Golgi staining of neurons: Oxidation-state dependent spread of chemical reaction identifies a testable property of the connectome

Camillo Golgi observed reticular nature of the nervous system by his staining method. Ramon Cajal modified this protocol to obtain staining restricted to individual neurons, in support of the cell theory. Close examination shows that Golgi used an oxidizing agent to pre-treat the brain tissue before the staining reaction and Cajal used an additional oxidizing agent for the same step. It shows that oxidation state of the tissue has a crucial role in determining the spread of Golgi chemical reaction between neurons. The correct structure-function mechanism of brain functions may reveal the nature of the route through which the staining reaction spreads between neurons under decreasing oxidation states. Present work examines the chemical reaction behind the staining, explores the role of oxidizing agents in limiting the stain to individual neurons, and discusses a probable property of the connectome that can provide a gateway for an oxidation state-dependent spread of staining reaction.

Cerebralcare Granule® enhances memantine hydrochloride efficacy in APP/PS1 mice by ameliorating amyloid pathology and cognitive functions

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by memory deficits and cognitive decline. Current drugs can only relieve symptoms, but cannot really cure AD. Cerebralcare Granule® (CG) is a Traditional Chinese medicine (TCM) containing a variety of biologically active compounds. In our previous studies, CG has shown a beneficial effect against memory impairment in mice caused by d-galactose. However, whether CG can be used as a complementary medicine for the treatment of AD remains unexplored. Here, we use a combination of CG and memantine hydrochloride (Mm) to treat Alzheimer-like pathology and investigate the effects and mechanisms in vivo. Methods The histology of brain was examined with Hematoxylin–eosin (HE) staining, Golgi staining and Thioflavin S staining. ELISA was applied to assess the expression levels or activities of CAT, SOD, GSH-Px, MDA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL) in serum, as well as the levels of IL-6, IL-1β, and TNF-α in the mice brain. Western blotting was used to assess the expression of β-secretase (BACE1), amyloid precursor protein (APP), APPβ, APPα, synaptophysin (SYN), growth-associated protein 43 (GAP43), and postsynaptic density 95 (PSD95). Results In the present study, the combination group (CG + Mm) significantly attenuated Alzheimer-like behavior without adverse effects in APP/PS1 mice, indicating its high degree of safety and efficacy after long-term treatment. CG + Mm reduced AD pathological biomarker Aβ plaque accumulation by inhibiting BACE1 and APP expression (P < 0.05 or P < 0.001). Besides, the combination group markedly inhibited the levels of IL-1β, IL-6, and TNF-α in hippocampus (P < 0.001), as well as activities of SOD, CAT, and GSH-Px in serum (P < 0.001). By contrast, the combination group improved synaptic plasticity by enhancing SYN, PSD95, and GAP43 expression. Conclusions Taken together, these data supported the notion that CG combined with Mm might ameliorate the cognitive impairment through multiple pathways, suggesting that CG could play a role as complementary medicine to increase anti-AD effect of chemical drugs by reducing Aβ deposition, neuroinflammation, oxidative damage, and improving synaptic plasticity.

Alcohol Co-Administration Changes Mephedrone-Induced Alterations of Neuronal Activity

Mephedrone (4-MMC), despite its illegal status, is still a widely used psychoactive substance. Its effects closely mimic those of the classical stimulant drug methamphetamine (METH). Recent research suggests that unlike METH, 4-MMC is not neurotoxic on its own. However, the neurotoxic effects of 4-MMC may be precipitated under certain circumstances, such as administration at high ambient temperatures. Common use of 4-MMC in conjunction with alcohol raises the question whether this co-consumption could also precipitate neurotoxicity. A total of six groups of adolescent rats were treated twice daily for four consecutive days with vehicle, METH (5 mg/kg) or 4-MMC (30 mg/kg), with or without ethanol (1.5 g/kg). To investigate persistent delayed effects of the administrations at two weeks after the final treatments, manganese-enhanced magnetic resonance imaging brain scans were performed. Following the scans, brains were collected for Golgi staining and spine analysis. 4-MMC alone had only subtle effects on neuronal activity. When administered with ethanol, it produced a widespread pattern of deactivation, similar to what was seen with METH-treated rats. These effects were most profound in brain regions which are known to have high dopamine and serotonin activities including hippocampus, nucleus accumbens and caudate-putamen. In the regions showing the strongest activation changes, no morphological changes were observed in spine analysis. By itself 4-MMC showed few long-term effects. However, when co-administered with ethanol, the apparent functional adaptations were profound and comparable to those of neurotoxic METH.

