Evaluation of an MRI receive head coil for use in transcranial MR guided focused ultrasound for functional neurosurgery.

BACKGROUND: Trans-cranial MR guided focused ultrasound (tcMRgFUS) ablation targets are <5mm from critical neurological structures, creating a need for improved MR imaging and thermometry. The purpose of this study was to evaluate the performance of a dual-channel radiofrequency receive-only head coil designed specifically for integrated use in tcMRgFUS. METHODS: Imaging used a 3 T MRI and the ExAblate Neuro System (INSIGHTEC Inc., Israel). Sensitivity maps determined receive-only coil uniformity. The head coil was compared to the volume body coil at the level of the thalamus using 1) T2-weighted imaging and 2) multi-echo MR thermometry of volunteers in the transducer helmet. Thermal sonications (40 W, 24s) were acquired in a heating phantom. Thermal maps in were constructed to evaluate temperature uncertainty, focal heating, and temperature evolution. RESULTS: The normalized signal intensity showed up to a 35% variation. On T2wFSE images the SNR with the head coil is improved by 4x in the axial plane, and 3x in sagittal and coronal planes. The head coil provided better visualization of the thalamus and globus pallidus (axial), and of the anterior/posterior commissure, and brain stem/cerebellum (sagittal) compared to the body coil. MR thermometry showed a 4x gain in SNR in the thalamus. Thermometry showed a preserved focal spot with 20 °C temperature rise. The average temperature uncertainty (mean ± std) was reduced from σ T = 0.96 °C ± 0.55 °C for the body coil to σ T = 0.41 °C ± 0.24 °C for the head coil. CONCLUSIONS: Greater SNR from the dual-channel head coil provides access to better treatment day visualization for treatment planning and higher precision intra-operative thermometry.

Technical Comparison of Treatment Efficiency of Magnetic Resonance-Guided Focused Ultrasound Thalamotomy and Pallidotomy in Skull Density Ratio-Matched Patient Cohorts.

OBJECTIVE: MR-guided focused ultrasound (MRgFUS) is increasingly being used to treat patients with essential tremor (ET) and Parkinson's disease (PD) with thalamotomy and pallidotomy, respectively. Pallidotomy is performed off-center within the cranium compared to thalamotomy and may present challenges to therapeutic lesioning due to this location. However, the impact of target location on treatment efficiency and ability to create therapeutic lesions has not been studied. This study aimed to compare the physical efficiency of MRgFUS thalamotomy and pallidotomy. METHODS: Treatment characteristics were compared between patients treated with thalamotomy (n = 20) or pallidotomy (n = 20), matched by skull density ratios (SDR). Aspects of treatment efficiency were compared between these groups. Demographic and comparative statistics were conducted to assess these differences. Acoustic field simulations were performed to compare and validate the simulated temperature profile for VIM and GPi ablation. RESULTS: Lower SDR values were associated with greater energy requirement for thalamotomy (R2 = 0.197, p = 0.049) and pallidotomy (R2 = 0.342, p = 0.007). The impact of low SDR on efficiency reduction was greater for pallidotomy, approaching significance (p = 0.061). A nearly two-fold increase in energy was needed to reach 50°C in pallidotomy (10.9kJ) than in thalamotomy (5.7kJ), (p = 0.002). Despite lower energy requirement, the maximum average temperature reached was higher in thalamotomy (56.7°C) than in pallidotomy (55.0°C), (p = 0.017). Mean incident angle of acoustic beams was lesser in thalamotomy (12.7°) than in pallidotomy (18.6°), (p < 0.001). For all patients, a lesser mean incident angle correlated with a higher maximum average temperature reached (R2 = 0.124, p = 0.026), and less energy needed to reach 50°C (R2=0.134, p = 0.020). Greater skull thickness was associated with a higher maximum energy for a single sonication for thalamotomy (R2 = 0.206, p = 0.045) and pallidotomy (R2 = 0.403, p = 0.003). An acoustic and temperature field simulation validated similar findings for thalamotomy and pallidotomy in a single patient. CONCLUSION: The centrally located VIM offers a more efficient location for therapeutic lesioning compared to GPi pallidotomy in SDR matched cohort of patients. The impact on therapeutic lesioning with lower SDR may be greater for pallidotomy patients. As newer off-center targets are investigated, these findings can inform patient selection and treatment requirements for lesion production.

