Cystatin C reduces the in vitro formation of soluble Abeta1-42 oligomers and protofibrils.

There are an increasing number of genetic and neuropathological observations to suggest that cystatin C, an extracellular protein produced by all nucleated cells, might play a role in the pathophysiology of sporadic Alzheimer's disease (AD). Recent observations indicate that small and large soluble oligomers of the beta-amyloid protein (Abeta) impair synaptic plasticity and induce neurotoxicity in AD. The objective of the present study was to investigate the influence of cystatin C on the production of such oligomers in vitro. Co-incubation of cystatin C with monomeric Abeta1-42 significantly attenuated the in vitro formation of Abeta oligomers and protofibrils, as determined using electron microscopy (EM), dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, thioflavin T (ThT) spectrofluorimetry and gel chromatography. However, cystatin C did not dissolve preformed Abeta oligomers. Direct binding of cystatin C to Abeta was demonstrated with the formation of an initial 1:1 molar high-affinity complex. These observations suggest that cystatin C might be a regulating element in the transformation of monomeric Abeta to larger and perhaps more toxic molecular species in vivo.

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers.

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.

Spontaneous in vitro formation of supramolecular beta-amyloid structures, "betaamy balls", by beta-amyloid 1-40 peptide.

The concentration of beta-amyloid peptide (Abeta), x-42 or x-40 amino acids long, increases in brain with the progression Alzheimer's disease (AD). These peptides are deposited extracellularly as highly insoluble fibrils that form densities of amyloid plaques. Abeta fibrillization is a complex polymerization process preceded by the formation of oligomeric and prefibrillar Abeta intermediates. In some of our in vitro studies, in which the kinetics of intermediate steps of fibril formation were examined, we used concentrations of synthetic Abeta that exceed what is normally employed in fibrillization studies, 300-600 microM. At these concentrations, in a cell free system and under physiological conditions, Abeta 1-40 peptide (Abeta40) forms fibrils that spontaneously assemble into clearly defined spheres, "betaamy balls", with diameters of approximately 20-200 microm. These supramolecular structures show weak birefringence with Congo red staining and high stability with prolonged incubation times (at least 2 weeks) at 30 degrees C, freezing, and dilution in H(2)O. At 600 microM, they are detected after incubation for approximately 20 h. Abeta peptide 1-42 (Abeta42) lacks the ability to form betaamy balls but accelerates Abeta40 betaamy ball formation at low stoichiometric levels (1:20 Abeta42:Abeta40 ratio). Abeta42 levels above this (=10-50% w/w) impede Abeta40 betaamy ball formation. Using light (LM) and electron microscopy (EM), this study examines the gross morphology and ultrastructure of Abeta40 betaamy balls and their time course of formation, in the absence and presence of Abeta42, along with some stability measures. As spheres of a misfolded protein, betaamy balls resemble both AD Abeta senile plaques and neuronal inclusion bodies associated with other neurodegenerative diseases.

The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation.

Several pathogenic Alzheimer's disease (AD) mutations have been described, all of which cause increased amyloid beta-protein (Abeta) levels. Here we present studies of a pathogenic amyloid precursor protein (APP) mutation, located within the Abeta sequence at codon 693 (E693G), that causes AD in a Swedish family. Carriers of this 'Arctic' mutation showed decreased Abeta42 and Abeta40 levels in plasma. Additionally, low levels of Abeta42 were detected in conditioned media from cells transfected with APPE693G. Fibrillization studies demonstrated no difference in fibrillization rate, but Abeta with the Arctic mutation formed protofibrils at a much higher rate and in larger quantities than wild-type (wt) Abeta. The finding of increased protofibril formation and decreased Abeta plasma levels in the Arctic AD may reflect an alternative pathogenic mechanism for AD involving rapid Abeta protofibril formation leading to accelerated buildup of insoluble Abeta intra- and/or extracellularly.

Stereoselective determination of amino acids in beta-amyloid peptides and senile plaques.

