Golgi-Cox Impregnation Research Articles

Dendritic morphology of motor neurons and interneurons within the compact, semicompact, and loose formations of the rat nucleus ambiguus.

Motor neurons (MNs) within the nucleus ambiguus innervate the skeletal muscles of the larynx, pharynx, and oesophagus. These muscles are activated during vocalisation and swallowing and must be coordinated with several respiratory and other behaviours. Despite many studies evaluating the projections and orientation of MNs within the nucleus ambiguus, there is no quantitative information regarding the dendritic arbours of MNs residing in the compact, and semicompact/loose formations of the nucleus ambiguus.. In female and male Fischer 344 rats, we evaluated MN number using Nissl staining, and MN and non-MN dendritic morphology using Golgi-Cox impregnation Brightfield imaging of transverse Nissl sections (15 μm) were taken to stereologically assess the number of nucleus ambiguus MNs within the compact and semicompact/loose formations. Pseudo-confocal imaging of Golgi-impregnated neurons within the nucleus ambiguus (sectioned transversely at 180 μm) was traced in 3D to determine dendritic arbourisation. We found a greater abundance of MNs within the compact than the semicompact/loose formations. Dendritic lengths, complexity, and convex hull surface areas were greatest in MNs of the semicompact/loose formation, with compact formation MNs being smaller. MNs from both regions were larger than non-MNs reconstructed within the nucleus ambiguus. Adding HBLS to the diet could be a potentially effective strategy to improve horses' health.

Antidepressant-like effects of the Punica granatum and citalopram combination are associated with structural changes in dendritic spines of granule cells in the dentate gyrus of rats.

Introduction: Natural products such as phytoestrogens-enriched foods or supplements have been considered as an alternative therapy to reduce depressive symptoms associated with menopause. It is known that the aqueous extract of Punica granatum (AE-PG) exerts antidepressant-like effects by activating β-estrogen receptors and facilitates the antidepressant response of the clinical drug citalopram (CIT). However, the effects on neuroplasticity are unknown. Objectvie investigated the antidepressant-like response of combining AE-PG and CIT at sub-optimal doses, analyzing their effects on the formation and maturation of dendrite spines in granule cells as well as on the dendrite complexity. Methods: Ovariectomized Wistar rats (3-month-old) were randomly assigned to one of the following groups: A) control (saline solution as vehicle of CIT and AE-PG, B) AE-PG at a sub-threshold dose (vehicle of CIT plus AE-PG at 0.125mg/kg), C) CIT at a sub-threshold dose (0.77mg/kg plus vehicle of AE-PG), and D) a combination of CIT plus AE-PG (0.125mg/kg and 0.77mg/kg, respectively). All rats were treated intraperitoneally for 14 days. Antidepressant-like effects were evaluated using the force swimming test test (FST). The complexity of dendrites and the number and morphology of dendrite spines of neurons were assessed in the dentate gyrus after Golgi-Cox impregnation. The expressions of the mature brain-derived neurotrophic factor (mBDNF) in plasma and of mBDNF and synaptophysin in the hippocampus, as markers of synaptogenesis, were also determined. Results: Administration of CIT combined with AE-PG, but not alone, induced a significant antidepressant-like effect in the FST with an increase in the dendritic complexity and the number of dendritic spines in the dentate gyrus (DG) of the hippocampus, revealed by the thin and stubby categories of neurons at the granular cell layer. At the same time, an increase of mBDNF and synaptophysin expression was observed in the hippocampus of rats that received the combination of AE-PG and CIT.

