Cortical layer 6b is considered by many to be a remnant of the subplate that forms during early stages of neocortical development, but its role in the adult is not well understood. Its neuronal complement has only recently become the subject of systematic studies, and its axonal projections and synaptic input structures have remained largely unexplored despite decades of research into neocortical function. In recent years, however, layer 6b (L6b) has attracted increasing attention and its functional role is beginning to be elucidated. In this review, I will attempt to provide an overview of what is currently known about the excitatory and inhibitory neurons in this layer, their pre- and postsynaptic connectivity, and their functional implications. Similarities and differences between different cortical areas will be highlighted. Finally, layer 6b neurons are highly responsive to several neuropeptides such as orexin/hypocretin, neurotensin and cholecystokinin, in some cases exclusively. They are also strongly controlled by neurotransmitters such as acetylcholine and norepinephrine. The interaction of these neuromodulators with L6b microcircuitry and its functional consequences will also be discussed. Layer 6b is thought to be a remnant of the subplate that develops during early stages of corticogenesis and persists, at least in part, into adulthood . Over thSublamina 6b of the neocortex was first described by Layer 6b has also been variously referred to as the “subgriseal” layer  or layerLayer 6b has also been identified in species belonging to different mammalian species belonging to different mammalian orders. For example, in carnivores  (Nurr1) (Nxph3) (Sulf2) (Cplx3), and monooxygenase Dbh-like 1 (MoxD1)  are expressed throughout the entire layer 6 of the entorhinal cortex, which belongs to the developmentally older periarchic cortex  , the orp (Nurr1) , neurexo (Nxph3) , heparin (Sulf2) , complex (MoxD1) . All the (MoxD1) . In ratsc cortex .In contrast, work focusing mainly on the morphology of L6b neurons in mice and rats demonstrated that the neuronal complement in the lower 30% of layer 6 differs markedly from those in the more superficial part, i.e., layer 6a . Moreovein situ hybridization studies have shown that the anatomically defined layer 6b in ferret and mouse ,1 a highly specific marker gene for layer 6b (see above).The axonal and dendritic domains of L6b excitatory neurons have been systematically investigated in the rat somatosensory barrel cortex . Five diy cortex  have shoy cortex . For visin vivo reconstructions of L6b neurons available and the degree of axonal truncation in brain slice preparations is likely to be high, it is difficult to assess the true L6b-L1 connectivity, which may be substantial. It should be noted that the L6b-L1 projecting neurons collateralize in two regions, layer 5/6 and 1/2, but pass through upper layer 5, 4 and lower layer 3 without branching .The proportion of GABAergic interneurons in the total population of L6b neurons has been found to be very low <5%; , even loFor mouse S1 cortex, L6b neuropeptide Y (NPY)-, fast-spiking (FS) parvalbumin (PV)-, and somatostation (Sst)-expressing interneuron types have been identified based on their electrophysiological properties, dendritic morphology and marker gene expression . L6b FS In mouse prefrontal cortex (PFC), L6b stFS interneurons possess an elongated axonal plexus . FurtherIn mouse primary visual cortex (V1), two distinct Sst+ interneuron subtypes were found in layer 6b ; howeverin vivo neuronal labeling will be required to address this issue.The main feature of most L6b interneurons is their dense local dendritic and axonal arbor with few projections outside layer 6b, predominantly to layer 6a and the white matter. However, some L6b Sst+, non-FS interneurons innervate supragranular layers 2 and 3 but only sparsely. Because truncation of axon collaterals, especially long-range ones, is common in brain slice preparations, this will inevitably lead to an underestimation of the translaminar intracortical connectivity of L6b interneurons. More detailed morphological analysis in slice preparations or This section attempts to describe the current knowledge of pre- and postsynaptic connectivity of L6b neurons and the level of local synaptic microcircuits (<50 μm), interlaminar synaptic connections and connections between cortical areas and across brain hemispheres.Drd1- and Ctgf-expressing L6b excitatory neurons  receive little synaptic input from other Drd1- and Ctgf-expressing L6b neurons, and if they do, this input is weak  and FS PV+ interneurons have a dense axonal plexus largely within layer 6b, but with some collaterals in layer 6a and the white matter . The axoDrd1+ CT PCs and Ctgf+ CC excitatory neurons receive inhibitory input from almost all cortical layers. Most of this input originates from PV+ and Sst+ interneurons, was relatively strong and showed short-term depression. Stimulation of VIP cells produced only small IPSPs that did not contribute to a disinhibitory mechanism as observed in L2/3 PCs. Finally, axonal afferents from layer 1 to layer 6b were identified and functional inhibitory synaptic input from L1 Ndnf-expressing interneuron was recorded; IPSPs at this connections were mediated by G protein-coupled GABAB receptors (L6b GABAergic interneurons (mainly of the PV+ and Sst+ subtype) are innervated by L6b excitatory neurons; an example of an L6b inverted PC-L6b FS interneuron connection is shown in eceptors .Early studies describing dense horizontal intracortical axons emanating from layer 6b suggested that this layer gives rise to long-range corticocortical connections ; howeverA first attempt to reveal the medium- and long-range synaptic connectivity of L6b neurons was made by Hay and coworkers  in rat pThis is indicative of a highly interconnected GABAergic network. Furthermore, an ipsilateral increase in spontaneous EPSP frequency was observed in L5 PCs, L1 interneurons and L2/3 PCs, although the effect in the latter was weak; no effect of orexin was found in L4 neurons. Thus, in the PtA L6b PCs provide strong ipsilateral inter-laminar synaptic input to layers 6, 5 and 1 but only weak and virtually no input to layers 2/3 and 4, respectively, in agreement with the morphological data . In addiDrd1-expressing PCs (In the primary somatosensory cortex (S1), it has been clearly demonstrated that higher order thalamic nuclei such as the POm in the somatosensory system are innervated by L6b CT excitatory neurons . Using asing PCs . Only vesing PCs . Furthersing PCs ; analogosing PCs .Drd1-Cre and Ctgf-Cre neurons as starter cells , layer location and axonal projection pattern; however, it should be noted that these terminologies are not fully interchangeable.The neuronal network providing synaptic input to layer 6b was only poorly understood until recently. Zolnik and coworkers identified the inter- and subcortical input to layer 6b in the primary somatosensory cortex using rabies monosynaptic retrograde tracing using ells see . Cell-tyells see . PostsynDrd1+) and CC (Ctgf+) excitatory neurons, respectively (Drd1+) CT PCs project. This suggests that layer 6b is central to a L6b-POm-L1 corticothalamocortical loop which is independent of the “lemniscal” TC innervation via the VPM. Apparently, this connection is already established in early postnatal animals by Cplx3-expressing subplate/L6b neurons projecting to layer 1  of the long-range intracortical afferents innervating layer 6b of S1 cortex came from the neocortex, with the largest proportion coming from the ipsilateral motor cortex, and here from layer 5; these inputs were particularly strong. Other cortical areas providing synaptic input to layer 6b were S2 cortex (9%), auditory cortex (7%), posterior parietal cortex , and visual and orbital cortex (<2% each). M1 and S2 inputs are already present in the subplate/layer 6b of the developing cortex and are likely to persist into adulthood . Low numSubcortical input (including TC connections) to S1 layer 6b was very sparse, accounting for only ∼1% of total presynaptic input. Most of the subcortical afferents originated in the thalamus, in particular from the second order nucleus POm, in line with previous findings in the rat showing that some axonal collaterals from this thalamic nucleus also ramify in layer 6b, albeit at a low density, but this was less than 1% of all presynaptic neurons, in line with the low density of POm and VPM axonal boutons in this layer . FurtherNtsr1 but rather subplate/L6b markers like Cplx3  which is not part of the neocortex proper but belongs also to the periallocortex which lies in the transition zone between the neocortex and hippocampus . This coke Cplx3 , Nxph4, and Ctgf . This suCplx3 has been used to label EC-L6b neurons (Cplx3-expressing neurons are in violet). Retrograde rabies virus tracing revealed that EC-L6b neurons predominantly innervate hippocampal CA3 PCs, in addition to the canonical (perforant) pathway from EC-L2 to CA3 and the recently identified L5a PC - CA3 connection  are characterized by a fast time course and short-term synaptic facilitation. In marked contrast, EPSCs elicited by optogenetic EC-L6b stimulation have a significantly slower time course and showed almost complete short-term depression indicating that the synaptic release probability at this connection is high. This establishes the EC-L6b connections as a source of substantial excitatory input to CA3 PCs. The EC-L6b-to-CA3 connection may play a role in spatial coding, learning and memory as the decreased performance in spatial orientation and learning following optogenetic inactivation of excitatory EC-L6b neurons suggests .Apart from the strong EC-L6b-CA3 connection, EC-L6b excitatory neurons provide strong synaptic input to CA1 PCs; in addition, dentate gyrus granule cells (DGCs) and hippocampal interneurons are also sparsely innervated by EC-L6b. This suggests that EC-L6b neurons are presynaptic to all hippocampal regions with a preference for CA3 and to a lesser extent CA1. Furthermore, EC-L6b axonal projections were found to target the anterior thalamic nuclei (ATN) and the lateral septal nucleus (LS).Prominent excitatory synaptic input to EC-L6b was found to originate from the claustrum (Cla) which may share a common developmental origin with the subplate/L6b . On the From the hippocampus, EC-L6b neurons receive excitatory input from the CA1 region and the subiculum, and inhibitory input from a population of CA1 stratum oriens and radiatum interneurons. Finally, major intrinsic excitatory EC synaptic connections originate predominantly from layers 5a and 6b itself, with other layers such as layer 5b providing low density input see also .Layer 6b is under the strong influence of neuromodulatory transmitters, many of which are neuropeptides. These neuromodulators are released from various subcortical brain regions and profoundly and often selectively alter L6b synaptic signaling. L6b neurons express many neuropeptide receptors, but are also strongly affected by acetylcholine and monoamines such as norepinephrine and dopamine. Here I will mainly discuss those neuromodulatory systems in layer 6b that have been investigated in some detail.The most prominent and best studied example of L6b-specific neuromodulation is that of the neuropeptide orexin/hypocretin. Orexinergic neurons are a subset of neurons located in the lateral hypothalamus that give rise to diffuse projection to many subcortical regions including the basal forebrain, the