Tetramethylrhodamine Dextran Amine Research Articles

Chondroitin Sulfate Expression in Perineuronal Nets After Goldfish Spinal Cord Lesion.

Perineuronal nets (PNNs) surrounding neuronal cell bodies regulate neuronal plasticity during development, but their roles in regeneration are unclear. In the PNNs, chondroitin sulfate (CS) is assumed to be involved in inhibiting contact formation. Here, we examined CS expression in PNNs in the ventral horn of a goldfish hemisected spinal cord in which descending axons regenerate beyond the lesion to connect with distal spinal neurons. In intact fish, chondroitin sulfate A (CS-A)–positive PNNs accounted for 5.0% of HuC/D-immunoreactive neurons, and 48% of choline acetyltransferase (ChAT)-immunoreactive neurons. At 2, 4 and 8 weeks after spinal hemisection, CS-A–positive PNNs accounted for 8.4%–9.9% of HuC/D-immunoreactive neurons, and 50%–60% of ChAT-immunoreactive neurons, which was not significantly different from intact fish. Chondroitin sulfate C (CS-C)–positive PNNs accounted for 6.4% of HuC/D-immunoreactive neuron, and 67% of ChAT-immunoreactive neurons in intact fish. At 2, 4 and 8 weeks after spinal hemisection, CS-C–positive PNNs accounted for 7.9%, 5.5% and 4.3%, respectively, of HuC/D-immunoreactive neurons, and 65%, 52% and 42%, respectively, of ChAT-immunoreactive neurons, demonstrating a significant decrease at 4 and 8 weeks after spinal hemisection. Among ventral horn neurons that received descending axons labeled with tetramethylrhodamine dextran amine (RDA) applied at the level of the first spinal nerve, CS-A–positive PNNs accounted for 53% of HuC/D-immunoreactive neurons. At 2 and 4 weeks after spinal hemisection, CS-A–positive PNNs accounted for 57% and 56% of HuC/D-immunoreactive neurons, which was not significantly different from intact fish. CS-C–positive PNNs, accounted for 48% of HuC/D-immunoreactive neurons that received RDA-labeled axons. At 2 and 4 weeks after spinal hemisection, CS-C–positive PNNs significantly decreased to 22% of the HuC/D-immunoreactive neurons, and by 4 weeks after spinal hemisection they had returned to 47%. These findings suggest that CS expression is maintained in the PNNs after spinal cord lesion, and that the descending axons regenerate to preferentially terminate on neurons not covered with CS-C–positive PNNs. Therefore, CS-C in the PNNs possibly inhibits new contact with descending axons, and plasticity in the spinal neurons might be endowed by downregulation of CS-C in the PNNs in the regeneration process after spinal hemisection in goldfish.

Retinal ganglion cell topography and spatial resolving power in the oriental fire-bellied toad Bombina orientalis.

The vertebrate visual system is determined by two main factors, a species' lifestyle and phylogenetic legacy. Studying the visual system in outgroup lineages may shed some light on the balance of these factors within a certain radiation. We studied the topography of retinal ganglion cells (RGCs) in the retina of the oriental fire-bellied toad Bombina orientalis. These toads belong to the ancient superfamily Discoglossoidea, a sister group to all extant Anura except for two small families. RGCs were retrogradely labeled with tetramethylrhodamine- dextran amine (TMR-DA) and examined in retinal wholemounts. RGCs occurred all over the retina except for the far periphery. Their total number was [Formula: see text] ([Formula: see text], [Formula: see text]). They comprised 73-77% of all cells in the ganglion cell layer. The spatial density of GCs increased gradually from the dorsal and ventral retinal periphery toward the equator to form a weak visual streak and a moderately pronounced area centralis. The minimum density was [Formula: see text], and the maximum, [Formula: see text]. The maximum density gradient was [Formula: see text]. The spatial resolution was minimum in the dorsal and ventral periphery ([Formula: see text] and [Formula: see text] cycles per degree in water and air, respectively). Intermediate values of spatial resolving power were found within the visual streak ([Formula: see text] and [Formula: see text] cycles per degree) and reached a peak in area centralis ([Formula: see text] and [Formula: see text] cycles per degree). This is sufficient for efficient prey location and capture. The relatively high RGC density and the presence of specialized retinal regions in oriental fire-bellied toads are consistent with their highly visual behavior. A brief review comparing the phylogeny and ecology of this with other anuran species suggests that the main factor shaping the RGC distribution in Anura is phylogenetic legacy; the environmental pressure results mainly in adjusting the maximum spatial density of RGCs (and hence the visual acuity) to meet the species' needs.