1147 MORPHO-FUNCTIONAL EVALUATION OF 3MG/KG ICV-STZ RAT SHOWED SPORADIC ALZHEIMER'S LIKE PATHOLOGY WITH PROGRESSIVE DEMENTIA

Abstract Background Intracerebroventricular streptozotocin (ICV-STZ) injection is among the best animal models to simulate sporadic Alzheimer’s disease (sAD). Abnormality in brain insulin signalling, neurodegeneration, neuroinflammation, cholinergic damage, mitochondrial dysfunction, genetic abnormality, respiratory problem, oxidative stress, gliosis, sleep disturbances are associated with cognitive abnormalities seen in ICV-STZ injected rats. Available experimental evidence has used varying doses of STZ (<1 to 3mg/kg) and studied its effect for different study durations, ranging from 14-21 (short), 30-42 (mild), 90-105 (moderate) and 250-270 (long) days. These studies indicated that 3mg/kg of body-weight is the optimum dose for inducing sAD in the rodents. However, studies on the pathological process with related the morphological and functional abnormalities reported were illusive. Objective/Method Hence in the present study, we have investigated the morpho-functional changes after 3mg/kg ICV-STZ treatment with a follow-up of two months in 54 male Wistar rats (ethical no. 937/IAEC/PhD-2016). Results Exhibited a spatial, episodic and avoidance memory decline and increase in anxiety (p<0.05) in ICV-STZ group progressively with time from 15th day to 60th day post-injection. Morphometry showed hippocampal atrophy with CA1, CA3 layer thinning (p ≤0.01) and loss of neurons (p<0.0001) associated with third ventricular enlargement (p= 0.007) in ICV-STZ rats versus sham, along-with extracellular amyloid plaque in AD rats with Congored staining. In addition, spine morphometry with Golgi-Cox impregnation of mossy fibre showed a reduction of spine density in AD group versus control and sham group (p<0.0001). Finally, immunohistochemistry of GSK3ß, PI3K and mtCOX-1 antigen in coronal sections revealed an increase in mean intensity of GSK3ß and decrease in PI3K and mtCox-1 in brain areas associated with limbic system in ICV-STZ group on 60th day. Conclusion These findings suggest progressive dementia and anxiety in 3mg/kg STZ treated rats, which may be due to hippocampal atrophy, amyloidopathy, ventricular enlargement, synaptic dysfunction and deficits in energy homeostasis of brain.

Neuroinflammation Underlies the Development of Social Stress Induced Cognitive Deficit in Sickle Cell Disease

