Perinatal iron deficiency alters apical dendritic growth in hippocampal CA1 pyramidal neurons

Iron deficiency early in life is associated with cognitive disturbances that persist beyond the period of iron deficiency. Within cognitive processing circuitry, the hippocampus is particularly susceptible to insults during the perinatal period. During the hippocampal growth spurt, which is predominantly postnatal in rodents, iron transport proteins and their messenger RNA stabilizing proteins are upregulated, suggesting an increased demand for iron import during this developmental period. Rat pups deprived of iron during the perinatal period show a 30-40% decrease in hippocampal metabolic activity during postnatal hippocampal development. We hypothesized that this reduced hippocampal neuronal metabolism impedes developmental processes such as neurite outgrowth. The goals of the current study were to investigate the effects of perinatal iron deficiency on apical dendritic segment growth in the postnatal day (P) 15 hippocampus and to determine if structural abnormalities persist into adulthood (P65) following iron treatment. Qualitative and quantitative immunohistochemical analyses of dendritic structure and growth using microtubule-associated protein-2 as an index showed that iron-deficient P15 pups have truncated apical dendritic morphology in CA1 and a persistence of an immature apical dendritic pattern at P65. These results demonstrate that perinatal iron deficiency disrupts developmental processes in the hippocampal subarea CA1 and that these changes persist despite iron repletion. These structural abnormalities may contribute to the learning and memory deficits that occur during and following early iron deficiency.     

Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons

We have used Golgi-impregnated tissue to demonstrate that apical dendritic spine density in CA1 hippocampal pyramidal cells undergoes a cyclic fluctuation as estradiol and progesterone levels vary across the estrous cycle in the adult female rat. We observed a 30% decrease in apical dendritic spine density over the 24-hr period between the late proestrus and the late estrus phases of the cycle. Spine density then appears to cycle back to proestrus values over a period of several days. In contrast, no significant changes in dendritic spine density across the estrous cycle occur in CA3 pyramidal cells or dentate gyrus granule cells. These results demonstrate rapid and ongoing dendritic plasticity in a specific population of hippocampal neurons in experimentally unmanipulated animals.     

The dendritic morphology of pyramidal neurons in the rat hippocampal CA3 area. I. Cell types

Pyramidal neurons from the hippocampal CA3 area of hooded rats were qualitatively and quantitatively described from Golgi-stained tissue. The most numerous of the pyramidal neurons, those with a single apical shaft, fell into two major categories. One category, termed short-shaft pyramidal neurons, is characterized by short apical shafts, a large number of thorny excrescences, and densely branched apical and basilar trees. The second category, long-shaft pyramidal neurons, is characterized by a long apical shaft, a small number of thorny excrescences and relatively less dendritic branching in both the apical and basilar trees. The somata of short-shaft pyramidal neurons tend to be located higher in stratum pyramidale than the somata of long-shaft neurons. Quantitative measurements, which included both analysis of dendritic branching and the distribution of dendritic material sampled at 10% intervals from the cell body, confirmed the qualitative observation that short-shaft neurons had more total dendritic length than long-shaft neurons. The difference in the total dendritic length observed between long- and short-shaft pyramidal neurons could be an indication that each type receives a different number of synapses per neuron. The less commonly observed variants of pyramidal neurons were briefly described but not quantified. This study demonstrates that CA3 pyramidal neurons are not a homogeneous group but that their heterogeneous characteristics fall into two major categories.     

Calcium-dependent alterations in dendritic architecture of hippocampal pyramidal neurons

Dendritic arbor formation and the underlying mechanisms are crucial for the functional connectivity and plasticity of neurons. We used a focal electric field to locally raise calcium levels in individual dendritic shafts of isolated hippocampal pyramidal neurons, in order to develop an accessible system for studying dendritic branch formation, and to test the role of calcium as an intrinsic signal that may participate in arborization. Filopodia were induced in a manner temporally and spatially related to induced calcium rises. Certain filopodia also thickened and were transformed into dendritic branches. These results suggest that calcium-mediated signaling can induce branching in dendrites, and describe an accessible system for studying the intracellular machinery that drives dendritic arborization.     

