Clozapine Induces Astrocyte-Dependent FDG-PET Hypometabolism

doi:10.21203/rs.3.rs-974963/v1

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Research Article

Clozapine Induces Astrocyte-Dependent FDG-PET Hypometabolism

https://doi.org/10.21203/rs.3.rs-974963/v1

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Journal Publication

published 05 Feb, 2022

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Purpose

Advances in functional imaging allowed us to visualize brain glucose metabolism in vivo and non-invasively with [¹⁸F]fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) imaging. In the past decades, FDG-PET has been instrumental in the understanding of brain function in health and disease. The source of the FDG-PET signal has been attributed to neuronal uptake, with hypometabolism being considered as a direct index of neuronal dysfunction or death. However, other brain cells are also metabolically active, including astrocytes. Based on the astrocyte-neuron lactate shuttle hypothesis, the activation of the glutamate transporter 1 (GLT-1) acts as a trigger for glucose uptake by astrocytes. With this in mind, we investigated glucose utilization changes after pharmacologically downregulating GLT-1 with clozapine (CLO), an antipsychotic drug.

Methods

Adult male Wistar rats (control, n = 14; CLO, n = 12) received CLO (25/35mg kg−1) for six weeks. CLO effects were evaluated in vivo with FDG-PET and cortical tissue was used to evaluate glutamate uptake, GLT-1 and GLAST levels. CLO treatment effects were also assessed in cortical astrocyte cultures (glucose and glutamate uptake, GLT-1 and GLAST levels) and in cortical neuronal cultures (glucose uptake).

Results

CLO markedly reduced in vivo brain glucose metabolism in several brain areas, especially in the cortex. Ex vivo analyses demonstrated decreased cortical glutamate transport along with GLT-1 mRNA and protein downregulation. In astrocyte cultures, CLO decreased GLT-1 density as well as glutamate and glucose uptake. By contrast, in cortical neuronal cultures, CLO did not affect glucose uptake.

Conclusion

This work provides in vivo demonstration that GLT-1 downregulation induces astrocyte-dependent cortical FDG-PET hypometabolism - mimicking the hypometabolic signature seen in people developing dementia - and adds further evidence that astrocytes are key contributors of the FDG-PET signal.

Nuclear Medicine & Medical Imaging

FDG-PET

astrocytes

GLT-1

glucose

glutamate

clozapine.

Regulation of brain glucose metabolism is key for supporting high-energy demands during neuronal firing and it involves multiple enzymes and transporters. Beyond neurons, other brain cells are also metabolically active such as the astrocytes. Indeed, astrocytes are known for their highly glycolytic metabolism [1–3] and positioning of their end-feet all around blood capillaries, which favors the uptake of glucose from the bloodstream [4].

Astrocytes take up glucose, via the glucose transporter 1 (GLUT1), and metabolize a substantial fraction into pyruvate through glycolysis. Then, pyruvate is converted to lactate, which is shuttled to feed neurons. This biochemical model is termed the astrocyte-to-neuron lactate shuttle (ANLS), a widely debated theoretical framework [5–7]. The ANLS model has been corroborated by a series of in vitro and in vivo studies [7–16]. Additionally, it was observed a neuronal preference for lactate oxidation and a predominant glycolytic metabolism of glucose by astrocytes in cellular models [1, 2]. In vivo studies have also unraveled that reduced astrocytic glutamate transport diminishes astrocyte glucose uptake [17, 18]. The glutamate transporter 1 (GLT-1) is the most abundant glutamate transporter in the mammalian brain, in which it has a dominant role in glutamate uptake (around 90%) [19] and is predominantly located on astrocytes [20]. Interestingly, glutamate transport via GLT-1 is considered as a triggering signal for glucose uptake by astrocytes [7], this biological process being vital for brain energetics. It is important to mention that there are reports challenging the ANLS concept [21], which makes this topic controversial and requiring further experimental evidence.

