Matured Myofibers in Bioprinted Constructs with In Vivo Vascularization and Innervation.

For decades, the study of tissue-engineered skeletal muscle has been driven by a clinical need to treat neuromuscular diseases and volumetric muscle loss. The in vitro fabrication of muscle offers the opportunity to test drug-and cell-based therapies, to study disease processes, and to perhaps, one day, serve as a muscle graft for reconstructive surgery. This study developed a biofabrication technique to engineer muscle for research and clinical applications. A bioprinting protocol was established to deliver primary mouse myoblasts in a gelatin methacryloyl (GelMA) bioink, which was implanted in an in vivo chamber in a nude rat model. For the first time, this work demonstrated the phenomenon of myoblast migration through the bioprinted GelMA scaffold with cells spontaneously forming fibers on the surface of the material. This enabled advanced maturation and facilitated the connection between incoming vessels and nerve axons in vivo without the hindrance of a scaffold material. Immunohistochemistry revealed the hallmarks of tissue maturity with sarcomeric striations and peripherally placed nuclei in the organized bundles of muscle fibers. Such engineered muscle autografts could, with further structural development, eventually be used for surgical reconstructive purposes while the methodology presented here specifically has wide applications for in vitro and in vivo neuromuscular function and disease modelling.

Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain.

The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin+neural precursor cells (NPCs) have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreERT2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP). eYFP+ midbrain cells were then characterized 1-32weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited a mature neuronal phenotype with large amplitude fast action potentials (APs), spontaneous post-synaptic currents (sPSCs), and expression of 'mature' neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2). This was the case even at the earliest time-point following tamoxifen (i.e. 1week). In comparison to neighboring eYFP- (control) cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP- cells by increased expression of 'immature' pro-neuronal genes (Pax6, Ngn2 and/or Msx1). However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin+NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.

Can Valproic Acid Regulate Neurogenesis from Nestin+ Cells in the Adult Midbrain?

Degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) causes the motor symptoms (e.g. tremor, muscle rigidity, bradykinesia, postural instability) of Parkinson's disease (PD). It is generally agreed that replacing these neurons will provide better motor symptom relief and fewer side effects than current pharmacotherapies. One potential approach to this is up-regulating endogenous DA neurogenesis in SNc. In the present study, we conducted bioinformatics analyses to identify signalling pathways that control expression of Pax6 and Msx1 genes, which have been identified as potentially important neurogenic regulators in the adult midbrain. From this Valproic acid (VPA) was identified as a regulator of these pathways, and we tested VPA for its ability to regulate midbrain neurogenesis in adult mice. VPA was infused directly into the midbrain of adult NesCreERT2/R26eYFP mice using osmotic pumps attached to implanted cannula. These mice enable permanent eYFP+ labelling of adult Nestin-expressing neural precursor cells and their progeny/ontogeny. VPA did not affect the number of eYFP+ midbrain cells, but significantly reduced the number of Pax6+, Pax6+/NeuN+, eYFP+/NeuN+ and eYFP-/NeuN+ cells. However, this reduction in NeuN expression was probably via VPA's Histone de-acetylase inhibitory properties rather than reduced neuronal differentiation by eYFP + cells. We conclude that Pax6 and Msx1 are not viable targets for regulating neurogenesis in the adult midbrain.

An investigation of gene expression in single cells derived from Nestin-expressing cells in the adult mouse midbrain in vivo.

