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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1G93A familial ALS mice. Six-week-old female SOD1G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2-treated mice survived around 3% longer than vehicle-treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2-treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline-treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.

Multiple system atrophy (MSA) exhibits widespread astrogliosis together with α-synuclein (α-syn) glial cytoplasmic inclusions (GCIs) in mature oligodendrocytes. We quantified astrocyte activation by morphometric analysis of MSA cases, and investigated the correlation to GCI proximity. Using Imaris software, we obtained "skinned" three-dimensional models of GFAP-positive astrocytes in MSA and control tissue (n=75) from confocal z-stacks and measured the astrocyte process length and thickness and radial distance to the GCI. Astrocytes proximal to GCI-containing oligodendrocytes (r25μm) had significantly (p, 0.05) longer and thicker processes characteristic of activation than distal astrocytes (r25μm), with a reciprocal linear correlation (m, 90μm(2)) between mean process length and radial distance to the nearest GCI (R(2), 0.7). In primary cell culture studies, α-syn addition caused ERK-dependent activation of rat astrocytes and perinuclear α-syn inclusions in mature (MOSP-positive) rat oligodendrocytes. Activated astrocytes were also observed in close proximity to α-syn deposits in a unilateral rotenone-lesion mouse model. Moreover, unilateral injection of MSA tissue-derived α-syn into the mouse medial forebrain bundle resulted in widespread neuroinflammation in the α-syn-injected, but not sham-injected hemisphere. Taken together, our data suggests that the action of localized concentrations of α-syn may underlie both astrocyte and oligodendrocyte MSA pathological features.

Type 2 diabetes is a chronic metabolic disorder that is becoming a leading cause of morbidity and mortality. The prolonged time-course of human type 2 diabetes makes modelling of the disease difficult and additional animal models and methodologies are needed. The goal of this study was to develop and characterise a new method that allows controlled, targeted and sustained induction of discrete stages of type 2 diabetes in rodents. Using adult, male rats, we employed a three-week high fat-diet regimen and confirmed development of obesity-associated glucose intolerance, a key feature of human type 2 diabetes. Next, we utilised osmotic mini-pumps to infuse streptozotocin (STZ; doses ranging 80–200 mg/kg) over the course of 14-days to decrease insulin-producing capacity thus promoting hyperglycemia. Using this new approach, we demonstrate a dose-dependent effect of STZ on circulating glucose and insulin levels as well as glucose tolerance, while retaining a state of obesity. Importantly, we found that insulin secretion in response to a glucose load was present, but reduced in a dose-dependent manner by increasing STZ. In conclusion, we demonstrate a novel method that enables induction of discrete stages of type 2 diabetes in rodents that closely mirrors the different stages of type 2 diabetes in humans.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647–661, 2013); Hegarat et al. (J Cell Biol 195:1103–1113, 2011); Lu et al. (J Biol Chem 283:31785–31790, 2008); Trakala et al. (Cell Cycle 12:1030–1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