A novel method for the determination of the enantiomeric composition of peptides is presented. In this paper, the focus has been on beta-amyloid peptides from deceased Alzheimer's disease patients. The peptides are hydrolyzed using mineral acid. The free amino acids are derivatized with the chiral reagent (+)- or (-)-1-(9-anthryl)-2-propyl chloroformate and subsequently separated using micellar electrokinetic chromatography (MEKC) and detected using laser-induced fluorescence (LIF) detection. The high separation efficiency of the MEKC-LIF system, yielding approximately 1 million theoretical plates/m for most amino acids, facilitates the simultaneous chiral determination of nine amino acids. The samples that have been analyzed were standard 1-40 beta-amyloid peptides, in vitro precipitated beta-amyloid fibrils, and human senile plaque samples.

Synthesis of novel 5-substituted 3-amino-3,4-dihydro-2H-1-benzopyran derivatives and their interactions with the 5-HT1A receptor.

A series of new enantiomerically pure 3-amino-3,4-dihydro-2H-1-benzopyrans (3-aminochromans) has been synthesized from (R)- and (S)-5-methoxy-3-amino-3,4-dihydro-2H-1-benzopyran. The absolute configuration of the respective (R)- and (S)-enantiomers was deduced from X-ray crystallography of (R)-3-(N-isopropylamino)-5-methoxy-3,4-dihydro-2H-1-benzopyran, (R)-9a. Various 5-substituents were introduced via palladium-catalyzed carbonylation of N-substituted 3-amino-5-trifluoromethanesulfonyloxy-3,4-dihydro-2H-1-benzopyran. The effect of N- and 5-substitution on affinity for the 5-HT1A receptor was evaluated in competition experiments using rat hippocampal membranes and [3H]8-OH-DPAT as radioligand. Selected compounds were also tested for their affinity to the D1 (rat striatum), D2 (rat striatum), D2A (human cloned), and 5-HT2A (rat cortex) receptors. The intrinsic activity of the compounds was evaluated by measuring their effect on VIP-stimulated cAMP production in GH4ZD10 cells stably transfected with the 5-HT1A receptor. High-affinity compounds with high selectivity for the 5-HT1A receptor were found among structures substituted with carboxylate esters, amides, and ketones in the 5-position. Primary and secondary amines bound with lower affinity than tertiary amines. Larger substituents were well-tolerated by the receptor, but the smaller N-ethyl-N-isopropyl bound with lower affinity. Generally, the (R)-enantiomers displayed higher affinity for the 5-HT1A receptor than the corresponding (S)-enantiomers. In the present series of compounds, both full and partial agonists were found.

Molecular mechanisms underlying forskolin-mediated up-regulation of human dopamine D2L receptors.

1. Human dopamine (DA) D2long (hD2L) receptors, expressed by Ltk- cells, can be up-regulated by treating the cells with forskolin for 16 hr (Johansson and Westlind-Danielsson, 1994). We have examined some of the molecular mechanisms underlying this forskolin-mediated up-regulation. 2. Forskolin (100 microM, 16 hr), but not 1,9-dideoxyforskolin, a forskolin analogue that is unable to activate adenylyl cyclase and raise intracellular cAMP concentrations, up-regulates the hD2L receptor population by 43%. The implication of a cAMP-dependent increase in the receptor up-regulation was further substantiated by treating the cells with 8-bromo-cAMP or prostaglandin E1 (PGE1). The forskolin-mediated rise in receptor number was blocked by cycloheximide or an antisense phosphorothioate oligodeoxynucleotide (ODN) directed toward the hD2L mRNA. KT5720, a specific protein kinase A (PKA) inhibitor, completely blocked the receptor rise, whereas pertussis toxin (PTX) attenuated the increase considerably. Forskolin also produced an increase in the level of the DA hD2short (hD2S) receptor expressed by Ltk- cells. This increase was 2.5-fold higher than that found for the hD2L receptor. 3. The forskolin-mediated hD2L receptor rise is dependent on de novo protein synthesis, a rise in cAMP levels, PKA activation, and, at least partially, PTX-sensitive G proteins. 4. Long-term increases in intracellular cAMP levels may change the sensitivity of a DA receptor expressing cell to DA by increasing D2 receptor density through enhanced cAMP-dependent transcription.

