Protection Effect of GDNF and Neurturin on Photosensitized Crayfish Neurons and Glial Cells

Neurons and glial cells can protect each other from stress and following death by mutual exchange with neurotrophins. In order to examine involvement of different neurotrophic factors in neuroglial interactions in a photosensitized crayfish stretch receptor, a simple model object consisting of only two sensory neurons enveloped by glial cells, we studied the influence of glial cell line-derived neurotrophic factor (GDNF), neurturin, and ciliary neurotrophic factor (CNTF) on its photodynamic injury. Photodynamic treatment, which causes strong oxidative stress, induced firing abolition and necrosis of neurons, necrosis, and apoptosis of glial cells. GDNF significantly reduced photoinduced neuronal necrosis and neurturin but not CNTF showed a similar tendency. Both of them significantly reduced necrosis and apoptosis of glial cells. At the ultrastructural level, neurons and glial cells treated with GDNF in the darkness contained large mitochondria with well-developed cristae, numerous ribosomes, polysomes, rough endoplasmic reticulum (ER), and dictyosomes. This indicated the high level of bioenergetic, biosynthetic, and transport processes. Photodynamic treatment caused swelling and vacuolization of mitochondria, dictyosomes, and ER. It also impaired formation of glial protrusions and double membrane vesicles that transfer glial material into the neuron. GDNF prevented photoinduced mitochondria swelling that disturbed the cellular bioenergetics and cytoplasm vacuolization associated with injury of intracellular organelles. It also preserved the structures involved in protein synthesis and transport: rough ER, dictyosomes, polysomes, microtubule bundles, submembrane cisterns, and double membrane vesicles. GDNF-mediated maintenance of metabolism and ultrastructure of photosensitized neurons and glial cells may be the basis of its neuro- and glia protective effects.     

Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.     

Developmental Changes in Neuronal Responsiveness to the CNS Myelin-associated Neurite Growth Inhibitor N I-35/250

The extent of fibre regeneration in the adult injured vertebrate nervous system appears to be primarily determined by the local environment. Thus, the failure of axon regrowth in the central nervous system (CNS) is crucially influenced by the presence of the myelin-associated neurite growth inhibitor Nl-35/250 and possibly also by molecules such as the myelin-associated glycoprotein and the proteoglycans. Developmental time course studies have shown that the capacity for regeneration declines sharply with the appearance of mature oligodendrocytes and myelin, which indicates a role of Nl-35/250 in restricting CNS regeneration and plasticity. However, recent in vitro and in vivo studies showed that embryonic neurons are capable of extending fibres on and in adult CNS tissue apparently unaffected by myelinated areas. A possible explanation is that very immature neurons have yet to express the appropriate receptors and response mechanisms for factors that normally induce growth inhibition at a later stage of development. Here we report that embryonic rat dorsal root ganglion and chick retinal ganglion cells display different sensitivity to bovine Nl-35/250 compared with mature neurons. In older neurons Nl-35/250 could evoke long-lasting collapse responses accompanied by a large increase in the intracellular calcium level, persisting for several minutes. In contrast, their embryonic counterparts collapsed only transiently when exposed to Nl-35/250, and increases in intracellular calcium concentration were small and transient. Calcium influx induced experimentally by the calcium ionophore A23187 revealed that it was not the maximal size of the calcium increase but rather the duration of elevated calcium concentration that was the most important determinant for subsequent morphological alterations of the growth cone. Our data further suggest that developing neurons acquire their complete sensitivity for Nl-35/250 around the time of myelination.     

Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro

Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.     

