Venlafaxine and L-Thyroxine Treatment Combination: Impact on Metabolic and Synaptic Plasticity Changes in an Animal Model of Coexisting Depression and Hypothyroidism

The clinical effectiveness of supportive therapy with thyroid hormones in drug-resistant depression is well-known; however, the mechanisms of action of these hormones in the adult brain have not been fully elucidated to date. We determined the effects of venlafaxine and/or L-thyroxine on metabolic parameters and markers involved in the regulation of synaptic plasticity and cell damage in an animal model of coexisting depression and hypothyroidism, namely, Wistar Kyoto rats treated with propylthiouracil. In this model, in relation to the depression model itself, the glycolysis process in the brain was weakened, and a reduction in pyruvate dehydrogenase in the frontal cortex was normalized only by the combined treatment with L-thyroxine and venlafaxine, whereas changes in pyruvate and lactate levels were affected by all applied therapies. None of the drugs improved the decrease in the expression of mitochondrial respiratory chain enzymes. No intensification of glucocorticoid action was shown, while an unfavorable change caused by the lack of thyroid hormones was an increase in the caspase-1 level, which was not reversed by venlafaxine alone. The results indicated that the combined administration of drugs was more effective in normalizing glycolysis and the transition to the Krebs cycle than the use of venlafaxine or L-thyroxine alone.


Introduction
Major depression, one of the most common mental illnesses, affects more individuals than all other psychiatric illnesses combined, and a significant number of patients do not remit after pharmacological or behavioral treatment. The low efficiency of the treatment of depression may be due to the poorly understood pathogenesis of this disease and the fact that most of the currently used drugs mainly affect serotonergic and noradrenergic transmission; while it is known that in this disease, metabolic, endocrine, and immune changes also occur [1][2][3][4]. In studies of the pathogenesis of depression, the focus has long been on disturbances in monoaminergic neurotransmission and the function of neurons [5]. However, as indicated by current studies, the disturbances of neurotransmission observed in depression may not be the primary cause but rather the consequence of metabolic alternations related mainly to the dysfunction of astrocytes, excessive action of pro-inflammatory cytokines released primarily from microglial cells [6,7], and the strong action of glutamate and glucocorticoids [8,9].
Currently, an increasing number of studies indicate that metabolic dysfunction and insufficient energy supply to neurons may be important in the pathogenesis of depression [10][11][12]. Glucose is the main source of energy for brain cells, and its metabolism

Behavioral Testing-Light-Dark Box (LDB) Test
In the last three days of the experiment, the animals underwent a light-dark box test (LDB). The light dark-box test was carried out according to the procedure described by Chocyk et al. [32] and Chamera et al. [33] in an apparatus consisting of a cage divided into two sections of equal size (one chamber was brightly illuminated by diodes (100 lux), whereas the other chamber was dark) by a partition with a door (10.6 cm × 10.4 cm). The apparatus was controlled with a computer system (TSE Systems, Bad Homburg, Germany). The whole experiment was carried out in a dark room, and the rats were kept in darkness for an hour before the test. At the beginning of each test session, the animal was placed in one corner of the light compartment, facing away from the gate. Then, the animal was allowed to move freely between the two chambers with the door open for 10 min. The behavioral response of rats during the trials was recorded by Fear Conditioning Software (TSE, Bad Homburg, Germany). The time spent in each compartment was recorded for each animal. After each trial, the chambers were cleaned to prevent bias based on olfactory cues.

Biochemical Analysis
The rats were sacrificed under nonstressed conditions by rapid decapitation. Trunk blood samples were collected into plastic test tubes containing EDTA as an anticoagulant. After centrifugation (3000 rpm, 20 min, 4 °C), separated plasma was gathered and frozen at −20 °C until use. Brain structures (frontal cortex and hippocampus) were dissected on ice-cold glass plates, frozen on dry ice, and stored at −80 °C until further analysis.

Behavioral Testing-Light-Dark Box (LDB) Test
In the last three days of the experiment, the animals underwent a light-dark box test (LDB). The light dark-box test was carried out according to the procedure described by Chocyk et al. [32] and Chamera et al. [33] in an apparatus consisting of a cage divided into two sections of equal size (one chamber was brightly illuminated by diodes (100 lux), whereas the other chamber was dark) by a partition with a door (10.6 cm × 10.4 cm). The apparatus was controlled with a computer system (TSE Systems, Bad Homburg, Germany). The whole experiment was carried out in a dark room, and the rats were kept in darkness for an hour before the test. At the beginning of each test session, the animal was placed in one corner of the light compartment, facing away from the gate. Then, the animal was allowed to move freely between the two chambers with the door open for 10 min. The behavioral response of rats during the trials was recorded by Fear Conditioning Software (TSE, Bad Homburg, Germany). The time spent in each compartment was recorded for each animal. After each trial, the chambers were cleaned to prevent bias based on olfactory cues.

