Development of a novel rodent rapid serial visual presentation task reveals dissociable effects of stimulant versus nonstimulant treatments on attentional processes.

The rapid serial visual presentation (RSVP) task and continuous performance tasks (CPT) are used to assess attentional impairments in patients with psychiatric and neurological conditions. This study developed a novel touchscreen task for rats based on the structure of a human RSVP task and used pharmacological manipulations to investigate their effects on different performance measures. Normal animals were trained to respond to a target image and withhold responding to distractor images presented within a continuous sequence. In a second version of the task, a false-alarm image was included, so performance could be assessed relative to two types of nontarget distractors. The effects of acute administration of stimulant and nonstimulant treatments for ADHD (amphetamine and atomoxetine) were tested in both tasks. Methylphenidate, ketamine, and nicotine were tested in the first task only. Amphetamine made animals more impulsive and decreased overall accuracy but increased accuracy when the target was presented early in the image sequence. Atomoxetine improved accuracy overall with a specific reduction in false-alarm responses and a shift in the attentional curve reflecting improved accuracy for targets later in the image sequence. However, atomoxetine also slowed responding and increased omissions. Ketamine, nicotine, and methylphenidate had no specific effects at the doses tested. These results suggest that stimulant versus nonstimulant treatments have different effects on attention and impulsive behaviour in this rat version of an RSVP task. These results also suggest that RSVP-like tasks have the potential to be used to study attention in rodents.

Mechanisms of mGluR-dependent plasticity in hippocampal area CA2.

Pyramidal cells in hippocampal area CA2 have synaptic properties that are distinct from the other CA subregions. Notably, this includes a lack of typical long-term potentiation of stratum radiatum synapses. CA2 neurons express high levels of several known and potential regulators of metabotropic glutamate receptor (mGluR)-dependent signaling including Striatal-Enriched Tyrosine Phosphatase (STEP) and several Regulator of G-protein Signaling (RGS) proteins, yet the functions of these proteins in regulating mGluR-dependent synaptic plasticity in CA2 are completely unknown. Thus, the aim of this study was to examine mGluR-dependent synaptic depression and to determine whether STEP and the RGS proteins RGS4 and RGS14 are involved. Using whole cell voltage-clamp recordings from mouse pyramidal cells, we found that mGluR agonist-induced long-term depression (mGluR-LTD) is more pronounced in CA2 compared with that observed in CA1. This mGluR-LTD in CA2 was found to be protein synthesis and STEP dependent, suggesting that CA2 mGluR-LTD shares mechanistic processes with those seen in CA1, but in addition, RGS14, but not RGS4, was essential for mGluR-LTD in CA2. In addition, we found that exogenous application of STEP could rescue mGluR-LTD in RGS14 KO slices. Supporting a role for CA2 synaptic plasticity in social cognition, we found that RGS14 KO mice had impaired social recognition memory as assessed in a social discrimination task. These results highlight possible roles for mGluRs, RGS14, and STEP in CA2-dependent behaviors, perhaps by biasing the dominant form of synaptic plasticity away from LTP and toward LTD in CA2.

Objective measures of reward sensitivity and motivation in people with high v. low anhedonia.

BACKGROUND: Anhedonia - a diminished interest or pleasure in activities - is a core self-reported symptom of depression which is poorly understood and often resistant to conventional antidepressants. This symptom may occur due to dysfunction in one or more sub-components of reward processing: motivation, consummatory experience and/or learning. However, the precise impairments remain elusive. Dissociating these components (ideally, using cross-species measures) and relating them to the subjective experience of anhedonia is critical as it may benefit fundamental biology research and novel drug development. METHODS: Using a battery of behavioural tasks based on rodent assays, we examined reward motivation (Joystick-Operated Runway Task, JORT; and Effort-Expenditure for Rewards Task, EEfRT) and reward sensitivity (Sweet Taste Test) in a non-clinical population who scored high (N = 32) or low (N = 34) on an anhedonia questionnaire (Snaith-Hamilton Pleasure Scale). RESULTS: Compared to the low anhedonia group, the high anhedonia group displayed marginal impairments in effort-based decision-making (EEfRT) and reduced reward sensitivity (Sweet Taste Test). However, we found no evidence of a difference between groups in physical effort exerted for reward (JORT). Interestingly, whilst the EEfRT and Sweet Taste Test correlated with anhedonia measures, they did not correlate with each other. This poses the question of whether there are subgroups within anhedonia; however, further work is required to directly test this hypothesis. CONCLUSIONS: Our findings suggest that anhedonia is a heterogeneous symptom associated with impairments in reward sensitivity and effort-based decision-making.

