FB-labeled Neurons Research Articles

Cytoarchitecture, morphology, and lumbosacral spinal cord projections of the red nucleus in cattle

To analyze the morphology, cytoarchitecture, and lumbosacral spinal cord projections of the red nucleus (RN) in cattle. 8 healthy Friesian male calves. Anesthetized calves underwent a dorsal laminectomy at L5. Eight bilateral injections (lateral to the midline) of the neuronal retrograde fluorescent tracer fast blue (FB) were administered into the exposed lumbosacral portion of the spinal cord. A postsurgical calf survival time of 38 to 55 days was used. Following euthanasia, the midbrain and the L5-S2 spinal cord segments were removed. Nissl's method of staining was applied on paraffin-embedded and frozen sections of the midbrain. The mean length of the RN from the caudal to cranial end ranged from 6,680 to 8,640 microm. The magnocellular and parvicellular components of the RN were intermixed throughout the nucleus, but the former predominate at the caudal portion of the nucleus and the latter at the cranial portion with a gradual transitional zone. The FB-labeled neurons were found along the entire craniocaudal extension of the nucleus, mainly in its ventrolateral part. The number of FB-labeled neurons was determined in 4 calves, ranging from 191 to 1,469 (mean, 465). The mean cross-sectional area of the FB-labeled neurons was approximately 1,680 microm2. In cattle, small, medium, and large RN neurons, located along the entire craniocaudal extension of the RN, contribute to the rubrospinal tract reaching the L6-S1 spinal cord segments. Thus, in cattle, as has been shown in cats, the RN parvicellular population also projects to the spinal cord.

The effects of delayed nerve repair on neuronal survival and axonal regeneration after seventh cervical spinal nerve axotomy in adult rats

It has been proposed clinically that delayed surgery after traumatic brachial plexus injury may adversely affect functional outcome. In the present experimental study the neuroprotective and growth-promoting effects of early and delayed nerve grafting following proximal seventh cervical spinal nerve (C7) axotomy were examined. The ventral branch of C7 spinal nerve was transected and axons projecting out of the proximal nerve stump were labelled with Fast Blue (FB). At the same time, the biceps brachii muscle was denervated by transecting the musculocutaneous nerve at its origin. Neuronal survival and muscle atrophy were then assessed at 1, 4, 8 and 16 weeks after permanent axotomy. In the experimental groups, a peripheral nerve graft was interposed between the transected C7 spinal nerve and the distal stump of the musculocutaneous nerve at 1 week [early nerve repair (ENR)] or 8 weeks [delayed nerve repair (DNR)] after axotomy. Sixteen weeks after nerve repair had been performed, a second tracer Fluoro-Ruby (FR) was applied distal to the graft to assess the efficacy of axonal regeneration. Counts of FB-labelled neurons revealed that axotomy did not induce any significant cell loss at 4 weeks, but 15% of motoneurons and 32% of sensory neurons died at 8 weeks after injury. At 16 weeks, the amount of cell loss in spinal cord and dorsal root ganglion (DRG) reached 29 and 50%, respectively. Both ENR and DNR prevented retrograde degeneration of spinal motoneurons and counteracted muscle atrophy, but failed to rescue sensory neurons. Due to substantial cell loss at 8 weeks, the number of FR-labelled neurons after DNR was significantly lower when compared to ENR. However, the proportion of regenerating neurons among surviving motoneurons and DRG neurons remained relatively constant indicating that neurons retained their regenerative capacity after prolonged axotomy. The results demonstrate that DNR could protect spinal motoneurons and reduce muscle atrophy, but had little effect on sensory DRG neurons. However, the efficacy of neuroprotection and axonal regeneration will be significantly affected by the amount of cell loss already presented at the time of nerve repair.

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2-4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 mum(2) for myenteric neurons, 750 mum(2) for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons.