locus coeruleus and the entire cerebral cortex. Orexin is involved in the regulation of appetite, sustained arousal and wakefulness . Direct 1R) and type 2 (OX2R) receptors, respectively. Early studies revealed that OX2Rs were highly expressed in neocortical layer 6b, but were also found in other cortical layers  and orexin-B (hypocretin-2), which bind predominantly to the two types of orexin receptors, the G-protein coupled type 1 .Cholecystokinin (CCK) was initially thought to be a gut peptide, with one of its primary roles being the regulation of digestion and food metabolism; it is found at high levels in the gastrointestinal tract. However, it was later discovered that CCK is even more abundant in the mammalian brain, particularly in the neocortex , 2021. CCCK/Cck gene is highly expressed in both hippocampal and neocortical excitatory neurons and GABAergic interneurons and here mainly in one specific interneuron subtype, the non-FS, CCK-positive large basket cell  is a classical neurotransmitter that is released from cholinergic neurons in the nucleus basalts of Meynert in the basal forebrain, which innervates every cortical area. ACh release is involved in wakefulness, arousal, REM sleep and attention . Dense c4, α5, and β2 subunit genes  were strong  to α4β2 heteromeric receptors  and nicotinic receptor channels (nAChRs). Information regarding mAChRs in layer 6b is scarce so that I will concentrate on nicotinic effects on L6b neurons. A cholinergic modulation of L6b neurons has already been suggested in early studies showing that the level of acetylcholinesterase was high in layer 6b  and thate strong . On the  neurons . The maj5 nAChRs . Importaeceptors .The ACh response in L6b excitatory neurons is strong and can lead to sustained AP firing but only when orexin-B is present (see above). This suggests an intracortical synergistic link between the orexin system and cholinergic modulation; it has been proposed that this mechanism serves to promote the transition to wakefulness and vigilance . More reIn the neocortex, dopamine is released by axonal afferents from the substantia nigra -ventral tegmental area (VTA) complex . Dopamins and Gi/o), respectively. D1-like receptors are prominently expressed in deep cortical layers including layer 6b  and the D2-like receptors (DRD2-4) which are coupled to excitatory and inhibitory G-proteins  act as neuromodulators in layer 6b, the gene of which is highly expressed in both adaptive spiking and FS L6b interneuron types ; however2R activation (Orexin appears to coordinate the release of several of the L6b targeting neurotransmitters/neuropeptides. In particular, the dopaminergic VTA-SNc complex is densely innervated by orexinergic neurons from the lateral hypothalamus , suggesttivation . Finallytivation . The acttivation . A more Thus, the orexin-neurotensin system is involved in the activation of cholinergic neurons in the basal forebrain and dopaminergic neurons in the VTA-SNc complex which show also direct interactions with one another. All of these systems appear to converge in layer 6b, making this layer a tightly controlled system. It has been argued that like L5b CT PCs, L6b CT neurons are also drivers of the POm . Howeverin vitro and in vivo in order to understand the functional role of layer 6b in the neocortex of rodents, humans and other animals.Layer 6b consists of a dense and long-range neural network that is highly specific for a given brain region. It integrates input from multiple cortical and/or subcortical regions and projects widely to adjacent areas in the neocortex and, in the case of the entorhinal cortex, to the hippocampus. Layer 6b is strongly modulated by several neuromodulatory transmitters and neuropeptides, all of which are released during different behaviors such as arousal and food intake. Further work is needed to dissect the input and output neuronal circuitry of layer 6b neurons in specific cortical areas, their similarities and differences, and to determine which neuromodulatory systems control their activity both DF: Conceptualization, Funding acquisition, Writing—original draft, Writing—review & editing.


Enterococcus faecalis in vitro. The vehicles evaluated alone and mixed with CTZ were Eugenol, propylene glycol (PG), super-oxidized solution (SOS), grapefruit-seed extract (GSE), and 0.9% saline solution as a negative control. A clinical isolate of E. faecalis was morphologically and biochemically characterized, and its antimicrobial susceptibility was tested using 20 antimicrobial agents. Once characterized, the clinical isolate was cultivated to perform the Kirby–Bauer disc diffusion method with paper discs embedded with the different vehicles mixed or used alone, and incubated at 37 °C for 24 h. Data were analyzed using one-way ANOVA, and the means were compared using Tukey test with a significance level of p < 0.05. For vehicles used alone, GSE presented the greatest inhibition showing a statistically significant difference with the rest of the vehicles. When vehicles were mixed with the CTZ paste, PG showed a greater inhibition with a statistically significant difference from the rest of the vehicles. In conclusion, the vehicle used to mix the CTZ paste plays an important role in the inhibition of E. faecalis in vitro; therefore, we consider that this can be an important factor to achieve success in the use of this technique.This study aimed to evaluate if the change of vehicle for CTZ  paste improves the inhibition of  Dental caries is an important disease in children, being the most prevalent disease in oral cavity at this stage of life . NowadayThis disease destroys dental hard tissue, affecting pulp vitality, with a high risk of the development of pulp and periapical lesions . To eradAntibiotic pastes have been proposed in endodontic therapy due to their antimicrobial capacity and low cost . If infeIn the clinic, several other types of pastes are used to disinfect the root canal system and create a favorable environment for endodontic regeneration. Triple Antibiotic Paste (TAP), composed of Ciprofloxacin, Metronidazole, and Minocycline ,22, is aEnterococcus faecalis  [Currently, it has been reported that antibiotic resistance decreases the antibacterial properties of endodontic filling pastes for root canal treatment , which haecalis) ,33,34.Nowadays, there is a tendency to evaluate natural products with antimicrobial activity against planktonic and biofilm microorganisms for therapeutic dentistry . Since wCitrus paradisi and mixing them with glycerin [Interestingly, the grapefruit-seed extract (GSE) is a natural product obtained by grinding the seeds, pulp, and white membranes from glycerin . This exglycerin  mainly aglycerin .E. faecalis, are present in endodontic infections. This one has been widely studied due to its resistance to conventional endodontic treatment [E. faecalis is a Gram-positive facultative anaerobe cocci bacterium that inhabits the human oral cavity, gastrointestinal tract, and vagina [Esp), producing a biofilm of polystyrene [2 solutions [E. faecalis, as adhesion and colonization, resistance to host defense, inhibition on other bacteria, tissue damage, and induction of inflammation summarized by Kayaoglu et al. [Several microorganisms, such as reatment , being oreatment . E. faecd vagina . This bad vagina , resist d vagina , and delystyrene , as wellystyrene , with thystyrene . It evenolutions , mostly olutions  and otheolutions . Hence, u et al. , emphasiE. faecalis by themselves or have the capacity to potentiate the antimicrobial effect of CTZ paste.Until now, no studies have been found in the literature evaluating if the change of vehicle in this paste can modify the drug delivery and potentiate its action. Thus, the aim of this study was to evaluate if these products inhibit ®, Mocorito, Sinaloa, México), SOS , and grapefruit-seed extract . Eugenol  and 0.9% saline solution (S.S.)  were used as positive and negative controls, respectively. The CTZ  was commercially obtained to avoid the variation in the formulation and the presence of excipients. The bacterium E. faecalis was donated by the Research Department of Endodontics of the Stomatology Faculty of the Autonomous University of San Luis Potosí where it was isolated from the root canal of patients with secondary endodontic infections employing sterile paper tips, and subsequently cultivated in thioglycolate tubes that were incubated in an anaerobic chamber [The evaluated vehicles were PG  for the determination of cell morphology and the classification in Gram-positive or Gram-negative bacteria using optical microscopy. Then, biochemical tests with twenty-six substrates and the antibiotic resistance of 20 drugs were determined using the kit Microscan pos combo panel type 33  following the manufacturer’s instructions.E. faecalis was cultivated in Tryptic Soy Broth (TSB)  medium evaluating absorbance at 600 nm using an Optizen pop  every hour for 12 h to establish growth kinetics. Additionally, serial dilutions were made in Trypto-casein Soy Agar plates (TSA) to evaluate colony forming units (CFUs), evaluating cell viability during the growth kinetics.An amount of 500 mg of CTZ was mixed with the vehicles, using a different amount of each , to obtaE. faecalis was adjusted to 0.5 McFarland standard by adding sterile distilled water, which corresponds to ~1.5 × 108 cells/mL. The bacterial suspension of 100 µL was distributed throughout the plate with Müller Hilton Agar  to place a triplicate of absorbent paper discs of 5 mm in diameter impregnated with each mixture or a triplicate of absorbent paper discs impregnated only with 5 µL of each different vehicle. And, as a control, discs impregnated with S.S. were employed. All plates were incubated 24 h at 37 °C. After incubation, the plates were observed, and the inhibition zone was measured with a digital vernier .The p < 0.05 using GraphPad prism v.8.For the data analysis, the means and standard deviation of the inhibition zone size were used. One-way ANOVA and Tukey tests were used with a significance level of E. faecalis showed the typical cell morphology of these bacteria as Gram-positive cocci in pairs and short chains on Gram stain  and GSE , with no significant difference to positive-control Eugenol (30.8 ± 0.1); however, PG did show a greater inhibition of bacterial growth  compared to the positive control (p = 0.1542), meaning that this vehicle does not promote a real antibacterial capacity of the CTZ paste.The results of the inhibition of bacterial growth were obtained by mixing CTZ paste with different vehicles, which, after 24 h, showed that the bacterial growth of the paste was not potentiated when mixed with SOS  has a similar effect to Eugenol on E. faecalis (14.2 ± 0.3), and a major inhibition was found using SOS  and GSE , the latter being the vehicle with the highest antibacterial effect  mixed with the paste, is due to its components, since used alone did not show any inhibition in the growth of E. faecalis (p < 0.0001), as well as SOS (p = 0.0011) and PG (p < 0.0001) increased its antibacterial potential once mixed with the paste. No significant differences were found when comparing GSE used alone or mixed with the paste (p = 0.9762). Therefore, it could be used alone as an irrigant solution since antibiotics are not necessary to improve its bactericidal capacity.The comparison of the inhibition of the bacterial growth of the vehicle alone and mixed with CTZ evidenced that the antibacterial effect, shown by the S.S.  has