Maternal creatine supplementation affects the morpho-functional development of hippocampal neurons in rat offspring.

Creatine supplementation has been shown to protect neurons from oxidative damage due to its antioxidant and ergogenic functions. These features have led to the hypothesis of creatine supplementation use during pregnancy as prophylactic treatment to prevent CNS damage, such as hypoxic-ischemic encephalopathy. Unfortunately, very little is known on the effects of creatine supplementation during neuron differentiation, while in vitro studies revealed an influence on neuron excitability, leaving the possibility of creatine supplementation during the CNS development an open question. Using a multiple approach, we studied the hippocampal neuron morphological and functional development in neonatal rats born by dams supplemented with 1% creatine in drinking water during pregnancy. CA1 pyramidal neurons of supplemented newborn rats showed enhanced dendritic tree development, increased LTP maintenance, larger evoked-synaptic responses, and higher intrinsic excitability in comparison to controls. Moreover, a faster repolarizing phase of action potential with the appearance of a hyperpolarization were recorded in neurons of the creatine-treated group. Consistently, CA1 neurons of creatine exposed pups exhibited a higher maximum firing frequency than controls. In summary, we found that creatine supplementation during pregnancy positively affects morphological and electrophysiological development of CA1 neurons in offspring rats, increasing neuronal excitability. Altogether, these findings emphasize the need to evaluate the benefits and the safety of maternal intake of creatine in humans.

Maternal dietary loads of alpha-tocopherol increase synapse density and glial synaptic coverage in the hippocampus of adult offspring.

An increased intake of the antioxidant α-Tocopherol (vitamin E) is recommended in complicated pregnancies, to prevent free radical damage to mother and fetus. However, the anti-PKC and antimitotic activity of α-Tocopherol raises concerns about its potential effects on brain development. Recently, we found that maternal dietary loads of α-Tocopherol through pregnancy and lactation cause developmental deficit in hippocampal synaptic plasticity in rat offspring. The defect persisted into adulthood, with behavioral alterations in hippocampus-dependent learning. Here, using the same rat model of maternal supplementation, ultrastructural morphometric studies were carried out to provide mechanistic interpretation to such a functional impairment in adult offspring by the occurrence of long-term changes in density and morphological features of hippocampal synapses. Higher density of axo-spinous synapses was found in CA1 stratum radiatum of α-Tocopherol-exposed rats compared to controls, pointing to a reduced synapse pruning. No morphometric changes were found in synaptic ultrastructural features, i.e., perimeter of axon terminals, length of synaptic specializations, extension of bouton-spine contact. Glia-synapse anatomical relationship was also affected. Heavier astrocytic coverage of synapses was observed in Tocopherol-treated offspring, notably surrounding axon terminals; moreover, the percentage of synapses contacted by astrocytic endfeet at bouton-spine interface (tripartite synapses) was increased. These findings indicate that gestational and neonatal exposure to supranutritional tocopherol intake can result in anatomical changes of offspring hippocampus that last through adulthood. These include a surplus of axo-spinous synapses and an aberrant glia-synapse relationship, which may represent the morphological signature of previously described alterations in synaptic plasticity and hippocampus-dependent learning.

Physical exercise and environment exploration affect synaptogenesis in adult-generated neurons in the rat dentate gyrus: possible role of BDNF.

A brief training in a pool maze, with or without cognitive tasks, modifies the synaptogenesis and maturation of newborn neurons in adult rat dentate gyrus. These types of trainings have many aspects, including physical activity and exploration. Therefore, to evaluate whether physical exercise and environment exploration are able to affect synapse formation and the maturation of adult-generated neurons, GFP-retrovirus infusion was performed on rats which, on the fourth day after injection, were housed under running conditions or allowed to explore an enriched environment briefly in the absence of exercise for the following three days. Afterward, at the end of the trainings, electrophysiological and morphological studies were conducted. Considering that neurotrophic factors increase after exercise or environment exploration, hippocampal BDNF levels and TrkB receptor activation were evaluated. In this study, we show that both spontaneous physical activity and enriched environment exploration induced synaptogenesis and T-type voltage-dependent Ca(2+) currents in very immature neurons. Hippocampal BDNF levels and TrkB receptor activation were determined to be increasing following physical activity and exploration. A possible contribution of BDNF signaling in mediating the observed effects was supported by the use of 7-8-dihydroxyflavone, a selective TrkB agonist, and of ANA-12, an inhibitor of TrkB receptors.

