Noradrenergic excitation of a subpopulation of GABAergic cells in the basolateral amygdala via both activation of nonselective cationic conductance and suppression of resting K+ conductance: a study using glutamate decarboxylase 67-green fluorescent protein knock-in mice.

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.

Expression of Hex mRNA in early murine postimplantation embryo development.

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Development of afferent fiber lamination in the infrapyramidal blade of the rat dentate gyrus.

In the rat dentate gyrus, the lateral perforant path, the medial perforant path, and the major part of the hilar projection to the molecular layer share the lamination domain, mainly in the outer one-third of the molecular layer, the middle one-third, and the inner one-third, respectively. To reveal the order of the afferent fiber lamination and to have an indication of how the synaptic sites on dendrites are determined, we investigated the ontogeny of afferent fiber lamination in the dorsal hippocampus by injecting 1, 1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the entorhinal cortex and hippocampus in vivo. Fibers from the contralateral hilar region were found under the pia mater of the infrapyramidal blade at postnatal day 3 (P3), whereas the entorhinal afferent fibers were absent in the infrapyramidal blade. Then the medial and the lateral perforant path appeared under the pia mater in the infrapyramidal blade as riding on top of the preexisting laminae by P7 and by P11, respectively. Based on the established knowledge that most entorhinal layer II neurons simultaneously innervate both the suprapyramidal blade and infrapyramidal blade by branching, it is assumed that the medial and lateral perforant path in the suprapyramidal blade await an appropriate timing for sprouting of interstitial branches into the infrapyramidal blade. The granule cells in the infrapyramidal blade had dendritic growth cones by P11. Calretinin immunohistochemistry revealed Cajal-Retzius cells in the infrapyramidal blade even at P14. Under the pia mater, axon growth cones of ingrowing afferent fibers may interact with the dendritic growth cones or the Cajal-Retzius cells, and determines the synaptic sites on the granule cell dendrites.

Crossing fiber arrays in the rat hippocampus as demonstrated by three-dimensional reconstruction.

The hippocampus is a neural substrate playing a key role in short-term memory. In order to achieve a better understanding of how the hippocampus functions in "learning and memory," we conducted an intracellular horseradish peroxidase (HRP) study of the CA3 pyramidal neurons and the granule cells of the fascia dentata. The axon of the CA3 pyramidal neurons has two components, the longitudinal association system and the Schaffer collateral system. The latter component is organized in a lamellar fashion and follows the alvear fiber stream. An electron microscopic analysis of myelinated fibers suggested that most myelinated fibers in the hippocampus are organized parallel to the alvear fibers. The mossy fibers of the granule cells, however, do not follow the alvear fiber stream. We propose a new model of the organization of the intrinsic excitatory circuitry of the rat hippocampus in which the distinct lamellar organization of the pyramidal and granule cells creates a crossing neural network.

Disposition of the slab-like modules formed by axon branches originating from single CA1 pyramidal neurons in the rat hippocampus.

The hippocampus is thought to be an area where the neuronal circuits for short-term memory or the cognitive map may reside. In order to advance theoretical studies and neuronal model simulations of such circuits, the projection of the CA1 pyramidal neurons in the rat dorsal hippocampus, especially in the subiculum, was studied by means of intracellular and extracellular HRP injection. The CA1 pyramidal neurons project principally to the subiculum where each forms a slab-like axonal field 2 mm long along the septotemporal axis, which may be regarded as a module for columnar organization, at a specific rostrocaudal level of the subiculum. The modules of the CA1a pyramidal neurons are disposed in the rostral part of the subiculum, those of the CA1c pyramidal neurons in the caudal part, and those of the CA1b pyramidal neurons in the middle part of the subiculum. The CA1 pyramidal neurons also participate in the construction of the lamellar organization in the hippocampus in that their axon branches run rostrocaudally following the stream of the alvear fibers. The CA1 pyramidal neurons in the dorsal rat hippocampus transfer the topographic map from field CA1 to the subiculum with reversed order in the lamellar direction. The topographical relationship is composed of partially shifted, overlapping slab-like modules. As a result, information conveyed through a lamella will diverge into the subiculum approximately 2 mm wide, and information through a group of lamellae 2 mm wide will converge upon single subicular neurons.

Preservation of topography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats.

In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV-VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex.

Morphology of physiologically identified retinal X and Y axons in the cat's thalamus and midbrain as revealed by intraaxonal injection of biocytin.

