Provincial Veterinary Services respond to drought in South Africa.

In 2018, Cape Town, South Africa, nearly ran out of water. That this has not yet happened is in large part due to the water-saving efforts of its citizens. It is highly likely that this situation will be repeated in Cape Town and that similar situations will be experienced by major cities in other parts of the world. Efforts to save water should thus continue and the lessons learned in Cape Town should be shared. The functioning of Veterinary Services during a drought is affected in the same way as any business, in terms of running an office, but veterinary professionals face an increased risk of exposure to pathogens, compared to that of many occupations, and of veterinary officials becoming disease vectors. One component of Veterinary Services is veterinary laboratory services. Laboratory procedures rely heavily on water and, without advance planning, a laboratory's function can be severely limited by a restricted water supply. In many cases, innovative water-saving techniques can be used to reduce water use substantially without compromising the quality of the services offered. Here, the authors share their experiences and some lessons learned while working in Veterinary Services in the Western Cape province of South Africa.

En 2018, la ville du Cap en Afrique du Sud a failli manquer d'eau. Si la pénurie totale a pu être évitée, ce fut en grande partie grâce aux efforts déployés par les habitants pour économiser l'eau. Or, il est très probable que cette situation se reproduise au Cap et que des situations analogues surviennent dans nombre de grandes métropoles d'autres régions du monde. C'est pourquoi il convient de poursuivre les efforts d'économie d'eau et de partager avec d'autres les enseignements tirés dans la ville du Cap. L'impact de la sécheresse sur le fonctionnement des Services vétérinaires est similaire à celui de toute organisation en termes de gestion administrative ; en revanche, par rapport à d'autres professionnels, les vétérinaires de terrain sont davantage exposés à des agents pathogènes et au risque de devenir eux-mêmes vecteurs de maladies. Les laboratoires vétérinaires sont l'une des composantes des Services vétérinaires. Les procédures de laboratoire sont amplement tributaires de l'eau ; or, en l'absence d'une planification préalable, les activités d'un laboratoire pourraient être gravement mises à mal par des restrictions de l'approvisionnement en eau. Dans bien des cas, il est possible d'utiliser des techniques innovantes pour économiser l'eau afin d'en diminuer la consommation sans pour autant compromettre la qualité des services rendus. Les auteurs font part de leur expérience et de certains enseignements tirés lorsqu'ils travaillaient dans les Services vétérinaires de la province du Cap-Occidental en Afrique du Sud.

En 2018 faltó poco para que Ciudad del Cabo (Sudáfrica) se quedara sin agua. Si las cosas aún no han llegado a este extremo es, en gran parte, gracias a los esfuerzos de los habitantes por economizar agua. Es muy probable que en el futuro Ciudad del Cabo vuelva a sufrir esta situación y que grandes metrópolis de otras partes del mundo conozcan dificultades parecidas. Por ello hay que perseverar en los esfuerzos de ahorro de agua y se deben compartir las enseñanzas extraídas en Ciudad del Cabo. Durante una sequía, el funcionamiento de los Servicios Veterinarios se ve afectado del mismo modo que cualquier otra actividad, por lo que respecta al trabajo de oficina, pero además los profesionales del ramo, en comparación con los de otros muchos sectores, corren mayor peligro de exposición a patógenos, lo que a su vez entraña el riesgo de que los propios veterinarios ejerzan de vectores de la enfermedad. Uno de los puntales de los Servicios Veterinarios son los laboratorios veterinarios, cuyo quehacer depende en gran medida del uso de agua. Por ello, cuando no se ha planificado con antelación la eventualidad de una penuria de agua, esta puede imponer graves cortapisas a las funciones de laboratorio. En muchos casos es posible emplear innovadoras técnicas de ahorro de agua para reducir sustancialmente las cantidades utilizadas sin menoscabo de la calidad de los servicios dispensados. Los autores comparten su experiencia y algunas de las lecciones que extrajeron de su trabajo en los Servicios Veterinarios de la provincia sudafricana del Cabo Occidental.

The Social, Historical, and Institutional Contingencies of Dam Removal.