Biochemical Analysis
The rats were sacrificed under nonstressed conditions by rapid decapitation. Trunk blood samples were collected into plastic test tubes containing EDTA as an anticoagulant. After centrifugation (3000 rpm, 20 min, 4 • C), separated plasma was gathered and frozen at −20 • C until use. Brain structures (frontal cortex and hippocampus) were dissected on ice-cold glass plates, frozen on dry ice, and stored at −80 • C until further analysis.

Thyroid Hormone Measurement in Plasma
Plasma concentrations of free T3, free T4 (both: DiaMetra, Perugia, Spello, Italy), and TSH (Demeditec, Kiel, Germany) were measured with enzyme-linked immunosorbent assays (ELISAs) and are presented as pg/mL, ng/dL, and ng/mL, respectively. Samples were homogenized in 2-mL plastic tubes filled with an appropriate buffer using a TissueLyser II (Qiagen Inc, Valencia, CA, USA) as described previously [34]. The protein level was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) [35].

Preparation of Mitochondria-Enriched and Cytosolic Fractions
To measure the concentrations and activities of mitochondrial enzymes, mitochondriaenriched membrane fractions were isolated from the frontal cortex and hippocampus according to the procedure described by Wernicke [36]. Brain tissue samples were homogenized in buffer containing 5 mol/L HEPES/NaOH, pH 7.4, 320 mmol/L sucrose, 1 mmol/L Na+/EDTA, and 0.5% protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) using a Teflon-glass homogenizer. Then, samples were centrifuged at 1300× g for 4 min at 4 • C, and supernatants were collected. The pellets were rinsed twice and centrifuged at 1500× g for 4 min at 4 • C. The combined supernatants were centrifuged at 17,000× g for 12 min at 4 • C, after which the mitochondria-containing pellets and supernatants (cytosolic fraction) were collected.

Pyruvate Measurement
The level of pyruvate in the cytosolic fraction isolated from the brain structures was measured using a fluorimetric assay kit (BioVision, Milpitas, CA, USA). Samples and pyruvate standards in 96-well plates were incubated with reaction mix for 30 min, after which fluorescence was measured at Ex/Em = 535/590 in a fluorometer (Tecan Infinite M1000, Männedorf, Switzerland). The concentration of pyruvate was calculated based on the standard curve and displayed as nmol of pyruvate per mg of protein.

Pyruvate Dehydrogenase (PDH) Expression and Activity Estimation
Pyruvate dehydrogenase levels in the mitochondria-enriched fraction of the frontal cortex and hippocampus were estimated using an ELISA method (Shanghai Qayee Biotechnology Co., Shanghai, China) according to the manufacturer's instructions. The absorbance was measured using a spectrophotometer (Tecan Infinite M200 Pro, Männedorf, Switzerland) at a wavelength of λ = 450. The concentration of PDH in the samples was calculated based on the standard curve and expressed as ng/mg protein.
PDH activity was measured with a colorimetric assay kit (BioVision, Milpitas, CA, USA) in a spectrophotometer (Tecan Infinite M200 Pro, Switzerland) according to the manufacturer's instructions. The absorbance was measured at 37 • C immediately after starting the reaction and after 1 h at a wavelength λ = 450 nm. The activity of PDH was calculated based on the standard curve and displayed as nanomoles per min per mg of protein.

Lactate Measurement
The levels of lactate in the mitochondria-enriched and cytosolic fractions obtained from the frontal cortex and hippocampus were measured with a colorimetric assay kit (BioVision, Milpitas, CA, USA). Samples and standards were mixed with the reaction mix on a 96-well plate, and after a 30-min incubation, the absorbance was measured at λ = 570 nm using a spectrophotometer (Tecan Infinite M200 Pro, Männedorf, Switzerland). The concentration of lactate was calculated based on the standard curve and displayed as nmol/mg protein. The tissue homogenates and mitochondria-enriched fractions were prepared as previously described [23,24,37]. The obtained samples of tissue homogenates were homogenized in 2% SDS (BioShop, Burlington, ON, Canada) in dH 2 O, whereas samples of isolated mitochondria-enriched membrane fraction were prepared through lysis in PBS containing 1% Triton X-100 and 0.1% SDS with phosphate and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated on 20% (w/v) Criterion™ TGX™ Precast Midi Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Sigma-Aldrich, Saint Louis, MO, USA). After semidry transfer (though for OXPHOS, wet transfer was conducted in CAPS buffer), some membranes were cut to allow simultaneous incubation with different antibodies. Then, membranes were blocked in 5% skim milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (Sigma-Aldrich, Saint Louis, MO, USA) for 1 h at room temperature (RT) and were incubated overnight at 4 • C with primary antibodies against the following proteins: Total OXPHOS Rodent WB Antibody Cocktail (Abcam, Cambridge, UK), MCT2, MCT4 (both Bioassay Technology Laboratory, Shanghai, China), HCAR1 (Sigma-Aldrich, Saint Louis, MO, USA), FKBP51, Bax (both ProteinTech, Manchester, UK), GR, beclin 1, and caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA). The next day, the membranes were extensively washed in TBST (TBS with 0.05% Tween 20) and incubated at RT with horse anti-mouse/goat anti-rabbit IgG HRP peroxidase-conjugated secondary antibody (Vector Laboratories, Peterborough, UK) for 1 h. After washing in TBST (Tris-buffered saline with 0.05% Tween 20), the bands were detected using BM Chemiluminescence Western Blotting Substrate (POD) (Roche, Mannheim, Germany) in a luminescent image analyzer with a Fujifilm LAS-1000 System (Fujifilm, Tokyo, Japan). The relative levels of immunoreactivity were densitometrically quantified using Fujifilm Multi Gauge software (Fujifilm, Tokyo, Japan). As an internal loading control, the amount of vinculin (Sigma-Aldrich, Saint Louis, MO, USA) was determined. Some membranes were stripped and reprobed.
2.5.9. OPA1, DNM1L, GLT-1, GLAST Protein Levels The levels of OPA1, DNM1L (both Bioassay Technology Laboratory, Shanghai, China), GLT-1, and GLAST (both ELK Biotechnology, Wuhan, China) in the tissue homogenates extracted from the frontal cortex and hippocampus in PBS buffer were estimated using commercially available ELISA kits. The absorbance was measured using a Tecan Infinite M200 Pro (TECAN, Männedorf, Switzerland) set to the wavelength λ = 450 nm. The concentration of measured protein in the samples was calculated based on the relevant standard curve and expressed as ng/mg protein.

