Dendritic Staining Research Articles

Abstract TP30: Exercise Postconditioning After Ischemic Stroke: Neuroprotection Through NGF/TRPV1-Mediated Astrocyte-Neuron Interaction

Introduction: Astrocytes activation and interaction with neurons potentially affect stroke outcomes. Transient receptor potential vanilloid 1 (TRPV1) participated in reducing brain injury and nerve growth factor (NGF) affected TRPV1 activation. The present study investigated neuroprotection induced by exercise postconditioning (PostE) and elucidated the mechanistic regulation of astrocytes-neuron crosstalk via NGF/TRPV1 pathway. Methods: Adult Sprague-Dawley rats received 2 h middle cerebral artery (MCA) occlusion, followed by 24 h of reperfusion. The treadmill exercise was then initiated 24 h after reperfusion for 3 days (PostE). Brain damage was determined by infarct volume, edema and apoptotic proteins expression (Bcl-2, BAX and Caspase-3). Neuroplasticity was determined by dendritic spine staining and protein expression of BDNF, GAP-43, PSD-95 and synaptophysin. Neuronal TRPV1 expression, astrocyte proliferation and astrocytic NGF expression was assessed with immunofluorescence. Primary astrocyte-neuron Transwell cultivation were used to determine the secretion of astrocytic NGF activating neural TRPV1. Interaction between TRPV1 and NGF or TRPV1 and A-kinase anchoring protein150 (AKAP150) were detected by immunofluorescence and Co-IP. Results: PostE significantly decreased brain damage and induced neuroplasticity in ischemic rats. PostE induced astrocyte proliferation and astrocytic NGF secretion, which, in turn, activated TRPV1 expression in neuron. Either NGF or TRPV1 inhibitor eliminated the neuroplasticity, while NGF inhibitor decreased TRPV1 expression, suggesting the potential regulating role of astrocytic NGF on TRPV1. Primary cells cultivation demonstrated a NGF/TRPV1 and TRPV1/AKAP150 interplays showing astrocyte-neuron communication. Conclusion: PostE induced neuroprotection, which was mediated by the activation of astrocytes and their interaction with neurons through the NGF/TRPV1 pathway, highlighting the significance of astrocyte-neuron crosstalk in post-stroke recovery processes.

The Duration of Oxygen and Glucose Deprivation (OGD) Determines the Effects of Subsequent Reperfusion on Rat Pheochromocytoma (PC12) Cells and Primary Cortical Neurons.

Reperfusion is the fundamental treatment for ischaemic stroke; however, many ischaemic stroke patients cannot undergo reperfusion treatment. Furthermore, reperfusion can cause ischaemic reperfusion injuries. This study aimed to determine the effects of reperfusion in an in vitro ischaemic stroke model-oxygen and glucose deprivation (OGD) (0.3% O2)-with rat pheochromocytoma (PC12) cells and cortical neurons. In PC12 cells, OGD resulted in a time-dependent increase in cytotoxicity and apoptosis, and reduction in MTT activity from 2 h onwards. Reperfusion following shorter periods (4 and 6 h) of OGD recovered apoptotic PC12 cells, whereas after 12 h, OGD increased LDH release. In primary neurons, 6 h OGD led to significant increase in cytotoxicity, reduction in MTT activity and dendritic MAP2 staining. Reperfusion following 6 h OGD increased the cytotoxicity. HIF-1a was stabilised by 4 and 6 h OGD in PC12 cells and 2 h OGD onwards in primary neurons. A panel of hypoxic genes were upregulated by the OGD treatments depending on the duration. In conclusion, the duration of OGD determines the mitochondrial activity, cell viability, HIF-1a stabilization, and hypoxic gene expression in both cell types. Reperfusion following OGD of short duration is neuroprotective, whereas OGD of long duration is cytotoxic.

Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons.

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount.

Novel localizations of TRPC5 channels suggest novel and unexplored roles: A study in the chick embryo brain.

Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca2+ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8 and 20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalohypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a paramedian raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development.

