Physiological, morphological, and cytochemical characteristics of a layer 1 neuron in cat striate cortex.

We have recorded from a small neuron in layer 1 of the striate visual cortex in a 34-day-old kitten. It had a simple, orientation-selective receptive field that was nondirectional and showed length summation. The neuron was injected intracellularly with horseradish peroxidase. Computer-aided reconstruction revealed that it had a dense axonal plexus confined to layer 1, elongated in the anteroposterior dimension. By means of an antibody directed against a GABA-like antigen, and postembedding immunocytohemistry, the neuron was found to be strongly immunoreactive. The main input to soma and dendrites of the neuron was from synapses that were not GABA-L-immunoreactive, and probably originated from pyramidal cells. The axon of the cell formed synapses on dendritic shafts and spines, whose most likely sources were the apical tufts of pyramidal cell dendrites. These data suggest that such neurons may be involved in local circuits that contribute to the formation of pyramidal cell receptive fields.

Anatomy and fine structure of neurons in the deutocerebral projection pathway of the crayfish olfactory system.

Golgi impregnation and neurobiotin injection were used to examine details of the neural pathways in the olfactory system of the freshwater crayfish, Procambarus clarkii. Deutocerebral projection neurons (globuli cells) were directly injected with neurobiotin. These neurons have dendritic arborizations in the ipsilateral olfactory and accessory lobes, and they project axons to the lateral protocerebrum, where they terminate in microglomeruli of the hemi-ellipsoid body. The axons of the deutocerebral projection neurons are readily impregnated by Golgi procedures, and they terminate as an expanded membranous knot about 5 microns in diameter. Electron microscopy on Golgi-stained terminals has revealed that each knot makes several hundred synapses with small spine-like or shaft-like processes of postsynaptic neurons. Injection of neurobiotin into local interneurons of the hemi-ellipsoid body and subsequent examination of stained preparations with the electron microscope reveals that these cells are a major postsynaptic target of the deutocerebral projection neurons. Furthermore, the local interneurons make extensive efferent synaptic connections with unidentified neurons in the terminal medulla.

Light perception in the vertebrate brain: an ultrastructural analysis of opsin- and vasoactive intestinal polypeptide-immunoreactive neurons in iguanid lizards.

Recent biochemical and immunocytochemical evidence indicates that a population of circadian and reproductive rhythm-entraining photoreceptors lies in the basal diencephalon of iguanid lizards. Here, we report the results of correlated light and electron microscopy of opsin-immunoreactive cells in the basal brain, and we discuss their ultrastructural relationship to known photoreceptors. Cerebrospinal fluid (CSF)-contacting bipolar neurons in the lizards Anolis carolinensis and Iguana iguana were immunolabeled with antisera generated against vertebrate retinal opsins and vasoactive intestinal polypeptide (VIP). Within the brain, opsin-immunoreactive cells were found exclusively in the ependyma of the basal region of the lateral ventricles (adjacent to nucleus paraolfactorius/nucleus ventromedialis and neostriatum/paleostriatum). Cells in the same anatomical location and with the same morphology were labeled with anti-VIP antisera. These cells possessed a dendritic process that extended toward the lateral ventricle, ending in a bulbous terminal that protruded into the ventricle. Axonal processes travelled ventrally and caudally. The entire cell, including the axonal process, exhibited opsin-like and VIP-like immunoreactivity. By light microscopy, opsin-like immunostaining appeared punctate, with immunoreactivity greatest in the bulbous terminal. Opsin- and VIP-immunostained thick sections were resectioned, and individual cells observed by light microscopy were then characterized using electron microscopy. We found that all immunostained cells were morphologically similar and that they were morphologically distinct from neighboring nonimmunoreactive cells. CSF-contacting opsin- and VIP-immunoreactive cells lacked the membranous stacks characteristic of retinal photoreceptors but were ciliated and contained numerous large electron-dense vesicles. Multiple synaptic contacts were made on the soma and putative dendritic processes of opsin- and VIP-immunoreactive CSF-contacting neurons. Our results provide the first ultrastructural characterization of opsin-immunostained encephalic CSF-contacting neurons in a vertebrate animal, and they indicate that these putative photoreceptors share structural features with pineal photoreceptors and with certain invertebrate extraretinal photoreceptors, but they are morphologically and biochemically distinct from visual photoreceptors of the retina.

Distribution of chicken-II gonadotropin-releasing hormone in mammalian brain.