Predicting final lesion characteristics during MR-guided focused ultrasound pallidotomy for treatment of Parkinson's disease.

OBJECTIVE: Magnetic resonance-guided focused ultrasound (MRgFUS) ablation of the globus pallidus interna (GPi) is being investigated for the treatment of advanced Parkinson's disease symptoms. However, GPi lesioning presents unique challenges due to the off-midline location of the target. Furthermore, it remains uncertain whether intraprocedural MR thermometry data can predict final lesion characteristics. METHODS: The authors first performed temperature simulations of GPi pallidotomy and compared the results with those of actual cases and the results of ventral intermediate nucleus (VIM) thalamotomy performed for essential tremor treatment. Next, thermometry data from 13 MRgFUS pallidotomy procedures performed at their institution were analyzed using 46°C, 48°C, 50°C, and 52°C temperature thresholds. The resulting thermal models were compared with resulting GPi lesions noted on postprocedure days 1 and 30. Finally, the treatment efficiency (energy per temperature rise) of pallidotomy was evaluated. RESULTS: The authors' modeled acoustic intensity maps correctly demonstrate the elongated, ellipsoid lesions noted during GPi pallidotomy. In treated patients, the 48°C temperature threshold maps most accurately predicted postprocedure day 1 lesion size, while no correlation was found for day 30 lesions. The average energy/temperature rise of pallidotomy was higher (612 J/°C) than what had been noted for VIM thalamotomy and varied with the patients' skull density ratios (SDRs). CONCLUSIONS: The authors' acoustic simulations accurately depicted the characteristics of thermal lesions encountered following MRgFUS pallidotomy. MR thermometry data can predict postprocedure day 1 GPi lesion characteristics using a 48°C threshold model. Finally, the lower treatment efficiency of pallidotomy may make GPi lesioning challenging in patients with a low SDR.

Assessment of the Effect of Autograft Orientation on Peripheral Nerve Regeneration Using Diffusion Tensor Imaging.

PURPOSE: Given no definite consensus on the accepted autograft orientation during peripheral nerve injury repair, we compare outcomes between reverse and normally oriented autografts using an advanced magnetic resonance imaging technique, diffusion tensor imaging. METHODS: Thirty-six female Sprague-Dawley rats were divided into 3 groups: sham-left sciatic nerve isolation without injury, reverse autograft-10-mm cut left sciatic nerve segment reoriented 180° and used to coapt the proximal and distal stumps, or normally oriented autograft-10-mm cut nerve segment kept in its normal orientation for coaptation. Animals underwent sciatic functional index and foot fault behavior studies at 72 hours, and then weekly. At 6 weeks, axons proximal, within, and distal to the autograft were evaluated using diffusion tensor imaging and choline acetyltransferase motor staining for immunohistochemistry. Toluidine blue staining of 1-µm sections was used to assess axon count, density, and diameter. Bilateral gastrocnemius/soleus muscle weights were compared to obtain a net wet weight. Comparison of the groups was performed using Mann-Whiney U or Kruskal-Wallis H tests to determine significance. RESULTS: Diffusion tensor imaging findings including fractional anisotropy, radial diffusivity, and axial diffusivity were similar between reverse and normally oriented autografts. Diffusion tensor imaging tractography demonstrated proximodistal nerve regeneration in both autograft groups. Motor axon counts proximal, within, and distal to the autografts were similar. Likewise, axon count, density, and diameter were similar between the autograft groups. Muscle net weight at 6 weeks and behavioral outcomes (sciatic functional index and foot fault) at any tested time point were also similar between reverse and normally oriented autografts. CONCLUSIONS: Diffusion tensor imaging may be a useful assessment tool for peripheral nerve regeneration. Reversing nerve autograft polarity did not demonstrate to have an influence on functional or regenerative outcomes.