Statistical Analysis
All graphs were prepared using GraphPad Prism 8 (San Diego, CA, USA). The results of the experiments are displayed as the means ± SEM. Statistical analysis was performed using Statistica 13.3 software (StatSoft, Palo Alto, CA, USA). The differences between WKY + PTU vs. WKY (control); WKY + PTU + VEN vs. WKY (control), WKY + PTU + L-T4 vs. WKY (control), and WKY + PTU + VEN + L-T4 vs. WKY (control) were analyzed by one-way analyses of variance (ANOVA), whereas WKY + PTU + VEN, WKY + PTU + L-T4, and WKY + PTU + VEN + L-T4 vs. WKY + PTU were analyzed by factorial ANOVA followed by the Duncan post hoc test when appropriate. Differences were considered statistically significant when the determined p-value was less than 0.05 (p < 0.05). The ANOVA results are reported as an F-statistic and its associated degrees of freedom.

Light-Dark Box Test
The light-dark box test was used to examine anxiety-like behavior, which is often observed during depression. The WKY rats treated with PTU were characterized by a decrease in the time spent in the light compartment (F 1,15 = 9.84, p < 0.05) and by an increase in the time spent in the dark compartment (F 1,15 = 8.09, p < 0.05) when compared to the WKY control rats. Applied drugs did not significantly regulate anxiety-like behavior in comparison to the WKY PTU-treated group (Figure 2A,B).
T4, and WKY + PTU + VEN + L-T4 vs. WKY + PTU were analyzed by factorial ANOV followed by the Duncan post hoc test when appropriate. Differences were considered st tistically significant when the determined p-value was less than 0.05 (p < 0.05). T ANOVA results are reported as an F-statistic and its associated degrees of freedom.

Light-Dark Box Test
The light-dark box test was used to examine anxiety-like behavior, which is oft observed during depression. The WKY rats treated with PTU were characterized by a d crease in the time spent in the light compartment (F1,15 = 9.84, p < 0.05) and by an increa in the time spent in the dark compartment (F1,15 = 8.09, p < 0.05) when compared to t WKY control rats. Applied drugs did not significantly regulate anxiety-like behavior comparison to the WKY PTU-treated group (Figure 2A,B).
The TSH level in the serum was elevated by PTU (PTU effect: F1,22 = 298.00, p < 0.0 Treatment with L-T4 in PTU animals led to significant reductions in the level of this ho mone in both groups: L-T4 alone and L-T4 + VEN (L-T4 effect: F1,44 = 369.81, p < 0.0 Treatment with VEN alone did not influence this change ( Figure 3C).
The TSH level in the serum was elevated by PTU (PTU effect: F 1,22 = 298.00, p < 0.05). Treatment with L-T4 in PTU animals led to significant reductions in the level of this hormone in both groups: L-T4 alone and L-T4 + VEN (L-T4 effect: F 1,44 = 369.81, p < 0.05). Treatment with VEN alone did not influence this change ( Figure 3C).
A significant enhancing effect of PTU was observed in the case of LDL (PTU effect: F 1,18 = 94.39, p < 0.05). L-T4 alone and together with VEN reversed the PTU-induced elevation in LDL (L-T4 effect: F 1,36 = 183.54, p < 0.05). Treatment with VEN alone did not influence the change evoked by PTU ( Figure 4D). PTU also significantly diminished the level of plasma triglyceride (PTU effect: F 1,18 = 21.48, p < 0.05), which was not normalized by VEN and/or L-T4 administration ( Figure 4E).

