Golgi-Cox Method Research Articles

PERBEDAAN PENGARUH PAPARAN MUSIK MOZART, BEETHOVEN, DAN CHOPIN SELAMA KEBUNTINGAN TERHADAP KEPADATAN DENDRIT DI CEREBELLUM Rattus norvegicus BARU LAHIR

Indonesia is categorized as middle human development index. One of the causes is the fluent ignorance of brain growth and development during 1000 days of life period. This period is the best opportunity to give proper stimulations to increase the maximal brain growth and development. Exposure to classical music such as Mozart, Beethoven, and Chopin music can increase the wave of brain activity. During pregnancy, Mozart music is proven to increase the dendritic density. Analyzing the difference of dendritic density of cerebrum and cerebellum of newborn baby of Rattus norvegicus that are exposed to the Mozart, Beethoven, and Chopin music, and that is not exposed to music during pregnancy. Laboratory experimental research, posttest-only control group design. Subjects were female pregnant Rattus norvegicus, grouped into 4 random groups: 1 control group and 3 treatments groups; with 6 samples each. Subjects were super ovulated, and 65 dB intensity of music was played for an hour at 20.00-21.00 on the subjects on 10th day of pregnancy. On 20th day of pregnancy, the mothers were dissected using caesarean section. 2 heaviest newborn babies of Rattus norvegicus were taken, and their brain tissues were taken as samples. The dendritic density of cerebrum and cerebellum were observed using GolgiCox method of silver impregnation procedure.Statistical test concluded there were significant differences of dendritic density of cerebrum and cerebellum of newborn babies of Rattus norvegicus among the exposure to Mozart, Beethoven, and Chopin music, and without the exposure to music with value of p=0,004 (<0,05) in cerebrum and p=0,003 (<0,05) in cerebellum. The dendritic density of cerebrum and cerebellum of newborn babies of Rattus norvegicus that were exposed to Mozart music during pregnancy were higher than that are exposed to Beethoven and Chopin music and that were not exposed to music. It is recommended for the next research to do further research to prove the dendritic density caused by the musical stimulation during pregnancy when Rattus norvegicus grows up.

Cytoarchitecture of the dorsal claustrum of the cat: a quantitative Golgi study.

The claustrum is a subcortical nucleus, found in the telencephalon of all placental mammals. Earlier Golgi studies have mostly focused on a qualitative description of the types of neurons. The aim of the present study was to describe the types of neurons found in the dorsal claustrum of the cat using the Golgi impregnation method and to perform a quantitative analysis of the following morphometric parameters: number of terminals (ends), total dendritic length, dendritic complexity, spine density (in spiny projection neurons), varicosity density (in aspiny interneurons). We used specimens from 5 healthy male cats stained according to the Golgi-Cox method. The dendritic trees of the studied neurons were then reconstructed through the Neurolucida software. Values of the studied quantitative parameters were obtained automatically and tested for statistically significant differences. Five types of spiny neurons were observed-large, medium-sized and small multipolar, bipolar and pyramidal-like. In addition, we described three types of aspiny neurons. The quantitative values and the statistical analysis were presented with tables and diagrams. In conclusion, we have presented a detailed analysis of the cytoarchitecture of the DC of the cat and have reported the first quantitative data on a number of morphometric parameters.

Step by Step Golgi-Cox Staining for Cryosection.

Golgi staining, though invented hundreds of years ago, is still a reliable method to study the cytoarchitecture of the brain. Almost all published Golgi staining protocols and methods were used for microtome, and rarely applied in cryosection, which restricted the application of this technique. Currently, several commercial Golgi-stain kits are available for both vibratome section and cryosection, but these kits are costly, and it is still challenging for researchers to obtain significant results. In the present study, we described a protocol of Golgi-Cox Staining for Cryosection, with the modified cryosection protection solution based on the Golgi-Cox method, which makes cryosection easy to apply in the section of the Golgi-Cox impregnated tissue. Our methods provide a low-cost and simple option for Golgi staining, and it will facilitate researchers to obtain useful Golgi staining results for neuronal architecture studies.

Golgi-Cox Staining of Neuronal Dendrites and Dendritic Spines With FD Rapid GolgiStain™ Kit.

