Docosahexaenoic acid protection in a rotenone induced Parkinson's model: Prevention of tubulin and synaptophysin loss, but no association with mitochondrial function.

Rotenone, a classic mitochondrial complex I inhibitor, leads to dopaminergic neuronal death resulting in a Parkinson's-like-disease. Docosahexaenoic acid (DHA) has shown neuroprotective effects in other experimental models of Parkinson's disease, but its effect on the rotenone-induced parkinsonism is still unknown. We tested whether DHA in vivo exerts a neuroprotective effect on rotenone-induced parkinsonism and explored the mechanisms involved, including mitochondrial function and ultrastructure as well as the expression of tubulin and synaptophysin. We pretreated eighty male Wistar rats with DHA (35 mg/kg/day) for seven days and then administered rotenone for eight days. We then measured rearing behavior, number of dopaminergic neurons, tyrosine hydroxylase content, tubulin and synaptophysin expression, mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential, ATP production activity and mitochondrial ultrastructure. We found that in vivo DHA supply exerted a neuroprotective effect, evidenced by decreased dopaminergic neuron cell death. Although we detected rotenone induced mitochondrial ultrastructure alterations, these were not associated with mitochondrial dysfunction. Rotenone had no effect on mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential or ATP production activity. DHA also prevented a rotenone-induced decrease in tubulin and synaptophysin expression. Our results support the neuroprotective effect of DHA on rotenone-induced parkinsonism, and a possible effect on early stage Parkinson's disease. This protective effect is not associated with mitochondrial function improvement, but rather with preventing loss of tubulin and synaptophysin, proteins relevant to synaptic transmission.

A Single Dose of Docosahexaenoic Acid Increases the Functional Recovery Promoted by Rehabilitation after Cervical Spinal Cord Injury in the Rat.

Task-specific rehabilitation has been shown to promote functional recovery after acute spinal cord injury (SCI). Recently, the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to promote neuroplasticity after SCI. Here, we investigated whether the combination of a single bolus of DHA with rehabilitation can enhance the effect of DHA or rehabilitation therapy in adult injured spinal cord. We found enhanced functional improvement with DHA in combination with rehabilitation compared with either treatment alone in a rat cervical lateral hemisection SCI model. This behavioral improvement correlated with a significant sprouting of uninjured corticospinal and serotonergic fibers. We also observed that the greatest increase in the synaptic vesicle protein, synaptophysin, and the synaptic active zone protein, Bassoon, occurred in animals that received both DHA and rehabilitation. In summary, the functional, anatomical, and synaptic plasticity induced by task-specific rehabilitation can be further enhanced by DHA treatment. This study shows the potential beneficial effects of DHA combined with rehabilitation for the treatment of patients with SCI.

Modification of hippocampal markers of synaptic plasticity by memantine in animal models of acute and repeated restraint stress: implications for memory and behavior.

Stress is any condition that impairs the balance of the organism physiologically or psychologically. The response to stress involves several neurohormonal consequences. Glutamate is the primary excitatory neurotransmitter in the central nervous system, and its release is increased by stress that predisposes to excitotoxicity in the brain. Memantine is an uncompetitive N-methyl D-aspartate glutamatergic receptors antagonist and has shown beneficial effect on cognitive function especially in Alzheimer's disease. The aim of the work was to investigate memantine effect on memory and behavior in animal models of acute and repeated restraint stress with the evaluation of serum markers of stress and the expression of hippocampal markers of synaptic plasticity. Forty-two male rats were divided into seven groups (six rats/group): control, acute restraint stress, acute restraint stress with Memantine, repeated restraint stress, repeated restraint stress with Memantine and Memantine groups (two subgroups as positive control). Spatial working memory and behavior were assessed by performance in Y-maze. We evaluated serum cortisol, tumor necrotic factor, interleukin-6 and hippocampal expression of brain-derived neurotrophic factor, synaptophysin and calcium-/calmodulin-dependent protein kinase II. Our results revealed that Memantine improved spatial working memory in repeated stress, decreased serum level of stress markers and modified the hippocampal synaptic plasticity markers in both patterns of stress exposure; in ARS, Memantine upregulated the expression of synaptophysin and brain-derived neurotrophic factor and downregulated the expression of calcium-/calmodulin-dependent protein kinase II, and in repeated restraint stress, it upregulated the expression of synaptophysin and downregulated calcium-/calmodulin-dependent protein kinase II expression.