Contrasting effects of adolescent and early-adult ethanol exposure on prelimbic cortical pyramidal neurons

BackgroundAdolescence and early-adulthood are vulnerable developmental periods during which binge drinking can have long-lasting effects on brain function. However, little is known about the effects of binge drinking on the pyramidal cells of the prelimbic cortex (PrL) during early and protracted withdrawal periods. MethodsIn the present study, we performed whole-cell patch clamp recordings and dendritic spine staining to examine the intrinsic excitability, spontaneous excitatory post-synaptic currents (sEPSCs), and spine morphology of pyramidal cells in the PrL from rats exposed to chronic intermittent ethanol (CIE) during adolescence or early-adulthood. ResultsCompared to chronic intermittent water (CIW)-treated controls, the excitability of PrL-L5 pyramidal neurons was significantly increased 21 days after adolescent CIE but decreased 21 days after early-adult CIE. No changes of excitability in PrL Layer (L) 5 were detected 2 days after either adolescent or early-adulthood CIE. Interestingly, decreases in sEPSC amplitude and increases in thin spines ratio were detected 2 days after adolescent CIE. Furthermore, decreased frequency and amplitude of sEPSCs, accompanied by a decrease in the density of total spines and non-thin spines were observed 21 days after adolescent CIE. In contrast, increased frequency and amplitude of sEPSCs, accompanied by increased densities of total spines and non-thin spines were found 21 days after early adult CIE. ConclusionCIE produced prolonged neuronal and synaptic alterations in PrL-L5, and the developmental stage, i.e., adolescence vs. early-adulthood when subjects receive CIE, is a key factor in determining the direction of these changes.

Protein phosphatase PP2A regulates microtubule orientation and dendrite pruning in Drosophila.

Pruning that selectively eliminates inappropriate projections is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, ddaC sensory neurons undergo dendrite-specific pruning in response to the steroid hormone ecdysone. However, the understanding of the molecular mechanisms underlying dendrite pruning remains incomplete. Here, we show that protein phosphatase 2A (PP2A) is required for dendrite pruning. The catalytic (Microtubule star/Mts), scaffolding (PP2A-29B), and two regulatory subunits (Widerborst/Wdb and Twins/Tws) play important roles in dendrite pruning. Functional analyses indicate that PP2A, via Wdb, facilitates the expression of Sox14 and Mical prior to dendrite pruning. Furthermore, PP2A, via Tws, governs the minus-end-out orientation of microtubules (MTs) in the dendrites. Moreover, the levels of Klp10A, a MT depolymerase, increase when PP2A is compromised. Attenuation of Klp10A fully rescues the MT orientation defects in mts or pp2a-29b RNAi ddaC neurons, suggesting that PP2A governs dendritic MT orientation by suppressing Klp10A levels and/or function. Taken together, this study sheds light on a novel function of PP2A in regulating dendrite pruning and dendritic MT polarity in sensory neurons.

Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix

Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber-reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage-gated sodium currents display a current-voltage relationship with a maximum peak current at -25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of Müller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of Müller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

MAP2 Splicing is Altered in Huntington's Disease.

Dendritic alteration of striatal medium spiny neurons is one of the earliest morphological abnormalities in Huntington's disease (HD). The main microtubule-associated protein in dendrites is MAP2. The low-molecular weight isoforms of MAP2 (LMW-MAP2) are the juvenile forms resulting from exclusion of the sequence encoded by exons E7-E9 and are downregulated after the early stages of neuronal development when E7-E9 exon-including high-molecular weight isoforms (HMW-MAP2) are favored. Splicing alteration has recently been proposed to contribute to HD in view of two pathogenic missplicing events resulting in a highly toxic N-terminal version of mutant huntingtin and in a detrimental imbalance in MAP Tau isoforms with three or four tubulin-binding repeats. Both splicing events are postulated targets of the SR splicing factor SRSF6 which has recently been reported to be dramatically altered in HD. SR proteins often regulate functionally related sets of genes and SRSF6 targets are enriched in genes involved in brain organogenesis including several actin-and tubulin-binding proteins. Here we hypothesized that MAP2 might be target of SRSF6 and altered in HD. By SRSF6 knockdown in neuroblastoma cells, we demonstrate that splicing of MAP2 E7-E9 exons is affected by SRSF6. We then show a disbalance in LMW and HMW MAP2 mRNA isoforms in HD striatum in favor of the juvenile LMW forms together with a decrease in total MAP2 mRNA. This is accompanied by a global decrease in total MAP2 protein due to almost total disappearance of HMW-MAP2 isoforms with preservation of LMW-MAP2 isoforms. Accordingly, the predominant dendritic MAP2 staining in striatal neuropil of control subjects is absent in HD cases. In these, MAP2-immunoreactivity is faint and restricted to neuronal cell bodies often showing a sharp boundary at the base of dendrites. Together, our results highlight the importance of splicing alteration in HD and suggest that MAP2 alteration contributes to dendritic atrophy.

