Neural Stem Cells and Neurospheres - Re-evaluating the Relationship

For most of the past century, the prospect of replacing lost or damaged cells in the central nervous system (CNS) was hampered by the opinion that the adult mammalian CNS was incapable of generating new nerve cells. This belief, Like most dogmas, was essentially founded on a lack of experimental evidence to the contrary. The overturning of this 'no new neuron' hypothesis began midway through the twentieth century with a series of reports documenting neurogenesis in the postnatal and adult brain(1), continued with the isolation and in vitro culture of neurogenic cells from the adult mammalian brain(2,3), and culminated in the discovery of a population of muttipotent, selfrenewing cells in the adult CNS (that is, bona fide neural stem cells)(3-5). Although a variety of techniques were initially used, the neurosphere assay (NSA)(3,6) rapidly emerged as the assay of choice and has since become a valuable toot for isolating, and understanding the biology of, embryonic and adult CNS stem cells. Like all technologies, it is not without its limitations. In this article we will hightight several shortcomings of the assay related to its application and interpretation that we believe have led to a significant body of research whose conclusions may well be misleading.

SK channels regulate excitatory synaptic transmission and plasticity in the lateral amygdala

At glutamatergic synapses, calcium influx through NMDA receptors (NMDARs) is required for long-term potentiation (LTP); this is a proposed cellular mechanism underlying memory and learning. Here we show that in lateral amygdala pyramidal neurons, SK channels are also activated by calcium influx through synaptically activated NMDARs, resulting in depression of the synaptic potential. Thus, blockade of SK channels by apamin potentiates fast glutamatergic synaptic potentials. This potentiation is blocked by the NMDAR antagonist AP5 (D(-)-2-amino-5-phosphono-valeric acid) or by buffering cytosolic calcium with BAPTA. Blockade of SK channels greatly enhances LTP of cortical inputs to lateral amygdala pyramidal neurons. These results show that NMDARs and SK channels are colocalized at glutamatergic synapses in the lateral amygdala. Calcium influx through NMDARs activates SK channels and shunts the resultant excitatory postsynaptic potential. These results demonstrate a new role for SK channels as postsynaptic regulators of synaptic efficacy.

Brain maps, great and small: lessons from comparative studies of primate visual cortical organization

In this paper, we review evidence from comparative studies of primate cortical organization, highlighting recent findings and hypotheses that may help us to understand the rules governing evolutionary changes of the cortical map and the process of formation of areas during development. We argue that clear unequivocal views of cortical areas and their homologies are more likely to emerge for 'core' fields, including the primary sensory areas, which are specified early in development by precise molecular identification steps. In primates, the middle temporal area is probably one of these primordial cortical fields. Areas that form at progressively later stages of development correspond to progressively more recent evolutionary events, their development being less firmly anchored in molecular specification. The certainty with which areal boundaries can be delimited, and likely homologies can be assigned, becomes increasingly blurred in parallel with this evolutionary/developmental sequence. For example, while current concepts for the definition of cortical areas have been vindicated in allowing a clarification of the organization of the New World monkey 'third tier' visual cortex (the third and dorsomedial areas, V3 and DM), our analyses suggest that more flexible mapping criteria may be needed to unravel the organization of higher-order visual association and polysensory areas.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1 beta responses using c-fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1 beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1 beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1 beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1 beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Temporal and spatial transcriptional programs in murine kidney development

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Hypothalamic control of bone formation: Distinct actions of leptin and Y2 receptor pathways

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2(-/-) anabolic pathways in modulating bone formation. Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient oblob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms. Materials and Methods: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2(-/-) oblob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2(-/-) mice. Cancellous bone volume and bone cell function were assessed. Results: Osteoblast activity was comparably elevated in oblob, Y2(-/-), and Y2(-/-) oblob mice. However, greater bone resorption in oblob and Y2(-/-) oblob mice reduced cancellous bone volume compared with Y2(-/-). Both wildtype and Y2(-/-) AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2(-/-) AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice. Conclusions: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2(-/-) pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.

