Postnatal maturational properties of rat parafascicular thalamic neurons recorded in vitro.

Thalamic relay neurons have homogeneous, adult-like firing properties and similar morphology by 12 days postnatally (PN 12). Parafascicular (Pf) neurons have a different morphology compared with typical thalamic relay neurons, but the development of their electrophysiological properties is not well studied. Intracellular recordings in PN 12-50 Pf neurons revealed several heterogeneous firing patterns different from those in thalamic relay neurons. Two types of cells were identified: Type I cells displayed a fast afterhyperpolarization (AHP) followed by a large-amplitude, slow AHP; whereas Type II cells had only a fast AHP. These cell types had overlapping membrane properties but differences in excitability. Some properties of Pf neurons were adult-like by PN 12, but, unlike thalamic relay neurons, there were significant maturational changes thereafter, including decreased action potential (AP) duration, increased fast AHP amplitude and increased excitability. Pf neurons did not exhibit rhythmic bursting and generally lacked low-threshold spike (LTS) responses that characterize thalamic relay neurons. Pf neurons exhibited nonlinear I-V relationships, and only a third of the cells expressed the time and voltage-dependent hyperpolarization activated (Ih) current, which declined with age. These results indicate that the morphological differences between Pf neurons and typical thalamic relay neurons are paralleled by electrophysiological differences, and that Pf membrane properties change during postnatal development.

Acute exposure to 25-hydroxy-cholesterol selectively reduces GABAb and not GABAa receptor-mediated synaptic inhibition.

Intracellular recording techniques were used to study the effects of the cholesterol oxide, 25-hydroxycholesterol (25-OH-Chol), on gamma-aminobutyric acid (GABA) receptor-mediated inhibitory postsynaptic potentials (IPSPs) in brain slices of the rat lateral septum. Superfusion of 25-OH-Chol increased the peak amplitude of the GABAa IPSP in more than half of the neurons tested, many of which exhibited a similar increase in the GABAb IPSP. However, some neurons exhibited a gradual decrease in input resistance and a selective reduction or blockade of the GABAb IPSP during prolonged exposure. Cholesterol partly mimicked the effects of 25-OH-Chol. These findings indicate that 25-OH-Chol can selectively reduce or block metabotropic GABAb while sparing ionotropic GABAa receptor-mediated synaptic inhibition. Our results indicate that brain slices can be used to study the effects of short term alterations in cholesterol on the excitability and synaptic integration properties of neurons.

Lack of feminization of the cremaster nucleus by prenatal flutamide administration in the rat and pig.

PURPOSE: The sexually dimorphic cremaster nucleus contains motoneurons that project via the genitofemoral nerve and theoretically direct androgen dependent testicular descent. The effects of flutamide on descent and masculinization of the cremaster nucleus were studied in the rat and pig. MATERIALS AND METHODS: Flutamide was given to pregnant rats and pigs on days 16 to 22 and 65 to 113 of gestation, respectively. Tissues were perfused and examined at birth (pigs) or at age 30 days (rats). Spinal cords were removed, sectioned and immunohistochemically stained for serotonin (rats) or substance P (pigs) to demarcate the position of the cremaster nucleus and allow the determination of cremaster motoneuron number. RESULTS: After exposure to flutamide testes were undescended in 6 of 9 rats and 7 of 10 pigs. Cremaster motoneuron number per nucleus were 288 +/- 22 in control versus 250 +/- 27 in flutamide treated rats, and 165 +/- 28 in control versus 148 +/- 24 in flutamide treated pigs. The decrease in motoneuron number by flutamide was significant in both species (p < 0.02) but it did not approach the levels in female rats (93 +/- 11) and pigs (57 +/- 12). Cremaster motoneuron number did not correlate with testicular position. Porcine undescended testes were associated with a significant increase in mean gubernacular volume. CONCLUSIONS: Unlike other sexually dimorphic spinal cord nuclei masculinization of the cremaster nucleus appears to be largely androgen independent and it does not correlate with ipsilateral testicular descent. These data suggest that androgens do not mediate descent of the testes via the efferent limb of the genitofemoral nerve.

Lack of feminization of the cremaster nucleus in cryptorchid androgen insensitive rats.

