Intact Central Nervous System Research Articles

Palytoxin evokes reversible spreading depolarization in the locust CNS.

Spreading depolarization (SD) describes the near-complete depolarization of central nervous system (CNS) neural cells as a consequence of chemical, electrical, or metabolic perturbations. It is well established as the central mechanism underlying insect coma and various mammalian neurological dysfunctions. Despite significant progress in our understanding, the question remains: which cation channel, if any, generates SD in the CNS? Previously, we speculated that the sodium-potassium ATPase (NKA) might function as a large-conductance ion channel to initiate SD in insects, potentially mediated by a palytoxin (PLTX)-like endogenous activator. In the current study, we evaluate the effectiveness and properties of PLTX as an SD initiator in Locusta migratoria. Whereas bath-applied PLTX failed to ignite SD, direct injection into the neuropil triggered SD in 57% of the preparations. Notably, PLTX-induced SD onset was significantly more rapid compared with ouabain (OUA) injection and azide controls, though their electrophysiological features remained similar. Furthermore, PLTX-induced SD was recoverable and resulted in a greater frequency of repetitive SD events compared with ouabain. Surprisingly, prior PLTX treatment disrupted the onset and recovery of subsequent SD evoked by other means. PLTX injection could attenuate the amplitude and even completely inhibit the onset of azide-induced SD at higher doses. These results show that PLTX can trigger repetitive and reversible SD-like events in locusts and simultaneously interfere with anoxic SD occurrence. We suggest that the well-documented NKA pump conversion into an open nonselective cationic channel is a plausible mechanism of SD activation in the locust CNS, warranting additional investigations.NEW & NOTEWORTHY Spreading depolarization (SD) is a critical mechanism underlying central nervous system (CNS) shutdown and injury under stress, yet the initiating ion channel remains unknown. Here, we used the marine poison palytoxin (PLTX), which converts the sodium-potassium ATPase (NKA) into an open channel, to initiate SD in intact locust CNS. We show for the first time that PLTX-induced SD is rapid and recoverable in vivo, providing support that NKA conversion to a channel may be the SD-initiating mechanism.

Cortical stimulation leads to shortened myelin sheaths and increased axonal branching in spared axons after cervical spinal cord injury.

Neural activity and learning lead to myelin sheath plasticity in the intact central nervous system (CNS), but this plasticity has not been well-studied after CNS injury. In the context of spinal cord injury (SCI), demyelination occurs at the lesion site and natural remyelination of surviving axons can take months. To determine if neural activity modulates myelin and axon plasticity in the injured, adult CNS, we electrically stimulated the contralesional motor cortex at 10 Hz to drive neural activity in the corticospinal tract of rats with sub-chronic spinal contusion injuries. We quantified myelin and axonal characteristics by tracing corticospinal axons rostral to and at the lesion epicenter and identifying nodes of Ranvier by immunohistochemistry. Three weeks of daily stimulation induced very short myelin sheaths, axon branching, and thinner axons outside of the lesion zone, where remodeling has not previously been reported. Surprisingly, remodeling was particularly robust rostral to the injury which suggests that electrical stimulation can promote white matter plasticity even in areas not directly demyelinated by the contusion. Stimulation did not alter myelin or axons at the lesion site, which suggests that neuronal activity does not contribute to myelin remodeling near the injury in the sub-chronic period. These data are the first to demonstrate wide-scale remodeling of nodal and myelin structures of a mature, long-tract motor pathway in response to electrical stimulation. This finding suggests that neuromodulation promotes white matter plasticity in intact regions of pathways after injury and raises intriguing questions regarding the interplay between axonal and myelin plasticity.

Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism.

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans. Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.

<em>In Situ</em> Visualization of Axon Growth and Growth Cone Dynamics in Acute <em>Ex Vivo</em> Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

Tonic and Phasic Amperometric Monitoring of Dopamine Using Microelectrode Arrays in Rat Striatum

Here we report a novel microelectrode array recording approach to measure tonic (resting) and phasic release of dopamine (DA) in DA-rich areas such as the rat striatum and nucleus accumbens. The resulting method is tested in intact central nervous system (CNS) and in animals with extensive loss of the DA pathway using the neurotoxin, 6-hydroxyDA (6-OHDA). The self-referencing amperometric recording method employs Nafion-coated with and without m-phenylenediamine recording sites that through real-time subtraction allow for simultaneous measures of tonic DA levels and transient changes due to depolarization and amphetamine-induced release. The recording method achieves low-level measures of both tonic and phasic DA with decreased recording drift allowing for enhanced sensitivity normally not achieved with electrochemical sensors in vivo.