Expression of ZNF804A in human brain and alterations in schizophrenia, bipolar disorder, and major depressive disorder: a novel transcript fetally regulated by the psychosis risk variant rs1344706.

The single-nucleotide polymorphism rs1344706 in the zinc finger protein 804A gene (ZNF804A) shows genome-wide association with schizophrenia and bipolar disorder. Little is known regarding the expression of ZNF804A and the functionality of rs1344706. To characterize ZNF804A expression in human brain and to investigate how it changes across the life span and how it is affected by rs1344706, schizophrenia, bipolar disorder, and major depressive disorder. Molecular and immunochemical methods were used to study ZNF804A messenger RNA (mRNA) and ZNF804A protein, respectively. ZNF804A transcripts were investigated using next-generation sequencing and polymerase chain reaction-based methods, and ZNF804A protein was investigated using Western blots and immunohistochemistry. Samples of dorsolateral prefrontal cortex and inferior parietal lobe tissue were interrogated from 697 participants between 14 weeks' gestational age and age 85 years, including patients with schizophrenia, bipolar disorder, or major depressive disorder. Quantitative measurements of ZNF804A mRNA and immunoreactivity, and the effect of diagnosis and rs1344706 genotype. ZNF804A was expressed across the life span, with highest expression prenatally. An abundant and developmentally regulated truncated ZNF804A transcript was identified, missing exons 1 and 2 (ZNF804AE3E4) and predicted to encode a protein lacking the zinc finger domain. rs1344706 influenced expression of ZNF804AE3E4 mRNA in fetal brain (P = .02). In contrast, full-length ZNF804A showed no association with genotype (P > .05). ZNF804AE3E4 mRNA expression was decreased in patients with schizophrenia (P = .006) and increased in those with major depressive disorder (P < .001), and there was a genotype-by-diagnosis interaction in bipolar disorder (P = .002). ZNF804A immunoreactivity was detected in fetal and adult human cerebral cortex. It was localized primarily to pyramidal neurons, with cytoplasmic as well as dendritic and nuclear staining. No differences in ZNF804A-immunoreactive neurons were seen in schizophrenia or related to rs1344706 (P > .05). rs1344706 influences the expression of ZNF804AE3E4, a novel splice variant. The effect is limited to fetal brain and to this isoform. It may be part of the mechanism by which allelic variation in ZNF804A affects risk of psychosis. ZNF804A is translated in human brain, where its functions may extend beyond its predicted role as a transcription factor.

Abstract T P69: Stroke Induces HDAC4 Nuclear Shuttling in Neurons

Background: Nuclear shuttling of class II histone deacetylases (HDACs) in neurons regulates apoptosis and facilitates neuronal recovery in response to injury. The present study examined the temporal profile of the subcellular localization of class II HDACs (4 and 5) in the peri-infarct cortex during brain recovery after stroke. Methods: Adult male Wistar rats were subjected to permanent middle cerebral artery occlusion (MCAo) and were sacrificed at 2, 7 and 14 days after MCAo. Sham operated rats were used as a control (n=6/group). To identify phenotypes of HDAC immunoreactive cells, double immunohistochemistry was performed with antibodies against each HDAC isoform (4 or 5) along with MAP-2 (a marker of dendrites), p-NFH (a marker of axons), APC (a marker of oligodendrocytes) or GFAP (a marker for astrocytes). Results: In cortical neurons of sham operated rats, HDACs 4 and 5 immunoreactivity was mainly localized to the cytoplasm and dendrites, but not axons. Stroke significantly (p&lt;0.05) increased the percentage of neurons with nuclear HDAC4 immunoreactivity in the peri-infarct cortex at 2 (55 ± 2%), 7 (65 ± 5%) and 14 (76 ± 2%) days after MCAo compared to sham (27 ± 3%). Stroke-induced nuclear expression of HDAC4 was accompanied by a significant (p&lt;0.05) decrease in HDAC4 staining in neuronal dendrites over the same period (7 ± 1% at 2d, 7 ± 2% at 7d, and 6 ± 1% at 14d vs. 19 ± 2% in sham). Neurons with nuclear HDAC4 were not TUNEL positive, indicating that these cells are not apoptotic. In parallel, augmentation of neurons expressing nuclear HDAC4 was significantly correlated to an increase in MAP-2 immunoreactive density after stroke (r=0.7, p&lt;0.05). Stroke-induced nuclear shuttling of HDAC4 is specific to neurons, since HDAC4 was not detected in nuclei of GFAP+ astrocytes or APC+ oligodendrocytes. Neuronal nuclear HDAC4 localization was not associated with a change in Histone H3 lysine 9 acetylation or methylation levels. Unlike HDAC4, stroke did not alter nuclear localization of HDAC5 in neurons. Conclusion: Our data show that stroke induces HDAC4 nuclear shuttling in neurons with increased dendritic density in the peri-infarct cortex, suggesting a role for HDAC4 in promoting neuronal remodeling after stroke.

