Mesostriatal projections, which arise from dopaminergic and non- dopaminergic neurons in the ventral tegmental area, substantia nigra, and retrorubral area, are compartmentally organized in the striatum. Anterograde axonal tract tracing with Phaseolus vulgaris- leucoagglutinin (PHA-L), combined with immunohistochemical localization of tyrosine hydroxylase (TH) and autoradiographic localization of mu- opiate receptor binding sites, shows that midbrain projections to the striatum are distributed to either the mu-opiate receptor-rich “patch” or the receptor-poor “matrix” striatal compartments. Three morphologically distinct mesostriatal afferent fiber types are labeled. The first type, type A, forms a plexus of relatively thin (0.1–0.4 micron), smooth fibers with small varicosities (0.3–0.6 micron). A second type, type B, is similar to the first in forming a plexus of fibers, but is slightly thicker (0.2–0.6 micron), with more frequent varicosities (0.4–1.0 micron) that give this fiber type a crinkled appearance. The third type, type C, constitutes a minority of striatal afferents and is characterized by its large caliber (0.4–0.7 micron) with large bulbous varicosities (1.2–2.0 micron). Projections of the ventral tegmental area (A10 cell group) are primarily dopaminergic type A fibers directed to the matrix of the ventromedial striatum, including the nucleus accumbens. The retrorubral area (A8 cell group) also provides predominantly dopaminergic type A fibers to the striatal matrix, but these are distributed dorsally. The substantia nigra contains a mixed population of neurons that project to the striatum. Some, located in the dorsal tier of the pars compacta (dorsal A9 cell group), provide dopaminergic type A fibers to the striatal matrix. Others, in the ventral tier of the pars compacta (ventral A9 cell group) and in the ventral tier of the pars reticulata (displaced A9 cells), provide dopaminergic type B fibers to the striatal patches. An additional set of substantia nigra neurons that are non-dopaminergic is the source of type C fibers to the striatal matrix. Thus, distinct dorsal and ventral sets of midbrain dopaminergic neurons project, respectively, to striatal matrix and patches, and there is a non- dopaminergic mesostriatal projection to the matrix.

In the previous paper (Gerfen et al., 1987) mesostriatal dopaminergic neurons were shown to be subdivided into dorsal and ventral tiers that project to the striatal matrix and patch compartments, respectively. The present study provides experimental evidence that these patch- matrix mesostriatal dopaminergic systems are biochemically and developmentally distinct. A 28 kDa calcium-binding protein (CaBP, or calbindin-D28 kDa) is expressed in dorsal tier mesostriatal dopaminergic neurons. The distribution of such neurons, located in the ventral tegmental area, dorsal tier of the substantia nigra pars compacta, and retrorubral area, matches that of dopaminergic neurons that project to the striatal matrix. Dopaminergic neurons that do not express CaBP--those in the ventral tier of the pars compacta and in the pars reticulata--are distributed in a pattern that matches the origin of the dopaminergic projection to the striatal patches. During development, dopaminergic afferents to the striatal patch compartment are in place prior to the development of those to the matrix. Injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the striatum of newborn rats result in a selective and long-lasting depletion of dopaminergic afferents in the striatal patches. The later- developing matrix projection is relatively spared by such lesions. The distribution of surviving dopaminergic neurons, labeled with tyrosine hydroxylase (TH) immunoreactivity, matches the pattern of dorsal tier neurons previously shown to provide inputs to the matrix. Surviving neurons also express CaBP immunoreactivity and have dendrites that spread mediolaterally, in the plane of the pars compacta. On the other hand, those neurons that project to the patches are selectively lesioned by the neonatal 6-OHDA striatal injections, do not express CaBP, and have dendrites that are directed ventrally into the pars reticulata.