Surface and inner cell behaviour along skeletal muscle cell in vitro differentiation.

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3-4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of "myosacs" containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell-cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

Bdnf expression in rat skeletal muscle after acute or repeated exercise.

Brain derived growth factor (BDNF) gene of rat has a complex structure: at least four 5' untranslated exons regulated by different promoters and one 3' exon containing the encoding region. BDNF is expressed by skeletal muscles in an activity-dependent manner. In this study, BDNF mRNA was analysed by RT-PCR in the soleus muscle following a single (acute) session of running or a training of five days of running (repetitive exercise). Moreover, the expression of the exons was quantitatively analysed by real time RT-PCR. Finally, muscle BDNF protein level was evaluated by western blotting. BDNF mRNA was found to increase over the second day after acute exercise; on the other hand, two peaks (2 and 24 hours after the last session, respectively) in BDNF mRNA level were found after repetitive exercise, but it was similar to that of controls 6 hours after the last session. BDNF protein level progressively increased also after the mRNA went back to the basal level, so suggesting that it cumulates within the cell after acute exercise, whereas it followed the mRNA level time course after repetitive exercise. These results point to the following conclusions: BDNF mRNA is up-regulated by activity, but this response is delayed to the second day after acute exercise; repetitive exercise transiently depresses the expression of BDNF mRNA, so that the over-expression due to the previous day's exercise completely disappears 6 hours after the last exercise session.

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus.

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.

The role of sensory input in motor neuron sprouting control.

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.

Glucose-6-phosphate dehydrogenase supports the functioning of the synapses in rat cerebellar cortex.

This study investigates heterogeneous glucose-6-phosphate dehydrogenase (G6PD) expression in the rat cerebellar cortex. G6PD activity and its electrophoretic pattern, evaluated on the cerebellar homogenate, were found to be similar to those of other brain areas. However, histochemical and immunohistochemical analyses revealed that the highest expression of G6PD activity and protein was in Purkinje's cells, followed by the molecular and granular layers. Electron microscopy analysis showed that, in Purkinje's cells, the G6PD reaction products were concentrated in the neurites while in the basket cells in the cell body. The granules showed a weaker activity everywhere. The quantitative distribution of G6PD is discussed in the light of the neurochemical function of these cells.

Alpha-tocopherol controls cell proliferation in the adult rat dentate gyrus.

The effect of alpha-tocopherol on cell proliferation and proliferated cell survival was investigated in the dentate gyrus of adult rats. Adult rats were supplemented with alpha-tocopherol, injected with 5-bromo-2'-deoxyuridine (BrdU), that is incorporated into DNA during the S-phase, and killed at different time after BrdU injection. The number of newborn cells decreased after alpha-tocopherol supplementation, confirming the hypothesis that alpha-tocopherol is able to depress cell proliferation in vivo. Most newborn cells die within few days; more newborn cells survive in alpha-tocopherol-treated rats, suggesting the hypothesis that alpha-tocopherol decreases cell death.

Spatial learning affects immature granule cell survival in adult rat dentate gyrus.

Neurogenesis occurs throughout life in mammalian dentate gyrus. The effect of learning on newborn cell survival was studied in rat. Rats were trained on a hippocampus-dependent spatial learning task by using Morris water maze. Neurogenesis was evaluated by 5-bromo-2'deoxyuridine administered before learning. Several newborn cells expressed the immature neuron marker TOAD-64. The main findings were as follows: (1) the survival of newborn cells was enhanced by learning at early stage of differentiation; (2) the newborn cells saved by learning were mainly located in the rostral part of external blade of granule cell layer and (3) there was a correlation between the actual individual learning and newborn cell survival.

Are there proliferating neuronal precursors in adult rat dorsal root ganglia?

The origin of new neurons in dorsal root ganglia of adult rat was investigated using an experimental model in which postnatal neurogenesis naturally occurring is enhanced and restricted in a brief period of life. Possible mitotic origin of new neurons was investigated by means of 5-bromo-2-deoxyuridine, anti-NF 200 antibody was used to detect if proliferated cells showed a neuronal phenotype. The results suggest that postnatal neurogenesis in dorsal root ganglia could depend only in part on precursor proliferation and that normally new neurons derive from the late differentiation of postmitotic cells.

Rat motor neuron plasticity induced by dorsal rhizotomy.

