Review: Spreading the word: precise animal models and validated methods are vital when evaluating prion-like behaviour of alpha-synuclein.

Synucleinopathies are characterized by abnormal proteinaceous aggregates, mainly composed of fibrillar α-synuclein (α-syn). It is now believed that α-syn can form small aggregates in a restricted number of cells, that propagate to neighbouring cells and seed aggregation of endogenous α-syn, in a 'prion-like manner'. This process could underlie the stereotypical progression of Lewy bodies described by Braak and colleagues across different stages of Parkinson's disease (PD). This prion-like behaviour of α-syn has been recently investigated in animal models of PD or multiple system atrophy (MSA). These models investigate the cell-to-cell transfer of α-syn seeds, or the induction and spreading of α-syn pathology in transgenic or wild-type rodent brain. In this review, we first outline the involvement of α-syn in Lewy body diseases and MSA, and discuss how 'prion-like' mechanisms can contribute to disease. Thereon, we debate the relevance of animal models used to study prion-like propagation. Finally, we review current main histological methods used to assess α-syn pathology both in animal models and in human samples and their relevance to the disease. Specifically, we discuss using α-syn phosphorylated at serine 129 as a marker of pathology, and the novel methods available that allow for more sensitive detection of early pathology, which has relevance for modelling synucleinopathies.

Adipocytes derived from PA6 cells reliably promote the differentiation of dopaminergic neurons from human embryonic stem cells.

The PA6 stromal cell line comprises a heterogeneous population of cells that can induce both mouse and human embryonic stem cells to differentiate into dopaminergic neurons. This ability of PA6 cells has been termed stromal cell-derived inducing activity (SDIA). The level of SDIA has been found to vary considerably between and within batches of PA6 cells. Not only are the molecular mechanisms that underlie SDIA unknown but also the cell type(s) within the heterogeneous PA6 cultures that underlie SDIA remain poorly defined. In this study, we reveal that adipocytes, which are present within the heterogeneous PA6 cell population, robustly release the factors mediating SDIA. Furthermore, we report that the coculture of human embryonic stem cells with PA6-derived adipocytes reliably induces their differentiation into midbrain dopaminergic neurons.

The future of cell therapies and brain repair: Parkinson's disease leads the way.

During the past 40 years brain tissue grafting techniques have been used both to study fundamental neurobiological questions and to treat neurological diseases. Motor symptoms of Parkinson's disease are largely due to degeneration of midbrain dopamine neurones. Because the nigrostriatal pathology is relatively focused anatomically, Parkinson's disease is considered the ideal candidate for brain repair by neural grafting and dopamine neurone transplantation for it has led the way in the neural transplantation research field. In this mini-review, we briefly highlight four important areas of development. First, we describe marked functional benefits up to 18 years after transplantation surgery in patients with Parkinson's disease. This is proof-of-principle that, using optimal techniques and patient selection, grafted dopamine neurones can work in humans and the duration of the benefit exceeds placebo effects associated with surgery. Second, we describe that eventually protein aggregates containing α-synuclein, identical to Lewy bodies, develop inside foetal dopamine neurones transplanted to patients with Parkinson's disease. This gives clues about pathogenetic mechanisms operating in Parkinson's disease, and also raises the question whether neural graft function will eventually decline as the result of the disease process. Third, we describe new emerging sources of transplantable dopamine neurones derived from pluripotent stem cells or reprogrammed adult somatic cells. Fourth, we highlight an important European Union-funded multicentre clinical trial involving transplantation of foetal dopamine neurones in Parkinson's disease. We describe the design of this ongoing trial and how it can impact on the overall future of cell therapy in Parkinson's disease.

Caspase inhibition reduces apoptosis and increases survival of nigral transplants.

