Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia.

The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC

The cannabinoid CB2 receptor as a target for the treatment of neuropathic pain

Cannabinoids are cannabis-like drugs that are moderately efficacious in the treatment of some types of chronic pain, including pain of a neuropathic origin. Limiting their therapeutic application, however, are adverse psychoactive effects as a result of cannabinoid receptor activation in the central nervous system (CNS). Cannabinoids act via two receptors: cannabinoid receptor I (CB1) and cannabinoid receptor II (CB2), which are respectively known as the central and peripheral cannabinoid receptors. While CB1 receptors are thought to mediate the antinociceptive effects of cannabinoids, they are also responsible for the adverse effects. Selective targeting of CB2 receptors has shown promise as a treatment in neuropathic pain models in animals, without the associated psychoactivity seen following CB1 receptor activation, however the cellular mechanisms involved have not been elucidated. While initially considered a peripheral receptor, recent evidence has suggested that CB2 receptors may be upregulated in the CNS following neuropathic pain. Using a well established rodent model of neuropathic pain, this study aimed to assess the efficacy of CB2 selective agonists in the treatment of allodynia (pain in response to a normally innocuous stimulus), and investigate the presence and function of CB2 receptors in the spinal cord, a key structure in nociceptive transmission and processing. In the chronic constriction injury (CCI) model of sciatic neuropathy, CB2 selective agonists were efficacious when delivered systemically at high doses, but not when delivered by intrathecal cannulation to the spinal cord. Using immunohistochemistry and Western blot, labeling was detected for CB2 receptors in the superficial dorsal horn of the spinal cord, which was not modulated by CCI surgery or drug administration. Attempted validation of the antibody used in these approaches, however, indicated that this antibody was not specific for CB2 protein, and detects at least one unspecified cytosolic protein in addition to CB2. These findings cast doubt on the validity of this primary antibody which has been widely used, both in these immunohistochemistry and Western blot studies, as well as in previous reports of CB2 receptor protein expression. To circumvent this issue, a functional receptor assay, the [35S]GTPγS assay, was employed to assess the presence of functional CB2 receptors in the spinal cord. Employing this assay on membrane preparations and tissue slices in situ, no evidence for functional CB2 receptors was found in sham or neuropathic spinal cords. This study found that while efficacious in the treatment of neuropathic pain in this model, CB2 selective agonists are not acting via spinal CB2 receptors. Furthermore, no evidence was found for functional CB2 receptors above the threshold for detection in the healthy or neuropathic spinal cord, suggesting that spinal CB2 receptors are not a rational target for the treatment of neuropathic pain following peripheral nerve injury

The cannabinoid CB2 receptor as a target for the treatment of neuropathic pain

Cannabinoids are cannabis-like drugs that are moderately efficacious in the treatment of some types of chronic pain, including pain of a neuropathic origin. Limiting their therapeutic application, however, are adverse psychoactive effects as a result of cannabinoid receptor activation in the central nervous system (CNS). Cannabinoids act via two receptors: cannabinoid receptor I (CB1) and cannabinoid receptor II (CB2), which are respectively known as the central and peripheral cannabinoid receptors. While CB1 receptors are thought to mediate the antinociceptive effects of cannabinoids, they are also responsible for the adverse effects. Selective targeting of CB2 receptors has shown promise as a treatment in neuropathic pain models in animals, without the associated psychoactivity seen following CB1 receptor activation, however the cellular mechanisms involved have not been elucidated. While initially considered a peripheral receptor, recent evidence has suggested that CB2 receptors may be upregulated in the CNS following neuropathic pain. Using a well established rodent model of neuropathic pain, this study aimed to assess the efficacy of CB2 selective agonists in the treatment of allodynia (pain in response to a normally innocuous stimulus), and investigate the presence and function of CB2 receptors in the spinal cord, a key structure in nociceptive transmission and processing. In the chronic constriction injury (CCI) model of sciatic neuropathy, CB2 selective agonists were efficacious when delivered systemically at high doses, but not when delivered by intrathecal cannulation to the spinal cord. Using immunohistochemistry and Western blot, labeling was detected for CB2 receptors in the superficial dorsal horn of the spinal cord, which was not modulated by CCI surgery or drug administration. Attempted validation of the antibody used in these approaches, however, indicated that this antibody was not specific for CB2 protein, and detects at least one unspecified cytosolic protein in addition to CB2. These findings cast doubt on the validity of this primary antibody which has been widely used, both in these immunohistochemistry and Western blot studies, as well as in previous reports of CB2 receptor protein expression. To circumvent this issue, a functional receptor assay, the [35S]GTPγS assay, was employed to assess the presence of functional CB2 receptors in the spinal cord. Employing this assay on membrane preparations and tissue slices in situ, no evidence for functional CB2 receptors was found in sham or neuropathic spinal cords. This study found that while efficacious in the treatment of neuropathic pain in this model, CB2 selective agonists are not acting via spinal CB2 receptors. Furthermore, no evidence was found for functional CB2 receptors above the threshold for detection in the healthy or neuropathic spinal cord, suggesting that spinal CB2 receptors are not a rational target for the treatment of neuropathic pain following peripheral nerve injury