Cognitive deficits have been observed in children and adults with sickle cell disease (SCD), with and without cerebral infarcts. Risk factors include systemic inflammation, neuroinflammation, and severe anemia, and sociological risk factors such as parental education level and income. Additionally, there is a reported relationship between plasma cytokine levels and cognitive function in children with SCD, suggesting a link between inflammation and cognitive deficit in SCD. This study aims to examine the mechanism underlying the development of cognitive deficit in SCD with exposure to social stress, by using the repeat social defeat (RSD) paradigm in sickle cell mice. We hypothesized that neuroinflammation is a major underlying mechanism mediating the relationship between social stress and development of cognitive deficit in SCD. RSD was done by introducing a male intruder mouse (an aggressor) into an established cage of 3, 6 months old male sickle cell or control mice, every day, for two hours, for six consecutive days. Age and sex-matched control (sickle cell and non-sickle cell mice) cages were set up but without aggressor mice. On the seventh day, mice were tested for cognitive/behavioral deficit using novel object recognition (NOR) and fear conditioning (FC) test paradigms. Except for the aggressors, all mice used were Townes humanized sickle cell (SS) and control (AA) mice. To test the hypothesis that neuroinflammation is an underlying mechanism, a second cohort of SS and AA nice were randomly assigned to oral (in drinking water) minocycline treatment (90mg/kg) or placebo (drinking water). Mice within treatment arms were randomly assigned to RSD exposure or no RSD exposure. Minocycline treatment was started one day prior to the day of commencing RSD and terminated on the same day as the final RSD session and the dose was kept constant by adjusting the amount administered daily, using the water consumption from the previous day. Cognitive/behavioral testing was performed as before, starting the next day after day 6 of RSD and day 7 of treatment. Mice were sacrificed 1-2 days after completion of behavioral testing and brains were extracted for histological analysis. Cellular evidence of neuroinflammation in the hippocampus/dentate gyrus was determined using immunohistochemistry to quantify peripheral immune cell infiltrates (CD45+ (bone marrow-derived microglia), CD3+ (T-cell density), B220+ (B-cell density), and Iba1+ (activated microglia). We used Golgi-Cox impregnation to quantify spine density and maturity in cortical and hippocampal pyramidal neurons. There were no differences between 6 months old AA and SS with regards to percent preference on NOR test (53.1 ± 4.4% vs. 56.1 ± 5.6%). However, SS mice exposed to RSD, had a lower percent preference (36.1 ± 6.7%, p = 0.06) for the novel object compared to SS mice not exposed to RSD (56.1 ± 5.6%), indicating the presence of a memory deficit in SS mice exposed to RSD. The SS mice exposed to RSD had worse performance on contextual FC test (p = 0.001), indicated by a lower percent freezing compared with SS mice not exposed to RSD. Additionally, SS mice exposed to RSD also had a higher density of activated microglia (260.2 ± 3.7 vs. 233.6 ± 4.0; p ≤ 0.0001) and bone marrow derived microglia (125.4 ± 6.2 vs. 93.7 ± 5.0; p = 0.0004) in the dentate gyrus, compared to SS mice not exposed to RSD, suggesting increased neuroinflammation after RSD exposure. There were no differences in B or T cell density. SS mice treated with minocycline during RSD exposure showed improvement in memory based on NOR (67.1 ± 4.5% vs 36.1 ± 6.7%, p = 0.0007) and contextual FC (p = 0.01) tests compared to SS mice exposed to RSD and not treated with minocycline. The treated mice had fewer activated microglia (168.4 ± 6.7 vs. 260.2 ± 3.7; p ≤ 0.0001) and bone marrow derived microglia (31.79 ± 2.2 vs. 125.4 ± 6.2; p≤0.0001) in the dentate gyrus compared to the non-treated SS mice exposed to RSD. Additionally, the treated SS mice exposed to RSD also showed increases in neuron progenitor stem cells density improvement in neurogenesis and a decrease in astrogliosis in granular zone of the dentate gyrus, compared to the SS mice exposed to RSD without minocycline treatment. We demonstrate for the first time that neuroinflammation is one of the underlying mechanisms of social stress related cognitive impairment in mice and may be applicable to children with SCD and might be treatable using anti-neuroinflammatory drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Golgi-Cox impregnation combined with fluorescence staining of amyloid plaques reveals local spine loss in an Alzheimer mouse model

BackgroundSpine loss is a hallmark of Alzheimer´s and other neurodegenerative diseases, and testing candidate therapeutic drugs needs quantitative analysis of dendritic spine densities. Golgi-Cox impregnation of neurons is a classical method to visualize dendritic spines in diseased brains. Importantly, at early disease stages spine loss occurs locally in the vicinity of amyloid plaques, and concomitant fluorescence labeling of amyloid plaques is required to detect local spine damage. New MethodBecause Golgi-Cox impregnation is done on unsectioned brains, whereas fluorescence staining is performed on sectioned material, the combination is technically challenging. We have now developed a novel combination of Golgi-Cox impregnation with methoxy-X04 fluorescence labeling of plaques that is performed on unsectioned brains. ResultsWe used this new combination method to quantify dendritic spine densities in mouse hippocampal CA1 pyramidal neurons. Comparison of neurons from wildtype and APP/PS1 mice revealed local spine loss in the vicinity of amyloid plaques in both male and female APP/PS1 mice. Comparison with existing methodGolgi-Cox impregnation of neurons combined with methoxy-X04 staining of amyloid plaques is a highly reliable, easy-to-use method for permanent visualization of spines as compared to the technically more sophisticated and less stable fluorescence imaging of spines. ConclusionOur novel combination method will be highly useful for testing potential therapeutic drugs in Alzheimer mouse models.

Size-Dependent Vulnerability of Lumbar Motor Neuron Dendritic Degeneration in SOD1G93A Mice.

