The C-terminal fragment of the heavy chain of the tetanus toxin (Hc-TeTx) improves motor activity and neuronal morphology in the limbic system of aged mice.

In the aging process, the brain presents biochemical and morphological alterations. The neurons of the limbic system show reduced size dendrites, in addition to the loss of dendritic spines. These disturbances trigger a decrease in motor and cognitive function. Likewise, it is reported that during aging, in the brain, there is a significant decrease in neurotrophic factors, which are essential in promoting the survival and plasticity of neurons. The carboxyl-terminal fragment of the heavy chain of the tetanus toxin (Hc-TeTx) acts similarly to neurotrophic factors, inducing neuroprotection in different models of neuronal damage. The aim here, was to evaluate the effect of Hc-TeTx on the motor processes of elderly mice (18 months old), and its impact on the dendritic morphology and density of dendritic spines of neurons in the limbic system. The morphological analysis in the dendrites was evaluated employing Golgi-Cox staining. Hc-TeTx was administered (0.5 mg/kg) intraperitoneally for three days in 18-month-old mice. Locomotor activity was evaluated in a novel environment 30 days after the last administration of Hc-TeTx. Mice treated with Hc-TeTx showed significant changes in their motor behavior, and an increased dendritic spine density of pyramidal neurons in layers 3 and 5 of the prefrontal cortex in the hippocampus, and medium spiny neurons of the nucleus accumbens (NAcc). In conclusion, the Hc-TeTx improves the plasticity of the brain regions of the limbic system of aged mice. Therefore, it is proposed as a pharmacological alternative to prevent or delay brain damage during aging.

Administering crocin ameliorates anxiety-like behaviours and reduces the inflammatory response in amyloid-beta induced neurotoxicity in rat.

Anxiety, hippocampus synaptic plasticity deficit, as well as pro-inflammatory cytokines, are involved in Alzheimer's disease (AD). The present study is designed to evaluate the possible therapeutic effect of crocin on anxiety-like behaviours, hippocampal synaptic plasticity and neuronal shape, as well as pro-inflammatory cytokines in the hippocampus using in vivo amyloid-beta (Aß) models of AD. The Aß peptide (1-42) was bilaterally injected into the frontal-cortex. Five hours after the surgery, the rats were given intraperitoneal (IP) crocin (30 mg/kg) daily up to 12 days. Elevated plus maze results showed that crocin treatment after bilateral Aß injection significantly increased the percentage of spent time into open arms, frequency of entries, and percentage of entries into open arms as compared with the Aß group. In the open field test, the Aß+crocin group showed a higher percentage of spent time in the centre and frequency of entries into central zone as compare with the Aß treated animals. Administering crocin increased the number of soma, dendrites and axonal arbores in the CA1 neurons among the rats with Aß neurotoxicity. Cresyl violet (CV) staining showed that crocin increased the number of CV-positive cells in the CA1 region of the hippocampus compared with the Aß group. Silver-nitrate staining indicated that crocin reduced neurofibrillary tangle formation induced by Aß. Crocin treatment attenuated the expression of TNF-α and IL-1ß mRNA in the hippocampus compared with the Aß group. Our results suggest that crocin attenuated Aß-induced anxiety-like behaviours and neuronal damage, and synaptic plasticity loss in hippocampal CA1 neurons may via its anti-inflammatory effects.

Determining factors for optimal neuronal and glial Golgi-Cox staining.