The Golgi-Cox method has been one of the most effective techniques for studying the morphology of neuronal dendrites and dendritic spines. However, the reliability and time-consuming process of Golgi-Cox staining have been major obstacles to the widespread application of this technique. To overcome these shortcomings and to promote this invaluable technique, we developed the FD Rapid GolgiStain™ Kit based on the principle of the methods described by Ramón-Moliner in 1970 and Glaser and Van der Loos in 1981. The kit significantly improves and simplifies the Golgi-Cox technique. This kit is reliable for visualizing morphological details of neurons, allowing for analysis of various parameters of dendritic morphology-such as dendritic length and branching pattern and dendritic spine number, shape, and size-in both animal and postmortem human brains. A 40-min instructional video for tissue freezing, cryosectioning, and staining is provided. © 2019 by John Wiley & Sons, Inc.

Adolescent social instability stress alters markers of synaptic plasticity and dendritic structure in the medial amygdala and lateral septum in male rats.

Much evidence indicates that experiences in adolescence can alter the development of social behaviour. We previously demonstrated that male rats exposed to social instability stress in adolescence (SS; 1h isolation and return to an unfamiliar cagemate daily from postnatal day [PND] 30-45) had reduced social interaction, impaired social recognition, reduced sexual performance, and increased aggression in competition for food reward compared with non-stressed control (CTL) rats. Here, we investigated whether SS affects stellate neuron morphology using the Golgi-Cox method and several markers of synaptic plasticity using western blotting in the medial amygdala (MeA) and lateral septum (LS), sites involved in social behaviour. On PND 46, 24h after the last stress exposure, SS rats had increased dendritic arborisation, a greater number of dendrite terminals, and a higher average dendrite branch order in the anterodorsal MeA compared with CTL rats. SS rats had reduced dendritic arborization and a reduced total length of dendrite matter in the anteroventral MeA and a reduced number of dendrite terminals in the posterodorsal MeA compared with CTL rats. Moreover, SS rats had a reduced number of dendritic spines in the dorsal LS compared with CTL rats. SS rats had less synaptophysin in the MeA and more CaMKII in the LS than did CTL rats, and did not differ in spinophilin, PSD95, or glucocorticoid receptor protein expression in the MeA and LS. We discuss how changes in neural structure and in markers of synaptic plasticity the MeA and LS of adolescent SS rats compared with CTL rats may underlie their differences in social behaviour.

Western-style diet induces object recognition deficits and alters complexity of dendritic arborization in the hippocampus and entorhinal cortex of male rats

Objective: Research demonstrates a link between diet-induced obesity and cognitive impairments; however, no studies have utilized the Sholl analysis to assess changes in dendritic arborization as a possible cause of obesity-induced memory deficits. Therefore, the purpose of this study was to examine the effect of a Western-style diet (WSD) on memory and dendritic complexity of male Sprague–Dawley rats.Methods: Male Sprague–Dawley rats (n = 18) were fed either a control or WSD. Spatial memory and episodic memory were assessed using the Morris Water Maze and novel object recognition (NOR) tasks, respectively. At termination, brains were removed and prepared with the Golgi-Cox method. Stained neurons in both the hippocampus and entorhinal cortex (EC) were imaged and digitally reconstructed.Results: Results indicated significant differences in percent body fat and TNFα levels between dietary conditions. WSD males also experienced reduced NOR exploration ratios, but no deficits in spatial memory were observed. Analysis of dendritic length and number of branch points revealed no significant differences in either the EC or the hippocampus; however, the Sholl analysis indicated that a WSD increased neuronal complexity in the EC.Discussion: Sholl analysis of the EC suggests a possible diet-induced dysfunction of pruning, which may contribute to reduced performance on the NOR task. Elevated TNFα levels indicate a putative role of inflammation in neuronal remodeling. The results demonstrate the importance of investigating mechanisms underlying obesity-related cognitive impairments.