Brains of nonmammalian vertebrates typically contain multiple forms of gonadotropin-releasing hormone (GnRH). Until recently, only the mammalian form of GnRH (mGnRH) had been isolated in placental mammals. Biochemical and histological data show that both mGnRH and chicken-II GnRH (cGnRH-II) are present in a primitive placental mammal, the musk shrew (Suncus murinus). Similar to the case in nonmammalian species, in the musk shrew, neurons that express cGnRH-II are located in a discrete cluster in the midbrain. We have used a combination of radioimmunoassay and immunocytochemistry, analyzed at the light level and with electron microscopy, to describe the distribution of cGnRH-II cell bodies and fibers in the musk shrew brain. All cGnRH-II-immunoreactive (ir) neurons reside in the midbrain, and this area contains the greatest concentration of cGnRH-II peptide in the brain. At the light and electron micrographic levels, we have identified synaptic terminals containing dense core vesicles that are immunoreactive for cGnRH-II in the medial habenula. Radioimmunoassay reveals that this region contains the second greatest concentration of cGnRH-II in the brain. Widely scattered cGnRH-II-ir fibers are present throughout the forebrain, particularly in the medial septum, hypothalamus, and midbrain central gray. Scant cGnRH-II fibers are present in the median eminence, arcuate nucleus, and infundibular stem, and only low concentrations of the peptide are detected in these areas. Finally, intravenous administration of mGnRH is ten times more effective than cGnRH-II in promoting ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular organization and growth-related plasticity of the crayfish olfactory midbrain.

Little knowledge is available concerning the detailed anatomy of the crusctacean central olfactory pathway. We are using radiolabeling, Golgi and biocytin/neurobiotin tracer methodologies, at the correlated light and electron microscopical levels, to study the olfactory midbrain of the freshwater crayfish. We have found that primary afferent fibers from the antennular olfactory receptor cells branch extensively throughout the length of the glomerular columns within the olfactory lobes in the midbrain. Globuli cells of the lateral cell clusters ramify as dendritic arborizations within both the olfactory and accessory lobes; their axons project out the olfactory-globular tracts to the lateral protocerebrum, often branching to both sides. Developmental plasticity involving the connections made by afferent fibers within the olfactory lobes may permit detailed examination of organizational changes within the midbrain as the animal grows and adds new afferent input from the periphery.

Serotonin-containing cell bodies in novel brain locations: effects of light input.

Cell bodies staining positively for serotonin (5HT) appear in the suprachiasmatic nuclei (SCN) of hamsters that have been held in constant darkness (DD) for several months but are otherwise untreated. No such cell bodies are found in the SCN of animals that have been bilaterally enucleated for the same amount of time; however, in enucleated hamsters 5HT-containing cell bodies appear in the superior colliculus. These data provide the first indication that changes in sensory input can modulate 5HT levels in cells bodies outside of the raphe nuclei.

Evidence for the coexistence of glutamate decarboxylase and Met-enkephalin immunoreactivities in axon terminals of rat ventral pallidum.

Evidence for the coexistence of glutamate decarboxylase (GAD) and Met-enkephalin (Met-Enk) in axon terminals of ventral pallidum was demonstrated by colocalization of anti-GAD and anti-Met-Enk immunoreactivities in alternate adjacent 1 micron serial sections. Conventional electron microscopy of immunostained ventral pallidum confirmed that the immunoreactive structures were boutons which made predominantly symmetrical synapses on ventral pallidal cell bodies and dendrites.

A corticotectal projection in the lizard Agama agama.

Following lesions in the dorsal cerebral cortex of the lizard Agama agama, terminal degeneration was observed by the Fink-Heimer technique in the mediodorsal cortex, septal area, nucleus periventricularis and area lateralis of the hypothalamus after 7 days survival time. Following longer survival times (up to 28 days) diffuse terminal degeneration was found in layers 7 to 13 laterally and medially in the optic tectum. Terminal degeneration of the electron-dense type was found in electron micrographs of the same areas of the optic tectum.

Neocortical transplants grafted into the newborn rat brain demonstrate a blood-brain barrier to macromolecules.

Vascular permeability was examined in fetal neocortical transplants grafted into the cerebral cortex of newborn rats. Methods based on the histochemical labeling of intravenously administered horseradish peroxidase or on the immunocytochemical demonstration of endogenous immunoglobulin showed the presence of a blood barrier within the transplants.

Morphological changes induced in turtle retinal neurons by exposure to 6-hydroxydopamine and 5,6-dihydroxytryptamine.

