Chronic membrane depolarization-induced morphological alteration of developing neurons.

During development of CNS, young neurons experience various stimuli, and thereafter differentiate to mature neurons in an activity-dependent manner. Membrane depolarization acts as an inducer of excitability and various signals in the neurons, which can be used as a model of neuronal activity. However, the mechanisms of the influence of membrane depolarization on neuronal differentiation have not been fully understood. Therefore, we investigated the effect of membrane depolarization on morphology of spines and generation of valid electrical activity. Using rat hippocampal cultures treated from the plating day with or without high KCl (35 mM, termed HK), we directly observed living neurons transfected with green fluorescence protein-expressing plasmid through a two-photon laser scanning confocal microscope and electrophysiological recording using a patch-clamp technique. Compared with controls, the neurons cultured with HK for 3 days in vitro (DIV) showed marked filopodia-like protrusions as well as an increase in the number of spines, but those cultured with HK for 6 DIV profoundly lost these spines, resulting in a small number of fine filopodia-like protrusions proximally and on the cell body, and a smooth surface of distal dendrites. Electrophysiological recordings showed no spontaneous responses in 6 DIV HK-treated neurons. Moreover, addition of an N-methyl-D-aspartate receptor (NMDAR) antagonist to HK-treated neurons blocked the shrinkage and decrease in the number of filopodia-like protrusions significantly. These findings suggest that membrane depolarization of developing neurons induces synaptogenesis in the early stages of development but chronic treatment with HK causes pathological changes through NMDAR, and that there may be alternative mechanisms for the physiological differentiation of neurons in later developmental stages.

Low-frequency depression of synaptic responses recorded from rat visual cortex.

To characterize the low-frequency depression (LFD) of synaptic transmission in the visual cortex, we recorded field potentials and minimal excitatory postsynaptic potentials (EPSPs) from layer II/III following intracortical stimulation at various frequencies in cortical slices of rats. Field potentials were stable at 0.017 Hz, but showed an amplitude depression at 0.033-0.1 Hz at stimulus intensity of 1.5 times the threshold for induction of the postsynaptic component and at 0.1-0.2 Hz at intensity of 1.2 times the threshold. The LFD was input-specific and its magnitude correlated with the stimulus frequency. An interruption of stimulation for 15 min yielded a nearly complete recovery from LFD. Minimal EPSPs tested at 0.1-1.7 Hz often showed LFD with similar features. However, some inputs were stable or even facilitated during repeated stimulation. At 0.1 and 0.2 Hz, >50% of inputs were stable, whereas 10% and 25% were depressed, respectively. At 0.5 and 1.7 Hz, LFD was observed in >60% and 80% of inputs, respectively. The magnitude of LFD strongly varied across inputs. In 3 of the 41 inputs analyzed, LFD was so strong that these inputs became virtually silent. Occurrence of responses to the second pulse in the paired-pulse paradigm when the first response was absent and recovery of depressed EPSPs following stimulus interruption or shift to a lower frequency suggest that these synapses were presynaptically silent due to a lowered probability of transmitter release. Altogether, the results indicate that testing intervals of <10 or even < or =30 s cannot be regarded as completely neutral. At the single-cell level, frequency-dependent changes were strongly heterogeneous across different inputs. LFD and its spontaneous recovery may underlie the previously described "post-rest" potentiation, and should be taken into account when considering information processing in cortical networks.

Brain-derived neurotrophic factor induces long-lasting potentiation of synaptic transmission in visual cortex in vivo in young rats, but not in the adult.

Brain-derived neurotrophic factor (BDNF) rapidly enhances excitatory synaptic transmission in cortical slices. To date, however, a question of how long such an action persists remains unanswered as it is hard to record synaptic responses longer than several hours in slice preparations. To address this question and to investigate possible age-dependency of the action, we analysed effects of a brief application of BDNF and nerve growth factor (NGF) on field potentials of visual cortex in rats of postnatal days 13-17 and 19-24 and in the adulthood for 10-24 h. Evoked potentials to stimulation of the lateral geniculate nucleus were recorded simultaneously from two cortical sites into which the neurotrophin and control solution were injected. An application of BDNF induced a slowly developing increase in the field potential amplitude in young rats. The amplitude attained a plateau level 3-4 h after the infusion; 139 +/- 26% (mean +/- SD) and 132 +/- 21% of the baseline in the rats at P13-17 and P19-24, respectively. This potentiation remained stable from 4 to 8 h, then gradually decreased to the baseline 15-16 h after the infusion. NGF applied in the same way did not induce potentiation. An inhibitor of BDNF receptors blocked the potentiation when it was applied immediately after the BDNF application, but was not effective about 2 h later. In the adults, BDNF did not potentiate field potentials. These results indicate that BDNF induces synaptic potentiation lasting for several hours only in the developing cortex through processes downstream of receptor activation.

