Chemogenetic inhibition of the lateral hypothalamus in rats: A simple but robust tool with high translational relevance to facilitate the development of central nervous system-targeted gene therapies

doi:10.21203/rs.3.rs-3620463/v1

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Chemogenetic inhibition of the lateral hypothalamus in rats: A simple but robust tool with high translational relevance to facilitate the development of central nervous system-targeted gene therapies

https://doi.org/10.21203/rs.3.rs-3620463/v1

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published 17 May, 2024

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Aims

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo assay in rats, which could support chemogenetic drug discovery. We also aimed to establish the proof-of-concept for the chemogenetic approach in obesity indication.

Methods

Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation.

Results

Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts for 1–3 hours post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-n-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint.

Conclusion

This study demonstrates that the chemogenetic silencing of the LH is a potential strategy in obesity treatment. The study also supports the development of chemogenetics by providing a “first in vivo” tool for the validation of new chemogenetic constructs.

DREADD

Obesity

Food-intake

Body weight

deschloroclozapine

clozapine-N-oxide

Chemogenetics is an emerging gene therapy method, by which genetically “designed” new receptors are expressed on specific neurons in the central nervous system (CNS), after a viral transfection [1–3]. Subsequently, by peripherally applying low doses of otherwise biologically inactive small molecule drugs (“actuators”), specific brain areas or neuronal networks can be “turned on or off”, on demand. This developing neuroscience method is not only providing a unique research tool for neuroscientists [4] but also has the potential to open a new class of novel CNS medicines in the near future [3, 5, 6], offering a superior therapeutic alternative to current pharmacotherapy and deep brain stimulation (DBS) methods, in several neurological and psychiatric diseases, including obesity.

One of the main reasons why chemogenetic methods have not reached human clinical research yet, against their very promising therapeutic potential, is the fact that they lack proper preclinical validation including the optimization of several important experimental and methodological factors. For example, one typical validity issue in the available animal studies is the general use of the actuator molecule clozapine-n-oxide (CNO) to stimulate chemogenetic receptors, which, surprisingly, turned out to be hardly brain penetrant, non-selective, and metabolically unstable, therefore it is not ideal for the specific activation of chemogenetic receptors in the brain [7, 8]. Other gaps in our knowledge include the lack of sufficient proof for the long term in vivo efficacy and safety of the chemogenetic approach, the pharmacodynamic and pharmacokinetic optimization of appropriate actuator molecules (i.e. deschloroclozapine, DCZ), the optimization of the generally applied Adeno-Associated Virus (AAV) gene transfer method (virus dose-, titer-, promoter- and serotype selection), the proper toxicological evaluation in the nervous tissue, and the validation of appropriate proof of concept (POC) and “first in vivo” methods to measure in vivo effectiveness quickly and reliably in rodent behavioral paradigms. Although the generation of in vivo chemogenetic POC studies has been started (e.g., in Parkinson's disease [9–11] or epilepsy [12–14]), the human translational validity of these pioneering studies is debatable, because of the above detailed methodological flaws. Moreover, several high unmet need CNS indications would be potentially suitable targets for chemogenetic drug discovery [2], and more that are completely lacking the proper preclinical in vivo POC studies, including obesity, as a potential future target indication for chemogenetic therapies.

There is a general hypothesis in the literature that presumes that one of the easiest ways to develop new chemogenetic therapies is to modify the existing DBS neurosurgical technology and leverage the fact that the surgical intervention that is applied in currently approved DBS methods is more or less the same as a chemogenetic application would require [3]. Moreover, chemogenetic surgical techniques might provide a safer option to patients because, in contrast to DBS, they do not require chronically implanted devices with the risk of developing postoperative complications [15].

If we take a closer look at the potential anti-obesity applications of future chemogenetic human therapies, there are encouraging human DBS results in the literature, and clinical trials, including the lateral hypothalamus (LH), as a target brain structure [16, 17]. A considerable amount of case studies that utilized hypothalamic DBS targets resulted in weight loss in obese patients [16]. Therefore, we hypothesize that a less risky surgical method (compared to DBS) accompanied by a potentially more effective (chemogenetic) modulation of LH or other obesity-related brain circuits might provide a new therapeutic alternative to severely obese patients next to pharmacotherapy and bariatric surgery.

There are several rodent studies in the literature that demonstrate the potential effectiveness of chemogenetic methods in reducing food intake [5, 18, 19], however none of the previous studies utilized methods that would be directly translatable to human therapeutic applications. Therefore, our aim in the current study was to describe a simple, however high translational value chemogenetic POC to support the development of potential future chemogenetic therapies to be applied in obesity indication in humans, by effectively reducing hunger and food intake without the usually observed post-drug rebound effects. Moreover, our additional goal was to aid the development of new chemogenetic systems towards human use, in general, by describing a simple but robust rodent behavioral assay by which the in vivo effectiveness and basic pharmacokinetic-pharmacodynamic properties of CNS-targeted chemogenetic treatments can be easily and reliably tested. We believe that the described method can serve as a “first in vivo” pharmacodynamic tool to aid early chemogenetic drug discovery, in any CNS indications.