Golgi staining allows for the analysis of neuronal arborisations and connections and is considered a powerful tool in basic and clinical neuroscience. The fundamental rules for improving neuronal staining using the Golgi-Cox method are not fully understood; both intrinsic and extrinsic factors may control the staining process. Therefore, various conditions were tested to improve the Golgi-Cox protocol for vibratome-cut rat brain sections. Optimal staining of cortical neurons was achieved after 72 h of impregnation. Well-stained neurons in both cortical and subcortical structures were observed after 96 h of impregnation. The dendritic arborisation pattern of cortical neurons derived from the 72-h impregnation group was comparable to those of the 96 and 168-h impregnation groups. The entire brain was stained well when the pH of the Golgi-Cox solution was 6.5 and that of the sodium carbonate solution was 11.2. Lack of brain perfusion or perfusion with 0.9% NaCl did not influence optimal neuronal staining. Perfusion with 37% formaldehyde, followed by impregnation, only resulted in glial staining, but perfusion with 4% formaldehyde facilitated both glial and neuronal staining. Whole brains required longer impregnation times for better staining. Although every factor had a role in determining optimal neuronal staining, impregnation time and the pH of staining solutions were key factors among them. This modified Golgi-Cox protocol provides a simple and economical procedure to stain both neurons and glia separately.

Caffeine consumption during development alters spine density and recovery from repetitive mild traumatic brain injury in young adult rats.

Caffeine is the most commonly used psychostimulant throughout the world, with its consumption being especially prevalent among adolescents and young adults, as over 75% of this group consumes caffeine daily. Similarly, the adolescent and young adult age group exhibit the highest incidence of traumatic brain injury (TBI). Given that both caffeine consumption and mild TBI (mTBI) are more prevalent among the late adolescent/young adult age group and that changes in dendritic spine morphology during this developmental period are poorly understood, this study sought to examine the effects of caffeine consumption during late adolescence/early adulthood on recovery from repetitive mTBI (RmTBI). The study specifically focused on changes to neuronal dendritic morphology as synaptic changes likely underlie long-term behavioral outcomes. The results demonstrate that during young adulthood caffeine consumption differentially affects the RmTBI outcomes of males and females, where the effects of caffeine and RmTBI were often additive in males while being equally detrimental, but rarely additive, in females. In general, caffeine and RmTBI induced the greatest impairments in males on cognitive and motor tasks whereas in females the most significant detriments were on pain-related tasks. Both caffeine and RmTBI increased spine density in the Cg3 (medial prefrontal cortex [mPFC]), AID (orbitofrontal cortex [OFC]), and nucleus accumbens (NAc), which is proposed to reflect an impairment in the normal pruning processes. Overall, despite caffeine's neuroprotective abilities among other age groups, this study offers concerning results regarding the detrimental effects of caffeine and RmTBI, in isolation, and especially in combination, in this susceptible population.

Juvenile stress causes reduced locomotor behavior and dendritic spine density in the prefrontal cortex and basolateral amygdala in Sprague-Dawley rats.

Little has been investigated about the effects of stress on synaptic communication at prepubertal age, a stage considered as juvenile. This period of development is related to socialization through play. Our group has studied the changes of neuronal morphology in limbic structures caused by stress at prenatal and at early postnatal ages (before weaning) in the rat. In the present study, we assessed the effect of restraint stress at juvenile ages. Male Sprague-Dawley rats from postnatal day (PD) 21 to PD35 were restrained (from movement) for 2 hrs. Locomotor activity in a novel environment was evaluated at three different ages, prepubertal PD38, pubertal PD50, and postpubertal PD68. Using the Golgi-Cox procedure, the dendritic morphology was evaluated in the pyramidal neurons of the prefrontal cortex (PFC), hippocampus, and basolateral amygdala (BLA). Juvenile stress caused a reduced locomotor activity at PD38 and PD68 together with reduction in dendritic spines after puberty in the PFC and at all the studied ages in the BLA. In addition, dendritic length was also reduced in the PFC at PD38 and PD68 and CA1 of the ventral hippocampus at PD50 and PD68. Our results suggest that stress in the juvenile stage can cause changes at the level of behavior and synaptic communication with an effect that remains until adulthood.

Intracerebroventricular administration of growth hormone induces morphological changes in pyramidal neurons of the hippocampus and prefrontal cortex in adult rats.