Based on the aforementioned data, the question whether astrocyte glucose metabolism contributes to functional imaging signals, such as for positron emission tomography (PET) imaging with [¹⁸F]fluoro-2-deoxyglucose (FDG) [22, 23], has been raised. Specifically, in 1996, Pellerin and Magistretti put forward a hypothesis based on compelling evidence supporting the notion of astrocytes as contributors of FDG-PET signal [24]. In the following years, additional findings have corroborated this proposal [25–27]. Still, for over 40 years, the FDG-PET signal has been used as an index of neuronal activity [28]. Recently, however, it has been demonstrated that targeting astrocytes alters the FDG-PET signal. More specifically, deletion of insulin receptors on astrocytes reduces FDG-PET signal [29] and activation of glutamate transport via GLT-1 on astrocytes causes widespread but graded increases in FDG-PET signal [30].

Recent important theoretical articles are already acknowledging the role of astrocytes in the brain FDG-PET signal [31–34]. Still, whether a reduction of glutamate uptake by astrocytes would cause in vivo FDG-PET hypometabolism – recapitulating what is seen in neurodegenerative disorders – remains unexplored. On the basis of the evidence presented above, we hypothesized that astrocyte GLT-1 downregulation would cause FDG-PET hypometabolism. To further test this hypothesis, we conducted in vivo and in vitro pharmacological challenges with CLO, a well-known anti-psychotic drug that reduces GLT-1 density [35] and glutamate transport [36], to assess its effects on cerebral FDG-PET signal in rats.

2.1 Animals

Adult male Wistar rats, 90 days old, were maintained under a 12 h light-dark cycle (lights on at 7 A.M.), at a controlled room temperature (22 ± 1◦C) and with free access to food and water. Animals’ weight was measured once a week and volume of liquid ingestion was measured three times a week.

2.2 Pharmacological treatment

Rats were randomly divided into two groups: control and CLO. Rats in the CLO group received the drug CLO (Cristália, Itapira/Brazil) in free drinking water (25-35mg kg⁻¹day⁻¹) for 6 weeks, starting at postnatal day (PND) 91, until euthanasia. The CLO solution was prepared as previously described by Terry et al. [37]. Treatment regimen, dose and length of treatment followed the procedure described by Melone et al [35].

2.3 [¹⁸F]FDG microPET scanning

Rats were scanned longitudinally before treatment (Baseline), at PND 90, and after six weeks of treatment (Follow-up), at PND 136, as previously described [38]. Briefly, after overnight fasting, animals received an intravenous injection (0.4 mL) of FDG (mean ± s.d.: 1.03 ± 0.07 mCi) into the tail vein. Then, each rat returned to its home cage for a 40 min period of awake uptake of FDG. After, they immediately underwent a 10 min static acquisition under anesthesia (2% isoflurane at 0.5 L/min oxygen flow). See supplementary material for detailed methodology.

2.4 Metabolic networks

Cross-correlation maps were built as previously described [38], using a mask containing 14 volumes of interest (VOIs): left (L)-right(R) frontal cortex (FtC), L-R temporoparietal cortex (TmPtC), L-R thalamus (Th), L-R Hypothalamus (Ht), L-R striatum (St), LR hippocampus (Hip) and L-R cerebellum (Cer). Stable metabolic brain networks, using an innovative multiple sampling scheme [39], were constructed by computing Pearson correlation coefficients based on 2000 bootstrap samples. Graph theoretical measures such as density, global efficiency, assortativity coefficient, average degree and average clustering coefficient were calculated for each of the bootstrap samples. Networks were corrected for multiple comparisons using the false discovery rate (P<0.005) [40].

2.5 Synaptosomal preparation and L-[³H]glutamate (L-[³H]Glut) potassium-stimulated release

This procedure was performed as previously described [41] using the whole cortex (left hemisphere) from 137-140 PND rats. See supplementary material for detailed methodology.

