Synaptic connections of neurones identified by Golgi impregnation: characterization by immunocytochemical, enzyme histochemical, and degeneration methods.

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi-impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi-impregnated neurones and their pre- and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre- and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP-filled neurones and Golgi-impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.

Mesencephalic innervation of the turtle retina by a single serotonin-containing neuron.

Co-localization of retrograde transported Nuclear yellow and serotonin immunoreactivity revealed the existence of a central serotonergic projection to the retina in the turtle Pseudemys scripta elegans. This serotonergic retinopetal system consists of only one fiber originating in the contralateral caudal mesencephalon. In the retina, the fiber arborizes exclusively in the temporal hemisphere, covering about one third of the total retinal surface. Interestingly this projecting area lies in the part of the retina, to which binocular perception can be attributed.

Thalamic projections to the hippocampal and entorhinal areas in the cat.

The thalamic projections to the hippocampal formation and to the subicular and entorhinal areas in the cat have been studied with retrograde transport of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) and anterograde transport of WGA-HRP. Retrograde transport tracers injected in various parts of these cortices resulted in labeled cells in the midline, anterior, and lateral dorsal nuclei. Injections into the hippocampal formation or the subiculum led to retrograde labeling of cells in the reuniens nucleus of the ipsilateral thalamus throughout its rostrocaudal extent, whereas the restricted injections into the dentate gyrus and the inferior region of the hippocampus led to no labeling. Following an injection into the pre- and parasubiculum, a large number of labeled cells were seen not only in the reuniens nucleus but in other midline nuclei. In addition, a substantial number of labeled cells were also detected in the anterior and lateral dorsal nuclei, particularly in the anterodorsal nucleus, which contained densely arranged labeled cells throughout almost the entire rostrocaudal extent. An injection into the medial entorhinal area labeled a number of cells in the anterior nuclei and in the reuniens nucleus, particularly its dorsal part. Injections into various subdivisions of the lateral entorhinal area yielded different patterns of distribution of labeled cells in the thalamic nuclei. An injection into the ventromedial division (VMEA) led to abundant labeling of cells in the paraventricular and reuniens nuclei. After an injection into the ventral division (VLEA), numerous labeled cells were detected in the reuniens nucleus and a lesser number in the paraventricular nucleus at anterior levels. When an injection was made into the dorsal division (DLEA), a large number of labeled cells were detected in the reuniens nucleus, and less numerous labeled cells were found in the central medial nucleus. There appears to be a topographic arrangement of cortical projections of the reuniens nucleus. The pre- and parasubiculum receive projections from the most medial part of the reuniens nucleus near the midline, and the DLEA receives projections from the medial part of the nucleus. The cells projecting to the VLEA and MEA are distributed in the central part of the reuniens nucleus, and those to the VMEA are distributed in the lateral part. Anterograde experiments were also performed; injections of WGA-HRP into the reuniens nucleus resulted in terminal labeling in the superficial layers of the subicular area and the neighboring hippocampus and in the entorhinal area.

Serotonergic efferent nerve fibers in the retinal plexiform layer of the abalone.

Serotonin (5-HT)-immunohistochemistries at light- and electron-microscopic levels, using rabbit anti-5-HT serum (#1234), were applied to the whole head and only to the eye of the abalone, respectively. Peroxidase-antiperoxidase and fluorescein isothiocyanate methods were used for the light-microscopic immunohistochemistries. Many immunoreactive nerve fibers were demonstrated in the outer zone of the retinal plexiform layer, small optic nerve fiber bundles, the optic nerve trunk and the cerebral ganglion. Immunoreactive somata were observed only in the cerebral ganglion. Accordingly the immunoreactive fibers in the retinal plexiform layer are considered to be efferent. Cored vesicles in the retinal plexiform layer demonstrated by both conventional chemofixation and a rapid-freeze-substitution method showed strong immunoreactivities localized within their limiting membrane. The same fibers also contained small clear vesicles. They are considered to be different from larger clear vesicles in non-immunoreactive fibers reportedly containing acetylcholine. The function of the efferent fibers remains to be elucidated.