Androgens may control rat testicular descent via effects on the genitofemoral nerve or cranial gonadal ligaments. Androgen-mediated release of calcitonin gene-related peptide from the genito-femoral nerve (whose motoneuron cell bodies reside in the sexually dimorphic cremaster nucleus) may stimulate cremaster sac formation and testicular descent. Alternatively, androgens may cause regression of cranial gonadal ligaments and thereby allow the testes to descend. To evaluate these theories testicular position, and the cremaster sac and nucleus were studied in Tfm (androgen insensitive) rats. Testes were abdominal, inguinal and scrotal in 20%, 67% and 13% of Tfm male rats, respectively, and cranial ligaments were present in all cases. Mean cremaster nucleus motoneuron number was lower in female rats (70 +/- 14) but not significantly different between normal male (256 +/- 44) and Tfm male (231 +/- 42) rats, and it correlated poorly with testicular position. Calcitonin gene-related peptide immunoreactivity was rarely observed in cremaster motoneurons. These data suggest that the cremaster nucleus is not androgen-dependent, calcitonin gene-related peptide release from cremaster motoneurons is not the likely mechanism of testicular descent and persistent cranial ligaments may cause cryptorchidism in the Tfm rat.

Biogenesis of presynaptic terminal proteins.

The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two of the major components of axonal transport, and the subsequent persistence and turnover of those proteins were examined in this study. Ganglion cell proteins were radiolabeled by intravitreal injection of radiolabeled amino acids and radioactive axonally transported proteins were analyzed in synaptosomes prepared from the superior colliculi. This procedure allowed examination of presynaptic components of ganglion cell synapses without having to compensate for postsynaptic or other unidentified contaminants. Each of the two major axonal transport components supplies a large number of proteins to the presynaptic terminal, in relative quantities similar although not identical to those seen in the axon. Proteins conveyed by the fast component of axonal transport reached the terminals by 3 h after intraocular injection, peaked by 24 h, and were largely undetectable by 15 days. Slow component b proteins reached the terminals by 12 days, peaked around 21 days, and persisted up to 63 days in the terminals. Proteins in both components demonstrated differential turnover relative to cotransported proteins once they reached the terminals. Differential turnover may account for change in relative concentration of a particular protein required to meet new functional demands on that protein once it enters the terminal.

Properties of the bovine striatal synaptic vesicles ATPase.

Synaptic vesicles prepared from bovine corpus striatum exhibit an ATPase activity that is insensitive to ouabain and specific inhibitors of mitochondrial ATPase, but that is stimulated by proton ionophores and inhibited by sulfhydryl reagents. Low concentrations of orthovanadate, DCCD and tributyl tin are also ineffective as inhibitors of the vesicle-associated activity. The properties of the synaptic vesicle enzyme suggest that this ATPase may be similar to that of clathrin-coated vesicles, and to one of the activities described in preparations of adrenal chromaffin granule membranes.

Reserpine labels the catecholamine transporter in synaptic vesicles from bovine caudate nucleus.

Tritiated reserpine binds to synaptic vesicles from bovine caudate with high affinity (Kappd = 1.25 nM, Bmax = 3.3 pmol/mg protein). This interaction is both ATP-dependent and sensitive to the protonophores CCCP and nigericin, suggesting that a proton electrochemical gradient is required for binding. Dopamine, epinephrine, norepinephrine and serotonin all inhibit reserpine binding at concentrations similar to those required for inhibition of dopamine uptake. Treatment with saponin to release vesicle contents results in complete loss of accumulated dopamine but retention of bound reserpine. These results indicate that reserpine binds to the catecholamine transport system of synaptic vesicles with high affinity and specificity.

Structural organization of filamentous proteins in postsynaptic density.

Actin is one of the major protein constituents of the postsynaptic density (PSD), a characteristic structural entity subjacent to the postsynaptic membrane in excitatory synapses of the vertebrate central nervous system. In isolated purified PSD preparations, it is present to the extent of 29 +/- 2 micrograms/mg of total protein, 90% of which is in the filamentous (F-actin) form. Iodination by a discriminatory labeling technique demonstrates that actin is located on the surface of the PSD from which it can be stripped by treatment with a mixture of strong anionic detergents, leaving behind an insoluble core held together by disulfide bridges, consisting in part of tubulin and "PSD protein".

Calcium-stimulated protein phosphorylation in synaptic membranes.