Generation of new dopamine (DA) neurons in the adult midbrain is a controversial issue in development of better treatments for Parkinson's disease (PD). Previous research suggests Nestin-expressing neural precursor cells (NPCs) have a propensity to differentiate into neurons here, including DA neurons. In the present study we sought confirmation of this by studying gene expression in single Nestin-expressing cells and their progeny/ontogeny within the adult mouse midbrain. Cells were identified by administering a pulse of Tamoxifen to adult Nestin-CreERT2×R26eYFP transgenic mice. Samples of cytoplasm were harvested 4 days to 8 months later from individual eYFP+ cells in acutely prepared midbrain slices and analysed by RT-qPCR for gene expression. Remarkably, most eYFP+ cells co-expressed genes associated with mature (including DA) neurons (i.e. NeuN, Gad1, Gad2, vGlut2, TH and/or D2R) and neurogenesis (i.e. Ki67, Dcx, Ncam, Pax6, Ngn2 and/or Msx1), and this was true at all time-points following Tamoxifen. Indeed, cell proliferation genes (Nestin, Ki67) were exclusively expressed by eYFP+ cells with mature neuronal morphology and gene expression, and only at early time-points after Tamoxifen. Expression of proneuronal genes (Pax6, Msx1, Ngn2) was, however, higher in eYFP+ cells with immature morphology compared with mature morphology. Gene expression bore no relationship to cell location indicating that, in contrast to development, Nestin-expressing cells arise throughout the midbrain parenchyma and do not migrate long distances. On the other hand, gene expression did change with time after Tamoxifen, although not in a way consistent with neurogenesis. Overall, our results suggest that Nestin expression in the adult midbrain occurs in mature neurons, casting doubt on the premise of neurogenesis from Nestin+ NPCs here.

Parallel processing of internal and external feedback in the spinocerebellar system of primates.

Cerebellar control of voluntary movements is achieved by the integration of external and internal feedback information to adjust and correct properly ongoing actions. In the forelimb of primates, rostral-spinocerebellar tract (RSCT) neurons are thought to integrate segmental, descending, and afferent sources and relay upstream a compound signal that contains both an efference copy of the spinal-level motor command and the state of the periphery. We tested this hypothesis by implanting stimulating electrodes in the superior cerebellar peduncle and recording the activity of cervical spinal neurons in primates. To dissociate motor commands and proprioceptive signals, we used a voluntary wrist task and applied external perturbations to the movement. We identified a large group of antidromically activated RSCT neurons located in deep dorsal sites and a smaller fraction of postsynaptically activated (PSA) cells located in intermediate and ventral laminae. RSCT cells received sensory input from broad, proximally biased receptive fields (RFs) and were not affected by applied wrist perturbations. PSA cells received sensory information from distal RFs and were more strongly related to active and passive movements. The anatomical and functional properties of RSCT and PSA cells suggest that descending signals converging on PSA cells contribute to both motor preparation and motor control. In parallel, RSCT neurons relay upstream an integrated signal that encodes the state of working muscles and can contribute to distal-to-proximal coordination of action. Thus the rostral spinocerebellar system sends upstream an efference copy of the motor command but does not signal abrupt errors in the performed movement.NEW & NOTEWORTHY Cerebellar coordination of voluntary movements relies on integrating feedback information to update motor output. With the use of a novel protocol, we identified spinal neurons constituting the ascending and descending components of the forelimb spinocerebellar system in behaving primates. The data suggest that descending information contributes to both motor preparation and execution, whereas ascending information conveys the spinal level motor command, such that internal and external feedback is relayed through parallel pathways.

Evidence of functional duplicity of Nestin expression in the adult mouse midbrain.