Chronic (-) deprenyl administration alters dendritic morphology of layer III pyramidal neurons in the prefrontal cortex of adult Bonnett monkeys

Chronic (-) deprenyl (0.2 mg/kg, b.wt; for 25 days) treatment induced alterations in the dendritic morphology of prefrontal cortical neurons in adult Bonnett monkeys were evaluated in the present study. The branching points and intersections in apical and basal dendrites were studied up to a distance of 400 and 200 micrometers, respectively, in Golgi impregnated layer III pyramidal neurons of the prefrontal cortex. Our results revealed a significant (p<0.001) increase in the number of branching points and intersections in both apical and basal dendrites in (-) deprenyl treated monkeys compared to controls. Such an enriched dendritic arborization in prefrontal cortical neurons may be responsible for the enhancement of cognitive functions in Alzheimer disease patients following (-) deprenyl treatment.     

Alterations in dendritic morphology of hippocampal neurons in adult rats after neonatal administration of N-omega-nitro-L-arginine

The dendritic length and dendritic-spine density of the pyramidal neurons of the prefrontal cortex and the CA1 hippocampus of rats using the nonselective nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NNA) at different postnatal day (P) periods of the brain development (P1-P3, P4-P6, and P7-P9) were assessed using Golgi-Cox staining after puberty (P60). At P4-P6, the L-NNA treatment produced a significant decrease of the dendritic length and dendritic-spine density of the pyramidal cells of the CA1 hippocampus. In addition, the dendritic length of the pyramidal neurons of the CA1 hippocampus decreased because of the L-NNA treatment at P1-P3. These data suggest that during a specific step in the development of the brain, the nitric oxide levels may play a critical role in the morphological modifications of the pyramidal neurons of the CA1 hippocampus at postpubertal age.     

The dendritic morphology of pyramidal neurons in the rat hippocampal CA3 area. II. Effects of gender and the environment

Sex differences in the dendritic structure of hippocampal CA3 pyramidal neurons were studied in Golgi-stained tissue from hooded rats that had been raised in either a relatively complex or an isolated environment from weaning for one month. Pyramidal neurons were sampled from both the short-shaft and long-shaft neuron categories as described by Fitch et al. The pattern of sex differences varies in different parts of the apical dendritic tree. In the apical tree proximal to the soma, females had more dendritic material than males. This pattern was attributable to the sex differences in the short-shaft neurons of rats from the more complex environment. The direction of sex differences was reversed in the distal apical dendritic tree where males had more dendritic material than females. As in the proximal dendritic tree, this pattern of sex differences stemmed from the short-shaft neurons of rats from the more complex environment. There were no sex differences in the basilar dendritic tree. Thus sex differences in the pyramidal neurons of hippocampal area CA3 vary with the portion of the dendritic tree examined, the type of pyramidal neuron, and the rearing environment of the animal.     

Effects of adult dysthyroidism on the morphology of hippocampal neurons

This study investigates the effect of thyroid hormones on the morphology of hippocampal neurons in adult rats. Hypo- and hyperthyroidism were induced by adding 0.02% methimazole and 1% l-thyroxine, in drinking water from 40 days of age, respectively. When the rats were 89 days old their brains were removed and stained by a modified Golgi method and blood samples were collected in order to measure T4 serum levels. Neurons were selected and drawn using a camera lucida. Our results show that methimazole administration reduces the dendritic branching of the apical shafts of CA3 and CA1 pyramidal neurons mainly by increasing the distance to the first branch point in both types of neurons, and reducing branch points in the radius of 50 microm from the soma in CA1 neurons. Nevertheless, it was observed an increase of apical spine density in CA3 neurons from this group. Thyroxine reduces apical and basal tree of CA3 pyramidal neurons increasing the distance to the first branch point, reducing branch points in the radius of 50 microm from the soma and increases their apical and basal spine density. In CA1 field, thyroxine reduces the number of basal branch points. Both treatments seems to provoke alterations in the same direction reducing the dendritic branching and increasing spine density, although no significances appeared in some of the parameters analyzed. The effects are more evident in thyroxine than methimazole group; and in CA3 neurons than in CA1 neurons. In discussion it is pointed that the increase of spine density could be a mechanism to compensate the functionality reduction that can be provoke by the treatment effect on dendritic branching.     