Central Hypothyroidism Impairs Heart Rate Stability and Prevents Thyroid Hormone-Induced Cardiac Hypertrophy and Pyrexia.

Background: Tachycardia, cardiac hypertrophy, and elevated body temperature are major signs of systemic hyperthyroidism, which are considered to reflect the excessive thyroid hormone (TH) action in the respective peripheral tissues. However, recent observations indicate that the central actions of TH also contribute substantially to cardiovascular regulation and thermogenesis. Methods: In this study, we dissect the individual contributions of peripheral TH action versus the central effects in body temperature regulation and cardiovascular functions by taking advantage of mice lacking the TH transporters monocarboxylate transporter 8 (MCT8) and organic anion transporting polypeptide 1C1 (OATP1C1) (M/O double knock-out [dko]), which exhibit elevated serum triiodothyronine (T3) levels while their brain is in a profoundly hypothyroid state. We compared these animals with wild-type (WT) mice that were treated orally with T3 to achieve similarly elevated serum T3 levels, but are centrally hyperthyroid. For the studies, we used radiotelemetry, infrared thermography, gene expression profiling, Western blot analyses, and enzyme linked immunosorbent assays (ELISA) assays. Results: Our analyses revealed mild hyperthermia and cardiac hypertrophy in T3-treated WT mice but not in M/O dko animals, suggesting that central actions of TH are required for these hyperthyroid phenotypes. Although the average heart rate was unaffected in either model, the M/O dko exhibited an altered heart rate frequency distribution with tachycardic bursts in active periods and bradycardic episodes during resting time, demonstrating that the stabilization of heart rate by the autonomic nervous system can be impaired in centrally hypothyroid animals. Conclusions: Our studies unravel distinct phenotypical traits of hyperthyroidism that depend on an intact central nervous system, and provide valuable insight into the cardiovascular pathology of the Allan-Herndon-Dudley syndrome, a condition caused by the lack of MCT8 in humans.

Investigating microglia during motor neuron degeneration using a zebrafish model

Many different types of pathologies can arise in the central nervous system (CNS), such as neurodegeneration. The incidence of neurodegenerative diseases continues to increase, yet the pathogenesis underlying most neurodegenerative diseases, notably in amyotrophic lateral sclerosis (ALS), remains elusive. Neuronal support cells, or glia, are known to play a crucial role in ALS. Microglia are the resident immune cells of the CNS and also have neurotrophic support functions. These cells have a disease-modifying function in ALS, yet this role is not well understood. A likely reason for this is that the intact CNS is particularly challenging to access for investigation in patients and in most animal models, which has impeded research in this field. The zebrafish is emerging as a robust model system to investigate cells in vivo, and offer distinct advantages over other vertebrate models for investigating neurodegenerative diseases. Live imaging in vivo is a powerful technique to characterize the role of dynamic cells such as microglia during neurodegeneration, and zebrafish provide a convenient means for live imaging. Here, we discuss the zebrafish as a model for live imaging, provide a brief overview of available high resolution imaging platforms that accommodate zebrafish, and describe our own in vivo studies on the role of microglia during motor neuron degeneration. Live in vivo imaging is anticipated to provide invaluable advancements to defining the pathogenesis underlying neurodegenerative diseases, which may in turn allow for more specifically targeted therapeutics.

Preclinical Pharmacokinetics of Complement C5a Receptor Antagonists PMX53 and PMX205 in Mice.

The cyclic hexapeptides PMX53 and PMX205 are potent noncompetitive inhibitors of complement C5a receptor 1 (C5aR1). They are widely utilized to study the role of C5aR1 in mouse models, including central nervous system (CNS) disease, and are dosed through a variety of routes of administration. However, a comprehensive pharmacokinetics analysis of these drugs has not been reported. In this study, the blood and CNS pharmacokinetics of PMX53 and PMX205 were performed in mice following intravenous, intraperitoneal, subcutaneous, and oral administration at identical doses. The absorption and distribution of both drugs were rapid and followed a two-compartment model with elimination half-lives of ∼20 min for both compounds. Urinary excretion was the major route of elimination following intravenous dosing with ∼50% of the drug excreted unchanged within the first 12 h. Oral bioavailability of PMX205 was higher than that of PMX53 (23% versus 9%), and PMX205 was also more efficient than PMX53 at entering the intact CNS. In comparison to other routes, subcutaneous administration of PMX205 resulted in high bioavailability (above 90%), as well as prolonged plasma and CNS exposure. Finally, repeated daily oral or subcutaneous administration of PMX205 demonstrated no accumulation of drug in blood, the brain, or the spinal cord, promoting its safety for chronic dosing. These results will be helpful in correlating the desired therapeutic effects of these C5aR1 antagonists with their pharmacokinetic profile. It also suggests that subcutaneous dosing of PMX205 may be an appropriate route of administration for future clinical testing in neurological disease.

