Age-dependent modifications of mitochondrial proteins in cerebral cortex and striatum of rat brain

The protein composition of free mitochondria purified from cerebral cortex and striatum during aging was analyzed by gel electrophoresis. Mitochondria were isolated from cerebral cortex and striatum of 4-, 12-, and 24-month-old rat brain. The percent amount of mitochondrial proteins after gel-electrophoretic separation was determined densitometrically. A significant decrease in the amount of two polypeptides (with molecular weights of 20 and 16 kDa, respectively) in both brain regions during aging was found. The decrease was higher in the striatum indicating a greater vulnerability of this brain area to the aging process. The age-dependent modifications of mitochondrial proteins observed may play an important role in several mitochondrial functions, such as energy transduction and transport processes as well as in structural changes occurring with age, causing altered membrane permeability and fluidity.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

大鼠海马—小脑皮层—小脑深部核团投射的电生理学和HRP研究

本文用电生理学和HRP示踪法,研究了大鼠海马-小脑皮层投射的空间分布,小脑皮层的海马投射区与其深部核团间的纤维联系。 电生理学的实验结果表明,刺激背侧海马CA_1/CA_3区,均可使小脑皮层第Ⅵ小叶的浦肯野细胞产生顺行多突触的诱发简单锋电位和复杂锋电位反应。提示背侧海马CA_1/CA_3区与小脑皮层之间有经苔状纤维和攀缘纤维的多突触投射。实验证明,大鼠的这一投射的终止区域,集中在小脑皮层第Ⅵ小叶中线外侧0.8—1.4mm的范围内;并且来自CA_1区的投射以对侧性为主,CA_3区的投射以同侧性为主。HRP示踪的实验表明,背侧海马CA_1/CA_3区在小脑皮层第Ⅵ小叶的投射区是小脑纵区组构的间位区,该区皮层与间位核之间存在着交互投射关系。     

隐神经C类纤维传入诱发小脑皮层电反应

当弱刺激只引起隐神经A类纤维传入时,小脑皮层出现A-CEP,由潜伏期为11.8±3.5ms的早成分和312.1±17.5ms的晚成分组成;当强刺激同时引起A类和C类纤维传入时,出现AC-CEP类似A-CEP;用极化电流选择性阻断A类纤维传导后,只让C类纤维传入时,出现潜伏期为134.2±18.4ms的C-CEP。在Ⅵ小叶蚓部原裂附近C-CEP以正波为主,幅值最大,并在深层位相倒转。C-CEP的潜伏期较长,频率响应较低,幅值较小,随C类纤维传入量而变化,且对镇痛剂较敏感。结果表明C-CEP是由单纯C类纤维传入引起的,在小脑皮层内产生,是小脑皮层对慢痛信息传入的反应。提示C类纤维传入可以到达小脑皮层,引起诱发电位。当A和C类纤维同时传入时,C-CEP不出现,可能是被A类纤维传入所抑制。     

Regulation of the spatial order of cortical microtubules in developing guard cells ofAllium

The organization of microtubules (MTs) in the cortex of cells at interphase is an important element in morphogenesis. Mechanisms controlling the initiation of MTs and their spatial ordering, however, are largely unknown. Our recent study concerning the generation of a radial array of MTs in stomatal guard cells inAllium showed that the MTs initiate in a cortical MT-organizing zone adjacent to the ventral wall separating the two young guard cells (Marc, Mineyuki and Palevitz, 1989, Planta179, 516, 530). In an attempt to detect MT-ordering mechanisms separate from the sites of MT initiation, we now employ various drugs to manipulate the geometry and integrity of the ventral wall and thereby also the associated MT-organizing zone. In the presence of cytochalasin D the ventral wall is tilted away from its normal mid-longitudinal anticlinal alignment, while treatments with the herbicide chloroisopropyl-N-phenylcarbamate (CIPC) induce the formation of a branched ventral wall. Nonetheless, in either case the MTs still form a radial array, although this is asymmetric as it is centered in accordance with the misaligned or branched ventral wall. Since the MTs maintain their original course undisturbed as they extend beyond the abnormal ventral wall, there is no evidence for the presence of an inherent MT-ordering mechanism at locations remote from MT-initiation sites. Following treatments with caffeine, which abolishes the formation of the ventral wall, the MTs revert to a transversely oriented cylindrical array as in normal epidermal cells. Thus the presence of the ventral wall, and presumably also the associated MT-organizing zone, is essential for the establishment of the radial array. The MT-organizing zone is therefore involved not only in the initiation of MTs, but also in determining their spatial order throughout the cell cortex. We thank Drs. Richard J. Cyr and Yoshi Mineyuki for providing valueable suggestions during the course of this work, and Ms. Elizabeth Bruce printing some of the figures. This research was supported by Funds from the National Science Foundation grants DCB-8703292 to B.A.P. and DCB-8803286 to B.A.P. and J.M.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Processing and transfer of epidermal growth factor in developing rat jejunum and ileum

Using everted sac technique we demonstrated the transfer of ¹²⁵I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of ¹²⁵I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced ¹²⁵I-des(53)mEGF and ¹²⁵I-des(49–53)mEGF, whereas ¹²⁵I-des(48–53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of ¹²⁵I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of ¹²⁵I-des(53)mEGF was higher than that of ¹²⁵I-mEGF, but those of ¹²⁵-des(49–53)mEGF and ¹²⁵I-des(48–53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Involvement of Presynaptic Histamine H3 Receptors in the Modulation of Somatostatin Binding and Its Effects on Adenylyl Cyclase Activity in the Rat Frontoparietal Cortex

Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H₃-receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with ( R )-α-methylhistamine (3.2 mg/kg, i.p.), a histamine H₃-receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H₁-receptor antagonist, or cimetidine, a histamine H₂-receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide-binding inhibitory protein (G_i protein) was not altered by thioperamide or ( R )-α-methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS-like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.