a subpopulation of IPN neurons that possess GAD immunoreactivity.

A comparison of the subnuclear and ultrastructural distribution of acetylcholinesterase and choline acetyltransferase in the rat interpeduncular nucleus.

The subnuclear and synaptic staining patterns for acetylcholinesterase (AChE) activity and choline acetyltransferase (ChAT) activity were studied in the rat interpeduncular nucleus (IPN) using histochemical and immunohistochemical methods. AChE reactivity was prominent in the neuropil of the rostral, lateral and dorsomedial subnuclei, whereas ChAT immunoreactivity was confined to axons and terminals in the rostral, intermediate and central subnuclei. AChE-positive somata were evident in all the subnuclear divisions of the IPN, and possessed reaction product in the rough endoplasmic reticulum and nuclear envelope. ChAT-positive somata were not present in the IPN. Characteristic axodendritic synapses in the rostral, intermediate and central subnuclei possessed ChAT immunoreactivity presynaptically, and AChE reactivity both pre- and postsynaptically. Other synaptic arrangements in the lateral subnucleus lacked ChAT-immunoreactive terminals, yet possessed prominent AChE reactivity. The results of the present study reveal that AChE reactivity and ChAT immunoreactivity are heterogeneously distributed among the subnuclear divisions of the rat IPN, and that AChE reactivity is present in both the cholinoceptive and noncholinoceptive subnuclei. Although neuronal colocalization of ChAT and AChE activity is not evident in the IPN, AChE-positive neurons are in receipt of putative cholinergic, as well as peptidergic, afferent inputs.

Synaptic organization of septal projections in the rat medial habenula: a wheat germ agglutinin-horseradish peroxidase and immunohistochemical study.

The synaptic organization of septal inputs to the rat habenular complex of the dorsal diencephalon was examined employing the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The cellular distribution of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivity was also studied at the light and electron microscopic level. Following placements of tracer within the entire septum, labeled axons were observed in the stria medullaris and in the medial and lateral subnuclei of the habenula. Following injections of tracer in the nuclei triangularis and septofimbrialis of the posterior septum, the medial subnucleus was heavily labeled, whereas the lateral subnucleus was devoid of peroxidase activity. The medial subnucleus possessed labeled myelinated axons and terminals that contained clear, spherical vesicles and formed asymmetric contacts with dendritic spines and shafts. Terminals possessing WGA-HRP activity also formed non-synaptic junctions with other labeled or unlabeled terminals. SP and ChAT immunoreactivity in normal and colchicine-treated animals was confined to dendrites and somata within the medial habenula. Terminals containing clear spherical vesicles formed asymmetric synaptic contacts with these immunoreactive somatic and dendritic profiles. Based on the combined anterograde tracing and immunohistochemical data, it is proposed that septal projections provide a direct innervation to habenular neurons that contain ChAT or SP activity. These septal inputs may play an important role in the facilitation of the ChAT- and SP-positive habenular neurons, both of which provide prominent afferent inputs to the interpeduncular nucleus. Thus, neurons of the habenula and interpeduncular nucleus are under the direct and indirect influence of septal neurons within the limbic forebrain circuit.

Axonal branching of the olivocerebellar projection in the rat: a double-labeling study.

Collateralization of olivocerebellar (climbing) fibers was studied in the rat by means of the fluorochrome double-labeling technique. Most of the olivocerebellar projection is crossed except for a minimal ipsilateral component which arises from the most rostal part of the inferior olivary nucleus (ION). ION neurons in the caudolateral part of the medial accessory olive (MAO) and the dorsal accessory olive (DAO) give off axons that branch to supply both hindlimb areas of the contralateral cerebellar cortex, i.e., the rostral anterior lobe and the caudal paramedian lobule. In addition, neurons in the middle one-third of the contralateral MAO and DAO send axons that divide to terminate in both the caudal part of the anterior lobe and the rostral part of the paramedian lobule (forelimb receiving areas). Neurons within the caudal part of the MAO, the lateral part of the DAO, the ventral lamella of the principal olive (PO), and the dorsomedial cell column (DMCC) send axonal branches that terminate within at least two different areas of the same sagittal zones throughout the contralateral cerebellar cortex. Thus, the ION contains specialized cells that provide a divergence of integrated information from the ION to at least two cerebellar regions.

Glutamate decarboxylase immunoreactivity in the rat interpeduncular nucleus: a light and electron microscope investigation.