Clinical and neuropathological findings in Hashimoto’s encephalopathy: a case report

Hashimoto’s encephalopathy (HE) is a controversial autoimmune condition of the central nervous system (CNS) characterized by focal and/or diffuse symptoms concomitant with raised levels of anti-thyroid antibodies, in particular anti-thyroperoxidase (TPO) and/or anti-thyroglobulin (TG) antibodies [1]. Hereby, we provide the full clinical and neuropathological description of an HE patient with a rapid and fatal course. A 67-year-old man with autoimmune hypothyroidism taking L-thyroxine 100 lg/day came to our observation because of 1-month history of apathy and speech difficulties. At admission in the neurological ward, fluent aphasia, disorientation, and spontaneous diffuse myoclonic jerks were present. Cranial CT and brain MRI were unremarkable. EEG evidenced rare diffuse theta-delta bursts. On laboratory investigations, we detected only a markedly raised titer of serum anti-TPO antibodies (3,790 UI/ml). CSF analysis disclosed very high protein concentration (126 mg/dL) with moderate alteration of blood–brain barrier permeability. Virological analyses were all negative. Total tau CSF concentration was normal, whereas the 14-33 protein was present in traces. To rule out a neurological paraneoplastic syndrome, the patient’s serum was analyzed by an indirect immunofluorescence assay on monkey cerebellum slides. We evidenced a Tr-like pattern, characterized by a prominent staining of cytoplasm and dendrites of Purkinje cells, but with no positive dots in the molecular layer (Fig. 1). No paraneoplastic antibody specificity (Hu, Yo, Ri, amphiphysin, Ma2, Ta) was detected by dot blot. Serum anti-VGKC and anti-NMDA-R antibodies were absent. A total body contrast-enhanced CT ruled out occult neoplasms. Given the subacute encephalopathy picture with no evident causes and the very raised serum titer of anti-TPO antibodies, a presumptive diagnosis of HE was made. A high-dose systemic methylprednisolone pulse (1,000 mg/ day) was started. After 5 days from steroid initiation, the patient was intubated and transferred to the intensive care unit (ICU) because of a refractory convulsive status epilepticus. Thiopentone was administered to get a suppression-burst pattern on EEG recordings. After 1 week the patient’s clinical conditions markedly ameliorated and no seizure relapses were observed. Therefore, he was readmitted to our neurologic ward. In the following 5 days, diffuse myoclonic jerks progressively increased, leading to convulsive status epilepticus. The patient entered again the ICU to employ intravenous thiopentone. A new brain MRI study evidenced diffuse hyperintensity of cortical white matter of parietal lobes in T2 and FLAIR scans with no contrast enhancement in T1 sequences. MRI signs of bilateral hippocampal sclerosis were also present. The patient’s neurologic status deteriorated in spite of a 10-day systemic methylprednisolone (1,000 mg/day) and 5-day intravenous human immunoglobulin pulse (400 mg/kg/ day). Exitus occurred from a multiorgan failure syndrome after 3 weeks. The autopsy procedure was limited to the intracranial content, since the remote suspect of a prion disease. On gross inspection, cortical gyri were slightly flattened. On microscopic examination, areas of neuropil vacuolation with congestion and swollen endothelial cells D. Imperiale (&) R. Testi A. Romito S. Taraglio Centro Regionale Diagnosi ed Osservazione Malattie Prioniche DOMP, ASL To2, Via Cibrario 72, 10144 Turin, Italy e-mail: daniele.imperiale@aslto2.piemonte.it