Whether or not neurogenesis occurs in the adult substantia nigra pars compacta (SNc) is an important question relevant for developing better treatments for the motor symptoms of Parkinson's disease (PD). Although controversial, it is generally believed that dividing cells here remain undifferentiated or differentiate into glia, not neurons. However, there is a suggestion that Nestin-expressing neural precursor cells (NPCs) in the adult SNc have a propensity to differentiate into neurons, which we sought to confirm in the present study. Adult (>8-weeks old) transgenic NesCreERT2/GtROSA or NesCreERT2/R26eYFP mice were used to permanently label Nestin-expressing cells and their progeny with ß-galactosidase (ß-gal) or enhanced yellow fluorescent protein (eYFP), respectively. Most ß-gal+ or eYFP+ cells were found in the ependymal lining of the midbrain aqueduct (Aq) and in the midline ventral to Aq. Smaller but significant numbers were in the periaqueductal gray (PAG), the ventral tegmental area (VTA), and in SNc. Low-level basal proliferation was evidenced by a modest increase in number of ß-gal+ or eYFP+ cells over time, fewer ß-gal+ or eYFP+ cells when mice were administered the anti-mitotic agent Cytarabine, and incorporation of the proliferation marker bromodeoxyuridine (BrdU) in a very small number of ß-gal+ cells. No evidence of migration was found, including no immunoreactivity against the migration markers doublecortin (DCX) or polysialic acid neural cell adhesion molecule (PSA-NCAM), and no dispersal of ß-gal+ or eYFP+ cells through the midbrain parenchyma over time. However, ß-gal+ or eYFP+ cells did increase in size and express higher levels of mature neuronal genes over time, indicating growth and neuronal differentiation. In mice whose SNc dopamine neurons had been depleted with 6-hydroxy-dopamine, a model of PD, there were ~2-fold more ß-gal+ cells in SNc specifically, although the proportion that were also NeuN+ was not affected. Remarkably, as early as 4days following putative Nestin-expression, many ß-gal+ or eYFP+ cells had mature neuronal morphology and were NeuN+. Furthermore, mature neuronal ß-gal+ cells were immunoreactive against the self-renewal or pluripotency marker sex determining region Y-box 2 (Sox2). Overall, our data support the notion that some Nestin-expressing, presumably NPCs, have a limited capacity for proliferation, no capacity for migration, and a propensity to generate new neurons within the microenvironment of the adult midbrain. However, our data also suggest that significant numbers of extant midbrain neurons express Nestin and other classical neurogenesis markers in contexts that are presumably not neurogenic. These findings foreshadow duplicitous roles for Nestin and other molecules that are traditionally associated with neurogenesis in the adult midbrain, which should be considered in future PD research.

Differences in Number of Midbrain Dopamine Neurons Associated with Summer and Winter Photoperiods in Humans.

Recent evidence indicates the number of dopaminergic neurons in the adult rodent hypothalamus and midbrain is regulated by environmental cues, including photoperiod, and that this occurs via up- or down-regulation of expression of genes and proteins that are important for dopamine (DA) synthesis in extant neurons ('DA neurotransmitter switching'). If the same occurs in humans, it may have implications for neurological symptoms associated with DA imbalances. Here we tested whether there are differences in the number of tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) and DA transporter (DAT) immunoreactive neurons in the midbrain of people who died in summer (long-day photoperiod, n = 5) versus winter (short-day photoperiod, n = 5). TH and DAT immunoreactivity in neurons and their processes was qualitatively higher in summer compared with winter. The density of TH immunopositive (TH+) neurons was significantly (~6-fold) higher whereas the density of TH immunonegative (TH-) neurons was significantly (~2.5-fold) lower in summer compared with winter. The density of total neurons (TH+ and TH- combined) was not different. The density of DAT+ neurons was ~2-fold higher whereas the density of DAT- neurons was ~2-fold lower in summer compared with winter, although these differences were not statistically significant. In contrast, midbrain nuclear volume, the density of supposed glia (small TH- cells), and the amount of TUNEL staining were the same in summer compared with winter. This study provides the first evidence of an association between environmental stimuli (photoperiod) and the number of midbrain DA neurons in humans, and suggests DA neurotransmitter switching underlies this association.

Environment- and activity-dependent dopamine neurotransmitter plasticity in the adult substantia nigra.

