Molecular analysis of the asense gene, a member of the achaete-scute complex of Drosophila melanogaster, and its novel role in optic lobe development.

The achaete-scute complex (AS-C) comprises five genetic regions: achaete, scute (sc) alpha, lethal of sc, sc beta and sc gamma. Each region promotes the determination and positional specification of different, but partially overlapping, subsets of neural elements of Drosophila. In this work, we report a molecular characterization of the sc gamma region. It comprises 22 kb of DNA and contains two transcription units, only one of which, named asense (ase), seems involved in neurogenesis. ase encodes a protein that shares with other three AS-C proteins a domain containing a helix--loop--helix motif characteristic of a group of DNA-binding proteins. In the embryo, ase is expressed in neural precursor cells, a pattern consistent with the known requirement of sc gamma for the development of the larval nervous system. In late third-instar larvae, the gene is expressed in developing structures of the central nervous system (CNS), namely the anlagen of the optic lobes and in many cells, including neuroblasts, of the central brain and ventral ganglia. Its removal leads to anatomical defects in the adult optic lobes. This is the first demonstration of a role for the AS-C in the development of the adult CNS.

Amino acid sequence of a low molecular weight, high affinity calcium-binding protein from the optic lobe of the squid Loligo pealei.

The amino acid sequence was determined for squid calcium-binding protein (CaBP), a low molecular weight, high affinity calcium-binding protein from squid optic lobe. The sequence shows this protein to consist of 149 amino acids with an N-acetylated N terminus. The protein has a molecular weight of 16,894 and is homologous to calmodulin, which is also present in squid optic lobe (Head, J. F., Spielberg, S., and Kaminer, B. (1983) Biochem. J. 209, 797-802). When the sequences of squid CaBP and bovine brain calmodulin are appropriately aligned, the proteins are found to share 68% identity, with a single residue insertion in squid CaBP, between domains III and IV. The four-domain structure of calmodulin appears to be retained in squid CaBP, which is consistent with the previously reported presence of four calcium-binding sites per molecule (Sheldon, A., and Head, J. F. (1988) J. Biol. Chem. 263, 14384-14389). The two tyrosines of squid CaBP are located in different halves of the molecule, one at the position corresponding to Tyr-138 in calmodulin, the other in an equivalent position in domain II. In addition, squid CaBP exhibits several differences in the region corresponding to the long central helix of calmodulin. These differences include the replacement of Lys-77 by glycine, Asp-78 by proline, and Ser-81 by proline. The sequence of this portion of the squid CaBP molecule suggests the protein is unlikely to possess the continuous long central helix found in calmodulin.

Dopamine-immunoreactive neurons in the blowfly visual system: light and electron microscopic immunocytochemistry.

Dopamine-immunoreactive (DA-IR) neurons were mapped in detail in the visual system of the blowfly, Calliphora erythrocephala. Three types of DA-IR neurons could be identified in the optic lobes. One type constitutes a population of several thousand columnar small field amacrine neurons in the second neuropil region, the medulla. The other two types are large field projection neurons innervating the next, more central, synaptic region comprising the lobula and the lobula plate, as well as centres of the midbrain. Their cell bodies are located latero-ventrally in the brain. No DA-IR neurons were seen in the most peripheral visual synaptic neuropil, the lamina. The two types of projection neurons form overlapping wide field arborizations in the lobula and lobula plate and cannot be distinguished from each other in this region. Their central connections are different, however. One type of projection neuron, BOD1, consists of two neurons that bilaterally connect the optic lobes and neuropil on each side of the oesophageal foramen in the posterior protocerebrum. The other type, BOD2, also consists of two bilateral neurons similar to BOD1, but with their central processes posteriorly in the lateral protocerebrum. The amacrine DA-IR neurons form lateral processes in three layers of the medulla synaptic neuropil. These neurons were also investigated by means of electron microscopical immunocytochemistry. They contain predominantly clear vesicles, but a few dense core vesicles could be resolved. The synaptic connections of the DA-IR amacrines suggest that they form centrifugal feedback circuits between the inner and the outer portion of the medulla. The present results indicate that dopamine may be a neurotransmitter in functionally different classes of neurons of the blowfly visual system: amacrines and projection neurons.