Effects of pro-depressant and immunomodulatory drugs on biases in decision-making in the rat judgement bias task.

Studies in human and non-human species suggest that decision-making behaviour can be biased by an affective state, also termed an affective bias. To study these behaviours in non-human species, judgement bias tasks (JBT) have been developed. Animals are trained to associate specific cues (tones) with a positive or negative/less positive outcome. Animals are then presented with intermediate ambiguous cues and affective biases quantified by observing whether animals make more optimistic or more pessimistic choices. Here we use a high versus low reward JBT and test whether pharmacologically distinct compounds, which induce negative biases in learning and memory, have similar effects on decision-making: tetrabenazine (0.0-1.0 mg/kg), retinoic acid (0.0-10.0 mg/kg), and rimonabant (0.0-10.0 mg/kg). We also tested immunomodulatory compounds: interferon-α (0-100 units/kg), lipopolysaccharide (0.0-10.0 µg/kg), and corticosterone (0.0-10.0 mg/kg). We observed no specific effects in the JBT with any acute treatment except corticosterone which induced a negative bias. We have previously observed a similar lack of effect with acute but not chronic psychosocial stress and so next tested decision-making behaviour following chronic interferon-alpha. Animals developed a negative bias which was sustained even after treatment was ended. These data suggest that decision-making behaviour in the task is sensitive to chronic but not acute effects of most pro-depressant drugs or immunomodulators, but the exogenous administration of acute corticosterone induces pessimistic behaviour. This work supports our hypothesis that biases in decision-making develop over a different temporal scale to those seen with learning and memory which may be relevant in the development and perpetuation of mood disorders.

Power failure: why small sample size undermines the reliability of neuroscience.

A study with low statistical power has a reduced chance of detecting a true effect, but it is less well appreciated that low power also reduces the likelihood that a statistically significant result reflects a true effect. Here, we show that the average statistical power of studies in the neurosciences is very low. The consequences of this include overestimates of effect size and low reproducibility of results. There are also ethical dimensions to this problem, as unreliable research is inefficient and wasteful. Improving reproducibility in neuroscience is a key priority and requires attention to well-established but often ignored methodological principles.

Optogenetic Stimulation of Prefrontal Glutamatergic Neurons Enhances Recognition Memory.

UNLABELLED: Finding effective cognitive enhancers is a major health challenge; however, modulating glutamatergic neurotransmission has the potential to enhance performance in recognition memory tasks. Previous studies using glutamate receptor antagonists have revealed that the medial prefrontal cortex (mPFC) plays a central role in associative recognition memory. The present study investigates short-term recognition memory using optogenetics to target glutamatergic neurons within the rodent mPFC specifically. Selective stimulation of glutamatergic neurons during the online maintenance of information enhanced associative recognition memory in normal animals. This cognitive enhancing effect was replicated by local infusions of the AMPAkine CX516, but not CX546, which differ in their effects on EPSPs. This suggests that enhancing the amplitude, but not the duration, of excitatory synaptic currents improves memory performance. Increasing glutamate release through infusions of the mGluR7 presynaptic receptor antagonist MMPIP had no effect on performance. SIGNIFICANCE STATEMENT: These results provide new mechanistic information that could guide the targeting of future cognitive enhancers. Our work suggests that improved associative-recognition memory can be achieved by enhancing endogenous glutamatergic neuronal activity selectively using an optogenetic approach. We build on these observations to recapitulate this effect using drug treatments that enhance the amplitude of EPSPs; however, drugs that alter the duration of the EPSP or increase glutamate release lack efficacy. This suggests that both neural and temporal specificity are needed to achieve cognitive enhancement.

The Affective Bias Test and Reward Learning Assay: Neuropsychological Models for Depression Research and Investigating Antidepressant Treatments in Rodents.