Myelin volume fraction imaging with MRI.

MRI is a valuable tool to assess myelin during development and demyelinating disease processes. While multiexponential T2 and quantitative magnetization transfer measures correlate with myelin content, neither provides the total myelin volume fraction. In many cases correlative measures are adequate; but to assess microstructure of myelin, (e.g. calculate the g-ratio using MRI), an accurate measure of myelin volume fraction is imperative. Using a volumetric model of white matter, we relate MRI measures of myelin to absolute measures of myelin volume fraction and compare them to quantitative histology. We assess our approach in control mice along with two models of hypomyelination and one model of hypermyelination and find strong agreement between MRI and histology amongst models. This work investigates the sensitivities of MRI myelin measures to changes in axon geometry and displays promise for estimating g-ratio from MRI.

Experimental studies of g-ratio MRI in ex vivo mouse brain.

This study aimed to experimentally evaluate a previously proposed MRI method for mapping axonal g-ratio (ratio of axon diameters, measured to the inner and outer boundary of myelin). MRI and electron microscopy were used to study excised and fixed brains of control mice and three mouse models of abnormal white matter. The results showed that g-ratio measured with MRI correlated with histological measures of myelinated axon g-ratio, but with a bias that is likely due to the presence of non-myelinated axons. The results also pointed to cases where the MRI g-ratio model simplifies to be primarily a function of total myelin content.

A novel conduit-based coaptation device for primary nerve repair.

BACKGROUND: Conduit-based nerve repairs are commonly used for small nerve gaps, whereas primary repair may be performed if there is no tension on nerve endings. We hypothesize that a conduit-based nerve coaptation device will improve nerve repair outcomes by avoiding sutures at the nerve repair site and utilizing the advantages of a conduit-based repair. METHODS: The left sciatic nerves of female Sprague-Dawley rats were transected and repaired using a novel conduit-based device. The conduit-based device group was compared to a control group of rats that underwent a standard end-to-end microsurgical repair of the sciatic nerve. Animals underwent behavioral assessments at weekly intervals post-operatively using the sciatic functional index (SFI) test. Animals were sacrificed at four weeks to obtain motor axon counts from immunohistochemistry. A sub-group of animals were sacrificed immediately post repair to obtain MRI images. RESULTS: SFI scores were superior in rats which received conduit-based repairs compared to the control group. Motor axon counts distal to the injury in the device group at four weeks were statistically superior to the control group. MRI tractography was used to demonstrate repair of two nerves using the novel conduit device. CONCLUSIONS: A conduit-based nerve coaptation device avoids sutures at the nerve repair site and leads to improved outcomes in a rat model. Conduit-based nerve repair devices have the potential to standardize nerve repairs while improving outcomes.

Loss of mTORC2 signaling in oligodendrocyte precursor cells delays myelination.

Myelin abnormalities are increasingly being recognized as an important component of a number of neurologic developmental disorders. The integration of many signaling pathways and cell types are critical for correct myelinogenesis. The PI3-K and mechanistic target of rapamycin (mTOR) pathways have been found to play key roles. mTOR is found within two distinct complexes, mTORC1 and mTORC2. mTORC1 activity has been shown to play a major role during myelination, while the role of mTORC2 is not yet well understood. To determine the role of mTORC2 signaling in myelinogenesis, we generated a mouse lacking the critical mTORC2 component Rictor in oligodendrocyte precursors (OPCs). Targeted deletion of Rictor in these cells decreases and delays the expression of myelin related proteins and reduces the size of cerebral white matter tracts. This is developmentally manifest as a transient reduction in myelinated axon density and g-ratio. OPC cell number is reduced at birth without detectable change in proliferation with proportional reductions in mature oligodendrocyte number at P15. The total number of oligodendrocytes as well as extent of myelination, does improve over time. Adult conditional knock-out (CKO) animals do not demonstrate a behavioral phenotype likely due in part to preserved axonal conduction velocities. These data support and extend prior studies demonstrating an important but transient contribution of mTORC2 signaling to myelin development.