Dendritic structural plasticity in the basolateral amygdala after fear conditioning and its extinction in mice

Previous research suggests that morphology and arborization of dendritic spines change as a result of fear conditioning in cortical and subcortical brain regions. This study uniquely aims to delineate these structural changes in the basolateral amygdala (BLA) after both fear conditioning and fear extinction. C57BL/6 mice acquired robust conditioned fear responses (70–80% cued freezing behavior) after six pairings with a tone cue associated with footshock in comparison to unshocked controls. During fear acquisition, freezing behavior was significantly affected by both shock exposure and trial number. For fear extinction, mice were exposed to the conditioned stimulus tone in the absence of shock administration and behavioral responses significantly varied by shock treatment. In the retention tests over 3 weeks, the percentage time spent freezing varied with the factor of extinction training. In all treatment groups, alterations in dendritic plasticity were analyzed using Golgi–Cox staining of dendrites in the BLA. Spine density differed between the fear conditioned group and both the fear extinction and control groups on third order dendrites. Spine density was significantly increased in the fear conditioned group compared to the fear extinction group and controls. Similarly in Sholl analyses, fear conditioning significantly increased BLA spine numbers and dendritic intersections while subsequent extinction training reversed these effects. In summary, fear extinction produced enduring behavioral plasticity that is associated with a reversal of alterations in BLA dendritic plasticity produced by fear conditioning. These neuroplasticity findings can inform our understanding of structural mechanisms underlying stress-related pathology can inform treatment research into these disorders.

Localization and developmental expression patterns of CSPG-cs56 (aggrecan) in normal and dystrophic retinas in two rat strains

Proteoglycans have a number of important functions in the central nervous system. Aggrecan (hyaluronan-binding proteoglycan, CSPG-cs56) is found in the extracellular matrix of cartilage as well as in the developing brain. We compared the postnatal distribution of CSPG-cs56 in Long Evans (LE) and Royal College of Surgeons (RCS) rat retinas to determine if this proteoglycan played a role in the development of dystrophic retinas. CSPG-cs56 expression was examined in rat retinas aged between birth (postnatal day 0, P0) and P150 using immunofluorescence and Western-blots. Immunofluorescence was quantified using ImageJ. GFAP staining was used to compare Müller cell labeling and the distribution of CSPG-cs56. Both rat strains showed a significant rise in total retinal CSPG-cs56 between P0 and P21; values peaked on P21 in LE rats and P14 in RCS rats. CSPG-cs56 then significantly decreased to lower levels (P35) in both strains before reaching significantly higher levels by P90–P150. CSPG-cs56 positive staining was present in the ganglion cell layer at birth and clear layering of the inner plexiform layer was seen between P7 and P21 due to dendritic staining of retinal ganglion cells. Staining was less intense and diffuse within the outer plexiform over a similar time-course. Light CSPG-cs56 labeling in the region of the outer segments was present at (P14) and became more intense as the retina approached maturity. CSPG-cs56 in the outer segments was the main contributor to the higher expression in older animals. Substantial differences in CSPG-cs56 labeling were not seen between LE and RCS rats. There was no evidence to suggest that Müller cells were the source of CSPG-cs56 in either rat strain, although their staining distributions had a degree of overlap. The lack of significant differences between LE and RCS rats indicates that CSPG-cs56 may not be involved in the degenerative process or the reorganization of the RCS rat retina. We suggest that the main role of CPSG-cs56 is to maintain retinal ganglion cell dendritic structure in the inner plexiform layer and is closely related to providing adequate support and flexibility for the photoreceptor outer segments, which is necessary to maintain their function.