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299.

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.

Dopamine D2 receptor upregulation in rat neostriatum following in vivo infusion of forskolin.

Intracerebroventricular (i.c.v.) forskolin infusion for 5 days resulted in a concentration-dependent increase in rat striatal dopamine (DA) D2 receptors measured with [3H]raclopride. In animals given 50 nmol/h forskolin, the highest concentration used, raclopride-mediated suppression of spontaneous locomotor activity was attenuated, and (+/-)-7-hydroxy-dipropyl-aminotetralin HBr (7-OH-DPAT)-mediated inhibition of striatal DA synthesis, as estimated by the accumulation of DOPA following inhibition of cerebral decarboxylase, was enhanced. These data suggest that the DA D2 receptor increase comprises receptors localized both post- and presynaptically. The density of striatal DA D1 receptors was also changed with the forskolin treatment, in a concentration-dependent fashion, but in the opposite direction to DA D2 receptors. These findings suggest that striatal DA receptor sensitivity can be changed by manipulation at the second messenger level (e.g. independent of direct neurotransmitter-receptor interactions) in vivo.

Derivatives of (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin: synthesis and interactions with 5-HT1A receptors.

Analogs of the 5-HT1A receptor antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin [(S)-1,(S)-UH301] have been prepared. The C8-substituent has been varied, and in some derivatives one of the N-propyl groups has been exchanged for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)-butyl group. The novel compounds have been evaluated for affinity to rat brain 5-HT1A receptors in competition experiments with [3H]-8-OH-DPAT. In addition, the efficacy of the compounds was assessed by their ability to inhibit the VIP-stimulated cAMP formation in GH4ZD10 cells expressing rat 5-HT1A receptors. Varying degrees of intrinsic activity was revealed among the compounds tested, i.e., the profiles ranged from full agonists to antagonists. All R-enantiomers are characterized as full agonists at 5-HT1A receptors, whereas partial agonists or antagonists were found among the corresponding S-enantiomers. Substitution of one of the N-propyl groups for a 4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group seems to increase efficacy as well as affinity for 5-HT1A receptors. A favorable interaction with an accessory binding site by the N-4-(8-aza-7,9-dioxospiro[4.5]decan-8-yl)butyl group may contribute to the increased affinity.

Remoxipride shows low propensity to block functional striatal dopamine D2 receptors in vitro.

The effect of remoxipride ((S)(-)3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,6-dimethoxybenzam ide) on dopamine D2 receptor-mediated inhibition of cAMP formation in rat striatal tissues pieces was established together with that of a number of other dopamine D2 receptor antagonists. The action of remoxipride, three other substituted benzamides, (-)-sulpiride, raclopride and NCQ 298 ((S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6-dimethoxysalicylam ide mesylate) and haloperidol, a butyrophenone, was studied in the presence of (I) (+/-) SKF 38393 (7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) hydrochloride (100 microM) plus pergolide (1 microM) or (II) forskolin (1 microM) plus dopamine (100 microM). In addition, four of the metabolites of remoxipride: FLA 797 ((S)-3-bromo-N[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-6- methoxybenzamide), NCR 181 ((S)(-)-5-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-6- methoxybenzamide tartrate), NCQ 436 ((S)(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6-dihydroxy-2- methoxybenzamide semioxalate) and NCQ 469 ((S)(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-hydroxy-2,6- dimethoxybenzamide hydrochloride), mainly found in rodents, were studied using test system I. The results demonstrate that remoxipride is significantly weaker in blocking functional striatal dopamine D2 receptors than either of the reference compounds studied and three of the four metabolites. The studies also demonstrate that dopamine D1 and D2 receptor interactions at the level of cAMP formation in the striatum are independent of action potentials or Ca2+.