Role of NMDA receptor functional domains in excitatory cell death.

The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death.

Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15-25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (Theta-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within approximately 5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex.

Brain-derived neurotrophic factor blocks long-term depression in rat visual cortex.

1. Brain-derived neurotrophic factor (BDNF) has been reported to play a role in long-term potentiation (LTP) in hippocampus, but whether it is involved also in long-term depression (LTD) is not yet known. In this study, we tested whether BDNF and its gene family, nerve growth factor (NGF), have any effect on synaptic transmission and LTD in visual cortical slices of young rats. 2. An application of BDNF at the concentration of 20 ng/ml did not significantly change layer II/III field responses evoked by layer IV stimulation at 0.1 Hz, although at 200 ng/ml it enhanced responses. BDNF at 20 ng/ml prevented LTD of field responses from being induced by low-frequency stimulation (1 Hz for 15 min) of layer IV. NGF did not have such effects in the same concentration range as that of BDNF. 3. The action of BDNF was antagonized by K252a, an inhibitor of receptor tyrosine kinases. When K252a alone was applied to slices, LTD of stronger magnitude than in control slices was induced by low-frequency stimulation. 4. These results suggest that endogeneous BDNF may prevent synapses from being depressed by low-frequency inputs in the developing visual cortex.

Postsynaptic calcium and calcium-dependent processes in synaptic plasticity in the developing visual cortex.

In this paper we describe some of the results obtained from recent experiments on mechanisms underlying long-term potentiation (LTP) and long-term depression (LTD) in the visual cortex of young rats. In particular, we focus on experiments which tested the hypotheses that the induction of LTP in the visual cortex is of Hebbian type and that an input-associated Ca2+ rise at postsynaptic sites and subsequent activation of protein kinases or protein phosphatases may play roles in the induction of LTP or LTD in the developing visual cortex.

Selective acid vulnerability of dopaminergic neurons and its recovery by brain-derived neurotrophic factor.

Among the pathogenetic phenomena of Parkinson's disease, the character of the selective degeneration of nigrostriatal system with severe gliosis is not fully understood. Here, we have shown that dopaminergic neurons may be exclusively sensitive to elevated acidity elicited after the addition of glial mitogenic factors such as epidermal growth factor and basic fibroblast growth factor or after the direct treatment with hydrochloric acid. The acid sensitivity was specific to dopaminergic neurons. The neurons other than dopaminergic neurons in culture from the ventral mesencephalon were not sensitive to acidity and the neurons from several brain areas were the same as above, except for the hippocampal neurons which had slight acid vulnerability. Choline acetyltransferase assay studies demonstrated that the cholinergic neuronal population in the septum and corpus striatum had no acid sensitivity. The vulnerability of dopaminergic neurons either elicited by glial mitogenic factor or derived from the direct acid exposure was inhibited by the addition of brain-derived neurotrophic factor (BDNF), but not by neurotrophin-3 or nerve growth factor. These findings suggest that dopaminergic neurons have selective acid vulnerability on which BDNF has a pronounced protective effect.

Interleukin-1 beta enhances survival and interleukin-6 protects against MPP+ neurotoxicity in cultures of fetal rat dopaminergic neurons.

To investigate the relationships between the central nervous system and interleukins, ventral mesencephalic cells from embryonic 17-day-old rats were cultured for 3 days in vitro (DIV) and exposed to interleukin-1 beta (IL-1 beta), interleukin-3 (IL-3), or interleukin-6 (IL-6) for the following 2 or 3 DIV with or without 2 microM 1-methyl-4-phenylpyridinium (MPP+). Thus, the survival of and the MPP+ neurotoxicity against the dopaminergic neurons immunostained with anti-tyrosine hydroxylase antibody were examined. For the survival studies, IL-1 beta has been shown to have a survival-promoting effect on dopaminergic neurons. This effect is initiated at a concentration between 0.1 and 1 ng/ml. In contrast to the effect of IL-1 beta, IL-3 and IL-6 failed to increase the survival of dopaminergic neurons. In MPP+ neurotoxicity analysis, only IL-6 among the three interleukins studied here has been shown to attenuate the MPP+ neurotoxicity against dopaminergic neurons in a dose-dependent manner; this neuro-protective action is apparent at a concentration of 10 ng/ml. In addition, these three interleukins did not promote glial proliferation. These findings suggest that the effects of IL-1 beta and IL-6 on dopaminergic neurons are not mediated by glial proliferation, that IL-1 beta acts as a neurotrophic factor on dopaminergic neurons, and that IL-6 is capable of protecting dopaminergic neurons from the neurotoxicity of MPP+.

