Effects of neonatal gamma-ray irradiation on rat hippocampus--II. Development of excitatory amino acid binding sites.

In the rat, neonatal irradiation produces a destruction of dentate granule cells and prevents the development of the mossy fibre-CA3 pyramidal cell synapse. The developmental increase of high affinity kainate binding sites in the stratum lucidum was reduced on the irradiated side as compared with the control side. This suggests that a proportion of high affinity kainate binding sites is associated with mossy fibres. In contrast, the developmental profile of N-methyl-D-aspartate binding sites, which are associated with associational and commissural synapses in CA3, was not affected by irradiation. The role that afferent fibres may play in the development of pyramidal cells is discussed in connection with the modulatory effects of glutamate receptors on the development of neurons.     

Postnatal maturation of the GABAergic system in rat neocortex.

1. The postnatal maturation of intracortical inhibitory circuitry and the development of responses to applied gamma-aminobutyric acid (GABA) and baclofen were studied in pyramidal and nonpyramidal neurons from layers II and III of the rat primary somatosensory and primary visual cortex, in vitro. 2. Depolarizing spontaneous inhibitory postsynaptic potentials (IPSPs) could be recorded in approximately 70% of the young (postnatal day 4-10; P4-10), juvenile (P11-16), and adult cells (P28-41), respectively, when they were loaded with nitrate. At all ages these spontaneous events could be blocked by application of the GABAA receptor antagonist bicuculline methiodide (BMI), indicating that they were mediated by activation of GABAA receptors. 3. In 122 of the 130 adult cells tested, standardized electrical stimulation of the white matter or layer VI evoked a brief excitatory postsynaptic potential (EPSP), followed by both a fast (f-) and a long-latency (l-)IPSP. Similar stimuli evoked a biphasic IPSP in only 51 of the 98 juvenile and in only 1 of the 56 young neurons studied. The mean peak conductance of the f-IPSP and the l-IPSP increased significantly from 50.2 and 7.5 nS, respectively, in juvenile cells to 84.2 and 18.0 nS, respectively, in adult neurons. 4. Application of the N-methyl-D-aspartate (NMDA) receptor antagonist D-amino-phosphonovaleric acid (D-APV) to juvenile cells induced a significant negative shift in the reversal potential of both the f-IPSP and l-IPSP. This effect was accompanied by a reduction in the peak conductance during these events by 31 and 48%, respectively, indicating that a prominent long-lasting NMDA receptor-mediated EPSP occurs concurrent with the early and late IPSP in immature neurons. In adult neurons, D-APV had no significant effect on the reversal potential of the f- and l-IPSP, although the peak conductance decreased by 20 and 5%, respectively, suggesting that there was a smaller concurrent activation of NMDA receptors in this age group. 5. The functional maturation of GABAA and GABAB receptors was studied using focal applications of GABA to the soma and the apical dendrite. Somatic GABA applications to adult neurons held at depolarized membrane potentials evoked a triphasic response, consisting of 1) a GABAA-mediated hyperpolarizing fast component (GABAhf; reversal potential, -76 mV), 2) a GABAA-mediated depolarizing phase (GABAd; -54 mV), and 3) a hyperpolarizing late response (GABAhl; -80 mV). The GABAd response could be demonstrated at all ages in almost every neuron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional maturation of neonatal spleen cells.

Maturation of neonatal spleen cells was studied in vitro with a cell population restricted with respect to functional properties. It is shown that the onset of the immune response to SRBc in post-natal mice was delayed because B and T cells were incompetent. It appears, however, that the development of these two cell populations does not occur in parallel. Since the addition of adult macrophages failed to overcome the incompetence of neonatal B cells in the presence of a T cell replacing factor, it is suggested that the late appearance of immune competence is due to the inability of B cells to process a T-cell signal.     