Deceleration affects anticipatory and reactive components of triggered postural responses

Understanding the physiological and psychological factors that contribute to healthy and pathological balance control in man has been made difficult by the confounding effects of the perturbations used to test balance reactions. The present study examined how postural responses were influenced by the acceleration-deceleration interval of an unexpected horizontal translation. Twelve adult males maintained balance during unexpected forward and backward surface translations with two different acceleration-deceleration intervals and presentation orders (serial or random). SHORT perturbations consisted of an initial acceleration (peak acceleration 1.3 m s(-2); duration 300 ms) followed 100 ms later by a deceleration. LONG perturbations had the same acceleration as SHORT perturbations, followed by a 2-s interval of constant velocity before deceleration. Surface and intra-muscular electromyography (EMG) from the leg, trunk, and shoulder muscles were recorded along with motion and force plate data. LONG perturbations induced larger trunk displacements compared to SHORT perturbations when presented randomly and larger EMG responses in proximal and distal muscles during later (500-800 ms) response intervals. During SHORT perturbations, activity in some antagonist muscles was found to be associated with deceleration and not the initial acceleration of the support surface. When predictable, SHORT perturbations facilitated the use of anticipatory mechanisms to attenuate early (100-400 ms) EMG response amplitudes, ankle torque change and trunk displacement. In contrast, LONG perturbations, without an early deceleration effect, did not facilitate anticipatory changes when presented in a predictable order. Therefore, perturbations with a short acceleration-deceleration interval can influence triggered postural responses through reactive effects and, when predictable with repeated exposure, through anticipatory mechanisms.

The effects of task complexity and practice on dual-task interference in visuospatial working memory

Although the n-back task has been widely applied to neuroimagery investigations of working memory (WM), the role of practice effects on behavioural performance of this task has not yet been investigated. The current study aimed to investigate the effects of task complexity and familiarity on the n-back task. Seventy-seven participants (39 male, 38 female) completed a visuospatial n-back task four times, twice in two testing sessions separated by a week. Participants were required to remember either the first, second or third (n-back) most recent letter positions in a continuous sequence and to indicate whether the current item matched or did not match the remembered position. A control task, with no working memory requirements required participants to match to a predetermined stimulus position. In both testing sessions, reaction time (RT) and error rate increased with increasing WM load. An exponential slope for RTs in the first session indicated dual-task interference at the 3-back level. However, a linear slope in the second session indicated a reduction of dual-task interference. Attenuation of interference in the second session suggested a reduction in executive demands of the task with practice. This suggested that practice effects occur within the n-back ask and need to be controlled for in future neuroimagery research using the task.

Test-retest reliability and practice effects of a rapid screen of mild traumatic brain injury

Test-retest reliabilities and practice affects of measures from the Rapid Screen of Concussion (RSC), in addition to the Digit Symbol Substitution Test (Digit Symbol), were examined. Twenty five male participants were tested three times; each testing session scheduled a week apart. The test-retest reliability estimates for most measures were reasonably good, ranging from .79 to .97. An exception was the delayed word recall test, which has had a reliability estimate of .66 for the first retest, and .59 for the second retest. Practice effects were evident from Times 1 to 2 on the sentence comprehension and delayed recall subtests of the RSC, Digit Symbol and a composite score. There was also a practice effect of the same magnitude found from Time 2 to Time 3 on Digit Symbol, delayed recall and the composite score. Statistics on measures for both the first and second retest intervals, with associated practice affects, are presented to enable the calculation of reliable change indices (RCI). The RCI may be used to assess any improvement in cognitive functioning after mild Traumatic Brain Injury.

The acute effects of mild traumatic brain injury on finger tapping with and without word repetition

This study aimed to investigate the acute effects of mild Traumatic Brain Injury (mTBI) on the performance of a finger tapping and word repetition dual task in order to determine working memory impairment in mTBI Sixty-four (50 male, 14 female) right-handed cases of mTBI and 26 (18 male and 8 female) right-handed cases of orthopaedic injuries were tested within 24 hours of injury. Patients with mTBI completed fewer correct taps in 10 seconds than patients with orthopaedic injuries, and female mTBI cases repeated fewer words. The size of the dual task decrement did not vary between groups. When added to a test battery including the Rapid Screen of Concussion (RSC; Comerford, Geffen, May, Medland T Geffen, 2002) and the Digit Symbol Substitution Test,finger tapping speed accounted for 1% of between groups variance and did not improve classification rates of male participants. While the addition of tapping rate did not improve the sensitivity and specificity of the RSC and DSST to mTBI in males, univariate analysis of motor performance in females indicated. that dual task performance might be diagnostic. An increase in female sample Size is warranted. These results confirm the view that there is a generalized slowing of processing ability following mTBI.