Time‐dependent differences in cortical measures and their associations with behavioral measures following mild traumatic brain injury

There is currently a critical need to establish an improved understanding of time‐dependent differences in brain structure following mild traumatic brain injury (mTBI). We compared differences in brain structure, specifically cortical thickness (CT), cortical volume (CV), and cortical surface area (CSA) in 54 individuals who sustained a recent mTBI and 33 healthy controls (HCs). Individuals with mTBI were split into three groups, depending on their time since injury. By comparing structural measures between mTBI and HC groups, differences in CT reflected cortical thickening within several areas following 0–3 (time‐point, TP1) and 3–6 months (TP2) post‐mTBI. Compared with the HC group, the mTBI group at TP2 showed lower CSA within several areas. Compared with the mTBI group at TP2, the mTBI group during the most chronic stage (TP3: 6–18 months post‐mTBI) showed significantly higher CSA in several areas. All the above reported differences in CT and CSA were significant at a cluster‐forming p < .01 (corrected for multiple comparisons). We also found that in the mTBI group at TP2, CT within two clusters (i.e., the left rostral middle frontal gyrus (L. RMFG) and the right postcentral gyrus (R. PostCG)) was negatively correlated with basic attention abilities (L. RMFG: r = ?.41, p = .05 and R. PostCG: r = ?.44, p = .03). Our findings suggest that alterations in CT and associated neuropsychological assessments may be more prominent during the early stages of mTBI. However, alterations in CSA may reflect compensatory structural recovery during the chronic stages of mTBI.     

Amyloid‐β interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in brain‐derived neurotrophic factor‐induced Arc expression in rat cortical neurons

The deposition of amyloid‐β (Aβ) contributes to the pathogenesis of Alzheimer's disease. Even at low levels, Aβ may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory. Brain‐derived neurotrophic factor (BDNF) is well known to be capable of inducing the synthesis of activity‐regulated cytoskeleton‐associated protein (Arc), which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar Aβ at a nonlethal level was sufficient to impair BDNF‐induced Arc expression in cultured rat cortical neurons. In this study, BDNF treatment alone induced the activation of the phosphatidylinositol 3‐kinase‐Akt‐mammlian target of rapamycin (PI3K‐Akt‐mTOR) signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the expression of Arc. Interrupting the PI3K‐Akt‐mTOR signaling pathway by inhibitors prevented the effects of BDNF, indicating the involvement of this pathway in BDNF‐induced 4EBP1 phosphorylation, p70S6K phosphorylation, eEF2 dephosphorylation, and Arc expression. Nonlethal Aβ pretreatment partially blocked these effects of BDNF. Double‐ immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2 dephosphorylation and Arc expression following BDNF treatment regardless of the presence of Aβ. These results reveal that, in cultured rat cortical neurons, Aβ interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in BDNF‐induced Arc expression. Moreover, this study also provides the first evidence that there is a close correlation between BDNF‐induced eEF2 dephosphorylation and BDNF‐induced Arc expression. © 2009 Wiley‐Liss, Inc.     

Basal ganglia‐cortical structural connectivity in Huntington's disease

Huntington's disease is an incurable neurodegenerative disease caused by inheritance of an expanded cytosine‐adenine‐guanine (CAG) trinucleotide repeat within the Huntingtin gene. Extensive volume loss and altered diffusion metrics in the basal ganglia, cortex and white matter are seen when patients with Huntington's disease (HD) undergo structural imaging, suggesting that changes in basal ganglia‐cortical structural connectivity occur. The aims of this study were to characterise altered patterns of basal ganglia‐cortical structural connectivity with high anatomical precision in premanifest and early manifest HD, and to identify associations between structural connectivity and genetic or clinical markers of HD. 3‐Tesla diffusion tensor magnetic resonance images were acquired from 14 early manifest HD subjects, 17 premanifest HD subjects and 18 controls. Voxel‐based analyses of probabilistic tractography were used to quantify basal ganglia‐cortical structural connections. Canonical variate analysis was used to demonstrate disease‐associated patterns of altered connectivity and to test for associations between connectivity and genetic and clinical markers of HD; this is the first study in which such analyses have been used. Widespread changes were seen in basal ganglia‐cortical structural connectivity in early manifest HD subjects; this has relevance for development of therapies targeting the striatum. Premanifest HD subjects had a pattern of connectivity more similar to that of controls, suggesting progressive change in connections over time. Associations between structural connectivity patterns and motor and cognitive markers of disease severity were present in early manifest subjects. Our data suggest the clinical phenotype in manifest HD may be at least partly a result of altered connectivity. Hum Brain Mapp 36:1728–1740, 2015. © 2015 Wiley Periodicals, Inc .     