Identification of neonatal rat hippocampal radial glia cells in vitro

Activation of central 5-HT₃ receptors by the selective agonist m-CPBG (1-(3-chlorophenyl)biguanide hydrochloride, 40 nM i.c.v.) produced stronger hypothermic effect in mice than activation of 5-HT_1A receptors by their agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propilamino)tetralin) injected by the same route at an equimolar dose. The hypothermic effect of m-CPBG was realized by influence on both the heat production and the heat loss: oxygen consumption and CO₂ expiration were decreased; heat dissipation determined by the tail skin temperature was increased. The heat loss effect of 5-HT₃ receptors was significantly shorter than the decrease in metabolism indicating the prevalent role of heat production decrease in 5-HT₃ receptor-induced deep and long-lasing hypothermia. In addition, the decrease in the respiratory exchange ratio (RER) was shown suggesting that the activation of the 5-HT₃ receptors switched metabolism to prevalent use of lipids as the main energetic substrate. 5-HT_1A receptor agonist 8-OH-DPAT (40 nM i.c.v.) produced less depressing effect on general metabolism: a decrease in oxygen consumption and CO₂ excretion began later and was not so deep as after m-CPBG administration. Heat-loss effect of 5-HT_1A receptors activation was not observed. In contrast to m-CPBG effect, RER after 5-HT_1A receptors activation raised immediately after injection and then gradually decreased to the values observed in m-CPBG-treated mice. Obtained results show that activation of central 5-HT₃ receptors are more effective in hypothermia induction due to marked decrease in thermogenesis and increase in heat loss.     

Postnatal maturation of mast cell subpopulations in the rat respiratory tract.

The distribution and enumeration of mast cell subpopulations within the respiratory tract of a high- and low-Ige responder rat strain was determined during postnatal development. Mast cells were identified in adjacent sections by the alcian blue (AB)/safranin (SAF) staining sequence, or using immunoperoxidase to detect the rat mast cell proteinases I (RMCPI) or II (RMCPII). At birth both mucosal mast cells (MMC) and connective tissue mast cells (CTMC) were represented in very low numbers at distinct locations throughout the respiratory tract. The total number of mast cells increased with age. MMC (AB+/RMCPII+ mast cells) were the predominant phenotype in the epithelium and lamina propria of the trachea and the major conducting airways of the lung in all age groups. In contrast, CTMC (AB+/RPMCPI+ and SAF+/RMCPI+ mast cells) predominated in the submucosa of the trachea and major conducting airways as well as in the parenchyma and visceral pleura of the peripheral lung. Both phenotypes co-exist in similar proportions in peribronchial adventitial tissue and adventitia surrounding large blood vessels in neonates as well as adults. In rats the tracheal epithelium is densely populated by MMC from around the time of weaning (3 weeks) and a small but generalized increase in the number of MMC at all sites within the respiratory tract is noted from this time. This increase in MMC frequency in tissue sections with increasing age is mirrored by the levels of circulating serum RMCPII. The number of bone marrow-derived MMC also increased with increasing age prior to weaning, with a significant drop (P less than 0.01) at 4 weeks of age before returning to the peak numbers in 3-week-old rats. The high-IgE responder Brown Norwegian (BN) rat strain constitutively produces significantly more IgE than the low-IgE responder White albino Glaxo (WAG) strain (P less than 0.001) at all ages studied. In contrast, only minor differences between the number and distribution of mast cells in the two strains were observed.     

Postnatal maturation of spindles in deafferented rat soleus muscles

Summary Whether the motor innervation can direct the morphological and histochemical differentiation of developing muscle spindles in the absence of sensory innervation was investigated by deafferentation of the soleus muscle in immature rats. Dorsal root ganglia containing the cell bodies of afferents from the soleus muscle were removed surgically at a stage of postnatal development when spindles already contain the full complement of intrafusal fibers innervated by both afferents and efferents, but when the fibers are histochemically and structurally immature. Experimental soleus muscles were excised one year after deafferentation and sectioned frozen at a thickness of 8 m. Sections were stained for enzymes indicative of types of muscle fibers and sites of neuromuscular junctions, and were examined by light microscopy. Spindles of muscles that matured in the absence of sensory innervation were abnormal. They lacked the periaxial fluid space and contained fewer intrafusal fibers than did normal spindles. The morphological and histochemical profiles of the encapsulated fibers present in the deafferented spindles more closely resembled extrafusal rather than intrafusal muscle fibers. These observations suggest that deafferentation of the immature spindles induces disintegration of some intrafusal fibers and alters maturation of others. Moreover, motor axons terminated less frequently along muscle fibers in deafferented spindles than on intrafusal fibers of normal spindles. Thus, maintenance of a full complement of intrafusal fibers in the developing spindle, emergence of histochemical profiles typical of normal intrafusal fibers, and development of adult pattern of fusimotor innervation require intact sensory innervation.     