demonstrated to improve the diffusing property of several drugs . Remarkafaecalis . In our acterium . This beacterium , proposiS. aureus, E. coli, and including E. faecalis in vitro [E. faecalis, showing intratubular decontamination in vitro [On the other hand, in the context of its function as a vehicle, PG mixed with endodontic medicament has shown antimicrobial activity against several bacteria, such as in vitro . Previouin vitro . In thisin vitro . In our in vitro . Therefo−), chlorine (Cl2), hydroxide (OH), and ozone (O3). However, controversial results have been described about the SOS antimicrobial capacity since this solution exhibits an erratic effect in the elimination of the E. faecalis from the root canal compared to NaOCl [E. faecalis; however, its properties as a vehicle are not remarkable compared to the other materials evaluated.In addition, super-oxidized solutions have been shown to be powerful antimicrobial agents and disinfectants through oxidative damage  since elto NaOCl ,34. Our Citrus paradisi by grinding its seeds, pulp, and white membranes and mixing them with glycerin [E. faecalis, showing low [E. faecalis clinical isolate, a great inhibition was observed, which could be related to GSE concentration since previous studies were performed at a concentration of 33%; meanwhile, we employed a higher concentration (46%). Although this extract by itself obtained the highest inhibition zone compared with the rest of the vehicles, the result was not the same when mixed with CTZ, showing a similar inhibition to the recommended vehicle Eugenol; however, until now, no studies have reported the evaluation of this extract as a vehicle for CTZ. We observed that it does not improve the antimicrobial activity of the paste; therefore, we consider that it is not the ideal vehicle for the application of these antibiotics.GSE is a natural product obtained from glycerin . This exglycerin  mainly aglycerin . This cawing low  to high wing low  antibactPorphyromonas endodontalis and Porphyromonas gingivalis, showing a promising antibiofilm activity. Also, a cytotoxicity assay with human gingival fibroblast was carried out observing high biocompatibility, absent characteristic in NaOCl, since it can cause a burning sensation in gingiva and periapical tissue necrosis [E. faecalis and other bacterial strains present in the oral microbiome associated with active dental infections. However, further experiments need to be performed inside the root canal.GSE could be used alone as an irrigation solution since it has demonstrated antimicrobial activity against Gram-negative and -positive bacteria and yeast . Previounecrosis . This finecrosis . TherefoE. faecalis in vitro, placing them as alternatives to sodium hypochlorite (NaOCl) to avoid severe injuries that might occur with NaOCl accidents or in allergic patients [Due to the latter, green technology extracts could be an excellent option against the oral microbiome associated with active dental infections. Furthermore, it is an excellent option to use plant or fruit extracts to clean the root canal system from the irrigation phase. Currently, the use of blueberry and wild strawberry extracts  and enzypatients .E. faecalis in vitro. And although this is not the only microorganism that the clinician faces during pulp therapy, it is one of the most resistant. Therefore, its eradication is extremely important. Our results showed that PG could be the best vehicle for the CTZ paste. Henceforth, it is necessary to evaluate if PG mixed with CTZ maintain a higher inhibition than Eugenol against other microorganisms related to pulpal pathologies. Furthermore, we proposed GSE as an irrigation solution although further experiments need to be performed to confirm this.The vehicle used to mix the CTZ paste plays an important role in the effect of inhibition against


Many people with Tourette syndrome are able to volitionally suppress tics, under certain circumstances. To understand better the neural mechanisms that underlie this ability, we used functional magnetic resonance neuroimaging to track regional brain activity during performance of an intentional inhibition task. On some trials, Tourette syndrome and comparison participants internally chose to make or withhold a motor action (a button press), while on other trials, they followed ‘Go’ and ‘NoGo’ instructions to make or withhold the same action. Using representational similarity analysis, a functional magnetic resonance neuroimaging multivariate pattern analysis technique, we assessed how Tourette syndrome and comparison participants differed in neural activity when choosing to make or to withhold an action, relative to externally cued responses on Go and NoGo trials. Analyses were pre-registered, and the data and code are publicly available. We considered similarity of action representations within regions implicated as critical to motor action release or inhibition and to symptom expression in Tourette syndrome, namely the pre-supplementary motor area, inferior frontal gyrus, insula, caudate nucleus and primary motor cortex. Strikingly, in the Tourette syndrome compared to the comparison group, neural activity within the pre-supplementary motor area displayed greater representational similarity across all action types. Within the pre-supplementary motor area, there was lower response-specific differentiation of activity relating to action and inhibition plans and to internally chosen and externally cued actions, implicating the region as a functional nexus in the symptomatology of Tourette syndrome. Correspondingly, patients with Tourette syndrome may experience volitional tic suppression as an effortful and tiring process because, at the top of the putative motor decision hierarchy, activity within the population of neurons facilitating action is overly similar to activity within the population of neurons promoting inhibition. However, not all pre-supplementary motor area group differences survived correction for multiple comparisons. Group differences in representational similarity were also present in the primary motor cortex. Here, representations of internally chosen and externally cued inhibition were more differentiated in the Tourette syndrome group than in the comparison group, potentially a consequence of a weaker voluntary capacity earlier in the motor hierarchy to suppress actions proactively. Tic severity and premonitory sensations correlated with primary motor cortex and caudate nucleus representational similarity, but these effects did not survive correction for multiple comparisons. In summary, more rigid pre-supplementary motor area neural coding across action categories may constitute a central feature of Tourette syndrome, which can account for patients’ experience of ‘unvoluntary’ tics and effortful tic suppression. On an intentional inhibition task, in which participants choose whether to move or not, participants with Tourette syndrome show greater representational similarity of action and inhibition. This occurs specifically in the pre-supplementary motor area, highlighting this region as a functional nexus of voluntary action control in tic disorders.https://doi.org/10.1093/braincomms/fcad237) for a scientific commentary on this article.See Martino and Ganos (  Tics are caused by aberrant interactions in cortico–striato–thalamo–cortical (CSTC) motor circuitry, likely through a combination of cortical hyperactivity, and direct pathway activation through the basal ganglia.7 Many patients report using volitional tic suppression as a coping strategy in public settings where they face uncomfortable social scrutiny and stigma.9 This capacity to withhold tics volitionally is also considered when differentiating a TS diagnosis from other hyperkinetic movement disorders such as myoclonus.7 However, many people with TS report tic suppression to be a tiring and cognitively demanding process.Another interesting feature of tics, also capitalized on by behavioural therapies, is that they are often suppressible.10 Moreover, in people with TS, hyperactivity is observed in the right IFG during ‘intentional inhibition’ tasks, in which participants actively choose to withhold actions.11 The IFG is strongly associated with motor inhibition processes, including during the stop signal task, which tests externally cued stopping of motor output.13 During stopping, the IFG amplifies the neural drive from the pre-supplementary motor area (preSMA) to the subthalamic nucleus (STN), pausing motor outflow to the primary motor cortex (M1), via a route known as the hyperdirect pathway.15 Notably, during both externally cued and volitionally chosen motor inhibition, M1 activity is suppressed in non-TS individuals but is not in TS participants.11 This may explain why enhanced IFG activity is observed during tic suppression, since greater recruitment of inhibitory processes must compensate for downstream hyperactivity in M1.Neuroimaging studies suggest a basis for this effortful nature of tic suppression in TS: The inferior frontal gyrus (IFG) is hyperactive during volitional tic withholding, compared to ‘free ticcing’.16 This feature again implicates the hyperdirect pathway in pausing motor outflow by blocking the competing tic action plan in the direct pathway but not removing it.17 In order to exert this blocking effect, the IFG works together with the preSMA to amplify midline cortical drive to the STN, preventing motor output.14 Correspondingly, lesions to the preSMA disrupt stopping, slowing motor inhibition and predisposing to impulsive action.19A second subjective experience reported by patients is that during tic suppression, the tic (and premonitory urge) often does not ‘go away’ completely.21 Thus, clearly differentiated signals from preSMA down the motor hierarchy may enhance the efficient control of voluntary action, including decisions to move, decisions not to move and whether to act or inhibit responses to external cues from the environment. However, it remains unknown whether actions are represented differently within prefrontal regions, including preSMA and IFG, in the brains of people with TS. For example, are the prefrontal signals driving action and inhibition less clearly differentiated in TS? Is the lack of M1 suppression during cued inhibition a sign that representations of action and inhibition here are more similar? Is there also less differentiation of action and inhibition in TS within the basal ganglia—in particular at the striatal entry point to the direct and indirect pathways?The preSMA has both a role in stopping action and as a cardinal site of voluntary action. Neurodegenerative conditions affecting the preSMA can engender either apathy at one end of the spectrum or alien limb phenomena and stimulus utilization behaviours at the other.22 How the insula might represent information relating to decisions to move, or to suppress movement, is speculative.In addition to cortical and subcortical motor regions, increasing evidence suggests that the insular cortex interacts with downstream motor areas to cue tics in TS. This likely occurs via the generation of premonitory sensations, which then foster the production of tics in order to relieve the uncomfortable urges.23 This task design enabled simultaneous investigation of voluntary action , intentional inhibition  and externally cued action (Go) and inhibition (NoGo). It also enabled direct comparison of action and inhibition processes between people with and without TS (while a tic suppression task can