The small heat shock protein Hsp27 protects cortical neurons against the toxic effects of β‐amyloid peptide

Neurofibrillary tangles and amyloid plaques are considered to be hallmarks of Alzheimer's disease (AD), and the toxic effects of amyloid‐β peptide (Aβ) lead to activation of stress‐related signaling and neuronal loss. The small heat shock protein Hsp27 is reported to be increased in AD brains and to accumulate in plaques, but whether this represents a potentially protective response to stress or is part of the disease process is not known. We hypothesized that increased expression of Hsp27 in neurons can promote neuronal survival and stabilize the cytoskeleton in the face of Aβ exposure. By using neonatal rat cortical neurons, we investigated the potential role of Hsp27 in neuronal cultures in the presence or absence of Aβ. We initially tested whether a heat stress (HS) would be sufficient to induce endogenous Hsp27 expression. HS not only did not result in neuronal Hsp27 up‐regulation but made the cells more vulnerable to Aβ exposure. We then used cDNA transfection to overexpress EGFP‐Hsp27 (or the empty vector) in cultures and then assessed neuronal survival and growth. Transfected neurons appeared healthy and had robust neuritic outgrowth. Aβ treatment induced significant cell death by 48–72 hr in nontransfected and empty‐vector‐expressing cultures. In contrast, cultures expressing Hsp27 did not display significant apoptosis. Our results show that Hsp27‐expressing neurons were selectively protected against the deleterious effects of Aβ treatment; neuronal degeneration was prevented, and Aβ‐induced alterations in mitochondrial size were attenuated. We also demonstrate that Hsp27 expression can enhance neurite growth in cortical neurons compared with control vector‐transfected cells. Overall, our study provides new evidence that Hsp27 can provide a protective influence in primary cortical neurons in the face of toxic concentrations of amyloid. © 2009 Wiley‐Liss, Inc.     

Long‐term lithium treatment increases intracellular and extracellular brain‐derived neurotrophic factor (BDNF) in cortical and hippocampal neurons at subtherapeutic concentrations

Objectives

The putative neuroprotective effects of lithium treatment rely on the fact that it modulates several homeostatic mechanisms involved in the neurotrophic response, autophagy, oxidative stress, inflammation, and mitochondrial function. Lithium is a well‐established therapeutic option for the acute and long‐term management of bipolar disorder and major depression. The aim of this study was to evaluate the effects of subtherapeutic and therapeutic concentrations of chronic lithium treatment on brain‐derived neurotrophic factor (BDNF) synthesis and secretion.

Methods

Primary cultures of cortical and hippocampal neurons were treated with different subtherapeutic (0.02 and 0.2 mM) and therapeutic (2 mM) concentrations of chronic lithium treatment in cortical and hippocampal cell culture.

Results

Lithium treatment increased the intracellular protein expression of cortical neurons (10% at 0.02 mM) and hippocampal neurons (28% and 14% at 0.02 mM and 0.2 mM, respectively). Extracellular BDNF of cortical neurons increased 30% and 428% at 0.02 and 0.2 mM, respectively and in hippocampal neurons increased 44% at 0.02 mM.

Conclusion

The present study indicates that chronic, low‐dose lithium treatment up‐regulates BDNF production in primary neuronal cell culture.     