Postnatal development of rat hippocampal gamma rhythm in vivo

Network oscillations in the gamma-frequency band (20-100 Hz) may have a central role in the timing and coordination of neural activity in the adult brain, yet their appearance in the course of development has remained unexplored. Moreover, electroencephalogram (EEG)-based classification of the vigilance states [active sleep (AS), quiet sleep (QS), or awake (W)] has been thought to be possible only after the second postnatal week. We now report the presence of spontaneous hippocampal gamma oscillations in the area CA3 of freely moving rats at postnatal days (P) 5-10. Initially, at P5, the gamma oscillations were seen in time-frequency analyses of intrahippocampal EEG recordings as brief (<500 ms) bursts at 20-30 Hz. The early gamma rhythmicity was most pronounced during periods of AS but was occasionally detected also during QS. Toward P10, the gamma oscillations gained amplitude and extended also to higher (相似文献   

Postnatal maturation of renal cortical peritubular fibroblasts in the rat

 The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5′nucleotidase (5′NT)-positive and lack alpha-smooth muscle actin (αSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5′NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5′NT and to the cytoskeletal proteins αSMA and vimentin. Both αSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display αSMA and vimentin, but lack 5′NT. During the second postnatal week, αSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5′NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5′NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and αSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5′NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function. Accepted: 18 July 1997     

Effects of histamine on hippocampal pyramidal cells of the rat in vitro

Summary The actions of bath applied histamine on CA1 pyramidal cells were investigated in hippocampal slices of the rat. Histamine caused a) a slight depolarization but no significant change in resting membrane conductance; b) an abbreviation of long afterhyperpolarizations after single action potentials, bursts of action potentials or TTX resistant spikes; c) a loss of accommodation of firing. In the presence of TEA or barium, histamine prolonged and increased the size and number of the slow TTX resistant spikes. A depolarizing plateau which follows such spikes was also increased by histamine. Evoked synaptic potentials were unaffected by histamine, but the population spike was increased. The frequency of spontaneous chloride dependent potentials, which reflect interneurone firing, was also increased. These effects considerably outlasted histamine application and were mimicked by the H₂-agonist impromidine but not the H₁-agonist thiazolethylamine, and blocked by the H₂-antagonists cimetidine and metiamide but not the H₁-antagonists mepyramine or the beta-antagonist propranolol. It is concluded that histamine, by activating H₂-receptors, antagonizes a calcium mediated potassium conductance in hippocampal pyramidal cells without affecting calcium current. By this mechanism histaminergic afferent fibres could effectively regulate cortical responsiveness by selectively potentiating large excitatory inputs of target neurones.     

Effects of acute wound environment on neonatal rat dermal multipotent cells

Dermal multipotent cells (DMCs) with the capacity to differentiate to osteocytes, chondrocytes, adipocytes and neurons were isolated from the dermis of newborn Wistar rats by their adherence to culture plastic dishes. After labeling with a fluorescent nuclear marker, 4',6-diamidino-2-phenylindole, neonatal DMCs were implanted into minced skin. The labeled cells were found in the regenerative skin 2 weeks after implantation. Topical transplantation of DMC suspension on full-thickness excision wounds made on the back of female rats enhanced the initial rate of contraction. Wound fluid collected using polyvinyl alcohol sponges from the incisions on the dorsa of adult rats on day 3 postwounding was used in the experiments. A methylthiazolyldiphenyl tetrazolium bromide assay showed that wound fluid with concentrations from 5 to 30% induced an increase in the number of cultured DMCs. The wound model of a monolayer of cells indicated that 10% wound fluid accelerated the migration rate of DMCs to fill in defects made in the layer of cultured cells. A colorimetric analysis also indicated that 10% wound fluid increased the hydroxyproline content in cultured cells. In conclusion, DMCs have been isolated from newborn rat dermis by their adherence to culture plastic dishes, and acute wound environment in the early stage promotes the viability, migration and collagen synthesis of these cells.     

Postnatal proliferation and maturation of olfactory bulb neurons in the rat

Mitral cells are formed prenatally whereas most granule cells originate postnatally. Material was taken from 2-day-old, 14-day-old, 28-day-old, and adult rat olfactory bulbs and processed for rapid Golgi or Cresyl Violet staining. We show that the number of granule cell bodies/mitral cell body increases from 7.0 to 46.3 during the first two weeks of life; most mitral cells appear morphologically functional during the first postnatal week; few granule cells appear to be functional until the second postnatal week; and the number of short axon interneurons increases dramatically during the second postnatal week.We conclude that newborn rats have an intact afferent pathway from olfactory receptors to primary cortex that lacks the extensive interneuronal circuitry characteristic of adults.     