Effects of GDNF on Axotomized Sensory and Motor Neurons in Adult Rats

Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor shown to rescue developing and adult motoneurons in vitro and in vivo from programmed and injury-induced cell death. To test whether GDNF would rescue adult mammalian sensory and motor neurons from physiological consequences of injury, the tibial nerve of rats was axotomized and, after a 10 day delay to permit injury processes to proceed, vehicle or GDNF was supplied directly to the nerve for 2 or 4 weeks or GDNF intrathecally for 2 weeks. Conduction velocity (CV) of both sensory and motor axons declined during the initial 10 days, and even more so if then treated with vehicle. Treatment with GDNF resulted in marginal improvement of sensory axon CV. CV of motor axons recovered significantly in a dose- and time-dependent manner. The results suggest that GDNF may have therapeutic potential in the treatment of peripheral neuropathies.     

Developmental Changes in Spontaneous GABAA-mediated Synaptic Events in Rat Hippocampal CA3 Neurons

Ongoing spontaneous postsynaptic potentials (SPSPs) were intracellularly recorded at 34-36°C from hippocampal CA3 neurons in slices obtained from postnatal days (P) 0 – 6 and 7 – 31. SPSPs occurred randomly, and their frequency distribution was fitted by a single exponential function. They were little affected by kynurenic acid, but were reversibly blocked by bicuculline, implying that they were mediated by GABA_A receptors. The mean amplitude was 4.53 ± 0.89 mV in control conditions and 4.07 ± 0.79 mV in kynurenic acid. In kynurenic acid (with CsCl-filled microelectrodes), SPSPs reversed polarity at 2.4 ± 2 mV. When tetrodotoxin (1 μM) was added to kynurenic acid solution, GABA_A-mediated miniature postsynaptic potentials (MPSPs) were recorded. Under these conditions large events disappeared. The mean amplitude of MPSPs was 2.51 ± 0.43 mV. The mean frequency decreased from 2.96 ± 1.04 Hz in kynurenic acid to 0.4 ± 0.15 Hz in kynurenic acid plus tetrodotoxin. In contrast with P0 – P6, at P7 – P31 SPSPs were significantly affected by kynurenic acid. The mean amplitude of SPSPs shifted from 4.71 ± 0.82 mV in control conditions to 3.79 ± 0.76 mV in kynurenic acid. At this developmental stage, the reversal potential of GABA_A-mediated SPSPs shifted towards more negative values (-23.7 ± 1.3 mV). Addition of tetrodotoxin to kynurenic acid solution abolished larger events and revealed GABAergic MPSPs. The mean amplitude of MPSPs was 2.72 ± 0.5 mV, a value very close to that observed at P0 – P6. Synaptic currents were recorded at 22 – 24°C from voltage-clamped CA3 pyramidal neurons (at P6) using the tight-seal whole-cell recording technique. Cells were dialysed with CsCl solution and held at -70 mV. Spontaneous GABA_A-mediated miniature postsynaptic currents (MPSCs) were recorded in the presence of kynurenic acid and tetrodotoxin. The decay time of MPSCs was fitted with a single exponential and was 29 ± 3 ms. No correlation was found between the peak amplitude of individual events and their rise or decay time constant. The mean amplitude distribution of MPSCs was 12 ± 4.3 pA. In outside-out patches from acutely dissociated CA3 hippocampal neurons, GABA (50 μM) activated single-channel events of 24 and 35 pS conductance. Therefore one quantal current represents the simultaneous opening of five to seven GABA_A receptor channels on the postsynaptic cell. These data show also that in the immediate postnatal period spontaneous glutamatergic potentials are poorly developed and start appearing towards the end of the first postnatal week concomitant with the shift of GABA from the depolarizing to the hyperpolarizing direction.     