A growing body of evidence suggests that growth hormone (GH) affects synaptic plasticity at both the molecular and electrophysiological levels. However, unclear is whether plasticity that is stimulated by GH is associated with changes in neuron structure. This study investigated the effect of intracerebroventricular (ICV) administration of GH on the morphology of pyramidal neurons of the CA1 region of the dorsal hippocampus and layer III of the prefrontal cortex. Male Wistar rats received daily ICV injections of GH (120 ng) for 7 days, and they were euthanized 21 days later. Changes in neuronal morphology were evaluated using Golgi-Cox staining and subsequent Sholl analysis. GH administration increased total dendritic length in the CA1 region of the dorsal hippocampus and prefrontal cortex. The Sholl analysis revealed an increase in dendritic length of the third to eighth branch orders in the hippocampus and from the third to sixth branch orders in the prefrontal cortex. Interestingly, GH treatment increased the density of dendritic spines in both brain regions, favoring the presence of mushroom-like spines only in the CA1 hippocampal region. Our results indicated that GH induces changes in the length of dendritic trees and the density of dendritic spines in two high-plasticity brain regions, suggesting that GH-induced synaptic plasticity at the molecular and electrophysiological levels may be associated with these structural changes in neurons.

The neuropeptide-12 improves recognition memory and neuronal plasticity of the limbic system in old rats.

Aging is a stage of life where cognitive and motor functions are impaired. This is because oxidative and inflammatory processes exacerbate neurodegeneration, which affects dendritic morphology and neuronal communication of limbic regions with memory loss. Recently, the use of trophic substances has been proposed to prevent neuronal deterioration. The neuropeptide-12 (N-PEP-12) has been evaluated in elderly patients with dementia, showing improvements in cognitive tasks due to acts as a neurotrophic factor. In the present work, we evaluated the effect of N-PEP-12 on motor activity and recognition memory, as well as its effects on dendritic morphology and the immunoreactivity of GFAP, Synaptophysin (SYP), and BDNF in neurons of the prefrontal cortex (PFC), dorsal hippocampus (DH) and nucleus accumbens (NAcc) of aged rats. The results show that N-PEP-12 improved the recognition memory, but the motor activity was not modified compared to the control animals. N-PEP-12 increases the density of dendritic spines and the total dendritic length in neurons of the PFC (layers 3 and 5) and in DH (CA1 and CA3). Interestingly NAcc neurons showed a reduction in the number of dendritic spines. In the N-PEP-12 animals, when evaluating the immunoreactivity for SYP and BDNF, there was an increase in the three brain regions, while the mark for GFAP decreased significantly. Our results suggest that N-PEP-12 promotes neuronal plasticity in the limbic system of aged animals, which contributes to improving recognition memory. In this sense, N-PEP-12 can be considered as a pharmacological alternative to prevent or delay brain aging and control senile dementias.

Ancestral Exposure to Stress Generates New Behavioral Traits and a Functional Hemispheric Dominance Shift.

In a continuously stressful environment, the effects of recurrent prenatal stress (PS) accumulate across generations and generate new behavioral traits in the absence of genetic variation. Here, we investigated if PS or multigenerational PS across 4 generations differentially affect behavioral traits, laterality, and hemispheric dominance in male and female rats. Using skilled reaching and skilled walking tasks, 3 findings support the formation of new behavioral traits and shifted laterality by multigenerational stress. First, while PS in the F1 generation did not alter paw preference, multigenerational stress in the F4 generation shifted paw preference to favor left-handedness only in males. Second, multigenerational stress impaired skilled reaching and skilled walking movement abilities in males, while improving these abilities in females beyond the levels of controls. Third, the shift toward left-handedness in multigenerationally stressed males was accompanied by increased dendritic complexity and greater spine density in the right parietal cortex. Thus, cumulative multigenerational stress generates sexually dimorphic left-handedness and dominance shift toward the right hemisphere in males. These findings explain the origins of apparently heritable behavioral traits and handedness in the absence of DNA sequence variations while proposing epigenetic mechanisms.