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

Origins of endomorphin-2 immunopositive fibers and terminals in the spinal dorsal horn of the rat

Endomorphin 2 (EM2) plays essential roles in regulating nociceptive transmission within the spinal dorsal horn, where EM2-immunopositive (EM2-IP) fibers and terminals are densely encountered. However, the origins of these EM2-IP structures are still obscure. Unilateral primary sensory afferents disruption (lumbar 3–6 dorsal roots rhizotomy) significantly decreased the density of EM2-IP fibers and terminals in the superficial laminae (laminae I and II) on the ipsilateral but not contralateral lumbar dorsal horn (LDH). Spinal hemisection at the 7th thoracic (T7) segment down-regulated bilateral EM2 expression, with a higher influence on the ipsilateral side of the LDH. Unilateral L3–6 dorsal roots rhizotomy combined with spinal transection but not with hemisection at T7 level completely obliterated EM2-IP fibers and terminals on the rhizotomized-side of the LDH. Disruption of bilateral (exposure to the primary afferent neurotoxin, capsaicin) primary sensory afferents combined with spinal hemisection at T7 decreased the EM2-IP density bilaterally but could obliterate it on neither side of the LDH. While in capsaicin plus transection rats, EM2 was depleted symmetrically and completely. In the colchicine treated rats, no EM2-IP neuronal cell bodies could be detected in the spinal gray matter. After injecting tetramethyl rhodamine dextran-amine (TMR) into the LDH, some of the TMR retrogradely labeled neurons in the nucleus tractus solitarii (NTS) showed EM2-immunoreactivities. The present results indicate that EM2-IP fibers and terminals in the spinal dorsal horn originate from the ipsilateral primary afferents and bilateral descending fibers from NTS.

Topographical distributions of endomorphinergic pathways from nucleus tractus solitarii to periaqueductal gray in the rat

In the central nervous system (CNS), endomorphin 1 (EM1)- and endomorphin 2 (EM2)-containing neuronal cell bodies have been found in the nucleus tractus sollitarii (NTS) and the hypothalamus, and EMergic fibers and terminals are distributed widely in many regions of the CNS, including the periaqueductal gray (PAG). The aim of the present study was to examine whether EM-expressing neurons in the NTS of the rat send their axons to the PAG, and determine whether the EMergic pathway from the NTS to the PAG is topographic by using. Immunofluorescent staining for EM1 or EM2 combined with retrograde and anterograde tract-tracing methods. The results showed that after injecting tetramethyl rhodamine dextran-amine (TMR) into the ventrolateral or lateral column of the PAG, some EM1- or EM2-immunoreactive (IR) neurons in the NTS were retrogradely labeled with TMR, and the majority of the EM-IR/TMR double-labeled neurons were mainly distributed in the medial and commissural subnuclei of the NTS. Following injection of biotinylated dextran amine (BDA) into the medial or commissural subnucleus of the NTS, EM1-IR/BDA and EM2-IR/BDA double-labeled fibers and terminals were mainly distributed in the ventrolateral or lateral column of the PAG, respectively. The results indicate that EMergic pathway from the NTS to PAG is topographically organized, and suggest that EMs released from NTS to PAG projecting terminals may bind to μ-opioid receptor on the PAG neurons, and thereby contribute to various functions.