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson disease. The main practical constraints of neural grafting are the shortage of human donor tissue and the poor survival of dopaminergic neurons grafted into patients, which is estimated at 5-10% (refs. 3,4). The required amount of human tissue could be considerably reduced if the neuronal survival was augmented. Studies in rats indicate that most implanted embryonic neurons die within 1 week of transplantation, and that most of this cell death is apoptotic. Modified peptides, such as acetyl-tyrosinyl-valyl-alanyl-aspartyl-chloro-methylketone (Ac-YVAD-cmk), that specifically inhibit proteases of the caspase family effectively block apoptosis in a plethora of experimental paradigms, such as growth factor withdrawal, excitotoxicity, axotomy, cerebral ischemia and brain trauma. Here we examined the effects of caspase inhibition by Ac-YVAD-cmk on cell death immediately after donor tissue preparation and on long-term graft survival. Treatment of the embryonic nigral cell suspension with Ac-YVAD-cmk mitigated DNA fragmentation and reduced apoptosis in transplants. It also increased survival of dopaminergic neurons grafted to hemiparkinsonian rats, and thereby substantially improved functional recovery.

Overexpressing Cu/Zn superoxide dismutase enhances survival of transplanted neurons in a rat model of Parkinson's disease.

A high survival rate of grafted dopamine neurons is crucial for reversing neurological deficits following brain tissue transplantation in Parkinson's disease. For unknown reasons the survival rate of transplanted dopamine neurons is only around 10% in experimental animals. The hypothesis that oxidative stress causes the loss of transplanted neurons was tested by grafting neurons from transgenic mice that overexpress Cu/Zn superoxide dismutase. Compared with the survival of those taken from non-transgenic littermates, the survival was 4 times higher for the transgenic dopamine neurons with a concomitant more extensive functional recovery. The results provide direct support for the free radical hypothesis of dopaminergic neuron death in brain tissue grafting.

Mutant huntingtin can paradoxically protect neurons from death.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a mutation in the gene huntingtin and characterized by motor, cognitive and psychiatric symptoms. Huntingtin contains a CAG repeat in exon 1. An expansion of this CAG repeat above 35 results in misfolding of Huntingtin, giving rise to protein aggregates and neuronal cell death. There are several transgenic HD mouse models that reproduce most of the features of the human disorder, for example protein inclusions, some neurodegeneration as well as motor and cognitive symptoms. At the same time, a subgroup of the HD transgenic mouse models exhibit dramatically reduced susceptibility to excitotoxicity. The mechanism behind this is unknown. Here, we review the literature regarding this phenomenon, attempt to explain what protein domains are crucial for this phenomenon and point toward a putative mechanism. We suggest, that the C-terminal domain of exon 1 Huntingtin, namely the proline rich domain, is responsible for mediating a neuroprotective effect against excitotoxicity. Furthermore, we point out the possible importance of this mechanism for future therapies in neurological disorders that have been suggested to be associated with excitotoxicity, for example Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Grafts of fetal dopamine neurons survive and improve motor function in Parkinson's disease.

Neural transplantation can restore striatal dopaminergic neurotransmission in animal models of Parkinson's disease. It has now been shown that mesencephalic dopamine neurons, obtained from human fetuses of 8 to 9 weeks gestational age, can survive in the human brain and produce marked and sustained symptomatic relief in a patient severely affected with idiopathic Parkinson's disease. The grafts, which were implanted unilaterally into the putamen by stereotactic surgery, restored dopamine synthesis and storage in the grafted area, as assessed by positron emission tomography with 6-L-[18F]fluorodopa. This neurochemical change was accompanied by a therapeutically significant reduction in the patient's severe rigidity and bradykinesia and a marked diminuation of the fluctuations in the patient's condition during optimum medication (the "on-off" phenomenon). The clinical improvement was most marked on the side contralateral to the transplant.

In vivo release of dopa and dopamine from genetically engineered cells grafted to the denervated rat striatum.

Fibroblastic 3T3 and endocrine RIN cells were genetically modified by infection with a recombinant retrovirus encoding the form I of human tyrosine hydroxylase (TH) and selection in tyrosine-free medium. These cells were grafted to rats unilaterally lesioned with 6-hydroxy-dopamine. Both cell types survived implantation into the striatum, expressed TH immunoreactivity, and as assessed by microdialysis 8-9 days after implantation, secreted high amounts of DOPA and/or dopamine into the surrounding host striatum. The modified 3T3 cells secreted large amounts of DOPA that was efficiently decarboxylated to dopamine by the host striatal tissue; the newly synthesized dopamine was stored only to a limited extent in the denervated striatum. The modified RIN cells synthesized dopamine that was stored intracellularly and released in a regulated fashion. The grafted DOPA-secreting cells produced 4-5 times higher extracellular dopamine levels than the dopamine-secreting cells, and they were more efficient in reducing apomorphine-induced rotation. No effect was observed with either cell type on amphetamine-induced turning behavior.