Learning-related Alterations in the Visual Responsiveness of Neurons in a Memory System of the Chick Brain

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain is known to be essential for the learning process of imprinting. The activity of neurons was recorded from the left IMHV of 2-day-old unanaesthetized chicks while the chicks were free to move in a running wheel. The chicks were either raised in complete darkness or visually trained (imprinted) with a set duration of exposure to a visual image. The first group of these birds was trained by exposure for 100 min to a rotating red box and the second was trained by similar exposure to a rotating blue cylinder. A third group was left untrained. Training more than doubled the proportion of sites that responded to the stimulus used to train the bird, relative to the proportion of sites responsive to the other stimulus and to the proportion of sites responsive in the untrained birds; the learning-related increase was selective and highly significant. Behavioural monitoring indicated that the enhanced responsiveness could not be explained by overt differences in the alertness, attentiveness or movements of the birds. No significant effect of training was found on the proportion of sites responsive to a rotating stuffed jungle fowl or to the sound of a maternal call. The response at certain sites selectively signalled the presence of the training stimulus, while at others the response showed generalization across stimulus shape or colour. There was a non-specific effect of training upon the pattern of spontaneous discharges of the neurons: the numbers of spikes occurring in clusters (bursts) was significantly reduced in trained birds compared with the dark reared controls.     

实验性癫癇大鼠海马内GDNFmRNA表达的变化

目的 :为探讨癫活动对大鼠GDNF基因表达的影响。方法 :应用同位素标记的原位杂交研究海人藻酸致后大鼠海马区GDNFmRNA表达时相的变化。结果 :正常大鼠海马未见GDNFmRNA表达 ,而癫大鼠海马神经元在致后 4h以后出现GDNFmRNA表达强烈的上调反应 ,12h达高峰 ,且各区均有表达 ,此后逐渐衰减 ,2 4h恢复正常。结论 :癫后内源性GDNFmRNA表达上调很可能是神经元对抗兴奋性损害的一种保护效应     

Chronic Hyperprolactinemia and Changes in Dopamine Neurons

The tuberoinfundibular dopaminergic (TIDA) system is known to inhibit prolactin (PRL) secretion. In young animals this system responds to acute elevations in serum PRL by increasing its activity. However, this responsiveness is lost in aging rats with chronically high serum PRL levels. The purpose of this study was to induce hyperprolactinemia in rats for extended periods of time and examine its effects on dopaminergic systems in the brain. Hyperprolactinemia was induced by treatment with haloperidol, a dopamine receptor antagonist, and Palkovits' microdissection technique in combination with high-performance liquid chromatography was used to measure neurotransmitter concentrations in several areas of the brain. After 6 months of hyperprolactinemia, dopamine (DA) concentrations in the median eminence (ME) increased by 84% over the control group. Nine months of hyperprolactinemia produced a 50% increase in DA concentrations in the ME over the control group. However, DA response was lost if a 9-month long haloperidol-induced hyperprolactinemia was followed by a 11

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month-long extremely high increase in serum PRL levels produced by implantation of MMQ cells under the kidney capsule. There was no change in the levels of DA, norepinephrine (NE), serotonin (5-HT), or their metabolites in the arcuate nucleus (AN), medial preoptic area (MPA), caudate putamen (CP), substantia nigra (SN), and zona incerta (ZI), except for a decrease in 5-hydroxyindoleacetic acid (5-HIAA) in the AN after 6-months of hyperprolactinemia and an increase in DA concentrations in the AN after 9-months of hyperprolactinemia. These results demonstrate that hyperprolactinemia specifically affects TIDA neurons and these effects vary, depending on the duration and intensity of hyperprolactinemia. The age-related decrease in hypothalamic dopamine function may be associated with increases in PRL secretion.     

Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia

CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler^J mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Individual Differences in Retronasal Odor Responsiveness: Effects of Aging and Concurrent Taste

Introduction

Individual differences in taste sensitivity have been considered the primary chemosensory factor in studies of chemical senses/ingestive behavior. Recent findings suggest, however, that retronasal odor perception is equally important in food preference and selection and, furthermore, the presence of a congruent taste can modulate responsiveness to retronasally perceived odors. The primary objective of this study was to measure individual differences in responsiveness to food odors in the presence and absence of a congruent taste. In order to achieve this goal, we experimentally manipulated the way taste and odor stimuli are presented. We hypothesized that when measured independently, variations across subjects in responsiveness to retronasal odors are greater than those of tastes, but that these variations are effectively reduced by the presence of a congruent taste, especially for the older cohort.