The control of peripheral structural plasticity of motor neurons by primary sensory neurons was studied in rat extensor digitorum longus (EDL) muscle. Polyinnervation of muscle fibers, sprouting and the motor neuron peripheral field size following L4 dorsal root cutting were evaluated using three different approaches: intracellular recording of end plate potentials, histochemical demonstration of sprouting and polyinnervation and in vivo recording of nerve-evoked twitch. Nodal sprouting was found in rhizotomized rats but not in controls and consistently muscle polyinnervation appeared. The muscle portion innervated by L3 ventral root was relatively reduced and that innervated by L5 was relatively enlarged: a trend to caudal shift of muscle innervation arose in rhizotomized rats. A control of motor neuron plasticity by primary sensory neurons is suggested.

Postnatal proliferation of DRG non-neuronal cells in vitamin E-deficient rats.

Changes in the number of satellite cells in neuron body sheaths in dorsal root ganglia (DRGs) were studied from 1 to 5 months of age in control and in vitamin E-deficient rats; furthermore, the satellite cell proliferation rate was detected in the same groups of animals with immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). The number of satellite cells in sheaths of DRG neurons increased in the period of life considered both in control and in vitamin E-deficient rats. Satellite cell proliferation was observed in both groups, but its rate was found to be higher in vitamin E-deficient rats. The results obtained in control rats confirm that mitotic ability is retained by satellite cells in adulthood and show that at least some of newborn satellite cells add to the pre-existing population. The results obtained in vitamin E-deficient rats suggest that a faster turnover in satellite cell population takes place in these animals and support the idea that vitamin E could be an exogenous factor controlling cell proliferation.

Neurogenesis in the adult rat dentate gyrus is enhanced by vitamin E deficiency.

Neurogenesis occurs throughout adult life in rat dentate gyrus. Factors and mechanisms of adult neurogenesis regulation are not well known. Vitamin E deficiency has been found to deliver a neurogenetic potential in rat dorsal root ganglia. To determine whether the role of tocopherols in adult neurogenesis may be generalized to the central nervous system, changes in adult rat dentate gyrus neurogenesis were investigated in vitamin E deficiency. Neurogenesis was quantitatively studied by determination of the density of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells and by determination of the total number of cells in the granule cell layer. The BrdU-labeled cells were immunocytochemically characterized by demonstration of neuronal marker calbindin D28K. The following results were found: (1) the volume of the granule layer increased in controls from 1 to 5 months of age, mainly due to cell density decrease; (2) the volume increased by a similar amount in vitamin E-deficient rats, mainly because of an increase in cell number; (3) BrdU-positive cells were more numerous in vitamin E-deficient rats in comparison to age-matched controls; (4) the increase in proliferated cells was located in the hilus and in the plexiform layer. This study confirms that neurogenesis occurs within adult dentate gyrus and demonstrates that this process is enhanced in vitamin E deficiency. This finding indicates that vitamin E may be an exogenous factor regulating adult neurogenesis.

Control of neuron outgrowth by NMDA receptors.

The role of N-methyl-D-aspartic acid receptors (NMDARs) of glutamate on neuritogenesis was studied in cultured neurons of chick embryo spinal cord using the NMDAR non-competitive antagonist dizocilpine maleate (MK-801). No cell population was fully prevented from neuritogenesis by MK-801. Different aspects of neuritogenesis were quantitatively evaluated. Neurite initiation, elongation and branching were depressed by MK-801. Inhibition was dose-dependent and reversible. A loss of responsiveness of neuritogenesis to MK-801 was found during the second day of treatment at a concentration of 10 microM, but not at higher concentrations. Our findings support the idea that Ca2+ influx through NMDAR associated channels is one of the possible triggers of a cascade resulting in neuritogenesis. The effects of NMDAR blocking on neuritogenesis occurred before synaptogenesis, suggesting a role of excitatory aminoacids in neuron morphological differentiation at early stages of development. Scanning electron microscopy confirmed a reduction in neurite tree complexity in MK-801 treated cells and showed a production of filopodium-like processes in some of these cells.

Neuron and glial cells in neocortex after methylazoxymethanol treatment in early development.