only be undertaken by patients).To understand better the neural mechanisms underlying tic suppression, we applied representational similarity analysis (RSA), a multivariate pattern analysis technique, to functional magnetic resonance neuroimaging (fMRI) data acquired as people with TS and comparison (non-TS) participants undertook an intentional inhibition task. In this paradigm, a modified Go/NoGo task, participants receive movement cues to either press a button (Go), withhold their button press (NoGo) or choose for themselves whether to press or inhibit (Choose).24 RSA is one multivariate technique, ideally suited to measuring the degree of similarity in neural responses to multiple stimuli classes or task conditions.25 In RSA, correlation coefficients across voxels are computed between conditions, such as externally cued or internally chosen action and inhibition.26Multivariate pattern analysis techniques can assess the granularity of neural activity within an area, to distinguish sub-populations of neurons associated with different task conditions.1 Furthermore, we predicted that the similarity of action and inhibition neural representations in TS may relate to tic and premonitory sensation severity. Poorer discrimination between action and inhibition representations in TS may underpin the heightened cognitive effort experienced by many patients when attempting to suppress tics volitionally, through less clear-cut differentiation of action plans throughout CSTC circuitry. This lack of strongly differentiated higher-order motor action plans may worsen premonitory sensations and tic expression.We hypothesized that people with TS would differ from non-TS individuals in the representation of action and inhibition and pre-registered our analyses. We examined six key regions previously implicated in motor action release or inhibition and to tic expression in TS: preSMA, IFG, bilateral insula, caudate nucleus and M1.11 . Twenty-three individuals with TS  and 21 individuals without TS and no history of major neurological or psychiatric disorder  participated. Clinical diagnosis of TS was made by a UK neurologist or psychiatrist specialized in the assessment of TS. Patients were recruited from the Sussex Partnership NHS Foundation Trust (SPFT) Neurodevelopmental Service (psychiatrist H.D.C.) and Tourettes Action UK . Obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) diagnoses were also recorded.The participants were identical to those described in a previous report27 fMRI analyses used the symptom severity score. Premonitory sensations were assessed using the Premonitory Urge for Tics Scale (PUTS);28 OCD severity using the Yale Brown Obsessive Compulsive Scale (YBOCS);29 and ADHD severity using the Adult ADHD Self-Report Scale (ASRS).30Tic severity was assessed using the Yale Global Tic Severity Scale .Two patients were taking dopaminergic medications, six were taking serotonergic medications and one was taking both dopaminergic and serotonergic medications. One patient on sertraline also took a benzodiazepine. One patient took melatonin as a sleep aid remedy . The remaining 13 patients and all participants in the comparison group were unmedicated.et al.11). Green Go cues indicated a button press to be made with the right index finger, red NoGo cues indicated the participant should withhold their button press and yellow ‘Choose’ cues indicated participants should choose whether to press the button or withhold. There were 864 trials: 432 Go (50%), 144 NoGo (17%) and 288 Choose (33%), presented in a pseudo-randomized order. The higher frequency of Go trials was designed to invoke a pre-potent tendency for action, as in traditional Go/NoGo tasks, to ensure that withholding on NoGo trials was sufficiently challenging and thus invoked inhibitory control.23 Participants were instructed to respond quickly on Go trials, withhold button presses on NoGo trials and choose quickly, making a fresh decision each time, on Choose trials.Participants performed a modified Go/NoGo task in which movement cues  were presented on a grey background for 800 ms (http://surfer.nmr.mgh.harvard.edu/optseq) for event-related design efficiency . The task was divided into three runs of 288 trials, 10 min 42 s in duration, with breaks in between to reduce fatigue and discomfort.A white fixation cross was displayed during intertrial intervals, jittered in duration and optimized using OptSeq , using independent sample t-tests and Bayesian equivalents (applying default priors) in JASP (https://jasp-stats.org).Motor behaviour  was compared between TS and comparison participants . Two hundred and fifty-five fMRI volumes were acquired per 10 min run . The first five volumes per run were discarded for steady-state magnetization. A T1-weighted image was acquired for fMRI pre-processing . Participants’ heads were tightly cushioned within the head coil to reduce head movements.Thttps://osf.io/94ybj/). These videos, recorded concurrently with acquisition of neuroimaging data, enabled us to identify the timings of tics. This information was then used to exclude the effects of tic generation and expression from neuroimaging analyses but was not used to rate tic severity.We did not instruct participants to suppress tics. This was essential to acquire intentional inhibition task fMRI data uncontaminated by simultaneous tic suppression in TS participants. Furthermore, not instructing participants to suppress tics reduced the likelihood of associated distress and fatigue over the imaging session. Instead, we removed fMRI signal relating to generation and expression of tics during the task. We video recorded tics, time locked to fMRI data, and included tic expression as a regressor in our general linear modelling (data and scripts available at www.mrc-systems.de), mounted on the head coil to view participants’ faces, and an out-of-bore camera to view limbs and body . Camera feeds and fMRI volume markers were simultaneously relayed to Spike2 physiological recording software . During fMRI acquisition, the researcher (C.L.R.) watched the live video feeds and recorded the fMRI volumes at which she observed tics within a written record. This provided corroborative information and cover in case the video recordings were interrupted, lost or failed in another way. Storage of the video recording failed for three participants; in these cases, the written records alone identified tic onsets and durations in relation to the fMRI time series.We acquired video data using both an in-bore MRI compatible camera , tics were identified in post hoc video assessment, using the written record as a supplementary guide. Initial tic ratings were conducted by two authors , before a second rater, familiar with each patient’s tic repertoire (C.L.R.), conducted a second rating, confirming or rejecting the status of each event as a tic and identifying any tics not previously flagged by L.P. or D.E.O.L. An in-house Spike2 script extracted tic onsets and durations, time locked to fMRI data. Phonic tics were often visible from facial movement, but we did not record sound. This means it is possible that not every single tic was captured by our method; however, we believe it was as comprehensive as possible.For the majority of participants with complete video recordings . The bodily locations at which tics were expressed were, on average, 40% facial, 8% head, 8% both face and head, 33% body or limbs and 11% combinations of face, head, body and limbs.www.fil.ion.ucl.ac.uk/spm; scripts available at https://osf.io/94ybj/). Pre-processing used default options, including realignment to the mean image, slice-time correction to the middle slice, co-registration with T1 structural and MNI normalization and 8 mm smoothing.fMRI data were pre-processed, and first-level general linear models fitted using SPM12  of (i) Go, (ii) NoGo-correct, (iii) Choose-Go and (iv) Choose-NoGo trials. If participants made Go omissions or NoGo errors, regressors were added for these trial types. The general linear model of TS participants contained a further regressor for observed onsets and durations of tics. The fMRI data from the three runs were concatenated (spm_concatenate.m), adding a constant (mean) column for each of the three runs, and a ‘run transitions’ regressor modelled the transition from end of one run to the start of the next. Six realignment parameter regressors modelled head movement.et al.11 for second-level statistical analysis and univariate brain activity results).Single-regressor T-contrasts were generated for (i) Go, (ii) NoGo-correct, (iii) Choose-Go and (iv) Choose-NoGo trials, with implicit baseline of intertrial interval fixation cross. These were the key task conditions entered to RSA, the focus of this manuscript . The masks were re-sliced (to an spmT image of one subject) using SPM co-registration, prior to application in RSA (mask files available at https://osf.io/6yknx/). We pre-registered the choice of these six ROIs prior to commencing RSA (https://osf.io/hx5ja/), but after data collection and fMRI univariate analysis, as the results in Rae, Parkinson, et al.11 informed ROI selection.We focused analyses on six key regions previously identified as strongly associated with intentional inhibition task performancer cortex . We defiet al.11 , using Mhttps://osf.io/hx5ja/), but after data collection and fMRI univariate analysis, as the results in Rae, Parkinson, et al.11 informed contrast selection. For some ROIs and contrasts (34%), we specified a directional hypothesis, predicting greater similarity in TS than non-TS participants (or vice versa), on the basis of prior literature (see https://osf.io/hx5ja/). For the remaining ROIs and contrasts (66%), we did not specify direction of effect, simply hypothesizing an exploratory group difference.We focused our RSA comparisons on four key contrasts that enable investigation of externally cued and internally chosen action and inhibition: (i) Go versus NoGo, (ii) Choose-Go versus Choose-NoGo, (iii) Go versus Choose-Go and (iv) NoGo versus Choose-NoGo. In (i) and (iv), we enter only NoGo trials in which participants successfully withheld their button press . The four relevant spmT images output from first-level models were merged into one 4D file for each subject, using the FSL command ‘fslmerge’. We pre-registered the choice of these four contrasts prior to commencing RSA (31 and custom scripts in MATLAB (data and scripts available at https://osf.io/6yknx/). The first-level contrasts (t-maps) reflecting activity during (i) Go, (ii) NoGo-correct, (iii) Choose-Go and (iv) Choose-NoGo trials were entered to RSA. We investigated the similarity between spatial patterns of fMRI activity using four different representational similarity analyses,25 testing the four contrasts outlined above. For all analyses, we computed the multivoxel spatial pattern similarity across pairs of trials using correlation. The Pearson correlation values were then Fisher transformed and weighted according to a contrast matrix that identified which task conditions were to be compared. A 4 × 4 contrast matrix  enabled the specific task conditions required for each contrast to be identified, applying a contrast weighting of (1) to the relevant row and column. Fisher-transformed similarity scores were then entered to statistical analysis.RSA was conducted using the CosMoMVPA toolboxt-tests and Bayesian equivalents (applying default priors) in JASP (https://jasp-stats.org). The JASP default priors are 0.707 (Cauchy) for a t-test; 1 (stretched beta prior width) for a correlation; and 1 (prior concentration) for a χ2 test. Because our four contrasts, tested across six ROIs, led to 24 independent statistical tests, we corrected P-values for multiple comparisons, across the six ROIs, per contrast, using false discovery rate (FDR)–adjusted P-values in MATLAB .We tested group differences in representational similarity between TS and comparison participants using two-tailed independent sample P-values for multiple correlations, across the six ROIs, per contrast and per symptom score, again using FDR-adjusted P-values.To investigate whether representational similarity is associated with symptom severity, we tested for two-tailed correlations between representational similarity and (i) tic severity, as measured by YGTSS (symptom severity score), and (ii) premonitory sensation severity, as measured by PUTS, for each of the four contrasts, in each of the six ROIs. Because this led to 48 independent statistical tests (24 per symptom score), we corrected https://osf.io/hx5ja/). However, the Bayes factors (BFs) were not originally pre-registered and were added post hoc, in order to give complementary insight into potential evidence for both the null (BF10 < 0.3) and alternative (BF10 > 3) hypothesis. The BFs should therefore be considered an exploratory approach. For each test, we reject the null hypothesis when the associated P-value is <0.05 and the BF10 > 3.We pre-registered the choice of statistical tests, number of tests and FDR correction for multiple tests prior to RSA  between preSMA representational similarity and the number of tics expressed during scanning, across each of the four task contrasts. Furthermore, we tested if medication status interacted with RSA scores in the TS participants, by comparing preSMA representational similarity scores between medicated and unmedicated participants, for each of the four task contrasts, using (frequentist and Bayesian) two-tailed t-tests and two-tailed χ2 tests  to identify whether these two sub-groups of TS individuals were significantly different with regard to (i) tic severity (YGTSS), (ii) premonitory sensation severity (PUTS), (iii) frequency of tic expression, (iv) medication status, (v) ADHD diagnosis, and (vi) OCD diagnosis.Also at the suggestion of a reviewer, we explored the possibility that there are characteristic features of the sub-groups of TS participants that were potentially driving significant group differences by scoring beyond the range of comparison participants. To do so, we identified the TS individuals who scored above the preSMA RSA comparison maximum , across the four task contrasts. In addition, we took a similar ‘sub-group’ approach as outlined above, using two-tailed t = −0.924, P = 0.361, BF10 = 0.420), nor did they make more NoGo errors . TS participants made slightly more Go omissions . Reaction times did not significantly differ between groups (TS participants did not choose to make or withhold actions (button presses) more often than the comparison group . Externally cued action and inhibition were not significantly different between TS and non-TS participants in any of the other ROIs .The first RSA computed similarity of externally cued action (Go) and externally cued inhibition (NoGo-correct), for the six ROIs . Represet = −2.721, P = 0.009, BF10 = 5.141; although this group difference was no longer significant after correcting for multiple comparisons across the six pre-registered ROIs, pFDR = 0.057). Chosen action and inhibition were not significantly different between TS and non-TS participants in any of the other ROIs .The second RSA computed similarity of internally chosen action (Choose-Go) and internally chosen inhibition (Choose-NoGo) . Represet = −2.647, P = 0.011, BF10 = 4.445; although this group difference was no longer significant after correcting for multiple comparisons across the six pre-registered ROIs, pFDR = 0.068). Externally cued and chosen action were not significantly different between TS and non-TS participants in any of the other ROIs .The third RSA computed similarity of externally cued (Go) and internally chosen (Choose-Go) action . Represet = −3.062, P = 0.004, BF10 = 10.466; and this group difference remained significant after correcting for multiple comparisons across the six pre-registered ROIs, pFDR = 0.023). In addition, there was a significant group difference in M1, in the opposite direction: representations of externally cued and chosen inhibition were more differentiated here in TS than in non-TS participants . Externally cued and chosen inhibition were not significantly different between TS and non-TS participants in the other four ROIs .The fourth RSA computed similarity of externally cued (NoGo) and internally chosen (Choose-NoGo) inhibition . Represer = 0.427, P = 0.042, pFDR = 0.252, BF10 = 1.817), and the greater the similarity within M1 between externally cued (Go) and internally chosen (Choose-Go) action, the worse the tic severity . However, neither of these correlations remained significant after correcting for multiple tests across the six pre-registered ROIs.In TS participants only, we tested for two-tailed correlations between similarity of representations in each of the four contrasts and tic severity, as measured by YGTSS . This idr = 0.513, P = 0.012, pFDR = 0.072, BF10 = 4.943), although this was no longer significant after correcting for multiple tests across the six pre-registered ROIs.In TS participants only, we tested for two-tailed correlations between similarity of representations in each of the four contrasts and premonitory sensation severity, as measured by PUTS . This id10 < 0.3). This suggests that frequency of tic expression did not relate to RSA scores.To examine if the propensity to tic frequently during the task affected RSA findings, we further tested for two-tailed correlations between preSMA representational similarity and the number of tics expressed during scanning, across each of the four task contrasts , although BFs for all four t-tests were inconclusive (between 0.3 and 3).Furthermore, we tested if preSMA representational similarity differed between medicated and unmedicated participants, for each of the four task contrasts . For thrt-tests or χ2 tests , indicating that the data are insufficient to conclude whether the TS participants scoring above the comparison participant RSA maximum formally differ from those scoring equal to or less than the comparison maximum.Also at the suggestion of a reviewer, we explored the possibility that sub-groups of TS participants, i.e. those with RSA scores greater than the maximum comparison group score, were potentially driving the significant preSMA group difference results. We compared ‘greater’ and ‘overlapping’ sub-groups of TS participants for differences in (i) tic severity (YGTSS), (ii) premonitory sensation severity (PUTS), (iii) frequency of tic expression, (iv) medication status, (v) ADHD diagnosis, and (vi) OCD diagnosis. None of the χ2 tests  were sigr = −0.421, P = 0.046), but this would not pass correction for multiple comparisons with FDR. Moreover, ∼40% of the BFs in this analysis suggested evidence for the null (BF10 < 0.3).We tested for two-tailed correlations between preSMA RSA scores and six behavioural measures , across the four task contrasts . Only ont-tests to identify whether the two sub-groups of TS individuals scoring above or less than the comparison maximum showed significantly different behavioural performance , but this would not pass correction for multiple comparisons with FDR, and all BFs in this analysis were inconclusive (between 0.3 and 3).We undertook an additional ‘sub-group’ approach similar to that outlined above, using two-tailed formance . Only onCollectively, these analyses indicate that there is no strong evidence for an association between behavioural performance and RSA results in TS participants.We compared representational similarity of action and inhibition between people with and without TS, finding that across every task condition, the preSMA showed greater representational similarity in TS. Whether comparing externally cued action and inhibition, internally chosen action and inhibition, externally cued and chosen action or externally cued and chosen inhibition, multivariate representations were more similar in TS. This highlights the preSMA as a nexus of less clearly differentiated action and inhibition plans in TS. We also observed an interesting reversal of this pattern in M1, with more differentiated representation of externally cued and chosen inhibition here in TS than in non-TS participants. However, it is worth noting that not all group differences survived correction for multiple comparisons. Finally, we investigated whether representational similarity was associated with symptom severity: three correlations suggested that a patient’s tics and premonitory sensations are worse when there is greater similarity in M1 and in the caudate nucleus, between externally cued action and inhibition, and between externally cued and chosen action .Overall, our findings suggest that while regions such as IFG and M1 may differ at the univariate level in general amount of activity, within preSMA, sub-populations of neurons show less clear-cut differentiation when coding for different action processes in TS. This blurring of action and inhibition within the preSMA of people with TS may explain, in part, the subjective experience of many patients that volitional tic suppression is a tiring and effortful process—because, at the top of the motor decision hierarchy, activity in the population of neurons signalling action is more similar to activity from the population of neurons signalling inhibition.11 However, RSA is insensitive to the overall average activity within a region, and thus, it is unlikely that these univariate activations are driving the similarity scores reported here.Our key finding that the preSMA showed elevated similarity of action representations in TS may arise through a number of mechanisms. Previously, we observed that the preSMA was differentially activated for different conditions , in both TS and comparison participants.per se. However, this would still suggest that the difficulty should be similar across conditions. Overall, these results suggest that the way in which volitional and externally driven action and inhibition plans are represented are less distinct, in terms of sub-populations of voxels, in TS, regardless of overall levels of activity.Our results suggest that action plans are more poorly distinguished in individuals with TS. This could be due to the preSMA having ‘poor eyesight’, i.e. is less well able to differentiate between the different conditions. However, it is unclear what led to this poorer discrimination across conditions. One possibility is that non-TS individuals exhibit similar brain pattern activations to the TS participants, just with more added noise. Additionally, higher similarity scores in TS participants could be driven by a sub-population of voxels responding to task difficulty, rather than representing the action plans 33 and electrical stimulation here famously elicits the urge to move.34 Given the preSMA’s role in intentional action and inhibition, it almost certainly underpins a TS patient’s decision to release, or to withhold, a tic.17The preSMA is critical to choosing whether to act or not. The preSMA has been identified in fMRI meta-analysis as a substrate for ‘whether’ action decisions,35 Some