The ability of neurons to change the amount or type of neurotransmitter they use, or 'neurotransmitter plasticity', is an emerging new form of adult brain plasticity. For example, it has recently been shown that neurons in the adult rat hypothalamus up- or down-regulate dopamine (DA) neurotransmission in response to the amount of light the animal receives (photoperiod), and that this in turn affects anxiety- and depressive-like behaviors (Dulcis et al., 2013). In this Chapter I consolidate recent evidence from my laboratory suggesting neurons in the adult mouse substantia nigra pars compacta (SNc) also undergo DA neurotransmitter plasticity in response to persistent changes in their electrical activity, including that driven by the mouse's environment or behavior. Specifically, we have shown that the amounts of tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) gene promoter activity, TH mRNA and TH protein in SNc neurons increases or decreases after ∼20h of altered electrical activity. Also, infusion of ion-channel agonists or antagonists into the midbrain for 2 weeks results in ∼10% (∼500 neurons) more or fewer TH immunoreactive (TH+) SNc neurons, with no change in the total number of SNc neurons (TH+ and TH-). Targeting ion-channels mediating cell-autonomous pacemaker activity in, or synaptic input and afferent pathways to, SNc neurons are equally effective in this regard. In addition, exposing mice to different environments (sex pairing or environment enrichment) for 1-2 weeks induces ∼10% more or fewer TH+ SNc (and ventral tegmental area or VTA) neurons and this is abolished by concurrent blockade of synaptic transmission in midbrain. Although further research is required to establish SNc (and VTA) DA neurotransmitter plasticity, and to determine whether it alters brain function and behavior, it is an exciting prospect because: (1) It may play important roles in movement, motor learning, reward, motivation, memory and cognition; and (2) Imbalances in midbrain DA cause symptoms associated with several prominent brain and behavioral disorders such as schizophrenia, addiction, obsessive-compulsive disorder, depression, Parkinson's disease and attention-deficit and hyperactivity disorder. Midbrain DA neurotransmitter plasticity may therefore play a role in the etiology of these symptoms, and might also offer new treatment options.

Environmental modulations of the number of midbrain dopamine neurons in adult mice.

Long-lasting changes in the brain or 'brain plasticity' underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in 'enriched environments' (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of 'DA' genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson's disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

Do sensorimotor ß-oscillations maintain muscle synergy representations in primary motor cortex?

Coherent ß-oscillations are a dominant feature of the sensorimotor system yet their function remains enigmatic. We propose that, in addition to cell intrinsic and/or local network interactions, they are supported by activity propagating recurrently around closed neural 'loops' between primary motor cortex (M1), muscles, and back to M1 via somatosensory pathways. Individual loops reciprocally connect individual muscle synergies ('motor primitives') with their representations in M1, and the conduction time around each loop resonates with the periodic spiking of its constituent neurons/muscles. During ß-oscillations, this resonance strengthens within-loop connectivity (via long-term potentiation, LTP), whereas non-resonance between different loops weakens connectivity (via long-term depression, LTD) between M1 representations of different muscle synergies. In this way, ß-oscillations help maintain accurate and discrete representations of muscle synergies in M1.

Herpes simplex encephalitis relapse with chorea is associated with autoantibodies to N-Methyl-D-aspartate receptor or dopamine-2 receptor.

BACKGROUND: Movement disorder relapses after herpes simplex virus 1 (HSV1) encephalitis have been hypothesized to be secondary to postviral autoimmunity. Recently, a proportion of patients with HSV1 encephalitis (HSE) were shown to produce autoantibodies against N-methyl-D-aspartate receptor (NMDAR). METHODS: We measured autoantibodies against NMDAR and dopamine-2 receptor (D2R) expressed at the cell surface in the stored acute serum of 9 children with HSE, 3 of whom had a relapsing course with chorea. RESULTS: The 3 patients with chorea had elevated autoantibodies against NMDAR (n = 1), D2R (n = 1), or both (n = 1), whereas patients without chorea were negative (n = 6). The prospectively identified patient with chorea and NMDAR autoantibodies improved after early treatment with steroids, intravenous immunoglobulin, and cyclophosphamide, with reduction in serum NMDAR antibody titers. CONCLUSIONS: These autoantibody findings lend support to the autoimmune hypothesis and the early use of immune suppression in post-HSE chorea.

Environmental and behavioral modulation of the number of substantia nigra dopamine neurons in adult mice.