The distribution of immunohistochemically demonstrable glutamate decarboxylase, the synthetic enzyme for GABA, was examined in the rat interpeduncular nucleus at the light and electron microscope levels. Immunoreactive perikarya were distributed in a characteristic pattern among the subnuclear divisions. The rostral, ventral and caudal portions of the nucleus possessed numerous immunoreactive perikarya, while few immunoreactive somata were observed in the subnuclei of the dorsal aspect. A dense field of immunostained axons and terminals was also present throughout. Ultrastructural examination of glutamate decarboxylase immunoreactivity revealed numerous labelled somata, dendritic processes, axons and boutons. Axodendritic and axosomatic synapses with immunoreactive postsynaptic profiles were numerous throughout those subnuclei with large numbers of immunoreactive somata. Immunostained terminals in contact with both immunoreactive and non-immunoreactive somatic and dendritic profiles were also present. An abundance of immunostained terminals was observed in the subnuclei that possessed a sparse population of immunoreactive somata. Immunoreactive myelinated axons of unknown origin were also present. This investigation demonstrates that the rat interpeduncular nucleus possesses a large population of glutamate decarboxylase-immunoreactive neurons coextensive with a plexus of immunostained axons and terminals. The results suggest that the immunoreactive neurons give rise to axons which contribute to an intrinsic circuit interconnecting the different subnuclear divisions. These immunoreactive neurons are in receipt of non-immunoreactive afferent inputs of variable morphology, as well as projections from intrinsic immunoreactive neurons.

Collateral axonal projections from the A1 noradrenergic cell group to the paraventricular nucleus and bed nucleus of the stria terminalis in the rat.

The A1 noradrenergic cell group in the caudal ventrolateral medullary reticular formation of the rat sends efferent projections to a number of regions in the basal forebrain and hypothalamus, but the extent to which these projections represent collateral branches of individual axons is not known. Immunohistochemical labeling of medullary neurons containing the catecholamine biosynthetic enzymes tyrosine hydroxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase was used to reveal the anatomical location of A1 noradrenergic neurons within the ventrolateral medulla. Subsequently, the retrograde fluorescence double-labeling technique was employed to investigate the collateralization of ascending A1 efferent axons. The subcommissural bed nucleus of the stria terminalis (BST) was injected with rhodamine-fluorescent latex microspheres and the ipsilateral left paraventricular nucleus of the hypothalamus (PVN) was injected with Fast blue. Within the ventrolateral medulla, single- and double-labeled neurons were identified in a distribution corresponding to that demonstrated for A1 noradrenergic perikarya. The results indicate that some ascending axons from cells within the A1 region collateralize to effect a simultaneous innervation of the BST and PVN. The innervation of multiple efferent targets by single neurons within the A1 region may have important implications with respect to A1's postulated role in central cardiovascular regulation.

Topographical and ultrastructural investigation of the habenulo-interpeduncular pathway in the rat: a wheat germ agglutinin-horseradish peroxidase anterograde study.

The topographical and ultrastructural organization of the habenular projection to the interpeduncular nucleus (IPN) of the rat was examined employing the anterogradely transported tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and the chromogen tetramethylbenzidine (TMB). Unilateral placements of WGA-HRP in the habenular complex resulted in heavy terminal labelling in the rostral, central, and intermediate subnuclei bilaterally, and in the lateral subnuclei ipsilaterally. The apical subnucleus possessed only a sparse amount of label. Placements confined to the medial habenula (mH) produced similar results to those observed when the entire habenula was filled, suggesting that the afferent contribution made by the lateral habenula (lH) to the IPN is small. Unilateral placements of WGA-HRP in the dorsal portion of the mH resulted in heavy, predominantly ipsilateral labelling in the lateral subnucleus and the dorsal cap of the rostral subnucleus. In the lateral subnucleus labelled habenular terminals consistently contacted single dendritic processes shared by one or more other boutons, possibly of nonhabenular origin. Labelled habenular terminals in the rostral subnucleus normally contacted one or two dendrites. Labelled terminals in both subnuclei possessed clear, spherical vesicles and a variable number of dense-core vesicles. Unilateral placements of WGA-HRP in the ventral portion of the mH resulted in heavy labelling in the rostral half of the rostral subnucleus with a slight ipsilateral predominance, and in the central and intermediate subnuclei bilaterally. Terminal labelling was observed in crest and S synapses in the intermediate and central subnuclei respectively. Crest synapses, which consist of two parallel habenular terminals contacting an attenuated dendritic process, normally possessed label in only one of the two boutons. In the central subnucleus labelled horizontal axons formed several en passant S synapses with dendritic processes of small and medium diameter. These synaptic specializations of habenular axons contained numerous clear, spherical vesicles. This study demonstrates that a major topographically organized projection to the IPN originates from two distinct subpopulations of habenular neurons which comprise a dorsal sector and a ventral sector of the mH. Ultrastructural examination demonstrated that axons originating from neurons in the ventral and dorsal mH form characteristic contacts in the various IPN subnuclei.

Ultrastructural study of remodeled rubral afferents following neonatal lesions in the rat.