evidence suggests that people with TS show readiness potentials prior to the release of tics,37 indicating, in line with many patients’ subjective reports, that (some) tics are ‘somewhat-voluntary’ responses to an involuntary premonitory urge to move. This has led some authors to describe tics as ‘unvoluntary’.3The readiness potential is a preSMA-generated event-related potential associated with voluntary decisions to move. It can be explained by an accumulation of neural evidence to threshold, at which point one commits to move.38 However, greater similarity of volitional action and inhibition signals in the preSMA, as observed here, may reduce the strength of signalling to basal ganglia during intentional tic suppression. As a result, the IFG compensates by amplifying its output, accounting for the IFG hyperactivity during intentional inhibition observed in our previous univariate analysis.11 We suggest that this IFG hyperactivity, compensating for the reduced granularity between intentional action and inhibition in the preSMA, underlies the feeling of cognitive effort reported by patients.We found that representations were more similar in the preSMA in TS for intentional action (Choose-Go) and intentional inhibition (Choose-NoGo). This less clear-cut differentiation of action plans to move, or to withhold, may lead to a noisier accumulation-to-threshold process, generating a subjective experience of ‘unvoluntariness’ during tics. Reduced differentiation of representations may also account for the subjective experience reported by many people with TS that volitional tic suppression is a tiring and cognitively demanding process. During motor inhibition, the preSMA works with the IFG to direct the basal ganglia to pause motor outflow.While most preSMA group differences did not survive correction for multiple comparisons, it is notable that, in addition to the comparison between volitional action (Choose-Go) and inhibition (Choose-NoGo), we also observed greater representational similarity in the preSMA for each of the other three pairs of response conditions examined, namely, externally cued action and inhibition (Go and NoGo), externally cued and chosen action (Go and Choose-Go) and externally cued and chosen inhibition (NoGo and Choose-NoGo). Thus, inflexible or homogeneous preSMA representations across movement categories may constitute a central feature of TS.40 Interestingly, unless there is comorbid ADHD, externally cued inhibition is not impaired in TS41, a finding replicated here, as TS participants made no more NoGo errors than non-TS individuals . Greater representational similarity between Go and NoGo trials in the preSMA therefore may not have significant ramifications for successful performance of the NoGo task in TS. Arguably, lateral prefrontal cortex resources are more important for environmentally signalled response inhibition .41Externally cued inhibition, as measured by contrasting Go versus NoGo, is supported by engagement of preSMA and dorsolateral prefrontal cortex.11 Speculatively, the experience of urge-driven movements over many years may erode the sense of agency and volition over one’s actions in general, leading to the attrition of distinct voluntary action representations in the preSMA. Alternatively, less representational distinction between internally chosen and externally cued movement plans may be a defining characteristic of this developmental condition, rather than arising through acquired adaptive processes during the life course. The testing and replication of our RSA results in children or adolescents with TS will help resolve such questions.Our final two contrasts (Go versus Choose-Go and NoGo versus Choose-NoGo) compared externally cued instructions with internally chosen action or inhibition. Here, the critical difference is an internal choice over whether to move or not versus the production or withholding of an action in response to an external cue . It is curious, therefore, that the preSMA, the cardinal site of voluntary action, differentiates less strongly between externally cued and internally chosen action and inhibition in TS. An explanation may be that TS patients have a developmental, chronic, sometimes lifelong, experience of tics as ‘unvoluntary’ actions that are partly cued by bodily associated premonitory urge sensations. Notably, we previously observed (in the same data set) that strength of connectivity between the preSMA and the basal ganglia  correlated with premonitory urge severity.42In addition to the preSMA, we observed one other group difference in representational similarity: Activity in M1 during externally cued and internally chosen inhibition was more similar in non-TS participants than TS. Potentially this is a consequence of aberrant preSMA representation, suggested above. However, this greater distinction in TS between inhibiting in response to environmental cues, versus chosen inhibition, may alternatively reflect many patients’ everyday practice of active tic suppression . M1 may become tonically tuned to suppress activity levels in this volitional state, in contrast to the more reflexive process of inhibiting in response to external cues. Indeed, applying TMS to M1 during tic suppression reveals reduced corticospinal excitability in TS.44 Varying developmental time courses in the plasticity of GABAergic interneurons within M1 may underpin this observation.46 We also observed an association between premonitory sensation severity and the similarity of representations within the caudate nucleus. Notably, all correlations were positive, such that more severe symptoms were linked to greater representational similarity. A lack of distinction between motor plans may increase the likelihood of tic expression, or, alternatively, the greater expression of tics may lead to the confusion of action plans. Unfortunately, correlational relationships cannot distinguish between these alternative accounts.Interestingly, it was in M1 that we also observed correlations between representational similarity and tic severity. Tic severity has previously been found to correlate with M1 structural and functional connectivity, highlighting a relationship between symptom expression and the integrity of this area.10 < 3) which, combined with the overall relative lack of symptom severity correlations across several ROIs and comparisons, indicates that representational similarity of action and inhibition has comparatively weak bearing on variability in symptom expression. Our limited sample size may also have constrained our power to detect weak relationships between RSA findings and symptom severity. Nevertheless, our sample had high symptom heterogeneity  targeted approach. However, recent promising neurostimulation interventions are directed at SMA (rather than preSMA)10 < 0.3) or experimental (BF10 > 3) hypothesis, which we therefore presented alongside corrected and uncorrected P-values. Notably, all four preSMA group differences showed a BF10 > 3. We also pre-registered a set of directional hypotheses based upon prior literature; none of these showed a significant difference. The group differences that we did observe were in regions and contrasts that we did not specify an a priori direction for (TS > comparison or comparison > TS). Thus, we note the exploratory nature of these results.With six (pre-registered) ROIs examined across four contrasts, we conducted a number of independent statistical tests and therefore pre-registered a FDR approach to adjust for multiple comparisons. When applying this correction, several results were no longer significant, although it is compelling that the preSMA was so consistently identified as altered in representational similarity across contrasts. Bayesian statistics (BFs) provide complementary information, indicating the weight of evidence for either the null  that might drive the overall group differences in preSMA. All tests returned inconclusive BFs (between 0.3 and 3), indicating that the data are insufficient to conclude if TS participants scoring above the comparison participant RSA maximum differ  to those scoring equal to or less than the comparison maximum. Given the limited sample size, it will be valuable in future investigations to enlarge the cohort, with a potentially stratified sample, in order to characterize sub-groups in greater depth. In addition, it will be valuable to seek replication of our RSA results in children or adolescents, to ascertain the extent to which greater similarity scores are driven by longer life experience of urge-driven ‘unvoluntary’ tics in adults with TS.Notably, group differences were seemingly driven by sub-groups of TS participants, who deviated from the non-TS participants more markedly than the rest of the group. RSA values in many TS individuals overlapped with values in the non-TS participants. This reflects the high patient heterogeneity that is characteristic of TS, although it is curious that there were relatively few correlations between RSA values and symptoms, suggesting that the group differences are not related to severity of TS 52 As mentioned above, larger samples are required to tease apart such potential effects of medication on RSA scores.It will also be valuable for future studies to assess the potential influence of medication on RSA scores. In an exploratory analysis, we saw that for one contrast (Go versus NoGo), patients taking psychoactive medications had higher preSMA RSA scores than unmedicated participants. As a canonical test of externally cued response inhibition, this contrast may relate most strongly to executive function, which is known to be impacted by noradrenergic and serotonergic medications.23 This factorial design gives maximum analytical potential to investigate contrasts amongst externally cued and internally chosen action and inhibition. In addition, this has the advantage of being a task that both TS and non-TS groups can do, enabling direct group comparison, which a tic suppression paradigm does not. However, our task has arguably lower ecological validity than other approaches to measuring intentional inhibition, such as withholding eye blinks, which capture an urge to blink akin to an urge to tic.53 However, successful trial numbers can vary between participants, and such tasks do not always include the ability to test response inhibition concurrently, as we do with NoGo trials. Nevertheless, it would be interesting to apply RSA to eye blink suppression fMRI data to examine how results replicate across paradigms. It would also be useful to apply RSA to other task-based fMRI studies of TS to understand how (dis)similarity of representations in other key areas such as insular cortex is altered.We adapted a Go/NoGo task, incorporating Choose trials on which participants decided whether to press the button or not.In TS, representations of action plans within the preSMA manifest heightened similarity across all four categories of externally cued and internally chosen action and inhibition. Our results suggest that in people with TS, signals from preSMA to subcortical nuclei to pause motor outflow are likely to be less well defined. This difference may explain why greater prefrontal activity is required in TS in order to withhold actions, such as those occurring during tic suppression, accounting for the subjective experience of many patients that the volitional withholding of tics is an effortful and cognitively demanding process.fcad224_Supplementary_DataClick here for additional data file.