Developmental ethanol exposure alters the morphology of mouse prefrontal neurons in a layer-specific manner

Chronic developmental exposure to ethanol can lead to a wide variety of teratogenic effects, which in humans are known as fetal alcohol spectrum disorders (FASD). Individuals affected by FASD may exhibit persistent impairments to cognitive functions such as learning, memory, and attention, which are highly dependent on medial prefrontal cortex (mPFC) circuitry. The objective of this study was to determine long-term effects of chronic developmental ethanol exposure on mPFC neuron morphology, in order to better-understand potential neuronal mechanisms underlying cognitive impairments associated with FASD. C57BL/6-strain mice were exposed to ethanol or an isocaloric/isovolumetric amount of sucrose (control) via oral gavage, administered both to the dam from gestational day 10–18 and directly to pups from postnatal day 4–14. Brains from male mice were collected at postnatal day 90 and neurons were stained using a modified Golgi-Cox method. Pyramidal neurons within layers II/III, V and VI of the mPFC were imaged, traced in three dimensions, and assessed using Sholl and branch structure analyses. Developmental ethanol exposure differentially impacted adult pyramidal neuron morphology depending on mPFC cortical layer. Neurons in layer II/III exhibited increased size and diameter of dendrite trees, whereas neurons in layer V were not affected. Layer VI neurons with long apical dendrites had trees with decreased diameter that extended farther from the soma, and layer VI neurons with short apical dendrite trees exhibited decreased tree size overall. These layer-specific alterations to mPFC neuron morphology may form a novel morphological mechanism underlying long-term mPFC dysfunction and resulting cognitive impairments in FASD.

A novel Golgi-Cox staining method for detecting and characterizing roles of the hepatic stellate cells in liver injury

The aim of this study was to investigate the utility of the Golgi-Cox method to characterize the distribution and morphological changes of the hepatic stellate cells (HSCs) in CCl4 liver damaged rats. Six-week-old male Wistar rats were injected with CCl4 for ten weeks. The livers were processed with the Golgi-Cox method, reticuline, and Massońs Trichrome stains, and analyzed under light microscopy. Histological evaluation of livers was made through the METAVIR score. In normal livers, the HSCs show stellate form with abundant thin cytoplasmic processes, distributed into hepatic lobule, mainly in zone 1. In addition, an intricate and broad network of fibers with radial distribution from the central vein to the periphery of the hepatic lobule was observed. In CCl4 damaged livers, with METAVIR score I and II, HSCs showed a moderate increase in the soma size, in the cytoplasmic processes and in density, distributed in zone 2 and 3; changes associated with a decrease in network fibers. In livers with METAVIR score III and IV, the morphology changes of the HSCs consisted of a significant increase in the soma size, cut and fraying appearance of the emerging cytoplasmic processes, and a decrease in HSCs density, distributed mainly in zone 3, with a significant depletion of network fibers. Results show that Golgi-Cox stain is able to impregnate the HSCs and could be an additional tool to study the morphological changes of the HSCs in the different experimental pathological conditions of the liver.

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method.

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 μm thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 μm working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritictrees and dendrite spines may be reliably visualized and quantified.

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The effect of maternal-induced diabetes on postnatal development of the paraventricular and ventromedial hypothalamic nuclei in albino rats: a histological, immunohistochemical, and morphometric study

BackgroundThe hypothalamus regulates body homeostasis by mediating endocrine, autonomic, and behavioral functions. Maternal diabetes constitutes an unfavorable environment for embryonic development. Aim of the workThe aim of the present study was to detect the effect of maternal-induced diabetes on the structural organization of the paraventricular and ventromedial hypothalamic nuclei during postnatal development. MethodsIn this study, a total of 60 adult female albino rats were used. They were divided into two groups – group I (control group) included 30 adult female rats, and group II (experimental group) included 30 adult female rats in which diabetes was induced by a single intraperitoneal injection of alloxan monohydrate. Hypothalamic specimens of the offspring were studied at day 1, 21 days, and 2 months of age postnatally. Several techniques were used in this study, including light microscopy, which included gallocyanine–chrom–alum staining and the Golgi–Cox method, transmission electron microscopy, and immunohistochemistry. In addition, a morphometric study was carried out to estimate the number of neurons in the paraventricular and ventromedial nuclei. ResultsIn the newborn, neurons in the studied hypothalamic nuclei showed presence of darkly stained nuclei. With age, these degenerative changes became more prominent. Marked reduction in the extension and branching of dendrites was observed. Electron microscopic examination of 2-month-old offspring of diabetic mothers showed the presence of chromatin condensation with many damaged mitochondria and a marked reduction in free ribosomes. Examination of the presynaptic terminals making contact with the neurons of the paraventricular and ventromedial nuclei showed marked reduction in synaptic vesicles and damaged mitochondria. In addition, an apparent increase in the number of astrocytes was found, indicating an increase in the expression of glial fibrillary acidic protein. The number of cells per area was highly decreased at 21 days and 2 months of age in the offsprings of diabetic mothers in comparison with the control group. ConclusionIn conclusion, this study indicates that maternal diabetes affects the development and structure of the ventromedial and paraventricular nuclei. These changes suggest that there should be strict control of diabetes before and during pregnancy.

