Non-glomerular Neuropil Research Articles

Antibodies against the PER protein of Drosophila label neurons in the optic lobe, central brain, and thoracic ganglia of the crickets Teleogryllus commodus and Teleogryllus oceanicus.

We describe labeling of neurons in the central nervous system of two cricket species, Teleogryllus commodus and T. oceanicus, with both mono- and polyclonal antibodies against the PER protein. Western blots reveal that the monoclonal antibodies recognize a single protein with a molecular weight of approximately 94 kDa, i.e., similar to that of the PER protein of the moth, Anterea pernii. Neurons and their processes are labeled both in the optic lobes and in the central brain. Processes occur in the accessory medulla, the medulla, and proximal lamina, in the central complex, in the non-glomerular neuropil, and in the retrocerebral complex, suggesting that PER-containing neurons form a widely distributed network. Neurons and processes were also labeled in the meso- and metathoracic ganglia. Four to six PER-immunoreactive (ir) neurons with processes in the accessory medulla were double labeled by an antibody against pigment-dispersion factor (PDF), a peptide that is implicated in circadian rhythmicity in Drosophila. In the central brain, projections of fibers labeled by the anti-PER and anti-PDF antibodies were mainly distinct, with overlap only in a few restricted regions. In most neurons, including those projecting into the accessory medulla, PER labeling was restricted to the cytoplasm and there was no indication of circadian variation in the intensity of staining.

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the non-glomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.

Abundant distribution of locustatachykinin-like peptide in the nervous system and intestine of the cockroach Leucophaea maderae.

An antiserum raised to the locust neuropeptide locustatachykinin I (LomTK I) was used for analysis of the distribution of tachykinin-related peptide in the cockroach Leucophaea maderae. Extracts of dissected brains, suboesophageal ganglia, thoracic ganglia and midguts were separated by high performance liquid chromatography and the fractions analysed in enzyme-linked immunosorbent assay with use of the LomTK antiserum. Each of the tissues was found to contain LomTK-like immunoreactive (LomTK-LI) components with retention times corresponding approximately to synthetic LomTK I and II and callitachykinins I and II. The LomTK antiserum was also used for immunocytochemical mapping of peptide in the nervous system and intestine of L. maderae. A large number of LomTK-LI interneurons were detected in the proto-, deuto- and tritocerebrum of the brain and in the suboesophaegeal ganglion. The immunoreactive neurons supply processes to most parts of the brain: the central body, protocerebral bridge, mushroom body calyces, antennal lobes, optic lobe and most regions of the non-glomerular neuropil. A few protocerebral neurons send LomTK-LI processes to the glandular lobe of the corpora cardiaca. In each of the thoracic ganglia there are six LomTK-LI interneurons and in each of the unfused abdominal ones there are two interneurons. The fused terminal ganglion contains some additional cell bodies in the posterior neuromers. LomTK-LI cell bodies were detected in the frontal ganglion and fibres were seen in this ganglion as well as in the hypocerebral ganglion. The frontal ganglion supplies LomTK-LI processes to the muscle layer of the pharynx. The muscle layer of the midgut is innervated by LomTK-LI fibres from the stomatogastric system (oesophageal nerve and associated ganglia). Additionally the midgut contains numerous LomTK-LI endocrine cells. A number of the pharyngeal dilator muscles were also found to be innervated by LomTK-LI fibres, probably derived from cell bodies in the suboesophageal ganglion. All the LomTK-LI neurons of the central nervous system appear to be interneurons, suggesting a neuromodulatory role of the endogenous tachykinins. The tachykinin-like peptides from peripheral ganglia may be involved in the control of foregut and midgut contractility and possibly the peptide of the endocrine cells in the midgut has additional actions related to intestinal function.

FMRFamide-like immunoreactivity in the brain of the honeybee ( Apis mellifera). A light- and electron microscopical study

Peptide-FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee Apis mellifera L. is demonstrated with the peroxidase-antiperoxidase technique. Immuno-reactivity is found in about 120 perikarya of the brain and in about 30 of the suboesophageal ganglion. These cells are distributed in 13 paired clusters representing neurons of different types including neurosecretory neurons projecting to neurohemal organs. Immunoreactivity of different intensity is found in the non-glomerular neuropil around the mushroom bodies, in the lateral protocerebrum, the central body, the optic tubercles, the lobula and medulla of optic lobe, the ocellar neuropil, in multiglomerular elements of the antennal lobes and in the dorsal deuterocerebrum. In the mushroom bodies, immuno-reactivity is located in layers of the lobes and stalks, corresponding to intrinsic fibre bundles of some Kenyon cell types. The somata of these intrinsic cells did not show FMRFamide-like immunoreactivity. Electron microscopy of immunostained somata and nerve fibres was performed employing a pre-embedding peroxidase-antiperoxidase technique. Fibres of optic lobes and the non-glomerular neuropil contain immunoreactive dense core vesicles (diameter 50–165 nm) accumulated in boutons besides small synaptic vesicles and synaptic membrane specializations. Immunoreactive layers of the mushroom body neuropil were analysed at the ultrastructural level. Axon profiles with dense-core vesicles of a small type (diameter 35–75 nm) show only faint immunoreactive products. Immunoreactivity of intrinsic mushroom body neurons does not appear to be specifically correlated with synapses and synaptic organelles. Our results indicate that FMRFamide or related peptides peptides may be neuroactive compounds in different classes of nerve cells in the bee brain.