Methods

Two groups of subjects (young vs. old cohorts) were asked to sample two tastants, four food odorants, and the congruent taste-odor pairs, and rate intensities for appropriate categories.

Results

Results showed that responsiveness to odors varied greatly across individuals compared to that of tastes and further that variations in odor responsiveness were greater for old compared to young cohort. In the presence of a congruent taste, however, the variations in responsiveness to the odors were significantly reduced, in particular for the old cohort.

Implication

The current data suggest that older individuals and those with low olfactory sensitivity may not recognize the reduced sensitivity when consuming foods.

     

Differences in NMDA Receptor Expression During Human Development Determine the Response of Neurons to HIV-Tat-mediated Neurotoxicity

HIV infection of the CNS can result in neurologic dysfunction in a significant number of infected individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV infection in neurons. Thus, neuronal damage and dropout are likely due to indirect effects of HIV infection of other CNS cells, through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivating protein tat. We and others demonstrated that tat induces apoptosis in differentiated mature human neurons. We now demonstrate that the high level of tat toxicity observed in human neurons involves specific developmental stages that correlate with N-Methyl-d-Aspartate receptor (NMDAR) expression, and that tat toxicity is also dependent upon the species being analyzed. Our results indicate that tat treatment of primary cultures of differentiated human neurons with significant amounts of NMDAR expression induces extensive apoptosis. In contrast, tat treatment induces only low levels of apoptosis in primary cultures of immature human neurons with low or minimal expression of NMDAR. In addition, tat treatment has minimal effect on rat hippocampal neurons in culture, despite their high expression of NMDAR. We propose that this difference may be due to low expression of the NR2A subunit. These findings are important for an understanding of the many differences among tissue culture systems and species used to study HIV-tat-mediated toxicity.     

Learning-dependent Changes in the Responses to Visual Stimuli of Neurons in a Recognition Memory System

When young chicks are trained by exposing them to a conspicuous object they learn its characteristics. The learning process is known as imprinting. In the present study neuronal activity in a region crucial for imprinting was shown to be affected by training and by the object on which the chicks had been trained. The region is the intermediate and medial part of the left hyperstriatum ventrale (left IMHV). No such effects were found in a visual projection area, the left hyperstriatum accessorium. Domestic chicks were imprinted on either a rotating red box (n=7 chicks) or a rotating blue box (n=8). When the chicks were approximately 48 h old they were anaesthetized and multiple-unit activity was recorded in simultaneous, single penetrations through each of the two regions. Records were also made from eight dark-reared chicks. Whilst recording, the red or blue box, placed in front of the contralateral eye, was switched on to give a total of 20 rotations, the interval between each rotation being 10 s. The alternative stimulus was then presented 20 times. Unit activity in the 3 s before and after stimulus onset was compared and the data for each of the 20 presentations were combined. In the left IMHV 18 out of a total of 115 recording sites (16%) responded significantly to the stimuli; in the left hyperstriatum accessorium 39 out of 126 recording sites (26%) did so. Measures of unit activity at each recording site were combined for a given penetration to provide a 'mean penetration response'. The response to the red box differed from the response to the blue box in the left IMHV of dark-reared chicks. After training with the blue box the response to both boxes was similar to the response to the blue box in dark-reared birds. After training with the red box the response to both boxes was similar to the response to the red box in dark-reared birds. No significant effects were found in the left hyperstriatum accessorium. The two training boxes were virtually identical apart from the differences in colour and brightness. Training appeared to stabilize the response of the visually naive left IMHV to the training stimulus whilst changing its response to the alternative, but similar stimulus. That is, one consequence of training is that the two stimuli are placed in the same category, and this neural change may provide a basis for stimulus generalization. The underlying neural system is modelled and a mechanism that allows such stimuli to be discriminated is proposed.