Burn injury can lead to abnormal sensory function at both the injury and at distant uninjured sites. Here, we used a mouse model to investigate return of nociceptive function and reinnervation of the skin at the wound and uninjured distant sites following a 3% total burn surface area full-thickness burn injury. We have previously shown that topical application of zinc-metallothionein-IIA (Zn 7 -MT-IIA) accelerates healing following burn injury, and here, we investigated the potential of Zn 7 -MT-IIA to enhance reinnervation and sensory recovery. In all burn-injured animals, there was a significant reduction in nociceptive responses (Semmes-Weinstein filaments) at locations near and distant to the wound up to 8 weeks following injury. Cutaneous nerve reinnervation (assessed using protein gene product 9.5 immunohistochemistry) of the wound center was slow in the epidermis but rapid in the dermis. In the dermis, nerves subsequently degenerated both at the wound center and in distant uninjured areas. In contrast, epidermal nerve densities in the distant uninjured areas returned to normal, uninjured levels. Zn 7 -MT-IIA did not influence return of nociceptive function nor reinnervation. We conclude that burn injury compromises nociceptive function and nerve regeneration both at the injury site and systemically; thus, therapies in addition to Zn 7 -MT-IIA should be explored to return normal sensory function.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ±1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Olfactory ensheathing cells (OECs) have been investigated extensively as a therapy to promote repair in the injured CNS, with variable efficacy in numerous studies over the previous decade. In many studies that report anatomical and functional recovery, the beneficial effects have been attributed to the ability of OECs to cross the PNS-CNS boundary, their production of growth factors, cell adhesion molecules and extracellular matrix proteins that promote and guide axon growth, and their ability to remyelinate axons. In this brief review, we focus on the interaction between OECs and astrocytes in vivo and in vitro, in the context of how OECs may be overcoming the deleterious effects of the glial scar. Drawing from a selection of different experimental models of spinal injury, we discuss the morphological alterations of the glial scar associated with OEC transplants, and the in vitro research that has begun to elucidate the interaction between OECs and the cell types that compose the glial scar. We also discuss recent research showing that OECs bear properties of immune cells and the consequent implication that they may modulate neuroinflammation when transplanted into CNS injury sites. Future studies in unraveling the molecular interaction between OECs and other glial cells may help explain some of the variability in outcomes when OECs are used as transplants in CNS injury and more importantly, contribute to the optimization of OECs as a cell-based therapy for CNS injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Neuronal growth-inhibitory factor, later renamed metallothionein-3, is one of four members of the mammalian metallothionein family. Metallothioneins are a family of ubiquitous, low-molecular-weight, cysteine-rich proteins. Although neuronal growth-inhibitory factor shares metal-binding and reactive oxygen species scavenging properties with the other metallothioneins, it displays several distinct biological properties. In this review, we examine the recent developments regarding the function of neuronal growth-inhibitory factor within the brain, particularly in response to brain injury or during neurodegenerative disease progression.

Olfactory nerves extend from the nasal cavity to the central nervous system and provide therefore, a direct route for pathogenic infection of the brain. Since actual infection by this route remains relatively uncommon, powerful endogenous mechanisms for preventing microbial infection must exist, but these remain poorly understood. Our previous studies unexpectedly revealed that the unique glial cells that ensheath olfactory nerves, olfactory ensheathing cells (OECs), expressed components of the innate immune response. In this study, we show that OECs are able to detect and respond to bacterial challenge via the synthesis of nitric oxide. In vitro studies revealed that inducible nitric oxide synthase (iNOS) mRNA and protein were present in Escherichia coli- and Staphylococcus aureus-incubated OECs, but were barely detectable in untreated OECs. Neuronal NOS and endothelial NOS were not expressed by OECs pre- and post-bacterial incubation. Nuclear translocation of nuclear factor kappa B (NFkappaB), detectable in the majority of OECs 1 h following bacterial incubation, preceded iNOS induction which resulted in the production of nitric oxide. N(G)-methyl-L-arginine significantly attenuated nitric oxide (P < 0.001) and nitrite production (P < 0.001) by OECs. In rat olfactory mucosa which was compromised by irrigation with 0.17M zinc sulfate or 0.7% Triton X-100 to facilitate bacterial infiltration, OECs contributed to a robust synthesis of iNOS. These data strongly support the hypothesis that OECs are an essential component of the innate immune response against bacterial invasion of the central nervous system via olfactory nerves.

The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

Severe injury to the epidermal barrier often results in scarring and life-long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn7-MT-IIA), a naturally occurring small cysteine-rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT-I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT-I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn7-MT-IIA significantly increased cell viability by up to 30% (p 0.05). Topical administration of exogenous Zn7-MT-IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9±0.3 mm diameter vs. MT-I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT-I/II, 3.6±1.0 mm, p

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate fifty years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Juan Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His group’s work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition. These topics are addressed in the second and third sections of this review by Dr Adrian West, and Drs Edward Levin and Donnie Eddins, respectively. Finally, Dr Michael Aschner examines the ability of MT to protect against a specific toxicant, methymercury, in the CNS.

Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.

This study examined the development of several physiological parameters that enable the development of endothermy during pouch life of the Tasmanian bettong (Bettongia gaimardi). By using several key age groups, we investigated the electromyography response of pouch young under different thermal conditions (35 or 20 1C), with and without injection of the b-agonist norepinephrine. We also used molecular techniques to examine the possible expression of uncoupling proteins 1, 2, and 3 (UCP1, 2, and 3), and if they were expressed, any association these may have with the timing of endothermic development. AT 6 weeks of age, pouch young were unable to thermoregulate via shivering or non-shivering mean, maintain a constant metabolic rate, or show any response to the b-agonist injection. When the animals were exposed to a cold stress (20 1C), 8-week-old pouch young initially shivered for 2–3 min before tapering off completely, causing body temperature to sharply decline. During the 10th week, cold-exposed pouch young began shivering, with the bout lasting approximately 10 min before ceasing and body temperature beginning to decline. It was also at this age that the expression of UCP2 was initially expressed. By the 12th week, cold exposed pouch young initially employed classical shivering; however, after approximately 12 min, this was replaced with an increase in muscular tone. This increase in muscular tone was also recorded in response to b-agonist injection at thermoneutral this age and was associated with an increase in metabolic rate. Also by this age, total body fat increased by approximately 300% from the levels expressed at 6 weeks of age and UCP2 was significantly upregulated. Although pouch young B. gaimardi did not show UCP1 expression at any time, UCP3 was expressed at every age investigated from 6 weeks of age. This study proposes that pouch young B. gaimardi use a mechanism of increased muscle tone as a source of heat production, and shows that UCP2 has an association with the onset of thermogenesis.

Chronic oxidative stress has been linked to the neurodegenerative changes characteristic of Parkinson's disease, particularly alpha-synuclein accumulation and aggregation. However, it remains contentious whether these alpha-synuclein changes are cytotoxic or neuroprotective. The current study utilised long-term primary neural culture techniques with antioxidant free media to study the cellular response to chronic oxidative stress. Cells maintained in antioxidant free media were exquisitely more vulnerable to acute exposure to hydrogen peroxide, yet exposure of up to 10 days in antioxidant free media did not lead to morphological alterations in neurones or glia. However, a subpopulation of neurones demonstrated a significant increase in the level of alpha-synuclein expressed within the cell body and at synaptic sites. This subset of neurones was also more resistant to apoptotic changes following exposure to antioxidant free media relative to other neurones. These data indicate that increased alpha-synuclein content is associated with neuroprotection from relatively low levels of oxidative stress.

Apoptotic-like changes in the neocortex of control, pathologically aged and Alzheimer's disease (AD) cases were investigated. There was no increase in labeling or change in localization of labeling that distinguished between these cases for active caspase-3, -8, -9, Bax, Bcl-2 or TRADD. Bax, Bcl-2 and TRADD mRNA levels also differed little between case types, although there were small but significant decreases in Bax mRNA levels in AD compared to control cases and Bcl-2 mRNA in AD cases compared to pathologically aged and control cases. There was no difference in the percentage of apoptotic-like nuclei between these cases, except for a small but significant decrease in the inferior temporal gyrus of AD cases relative to controls. Nuclei observed within or adjacent to beta-amyloid plaques were rarely abnormal, and neurons bearing neurofibrillary tangles (NFTs) did not have abnormal nuclei. Apoptosis may not play a major role in the pathogenesis of neuronal degeneration of AD.

This study investigates proliferation and apoptosis of olfactory ensheathing cells in cocultures with spinal cord tissue. Proliferation of ensheathing cells was significantly increased when cocultured with explants from uninjured spinal cord, and spinal cord that had been subjected to chronic contusion or chronic needle stab injury, but not to acute needle stab injury. Proliferation rate was highest in cocultures with chronically stabbed cord tissue. Contaminating (p75NGFR-negative) cells in the cultures showed a significantly higher proliferation rate than ensheathing cells. Apoptosis of ensheathing cells was significantly increased in cocultures with acutely stabbed spinal cord explants compared with chronically contused spinal cord explants. These results suggest that delaying transplantation after spinal cord injury may be beneficial to ensheathing cell survival.