Effect of lipopolysaccharide-mediated infection during pregnancy on the expression of mineralocorticoid receptor and dendritic spines in hippocampus of rat offspring

Objective To observe the effect of lipopolysaccharide (LPS)-mediated infection during pregnancy on the expression of mineralocorticoid receptor (MR) and density of dendritic spines in CA1 region of the dorsal hippocampus of rat offspring, so as to explore the mechanisms for learning and memory injury of rat offspring which were infected during prenatal period, then to provide scientific experimental evidence for the prevention of prenatal infection-induced delayed neuropsychiatric sequelae which contributed to learning and memory dysfunction. Methods Ten-week-old female Sprague-Dawley rats (n=30) were matched with male rats (11). Pregnant rats were randomly divided into a control group (n=10) and an experimental group (n=20). The pregnant rats in experimental group were treated with LPS (66 μg/kg, intraperitoneally), and the pregnant rats in control group were intraperitoneally injected with same volume of saline on gestational day 10.On postnatal day 48, Morris water maze was used to estimate the ability of learning and memory; the brain tissues of offspring were taken and paraffin sections were stained with hemato-xylin eosin (HE) for histological observation of CA1 region of the dorsal hippocampus; frozen sections were treated with indirect immunofluorescence to observe the expression of MR in CA1 region of the dorsal hippocampus; Golgi-Cox method was used to observe the density of dendritic spines of CA1 region. Results In Morris water maze test, from the third day the time of escape latency in experimental group [the 3rd day: (42.603±9.837) s; the 4th day: (30.222±9.789) s; the 5th day: (28.808±12.526) s] was significantly higher than that of the control group [the 3rd day: (28.078±14.088) s; the 4th day: (20.692±13.099) s; the 5th day: (14.632±11.624) s] (the 3rd day: t=-3.274, P<0.01; the 4th day: t=-2.257, P<0.05; the 5th day: t=-3.213, P<0.01); the swimming time in the target quadrant [(14.660±7.337) times] and the number of crossing platform [(0.933±0.704) times] in experimental group were significantly decreased compared with those of the control group [time: (23.820±6.321) s; numbers: (2.000±0.756) times](t=3.663, 4.000, all P<0.01). Hematoxylin eosin staining showed that the nerve cells of the hippocampus in the control group distributed in order, nucleuses were round or oval, nucleoli were obvious, and chromatins were homogeneous; but the cells in the experimental group distributed in disorder and pathological changes were detected, such as cellular swelling, necrosis and obvious nuclear pyknosis.By immunofluorescence staining, the average optical density (AOD) of MR in CA1 region decreased significantly in the experimental group (0.067±0.017) compared with that of the control group (0.083±0.009)(t=2.644, P<0.05). In Golgi-Cox method, the density of dendritic spines in experimental group [(7.705±0.791)/10 μm] was below that of the control group [(9.655±1.391)/10 μm](t=3.852, P<0.01). Conclusions LPS-mediated infection during pregnancy might lead to hippocampus-dependent learning and memory dysfunction which might be associated with the reduced expression of MR and the low density of dendritic spines in CA1 region of the dorsal hippocampus. Key words: Lipopolysaccharide; Hippocampus; Mineralocorticoid receptor; Dendritic spine

Optimising Golgi–Cox staining for use with perfusion-fixed brain tissue validated in the zQ175 mouse model of Huntington's disease