2.6 Primary cortical astrocyte cultures

Newborn (1-2 days old) male Wistar rats’ cortices were aseptically dissected out. The tissue was dissociated enzymatically - with trypsin 0.05% (Gibco®, Thermo Fischer Scientific) and DNase 0.003% (Sigma Aldrich, Saint Louis-MO/USA) - and mechanically (Pasteur pipet). Samples were incubated for 15 min at 37°C for enzymatic action, followed by the addition of 10 % fetal bovine serum (FBS) and centrifugation at 1000 g for 5 min. Pellet was resuspended in HBSS (Gibco®) containing DNase 0.003 % and centrifuged for 7 min at 1000 g. The cells pellet was resuspended in DMEM (Gibco®) supplemented with 10 % FBS, 15 mM HEPES, 14.3 mM NaHCO₃, 1 % Fungizone and 0.04 % gentamicin, and plated onto 24-well plates at a density of 3–5×10⁵ cells/cm². Cells were cultured at 37°C in an atmosphere with 5% CO₂. The first medium exchange occurred 24 h after plating cells. The following medium changes occurred once every four days. After cells reached confluence, the protocol followed Vallejo-Illarramendi et al [36]. First, 0.2 mM dibutyryl-cAMP (dBcAMP) was added for 6 days to induce GLT-1 expression. Subsequently, dBcAMP was removed and astrocytes were treated with 50 µM CLO (Sigma Aldrich), in medium supplemented with 1 % FBS, for 48 h and used for immunofluorescence, western blot or uptake procedures.

2.7 Primary cortical neuronal cultures

Neuronal cultures were prepared as previously described [42] with modifications. Newborn (0-1 days old) male Wistar rats’ cortices were aseptically dissected out. Tissue was dissociated enzymatically - with trypsin 0.05 % (Gibco®) and DNase 0.006 % (Sigma Aldrich) - and mechanically (Pasteur pipet). Samples were incubated for 20 min at 37°C for enzymatic action and again mechanically dissociated for 5min. After, 20 % FBS was added and samples were centrifuged at 1500 g for 5 min. The pellet was resuspended in Neurosabal medium (Gibco®) supplemented with 0.5 % B27 (Gibco®), 10 mM glutamine and 0.02 % penicillin/streptomycin (10.000 U/mL - Gibco®). Cells were plated at a density of 3–5×10⁵ cells/cm² into 24-well plates pre-coated with poly-L-lysine and cultured at 37°C in an atmosphere with 5 % CO₂. .Half of the medium was replaced 1 h after plating cells. Medium changes, also of half of the medium volume, occurred once every four days. After cells reached confluence, neurons were treated with 50 µM CLO (Sigma Aldrich) for 48 h and used for immunofluorescence or uptake procedures.

2.8 Sodium-dependent D-[2,3-³H]-aspartate (D-[³H]Asp) uptake

D-[³H]Asp is a substrate for the high-affinity sodium-dependent glutamate transport system, therefore, its uptake was used as a proxy of glutamate uptake [43, 44]. The sodium-dependent D-[3H]Asp uptake was assessed in adult cortical slices (137-140 PND rats) incubated with 100 µM L-glutamate and in primary cultured astrocytes (P1-2 rats) incubated with 10 µM (basal) or 100 µM (stimulated) L-glutamate. The procedures followed protocols previously described [44, 45] with modifications. See supplementary material for detailed methodology.

2.9 2-Deoxy-D-[³H]glucose ([³H]2DG) uptake

[³H]2DG uptake was assessed in neuronal (P0-1 rats) and astrocyte (P1-2 rats) cultures incubated with with 10 µM (basal) or 100 µM (stimulated) L-glutamate. The procedures followed protocols previously described [38] with modifications. See supplementary material for detailed methodology.

2.10 SDS-PAGE

Whole cortex (left hemisphere) from 137-140 PND rats or astrocyte cultures were analyzed by SDS-PAGE. as previously described [38]. The following antibodies: GLT-1, glutamate-aspartate transporter – GLAST, β-actin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:4000 dilution, abcam). See supplementary material for detailed methodology.

2.11 RNA extraction and quantitative real time PCR (qRT-PCR)

Whole cortex (right hemisphere) from 137-140 PND rats was used for the qRT-PCR analysis on which the mRNAs of GLT-1 and GLAST were quantified as previously described [46]. See supplementary material for detailed methodology.

2.12 High-performance liquid chromatography (HPLC) procedures

High-performance liquid chromatography (HPLC) procedure was performed as previously described [47] using cerebrospinal fluid (CSF) to quantify three amino acids: glutamate (Glut), aspartate (Asp) and glutamine (Gln). Additionally, a HPLC with ultraviolet detection (UV) method was applied to verify CLO presence and stability in the animals drinking solution. See supplementary material for detailed methodology.