Axin  is a central negative regulator of the proto-oncogenic Wnt/β-catenin signaling pathway, as axin condensates provide a scaffold for the assembly of a multiprotein complex degrading β-catenin. Axin, in turn, is degraded through tankyrase. Consequently, tankyrase small-molecule inhibitors block Wnt signaling by stabilizing axin, revealing potential for cancer therapy. Here, we discovered that axin is phosphorylated by casein kinase 1 alpha 1  at an N-terminal casein kinase 1 consensus motif, and that this phosphorylation is antagonized by the catalytic subunit alpha of protein phosphatase 1 . Axin condensates promoted phosphorylation by enriching CK1α over PP1. Importantly, the phosphorylation took place within the tankyrase-binding site, electrostatically and/or sterically hindering axin–tankyrase interaction, and counteracting tankyrase-mediated degradation of axin. Thus, the presented data propose a novel mechanism regulating axin stability, with implications for Wnt signaling, cancer therapy and self-organization of biomolecular condensates. Highlighted Article: Within biomolecular condensates the Wnt pathway regulator axin is N-terminally phosphorylated by CK1α, which impairs axin–tankyrase interaction and protects axin from tankyrase-mediated degradation. The Wnt/β-catenin signaling pathway controls patterning of body axes during embryonic development as well as stem cell fate in diverse adult epithelia, explaining its pivotal roles in tissue homeostasis and regeneration . Not surAxin has a highly related homolog, axin2 , similarly promoting β-catenin degradation . In contHere, we used human cell lines to study a variant of phosphorylated axin (hereafter referred to as phospho-axin) that became apparent as distinct second band with reduced electrophoretic mobility on western blots. Axin was phosphorylated by casein kinase 1 alpha 1  and dephosphorylated by the catalytic subunit alpha of protein phosphatase 1 , and axin condensates favored phosphorylation by enriching CK1α over PP1. We then mapped the phosphorylation site to an N-terminal CK1 phosphorylation consensus motif overlapping with the tankyrase-binding site, which was not conserved in axin2. Importantly, axin phosphorylation sterically and/or electrostatically hindered tankyrase binding, counteracted tankyrase-mediated degradation and increased axin stability.In cell culture assays with transiently expressed axin, we noticed a distinct double band for axin on western blots . The axiRGS) with diffuse cellular distribution  also abolished phosphorylation, supporting a role of condensates in phosphorylation. Interestingly, the upper axin band also disappeared upon co-expression of the positive Wnt pathway regulator dishevelled 2 , which is indicative of decreased phosphorylation  showed only a single band on western blots, its molecular mass being similar to that of calf intestinal phosphatase-treated, dephosphorylated wild-type (WT) axin, indicating that axin M3 was not phosphorylated . Howeverribution  because ribution . It is nrylation . Like axrylation , 2005. Arylation , as had rylation . Notablyrylation , potentirylation , Dvl2 miTo map the crucial site(s) within axin that are phosphorylated by CK1, we used axin mutants comprising N-terminal deletions, which did not compromise condensate formation through the C-terminal DIX domain . An axinRGS (1-75) completely abolished phosphorylation  and Lipofectamine 2000  and transfection of small interfering RNAs (siRNAs) was performed with Oligofectamine (Thermo Fisher Scientific). The CK1 inhibitors D4476 (ab120220) and IC261 (ab145189) were obtained from Abcam  the GSK3 inhibitor BIO (B1686), the PP1 and PP2A inhibitor okadaic acid (O7885), the tankyrase inhibitor G007-LK (5.04907) and the protein synthesis inhibitor cycloheximide (239765) from Sigma-Aldrich . For treatment of cells, the inhibitors were added to the cell culture medium. Inhibitor concentrations and treatment times are stated in the figure legends.Cells of the human cell lines HEK293T, SW480 and U2OS were cultivated in Dulbecco's modified Eagle Medium supplemented with fetal bovine serum and antibiotics at 37°C in a humidified 10% COCells were seeded and transfected the following day with a combination of plasmid DNA and siRNA, as indicated in the relevant figure legends, using Lipofectamine 2000 according to the instructions of the manufacturer . siRNA sequences are provided in the ‘Antibodies and siRNAs’ section below. The cells were lysed 48 h after transfection and subjected to western blotting (see below).Cells were seeded, transfected the following day, when required, and lysed at day three in hypotonic lysis buffer . Treatments of cells or cell extracts are stated in the figures and figure legends. For calf intestinal phosphatase (CIP) treatment, cell extracts were treated at 37°C for 1 h with 0.5 U/µl CIP . Extracts referred to as ‘untreated' in the figure legends were kept at 4°C on ice, as routinely done during cell lysis. For immunoprecipitation , Flag-TNCells were seeded, transfected the following day and stained on day three. For this, cells were fixed in ice-cold methanol  or 3% paRGS (GFP-AC69-196), GFP-cAxinDIX (GFP-AC700-840), GFP-cAxin1-75 (GFP-AC1-68), GFP-axin2 , GFP-axin2 M3 , CFP-Dvl2 (mouse) and HA-Dvl2 (mouse), as previously described , cyan fluorescent protein (CFP) or the Flag-tag, were: GFP-axin , GFP-axin M3, Flag-axin, Flag-axin M3, GFP-axin 89-827, GFP-cAxinescribed . GFP-axiemplates . GFP-axiPrimary antibodies were rabbit anti-axin1 ; rabbit anti-phosphorylated-β-catenin , mouse anti-GFP , rat anti-HA High Affinity , rat anti-α-tubulin , rabbit anti-Flag .Antibodies against endogenous proteins (anti-axin1 and anti-phosphorylated β-catenin) were validated at our laboratory by knockdown experiments. Antibodies against tags  were validated by the absence of signal without the transient expression of tagged proteins.Secondary antibodies were goat anti-rabbit-Cy3 (1:300), goat anti-rat-Cy2 (1:200) goat anti-mouse/rabbit/rat-HRP (between 1:1000 and 1:2000), all obtained from Jackson ImmunoResearch.siRNAs targeting human CK1α , CK1δ  and CK1ε  have been described previously .CSNK1A1 (CK1α), CSNK1D (CK1δ), CSNK1E (CK1ε) and GAPDH (housekeeping gene) was determined by SYBR Green-based qPCR  with gene-specific primer pairs in technical triplicates . The expression of the CK1 isoforms is presented relative to GAPDH.Cells were lysed 48 h after siRNA transfection, total RNA was extracted using the RNeasy Mini Kit following the manufacturer's instructions  and transcribed into cDNA . The relative abundance of CK1 transcripts, i.e. of For the alignment of protein sequences, the Clustal Omega multiple sequence alignment program was used . The UniThe crystal structure of the axin–tankyrase complex  was analt-tests were performed in a paired fashion owing to the experimental setup. Based on the nature of the assays and graphical assessment, we assumed normal distribution of the data, which was not formally tested owing to the small sample sizes. All n values are explicitly stated in the figure legends, and statistical significance is indicated by asterisks in the figures , when required. Sample sizes between three and four were chosen depending on the differences in exploratory experiments and the variability of the assay. The sample sizes are within the range found in published literature with similar methodologies, and no data of decent experimental quality was excluded. Samples, i.e. seeded cells, were allocated randomly into experimental groups. The investigator was blinded when quantifying phenotypes at the microscope (To probe the datasets for statistical significance two-tailed Student's croscope .Click here for additional data file.10.1242/joces.261214_sup1Supplementary informationClick here for additional data file.