Environmental managers in the United States and elsewhere are increasingly perceiving dam removal as a critical tool for river restoration and enhancing watershed resilience. In New England, over 125 dams have been dismantled for ecological and economic rationales. A surprising number of these removals, including many that are ongoing, have generated heated conflicts between restoration proponents and local communities who value their dammed landscapes. Using a comparative case study approach, we examine the environmental conflict around efforts to remove six dams in New England. Each of these removal efforts followed quite different paths and resultant outcomes: successful removal, stalled removal, and failure despite seemingly favorable institutional conditions. Lengthy conflicts often transpired in instances where removals occurred, but these were successfully arbitrated by paying attention to local historical-geographical conditions conducive to removal and by brokering effective compromises between dam owners and the various local actors and stakeholders involved in the removal process. Yet our results across all cases suggest that these are necessary, but not sufficient conditions for restoration through dam removal since a similar set of conditions typified cases where removals are continuously stalled or completely halted. Scholars examining the intersection between ecological restoration and environmental politics should remain vigilant in seeking patterns and generalities across cases of environmental conflict in order to promote important biophysical goals, but must also remain open to the ways in which those goals are thwarted and shaped by conflicts that are deeply contingent on historical-geographical conditions and broader institutional networks of power and influence.

Influences of the cell cycle on silencing.

Silencing in Saccharomyces cerevisiae is a form of transcriptional repression that involves the assembly of a specialized and heritable structure of chromatin. The HML and HMR loci, which contain copies of the genes found at the yeast mating-type locus, are silenced, as are telomeres. These examples share several features which are also found in position-effect variegation in flies and X-chromosome inactivation and genomic imprinting in mammals. Silenced chromatin is confined to a few special domains of the yeast genome, and active genes inserted into these domains become silenced. Molecular and genetic evidence has suggested that the establishment of silenced chromatin requires some S phase specific function. Recent experiments indicate that the assembly and maintenance of silenced chromatin can also be influenced at other phases of the cell cycle.

The origin recognition complex, SIR1, and the S phase requirement for silencing.

Silencing of transcription in Saccharomyces cerevisiae has several links to DNA replication, including a role for the origin recognition complex (ORC), the DNA replication initiator, in both processes. In addition, the establishment of silencing at the HML and HMR loci requires cells to pass through the S phase of the cell cycle. Passage through S phase was required for silencing of HMR even under conditions in which ORC itself was no longer required. The requirement for ORC in silencing of HMR could be bypassed by tethering the Sir1 protein to the HMR-E silencer. However, ORC had a Sir1-independent role in transcriptional silencing at telomeres. Thus, the role of ORC in silencing was separable from its role in initiation, and the role of S phase in silencing was independent of replication initiation at the silencers.

The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.

Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications.

The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization.

A region of the Sir1 protein dedicated to recognition of a silencer and required for interaction with the Orc1 protein in saccharomyces cerevisiae.

Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.

Forkhead genes in transcriptional silencing, cell morphology and the cell cycle. Overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae.

The SIR1 gene is one of four specialized genes in Saccharomyces cerevisiae required for repressing transcription at the silent mating-type cassettes, HMLalpha and HMRa, by a mechanism known as silencing. Silencing requires the assembly of a specialized chromatin structure analogous to heterochromatin. FKH1 was isolated as a gene that, when expressed in multiple copies, could substitute for the function of SIR1 in silencing HMRa. FKH1 (Forkhead Homologue One) was named for its homology to the forkhead family of eukaryotic transcription factors classified on the basis of a conserved DNA binding domain. Deletion of FKH1 caused a defect in silencing HMRa, indicating that FKH1 has a positive role in silencing. Significantly, deletion of both FKH1 and its closest homologue in yeast, FKH2, caused a form of yeast pseudohyphal growth, indicating that the two genes have redundant functions in controlling yeast cell morphology. By several criteria, fkh1Delta fkh2Delta-induced pseudohyphal growth was distinct from the nutritionally induced form of pseudohyphal growth observed in some strains of S. cerevisiae. Although FKH2 is redundant with FKH1 in controlling pseudohyphal growth, the two genes have different functions in silencing HMRa. High-copy expression of CLB2, a G2/M-phase cyclin, prevented fkh1Delta fkh2Delta-induced pseudohyphal growth and modulated some of the fkhDelta-induced silencing phenotypes. Interestingly, deletions in either FKH1 or FKH2 alone caused subtle but opposite effects on cell-cycle progression and CLB2 mRNA expression, consistent with a role for each of these genes in modulating the cell cycle and having opposing effects on silencing. The differences between Fkh1p and Fkh2p in vivo were not attributable to differences in their DNA binding domains.