2.13 Behavioral Analysis

At the end of the six weeks of treatment, animals were submitted to the Open Field and to the Novel object recognition (NOR) tasks. See supplementary material for detailed methodology.

3.1 CLO treatment causes brain glucose hypometabolism in vivo

MicroPET analysis after CLO pharmacological intervention showed a clear FDG SUVr reduction mainly in cortical brain regions (Fig. 1a, 1b). Specifically, the CLO group (n = 13) presented a widespread FDG cortical hypometabolism, ranging from 10-20 % (Fig. 1c). By contrast, the control group (n = 11) presented only a few small clusters of glucose metabolism reduction, comprising values between 1- 6 % (Fig. 1c). A voxel-wise t-statistical analysis showed a significant glucose hypometabolism mostly in the cortical regions of the CLO group, with a peak effect in the parietal cortex (peak t₍₂₃₎ = 7.62, p < 0.0001; Fig. 1d). On the other side, control group presented only negligible reductions in a few isolated voxels (Fig. 1d). The analysis of the whole brain FDG mean SUVr showed a reduction effect of time (p = 0.0029; F(1, 22) = 11.2 Fig. 1f) but not of group (p = 0.86; F(1, 22) = 0.035) or interaction (p = 0.89; F(1, 22) = 0.016). A brain mask with 14 VOIs (Fig. 1e), based on the Paxinos and Watson Rat Brain Atlas, was used to obtain mean regional FDG SUVr values. In the regional analysis, a Two-Way ANOVA identified a significant effect of time and an interaction in the frontal cortex (time p < 0.0001; interaction p = 0.02; Fig. 1g), temporoparietal cortex (time p < 0.0001; interaction p = 0.003; Fig. 1h) and hippocampus (time p < 0.0001; interaction p = 0.04; Fig. 1l). The striatum (Fig. 1j) and thalamus (Fig. 1k) presented only an effect of time (striatum p = 0.008, thalamus p = 0.007). The hypothalamus (Fig. 1l) and cerebellum (Fig. 1m) showed no significant differences in the Two-Way ANOVA analysis. Following the Two-way ANOVA, a Sidak’s multiple comparison test showed FDG hypometabolism in the frontal cortex (p < 0.0001; t₍₂₂₎ = 5.8), temporoparietal cortex (p < 0.0001; t₍₂₂₎ = 6.6) and hippocampus (p < 0.0001; t₍₂₂₎ = 5.49) on the follow-up CLO group in comparison to its baseline. By contrast, no regional changes were found in the control group (Baseline vs Follow-up). The individual dynamic raster plot reinforces the observation that CLO-induced FDG hypometabolism is more pronounced in cortical areas, and that FDG metabolism remains stable in the control group (Fig. 1n, 1o). Additionally, a multiple sampling scheme metabolic network analysis was performed to identify brain reorganization patterns in response to CLO. In comparison to the control group (Fig. 2a), the CLO group showed multiple altered connections within the metabolic network, presenting a widespread hyposynchronicity (p < 0.005, FDR-corrected, Fig. 2b). These findings were corroborated by consistent changes in graph measures, including reduced global efficiency (p < 0.0001, Fig. 2c), density (p < 0.0001, Fig. 2d), degree (p < 0.0001, Fig. 2e) and clustering coefficient (p < 0.0001, Fig. 2f). The assortativity coefficient was not significantly different between groups (p = 0.31, Fig. 2g).

