Binding of the monoacylglycerol lipase (MAGL) radiotracer [ 3 H]T-401 in the rat brain after status epilepticus

Objectives: Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase considered a potential novel drug target for the treatment of CNS disorders including epilepsy. Here we examined MAGL levels in a rat model of epilepsy. Methods: Autoradiography has been used to validate the binding properties of the MAGL radiotracer, [ 3 H]T-401, in the rat brain, and to explore spatial and temporal changes in binding levels in a model of temporal lobe epilepsy model using unilateral intra-hippocampal injections of kainic acid (KA) in rats. Results: Specific and saturable binding of [ 3 H]T-401 was detected in both cortical grey and subcortical white matter. Saturation experiments revealed a K D in the range between 15 nM and 17 nM, and full saturation was achieved at concentrations around 30 nM. The binding could be completely blocked with the cold ligand (K i 44.2 nM) and at higher affinity (K i 1.27 nM) with another structurally different MAGL inhibitor, ABD 1970. Bilateral reduction in [ 3 H]T-401 binding was observed in the cerebral cortex and the hippocampus few days after status epilepticus that further declined to a level of around 30% compared to the control. No change in binding was observed in either the hypothalamus nor the white matter at any time point. Direct comparison to [ 3 H]UCB-J binding to synaptic vesicle glycoprotein 2 A (SV2A), another protein localized in the pre-synapse, revealed that while binding to MAGL remained low in the chronic phase, SV2A was increased significantly in some cortical areas. Significance: These data show that MAGL is reduced in the cerebral cortex and hippocampus in a chronic epilepsy model and indicate that MAGL inhibitors may further reduce MAGL activity in the treatment resistant epilepsy patient.

The lipid-derived signaling endocannabinoid molecule 2-AG functions as a retrograde neurotransmitter and activate presynaptic cannabinoid receptors, which inhibit presynaptic neurotransmitter release (Alger, 2002;Bisogno et al., 1999).Notably, as 2-AG is lipophilic and can penetrate membranes and not concentrated in vesicles, MAGL regulates not only 2-AG concentrations but also 2-AG dependent neurotransmission directly (Long, et al., 2009;Schlosburg et al., 2010).Endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice (Monory et al., 2006).This mechanism is considered to play a significant role in neuronal excitation and seizures and perhaps also in downstream events including cell loss and comorbidities (Rosenberg et al., 2017).
Based on these neurobiological observations, attempts have been made in developing MAGL inhibitors that enhance endocannabinoid concentrations and produces anti-convulsant effects in animal models of epilepsy (Sugaya and Kano, 2018;Terrone et al., 2018;von Ruden et al., 2015).One such modulator, CPD-4645 reduced spike frequencies and shortening status epilepticus in mice intracerebrally injected with KA (Terrone, et al., 2018).Another inhibitor ABX-1413 had anticonvulsant effect against hyperthermia-induced seizure in an animal model of Dravets syndrome (Anderson et al., 2022).Finally, JZL184 delayed the development of generalized seizures, decreased seizures, and reduced after-discharge duration in the kindling model of temporal lobe epilepsy, but caused only modest effects in fully kindled mice via the cannabinoid (CB) receptor 1 (CB1R) (von Ruden, et al., 2015).
The endocannabinoids, the molecular target(s), and the cellular substrate mediating the action of MAGL inhibitors are not fully understood.Also, as to whether MAGL expression and activity are changed under seizures has not been investigated.In this study, we aimed to analyze the level of MAGL in the brain from rats injected with KA in the hippocampus that behaviorally entered into status epilepticus both acutely and chronically.
[ 18 F]T-401 is a novel radiotracer that binds to MAGL and has been used in positron emission tomography (PET) in rat, monkey and human (Hattori et al., 2019(Hattori et al., , 2022;;Takahata et al., 2022).The radioligand can be displaced by pretreatment with JW642 in the monkey brain, and is considered suitable for evaluations of target engagement in the CNS (Hattori, et al., 2022).In line with these observations, we tritiated the same compound in order to determine binding levels as a marker of MAGL concentrations in the rat brain at high resolution.First, the binding characteristics of this novel radiotracer was evaluated for in vitro autoradiography and next we applied the method to determine the binding levels in various regions of the brains after status epilepticus and under epileptogenesis.