The radial fibers in the globus pallidus.

In our Golgi collection of adult monkey brains the striatal efferents, i.e., the radial fibers in the globus pallidus and the "comb" bundle fibers in the internal capsule and in the cerebral peduncle, are well impregnated in the horizontally sectioned brain and in a sagittal sectioned brain. Since collaterals emerging from radial fibers are seen only in the horizontal series and not in the saggittal series, the interpretation is that they proceed anteriorly and posteriorly only, following the curvature of the pallidal segments, and do not run superiorly or inferiorly as they emerge. Although radial fibers emitting collaterals in the lateral segment and in the medial segment of the globus pallidus have been observed, it has not been possible to observe the same radial fiber emitting collaterals in both pallidal segments and the prospects of ever doing so are not good. The radial fibers converging in the globus pallidus pursue many radii and there is little coincidence between the plane of section and the planes in which they travel. At most only severed radial fiber segments 100-150 microns in length can be found in the horizontal sections needed to observe the collaterals. Moreover, sagittal sections trodorsally, as they pass through the internal medullary lamina to enter the medial segment of the globus pallidus. The radial fibers in the medial segment of the globus pallidus are continuous with the "comb" bundle fibers and appear to be thinner than the radial fibers in the lateral segment of the globus pallidus. It is not proved; nonetheless, the view expressed here is that the radial fibers are thinner in the medial segment of the globus pallidus because they may be the same fibers that gave off collaterals in the lateral segment of the globus pallidus. This is discussed in the light of the electrophysiological disclosure of Yoshida et al. ('71, '72) that caudatopallidal fibers are collaterals off caudatonigral fibers. The afferent plexuses of fine, "bouton en passage" fibers, which completely ensheath the long radiating dendrites in the globus pallidus (Fox et al., '66) are well impregnated in the horizontal series. Obviously, they are formed by a number of ultimate branches converging from the collateral brances of a number of different radial fibers. The divergence, too, in this system must be considerable; however, its true extent can only be surmised from the several radial fibers and radial fiber collaterals seen in the incompletely impregnanted Golgi section. Continued.

Afferent fibers in the substantia gelatinosa of the adult monkey (Macaca mulatta): a Golgi study.

In horizontally and sagittally sectioned Golgi preparations of the adult monkey spinal cord the afferent fibers in the substantia gelatinosa (laminae II and III of Rexed, '52) were studied with the hope of finding clues that would make it possible to correlate them with the afferent fibers that Ramón y Cajal ('09) traced from the white matter into the substantia gelatinosa in Golgi preparations of the newborn cat. Throughout the substantia gelatinosa there are fibers with fine terminal branches, bearing small "bouton en passant" endings. These fine terminal branches and the collaterals of the axons of the neurons in the substantia gelatinosa are similar; hence, the fine terminal branches are interpreted as the terminals of the proprius bundles generated by the axons of the neurons in the substantia gelatinosa. Also, there are fibers in lamina II that can be followed for relatively long distances (900 microns) before they are cut off in section. They have collaterals bearing large irregular endings, which usually consist of several or more elongated swelling connected by constricted regions. Some of these endings have holes within them and their surfaces have prominent indentations. The fibers with fine terminal branches and the fibers with large irregular endings are interpreted here as the two varieties of fine or superficial collaterals of the substance of Rolando described by Ramóm y Cajal ('09). The most elaborate afferent formations in the present material are found in horizontal sections. They are designated "confined ansiform axonal complexes" and are confined to elongated, oblong blocks of substantia gelatinosa that approximate each other in length, width and depth. Their branches, running rostrally and caudally, loop back and forth, interweave in and intricate pattern and bear large synaptic endings. Because these formations are elaborate and because their parent fibers and initial branches are thicker in lamina III than they are in lamina II, these complexes are interpreted here as the large or deep collaterals of the substance of Rolando described by Ramón y Cajal ('09). Considering their shapes, sizes and surface contours, it is probable that the large endings on the ansiform axonal complexes and the large irregular endings on the long fibers in lamina II are the central terminals in the glomeruli of the substanta gelatinosa that have been revealed by electron microscopy. Finally, there are fine collaterals from fibers in Lissauer's fasciculus which distribute exclusively to the marginal zone (lamina I) of the dorsal horn. Their endings are ovoid or bulbous and are almost uniformly approximately 2 microns in diameter. Undoubtedly, these collaterals are the marginal collaterals Ramón y Cajal ('09) demonstrated in Ehrlich's methylene blue preparations of the 8-day old cat.