Network inhomogeneity supports burst initiation in vitro

The emergence of spontaneous bursting events in developing neuronal networks likely depends on the evolving network connectivity. Theoretical models have shown that hierarchical network structures embedding clusters of strongly inter-connected neurons are optimal for initiating and sustaining spontaneous activity [1]. It is conceivable that activity-dependent wiring could innately support the formation of similar network structures. To test this we chronically manipulated activity-dependent structural plasticity by inhibition of protein kinase C (PKC) in developing networks of cortical neurons in vitro. Previous studies showed that PKC inhibition in developing cerebellum promotes dendritic outgrowth and arborization of Purkinje cells and impairs pruning of climbing fibers. We found that developmental inhibition of PKC in cortical cell cultures increased dendritic outgrowth, impaired neurite fasciculation and clustering and abolished network pruning. This resulted in more homogeneous and potentially better connected networks (fig. 1A-B). As a result, propagation of activity within bursts was faster and occurred in strongly isotropic waves (fig. 1C-D). Interestingly, bursts in these networks were triggered from fewer sites and at much lower rates suggesting that the homogeneous networks forming under blockade of activity-dependent wiring processes embed fewer burst initiation zones. Figure 1 MAP2 staining of dendrites and somata: control networks (A) display characteristic features of an activity-dependent wiring process including fasciculation and ramification of dendrites within clusters. Developmental inhibition of PKC (B) generates more ...

A-type K+ currents in intralaminar thalamocortical relay neurons

Transient A-type K+ currents (I(A)) are known to influence the firing pattern of a number of thalamic cell types, but have not been investigated in intralaminar thalamocortical (TC) relay neurons yet. We therefore combined whole-cell patch-clamp techniques, PCR analysis, and immunohistochemistry to investigate the voltage-dependent and pharmacological properties of I(A) and to determine its molecular basis in TC neurons from the centrolateral, paracentral, and centromedial thalamic nuclei. I(A) revealed half-maximal (V (h)) activation and inactivation at about -17 and -67 mV, respectively. At a concentration of 5-10 mM 4-aminopyridine (4-AP) completely blocked I(A). Furthermore, I(A) was nearly unaffected by two sea anemone toxins (blood depressing substances 1 and 2, BDS1 and BDS2; 6-8% block at a concentration of 1 μM) but strongly sensitive to the K(V)4 channel blocker Heteropoda venatoria toxin 2 (HpTx2; about 45% block at a concentration of 5 μM). PCR screening revealed the expression of K(V)4.1-4.3, with strongest expression for K(V)4.2 and weak expression for K(V)4.1. Accordingly K(V)4.1 was not detected in immunohistochemical staining. Furthermore, K(V)4.2 and K(V)4.3 revealed mainly dendritic and somatic staining, respectively. Together with current clamp recordings, these findings point to a scenario where the fast transient I(A) in intralaminar TC neurons has a depolarized threshold at potentials negative to -50 mV, is substantially generated by K(V)4.2 and K(V)4.3 channels, allows prominent burst firing at hyperpolarized potentials, prevents the generation of high-threshold potentials, generates a delayed onset of firing at more depolarized potentials, and allows fast tonic firing.

Dendritic spine density deficits in the hippocampal CA1 region of young Tg2576 mice are ameliorated with the PDE9A inhibitor PF-04447943

Many forms of synaptic plasticity are critically dependent upon production of cGMP to trigger activity-dependent increases in synaptic size and strength. Phosphodiesterase 9A (PDE9A) is a high affinity, cGMP-specific phosphodiesterase with widespread distribution in the central nervous system. Inhibition of PDE9A results in significant accumulation of cGMP in brain tissue and CSF of rodents and also increases CSF cGMP in human volunteers (Schmidt et al., 2009 ICAD; Nicholas et al 2009 ICAD). We hypothesize that chronic exposure to a PDE9 inhibitor, and the associated elevations in brain cGMP could provide a therapeutic benefit to vulnerable synapses chronically exposed to Abeta in transgenic amyloid overexpressing mice. A total of N = 20 animals per group of 4 month old Tg2576+ and non-transgenic animals were implanted with Alzet osmotic minipumps to deliver vehicle or the PDE9A inhibitor PF-4447943, currently in clinical trials for the treatment of mild to moderate Alzheimer's Disease. Neurobehavioral outcomes were measured as conditioned fear response after 30 days of treatment and subsequently the brains were harvested for measurement of Abeta, gene expression profiling or synaptic density as assessed by Golgi staining of dendrites. Dendritic spine density on apical dendrites of CA1 neurons exhibited a small but significant deficit in the density of dendritic spines in vehicle treated transgenic animals as compared to non-transgenic animals. This deficit was ameliorated by 30 days of exposure to PF-04447943. No significant drug effect was observed in the non-transgenic animals. No significant effects of drug treatment were observed on Abeta levels in Tg2576 mice. Behavioral analysis of transgenic animals showed deficits in fear conditioning as early as 2 months of age, and therefore were considered unlikely to be due to the accumulation of oligomeric Abeta. These deficits were not affected by drug treatment. Transcriptional profiles of animals treated with drug compared to vehicle showed evidence of regulation of pathways related to synaptic plasticity and remodeling of the dendritic cytoskeleton, consistent with stabilization of vulnerable spine structure. This data supports the hypothesis that PDE9 inhibition can stabilize vulnerable synapses early in the Alzheimer's disease process.

