Recent Developments in Molecular Brain Imaging of Neuropsychiatric Disorders

Recent Developments in Molecular Brain Imaging of Neuropsychiatric Disorders Mark Slifstein, PhD,*,‡,§ and Anissa Abi-Dargham, MD*,†,‡,§ Molecular imaging with PET or SPECT has been an important research tool in psychiatry for as long as these modalities have been available. Here, we discuss two areas of neuroimaging relevant to current psychiatry research. The first is the use of imaging to study neurotransmission. We discuss the use of pharmacologic probes to induce changes in levels of neurotransmitters that can be inferred through their effects on outcome measures of imaging experiments, from their historical origins focusing on dopamine transmission through recent developments involving serotonin, GABA, and glutamate. Next, we examine imaging of neuroinflammation in the context of psychiatry. Imaging markers of neuroinflammation have been studied extensively in other areas of brain research, but they have more recently attracted interest in psychiatry research, based on accumulating evidence that there may be an inflammatory component to some psychiatric conditions. Furthermore, new probes are under development that would allow unprecedented insights into cellular processes. In summary, molecular imaging would continue to offer great potential as a unique tool to further our understanding of brain function in health and disease. Semin Nucl Med 47:54-63 C 2017 Elsevier Inc. All rights reserved.

Introduction

I

n vivo imaging of brain neurochemistry with PET or SPECT has been used in psychiatry research as these tools became available, and continues to be a widely used methodology for gaining insight into the neuropathology of psychiatric conditions. Seminal studies examined receptor and transporter availability, demonstrated target engagement by drugs, and provided evidence of dysregulated neurotransmission. The methodological principles established by these early studies continue to provide the framework for ongoing research, albeit with considerable refinement and expansion of the approaches to analysis, the molecular targets, and the imaging probes available. Here, we examine two areas of particular interest in psychiatric neuroimaging. Following a brief introduction to commonly used outcome measures, we discuss approaches to *Department of Psychiatry, Columbia University Medical Center, New York, NY. †Department of Radiology, Columbia University Medical Center, New York, NY. ‡New York State Psychiatric Institute, New York, NY. §Department of Psychiatry, Stony Brook University, New York, NY. Address reprint requests to Mark Slifstein, PhD, Department of Psychiatry, Stony Brook University, HSC T10-41-I, Stony Brook, NY, 11794. E-mail: [email protected]

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http://dx.doi.org/10.1053/j.semnuclmed.2016.09.002 0001-2998/& 2017 Elsevier Inc. All rights reserved.

making inferences about neurotransmission using imaging, from the inception of these methods through recent developments and refinements. Next, we examine the methodology and recent results for imaging neuroinflammatory markers, a branch of brain imaging that has an extensive literature in the context of other conditions but has more recently become of interest in psychiatry, owing to accumulating evidence suggesting a role of inflammation in psychiatric conditions.

Methodological considerations Many of the tracers used in psychiatric imaging bind reversibly to their target molecules. To extract biologically relevant parameter values, data are fitted to mathematical models, frequently referred to as compartment models, that combine equations for transport across the blood-brain barrier with mass action laws governing the association and dissociation of the tracer with its target. Compartment models have been reviewed in detail elsewhere,1-3 but the most relevant outcome measures are described briefly here. These are binding potentials (BP) and distribution volumes (V). Both represent ratios of the concentrations of different pools, or compartments, of the radioligand to each other at equilibrium, but can also be expressed in terms of pharmacokinetic parameters. BP is proportional to the equilibrium ratio between the