Animals and tissues
Sprague-Dawley adult male rats (200-250 g) breeding in our facility (Izmir Katip Celebi University, Izmir, Turkey) were used in this investigation.The animals were kept in cages (464 × 300 mm) with up to 4 animals pr cage and with free access to standard food and water under 12 L:12D conditions.At the day of the experiment, the rats were under ketamine (75-100 mg/kg i. p.)/xylazine (10 mg/kg i. p.) anesthesia, placed in a stereotaxic frame; and using bregma and lambda as reference points and the following coordinates: Anteroposterior − 2.04 mm; Mediolateral 1.10 mm; and Dorsoventral 3.81 mm were applied for an injection into the right dorsal hippocampus.A single 2 μl dose of either 1.9 mM kA (purchased from Tocris, cat# 0222) dissolved in sterile saline or saline alone, was injected by a stereotaxic robot (Neurostar GmbH, Germany) with a 1.0-μl Hamilton microsyringe (30G cannula and a flow rate of 1 μl/min).Repulse of the injected solution was prevented by waiting for 5 min in the injection area before withdrawal.The animals then returned to a novel cage and were kept individually and observed until full recovery.Post-surgical care of animals within the first three days included treatment with lidocaine as an analgetic and ceftriaxone as antibiotic.
Rats were video-monitored for 10 h during the light period during 90 days and convulsive behaviors of the treated rats were scored based on the Racine's Scale.Rats displaying at least 4th degree seizures within the first 4 h after treatment were included in this study.In addition, rats exhibiting spontaneous recurrent seizures (SRSs) after a month were included in the chronic group.Only the rats that fulfilled these criteria were included in the longitudinal autoradiography study which allowed 6 animals/per group.
To construct a time-course study, experimental and control animals were randomly divided into 8 different groups according to brain sampling time points (baseline, 1, 3, 5, 10, 15, and a chronic group more than 30 days after surgery).The animals were killed by cervical dislocation under ketamine (75-100 mg/kg i. p.) -xylazine (10 mg/kg i. p.) anesthesia, and the brains rapidly removed from the skull, frozen on dryice and kept at − 80 • C until further processing.
For the saturation studies, duplicate sections were incubated in increasing concentrations of [ 3 H]T-401.Adjacent duplicate sections were incubated with the same concentration of the radioligand in the presence of either 10 μM cold ligand (T-401) or 10 μM ABD (Clapper et al., 2018;Hattori et al., 2019) (obtained from Lundbeck A/S).
For the displacement study, 50 nM [ 3 H]T-401 was mixed with either increasing concentrations of T-401 or with ABD 1970.The concentrations used for the displacement study ranged from 0.1 nM to 10 μM for both compounds.For these initial validation experiments, all experiments were replicated twice.
For all autoradiography experiments with [ 3 H]T-401, slides were washed for 3 × 5 min with ice cold pre-incubation buffer followed by a quick dip in ice cold distilled water, air-dried and placed overnight in a paraformaldehyde vapor chamber at 4 • C.After fixation the glass slides were kept in a silica gel desiccator for 45 min to remove any leftover moisture before exposing them to the imaging plate.Then, glass slides were put together with tritium standards ([ 3 H] microscale ART0123 (0-489.1 nCi/mg) and ART0123B (3-109.4nCi/mg) American Radiolabeled Chemicals, Inc., St. Louis, USA) in a radiation-shielded imaging plate cassette (Fuji casette 2 BAS-TR, 2040; Fujifilm, Tokyo, Japan) with a tritium-sensitive imaging plate (Fuji IP BAS-TR, 2040; Fujifilm, Tokyo, Japan) The glass slides were exposed to image plates overnight at 4 • C.After exposure the imaging plate was then scanned using Amersham™ Typhoon™ IP Biomolecular Imager (GE healthcare, Chicago, USA) at pixel size 25 μm.