Neuroinflammation in the generation of post-transplantation dyskinesia in Parkinson's disease.

The observation that neural grafts can induce dyskinesias has severely hindered the development of a transplantation therapy for Parkinson's disease (PD). We addressed the hypothesis that inflammatory responses within and around an intrastriatal graft containing dopamine neurons can trigger dyskinetic behaviors. We subjected rats to unilateral nigrostriatal lesions with 6-hydroxydopamine (6-OHDA) and treated them with L-DOPA for 21 days in order to induce abnormal involuntary movements (AIMs). Subsequently, we grafted the rats with allogeneic embryonic ventral mesencephalic tissue in the dopamine-denervated striatum. In agreement with earlier studies, the grafted rats developed dyskinesia-like AIMs in response to amphetamine. We then used two experimental approaches to induce an inflammatory response and examined if the amphetamine-induced AIMs worsened or if spontaneous AIMs developed. In one experiment, we challenged the neural graft hosts immunologically with an orthotopic skin allograft of the same genetic origin as the intracerebral neural allograft. In another experiment, we infused the pro-inflammatory cytokine interleukin 2 (IL-2) adjacent to the intrastriatal grafts using osmotic minipumps. The skin allograft induced rapid rejection of the mesencephalic allografts, leading to disappearance of the amphetamine-induced AIMs. Contrary to our hypothesis, the rejection process itself did not elicit AIMs. Likewise, the IL-2 infusion did not induce spontaneous AIMs, nor did it alter L-DOPA-induced AIMs. The IL-2 infusions did, however, elicit the predicted marked striatal inflammation, as evidenced by the presence of activated microglia and IL2Ralpha-positive cells. These results indicate that an inflammatory response in and around grafted dopaminergic neurons is not sufficient to evoke dyskinetic behaviors in experimental models of PD.

Dopamine release from nigral transplants visualized in vivo in a Parkinson's patient.

Synaptic dopamine release from embryonic nigral transplants has been monitored in the striatum of a patient with Parkinson's disease using [11C]-raclopride positron emission tomography to measure dopamine D2 receptor occupancy by the endogenous transmitter. In this patient, who had received a transplant in the right putamen 10 years earlier, grafts had restored both basal and drug-induced dopamine release to normal levels. This was associated with sustained, marked clinical benefit and normalized levels of dopamine storage in the grafted putamen. Despite an ongoing disease process, grafted neurons can thus continue for a decade to store and release dopamine and give rise to substantial symptomatic relief.

Working memory training decreases hippocampal neurogenesis.

The relationship between adult hippocampal neurogenesis and cognition appears more complex than suggested by early reports. We aimed to determine if the duration and task demands of spatial memory training differentially affect hippocampal neurogenesis. Adult male rats were trained in the Morris water maze in a reference memory task for 4 days, or alternatively working memory for either 4 or 14 days. Four days of maze training did not impact neurogenesis regardless of whether reference or working memory paradigms were used. Interestingly, 2 weeks of working memory training using a hidden platform resulted in fewer newborn hippocampal neurons compared with controls that received either cue training or no maze exposure. Stress is a well-established negative regulator of hippocampal neurogenesis. We found that maze training in general, and a working memory task in particular, increased levels of circulating corticosterone after 4 days of training. Our study indicates that working memory training over a prolonged period of time reduces neurogenesis, and this reduction may partially be mediated by increased stress.

High-fat diet impairs hippocampal neurogenesis in male rats.

High fat diets and obesity pose serious health problems, such as type II diabetes and cardiovascular disease. Impaired cognitive function is also associated with high fat intake. In this study, we show that just 4 weeks of feeding a diet rich in fat ad libitum decreased hippocampal neurogenesis in male, but not female, rats. There was no obesity, but male rats fed a diet rich in fat exhibited elevated serum corticosterone levels compared with those fed standard rat chow. These data indicate that high dietary fat intake can disrupt hippocampal neurogenesis, probably through an increase in serum corticosterone levels, and that males are more susceptible than females.

