Toshiro Sakamoto

The therapeutic potential of human umbilical cord blood-derived mesenchymal stem cells in Alzheimer's disease

The neuropathological hallmarks of Alzheimers disease (AD) include the presence of extracellular amyloid-beta peptide (Abeta) in the form of amyloid plaques in the brain parenchyma and neuronal loss. The mechanism associated with neuronal death by amyloid plaques is unclear but oxidative stress and glial activation has been implicated. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are being scrutinized as a potential therapeutic tool to prevent various neurodegenerative diseases including AD. However, the therapeutic impact of hUCB-MSCs in AD has not yet been reported. Here we undertook in vitro work to examine the potential impact of hUCB-MSCs treatment on neuronal loss using a paradigm of cultured hippocampal neurons treated with Abeta. We confirmed that hUCB-MSCs co-culture reduced the hippocampal apoptosis induced by Abeta treatment. Moreover, in an acute AD mouse model to directly test the efficacy of hUCB-MSCs treatment on AD-related cognitive and neuropathological outcomes, we demonstrated that markers of glial activation, oxidative stress and apoptosis levels were decreased in AD mouse brain. Interestingly, hUCB-MSCs treated AD mice demonstrated cognitive rescue with restoration of learning/memory function. These data suggest that hUCB-MSCs warrant further investigation as a potential therapeutic agent in AD.

Differential effects of site-specific knockdown of estrogen receptor α in the medial amygdala, medial pre-optic area, and ventromedial nucleus of the hypothalamus on sexual and aggressive behavior of male mice.

Testosterone is known to play an important role in the regulation of male‐type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol‐induced activation of estrogen receptor α (ERα) may be crucial for the induction of these behaviors in male mice. However, the importance of ERα expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno‐associated virus vector expressing a small hairpin RNA targeting ERα to site‐specifically knockdown ERα expression. We stereotaxically injected either a control or ERα targeting vector into the medial amygdala, medial pre‐optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ERα in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ERα in the medial amygdala did not alter either behavior. Additionally, it was found that ERα knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase‐expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ERα in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ERα in only the VMN.

Inducible cAMP Early Repressor Acts as a Negative Regulator for Kindling Epileptogenesis and Long-Term Fear Memory

Long-lasting neuronal plasticity as well as long-term memory (LTM) requires de novo synthesis of proteins through dynamic regulation of gene expression. cAMP-responsive element (CRE)-mediated gene transcription occurs in an activity-dependent manner and plays a pivotal role in neuronal plasticity and LTM in a variety of species. To study the physiological role of inducible cAMP early repressor (ICER), a CRE-mediated gene transcription repressor, in neuronal plasticity and LTM, we generated two types of ICER mutant mice: ICER-overexpressing (OE) mice and ICER-specific knock-out (KO) mice. Both ICER-OE and ICER-KO mice show no apparent abnormalities in their development and reproduction. A comprehensive battery of behavioral tests revealed no robust changes in locomotor activity, sensory and motor functions, and emotional responses in the mutant mice. However, long-term conditioned fear memory was attenuated in ICER-OE mice and enhanced in ICER-KO mice without concurrent changes in short-term fear memory. Furthermore, ICER-OE mice exhibited retardation of kindling development, whereas ICER-KO mice exhibited acceleration of kindling. These results strongly suggest that ICER negatively regulates the neuronal processes required for long-term fear memory and neuronal plasticity underlying kindling epileptogenesis, possibly through suppression of CRE-mediated gene transcription.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate–deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10–15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Impairment of conditioned freezing to tone, but not to context, in Fyn‐transgenic mice: relationship to NMDA receptor subunit 2B function

We previously demonstrated that transgenic mice overexpressing Fyn tyrosine kinase exhibit higher seizure susceptibility and enhanced tyrosine phosphorylation of several proteins, including the N‐methyl‐D‐aspartate (NMDA) receptor subunit 2B (NR2B). In the present study, we analysed behavioural phenotypes, especially conditioned fear responses, of Fyn‐transgenic (TG) mice to better understand the role of Fyn in learned emotional behaviour. Tone‐dependent conditioned freezing was significantly attenuated in Fyn‐TG mice, whereas context‐dependent freezing was unaffected. Neither massed nor spaced conditioning ameliorated the attenuation of tone‐dependent freezing. However, the selective NR2B antagonist ifenprodil, when administered before conditioning, restored tone‐dependent freezing in Fyn‐TG mice at a dose that did not affect freezing in wild‐type (WT) mice. These results suggest that impairment of tone‐dependent conditioned freezing in Fyn‐TG mice is caused by disruption of the NR2B‐containing NMDA receptor function. Tyrosine phosphorylation of brain proteins, including NR2B, was enhanced in Fyn‐TG mice compared with that in WT mice. We also found that ifenprodil significantly suppressed the enhanced tyrosine phosphorylation. Thus, our data support the notion that NMDA receptor activity is tightly correlated with protein tyrosine phosphorylation, and Fyn might be one key molecule that controls tone‐dependent conditioned freezing through the regulation of NMDA receptor function.