Survival of and 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity against dopaminergic neurons in coculture of rat mesencephalon with their target on non-target regions.

It is known that dopaminergic neurons in the substantia nigra of the mesencephalon mainly project to the corpus striatum and neocortex, while the hippocampus receives major cholinergic projection from the septum. In the present study, the ventral mesencephalon was cocultured with target regions of its dopaminergic neurons, the striatum and neocortex, and with non-target regions, the hippocampus, thalamus, colliculus and cerebellum, using embryonic day-17 (E17) rats. Thus, the effects of coculture on the survival and the 1-methyl-4-phenylpyridnium (MPP+) neurotoxicity of dopaminergic neurons were investigated. The numbers of viable dopaminergic neurons were enhanced in coculture not only with corpus striatum or neocortex, but also with hippocampus or cerebellum. However, the survival of dopaminergic neurons cocultured with thalamus and colliculus were almost the same as those of controls. These findings suggest that putative factor(s), possibly target-derived neurotrophic factor(s), emerging from the regions cocultured with ventral mesencephalon can influence the dopaminergic neurons resulting in the augmentation of survival. Cocultivation with all the regions studied failed to protect dopaminergic neurons from MPP+ neurotoxicity. The results suggest that even though the survival of dopaminergic neurons was supported by coculture, the action of MPP+, an exogeneous substance, surpassed the supporting capacity of the coculture conditions.

Involvement of free radicals in MPP+ neurotoxicity against rat dopaminergic neurons in culture.

To examine the mechanisms of the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+) against dopaminergic neurons, ventral mesencephalic cells from embryonic rats were cultured and exposed to MPP+ with various antioxidants or glutamate receptor antagonists to investigate the participation of free radicals and glutamate, respectively. Such antioxidants as vitamin E, vitamin C, coenzyme Q10, and catalase, but neither allopurinol nor superoxide dismutase, alleviated the MPP(+) -induced death of dopaminergic neurons, while glutamate receptor antagonists did not alter MPP+ neurotoxicity. These findings suggest the participation of free radicals, particularly hydroxyl radicals rather than superoxides, in the process of dopaminergic neuronal death evoked by MPP+.

Death of cultured postnatal rat CNS neurons by in vitro hypoxia with special reference to N-methyl-D-aspartate-related toxicity.

We have established an in vitro hypoxia model using cultured central nervous system neurons from postnatal 4-day-old (P4) rats, in which death may be correlated with N-methyl-D-aspartate (NMDA)-related toxicity. P4 rat hippocampal and neocortical neurons in culture were prevented from death by the addition of MK-801, an NMDA receptor antagonist, and also partially by the removal of calcium ions from the medium, suggesting that NMDA receptors were associated with neuronal death in this in vitro hypoxia model. The neuronal death induced by the model was attenuated by the addition of alpha-tocopherol, indicating that free radicals emerged after hypoxia. This event seems similar to the hypoxia-reoxygenation phenomenon in in vitro hypoxia. Continuous treatment with basic fibroblast growth factor (bFGF) during hypoxia, and bFGF pretreatment for 6 h and removal before hypoxia induced the resistance to hypoxia-mediated cell death.

Enhancement of choline acetyltransferase activity in coculture of rat septal and hippocampal neurons.

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.

In vitro model of hypoxia: basic fibroblast growth factor can rescue cultured CNS neurons from oxygen-deprived cell death.

We established an in vitro hypoxia model and investigated the protective effect of basic fibroblast growth factor (bFGF) against neuronal cell death caused by hypoxia. Hippocampal neurons obtained from rats on embryonic day (E) 17 and 20 and on postnatal day (P) 4 were cultured for 6-24 h in an oxygen-deprived state. This in vitro hypoxia study showed that the cultured neurons were sensitive to the oxygen deprivation. The cultured P4 rat hippocampal neurons seemed to be weaker in the hypoxia condition than those of E17 and E20 rats, suggesting that the cultured postnatal cells might be sensitive to hypoxia. bFGF, but not nerve growth factor, prevented the neuronal cell death caused by hypoxia in a dose-dependent manner.

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture.

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.