The basilar pontine gray in the adult monkey (Macaca mulatta): a Golgi study.

In Golgi preparations of the adult monkey (Macaca mulatta) two types of neurons are distinguished in the pontine gray: (1) larger neurons which impregnate most frequently and (2) smaller neurons which impregnate rarely. The former are judged to be projection neurons with myelinated axons because only the initial segments of their axons impregnate, while the latter are judged to be intrinsic neurons since they appear to participate only in the local circuitry of the pontine gray. The projection neurons show a variety of sizes and shapes and are the equivalent of the large, medium and small neurons that Ramón y Cajal ('09) illustrated in the pons of the 5-day-old infant. Their cell bodies are rounded, polygonal, triangular, egg-, pear-, and spindle-shaped. Some have somatic spines. Usually four to seven dendrites issue from the cell body and as they branch they attentuate. The dendrites have knobby, nodular protuberances which give them a gnarled appearance. Also the dendrites have a few scattered spines. In most instances the dendrites have a wavy recurring pattern. Neurons pressed against the corticospinal, corticopontine fiber bundles frequently have elongated cell bodies and the dendrites sprouting from them form, tight, brush-like arrays. The intrinsic neurons have small ellipssoid or pear-shaped cell bodies and two or three long dendrites, which do not taper. In some impregnations short axons issuing from the cell body were found and in other impregnations several widely separated, shor axon-like processes were found on dendrites. A striking feature of the intrinsic neurons is the presence of stalked dendritic appendages bearing one or more bulbous bodies, 1 to 3 microns in diameter. The intrinsic neurons in no way resemble the hairy or mossy cells with short axis cylinders that Ramón y Cajal (09) described in the pons of the 5-day-old infant. The latter were not found in the present material.

The neurons in the primate subthalamic nucleus: a Golgi and electron microscopic study.

In Golgi preparations of the adult monkey (Macaca mulatta) local interneurons and two varieties of principal neurons, radiating and elongated fusiform, are found in the subthalamic nucleus. The cell bodies of the radiating neurons have a few delicate, somatic spines some of which are occasionally bilobed and trilobed. Five to eight dendritic trunks give rise to branching, tapering dendrites, which may extend for over 400 microns. These dendrites are much thinner than the dendrites in the globus pallidus and the substantia nigra. Some neurons have many and some neurons have few dendritic spines. When numerous the dendritic spines are concentrated on the dendritic trunks and proximal dendrites. The relatively few elongated fusiform neurons are found not only in the capsule but also in the center of the nucleus. Most dendrites emerge from the opposite poles of their smooth surfaced cell bodies. They have a few dendritic spines. Some of these dendrites extend for more than 750 microsn. In 1-micron thick plastic sections lipofuscin granules are present in some but not all principal neuron cell bodies of the monkey (Macaca mulatta); but these granules are present in all principal neuron cell bodies of the pig-tail monkey (Macaca nemestrina) and of the squirrel monkey (Saimiri sciureus). The local interneurons have small cell bodies and a few relatively long undulating dendrites. The dendrites have bulbous dendritic appendages of varying complexity and beaded axon-like processes. The dendritic appendages and axon-like processes are more numerous distally and on the distal ends of the dendrites they form complex entanglements. Axons coming from the cell body have not been observed. The cell bodies of the local interneurons are identified in cresyl violet stained sections of the monkey (Macaca mulatta), in 1-micron thick plastic sections and electron micrographs of the squirrel monkey (Saimiri sciureus). They have relatively large nuclei surrounded by a thin rim of cytoplasm rich in polyribosomes.

Computer measurements of axis cylinder diameters of radial fibers and "comb" bundle fibers.