Molecular brain imaging concentration of radioligand specifically bound to its target and unbound, or free, tracer. It can also be represented as proportional to the product of the target density and the affinity of the radiotracer for the target, or Bavail/KD, where Bavail is the concentration of target available for binding to the radioligand and KD is the equilibrium dissociation constant of the reaction. The constant of proportionality depends on how the free tracer is represented in the model, but the most frequently reported version is BPND ¼ fNDBavail/KD, where fND is the free fraction of free and nonspecifically bound radiotracer (the nondisplaceable compartment) in brain tissue. If the free concentration is measured in arterial plasma, the constant is fp, the portion of tracer not bound to plasma proteins, and the binding potential is BPP ¼ fpBavail/KD. If fp is measured and corrected for, the constant is 1 and the binding potential is BPF ¼ Bavail/KD. Below, we will use the specific forms when referring to studies in which they were used, and the generic BP for properties that apply equally to all versions. The equilibrium ratio of the nondisplaceable compartment to arterial plasma is VND and is usually assumed to be equal throughout the brain. The equilibrium ratio of all tracer in a brain region to the arterial plasma concentration is VT, the total distribution volume, VT ¼ VND þBPP. Reliable methods of deriving BP usually require either an estimate of VND that can be subtracted from VT, or use of a reference tissue model in which a direct relationship is formed between the target-rich region and a region with negligible concentration of the target molecule (the reference tissue). VND can be estimated either by measuring it in the reference tissue or by performing separate pharmacologic blocking experiments. When none of these options are available but radiotracer concentration in arterial plasma is measured, VT can be used to make inferences about neurotransmission, although it differs from proportionality to the target concentration by the constant VND. Importantly, the radioligand is usually administered at tracer dose, a concentration too low to occupy more than a small percentage of the target molecules. The net effect is that inferences about target occupancy by endogenous or exogenously administered ligands are independent of radiotracer concentration. In particular, fractional changes in BP following experimental manipulations that cause changes in concentrations of competing ligands can be interpreted solely in terms of the properties of the competing ligand.

Neurotransmission Imaging A general approach to imaging neurotransmission is to assess BP under a baseline condition and then again following a perturbation of the system. Inferences are made by calculating the percentage change, ΔBP, of the binding potential. An essential requirement is the fact that the binding is sensitive to changes in the concentration of endogenous ligand, a property that is not observed for all radiotracers. If the perturbation increases the concentration of the endogenous transmitter at the level of the target receptor and the tracer and the transmitter compete for the same binding site, ΔBP would be a proxy for receptor occupancy by the transmitter. In reality, the

55 interaction may be more complicated than pure competition, involving other phenomena such as receptor trafficking or the contribution of baseline receptor occupancy by the transmitter. Still, at least for some systems, validating studies have been performed showing clear dose responses between measured endogenous transmitter release and the magnitude of ΔBP. If the perturbation depletes endogenous transmitter, ΔBP would be indicative of the baseline level of transmitter, and under the assumption of complete or near-complete depletion, ΔBP/(1 þ ΔBP) provides an estimate of baseline receptor occupancy. If the tracer binds to an allosteric site, changes in the level of endogenous transmitter would affect the affinity, 1/KD; theoretical predictions suggest that increased transmitter levels should decrease BP of allosteric antagonist radiotracers and increase BP of allosteric agonists, although again, other processes such as receptor trafficking may come into play. Finally, in some cases, metabolism radiotracers that partially follow the same metabolic pathway as the precursor to the endogenous transmitter have been used to make inferences about transmitter synthesis and storage capacity. Here, we discuss neurotransmitter imaging as it relates to imaging applications in psychiatry. In some cases, this involves paradigms that have been in use for years to study psychiatric populations. In others, the methods are just being developed or have been less successful despite much research.

Dopamine Dopamine Transmission: Transmitter Release and Reuptake Blockade The most robust paradigms for imaging dopamine transmission have been the combination of D2/D3 receptor radiotracers with pharmacologic challenges such as D-amphetamine that extrudes dopamine from synaptic vesicles and releases it through reversal of the dopamine reuptake transporter,4 or methylphenidate that blocks the reuptake transporter, causing accumulation of dopamine released via synaptic transmission.5 The approach was also tested with D1 receptor radioligands6 without success. However, D2/D3 tracers produced clear results and have been used in release and reuptake blockade paradigms. In the 1990s, studies used the SPECT D2/ D3 radiotracer [123I]IBZM in combination with amphetamine,7 or the PET D2/D3 radiotracer [11C]raclopride with either amphetamine8 or methylphenidate9 to examine dopamine transmission in the striatum. Using [123I]IBZM, it was shown that amphetamine-induced dopamine release was higher, on average, in unmedicated patients with schizophrenia than matched controls, and that the magnitude of release was correlated with the level of symptom exacerbation in patients,10 an observation that fits well with the fact that virtually all antipsychotic drugs block a substantial portion of dopamine binding to D2 receptors. Similar results were obtained independently using amphetamine in combination with PET and [11C]raclopride.8 Studies in nonhuman primates correlating these results with microdialysis measurement of dopamine levels showed a dose response to amphetamine of both dopamine levels and the magnitude of ΔBP,11 but also