3.2 Cortical GLT-1 and D-[3H]Asp uptake are reduced after CLO treatment

The astrocyte glutamate transporters immunocontent analysis on the cortical tissue revealed a significant reduction, around 18%, of GLT-1 levels (p = 0.0043; t₍₂₄₎ = 3.16, Fig. 3a, b) in the CLO group (Control, n = 14 and CLO n = 12), but no differences on GLAST levels (p = 0.74; t₍₂₄₎ = 0.33; Fig. 3d, e). In addition, GLT-1 mRNA expression was also reduced (p = 0.025; t₍₁₃₎ = 2.53; Control, n = 7 and CLO = 8; Fig. 3c). Consistently, GLAST expression was not altered (p = 0.27; t₍₁₃₎ = 1.15; Fig. 3f). Glutamatergic transport was also evaluated by using a D-[³H]Asp uptake assay on fresh cortical brain slices. The CLO group (n = 5) showed a significant reduction (p = 0.048; t₍₉₎ = 2.27; Fig. 3g) of D-[³H]Asp uptake when compared to the control group (n = 6). In parallel, potassium-stimulated L-[³H]Glut release was assessed in synaptosomal preparations and no differences were observed (p = 0.76; t₍₁₄₎ = 0.29; Fig. 3h). Finally, cerebrospinal fluid (CSF) levels of glutamate (p = 0.62; t₍₁₉₎ = 0.49), aspartate (p = 0.48; t₍₁₉₎ = 0.71) and glutamine (p = 0.19; t₍₁₁₎ = 1.38) were not altered as well (Fig. 3i-k).

3.3 Effects of CLO on primary cultures of cortical astrocytes

To assess CLO specific effects on astrocytes, we treated cortical astrocyte cultures with 50 mM CLO for 48 h. Primary astrocyte cultures were immunostained with anti-GFAP (red) and DAPI (blue) (Fig. 4a). We found a significant decrease of GLT-1 (~36%, p = 0.0019; t₍₇₎ = 4.81; Fig. 4b, c) and GLAST (~18%, p=0.011; t₍₇₎=3.38; Fig. 4d, e) immunocontents in the CLO group (n = 8, per group). In parallel, the CLO group showed a reduction of [³H]D-Asp uptake in basal (p = 0.0005; t₍₅₎ = 7.97; Fig. 4f) and stimulated assays (p = 0.023; t₍₅₎ = 3.21; Fig. 4g). When comparing basal and stimulated [³H]D-Asp uptake from control and CLO groups (Fig. 4h), we identified significant effects of treatment [p=0.0002; F(1, 24)=18.91] and glutamate levels [p < 0.0001; F(1, 24) = 48.14] but no interaction [p = 0.12; F(1, 24) = 2.49]. A Sidak’s multiple comparison analysis demonstrated that the control group stimulated [³H]D-Asp uptake was higher than the basal uptake for both control (p < 0.0001; t₍₈₎ = 5.36) and CLO groups (p = 0.005; t₍₈₎ = 5.45). In contrast, the CLO group stimulated [³H]D-Asp uptake did not differ in comparison to the basal uptake (vs. basal control, p = 0.77; vs. basal CLO, p = 0.82) and was lower than the control group stimulated [³H]D-Asp uptake (p = 0.003; t₍₈₎ = 6.623). Also, results of [³H]2DG uptake revealed no differences for basal uptake (p = 0.6054; t₍₇₎ = 0.54; Fig. 4i) but a decrease of stimulated uptake for the CLO group (p = 0.0002; t₍₇₎ = 7.19; Fig. 4j). An analysis comparing [³H]2DG uptake in these four experimental conditions (Fig. 4k) showed effects of treatment [p < 0.0001; F(1, 28)=22.54], glutamate level [p = 0.0061; F(1, 28) = 8.79], and an interaction [p < 0.0001; F(1, 28) = 21.34]. A Sidak’s multiple comparison analysis demonstrated that the control group stimulated [³H]2DG uptake was higher than control and CLO groups in the basal condition (p < 0.0001). Also, the CLO group stimulated [³H]2DG uptake was reduced if compared to the stimulated control group (p < 0.0001).

3.4 Effects of CLO treatment on primary cultures of cortical neurons

Additionally, we evaluated the effect of CLO on neuronal cultures. Primary neuronal cultures were immunostained with anti-GFAP (red), anti-β-Tubulin III (green) and DAPI (blue) (Fig. 5a). We found no differences between control and CLO groups for [³H]2DG uptake under basal (p = 0.70; t₍₄₎ = 0.40; Fig. 5b) and stimulated conditions (p = 0.54; t₍₄₎ = 0.66; Fig. 5c). An analysis comparing [³H]2DG uptake in these four experimental conditions (Fig. 5d) showed no effects of treatment [p = 0.7284; F(1, 16) = 0.12], glutamate levels [p = 0.5407; F(1, 16) = 0.39] or interaction [p = 0.9787; F(1, 16) = 0.0007].

