Tohru Tatsuno

BDNF Prevents and Reverses Adult Rat Motor Neuron Degeneration and Induces Axonal Outgrowth

To assess the therapeutic potential of brain-derived neurotrophic factor (BDNF) in clinics, we extensively investigated the effects of BDNF on adult motor neurons in a rat spinal root avulsion model. Intrathecal administration of BDNF immediately after the spinal root avulsion greatly protected against the motor neuron cell death. BDNF also showed a protective effect on the atrophy of soma and on the reduction of transmitter-related enzymes such as choline acetyl transferase and acetylcholine esterase. Very interestingly, BDNF induced axonal outgrowth of severely damaged motor neurons at the avulsion site. The BDNF administration following 2-week treatment with phosphate-buffered saline after avulsion prevented further augmentation of cell death and reversed cholinergic transmitter-related enzyme deficiency. BDNF was demonstrated to possess a wide variety of biological effects on survival, soma size, cholinergic enzymes, and axonal outgrowth of adult motor neurons. These results provide a rationale for BDNF treatment in motor neuron diseases such as spinal cord injury and amyotrophic lateral sclerosis.

Analysis of effects and pharmacokinetics of subcutaneously administered BDNF

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and has been shown to be a potent and effective trophic factor for motor neurons and other neurons of the peripheral and central nervous. Little is known, however, about the relationship between the efficacy and pharmacokinetics of s.c. administered BDNF. In this study, the efficacy of BDNF on motor neuron protection in sciatic or facial nerve axotomy models was examined and compared with the concommitant concentrations of BDNF in plasma. Delayed treatment (started at 1 week after surgery) of BDNF was also shown to retard choline acetyltransferase reduction in sciatic nerve axotomy models.

Characterization of the putative anxiolytic SM-3997 recognition sites in rat brain.

In order to clarify the mechanism of action of the putative nonbenzodiazepine anxiolytic SM-3997 [3a alpha,4 beta,7 beta,7a alpha)-Hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3 (2H)-dione dihydrogen citrate), in vitro binding studies with radiolabeled compound were performed. 3H-SM-3997 bound rapidly, reversibly and in a saturable manner with high affinity to rat brain hippocampal membranes (Kd = 9.4 nM, Bmax = 213 fmol/mg protein). This specific binding was displaced by 5-hydroxytryptamine (5-HT) and related compounds. Especially, 8-OH-DPAT, a 5-HT-1A selective agonist, bound with the highest affinity to these binding sites. 3H-SM-3997 binding, however, was not displaced by a variety of other neurotransmitters, neuropeptides and some other drugs. EDTA and physiological concentration of Na+ inhibited this specific binding, but several divalent cations, Mn2+, Ca2+ and Mg2+, enhanced this binding. GTP decreased the affinity of these binding sites for 3H-SM-3997 without changing the number of binding sites, but GMP and ATP did not influence 3H-SM-3997 binding. Furthermore, 3H-SM-3997 bound with marked regional selectivity to hippocampal membranes. These characteristics and the regional distribution of 3H-SM-3997 binding sites were very similar to those of 3H-8-OH-DPAT binding sites (5-HT-1A receptors). Therefore, these results indicate that SM-3997 binds selectively and with high affinity to 5-HT-1A receptors in rat brain and may be an agonist.

Modulation by Unsaturated Fatty Acids of Norepinephrine- and Adenosine-Induced Formation of Cyclic AMP in Brain Slices

Abstract: The effect of linoleic acid on the formation of cyclic AMP in the slices of guinea pig cerebral cortex was examined. Treatment of the slices with linoleic acid resulted in an increase of basal and of norepinephrine‐stimulated formation of cyclic AMP. The stimulatory effect on the basal level of cyclic AMP was not specific for linoleic acid: the potency of the fatty acid was related to the magnitude of unsaturation. In contrast, the enhancement of norepinephrine‐stimulated formation of cyclic AMP seemed relatively specific for linoleic acid and arachidonic acid. Linoleic acid markedly enhanced the stimulated formation of cyclic AMP by histamine and adenosine, as well that by norepinephrine, without affecting that by excitatory amino acids and veratridine. Theophylline, adenosine deaminase, and 2′‐deoxyadenosine antagonized the effect of linoleic acid. Linoleic acid enhanced the maximum responses to norepinephrine and adenosine without altering the ED50 values for these agonists. When linoleic acid‐treated slices were washed with Krebs‐Ringer containing defatted bovine serum albumin, both enhancement of the response to norepinephrine and the amount of [14C]linoleic acid incorporated in a free form significantly diminished.

Membrane-bound lipoxygenase of rat cerebral microvessels

The microvessels isolated from rat cerebral cortex has arachidonate lipoxygenase activity, which was not due to possible contamination of the platelets. The major product was identified to be 12-hydroxyeicosatetraenoic acid. After homogenization and sonication of the microvessel preparations, the lipoxygenase activity was recovered both in the membrane- and the cytosol-fractions, whereas that in the platelets was recovered in the cytosol fraction. Membrane-bound lipoxygenase of the microvessels has apparent Km value of 3.8 microM for arachidonic acid, which was corresponded to 1/5 of that in the platelet enzyme. Microvessel lipoxygenase was inhibited by nordihydroguaiaretic acid but not by indomethacin.