2.1. Animals

Young male Lister hooded rats (Envigo, Horst, Netherlands) weighing 320 to 360 g at surgery were pair-housed under a 12/12 h daily light/dark cycle with controlled temperature and humidity in the animal house of the Department of Neurobiology, Faculty of Sciences, University of Pécs, Hungary. Water and food (Ssniff Rat/Mouse Maintenance chow, ssniff Spezialdiäten GmbH, Soest, Germany) were available ad libitum except for the day before food intake experiments. The experiments were approved by the Animal Welfare Committee of the University of Pécs, and the National Scientific Ethical Committee on Animal Experimentation (ÁTET) at the Ministry of Agriculture (ethical license no.: BA02/2000-40/2021). All procedures fully complied with Decree No. 40/2013. (II. 14.) of the Hungarian Government and the EU Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes.

2.2. Intracranial injection of AAVs

The rat was anesthetized with a combination of ketamine (100 mg/kg, intraperitoneal, i.p.) and xylazine (25 mg/kg, i.p.) and was placed in a stereotaxic frame. An incision was made in the skin of the head and soft tissues were eliminated to expose the skull. The skull was washed two times with hydrogen peroxide, then holes were drilled in the skull bilaterally over the LH. A Hamilton (Reno, NV) microsyringe was filled with PBS or a formulation of viral vectors (Addgene, Watertown, MA) carrying sequences of the human synapsin promoter, the designer receptor exclusively activated by designer drug (DREADD, modified human muscarinic receptor 4, hM4D(Gi)), and the mCherry reporter gene. Two different serotypes were used in separate groups of animals: serotype AAV5 was applied at 7×10¹² vg/ml and AAV9 was applied at 1×10¹³ vg/ml. A group of animals received an infusion with PBS into the LH as a control group. The microsyringes were inserted in a Stoelting Quintessential Stereotaxic injector (Wood Dale, IL) and were introduced into the brain to the stereotaxic coordinates AP: -3.0, ML: 1.8, DV: 8.3 mm from Bregma according to the rat stereotaxic atlas of Paxinos and Watson [20]. After the correct positioning of the microsyringe, 1000 nl of the viral formulation was infused at 100 nl/min speed. After the end of the infusion, we waited an additional 10 minutes, then the microsyringe was slowly removed from the brain. Injections were made bilaterally into the LH. After injections, the skin of the head was closed using stainless steel wound clips. The weight, food and water intake, and general well-being of the rats were monitored daily after the surgery for 2 weeks. The first food intake experiment started 7 weeks after the surgery.

2.3. Experimental design

The first experiment was designed to validate our food intake measurement protocol using exenatide, a potent GLP-1 agonist compound known for its inhibitory effect on food intake [21, 22]. Exenatide was administered to naïve (non-AAV-injected) rats as a pharmaceutical formulation (Byetta, Eli Lilly, Netherlands) at doses of 2.5, 10, and 50 µg/kg s.c. The effects of exenatide were compared to its vehicle which was 0.1 N acetate buffer (pH = 5.0). Further experiments were designed to demonstrate the effects of chemogenetic silencing of the LH on the food intake of the animals that were previously intracranially injected with DREADD-carrying AAV. First, we tested the effects of subcutaneously (s.c.) applied CNO at doses of 100, 300, and 1000 µg/kg). Next, another DREADD actuator, DCZ was tested in the food intake experiments at doses of 30, 100, and 300 µg/kg (s.c.). The vehicle of CNO and DCZ was 0.01 M phosphate-buffered saline (pH = 7.4). Later, DCZ was also administered via oral route (gavage) at doses of 100, 300, and 1000 µg/kg. Subcutaneous injections were applied 30 min prior to testing (re-feeding), while oral drug administrations were applied 60 min prior to re-feeding. However, in an additional experiment, we also tested the time window of the effects of DCZ on the food intake of the animals. Thus, we administered 100 µg/kg DCZ (s.c.) to animals 0.5, 1, 3, or 16 h prior to re-feeding. All food intake experiments were performed in a within-subject design thus, the different treatments (doses or pre-treatment times) were applied to all animals on different days according to a counterbalanced Latin-square design.

2.4. Food intake measurements

Food was taken away from the rats 16 h prior to the start of the food intake experiments to achieve a similar level of hunger in the animals. At the start of the experiment, animals were re-fed with 100 g of the same laboratory chow used as a general maintenance food. The residual amount of laboratory chow was first measured 30 min after re-feeding. Then, the second measurement was performed at 1 h after re-feeding, and further measurements were taken every hour until 8 h after re-feeding. One exception was the experiment assessing the effects of CNO in which the food intake was not measured between 3 and 8 hours after re-feeding. The cumulative food intake of the animals in a given measurement time point was calculated as 100 g minus the residual amount of food measured in their feeder in a given time point.