Alaproclate acts as a potent, reversible and noncompetitive antagonist of the NMDA receptor coupled ion flow.

We studied the effect of alaproclate [2-(4-chlorophenyl)-1,1-dimethyl 2-aminopropanoate] on the N-methyl-D-aspartate (NMDA)-induced changes in membrane potential and intracellular free Ca++ in cerebellar granule cells by using the fluorescent indicators DiBaC4(3) and fura-2, respectively. The NMDA-induced responses were blocked by the 5-hydroxytryptamine reuptake blocker alaproclate in a noncompetitive manner with an IC50 value of 0.3 microM. The effect of alaproclate was stereoselective because the S-(-)-enantiomer was more potent than the R-(+)-enantiomer. The inhibitory response was rapidly reversed if alaproclate was removed by perfusion. The same was the case with the reversible noncompetitive NMDA receptor antagonists dextromethorphan, dextrorphan, amitriptyline and desipramine. The inhibition caused by the noncompetitive antagonist dizolcipine could not be reversed by perfusion. The glycine-sensitivity of the NMDA response was unaffected by alaproclate, and high concentrations of glycine were unable to reverse the inhibition of alaproclate. Alaproclate also did not affect the sensitivity of the responses to Mg++. The results suggest that alaproclate, which has previously been in clinical trials for depressive illness, acts as a reversible noncompetitive antagonist of the NMDA receptor.

Forskolin-induced up-regulation and functional supersensitivity of dopamine D2long receptors expressed by Ltk- cells.

Mouse fibroblast Ltk- cells, stably expressing the human dopamine D2long receptor, were grown in the presence of forskolin (100 microM) for 4 or 16 h. The 16 h treatment resulted in a significant up-regulation of the dopamine D2long receptors by 43-96% as measured with [3H]raclopride with no change in the Kd value. A significant increase in the maximal inhibition of acute forskolin-stimulated cAMP accumulation by dopamine (0.1 nM-3 microM) was found both at 4 and 16 h. No such D2long-receptor-coupled response to dopamine could be detected in wild-type, untransfected, Ltk- cells with or without forskolin treatment. Furthermore, basal cAMP levels as well as the maximal response to acute forskolin stimulation decreased in the D2long receptor expressing cells with the treatment, by 33% and 23% respectively. The results indicate that persistent maintenance of high cAMP levels in transfected Ltk- cells may lead to adaptive quantitative and functional changes of the dopamine D2long receptor reminiscent of receptor supersensitivity induced by chronic antagonist treatment in vivo where the receptor targeted is inhibitorily coupled to adenylyl cyclase, as is the D2long receptor. This may provide a model for studying mechanisms underlying dopamine D2 receptor up-regulation and receptor supersensitivity, not readily induced in cell lines.

Dopamine receptors: molecular biology, biochemistry and behavioural aspects.

The description of new dopamine (DA) receptor subtypes, D1-(D1 and D5) and D2-like (D2A, D2B, D3, D4), has given an impetus to DA research. While selective agonists and antagonists are not generally available yet, the receptor distribution in the brain suggests that they could be new targets for drug development. Binding characteristics and second messenger coupling has been explored in cell lines expressing the new cloned receptors. The absence of selective ligands has meant that in vivo studies have lagged behind. However, progress has been made in understanding the function of DA-containing discrete brain nuclei and the functional consequence of the DA's interaction with other neurotransmitters. This review explores some of the latest advances in these various areas.

High concentrations of the neuroleptic remoxipride block voltage-activated Na+ channels in central and peripheral nerve membranes.