Voltage gated sodium channels as therapeutic targets for chronic pain.

Being maladaptive and frequently unresponsive to pharmacotherapy, chronic pain presents a major unmet clinical need. While an intact central nervous system is required for conscious pain perception, nociceptor hyperexcitability induced by nerve injury in the peripheral nervous system (PNS) is sufficient and necessary to initiate and maintain neuropathic pain. The genesis and propagation of action potentials is dependent on voltage-gated sodium channels, in particular, Nav1.7, Nav1.8 and Nav1.9. However, nerve injury triggers changes in their distribution, expression and/or biophysical properties, leading to aberrant excitability. Most existing treatment for pain relief acts through non-selective, state-dependent sodium channel blockage and have narrow therapeutic windows. Natural toxins and developing subtype-specific and molecular-specific sodium channel blockers show promise for treatment of neuropathic pain with minimal side effects. New approaches to analgesia include combination therapy and gene therapy. Here, we review how individual sodium channel subtypes contribute to pain, and the attempts made to develop more effective analgesics for the treatment of chronic pain.

Towards High-Throughput Chemobehavioural Phenomics in Neuropsychiatric Drug Discovery.

Identifying novel marine-derived neuroactive chemicals with therapeutic potential is difficult due to inherent complexities of the central nervous system (CNS), our limited understanding of the molecular foundations of neuro-psychiatric conditions, as well as the limited applications of effective high-throughput screening models that recapitulate functionalities of the intact CNS. Furthermore, nearly all neuro-modulating chemicals exhibit poorly characterized pleiotropic activities often referred to as polypharmacology. The latter renders conventional target-based in vitro screening approaches very difficult to accomplish. In this context, chemobehavioural phenotyping using innovative small organism models such as planarians and zebrafish represent powerful and highly integrative approaches to study the impact of new chemicals on central and peripheral nervous systems. In contrast to in vitro bioassays aimed predominantly at identification of chemicals acting on single targets, phenotypic chemobehavioural analysis allows for complex multi-target interactions to occur in combination with studies of polypharmacological effects of chemicals in a context of functional and intact milieu of the whole organism. In this review, we will outline recent advances in high-throughput chemobehavioural phenotyping and provide a future outlook on how those innovative methods can be utilized for rapidly screening and characterizing marine-derived compounds with prospective applications in neuropharmacology and psychosomatic medicine.

Stimulation of Single, Possible CHX10 Hindbrain Neurons Turns Swimming On and Off in Young Xenopus Tadpoles

Vertebrate central pattern generators (CPGs) controlling locomotion contain neurons which provide the excitation that drives and maintains network rhythms. In a simple vertebrate, the developing Xenopus tadpole, we study the role of excitatory descending neurons with ipsilateral projecting axons (descending interneurons, dINs) in the control of swimming rhythms. In tadpoles with both intact central nervous system (CNS) and transections in the hindbrain, exciting some individual dINs in the caudal hindbrain region could start swimming repeatedly. Analyses indicated the recruitment of additional dINs immediately after such evoked dIN spiking and prior to swimming. Excitation of dINs can therefore be sufficient for the initiation of swimming. These “powerful” dINs all possessed both ascending and descending axons. However, their axon projection lengths were not different from those of other excitatory dINs at similar locations. The dorsoventral position of dINs, as a population, significantly better matched that of cells marked by immunocytochemistry for the transcription factor CHX10 than other known neuron types in the ventral hindbrain and spinal cord. The comparison suggests that the excitatory interneurons including dINs are CHX10-positive, in agreement with CHX10 as a marker for excitatory neurons with ipsilateral projections in the spinal cord and brainstem of other vertebrates. Overall, our results further demonstrate the key importance of dINs in driving tadpole swimming rhythms.