NR2B-Tyr phosphorylation regulates synaptic plasticity in central sensitization in a chronic migraine rat model.

BACKGROUND: Although the mechanism of chronic migraine (CM) is unclear, it might be related to central sensitization and neuronal persistent hyperexcitability. The tyrosine phosphorylation of NR2B (NR2B-pTyr) reportedly contributes to the development of central sensitization and persistent pain in the spinal cord. Central sensitization is thought to be associated with an increase in synaptic efficiency, but the mechanism through which NR2B-pTyr regulates synaptic participation in CM-related central sensitization is unknown. In this study, we aim to investigate the role of NR2B-pTyr in regulating synaptic plasticity in CM-related central sensitization. METHODS: Male Sprague-Dawley rats were subjected to seven inflammatory soup (IS) injections to model recurrent trigeminovascular or dural nociceptor activation, which is assumed to occur in patients with CM. We used the von Frey test to detect changes in mechanical withdrawal thresholds, and western blotting and immunofluorescence staining assays were performed to detect the expression of NR2B-pTyr in the trigeminal nucleus caudalis (TNC). NR2B-pTyr was blocked with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo [3,4-d] pyrimidine (PP2) and the protein tyrosine kinase inhibitor genistein to detected the changes in calcitonin gene-related peptide (CGRP), substance P (SP), and the synaptic proteins postsynaptic density 95 (PSD95), synaptophysin (Syp), synaptotagmin1 (Syt-1). The synaptic ultrastructures were observed by transmission electron microscopy (TEM), and the dendritic architecture of TNC neurons was observed by Golgi-Cox staining. RESULTS: Statistical analyses revealed that repeated infusions of IS induced mechanical allodynia and significantly increased the expression of NR2B Tyr-1472 phosphorylation (pNR2B-Y1472) and NR2B Tyr-1252 phosphorylation (pNR2B-Y1252) in the TNC. Furthermore, the inhibition of NR2B-pTyr by PP2 and genistein relieved allodynia and reduced the expression of CGRP, SP, PSD95, Syp and Syt-1 and synaptic transmission. CONCLUSIONS: These data indicate that NR2B-pTyr might regulate synaptic plasticity in central sensitization in a CM rat model. The inhibition of NR2B tyrosine phosphorylation has a protective effect on threshold dysfunction and migraine attacks through the regulation of synaptic plasticity in central sensitization.

Biphasic plasticity of dendritic fields in layer V motor neurons in response to motor learning.

Motor learning is associated with plastic reorganization of neural networks in primary motor cortex (M1) that advances through stages. An initial increment in spine formation is followed by pruning and maturation one week after training ended. A similar biphasic course was described for the size of the forelimb representation in M1. This study investigates the evolution of the dendritic architecture in response to motor skill training using Golgy-Cox silver impregnation in rat M1. After learning of a unilateral forelimb-reaching task to plateau performance, an increase in dendritic length of layer V pyramidal neurons (i.e. motor neurons) was observed that peaked one month after training ended. This increment in dendritic length reflected an expansion of the distal dendritic compartment. After one month dendritic arborization shrinks even though animals retain task performance. This pattern of evolution was observed for apical and basal dendrites alike - although the increase in dendritic length occurs faster in basal than in apical dendrites. Dendritic plasticity in response to motor training follows a biphasic course with initial expansion and subsequent shrinkage. This evolution takes fourth as long as the biphasic reorganization of spines or motor representations.

Sleep deprivation induces differential morphological changes in the hippocampus and prefrontal cortex in young and old rats.