The light and electron microscopic characterisation of identified striato-ventrotegmental projection neurons in the domestic chick ( Gallus domesticus)

A major projection of the medial striatum (lobus parolfactorius, LPO) of birds is the striato-ventrotegmental pathway projecting to the area ventralis tegmentalis. In the present study, we investigated the morphology and connectivity of striato-ventrotegmental neurons in the medial LPO. The neurons were identified by injecting the fluorescent retrograde tracer fast blue (FB) into the area ventralis tegmentalis. FB-labelled neurons in the LPO were targeted and iontophoretically injected with lucifer yellow (LY) in paraformaldehyde fixed slices. The fluorescent LY label in the filled neurons was then photoconverted, and the ultrastructure of cells was investigated. According to our results, the soma of striato-ventrotegmental neurons is rich in organelles, in particular rough and smooth endoplasmic reticula and they possess a large, unindented and slightly eccentric nucleus. The LY-labelled cells possess relatively few, sparsely spiny dendrites, and represent a type of medium-sized spiny projection neuron characteristic of the striata of birds. Axospinous synapses on the labelled cells are asymmetric and correspond morphologically to the glutamatergic excitatory type of terminals described previously in the LPO. Both symmetric and asymmetric axodendritic and axosomatic synapses were detected. Some symmetric synapses were GABA immunolabelled, whereas some asymmetric synapses were immunopositive to glutamate. Axon collaterals of labelled cells formed symmetric or asymmetric axodendritic synapses. Direct contact without interposing glial processes was observed between one of the FB-labelled neurons and an adjacent neuronal soma. There was also a microneuron attached to one of the labelled cells, which we identified as a neurogliaform ‘dwarf’ cell.

Neural circuitry of the adult rat central nervous system after spinal cord injury: a study using fast blue and the Bartha strain of pseudorabies virus.

The distribution of retrogradely and transneuronally labeled neurons in the adult rat brain and spinal cord after contusive mid-thoracic spinal cord injury (SCI) was studied using Fast Blue (FB) and the Bartha strain of pseudorabies virus (PRV), respectively. When FB was injected into the distal spinal cord at 2 days after graded SCI at the 10th vertebral level, labeled neurons were consistently found 7 days later in supraspinal areas that normally project to the spinal cord. The number of FB-labeled neurons decreased as the injury severity increased. An inverse correlation between the number of FB-labeled neurons and injury severity was seen in most investigated brain nuclei with coefficient of correlations (r) ranging from -0.84 in the red nucleus to -0.92 in the raphe nuclei. The coefficient of correlation was relatively poor in the motor cortex (r = -0.63), where a mild injury (6.25 g.cm) resulted in a 99% damage of the corticospinal tract. Such a prominent difference between the corticospinal tract and other descending pathways can be related to the difference in location of these pathways within the adult rat spinal cord. When PRV was injected into the right sciatic nerve one month after the injury, labeled cells were consistently identified 5 days later in the spinal cord rostral to the injury and in certain supraspinal regions that regulate autonomic outflow. In these nuclei, the distribution and number of PRV-labeled neurons markedly decreased after SCI as compared to the control group. In contrast, PRV-labeled neurons were inconsistently found in the supraspinal nuclei that contribute to somatic motor outflow in normal controls and no labeling was observed in these nuclei after injury. These results demonstrate that (1) a proportion of neural network across the injured spinal cord has been spared after acute contusive SCI, (2) the proportion of spared axons of a particular pathway is closely correlated to the injury severity and the position of that pathway, and (3) the transneuronal labeling method using PRV may provide a unique approach to investigate multi-synaptic neural circuitry of the central autonomic control after SCI, but its application to the somatic motor system is limited.