Since its initial discovery more than 40 years ago, a series of significant discoveries and innovative research established a central role for glycogen synthase kinase 3 (GSK3) in many critical cellular processes, including gene expression, metabolism, cellular transport, cell proliferation and apoptosis. GSK3 is a highly conserved serine/threonine protein kinase that is present in all eukaryotes. GSK3 has a broad substrate range and is ubiquitously expressed. Interestingly, GSK3 is present at high levels in brain and specifically phosphorylates numerous neuronal proteins implicating it in a variety of neurobiological functions such as axonal transport, neuronal development, neuronal polarization as well as in the regulation of synaptic plasticity and memory formation , the RNA-binding protein FXR1, kinesin subunits, and the cytoskeletal regulator CRMP2. Indeed, understanding the mechanistic link between GSK3 inhibition and the therapeutic effects of lithium may prove critical to identify novel, more specific drug targets and to provide a rationale for the inhibitory action of ketamine and other antipsychotics toward GSK3.Over the years, an enduring and far-reaching effort to develop specific GSK3 inhibitors has been driven by a number of critical discoveries. In contrast to most protein kinases, GSK3 can be active in its basal state and is inhibited in response to various physiological stimuli. Consistently, dysregulation and hyperactivation of GSK3 have been associated with numerous, diverse pathophysiological changes, which continuously prompted the demand for diverse, selective, and potent GSK3 inhibitors. The first GSK3 inhibitor discovered, was the cation lithium , a molecule that has been critically implicated in Alzheimer's disease amongst other CNS disorders. This study also uncovered differential effects of the two tested GSK3 inhibitors on some of the read-outs, highlighting common complications encountered with pharmacological inhibitors. Functional differences between inhibitors may be due to a variety of factors, such as different specificity, off-target effects and altered bioavailability. Many commonly used inhibitors are directed toward the ATP binding site of a specific kinase. However, as ATP binding domains exhibit a high degree of structural homology between kinases, confounding side effects due to cross-reactivity are a common problem of this type of inhibitors.Taking advantage of an animal model for Fragile X Syndrome, a genetic condition causing intellectual disability, Di Re et al. examined the effect of pharmacological inhibition of GSK3 and of its inhibitory upstream regulator, Akt, on the functionality of the axon initial segment (AIS) that is required for neuronal firing. GSK3 inhibition induced no major changes, whereas Akt inhibition resulted in increased excitability in primary hippocampal neurons and altered subcellular localization of βIV spectrin.Lee et al. investigated the effect of pharmacological GSK3 inhibition on synaptic plasticity and memory formation in vivo in this Topic. Systemic administration of a selective GSK-3 inhibitor, CT99021, reversibly blocked NMDAR-dependent long-term depression (LTD) in the CA1 region of the hippocampus and facilitated learning in the Morris water maze (MWM) and T-maze. These data suggest that GSK3 may be involved in the fine-tuning of spatial memory acquisition and recall.The GSK3 inhibitor, CT99021, while selective for GSK3, does not discriminate between the two GSK3 isozymes that are generated from distinct genes. The two GSK3 isoforms, termed α and β, share high levels of homology, feature near identical substrate specificity and hence may have largely a common substrate range. Nevertheless, it has also become evident that there are GSK3 isoform-specific substrates and functions. Indeed, GSK3 isoform-inhibitors have been developed. In addition, there might also be functional interplay between the two GSK3 isoforms, which adds an additional layer of complexity to the analysis of GSK3 functions in neurobiological and pathophysiological processes.Amini et al. evaluated long-term potentiation (LTP) in CA3-CA1 of the hippocampus from conditional GSK3α and GSK3β knockout mice in this Topic. Deletion of GSK3α in CA1 pyramidal neurons resulted in facilitation of CA3-CA1 LTP, whereas GSK3β deletion had no effect on LTP. Basal synaptic properties were not affected by either GSK3 deletion. These results support the notion that GSK3α plays a specific role in regulating CA1 LTP and adds a further example of diverging functions between GSK3 isoforms.In order to gain insight on GSK3 isoform-specific roles in synaptic plasticity, But as occurs so often in research and is highlighted by the articles in the Topic, finding a novel answer raises a myriad of new, unsolved questions. In fact, despite a considerable research effort the neurobiological and pathophysiological roles of GSK3 remain still poorly understood. The complex biology of GSK3, including two isoforms, diverse regulatory mechanisms, countless substrates and its involvement in many different cellular functions, makes this a challenging research field and progress is hard to come by. Over the last few years, progress has been further hampered due to a shift in research focus away from investigating the mechanistic basis of physiological and pathophysiological processes as well as to changing funding priorities by sponsoring bodies.As we reflect on the past 40 years, there have been tremendous advances that single out GSK3 as a relevant, druggable target. Future research will unequivocally progress our mechanistic understanding of GSK3 functions and promote the development of GSK3-based therapeutic applications.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.


Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised individuals. A. baumannii forms biofilm and produces extracellular matrix, which supports bacteria to survive under harsh conditions and be resistant to antibacterial treatments. In the present study, we investigated the biofilm and quorum-sensing inhibitory effects of antimicrobial peptide, octopromycin in A. baumannii. Field emission-scanning electron microscopy results clearly showed significantly reduced biofilm mass and caused a collapse in biofilm architecture at the minimum inhibitory concentration (50 µg/mL) and minimum bactericidal concentration (200 µg/mL) of octopromycin. Antibiotic-resistant persister cells of A. baumannii were successfully killed by octopromycin treatment, and it inhibited violacein production in Chromobacterium violaceum in a concentration-dependent manner. Octopromycin also inhibited alginate production, surface movements (swarming and swimming), and twitching motility of A. baumannnii, confirming its anti-quorum-sensing activity. Multiple metabolic pathways, two-component regulation systems, quorum-sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were strongly affected by octopromycin treatment. The collective findings indicate that the antibacterial peptide octopromycin may control A. baumannii biofilms through multi-target interactions. Octopromycin could be a desirable therapeutic option for the prevention and control of A. baumannii infections. Acinetobacter baumannii is a short, round, rod-shaped, Gram-negative, opportunistic pathogen -l-homoserine lactone . Twenty microliters of octopromycin at MIC (50 μg/mL) and MBC (200 μg/mL) levels were loaded on the sterile discs, dried, placed on the agar plates, and incubated at 25 °C. The plates were observed for violacein inhibition, which was evident as obscure, colorless areas around the discs. Sterilized distilled water and CHL (100 µg/mL) were used as the negative and positive controls, respectively.o operon . The QS  et al.,  with sliC. violaceum was quantitatively analyzed using the MTP method. Briefly, 1% of the overnight grown culture was inoculated into TSB supplemented with 10 μM OHL and plated in a 96-well microtiter plate. Wells were added with octopromycin at different concentrations (0–300 μg/mL), incubated at 25 °C for 24 h, and extracted for violacein pigment [g for 5 min to precipitate violacein. The obtained pellet was dissolved in dimethyl sulfoxide (1 mL) and vortexed robustly to completely solubilize violacein. The mixture was centrifuged to remove the bacterial cells and quantified at 585 nm using a microplate reader . The experiment was repeated in triplicate. The percentage of inhibition was calculated using the following formula:Violacein inhibition by octopromycin in  pigment . One milA. baumannii was subcultured in fresh TSB that was not supplemented or supplemented with octopromycin (0–300 μg/mL). The inoculated broths were incubated overnight at 25 °C in a shaker incubator (180 rpm). After incubation, alginate production was estimated. Six hundred microliters of boric acid-sulfuric acid solution in a 4:1 ratio was slowly added to 70 μL of the test sample and placed in an ice bath. The mixture was vortexed for 10 s and placed in an ice bath. To this mixture, 20 μL of 0.2% carbazole  dissolved in ethanol was added and vortexed for 10 s. The mixture was incubated at 55 °C for 30 min. Absorbance was measured at 530 nm using a microplate reader . The percentage of alginate inhibition was calculated using the following formula:The effect of octopromycin on the disruption of biofilms due to the inhibited production of low molecular weight alginate oligosaccharide was determined as described by Gholami et al.  with somA. bauamnnii motility was determined by swimming and swarming motility assays, as described by Gholami et al. [A. baumannii was point inoculated at the center of swimming agar  and swarming agar . The agar also contained MIC (50 μg/mL) and MBC (200 μg/mL) levels of octopromycin. Inoculated plates were incubated at 25 °C for 24 h. Agar media without octopromycin and CHL (100 µg/mL) were the negative and positive controls, respectively. The twitching motility assay was conducted as described by Weiwei et al. [A. baumannii was picked from a normal TSA agar plate and inoculated vertically at the bottom of the plate to facilitate growth at the intersection of the bottom of the agar and the culture plate. The plates were incubated at 25 °C for 20 h. Agar medium without octopromycin and CHL (100 µg/mL) were used as negative and positive controls, respectively.The inhibition of i et al. . For suri et al.  with somp ≤ 0.05.Data were analyzed using GraphPad Prism software version 5 . Data analysis was performed using one-way and/or two-way analysis of variance (ANOVA) to determine the significance between the experimental groups and/or time points. Bonferroni post hoc and/or unpaired two-tailed t-tests were conducted to compare the average means of the controls and treatments. The cutoff for the significance level was set at A. baumannii. FE-SEM analysis results revealed significant inhibition of biofilm mass, bacterial density, and structure at low doses (50 and 200 µg/mL) of octopromycin. The formation of persister cells in biofilms involves adaptation to environmental changes. Regarding the various virulence factors present in Gram-negative bacteria, QS systems protect against antibiotic pressure to form biofilms. The anti-QS activity of octopromycin was confirmed through violacein production in C. violaceum as part of the biofilm inhibition by affecting bacterial cross-talk. Moreover, anti-QS activity was further shown to involve inhibition of alginate production, and swimming, swarming, and twitching motility behaviors in A. baumannii treated with octopromycin. The collective findings indicate the potential of octopromycin as an AMP in the inhibition of biofilm formation by pathogenic bacteria.In this study, we systematically investigated the inhibitory effects of biofilm formation and QS using in vitro assays to reveal the antibiofilm potential of octopromycin against MDR