Cerebralcare Granule® enhances memantine hydrochloride efficacy in APP/PS1 mice by ameliorating amyloid pathology and cognitive functions

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by memory deficits and cognitive decline. Current drugs can only relieve symptoms, but cannot really cure AD. Cerebralcare granule® (CG) is a Traditional Chinese medicine (TCM) containing a variety of biologically active compounds. In our previous studies, CG has shown a beneficial effect on memory impairment in mice caused by D-galactose. However, whether CG can be used as a complementary medicine for the treatment of AD remains unexplored. Here, we use a combination of CG and memantine hydrochloride (Mm) to treat Alzheimer-like pathology and investigate the effects and mechanisms in vivo. Methods The histology of brain was examined with Hematoxylin-eosin (HE) staining, Golgi staining and Thioflavin S staining. ELISA was applied to assess the expression levels or activities of CAT, SOD, GSH-Px, MDA, alanine aminotr-ansferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL) in serum, as well as the levels of IL-6, IL-1β, and TNF-α in the mice brain. Western blotting was used to assess the expression of β-secretase (BACE1), amyloid precursor protein (APP), APPβ, APPα, synaptophysin (SYN), growth-associated protein 43 (GAP43), and postsynaptic density 95 (PSD95). Results In the present study, the combination group (CG + Mm) significantly attenuated Alzheimer-like behavior without adverse effects in APP/PS1 mice, indicating its high degree of safety and efficacy after long-term treatment. CG + Mm reduced AD pathological biomarker Aβ plaque accumulation by inhibiting BACE1 and APP expression, while Mm has no similar effect. Besides, the combination group markedly inhibited the levels of IL-1β, IL-6, and TNF-α in the hippocampus, as well as activities of SOD, CAT, and GSH-Px in serum. By contrast, the combination group improved synaptic dysfunction by enhancing SYN, PSD95, and GAP43 expression. Conclusions Taken together, these data supported the notion that CG might ameliorate the cognitive impairment through multiple pathways, suggesting that CG could play a role as complementary medicine to increase anti-AD effect of chemical drugs by reducing Aβ deposition, neuroinflammation, oxidative damage, and improving synaptic.

Oral Administration of the Food-Derived Hydrophilic Antioxidant Ergothioneine Enhances Object Recognition Memory in Mice

Background: The enhancement of learning and memory through food-derived ingredients is of great interest to healthy individuals as well as those with diseases. Ergothioneine (ERGO) is a hydrophilic antioxidant highly contained in edible golden oyster mushrooms (Pleurotus cornucopiae var. citrinopileatus), and systemically absorbed by its specific transporter, carnitine/organic cation transporter OCTN1/SLC22A4. Objective: This study aims to examine the possible enhancement of object recognition memory by oral administration of ERGO in normal mice. Method: Novel object recognition test, spatial recognition test, LC-MS/MS, Golgi staining, neuronal culture, western blotting, immunocytochemistry, and quantitative RT-PCR were utilized. Result: After oral administration of ERGO (at a dose of 1–50 mg/kg) three times per week for two weeks in ICR mice, the novel object recognition test revealed a longer exploration time for the novel object than for the familiar object. Oral administration of ERGO also revealed a longer exploration time for the moved object in the spatial recognition test in mice fed ERGO-free diet. The discrimination index was significantly higher in the ERGO-treated group than the control in both behavioral tests. ERGO administration led to an increase in its concentration in the plasma and hippocampus. The systemic concentration reached was relevant to those found in humans after oral ERGO administration. Golgi staining revealed that ERGO administration increased the number of matured spines in the hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I. This elevation of synapsin I was inhibited by the tropomyosin receptor kinase inhibitor, K252a. Treatment with ERGO also increased the expression of neurotrophin-3 and -5, and phosphorylated mammalian target of rapamycin in hippocampal neurons. Conclusion: Oral intake of ERGO which provides its plasma concentration achievable in humans may enhance object recognition memory, and this enhancement effect could occur, at least in part, through the promotion of neuronal maturation in the hippocampus.