Long-term observation on the changes of somatotopy in the facial nucleus after nerve suture in the cat: Morphological studies using retrograde labeling

To examine the time course of plasticity of the cranial nucleus during axonal regeneration, we followed the topographical reorganization of the cat facial nucleus (FN) up to 24 months after facio-facial nerve suture using retrograde labeling methods. The trunk of the temporal-zygomatico-orbital and both superior and inferior buccolabial branches (defined as main branch) of the facial nerve was cut and sutured again under ketamine hydrochloride anesthesia. At 11–722 days after nerve suture, Fast Blue (FB) and 1,1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate (DiI) or horseradish peroxidase (HRP) were injected into the distal part of the sutured main branch and the unoperated posterior auricular branch, respectively. Until about 3 months after suture, the topographical pattern in FN was similar to that observed in normal cats. At about 4 months after suture, FB-labeled motoneurons were distributed not only in the lateral part (including intermediate, dorsal and ventrolateral divisions) but also in the medial subdivision of FN. After a survival period of 18–24 months, FB-labeled neurons were found all over the FN, and their number increased significantly. Interestingly, in the longer survival cases, we noticed that the DiI- or HRP-labeled posterior auricular branch motoneurons also showed a tendency to distribute outside the medial region. The present study showed that somatotopic disorganization starts at around 4 months after suture, which seems to be somewhat slower than that in rats, and continues until a much later postoperative period. Furthermore, we suggested a possibility that the regeneration of one branch may affect the somatotopy of the unoperated nerve branch. These phenomena may contribute to aberrant facial nerve functions such as abnormal associated movement and facial spasm observed after nerve injury.

The neurotrophins NGF and NT-3 reduce sensory neuronal loss in adult rat after peripheral nerve lesion

The effect of three different neurotrophins on axotomy-induced death of adult rat sensory neurons was examined. The ventral branch of the 13th spinal nerve was transected and the corresponding neurons in the 13th thoracic (T13) dorsal root ganglion (DRG) were pre-labelled with Fast Blue (FB). For a period of 4 weeks, animals received either no treatment, continuous intrathecal infusion of phosphate buffer, nerve growth factor (NGF), neurotrophin-3 (NT-3), or brain-derived neurotrophic factor (BDNF). Labelled neurons remaining after this period were counted. Inert, or no treatment, resulted in extensive loss of the DRG neurons. BDNF application was virtually non-effective, while NGF or NT-3 resulted in a greater number of FB-labelled neurons compared to normal controls. This suggests that NGF and NT-3 are survival factors for adult sensory neurons with a therapeutic potential in peripheral nerve injuries.

Connections of the rostral ventral respiratory neuronal cell group: an anterograde and retrograde tracing study in the rat

The connections of the rostral ventral respiratory cell group (VRG) were retrogradely and anterogradely determined after discrete injections of a mixture of the fluorescent tracers Fast Blue (FB) and Fluoro Ruby (FR) into the physiologically identified rostral inspiratory cell group. Retrogradely FB-labeled neurons and/or anterogradely FR-labeled fibers and terminal fields were located bilaterally in a variety of brain areas. Both retrograde and anterograde labelings were mainly found in: 1) the deep cerebellar nuclei; 2) the lateral lemniscus and paralemniscal nuclei, deep gray, and white intermediate layers of the superior colliculus, tegmental (laterodorsal and microcellular) nuclei, and central gray; and 3) the septohypothalamic nucleus, and lateral and posterior hypothalamic areas. The FR-labeled terminal-like elements were found in: 1) Crus 2 of the ansiform lobule, and the simple, 2, and 3 cerebellar lobules; 2) the subcoeruleus, deep mesencephalic, and Edinger-Westphal nuclei; and 3) the premammillary, lateral, and medial mammillary nuclei, retrochiasmatic part of the supraoptic nucleus, and the zona incerta. The FB-labeled neurons were found in: 1) the parapedunculopontine tegmental and cuneiform nuclei, caudal linear nucleus of the raphe, and adjacent area of the cerebral peduncle; 2) the thalamic posterior nuclear group and subparafascicular, parafascicular, and gelatinosus thalamic nuclei; 3) the parastrial amygdaloid and subthalamic nuclei; and 4) the olfactory tubercle, granular, and agranular insular cortex, parietal and lateral orbital cortices. The connections of the rostral VRG with several cerebellar, midbrain, diencephalic, and telencephalic regions could provide an anatomical substrate for a role of these regions in the control of respiratory-related functions.