This study investigated the role of alpha-internexin in the neuronal alterations associated with beta-amyloid plaque formation in Alzheimer's disease (AD). Cortical neurons could be defined by their variable content of neurofilament (NF) triplet and alpha-internexin proteins, with a distinct population of supragranular pyramidal cells containing alpha-internexin alone. Both NF triplet and alpha-internexin were localized to reactive axonal structures in physically damaged neurons in experimental trauma models. Similarly, NF triplet and alpha-internexin immunoreactive neurites were localized to plaques densely packed with beta-amyloid fibrils in preclinical AD cases, indicating that certain plaques may cause structural injury or impediment of local axonal transport. However, alpha-internexin, and not NF triplet, ring-like reactive neurites were present in end-stage AD cases, indicating the relatively late involvement of neurons that selectively contain alpha-internexin. These results implicate the expression of specific intermediate filament proteins in a distinct hierarchy of differential neuronal vulnerability to AD.

In recent years metallothionein (MT) biology has moved from investigation of its ability to protect against environmental heavy metals to a wider appreciation of its role in responding to cellular stress, whether as a consequence of normal function, or following injury and disease. This is exemplified by recent investigation of MT in the mammalian brain where plausible roles for MT action have been described, including zinc metabolism, free radical scavenging, and protection and regeneration following neurological injury. Along with other laboratories we have used several models of central nervous system (CNS) injury to investigate possible parallels between injury-dependent changes in MT expression and those observed in the ageing and/or degenerating brain. Therefore, this brief review aims to summarise existing information on MT expression during CNS ageing, and to examine the possible involvement of this protein in the course of human neurodegenerative disease, as exemplified by Alzheimer's disease.

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

Olfactory ensheathing cells (OECs), also known as olfactory neuroglia, are widely accepted to play a role in the ontogeny of the primary olfactory pathway. Their expression of numerous growth factors and extracellular matrix molecules is consistent with a role in guiding elongating olfactory axons from the nasal cavity to their termination in the olfactory bulb. In recent years, researchers have attempted to exploit these phenotypic features by utilizing these cells as a therapeutic agent to induce repair of the injured nervous system. However, their strategic placement along olfactory nerves, spanning the region between the external nasal cavity and the olfactory bulb (part of the central nervous system), and their expression profile of immunomodulatory proteins support a possible involvement in the protection of the brain against infection. This commentary will briefly outline the relationship between OECs and developing olfactory nerves, and recent experimental evidence that suggests a remarkable functional plasticity of OECs including a possible active role in the immune system.

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T(8-9) and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P=0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs (P=0.07).

Recent data suggests that metallothioneins (MTs) are major neuroprotective proteins within the CNS. In this regard, we have recently demonstrated that MT-IIA (the major human MT-I/-II isoform) promotes neural recovery following focal cortical brain injury. To further investigate the role of MTs in cortical brain injury, MT-I/-II expression was examined in several different experimental models of cortical neuron injury. While MT-I/-II immunoreactivity was not detectable in the uninjured rat neocortex, by 4 days, following a focal cortical brain injury, MT-I/-II was found in astrocytes aligned along the injury site. At latter time points, astrocytes, at a distance up to several hundred microns from the original injury tract, were MT-I/-II immunoreactive. Induced MT-I/-II was found both within the cell body and processes. Using a cortical neuron/astrocyte co-culture model, we observed a similar MT-I/-II response following in vitro injury. Intriguingly, scratch wound injury in pure astrocyte cultures resulted in no change in MT-I/-II expression. This suggests that MT induction was specifically elicited by neuronal injury. Based upon recent reports indicating that MT-I/-II are major neuroprotective proteins within the brain, our results provide further evidence that MT-I/-II plays an important role in the cellular response to neuronal injury.