The motor neuron (MN) soma surface area is correlated with motor unit type. Larger MNs innervate fast fatigue-intermediate (FInt) or fast-fatiguable (FF) muscle fibers in type FInt and FF motor units, respectively. Smaller MNs innervate slow-twitch fatigue-resistant (S) or fast fatigue-resistant (FR) muscle fibers in type S and FR motor units, respectively. In amyotrophic lateral sclerosis (ALS), FInt and FF motor units are more vulnerable, with denervation and MN death occurring for these units before the more resilient S and FR units. Abnormal MN dendritic arbors have been observed in ALS in humans and rodent models. We used a Golgi-Cox impregnation protocol to examine soma size-dependent changes in the dendritic morphology of lumbar MNs in SOD1G93A mice, a model of ALS, at pre-symptomatic, onset and mid-disease stages. In wildtype control mice, the relationship between MN soma surface area and dendritic length or dendritic spine number was highly linear (i.e., increased MN soma size correlated with increased dendritic length and spines). By contrast, in SOD1G93A mice, this linear relationship was lost and dendritic length reduction and spine loss were observed in larger MNs, from pre-symptomatic stages onward. These changes correlated with the neuromotor symptoms of ALS in rodent models. At presymptomatic ages, changes were restricted to the larger MNs, likely to comprise vulnerable FInt and FF motor units. Our results suggest morphological changes of MN dendrites and dendritic spines are likely to contribute ALS pathogenesis, not compensate for it. Anat Rec, 303:1455-1471, 2020. © 2019 American Association for Anatomy.

Direct Intracellular Signaling by the Carboxy terminus of NMDA Receptor GluN2 Subunits Regulates Dendritic Morphology in Hippocampal CA1 Pyramidal Neurons

N-methyl-d-aspartate receptors (NMDARs) are glutamatergic receptors that take part in excitatory synaptic transmission and drive functional and structural neuronal plasticity, including activity-dependent changes in dendritic morphology. Forebrain NMDARs contribute to neuronal plasticity in at least two ways: through calcium-mediated processes or via direct intracellular postsynaptic signaling. Both properties are regulated by the GluN2 subunits. However, the separate contributions of these properties to the regulation of dendritic morphology are unknown. We created transgenic mice that express chimeric GluN2 subunits and examined the impact on pyramidal cell dendritic morphology in hippocampal region CA1. Golgi-Cox impregnation and transgenic expression of green fluorescent protein were employed to visualize dendritic arbors. In adult mice with a predominantly native GluN2A background, overexpression of the GluN2B carboxy terminus increased the total path of the dendritic arbor without affecting branch number or tortuosity. Overexpressing the amino terminus and transmembrane domains of GluN2B had little effect. It may be inferred from these results that NMDAR-dependent intracellular signaling regulates dendritic morphology of hippocampal pyramidal cells more so than calcium conductance dynamics. The findings add to the understanding of NMDAR-mediated signaling in hippocampal neurons and support re-investigation of the molecular underpinnings of NMDAR involvement in postnatal dendrite maturation.

Towards A Structural Characterization of The Leopard Gecko Cerebellum: Purkinje Cells And Cells of The External Granular Layer

The cerebellum is a major component of the hindbrain, and plays important roles in motor coordination, posture, and cognitive functions. Although the gross anatomy and histological organization of the cerebellum has been documented across many species, less is known about the contributing cell types, especially among non‐mammals. Here we focus on Purkinje cells (PC) and cells of the External Granular Layer (EGL) in a representative lizard, the leopard gecko (Eublepharis macularius). PC serve as the major motor output of the cerebellum. Similar to other vertebrates, gecko PC are large, calbindin‐immunopositive neurons located at the junction between the molecular layer and the granular layer. Golgi‐cox impregnation reveals a dendritic arborization broadly comparable with that of alligators (and distinct from that of mammals). There is typically a single, lengthy primary dendrite that passes towards the molecular layer. This primary dendrite divides into multiple short dendritic branches that conclude in thin spiny branchlets with abundant, closely packed spines. Adult geckos also retain an EGL, a progenitor cell population that characteristically disappears prior to adulthood in mammals. Cells of the gecko EGL are immunopositive for the transcription factor SOX2, a hallmark protein of neural stem/progenitor cells, and the intermediate filament (and radial glia marker) glial fibrillary acid protein, but are immunonegative for NeuN, a marker of mature neurons. Using a bromodeoxyuridine pulse‐chase strategy, we determined that a subset of gecko EGL cells were label retaining after a 140 day chase period. Combined, these data provide important new information about the evolutionary variability of the cerebellum, with implications for functional adaptations in a species capable of tail loss.Support or Funding InformationNatural Sciences and Engineering Research Council Discovery Grant #400358 (MKV), #436190 (CDCB) and The Canada Foundation for Innovation #25371 (MKV) and #30381 (CDCB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuroanatomical and electrophysiological recovery in the contralateral intact cortex following transient focal cerebral ischemia in rats

