Philip W. Brownjohn

Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

Spinal cannabinoid CB2 receptors as a target for neuropathic pain: an investigation using chronic constriction injury

Agonists for the cannabinoid CB2 receptor are antinociceptive in several rodent models and several reports have suggested that the target for these drugs is CB2 expressed in the spinal cord pain pathway. After confirming the efficacy of a systemically delivered CB2-selective agonist, GW405833, we tested this hypothesis by administering the CB2 agonists GW405833 and JWH-133, via intrathecal cannulation, to the lumbar spinal cord of rats that had undergone chronic constriction injury to induce mechanical allodynia. We found that although the non-selective CB1/CB2 cannabinoid receptor agonist WIN55,212-2 reversed mechanical allodynia in both ipsilateral and contralateral hind paws, neither GW405833 nor JWH-133 reversed mechanical allodynia. In addition, we investigated the expression of CB2 receptors in the neuropathic spinal cord using immunohistochemistry, Western blot and CB2 agonist stimulated [(35)S]GTPγS binding. Although protein-based analysis of CB2 partially matched the results of earlier studies using the same antibody, we found evidence that this antibody may be insufficiently specific for the detection of CB2 in native tissue. Using [(35)S]GTPγS binding assays, we found no evidence of functional CB2 in the spinal cord, in sham or surgery-treated tissue. However, WIN55,212-2 stimulated [(35)S]GTPγS binding showed clear evidence of functional CB1 receptors consistent with the known distribution of elements of the pain pathway, and we concluded that spinal CB2 receptors are not a likely target for cannabinoid-mediated antinociception in this model.

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 ( < ∼ 10 ms) and long ( > ∼ 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’s electroencephalogram. Moreover, the model can be used to make predictions about protocols that may lead to improvements in repetitive TMS outcomes.

The atypical cannabinoid O-1602 increases hind paw sensitisation in the chronic constriction injury model of neuropathic pain

O-1602 is an atypical cannabinoid that acts as an agonist at GPR55, a g protein-coupled receptor that previous studies have indicated may have a pronociceptive role in neuropathic pain. We administered O-1602 to both naive rats and rats that had undergone chronic constriction injury surgery. O-1602 did not cause any changes in hind paw responses to Von Frey hair testing in naive rats. However, O-1602 reversed the desensitising effects of ETOH, which was used as an active and opposing vehicle. Our results are consistent with the hypothesis that GPR55 has a pronociceptive role in neuropathic pain.

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.

Effect of styrene maleic acid WIN55,212-2 micelles on neuropathic pain in a rat model

Abstract Cannabinoid receptor agonists are moderately effective at reducing neuropathic pain but are limited by psychoactivity. We developed a styrene maleic acid (SMA) based on the cannabinoid WIN 55,212-2 (WIN) and tested in a rat model of neuropathic pain and in the rotarod test. We hypothesized that miceller preparation can ensure prolonged plasma half-life being above the renal threshold of excretion. Furthermore, SMA-WIN could potentially reduce the central nervous system effects of encapsulated WIN by limiting its transport across the blood–brain barrier. Using the chronic constriction injury model of sciatic neuropathy, the SMA-WIN micelles were efficacious in the treatment of neuropathic pain for a prolonged period compared to control (base WIN). Attenuation of chronic constriction injury-induced mechanical allodynia occurred for up to 8 h at a dose of 11.5 mg/kg of SMA-WIN micelles. To evaluate central effects on motor function, the rotarod assessment was utilized. Results showed initial impairment caused by SMA-WIN micelles to be identical to WIN control for up to 1.5 h. Despite this, the SMA-WIN micelle formulation was able to produce prolonged analgesia over a time when there was decreased impairment in the rotarod test compared with base WIN.

Microglial encapsulation of motor neurons in models of neuropathic pain: A confound in pain assessment?