Concentration and Activity of PDH in the Brain
In the frontal cortex, PTU administration diminished the level of PDH (PTU effect: F 1,14 = 8.46, p < 0.05), while VEN and L-T4 co-treatment reversed the PTU-induced effect (VEN x L-T4 interaction: F 1,33 = 5.03, p < 0.05). Treatment with VEN or L-T4 alone did not affect the PDH level in this brain structure in groups treated with PTU ( Figure 6A). In the hippocampus, VEN in rats treated with PTU significantly upregulated the level of PDH (VEN effect: F 1,34 = 6.50, p < 0.05) ( Figure 6B).
Measurements of PDH activity in the frontal cortex revealed that animals treated with PTU and VEN or L-T4 displayed reduced activity of this enzyme vs. WKY animals (PTU VEN effect: F 1,14 = 7.82, PTU L-T4 effect: F 1,14 = 5.71, p < 0.05). In PTU-treated animals, an effect of co-treatment was also shown (VEN x L-T4 interaction: F 1,28 = 5.47, p < 0.05); animals that underwent combined treatment showed an increase in the activity of PDH compared to animals treated only with VEN ( Figure 6C). In the hippocampus, treatment with PTU did not change the activity of PDH; however, a decreased enzyme activity was observed in the PTU + L-T4 group in comparison to the control (PTU + L-T4 effect: F 1,14 = 4.82, p < 0.05). Rats receiving PTU and drugs showed the impact of L-T4 (F 1,28 = 4.36, p < 0.05) and reduced PDH activity was observed in the hippocampi of rats treated with L-T4 alone or with VEN ( Figure 6D).
with PTU did not change the activity of PDH; however, a decreased enzyme activity w observed in the PTU + L-T4 group in comparison to the control (PTU + L-T4 effect: F1,1 4.82, p < 0.05). Rats receiving PTU and drugs showed the impact of L-T4 (F1,28 = 4.36, 0.05) and reduced PDH activity was observed in the hippocampi of rats treated with Lalone or with VEN ( Figure 6D).

Lactate Level in the Brain
In both examined brain areas, PTU decreased the levels of lactate in the cytosolic fraction (PTU effect: F 1,12 = 35.52 in FCx, F 1,12 = 10.86 in Hp; p < 0.05) ( Figure 7A,B). Furthermore, in the frontal cortex administration of VEN, L-T4, and co-treatment with these drugs normalized the observed PTU-induced change (VEN × L-T4 interaction: F 1,26 = 16.63, p < 0.05) ( Figure 7A), whereas in the hippocampus, a normalization effect was observed only in the case of VEN (VEN effect: F 1,26 = 5.96, p < 0.05, in groups treated with VEN and VEN + L-T4) ( Figure 7B). In the frontal cortex, in WKY PTU rats treated with VEN alone, cytosolic lactate was increased in comparison to control WKY animals (F 1,14 = 7.23, p < 0.05) ( Figure 7A), whereas in the hippocampus in the WKY + PTU + L-T4 group, lactate was reduced, as in PTU-treated animals ( Figure 7B).
Taking into account the mitochondria-enriched fraction in the frontal cortex, PTU treatment led to an elevation in lactate levels (PTU effect: F 1,13 = 8.00, p < 0.05) ( Figure 7C). This effect dependence was still present after VEN or L-T4 administration. In the hippocampus, no changes in the concentration of lactate in the mitochondria-enriched fraction were detected ( Figure 7D).
Taking into account the mitochondria-enriched fraction in the frontal cortex, PT treatment led to an elevation in lactate levels (PTU effect: F1,13 = 8.00, p < 0.05) (Figure 7 This effect dependence was still present after VEN or L-T4 administration. In the hipp campus, no changes in the concentration of lactate in the mitochondria-enriched fracti were detected ( Figure 7D).

Hydroxycarboxylic Acid Receptor 1 (HCAR1) Level in the Brain
There was no impact of either PTU or applied treatment on HCAR1 levels in t frontal cortex and hippocampus (Table 1).

Hydroxycarboxylic Acid Receptor 1 (HCAR1) Level in the Brain
There was no impact of either PTU or applied treatment on HCAR1 levels in the frontal cortex and hippocampus (Table 1).      There were no changes in the protein levels of MCT2 and MCT4 in either examined brain structure (Table 1).

Monocarboxylate Transporters MCT2 and MCT4 Levels in the Brain
There were no changes in the protein levels of MCT2 and MCT4 in either examined brain structure (Table 1).