3.5 Behavioral effects and treatment measures

Total distance traveled (p = 0.17; t₍₂₆₎ = 1.42) and total immobile time (p = 0.13; t₍₂₆₎ = 1.56 ) during the open field task were not different between groups (Control, n = 14 and CLO n =12). Therefore, suggesting no sedative side effects of CLO [48] (Supplementary Fig. 1a-c). Analysis of the time spent exploring objects during the NOR task revealed that both groups (Control, n = 7 and CLO, n = 5) equally explored the two identical objects during the training session and spent more time exploring the new object during the test session (control p = 0.0004; t₍₆₎ = 7.22; CLO p = 0.0004; t₍₄₎ = 10.86), indicating no impairment of short-term recognition memory (Supplementary Fig. 1d-f). Animals’ body weight was measured once a week during treatment. Results showed time effect, as animals gained weight due to age-dependent normal body growth (p < 0.0001; F(7, 196) = 336.6; Supplementary Fig. 2a), but no group effect (p = 0.35; F(1, 28) = 0.89). The liquid intake showed an increase over time (p < 0.0001; F(11, 121) = 7.85; Supplementary Fig. 2b), and a lower volume intake for the CLO group [group effect, p = 0.036; F(1, 11) = 11.73], probably due to differences in the drinking water palatability. In future experiments, the addition of small quantities of a compound such as sucrose, in order to make the drinking water containing the experimental drug more palatable, should be considered. Finally, a high-performance liquid chromatography (HPLC) analysis confirmed that the animals drinking solution preparation was yielding the expected concentration with high reproducibility, presenting a relative standard deviation (RSD) of 1.86% (Supplementary Fig. 2c). The experimental drinking solution also showed a high stability, with no noticeable modifications of CLO concentration in a 24 h period at room temperature (Supplementary Fig. 2d).

In this study, we demonstrated that CLO causes in vivo brain glucose hypometabolism and alters the brain metabolic network. We also found that CLO decreases GLT-1 immunocontent and mRNA expression along with functional changes, including a reduction in glutamate uptake in cortical slices, and a decline in glucose and glutamate uptake in astrocyte cortical cultures. Importantly, no differences were found in cultures of cortical neurons.

Animals treated with CLO exhibited a significant widespread reduction in FDG-PET signal in the cortex (~20%) and hippocampus. Consistently, those brain regions are the ones presenting a high content of GLT-1 [49]. Additionally, we found that CLO selectively reduced cortical GLT-1 protein levels, but did not alter GLAST levels, confirming the effect originally seen by Melone et al [50]. We also identified a reduction in cortical GLT-1 mRNA expression, suggesting that CLO may act at the transcriptional level. In parallel, CLO reduced GLT-1 and GLAST protein levels in cortical astrocyte cultures. Quite importantly, we corroborated these findings with the functional reduction of astroglial glutamate transport, measured by D-[³H]Asp uptake, in cortical slices and astrocyte primary cultures. We did not find changes in synaptosomal glutamate release from cortical preparations, or in glutamate, aspartate and glutamine concentrations in the CSF, which suggests that the glutamate-glutamine recycling process remains functional.

The in vitro evaluation using primary astrocyte cultures treated with CLO also showed a reduction of glucose uptake, while no difference was found in cultures of cortical neurons. These results suggest that astrocytes, not neurons, are the main target cells responsible for the phenomenon observed in vivo, i.e CLO-induced FDG-PET signal reduction. Importantly, the reduction of [³H]2DG uptake by CLO in cortical astrocyte cultures was only observed when stimulated by glutamate. Astrocytes increase their glucose uptake in situations of neuronal activation [5], i.e, during high energetic demand. Accordingly, a similar outcome was also demonstrated in vivo in a two-photon microscopy study in mice, which also showed that glucose uptake is elevated in astrocytes during activation but remains close to basal levels in neurons [14]. Our data showing that reducing GLT-1 expression decreases activity-dependent [³H]2DG accumulation is also in agreement with another study performed with GLT-1 KO mice showing that invalidation of GLT-1 prevents the whisker-stimulated increase in [¹⁴C]-2-deoxyglucose accumulation in the somatosensory cortex of developing animals [18].