The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsi- RNA (16) and immunoprecipitation of synthesized UCP were carried out as described (9,10).cDNA Synthesis and Cloning. Poly(A+) RNA from thermogenic brown fat was enriched for UCP mRNA by sucrose gradient centrifugation. The mRNA fraction sedimenting around 15S was used to synthesize double stranded (ds) cDNA according to standard procedures (17). Both first and second strands were prepared by using avian myeloblastosis virus reverse transcriptase. After hairpin loop cleavage with S1 nuclease and deproteinization with phenol and chloroform extractions, ds cDNA was size-fractionated by sedimentation on a sucrose gradient. Ten nanograms of a ds cDNA fraction (500 base pairs long) was tailed with calf thymus terminal deoxynucleotidyl transferase and dCTP (18) and was hybridized with 100 ng of dG-tailed, 20). Chimeric vectors were used to transform CaCl2-treated E. coli C600, and transformant clones were selected on L medium agar plates containing 10 mg of tetracycline per liter. About 2000 recombinant clones were obtained and further characterized. A second library of recombinant clones bearing longer cDNA inserts were prepared from a Abbreviations: UCP, uncoupling protein; ds, double stranded; kb, kilobase.

The present study describes the distribution and morphological characteristics of neurons and nerve fibers containing the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase, in the sheep brainstem and diencephalon on the basis of immunohistochemical procedures. Neurons and fibers were considered to be dopaminergic if they showed anti-tyrosine hydroxylase immunoreactivity, without corresponding anti-dopamine-beta-hydroxylase immunoreactivity. The structures labeled with both antisera were considered noradrenergic or adrenergic. The distribution of catecholaminergic neurons corresponds to that described by other authors with similar methods in the rat and in primates. The noradrenergic neurons belong to cell groups A1 to A7 and the dopaminergic neurons to cell groups A8 to A15. In almost all studied areas, the catecholaminergic innervation is similar to that observed in the other species. However, the central catecholaminergic systems of the sheep showed some specific characteristics: (1) groups A3 and A4, described in the rat, were not found, (2) group A14 contains fewer neurons than in the rat, (3) group A15 does not contain a dorsal but only a ventral portion, (4) there is a larger dispersion of neurons within each group, especially A6 and A7, than in rodents, and (5) there is a larger noradrenergic innervation of the catecholaminergic groups than in the other species.

SynopsisThe free fractions of monovalent and divalent counterions were determined on saltfree solutions of pectins. The effects of charge density, distribution of the carboxyl groups, polymer concentration, and the nature of the counterion were investigated by determinating the calcium and sodium activity coefficients (with specific electrodes) and by measuring the transport parameters (by conductimetry). Poor agreement for calcium ions was found with the Manning theory. The strong binding of these ions to highly charged polymers, which is ascribed to a dimerization process was demonstrated in very dilute solutions. INTRODUCTIONPectins are ionic polysaccharides extracted from plant cell walls used as gelling po1ymers.l They are composed mainly of a linear 1,4-linked a-D-galacturonic acid unit backbone, with some interruptions by 1,2-linked L-rhamnose residues and some neutral sugars, typically galactose and arabinose, as side chains2 In nature, they generally occur as the partial methyl ester with a degree of esterification, the fraction of galacturonate residues that are methyl-esterified being of about 70%.l These "high-methoxyl" pectins form gels in an acidic medium on addition of sucrose. Pectins of lower ester content are prepared by controlled deesterification, and the resulting pectins can gel in the presence of calcium ions. ' The nature of conformational ordering and calcium binding was investigated by Rees and coworker^,^-^ mainly by equilibrium dialysis and CD. They proposed a mechanism of calcium binding similar to the "egg-box" model given for alginates7 and postulated a two-stage process: initial dimerization of the molecules and subsequent aggregation of these preformed dimers. This intermolecular binding of the calcium counterion was confirmed for polygalacturonic acid by Kohn and LuknarB and by Ravanat and Rinaudog using methods such as measurement of calcium activity coefficient. THIBAULT AND RINAUDOThe purpose of this paper is to study the binding of cations, especially calcium ions, to pectin molecules with different levels and patterns of esterification. Well-characterized samples of pectin were prepared, and the degree of binding of the counterions was determined from conductimetric and potentiometric data in salt-free solutions. This study should contribute to an understanding of the polyelectrolyte behavior of pectins and may help to complete our knowledge of gel formation of these polysaccharides. EXPERIMENTAL MaterialsThe starting pectin was a commercial apple pectin ("RS" pectin, Unipectine, Redon, France). It was purified by precipitation with cupric ions.IO A pectin sample from apple juice was also used.ll Purity was tested as described elsewhere.12 DGalacturonic acid was a Baker sample. All the hydroxide solutions were carbonate-free. Methods Chemical Characterization of the SamplesPectin samples were characterized by their galacturonic acid content and by their degree of esterification, as determined by c o n d~c t i m e t r y .~~ This method involves three or four steps on 10 mL...

