Excessive sucrose consumption reduces synaptic density and increases cannabinoid receptors in Göttingen minipigs.

Diets high in sucrose and fat are becoming more prevalent the world over, accompanied by a raised prevalence of cardiovascular diseases, cancers, diabetes, obesity, and metabolic syndrome. Clinical studies link unhealthy diets with the development of mental health disorders, particularly depression. Here, we investigate the effects of 12 days of sucrose consumption administered as 2 L of 25% sucrose solution daily for 12 days in Göttingen minipigs on the function of brain receptors involved in reward and motivation, regulating feeding, and pre- and post-synaptic mechanisms. Through quantitative autoradiography of cryostat sections containing limbic brain regions, we investigated the effects of sucrose restricted to a 1-h period each morning, on the specific binding of [3H]raclopride on dopamine D2/3 receptors, [3H]UCB-J at synaptic vesicle glycoprotein 2A (SV2A), [3H]MPEPγ at metabotropic glutamate receptor subtype 5 (mGluR5) and [3H]SR141716A at the cannabinoid receptor 1 (CB1). Compared to control diet animals, the sucrose group showed significantly lower [3H]UCB-J and [3H]MPEPγ binding in the prefrontal cortex. The sucrose-consuming minipigs showed higher hippocampal CB1 binding, but unaltered dopamine D2/3 binding compared to the control group. We found that the sucrose diet reduced the synaptic density marker while increasing CB1 binding in limbic brain structures, which may subserve maladaptive changes in appetite regulation and feeding. Further studies of the effects of diets and lifestyle habits on brain neuroreceptor and synaptic density markers are warranted.

Subanesthetic S-ketamine does not acutely alter striatal dopamine transporter binding in healthy Sprague Dawley female rats.

Major depressive disorder is one of the most prevalent mental health disorders, posing a global socioeconomic burden. Conventional antidepressant treatments have a slow onset of action, and 30% of patients show no clinically significant treatment response. The recently approved fast-acting antidepressant S-ketamine, an N-methyl-D-aspartate receptor antagonist, provides a new approach for treatment-resistant patients. However, knowledge of S-ketamine's mechanism of action is still being established. Depressed human subjects have lower striatal dopamine transporter (DAT) availability compared to healthy controls. Rodent studies report increased striatal dopamine concentration in response to acute ketamine administration. In vivo [18F]FE-PE2I ([18F]-(E)-N-(3-iodoprop-2-enyl)-2ß-carbofluoroethoxy-3ß-(4'-methyl-phenyl) nortropane) positron emission tomography (PET) imaging of the DAT has not previously been applied to assess the effect of acute subanesthetic S-ketamine administration on DAT availability. We applied translational in vivo [18F]FE-PE2I PET imaging of the DAT in healthy female rats to evaluate whether an acute subanesthetic intraperitoneal dose of 15 mg/kg S-ketamine alters DAT availability. We also performed [3H]GBR-12935 autoradiography on postmortem brain sections. We found no effect of acute S-ketamine administration on striatal DAT binding using [18F]FE-PE2I PET or [3H]GBR-12935 autoradiography. This negative result does not support the hypothesis that DAT changes are associated with S-ketamine's rapid antidepressant effects, but additional studies are warranted.

A single dose of cocaine raises SV2A density in hippocampus of adolescent rats.

OBJECTIVE: Cocaine is a highly addictive psychostimulant that affects synaptic activity with structural and functional adaptations of neurons. The transmembrane synaptic vesicle glycoprotein 2A (SV2A) of pre-synaptic vesicles is commonly used to measure synaptic density, as a novel approach to the detection of synaptic changes. We do not know if a single dose of cocaine suffices to affect pre-synaptic SV2A density, especially during adolescence when synapses undergo intense maturation. Here, we explored potential changes of pre-synaptic SV2A density in target brain areas associated with the cocaine-induced boost of dopaminergic neurotransmission, specifically testing if the effects would last after the return of dopamine levels to baseline. METHODS: We administered cocaine (20 mg/kg i.p.) or saline to rats in early adolescence, tested their activity levels and removed the brains 1 hour and 7 days after injection. To evaluate immediate and lasting effects, we did autoradiography with [3H]UCB-J, a specific tracer for SV2A, in medial prefrontal cortex, striatum, nucleus accumbens, amygdala, and dorsal and ventral areas of hippocampus. We also measured the striatal binding of [3H]GBR-12935 to test cocaine's occupancy of the dopamine transporter at both times of study. RESULTS: We found a significant increase of [3H]UCB-J binding in the dorsal and ventral sections of hippocampus 7 days after the cocaine administration compared to saline-injected rats, but no differences 1 hour after the injection. The [3H]GBR-12935 binding remained unchanged at both times. CONCLUSION: Cocaine provoked lasting changes of hippocampal synaptic SV2A density after a single exposure during adolescence.