Autoradiography for the epileptic rats
Sections of the brains of animals exposed to KA or control were cut on a cryostat in 20 μm sections.The sections were selected along the rostro-caudal axis according to different distances to the bregma and content of regions of interest (ROI).To reduce possible variations between the tissue, experiments were performed on sections adjacent to each other.At least 3 technical replicates (slice/slide) were used.The sections were first pre-incubated and then incubation for 120 min in nM [ 3 H]T-401 as described above.Adjacent sections were incubated in 6 nM of [ 3 H]UCB-J (generously supplied by UCB Pharma, Belgium) according to a earlier described protocols (Pazarlar et al., 2022a).Glass slides from the same experiment were exposed to the same FUJI imaging phosphor plate for 3 day at 4 • C together with tritium standard ARC (American Radiolabeled Chemicals, Inc, USA) and tritium microscale Batch 21 A (GE Healthcare, UK).

Data analysis and statistics
Quantitative analysis of receptor binding in cortex slices was performed by measuring the mean of optical density (OD) in the region of interest (ROI).The grey values from tritium standards were used to interpolate the grey values from each ROI to obtain radioactivity values using Image J Rodbard (NIH Image) curve.The decay-corrected specific activity of the radioligand is used to convert binding values.
Determination of at least two sections, and the experiments were replicated three times.The interpolated values were then calculated into the amount of bound radioligand [fmol/mg tissue equivalent] in the tissue, and the mean of these values for the different sections was used for each animal.
Statistical analysis from the autoradiography data calculation was performed using the nonlinear regression function program of Graphpad Prism (version 9.2.0;GraphPad Software, San Diego, USA).Data points on graphs are expressed as mean ± standard deviation, and P < 0.05 was considered significant.
To evaluate the time course change in binding density of [ 3 H]T-401 after induction of epileptogenesis with KA, One-Way ANOVA was used and differences in binding between time sampling points were tested using an unpaired t-test.Parametric unpaired t-test was used to determine the differences in binding density between ipsilateral and contralateral sites of same section.To test the differences in the extent of % change in binding according to control between [ 3 H]T-401 and [ 3 H] UCB-J parametric paired t-test was used.

Validation of the autoradiographic method in rat brain
Frozen brain sections from naïve adult rats were used in several pilot experiments to validate the binding characteristics of the selective MAGL radiotracer [ 3 H]T-401 using in vitro autoradiography.This method has not been applied before for this particular radiotracer and it is therefore relevant to investigate some basic physiochemical binding properties.At first, experiments were carried out to determine the optimal temperatures and incubation time for the radioligand exposure and subsequent washing steps.The optimal temperature for the incubation was found to be room temperature, and for the washing steps 4 • C. It was determined that the optimal time frame for the incubation producing the best signal to noise ratio was between 30 min and 4 h.Based on these initial pilot experiments, the autoradiography was carried out with 120 min incubations at room temperature, and washing was performed by subsequent short washes for a total of 15 min at 4 • C.

[ 3 H]T-401 binding could be saturated and displaced in the rat brain
As illustrated in Fig. 1A the radiotracer bound predominantly to the grey matter i.e. in the cerebral cortex, hippocampus and thalamus.Interestingly, lower levels of binding were seen in the piriform cortex compared to other cortical areas.Detectable binding could also be observed in other subcortical structures, but at lower levels.Specific and saturable binding was detected in the white matter such as the corpus callosum, but in much lower density than seen in most grey matter areas (Fig. 1E).
Saturation experiments were performed and full saturation could be achieved at low nM concentrations (Fig. 1D and E).As illustrated in Fig. 1A binding of [ 3 H]T-401 reached full saturation in the grey matter, and semi-quantitative determination of binding intensities revealed saturation of binding in low nM concentrations in the grey matter.Coincubation with 10 μM of either the cold ligand (Fig. 1B), or another MAGL inhibitor ABD 1970 (Fig. 1C) revealed in both cases a complete block in binding up to 25 nM [ 3 H]T-401.Incubation at concentrations of 100 nM and higher suggested binding to another low affinity nonspecific site.Saturation of binding with increasing concentration of the radioligand [ 3 H]T-401 to rat brain and with the two different compounds for displacement revealed a calculated K D at 17.5 nM (95% CI: 15.9 nM-19.1 nM) for displacing with the cold ligand and 15.6 nM (95% CI: 13.8 nM-17.6 nM) for displacing with ABD1970 when using the whole brain sections as the reference (Fig. 1D).Saturation analysis over the white matter also produced a saturation with full inhibition of binding with the same kinetics (Fig. 1E).
A displacement study was also carried out using a fixed 50 nM concentration of the ligand together with increasing concentrations of the two compounds, T-401 and ABD 1970 (Fig. 2A and B).Displacement of the radiotracer with increasing concentrations of either the cold ligand or ABD1970 revealed full blockage in the grey matter in both cases (Fig. 2A).Both ligands were able to reach full displacement of [ 3 H] T-401 at high concentrations up to 1 μM, and the calculated best fit for IC 50 was 44.2 nM (95% CI: 32.6 nM-60.3nM) for T-401 and 1.27 nM (95% CI: 1.11 nM-1.44 nM) for ABD1970 for both the entire sections and the hippocampus (Fig. 2C).