Although olfactory ensheathing cells (OECs) are used to promote repair in the injured spinal cord, little is known of their phenotype in this environment. In this study, using quantitative reverse transcriptase-polymerase chain reaction RT-PCR, expression of neuregulin-1 mitogen/survival factors and the axonal growth regulator Nogo was quantified in OECs and compared with other non-neuronal cells. Their expression was also compared with OECs which had previously been encapsulated in a porous polymer tube and implanted into the injured spinal cord. Similar to astrocytes and fibroblasts, OECs expressed various neuregulin subtypes including neu differentiation factor, glial growth factor and sensory and motorneuron-derived factor. Implanted OECs upregulated neu differentiation factor and secreted neuregulin, but downregulated expression of all other variants. OECs and oligodendrocytes expressed Nogo-A, -B and -ABC and were immunopositive for Nogo-A protein. The Nogo-A protein in OECs was found to be cytoplasmic rather than nuclear or cell surface associated. Unlike oligodendrocytes, OECs expressed Nogo-66 receptor (NgR) mRNA. Implanted OECs upregulated Nogo-A and -B, but downregulated Nogo-ABC and NgR.

The presence of, and the physiological mechanisms behind, non-shivering thermogenesis (NST) were investigated in the Tasmanian Devil (Sarcophilus harrisii, Boitard 1841). This was performed by measuring metabolic rate in response to cold exposure with and without an injection of norepinephrine. We also attempted to identify the presence of uncoupling protein 1 (UCP1) and its involvement (if any) in NST in S. harrisii, prior to and after cold exposure. UCP1 is a specialized protein channel found within the mitochondria of brown adipose tissue that is known to be involved in eutherian NST. Increase in resting oxygen consumption (VO2), were measured under constant conditions at 20°C after various stimuli; long-term cold exposure (14 days at 2–3°C), and with or without subsequent norepinephrine injection was used to indicate the presence of NST. Concurrent with the functional studies, molecular level studies including Western blots and RT-PCR were used to identify the expression of UCP1. We found that injection of norepinephrine significantly increased NST after cold exposure in S. harrisii. The post-acclimation resting (VO2) increased by 11%, whereas the combination of cold exposure and injection of NE elicited an approximately 30% increase in metabolic rate. However, expression of UCP1 in S. harrisii was not identified by the molecular techniques employed, in either the pre- or post-cold-acclimated tissues. These data suggest that S. harrisii shows NST ability and that it is accomplished in the absence of UCP1.

Olfactory ensheathing cells (ECs) are a promising tool for the repair of injury in the adult central nervous system. However, important aspects of the cell biology of ECs remain unclear, such as whether ECs exist as a single population or as two subpopulations with Schwann cell-like and astrocyte-like characteristics. The morphologies of these subpopulations are used as defining characteristics, yet ECs are known to be morphologically plastic. To elucidate this apparent inconsistency, we investigated the morphological plasticity of ECs in culture. We defined purified ECs as immunopositive for both p75 neurotrophin receptor and glial fibrillary acidic protein. In MEM (D)-valine modification + 10% dialyzed fetal calf serum, 87%-90% of ECs displayed a flat morphology. In three different serum-free media (N2 medium, neurobasal medium + B27 supplement, and DMEM/F-12 medium + G5 supplement), 78%-84% of ECs displayed process-bearing morphology. Ensheathing cells switched reversibly between these morphologies within a day of the serum conditions being changed. Exposure to 1 nM endothelin-1 in serum-free medium prevented the switch from flat to process-bearing morphology, while 1 mM dibutyryl cAMP accelerated this change. The effects of both agents were completely reversible and similar to that reported for astrocytes. Both flat and process-bearing ECs were immunopositive for brain-derived neurotrophic factor, nerve growth factor, neurotrophin-4, and TrkB but not TrkA. Together, these results suggest that ECs exist as a single morphologically plastic population.