Prior morphological studies of individual retinal X and Y axon arbors based on intraaxonal labeling with horseradish peroxidase have been limited by restricted diffusion or transport of the label. We used biocytin instead as the intraaxonal label, and this completely delineated each of our six X and 14 Y axons, including both thalamic and midbrain arbors. Arbors in the lateral geniculate nucleus appeared generally as has been well documented previously. Interestingly, all of the labeled axons projected a branch beyond thalamus to the midbrain. Each X axon formed a terminal arbor in the pretectum, but none continued to the superior colliculus. In contrast, 11 of 14 Y axons innervated both the pretectum and the superior colliculus, one innervated only the pretectum, and two innervated only the superior colliculus. Two of the Y axons were quite unusual in that their receptive fields were located well into the hemifield ipsilateral with respect to the hemisphere into which they were injected. These axons exhibited remarkable arbors in the lateral geniculate nucleus, diffusely innervating the C-laminae and medial interlaminar nucleus, but, unlike all other X and Y arbors, they did not innervate the A-laminae at all. In addition to these qualitative observations, we analyzed a number of quantitative features of these axons in terms of numbers and distributions of terminal boutons. We found that Y arbors contained more boutons than did X arbors in both thalamus and midbrain. Also, for axons with receptive fields in the contralateral hemifield (all X and all but two Y axons), 90-95% of their boutons terminated in the lateral geniculate nucleus; the other two Y axons had more of their arbors located in midbrain.

Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex.

The chemical organization of excitatory axon terminals in the rat cerebellar cortex was examined by immunocytochemistry and in situ hybridization histochemistry of vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2). Chemical depletion of the inferior olivary complex neurons by 3-acetylpyridine treatment almost completely removed VGluT2 immunoreactivity from the molecular layer, leaving VGluT1 immunoreactivity apparently intact. On the other hand, neuronal deprivation of the cerebellar cortex by kainic acid injection induced a large loss of VGluT1 immunoreactivity in the molecular layer. In the cerebellar granular layer, both VGluT1 and VGluT2 immunoreactivities were found in mossy fiber terminals, and the two immunoreactivities were mostly colocalized in single-axon terminals. Signals for mRNA encoding VGluT2 were found in the inferior olivary complex, and those for VGluT1 and VGluT2 mRNAs were observed in most brainstem precerebellar nuclei sending mossy fibers, such as the pontine, pontine tegmental reticular, lateral reticular and external cuneate nuclei. These results indicate that climbing and parallel fibers selectively use VGluT2 and VGluT1, respectively, whereas mossy fibers apply both VGluT1 and VGluT2 together to accumulate glutamate into synaptic vesicles. Since climbing-fiber and parallel-fiber terminals are known to make depressing and facilitating synapses, respectively, VGluT1 and VGluT2 might have distinct properties associated with those synaptic characteristics. Thus, it would be the next interesting issue to determine whether mossy-fiber terminals co-expressing VGluT1 and VGluT2 show synaptic facilitation or depression.

In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins.

A new recombinant virus which labeled the infected neurons in a Golgi stain-like fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (palGFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.

A procedure for in situ hybridization combined with retrograde labeling of neurons: application to the study of cell adhesion molecule expression in Dil-labeled rat pyramidal neurons.

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer Dil. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry.

Cell migration from the corticostriatal angle to the basal telencephalon in rat embryos.

Lateral cortical stream (LCS) has been described as a flow of cell migration found in the lateral cortex of the embryonic telencephalon of mammals. The destinations of the cell migration were reported as the ventrolateral neocortex, the pyriform cortex, endopyriform nucleus and the claustrum. At the same time, however, other destinations of LCS have been suggested in the ventral telencephalon. Therefore, we investigated the additional destinations of the LCS using a combination of several molecular biological techniques. Using an expression vector of modified green fluorescent protein (GFP) introduced into the ventricular zone (VZ) around the corticostriatal angle, both tangential and radial cell migration was revealed in the LCS. The radial cell migration in the LCS supplied cells to the ventro-lateral neocortex, pyriform cortex and to the level of the lateral olfactory tract. In the second experiment, we injected COS-1 cells transfected with a Sema3A expression vector into one side of the neocortex. The cell supply to the destination of the LCS ceased due to the formation of a large necrosis in the LCS, which was triggered by the Sema3A-COS cell injection, and the dense cell layer in the olfactory tubercle shrunk on the side where COS cells were injected. These data indicated that the majority of neurons in the dense cell layer of the olfactory tubercle reached this point through the LCS. One of the origins of the cells in the LCS would be in the corticostriatal angle.

Arborization pattern of sympathetic preganglionic axons in the rat superior cervical and stellate ganglia.

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600-700 microns in the rostrocaudal direction, and about 200 microns in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 microns in the rostrocaudal direction and about 400 microns in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.

Radial glia is a progenitor of neocortical neurons in the developing cerebral cortex.