In electron micrographs of the squirrel monkey (Saimiri sciureus) brain in the striatal efferents were observed at two different levels in their course: (1) in cross-sectioned radial fiber bundles just before they enter the globus pallidus; (2) in cross-sectioned "comb" bundle fibers just before they enter the substantia nigra. In the radial bundles nearly all of the fibers are myelinated; in the "comb" bundle most are unmyelinated. The polarity of all the "comb" bundle fibers is descending. None of them degenerate following a large lesion in the substantia nigra but they do degenerate following a large lesion in the striatum. Also following this latter lesion the endings with large synaptic vesicles, which make up most of the endings in the globus pallidus and the substantia nigra, degenerate. For computer measurements, electron micrographs of the radial bundle were enlarged photographically to a final magnification of 20,000; those of the "comb" bundle to times 50,000. Measurements of 1309 radial fibers revealed a mean axis-cylinder diameter of 0.68 microns, and measurements of 749 unmyelinated "comb" bundle fibers gave a mean axis-cylinder diameter of 0.21 microns. Myelinated fibers were not included in the "comb" bundle measurements because it contains myelinated fibers of pallidal origin in addition to myelinated fibers of striatal origin. The results here indicate that the striatal efferents undergo a decided decrease in axis-cylinder diameter during their transit through the globus pallidus. It is suggested that the large non-spine bearing neurons in the striatum are the source of the striatal efferents and that they send their axons into the substantia nigra and enroute spend a great quantity of their axoplasm by extending extensive collaterals in both segments of the globus pallidus. This could account for the decreased caliber of the striatal efferents in the "comb" bundle and other findings in the striatum, globus pallidus and substantia nigra.

The neurons in the centromedian-parafascicular complex of the monkey (Macaca mulatta): a Golgi study.

The neurons of the nucleus centrum medianum and the neurons of the nucleus parafascicularis were studied in Golgi preparations of the adult monkey (Macaca mulatta). The cell bodies of the prinicipal neurons in the nucleus centrum medianum have a few somatic spines and vary in shape: some are cubical with protruding angles; some are egg-shaped; some are elongated and sausage-shaped. Four to six slightly branched dendrites of unequal thickness radiate from the cell body. Some dendrites extend for nearly 500 microns; all have dendritic spines. In the nucleus parafascicularis there are two varieties of principal neurons: (1) neurons with somatic spines and (2) neurons without somatic spines. The neurons with somatic spines are most numerous. They have polygonal-shaped cell bodies, prominent somatic spines and processes, larger than spines but considerably smaller than dendrites. These processes bear spines and are designated here "microdendrites." Spines and occasionally a "microdendrite" are found on the axon-hillocks. Five to six dendrites of unequal thickness emerge from the cell bodies. Some extend for more than 500 microns; all have prominent dendritic spines. The neurons without somatic spines are relatively few. Usually three exceptionally long, slightly branched dendrites, one apical and two basal, emerge from their elongated, slim cell bodies. Some dendrites extend for more than 800 microns; all have few scattered spines. The Golgi type II neurons found in both of these intralaminar nuclei have small cell bodies and a few, relatively long, undulating dendrites, which bear bulbous dendritic appendages and beaded axon-like processes. Distally on these dendrites, where the appendages and processes are more numerous, the dendritic appendages and axon-like processes form complex entanglements. Distally on these dendrites, where the appendages and processes are more numerous, the dendritic appendages and axon-like processes form complex entanglements. Beaded axons are found on some but not all of the cell bodies. Morphologically these neurons resemble the local interneurons that have been described in various thalamic nuclei.

Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study.