An explant culture system was used to test the effect of nerve growth factor (NGF) on olfactory axonal elongation. Statistical analysis showed that exogenously applied NGF (50 ng/ml) significantly enhanced olfactory neurite elongation from E14 rat olfactory epithelial explants (p = 0.025). Immunostaining showed that the neurites expressed active TrkA receptors and that S-100-positive ensheathing cells were also present. In a separate experiment, immunoassay confirmed that following a growth period of 72 h, E14 presumptive olfactory bulb expressed and secreted NGF into the culture medium. The results indicate that during ontogeny, the olfactory bulb secretes NGF which binds to olfactory axons and facilitates their elongation.

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression. Methods We examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1G93A) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1G93A and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene’s test showed that the variance in WT and SOD1G93A measurements was substantially different. Results Disease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1G93A mice. The ventral horn of the SOD1G93A spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1G93A mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1G93A mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1G93A mice, suggesting that Arg1 may have a neuroprotective function. Conclusions This study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.

Metallothionein (MT) is thought to have an antioxidant function and is strongly expressed during activation of thermogenesis and increased oxidative stress in brown adipose tissue (BAT). Localization and regulation of MT expression in BAT was therefore investigated in rats and mice. Immunohistochemical analysis of BAT from rats exposed to 4°C for 24 h showed that MT and uncoupling protein 1 (UCP1) were coexpressed in differentiated adipocytes, and both cytoplasmic and nuclear localization of MT was observed. Cold induction of MT-1 expression in BAT was also observed in mice. Administration of norepinephrine to rats and isoproterenol to mice stimulated MT and UCP1 expression in BAT, implying a sympathetically mediated pathway for MT induction. In mice, zinc, and particularly dexamethasone, induced MT-2 expression in BAT and liver. Surprisingly, zinc also induced UCP1 in BAT, suggesting that elevated zinc may induce thermogenesis. We conclude that expression of MT in mature brown adipocytes upon β-adrenoceptor activation is consistent with a role in protecting against physiological oxidative stress or in facilitating the mobilization or utilization of energy reserves.

The Tasmanian bettong (Bettongia gaimardi, a marsupial) is a rat-kangaroo that increases nonshivering thermogenesis (NST) in response to norepinephrine (NE). This study attempted to assess whether brown adipose tissue (BAT), a specialized thermogenic effector, is involved in NST in the bettong. Regulatory NST, indicated by resting oxygen consumption (Vo2) of the whole body, was measured under conscious conditions at 20 degrees C with various stimuli: cold (4 degrees -5 degrees C) or warm (25 degrees C) acclimation, NE injection, and the beta3-adrenoceptor agonist (BRL) 37344. In line with the functional studies in vivo, the presence of BAT was evaluated by examining the expression of the uncoupling protein 1 (UCP1) with both rat cDNA and oligonucleotide probes. Both NE and BRL 37344 significantly stimulated NST in the bettong. After cold acclimation of the animals (at 4 degrees -5 degrees C for 2 wk), the resting Vo2 was increased by 15% and the thermogenic effect of NE was enhanced; warm-acclimated animals showed a slightly depressed response. However, no expression of UCP1 was detected in bettongs either before or after cold exposure (2 wk). These data suggest that the observed NST in the marsupial bettong is not attributable to BAT.

Metallothioneins are small cysteine-rich proteins that bind heavy metals. In higher mammals there are complex families of metallothionein isoforms, which are well characterised at the DNA level but less so in terms of their cellular expression and function. In particular, little is known about the localisation of metallothionein in the developing mammalian brain. In this study, using sheep fetuses, we have shown that metallothionein 1 and 2 isoform expression undergoes shifts in regional and cellular localisation during development of the brain. Metallothionein l and 2 expression is first detected by embryonic days E72–E73 (gestation is 150 days) at the mRNA level and the metallothionein protein is observed in cells of the proliferating ventricular zones. Subsequent expression is detected in radial glial cells, oligodendrocytes and astrocytes in several regions of the brain, most notably the cerebral cortex. In the adult brain, metallothionein is expressed in astrocytes but not in oligodendrocytes. Double-labelling immunohistochemistry using the glial fibrillary acidic protein (GFAP), an astrocyte marker, and metallothionein revealed that although there is an overlap in the profiles of the two proteins, there is no simple correlation in their expression. These observations are consistent with metallothionein, under physiological conditions, being regulated mainly by intracellular factors.