Level of [ 3 H]T-401 binding in brain sections from rats with status epilepticus
With these basic pharmacokinetic properties defined for the procedure, we then analysed the binding levels of [ 3 H]T-401 in brain sections from rats exposed to intrahippocampal KA at different timepoints after the injection.Status epilepticus was observed in all animals that were then kept at various time points until they were euthanized and brains removed, sectioned and exposed to an autoradiographic analysis simultaneously.As illustrated in Fig. 3, the autoradiographic images revealed changes in density over time.In particular, reduction in binding intensity is seen in the cerebral cortex and the hippocampus.The reduction in binding was evident after 72 h, and the level was reduced further until 1 months after status epilepticus (Fig. 3).Notably, the magnitude of reductions were dependent on the brain region.
The entire cerebral cortex showed a reduction in binding over time that reached a strong statistical significance in both hemispheres 5 days after status epilepticus, and remained gradually lower over time (Fig. 4A).There was no difference between the binding intensity in the two hemispheres at any time point in the cerebral cortex as well as in other brain regions examined (Fig. 4A-D).When analyzing subregions of the cerebral cortex, such as the frontal cortex (Fig. 4B) or the parietal cortex (Fig. 4C), the profile in binding levels over time was similar.In both subregions, a gradual reduction was seen over the first two weeks reaching around 20% reduction after 10 days, and not declining significantly more (Fig. 4B and C).In the hippocampal formation a major reduction was seen earlier than in the cerebral cortex and after the first 72 h the decline was prominent, and this level was not further declining during the entire observation period (Fig. 4D).
Also, in the thalamus a significant reduction after 72 h was observed (Fig. 4E).In contrast to the cortical structures, the changes were not stable over time even though reduction was evident in the chronic phase (Fig. 4E).Binding intensity in the hypothalamus was relatively low and showed no difference in the observation period (Fig. 4E).Similarly, binding in the corpus callosum representing the white matter was also low and was not affected by the treatment (Fig. 4E).
We next analysed the [ 3 H]T-401 binding in subparts of the hippocampus, with special emphasis on differences between the two hemispheres.In all subregions the level of binding was evaluated at high resolution, and in no cases a significant difference between the two hemisperes was observed (Fig. 5).The temporal profile between the dentate gyrus (Fig. 5A), the CA1 (Fig. 5B), and the CA3 (Fig. 5C) was rather identical.In all subregions, significant reduction was observed after the first day and further reduction was found after 3 days, where after the binding level remained stable during the observation period in all subregions (Fig. 5).