BACKGROUND: Recent evidence indicates that hypothalamic neurons acquire or lose the capacity to synthesize and release dopamine (DA) in response to environmental stimuli, and this has functional and behavioral consequences for adult rats. We have evidence that neuronal activity, including that driven by afferent input, regulates acquisition and loss of the DA phenotype by substantia nigra pars compacta (SNc) neurons in adult mice. Hypotheses The aims of the present study were to determine whether the environment or behavior regulates the number of SNc DA neurons in adult mice, and whether this is mediated by afferent input. METHODS: ADULT MICE WERE SUBJECT TO TWO DIFFERENT ENVIRONMENTS/BEHAVIORS: "mating" for 1 week or "environment enrichment" (EE) for 2 weeks; then the numbers of tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis) immunopositive (TH+) and immunonegative (TH-) SNc neurons were counted. RESULTS: More TH+ neurons were present in mated males whereas less TH+ neurons were present in mated females. Also, more TH+ neurons were present in EE males, and this increase was completely abolished by concurrent local infusion of GABAA receptor antagonists. CONCLUSIONS: The number of DA neurons in the adult SNc is not fixed, but readily increases and decreases in response to environmental stimuli and/or behaviors. These changes are mediated by afferent input relaying information about the environment or behavior to SNc neurons.

Investigation of sequential growth factor delivery during cuprizone challenge in mice aimed to enhance oligodendrogliogenesis and myelin repair.

Repair in multiple sclerosis involves remyelination, a process in which axons are provided with a new myelin sheath by new oligodendrocytes. Bone morphogenic proteins (BMPs) are a family of growth factors that have been shown to influence the response of oligodendrocyte progenitor cells (OPCs) in vivo during demyelination and remyelination in the adult brain. We have previously shown that BMP4 infusion increases numbers of OPCs during cuprizone-induced demyelination, while infusion of Noggin, an endogenous antagonist of BMP4 increases numbers of mature oligodendrocytes and remyelinated axons following recovery. Additional studies have shown that insulin-like growth factor-1 (IGF-1) promotes the survival of OPCs during cuprizone-induced demyelination. Based on these data, we investigated whether myelin repair could be further enhanced by sequential infusion of these agents firstly, BMP4 to increase OPC numbers, followed by either Noggin or IGF-1 to increase the differentiation and survival of the newly generated OPCs. We identified that sequential delivery of BMP4 and IGF-1 during cuprizone challenge increased the number of mature oligodendrocytes and decreased astrocyte numbers following recovery compared with vehicle infused mice, but did not alter remyelination. However, sequential delivery of BMP4 and Noggin during cuprizone challenge did not alter numbers of oligodendrocytes or astrocytes in the corpus callosum compared with vehicle infused mice. Furthermore, electron microscopy analysis revealed no change in average myelin thickness in the corpus callosum between vehicle infused and BMP4-Noggin infused mice. Our results suggest that while single delivery of Noggin or IGF-1 increased the production of mature oligodendrocytes in vivo in the context of demyelination, only Noggin infusion promoted remyelination. Thus, sequential delivery of BMP4 and Noggin or IGF-1 does not further enhance myelin repair above what occurs with delivery of Noggin alone.

Antibodies to surface dopamine-2 receptor in autoimmune movement and psychiatric disorders.

Recent reports of autoantibodies that bind to neuronal surface receptors or synaptic proteins have defined treatable forms of autoimmune encephalitis. Despite these developments, many cases of encephalitis remain unexplained. We have previously described a basal ganglia encephalitis with dominant movement and psychiatric disease, and proposed an autoimmune aetiology. Given the role of dopamine and dopamine receptors in the control of movement and behaviour, we hypothesized that patients with basal ganglia encephalitis and other putative autoimmune basal ganglia disorders harboured serum autoantibodies against important dopamine surface proteins. Basal ganglia encephalitis sera immunolabelled live surface cultured neurons that have high expression of dopamine surface proteins. To detect autoantibodies, we performed flow cytometry cell-based assays using human embryonic kidney cells to express surface antigens. Twelve of 17 children (aged 0.4-15 years, nine males) with basal ganglia encephalitis had elevated immunoglobulin G to extracellular dopamine-2 receptor, compared with 0/67 controls. Immunofluorescence on wild-type mouse brain showed that basal ganglia encephalitis sera immunolabelled microtubule-associated protein 2-positive neurons in striatum and also in cultured striatal neurons, whereas the immunolabelling was significantly decreased in dopamine-2 receptor knock-out brains. Immunocytochemistry confirmed that immunoreactivity localized to the surface of dopamine-2 receptor-transfected cells. Immunoabsorption of basal ganglia encephalitis sera on dopamine-2 receptor-transfected human embryonic kidney cells decreased immunolabelling of dopamine-2 receptor-transfected human embryonic kidney cells, neurons and wild-type mouse brain. Using a similar flow cytometry cell-based assay, we found no elevated immunoglobulin G binding to dopamine 1, 3 or 5 receptor, dopamine transporter or N-methyl-d-aspartate receptor. The 12 dopamine-2 receptor antibody-positive patients with encephalitis had movement disorders characterized by parkinsonism, dystonia and chorea. In addition, the patients had psychiatric disturbance with emotional lability, attention deficit and psychosis. Brain magnetic resonance imaging showed lesions localized to the basal ganglia in 50% of the patients. Elevated dopamine-2 receptor immunoglobulin G was also found in 10/30 patients with Sydenham's chorea, 0/22 patients with paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection and 4/44 patients with Tourette's syndrome. No dopamine-1 receptor immunoglobulin G was detected in any disease or control groups. We conclude that assessment of dopamine-2 receptor antibodies can help define autoimmune movement and psychiatric disorders.