The Affective Bias Test (ABT) quantifies acute changes in affective state based on the affective biases they generate in an associative reward learning task. The Reward Learning Assay (RLA) provides a control assay for the ABT and reward-induced biases generated in this model are sensitive to changes in core affective state. Both tasks involve training animals to associate a specific digging substrate with a food reward. Animals learn to discriminate between two digging substrates placed in ceramic bowls, one rewarded and one unrewarded. In the ABT, the animal learns two independent substrate-reward associations with a fixed reward value following either an affective state or drug manipulation, or under control conditions. Affective biases generated are quantified in a choice test where the animals exhibit a bias (make more choices) for one of the substrates which is specifically related to affective state at the time of learning. The ABT is used to investigate biases generated during learning as well as modulation of biases associated with past experiences. The RLA follows a similar protocol, but the animal remains in the same affective state throughout and a reward-induced bias is generated by pairing one substrate with a higher value reward. The RLA provides a control to determine if drug treatments affect memory retrieval more generally. Studies in depression models and following environmental enrichment suggest that reward-induced biases are sensitive to core changes in affective state. Each task offers different insights into affective processing mechanisms and may help improve the translational validity of animal studies and benefit pre-clinical drug development. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bowl digging and discrimination training Basic Protocol 2: The reward learning assay Basic Protocol 3: The affective bias test - new learning Basic Protocol 4: The affective bias test - modulation of affective biases associated with past experiences.

Analysis of hippocampal synaptic function in a rodent model of early life stress.

Background: Early life stress (ELS) is an important risk factor in the aetiology of depression. Developmental glucocorticoid exposure impacts multiple brain regions with the hippocampus being particularly vulnerable. Hippocampal mediated behaviours are dependent upon the ability of neurones to undergo long-term potentiation (LTP), an N-methyl-D-aspartate receptor (NMDAR) mediated process. In this study we investigated the effect of ELS upon hippocampal NMDAR function. Methods: Hooded Long-Evans rat pups (n=82) were either undisturbed or maternally separated for 180 minutes per day (MS180) between post-natal day (PND) 1 and PND14. Model validation consisted of sucrose preference (n=18) and novelty supressed feeding (NSFT, n=34) tests alongside assessment of corticosterone (CORT) and paraventricular nucleus (PVN) cFos reactivity to stress and hippocampal neurogenesis (all n=18). AMPA/NMDA ratios (n=19), miniEPSC currents (n=19) and LTP (n=15) were assessed in whole-cell patch clamp experiments in CA1 pyramidal neurones. Results: MS180 animals showed increased feeding latency in the NSFT alongside increased overall CORT in the restraint stress experiment and increased PVN cFos expression in males but no changes in neurogenesis or sucrose preference. MS180 was associated with a lower AMPA/NMDA ratio with no change in miniEPSC amplitude or area. There was no difference in short- or long-term potentiation between MS180 and control animals nor were there any changes during the induction protocol. Conclusions: The MS180 model showed a behavioural phenotype consistent with previous work. MS180 animals showed increased NMDAR function with preliminary evidence suggesting that this was not concurrent with an increase in LTP.

Highly stressful early life events are the biggest risk factor for developing depression in adulthood. The hippocampus is a brain region that is highly susceptible to this stress and is crucial for coordinating learning and memory which underpins many aspects of cognitive function. Our study investigated if changes in the way that the neurons in the hippocampus communicate could provide explanations as to why early life stress predisposes to depression. We used an animal model of early life stress where rat pups are separated from their mother for three hours per day during their early life. Upon adulthood this resulted in the rats being slower to eat food in a new environment, a standard test of anxiety behaviour. We then used a technique called ex-vivo patch clamp electrophysiology to study how the individual neurons in their hippocampi and their connections functioned after early life stress. We measured the relative power of the signals from two key synaptic receptors essential for communication between neurons: AMPA and NMDA receptors. AMPA receptors are the key receptors enabling communication between neurons at synapses whereas NMDA receptors allow a neuron to become more sensitive to input signals and adapt synaptic strength. Animals with early life stress had more NMDA receptor function relative to AMPA function compared to control animals. We used a technique called miniEPSC recordings to rule out any change in AMPA receptor function in ELS animals meaning an effect specific to NMDA receptors. However, we found no changes to the ability for synapses to adapt their strength between groups. This work presents evidence for changes in hippocampal neurons and synapses caused by early life stress but further work is needed to understand how this relates to depression.

Characterisation of behaviours relevant to apathy syndrome in the aged male rat.