Activated natural killer cells adhere to cultured hippocampal neurons and affect the dendritic morphology

To examine the manner of interactions between immune cells and central nervous system (CNS) neurons, mouse hippocampal neurons were co-cultured with lymphokine (IL-2)-activated killer (LAK) cells. Immunocytochemical and time-lapse observations indicated that LAK cells migrated along neuronal processes and made adhesive contacts with them. In addition to the direct physical effects, LAK cells released glutamate, induced the formation of beads-like structure in the dendrites of about 14% of hippocampal neurons and caused the reduction of dendritic protrusions. These results suggest that infiltrating immune cells can form direct adhesive connections with CNS neurons and affect their dendritic morphology.     

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca2+‐permeable TRPC6 channels

The standardized extract of the St. John's wort plant (Hypericum perforatum ) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na⁺ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca²⁺‐permeable, resulting in intracellular Ca²⁺ elevations. Indeed, hyperforin activates TRPC6‐mediated currents and Ca²⁺ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca²⁺ transients and depolarizing inward currents sensitive to the TRPC channel blocker La³⁺, thus resembling the actions of the neurotrophin brain‐derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF. © 2012 Wiley Periodicals, Inc.     

Glutamate-stimulated protein phosphorylation in cultured hippocampal pyramidal neurons.

The regulation of second-messenger production and protein phosphorylation by glutamate has been investigated in primary cultures of pure hippocampal pyramidal neurons. Embryonic rat pyramidal neurons were prepared according to the procedures of Bartlett and Banker (1984) and studied 1-21 d after plating. Glutamate caused a transient increase in intracellular free [Ca2+], determined with fura-2, in the presence of 1.26 mM extracellular Ca2+, but not in 50 nM free Ca(2+)-containing solution. Glutamate also transiently increased cellular diacylglycerol content in both normal and low-[Ca2+] media. Neurons were prelabeled with 32P-orthophosphate to label intracellular ATP, then stimulated with glutamate (100 microM). A rapid transient incorporation of 32P into primarily three proteins of 120, 87, and 48 kDa was found by analysis of two-dimensional gels. At 30 sec after glutamate stimulation, 32P incorporation into the 87-kDa and 48-kDa proteins peaked (240% and 170% basal levels, respectively), and by 2 min, phosphorylation of the 87-kDa protein had returned to basal levels, while that of the 48-kDa protein decreased but remained above control levels. The phosphorylation of these proteins appeared to be mediated by protein kinase C (PKC) because all three showed an increase in phosphorylation after phorbol ester treatment of cultures. Phosphate incorporation was accompanied by an acidic shift in the isoelectric point of both 87- and 48-kDa proteins. Glutamate stimulation resulted in phosphorylation in the presence and absence of Ca2+ influx. Antibody recognition and biochemical characteristics indicated that the 87-kDa phosphoprotein is the PKC substrate MARCKS (myristoylated, alanine-rich C-kinase substrate). The 48-kDa protein, though very similar to GAP-43, was not recognized by specific antibodies raised against GAP-43. These results suggest that glutamate stimulates the transient generation of second messengers that activate PKC in hippocampal neurons, resulting in a significant increase in the phosphorylation of three specific proteins.     

Hypobaric hypoxia induces dendritic plasticity in cortical and hippocampal pyramidal neurons in rat brain

Hypobaric hypoxia (HH), a predisposing environmental condition at high altitude (HA) encountered by many mountaineers jeopardizes their normal physiology like motor coordination and cognitive functions. Our previous studies revealed that the HH induces oxidative stress and neurodegeneration, which is associated with spatial memory impairment in rats. However, the dendritic changes after exposure to different duration of HH remain largely unknown. The aim of the present study was to investigate the duration-dependent dendritic changes in CA1, CA3 and entorhinal cortex (EC) of hippocampus and layer II of prefrontal cortex (PFC) with spatial memory functions in rats on exposure to different duration of HH. The rats were exposed to simulated HA of 6100 m for 3, 7, 14 and 21 days and the spatial reference memory was investigated using Morris water maze (MWM) and the morphological alteration of CA1, CA3, EC and layer II of PFC were investigated. There was a significant decrease in dendritic arborization and spine number along with increased number of damaged neurons, after 3, 7 and 14 days of HH but after 21 days of HH exposure the structural recovery was noted in all the regions. There was impairment of spatial memory after 3 and 7 days of exposure, but slight improvement of spatial memory was noted after 14 and 21 days of exposure. Our studies suggested that HH induces dendritic plasticity of PFC and hippocampal pyramidal neurons of rat brain, which might be associated with improvement of spatial memory function after 21 days of HH exposure.     