Light and electron microscopic immunocytochemistry of neurons in the blowfly optic lobe reacting with antisera to RFamide and FMRFamide.

Different antisera to the molluscan cardioexcitatory peptide FMRFamide, and its fragment, RFamide (Arg-Phe-NH2), label a distinct population of neurons in the optic lobe of the blowfly, Calliphora erythrocephala. Seven morphological types of RFamide/FMRFamide-like immunoreactive neurons could be distinguished in the optic lobes based on the locations of their cell bodies, their axonal projections and the distribution of their processes. Of these, two types could be resolved in their entire extent, the others were labeled only in their cell bodies and terminal processes or were partly obscured by other immunoreactive processes. The RF-like immunoreactive neurons in the optic lobes are of two main classes: (1) two types of large field projection neurons and (2) five types of local neurons. One type of projection neurons (five in each lobe) connects the entire projected retinal mosaic of the medulla and lobula in the optic lobe with protocerebral centres associated with the mushroom body calyx. The other type (2-3 invading each lobe) has cell bodies in the protocerebrum and contralateral processes invading optic lobes. Of the class of local neurons there are two amacrine RF-like immunoreactive neurons in each medulla. Each of these amacrines supplies the entire mosaic with fine processes. The remaining local RF-like immunoreactive neurons are present in relatively large numbers (one type in more than 2000 copies in each medulla) and-supply the medulla, lobula and lobula plate neuropils with fine varicose processes. In the medulla the RF-like immunoreactive processes are arranged in strict layers whereas in the lobula complex the distribution is diffuse. Electron microscopic immunocytochemistry, using both pre-embedding immuno peroxidase-antiperoxidase and post-embedding protein A-gold labeling, was employed for analysis of cytology and synaptic connections of RF-like immunoreactive neurons in the medulla. The varicosities of the processes of the large field projection neurons were not found to make chemical synapses with other neurons in the medulla. The spines of the RF-like immunoreactive processes of the large medulla amacrines, however, make pre- and postsynaptic contacts with other neural elements. Our findings indicate that an RFamide/FMRFamide-like substance may be used as a neurotransmitter or neuromodulator by optic lobe neurons of different types. The local and projection RF-like immunoreactive pathways probably play different roles in visual processing.

Immunohistological demonstration of a substance related to neuropeptide Y and FMRFamide in the cephalic and thoracic nervous systems of the locust Locusta migratoria.

A neuropeptide related to the mammalian neuropeptide Y (NPY) is present in various neurosecretory cells (NSC) of the cephalic and thoracic nervous systems of the insect Locusta migratoria. Immunoreactive perikarya are detected in the protocerebrum, tritocerebrum, optic lobes and the suboesophageal and thoracic ganglia. They give rise to many immunoreactive processes that ramify extensively throughout the neuropiles. In the brain, prominent axon bundles tightly surround the tractus I to the corpora cardiaca. This fiber pattern suggests that the NPY-like substance may have a neuromodulator and/or neurotransmitter function. This substance may also have a neurohormonal role, since some immunoreactive tracts penetrate into neurohaemal organs via the nervi corporis cardiaci II and the thoracic median nerves. NCS containing NPY-like neuropeptide also display an FMRFamide-like immunoreactivity (except for the abdominal part of the metathoracic ganglion). NPY or FMRFamide antisera are not inactivated after preabsorption with FMRFamide or NPY, respectively. It might therefore be inferred that in locust NSC these two antisera recognize two distinct antigenic sites belonging either to a large polypeptide, or to two distinct neuropeptides.

Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm.

Axoplasmic RNA from the giant axon of the squid (Loligo pealii) comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/microgram of total RNA) suggests that the poly(A)+ RNA population makes up approximately 0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments greater than 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower-reacting component (moderately abundant and rare sequences). The latter component accounts for approximately 56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of approximately 2.7 kilobases and 3.1 X 10(2) kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents approximately 0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules.

Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP-sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility.