Sleep is a fundamental state necessary for maintenance of physical and neurological homeostasis throughout life. Several studies regarding the functions of sleep have been focused on effects of sleep deprivation on synaptic plasticity at a molecular and electrophysiological level, and only a few studies have studied sleep function from a structural perspective. Moreover, during normal aging, sleep architecture displays some changes that could affect normal development in the elderly. In this study, using a Golgi-Cox staining followed by Sholl analysis, we evaluate the effects of 24 h of total sleep deprivation on neuronal morphology of pyramidal neurons from Layer III of the prefrontal cortex (PFC) and the dorsal hippocampal CA1 region from male Wistar rats at two different ages (3 and 22 months). We found no differences in total dendritic length and branching length in both analyzed regions after sleep deprivation. Spine density was reduced in the CA1 of young-adults, and interestingly, sleep deprivation increased spine density in PFC of aged animals. Taken together, our results show that 24 h of total sleep deprivation have different effects on synaptic plasticity and could play a beneficial role in cognition during aging.

Quantification of Neuronal Dendritic Spine Density and Lengths of Apical and Basal Dendrites in Temporal Lobe Structures Using Golgi-Cox Staining.

The objective of this chapter is to provide an overview of the methods used to investigate the connectivity and structure of the nervous system. These methods allow neuronal cells to be categorized according to their location, shape, and connections to other cells. The Golgi-Cox staining gives a thorough picture of all significant neuronal structures found in the brain that may be distinguished from one another. The most significant characteristic is its three-dimensional integrity since all neuronal structures may be followed continuously from one part to the next. Successions of sections of the brain's neurons are seen with the Golgi stain. The Golgi method is used to serially segment chosen brain parts, and the resulting neurons are produced from those sections.

Increased Brain-Derived Neurotrophic Factor and Hippocampal Dendritic Spine Density Are Associated with the Rapid Antidepressant-like Effect of Iron-citalopram and Iron-Imipramine Combinations in Mice.

Iron supplementation previously demonstrated antidepressant-like effects in post-partum rats. The present study evaluates the possible synergistic antidepressant effect of sub-therapeutic dose of iron co-administered with citalopram or imipramine in female Institute of Cancer Research mice. Depression-like symptoms were induced in the forced swim (FST), tail suspension (TST), and open space swim (OSST) tests while open field test (OFT) was used to assess locomotor activity. Mice (n = 8) received iron (0.8-7.2 mg/kg), citalopram (3-30 mg/kg), imipramine (3-30 mg/kg), desferrioxamine (50 mg/kg) or saline in the single treatment phase of each model and subsequently a sub-therapeutic dose of iron co-administered with citalopram or imipramine. Assessment of serum brain derived neurotrophic factor (BDNF) and dendritic spine density was done using ELISA and Golgi staining techniques respectively. Iron, citalopram and imipramine, unlike desferrioxamine, reduced immobility score in the TST, FST and OSST without affecting locomotor activity, suggesting antidepressant-like effect. Sub-therapeutic dose of iron in combination with citalopram or imipramine further enhanced the antidepressant-like effect, producing a more rapid effect when compared to the iron, citalopram or imipramine alone. Iron, citalopram and imipramine or their combinations increased serum BDNF concentration, hippocampal neuronal count and dendritic spine densities. Our study provides experimental evidence that iron has antidepressant-like effect and sub-therapeutic dose of iron combined with citalopram or imipramine produces more rapid antidepressant-like effect. We further show that iron alone or its combination with citalopram or imipramine attenuates the neuronal loss associated with depressive conditions, increases dendritic spines density and BDNF levels. These finding suggest iron-induced neuronal plasticity in the mice brain.

Dendritic reorganization in the hippocampus, anterior temporal lobe, and frontal neocortex of lithium-pilocarpine induced Status Epilepticus (SE).