Immediate Enhancement of Nerve Function Using a Novel Axonal Fusion Device After Neurotmesis.

BACKGROUND: The management of peripheral nerve injuries remains a large challenge for plastic surgeons. With the inability to fuse axonal endings, results after microsurgical nerve repair have been inconsistent. Our current nerve repair strategies rely upon the slow and lengthy process of axonal regeneration (~1 mm/d). Polyethylene glycol (PEG) has been investigated as a potential axonal fusion agent; however, the percentage of axonal fusion has been inconsistent. The purpose of this study was to identify a PEG delivery device to standardize outcomes after attempted axonal fusion with PEG. MATERIALS AND METHODS: We used a rat sciatic nerve injury model in which we completely transected and repaired the left sciatic nerve to evaluate the efficacy of PEG fusion over a span of 12 weeks. In addition, we evaluated the effectiveness of a delivery device's ability to optimize results after PEG fusion. RESULTS: We found that PEG rapidly (within minutes) restores axonal continuity as assessed by electrophysiology, fluorescent retrograde tracer, and diffusion tensor imaging. Immunohistochemical analysis shows that motor axon counts are significantly increased at 1 week, 4 weeks, and 12 weeks postoperatively in PEG-treated animals. Furthermore, PEG restored behavioral functions up to 50% compared with animals that received the criterion standard epineurial repair (control animals). CONCLUSIONS: The ability of PEG to rapidly restore nerve function after neurotmesis could have vast implications on the clinical management of traumatic injuries to peripheral nerves.

Bridging the Gap: Engineered Porcine-derived Urinary Bladder Matrix Conduits as a Novel Scaffold for Peripheral Nerve Regeneration.

PURPOSE: This study aims to compare engineered nerve conduits constructed from porcine-derived urinary bladder matrix (UBM) with the criterion-standard nerve autografts, for segmental loss peripheral nerve repairs. METHODS: Forty-eight Sprague-Dawley rats were divided into 2 groups. All underwent a 10-mm sciatic nerve gap injury. This was repaired using either (1) reverse autograft-the 10-mm cut segment was oriented 180 degrees and used to coapt the proximal and distal stumps or (2) UBM conduit-the 10-mm nerve gap was bridged with UBM conduit. Behavior assessments such as sciatic function index and foot fault asymmetry scores were performed weekly. At 3- or 6-week time endpoints, the repaired nerves and bilateral gastrocnemius/soleus muscles were harvested from each animal. Nerves were evaluated using immunohistochemistry for motor and sensory axon staining and with diffusion tensor imaging. The net wet muscle weights were calculated to assess the degree of muscle atrophy. RESULTS: The UBM group demonstrated significantly improved foot fault asymmetry scores at 2 and 4 weeks, whereas there was no difference in sciatic function index. The net muscle weights were similar between both groups. Motor axon counts proximal/inside/distal to the conduit/graft were similar between UBM conduits and reverse autografts, whereas sensory axon counts within and distal to the conduit were significantly higher than those of the autograft at 6 weeks. Sensory axonal regeneration seemed to be adherent to the inner surface of the UBM conduit, whereas it had a scattered appearance in autografts. Diffusion tensor imaging parameters between groups were similar. CONCLUSIONS: Urinary bladder matrix conduits prove to be at least similar to nerve autografts for the repair of peripheral nerve injuries with a short gap. The matrix perhaps serves as a scaffold to augment sensory nerve growth. CLINICAL RELEVANCE: In a clinical setting, UBM may eliminate the donor site morbidity and increased operative time associated with nerve autografting.

A novel therapy to promote axonal fusion in human digital nerves.