The mu, delta, and kappa opioid receptors are the three main types of opioid receptors found in the central nervous system (CNS) and periphery. These receptors and the peptides with which they interact are important in a number of physiological functions, including analgesia, respiration, and hormonal regulation. This study examines the expression of mu, delta, and kappa receptor mRNAs in the rat brain and spinal cord using in situ hybridization techniques. Tissue sections were hybridized with 35S-labeled cRNA probes to the rat mu (744-1,064 b), delta (304-1,287 b), and kappa (1,351-2,124 b) receptors. Each mRNA demonstrates a distinct anatomical distribution that corresponds well to known receptor binding distributions. Cells expressing mu receptor mRNA are localized in such regions as the olfactory bulb, caudate-putamen, nucleus accumbens, lateral and medial septum, diagonal band of Broca, bed nucleus of the stria terminalis, most thalamic nuclei, hippocampus, amygdala, medial preoptic area, superior and inferior colliculi, central gray, dorsal and median raphe, raphe magnus, locus coeruleus, parabrachial nucleus, pontine and medullary reticular nuclei, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis and cuneatus, dorsal motor nucleus of vagus, spinal cord, and dorsal root ganglia. Cellular localization of delta receptor mRNA varied from mu or kappa, with expression in such regions as the olfactory bulb, allo- and neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, ventromedial hypothalamus, hippocampus, amygdala, red nucleus, pontine nuclei, reticulotegmental nucleus, motor and spinal trigeminal, linear nucleus of the medulla, lateral reticular nucleus, spinal cord, and dorsal root ganglia. Cells expressing kappa receptor mRNA demonstrate a third pattern of expression, with cells localized in regions such as the claustrum, endopiriform nucleus, nucleus accumbens, olfactory tubercle, medial preoptic area, bed nucleus of the stria terminalis, amygdala, most hypothalamic nuclei, median eminence, infundibulum, substantia nigra, ventral tegmental area, raphe nuclei, paratrigeminal and spinal trigeminal, nucleus of the solitary tract, spinal cord, and dorsal root ganglia. These findings are discussed in relation to the physiological functions associated with the opioid receptors.

Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA.

The existence of opioid receptors within glial cell membranes has been proposed by several laboratories based on biochemical and radioligand binding data. The recent cloning of the mu, delta and kappa receptors has enabled us to directly examine the issue of opioid receptor expression in rat brain astroglia by using solution hybridization/ribonuclease protection assays to analyze the total RNA obtained from primary cultures of cortical, striatal, cerebellar, hippocampal and hypothalamic astrocytes. The results indicate that all five glial cultures expressed mu, delta and kappa receptor mRNA. The rank order of receptor mRNA abundance, expressed collectively across all five cultures, was determined to be delta > or = kappa >> mu. An analysis of the glial distribution profile for each receptor type revealed that mu receptor mRNA levels were the most abundantly expressed in cortical cultures, while the greatest levels of delta receptor mRNA were found in the cortical and hypothalamic cultures, and significant kappa receptor mRNA levels were produced by the cortical, hypothalamic and cerebellar cultures. Furthermore, the five glial cultures each expressed different levels of total opioid receptor (mu + delta + kappa) mRNA. The rank order of total opioid receptor mRNA expression across different astroglial cultures was found to be cortex > hypothalamus > cerebellum = hippocampus > striatum. An analysis of the relative expression profiles for mu, delta and kappa receptor mRNA within each culture revealed that all cultures manifested relatively high levels of delta and kappa receptor mRNA, but relatively low levels of mu receptor mRNA. Generally, cortical, hippocampal and hypothalamic cultures were characterized by comparable levels of delta and kappa receptor mRNA, and little, if any, mu receptor mRNA. However, striatal cultures were characterized by a high level of delta receptor mRNA which was approximately twice and four times that of the kappa and mu receptor mRNA, respectively. In contrast, cerebellar cultures expressed predominantly kappa receptor mRNA at a level which was almost twice that of the delta receptor mRNA, and expressed very little mu receptor mRNA. These data show that primary astroglial cultures not only express mu, delta and kappa receptor mRNAs, but they do so in a manner dependent upon receptor type and brain region. This suggests a regional heterogeneity of astrocytes with respect to opioid receptor expression, a characteristic previously described only for neurons. Furthermore, it suggests the existence of an additional anatomical component in CNS opioid systems.

The distribution of dopamine D2 receptor heteronuclear RNA (hnRNA) in the rat brain.