Xiao-Yao-San Formula Improves Cognitive Ability by Protecting the Hippocampal Neurons in Ovariectomized Rats

Xiao-Yao-San (XYS) decoction is a traditional Chinese medicine formula. This study aimed to investigate the effect of XYS on cognitive abilities and its underlying mechanism in ovariectomized rats. Female Sprague-Dawley rats were ovariectomized and treated with XYS (3 g/kg or 9 g/kg) by gavage, with subcutaneous injection of 17-β estradiol (E2, 2 μg/kg) as a positive drug control and gavage of 1 ml saline (0.9%) as a placebo control. After 6 weeks of treatment, rats were examined using the Morris water maze test. The estradiol level in the serum and hippocampus was measured by ELISA. Golgi staining was performed to observe neuronal morphology in the hippocampus. Apoptosis of hippocampal cells was observed by TUNEL staining. The protein content of N-methyl-D-aspartate receptor (NMDAR) 2A and 2B in the hippocampal CA1 region was determined by Western blot and immunohistochemistry. Expression of estrogen receptor (ER) and PI3K signaling was detected by Western blot. Compared with the sham group, both learning and memory were impaired in ovariectomized rats. Rats treated with E2 or high-dose XYS showed better learning and memory compared with the saline-treated rats. High-dose XYS significantly reduced escape latency in the spatial acquisition trial; meanwhile, the cross times and duration in the probe quadrant were increased in the spatial probe trial. High-dose XYS promoted the de novo synthesis of E2 content in the hippocampus but had no significant effect on the serum E2 level. Golgi staining indicated that high-dose XYS could increase the branch number and density of dendritic spines in the hippocampal CA1 area. TUNEL staining showed that high-dose XYS alleviated ovariectomy-induced neuronal apoptosis. The expression level of NMDAR2A and NMDAR2B in hippocampal CA1 was upregulated by XYS treatment. The beneficial effect of XYS was through activating ERα-PI3K signaling. In conclusion, high-dose XYS treatment can improve the cognitive abilities of ovariectomized rats by protecting the hippocampal neurons and restoring the hippocampal E2 level.

Visualization of Purkinje Neurons of Hypothyroid Brain under Golgi Cox Stain

Background: Golgi staining was invented hundreds of years ago but it is still a reliable method to study the anatomy of neurons.Objective: This experiment was conducted to study the structure of Purkinje neurons of cerebellum of neonate rats born from hypothyroid dams under golgi cox stain and compare it to the control group.Material and Methods: For this purpose 10 female wistar rats were divided equally into 2 groups control (A) and hypothyroid (B) groups and allowed to conceive. For inducing hypothyroidism in dams, Propylthiouracyl (PTU) was administered in a dose of 15mg/kg/day orally mixed with chow daily a week before mating and throughout the period of gestation and weaning uptil 22nd day after delivery. On the 23rd day, 10 neonatal rats from each group were sacrificed and blood samples were immediately collected for evaluating serum levels of T3, T4 and TSH. The freshly extracted brains were placed in Golgi Cox solution in complete darkness for 18 to 21 days and the solution waschanged every alternate day. The brains were then removed from this stain, processed, infiltrated with parafin, cut into 80ìm thick sections by a microtome and mounted on gelatin coated slides, which were now incubated in 20% ammonium hydroxide for ten minutes.They were then washed in distilled water for 2 minutes and passed through ascending ethanol series 70%, 95%, 100% (five minutes each) and two times xylene (10 minutes each). The sections were now coverslipped, visualized and photographed under a research microscope.Results: Serum enzyme analysis revealed that group PP pups had significantly increased serum levels of TSH and control CC pups showed normal levels of this hormone. T3 and T4 levels were not significantly altered. The number of dendrites seen in purkinje neurons were less in group PP as compared to CC. Normal Purkinje cell count in both the groups were not significantly variable, but the apoptotic cells were significantly more in PP group.Conclusion; Hypothyroidism decreases the number of dendrites ofpurkinje neurons in cerebellum, causing impaired neuronalconnectivity.

The Recent Advancement in Rapid Golgi Method and Result Interpretation

The foundation knowledge of recent advancements of neuroscience was based on the Golgi staining observations. This is one of the best approaches to visualise the neuronal cytoarchitecture and complete morphology of neurons with incomparable clarity. This technique is based on the principle of heavy metal impregnation. There are many modifications and advancement occurred to improve the visualization. This chapter will provide the recently used protocols to visuals the neuronal architecture, dendritic arborization and spine density in different brain regions. Along with the manual observation, the present chapter also describes the currently used tools and software for the better understanding and visualisation of neurons.