Therapeutic potential of controlled drug delivery systems in neurodegenerative diseases.

Several compounds that exhibit a therapeutic effect in experimental models of neurodegenerative diseases have been identified over recent years. Safe and effective drug delivery to the central nervous system is still one of the main obstacles in translating these experimental strategies into clinical therapies. Different approaches have been developed to enable drug delivery in close proximity to the desired site of action. In this review, we describe biodegradable polymeric systems as drug carriers in models of neurodegenerative diseases. Biomaterials described for intracerebral drug delivery are well tolerated by the host tissue and do not exhibit cytotoxic, immunologic, carcinogenic or teratogenic effects even after chronic exposure. Behavioral improvement and normalization of brain morphology have been observed following treatment using such biomaterials in animal models of Parkinson's, Alzheimer's and Huntington's diseases. Application of these devices for neuroactive drugs is still restricted due to the relatively small volume of tissue exposed to active compound. Further development of polymeric drug delivery systems will require that larger volumes of brain tissue are targeted, with a controlled and sustained drug release that is carefully controlled so it does not cause damage to the surrounding tissue.

An enzyme in the kynurenine pathway that governs vulnerability to suicidal behavior by regulating excitotoxicity and neuroinflammation.

Emerging evidence suggests that inflammation has a key role in depression and suicidal behavior. The kynurenine pathway is involved in neuroinflammation and regulates glutamate neurotransmission. In the cerebrospinal fluid (CSF) of suicidal patients, levels of inflammatory cytokines and the kynurenine metabolite quinolinic acid (QUIN), an N-methyl-d-aspartate receptor agonist, are increased. The enzyme amino-ß-carboxymuconate-semialdehyde-decarboxylase (ACMSD) limits QUIN formation by competitive production of the neuroprotective metabolite picolinic acid (PIC). Therefore, decreased ACMSD activity can lead to excess QUIN. We tested the hypothesis that deficient ACMSD activity underlies suicidal behavior. We measured PIC and QUIN in CSF and plasma samples from 137 patients exhibiting suicidal behavior and 71 healthy controls. We used DSM-IV and the Montgomery-Åsberg Depression Rating Scale and Suicide Assessment Scale to assess behavioral changes. Finally, we genotyped ACMSD tag single-nucleotide polymorphisms (SNPs) in 77 of the patients and 150 population-based controls. Suicide attempters had reduced PIC and a decreased PIC/QUIN ratio in both CSF (P<0.001) and blood (P=0.001 and P<0.01, respectively). The reductions of PIC in CSF were sustained over 2 years after the suicide attempt based on repeated measures. The minor C allele of the ACMSD SNP rs2121337 was more prevalent in suicide attempters and associated with increased CSF QUIN. Taken together, our data suggest that increased QUIN levels may result from reduced activity of ACMSD in suicidal subjects. We conclude that measures of kynurenine metabolites can be explored as biomarkers of suicide risk, and that ACMSD is a potential therapeutic target in suicidal behavior.

Platelet-derived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions.

The effects of i.c.v. infused platelet-derived growth factor and brain-derived neurotrophic factor on cell genesis, as assessed with bromodeoxyuridine (BrdU) incorporation, were studied in adult rats with unilateral 6-hydroxydopamine lesions. Both growth factors increased the numbers of newly formed cells in the striatum and substantia nigra to an equal extent following 10 days of treatment. At 3 weeks after termination of growth factor treatment, immunostaining of BrdU-labeled cells with the neuronal marker NeuN revealed a significant increase in newly generated neurons in the striatum. In correspondence, many doublecortin-labeled neuroblasts were also observed in the denervated striatum following growth factor treatment. Further evaluation suggested that a subset of these new neurons expresses the early marker for striatal neurons Pbx. However, no BrdU-positive cells were co-labeled with DARPP-32, a protein expressed by mature striatal projection neurons. Both in the striatum and in the substantia nigra there were no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment, such that BrdU-labeled cells never co-expressed tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. In conclusion, our results suggest that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats.

Cell survival and clinical outcome following intrastriatal transplantation in Parkinson disease.