ObjectivesFocal cerebral ischemia may induce synaptic, electrophysiological, and metabolic dysfunction in remote areas. We have shown that the remote dendritic spine density changes and electrophysiological diaschisis in the acute and subacute stages after stroke previously. Here, we further evaluated electrophysiological outcomes and synapto-dendritic plasticity in long-term recovery in the contralateral cortex following focal cerebral ischemia.MethodsMale Sprague–Dawley rats were subjected to intraluminal suture occlusion for 90 min or sham-occlusion. Somatosensory electrophysiological recordings (SSEPs) and neurobehavioral tests were recorded each day for 28 days. Postmortem brains were sectioned and subjected to Nissl staining and Golgi–Cox impregnation through a 28-day period following ischemic stroke.ResultsIn the ipsilateral cortex, infarct size in the cortex and striatum was decreased after the subacute stage; the brains showed reduced swelling in the cortex and stratum 3 days after ischemic insults. Dendritic spine density and SSEP amplitude decreased significantly during a 28-day recovery period. In the contralateral cortex, dendritic spine density and SSEP amplitude decreased significantly for 21 days after ischemic stroke, but recovered to baseline by day 28. The deterioration of the dendritic spine (density reduction) in the ischemic cortex was observed; however, this increased neuroplasticity in the contralateral cortex in the subacute stage.DiscussionFocal cerebral ischemia–reperfusion induces time-dependent reduction of dendritic spine density and electrophysiological depression in both the ipsilateral and contralateral cortices and intact brain. This neuroanatomical and electrophysiological evidence suggests that neuroplasticity and functional re-organization in the contralateral cortex is possible following focal cerebral ischemia.

Obesity-Induced Structural and Neuronal Plasticity in the Lateral Orbitofrontal Cortex.

The orbitofrontal cortex (OFC) integrates sensory information with the current value of foods and updates actions based on this information. Obese humans and rats fed a cafeteria diet have impaired devaluation of food rewards, implicating a potential obesity-induced dysfunction of the OFC. We hypothesized that obesity alters OFC pyramidal neuronal structure and function and reduces conditioned suppression of feeding. Rats were given restricted (1 h/day), extended (23 h/day) or no (chow only) access to a cafeteria diet and tested for a conditioned suppression of feeding. Golgi-cox impregnation and whole-cell patch clamp experiments were performed in lateral OFC pyramidal neurons of rats from the 3 feeding groups. Rats with 40 days of extended, but not restricted, access to a cafeteria diet became obese and continued to feed during foot shock-predicting cues. Access to a cafeteria diet induced morphological changes in basilar dendrites of lateral OFC pyramidal neurons. While there were no alterations in excitatory synaptic transmission underlying altered spine density, we observed a more depolarized resting membrane potential. This was accompanied by decreased inhibitory synaptic transmission onto lateral OFC pyramidal neurons due to decreased release probability at GABAergic inputs. These changes could underlie the inability of the OFC to encode changes in the motivation value of food that is observed in obese rodents and humans.

Effects of long-term agomelatine treatment on the cognitive performance and hippocampal plasticity of adult rats.