Brain-derived neurotrophic factor shows a protective effect and improves recovery of the ERG b-wave response in light-damage

We investigated the neuroprotective effects of brain‐derived neurotrophic factor (BDNF) and its influence on the functional recovery of the retina following light‐induced retinal damage by electroretinogram (ERG). Rats were exposed to constant fluorescent light for 2, 5, 7, or 14 days, then returned to a cyclic light environment for 14 days. The result indicated that BDNF had few effects on the a‐wave amplitude, but there was a statistically significant difference in the b‐wave amplitudes between BDNF‐treated and control eyes from day 0–14 of the recovery period following 2 days of light exposure (p < 0.05). Our findings suggest that BDNF not only protects the retinal neuronal function but also enhances the recovery from retinal light damage.

Effects of tandospirone on second messenger systems and neurotransmitter release in the rat brain

1. We studied the effects of tandospirone, a novel serotonin (5-HT)1A receptor-related anxiolytic, on the intracellular second messenger systems and neurotransmitter release. 2. Tandospirone inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes by activation of 5-HT1A receptors and had high efficacy comparable to 5-HT1A receptor agonists such as 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). 3. Tandospirone suppressed carbachol-stimulated phosphatidyl-inositol metabolism (PI response), which was shown to be a 5-HT1A receptor-mediated event. 4. Tandospirone did not affect the release of 5-HT, norepinephrine (NE), dopamine (DA) and acetylcholine (ACh) from rat brain slice preparations. 5. These findings suggested that tandospirone shows high agonistic efficacy on the postsynaptic 5-HT1A receptors but does not affect the presynaptic autoreceptors located on nerve endings. The modulation of the second messenger system via postsynaptic 5-HT1A receptors might be involved in the anxiolytic efficacy of tandospirone.

Autoradiographic localization and pharmacological characterization of [3H]tandospirone binding sites in the rat brain

The regional distribution and pharmacological properties of [3H]tandospirone binding sites in the rat brain were investigated using quantitative autoradiography. [3H]Tandospirone binding was notably high in the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus. The distribution profiles of [3H]tandospirone binding sites significantly correlated with that of serotonin (5-HT)1A receptors identified using [3H]8-OH-DPAT. In competitive binding studies, [3H]tandospirone binding was inhibited by 5-HT, 8-OH-DPAT, pindolol, buspirone and N-(a,a,a-trifluoro-m-tolyl)-piperazine. The potencies of these ligands correlated with their affinities for 5-HT1A receptors. In addition, there was no significant difference in the dissociation constant of [3H]tandospirone binding between the dentate gyrus, CA1 area, dorsal raphe nucleus, lateral septum and entorhinal cortex (about 10 nM) suggesting that [3H]tandospirone binds to 5-HT1A receptors with same affinities in these brain structures. The distribution pattern of binding sites for [3H]tandospirone was also compared with that of benzodiazepine receptors identified using [3H]fludiazepam to find common effector sites for different types of anxiolytics. Some similarities were observed. It is evident in the hippocampal formation that an overlap of intense binding occurred. 5-HT1A receptors in the hippocampus may participate in the anxiolytic effects of tandospirone.

Effects of the putative anxiolytic SM-3997 on central monoaminergic systems

The effects of SM-3997 on central monoaminergic systems were evaluated by ex vivo measurement of monoamines and their metabolite levels in rat brain after intraperitoneal treatment of drugs and by in vitro measurement of monoamine uptake into rat brain slices. The effects of SM-3997 were also compared with those of other new nonbenzodiazepine anxiolytic compounds. SM-3997, buspirone, gepirone and ipsapirone showed no effects on serotonin uptake and dopamine uptake, and a weak inhibition of norepinephrine uptake at the concentration of 100 microM. SM-3997 decreased the serotonin metabolite (5-hydroxyindole-3-acetic acid) level without changing the serotonin level in hippocampus and increased dopamine metabolite (3,4-dihydroxyphenylacetic acid, homovanillic acid) level with no effect on the dopamine level in striatum. SM-3997 also produced an increase in the norepinephrine metabolite (3-methoxy-4-hydroxyphenylglycol) level with a decrease in the norepinephrine levels in hippocampus. Similar effects on serotonin metabolites and norepinephrine metabolites were observed in several other regions. Although the serotonergic effect of SM-3997 was similar to that of buspirone, gepirone and ipsapirone, the dopaminergic effect of SM-3997 was much weaker than that of buspirone.

Ciliary neurotrophic factor protects rat retina cells in vitro and in vivo via PI3 kinase

Purpose. Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function. Methods. Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 µg/eye) or PI3K (LY294002, 10 µg/eye) pathways were injected into the vitreous together with CNTF (1 µg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF. Results. CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnets test, p < 0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 µg/eye LY294002 (Dunnets test, p < 0.05). Conclusions. These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.