Ultrastructural organisation of the projection from the superior colliculus to the ventral lateral geniculate nucleus of the rat.

Retinorecipient regions of the ventral lateral geniculate nucleus of the thalamus and the superior colliculus of the midbrain are linked by reciprocal axonal projections. In this study we have investigated the ultrastructural characteristics, the distribution, and the postsynaptic targets of the terminals of axons projecting to the ventral lateral geniculate nucleus from the superior colliculus. Horseradish peroxidase was injected into the superior colliculi of adult albino rats, and the Hanker-Yates method was used to visualize anterogradely and retrogradely transported peroxidase in the ventral lateral geniculate nuclei 24 hours following the injection. Labelled terminals were found in the lateral and ventrolateral parts of the external division of the ipsilateral ventral lateral geniculate nucleus. The labelled terminals were confined to areas of simple, nonglomerular neuropil. They were 0.45-1.5 micron in diameter; contained small, dark mitochondria and spherical synaptic vesicles; and established Gray type I (asymmetrical) synaptic contacts with the dendritic shafts, dendritic spines, and occasionally cell bodies of cells with the ultrastructural characteristics of projection cells. A few labelled terminals established synaptic contact with retrogradely labelled cells. Thus, in the rat, the projection from the superior colliculus gives rise to a uniform population of axon terminals in the nonglomerular neuropil of the lateral portion of the ventral lateral geniculate nucleus, which synapse with, and are probably excitatory to, geniculocollicular and other projection cells.

Axon terminals of the projection from the superior colliculus to the olivary pretectal nucleus in the rat

The terminals of axons projecting to the olivary pretectal nucleus have been identified by electron microscopy following injections of horseradish peroxidase into the superior colliculus of adult albino rats. The labelled terminals were equated with RD-terminals described in previous studies of this nucleus. They were 0.3–1.3 μm in diameter and contained round synaptic vesicles. Most also contained small dark mitochondria. They established Gray type 1 synaptic contacts with the dendrites of presumptive projection cells. Most terminated within non-glomerular neuropil, chiefly in the peripheral ‘shell’ of the nucleus; a few terminated in regions of glomerular neuropil.

Serotonin-immunoreactive neurons in the brain of the honeybee.

The distribution of serotonin-immunoreactive neurons in the brain of the worker honey bee Apis mellifera was studied by means of immunocytochemical staining by using a well-characterized antibody to serotonin (5-HT). About 75 immunoreactive perikarya are grouped into clusters in the optic lobe and in the median and dorsal protocerebrum. Immunoreactive fibers were resolved in all areas of the brain. The optic lobe shows restricted layers of 5-HT-immunoreactive fibers in the lamina and medulla organized perpendicular to the retinotopic elements. Immunoreactive fibers in the lobula represent invasions of protocerebral giant wide-field neurons. The nonglomerular neuropil of the brain exhibits a meshwork of immunoreactive fibres invading glomerular neuropil of the mushroom bodies, central body complex, and antennal lobes. Mushroom body stalks and lobes contain immunoreactive fibers arranged perpendicular to the Kenyon cell fibers and matching subcompartments of these corpora pedunculata areas. The calyces are devoid of immunofluorescence. Serotonin-positive fibres in the central body complex are arranged in its subcompartments. No 5-HT immunoreactivity was found in the pons. Antennal glomeruli contain immunoreactive fibers restricted around the margin of the glomeruli. The selective mapping of 5-HT-immunoreactive neurons complements studies on the distribution of monoamine-containing neurons in the bee brain. Serotonin- and catecholamine-containing neurons often occur together in the same brain areas and subcompartments. The immunohistochemical approach in chemoneuroanatomy gives new evidence for a more complicated architecture of the brain than could be deduced from the classical neuroanatomical studies.

EM-autoradiography of cerebellar nucleocortical terminals in the cat.

The findings from the present EM-autoradiography experiments identify the cerebellar nucleocortical projection as a mossy fiber-type pathway. In these studies orthogradely labelled axons and terminals were observed in the folial white matter and granule cell layer, with only background levels of silver grains over the Purkinje cell and molecular layers. Most nucleocortical axons synapsed within cerebellar glomeruli either at en passant contacts along dilated segments of unmyelinated axons or at multilobulated (terminal) expansions of the axons. Typically these synaptic junctions were of the asymmetrical type containing clear round vesicles. Usually, each labelled mossy fiber rosette synapsed on a large number of small terminal dendrites of granule cells; however, occasionally a series of 4-8 asymmetric contacts were noted on a single large dendrite. Labelled nucleocortical terminals were occasionally present in the nonglomerular neuropil between granule cell bodies where they contacted large dendrites of Golgi cells.