Complexity of nuclear and polysomal RNA from squid optic lobe and gill.

The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 X 10(8) and 8.8 X 10(7) nucleotides, respectively. Approximately 80-85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single-copy DNA, values that were 2.5-fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Insect optic lobe neurons identifiable with monoclonal antibodies to GABA.

Five monoclonal antibodies against GABA were tested on glutaraldehyde fixed sections of optic lobes of three insect species, blowflies, houseflies and worker bees. The specificity of these antibodies was analyzed in several tests and compared with commercially available anti-GABA antiserum. A very large number of GABA-like immunoreactive neurons innervate all the neuropil regions of these optic lobes. Immunoreactive processes are found in different layers of the neuropils. The immunoreactive neurons are amacrines and columnar or noncolumnar neurons connecting the optic lobe neuropils. In addition some large immunoreactive neurons connect the optic lobes with centers of the brain. Some neuron types could be matched with neurons previously identified with other methods. The connections of a few of these neuron types are partly known from electron microscopy or electrophysiology and a possible role of GABA in certain neural circuits can be discussed.

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.

Axoplasm from the squid giant axon contains a soluble protein translocator that induces movement of microtubules on glass, latex beads on microtubules, and axoplasmic organelles on microtubules. We now report the partial purification of a protein from squid giant axons and optic lobes that induces these microtubule-based movements and show that there is a homologous protein in bovine brain. The purification of the translocator protein depended primarily on its unusual property of forming a high affinity complex with microtubules in the presence of a nonhydrolyzable ATP analog, adenylyl imidodiphosphate. The protein, once released from microtubules with ATP, migrates on gel filtration columns with an apparent molecular weight of 600 kilodaltons and contains 110-120 and 60-70 kilodalton polypeptides. This protein is distinct in molecular weight and enzymatic behavior from myosin or dynein, which suggests that it belongs to a novel class of force-generating molecules, for which we propose the name kinesin.

Histochemical detection of anionic components in the cephalopod brain.

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.

Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine--Sepharose affinity chromatography.

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.

Neurological disease and herpes simplex virus. An immunohistochemical study.

The brains of 43 patients, some with various neurological disorders, other controls, were examined for herpes simplex virus (HSV) antigen using immunoperoxidase technique. The three patients with herpes simplex encephalitis shared a pattern of staining, consistent with that reported previously. However, of the other 40 patients, only two (one a patient with Alzheimer's disease, the other a control patient) showed areas of brain positive for HSV antigen (VA). In the patient with Alzheimer's disease VA was present within nerve and glial cells of the amygdala, within oligodendrocytes of the optic and olfactory tracts and in macrophages within the temporal cortex hippocampus and cerebellum. In the control patient VA was seen only in oligodendrocytes of optic chiasma and olfactory tract. The scarcity of these findings suggests "coincidental disease" processes within these two patients and means that any hypothesis implicating HSV as an aetiological agent in degenerative disease must still remain extremely speculative.

Immunohistochemical evidence of methionine enkephalin-like material in the brain of the migratory locust.

Four neurons in the brain of the migratory locust were immunohistologically identified with an anti-met-enkephalin antiserum. The perikarya of two of these cells are located in the center of each of the two groups of lateral protocerebral neurosecretory cells. The fibres coming from these perikarya terminate in numerous immunoreactive ramifications visible at the periphery of both tractus I to the corpora cardiaca, through which pass the neurosecretory products of the pars intercerebralis. The other two cell bodies are located at the bases of the two optic lobes; their fibres enter the posterior part of the protocerebrum and ramify around the root of the nervus corporis cardiaci II, another area through which neurosecretory products pass. The topographic distribution of these met-enkephalin arborizations suggests that these four neurons may act a s neuromodulators of the activity of the major neurosecretory cells in the brain of this insect.

Octopus calmodulin. Structural comparison with bovine brain calmodulin.