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To investigate the existence of a dopaminergic preoptico-hypophyseal pathway in the goldfish, electrolytic lesions were placed in the rostral preoptic area and their effects on gonadotrophin levels and pituitary innervation examined. In a first experiment, the fish were sacrificed 2 days after surgery and the pituitary studied by electron microscopy. Numerous exocytosis profiles were observed in the gonadotrophs, confirming the large increase in serum gonadotrophin levels measured in the animals. In addition, type A and B degenerating fibers were detected in the neurohypophysis and the pars distalis, in particular at the level of the gonadotrophs. In the second experiment, the distribution of tyrosine hydroxylase-immunopositive fibers was studied in the pituitary of controls and lesioned animals. It was found that lesioning the anterior ventral preoptic region resulted in the disappearance of all positive fibers in the pars distalis, while those in the neurointermediate lobe appeared unaffected. The presence of a large group of catecholaminergic perikarya in the destroyed area was confirmed in control animals. These results and other data strongly support the existence of a dopaminergic preoptico-hypophyseal pathway, providing a morphological support for the inhibitory effect of dopamine on the release of anterior pituitary hormones in teleosts, in particular gonadotrophin.

The preoptic area contains most of the luteinizing hormone releasing hormone immunoreactive neurons and numerous monoaminergic afferents whose cell origins are unknown in sheep. Using tract tracing methods with a specific retrograde fluorescent tracer, fluorogold, we examined the cells of origin of afferents to the medial preoptic area in sheep. Among the retrogradely labeled neurons, immunohistochemistry for tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and serotonin was used to characterize catecholamine and serotonin fluorogold labeled neurons. Most of the afferents came from the ipsilateral side to the injection site. It was observed that the medial preoptic area received major inputs from the diagonal band of Broca, the lateral septum, the thalamic paraventricular nucleus, the lateral hypothalamus, the area dorsolateral to the third ventricle, the perimamillary area, the amygdala, and the ventral part of the hippocampus. Other numerous, scattered, retrogradely labeled neurons were observed in the ventral part of the preoptic area, the vascular organ of the lamina terminalis, the ventromedial part of the hypothalamus, the periventricular area, the area lateral to the interpeduncular nucleus, and the dorsal vagal complex. Noradrenergic afferents came from the complex of the locus coeruleus (A6/A7 groups) and from the ventro-lateral medulla (group A1). However, dopaminergic and adrenergic neuronal groups retrogradely labeled with fluorogold were not observed. Serotoninergic fluorogold labeled neurons belonged to the medial raphe nucleus (B8, B5) and to the serotoninergic group situated lateral to the interpeduncular nucleus (S4). In the light of these anatomical data we hypothesize that these afferents have a role in the regulation of several functions of the preoptic area, particularly those related to reproduction. Accordingly these afferents could be involved in the control of luteinizing hormone releasing hormone (LHRH) pulsatility or of preovulatory LHRH surge.