Vestibular afferents to the motoneurons of glossopharyngeal and vagus nerves in the frog, Rana esculenta

The aim of this work was to study whether the vestibular afferent fibers establish direct connections with the motoneurons of glossopharyngeal and vagus nerves of the frog, Rana esculenta. In anaesthetized animals the vestibulocochlear nerve and the common root of glossopharyngeal-vagus and accessory (IX-X-XI) nerves were simultaneously labeled with fluorescein dextran amine (vestibulocochlear nerve) and tetramethylrhodamine dextran amine (IX-X-XI). With a confocal laser scanning microscope we could detect close appositions between the vestibular afferent fibers and somatodendritic components of the general and special visceral motoneurons of the ambiguus nucleus of IX-X nerves. The direct impulse transmission may provide a quick and immediate response of cardiovascular and gastrointestinal system upon body displacement.

Endomorphin 1‐ and Endomorphin 2‐Containing Neurons in Nucleus Tractus Solitarii Send Axons to the Parabrachial Nuclei in the Rat

Endomorphin-1 (EM1) and endomorphin-2 (EM2) are the selective endogenous ligands for the mu-opioid receptor (MOR). Since EMs-expressing neuronal cell bodies or axonal components have been observed, respectively, in the nucleus tractus solitarii or the parabrachial nuclei, we examined if EMs-expressing neurons in the NTS of the rat might send their axons to the PBN. Immunofluorescent stainings for EM1 or EM2 were combined with retrograde or anterograde tract-tracing method. After injecting tetramethyl rhodamine dextran-amine (TMR) into the parabrachial nuclei of rats, some EM1- or EM2-immunoreactive neurons in the nucleus tractus solitarii were labeled retrogradely with TMR. The majority of the EM1/TMR and EM2/TMR double-labeled neurons were observed in the medial, commissural, and dorsolateral subnuclei of the nucleus tractus solitarii. Following injection of biotinylated dextran amine (BDA) into the medial, commissural, or dorsolateral subnuclei of the nucleus tractus solitarii, EM1- or EM2-immunopositive axons and axon terminals were anterogradely labeled with BDA mainly in the lateral parabrachial nucleus. The present results have indicated that endomorphinergic neurons in the nucleus tractus solitarii project to the parabrachial nuclei. This suggests that EMs released from NTS-PBN projection fibers may bind to MOR on the PBN neurons to be implicated in processing of visceral information within the parabrachial nuclei.

Vestibulotrigeminal pathways in the frog, Rana esculenta

The aim of this study was to investigate whether primary vestibular afferent fibers establish direct connections with the motor and sensory trigeminal system in the brainstem of the frog. The experiments were carried out on Rana esculenta. In anaesthetized animals the trigeminal and vestibular nerves were prepared, and their proximal stumps were labeled either with fluorescein binding dextran amine (trigeminal nerve) or tetramethylrhodamine dextran amine (vestibulocochlear nerve). With a confocal laser scanning microscope we could detect close connections between the vestibular fibers and branches of the dorsal dendritic array of the jaw-closing motoneurons, suggestive of monosynaptic contacts. In the other parts of the brainstem, vestibular terminals were detected in the termination areas of the mesencephalic trigeminal nucleus and of the Gasserian (Vth) ganglion and they were probably involved in polysynaptic connections. In agreement with the results obtained in mammalian species, the present findings suggest that the vestibulotrigeminal relationship is quite complex and uses multiple pathways to connect the vestibular apparatus with the motor and sensory nuclei of the trigeminal nerve in the anurans as well.