A SPECT-based method for dynamic imaging of the glymphatic system in rats.

The glymphatic system is a brain-wide waste drainage system that promotes cerebrospinal fluid circulation through the brain to remove waste metabolites. Currently, the most common methods for assessing glymphatic function are ex vivo fluorescence microscopy of brain slices, macroscopic cortical imaging, and MRI. While all these methods have been crucial for expanding our understanding of the glymphatic system, new techniques are required to overcome their specific drawbacks. Here, we evaluate SPECT/CT imaging as a tool to assess glymphatic function in different anesthesia-induced brain states using two radiolabeled tracers, [111In]-DTPA and [99mTc]-NanoScan. Using SPECT, we confirmed the existence of brain state-dependent differences in glymphatic flow and we show brain state-dependent differences of CSF flow kinetics and CSF egress to the lymph nodes. We compare SPECT and MRI for imaging glymphatic flow and find that the two imaging modalities show the same overall pattern of CSF flow, but that SPECT was specific across a greater range of tracer concentrations than MRI. Overall, we find that SPECT imaging is a promising tool for imaging the glymphatic system, and that qualities such as high sensitivity and the variety of available tracers make SPECT imaging a good alternative for glymphatic research.

Synaptic Vesicle Glycoprotein 2A: Features and Functions.

In recent years, the field of neuroimaging dramatically moved forward by means of the expeditious development of specific radioligands of novel targets. Among these targets, the synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles, present in all synaptic terminals, irrespective of neurotransmitter content. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. The ubiquitous expression in gray matter regions of the brain is the basis of its candidacy as a marker of synaptic density. Following the development of molecules derived from the structure of the anti-epileptic drug levetiracetam, which selectively binds to SV2A, several radiolabeled markers have been synthetized to allow the study of SV2A distribution with positron emission tomography (PET). These radioligands permit the evaluation of in vivo changes of SV2A distribution held to be a potential measure of synaptic density in physiological and pathological conditions. The use of SV2A as a biomarker of synaptic density raises important questions. Despite numerous studies over the last decades, the biological function and the expressional properties of SV2A remain poorly understood. Some functions of SV2A were claimed, but have not been fully elucidated. While the expression of SV2A is ubiquitous, stronger associations between SV2A and Υ amino butyric acid (GABA)-ergic rather than glutamatergic synapses were observed in some brain structures. A further issue is the unclear interaction between SV2A and its tracers, which reflects a need to clarify what really is detected with neuroimaging tools. Here, we summarize the current knowledge of the SV2A protein and we discuss uncertain aspects of SV2A biology and physiology. As SV2A expression is ubiquitous, but likely more strongly related to a certain type of neurotransmission in particular circumstances, a more extensive knowledge of the protein would greatly facilitate the analysis and interpretation of neuroimaging results by allowing the evaluation not only of an increase or decrease of the protein level, but also of the type of neurotransmission involved.

Validation and optimisation of an automatic blood sampler for preclinical positron emission tomography research in domestic pigs.

In preclinical positron emission tomography animal studies, continuous blood sampling is used to measure the time course of the activity concentration in arterial blood. However, pigs have hypercoagulable blood that tends to clot inside plastic tubes. We tested several tube materials and lengths and the use of three-way connectors. We validated set-ups for automated blood sampling with and without blood recirculation that could run for 90 minutes without problematic clots and without any evidence of emboli formation during necropsy.