Synaptic Activation of T-Type Ca2+Channels Via mGluR Activation in the Primary Dendrite of Mitral Cells

Mitral cells are the primary output of the olfactory bulb, projecting to many higher brain areas. Understanding how mitral cells process and transmit information is key to understanding olfactory perception. Mitral dendrites possess high densities of voltage-gated channels, are able to initiate and propagate orthodromic and antidromic action potentials, and release neurotransmitter. We show that mitral cells also possess a low-voltage-activated T-type Ca(2+) current. Immunohistochemistry shows strong Cav3.3 labeling in the primary dendrite and apical tuft with weaker staining in basal dendrites and no staining in somata. A low-voltage-activated Ca(2+) current activates from -68 mV, is blocked by 500 microM Ni(2+) and 50 microM NNC 55-0396, but is insensitive to 50 microM Ni(2+) and 500 microM isradipine. 2-photon Ca(2+) imaging shows that T channels are functionally expressed in the primary dendrite where their activity determines the resting [Ca(2+)] and are responsible for subthreshold voltage-dependent Ca(2+) changes previously observed in vivo. Application of the group 1 mGluR agonist dihydroxyphenylglycine (DHPG) (50 microM) robustly upregulates T-channel current in the primary and apical tuft dendrite. Olfactory nerve stimulation generates a long-lasting depolarization, and we show that mGluRs recruit T channels to contribute approximately 36% of the voltage integral of this depolarization. The long-lasting depolarization results in sustained firing and block of T channels decreased action potential firing by 84.1 +/- 4.6%. Therefore upregulation of T channels by mGluRs is required for prolonged firing in response to olfactory nerve input.

Amyloid and Tau accumulate in the brains of aged hydrocephalic rats

AD pathology is often seen in cortical biopsies of NPH patients. It remains unclear whether these findings are coincidental or causally related. In an aged animal model of NPH, we quantify Aβ and pTau accumulation and describe its temporal and spatial distribution. One-year-old male Sprague–Dawley rats had hydrocephalus induced by cisternal kaolin injection. Immunohistochemistry (IMHC) for AβPP, Aβ40, Aβ42 and pTau (epitope pT231) and ELISA for Aβ40, Aβ42 and pT231 were performed on controls and after 2, 6 and 10 weeks of hydrocephalus. Rats had double-label fluorescence IMHC for localization of Aβ42 and pT231. IMHC showed no change in neuronal AβPP expression following hydrocephalus. Aβ42 appeared earliest in CSF clearance pathways, p < 0.05, and also showed significant rises in perivascular spaces and in cortical parenchyma. Mean ELISA values for Aβ40 and Aβ42 increased three- to four-fold in hydrocephalic rats at 6 and 10 weeks. Aβ40 increased between 2 and 6 weeks ( p = 0.0001), and remained stable at 10 ( p = 0.0002); whereas Aβ42 was elevated at 2 weeks ( p < 0.04) and remained at 6 ( p = 0.015). PTau at 6 and 10 weeks showed AD-like increased neuronal somatic staining and loss of dendritic staining. ELISA demonstrated increased pT231 in hydrocephalic rats at 10 weeks ( p < 0.0002). Double-label fluorescence for Aβ42 and pT231 revealed intraneuronal co-localization. Hydrocephalus in the elderly rat, therefore, can induce both Aβ and pTau accumulation. As distinct from brain injury models, no increase in AβPP expression was demonstrated. Rather, altered CSF dynamics appears to impair Aβ clearance in this NPH model.