Agomelatine is an antidepressant with a distinct pharmacological mechanism of action as an MT1 and MT2 receptor agonist and as a 5-HT2C receptor antagonist. We evaluated the chronic effects of agomelatine administration (40 mg/kg, 20 weeks) on the cognitive performance of rats in the Morris water maze task. We applied unbiased stereological quantification methods to estimate the total numbers of granular and pyramidal neurons located in the dorsal hippocampus. We also analyzed the dendritic spines of pyramidal neurons in the CA1 region using the Golgi-Cox impregnation method. The agomelatine-treated group found the hidden platform more quickly than did the control group and spent significantly more time in the target quadrant. Agomelatine administration caused significant volumetric and numerical enhancements in granular and pyramidal neurons in the dentate gyrus and CA1-3 subregions, respectively. Increased densities of the mushroom and stubby types of spines, with no alteration in the thin-shaped spines, were observed in the agomelatine-treated group. These results showed that long-term agomelatine administration induced a nootropic effect supported by structural changes. Enhancement of the more stable types of dendritic spines might indicate improved adaptive capacity in hippocampal neurons. Future studies will provide a better understanding of the effect of this drug on synaptic plasticity.

Book Review

Maternal infection during pregnancy increases the risk for neurodevelopmental disorders in offspring. Rodent models have played a critical role in establishing maternal immune activation (MIA) as a causal factor for altered brain and behavioral development in offspring. We recently extended these findings to a species more closely related to humans by demonstrating that rhesus monkeys (Macaca mulatta) prenatally exposed to MIA also develop abnormal behaviors. Here, for the first time, we present initial evidence of underlying brain pathology in this novel nonhuman primate MIA model. Pregnant rhesus monkeys were injected with a modified form of the viral mimic polyI:C (poly ICLC) or saline at the end of the first trimester. Brain tissue was collected from the offspring at 3.5 years and blocks of dorsolateral prefrontal cortex (BA46) were used to analyze neuronal dendritic morphology and spine density using the Golgi-Cox impregnation method. For each case, 10 layer III pyramidal cells were traced in their entirety, including all apical, oblique and basal dendrites, and their spines. We further analyzed somal size and apical dendrite trunk morphology in 30 cells per case over a 30 μm section located 100 ± 10 μm from the soma. Compared to controls, apical dendrites of MIA-treated offspring were smaller in diameter and exhibited a greater number of oblique dendrites. These data provide the first evidence that prenatal exposure to MIA alters dendritic morphology in a nonhuman primate MIA model, which may have profound implications for revealing the underlying neuropathology of neurodevelopmental disorders related to maternal infection.

Influence of pregnant rats' prenatal chronic stress on hippocampal dendritic spines and the function of learning and memory in their offsprings

Objective To study the influence of pregnant rats' prenatal chronic stress (PS) on learning and memory of their offspring rats and its possible molecular mechanisms. Methods Pregnant females were individually restrained for 45 min 3 times a day during pregnancy from day 14 to day 21.Control pregnant females were left undisturbed in their home cages.The rat offsprings were randomly assigned to PS group or control group.Males and females were kept for the study separately.The learning and memory of the developing rat offspring in the Morris water maze were examined.The basal levels of corticosterone(COR) and adreno-cortico-tropic-hormone(ACTH) were analyzed by using radioimmunoassay.The Golgi-Cox impregnation technique was used to compare density and morphology of the CA1 hippocampal dendritic spines. Results The escape latency(EL) to find the platform in the control group was significantly less than that in the PS group in female rat offspring (F=4.533, P 0.05). The basal levels of the serum COR in the PS group were higher than those in the control group of female rat offspring(t=3.658, P<0.01)and the basal levels of the serum ACTH in the PS group were higher than those in the control group of male rat offsprings(t=2.319, P<0.05). A simplified pattern was observed in the CA1 hippocampal dendritic spines in the PS group, showing a less extent of dendritic arborization and the density was significantly lower than that in the control group(t=-3.072, P<0.01). Conclusions Altered function of the hypothalamic-pituitary-adrenal axis in the offspring mediates the cognitive alterations observed following prenatal stress should to be associated with the lower density and simplified pattern of CA1 dendritic spines. Key words: Prenatal chronic stress; Dendritic spine; Hippocampus; Learning and memory; Offspring rat

Preliminary evidence of neuropathology in nonhuman primates prenatally exposed to maternal immune activation.