56 that the duration of reduced binding potential lasted many hours longer than the surge of dopamine, prompting the conjecture that receptor trafficking may play a role, in which high concentrations of synaptic dopamine induce receptor internalization, with tracers having lower affinity for internalized receptors than for surface-bound receptors.12 More recently, evidence has been provided in support of the internalization model in the form of in vitro and ex vivo binding assays showing lowered binding affinity of many D2/ D3 radiotracers for internalized receptors in cell or membrane preparations,13,14 and in a mutant mouse model lacking a protein necessary for D2/D3 receptor internalization that does not display as prolonged an effect of amphetamine on [18F] fallypride binding as observed in wild-type mice.15 Based on the available data, the most parsimonious explanation for the amphetamine effect in striatum appears to be that the initial decrease in tracer binding following amphetamine is due to competition between the tracer and dopamine, whereas the continued suppression of BP for hours following amphetamine administration is due to reduced affinity associated with receptor trafficking. Additional evidence has been provided for striatal hyperdopaminergia by studies using [18F]DOPA. The uptake of [18F]DOPA, a substrate for amino acid decarboxylase (AADC), partially follows the synthesis and storage pathway of endogenous dopamine and its precursors, and its steady state uptake rate constant, Ki, has been interpreted as an indicator of the capacity for dopamine synthesis. [18F]DOPA Ki has been measured in a number of studies comparing patients with schizophrenia to controls,16-20 and in most of these it was found to be elevated in the striatum. More recently, Kcer i , a reference tissue-based version of the uptake rate constant, was shown to be increased in the striatum of patients with schizophrenia compared to matched controls, and intermediately elevated in ultra-high-risk subjects who have attenuated schizophrenia-like symptoms and are at increased risk of developing the full condition.21 Additionally, in UHR, the was predictive of the probability of magnitude of Kcer i subjects more likely to develop conversion, with high Kcer i schizophrenia.22 It has long been posited that in schizophrenia, excess dopamine transmission in striatum may coexist with a cortical dopamine deficit based on evidence from postmortem data and performance on cognitive tasks that engage brain areas such as dorsolateral prefrontal cortex.23 Tracers such as [11C] raclopride and [123I]IBZM have proven useful for studying dopamine transmission in the striatum, but are not useful for studying cortical dopamine transmission, where the D2/D3 receptor density is an order of magnitude lower, and specific binding of these tracers barely rises above background level. Higher-affinity D2/D3 radiotracers such as [18F]fallypride and [11C]FLB457 provide a stronger signal in extrastriatal brain regions and have both been shown to be sensitive to amphetamine-induced dopamine release.24-28 Preclinical studies using PET imaging with [11C]FLB457 and simultaneous microdialysis in nonhuman primates demonstrated a clear dose response to amphetamine in frontal cortex of both dopamine release and ΔBP that were highly correlated with

M. Slifstein and A. Abi-Dargham each other,29 validating this paradigm. Cortical dopamine release was lower in magnitude and displayed a different temporal profile than in striatum,30 but this result was not entirely unexpected, given the differences in dopaminergic innervation and regulation between the two brain regions. Recently, we performed a study using [11C]FLB457 imaging with an amphetamine challenge in a cohort of unmedicated patients with schizophrenia and matched healthy controls and found a generalized blunting of dopamine release in the cortex of the patients, especially in the dorsolateral prefrontal cortex, providing in vivo evidence in support of the cortical hypodopaminergia hypothesis.31 The radiotracers used in all the studies described above are D2/D3 receptor antagonists. For some time, there has been discussion within the field about the possibility that agonist radiotracers may be more sensitive to changes in dopamine levels because, in theory, they compete with dopamine only at the receptors configured in the high affinity, that is, G proteincoupled state, whereas antagonists bind with equal affinity to uncoupled receptors where dopamine affinity is also low. Although there has been some controversy as to whether affinity state is detectable with in vivo imaging,32 empirical evidence supporting the increased sensitivity of agonist tracers has been published. [11C]-(þ)-PHNO is a strongly D3-preferring, D2/D3 agonist radiotracer that is suitable for imaging the dorsal striatum, but also has high binding potential in extrastriatal regions including midbrain, thalamus, and globus pallidus, owing to its high affinity for D3 receptors.33-35 The heterogeneous binding to the two receptor types complicates quantification and interpretation of [11C]-(þ)-PHNO BP, but in dorsal striatum, BP is predominant owing to D2 receptor binding and its interpretation is straightforward. To date, several amphetamine challenge studies have been performed using [11C](þ)-PHNO,36-38 and the reported magnitude of ΔBP in all regions including dorsal striatum is consistently larger than in similar studies performed with [11C]raclopride or [123I]IBZM, providing support for the idea that agonist tracers may be more sensitive to endogenous transmitter changes than antagonists.