BACKGROUND: Peripheral nerve injury can have a devastating impact on our military and veteran population. Current strategies for peripheral nerve repair include techniques such as nerve tubes, nerve grafts, tissue matrices, and nerve growth guides to enhance the number of regenerating axons. Even with such advanced techniques, it takes months to regain function. In animal models, polyethylene glycol (PEG) therapy has shown to improve both physiologic and behavioral outcomes after nerve transection by fusion of a portion of the proximal axons to the distal axon stumps. The objective of this study was to show the efficacy of PEG fusion in humans and to retrospectively compare PEG fusion to standard nerve repair. METHODS: Patients with traumatic lacerations involving digital nerves were treated with PEG after standard microsurgical neurorrhaphy. Sensory assessment after injury was performed at 1 week, 2 weeks, 1 month, and 2 months using static two-point discrimination and Semmes-Weinstein monofilament testing. The Medical Research Council Classification (MRCC) for Sensory Recovery Scale was used to evaluate the level of injury. The PEG fusion group was compared to patient-matched controls whose data were retrospectively collected. RESULTS: Four PEG fusions were performed on four nerve transections in two patients. Polyethylene glycol therapy improves functional outcomes and speed of nerve recovery in clinical setting assessed by average MRCC score in week 1 (2.8 vs 1.0, p = 0.03). At 4 weeks, MRCC remained superior in the PEG fusion group (3.8 vs 1.3, p = 0.01). At 8 weeks, there was improvement in both groups with the PEG fusion cohort remaining statistically better (4.0 vs 1.7, p = 0.01). CONCLUSION: Polyethylene glycol fusion is a novel therapy for peripheral nerve repair with proven effectiveness in animal models. Clinical studies are still in early stages but have had encouraging results. Polyethylene glycol fusion is a potential revolutionary therapy in peripheral nerve repair but needs further investigation. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Multi-compartment microscopic diffusion imaging.

This paper introduces a multi-compartment model for microscopic diffusion anisotropy imaging. The aim is to estimate microscopic features specific to the intra- and extra-neurite compartments in nervous tissue unconfounded by the effects of fibre crossings and orientation dispersion, which are ubiquitous in the brain. The proposed MRI method is based on the Spherical Mean Technique (SMT), which factors out the neurite orientation distribution and thus provides direct estimates of the microscopic tissue structure. This technique can be immediately used in the clinic for the assessment of various neurological conditions, as it requires only a widely available off-the-shelf sequence with two b-shells and high-angular gradient resolution achievable within clinically feasible scan times. To demonstrate the developed method, we use high-quality diffusion data acquired with a bespoke scanner system from the Human Connectome Project. This study establishes the normative values of the new biomarkers for a large cohort of healthy young adults, which may then support clinical diagnostics in patients. Moreover, we show that the microscopic diffusion indices offer direct sensitivity to pathological tissue alterations, exemplified in a preclinical animal model of Tuberous Sclerosis Complex (TSC), a genetic multi-organ disorder which impacts brain microstructure and hence may lead to neurological manifestations such as autism, epilepsy and developmental delay.

A novel technique using hydrophilic polymers to promote axonal fusion.

The management of traumatic peripheral nerve injury remains a considerable concern for clinicians. With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury, outcomes of surgical intervention have been unpredictable. The inability to manipulate the pathophysiology of nerve injury (i.e., Wallerian degeneration) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration (~1 mm/day). When axons are severed, the endings undergo calcium-mediated plasmalemmal sealing, which limits the ability of the axon to be primarily repaired. Polythethylene glycol (PEG) in combination with a bioengineered process overcomes the inability to fuse axons. The mechanism for PEG axonal fusion is not clearly understood, but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion. The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur. This review highlights PEG fusion, its past and current studies, and future directions in PEG fusion.

Evaluation of diffusion kurtosis imaging in ex vivo hypomyelinated mouse brains.

Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and DKI-derived white matter tract integrity metrics (WMTI) were experimentally evaluated ex vivo through comparisons to histological measurements and established magnetic resonance imaging (MRI) measures of myelin in two knockout mouse models with varying degrees of hypomyelination. DKI metrics of mean and radial kurtosis were found to be better indicators of myelin content than conventional DTI metrics. The biophysical WMTI model based on the DKI framework reported on axon water fraction with good accuracy in cases with near normal axon density, but did not provide additional specificity to myelination. Overall, DKI provided additional information regarding white matter microstructure compared with DTI, making it an attractive method for future assessments of white matter development and pathology.

A revised model for estimating g-ratio from MRI.