Neocortical neurons are produced by cell division of neural stem cells in the ventricular zone of the cerebral cortex. We investigated the production of neurons by infecting neuroepithelial cells with a modified GFP-recombinant adenovirus. The adenovirus DNA is inherited by only one daughter cell at each cell division and travels one way from the progenitor to the progeny. Since the ventricular zone (VZ) of the embryo neocortex expressed an adenovirus receptor, CAR ubiquitously, morphology and cell-lineage of cells in the VZ could be revealed by the adenovirus infection. Radial glias, cells with a bipolar shape, and spherical cells were found as modified-GFP-positive (mGFP+) in the VZ. The bipolar cells (radial cells) had a radial process not in contact with the pia mater and a growth-cone-like structure at the edge of their radial process, while the radial glias had a process spanning all the cortical layers. Ten hours after viral infection, most mGFP+ cells were radial cells. In the following 8 h, the percentage of mGFP+ radial glias in mGFP+ neocortical cells increased from 18 to 50%, while that in radial/spherical cells decreased from 75 to 19%. The radial glias often divided asymmetrically and produced spherical cells and neuronal precursors. The spherical cells seemed to become radial cells by extending a radial process. The spherical cells, radial cells and radial glias seemed to constitute a proliferating cell cycle during which postmitotic neuronal precursors are produced. The neuronal precursors that inherited the radial processes migrated radially and developed into neocortical neurons. Four days after the viral infection, 97% of mGFP+ cells were neocortical neurons. Here, we propose that the radial glia is a progenitor of neocortical neurons, and that a significant number of radially migrating neurons is guided by their own radial processes connected to the pia mater.

Cell migration from the ganglionic eminence to the neocortex investigated by labeling nuclei with UV irradiation via a fiber-optic cable.

Recent studies have shown that the ganglionic eminence is one of the sources of tangentially migrating cells in the developing neocortex. Since the migration of the DiI-labeled cells from the ganglionic eminence to the neocortex was not monitored by videomicroscopy in these reports, we devised a novel method to study cell migration in vitro and in vivo. The new method involves ultraviolet (UV) irradiation of the cells through a fiber-optic cable and subsequent identification of the irradiated cells on the basis of the formation of thymine dimers in the nuclei. First, we tested the new method (UV-thymine dimer-labeling method) by applying it to monitor the cell migration of neuronal precursor cells in the rostral migratory stream in the neonatal rat telencephalon. In vitro, UV irradiation for 1 s through the fiber-optic cable resulted in the formation of sufficient thymine dimers as to allow immunohistochemical detection after 6 h of incubation; a significant proportion of the irradiated cells continued to migrate in the same direction and at the same speed as those before irradiation. There was no significant difference in the cell migration distance over 6 h between cells exposed and not exposed to the UV irradiation in vitro. In vivo, this method revealed that three times as many cells in the subventricular zone of the olfactory bulb migrated rostrally as caudally. The new method also allowed us to measure the speed of cell migration, which was estimated to be about 70 microm/h at the maximum in the rostral direction. After these examinations of reliability of the method, we applied it to the rat embryo brain. One day after UV irradiation of the ganglionic eminence, labeled migrating cells were found in the striatum, in the internal capsule, and in the intermediate zone of the neocortex. The observation period of cell migration to the neocortex was extended by the use of a xeroderma pigmentosum group A gene mutant mouse, which lacked an ability to remove thymine dimer from the UV-irradiated nuclei. Two days after the UV irradiation, labeled migrating cells from the ganglionic eminence of the mutant mouse embryos were found both in the cortical plate and in the intermediate zone of the neocortex.

Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo.

Neurons in the adult brain have a very complex morphology with many processes, including tremendously long axons. Since dendrites and axons play key roles in the input and output of neural information, respectively, the visualization of complete images of these processes is necessary to reveal the mechanism of neural information processing. Here we made a recombinant adenovirus vector which encodes green fluorescent protein (GFP) tagged with a palmitoylation site, a membrane-targeting signal, produced specific antibodies to GFP, and used them as probes for staining the nervous system. In the neocortex, after injection of the recombinant virus and immunoperoxidase staining with the antibodies, many different types of cells were labeled in a Golgi stain-like fashion. Although the number of labeled cells varied depending on the amount of virus injected, the recombinant virus was considered to be infectious to cortical neurons of all cell types without selectivity. In contrast, the viral infection in the cerebellar cortex and superior cervical ganglion showed some selectivity toward the cell type. It is expected that this recombinant virus will be a useful tool for the morphological analysis of neuronal connections, especially the analysis of microcircuitry in the cerebral cortex.

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion in the rat: WGA-HRP anterograde labeling study.