It is important to emphasize that the FDG-PET uptake phase in our experiments was conducted in freely moving animals and thus, FDG-PET signal here represents glucose metabolism in an awake brain. Our present data should be also put in perspective with previous results obtained with a completely different drug. Ceftriaxone is an antibiotic that was shown to cause an increase in astrocyte GLT-1 expression and activity [51]. Treatment of primary cultures of cortical astrocytes with ceftriaxone was shown to enhance both glutamate transport and glutamate-stimulated glucose uptake through its effect on GLT-1 [30]. But quite strikingly, ceftriaxone increased brain FDG-PET signal in rats proportionately to the density of GLT-1 expression in different brain regions [30]. These results are simply a mirror image of the data obtained with CLO. Altogether, results with CLO and ceftriaxone reinforce the notion that astrocytes, through glutamate-stimulated glucose uptake, represent an important source of the FDG-PET signal in vivo, as postulated exactly 25 years ago [24].

Besides its effects on glutamate transport presented above, it is also known that CLO interacts with serotonin, dopamine and histamine receptors [52]. However, serotoninergic, dopaminergic and histaminergic systems contribute very little to brain energetics [53]. Therefore, it is very unlikely that changes in glucose uptake result from these interactions. In fact, there are several studies showing that brain energy metabolism is driven predominantly by glutamatergic and, to a minor extent, GABAergic systems (for review see [54, 55]), with contributions of other neurotransmitters being so small that it can be disregarded in the overall scenario. However, we could not ignore the possibility of a direct neuronal effect independent of the influence of serotoninergic, dopaminergic and histaminergic systems. To rule out a direct effect on neuronal energy metabolism, we tested CLO impact on neuronal cortical cultures under resting and stimulated conditions. Clearly, CLO did not change neuronal glucose metabolism in both situations, thus, strengthening the conclusion of astrocytes being at the origin of the observed hypometabolism. It is important to mention that cortical FDG-PET hypometabolism is a signature of neurodegenerative disorders such as Alzheimer’s disease and frontotemporal dementia, which is interpreted as an index of neurodegeneration [34]. Since CLO does not induce neuronal death [56–59], one could argue we recapitulated cortical FDG-PET hypometabolism seen in people with dementia by only reducing glutamate transport in astrocytes.

A potential limitation to this conclusion would pertain to behavioral changes such as sedation or freezing that could have contributed to the effects observed with CLO and might jeopardized our interpretation of the cellular origin of the FDG-PET signal [60]. However, we did not observe changes neither in the open field task nor in a more complex behavioral task such as the NOR. Thus, it seems that CLO-induced FDG hypometabolism is not related to major behavioral changes.

In summary, our results provide microPET evidence that a treatment with CLO causes a reduction in FDG-PET signal. Primary culture data indicate that astrocytes, not neurons, are the cells responsible for this phenomenon. These results corroborate the notion that FDG-PET signal might reflect not only neuronal activity but also astrocyte metabolism. These findings reinforce the need for a renewed interpretation of FDG-PET data in brain imaging studies. Finally, as a clinical perspective, CLO is an important antipsychotic drug widely used in patients with schizophrenia who are either intolerant or refractory to classical neuroleptics [61]. CLO mechanisms of action are yet not fully understood; therefore, the effect on brain energetics observed in our study might be of importance and should be better studied in this perspective. As matter of fact, a FDG-PET hypometabolism in the frontal cortex of CLO-responsive patients has been reported a few years ago [62–64]. We believe this phenomenon deserves further investigation in clinical research.