The actions of the neuroleptic compounds remoxipride, haloperidol and (-)-sulpiride on Na+ and K+ ion current flow were examined in rat CNS and frog PNS, using 86Rb+ ion flux and voltage-clamp techniques, respectively. By combining veratridine and high K(+)-evoked 86Rb+ efflux, it was determined that remoxipride blocked Na+ current flow in a concentration-dependent fashion in tissue pieces from either cerebral cortex or striatum (IC50 approximately 20 microM), leaving K+ current flow virtually unaffected. Similarly, haloperidol concentration dependently blocked Na+ current flow in both tissues (IC50 approximately 50 microM). (-)-Sulpiride did not have a significant effect. Direct actions of the compounds on voltage-gated Na+ and K+ channels were determined in voltage-clamp experiments. The findings confirmed the results of the ion flux measurements in that remoxipride (Kd approximately 300 microM) and haloperidol (Kd approximately 1.5 microM) reduced mainly the Na+ current, having little effect on the K+ current, whereas (-)-sulpiride did not have a measurable action. The relatively high concentrations of remoxipride or haloperidol needed to alter the Na+ current makes it unlikely that these actions are of importance at clinically relevant doses.

Steroid hormones as mediators of neural plasticity.

Steroid and thyroid hormone receptors are expressed in the developing brain and persist throughout adult life. They mediate a variety of effects on the brain, ranging from developmental effects of thyroid hormone and the process of sexual differentiation to the cyclic changes during reproductive cycles in adult female animals. This review summarizes data from the author's laboratory on three topics: (1) actions of extradiol and progesterone on the ventromedial nucleus of the hypothalamus in adult female and male rats, showing both the cyclicity and the consequences of brain sexual differentiation; (2) actions of estradiol on the cholinergic neurons of the basal forebrain of the female and male rat, reflecting the plasticity of the adult cholinergic system as well as sex differences which are developmentally programmed; and (3) diverse actions of estrogens, thyroid hormone and glucocorticoids on the morphology of hippocampal neurons. The review concludes by discussing the interactions between "organizational" (i.e. developmental) effects and the "activational" effects of steroids on the mature nervous system in relation to the environmental control of brain gene expression.

Neonatal hyperthyroidism disrupts hippocampal LTP and spatial learning.

Excess thyroid hormone at an early stage of development produces marked neurochemical and morphological alterations in the rat hippocampal formation. In order to better understand the functional significance of these changes, we tested adult rats treated neonatally with triiodothyronine (T3), and their control litter mates, in a spatial learning task and for the induction of long-term potentiation (LTP) in the dentate gyrus (DG) of the hippocampal formation. The T3-treated rats were significantly impaired in their performance on the spatial task in comparison to their matched controls. Similarly, the efficacy of LTP induction was significantly attenuated in the T3-treated animals. Further, a significant correlation was obtained between LTP induction and performance on the spatial learning task. Thus, a brief neonatal excess of thyroid hormone produces impairments in spatial learning along with decreases in LTP, long held as a model of learning and memory. This relationship provides a unique opportunity to study associations between behavioral, physiological, pharmacological and morphological processes intimately associated with the hippocampal formation.

Thyroid hormone causes sexually distinct neurochemical and morphological alterations in rat septal-diagonal band neurons.

Sex differences were investigated in cholinergic neurons of the septal-diagonal band region of adult rats subjected to neonatal treatment with 3,3',5-triiodo-L-thyronine (T3). Neonatal hyperthyroidism resulted in a 44% increase in specific activity of choline acetyltransferase (ChAT; EC 2.3.1.6) in adult male rat septal-diagonal band region, whereas no change in ChAT activity could be detected in either dorsal or ventral hippocampus. An increase in muscarinic cholinergic receptors, as measured by [3H]quinuclidinyl benzilate [( 3H]QNB) binding, was discovered in both septum-diagonal band and dorsal hippocampus of the T3-treated male rats. Immunohistochemistry in the septal-diagonal band region indicated a more intense staining in the neonatally T3-treated adult male rats than in controls, with larger and more abundant ChAT-positive and nerve growth factor receptor (NGF-R)-positive varicosities. ChAT immunocytochemistry showed a substantial decrease in cell body area in the medial septum and in the vertical limb of the diagonal band of T3-treated male rats, while cell density increased twofold. Female littermates subjected to the same treatment showed no changes in any of the biochemical or immunohistochemical cholinergic markers. Only in the medial septum was morphology significantly altered in the female T3-treated rats in that ChAT-positive cell body area increased. These results indicate a marked sexual variation in the septal-diagonal band region with respect to the sensitivity of postnatally developing cholinergic neurons to the actions of excess thyroid hormone.