Autocrine motility factor receptor is involved in the process of learning and memory in the central nervous system.

The autocrine motility factor receptor (AMFR) is a multifunctional protein involved in cellular adhesion, proliferation, motility and apoptosis. Our study showed that increased AMFR protein expression in the hippocampus of KM mice correlated with enhanced capacity for learning and memory following the shuttle-box test and was significantly elevated in the highest score group. Also, AMF and AMFR mRNA expression positively correlates with the mRNA expression of the synapse marker synaptophysin (Syp). Aging studies in the senescence-accelerated mouse strain (SAM) prone/8 (SAMP8), an animal model of Alzheimer's disease (AD), revealed significantly decreased mRNA and protein expression of AMF and AMFR in the hippocampus. This is especially true for AMFR and AMF protein expression compared with age-matched SAM resistant/1 (SAMR1) mouse strain as the control. Additionally, the low mRNA expression of AMFR could be up-regulated by the four nootropic traditional Chinese medicinal prescriptions (TCMPs): Ba-Wei-Di-Huang decoction (BW), Huang-Lian-Jie-Du decoction (HL), Dang-Gui-Shao-Yao-San (DSS) and Tiao-Xin-Fang decoction (TXF). AMFR protein expression could be up-regulated by two TCMPs, Liu-Wei-Di-Huang decoction (LW) and BW. This indicated that AMFR is involved in the process of learning and memory in the central nervous system. These results may provide useful clues for understanding the etiology of AD.

Recurrent hypoglycemia alters hypothalamic expression of the regulatory proteins FosB and synaptophysin.

A limiting factor to the clinical management of diabetes is iatrogenic hypoglycemia. With multiple hypoglycemic episodes, the collective neuroendocrine response that restores euglycemia is impaired. In our animal model of recurrent hypoglycemia (RH), neuroendocrine deficits are accompanied by a decrease in medial hypothalamic activation. Here we tested the hypothesis that the medial hypothalamus may exhibit unique changes in the expression of regulatory proteins in response to RH. We report that expression of the immediate early gene FosB is increased in medial hypothalamic nuclei, anterior hypothalamus, and posterior paraventricular nucleus of the thalamus (THPVN) of the thalamus following RH. We identified the hypothalamic PVN, a key autonomic output site, among the regions expressing FosB. To identify the subtype(s) of neuronal populations that express FosB, we screened candidate neuropeptides of the PVN for coexpression using dual fluorescence immunohistochemistry. Among the neuropeptides analyzed [including oxytocin, vasopressin, thyrotropin-releasing hormone, and corticotropin-releasing factor (CRF)], FosB was only identified in CRF-positive neurons. Inhibitory gamma-aminobutyric acid-positive processes appear to impinge on these FosB-expressing neurons. Finally, we observed a significant decrease in the presynaptic marker synaptophysin within the PVN of RH-treated vs. saline-treated rats, suggesting that rapid alterations of synaptic morphology may occur in association with RH. Collectively, these data suggest that RH stress triggers cellular changes that support synaptic plasticity, in specific neuroanatomical sites, which may contribute to the development of hypoglycemia-associated autonomic failure.

Pueraria mirifica, phytoestrogen-induced change in synaptophysin expression via estrogen receptor in rat hippocampal neuron.

OBJECTIVE: To examine Pueraria mirifica (Leguminosae) containing-phytoestrogen effect on synaptic density and involvement of estrogen receptor. MATERIAL AND METHOD: The level of synaptophysin, a presynaptic vesicle protein, was measured using Western blot analysis and immunocytochemistry in hippocampal primary cell cultures at 6 days in vitro. RESULTS: P. mirifica and 17beta-estradiol (0.1 microM) treatment for 4 days, but not for 2 days, significantly increased synaptophysin immunoreactivity and level of synaptophysin. P. mirifica up to 60 microg/ml resulted in a dose related increase in the level of synaptophysin immunoreactivity. The classical estrogen receptor antagonist, ICI 182 780, significantly blocked P. mirifica-induced increase in synaptophysin. CONCLUSION: P. mirifica-containing phytoestrogen affects synaptic density by inducing synaptophysin expression via estrogen receptor.