Comparison of binding to MAGL and to the synaptic vesicle protein 2 A (SV2A)
Given MAGL to be dominantly expressed in the pre-synapse, we compared the binding level to [ 3 H]UCB-J that bind to SV2A, which is another presynaptic protein in the neighboring sections from the same animals (Fig. 6A).While both tracers bind to the grey matter i.e. in the hippocampus, they also decline in the first week (Fig. 6A).As earlier reported SV2A binding increases in the hippocampus and the cerebral cortex from the first weeks until about a month after status epilepticus (Pazarlar et al., 2022b), but MAGL binding does not.Interestingly, the image analysis revealing the change in binding over time profiles revealed different binding intensities for the two tracers dependent on the region.In the hippocampus and the temporal cortex (Fig. 6B and C) the level of SV2A binding declined around 50% and MAGL binding about 30%.But whereas SV2A binding increased significantly in the chronic phase, the MAGL binding was unchanged.By contrast in the  Fig. 4. Measurements of level of binding in major areas of both hemispheres including the entire cerebral cortex (A), the temporal/parietal cortex (B), the frontal cortex (C), and the hippocampus (D).For the cortical structures the measurements are performed on both ipsilateral (grey) and contralateral (black) sides.There is no difference between the two sites in any region.In all cortical areas significant reduction is determined 3 days after, and further reduction at later time points in the chronic phase.In the hippocampus significant reduction is seen the day after, and further reduction is seen after 3 days after, and remained stable until the chronic phase.In the thalamus the profile also displayed some reduction over time with more variability.By contrast, no change was seen in the hypothalamus and white matter (E).*p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001.One-Way ANOVA, and differences in between time sampling points were tested by an unpaired t-test.

Fig. 5. Levels of [ 3 H]
T-401 binding in different subparts of the hippocampus in both the ipsilateral and contralateral side to the KA injection.Detailed analysis of the binding in subparts of the hippocampus such as the dentate gyrus (A), CA1 (B), and CA3 (C) showed a gradual reduction in both sides.The pattern in binding over time is rather identical between the structures, and there was seen no significant difference between the two sites at any time point.Significant reduction was seen in both sides within the first 3 days after status epilepticus.One-Way ANOVA, and differences between individual time points were tested compared to control by an unpaired t-test.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.frontal cortex, binding to both presynaptic proteins were reduced in the first week to about 20 % under control level (Fig. 6D).Butwhile SV2A level increased significantly to a level not different from baseline, MAGL binding remained low (Fig. 6C).