Dorsal striatal dopamine induces fronto-cortical hypoactivity and attenuates anxiety and compulsive behaviors in rats.

Dorsal striatal dopamine transmission engages the cortico-striato-thalamo-cortical (CSTC) circuit, which is implicated in many neuropsychiatric diseases, including obsessive-compulsive disorder (OCD). Yet it is unknown if dorsal striatal dopamine hyperactivity is the cause or consequence of changes elsewhere in the CSTC circuit. Classical pharmacological and neurotoxic manipulations of the CSTC and other brain circuits suffer from various drawbacks related to off-target effects and adaptive changes. Chemogenetics, on the other hand, enables a highly selective targeting of specific neuronal populations within a given circuit. In this study, we developed a chemogenetic method for selective activation of dopamine neurons in the substantia nigra, which innervates the dorsal striatum in the rat. We used this model to investigate effects of targeted dopamine activation on CSTC circuit function, especially in fronto-cortical regions. We found that chemogenetic activation of these neurons increased movement (as expected with increased dopamine release), rearings and time spent in center, while also lower self-grooming. Furthermore, this activation increased prepulse inhibition of the startle response in females. Remarkably, we observed reduced [18F]FDG metabolism in the frontal cortex, following dopamine activation in the dorsal striatum, while total glutamate levels- in this region were increased. This result is in accord with clinical studies of increased [18F]FDG metabolism and lower glutamate levels in similar regions of the brain of people with OCD. Taken together, the present chemogenetic model adds a mechanistic basis with behavioral and translational relevance to prior clinical neuroimaging studies showing deficits in fronto-cortical glucose metabolism across a variety of clinical populations (e.g. addiction, risky decision-making, compulsivity or obesity).

Blocking of efflux transporters in rats improves translational validation of brain radioligands.

BACKGROUND: Positron emission tomography (PET) is a molecular imaging technique that can be used to investigate the in vivo pharmacology of drugs. Initial preclinical evaluation of PET tracers is often conducted in rodents due to the accessibility of disease models as well as economic considerations. Compared to larger species, rodents display a higher expression and/or activity of efflux transporters such as the P-glycoprotein (P-gp). Low brain uptake could, therefore, be species-specific and uptake in rodents not be predictive for that in humans. We hypothesized that a better prediction from rodent data could be achieved when a tracer is evaluated under P-gp inhibition. Consequently, we compared the performance of eight neuroreceptor tracers in rats with and without P-gp inhibition including a specific binding blockade. This data set was then used to predict the binding of these eight tracers in pigs. METHODS: PET tracers targeting serotonin 5-HT2A receptors ([18F]MH.MZ, [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin), serotonin 5-HT7 receptors ([11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10) and dopamine D2/3 receptors ([18F]Fallypride) were used in the study. The brain uptake and target-specific binding of these PET radiotracers were evaluated in rats with and without inhibition of P-gp. Rat data were subsequently compared to the results obtained in pigs. RESULTS: Without P-gp inhibition, the amount of target-specific binding in the rat brain was sufficient to justify further translation for three out of eight evaluated tracers. With P-gp inhibition, results for five out of eight tracers justified further translation. The performance in pigs could correctly be predicted for six out of eight tracers when rat data obtained under P-gp inhibition were used, compared to four out of eight tracers without P-gp inhibition. CONCLUSIONS: P-gp strongly affects the uptake of PET tracers in rodents, but false prediction outcomes can be reduced by evaluating a tracer under P-gp inhibition.

Preclinical PET Studies of [11C]UCB-J Binding in Minipig Brain.