Levels of Oxidative Phosphorylation (OXPHOS) Complexes in the Mitochondria-Enriched Fraction of the Brain
In the frontal cortex in groups treated with PTU, significant reductions in the expression of oxidative phosphorylation complexes I, III, and IV were observed (PTU effect: F 1,14 = 8.86, complex I; PTU effect: F 1,14 = 9.73, complex III; PTU effect: F 1,14 = 6.82, complex IV, p < 0.05). Injections with VEN and/or L-T4 did not modulate the impact of PTU on these proteins. In complex II, a PTU effect was not observed, but in the PTU + L-T4and PTU + VEN + L-T4-treated groups, a reduction in the level of this factor was shown ( Figure 8A). In both examined brain areas, the expression of complex V did not differ between the groups ( Figure 8A,B). Furthermore, no changes in the hippocampus were demonstrated in the case of complexes I, II, and IV, but in complex III, an L-T4 effect was observed in PTU + VEN + L-T4 vs. PTU-treated animals (F 1,28 = 8.88, p < 0.05). These animals had also diminished level of complex III in comparison to control WKY rats (PTU + VEN + L-T4 effect, F 1,14 = 9.26).

Levels of Mitochondrial Fusion Factor, OPA-1, and Fission Factor, DNM1L
There were no significant changes in OPA-1 expression in the frontal cortices between the examined groups ( Figure 9A). In the hippocampi of WKY PTU-treated rats, there was a decrease in the level of OPA-1 (PTU effect: F1,17 = 10.03, p < 0.05), but neither VEN nor L-T4 separately administered evoked any impact on this change, whereas in cotreatment, no difference vs. WKY was observed ( Figure 9B).
In the frontal cortex, no changes in the levels of the mitochondrial fission marker DNM1L were shown ( Figure 9C); however, in the hippocampus, PTU administration resulted in the DNM1L downregulation (PTU effect: F1,16 = 7.26, p < 0.05), and applied ther-

Levels of Mitochondrial Fusion Factor, OPA-1, and Fission Factor, DNM1L
There were no significant changes in OPA-1 expression in the frontal cortices between the examined groups ( Figure 9A). In the hippocampi of WKY PTU-treated rats, there was a decrease in the level of OPA-1 (PTU effect: F 1,17 = 10.03, p < 0.05), but neither VEN nor L-T4 separately administered evoked any impact on this change, whereas in co-treatment, no difference vs. WKY was observed ( Figure 9B).

Levels of Glucocorticoid Receptor (GR) and FKBP51-Factor Regulating GR Brain
The level of GR was not changed by either PTU or the drugs in either ( Figure 10A,B).
An analysis of the frontal cortex homogenates revealed an increased c FKBP51 in rats receiving PTU together with combined treatment (VEN + L ison to the PTU group and to PTU animals that were administered VEN creased level of FKBP51 in this group was induced by L-T4 (L-T4 effect 0.05) ( Figure 10C). In the hippocampus, no changes between the groups ( Figure 10D). In the frontal cortex, no changes in the levels of the mitochondrial fission marker DNM1L were shown ( Figure 9C); however, in the hippocampus, PTU administration resulted in the DNM1L downregulation (PTU effect: F 1,16 = 7.26, p < 0.05), and applied therapy did not reverse the induced effect in this brain structure ( Figure 9D).

Levels of Glucocorticoid Receptor (GR) and FKBP51-Factor Regulating GR-Function in the Brain
The level of GR was not changed by either PTU or the drugs in either brain structure ( Figure 10A,B).
An analysis of the frontal cortex homogenates revealed an increased concentration of FKBP51 in rats receiving PTU together with combined treatment (VEN + L-T4) in comparison to the PTU group and to PTU animals that were administered VEN alone. An increased level of FKBP51 in this group was induced by L-T4 (L-T4 effect: F 1,28 = 8.83, p < 0.05) ( Figure 10C). In the hippocampus, no changes between the groups were observed ( Figure 10D).

Levels of the Cell Damage Markers Caspase-1 p20, Bax, and Beclin1 in the
In both tested brain structures, PTU induced elevations in caspase-1 effect: F1,12 = 7.64 in FCx; F1,13 = 5.72 in Hp, p < 0.05, Figure 11A No changes in Bax and beclin 1 between the examined groups were d the frontal cortex or the hippocampus (Table 2).
No changes in Bax and beclin 1 between the examined groups were detected in either the frontal cortex or the hippocampus ( Table 2).

Concentrations of the Glutamate Transporters GLT-1 and GLAST in the Brain
In the frontal cortex, no changes in GLT-1 ( Figure 12A) or GLAST between the examined groups were detected ( Figure 12C).
Similarly, neither PTU nor VEN and/or L-T4 affected the level of GLT-1 in the hippocampus ( Figure 12B). However, PTU administration resulted in a reduction in GLAST-1, which was intensified by L-T4 administered alone but not by co-treatment with these two compounds (VEN x L-T4 interaction: F1,35 = 4.82) ( Figure 12D).

Concentrations of the Glutamate Transporters GLT-1 and GLAST in the Brain
In the frontal cortex, no changes in GLT-1 ( Figure 12A) or GLAST between the examined groups were detected ( Figure 12C).
Similarly, neither PTU nor VEN and/or L-T4 affected the level of GLT-1 in the hippocampus ( Figure 12B). However, PTU administration resulted in a reduction in GLAST-1, which was intensified by L-T4 administered alone but not by co-treatment with these two compounds (VEN x L-T4 interaction: F1,35 = 4.82) ( Figure 12D).