Medullary dorsal horn neurons providing axons to both the parabrachial nucleus and thalamus

It has often been suggested that the trigemino- and spino-thalamic pathways are highly implicated in sensory-discriminative aspects of pain, whereas the trigemino- and spino-parabrachial pathways are strongly implicated in affective/emotional aspects of pain. On the other hand, the superficial laminae of the spinal dorsal horn, where many nociceptive neurons are distributed, have been reported to contain projection neurons innervating both the parabrachial nucleus (PBN) and thalamus by way of axon collaterals (Hylden et al., 1989). For the medullary dorsal horn (caudal subnucleus of spinal trigeminal nucleus: Vc), however, the existence of such neurons has not been reported. Thus, in the present study, we examined whether the Vc might contain projection neurons sending their axons to both the thalamus and PBN. Dual retrograde labeling with fluorescence dyes was attempted. In each rat, tetramethylrhodamine-dextran amine and Fluoro-gold were stereotaxically injected into the PBN and thalamic regions, respectively. The proportion of the dually labeled Vc cells in the total population of all labeled Vc cells was about 20%. More than 90% of the dually labeled neurons were distributed in lamina I (marginal zone), less than 10% of them were located in lamina II (substantia gelatinosa), and only a few (about 1%) were found in lamina III (magnocellular zone). The results indicate that some Vc neurons in the superficial laminae mediate nociceptive information directly to the PBN and thalamus by way of axon collaterals and that the vast majority of them project to the ipsilateral PBN and contralateral thalamus.

Identification of roughskin newt medullary vasotocin target neurons with a fluorescent vasotocin conjugate

Arginine8 vasotocin (AVT), a neurohypophyseal peptide in nonmammalian vertebrates, plays a key role in the regulation of social behaviors related to reproduction. In male roughskin newts (Taricha granulosa), AVT is an important facilitator of several reproductive behaviors, including courtship clasping of females. Although AVT is known to act in certain brain regions and AVT receptors have been localized to some extent, specific target neurons for this peptide have not been identified in any species. Internalization of a receptor-specific conjugate of AVT and the fluorescent dye Oregon green was used to identify AVT target cells in the medulla of male roughskin newts. Medullary neurons are of interest because they appear to mediate facilitation of clasping by AVT. Direct application of AVT-Oregon green to the fourth ventricular surface of the medulla in vivo resulted in conjugate internalization by a widespread population of medullary neurons, particularly in the medial reticular formation and nuclei of cranial nerves V, VII, VIII, IX, and X. Some fourth-ventricle ependymal cells were also labeled. Reticulospinal neurons, which play an important role in clasping, were identified by retrograde labeling with tetramethylrhodamine dextran amine. AVT-Oregon green was internalized by 72% of these neurons. These results show that AVT can directly affect a very large and diverse medullary neuronal population, which may underlie the peptide's role in multiple neuroendocrinological processes, including autonomic and behavioral regulation. Selectivity of the AVT action may arise through interactions between AVT and steroids such as corticosterone.

Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: Projections from the heart preferentially express vesicular glutamate transporter 1

The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.

Projections from the parabrachial nucleus to the vestibular nuclei: potential substrates for autonomic and limbic influences on vestibular responses

Previous anatomical studies in rabbits and rats have shown that the superior vestibular nucleus (SVN), medial vestibular nucleus (MVN) and inferior vestibular nucleus (IVN) project to the parabrachial nucleus (PBN) and Kölliker–Fuse (KF) nucleus. Adult male albino rabbits and Long–Evans rats received iontophoretic injections of biotinylated dextran amine, Phaseolus vulgaris leucoagglutinin, Fluoro-Gold or tetramethylrhodamine dextran amine into either the vestibular nuclei or the PBN and KF nuclei. The results were similar in both rats and rabbits. Injections of retrograde tracers into the vestibular nuclei produced retrogradely labeled neurons bilaterally in caudal third of the medial, external medial, and external lateral PBN in both species, with more variable labeling in KF. Rats also had consistent bilateral (predominantly contralateral) labeling in the ventrolateral PBN. The most prominent labeling was produced from injections that included the SVN, with fewer labeled neurons observed from injections in the caudal MVN and the IVN. Anterograde transport of BDA from injections into the PBN and KF nuclei of rabbits revealed prominent projections to the SVN, dorsal aspect of the rostral MVN, caudal MVN, pars beta of the LVN and IVN. These connections appear to contain a component that is reciprocal to the vestibulo-parabrachial pathway and a non-reciprocal component to regions connected with the vestibulocerebellum and vestibulo-motor reflex pathways. These connections support the concept that a synthesis of autonomic, vestibular and limbic information is an integral property of pathways related to balance control in both the brain stem and forebrain. It is suggested that these projections may contribute broadly to both performance tradeoffs in vestibular-related pathways during variations in the behavioral context and affective state and the close association between anxiety and balance function.