Acetylcholine induced modulation of hippocampal pyramidal neurons

Applications of acetylcholine to hippocampal slices maintained in vitro resulted in slow depolarizations and simultaneous increases in membrane resistance (R_N) in hippocampal pyramidal neurons. Increases in R_N had both voltage dependent and voltage independent components. These effects were associated with increases in cell discharge frequency, and development of spontaneous as well as synaptically and directly evoked burst discharges. The increase in R_N and burst firing lasted for hours. Muscarinic antagonists blocked these actions and in addition, produced a decrease in membrane resistance, which appeared to be due to blockade of a tonic effect of acetylcholine on postsynaptic membrane properties. These findings suggest that ACh acts as a neuromodulator in the hippocampus.     

Alterations in the dendritic morphology of prefrontal pyramidal neurons in adult rats after blockade of NMDA receptors in the postnatal period

The present study assessed whether the blockade of NMDA receptors in the postnatal period, used to model the symptoms of schizophrenia altered morphology of pyramidal neurons in the medial prefrontal cortex of rats. CGP 40116, an antagonist of NMDA receptors, was given postnatally (days 1-21 after birth). The analysis of the morphology of pyramidal neurons visualized by the Golgi-Cox technique revealed that the exposure to an antagonist of NMDA receptors in the postnatal period diminished the length of basilar dendrites, while that of apical dendrites remained unchanged. The number of dendritic branches and the spine density remained unchanged. It is concluded that the blockade of NMDA receptors in the postnatal period only partially models morphological changes in pyramidal neurons of the medial prefrontal cortex, which are observed in some cases of schizophrenia.     

Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons.

The shape of dendritic trees and the density of dendritic spines can undergo significant changes during the life of a neuron. We report here the function of the small GTPases Rac and Rho in the maintenance of dendritic structures. Maturing pyramidal neurons in rat hippocampal slice culture were biolistically transfected with dominant GTPase mutants. We found that expression of dominant-negative Rac1 results in a progressive elimination of dendritic spines, whereas hyperactivation of RhoA causes a drastic simplification of dendritic branch patterns that is dependent on the activity of a downstream kinase ROCK. Our results suggest that Rac and Rho play distinct functions in regulating dendritic spines and branches and are vital for the maintenance and reorganization of dendritic structures in maturing neurons.     

慢性应激对大鼠海马CA3区锥体细胞形态结构的效应

目的　探讨慢性应激对大鼠海马CA3区锥体细胞形态结构的效应。方法　将 2 6只雄性Sprague Dawley大鼠随机分为对照组和应激组 ,每组 13只。采用尼氏 (Nissl)染色法、高尔基 (Golgi)镀染法和透射电镜技术 ,观察慢性强迫游泳应激对大鼠海马CA3区锥体细胞形态结构的效应。结果应激组大鼠海马CA3区锥体细胞数 (35 1± 3 9)较对照组 (38 7± 3 5 )明显减少 (P <0 0 5 ) ;顶树突的总长度为 (15 5 7± 33 3) μm ,较对照组 (195 6± 34 6 ) μm明显缩短 (P <0 0 5 )。应激组大鼠海马CA3区锥体细胞出现超微结构的改变 ,包括细胞固缩、体积缩小、核膜皱缩、线粒体变性和粗面内质网模糊不清。结论　慢性应激可引起海马CA3区锥体细胞形态和微细结构的改变及细胞丧失。     

Insulin inhibits pyramidal neurons in hippocampal slices

The effect of bombesin, applied intraventricularly in the rat, was examined with regard to the central control of behavior and arousal. Infusion of 1.0 micrograms bombesin produced stereotypic grooming activity and completely eliminated observable sleep. EEG frequency analysis confirmed the absence of sleep and demonstrated normal waking patterns during behavioral stereotypy. These results support a possible physiologic function for bombesin-like peptides in grooming and the sleep-waking cycle.