Concentrations of the Glutamate Transporters GLT-1 and GLAST in the Brain
In the frontal cortex, no changes in GLT-1 ( Figure 12A) or GLAST between the examined groups were detected ( Figure 12C).

Discussion
In this study, a hypothyroidism model was verified by measuring the levels T3 and T4 forms as well as TSH in the blood. In animals treated with PTU, the lev fT3 and fT4 decreased significantly, while the concentration of TSH increased, indi severe hypothyroidism. Administration of venlafaxine alone did not affect the lev either hormone, while in animals receiving L-T4 alone and L-T4 with venlafaxine, no were the PTU-induced changes reversed, but fT3, fT4, and TSH levels also show appearance of hyperthyroidism. L-T4 was used at a dose higher than the one that re euthymia because this hormone is effective in the treatment of depression in hum supraphysiological doses [38]. In our previous studies, we have shown that adminis PTU for three weeks to WKY rats lowers their body weight while not affecting shor memory and spatial memory, as determined in novel object recognition and novel location tests, respectively, probably because the discrimination/preference inde these tests are much lower in this strain of rats than in Wistar animals [23,24]. Sim PTU did not affect the anxiety behavior evaluated in the elevated plus maze test pression-like behavior assayed in the forced swim test, perhaps because the param Similarly, neither PTU nor VEN and/or L-T4 affected the level of GLT-1 in the hippocampus ( Figure 12B). However, PTU administration resulted in a reduction in GLAST-1, which was intensified by L-T4 administered alone but not by co-treatment with these two compounds (VEN x L-T4 interaction: F 1,35 = 4.82) ( Figure 12D).