Discussion
The present autoradiography study validates the binding characteristics of the novel MAGL radiotracer [ 3 H]T-401 in the rat brain.Previously, the same molecule has been radiolabelled with fluoride-18 and used for in vivo PET studies in the brain of primates including man (Hattori, et al., 2019;Hattori et al., 2022).It is therefore of interest to further validate the binding properties of this ligand both in vivo and ex vivo in different species as well as studying changes in binding in models of disease.
The present study shows the specificity and saturability of the MAGL ligand [ 3 H]T-401 in the rat brain.The radioligand saturated to rat brain sections at K D around 15 nM and complete inhibition was achieved not only with the cold molecule ifself, but also with another structurally distinct small molecule ABD 1970 (Clapper, et al., 2018).The measured K i for ABD1970 is in accordance with the pharmacological characteristics of this compound (Cisar et al., 2018;Clapper et al., 2018;Jiang and van der Stelt, 2018).
The present study also reveals the distribution of MAGL binding sites in the rat brain.The distribution pattern of binding sites shown here completely overlaps with the distribution of magl mRNA as shown by in situ hybridization (Dinh et al., 2002) not only emphasizing the specificity of the radioligand, but also adding to our understanding of neuronal circuits influenced by MAGL activity.The distribution of MAGL binding is also in accordance with the regional changes in CBR1 desensitization after chronic MAGL inhibition (Schlosburg, et al., 2010).Overall, [ 3 H]T-401 binding was mainly found in the grey matter though detectable levels were also observed in the white matter.Interestingly, significantly more binding was found in the frontal and parietal cortices than in the piriform and inferior temporal cortices.High levels were also observed in the hippocampus and in some parts of the thalamus, whereas lower levels were found in the striatum and hypothalamus.Based on this topography it is likely that MAGL is not expressed in all neurons of the brain.
Histologically and functional data showed the presence of MAGL in presynaptic terminals (Dinh, et al., 2002;Gulyas et al., 2004;Ludanyi et al., 2011).On a cellular level, hippocampal granule and interneuron axonal terminals have been demonstrated to contain MAGL-immunoreactivity (Gulyas, et al., 2004).Autoradiographic images are not sufficient to conclude any cellular distribution, but the high expression in the grey matter supports a neuronal and presynaptic localization of MAGL.
Functional knock out studies have demonstrated that MAGL is also expressed in astrocytes (Hu et al., 2022).The specific and saturable binding in the white matter reported here is in accordance with an expression in non-neuronal cells i.e. astrocytes.
Endogeneous cannabinoids and CBR1 agonists have anticonvulsant effects in a number of electroshock and KA seizure models (Hofmann and Frazier, 2013;Marsicano et al., 2003;Rosenberg et al., 2017;Wallace et al., 2003).The event-specific activation of the endocannabinoid system by inhibition of the endocannabinoid degrading enzymes, such as MAGL, represents a novel and promising strategy, more than direct activation of CB receptors, to selectively activate cannabinoid receptors at the site of excessive neuronal activation with the overall objective to prevent epilepsy to develop (Chen, 2023).
Two-arachidoylglycerol (2-AG) is an endocannabinoid that under physiological conditions is released from neurons and acts retrogradely to modulate glutamate release via presynaptic CB receptors (Alger, 2002;Katona and Freund, 2012;Kendall and Yudowski, 2016).MAGL plays a role in seizure-induced cell loss and comorbidities in epilepsy (Rojas et al., 2014;Rosenberg et al., 2017) and support for this mechanism is that 2-AG, which accumulates as a result of MAGL inhibition, inhibits seizures, delay kindling, and decrease after discharges in animal models (Kow et al., 2014;Sugaya et al., 2016).Arachidonic acid (AA) is converted to prostaglandin H by COX-2 and prostaglandin H is proinflammatory and acts on microglia cells to promote inflammatory processes in the brain (Rojas, et al., 2014).Thus, MAGL inhibitors lowering AA synthesis may also reduce inflammation via this mechanism that does not involve CBR1 (Carlisle et al., 2002;Schmole et al., 2015).This mechanism might be important because anti-epileptic activity of MAGL inhibition remained in CBR1 KO animals (Terrone, et al., 2018).Alternatively, CB2R may play a role, but this is yet not explored.
Here we also demonstrate that binding to MAGL probably reflecting a reduction in the enzyme concentration, is reduced in some brain grey matter structures in the latent phase in one model of status epilepticus.SV2A) at controls and two other time points [10 days and >30 days (chronic)] after status epilepticus (A).Measures from three regions are illustrated relative to control.In the hippocampus (B) the relative reduction after 10 days is larger for [ 3 H]UCB-J than for [ 3 H]T-401 whereas the relative reduction is the same in the chronic phase, because the level of [ 3 H]UCB-J binding increases in the latent phase after 10 days.In the temporal (C) and frontal cortex (D) both [ 3 H]T-401 and [ 3 H]UCB-J was also lower compared to control 10 days after status epilepticus, and as for the hippocampus [ 3 H]T-401 that remained low in the chronic phase, whereas [ 3 H]UCB-J increased.
The reduction is seen not only in the hippocampus in which KAis injected but also in the contralateral hippocampus as well as in extrahippocampal areas such as throughout the cerebral cortex.
The concentration of 2-AG is affected by acute seizures (Magloczky et al., 2010;Sugaya and Kano, 2018), and could inhibit excitatory neurotransmission.It is worth mentioning that lower MAGL as seen in the latent phase in this study, that would lead to sustained increase in 2-AG and thus decrease neuronal activity in the cerebral cortex and the hippocampus.Pharmacological blockade by MAGL inhibitors will further increase 2-AG but the effect may be different dependent on the reduction in MAGL under uncontrolled seizures.This suggesting that changes in the cannabinoid signaling occur in the epileptic brain and effect of pharmacologically induced MAGL inhibition may be different between the epileptic and healthy brain.
In contrast to the grey matter, no difference in MAGL binding was found in the white matter.Because the cellular target of MAGL inhibition in models of epilepsy is suggested to be non-neuronal cells it is interesting to note that no change is seen in the concentration in the white matter.Nevertheless, microglia and astrocytes in the grey matter may be affected.
It is also noteworthy that the reduction in MAGL binding in the hippocampus is accompanied by reduction in SV2A, a presynaptic protein localized in the vesicular membrane and shown to be reduced in several animal models of epilepsy and in human epilepsy brains (Carson et al., 2022;Finnema et al., 2016;Mikkelsen et al., 2022;Pazarlar et al., 2022a,b).We suggest that the loss of binding occurs in neurons due to neurochemical changes rather than loss or formation of mature synapses because reduction is seen not only in the hippocampus, which is the primary target for neurodegeneration, but also throughout the cerebral cortex where no neurodegeneration occurs.
Epileptogenesis is the process mostly affecting the temporal lobe by which a normal network alters leading to an enhanced probability of seizures (Goldberg and Coulter, 2013;Pitkanen et al., 2015).The change in SV2A as a marker of synaptic density might affect the epileptic process and reorganization of neural circuits in the brain.MAGL and SV2A binding are both reduced in the hippocampus and the cerebral cortex in the latent phase of status epilepticus.But while SV2A binding increases, this is not seen for the MAGL concentration that remains low.Thus, the two drug target for epileptic drug therapy and the two presynaptic proteins are not co-regulated and cannot both be regarded markers of synaptic densities.
In conclusion, this study reports the validation of the binding properties of a novel radiotracer for MAGL in the rat brain, and show specificity and relatively high affinity in the low nM range.Further, we report a reduction of MAGL binding probably reflecting lower MAGL concentrations throughout the cerebral cortex and hippocampus after status epilepticus.These results emphasize changes in the endocannabinoid system in the epileptic brain that likely have impact on targeting this enzyme for novel drugs.