Apathy is a complex psychiatric syndrome characterised by motivational deficit, emotional blunting and cognitive changes. It occurs alongside a broad range of neurological disorders, but also occurs in otherwise healthy ageing. Despite its clinical prevalence, apathy does not yet have a designated treatment strategy. Generation of a translational animal model of apathy syndrome would facilitate the development of novel treatments. Given the multidimensional nature of apathy, a model cannot be achieved with a single behavioural test. Using a battery of behavioural tests we investigated whether aged rats exhibit behavioural deficits across different domains relevant to apathy. Using the effort for reward and progressive ratio tasks we found that aged male rats (21-27 months) show intact reward motivation. Using the novelty supressed feeding test and position-based object exploration we found aged rats showed increased anxiety-like behaviour inconsistent with emotional blunting. The sucrose preference test and reward learning assay showed intact reward sensitivity and reward-related cognition in aged rats. However, using a bowl-digging version of the probabilistic reversal learning task, we found a deficit in cognitive flexibility in aged rats that did not translate across to a touchscreen version of the task. While these data reveal important changes in cognitive flexibility and anxiety associated with ageing, aged rats do not show deficits across other behavioural domains relevant to apathy. This suggests that aged rats are not a suitable model for age-related apathy syndrome. These findings contrast with previous work in mice, revealing important species differences in behaviours relevant to apathy syndrome in ageing.

Rapid-acting antidepressant drugs modulate affective bias in rats.

How rapid-acting antidepressants (RAADs), such as ketamine, induce immediate and sustained improvements in mood in patients with major depressive disorder (MDD) is poorly understood. A core feature of MDD is the prevalence of cognitive processing biases associated with negative affective states, and the alleviation of negative affective biases may be an index of response to drug treatment. Here, we used an affective bias behavioral test in rats, based on an associative learning task, to investigate the effects of RAADs. To generate an affective bias, animals learned to associate two different digging substrates with a food reward in the presence or absence of an affective state manipulation. A choice between the two reward-associated digging substrates was used to quantify the affective bias generated. Acute treatment with the RAADs ketamine, scopolamine, or psilocybin selectively attenuated a negative affective bias in the affective bias test. Low, but not high, doses of ketamine and psilocybin reversed the valence of the negative affective bias 24 hours after RAAD treatment. Only treatment with psilocybin, but not ketamine or scopolamine, led to a positive affective bias that was dependent on new learning and memory formation. The relearning effects of ketamine were dependent on protein synthesis localized to the rat medial prefrontal cortex and could be modulated by cue reactivation, consistent with experience-dependent neural plasticity. These findings suggest a neuropsychological mechanism that may explain both the acute and sustained effects of RAADs, potentially linking their effects on neural plasticity with affective bias modulation in a rodent model.

Neuropsychological Effects of Antidepressants: Translational Studies.

Pharmacological treatments that improve mood were first identified serendipitously, but more than half a century later, how these drugs induce their antidepressant effects remains largely unknown. With the help of animal models, a detailed understanding of their pharmacological targets and acute and chronic effects on brain chemistry and neuronal function has been achieved, but it remains to be elucidated how these effects translate to clinical efficacy. Whilst the field has been dominated by the monoamine and neurotrophic hypotheses, the idea that the maladaptive cognitive process plays a critical role in the development and perpetuation of mood disorders has been discussed since the 1950s. Recently, studies using objective methods to quantify changes in emotional processing found acute effects with conventional antidepressants in both healthy volunteers and patients. These positive effects on emotional processing and cognition occur without a change in the subjective ratings of mood. Building from these studies, behavioural methods for animals that quantify similar cognitive affective processes have been developed. Integrating these behavioural approaches with pharmacology and targeted brain manipulations, a picture is beginning to emerge of the underlying mechanisms that may link the pharmacology of antidepressants, these neuropsychological constructs and clinical efficacy. In this chapter, we discuss findings from animal studies, experimental medicine and patients investigating the neuropsychological effects of antidepressant drugs. We discuss the possible neural circuits that contribute to these effects and discuss whether a neuropsychological model of antidepressant effects could explain the temporal differences in clinical benefits observed with conventional delayed-onset antidepressants versus rapid-acting antidepressants.

Pharmacological characterisation of the effort for reward task as a measure of motivation for reward in male mice.