Conventional in situ hybridization methods have been useful in characterizing the anatomical distribution of cells in the central nervous system that express dopamine D2 receptor mRNA. However, due to the large size of the D2 mRNA pool, this method may be insensitive to changes in D2 gene transcription. We have developed a method of hybridizing a 35S-labelled cRNA probe to an intron in the D2 receptor gene in order to measure the amount of primary transcript or heteronuclear RNA (hnRNA) in D2-expressing cells. Introns are found uniquely in hnRNA and are thought to be short-lived intermediates. Thus, monitoring introns could represent a more direct measure of D2 gene transcription. The anatomical distribution of the D2 hnRNA is similar to the distribution of D2 mRNA in the rat brain. D2 heteronuclear RNA was found in the nuclei of cells in the caudate putamen, nucleus accumbens, hippocampus, olfactory tubercle, substantia nigra, ventral tegmental area, and zona incerta. Other regions that contain D2 mRNA, but do not demonstrate intronic signal, include the globus pallidus, prefrontal, cingulate, entorhinal, and piriform cortex, septum, and amygdala. However, these areas have low amounts of D2 mRNA and may contain levels of D2 hnRNA that are below detection. Heteronuclear RNA quantitation by solution hybridization followed by RNase protection was performed on striatum, substantia nigra, cerebral cortex, hippocampus, hypothalamus, and pituitary using a D2 intron 7/exon 8 border probe. These results corroborate the distribution of hnRNA revealed with intronic in situ hybridization. In addition, protection assays were able to detect hnRNA in areas that express low levels of D2 like the cortex, hippocampus and hypothalamus. hnRNA/mRNA ratios calculated from intron/exon border probe protection assays were not equivalent for all the tissue areas studied, indicating that transcription and/or hnRNA half lives may differ between tissues that express D2 receptors. The combined use of intronic in situ hybridization and intron/exon border protection assay as an index of D2 gene transcription and RNA processing provides more information than measuring the mRNA pool alone. It may also prove to be a more useful measure of gene regulation, allowing for evaluation of gene responses to acute treatments.

Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS.

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined mu, delta and kappa 1 receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned mu receptor (335-398) to examine the distribution of the mu receptor-like protein with immunohistochemical techniques. mu receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known mu receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.

Localization of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain.

The Brazilian opossum (Monodelphis domestica) is a small, pouchless marsupial whose young are born in an immature, sexually undifferentiated state. Etgen and Fadem, and Handa and coworkers have biochemically detected and characterized estrogen receptors in the forebrain of the Brazilian opossum. In this study, we have examined the distribution of estrogen receptor-like immunoreactive (ER-LI) cells in the brains of gonadectomized male and female Brazilian opossums using Abbott H222 rat monoclonal estrogen receptor antibody (H222 is a gift of Abbott Labs). An indirect immunohistochemical procedure employing the Vectastain Elite system and a nickel-enhanced DAB chromogen was used. A large number of ER-LI cell nuclei were observed in the medial preoptic area, ventral septal nucleus, medial division of the bed nucleus of the stria terminalis, lateral part of the ventromedial hypothalamus, premammillary nucleus, arcuate nucleus, posterior amygdaloid nucleus, and the midbrain central grey. Lower numbers of ER-LI cell nuclei were observed in the intermediate subdivision of the lateral septal nucleus, and in the anterior, medial, and posterior cortical amygdaloid nuclei. The anatomical distribution of ER-LI in the Brazilian opossum brain is similar to that which has been reported for estrogen binding sites following biochemical analysis. Based on these findings, we believe specific regions of the Brazilian opossum brain may serve as substrata for the action of estrogen in the adult. In addition, these results are supportive of the use of this animal model to investigate the organizational effects of estrogen on the developing central nervous system.

Characterization and distribution of estrogen receptors in the diencephalon of the gray short-tailed opossum.

The Brazilian gray short-tailed opossum is a pouchless marsupial whose young are born sexually undifferentiated making this animal ideal for developmental studies. Previously, Etgen and Fadem (Dev. Brain Res., 49 (1989) 131-133; Gen. Comp. Endocrinol., 66 (1987) 441-446) detected estrogen receptor (ER) in the hypothalamus-preoptic area, and compared males and females in the adult and during development. In this study we characterized the ER and determined its distribution in specific diencephalic regions in the brains of adult male and female opossums. ER were measured by the in vitro binding of [3H]estradiol to cytosol of microdissected brain nuclear regions. Radioinert moxestrol (R2858) was used to define non-specific binding. Saturation analysis showed a single high-affinity binding site. Binding was displaced by estradiol (E2), diethylstilbestrol (DES) and R2858, but not by non-estrogenic steroids. Ligand bound receptor adhered to DNA-cellulose and was eluted as a single peak with 0.2-0.3 M NaCl. High levels of ER were found in the medial preoptic-periventricular area. Intermediate levels were seen in the ventromedial hypothalamic nucleus, medial amygdala and arcuate nucleus. No sex differences were observed. The presence of a neural ER and its similarity of distribution to that of the laboratory rat support the use of this animal model in studies examining steroid dependent organization of the hypothalamus.