Differences in motor evoked potentials induced in rats by transcranial magnetic stimulation under two separate anesthetics: implications for plasticity studies

Repetitive transcranial magnetic stimulation (rTMS) is primarily used in humans to change the state of corticospinal excitability. To assess the efficacy of different rTMS stimulation protocols, motor evoked potentials (MEPs) are used as a readout due to their non-invasive nature. Stimulation of the motor cortex produces a response in a targeted muscle, and the amplitude of this twitch provides an indirect measure of the current state of the cortex. When applied to the motor cortex, rTMS can alter MEP amplitude, however results are variable between participants and across studies. In addition, the mechanisms underlying any change and its locus are poorly understood. In order to better understand these effects, MEPs have been investigated in vivo in animal models, primarily in rats. One major difference in protocols between rats and humans is the use of general anesthesia in animal experiments. Anesthetics are known to affect plasticity-like mechanisms and so may contaminate the effects of an rTMS protocol. In the present study, we explored the effect of anesthetic on MEP amplitude, recorded before and after intermittent theta burst stimulation (iTBS), a patterned rTMS protocol with reported facilitatory effects. MEPs were assessed in the brachioradialis muscle of the upper forelimb under two anesthetics: a xylazine/zoletil combination and urethane. We found MEPs could be induced under both anesthetics, with no differences in the resting motor threshold or the average baseline amplitudes. However, MEPs were highly variable between animals under both anesthetics, with the xylazine/zoletil combination showing higher variability and most prominently a rise in amplitude across the baseline recording period. Interestingly, application of iTBS did not facilitate MEP amplitude under either anesthetic condition. Although it is important to underpin human application of TMS with mechanistic examination of effects in animals, caution must be taken when selecting an anaesthetic and in interpreting results during prolonged TMS recording

Differences in Motor Evoked Potentials Induced in Rats by Transcranial Magnetic Stimulation under Two Separate Anesthetics: Implications for Plasticity Studies

Repetitive transcranial magnetic stimulation (rTMS) is primarily used in humans to change the state of corticospinal excitability. To assess the efficacy of different rTMS stimulation protocols, motor evoked potentials (MEPs) are used as a readout due to their non-invasive nature. Stimulation of the motor cortex produces a response in a targeted muscle, and the amplitude of this twitch provides an indirect measure of the current state of the cortex. When applied to the motor cortex, rTMS can alter MEP amplitude, however, results are variable between participants and across studies. In addition, the mechanisms underlying any change and its locus are poorly understood. In order to better understand these effects, MEPs have been investigated in vivo in animal models, primarily in rats. One major difference in protocols between rats and humans is the use of general anesthesia in animal experiments. Anesthetics are known to affect plasticity-like mechanisms and so may contaminate the effects of an rTMS protocol. In the present study, we explored the effect of anesthetic on MEP amplitude, recorded before and after intermittent theta burst stimulation (iTBS), a patterned rTMS protocol with reported facilitatory effects. MEPs were assessed in the brachioradialis muscle of the upper forelimb under two anesthetics: a xylazine/zoletil combination and urethane. We found MEPs could be induced under both anesthetics, with no differences in the resting motor threshold or the average baseline amplitudes. However, MEPs were highly variable between animals under both anesthetics, with the xylazine/zoletil combination showing higher variability and most prominently a rise in amplitude across the baseline recording period. Interestingly, application of iTBS did not facilitate MEP amplitude under either anesthetic condition. Although it is important to underpin human application of TMS with mechanistic examination of effects in animals, caution must be taken when selecting an anesthetic and in interpreting results during prolonged TMS recording

