Hiroki Yasuda

Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex

To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application of BDNF for 10-15 days on autapses (synapses) of solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. Neurons were confirmed to be GABAergic pharmacologically with bicuculline, a selective antagonist for GABAA receptors and immunocytochemically with antibody against glutamic acid decarboxylase 65, a GABA synthesizing enzyme. To evaluate GABAergic synaptic function, evoked and/or miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The treatment with BDNF at a concentration of 100 ng/ml enhanced the amplitude of evoked IPSCs and the frequency of miniature IPSCs. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. To evaluate morphological changes, neurons were immunocytochemically stained with antibodies against microtubule-associated protein 2, to visualize somatodendritic region and synapsin I, to visualize presynaptic sites. The quantitative analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. Such an action of BDNF was seen in both subgroups of GABAergic neurons, parvalbumin-positive and -negative neurons. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of stained sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.

Modes of action of local hypothalamic and skin thermal stimulation on salivary secretion in rats.

1. In urethane or ketamine‐anaesthetized rats, salivary secretion was observed when local brain sites or trunk skin were stimulated thermally or electrically. 2. Salivary secretion was facilitated by bilateral local brain warming. Sensitive sites were restricted to the preoptic area and anterior hypothalamus, but in a region distinct from a previously reported sensitive site for producing saliva‐spreading behaviour. 3. Unilateral warming of the preoptic area produced greater salivary secretion from the ipsilateral submandibular/sublingual salivary glands than from the contralateral glands. Electrical stimulation of the same sites elicited salivation only from the ipsilateral glands. 4. Trunk skin, not including the scrotum, was unilaterally cooled when spontaneous salivary secretion was observed in a hot environment. Salivary secretion from both sides was equally suppressed in response to the unilateral skin cooling. 5. We conclude that efferent signals from the anterior part of the hypothalamus project dominantly to the ipsilateral salivary gland for thermally induced salivary secretion. Thermal signals from the skin of either side of the trunk, on the other hand, appear to be integrated and to affect salivary secretion bilaterally.

Long-term depression in rat visual cortex is associated with a lower rise of postsynaptic calcium than long-term potentiation

To test the hypothesis that an input-associated rise of Ca2+ at postsynaptic sites beyond a certain threshold leads to the induction of long-term potentiation (LTP) while a lower rise below the threshold leads to long-term depression (LTD), the method of microscopic Ca2+ fluorometry was employed simultaneously with recordings of synaptic activity from layer II/III of visual cortical slices prepared from young rats. The conventional Ca2+ indicators, such as fura-2 or fluo-3, may interfere with intracellular processes for the induction of LTP/LTD because of their strong Ca(2+)-chelating action. To minimize such a problem, another Ca2+ indicator, rhod-2, was used since it has a much weaker Ca(2+)-chelating action than those indicators. In 16 slices loaded with rhod-2 through the perfusion medium, tetanic stimulation of theta-burst type was applied to layer IV of the cortex and changes in Ca2+ concentration were analyzed in layer II/III from which field potentials to test stimulation of layer IV were recorded simultaneously. In 7 slices in which weak tetanic stimulation consisting of 0.1 ms duration pulses was applied to layer IV, LTD of field responses was induced, while LTP was induced in 6 of the 9 slices in which strong tetanus consisting of 0.2 ms pulses was applied. In the 6 slices in which LTP was induced, the peak rise of fluorescence intensity during tetanus was 13.9 +/- 0.2 (S.E.M.) %, which was significantly (t-test, P < 0.01) higher than that (10.4 +/- 0.3%) in the 9 slices in which LTD was induced. In another series of experiments, rhod-2 was injected directly into 12 pyramidal cell-like neurons in layer II/III through patch pipettes, and changes in Ca2+ concentration in apical dendritic areas during tetanus were measured simultaneously with recordings of excitatory postsynaptic potentials (EPSPs) evoked by test stimulation of layer IV. It was found that LTP of EPSPs was induced in 4 cells which exhibited a strong rise of dendritic Ca2+ signal (197.1 +/- 18.5%) while LTD was induced in other 5 cells which showed a weak rise of the signal (31.0 +/- 4.1%). These results seem consistent with the above-mentioned, Ca(2+)-switching hypothesis for the induction of LTP and LTD in visual cortex.