Atropine treatment induced cholinergic supersensitivity at receptor and second messenger levels in the rat salivary gland.

Physiological, biochemical and morphological correlates of chronic treatment of rats with the classical muscarinic antagonist atropine for 14 days (20 mg kg-1 day-1 s.c.) were studied in submandibular salivary glands. The amount of saliva collected from submandibular glands following a single injection of isoproterenol (30 mg kg-1 i.p.) was significantly larger and had higher protein concentration in rats treated with atropine than in saline-treated animals. In the glands of atropine-treated rats a conspicuous increase in the amount of rough endoplasmic reticulum (RER) along with a decrease in the mucous volume was observed in the acinus when examined by light microscopy. Several biochemical changes were observed in an enriched plasma membrane fraction from the submandibular gland of the atropine-treated rats: (1) an increase in the number of muscarinic antagonist binding sites (31 + 3.4%), (2) a decrease in the specific activity of basal adenylate cyclase, (3) a significantly lower Vmax of the adenylate cyclase in the presence of GTP (10 microM) and varying concentrations of Mg2+ (0-22.5 mM) with no apparent change in affinity of the enzyme for Mg2+ but (4) higher magnitude of stimulation in the presence of GTP (100 microM), vasoactive intestinal polypeptide (5 microM), isoproterenol (100 microM), NaF (10 microM) and forskolin (10 microM). There was however no change in the density of beta-adrenergic receptors upon atropine treatment. In tissue slices from the submandibular glands of atropine-treated rats we found lower basal cAMP levels (decrease 29 +/- 6.9%) and no significant change in the phosphatidylinositol breakdown stimulated by carbachol (10(-6) to 10(-4) M). It appears that chronic blockade of an inhibitory muscarinic input to the adenylate cyclase system is compensated by lowered adenylate cyclase activity. Phosphoinositide metabolism is not subject to the same adaptation, suggesting that cAMP may be the pivotal second messenger in the supersensitive salivary response.

Sex differences and thyroid hormone sensitivity of hippocampal pyramidal cells.

In an effort to determine if sex differences exist in the morphologic characteristics of pyramidal cells and granule cells of the hippocampal formation and whether sex plays a role in determining thyroid hormone sensitivity of these neuronal populations, we used single-section Golgi impregnation to examine the effects of neonatal thyroid hormone administration on hippocampal cells from the brains of adult rats of both sexes. Quantitative analyses of control brains revealed sex differences in the number of primary dendrites and the number of spines on the apical dendritic shaft of CA3 pyramidal cells. These differences showed opposite trends; females possessed more primary dendrites, whereas males showed more apical excrescences. Neonatal treatment with thyroid hormone resulted in long-lasting and dramatic changes of the entire CA3 pyramidal cell. CA3 pyramidal cells from thyroid hormone-treated animals showed significantly larger cell body areas, greater numbers of dendritic branchpoints, and longer dendrites. In addition, CA3 pyramidal cells from thyroid hormone-treated animals showed changes in the morphological characteristics which were shown to be sexually dimorphic; treatment resulted in significantly greater numbers of both primary dendrites and apical excrescences. These treatment differences occurred in both sexes and were of equal magnitude, regardless of sex. On the other hand, no sex differences in the morphologic parameters examined were detected for pyramidal cells in the CA1 region. Moreover, neonatal thyroid hormone treatment did not affect the cell body area, dendritic branch points, or the length of dendrites of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)