Dopamine Transmission: Depletion Studies The increased presynaptic dopamine tone observed in imaging studies of patients with schizophrenia suggests the possibility that average baseline occupancy of D2/D3 receptors by dopamine might also be high, and depletion studies have confirmed this. The tyrosine hydroxylase inhibitor alphamethyl-para-tyrosine (AMPT) disrupts the dopamine synthesis pathway, and oral administration over the course of 48 hours significantly depletes dopamine in the brain. Imaging before and after AMPT administration unmasks receptors bound by dopamine at baseline, and imaging studies using [123I]IBZM showed that unmedicated patients with schizophrenia did have higher baseline dopamine binding in striatum. Further, in a subset of drug-naïve patients and controls who were scanned using both the amphetamine and AMPT paradigms, patients were increased compared to controls according to both

Molecular brain imaging measures, and also showed high correlation between the two measures, lending support to the model of presynaptic dopaminergic hyperactivity in schizophrenia. More recently, these results were replicated in a different cohort using [11C] raclopride with PET imaging.39 The higher resolution of the PET scanner compared to the previous SPECT study also allowed for examination of striatal subdivisions, demonstrating that the effect was most pronounced in the head of the caudate, a brain region that integrates cortical and limbic information and is likely involved in functions and behavior affected in schizophrenia.

Serotonin It would be of great interest to psychiatry researchers to be able to image serotonin transmission, especially given the demonstrated role of serotonin in affective disorders. The development of imaging methods to measure neurotransmission has proven to be more difficult for serotonin than for dopamine. This is partly because of limited methods for robustly releasing serotonin that are suitable for use in humans. Compounds such as fenfluramine, which does induce substantial serotonin release, cannot be used in humans due to cardiac effects,40 limiting available methods to reuptake blockade with selective serotonin reuptake inhibitors (SSRIs) that cause more modest increases in serotonin concentration than fenfluramine. But it has also been difficult to identify radiotracers that are sensitive enough to changes in serotonin levels to provide a reliable signal, even in preclinical species where more direct pharmacologic challenges can be used.41 The 5-HT1A antagonist tracer [18F]MPPF showed some initial promise in rodent studies,42,43 but studies in nonhuman primates with fenfluramine challenge44 or in humans with a serotonin depletion paradigm45 failed to detect significant changes in binding potential in response to manipulations of serotonin levels. More recently, the 5-HT1A partial agonist [11C]CUMI-101 showed decreased binding in response to fenfluramine and citalopram in nonhuman primates.46 Subsequently, one study used this tracer in humans with a citalopram challenge and showed modestly increased binding across all serotonergic projection regions compared to placebo, but not in the raphe nuclei.47 These investigators interpreted their results as decreased serotonin in the terminal fields subsequent to SSRI-induced increased serotonin in the raphe nuclei, leading in turn to 5-HT1A autoreceptor stimulation and resultant reduced serotonin transmission. Another study, on the contrary, detected no effect of citalopram on [11C]CUMI-101 binding in humans.48 Also recently, two radiotracers that bind to 5-HT1B receptors, [11C]AZ10419369 and [11C]P943, have shown consistent responses to serotonin challenges in nonhuman primates.49-51 BP of both tracers decreased substantially in response to fenfluramine challenge, and [11C]P943 binding was also displaced by reuptake blockade with citalopram. Although studies in humans have been performed with these tracers to validate quantitative procedures, examine receptor availability or to measure 5-HT1B receptor occupancy by drugs, only one study examining transmitter release has been