Discussion
In this study, a hypothyroidism model was verified by measuring the levels of free T3 and T4 forms as well as TSH in the blood. In animals treated with PTU, the levels of fT3 and fT4 decreased significantly, while the concentration of TSH increased, indicating severe hypothyroidism. Administration of venlafaxine alone did not affect the levels of either hormone, while in animals receiving L-T4 alone and L-T4 with venlafaxine, not only were the PTU-induced changes reversed, but fT3, fT4, and TSH levels also showed the appearance of hyperthyroidism. L-T4 was used at a dose higher than the one that restores euthymia because this hormone is effective in the treatment of depression in humans at supraphysiological doses [38]. In our previous studies, we have shown that administering PTU for three weeks to WKY rats lowers their body weight while not affecting short-term memory and spatial memory, as determined in novel object recognition and novel object location tests, respectively, probably because the discrimination/preference indexes in these tests are much lower in this strain of rats than in Wistar animals [23,24]. Similarly, PTU did not affect the anxiety behavior evaluated in the elevated plus maze test or depression-like behavior assayed in the forced swim test, perhaps because the parameters determined in these tests were already different at baseline in WKY rats compared to Wistar rats, i.e., showing pro-depressive and pro-anxiety behaviors in WKY individuals [23,24]. Therefore, in the present study, we determined anxiety behavior using a different methodology, a light-dark box test, in which the anxiety-inducing action of PTU was observed. The applied therapies, especially the administration of L-T4 together with venlafaxine, tended to decrease the anxiolytic effect of PTU, but due to large interindividual variability, these differences did not reach significance. The hypothyroidism model used was also verified by determining the impacts of PTU and applied treatment on blood glucose and lipid metabolism markers. According to the results obtained previously [23], the present results also showed that administration of PTU increased the levels of total cholesterol and its HDL and LDL fractions and simultaneously decreased the level of triglycerides. Of these parameters, only LDL levels were normalized by L-T4 and L-T4 administered together with venlafaxine, while venlafaxine treatment alone did not show such a beneficial effect. Additionally, in the case of HDL, a favorable impact in animals receiving L-T4 alone and with venlafaxine, i.e., an increase in this protective cholesterol fraction, was demonstrated. In contrast to the beneficial effect of L-T4 alone and combined with venlafaxine on LDL and HDL levels, these animals experienced an increase in glucose levels.
Our results indicate that in the applied animal model of coexisting depression with anxiety and hypothyroidism, glycolysis was reduced compared to that in the depression model itself. In the frontal cortex, it was evidenced by the decreased levels of pyruvate and lactate in the cytosolic fraction, i.e., both end products of the glycolysis process. What is important, the decreased cytosolic lactate level was normalized in a similar way by venlafaxine, L-T4, and by the co-treatment, while the decreased level of pyruvate was increased more strongly in the frontal cortex of animals receiving venlafaxine plus L-T4 than those receiving venlafaxine alone, which indicated that L-T4 is able to potentiate the effect of venlafaxine. In the frontal cortex, there was not only a decrease in the level of glycolysis products but also a decrease in the level of pyruvate dehydrogenase in the mitochondrial fraction, which suggests a decrease in the supply of substrates for the Krebs cycle and therefore possibly also the limitations of this cycle. As with pyruvate levels, decreased expression of pyruvate dehydrogenase, an enzyme that connects the glycolysis process with the Krebs cycle, was normalized in WKY rats treated with L-T4 and venlafaxine. However, in the case of this enzyme, administration of either venlafaxine or L-T4 alone did not affect its level at all, which indicated that the antidepressant used had an effect on this parameter only after thyroid hormone deficiency had been compensated. In the hippocampus, lower levels of lactate in the cytosolic fraction in PTU-treated WKY rats in comparison to animals of this strain without hypothyroidism support the idea of glycolysis reduction. Lactate levels were normalized by both the administration of venlafaxine and venlafaxine together with L-T4, while in the case of pyruvate dehydrogenase, the tested drugs did not have a significant effect. In both the frontal cortex and the hippocampus, lactate levels were much higher than pyruvate levels, which is consistent with the present data showing that as a result of glycolysis, which is performed mostly in astrocytes, lactate is released from these cells into the extracellular fluid and delivered to neurons [39]. Neurons, as brain cells, need a large amount of energy stored in ATP to maintain their function and mainly carry out the process of oxidative phosphorylation. Lactate shuttling from astrocytes to neurons [40][41][42] is an important process that ensures the proper production of ATP by neurons, and disturbance of this process, which plays an important role not only in the pathogenesis of neurodegenerative diseases but also in depression, may also be due to altered expression of lactate transporters. Since we did not show differences in the expression of monocarboxylate transporter 2 (MCT2), which is involved in the transport of lactate to neurons, or MCT4, which is responsible for the transport of lactate to astrocytes, in any of the study groups, the metabolic changes observed in the model of coexisting depression and hypothyroidism were not due to changes in lactate transport. Since lactate is not only an end-product of glycolysis but may also act as an intercellular signaling molecule via G-protein-coupled receptor 81 (GPR81), also known as hydroxycarboxylic acid receptor 1-HCAR1 [43,44], we measured the level of this receptor in the brain; however, no changes were detected, which suggested that the observed effects were mainly metabolic.
Contrary to the effective reversal of hypothyroidism-induced decreases in lactate and pyruvate levels and pyruvate dehydrogenase expression in the frontal cortex of WKY rats by venlafaxine and/or L-T4, these drugs did not have any effect on respiratory chain complexes. The administration of PTU significantly decreased the expression of complexes I, III, and IV, while all the applied therapies (venlafaxine and L-T4 separately and in combination) not only did not inhibit these changes, but even in the case of complexes I and IV, there was a tendency to intensify the decrease. The reduction in the level of mitochondrial complexes in the model of depression and hypothyroidism occurred only in the frontal cortex and not in the hippocampus, which is consistent with our previous data and indicates a greater sensitivity of the frontal cortex to thyroid hormone deficiencies. Thus, among the metabolic disturbances observed in the model of coexisting depression and hypothyroidism, changes in the process of glycolysis and the transition from glycolysis to the Krebs cycle were normalized under the influence of T4 and/or venlafaxine administration, while the applied therapy did not affect the reduction in the expression of mitochondrial respiratory chain enzymes in the frontal cortex. Since not only the expression of particular enzymes but also mitochondrial dynamics are important in mitochondrial metabolism, the effects of hypothyroidism on the markers of mitochondrial fission and fusion were determined [45]. The reduction in the concentration of OPA-1, a fusion marker, in the hippocampus of WKY rats receiving PTU, indicates that in the model of depression and hypothyroidism, the process of mitochondrial dynamics could also be disrupted; however, this change was not normalized by L-T4 and venlafaxine but was attenuated only in the animals receiving the combined T4 and venlafaxine treatment. However, the influence of PTU and the therapy used on the dynamics of mitochondria in the hippocampus is difficult to explain and its physiological significance is difficult to evaluate because the expression levels of not only OPA-1 but also the fission marker DNM1L were reduced.