PURPOSE: Loss of neuronal synapse function is associated with a number of brain disorders. The [11C]UCB-J positron emission tomography (PET) tracer allows for in vivo examination of synaptic density, as it binds to synaptic vesicle glycoprotein 2A (SV2A) expressed in presynaptic terminals. Here, we characterise [11C]UCB-J imaging in Göttingen minipigs. PROCEDURES: Using PET imaging, we examined tracer specificity and compared kinetic models. We explored the use of a standard blood curve and centrum semiovale white matter as a reference region. We compared in vivo [11C]UCB-J PET imaging to in vitro autoradiography, Western blotting and real-time quantitative polymerase chain reaction. RESULTS: The uptake kinetics of [11C]UCB-J could be described using a 1-tissue compartment model and blocking of SV2A availability with levetiracetam showed dose-dependent specific binding. Population-based blood curves resulted in reliable [11C]UCB-J binding estimates, while it was not possible to use centrum semiovale white matter as a non-specific reference region. Brain [11C]UCB-J PET signals correlated well with [3H]UCB-J autoradiography and SV2A protein levels. CONCLUSIONS: [11C]UCB-J PET is a valid in vivo marker of synaptic density in the minipig brain, with binding values close to those reported for humans. Minipig models of disease could be valuable for investigating the efficacy of putative neuroprotective agents for preserving synaptic function in future non-invasive, longitudinal studies.

Dexmedetomidine enhances glymphatic brain delivery of intrathecally administered drugs.

Drug delivery to the central nervous system remains a major problem due to biological barriers. The blood-brain-barrier can be bypassed by administering drugs intrathecally directly to the cerebrospinal fluid (CSF). The glymphatic system, a network of perivascular spaces promoting fluid exchange between CSF and interstitial space, could be utilized to enhance convective drug delivery from the CSF to the parenchyma. Glymphatic flow is highest during sleep and anesthesia regimens that induce a slow-wave sleep-like state. Here, using mass spectrometry and fluorescent imaging techniques, we show that the clinically used α2-adrenergic agonist dexmedetomidine that enhances EEG slow-wave activity, increases brain and spinal cord drug exposure of intrathecally administered drugs in mice and rats. Using oxycodone, naloxone, and an IgG-sized antibody as relevant model drugs we demonstrate that modulation of glymphatic flow has a distinct impact on the distribution of intrathecally administered therapeutics. These findings can be exploited in the clinic to improve the efficacy and safety of intrathecally administered therapeutics.

Development and Evaluation of Two Potential 5-HT7 Receptor PET Tracers: [18F]ENL09 and [18F]ENL10.

The latest addition to the serotonin (5-HT) receptor family is the 5-HT7 receptor (5-HT7R). This receptor has gained interest as a drug target due to its involvement in various disorders such as depression or schizophrenia. There is currently no clinically validated positron emission tomography (PET) tracer for the 5-HT7R available. But, the (arylpiperazinyl-butyl)oxindole scaffold provides a promising lead structure for this purpose. Here, we synthesized 12 (arylpiperazinyl-butyl)oxindole derivatives and in vitro affinity screening identified two structures with suitable affinity and selectivity to be radiolabeled and tested as 5-HT7R selective PET tracers. Next, the radiolabeled products [18F]ENL09 and [18F]ENL10 were evaluated as PET tracers in rats. Both tracers were found to be P-gp substrates, but after P-gp inhibition the brain uptake showed a regional distribution in line with the known 5-HT7R distribution.  The [18F]ENL10 brain binding was displaceable with a 5-HT7R selective ligand, whereas [18F]ENL09 was not. We find that [18F]ENL10 is a promising 5-HT7R selective PET tracer candidate that should be investigated in higher species.

The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels.

Chemogenetic studies with the ligand clozapine N-oxide (CNO) are predicated upon the assumption that CNO is devoid of actions at natural neuroreceptors. However, recent evidence shows that CNO may be converted back to clozapine (CLZ) in vivo, which could yield plasma concentrations that may be sufficient to occupy inter alia dopamine D2/3 and serotonin 5HT2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D2/3 receptor occupancy with [18F]fallypride PET and serotonin 5HT2A occupancy ex vivo using [18F]MH.MZ. We found a CNO dose-dependent effect on the availability of both neuroreceptor sites. In parallel MR spectroscopy experiments, we found that CNO reduced creatine + phosphcreatine (Cr+PCr) and increased N-acetylaspartate + N-acetylaspartylglutamate (NAA+NAAG) signals in the prefrontal cortex, and also reduced the glutamate signal in dorsal striatum, with peak effect at 2 mg/kg. Thus, our findings suggest that conversion of CNO to CLZ in living rats imparts significant occupancy at endogenous neuroreceptors and significant changes to neurometabolite levels.