57 published to date.52 These investigators imaged humans’ response to oral administration of the SSRI escitalopram using [11C]AZ10419369 as well as the response of nonhuman primates to i.v. administration of escitalopram. They observed decreased binding potential across the brain in the monkeys, in line with previous results. In the humans, where the doses and resulting plasma concentrations of escitalopram were much lower, binding potential was decreased in the raphe nuclei but modestly increased throughout the rest of the brain, similar to the observation in the one [11C]CUMI-101 study where increased binding was observed in humans following citalopram.47 This also agrees with earlier rodent studies showing increased binding of [18F]MPPF in hippocampus in response to pharmacologically induced serotonin depletion.43 These findings all suggest the need for serotonin depletion studies in humans to demonstrate baseline occupancy by serotonin of 5-HT1B or 5-HT1A receptors in the projection fields that can be reliably detected with the more recently developed radiotracers.

Inferred Neurotransmission Through Allosteric Interactions In all cases described so far, the interaction between the radiotracer and the neurotransmitter was assumed to involve direct competition at the orthosteric binding site, albeit potentially complicated by receptor trafficking. Some radiotracers bind to an allosteric site on receptors or other targets. If the radiotracer itself is capable of acting as a positive or negative allosteric modulator through alteration of the affinity of the orthosteric site for the endogenous neurotransmitter, thermodynamic considerations suggest that the neurotransmitter should also affect the affinity of the allosteric site for the radiotracer in the same direction that the positive or negative allosteric modulator affects the neurotransmitter’s affinity for the orthosteric site.53 Currently, available PET radiotracers for imaging amino acid neurotransmitters bind to allosteric sites on receptors. These are [11C]flumazenil (originally known as Ro15-1788) and [11C]Ro15-4513 that bind to the benzodiazepine site on the GABA-A receptor, and [11C]ABP688 or [18F] FPEB that bind to a transmembrane domain site on the metabotropic glutamate receptor 5 (mGluR5).

Imaging GABA Transmission GABA-A receptors are inhibitory ionotropic receptors formed from multiple subunits termed α, β, γ, δ, and ρ that together form an anion channel permeable to chloride when stimulated by GABA. There are six types of α subunits (α1-α6). Ligands for the benzodiazepine site on the receptor can enhance or inhibit the action of GABA. [11C]Ro15-4513 is a partial reverse agonist that binds to the benzodiazepine site on receptors containing α1 or α5 subunits, but preferentially to those containing α5 subunits. These are most abundantly expressed in hippocampus, anterior cingulate, and ventral striatum. [11C] flumazenil is a weak partial agonist that binds to the benzodiazepine site with similar affinity to receptors containing