Declaration of competing interest
The authors declare no conflicts of interest relevant for this article.

Fig. 1 .
Fig. 1.Saturation experiments.The figure shows representative consecutive colored autoradiograms of adjacent naïve rat brain sections incubated with increasing concentrations of [ 3 H]T-401 (A).Labelling can be seen mostly in the grey matter with most extensive binding in the cerebral cortex, the hippocampus, and thalamus.Lower levels of binding are observed in the temporal lobe, i. e in the piriform cortex and medial amygdala, as well as in the hypothalamus.(B) Shows adjacent sections incubated with the same concentration of [ 3 H]T-401 together with 10 μM T-401.(C) Similarly, adjacent sections incubated with [ 3 H]T-401 together with 10 μM ABD 1970.In both experiments, the binding could be completely blocked up to around 50 nM of the [ 3 H]T-401.(D) Illustrates the quantitative analysis of the saturation of the labelling in the entire section after blocking with either the cold ligand itself (left) or ABD 1970 (right).(E) Quantitative analysis of binding in the white matter illustrates specific binding and saturation also in this compartment.

Fig. 2 .
Fig. 2. Inhibition experiments Representative autoradiograms of displacement of 50 nM [ 3 H]T-401 with either increasing concentrations of the cold ligand T-401 (A, upper row) or the other MAGL inhibitor, ABD 1970 (B, lower row).The quantitative analysis of the image as shown in (C) illustrates displacement of the cold ligand (circles) and ABD 1970 (squares).

Fig. 3 .
Fig. 3.Representative autoradiograms of brain sections from individual rats injected unilaterally with KA in the dorsal hippocampus.The images represent sections at the same level of the dorsal hippocampus from brains taken from representative animals taken at various time points after their status epilepticus.Over the first days, lower color intensities can be seen bilaterally in the hippocampus, throughout the cerebral cortex and the thalamus peaking around 10 days after status epilepticus.

Fig. 6 .
Fig.6.Representative high resolution autoradiograms of neighboring hippocampal sections incubated with either of [ 3 H]T-401 (MAGL) or [ 3 H]UCB-J (SV2A) at controls and two other time points [10 days and >30 days (chronic)] after status epilepticus (A).Measures from three regions are illustrated relative to control.In the hippocampus (B) the relative reduction after 10 days is larger for [ 3 H]UCB-J than for [ 3 H]T-401 whereas the relative reduction is the same in the chronic phase, because the level of [ 3 H]UCB-J binding increases in the latent phase after 10 days.In the temporal (C) and frontal cortex (D) both [ 3 H]T-401 and [ 3 H]UCB-J was also lower compared to control 10 days after status epilepticus, and as for the hippocampus [ 3 H]T-401 that remained low in the chronic phase, whereas [ 3 H]UCB-J increased.