Paw withdrawal reflexes are commonly used to assess allodynia and hyperalgesia in rodent models of neuropathic pain, despite suggestions that these tests may partially reflect motor reflex responses and not exclusively pain behaviour (Baliki et al., 2005). Gliosis in the spinal cord dorsal horn has been extensively studied as a mechanism of central sensitization in neuropathic pain models. Although gliosis has also been reported in the ventral horn proximate to motor neurons in a number of these models, to our knowledge, this has not been previously proposed as a confounding factor in paw withdrawal tests. This is despite a growing body of evidence that, taken together, strongly suggest that there may be a link between motor dysfunction and pain behaviours in models of neuropathic pain. Specifically, Hu et al. (2007) showed that motor neuron axons are damaged at the peak of allodynia in the chronic constriction injury (CCI) model but recover by week 10, which correlates with the resolution phase of allodynia in CCI (Kim et al., 1997). In complimentary fashion, deficits and recovery of motor function are closely correlated with the development and resolution of hyperalgesia in spinal cord injury (Cho et al., 2011). The characteristic hind paw ventroflexion exhibited by animals following CCI and other models such as spinal nerve ligation has also been suggested as evidence of motor dysfunction (Walczak et al., 2005). Using high-magnification microscopy of Iba-1 immunolabeling in lumbar spinal cord sections from rats dissected 10 days after CCI, we have found that large numbers of motor neurons in the ipsilateral ventral horn were encapsulated by a mantle of activated microglia (Fig. 1). Although previous research has shown some association of motor neurons with activated microglia following CCI or axotomy of the sciatic nerve (Zhang et al., 2003; Hu et al., 2007), the pattern we describe most precisely matches the pattern of gliosis that occurs after axotomy of motor neurons (Köbbert and Thanos, 2000). Critically, Li et al. (2002) have shown that axotomy of ventral nerve roots, which lack sensory fibres, is able to produce robust neuropathic pain. Obata et al. (Obata et al., 2004) have argued that motor neuron damage contributes to sensory neuropathy by neurotrophin signalling, although a direct effect on paw withdrawal behaviours by alteration of motor neuron responses has not been ruled out.

What can be concluded from blocking peptide controls

To the Editor: Preincubation with immunizing (blocking) peptide is routinely used in antibody quality control checks for both immunohistochemical analysis and Western blot. Ideally, binding of the antibody to the peptide causes competitive inhibition of binding of the antibody to the antigen of interest. When the high-affinity binding domains of antibodies are blocked, this tests for nonspecific binding by the constant domains. However, although this is a valid justification for immunohistochemical analysis, it is probably incorrect for Western blot. Moreover, although it is increasingly recognized that blocking peptide control is not a test of antibody specificity, blocking peptide controls continue to be uncritically used as tests for antibody specificity in the absence of better controls, such as gene knockout controls. If a blocking peptide control produces staining in immunohistochemical analysis that is similar to that seen with the “unblocked” primary antibody and if this staining does not appear in a no-primary control (secondary antibody only), then the staining may be attributed to nonspecific binding by the primary antibody. For instance, Burry showed that antibodies can bind by their constant domains in native tissue during immunohistochemical analysis to Fc receptors on macrophages or other immune cells. However, for SDS-PAGE Western blot, proteins are largely denatured and distributed over the length of the gel. Therefore, it seems unlikely that the constant domains of a primary antibody would cause nonspecific bands in a Western blot in a way that would confound specific binding to the protein of interest. Moreover, if any such effect was present, then it should follow a similar pattern for a wide variety of antibodies generated from a given species, something that does not seem to be the case. Indeed, evidence for this also seems to be lacking in the case of immunohistochemical analysis. What the blocking peptide control can provide for both immunohistochemical analysis and Western blot is a quality assurance check on the ability of the protein to bind to the epitope of interest (assuming the epitope is exposed to the antibody). This is not a true positive control, which is a check on falsenegative results (ie, the failure to detect a band with an antibody— whereas blocking peptide controls are used for when bands are detected). However, inhibition of staining by a blocking peptide does show (a) that the labeling is because of the highaffinity binding domains of the antibody and (b) that the antibody is capable of binding to the epitope used during its manufacture. As (a) is already likely to be true based on the improbability of binding through the conserved antibody domain in Western blot as discussed above, the only useful information garnered from a blocking peptide control in this case is an answer to (b). Therefore, the blocking peptide control in Western blot can only be considered to be the test of an antibody’s ability to recognize the antigen under ideal conditions, that is, a quality control check. An antibody that passes the blocking peptide control test may still cross-react with other proteins when its high-affinity binding domains are exposed. At best then, in Western blot, the blocking peptide control test provides the researcher with only a little more information than a noprimary antibody control—namely that the manufacturing process for the particular lot or batch of the antibody was sound.

The Effect of Paired Muscle Stimulation on Preparation for Movement

ABSTRACT 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.