Following intraocular injection of the dopamine neurotoxin 6-hydroxydopamine (10-50 micrograms on two successive days in a Ringer vehicle containing ascorbate and pargyline) and an incubation period of 1 to 18 days, degeneration was noted in presumptive amacrine cells in the retina of the turtle, Pseudemys scripta elegans. Injection of vehicle alone produced no effect. Affected perikarya initially showed swollen mitochondria, lysosomes and distended cisternae. At later stages the cells took on a darkened appearance. In contrast, affected amacrine processes in the inner plexiform layer became markedly distended and lost their cytoplasmic contents, resulting in empty, very swollen profiles. No degeneration was noted distal to the affected cell bodies, i.e. the affected cells were not interplexiform neurons. Cells lesioned by 6-hydroxydopamine were shown to accumulate [3H]dopamine. Intraocular administration of 5,6-dihydroxytryptamine (a single dose of 10-40 micrograms in the same vehicle) followed by 4-6 days incubation resulted in a marked darkening of certain bipolar cell axon terminals, cell bodies and Landolt's clubs. The toxic effects of 5,6-dihydroxytryptamine were blocked by zimelidine, a serotonin uptake blocker. Thus, these two neurotoxins have different targets in the turtle retina. At the highest dose tested, however, 6-hydroxydopamine did produce degenerative changes in the presumed serotonergic bipolar cell.

Coexpression of opsin- and VIP-like-immunoreactivity in CSF-contacting neurons of the avian brain.

Cerebrospinal fluid-contacting (CSF) cells in both the septal and the tuberal areas in the brain of the ring dove are labeled by RET-P1, a monoclonal antibody to opsin that reacts with inner and outer segment membranes of rod photoreceptors in a variety of vertebrates. Immunoblot analysis of proteins from diverse brain regions, however, revealed bands of anti-RET-P1 immunoreactivity that did not correspond to opsin. Binding of RET-P1 to opsin-containing membranes, was not inhibited by membranes rich in muscarinic and beta-adrenergic receptor proteins (red blood cells, heart, lung) taken from doves. RET-P1-immunoreactive CSF-contacting cells emit a dendritic process that penetrates the ependyma and ends in a knob-like terminal suspended in the ventricle. These cells also possess other processes that penetrate more or less deeply into the neuropil. Additionally, a band of labeled fibers occurs in the external layer of the median eminence. A double-label technique demonstrated that RET-P1-positive cells coexpress VIP-like immunoreactivity. VIP-positive cells in other brain areas are not RET-P1-positive.

Terminal degeneration in the mediodorsal cerebral cortex of the lizard Agama agama: Light and electron microscopy.

The degeneration of axon terminals in the small-celled part of the mediodorsal cortex (sMDC) of the lizard Agama agama has been studied after lesions in the dorsal cortex at various survival periods. The Fink-Heimer stain was used to map and demonstrate terminal degeneration with the light and electron microscope. Electron microscopy was used to identify and describe degenerating boutons ultrastructurally. One sham-operated and three unoperated animals served as controls. Between 6 and 21 days postsurgically, degenerating terminals can be seen through 80% of the superficial plexiform layer, the zone adjacent to the cellular layer remaining free of degeneration. Swelling of dendrites in the outer part of the superficial plexiform layer and increased numbers of vacuolar invaginations, both present at short (24 hr-6 days; peak at 48-54 hr) survival periods, can be regarded as reaction to the surgical trauma. Degeneration of axon terminals takes three forms, all of the electron-dense type: gray boutons, degenerating bouton-dendritic spine complexes surrounded or engulfed by glia, and degeneration debris inside glial processes. Several forms of terminal degeneration occur concomitantly at any short (3-12 days) survival time. At longer survival times (15-21 days) only debris is present. From 6 days on, considerable numbers of degenerating structures are present, but the majority of degenerating boutons and debris are associated with reactive glia rather than with dendrites. From these observations it is concluded that in this lizard application of the combined degeneration-Golgi-EM technique would probably lead to little success. Electron microscopy of Fink-Heimer-stained sections suggests that degenerating bouton-dendritic spine complexes and degeneration debris accumulate silver particles, whereas gray boutons do not.

Pineal gland transplants into the cerebral hemisphere of newborn rats: a study of the blood brain barrier and innervation.

Pineal glands from neonatal (0-1 day) Long-Evans black-hooded rats were transplanted into the cerebral hemispheres of litter mates for periods of 1 to 5.5 months. Grafts exhibited differentiated pinealocytes that were intensely immunoreactive for serotonin. Transplant vasculature was permeable to endogenous IgG, comprised fenestrated endothelia with wide pericapillary spaces typical of in situ glands, and had a volume density intermediate to that of surrounding cortex and in situ pineals. Along the periphery, transplant capillaries tended to have continuous endothelia similar to those of host cortex. This peripheral zone was impermeable to endogenous IgG and appeared to increase in size in older grafts. The presence of noradrenergic-like fibers within the perivascular compartment suggested that transplants were innervated by peripheral sympathetic neurons from the superior cervical ganglia. In animals which had been superior cervical ganglionectomized, noradrenergic-like fibers were absent or degenerating. Neural regulation of transplant metabolic activity was suggested by the increased frequency of pinealocyte synaptic ribbons in denervated grafts. These findings are consistent with the hypothesis that factors from both graft and host influence vasculature physiology and differentiation in neural transplants. Furthermore, grafts appeared to receive appropriate neural input from the peripheral sympathetic system.