Differential morphology of pyramidal tract-type and intratelencephalically projecting-type corticostriatal neurons and their intrastriatal terminals in rats.

Two types of corticostriatal projection neurons have been identified: 1) one whose intrastriatal arborization arises as a collateral of a projection to the ipsilateral brainstem via the pyramidal tract (PT-type); and 2) one that projects intratelencephalically to the cortex and striatum, in many cases bilaterally, but not extratelencephalically (IT-type). To assess possible functional differences between these two neuron types, we characterized their laminar location in the cortex, their perikaryal size, and the morphology of their intrastriatal terminals. IT-type neurons were retrogradely labeled by tetramethylrhodamine-dextran amine (RDA)3k injection into the contralateral striatum, whereas their intrastriatal terminals were labeled anterogradely by biotinylated dextran amine (BDA)10k injection into the contralateral motor or primary somatosensory cortex. To label PT-type neurons and their ipsilateral intrastriatal terminals retrogradely, BDA3k was injected into the pontine pyramidal tract. We found that IT-type neuronal perikarya are medium-sized (12-13 microm) and located in layer III and upper layer V, whereas PT-type perikarya are larger (18-19 microm) and most commonly located in lower layer V. At the electron microscopic level, the intrastriatal terminals of both corticostriatal neuron types made asymmetric synaptic contact with spine heads and less frequently with dendrites. IT-type axospinous terminals were characteristically small (0.4-0.5 microm) and regular in shape, whereas PT-type terminals were typically large (0.8-0.9 microm) and often irregular in shape. Perforated postsynaptic densities were common for PT-type terminals, but not IT-type. The clear differences between these two corticostriatal neuron types in perikaryal size and laminar location in the cortex, and in the size and shape of their intrastriatal terminals, suggest that they may differ in the nature of their influence on the striatum.

Corticonuclear projections of the cerebellum preserve both anteroposterior and mediolateral pairing patterns.

The aim of the present study was to establish whether a diverging arrangement of the corticonuclear cerebellar projections exists and, if so, what relation it has with the inferior olivary complex. Iontophoretic injections of a 1 : 1 mixture of tetramethylrhodamine dextran amine and biotinylated dextran amine into the cerebellar cortex orthogradely labelled fibre terminals in the cerebellar nuclei and retrogradely labelled cell bodies in the inferior olivary complex. The injections were into A, B, C2, C3, D1 and D2 bands. These injections showed diverging projections to the cerebellar nuclei, with 'primary projections' directed to the nuclear region previously reported to be specifically connected with the injected band and 'secondary projections' directed to other nuclear regions. Secondary projections from the A, C2 and C3 bands diverged to nuclear regions primarily controlled by cortical bands lateral to those injected. Secondary projections from the D1, and D2 bands diverged to nuclear regions primarily controlled by cortical bands medial to those injected. Moreover, injections distributed along the D1 and D2 bands showed similar sets of nuclear targets, while those distributed along the A, C2 and C3 bands showed two sets of nuclear targets in relation to the anteroposterior location of the injected area within these bands. The cortical areas that projected to the same set of nuclear targets were innervated from single olivary regions, while those that projected to different sets of nuclear targets were innervated from different subsets of single regions of the inferior olive. The results suggest that the olivary bands of the cerebellar cortex project to the cerebellar nuclei with a diverging pattern that is organized in both the mediolateral and the anteroposterior axes.