Increasing cDNA yields from single-cell quantities of mRNA in standard laboratory reverse transcriptase reactions using acoustic microstreaming.

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50 pg total RNA, or 0.01-2.5 pg mRNA, per cell) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: greater numbers of genes to be analyzed per cell; more quantitative analysis of gene expression; and better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

Remyelination is altered by bone morphogenic protein signaling in demyelinated lesions.

Remyelination of the CNS involves the regeneration of mature oligodendrocytes by endogenous oligodendrocyte progenitor cells (OPCs). Previous studies have shown that bone morphogenic proteins (BMPs) inhibit the production of oligodendrocytes in the healthy CNS. However, there is currently no information on the influence of BMP signaling in vivo within demyelinated lesions of the brain or on subsequent remyelination. Here, we determine a role for BMP signaling in modulating oligodendrogliogenesis and remyelination in the brain following cuprizone-induced demyelination. We identified that BMP signaling is active in oligodendroglia and astrocytes within the demyelinated corpus callosum. Intraventricular infusion of BMP4 into the brains of mice during demyelination increased the proliferation of OPCs and, to a lesser extent, microglia and astrocytes in the corpus callosum. In contrast, infusion of Noggin, an extracellular antagonist of BMP4, increased the density of mature oligodendrocytes in the remyelinating corpus callosum. Additional evidence from myelin staining and electron microscopy indicates there is an increase in remyelinated axons in the corpus callosum of Noggin-infused mice. Thus, inhibition of endogenous BMP signaling during demyelination promotes mature oligodendrocyte regeneration and remyelination.

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons.

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1 µM tetrodotoxin decreased TH expression beginning around 20 h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300 nM apamin increased TH mRNA and protein between 12 and 24 h later in slices of adult midbrain. Two-week infusions of 300 nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ∼20 h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.

Modulation of bone morphogenic protein signalling alters numbers of astrocytes and oligodendroglia in the subventricular zone during cuprizone-induced demyelination.

The adult subventricular zone (SVZ) is a potential source of precursor cells to replace neural cells lost during demyelination. To better understand the molecular events that regulate neural precursor cell responsiveness in this context we undertook a microarray and quantitative PCR based analysis of genes expressed within the SVZ during cuprizone-induced demyelination. We identified an up-regulation of the genes encoding bone morphogenic protein 4 (BMP4) and its receptors. Immunohistochemistry confirmed an increase in BMP4 protein levels and also showed an increase in phosphorylated SMAD 1/5/8, a key component of BMP4 signalling, during demyelination. In vitro analysis revealed that neural precursor cells isolated from demyelinated animals, as well as those treated with BMP4, produce more astrocytes. Similarly, there were increased numbers of astrocytes in vivo within the SVZ during demyelination. Intraventricular infusion of Noggin, an endogenous antagonist of BMP4, during cuprizone-induced demyelination reduced pSMAD1/5/8, decreased astrocyte numbers and increased oligodendrocyte numbers in the SVZ. Our results suggest that lineage commitment of SVZ neural precursor cells is altered during demyelination and that BMP signalling plays a role in this process.