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion (SCG) in the rat were investigated by means of anterograde labeling following the injection of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the unilateral SCG. The trajectory of the sympathetic fiber innervating the pineal gland and its continuing structures was found advancing along the abducent nerve, through the cavernous plexus, then along the trochlear nerve. Labeled sympathetic fibers showed two patterns of distribution in the blood vessels on the basal surface of the brain. The sympathetic fibers originating in the unilateral SCG were intermingled with those fibers from the contralateral SCG in the pineal gland, its continuing structures and the choroid plexus of the third ventricle as well as in the cerebral blood vessels.

A whole image of the hippocampal pyramidal neuron revealed by intracellular pressure-injection of horseradish peroxidase.

The intracellular pressure-injection of HRP was applied to the rat hippocampus and has brought an excellent presentation of the Golgi-like image of the pyramidal neuron. Rats were allowed to survive for 3 days and brain sections were treated with the PAP-immunohistochemical technique to enhance the sensitivity of HRP neurohistochemistry. The pyramidal neuron densely developed axon branches in the ipsilateral hippocampus and sent the commissural axon to the contralateral hippocampus. Moreover, short axon branches diverged from the commissural axon to the bilateral septal nuclei.

Columnar organization in the subiculum formed by axon branches originating from single CA1 pyramidal neurons in the rat hippocampus.

An intracellular horseradish peroxidase study combined with immunoperoxidase techniques was carried out on hippocampal CA1 pyramidal neurons in the rat. Most axon branches originating from a single CA1 pyramidal neuron ran caudally and terminated in the subiculum. The individual axon branches of the single pyramidal neurons bifurcated repeatedly in the subiculum and finally formed a slab-like or columnar terminal arborization (250-300 microns wide, 500-550 microns high and 1.8-2.2 mm long). The present results suggest, in association with other data, that the CA1 pyramidal neurons receive afferent information through lamellar organized connections and they send efferent information to the subiculum through columnar organized connections.

Three-dimensional analysis of the whole axonal arbors originating from single CA2 pyramidal neurons in the rat hippocampus with the aid of a computer graphic technique.

The axonal arborization of single pyramidal neurons in field CA2 and the rostral adjacent area of the rat hippocampus was studied with intracellular staining following the pressure microinjection of horseradish peroxidase (HRP) in combination with the immunoperoxidase technique, and was analyzed three-dimensionally with the aid of a computer system. The axonal arbors were composed of two types of axon branches, which were distinguished as the primary and secondary axon branches on the basis of morphological criteria. The axon branches in the ipsilateral hippocampus exhibited almost the contour of the dorsal hippocampus. The large amount of axon branches labeled with HRP in the stratum (str.) oriens of field CA1 was comparable to that in the str. radiatum of the field. The labeled axon branches in the dorsal hippocampus were not distributed uniformly in terminal regions but were focused on the caudolateral CA1a-b subfields. Most primary axon branches ran to a focus along the alvear fibers. The lamellar organization in the CA2 pyramidal neurons may be composed of axon branches running caudally and terminal branches forming a focus. The dense association fibers along the septotemporal axis may connect the lamellar organized circuits to each other. Axon branches in the septal nuclei of each hemisphere formed a rather flat plane. The commissural fibers of the CA2 pyramidal neurons seemed to form a symmetrical projection field in the contralateral side against the median plane. The axonal arbors and dendritic expansion of the pyramidal neurons shown in this study appeared to reveal the whole image of the single CA2 pyramidal neuron.

The trajectory of the sympathetic nerve fibers to the rat cochlea as revealed by anterograde and retrograde WGA-HRP tracing.

Wheat germ agglutinin-horseradish peroxidase conjugate was injected in the unilateral superior cervical ganglion (SCG), and the projection pathways of postganglionic sympathetic nerve fibers innervating the cochlea were traced in the rat. The labeled axons advanced along the internal carotid artery (ICA), and a few advanced caudally in the major petrosal nerve (MPN) and entered the facial nerve, while the majority ran rostral to the pterygopalatine ganglion at the point where they crossed the MPN in the carotid canal. The rest of the labeled fibers remained on the surface of the ICA and advanced to the cranial cavity. Most of the labeled fibers along the facial nerve joined the cochlear nerve and finally reached the osseous spiral lamina through the spiral ganglion. Some of the labeled fibers ran along the anterior inferior cerebellar artery from the basilar artery which was previously thought to have been the only pathway. We could not find any labeled fiber on the modiolar artery from anterior inferior cerebellar artery in the cochlea. These observations are consistent with our hypothesis that the sympathetic fibers innervating the neural tissues or related structures follow nerve fibers and meninges as matrices of projection pathways rather than arteries.