Synaptic membranes from rat brain contain several calcium-requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (CaH-PK) effective at high concentrations of Ca2+ ion in the absence of Mg2+ (active on class F substrates); (b) a (Ca + Mg)-PK activity that is mediated by Ca2+ ion in the presence of Mg2+ (active on class B substrates); (c) (Ca-CaM)-PK activities that exhibit simultaneous requirements for both Ca2+ ion and CaM (for class C and D substrates). Also described are three activities (d-f) that do not require Ca2+ ion: (d) a Mg-PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ ion alone (Mg-PK activity) and is unaffected by the addition of Ca2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP-dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: among them are P43 (class A), as the alpha subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75-P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca2+ and CaM. The half-maximal stimulation (K0.5) for Ca2+ in the presence of CaM was found to be 1.0 microM Ca2+F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by greater than or equal to 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ ion was found to equal 0.4 microgram per reaction mixture (8 micrograms/ml) or 1.35 micrograms per reaction mixture (27 micrograms/ml), for the untreated and EGTA-treated membranes, respectively.

Functional domains in introns. RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b.

The penultimate intron of the split mitochondrial gene (cob) for apocytochrome b of Saccharomyces cerevisiae is of particular interest; it contains a long unassigned reading frame, is present in both long form (six exons) and short form (three exons) of the gene, and a product expressed from it is required for the removal of its transcript and that of an intron in the transcript of the oxi3 gene. Complementation analysis shows mutants in this intron to be either cis-dominant or transrecessive. Cis-dominant mutants are located in the first third (approximately 350 base pairs) of the open and near the 3'-end of the closed reading frame, while trans-recessive mutants are scattered throughout the remaining two-thirds (approximately 750 base pairs) of the open frame. Mutants in both classes exhibit the same pattern of splicing defects in their transcripts, but for different reasons. Those in the trans-recessive class lack a functional maturase (probably a protein of Mr = 27,000) encoded wholly within the 3'-terminal segment of the intron, and for this reason also fail to express oxi3. In contrast, cis-dominant mutants are incapable of providing the splicing complex with a substrate of appropriate 2 degrees structure. They also accumulate a novel transcript, 1900 nucleotides long, which contains the intron fused to the downstream (3') exons. This may reflect an inability of the splicing complex to complete the normal sequence of cleavage of the intron at its downstream junction and the ligation of the two exonic moieties.

Genetics and biogenesis of cytochrome b.

We have reviewed here the genetic methods used for isolating and manipulating nuclear and mitochondrial mutants of bakers' yeast that affect the function and biogenesis of complex III of the mitochondrial respiratory chain. All the methods have been used with success in the past, and it is hoped that this compilation will aid biochemists in using these techniques to study electron transfer.

Localization of endogenous ATPases at the nerve terminal.

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg2+-ATPase; (b) an ATPase active at high concentrations of Ca2+ in the absence of Mg2+ (CaH-ATPase); (c) a Ca2+ requiring Mg2+-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca2+-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and CaH-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.

Properties and possible functions of the adenylate cyclase in plasma membranes of Saccharomyces cerevisiae.

We have examined the possible role of adenosine 3',5'-phosphate (cAMP) in functions associated with the plasma membranes of Saccharomyces cerevisiae. Purified membranes from this source contained an adenylate cyclase which was insensitive to activation by fluoride or guanine nucleotides, only weakly responsive to changes of carbon source in the growth medium, and strongly stimulated by vanadate. They also contained at least two classes of receptor proteins for guanine nucleotides (as measured by binding of labeled 5'-guanylyl methylene diphosphate) with apparent dissociation constants equal to 1.0 x 10(-7) and 3 x 10(-6) M, a protein kinase capable of phosphorylating added histones, the activity of which was stimulated by cAMP, and cAMP receptors that may function as regulatory subunits for this kinase. Membrane proteins were also susceptible to phosphorylation by endogenous kinase(s), with polypeptides of apparent molecular weights equal to 160 x 10(3), 135 x 10(3), 114 x 10(3), and 58 x 10(3) as the major targets. Of these, the 114,000-molecular-weight polypeptide was probably identical to the proton-translocating ATPase of the membranes. However, the cAMP-dependent protein kinase did not appear to be involved in these reactions. Intact (rho+ or rho0) cells responded to dissipation of the proton electrochemical gradient across their plasma membranes by rapid and transient changes in their intracellular level of cAMP, as suggested earlier (J. M. Trevillyan and M. L. Pall, J. Bacteriol., 138:397-403, 1979). Thus, although yeast plasma membranes contain all the essential components of a stimulus-responsive adenylate cyclase system, the precise nature of the coupling device and the targets involved remain to be established.