Calcium imaging method to visualize the spatial patterns of neural responses in the pygmy squid, Idiosepius paradoxus, central nervous system

BackgroundCephalopods exhibit unique behaviors such as camouflage and tactile learning. The brain functions correlated to these behaviors have long been analyzed through behavioral observations of animals subject to surgical manipulation or electrical stimulation of brain lobes. However, physiological methods have rarely been introduced to investigate the functions of each individual lobe, though physiological work on giant axons and slices of the vertical lobe system of the cephalopods have provided deep insights into ion conductance of nerves and long-term synaptic plasticity. The lack of in vivo physiological work is partly due to difficulties in immobilizing the brain which is contained within the soft body and applying calcium indicators to the cephalopod central nervous system. New methodWe here present a calcium imaging method to visualize neural responses in the central nervous system of the smallest squid, Idiosepius paradoxus. ResultsWe injected calcium indicator Cal-520 into the brachial lobes and revealed a spatiotemporal pattern of neural responses to the electrical stimulations of the axial nerve cord in the first arm. Comparison with existing methodsWe established a method to immobilize the central nervous system which is contained within the soft body and record the calcium responses from the intact central nervous system. ConclusionsOur method provides a novel approach to investigate the mechanisms of how the characteristic organization of the cephalopod brain functions to induce their unique behaviors.

Epothilone B impairs functional recovery after spinal cord injury by increasing secretion of macrophage colony-stimulating factor

The microtubule-stabilizing drug epothilone B (epoB) has shown potential value in the treatment of spinal cord injury (SCI) through diverse mechanisms. However, it remains elusive why a limited overall effect was observed. We aim to investigate the limiting factors underlying functional recovery promoted by epoB. The same SCI model treated by epoB was established as discussed previously. We used a cerebrospinal fluid (CSF) sample to assess the changes in cytokines in milieu of the SCI lesion site after epoB treatment. We then analyzed the source of cytokines, the state of microglia/macrophages/monocytes (M/Ms), and the recruitment of neutrophil in the lesion site by using the results of antibody array. Following these findings, we further evaluated the motor functional recovery caused by the reshaped microenvironment. Systemic administration of epoB significantly increased levels of several cytokines in the CSF of the rat SCI model; macrophage colony-stimulating factor (M-CSF) secreted by intact central nervous system (CNS) cells was one of the cytokines with increased levels. Along with epoB and other cytokines, M-CSF reshapes the SCI milieu by activating the microglias, killing bone marrow-derived macrophages, polarizing the M/M to M1 phenotype, and activating downstream cytokines to exacerbate the SCI injury, but it also increases the expression of neurotrophic factors. Anti-inflammatory therapy using a neutralizing antibody mix shows encouraging results. Using in vivo experiments, our findings indicate that epoB inhibits the SCI functional recovery in many ways by reshaping the milieu, which counteracts the therapeutic efficacy that led to the limited overall effectiveness.

Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.

Identification of Intrinsic Axon Growth Modulators for Intact CNS Neurons after Injury

SUMMARYFunctional deficits persist after spinal cord injury (SCI) because axons in the adult mammalian central nervous system (CNS) fail to regenerate. However, modest levels of spontaneous functional recovery are typically observed after trauma and are thought to be mediated by the plasticity of intact circuitry. The mechanisms underlying intact circuit plasticity are not delineated. Here, we characterize the in vivo transcriptome of sprouting intact neurons from Ngr1 null mice after partial SCI. We identify the lysophosphatidic acid signaling modulators LPPR1 and LPAR1 as intrinsic axon growth modulators for intact corticospinal motor neurons after adjacent injury. Furthermore, in vivo LPAR1 inhibition or LPPR1 overexpression enhances sprouting of intact corticospinal tract axons and yields greater functional recovery after unilateral brainstem lesion in wild-type mice. Thus, the transcriptional profile of injury-induced sprouting of intact neurons reveals targets for therapeutic enhancement of axon growth initiation and new synapse formation.

Neurogenic lower urinary tract dysfunction: evaluation and management.

The lower urinary tract (LUT) in health is regulated by coordinated multi-level neurological inputs which require an intact central and peripheral nervous system. Lower urinary tract dysfunction is, therefore, a common sequelae of neurological disease and the patterns of bladder storage and voiding dysfunction depend upon the level of neurological lesion. Evaluation includes history taking, bladder diary, urological examination when relevant, ultrasonography and urodynamic testing when indicated. Antimuscarinic agents are the first line treatment for patients with storage dysfunction. Alternative treatments include intradetrusor injection of onabotulinumtoxinA, which has been shown to be of benefit in patients with neurogenic detrusor overactivity (NDO), and neuromodulation. Intermittent catheterization remains the option of choice in patients with significant voiding dysfunction resulting in high post-void residual volumes.