Depth‐dependent abnormal cortical myelination in first‐episode treatment‐naïve schizophrenia

Myelination is key to effective message passing in the central nervous system and is likely linked to the pathogenesis of schizophrenia (SZ). Emerging evidence indicates that a large portion of intracortical myelin insulates inhibitory interneurons that are highly relevant to pathogenesis of schizophrenia. Here for the first time, we characterized intracortical myelination across the entire cortical surface in first‐episode treatment‐naïve patients with schizophrenia (FES) using T1w/T2w ratio of structural MRI, FES patients exhibited significantly higher myelin content in the left inferior parietal lobe, supramarginal gyrus, and superior temporal gyrus in the superficial layer, as well as left IPL in the middle layer, but significantly lower myelin content in the left middle insula and posterior cingulate gyrus. Years of education, a proxy for onset of functional decline, significantly altered the relationship between abnormal parietal and posterior cingulate myelination and clinical symptoms, indicating that the pathoplastic role of myelination hinges on the age of onset of functional decline. In addition, higher myelination generally related to better cognitive function in younger subjects but worse cognitive function in older subjects. We conclude that FES is characterized by increased myelination of the superficial layers of the parietal–temporal association cortex, but reduced myelination of the cingulo‐insular midcortical layer cortex. Intracortical myelin content affects both cognitive functioning and symptom burden in FES, with the effect conditional upon age and timing of onset of functional decline. These results suggest myelination might be a critical biological target for procognitive interventions in SZ.     

Axonal fasciculation and the role of polysialic acid‐neural cell adhesion molecule in rat cortical neurons

Axonal fasciculation is a mechanism deployed by growing axons to reach their targets during development of the nervous system. Published data have suggested the involvement of neuronal cell adhesion molecules (NCAM) in axonal fasciculation. We have characterized the formation of axonal fascicles in serum‐free, primary cultures of cortical neurons from embryonic rat brains. Unlike the published data, axonal fascicles in our system have a unique morphology: they are waveform, are rarely thicker than 20 μm, and can reach up to several millimeters in length. We observed an age and time dependence in the formation of fascicles. They formed only in cultures from embryonic day 15–17 brain and only between 4 days in vitro (DIV) and 11 DIV. Electron microscopy showed that the fascicles consisted of mostly axonal processes. Immunocytochemical staining confirmed that the fascicles were positive for the 66‐kDa neurofilament protein, NF66, but they contained few, if any, microtubule‐associated protein‐2‐positive or glial fibrillary acidic protein‐positive processes. Polysialic acids appeared to be critical in the formation of fascicles. Neuraminidase treatment prevented the formation of fascicles when added before 5 DIV. Addition of a specific inhibitor blocked the effect of neuraminidase. The cortical neurons in our model shared several important features with axon fasciculation in vivo and may provide a unique system for studying the molecular mechanisms involved in the formation of axonal tracts in the brain. © 2013 Wiley Periodicals, Inc.     

Learning‐based subject‐specific estimation of dynamic maps of cortical morphology at missing time points in longitudinal infant studies

Longitudinal neuroimaging analysis of the dynamic brain development in infants has received increasing attention recently. Many studies expect a complete longitudinal dataset in order to accurately chart the brain developmental trajectories. However, in practice, a large portion of subjects in longitudinal studies often have missing data at certain time points, due to various reasons such as the absence of scan or poor image quality. To make better use of these incomplete longitudinal data, in this paper, we propose a novel machine learning‐based method to estimate the subject‐specific, vertex‐wise cortical morphological attributes at the missing time points in longitudinal infant studies. Specifically, we develop a customized regression forest, named dynamically assembled regression forest (DARF), as the core regression tool. DARF ensures the spatial smoothness of the estimated maps for vertex‐wise cortical morphological attributes and also greatly reduces the computational cost. By employing a pairwise estimation followed by a joint refinement, our method is able to fully exploit the available information from both subjects with complete scans and subjects with missing scans for estimation of the missing cortical attribute maps. The proposed method has been applied to estimating the dynamic cortical thickness maps at missing time points in an incomplete longitudinal infant dataset, which includes 31 healthy infant subjects, each having up to five time points in the first postnatal year. The experimental results indicate that our proposed framework can accurately estimate the subject‐specific vertex‐wise cortical thickness maps at missing time points, with the average error less than 0.23 mm. Hum Brain Mapp 37:4129–4147, 2016. © 2016 Wiley Periodicals, Inc.     