A quantitative electron-microscopical study of the postnatal development of the lateral geniculate nucleus in normal kittens and in kittens with eyelid suture.

The postnatal development of the lateral geniculate nucleus has been studied quantitatively with the electron microscope in normal kittens and in kittens with eyelid closure. The maturation of the synaptic organization of glomeruli in the normal kitten occurs during the period of susceptibility to eyelid closure and is due predominantly to a logarithmic increase in the number of symmetric presynaptic dendritic synapses. In contrast, the proportion of symmetric synapses falls with age in non-glomerular neuropil over this period. Unilateral and bilateral eyelid suture do not interfere with the normal development of the lateral geniculate nucleus.

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. I. preservation of large fragments of the cerebellar glomeruli

In view of isolating synaptosomes of a single type from rat cerebellum, a procedure was developed which led to the preservation of morphologically characteristic structures with a more complex level of organization than is usually encountered in disrupted brain tissue. The method involved manual fragmentation of the cerebellum, by using a Dounce type of homogenizer, in0.3 M sucrose solution containing1m M MgSO 4 . The following complex structures were obtained in sufficient yield: large fragments of the cerebellar glomeruli (glomerulus particles), non-glomerular neuropil fragments probably derived from the molecular layer, and large myelinated axon segments with enclosed axoplasm. The glomerulus particles contained large, relatively intact portions of the mossy fibre terminals with numerous synaptic vesicles and mitochondria and dendritic digits of the granule cells, which remained attached to the giant terminals. Due to their size, these complex structures, together with the well preserved nuclei, could be separated by differential centrifugation from other organelles, such as synaptosomes of conventional size and free mitochondria (fractions) N and M respectively). Fraction N contained about 60% of the tissue proteins, whereas fraction M contained only about 10%. There were marked differences in the enzyme composition of the primary fractions. The fractional distribution ratio of the two mitochondrial enzymes, glutamate dehydrogenase (GDH) to succinate dehydrogenase (SDH), was about 2 in fraction M and 1 in fraction N. These results were consistent with the view that free mitochondria are enriched in GDH relative to SDH. The fractional distribution ratio of the two ‘transmitter’ enzymes, choline acetyltransferase to glutamate decar☐ylase, was about 2.7 in fraction M and 1 in fraction N. Since the the relative specific activity of acetylcholinesterase was also high (2.0) in fraction M, the results suggested an enrichment of cholinergic synaptosomes in this fraction.

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. II. Resolution of morphologically characterised fractions

Summary Subcellular fractions with relatively homogeneous morphological composition were separated from fragmented rat cerebellum. The tissue was gently disrupted, and the low speed sediment (fraction N) was further fractionated by centrifugation on a discontinuous sucrose gradient: all the sucrose solutions contained 1 m M MgSO 4 . The main constituents of fraction ‘A’ (0.3/0.8 M sucrose interface) were large myelinated axon segments with enclosed axoplasm. The myelin sheaths were swollen which may have contributed to the high purity of this preparation. Axon segments with less damaged myelin sheath penetrated to a denser region of the gradient (fraction ‘B’, 0.8/1.2 M sucrose interface) and they were contaminated by other particles. Fraction ‘A’ had a characteristic enzyme composition: the relative specific activities (RSA) of the two mitochondrial marker enzymes, succinate- and glutamate dehydrogenases (SDH and GDH), were comparable, and these values were significantly higher than the RSAs for the ‘transmitter’ enzymes, choline acetyltransferase (ChAc) and glutamate decar☐ylase (GAD). The RSA of ChAc was 60% higher than that of GAD. Fraction ‘C’ (1.2/1.4 M sucrose interface) was mainly composed of neuropil fragments. Quantitative electron microscopic investigations indicated that large pieces of the cerebellar glomeruli (glomerulus particles) constituted approximately half of the total tissue volume in this fraction. The relatively intact mossy fibre terminals accounted for about 60% of the area of the glomerulus particles. The attached dendritic digits of the granule cells and synaptic specializations could be recognized. It seems that the non-glomerular neuropil fragments in fraction ‘C’ originated mainly from the molecular layer. In comparison with the glomerulus particles, they accounted for about the same proportion of the total tissue in fraction ‘C’, and their average size was also similar. Nevertheless, about 95% of the presynaptic area in fraction ‘C’ was attributable to the mossy fibre terminals. Fraction ‘C’ contained about half of the protein of fraction N, but the enzyme profile did not indicate unique characteristics. The RSAs of the various enzymes were about 1, with the exception of GAD (1.2) and SDH (1.8) which was significantly higher than 1. Fraction ‘E’ was a relatively pure preparation of nuclei containing approximately 90% of the DNA of the filtered homogenate. The nuclei were well preserved, and the most common contaminants among the occasional non-nuclear structures were neuropil fragments, especially glomerulus particles.