RATIONALE: Motivational deficits are a common symptom shared across multiple psychiatric and neurodegenerative disorders. Effort-based decision-making tasks are a translatable method for assessing motivational state. Much of the preclinical validation of the task derives from acute pharmacological manipulations in rats. However, mice currently offer a greater genetic toolkit to study risk genes and phenotypic models. Despite this, there is limited characterisation of their behaviour in this type of motivation task. OBJECTIVES: Here, we investigate the effort for reward (EfR) task as a measure of motivational state in mice using drugs previously shown to modulate effort-based decision-making in rats and humans. METHOD: Using male C57bl/6j mice, we test the effects of drugs which modulate DA transmission. We also test the effects of CP101-606 which does not act directly via DA modulation but has been shown to exert beneficial effects on motivational state. Finally, we test the sensitivity of the task to a chronic corticosterone (CORT) treatment. RESULTS: Amphetamine, methylphenidate, and CP101606 in mice increased high-effort responses for high-value reward, while administration of haloperidol decreased high-effort responses. Surprisingly, tetrabenazine had no effect at the doses tested. Chronic, low-dose CORT consumption did not alter task performance. CONCLUSION: These data suggest that the EfR task is sensitive to acute dopaminergic modulation and NR2B selective antagonism in mice. However, it may lack sensitivity to non-acute phenotypic models. Further work is required to demonstrate the utility of the task in this context.

Correction: Davies et al. Impact of Refinements to Handling and Restraint Methods in Mice. Animals 2022, 12, 2173.

In the original publication [...].

Distinct hippocampal-prefrontal neural assemblies coordinate memory encoding, maintenance, and recall.

Short-term memory enables incorporation of recent experience into subsequent decision-making. This processing recruits both the prefrontal cortex and hippocampus, where neurons encode task cues, rules, and outcomes. However, precisely which information is carried when, and by which neurons, remains unclear. Using population decoding of activity in rat medial prefrontal cortex (mPFC) and dorsal hippocampal CA1, we confirm that mPFC populations lead in maintaining sample information across delays of an operant non-match to sample task, despite individual neurons firing only transiently. During sample encoding, distinct mPFC subpopulations joined distributed CA1-mPFC cell assemblies hallmarked by 4-5 Hz rhythmic modulation; CA1-mPFC assemblies re-emerged during choice episodes but were not 4-5 Hz modulated. Delay-dependent errors arose when attenuated rhythmic assembly activity heralded collapse of sustained mPFC encoding. Our results map component processes of memory-guided decisions onto heterogeneous CA1-mPFC subpopulations and the dynamics of physiologically distinct, distributed cell assemblies.

A mild impairment in reversal learning in a bowl-digging substrate deterministic task but not other cognitive tests in the Dlg2+/- rat model of genetic risk for psychiatric disorder.

Variations in the Dlg2 gene have been linked to increased risk for psychiatric disorders, including schizophrenia, autism spectrum disorders, intellectual disability, bipolar disorder, attention deficit hyperactivity disorder, and pubertal disorders. Recent studies have reported disrupted brain circuit function and behaviour in models of Dlg2 knockout and haploinsufficiency. Specifically, deficits in hippocampal synaptic plasticity were found in heterozygous Dlg2+/- rats suggesting impacts on hippocampal dependent learning and cognitive flexibility. Here, we tested these predicted effects with a behavioural characterisation of the heterozygous Dlg2+/- rat model. Dlg2+/- rats exhibited a specific, mild impairment in reversal learning in a substrate deterministic bowl-digging reversal learning task. The performance of Dlg2+/- rats in other bowl digging task, visual discrimination and reversal, novel object preference, novel location preference, spontaneous alternation, modified progressive ratio, and novelty-suppressed feeding test were not impaired. These findings suggest that despite altered brain circuit function, behaviour across different domains is relatively intact in Dlg2+/- rats, with the deficits being specific to only one test of cognitive flexibility. The specific behavioural phenotype seen in this Dlg2+/- model may capture features of the clinical presentation associated with variation in the Dlg2 gene.

Study of novel selective mGlu2 agonist in the temporo-ammonic input to CA1 neurons reveals reduced mGlu2 receptor expression in a Wistar substrain with an anxiety-like phenotype.