A well-described change in depression is the excessive concentration or action of glucocorticoids [46,47]. Many studies have shown that enhanced activity of the hypothalamicpituitary-adrenal (HPA) axis in depression can be evoked by a reduction in the expression of glucocorticoid receptors (GRs) in the hippocampus or frontal cortex and, as a consequence, a weakening of the inhibitory feedback mechanism [4,48,49]. It has also been shown that in depression, there may be changes in the factors regulating GR functions and, consequently, the impaired potency of glucocorticoids [50]. No changes in GR expression were observed in any of the groups tested in either the hippocampus or the frontal cortex or in the level of FKBP51 in the hippocampus. The level of FKBP51 did not change in the frontal cortex in the model of coexisting depression and hypothyroidism, but an interesting change is the increase in levels of this protein in animals receiving both venlafaxine and L-T4 but not in rats treated with venlafaxine or L-T4 alone. FKBP-51 is a co-chaperone of GR that decreases GR translocation from the cytoplasm to the nucleus and thereby inhibits GR function [51]. The weakening of GR function in depression is considered to be a beneficial change because it may limit the adverse effects of glucocorticoids on the morphology and function of nerve cells. However, venlafaxine exerts this beneficial effect in the model of depression and hypothyroidism comorbidity only in the presence of thyroid hormone replacement. It is worth noting that both thyroid hormones and some antidepressants have neuroprotective effects. This fact may be important in their antidepressant properties because disturbances in synaptic plasticity in the frontal cortex and hippocampus in depression are well recognized [52]. Due to the neuroprotective effects of thyroid hormones, we checked whether lowering their levels in WKY rats would cause changes in selected markers of neurodegenerative processes. It was shown that PTU did not change the level of the major pro-apoptotic protein (Bax) or the marker of the autophagy process (beclin-1); conversely, it strongly increased the level of caspase-1, the enzyme involved in the activation of pro-inflammatory cytokine release. We have previously shown that the level of caspase-1 in the hippocampus is higher in WKY rats than in their control strain (Wistar rats) and that only in WKY rats does administration of PTU increase the expression of this enzyme [24]. In the present study, we confirmed an increase in caspase-1 levels in the hippocampus of WKY rats with hypothyroidism and found the same effect in the frontal cortex. The important roles of caspase-1, which is activated mainly by the NLRP3 inflammasome, involve the activation of inflammatory cytokines and the induction or intensification of neuroinflammation. In addition to immunoactivation, it has been shown that this enzyme, by affecting the function of the AMPA receptor, weakens long-term memory [53]. Interestingly, as in the brain structures in the present model, it has been found that in the heart, hypothyroidism also increases the concentration of caspase-1 protein, which suggests that this action may be one of the important mechanisms of hypothyroidism-induced tissue damage [54]. Importantly, the effects of PTU on the levels of caspase-1 in both examined brain structures were normalized by L-T4 and the combined administration of L-T4 and venlafaxine, whereas administration of venlafaxine alone was not effective.
It has been demonstrated that one of the neuroprotective mechanisms of action of thyroid hormones is decreasing the extracellular glutamate concentration and, thus, its toxic effects by enhancing its uptake into astrocytes [55]. However, there was no significant reduction in GLT-1, the main astrocytic glutamate transporter, which is responsible for more than 90% of glutamate uptake activity, in any of the examined brain structures in animals receiving PTU. The expression level of the GLAST transporter did not change in the frontal cortex, while in the hippocampus, its expression decreased in animals receiving PTU, and none of the applied treatments normalized its level, a result that is difficult to explain. Administration of L-T4 alone even enhanced this change. However, given the much smaller contribution of GLAST than GLT-1 to glutamate uptake, it seems that lowering hippocampal GLAST should not lead to a significant increase in extracellular glutamate levels, at least not by decreasing the expression levels of these transporters.
In summary, the obtained data demonstrated that in the model of coexisting depression and hypothyroidism, some metabolic changes, such as a reduction in the level of pyruvate dehydrogenase in the frontal cortex, were normalized only by the combined use of L-T4 with venlafaxine, and such treatment also inhibited changes in the pyruvate level in this brain structure more strongly than using L-T4 or venlafaxine alone. Contrary to the beneficial effects of L-T4 and/or venlafaxine on glycolysis and the transition from glycolysis to the Krebs cycle, neither of these drugs improved the changes in the expression of mitochondrial respiratory chain enzymes in the frontal cortex in WKY rats with hypothyroidism. An unfavorable change caused by the lack of thyroid hormones in WKY rats was the increase in the active form of caspase-1 in both examined brain structures, which indicated the possibility of intensifying proinflammatory processes, and this change was not normalized by venlafaxine alone without improvement of thyroid hormone deficiency. It should be noted, however, that the WKY rats used in this study as a model of depression, apart from depression-like behavior, also exhibit anxiety-like behavior, often observed in depression, and administration of PTU intensifies the anxiety-like behavior, therefore, it cannot be unequivocally determined whether the metabolic changes in the brain observed in the WKY + PTU model are more related to depression or anxiety.
Our results indicate that the use of thyroxine as an adjunctive therapy for depression may normalize neurotransmission not only by acting on neurons, as other authors suggest [13,25], but also by influencing the function of astrocytes and microglial cells, weakening disturbances in the glycolysis process and possibly also by reducing expression of the active form of caspase-1 to regulate the process of neuroinflammation, but this potential effect requires further research.

Conclusions
In recent years, studies on depression and anxiety disorders are increasingly focusing on defining the role of glial cells and metabolic disturbances in the brain in the pathogenesis of these diseases. The influence of thyroid hormones on biochemical processes in the brain affected in these diseases is only beginning to be studied, and getting to know them could further help to advance the diagnostic, as well as the therapy of these diseases.
Our results indicate that in the treatment of patients with depression or anxiety disorders, it is important to determine the level of thyroid hormones and in the case of hypothyroidism, simultaneous administration of L-T4 with an antidepressant. An important issue is also the selection of an appropriate antidepressant in the treatment of depression with hypothyroidism, because as our study showed, although L-T4 enhances many of the effects of venlafaxine, none of these drugs normalized the reduction of the expression of the respiratory chain complexes in the frontal cortex, so this issue requires further study.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request from qualified researchers.