58 several different α subunits including α1. Its binding pattern coincides with the distribution of GABA-A receptors that contain α1 subunits, as these are the most highly expressed type throughout the brain. Investigators have used both radiotracers in conjunction with tiagabine, an inhibitor of the GAT1 GABA uptake transporter, to increase synaptic GABA and detect an allosteric interaction between the GABA and benzodiazepine binding sites, an effect sometimes referred to as the “GABA shift.” Early in vitro and preclinical literature referred to Ro15-1788 as an antagonist, but some reports showed that it acted as a partial agonist, especially when administered at high doses.54-56 Regardless of its pharmacologic action, experimental paradigms that increased GABA concentration also caused increases in Ro15-1788 binding in preclinical models.57,58 Thus, investigators using a tiagabine challenge in conjunction with [11C]flumazenil imaging predicted that tiagabine would cause increased binding of the radiotracer in humans, and this has turned out to be the case. In an initial study in healthy volunteer subjects, [11C]flumazenil BPND increased approximately 15% on average across most measured brain regions following 16 mg of orally administered tiagabine.59 Additionally, γ band synchrony, an electroencephalogram-derived parameter indicative of the ability to entrain cortical networks, measured during a cognitive task, was strongly correlated with the change in binding potential. In a follow-up study performed in healthy volunteer subjects in which two doses of tiagabine were tested (0.15 mg/kg and 0.25 mg/kg), the higher, but not the lower dose, also lead to increased radiotracer binding and correlation with electroencephalogram-detected γ band synchrony.60 The second study used VT as an outcome measure rather than BPND, based on the observation that in the reference tissue used in the previous study, the pons, [11C]flumazenil had been shown to have substantial specific binding. The percentage increase reported in VT, 5%-7% across brain regions, likely represented a similar magnitude of effect as the BPND changes reported earlier. Most recently, these investigators applied their method to a cohort of 17 unmedicated patients with schizophrenia and 22 matched healthy controls, 10 of whose data were drawn from the previous two studies.61 They again observed increased [11C]flumazenil VT following an average dose of 0.2 mg/kg tiagabine and correlation with γ band synchrony during a cognitive task in the expanded sample of healthy controls, but not in the patients. Closer examination showed that the lack of response at the group level in the patients was driven mainly by the drug naïve, rather than the previously treated patients. They interpreted their observation as evidence of dysregulated GABA transmission in untreated schizophrenia. Quantification of [11C]Ro15-4513 binding is complicated to some degree by its mixed binding profile. Using a data-driven analysis method called spectral analysis,62 one group of investigators decomposed the shape of [11C]Ro15-4513 time activity curves into a sum of components that included a slowclearing specific-binding component and a fast-clearing specific-binding component which they inferred were associated with binding to α5- and α1-containing subtypes, respectively, based on anatomical distribution and the higher affinity of

M. Slifstein and A. Abi-Dargham [11C]Ro15-4513 for the α5-containing subtype. In a subsequent study from the same laboratory, [11C]Ro15-4513 imaging was performed in healthy human volunteers in a crossover design comparing 15 mg tiagabine to placebo. Using the spectral analysis approach, the slow component, attributed to the α5-containing subtype was increased in some, but not all brain regions following tiagabine compared to placebo, whereas the fast component, attributed to the α1-containing subtype, was widely decreased. This result was interpreted as a GABA shift at the α1-containing GABA-A receptors leading to reduced binding, with the conjecture that increased binding at the α5-containing receptors may have been caused by increased concentration of radiotracer available to bind to these. These results should be interpreted cautiously, as there has been limited validation that the spectral components correspond closely to actual binding to the distinct receptor types even if their composite fits the total time activity curve well, or that the components retain their identity across conditions, that is, the apparent α1-related component posttiagabine is associated with the same binding sites as the apparent α1-related component post-placebo. It should also be noted that the explanation of increased binding at α5containing receptors does not comport well with the assumptions of tracer dose models, which would predict that distribution volumes of the spectral components should be independent of these relatively small changes in concentration, and only change in response to changes in receptor availability or affinity. Nonetheless, the results and the method are intriguing, and it would be of great interest to see additional characterization of [11C]Ro15-4513 imaging.

Imaging Glutamate Transmission [11C]ABP688 and [18F]FPEB are both tracers for mGluR5 that have chemical structures similar to the noncompetitive antagonist MPEP,63 which binds to the transmembrane domain of mGluR5.64 Both [11C]ABP688 and [18F]FPEB have been shown to bind to the same site as MPEP63,65 and APB688 has been shown to act as an antagonist. Several studies have been published examining glutamate transmission with [11C] ABP688 imaging using pharmacologic challenges. The more recently developed [18F]FPEB has higher signal-to-noise ratio than [11C]ABP688 (higher BPND), but as yet no studies examining glutamate transmission using [18F]FPEB have been published. MGluR5 receptors are expressed extrasynaptically on postsynaptic terminals.66 Their exposure to synaptic release of glutamate is normally limited by reuptake by glutamate transporter 1 (GLT1) located on glia proximal to synapses. Glia also contain an exchange pump, the cystine-glutamate antiporter, that controls levels of extracellular, extrasynaptic glutamate.67 N-acetylcysteine (NAC) is a prodrug that is deacetylated after the administration and dimerizes to cystine,66 stimulating the activity of the cystine-glutamate antiporter, thereby increasing extracellular glutamate. Thus, it has been hypothesized that NAC administration may induce a detectable change in [11C]ABP688. An initial preclinical study in a small cohort of nonhuman primates that were administered a 50 mg/kg infusion of NAC over 1 hour was promising,