Maternal infection during pregnancy increases the risk for neurodevelopmental disorders in offspring. Rodent models have played a critical role in establishing maternal immune activation (MIA) as a causal factor for altered brain and behavioral development in offspring. We recently extended these findings to a species more closely related to humans by demonstrating that rhesus monkeys (Macaca mulatta) prenatally exposed to MIA also develop abnormal behaviors. Here, for the first time, we present initial evidence of underlying brain pathology in this novel nonhuman primate MIA model. Pregnant rhesus monkeys were injected with a modified form of the viral mimic polyI:C (poly ICLC) or saline at the end of the first trimester. Brain tissue was collected from the offspring at 3.5 years and blocks of dorsolateral prefrontal cortex (BA46) were used to analyze neuronal dendritic morphology and spine density using the Golgi-Cox impregnation method. For each case, 10 layer III pyramidal cells were traced in their entirety, including all apical, oblique and basal dendrites, and their spines. We further analyzed somal size and apical dendrite trunk morphology in 30 cells per case over a 30 μm section located 100±10 μm from the soma. Compared to controls, apical dendrites of MIA-treated offspring were smaller in diameter and exhibited a greater number of oblique dendrites. These data provide the first evidence that prenatal exposure to MIA alters dendritic morphology in a nonhuman primate MIA model, which may have profound implications for revealing the underlying neuropathology of neurodevelopmental disorders related to maternal infection.

Morphology and Chemical Phenotype of the Ovarian Intrinsic Neurons in Neonate and Sexually Mature Reproductive Guinea Pig

Introduction: The existence of ovarian intrinsic neurons is well established. However, the morphology and chemical phenotype are not completely characterized and are even unknown for some species used in medical research. The purpose of this work was to determine the morphology and chemical phenotype of intrinsic neurons of the guinea pig ovary at two ages: neonates (0 days old) and sexually mature reproductive animals (90 days old). Materials and Methods: For the morphological analysis, we employed the modified Golgi-Cox impregnation technique. For the chemical phenotype, we used immunohistochemistry and the following antibodies; tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), transient receptor potential type 1 (TRPV1), neuron-specific nuclear protein (NeuN) and proto-oncogene product of the cFos gene (cFos). We also used enzyme histochemistry for NADPH-diaphorase detection. Results: The number of intrinsic neurons in the neonate ovary was low in comparison to the adult guinea pig ovary. The intrinsic neurons were located in the cortex and the ovarian medulla; some were isolated or clustered, forming ganglia, and others were interconnected and formed networks. The neurons were small, medium or large. In the cortex of neonate vs adult ovaries, the small and medium neurons comprised 23% vs 36% and 5.2% vs 11.6%, respectively. In the medulla, the percent of the same neurons was 10.1% vs 10.1% and 1.1% vs 2.2% in the neonate and adult, respectively. In both cortex and medulla < 1% were large neurons at two ages. Also, the neurons were rounded, fusiform or multipolar. In the cortex, they were 12.7% vs 20.9%, 14.9% vs 24.2% and 1.1% vs 3.0%, respectively. In the medulla, the percent of small vs medium neurons was 6% vs 7.1% and 4.1% vs 4.8% in the neonate and adult ovary, respectively, and <1% were large neurons at both ages. The chemical phenotypes were in the neonate and adult: TH/NeuN-positive neurons, 16.3% vs 26.5%; CGRP/NeuN, 13.5% vs 35.8%; TRPV1/NeuN, 10.2% vs 38.6%; and cFos/NeuN, 4.6% vs 5.4%, respectively.The percent of NADPHd-positive cells in the cortex was 9.5% vs 25.1% and 3.2% vs 62.2% in the medulla in the neonate and adult, respectively. Conclusion: Altogether, these data showed that the number of ovarian intrinsic neurons was low at birth and increased in the sexually mature reproductive guinea pig. The chemical phenotype was rich and peptidergic, catecholaminergic and nitrergic in nature and positive for cFos immunoreactivity. Therefore, intrinsic neurons can be chemical sensors inside of the gonad and transmit signal to the central nervous system.