We have examined the expression of the MT1 and MT2 isoforms of metallothionein in the mouse olfactory mucosa. In untreated mice, metallothionein was strongly expressed in supporting cells, acinar cells of the Bowman's glands, and olfactory neurons. Expression was however restricted to a subset of cells within each type, and to zones within the olfactory system. Irrigation with ZnSO4 solution caused exfoliation of the olfactory epithelium and during the resultant regeneration, metallothionein immunoreactivity was associated with the proliferating basal cells. The ability to express MTs 1 and 2 did not appear to be obligatory for the early stages of regeneration since mice which do not express these isoforms responded similarly to wild type mice. Strong nuclear expression of metallothionein was noted in the untreated olfactory chamber following unilateral irrigation.

Exposure to cadmium (Cd) is currently monitored by measurement of the metal in blood or urine, or by observation of excreted compounds such as beta 2-microglobulin or N-acetyl-beta-D-glucose. Whilst these approaches are useful for the detection of acute exposure to Cd, their applicability in the management of long-term, low-level exposure is less clear. Metallothioneins are ubiquitous proteins that are synthesized in response to heavy metal ions and may offer themselves as being a biologically sensitive indicator of Cd exposure. We have examined both basal and Cd-induced metallothionein mRNA levels in cultured lymphocytes from groups with different exposures to Cd, attempting to assess their potential as an indicator of Cd exposure and the suitability of such an assay for routine analysis. We found that induced metallothionein mRNA levels, rather than basal mRNA levels, increased in groups known to have received elevated body burdens of Cd, although these increases were not significant between groups. There was, however, a significant correlation between induced metallothionein mRNA levels and urinary beta 2-microglobulin. These results suggest that further work on the in vitro lymphocyte response to Cd as a diagnostic tool is warranted.

Background: The beta-amyloid (Aβ) peptide comprises the amyloid plaques that characterise Alzheimer's disease (AD), and is thought to significantly contribute towards disease pathogenesis. Oxidative stress is elevated in the AD brain, and there is substantial evidence that the interaction between Aβ and redox-active copper is a major contributing factor towards oxidative stress in AD. Results: The major findings of this study are that redox-active Cu(II)–Aβ causes pronounced axonal pathology in long-term neuronal cultures, including axonal fragmentation and the formation of hyperphosphorylated tau-immunoreactive axonal swellings. Notably, MAP-2 expressing dendritic processes remain largely un-affected by Cu(II)–Aβ treatment. These dystrophic axonal manifestations resemble some of the characteristic neuritic pathology of the AD brain. We show that Cu(II)–Aβ directly causes formation of intra-axonal swellings via the generation of free radicals and subsequent efflux of K + out of neurons. Conclusion: In summary, we report that redox-active Cu(II)–Aβ can induce substantial neurodegenerative changes in mature neurons, and may have an important role to play in the slowly progressing pathogenesis of AD.

There are currently few clinical strategies in place, which provide effective neuroprotection and repair, despite an intense international effort over the past decades. One possible explanation for this is that a deeper understanding is required of how endogenous mechanisms act to confer neuroprotection. This mini-review reports the proceedings of a recent workshop >Neuroprotection and Neurorepair: New Strategies> (Iguazu Falls, Misiones, Argentina, April 11-13, 2011, Satellite Symposium of the V Neurotoxicity Society Meeting, 2011) in which four areas of active research were identified to have the potential to generate new insights into this field. Topics discussed were (i) metallothionein and other multipotent neuroprotective molecules; (ii) oxidative stress and their signal mediated pathways in neuroregeneration; (iii) neurotoxins in glial cells, and (iv) drugs of abuse with neuroprotective effects. © 2011 Springer Science+Business Media, LLC.