Sodium‐dependent Vitamin C transporter 2 deficiency impairs myelination and remyelination after injury: Roles of collagen and demethylation

Peripheral nerve myelination involves rapid production of tightly bound lipid layers requiring cholesterol biosynthesis and myelin protein expression, but also a collagen‐containing extracellular matrix providing mechanical stability. In previous studies, we showed a function of ascorbic acid in peripheral nerve myelination and extracellular matrix formation in adult mice. Here, we sought the mechanism of action of ascorbic acid in peripheral nerve myelination using different paradigms of myelination in vivo and in vitro. We found impaired myelination and reduced collagen expression in Sodium‐dependent Vitamin C Transporter 2 heterozygous mice (SVCT2^+/‐) during peripheral nerve development and after peripheral nerve injury. In dorsal root ganglion (DRG) explant cultures, hypo‐myelination could be rescued by precoating with different collagen types. The activity of the ascorbic acid‐dependent demethylating Ten‐eleven‐translocation (Tet) enzymes was reduced in ascorbic acid deprived and SVCT2^+/‐ DRG cultures. Further, in ascorbic acid‐deprived DRG cultures, methylation of a CpG island in the collagen alpha1 (IV) and alpha2 (IV) bidirectional promoter region was increased compared to wild‐type and ascorbic acid treated controls. Taken together, these results provide further evidence for the function of ascorbic acid in myelination and extracellular matrix formation in peripheral nerves and suggest a putative molecular mechanism of ascorbic acid function in Tet‐dependent demethylation of collagen promoters.     

Age‐dependent differences in dopamine transporter and vesicular monoamine transporter‐2 function and their implications for methamphetamine neurotoxicity

The abuse of methamphetamine (METH) is a serious public health problem because METH can cause persistent dopaminergic deficits in the brains of both animal models and humans. Surprisingly, adolescent postnatal day (PND)40 rats are resistant to these METH‐induced deficits, whereas young adult PND90 rats are not. Studies described in this report used rotating disk electrode voltammetry and western blotting techniques to investigate whether there are age‐dependent differences in monoamine transporter function in PND38–42 and PND88–92 rats that could contribute to this phenomenon. The initial velocities of dopamine (DA) transport into, METH‐induced DA efflux from, and DA transporter (DAT) immunoreactivity in striatal suspensions are greater in PND38–42 rats than in PND88–92 rats. DA transport velocities into vesicles that cofractionate with synaptosomal membranes after osmotic lysis are also greater in PND38–42 rats. However, there is no difference in vesicular monoamine transporter‐2 (VMAT‐2) immunoreactivity between the two age groups in this fraction. This suggests that younger rats have a greater capacity to sequester cytoplasmic DA into membrane‐associated vesicles due to kinetically upregulated VMAT‐2 and also have increased levels of functionally active DAT. In the presence of METH, these may provide additional routes of cellular efflux for DA that is released from vesicles into the cytoplasm and thereby prevent cytoplasmic DA concentrations in younger rats from rising to neurotoxic levels after drug administration. These findings provide novel insight into the age‐dependent physiological regulation of neuronal DA sequestration and may advance the treatment of disorders involving abnormal DA disposition including substance abuse and Parkinson's disease. Synapse 63:147–151, 2009. © 2008 Wiley‐Liss, Inc.     

Exchange protein directly activated by cAMP 2 is required for corticotropin‐releasing hormone‐mediated spine loss

Corticotropin‐releasing hormone is produced in response to acute and chronic stress. Previous studies have shown that activation of the corticotropin‐releasing hormone receptor 1 (CRHR1) by corticotropin‐releasing hormone results in the rapid loss of dendritic spines which correlates with cognitive dysfunction associated with stress. Exchange protein directly activated by cAMP (EPAC2), a guanine nucleotide exchange factor for the small GTPase Rap, plays a critical role in regulating dendritic spine morphology and has been linked with CRHR1 signalling. In this study, we have tested whether EPAC2 links corticotropin‐releasing hormone with dendritic spine remodelling. In primary rat cortical neurons, we show that CRHR1 is highly enriched in the dendritic spines. Furthermore, we find that EPAC2 and CRHR1 co‐localize in cortical neurons and that acute exposure to corticotropin‐releasing hormone induces spine loss. To establish whether EPAC2 was required for corticotropin‐releasing hormone–mediated spine loss, we knocked‐down EPAC2 in cortical neurons using a short hairpin RNA‐mediated approach. In the presence of Epac2 knocked‐down, corticotropin‐releasing hormone was no longer able to induce spine loss. Taken together, our data indicate that EPAC2 is required for the rapid loss of dendritic spines induced by corticotropin‐releasing hormone and may ultimately contribute to responses to acute stress.     