The differential ascending projections from the anterior, central and posterior regions of the lateral hypothalamic area: an autoradiographic study.

The ascending efferent projections of the neurons of the anterior (LHAa), central (LHAc) and posterior (LHAp) parts of the lateral hypothalamic area (LHA) have been studied using an autoradiographic analysis of the anterograde axonal transport after local injection of tritiated amino acids. Our results show that LHA regions have common projections particularly to the thalamus and to the hypothalamus. They also demonstrate the existence of differential projections: i.e. an anteroposterior gradient of projection sites according to anterior, central and posterior localisation of the LHA neurons. The LHAa neuronal projections terminate in the lateral septal area; the LHAc projections innervate the frontal cortex while LHAp neurons send their projections to the olfactory bulb and innervate both the cerebral cortex and the hippocampus. Only the LHAp neurons project measurably to the globus pallidus, the caudate putamen and the nucleus accumbens.

The olivonodular projection: a re-examination based on folial cerebellar implants.

In 5 cats implants of crystalline wheat germ agglutinin-horseradish peroxidase complex were placed in one or two folia of the nodulus with no contamination of adjacent cerebellar lobules. In the inferior olive only the dorsal cap with the adjacent ventrolateral outgrowth and the caudalmost part of nucleus beta were found to project to the nodulus. Negative findings were consistently made in all other parts of the inferior olive, some of which have previously been described to contribute to the olivonodular projection.

Substance P-containing medullary projections to the intermediolateral cell column: identification with retrogradely transported rhodamine-labeled latex microspheres and immunohistochemistry.

Substance P (SP)-containing medullary neurons that project to the intermediolateral cell column (IML) of the rat were studied. Neurons were retrogradely labeled with rhodamine-labeled latex microspheres (RITC-M) injected into the T-3 IML, and SP-immunoreactive neurons were identified with immunocytochemistry. RITC-M labeled cells occurred in the nucleus reticularis paragigantocellular lateralis (RPgcl), adjacent and lateral to the pyramidal tract at the level of the rostral inferior olivary nucleus and extended to the mid-facial nucleus in the medulla. Cells were also labeled caudal to the RPgcl, in the nucleus reticularis ventralis, pars alpha (RVa), rostral to the RVa, in the nucleus reticularis gigantocellularis (RGc), and in the raphe nuclei. SP immunoreactivity (SP-IR) was seen in cells that were also retrogradely labeled. These double-labeled cells were observed in the RPgcl, RVa and the raphe pallidus. These data show that the IML receives SP-neuronal projections from multiple locations in the medulla. The SP-neuronal projections from the RPgcl of the ventral medulla to the IML likely represent one component of the ventral medullary region that influences cardiovascular functions.

Vestibular and cochlear efferent neurons in the monkey identified by immunocytochemical methods.

Attempts were made to identify vestibular (VEN) and cochlear (CEN) efferent neurons in the squirrel monkey using retrograde transport of horseradish peroxidase (HRP) and immunocytochemical methods. HRP implants in the ampulla of the lateral semicircular duct retrogradely labeled cells of VEN bilaterally and some cells of CEN. VEN located lateral to the rostral part of the abducens nucleus formed a compact collection of cells, all of which were immunoreactive only to antisera for choline acetyltransferase (ChAT). CEN, identified by immunoreactivity to ChAT were located at the hilus of the lateral superior olive (LSO), along the lateral border of the LSO and sparsely near lateral parts of the ventral trapezoid nucleus (VTN). A small number of cells and fibers near the border of the VTN and lateral to the LSO were immunoreactive for leucine enkephalin (L-ENK). Fibers immunoreactive for L-ENK also were identified in the hilus of the LSO. No cells of the superior olivary complex were immunoreactive for antisera to ChAT, L-ENK, substance P, gamma-aminobutyric acid or glutamic acid decarboxylase. Cells of VEN and CEN can be identified by their immunoreactivity to ChAT, and some cells and fibers of CEN also contain L-ENK.