Status Epilepticus (SE) is a distributed network disorder, which involves the hippocampus and extra-hippocampal structures. Epileptogenesis in SE is tightly associated with neurogenesis, plastic changes and neural network reorganization facilitating hyper-excitability. On the other hand, dendritic spines are known to be the excitatory synapse in the brain. Therefore, dendritic spine dynamics could play an intricate role in these network alterations. However, the exact reason behind these structural changes in SE are elusive. In the present study, we have investigated the aforementioned hypothesis in the lithium-pilocarpine treated rat model of SE. We have examined cytoarchitectural and morphological changes using hematoxylin-eosin and Golgi-Cox staining in three different brain regions viz. CA1 pyramidal layer of the dorsal hippocampus, layer V pyramidal neurons of anterior temporal lobe (ATL), and frontal neocortex of the same animals. We observed macrostructural and layer-wise alteration of the pyramidal layer mainly in the hippocampus and ATL of SE rats, which is associated with sclerosis in the hippocampus. Sholl analysis exhibited partial dendritic plasticity in apical and basal dendrites of pyramidal cells as compared to the saline-treated weight-/age-matched control group. These findings indicate that region-specific alterations in dendritogenesis may contribute to the development of independent epileptogenic networks in the hippocampus, ATL, and frontal neocortex of SE rats.

A report on digitised neuronal tracing method to study neurons in their entirety.

Conventional camera lucida (CL) aided neuronal tracing technique for studying neural plasticity is a demanding procedure. Stereo Investigator-Neurolucida enabled neuronal tracing system is not accessible to all researchers. This necessitates alternate simple and less challenging digitised neuronal tracing methods. This report describes a novel digitised neuronal tracing method using widefield microscopy, and its effectiveness is compared with the traditional camera lucida aided neuronal tracing method. Golgi-Cox stained hippocampal cornu ammonis area-3 (CA3) pyramidal neuron photomicrographs were serially captured at a depth of every 2µm in the z-axis by a wide field microscope from the point of appearance to the disappearance. These images were stacked along the axis perpendicular to the image plane to reconstruct the neuron in its entirety, digitally traced and dendritic quantification was performed using open source software. The same neurons were manually traced using camera lucida, and Sholl analysis was done manually to quantify the dendritic arborisation pattern. The dendritic quantification data were not significantly different in both methods. Hence, the technology-enabled, less demanding, and equally accurate neuronal tracing can be adopted instead of manual tracing and analysis of neurons. •A simple digitised neuronal tracing method is described.•It is fast, rigorous, and comparable to traditional tracing techniques.•Helps the researcher to repeatedly probe data to reduce errors.

Progression of Alzheimer's disease parallels unusual structural plasticity of human dentate granule cells.

Alzheimer´s disease (AD), the most common form of dementia in industrialized countries, severely targets the hippocampal formation in humans and mouse models of this condition. The adult hippocampus hosts the continuous addition of new dentate granule cells (DGCs) in numerous mammalian species, including humans. Although the morphology and positioning of DGCs within the granule cell layer (GCL) match their developmental origin in rodents, a similar correlation has not been reported in humans to date. Our data reveal that DGCs located in inner portions of the human GCL show shorter and less complex dendrites than those found in outer portions of this layer, which are presumably generated developmentally. Moreover, in AD patients, DGCs show early morphological alterations that are further aggravated as the disease progresses. An aberrantly increased number of DGCs with several primary apical dendrites is the first morphological change detected in patients at Braak-Tau I/II stages. This alteration persists throughout AD progression and leads to generalized dendritic atrophy at late stages of the disease. Our data reveal the distinct vulnerability of several morphological characteristics of DGCs located in the inner and outer portions of the GCL to AD and support the notion that the malfunction of the hippocampus is related to cognitive impairments in patients with AD.

Dendritic spine loss deep in the neocortex and dendrite distortion with diffusion disturbances occur early in experimental pneumococcal meningitis.