Characterization of voltage-sensitive Na+ and K+ currents recorded from acutely dissociated pelvic ganglion neurons of the adult rat.

1. Electrophysiological properties of acutely dissociated neurons from the major pelvic ganglion (MPG) of the adult male rat were studied with whole cell patch-clamp recording techniques. The MPG neurons innervating the urinary bladder were labeled by retrograde axonal tracing methods with the use of a fluorescent dye, Fast Blue (FB) injected into the bladder wall and identified with a fluorescent microscope. 2. Passive and active membrane properties such as resting membrane potential, input resistance, duration of action potentials, thresholds for spike activation, or duration of afterhyperpolarization in unidentified MPG neurons were comparable with those of FB-labeled neurons innervating the urinary bladder. The action potential in both unidentified and bladder efferent MPG neurons was reversibly abolished by tetrodotoxin (TTX, 1 microM). The afterhyperpolarization of the TTX-sensitive action potential in both groups was reduced by application of Cd2+ (0.1 mM) and further suppressed by tetraethylammonium (TEA, 10 mM). Extracellularly applied TEA increased the duration of the action potential, and 4-aminopyridine (4-AP, 1 or 2 mM) also reduced the spike afterhyperpolarization and increased the spike duration. The duration of the action potential was decreased and the rate of spike repolarization was increased by approximately 2.5-fold with negative shift of membrane potential from -40 to -80 mV. 3. The isolated Na+ current was reversibly blocked by 1 microM TTX and had a mean peak amplitude of 127.3 pA/pF when activated from a holding potential of -70 mV in the external solution containing 100 mM Na+. The Na+ conductance reached half-maximal activation at a membrane potential of -21.5 mV with a slope factor of 4.9 mV. The steady-state inactivation of Na+ conductance occurred at membrane potentials more depolarized than -90 mV, and the half-maximal inactivation was obtained at -57.5 mV with a slope factor of 8.8 mV. 4. The fast-transient A-type K+ current (IA) was activated at membrane potentials more depolarized than -60 mV from a holding membrane potential of -100 mV, reached a peak amplitude within 10 ms after the onset of depolarizing voltage steps, and decayed within 20-30 ms at membrane potential depolarizations to +20 to +30 mV. The IA current activated by a voltage step to +20 mV from a holding potential of -100 mV averaged 102.1 pA/pF. The half-maximal activation of the IA conductance was obtained at a membrane potential of -21.2 mV with a slope factor of 9.9 mV. In steady-state inactivation of IA current, the half-maximal inactivation occurred at -76.5 mV and the slope factor was 8.0 mV. 5. The delayed K+ current was reduced by 25-35% by bath application of Cd2+ or the elimination of extracellular Ca2+ ions. The bath application of 4-AP (2 mM) suppressed the IA current by 75% and the delayed K+ current by 60%. Extracellularly applied TEA (10 mM) suppressed the delayed K+ current by 90%, but suppressed the IA current by only 16%. 6. These results indicate that bladder neurons and unidentified neurons in the MPG have similar properties including a TTX-sensitive Na+ current and three distinct types of voltage-sensitive K+ currents-IA current, Ca(2+)-activating K+ current, and delayed rectifier K+ current-that contribute to the repolarization phase of the action potential. These electrical properties of the MPG neurons resemble those of sympathetic neurons in the superior cervical and inferior mesenteric ganglia.

Organization of the vagal preganglionic neurons innervating the stomach in the Japanese quail (Conturinx japonica) double labeling fluorescent method.