Amino acid labeling patterns in the efferent innervation of the cochlea: an electron microscopic autoradiographic study.

Light microscopic autoradiography and electron microscopic autoradiography were used to study the distribution of label in the cochlear efferents following in vivo incubation with tritiated amino acids. Two basic patterns of labeling were observed. These patterns correspond closely to the lateral and medial superior olivary complex (SOC) olivocochlear systems identified by Warr and Guinan ('79, Brain Res. 173:152-155). Our electron microscopic observations suggest that, at least in the gerbil, the complete separation of outer hair cell (OHC) versus inner hair cell (IHC) efferent innervation proposed by these investigators based upon light microscopic data does not occur. Rather, our data suggest that while the lateral SOC system supplies endings only to the region under the IHC, the medial SOC system may supply endings beneath both the IHCs and OHCs.

Substance P- and enkephalin-containing projections from the interpeduncular nucleus to the dorsal tegmental region in the rat.

A combination of fluorescent retrograde tracing and immunohistochemical staining for substance P (SP) and Leu-enkephalin (Enk) was used to study the projections from the interpeduncular nucleus (IP) to the dorsal tegmental region, i.e. the dorsal tegmental nucleus of Gudden, the dorsolateral tegmental nucleus and the caudal extension of the dorsal raphe nucleus. After injections of 'Granular Blue' (GB) in the dorsal tegmental region, followed one or two days later by a colchicine injection near the IP, and subsequently two days later by the immunohistochemical procedure, populations of neurons double labeled for Enk and GB, or SP and GB, cells that only showed GB labeling, and a number of cells stained only for one of the peptides could be identified in the rostral subnucleus of the IP. This study demonstrates the existence of both Enk- and SP-containing projections from the IP to the dorsal tegmental region.

Autoradiographic localization of newly synthesized octopamine to retinal efferents in the Limulus visual system.

The biogenic amine octopamine is synthesized from both tyrosine and tyramine in the lateral, median, and ventral eyes of Limulus. The autoradiographic studies presented here were designed to locate the sites of octopamine synthesis in the ventral and lateral eyes. We found that efferent fibers, which project to ventral and lateral eyes from the central nervous system, became intensely and selectively labeled during in vitro incubations with 3H-tyramine. In the ventral eye, more than 95% of the efferent fibers were labeled. Results of biochemical analyses suggested that most of the radioactive substance within these efferent fibers was newly synthesized octopamine. The selective labeling of efferent fibers during incubation with 3H-tyramine was used as an anatomical tool to study the number and distribution of efferent fibers within the ventral eye. Light microscopic (LM) reconstructions of the distribution of label in serial longitudinal sections through ventral optic nerves together with electron microscopic (EM) autoradiographic analyses revealed between 70 and 200 efferent axons. The results of these studies and of reconstructions of efferent innervation to photoreceptor somata suggest that each ventral photoreceptor cell or each small cluster of cells is innervated by a separate efferent fiber. Both LM reconstructions and EM analyses showed that efferent fibers ramify extensively and specifically in and near the internal rhabdom of ventral photoreceptor cells. In EM autoradiographs of lateral eyes incubated with 3H- tyramine, the silver grains that were located over ommatidia were concentrated exclusively over efferent fibers. All of these efferent fibers, which lay near rhabdoms and in partitions between retinular cells, were labeled. The results of our present studies support our hypothesis that octopamine is a neurotransmitter in Limulus retinal efferent fibers. This amine may modulate the biochemistry and physiology of ventral photoreceptor cells and may mediate many of the known effects of circadian efferent innervation to the lateral eye.

The corticopontocerebellar pathway to crus I in the cat as studied with anterograde and retrograde transport of horseradish peroxidase.