Funding

ASR is supported by CNPq [165626/2017-8 and 141254/2019-0]. BB receives financial support from CAPES [88887.336490/2019-00]. DGS is supported by CNPq [152189/2020-3]. ICF is supported by CAPES [88887.185806/2018-00]. LSM is supported by CAPES [88887.481222/2020-00]. DOS is supported by CNPq/INCT [465671/2014-4], CNPQ/ZIKA [440763/2016-9], CNPQ/FAPERGS/PRONEX [16/2551-0000475-7], FAPERGS [19/2551-0000700-0], CAPES [88887.507218/2020-00] [88887.507161/2020-00]. LP is supported by Inserm and Université de Poitiers. ERZ is supported by CNPq [435642/2018-9], [312410/2018-2] and Instituto Serrapilheira [Serra-1912-31365].

Acknowledgements

We would like to thank Prof. Dr. Elaine Elisabetsky for donating CLO for cell cultures and HPLC experiments.

Conflict of interest

The authors report no biomedical financial interests or potential conflicts of interest.

Ethics approval

All the experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the Universidade Federal do Rio Grande do Sul (approval number 32983).

Authors contributions

AR, BB, DGS, DS and ERZ participated on the conceptualization, design and interpretation of the experiments. DGS, BB an AR carried out the cultures preparation and maintenance. BB performed the qRT-PCR procedure. GTV and SG performed the PET imaging procedure. GS performed the metabolic network analysis. FUF and DM performed the HPLC procedures. AR carried out all the remaining experiments with ICF, MLS and LSM assistance. AR and ERZ wrote the manuscript. All authors critically revised and approved the final version of the manuscript.

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You are reading this latest preprint version

Clozapine Induces Astrocyte-Dependent FDG-PET Hypometabolism

Status:

Journal Publication

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Pharmacological treatment

2.3 [¹⁸F]FDG microPET scanning

2.4 Metabolic networks

2.5 Synaptosomal preparation and L-[³H]glutamate (L-[³H]Glut) potassium-stimulated release

2.6 Primary cortical astrocyte cultures

2.7 Primary cortical neuronal cultures

2.8 Sodium-dependent D-[2,3-³H]-aspartate (D-[³H]Asp) uptake

2.9 2-Deoxy-D-[³H]glucose ([³H]2DG) uptake

2.10 SDS-PAGE

2.11 RNA extraction and quantitative real time PCR (qRT-PCR)

2.12 High-performance liquid chromatography (HPLC) procedures

2.13 Behavioral Analysis

3. Results

3.1 CLO treatment causes brain glucose hypometabolism in vivo

3.2 Cortical GLT-1 and D-[3H]Asp uptake are reduced after CLO treatment

3.3 Effects of CLO on primary cultures of cortical astrocytes

3.4 Effects of CLO treatment on primary cultures of cortical neurons

3.5 Behavioral effects and treatment measures

4. Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Clozapine Induces Astrocyte-Dependent FDG-PET Hypometabolism

Status:

Journal Publication

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Pharmacological treatment

2.3 [18F]FDG microPET scanning

2.4 Metabolic networks

2.5 Synaptosomal preparation and L-[3H]glutamate (L-[3H]Glut) potassium-stimulated release

2.6 Primary cortical astrocyte cultures

2.7 Primary cortical neuronal cultures

2.8 Sodium-dependent D-[2,3-3H]-aspartate (D-[3H]Asp) uptake

2.9 2-Deoxy-D-[3H]glucose ([3H]2DG) uptake

2.10 SDS-PAGE

2.11 RNA extraction and quantitative real time PCR (qRT-PCR)

2.12 High-performance liquid chromatography (HPLC) procedures

2.13 Behavioral Analysis

3. Results

3.1 CLO treatment causes brain glucose hypometabolism in vivo

3.2 Cortical GLT-1 and D-[3H]Asp uptake are reduced after CLO treatment

3.3 Effects of CLO on primary cultures of cortical astrocytes

3.4 Effects of CLO treatment on primary cultures of cortical neurons

3.5 Behavioral effects and treatment measures

4. Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

2.3 [¹⁸F]FDG microPET scanning

2.5 Synaptosomal preparation and L-[³H]glutamate (L-[³H]Glut) potassium-stimulated release

2.8 Sodium-dependent D-[2,3-³H]-aspartate (D-[³H]Asp) uptake

2.9 2-Deoxy-D-[³H]glucose ([³H]2DG) uptake