亚低温对缺氧缺血新生大鼠海马星形胶质细胞增殖和凋亡的影响

目的观察亚低温对新生大鼠海马星形胶质细胞增殖和凋亡的影响,以探讨亚低温对缺氧缺血脑损伤保护作用的机制。方法①体外实验：取3日龄大鼠海马脑片,培养至第4天用氧糖剥夺法制备标本,于33℃（亚低温组）和37℃培养48h（常温组）;对照组脑片不进行氧糖剥夺处理,37℃培养7d。采用免疫荧光染色方法观察培养脑片星形胶质细胞的活化和增殖。②体内实验：取7日龄大鼠,分手术组和假手术组。手术组永久性结扎左侧颈总动脉,然后置于含8%O2＋92%N2中缺氧2h,制备缺氧缺血模型,分为手术亚低温亚组和手术常温亚组。假手术组仅分离左侧颈总动脉,不结扎,不予缺氧处理,分为假手术常温亚组和假手术亚低温亚组。各组于术后3和7d处死取材,采用免疫荧光染色方法检测海马星形胶质细胞的活化、增殖和凋亡。结果①体外实验：氧糖剥夺后3d常温组较对照组胶质纤维酸性蛋白（GFAP）阳性细胞数量明显增多;亚低温组与常温组比较,GFAP阳性细胞数量明显减少。②体内实验：术后3和7d亚低温组GFAP阳性细胞数量较常温组显著降低,较常温对照组和亚低温对照组显著增高。GFAP与caspase-3免疫荧光双标记结果显示,术后3d常温组海马区84.5%GFAP阳性细胞表达caspase-3,亚低温组仅32.3%GFAP阳性细胞表达caspase-3,差异有统计学意义。结论亚低温能减轻新生大鼠缺氧缺血后海马星形胶质细胞的活化增殖和凋亡。     

Effects of phorbol ester on gap junctions of neonatal rat heart cells

Myocytes were isolated from the ventricles of neonatal rat hearts and cultured for 1–3 days. Newly formed cell pairs were used to examine the conductance of gap junctions, g _j. Measurements were performed using a dual voltage-clamp method in conjunction with a whole-cell, tight-seal recording. Exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 100–160 nM) led to a decrease in g _j. Single-channel events recorded immediately before complete uncoupling yielded a single-channel conductance, _j, of 40.5 pS, implying that TPA affects the channel kinetics rather than _j. TPA-induced uncoupling was observed at subphysiological levels of cytosolic Ca²⁺ (pipette solution=18 nM), not at physiological levels (pipette solution=170 nM). The effects of TPA could not be mimicked by 250 M 1-oleoyl-2-acetyl-glycerol (OAG). Preincubation with TPA (up to 24 h) revealed no changes in g _j attributable to down-regulation of protein kinase C, PKC. Pretreatment with PKC inhibitors, staurosporine or PKCI, prevented the TPA-dependent decrease in g _j. TPA-dependent uncoupling was not impaired by 4-bromophenacyl bromide, an inhibitor of phospholipase A₂, PLA₂; conversely, an arachidonic acid-dependent decrease in g _j was not prevented by PKCI. This suggests that g _j regulation does not involve an interaction between PLA₂ and PKC.     

丹酚酸B对培养的新生大鼠海马神经元的影响

目的:本研究旨在观察丹酚酸B对体外培养新生大鼠海马神经元的影响,为丹酚酸B对中枢神经系统的作用提供实验依据。方法:体外培养新生大鼠海马神经元,神经元微管相关蛋白-2(MAP-2)免疫荧光染色对培养细胞进行鉴定,实验分为对照组和给药组(终浓度分别为5 mg/L和10 mg/L),荧光显微镜下观察细胞形态,MTT比色法检测丹酚酸B对海马神经细胞的活性,细胞核染色(DAPI)观察丹酚酸B对海马神经细胞增殖的影响。结果:免疫荧光染色显示细胞呈MAP-2阳性,丹酚酸B作用7 d后,能明显促进海马神经细胞的存活与增殖,且10 mg/L的丹酚酸B作用更明显。结论:丹酚酸B能明显促进海马神经细胞的存活与增殖,提示其具有改善脑功能的重要作用。     

脑源性神经营养因子促进低血糖幼鼠海马神经干细胞的定向分化

背景：有研究表明脑源性神经营养因子可以维持神经元的存活、影响神经元的迁移,在体外可以促进神经干细胞的存活和分化。 目的：探讨脑源性神经营养因子对低血糖幼鼠海马神经干细胞定向分化的作用。 方法：取新生1 d低血糖模型大鼠脑海马组织进行原代、传代及单细胞克隆培养。培养的细胞一部分进行神经干细胞鉴定,另一部分依据培养液中脑源性神经营养因子质量浓度的不同将单克隆细胞分为0,100,200 μg/L组,取第4代细胞进行诱导分化,行神经元特异性烯醇化酶免疫荧光染色,计数阳性细胞比例。 结果与结论：单克隆培养后3组细胞均呈巢蛋白阳性,诱导分化后细胞呈行神经元特异性烯醇化酶和胶质纤维酸性蛋白阳性;100,200 μg/L组神经干细胞生长较快,且分化为神经元特异性烯醇化酶阳性细胞比例较高(P < 0.05),但两组神经干细胞之间差异无显著性意义(P > 0.05)。提示脑源性神经营养因子促进低血糖幼鼠海马神经干细胞向神经元定向分化。     