A key measure of white matter health is the g-ratio, which is defined as the ratio between the inner axon radius and the outer, myelinated, axon radius. Recent methods have been proposed to measure the g-ratio non-invasively using the relationship between two magnetic resonance imaging (MRI) measures. While this relationship is intuitive, it predicates on the simplifying assumption that g-ratio is constant across axons. Here, we extend the model to account for a distribution of g-ratio values within an imaging voxel, and evaluate this model with quantitative histology from normal and hypomyelinated mouse brains.

Quantitative analysis of mouse corpus callosum from electron microscopy images.

This article provides morphometric analysis of 72 electron microscopy images from control (n=4) and hypomyelinated (n=2) mouse corpus callosum. Measures of axon diameter and g-ratio were tabulated across all brains from two regions of the corpus callosum and a non-linear relationship between axon diameter and g-ratio was observed. These data are related to the accompanying research article comparing multiple methods of measuring g-ratio entitled 'A revised model for estimating g-ratio from MRI' (West et al., NeuroImage, 2015).

4.7-T diffusion tensor imaging of acute traumatic peripheral nerve injury.

Diagnosis and management of peripheral nerve injury is complicated by the inability to assess microstructural features of injured nerve fibers via clinical examination and electrophysiology. Diffusion tensor imaging (DTI) has been shown to accurately detect nerve injury and regeneration in crush models of peripheral nerve injury, but no prior studies have been conducted on nerve transection, a surgical emergency that can lead to permanent weakness or paralysis. Acute sciatic nerve injuries were performed microsurgically to produce multiple grades of nerve transection in rats that were harvested 1 hour after surgery. High-resolution diffusion tensor images from ex vivo sciatic nerves were obtained using diffusion-weighted spin-echo acquisitions at 4.7 T. Fractional anisotropy was significantly reduced at the injury sites of transected rats compared with sham rats. Additionally, minor eigenvalues and radial diffusivity were profoundly elevated at all injury sites and were negatively correlated to the degree of injury. Diffusion tensor tractography showed discontinuities at all injury sites and significantly reduced continuous tract counts. These findings demonstrate that high-resolution DTI is a promising tool for acute diagnosis and grading of traumatic peripheral nerve injuries.

Hypomyelination following deletion of Tsc2 in oligodendrocyte precursors.

OBJECTIVE: While abnormalities in myelin in tuberous sclerosis complex (TSC) have been known for some time, recent imaging-based data suggest myelin abnormalities may be independent of the pathognomonic cortical lesions ("tubers"). Multiple mouse models of TSC exhibit myelination deficits, though the cell types responsible and the mechanisms underlying the myelin abnormalities remain unclear. METHODS: To determine the role of alterations in mTOR signaling in myelination, we generated a conditional knockout (CKO) mouse model using Cre-recombinase and the Olig2 promoter to inactivate the Tsc2 gene in oligodendrocyte precursor cells. RESULTS: Characterization of myelin and myelin constituent proteins demonstrated a marked hypomyelination phenotype. Diffusion-based magnetic resonance imaging studies were likewise consistent with hypomyelination. Hypomyelination was due in part to decreased myelinated axon density and myelin thickness as well as decreased oligodendrocyte numbers. Coincident with hypomyelination, an extensive gliosis was seen in both the cortex and white matter tracks, suggesting alterations in cell fate due to changes in mTOR activity in oligodendrocyte precursors. Despite a high-frequency appendicular tremor and altered gait in CKO mice, no significant changes in activity, vocalizations, or anxiety-like phenotypes were seen. INTERPRETATION: Our findings support a known role of mTOR signaling in regulation of myelination and demonstrate that increased mTORC1 activity early in development within oligodendrocytes results in hypomyelination and not hypermyelination. Our data further support a dissociation between decreased Akt activity and increased mTORC1 activity toward hypomyelination. Thus, therapies promoting activation of Akt-dependent pathways while reducing mTORC1 activity may prove beneficial in treatment of human disease.

Antipsychotic drug-like effects of the selective M4 muscarinic acetylcholine receptor positive allosteric modulator VU0152100.

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.