In 13 cats injections of horseradish peroxidase (HRP) in various parts of the cerebral cortex were combined with injections of HRP in the cerebellar crus I in the same animal in order to study the cortical regions which may influence the crus I via the pontine nuclei. In the pons terminal regions anterogradely labeled from the cerebral cortex and cell groups retrogradely labeled from crus I were carefully plotted. Overlap between sites of ending of cortical fibers and sites of origin of fibers to crus I was relatively modest in all experiments. On the other hand, partial overlap was usually found at multiple sites. The largest single input to crus I appears to come from the parietal region, particularly the anterior part of the suprasylvian gyrus, while the sensorimotor region contributes much less. Area 6, the second somatosensory cortex and the orbital gyrus, all seem able to influence pontine cells projecting to crus I. Least overlap is found after injections of the visual cortex. The size and orientation of the dendritic fields of pontine cells were studied in Golgi-impregnated material. The dendritic fields average 187 X 339 microns in the transverse plane and are so small that they will only moderately increase the overlap. It is concluded that small subgroups of neurons projecting to crus I receive somewhat different sets of cortical afferents. The input to crus I must originate in wide areas of the cerebral cortex, and probably exhibits a high degree of spatial order, with an intricate pattern of specific divergence and convergence. The present results are compatible with previous physiological evidence from micromapping studies of a precise and complicated mosaic pattern of connections to the cerebellar hemispheres.

LHRH systems in brain of platyfish.

The luteinizing hormone-releasing hormone (LHRH) system of the platyfish Xiphophorus has been studied using immunohistochemistry and retrograde transport of horseradish peroxidase (HRP). Three different populations of LHRH-positive cell bodies are present in the brain, one in the ventral telencephalon at the border to the olfactory bulb (nucleus of olfactoretinalis), one lateral to the n. preopticus (nucleus preopticus basalis lateralis) and one in the midbrain. LHRH neurons from the nucleus olfactoretinalis project via the medial olfactory tract to the olfactory bulb and to olfactory nerves. A second projection from this nucleus enters the optic tract, crosses in the optic chiasm, and courses rostrally in the outer layer of the optic nerve to the retina, where LHRH-positive nerve fibers terminate near amacrine and bipolar cells. HRP injections into the eye or into the cut optic nerve result in retrograde transport of the enzyme to the contralateral LHRH nucleus olfactoretinalis. Projections from LHRH neurons in the lateral preoptic region can be followed medially to surround the interhemispheric ventricle and laterally to border the optic tract. At the level of the postoptic commissure, LHRH fibers condense to form a fascicle which reaches the pituitary stalk to arborize throughout the hypophysis. LHRH fibers, probably in part from the midbrain LHRH neurons, project to the optic tectum, torus semicircularis, corpus and valvula of the cerebellum, as well as to the medulla oblongata. Associations of LHRH projections with sensory systems and with endocrine-autonomic systems in hypothalamus-pituitary and lower brain stem suggest a role in the modulation and integration of sensory, autonomic, behavioral and hypophyseotrophic functions.

Glycine receptor: light microscopic autoradiographic localization with [3H]strychnine.

Glycine receptors have been localized by autoradiography in the rat central nervous system (CNS) using [3H]strychnine. The gross distribution of receptors is in excellent accord with the distribution determined by filtration binding assays. Specifically, the density of glycine receptors is greatest in the gray matter of the spinal cord and decreases progressively in regions more rostral in the neuraxis. Glycine receptors were found to be associated with both sensory and motor systems in the CNS. Moreover, there is a striking correlation between areas of high strychnine binding site density and areas in which glycine has been found to be electrophysiologically active. Finally, the anatomic localization of strychnine binding sites may help explain many of the signs and symptoms of strychnine ingestion. For example, individuals consuming subconvulsive doses of strychnine frequently experience altered cutaneous and auditory sensation. We have localized strychnine receptors in areas of the acoustic system known to influence discriminative aspects of audition and in areas of the spinal cord and trigeminal nuclei which modulate discriminative aspects of cutaneous sensation. The alteration of visceral functions (e.g., blood pressure and respiratory rate) associated with strychnine ingestion may be accounted for in a similar manner.