Group II metabotropic receptors (mGluRs) regulate central synaptic transmission by modulating neurotransmitter release. However, the lack of pharmacological tools differentiating between mGlu2 and mGlu3 receptors has hampered identification of the roles of these two receptor subtypes. We have used LY395756 [(1SR,2SR,4RS,5RS,6SR)-2-amino-4-methylbicyclo[3.1.0]-hexane2,6-dicarboxylic], an agonist at mGlu2 receptors and an antagonist at mGlu3 receptors in cell lines, to investigate the roles of these receptors in the temporo-ammonic path from entorhinal cortex to CA1-stratum lacunosum moleculare in rat hippocampal slices. Surprisingly, the degree of inhibition of the field EPSP induced by LY395756 fell into two distinct groups, with EC(50) values of <1 µm and >100 µm. In "sensitive" slices, LY395756 had additive actions with a mixed mGlu2/mGlu3 agonist, DCG-IV [(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine], whereas in "insensitive" slices, LY395756 reduced the effect of DCG-IV, with an IC(50) of ∼1 µm. This separation into sensitive and insensitive slices could be explained by LY395756 acting as an mGlu2 agonist and mGlu3 antagonist, respectively, a finding supported by data from mice lacking these receptors. The heterogeneity was correlated with differences in expression levels of mGlu2 receptors within our Wistar colony and other Wistar substrains. The initial search for a behavioral correlate indicated that rats lacking mGlu2 receptors showed anxiety-like behavior in open-field and elevated plus maze assays. These findings have implications for rat models of psychiatric disease and are especially pertinent given that mGlu2 receptors are targets for compounds under development for anxiety.

Evaluation of a novel translational task for assessing emotional biases in different species.

Changes in the processing of emotional information are key features of affective disorders. Neuropsychological tests based on emotional faces or words are used to detect emotional/affective biases in humans, but these tests are not applicable to animal species. In the present study, we investigated whether a novel affective tone discrimination task (ATDT), developed to study emotion-related behaviour in rats, could also be used to quantify changes in affective states in humans. To date, the methods used in human neuropsychology have not been applicable to animal experiments. Participants completed a training session in which they learnt to discriminate specific tone frequencies and to correctly respond in order to gain emotionally valenced outcomes, to obtain rewards (money), or to avoid punishment (an aversive sound clip). During a subsequent test session, additional ambiguous probe tones were presented at frequencies intermediate between the reward and avoidance paired tones. At the end of the task, participants completed self-report questionnaires. All participants made more avoidance responses to the most ambiguous tone cues, suggesting a bias towards avoidance of punishment. Individual differences in the degrees of bias observed were correlated with anxiety measures, suggesting the task's sensitivity to differences in state anxiety within a healthy population. Further studies in clinical populations will be necessary to assess the task's sensitivity to pathological anxiety states. These data suggest that this affective tone discrimination task provides a novel method to study cognitive affective biases in different species, including humans, and offers a novel assessment to study anxiety.

Evaluation and initial in vitro and ex vivo characterization of the potential positron emission tomography ligand, BU99008 (2-(4,5-dihydro-1H-imidazol-2-yl)-1- methyl-1H-indole), for the imidazoline2 binding site.

The density of the Imidazoline2 binding site (I2BS) has been shown to change in psychiatric conditions such as depression and addiction, along with neurodegenerative disorders such as Alzheimer's disease and Huntington's chorea. The presence of I2BS on glial cells and the possibility that they may in some way regulate glial fibrillary acidic protein has led to increased interest into the role of I2BS and I2BS ligands in conditions characterized by marked gliosis. In addition, it has been suggested that I2BS may be a marker for human glioblastomas. Therefore, the development of a positron emission tomography (PET) radioligand for the I2BS would be of major benefit in our understanding of these conditions. We now report the successful synthesis and initial pharmacological evaluation of potential PET radioligands for the I2BS as well as the tritiation and characterization of the most favorable of the series, BU99008 (6), both in vitro and ex vivo in rat. The series as a whole demonstrated excellent affinity and selectivity for the I2BS, with BU99008 (6) selected as the lead candidate to be taken forward for in vivo assessment. BU99008 (6) showed very good affinity for the I2BS (K(i) of 1.4 nM; K(d) = 1.3 nM), good selectivity compared with the α2 -adrenoceptor (909-fold). In addition, following peripheral administration, [³H]BU99008 demonstrated a heterogenous uptake into the rat brain consistent with the known distribution of the I2BS in vivo. This, and the amenability of BU99008 (6) to radiolabeling with a positron-emitting radioisotope, indicates its potential as a PET radioligand for imaging the I2BS in vivo.