The Effect of Paired Muscle Stimulation on Preparation for Movement

Paired muscle stimulation is used clinically to facilitate the performance of motor tasks for individuals with motor dysfunction. However, the optimal temporal relationship between stimuli for enhancing movement remains unknown. We hypothesized that synchronous, muscle stimulation would increase the extent to which stimulated muscles are concurrently prepared for movement. We validated a measure of muscle-specific changes in corticomotor excitability prior to movement. We used this measure to examine the preparation of the first dorsal interosseous (FDI), abductor digiti minimi (ADM), abductor pollicis brevis (APB) muscles prior to voluntary muscle contractions before and after paired muscle stimulation at four interstimulus intervals (0, 5, 10, and 75 ms). Paired muscle stimulation increased premovement excitability in the stimulated FDI, but not in the ADM muscle. Interstimulus interval was not a significant factor in determining efficacy of the protocol. Paired stimulation, therefore, did not result in a functional association being formed between the stimulated muscles. Somatosensory potentials evoked by the muscle stimuli were small compared to those commonly elicited by stimulation of peripheral nerves, suggesting that the lack of functional association formation between muscles may be due to the small magnitude of afferent volleys from the stimulated muscles, particularly the ADM, reaching the cortex

The effects of individualized theta burst stimulation on the excitability of the human motor system

Background: Theta burst stimulation (TBS) is a pattern of repetitive transcranial magnetic stimulation that has been demonstrated to facilitate or suppress human corticospinal excitability when applied intermittently (iTBS) or continuously (cTBS), respectively. While the fundamental pattern of TBS, consisting of bursts of 50 Hz stimulation repeated at a 5 Hz theta frequency, induces synaptic plasticity in animals and in vitro preparations, the relationship between TBS and underlying cortical firing patterns in the human cortex has not been elucidated. Objective: To compare the effects of 5 Hz iTBS and cTBS with individualized TBS paradigms on corticospinal excitability and intracortical inhibitory circuits. Methods: Participants received standard and individualized iTBS (iTBS 5; iTBS I) and cTBS (cTBS 5; cTBS I), and sham TBS, in a randomised design. For individualized paradigms, the 5 Hz theta component of the TBS pattern was replaced by the dominant cortical frequency (4-16 Hz; upper frequency restricted by technical limitations) for each individual. Results: We report that iTBS 5 and iTBS I both significantly facilitated motor evoked potential (MEP) amplitude to a similar extent. Unexpectedly, cTBS 5 and cTBS I failed to suppress MEP amplitude. None of the active TBS protocols had any significant effects on intracortical circuits when compared with sham TBS. Conclusion: In summary, iTBS facilitated MEP amplitude, an effect that was not improved by individualizing the theta component of the TBS pattern, while cTBS, a reportedly inhibitory paradigm, produced no change, or facilitation of MEP amplitude in our hands

Numerical modelling of plasticity induced by transcranial magnetic stimulation

We use neural field theory and spike-timing dependent plasticity to make a simple but biophysically reasonable model of long-term plasticity changes in the cortex due to transcranial magnetic stimulation (TMS). We show how common TMS protocols can be captured and studied within existing neural field theory. Specifically, we look at repetitive TMS protocols such as theta burst stimulation and paired-pulse protocols. Continuous repetitive protocols result mostly in depression, but intermittent repetitive protocols in potentiation. A paired pulse protocol results in depression at short ( \u3c ∼ 10 ms) and long ( \u3e ∼ 100 ms) interstimulus intervals, but potentiation for mid-range intervals. The model is sensitive to the choice of neural populations that are driven by the TMS pulses, and to the parameters that describe plasticity, which may aid interpretation of the high variability in existing experimental results. Driving excitatory populations results in greater plasticity changes than driving inhibitory populations. Modelling also shows the merit in optimizing a TMS protocol based on an individual\u27s electroencephalogram. Moreover, the model can be used to make predictions about protocols that may lead to improvements in repetitive TMS outcomes