Postnatal maturation of tissue kallikrein-producing cells (connecting tubule cells) in the rat kidney: a morphometric and immunohistochemical study

The effects of a sympathetic neurotoxin, 6-hydroxydopamine (6-OHDA), on hair growth in neonatal mice were examined. Newborn mice were injected once subcutaneously in the mid-dorsal region with 6-OHDA (0.3 mg/g body weight) and bovine serum albumin (BSA) or with BSA only (controls) on day 0. By day 10, distinct areas of hairless skin were observed surrounding the sites treated with 6-OHDA. The areas of hairless skin were smaller at 15 days of age and were covered with hair by 20 days of age. Control sites injected with BSA only were indistinguishable from the surrounding skin at all ages examined. Microscopic and morphometrical analyses of skin obtained from the neonatal mice at various ages showed that the hairless skin in 6-OHDA-treated mice was thinner than the skin of control animals. The thinning of the skin and delay in hair growth induced by 6-OHDA treatment showed a significant difference from the skin of control animals injected with BSA only. Immunohistochemical experiments demonstrated that the administration of 6-OHDA had caused the complete depletion of tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) around blood vessels and piloerector muscles and in nerve bundles throughout the dermis and subcutaneous tissue. These findings indicate that the sympathetic neurons are associated with skin thickness and hair growth in neonatal mice.     

Effects of arachidonic acid on the gap junctions of neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in tissue-culture dishes for 1–2 days. Spontaneously formed cell pairs were used to study the conductance of gap junctions. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Exposure to arachidonic acid (AA) produced a quasi dose-dependent decrease in junctional conductance, g _j (binding constant, K _d=4 M; Hill coefficient, n = 0.75). AA-dependent uncoupling was reversible. Addition of 1 mg/ml albumin to the bath solution accelerated the recovery. During control, cell pairs exhibited a gradual decrease in g _j (16.4 % in 6 min). Exposure to 20 M 4-bromophenacyl bromide, a phospholipase inhibitor, suppressed the decay in g _j (1.8% in 6 min), suggesting that endogenous AA may be involved in spontaneous uncoupling. The effect of AA on g _j was specific. Arachidic acid (100 M) and arachidonamide (10 M), structural analogues of AA, had no effect on g _j. Currents recorded shortly before complete uncoupling caused by AA, or early during recovery from uncoupling, revealed random opening and closing of single channels. The single channel conductance, _j, was not affected by the concentration of AA (1 M–100 M). The mean _j turned out to be 33.5 pS. The results suggest that AA-dependent uncoupling was caused via decrease in open channel probability, presumably mediated by a direct action on channel proteins.     

右美托咪定对乳大鼠心肌细胞线粒体活性氧和细胞凋亡的影响

目的:探讨右美托咪定(Dexmedetomidine,DEX)对线粒体介导的乳大鼠心肌细胞氧化应激通路的作用。方法:取新生乳大鼠(3~4天)的心肌进行原代培养,培养24h后分为对照组(C组)、H_2O_2组(终浓度500μM,H组)、右美托咪定组(5μM DEX,D组)、联合用药组(500μM H_2O_2+5μM DEX,DH组)。各组细胞分别给予相应药物刺激6h,采用流式细胞仪测定活性氧自由基(Reactive oxygen species,ROS)水平及心肌细胞凋亡变化;并通过ELISA法测定各组乳大鼠心肌细胞线粒体介导的凋亡因子Caspase-3,Caspase-9表达的变化。结果:与H_2O_2组相比,DEX明显抑制H_2O_2介导的乳大鼠心肌细胞的ROS水平增加(P0.05);下调线粒体介导的下游凋亡因子Caspase-3,Caspase-9的表达(P0.05),从而减少H_2O_2诱导的凋亡(P0.05)。结论:右美托咪定通过抑制心肌细胞活性氧水平以及下调线粒体介导的Caspase-3和Caspase-9的表达发挥抑制凋亡作用,对心肌细胞有一定的保护作用。