Cytoarchitectural study indicated that the dorsal motor nucleus of the vagal nerve (nX) was formed by two subnuclei in a coronal section. One of them had large and round neurons, and occupied the dorsal part of the nX (Xd). The second one had small and oval neurons, and occupied the ventral part of the nX (Xv). The double labeling fluorescent method was performed to determine the organization of preganglionic neurons in the nX innervating different muscles of the quail stomach. Injections of nuclear yellow (NY) into the proventriculus, and of fast blue (FB) into the gizzard showed that there were no double-labeled neurons in the nX. NY- and FB-labeled neurons were found throughout the nX. After separate injections of NY and FB into both the cranioventral and caudodorsal thick muscles, and the craniodorsal and caudodorsal thin muscles, many NY- and FB-labeled neurons were distributed and intermingled in the middle two thirds of the nX. Considerable numbers of double-labeled neurons were also found at the middle level of the nX. There was no side-predominancy of the population of the preganglionic neurons innervating the quail stomach. These results show that, many neurons of both sides of the nX are separately innervating both the proventriculus and gizzard, and considerable numbers of single neurons of the nX also innervate a pair of thin or thick muscles with collateral axons.

Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

Calcium-binding proteins and neuropeptides were examined in trigeminal neuronal cell bodies retrogradely labeled with Fast blue (FB) from the maxillary molar tooth pulp of the rat. FB-labeled cells were located in the maxillary division of the trigeminal ganglion. ∼ 30 and 50% of the labeled cells were immunoreactive for parvalbumin and calcitonin gene-related peptide (CGRP), respectively. The coexpression of these substances was observed in 9.5% of FB-labeled cells. On the other hand, 2.4% of FB-labeled cells exhibited calretinin-immunoreactivity (CR-ir) and 20% tachykinin (TK)-ir. The coexpression of CR and TK was observed in 1.9% of FB-labeled cells, i.e., most of CR-ir FB-labeled neurons coexpressed TK-ir. An immuno-EM method revealed that all parvalbumin-ir nerve fibers in the root pulp were myelinated and that CGRP-ir nerve fibers were both myelinated (15%) and unmyelinated (85%). The present study indicated that primary nociceptors innervating the rat molar both pulp contained parvalbumin and CR and coexpressed these calcium-binding proteins and neuropeptides. It was suggested that peripheral axons of parvalbumin-ir tooth pulp primary neurons are all myelinated. Most peripheral CR-ir axons are probably unmyelinated because TK-ir myelinated axons have never been demonstrated in any peripheral organ.

Spinal projection neurons to the laterodorsal pontine tegmental nucleus: relationship to preganglionic neurons and nitric oxide synthase.

The region of the rat sacral parasympathetic nucleus (SPN) contains distinct subpopulations of neurons that project supraspinally or are preganglionic neurons. Some preganglionic neurons in the SPN serve as the motor outflow for urinary bladder contraction; other neurons in the SPN project to regions of the rostral pons that subserve micturition reflexes. Previous studies utilizing immunohistochemistry or staining for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) have demonstrated that numerous neurons in the SPN contain nitric oxide synthase (NOS), the enzyme for nitric oxide synthesis. Thus, the objectives of this study were to determine 1) the distribution of neurons in the region of the SPN that project to the laterodorsal tegmentum (LDT) of the pons, 2) whether spinal neurons projecting to a peripheral autonomic ganglion also project to the LDT, and 3) whether NOS or NADPH-d is present in LDT projection neurons. Preganglionic neurons were identified by injecting the retrograde tracer fluorogold (FG) into the major pelvic ganglion (MPG). Supraspinally projecting neurons were identified by injecting the retrograde tracer fast blue (FB) into the LDT. Numerous FB-labeled neurons were present in the ipsi- and contralateral SPN and were immediately dorsal to FG-labeled preganglionic neurons. Neurons containing both tracers were not observed. Approximately 20% of preganglionic neurons, but no LDT projection neurons, were reactive for NOS and NADPH-d. These data suggest that the region of the SPN is a site for distinct subpopulations of neurons that project to the LDT and to the MPG and that NOS is contained in some preganglionic neurons, but is not a marker for LDT projection neurons.