Functional domains in introns: trans-acting and cis-acting regions of intron 4 of the cob gene.

We have sequenced the mutational changes in eight mutants in the open reading frame of intron 4 of the cob gene on yeast mitochondrial DNA. Three have a cis-acting splicing defect, while the other inactivate a trans-recessive intron domain that specifies a trans-acting splicing factor. From phenotypic evidence, including analyses of the allele-specific extra proteins, we have identified a protein (P27) encoded wholly within the intron that appears to be the intron 4 splicing factor (maturase). The evidence suggests that P27 is a secondary translation product resulting from the proteolytic cleavage of a larger precursor encoded by exon and intron sequences, but an alternative model, in which P27 is a primary translation product, has not been ruled out.

Guanine nucleotides do not inhibit tritiated dopamine agonist binding to bovine striatal membranes.

The addition of GTP (50 muM), MnCl2 (1 mM) or EDTA (2 mM) had no effect on the affinity or capacity of bovine striatal plasma membranes for [3H]spiperone. However, GTP caused a decrease in the potency of dopamine as an inhibitor of [3H]spiperone binding under all conditions tested. Manganese enhanced the potency of dopamine both in the presence and absence of GTP, but NaCl (100 mM) had no effect. Neither manganese nor GTP caused any change in the affinity or capacity of bovine striatal membranes for the tritiated agonists dopamine, apomorphine or ADTN. GPPNHP, a nonhydrolyzable analog of GTP, was also ineffective. However, in identical experiments using rat striatal membranes, 50 muM GTP caused a decrease in affinity for all three tritiated agonists and this effect was observed both in the presence and absence of manganese (1 mM). In addition, binding capacities for [3H]dopamine and [3H]ADTN were doubled when manganese was present. In light of this and other reports that GTP inhibits tritiated agonist binding in rat striatum, it is suggested that the absence of such inhibition in bovine striatal membranes may reflect a fundamental difference between the two species with regard to their receptors for dopamine agonists.

Evidence for translated intervening sequences in the mitochondrial genome of Saccharomyces cerevisiae.

In yeast, the mitochondrial genes for subunit I of cytochrome oxidase (oxi3) and for apocytochrome b (cob) are known to be split. In some strains, the latter contains five intervening sequences, three of which coincide with clusters of mutational sites referred to in their order of transcription as the loci box3, 10, and 7, respectively. Mutations at the first of these result in the accumulation of novel, large polypeptides (apparent Mr congruent to 43,000) believed to originate from a fusion of sequences found in the NH2-terminal segment of apocytochrome b to others encoded in the intervening sequence itself. We now provide evidence for close similarities of at least a part of translated intron sequences between (a) mutants in box7 in "long" form and "short" form strains (which lack the first three introns including the one for the box3 locus); (b) mutants in a subset of box7 mutants and those in box3, and (c) between intron sequences in box7 and a sequence presumably encoded in oxi3. These structural homologies have been analyzed and shown to be referable to sequence homologies in two proteins, one derived from the second intron (box3) in cob and the other from oxi3. The accumulation in certain cob mutants of proteins and of a transcript containing a sequence specified by oxi3 provides additional strong evidence for the previously suggested regulation of oxi3 by the penultimate, box7-containing intron of cob.

Specific proteolysis of a brain membrane phosphoprotein (B-50): effects of calcium and calmodulin.

The membrane bound phosphoprotein B-50 (MW 48K) was isolated from rat brain tissue. The fraction containing the highest endogenous B-50 phosphorylating activity (ASP 57-82%) contains protease activity. In the absence of calcium a time-dependent decrease of the protein B-50 is observed. Under these conditions another phosphoprotein B-60 (MW 46K) appears in the incubation medium. Addition of calcium and/or calmodulin enhances the protease activity whereas the substrate specificity is lost. Results of both isoelectric focussing and peptide mapping indicate the B-50 and B-60 are related proteins. These data support our hypothesis that the recently isolated behaviorally active peptide PIP (MW approx. 1600 D) is the smaller cleavage product of the proteolytic degradation of B-50 to B-60.