Molecular brain imaging showing a decrease in BPND in cortex and striatum between 10% and 15% compared with baseline.68 A second study in nonhuman primates was less conclusive, with average decreases of BPND that were of similar magnitude to those observed in the initial study, following 50 mg/kg and 100 mg/kg NAC, but not statistically significant.69 These two studies were both performed in animals under ketamineinitiated, isoflurane-maintained anesthesia, a setting that may lead to very different results than in awake humans. To date, studies using [11C]ABP688 imaging with NAC challenge in humans have not been published, but one study using [11C] ABP688 imaging in humans with ketamine, an NMDA receptor antagonist and itself a potent glutamate releaser,70,71 has been performed.72 Subjects were scanned at baseline and again during a subanesthetic ketamine infusion. Distribution volumes measured during the ketamine condition were decreased compared with baseline, on average by 20%, across all measured brain regions. A factor tempering the interpretation of these studies as evidence of an allosteric affinity shift similar to the GABA shift is that in vitro binding assays have failed to detect an effect of the presence of glutamate on [11C] ABP688 affinity for mGluR5 (Javitch and Lin, personal communication). MgluR5 receptors are known to internalize readily in response to agonists,73,74 and this, combined with the observation that in cell preparations, ABP688 does not easily cross the plasma membrane (Javitch and Lin, personal communication), suggests that receptor trafficking, rather than allosteric affinity shift, may provide an explanation for the observed effects of glutamate releasers on [11C]ABP688 binding. Thus, at this time, the mechanism and quantitative interpretation of glutamate-stimulated changes in binding of mGluR5 radiotracers requires further clarification.

Imaging Neuroinflammation Imaging markers of neuroinflammation have been studied extensively in infection, brain injury, autoimmune, neurologic, and neurodegenerative disorders.75-77 Recently, there has also been interest in application to psychiatric conditions, as increasing evidence has been gathered showing that inflammation may play a role in these.78,79 Evidence from animal models has shown that microglia, the resident immune cells in brain, may play a part in synaptic pruning in addition to clearing cellular debris from injured tissue, suggesting a role in neurodevelopment.80 There is also evidence of increased expression of several inflammatory cytokines in schizophrenia81 and in major depression.82 The 18 kD translocator protein (TSPO) is expressed in activated microglia and reactive astrocytes,83 and this is the binding target of the radiotracers currently in use for imaging neuroinflammation. Quantification of neuroinflammation presents some unique challenges. There are no locations within brain where TSPO expression is expected to be elevated a priori, nor are there obvious reference tissues. The PET radiotracer that, until recently, was the only radioligand available for in vivo imaging in humans is [11C] PK11195, which has low signal-to-noise ratio (low BPND).84 More recently, a number of “second generation” TSPO

59 radiotracers have been developed; some of these are [11C] PBR28, [18F]PBR06, [18F]PBR111, [18F]FEPPA, and [11C] DA1106. Unlike [11C]PK11195, all of these tracers have turned out to be sensitive to a single nucleotide polymorphism (rs6971) in TSPO, leading to distinct subgroups of subjects with different binding affinities for TSPO.85,86 These are referred to as high-affinity binders, mixed-affinity binders, and low-affinity binders (LAB). The specific binding in LABs is generally too low for quantification and their population prevalence is low,85 so the strategy employed by investigators has been to exclude LABs from their studies and to include genotype as a covariate in group comparisons that include both high-affinity binders and mixed-affinity binders.