Extension and Regeneration of Corticospinal Axons after Early Spinal Injury and the Maintenance of Corticospinal Topography

It has been established that neonatal corticospinal (CS) axons are able to grow around lesions of the spinal cord early in neonacy (Bernstein and Stelzner, J. Comp. Neurol. 221:382-400; Firkins, Bates, and Stelzner, Exp. Neurol. , 120:1-15). To determine if these corticospinal axons include regenerating as well as late developing axons a double-labeling paradigm is used in which CS neurons are retrogradely labeled from the cervical spinal cord by injections of fast blue (FB) on Postnatal Day (PND) 2, 4, or 10. Two days later, the FB is aspirated along with the left dorsal funiculus and the right hemicord (CHR). As adults, the animals receive an injection of diamidino yellow (DY) or rhodamine into the spinal cord caudal to the lesion site. Thus, FB neurons are those that originally projected to the spinal cord before the lesion and which survived axotomy, DY neurons are those whose axons reached the spinal cord after the lesion, and double-labeled neurons (FB/DY) are cells which projected to the spinal cord prior to the lesion and regenerated a spinal axon postlesion. Animals FB injected on PND 2 have a widespread distribution of FB-labeled neurons in cortex, including areas outside of sensorimotor cortex. These animals also had both DY- and FB/DY-labeled cells within sensorimotor cortex, indicating that the population of axons growing caudal to neonatal spinal lesions consists of both late growing and regenerating axons. In animals FB injected on PND 10, the FB neurons were all located in sensorimotor cortex. Very few DY and no FB/DY neurons were present. We have also looked at the topography of the CS neurons which project caudal to early spinal lesions. Rat pops received a CHR on PND 0, 3, 6, or 12. As adults, horseradish peroxidase was injected into the cervical or lumbar enlargement of the spinal cord and the distribution of labeled cells in the cerebral cortex was plotted and compared to normal and lesioned adult controls. In all experimental animals, the distribution of retrogradely labeled cells was restricted to the area containing CS projection neurons in the normal animal. This is despite the fact that the number of CS projection neurons is greatly reduced from normal and the normal pathway for CS axonal outgrowth has been completely disrupted by the neonatal lesion.

Hypertrophy of rat sensory ganglion neurons following intestinal obstruction

Background: Neuroplastic changes following ileum hypertrophy have been reported in intrinsic enteric neurons. The hypothesis in the present study was that intestinal hypertrophy induces neuronal changes in dorsal root ganglia (DRG). Methods: Under sodium pentobarbital anesthesia, partial obstruction was produced in the rat by tying a plastic ring around the terminal loop of ileum. Fast Blue (FB)(Sigma, St. Louis, MO) was injected into the obstructed ileum wall, and the rat was perfused after 8 days. DRG were immunostained and examined to identify and measure sizes of perikarya containing FB and/or calcitonin gene-related peptide (CGRP) or FB and/or substance P (SP). Results: Of the DRG neurons that projected to the ileum in control or obstructed animals, approximately 50% were CGRP-immunoreactive (IR) and 30% were SP-IR (colchicine pretreatment was not used). Neurons that projected to the obstructed ileum were increased in size compared with neurons in nonobstructed controls. Some of these neurons were CGRP-IR or SP-IR; some were large FB-labeled neurons that were not SP-IR or CGRP-IR. Conclusions: The morphology of sensory autonomic neurons in adult animals is influenced by dynamic interactions with the targets they innervate, whether directly or transneuronally.