BackgroundThe Golgi–Cox stain is an established method for characterising neuron cell morphology. The method highlights neurite processes of stained cells allowing the complexity of dendritic branching to be measured. New methodsConventional rapid Golgi and Golgi–Cox methods all require fresh impregnation in unfixed brain blocks. Here, we describe a modified method that gives high quality staining on brain tissue blocks perfusion-fixed with 4% paraformaldehyde (PFA) and post-fixed by immersion for 24h. ResultsTissue perfused with 4% PFA and post fixed for 24h remained viable for the modified Golgi–Cox silver impregnation staining of mouse striatum from perfused wild type and zQ175. It was not found necessary to impregnate tissue blocks with Golgi solutions prior to sectioning, as post-sectioned tissues yielded equally good impregnation. Impregnation for 14 days resulted in optimal visualisation of striatal neuron and dendritic morphology. Although no modifications applied to the rapid Golgi method were reliable, the modified Golgi–Cox method yielded consistently reliable high-quality staining. Comparison with existing methodsThe current method used fixed tissues to reduce damage and preserve cell morphology. The revised method was found to be fast, reliable and cost effective without the need for expensive staining kits and could be performed in any neuroscience lab with limited specialist equipment. ConclusionsThe present study introduces a robust reproducible and inexpensive staining method for identifying neuronal morphological changes in the post fixed mouse brain, and is suitable for assessing changes in cell morphology in models of neurodegeneration and in response to experimental treatment.

Consistent and reproducible staining of glia by a modified Golgi–Cox method

BackgroundGolgi–Cox staining is a powerful histochemical approach which has been used extensively to visualize the morphology of neurons and glia. However, its usage as a first-choice method is hindered by its uncertain nature, diminished consistency and lengthy staining duration. The FD Rapid GolgiStain™ Kit (FD Neurotechnologies, Inc., USA) has been developed by employing the Golgi–Cox approach. It is a simple, reliable and reproducible way of performing Golgi impregnation for the analysis of neuronal morphology. New methodWe report here simple modifications to the manufacturer's protocol which enable reproducible and reliable staining of glial cells. ResultsExposure of brain tissue to 4% paraformaldehyde (PFA) during perfusion followed by postfixation with 8% glutaraldehyde in 4% PFA led to only glial cells being stained, whereas in the absence of postfixation both neurons and glia were stained with unclear morphology. Additionally, we found that impregnation at 26°C±1 was critical to attain uniform staining. Comparison with existing methodOur modified Golgi–Cox approach is consistent and reproducible and affords uniform glial staining throughout the brain. ConclusionAs this protocol stains only a small percentage of cells, it is suitable for the analysis of individual cells.

Ethanol-Induced Alterations in Purkinje Neuron Dendrites in Adult and Aging Rats: a Review.

Uncomplicated alcoholics suffer from discrete motor dysfunctions that become more pronounced with age. These deficits involve the structure and function of Purkinje neurons (PN), the sole output neurons from the cerebellar cortex. This review focuses on alterations to the PN dendritic arbor in the adult and aging Fischer 344 rat following lengthy alcohol consumption. It describes seminal studies using the Golgi-Cox method which proposed a model for ethanol-induced dendritic regression. Subsequent ultrastructural studies of PN dendrites showed dilation of the extensive smooth endoplasmic reticulum (SER) which preceded and accompanied dendritic regression. The component of the SER that was most affected by ethanol was the sarco/endoplasmic reticulum Ca(2+) ATPase pump (SERCA) responsible for resequestration of calcium into the SER. Ethanol-induced decreases in SERCA pump levels, similar to the finding of SER dilation, preceded and occurred concomitantly with dendritic regression. Discrete ethanol-induced deficits in balance also accompanied these decreases. Ethanol-induced ER stress within the SER of PN dendrites was proposed as an underlying cause of dendritic regression. It was recently shown that increased activation of caspase 12, inherent to the ER, occurred in PN of acute slices in ethanol-fed rats and was most pronounced following 40 weeks of ethanol treatment. These findings shed new light into alcohol-induced disruption in PN dendrites providing a new model for the discrete but critical changes in motor function in aging, adult alcoholics.

Staining neurons with Golgi techniques in degenerative diseases of the brain.

Staining neurons with Golgi techniques in degenerative diseases of the brain.

Structural changes in the adult rat auditory system induced by brief postnatal noise exposure.