Intrastriatal transplantation of embryonic dopaminergic neurons is currently explored as a restorative cell therapy for Parkinson disease (PD). Clinical results have varied, probably due to differences in transplantation methodology and patient selection. In this review, we assess clinical trials and autopsy findings in grafted PD patients and suggest that a minimum number of surviving dopaminergic neurons is required for a favorable outcome. Restoration of [18F]-fluorodopa uptake in the putamen to about 50% of the normal mean seems necessary for moderate to marked clinical benefit to occur. Some studies indicate that this may require mesencephalic tissue from 3-5 human embryos implanted into each hemisphere. The volume, density and pattern of fiber outgrowth and reinnervation, as well as functional integration and dopamine release. are postulated as additional important factors for an optimal clinical outcome. For neural transplantation to become a feasible therapeutic alternative in PD, graft survival must be increased and the need for multiple donors of human embryonic tissue substantially decreased or alternate sources of donor tissue developed. Donor cells derived from alternative sources should demonstrate features comparable to those associated with successful implantation of human embryonic tissue before clinical trials are considered.

Effect of Prior Dopamine Denervation on Survival and Fiber Outgrowth from Intrastriatal Fetal Mesencephalic Grafts.

[3H]Dopamine (DA) uptake radioautography and tyrosine hydroxylase (TH) immunocytochemistry were used to assess quantitatively the effects of the presence or absence of host mesostriatal DA afferents on the survival and fiber outgrowth from fetal ventral mesencephalic DA neurons grafted into the neostriatum of adult recipient rats. Rats received bilateral intrastriatal transplants of fetal ventral mesencephalic tissue 1 month after a unilateral injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle (denervated side). Five to six months later, some of the grafted rats received a second 6-OHDA injection in the left nigrostriatal bundle (acutely denervated or 'intact' side). After a further 7 days, slices of each hemisphere from the latter rats were incubated with [3H]DA and processed for film and high resolution radioautography. The density of the film radioautographs was measured with a computerized image analysis system and calibrated by silver grain cluster (i.e. DA terminal) counting over selected areas of the same sections in light microscope radioautographs. The brains of the remaining grafted rats were processed for TH immunoreactivity 6 - 12 months after graft surgery. Neither the size of the grafts, nor the number of surviving TH-positive graft neurons showed any significant difference between the nondenervated and the denervated sides. However, the size of the TH-positive cell bodies was significantly greater in the grafts on the denervated side. In the [3H]DA uptake radioautographs, considerable outgrowth of DA fibers was evident in the neostriatum on the 'intact' side in spite of the presence of an intact host DA innervation until 7 days before sacrifice. The overall DA fiber outgrowth was nevertheless almost two-fold greater on the denervated side, and extended deeper into the host neostriatum than on the 'intact' side; only 7% of the total neostriatal area, on average, was at background level compared to 30% on the 'intact' side, and the overall density of neostriatal DA innervation amounted to 36% of normal as compared to 20% on the 'intact' side. The correlation between the overall density of graft-derived DA innervation and the size of the grafts was linear on the 'intact' side, but reached a plateau with relatively small grafts on the denervated side. However, the ventral striatum on both sides was very poorly innervated by these grafts. These findings demonstrate that the mature neostriatal tissue can support axonal growth and innervation from grafted fetal DA neurons even in the presence of a normal complement of endogenous DA fibers. Prior removal of the host striatal DA innervation does not influence the overall size of the grafts nor the number of surviving DA neurons, but induces an increase in the cell body size and fiber outgrowth of the grafted DA neurons.

Long distance directed axonal growth from human dopaminergic mesencephalic neuroblasts implanted along the nigrostriatal pathway in 6-hydroxydopamine lesioned adult rats.