Zonal organization of flocculo–vestibular connections in rats

Anatomical and electrophysiological evidence has contributed to the hypothesis that microzones in the mammalian flocculus are organized to reflect control of eye movements in the planes of semicircular canals. Adult male Long–Evans rats received iontophoretic injections of FluoroGold and/or tetramethylrhodamine dextran amine (10,000 molecular weight, “FluoroRuby”) into the vestibular nuclei. The distribution of retrogradely labeled Purkinje cells revealed that efferent projections from the dorsal surface of the flocculus and the ventral paraflocculus to the superior vestibular nucleus, rostral medial vestibular nucleus, ventral lateral vestibular nucleus, and caudal aspect of the vestibular nuclear complex (caudal medial vestibular nucleus, inferior vestibular nucleus and nucleus prepositus hypoglossi) tended to correspond to previously identified climbing fiber zones [Ruigrok et al. (1992) J. comp. Neurol. 316, 129–150] in a manner consistent with other mammals. However, vestibular nucleus projections from the ventral surface of the flocculus did not appear to respect climbing fiber zonal boundaries. Rather, climbing fiber zones each contained interdigitated groups of Purkinje cells that project to different vestibular nuclear regions. It is suggested that this pattern of flocculus efferent organization is a specialization for controlling the activity of primary and accessory extraocular muscle pairs to confine vestibulo-ocular reflexes within semicircular canal planes when the “center of regard” is located at different eccentricities.

Spinocerebellar projections in the pigeon with special reference to the neck region of the body

Neck sensory information is important for control of head and body movements in all vertebrates. Neuroanatomic tracing methods were used to study the pathways of neck afferent systems. Both the projection of primary afferent fibers and of secondary afferent pathways to brainstem and cerebellum were investigated with the anterograde transport of dextran amines as tracers (biotinylated dextran amine and tetramethyl rhodamine dextran amine). For comparison, the projections of spinocerebellar systems of wing and leg were studied also. Complementary experiments using retrograde tracers (Fast Blue, tetramethyl rhodamine dextran amine, rhodamine isothiocyanate) injected into the cerebellum served to corroborate the results of the anterograde tracing experiments. Primary neck afferent fibers terminated in the spinal gray substance with dense terminal fields in laminae I to V of the dorsal horn and lamina IX of the ventral horn as well as in the marginal nuclei located at the lateral border of the spinal cord. In the brainstem, dense terminal fields were seen in deep layers of the medullary dorsal horn, in the external cuneate nucleus, and in group x. Secondary neck afferents arising from ventral horn cells showed a significant projection to the descending and medial vestibular nuclei and to the medial cerebellar nucleus. Terminals were found both in the anterior and the posterior cerebellum. A quantitative evaluation disclosed that most terminals of neck afferents distributed in lobules II-IV of the anterior cerebellum and lobule IX of the posterior cerebellum. With injections aimed at spinocerebellar neurons located into the cervical and lumbosacral enlargements, no projections were found in the vestibular or deep cerebellar nuclei. Projections from the cervical enlargement were concentrated in lobules III-V and those from the lumbosacral enlargement in lobules III-VI. This points to a rostrocaudal somatotopic representation of neck, wing, and leg in the anterior cerebellum. The results of the retrograde tracing experiments support such a somatotopic organization.

Basic fibroblast growth factor applied to the optic nerve after injury increases long-term cell survival in the frog retina

The neuroprotective effects of basic fibroblast growth factor (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and 16 weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Müller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Müller cells. FGF receptor-LI was also observed in optic nerve glia.

Calcium-binding protein-immunoreactive projection neurons in the caudal subnucleus of the spinal trigeminal nucleus of the rat.

It has been reported that calcium-binding proteins are good markers for different sets of neurons in various brain regions. We examined expression of the main calcium-binding proteins in projection neurons in the rat medullary dorsal horn (MDH) by combining immunofluorescence histochemistry for calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) with the retrograde tract-tracing method. A fluorescence tracer, tetramethylrhodamine-dextran amine (TMR-DA), was injected into the parabrachial, thalamic or hypothalamic region. After such injections, a number of PV-, CR-, and/or CB-immunoreactive MDH neurons were labeled retrogradely with TMR-DA. Triple-immunofluorescence histochemistry further revealed that a number of CB-, CR-, or PV-immunoreactive TMR-DA-labeled MDH neurons showed immunoreactivity for substance P receptor (NK1), and that they expressed immunoreactivity for c-fos protein in the rats which were injected with formalin into the lips. Thus, it was indicated that some of CB-, CR-, or PV-containing projection neurons in the MDH might be involved in the transmission of nociceptive stimuli.