The quantitative changes were investigated in neuron and glia density in the different cortical layers of the frontal cortex of 3 and 12 month old mice, exposed to methylazoxymethanol on embryonic day 13 (MAM13). No loss of cortical neurons was found between young and adult animals. MAM exposure on the 13th day of development induced a neuron density decrease throughout on the entire cortical depth and did not produce changes in the density of glial cells with respect to the controls and to age. Consequently, at 3 months of age we observe a glia/neuron ratio greater than that of controls and at 12 months a similar value. In the neocortex of MAM-mice at this numerical uniformity of glial cell density, did not correspond to a similar proportional composition: the frequency of the astrocytes is lower, adapting to the decreased neuron density; the greater oligodendrocyte percentage may be related to disturbed layering and to the hyperinnervation of the hypoplastic cortex; the microglia shows a trend similar to that of the controls. These results, together with those of other studies, suggest that prenatal exposure to MAM causes a cortical compensatory response regulating glial cells proliferation.

Enlargement of motoneuron peripheral field following partial denervation with or without dorsal rhizotomy.

In partially denervated skeletal muscle, spared motor fibres sprout, enlarging motor unit size. Neuritogenesis and sprouting are known to depend on the synaptic input to the neurons. This suggests that spared motoneuron reaction to partial muscle denervation might be controlled by primary sensory neurons which directly or indirectly project to motoneurons. In two groups of rats, different surgical procedures were carried out: partial denervation of the extensor digitorum longus muscle without or with homolateral dorsal rhizotomy. Spared motoneuron peripheral field was evaluated by nerve-evoked tension measures. Following partial muscle denervation, spared motoneurons enlarged their projection peripheral field five to six times, innervating most of the denervated portion of the muscle. When dorsal rhizotomy was carried out together with partial denervation, the enlargement of the motoneuron's peripheral field occurred later; however, the peripheral field size was the same or greater than that found in partially denervated muscles without dorsal rhizotomy in the long term. Excitatory postsynaptic potential recordings at neuromuscular junctions consistently showed that innervation of denervated muscle cells by spared motoneurons was impaired when the dorsal roots were cut. Finally, in both groups of operated rats an increase in motor unit number occurred early after surgery, anticipating a process normally occurring in the same age range. These findings are consistent with the idea that sensory input trans-synaptically controls motoneuron peripheral field size.

Developmental changes in innervation of rat extensor digitorum longus muscle.

Motor neurons and ventral root motor fibres innervating extensor digitorum longus (EDL) muscle in rats of 0.5-3.5 months of age were studied by HRP-retrograde labelling and in vivo muscle tension recording, respectively. EDL nucleus size increased with age but motor neurons number and size did not change. Twitch and tetanus tension increased with age proportionally to muscle mass. At 0.5 months of age, but not at subsequent ages, the muscle was incompletely innervated functionally. The contribution to EDL muscle innervation came from L3, L4 (pre-eminently), and L5 ventral roots. The number of motor fibres running in L3-L5 ventral roots and innervating EDL muscle increased from 41.7 +/- 2.2 (mean +/- S.E.M.) at 0.5 months to 68.6 +/- 1.9 at 3.5 months (P < 0.001). The greatest changes in m.w., in tension and in number of alpha nerve fibres innervating the muscle occurred from 0.5 to 1.5 months of age; afterwards changes occurred, but at a slower rate. HRP labels all neurons innervating EDL muscle, while tension recordings allow the counting of alpha nerve fibres (not gamma) running in ventral roots; these differences may account for the different results obtained.

Quantitative changes in neuron and glial cells of neocortex following prenatal exposure to methylazoxymethanol.

The quantitative changes in neuron and glial cell density were analyzed in different cortical layers from frontal cortex of 3 and 12 months old mice exposed to Methylazoxymethanol (MAM) on embryonic day 15 (MAM 15) or 17 (MAM 17). An increase was found in glial cell density in all cortical layers from 3 months old MAM 17 compared to the age-matched controls. In particular, electron microscopic analysis showed that the only cells to increase in all cortical layers were the microglia cells. When the young and adult mice were compared it was noted that the glia increased significantly in all cortical layers of controls, whereas it did not change the older MAM 17. In the older MAM 15 the increase in glial density was similar to that observed in controls. No loss of cortical neurons was found between young and adult animals. In addition, the different trend of glial cell density between control and MAM 17 mice during aging was accompanied by a decline in the mean nuclear area in neurons of treated mice with respect to the controls. The hypothesis that MAM treatment seems to produce events similar to those normally occurring in aging finds support in the data of glial density and neuron size.