Extinction of opiate reward reduces dendritic arborization and c-Fos expression in the nucleus accumbens core

Recurrent opiate use combined with environmental cues, in which the drug was administered, provokes cue-induced drug craving and conditioned drug reward. Drug abuse craving is frequently linked with stimuli from a prior drug-taking environment via classical conditioning and associative learning. We modeled the conditioned morphine reward process by using acquisition and extinction of conditioned place preference (CPP) in C57BL/6 mice. Mice were trained to associate a morphine injection with a drug context using a classical conditioning paradigm. In morphine conditioning (0, 0.25, 0.5, 1, 5, or 10mg/kg) experimental mice acquired a morphine CPP dose response with 10mg/kg as most effective. During morphine CPP extinction experiments, mice were divided into three test groups: morphine CPP followed by extinction training, morphine CPP followed by sham extinction, and saline controls. Extinction of morphine CPP developed within one extinction experiment (4 days) that lasted over two more trials (another 8 days). However, the morphine CPP/sham extinction group retained a place preference that endured through all three extinction trials. Brains were harvested following CPP extinction and processed using Golgi-Cox impregnation. Changes in dendritic morphology and spine quantity were examined in the nucleus accumbens (NAc) Core and Shell neurons. In the NAcCore only, morphine CPP/extinguished mice produced less dendritic arborization, and a decrease in neuronal activity marker c-Fos compared to the morphine CPP/sham extinction group. Extinction of morphine CPP is associated with decreased structural complexity of dendrites in the NAcCore and may represent a substrate for learning induced structural plasticity relevant to addiction.

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

Establishment of an optimised protocol for a Golgi–electron microscopy method based on a Golgi–Cox staining procedure with a commercial kit

The introduction of the FD Rapid GolgiStain Kit has enabled many researchers to easily examine the morphology of neurons. Owing to an improved Golgi–Cox impregnation method, both stable and highly sensitive stained images of neurons can be acquired using this kit. To analyse microstructures such as dendritic spines and growth cones at detail, we attempted to create a useful method combining Golgi–Cox staining and electron microscopy (Golgi–EM method) so that the prepared Kit-stained samples would be appropriate for use in electron microscopy (EM) studies. Results from the present study suggest the importance of the glutaraldehyde fixation step in the Golgi–EM method. Here, we introduce the recommended protocol for the Golgi–EM method, which is available for ultrahigh-voltage electron microscopy and transmission electron microscopy. We are confident that our Golgi–EM method may become a reliable and important method that can contribute to new discoveries in neuronal morphology.

β2 Adrenergic Receptor, Protein Kinase A (PKA) and c-Jun N-terminal Kinase (JNK) Signaling Pathways Mediate Tau Pathology in Alzheimer Disease Models

Alzheimer disease (AD) is characterized by neurodegeneration marked by loss of synapses and spines associated with hyperphosphorylation of tau protein. Accumulating amyloid β peptide (Aβ) in brain is linked to neurofibrillary tangles composed of hyperphosphorylated tau in AD. Here, we identify β2-adrenergic receptor (β2AR) that mediates Aβ-induced tau pathology. In the prefrontal cortex (PFC) of 1-year-old transgenic mice with human familial mutant genes of presenilin 1 and amyloid precursor protein (PS1/APP), the phosphorylation of tau at Ser-214 Ser-262 and Thr-181, and the protein kinases including JNK, GSK3α/β, and Ca(2+)/calmodulin-dependent protein kinase II is increased significantly. Deletion of the β2AR gene in PS1/APP mice greatly decreases the phosphorylation of these proteins. Further analysis reveals that in primary PFC neurons, Aβ signals through a β2AR-PKA-JNK pathway, which is responsible for most of the phosphorylation of tau at Ser-214 and Ser-262 and a significant portion of phosphorylation at Thr-181. Aβ also induces a β2AR-dependent arrestin-ERK1/2 activity that does not participate in phosphorylation of tau. However, inhibition of the activity of MEK, an upstream enzyme of ERK1/2, partially blocks Aβ-induced tau phosphorylation at Thr-181. The density of dendritic spines and synapses is decreased in the deep layer of the PFC of 1-year-old PS1/APP mice, and the mice exhibit impairment of learning and memory in a novel object recognition paradigm. Deletion of the β2AR gene ameliorates pathological effects in these senile PS1/APP mice. The study indicates that β2AR may represent a potential therapeutic target for preventing the development of AD.