We have investigated the expression of the protooncogene c- myc in rat hearts following exposure to norepinephrine, both in vivo and in isolated perfused preparations. Both chronic and acute norepinephrine treatment produced a rapid, transient elevation of c- myc mRNA in adult rat hearts, but chronic infusion produced a second, larger increase. This expression profile was characteristic for c- myc since it was not found for four other protooncogenes. In the isolated, perfused heart, addition of norepinephrine to the perfusion buffer and elevation of perfusion pressure separately increase c- myc mRNA suggesting both direct hormonal and hemodynamic factors might be important in vivo. Immunocytochemistry showed that Myc protein accumulated predominately in the nuclei of non-myocyte cells following norepinephrine treatment indicating that expression at the mRNA level culminated in protein synthesis. These findings suggest that the c- myc expression observed in the hypertrophying adult heart following exposure to norepinephrine may be associated with proliferating cells like fibroblasts rather than cardiomyocytes.

Background Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system. Methods Wild type and MT-I/II-/- mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay. Results Comparison of MT-I/II-/- mice with wild type controls following cryolesion brain injury revealed that the MT-I/II-/- mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II-/- mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II-/- mice that was independent of brain injury. Conclusions These results contribute to the evidence that MT-I/II-/- mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II-/- mice after brain injury relative to wild type mice.

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.

BackgroundOne of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time.ResultsIn this study, we report that daily exposure to a sublethal concentration of Aβ(1-40) (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ(1-40) treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K(+) homeostasis following Aβ treatment. Furthermore, blocking K(+) efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ(1-40) caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions.ConclusionsOur data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K(+)-dependent neurodegeneration that has pathological characteristics similar to AD.

Metallothionein has a well-documented protective and proregenerative effect in the mammalian brain, particularly following physical trauma and ischemia or during the onset of neurodegenerative disease. A range of mechanisms have been established for this, including metallothionein’s metal binding properties and its ability to scavenge free radicals. In recent years it has become apparent that metallothionein is present in the extracellular compartment of the central nervous system and that it can interact with cell surface receptors of the lipoprotein-receptor-related protein family, including lipoprotein-receptor-related protein 1 (LRP1) and megalin. These interactions activate intracellular pathways which are consistent with many of the observed effects of metallothionein in the central nervous system, including its effects on neurons, glial cells, and cells of the immune system. The evidence describing the release, receptor interactions, and subsequent physiological consequences of metallothionein is discussed in this review.

We have investigated the cellular and regional localization of Fos-like immunoreactivity (FLI) in the rat heart in response to the hypertrophic hormone norepinephrine (NE). Previous studies have demonstrated elevated c-fos mRNA levels in the rat heart following this treatment but have not shown which cell type(s) or specific chamber(s) of the heart contribute to the response in vivo, or whether Fos protein is actually produced. Administration of a single injection of NE (2.5 mg/kg) or chronic infusion of NE (100 micrograms/kg/h) led to an increase in Fos-like immunoreactivity in the nuclei of cardiac myocytes and vascular smooth muscle cells compared to control tissue. The response was transient with maximal immunoreactivity observed 2-3h following treatment, falling to near basal levels in most regions of the heart after 6 h. Although all chambers of the heart contributed to the response, greatest Fos-like immunoreactivity was observed in the left atrium and left ventricle, with intermediate levels found in the septum and right ventricle, and lowest levels in the right atrium. Fos-like immunoreactivity observed in the left atrium was accompanied by elevated mRNA levels of fra-1 and fra-2 but not c-fos itself indicating that a related gene product other than Fos contributed to the observed response. Experiments with the Langendorff perfused rat heart showed that NE and elevated perfusion pressure independently increased both c-fos mRNA and FLI. This work is the first evidence for a direct action of NE on Fos expression in adult, as opposed to neonatal, cardiomyocytes. These results lend further support to the notion that Fos and related gene products mediate some of the hypertrophic actions of norepinephrine.

Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p

Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 micrograms/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2 h. The response of both cellular oncogenes to norepinephrine was reduced significantly by alpha blockade but beta blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2 x 10(-7) M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.