Inflammation Imaging in Schizophrenia Two initial studies examining [11C]PK11195 binding in small cohorts of patients and matched controls were published. Both observed substantial elevation of BP in patients with schizophrenia—50% increased BPND in hippocampus in one case (seven medicated patients with active psychotic symptoms and eight controls)87 and 17% increased BPP across all gray matter in the others (10 medicated patients who had schizophrenia for 5 years or less and 10 controls).88 More recently, several studies have been published using second generation tracers in larger cohorts. A study using [11C]DA1106 in 14 medicated patients and 14 matched controls found no difference in mean BPND between groups either in regions of interest or total cortex,89 although there was some association between BPND and positive symptom scores. However, this study was performed before characterization of the multiple binding types, and rs6971 genotype was not measured or controlled for. A more recent study examined 16 medicated patients with ongoing psychotic symptoms and 27 healthy controls with [18F]FEPPA while controlling for rs6971 genotype and found no group level differences across a large set of gray and white matter regions.90 Most recently, a study was performed using [18F]PBR28 imaging to compare 14 UHR subjects (12 drug naïve) to 14 age-matched healthy controls and 14 medicated patients with schizophrenia to an additional set of 14 agematched controls.91 The analysis approach had two unique features, first by including a vascular endothelial cell binding compartment,92 and second, by normalizing regional VT to whole brain VT (distribution volume ratio [DVR]). Direct comparison of VT between groups detected no significant differences in any region. However, DVR was significantly elevated in both UHR and schizophrenia relative to their respective controls in temporal lobe, frontal cortex, and total gray matter. In summary, studies in schizophrenia and related conditions are thus far inconclusive as to whether TSPO is elevated in these patients. The studies have differed in radioligand, analysis approach, and phase of illness. All these factors may have influenced outcomes and would need to be examined carefully in future research. It may be the case that elevated inflammatory markers only occur at a particular phase of the illness. All of these studies measured TSPO in antipsychotic-medicated patients, which may have affected microglial activation.93-95

M. Slifstein and A. Abi-Dargham

60 The DVR approach, by normalizing to whole brain, only provides information about the relative expression of TSPO, which may be less variable and possibly more sensitive to group differences than VT, but limits interpretation as to whether activated microglial density is elevated or just differently distributed but within normal range. Neuroinflammation imaging in schizophrenia is in its early stages, and there are many questions to be addressed.

Inflammation Imaging in Depression Two studies have been performed with TSPO imaging in major depression. In one, 10 subjects undergoing a major depressive episode, two of whom were medicated with SSRIs, and 10 matched controls were scanned with [11C]PBR28.96 Although binding genotype was measured, it was not included in the statistical model, but the groups were well matched for genotype. No group differences in VT were observed in any brain region. In the second study, [18F]FEPPA imaging was performed in 20 subjects undergoing a major depressive episode who had been medication-free for at least 9 weeks and 20 matched controls.97 Binding genotype was measured and controlled for in the statistical model. VT was increased in patients by 25%-35% across all measured brain regions. Thus, in depression as in schizophrenia, early results do not yet show a consistent pattern. Neuroinflammation imaging in psychiatry is in an early stage, and there is much more to investigate. There are open questions as to whether inflammatory markers are elevated at certain phases of the illnesses and not others, if inflammatory processes are causal or consequential (if they do in fact have a role), and how they are involved in conditions as different as schizophrenia and depression. Also, while all imaging agents to date have been TSPO ligands, it may be that other target molecules such as the cyclooxygenases (COX-1 and COX-2) would also be informative about microglia expression and inflammatory processes, and there has been some recent work to develop COX-1 and COX-2 radiotracers.98,99

Conclusions and Future Directions Although considerable research effort has been dedicated to expanding the tools to measure neurotransmission into systems other than dopamine, much remains to be done. There is not a robust method for measuring serotonin transmission, although there are numerous radiotracers for measuring many 5-HT receptor types and reuptake transporters. The recently developed 5-HT1B radiotracers show great promise in this area, but have yet to be thoroughly tested in humans. Reliable tracers for the amino acid transmitter systems have been difficult to develop, and the few successful ones to date bind to allosteric sites, so that inferences about neurotransmission can only be detected through indirect, noncompetitive interactions. Currently available tracers for imaging neuroinflammation all bind to TSPO, which may provide limited information about inflammatory processes.

Psychiatry research would benefit from the continued development of tracers that provide more specific information in all of these areas. Conversely, tracers for targets that are not specific to one transmitter system or process, such as recently developed tracers for histone deacetylaces100,101 or synaptic vesicle glycoprotein 2A102,103 may provide new perspectives on brain function in many research areas including psychiatry. With these new developments and the current ongoing work in the new areas highlighted here, we expect that molecular imaging will continue to be a powerful tool to investigate brain function in health and disease.

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