Organization of geniculate and trigeminal ganglion cells innervating single fungiform taste papillae: a study with tetramethylrhodamine dextran amine labeling

Single gustatory nerve fibers branch and innervate several taste buds. In turn, individual taste buds may receive innervation from numerous gustatory nerve fibers. To evaluate the pattern of sensory innervation of fungiform papilla-bearing taste buds, we used iontophoretic fluorescent injection to retrogradely label the fibers that innervate single taste papillae in the hamster. For each animal, a single taste papilla was injected through the gemmal pore with 3.3% tetramethylrhodamine dextran amine. Fungiform papillae either at the tongue tip (0.5–1.5 mm from the tip) or more posteriorly (1.5–3.0 mm from the tip) were injected. After one to seven days survival, the geniculate and trigeminal ganglia and the tongue were sectioned and examined for labeled cells and fibers, respectively. Analysis of the number and topographic distribution of geniculate cells innervating single taste papillae, shows that: (i) 15±4 (S.D.) ganglion cells converge to innervate a single fungiform taste bud; (ii) more ganglion cells innervate anterior- (range: 13–35 cells) than posterior-lying buds (range: five to 12 cells), which, in part, may be related to bud volume (μm 3); and (iii) ganglion somata innervating a single taste bud are scattered widely within the geniculate ganglion. Analysis of labeled fibers in the tongue demonstrated that two to eight taste buds located within 2 mm of the injected taste bud share collateral innervation with the injected taste bud. Since all buds with labeled fibers were located in close proximity (within a 2-mm radius), widely dispersed geniculate ganglion cells converge to innervate closely spaced fungiform taste buds. Trigeminal ganglion (mandibular division) cells were also labeled in every case and, as with the geniculate ganglion, a dispersed cell body location and collateralization pattern among papillae were observed. This study shows that iontophoresis of tetramethylrhodamine dextran amine, selectively applied to individual peripheral receptor end-organs, effectively locates sensory ganglion cells in two different ganglia that project to these sites. Moreover, the marker demonstrates collateral branches of sensory afferents associated with the labeled fibers and the nearby receptor areas innervated by these collaterals. The labeling of single or clusters of receptor cells, as well as identified sensory afferents, affords future possibilities for combining this technique with immunocytochemistry to establish the relationships of innervation patterns with neurotransmitters and neurotropic substances within identified cells.

Fluorescent tracers as potential candidates for double labeling of descending brain neurons in larval lamprey

In larval lamprey, seven fluorescent tracers were tested as potential candidates for retrograde double labeling of descending brain neurons: Fluoro–Gold (FG); fluorescein dextran amine (FDA); True Blue (TB); cascade blue dextran amine (CBDA); Fast Blue (FB); Texas red dextran amine (TRDA); and tetramethylrhodamine dextran amine (RDA). The first tracer (FG, TB, FB, or CBDA) was applied to the spinal cord at 40% body length (BL). In separate experiments, the second tracer (TRDA or RDA) was applied to the spinal cord at 20% BL. The tracer combination FG/TRDA was found to have the best optical properties for double labeling. However, application of FG to the spinal cord with the method used for the other tracers resulted in labeling of ‘lateral cells’ along the sides of the rhombencephalon that were presumed to be non-neuronal and that obscured some of the descending brain neurons. Control experiments suggested that FG was transported in the circulation to the brain, where the tracer was taken up by lateral cells. Therefore, a special technique was developed for applying FG to the spinal cord without allowing the tracer to enter the circulation. In larval lamprey, this important double-labeling technique that was developed for TRDA and FG is being used to examine axonal regeneration and projection patterns of descending brain neurons.