A protein previously isolated from octopus optic lobe is shown to have the biochemical characteristics of a calmodulin-like protein. The amino acid composition of the octopus calmodulin is similar to that of another sea invertebrate calmodulin, from Renilla reniformis, in that both contain a single residue of tyrosine which distinguishes them from the vertebrate calmodulins which contain two tyrosines. The 1H NMR spectra of the octopus calmodulin and bovine brain calmodulin are compared in their apo- and calcium-saturated conformations. A comparison of these spectra indicates that the single tyrosine of the octopus calmodulin is in a structurally homologous position to tyrosine-138 of bovine brain calmodulin. 1H NMR and UV difference spectroscopy also demonstrate that the solution conformations of the apo- and calcium-saturated forms of octopus calmodulin are very similar to those of bovine brain calmodulin. It is concluded that both proteins undergo similar calcium-induced changes in tertiary structure, which result in near identical solution conformations.

Immunocytochemical localization of somatostatin-containing neurons in the brain of Rana temporaria.

The brain of the frog Rana temporaria was studied at the light microscopic level with the use of a double immunocytochemical staining method. The telencephalon, diencephalon and rhombencephalon contain somatostatin perikarya and fibers. In the telencephalon, the location of the somatostatin neurons largely corresponds to that of mammals. In the hypothalamus, the somatostatin perikarya are located in and near the magnocellular preoptic nucleus and also in the pars ventralis of the tuber cinereum. Like the somatostatin neurons of the rat hypothalamus, they form a separate subpopulation, different from the neurons producing neurohypophysial hormones. In Rana, somatostatin neurons are also present in (as well as in the vicinity of) the subfornical organ, in the thalamus, the tectum opticum, the interpeduncular nucleus and the caudal end of the roof of the calamus scriptorius. A precise localization of the perikarya of most somatostatin fibers, including those found in the median eminence and the neural lobe, was not attained.

Effects of eye removal on receptors of alpha-bungarotoxin in the optic lobes of chick brain.

The existence of receptors with high affinity for alpha-bungarotoxin and the pharmacologic properties of a nicotinic ACh receptor has been shown in the optic tectum of chick brain. In the present study eyes were removed from chicks at embryonic day 3 (E3), or at post-hatching day 3 (P3). In both cases, the total number of receptors for I125-alphaBT which developed in the deafferented optic lobe was about 50% of the number in normal lobes.

Biochemical and immunological studies with human optic and olfactory tracts.

Clinical and pathological studies have revealed that in multiple sclerosis (MS) the involvement of the optic tracts is much more frequent than that of the olfactory tracts. To investigate the possible reasons for this difference in involvement of these two adjacent structures, both containing a central type myelin, we have studied optic and olfactory tracts obtained at autopsy from 7 adult males ranging in age from 54 to 64 years. White matter from the frontal poles of the same individuals was used for reference. These tissues were compared with respect to the relative content of a) water, b) soluble proteins, c) 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity, and d) immunologically precipitable basic protein (BP). Homogenates from these tissues were further compared by disc gel electrophoresis in two systems; phenolformic acid-water and SDS-urea gels. Results indicate that while the optic tracts and the frontal pole white matter were similar with respect to their water, total protein content and BP content, the optic tracts had lower CNP activity than the frontal poles. The olfactory tracts contained more water and less BP and the CNP activity of these structures was lower than that of the frontal pole white matter. Assuming the CNP activity and the BP content are true measures of the total myelin content of a given tissue, it appears that olfactory tracts have smaller amounts of myelin. On the other hand, the optic tracts contain only half as much CNP-activity with a disproportionately greater amount of BP. The possible significance of these findings is discussed.

Biochemical maturation of the non-innervated chick optic lobe.

The development of the chick optic lobe was impaired following removal of the optic cup of the early embryo. Tectal cell number is reduced but cell size may be relatively normal. Ther was evidence of neuronal cell death and several neuron-associated proteins and enzymes (nerve-specific protein and acetylcholinesterase) showed selectively impaired maturation. However, other nerve-specific enzymes (choline acetyltransferase, tyrosine hydroxylase), develop normally on a per cell basis. The noninnervated optic lobe had a normal blood-brain barrier but a depressed ability to accumulate amino acids from plasma. Levels of 3':5'-cyclic GMP were also reduced in the nonafferented lobe.