In previous studies (Grécová et al., Eur J Neurosci 29:1921-1930, 2009; Bures et al., Eur J Neurosci 32:155-164, 2010), we demonstrated that after an early postnatal short noise exposure (8min 125dB, day 14) changes in the frequency tuning curves as well as changes in the coding of sound intensity are present in the inferior colliculus (IC) of adult rats. In this study, we analyze on the basis of the Golgi-Cox method the morphology of neurons in the IC, the medial geniculate body (MGB) and the auditory cortex (AC) of 3-month-old Long-Evans rats exposed to identical noise at postnatal day 14 and compare the results to littermate controls. In rats exposed to noise as pups, the mean total length of the neuronal tree was found to be larger in the external cortex and the central nucleus of the IC and in the ventral division of the MGB. In addition, the numerical density of dendritic spines was decreased on the branches of neurons in the ventral division of the MGB in noise-exposed animals. In the AC, the mean total length of the apical dendritic segments of pyramidal neurons was significantly shorter in noise-exposed rats, however, only slight differences with respect to controls were observed in the length of basal dendrites of pyramidal cells as well as in the neuronal trees of AC non-pyramidal neurons. The numerical density of dendritic spines on the branches of pyramidal AC neurons was lower in exposed rats than in controls. These findings demonstrate that early postnatal short noise exposure can induce permanent changes in the development of neurons in the central auditory system, which apparently represent morphological correlates of functional plasticity.

Sleep deprivation induce morphology changes in the hippocampus and prefrontal cortex in young and old rats

Introduction During normal aging several changes in sleep/wake patterns are observed, which include frequent awakenings during sleep and increased daytime naps, among others. Likewise, aging has also been associated with a deterioration of cognitive function, learning and memory, although widespread loss of nerve cells does not occur, the most of age-related structural changes observed in nerve cells are modifications in dendrites, dendritic spines or even axons. Evidence accumulated over the last years indicates that these functional changes observed during sleep loss, aging, or both could be due to modifications in synaptic connectivity and intracellular signaling; for example, excitatory synaptic transmission at the hippocampal CA1 region is affected by sleep deprivation; in the locus coeruleus (involved in both arousal system and cognitive performance) the number of neurons projecting to areas such as the cortex and the hippocampus declines with age. Therefore, it has been hypothesized that sleep deprivation may compromise neurophysiological and behavioral events; however, relatively few studies have investigated links between sleep loss and structural changes in neurons and, despite the seemingly similar effects of age and sleep deprivation on cognition and the prevalence of sleep changes with age, little is known about the impact of sleep loss on cellular morphology in aging neurons. For that reasons, the aim of this study was to evaluate the effects of total sleep deprivation on neuronal morphology in the hippocampus and prefrontal cortex of both young and aged animals. Materials and methods A total of 28 male Wistar rats (14 “young-adult” rats, 3–4 months old; 14 “aged rats”, 22–23 months old; 7 for control and 7 for sleep deprivation for each age) were used in this study. Total sleep deprivation was carried out in both experimental groups (young-adult and aged, n = 7 per group) by gentle handling: once sleep-behaviour was observed or low amplitude waves first appear in sleep recording, rats were softly touched in their tails, whiskers or handling them to prevent falling asleep during 24 h. Immediately after sleep deprivation finished, animals were deeply anesthetized with sodium pentobarbital (75 mg/kg, i.p.) and then perfused intracardially with 0.9% saline solution. Brains were removed and stained by modified Golgi-Cox method. Pyramidal neurons from layer 3 of prefrontal cortex and hippocampus (CA1 area) were selected for study. Five neurons from each region of each brain hemisphere per animal were drawn using a camera lucida. Basal dendrites, including all branches, were reconstructed for each neuron and their dendritic tracings were quantified by Sholl analysis. Results Results showed that total dendritic length of prefrontal cortex and hippocampus was not affected either age or after 24 h of sleep deprivation compared to their corresponding control group. However, after 24 h of sleep deprivation (SD) aged animals had an increase in spine density in prefrontal cortex but not in hippocampus. Conclusion Sleep deprivation could be considered a factor that induces neuronal plasticity, which may depend on age. Acknowledgement This study was supported by CONACYT grant 133178.