Introduction: Streptococcus pneumoniae (pneumococcus) meningitis is a serious disease with substantial lethality and long-term disability in survivors. Loss of synaptic staining in the superficial layers of the neocortex in rodent models and in humans, and pneumolysin (a major pneumococcal toxin)-dependent dendritic spine collapse in brain slices have been described. It remains unclear how deep in the neocortex more discrete changes are present, how soon after disease onset these changes occur, and whether other properties of dendrites are also affected. Methods: Using a mouse model of pneumococcal meningitis, we studied changes in the neocortex shortly (3-6 h) after the onset of clinical symptoms via modified Golgi-Cox silver staining. Results: Dendritic changes were present in areas with otherwise unchanged cell numbers and no signs of necrosis or other apparent neuronal pathology. Mature dendritic spines were reduced in the pyramidal neurons running through layers 1-5. Additionally, spine morphology changes (swelling, spine neck distortion), were also observed in the deeper layers 4 and 5 of the neocortex. Immature spines (filopodia) remained unchanged between groups, as well as the dendritic arborization of the analyzed neurons. In a third of the animals with meningitis, massive mechanical distortion of the primary dendrites of most of the pyramidal neurons through layers 1-5 was observed. This distortion was reproduced in acute brain slices after exposure to pneumolysin-containing bacterial lysates (S. pneumoniae D39 strain), but not to lysates of pneumolysin-deficient bacteria, which we explain by the tissue remodeling effect of the toxin. Experimental mechanical dendrite distortion in primary neural cultures demonstrated diminished FRAP diffusion of neuronally-expressed enhanced green fluorescent protein (eGFP), indicative of disturbed dendritic diffusion. Discussion: Our work extends earlier knowledge of synaptic loss in the superficial cortical layers during meningitis to deeper layers. These changes occurred surprisingly early in the course of the disease, substantially limiting the effective therapeutic window. Methodologically, we demonstrate that the dendritic spine collapse readout is a highly reliable and early marker of neural damage in pneumococcal meningitis models, allowing for reduction of the total number of animals used per a group due to much lower variation among animals.

An improved and simplified protocol to combine Golgi-Cox staining with immunofluorescence and transmission electron microscopy techniques.

Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.

Unveiling Hg-binding protein within black deposit formed on Golgi-Cox-stained brain neuron.

BACKGROUND: Golgi-Cox staining has been conventionally used for investigating neuronal development. After the brain tissue is subject to Golgi-Cox staining, black deposits are formed on the surface of the stained neurons because of mercuric sulfide, which does not show a fluorescence response under two-photon excitation. However, we unexpectedly observed fluorescence emitted by these black deposits during two-photon fluorescence measurements. Further, the in-depth of physical and chemical methods analysis revealed that the black deposits on the stained neurons are composed of Hg-binding proteins. METHODS: We studied black deposits present in the Golgi-Cox-stained mouse brain neurons using techniques such as multiple-photon microscopy, scan electron microscopy, micro-Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. RESULTS: The emitted fluorescence was because of the fluorescence groups of Hg-binding protein present within the Golgi-Cox deposits on the neuronal surface. CONCLUSIONS: The presence of Hg-binding proteins within black deposits on the surface of Golgi-Cox-stained neurons was proven for the first time. The novel interaction between the neurons and Hg2+ ions during Golgi-Cox staining help to understand the mechanism of Golgi-Cox staining.

Optimized Golgi-Cox Staining Validated in the Hippocampus of Spared Nerve Injury Mouse Model.

Golgi-Cox staining has been used extensively in neuroscience. Despite its unique ability to identify neuronal interconnections and neural processes, its lack of consistency and time-consuming nature reduces its appeal to researchers. Here, using a spared nerve injury (SNI) mouse model and control mice, we present a modified Golgi-Cox staining protocol that can stain mouse hippocampal neurons within 8 days. In this improved procedure, the mouse brain was fixed with 4% paraformaldehyde and then stored in a modified Golgi-Cox solution at 37 ± 2°C. The impregnation period was reduced from 5-14 days to 36-48 h. Brain slices prepared in this way could be preserved long-term at -80°C for up to 8 weeks. In addition to minimizing frequently encountered problems and reducing the time required to conduct the method, our modified protocol maintained, and even improved, the quality of traditional Golgi-Cox staining as applied to hippocampal neuronal morphology in SNI mice.