92. Detection and Characterization of Immune Response Against AAVrh10 Vector After Intrathecal Vector Delivery in Non-Human Primates

Immune responses against recombinant AAV (rAAV) capsid are a major concern in clinical trials. It was first described in hemophilic patients injected by intrahepatic delivery that AAV capsids elicited a cytotoxic lymphocyte response leading to clearance of transduced hepatocytes. On the other hand, intramuscular delivery of rAAV1 in humans induced a tolerogenic response against rAAV capsid mediated by regulatory T cells leading to stable expression in patients for more than 3 years. Several parameters could modulate this response and among them the mode of delivery. While transfer to the intact CNS has been shown to elicit a minimal T cell-mediated response without a salient plasma cell-mediated immune response in preclinical animal studies, these data are lacking in primate studies after intrathecal (IT) injection. In our study, African Green primates were injected intrathecally with a serotype 10 AAV vector (AAVrh10), described to transduce efficiently the central nervous system (CNS), expressing artificial miRNAs against superoxide dismutase 1 (SOD1) as a therapy for familial amyotrophic lateral sclerosis (ALS). This vector was administered at a dose of 6e12 viral genomes per kilogram modeled directly off the anticipated dose per patient in the clinical trial and based on IT rAAV delivery in primate studies showing robust cortical and spinal cord transduction. We are currently characterizing what type of immune response is elicited against the AAV capsid after IT delivery. A particular interest will be given to cytokine secretion mediated by restimulated T lymphocytes in vitro and to neutralizing antibodies directed against AAVrh10 and their potential cross-reactivity to other serotypes. In addition to the proof-of-concept of knockdown and spread of AAV vector after delivery to the CNS of primates, this study is important for the safety of IT delivery in large animal models before translation to human clinical trials.

Brain-mediated antidiabetic, anorexic, and cardiovascular actions of leptin require melanocortin-4 receptor signaling.

We previously demonstrated that leptin has powerful central nervous system (CNS)-mediated antidiabetic actions. In this study we tested the importance of melanocortin-4 receptors (MC4Rs) for leptin's ability to suppress food intake, increase blood pressure (BP) and heart rate (HR), and normalize glucose levels in insulin-dependent diabetes. MC4R knockout (MC4R-KO) and control wild-type (WT) rats were implanted with intracerebroventricular (ICV) cannula and BP and HR were measured 24 h/day by telemetry. After 5-day control period, an injection of streptozotocin (50 mg/kg, ip) was used to induce diabetes. Eight days after injection, an osmotic pump was implanted subcutaneously and connected to the ICV cannula to deliver leptin (15 μg/day) for 7 days. At baseline, MC4R-KO rats were hyperphagic and 40% heavier than WT rats. Despite obesity, BP was similar (112 ± 2 vs. 111 ± 2 mmHg) and HR was lower in MC4R-KO rats (320 ± 6 vs. 347 ± 5 beats/min). Induction of diabetes increased food intake (30%) and reduced BP (∼17 mmHg) and HR (∼61 beats/min) in WT rats, while food intake, BP, and HR were reduced by ∼10%, 7 mmHg, and 33 beats/min, respectively, in MC4R-KO rats. Leptin treatment normalized blood glucose (437 ± 10 to 136 ± 18 mg/dl), reduced food intake (40%), and increased HR (+60 beats/min) and BP (+9 mmHg) in WT rats. Only modest changes in blood glucose (367 ± 16 to 326 ± 23 mg/dl), food intake (5%), HR (+16 beats/min) and BP (+4 mmHg) were observed in MC4R-KO rats. These results indicate that intact CNS MC4R signaling is necessary for leptin to exert its chronic antidiabetic, anorexic, and cardiovascular actions.

Dynamic expression of ATF3 as a novel tool to study activation and migration of endogenous spinal stem cells and their role in neural repair.

One of the major problems of modern neurobiology is how to replace dead or damaged neurons in the human brain or spinal cord after injury or as a consequence of neurodegenerative diseases. In fact, because adult mammalian neurons are post-mitotic cells that cannot divide to replace dead cells, loss due to lesion or disease is permanent. Furthermore, surviving neurons have modest capacity to regenerate their damaged axons and re-establish functional connections. Thus, a gradual neurodegenerative scenario with certain similarities in stroke, brain or spinal cord injuries and neurological diseases like Alzheimer's disease is produced. These conditions represent the major disease burden of the modern world in terms of mortality, disability, productivity loss and health-care costs (World Health Organization, 2008). While much effort has been directed to understand the molecular and cellular mechanisms involved in the pathology of these diseases to set new effective treatments, many neuroprotective and regenerative approaches, although showing positive results in preclinical studies, have so far failed to provide strong benefit to patients.