Uptake of horseradish peroxidase at isolated nerve terminals in relationship to transmitter release; Biochemical results

The uptake of horseradish peroxidase (HRP) into isolated nerve terminals (synaptosomes) has been studied by using a spectrophotometric method to determine the enzyme activity. HRP is rapidly taken up by synaptosomes, it is not removed by multiple washes in iso-osmotic medium but is lost if the particles are ruptured by hypo-osmotic conditions. The uptake is not affected by metabolic poisons, is reduced at lower temperature and is not with any significant release of cytoplasmic lactate dehydrogenase suggesting an endocytotic mechanism. Intra-synaptosomal HRP can be released by a process that is similar to uptake and also not accompanied by any loss of synaptosomal lactate dehydrogenase exocytosis. Depolarization of synaptosomes (by high potassium concentrations) was found to release [¹⁴C]ACh but to have no effect on HRP uptake either simultaneouly or after recovery in an non-depolarizing medium. Absence of Ca²⁺ prevented depolarization evoked release of [¹⁴C]ACh but had no effect on the uptake of HRP. The release of HRP was not increased by depolarization even though [¹⁴C]choline taken up during the same period wasreleased as [¹⁴C]ACh. It is concluded that the endo-exocytotic cycle that transport HRP across the synaptosomal membrane is unrelated to tranmitter release. A discrete vesicular localization of HRP reaction product was only occasionally in the EM nor could consistent differences resulting from depolarization be observed. However, nthe untrastructural localization was found to be unreliable because glutaradehyde fixation irreversiy inactivated 80–90% of the HRP even when it was sequestered within synaptosomes and the insoluble reaction product precipitated from a supersaturated solution onto membranes.     

Proteinase‐activated receptor‐2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin‐sensitive and neurokinin‐2 receptor‐dependent pathway

Background Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin‐sensitive neurons. The aim of this study was to determine whether proteinase‐activated receptor‐2 (PAR‐2) activation evokes reflex LSM contraction via similar mechanisms. Methods Tension recording studies were performed using opossum oesophageal LSM strips in the presence and absence of pharmacological agents. In addition, the effect of trypsin on single isolated LSM cells was determined using videomicroscopy, and the expression of PAR‐2 in oesophageal tissue was examined using immunohistochemistry. Key Results The PAR‐2 agonist trypsin evoked sustained, concentration‐dependent contraction of LSM muscle strips, but had no effect on isolated LSM cells. The trypsin‐induced contraction was blocked by capsaicin desensitization, substance P (SP) desensitization or application of the selective neurokinin‐2 (NK‐2) receptor antagonist MEN 10376. Immunohistochemistry revealed co‐localization of SP, calcitonin gene‐related peptide and PAR‐2 in axons of opossum oesophageal LSM. Conclusions & Inferences Longitudinal smooth muscle contraction induced by trypsin involves capsaicin‐sensitive neurons and subsequent activation of NK‐2, which is identical to the pathway involved in acid‐induced LSM contraction and oesophageal shortening. This suggests that acid‐induced LSM contraction may involve mast cell‐derived mediators that activate capsaicin‐sensitive neurons via PAR‐2.     

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin‐dependent protein kinase II (CaMKII) is associated with memory and its α isoform is critical for development of activity‐induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague–Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its α isoform (pCaMKIIα) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIα‐positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIα/N52 colabeled neurons but decreased the percentage of N52‐negative nonmyelinated neurons that expressed pCaMKIIα from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIα/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIα levels following peripheral injury is due to the loss of IB4‐positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIα. J. Comp. Neurol. 518:64–74, 2010. © 2009 Wiley‐Liss, Inc.