Salivary secretion and grooming behaviour during heat exposure in freely moving rats

1. At neutral (24 degrees C) and at hot (40 degrees C) ambient temperatures (Ta) salivary secretion from the submandibular gland of freely moving rats was recorded, together with simultaneous observation of saliva‐spreading behaviour (grooming). 2. At a Ta of 24 degrees C, basal salivary flow was less than 2 microliters/min. When rats were first placed in the experimental chamber, brief grooming bouts often occurred. Transient secretion at more than 10 microliters/min was associated with this grooming, and the rate of salivary flow was positively correlated with the duration of grooming activity. 3. At a Ta of 40 degrees C, grooming appeared frequently and salivary secretion at more than 20 microliters/min continued even between grooming bouts. Threshold rectal temperatures (Tre) for thermally induced grooming (38.2 +/‐ 0.2 degrees C) and for salivary secretion (38.2 +/‐ 0.1 degrees C) were similar, and for eleven of sixteen rats the two thresholds coincided. 4. At the threshold Tre both the rate of salivary secretion and the duration of grooming increased in a stepwise fashion. Above the threshold, there was no correlation between the duration of grooming and the rate of salivary flow. 5. Thermally induced salivary secretion and grooming behaviour appear to be controlled by independent mechanisms.

BRAIN-DERIVED NEUROTROPHIC FACTOR ACUTELY DEPRESSES EXCITATORY SYNAPTIC TRANSMISSION TO GABAERGIC NEURONS IN VISUAL CORTICAL SLICES

Brain‐derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67‐GFP knock‐in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layeru2003IV stimulation in most GFP‐positive neurons in layeru2003II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti‐TrkB IgG, indicating that the acute inhibitory action of BDNF is activity‐dependent and mediated through TrkB. Paired‐pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10–100u2003ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca2+ chelator, suggesting that a rise in Ca2+ is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)‐positive neurons, but in only 19% of PV‐negative neurons, indicating that the depressive action is biased to PV‐positive GABAergic neurons.

Localized contribution of N-methyl-d-aspartate receptors to synaptic input-induced rise of calcium in apical dendrites of layer II/III neurons in rat visual cortex

To examine the role of N-methyl-D-aspartate receptors in the synaptic input-induced increase in Ca2+ in layer II/III neurons of visual cortex, Ca2+ imaging with a relatively low-affinity indicator was carried out simultaneously with whole-cell recordings of synaptic activity in cortical slices of young rats. Electrical stimulation of layer IV elicited excitatory postsynaptic potentials that generated action potentials concomitantly with a marked rise of Ca2+ signal in apical dendrites of 24 pyramidal cell-like neurons. Dendritic regions about 50 microm from the soma (peak regions) consistently showed the strongest increase in signal. The application of an N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovalerate, reduced the Ca2+ signal in peak regions but did not in other regions in nine of the 15 neurons tested. In all the dendritic regions in which Ca2+ signal was measured, the magnitude of the increase in signal was related linearly to the number of action potentials, suggesting that voltage-dependent Ca2+ channels activated by action potentials may also be involved in the increase. In 25 of 33 neurons which were voltage-clamped at 80 mV and perfused with the Mg2+-free solution, layer IV stimulation could induce a local rise in Ca2+ signal in apical dendrites. This rise was blocked by the N-methyl-D-aspartate receptor antagonist almost completely. Such an antagonist-sensitive Ca2+ rise was still seen even when the membrane potential was held at +10 mV when voltage-dependent Ca2+ channels were expected not to be activated by excitatory synaptic inputs. These results suggest that N-methyl-D-aspartate receptors are involved in the synaptic input-induced rise in Ca2+ at postsynaptic sites in apical dendrites of layer II/III neurons, and the increase may be boosted through voltage-dependent Ca2+ channels activated by action potentials.

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.

Effect of Reverse Voltage on Metal/Glass Anodically-Bonded Interface

A voltage in the opposite direction to that of anodic bonding was applied to anodically-bonded joints of Fe and Kovar alloy (Fe-Ni-Co alloy) to borosilicate glass at a temperature at which thermal diffusion of the alkali ions in the glass was activated. Disjunction of the bond interfaces occurred in the Kovar/glass joints. On the other hand, the Fe/glass joints retained cohesion of their bond interface after a long application of reverse voltage. At the bond interface in an as-bonded Kovar/glass joint, a reaction layer of a crystalline Fe oxide formed by the reaction between Fe from Kovar alloy and O from glass. The content of Fe in Kovar alloy adjacent to the bond interface decreased. In an Fe/glass joint the reaction layer of amorphous Fe-Si oxide formed. This layer was much thicker than the reaction layer in the Kovar/glass joint. The Kovar alloy adjacent to the bond interface in the Kovar/glass joint was rich in Ni and Co content. This result suggested that properties of the Kovar/glass bond interface were similar to those of Ni/glass or Co/glass bond interface in some instances. For this reason, effects of reverse voltage on anodically-bonded Ni/glass and Co/glass joints were examined. Disjunction occurred in both joints, and it was suggested that the disjunction of anodically-bonded Kovar/glass interface was caused by alloying elements of Ni and Co.