Amygdala, deep cerebellar nuclei and red nucleus contribute to delay eyeblink conditioning in C57BL ⁄6 mice

That the cerebellum plays an essential role in delay eyeblink conditioning is well established in the rabbit, but not in the mouse. To elucidate the critical brain structures involved in delay eyeblink conditioning in mice, we examined the roles of the deep cerebellar nuclei (DCN), the amygdala and the red nucleus (RN) through the use of electrolytic lesions and reversible inactivation. All mice received eyeblink training of 50 trials during a daily session in the higher‐intensity conditioned stimulus (CS) condition (10 kHz, 70 dB). DCN lesions caused severe ataxia; nonetheless, the mice acquired conditioned responses (CRs). Reversible inactivation of DCN, by muscimol (MSC) injection, led to a severe CR impairment in the early sessions of conditioning; however, in later sessions, the mice acquired CRs. Amygdala lesions impaired the acquisition of CRs, which did not reach the level of sham‐operated mice, even after prolonged training sessions. MSC injections into the lateral amygdala severely impaired CRs, which began to recover after the removal of MSC. RN inactivation with MSC completely abolished CRs, and removal of MSC immediately restored CRs to the level of control mice. The results indicate that: (i) the DCN are important, but not essential, at least for the late acquisition in mouse eyeblink conditioning; (ii) the amygdala plays an important role in the acquisition and expression of CRs; and (iii) the RN is essential for the expression of CRs. Our findings reveal the various brain areas critically involved in mouse eyeblink conditioning, which include the cerebellum, amygdala and RN.

Acoustic priming lowers the threshold for electrically induced seizures in mice inferior colliculus, but not in the deep layers of superior colliculus.

Mice become highly susceptible to audiogenic seizures (AGS) after exposing them to an intense noise in their early life (priming). To elucidate the brain mechanisms for this priming effect of AGS, we compared the threshold current intensities inducing AGS syndromes between primed (n=88) and non-primed (n=84) mice by electrically stimulating the central nucleus and external cortex of the inferior colliculus (CIC and ECIC), and the deep layers of the superior colliculus (DLSC). The threshold for wild running was significantly lower for the primed mice than for the control mice in the case of the CIC and ECIC, but not the DLSC. The current intensity for inducing clonic seizure was lower for the primed mice than for the control mice in the case of the ECIC. These results show that the inferior colliculus (IC) plays an important role in the priming effect of AGS in mice, but that the DLSC does not.

GABAA receptors in deep cerebellar nuclei play important roles in mouse eyeblink conditioning

The neural circuitry of eyeblink conditioning in rabbits has been studied in detail, however, the basic knowledge of eyeblink conditioning in mice remains limited. In the present study, we examined the role of the deep cerebellar nuclei (DCN) in mice in delay eyeblink conditioning and rotor rod test performance by using the gamma-aminobutyric acidA (GABA(A)) receptor agonist muscimol (MSC) and the GABA(A) receptor antagonist picrotoxin (PTX). Bilateral injections of MSC and PTX into the DCN significantly impaired motor coordination in the rotor rod test, however the performance recovered within 24 h after the injections. Bilateral injection of MSC and PTX significantly impaired learned eyeblink responses (LER) during the acquisition test. MSC-injected mice could not acquire LER, however, PTX-injected mice acquired LER latently, suggesting the distinctive effect of these drugs in DCN. Bilateral injection of MSC and PTX also impaired the retention of acquired LER during a 7-day performance test. Furthermore, ipsilateral injections of MSC and PTX impaired the acquired LER as much as bilateral injection of them. Contralateral MSC injections also impaired the expression of LER, but contralateral PTX injections only partially impaired eyeblink conditioning. These results suggest that GABA(A) receptors in bilateral DCN play important roles in both the acquisition and the expression of mouse eyeblink conditioning, and that GABA(A) receptors not only in ipsilateral but also in contralateral DCN are critical for the expression of LER.

Mutation of NMDA receptor subunit epsilon 1: effects on audiogenic-like seizures induced by electrical stimulation of the inferior colliculus in mice.

It has been shown that the N-methyl-D-asparate (NMDA) receptor in the inferior colliculus is involved in the induction of audiogenic seizures (AGS). In the present study we examined audiogenic-like seizure susceptibility in GluR epsilon 1 null KO adult mice (n=32) and wild-type adult mice (n=28) by electrically stimulating the inferior colliculus (IC). Threshold current intensities of the GluR epsilon 1 KO mice for wild running, clonic and tonic seizures were higher than those of wild-type mice. In addition, the incidence rates of each seizure syndrome in GluR epsilon 1 KO mice were lower than in wild-type mice at each current intensity. These results show that GluR epsilon 1 KO mice were more resistant to audiogenic-like seizures induced by stimulating the IC. Thus, our findings suggest that the GluR epsilon 1 subunit plays an important role in regulating AGS.

Deep Cerebellar Nuclei Play an Important Role in Two-Tone Discrimination on Delay Eyeblink Conditioning in C57BL/6 Mice

Previous studies have shown that deep cerebellar nuclei (DCN)-lesioned mice develop conditioned responses (CR) on delay eyeblink conditioning when a salient tone conditioned stimulus (CS) is used, which suggests that the cerebellum potentially plays a role in more complicated cognitive functions. In the present study, we examined the role of DCN in tone frequency discrimination in the delay eyeblink-conditioning paradigm. In the first experiment, DCN-lesioned and sham-operated mice were subjected to standard simple eyeblink conditioning under low-frequency tone CS (LCS: 1 kHz, 80 dB) or high-frequency tone CS (HCS: 10 kHz, 70 dB) conditions. DCN-lesioned mice developed CR in both CS conditions as well as sham-operated mice. In the second experiment, DCN-lesioned and sham-operated mice were subjected to two-tone discrimination tasks, with LCS+ (or HCS+) paired with unconditioned stimulus (US), and HCS− (or LCS−) without US. CR% in sham-operated mice increased in LCS+ (or HCS+) trials, regardless of tone frequency of CS, but not in HCS− (or LCS−) trials. The results indicate that sham-operated mice can discriminate between LCS+ and HCS− (or HCS+ and LCS−). In contrast, DCN-lesioned mice showed high CR% in not only LCS+ (or HCS+) trials but also HCS− (or LCS−) trials. The results indicate that DCN lesions impair the discrimination between tone frequency in eyeblink conditioning. Our results suggest that the cerebellum plays a pivotal role in the discrimination of tone frequency.