D-cycloserine improves functional recovery and reinstates long-term potentiation (LTP) in a mouse model of closed head injury.

Traumatic brain injury triggers a massive glutamate efflux, activation of NMDA receptor channels, and cell death. Recently, we reported that NMDA receptors in mice are down-regulated from hours to days following closed head injury (CHI), and treatment with NMDA improved recovery of motor and cognitive functions up to 14 d post-injury. Here we show that a single injection of a low dose of D-cycloserine (DCS), a partial NMDA receptor agonist, in CHI mice 24 h post-injury, resulted in a faster and greater recovery of motor and memory functions as assessed by neurological severity score and object recognition tests, respectively. Moreover, DCS treatment of CHI mice led to a significant improvement of hippocampal long-term potentiation (LTP) in the CA1 region that was completely blunted in CHI control mice. However, DCS did not improve CHI-induced impairment in synaptic glutamate release measured by paired pulse facilitation (PPF) ratio in hippocampal CA1 region. Finally, CHI-induced reduction of brain-derived neurotrophic factor (BDNF) was fully restored following DCS treatment. Since DCS is in clinical use for other indications, the present study offers a novel approach to treat human brain injury.

Repair of amyloid beta(25-35)-induced memory impairment and synaptic loss by a Kampo formula, Zokumei-to.

Although Zokumei-to (ZMT), a Kampo formula, has been used for postapopletic sequelae such as paralysis and logopathy, only few studies of this drug have been carried out. We hypothesized that ZMT may affect neuronal plasticity and investigated whether or not this drug is capable of improving learning impairment and synaptic loss observed in patients with Alzheimer's disease (AD). Amyloid beta(25-35) [Abeta(25-35)] (4.7 nmol) was intracerebroventricularly injected into ddY mice (male, 6 weeks old). Fourteen days after the injection, mice were given ZMT extract (500 mg/kg/day) per os for 15 days. In a memory acquisition test, the Abeta(25-35)-injected mice required more time to master this task than did mice in the saline- or reverse peptide Abeta(35-25)-treated groups. ZMT-treated mice shortened escape latencies during trial days 3-5, but not significantly. Three days after the last drug treatment, a retention test was performed. Following ZMT, the number of crossings over a platform was significantly decreased in Abeta(25-35)-injected mice compared with those in the control groups. However, ZMT-treated mice showed complete recovery of this number. Although Abeta(25-35) injection decreased synaptophysin expression in the cerebral cortex and the hippocampus, ZMT treatment significantly increased the level of expression of synaptophysin up to the control level. Donepezil hydrochloride (DNP, 0.5 mg/kg/day, p.o.) clinically used for AD had no effect on memory retention and synaptophysin levels. Abeta(25-35)-induced neuronal loss was not observed in any region of the brain. The present results suggest that memory impairment and synaptic loss in AD patients may be improved by treatment with ZMT, even after such impairment has already progressed.

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes.

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l-glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l-glutamate in synaptic-like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l-glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT-PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co-localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l-glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.

Low levels of estrogen significantly diminish axonal sprouting after entorhinal cortex lesions in the mouse.

This study tested the hypothesis that estrogen enhances axonal sprouting in the hippocampal formation in the female mouse. The entorhinal cortex was unilaterally lesioned with ibotenic acid in control mice and in ovariectomized mice that were treated with a high dose of, a moderate dose of, or zero estrogen supplementation pellets. Four weeks later the density of staining for synaptophysin immunoreactivity and acetylcholinesterase (AChE) histochemistry was measured in the molecular layer of the dentate gyrus. In control mice, lesions of the lateral part of the entorhinal cortex increased synaptophysin and acetylcholinesterase staining (i.e., indicative of axonal sprouting) in the outer one-third of the molecular layer of the dentate gyrus. Mice receiving high and moderate estrogen supplementation displayed the same sprouting response; however, in ovariectomized mice the sprouting response was significantly reduced (to nearly nothing). Thus, in ovariectomized compared with control mice the lesion-induced sprouting response is severely blunted, and this effect is reversed by estrogen supplementation. Together, these findings suggest that estrogen plays a prominent role in promoting neuronal plasticity and remodeling in the dentate gyrus.