Following neonatal hemicerebellectomy, an aberrant ipsilateral cerebellorubral projection develops that maintains the topographic specificity of the normal contralateral projection. Similarly, neonatal lesions of the sensorimotor cortex lead to the appearance of an aberrant contralateral corticorubral projection that mirrors the topographic specificity of the normal ipsilateral input. The specificity of synaptic localization in these aberrant projections was studied by use of ultrastructural visualization of anterogradely transported HRP-WGA. Following neonatal ablations, adults received HRP-WGA injections in the unablated deep cerebellar nuclei or sensorimotor cortex. After 48 hours, animals were sacrificed and processed for ultrastructural localization of anterogradely transported HRP-WGA. In hemicerebellectomized animals, both the contralateral and ipsilateral interpositorubral projections terminated on the somatic and proximal dendritic membrane of magnocellular neurons. Some of these labeled synaptic terminals were located on somatic and dendritic spines. Following HRP-WGA injection in the unablated nucleus lateralis, anterogradely labeled synaptic terminals were located bilaterally on small- to medium-sized dendrites of parvicellular neurons. Injection of HRP-WGA in the remaining sensorimotor cortex of animals that had undergone neonatal unilateral ablation of the sensorimotor cortex resulted in labeled corticorubral synaptic terminals that contacted distal dendrites of ipsilateral and contralateral parvicellular neurons. These results demonstrate that, following neonatal deafferentation of the rat red nucleus, the topographic specificity of the aberrant rubral afferents is accompanied by a specificity of synaptic localization on discrete membrane areas of rubral neurons.

Double-labeling study of axonal branching within the lateral reticulocerebellar projection in the rat.

Collateral axonal branching to the cerebellum from the lateral reticular nucleus (LRN) was studied in the rat by using the fluorescent double-labeling technique. Following injection of Fast Blue (FB) into the cerebellar cortex, followed 3 days later by injection of Nuclear Yellow (NY) into a different region of the cortex, single- and double-labeled cells were found within the LRN. Most LRN-cerebellar projections were bilateral with ipsilateral preponderance, except for the projection to the paramedian lobule, which was completely ipsilateral. The dorsolateral area of the magnocellular division of the LRN contained cells whose axons branch to terminate in the rostral anterior lobe and the caudal part of the ipsilateral paramedian lobule (hindlimb areas of the cerebellar cortex), while the medial area of the LRN contained cells that supply, via collateral axonal branching, the caudal area of the contralateral anterior lobe and the rostral part of the ipsilateral paramedian lobule (forelimb areas of the cerebellum). Branched LRN-cerebellar axons projected to both hemispheres and to both sides of the caudal anterior lobe. No axonal branching was evident in the LRN-cerebellar projection to the rostral anterior lobe. The projection to the anterior and posterior lobe vermis also contained collateral axonal branching.

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons.

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T4 or T7 levels centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T2 level were observed throughout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.

Quantitative analysis of rubral degeneration following neonatal deafferentation.

The effect of neonatal hemicerebellectomy on the cytoarchitecture of the red nucleus was investigated in the rat. Quantitative analysis revealed a 31% loss of neurons in the magnocellular red nucleus contralateral to the lesion. This cell loss was accompanied by a similar decrease in the cross-sectional area of the red nucleus. To provide insight into this degenerative process, the neonatal status of the normal cerebellorubral projection was determined using anterograde transport of HRP-WGA which was injected into the deep cerebellar nuclei at various times during the first 10 postnatal days. A definitive cerebellorubral projection was not detectable before postnatal day 8. The data indicate that the rubral afferent fibers from the cerebellum were removed before they reached their target. These results are significant in light of the aberrant projections that develop after neonatal lesions.

Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projection.

Unilateral neonatal cortical ablation induces the development of a bilateral corticorubral projection from the remaining sensorimotor cortex. The retrograde fluorescent tracers Fast blue (FB) and Nuclear yellow (NY) were used to determine if the aberrant contralateral projection arises from axon collaterals of the normal uncrossed projection. Six to 8 weeks after unilateral cortical ablation in neonatal rats, the red nuclei were injected with FB on one side and NY on the other to study the source of the normal and aberrant afferents from the cerebral cortex. In control animals, many neurons in layer V of the sensorimotor cortex were retrogradely labeled with the tracer that had been injected into the ipsilateral red nucleus. In animals with unilateral ablations, many neurons throughout the remaining sensorimotor cortex were retrogradely labeled with FB or NY. No cortical neurons were doubly labeled. In addition to demonstrating the bilaterality of the corticorubral projection in animals which had received neonatal lesions, these results indicate that the aberrant contralateral corticorubral projection does not consist of axon collaterals of the normal ipsilateral fibers.