Skin-visceral divergent projection of cholecystokinin — containing dorsal root ganglion neurons: A tri-labelling study with fluorescent tracers and immunohistochemistry

Skin-visceral divergent projections of cholecystokinin (CCK)-containing dorsal root ganglion neurons were studied by combined technique of fluorescent double-labelling and immunohistochemistry. Fast blue (FB) and nuclear yellow (NY) were injected into the coeliac ganglion and the cutaneous branches of left 9th-11th intercostal nerves, respectively. Three kinds of neurons labelled with fluorescein were observed in T9-11 dorsal root ganglia: FB-labelled neurons with blue-fluorescent cytoplasm; NY-labelled neurons with yellow-fluorescent nucleus and double-labelled neurons with blue cytoplasm and yellow nucleus. The double-labelled neurons were found to account for 2.8% of total labelled neurons. The sections containing neurons labelled with fluorescein were stained by CCK-immunohistochemical procedure. Four kinds of neurons could be identified: NY-neurons with CCK-immunoreactivity (NY+CCK); FB-neurons with CCK-immunoreactivity (FB+CCK); NY+FB neurons with CCK-immunoreactivity (NY+FB+CCK); and neurons only CCK-positive. NY+FB+CCK tri-labelled neurons accounted for approximately 11.5% of NY+FB double-labelled neurons, and for 0.4% of all CCK-positive neurons. The findings clearly indicated that the peripheral processes of some sensory dorsal root ganglion neurons divergently project to both skin and visceral structure and contain CCK.

Thalamic projection to motor area and area 3a of the cat cerebral cortex

The distributions of thalamic neurons projecting to the motor cortex and cortical area 3a were studied in cat by means of the retrograde double-labeling technique using Nuclear Yellow (NY) and Fast Blue (FB) as tracers. Following injection of NY and FB into the motor cortex and area 3a respectively, the NY-labeled neurons were found to be mainly located in ventrolateral (VL) nucleus and FB-labeled neurons in ventro-posterolateral nucleus (VPL). However, these two kinds of neurons were intermingled with each other in the border area between VL and VPL. A small number of neurons were double-labeled by both NY and FB. They were also distributed in the border area. Some of them could often be found in centromedian and parafascicular nuclei.

A reevaluation of midbrain and diencephalic projections to the inferior olive in rat with particular reference to the rubro-olivary pathway.

Projections from the midbrain and caudal diencephalon to the inferior olivary nucleus (ION) in the rat were investigated by using anterograde and retrograde tracing techniques. Particular attention was directed to studying the projection from the red nucleus (RN) to the ION. Tritiated leucine was stereotaxically injected into the RN in six animals. The injection sites and the portion of the medulla containing the ION were processed for autoradiography. In three cases, the injections were largely confined to the RN. In these instances, no terminal labeling was noted in the ION. In the other three animals, the injections spread beyond the RN to include, in one or another of the rats, such areas as the mesencephalic reticular formation (MRF) between the RN and the periaqueductal gray (PAG), the region around the fasciculus retroflexus (FR), the prerubral area, the MRF dorsolateral to the RN, or the region dorsal to the medial geniculate body. In these animals, terminal labeling was observed ipsilaterally in the ION, chiefly in the caudal one-half of the medial accessory olive and in the dorsal lamella and lateral bend of the principal olive. Retrograde transport of lectin-conjugated horseradish peroxidase (WGA-HRP) or the fluorescent dye fast blue (FB), injected into the ION, was used to localize regions in the midbrain and caudal diencephalon containing neurons which project to the inferior olive. Following WGA-HRP injections into the ION, retrogradely labeled neurons were found in the ipsilateral ventral PAG, the MRF between the PAG and the RN, the MRF dorsolateral to the RN, and the area dorsal to the medial geniculate body. Large numbers of heavily labeled neurons were found in the area surrounding the FR. No labeled cells were noted in the RN or the nucleus of Darkschewitsch (ND). Subsequent to FB injections into the ION, fluorescent neurons were observed in the same regions which contained HRP-labeled neurons in the experiments using the enzyme as a tracer. Additionally, very small numbers of FB-labeled neurons were found in the RN and the ND. These results indicate that, in the rat, descending input from the midbrain and caudal diencephalon to the ION arises chiefly from a medially located column of neurons extending from the level of the rostral RN to the region of the FR. Little, if any, input to the ION is derived from the ND or RN in this species, unlike the cat, in which a substantial projection to the ION arises from the ND and the RN.