Dissociated ventral mesencephalon of 6 to 8-week-old human embryos were implanted by stereotaxic injection at different sites along the nigrostriatal pathway in adult rats, previously subjected to a 6-hydroxydopamine lesion of the intrinsic mesotelencephalic dopamine pathways. The recipients were immunosuppressed by daily injections of cyclosporin A to prevent rejection. At 13-20 weeks after transplantation, the implanted human neurons and their associated fiber outgrowths were visualized with a species-specific antibody recognizing human, but not rat, intermediary neurofilaments (HNF). From implants placed in the host rostral mesencephalic region, HNF-positive axonal projections were seen to extend in large numbers rostrally along the medial forebrain bundle and the internal capsule, and ramify within the caudate putamen, the ventral striatum and the amygdaloid nuclei (a distance of about 5-6 mm), and more sparsely in the frontal cortex and the olfactory bulb (a distance of about 10 mm). From implants placed in the internal capsule, abundant HNF-positive axons extended in the rostral, but not caudal, direction along the myelinated fiber bundles into the caudate putamen and the ventral striatum. Tyrosine hydroxylase (TH) immunohistochemistry revealed that the vast majority of the rostrally projecting HNF-positive axons were also TH-positive, and that the graft-derived axons gave rise to dense TH-positive terminal networks, above all in large areas of the previously denervated caudate putamen. From control implants of cortical neuroblasts, axonal projections were seen along the medial forebrain bundle and the internal capsule, but the axons were TH-negative and showed only sparse projections to the striatal areas. Instead, dense projections were seen, e.g., in the frontal cortex. The results demonstrate a remarkable ability of human mesencephalic neuroblasts to extend axons along the trajectories of the nigrostriatal and mesolimbocortical pathways to reach and innervate the principal striatal and limbic target areas in the forebrain. This shows that the basic requirements for the formation of long axonal pathways may be present in the adult mammalian central nervous system (CNS) at least for certain types of projection neurons. Furthermore, it implies that the developing human neuroblasts can escape the inhibitory features known to be present along myelinated growth trajectories in the adult mammalian brain. In addition, the present approach may offer new possibilities for functional neural grafting in the rat Parkinson model, since transplanted nigral neurons placed in their natural position within the rostral mesencephalon could provide an anatomically and functionally more integrated system than the standard model with ectopically placed intrastriatal nigral grafts.

Comparison of growth and reinnervation properties of cholinergic neurons from different brain regions grafted to the hippocampus.

Grafts of five different types of central cholinergic neurons, from the septal-diagonal band region, the nucleus basalis magnocellularis region (NBM), the striatum, the pontomesencephalic tegmentum of the brainstem, and the spinal cord, were compared with respect to their ability to grow and to reinnervate the cholinergically denervated hippocampal formation of adult rats. The areas were dissected from 14 to 15-day-old rat fetuses, and the same number of viable cells (35 X 10(4) from each of the different regions were stereotaxically injected as cell suspensions into the hippocampus of rats subjected to a transection of the intrinsic septo-hippocampal cholinergic pathways. At 17-19 weeks after transplantation, the various graft types differed considerably in their volume, the total amount of acetylcholinesterase (AChE)-positive fiber outgrowth, and the innervation pattern and morphology of the AChE-positive fibers growing into the host hippocampus. On average the NBM and spinal cord grafts had grown to become three to four times larger than the septal and the brainstem grafts, and 15-20 times larger than the striatal grafts. By contrast, the total ingrowth score of AChE-positive fibers in the host hippocampus from the septal grafts was about twice that of the NBM and brainstem grafts, about five times greater than the striatal grafts, and about six times greater than that of the spinal cord grafts. The large NBM grafts thus exhibited similar fiber outgrowth to the much smaller brainstem grafts, and the AChE-positive neurons of the grafted spinal cord grew very poorly into the hippocampus despite the fact that they survived very well. The innervation pattern and morphological features of the ingrowing AChE-positive fibers in the host hippocampus proper and in the dentate gyrus resembled those of normal rats in animals with grafts from any of the three forebrain regions (i.e., septum, NBM, or striatum), whereas ingrowth from the brainstem and spinal cord grafts were markedly abnormal with respect to both innervation pattern and fiber morphology. These results provide further evidence that the overall survival, growth, and fiber outgrowth of intracerebral neural grafts depend on interactions with the surrounding host tissue. Since the ability to reinnervate the previously denervated host target was greatest for the neuron type normally innervating that area, i.e., the septal-diagonal band neurons, we conclude that neuronal properties beyond the transmitter type are essential for the optimal performance of implanted neurons in intracerebral grafting experiments.