2.5. Histology

At the end of the project, rats were sacrificed by the administration of an overdose of pentobarbital anesthetic (800 mg/kg, i.p.). Brains were rapidly removed and immersed into 4% paraformaldehyde (Sigma) solution for a few days. Then, the brains were put into 30% sucrose (Sigma) solution until they sank to the bottom of the containers indicating that the brains were fully saturated with sucrose. Then, the brains were sectioned using a freezing microtome (Leica, Wetzlar, Germany), and sections were mounted on microscope slides and covered using Vectashield with DAPI (Vector Laboratories, Newark, CA). The slides were observed and scanned using a Zeiss LSM 710 confocal microscope (Oberkochen, Germany) with lasers at 405 nm, and 543 nm wavelengths visualizing cell nuclei (DAPI) and reporter gene expression (mCherry), respectively.

2.6. Data analysis

Data and statistical analyses were carried out in Microsoft Excel (part of Microsoft 365, version 18.2306.1061.0, Microsoft Corporation, Redmond, WA, USA) and Statistica 6.1 (StatSoft. Inc., Tulsa, OK, USA) software. An effect was determined significant if both the one-way or factorial ANOVA p-values and post hoc Tukey Honest Significant Difference (HSD) test p-values were smaller than 0.05.

3.1. Effect of exenatide on the food intake of control rats

As a validation of our current food intake protocol, s.c. application of exenatide resulted in a robust food intake decrease at 10 and 50 µg/kg doses (Fig. 1). This effect was sustained and remained highly significant throughout the whole 8h post-re-feeding period. Statistical analysis yielded highly significant (p < 0.001) one-way ANOVA results if we compared the experimental groups against each other, in all time points, for example at the 30 min time point it was F_3,24 = 56.729 (p < 0.001), while at the end of the measurements at the 8h time point it remained significant (F_3,24 = 100.322, p < 0.001). Post-hoc Tukey HSD analyses similarly revealed highly significant (p < 0.001) differences between the vehicle-treated and the low dose (2 µg/kg) exenatide vs. the 10 and 50 µg/kg exenatide groups, while there was no significant difference between the 10 and 50 µg/kg, nor the vehicle-treated and the low dose groups (Fig. 1).

3.2. Effects of LH-hM4D(Gi) activation by CNO on the food intake of rats

Subcutaneously applied CNO resulted in a significant food intake decrease only in the case of the AAV5 serotype, and only in the larger doses (300 and 1000 µg/kg, Fig. 2/B), while in the case of the AAV9 serotype, the effects of CNO did not reach statistical significance (Fig. 2/C). In detail, factorial ANOVA on DOSE and SEROTYPE, as the two main factors, yielded a significant effect for the SEROTYPE factor (F_10,48 = 2.784, p < 0.01), however nor the DOSE nor the interaction between SEROTYPE and DOSE were statistically significant. The subsequent one-way ANOVA tests showed significant differences in the case of AAV5, at 4 out of the 5 time points (30 min: F_3,11 = 12.451, p < 0.001; 1h: F_3,11 = 7.362, p < 0.01; 2h: F_3,11 = 6.134, p < 0.05; 3h: F_3,11 = 2.617, p = 0.104; 8h: F_3,11 = 4.286, p < 0.05), but were not statistically significant in the case of SHAM and AAV9 tests, at any time points. Post-hoc Tukey HSD analyses revealed the largest differences at the 30-min time point, while subsequently these differences either vanished, or their significance decreased (Fig. 2/B).

3.3. Effects of s.c. DCZ stimulation on the food intake of LH-hM4D(Gi) rats

Subcutaneously applied DCZ resulted in a strong, sustained and highly significant food intake decrease in both the AAV5 and the AAV9 serotype transfected animals (Fig. 3/A-C). In detail, factorial ANOVA on the two key factors DOSE and SEROTYPE, showed significant effects for both (dose: F_27,71 = 2.314, p < 0.01; serotype: F_18,48 = 2.103, p < 0.05), nonetheless their interaction was not significant (p = 0.076). One-way ANOVA tests showed significant differences in the case of AAV5, at 8 out of the 9 food intake time points (30 min: F_3,12 = 6.858, p < 0.01; 1h: F_3,12 = 5.531, p < 0.05; 2h: F_3,12 = 4.783, p < 0.05; 3h: F_3,12 = 5.311, p < 0.05; 4h: F_3,12 = 4.494, p < 0.05; 5h: F_3,12 = 3.597, p < 0.05; 6h: F_3,12 = 3.972, p < 0.05; 7h: F_3,12 = 3.187, p = 0.063; 8h: F_3,12 = 4.364, p < 0.05). Similarly, we found a significant DOSE main effect, in the case of AAV9, at all time points (30 min: F_3,12 = 20.813, p < 0.001; 1h: F_3,12 = 11.936, p < 0.001; 2h: F_3,12 = 10.240, p < 0.01; 3h: F_3,12 = 11.324, p < 0.001; 4h: F_3,12 = 7.531, p < 0.01; 5h: F_3,12 = 9.259, p < 0.01; 6h: F_3,12 = 5.380, p < 0.05; 7h: F_3,12 = 7.862, p < 0.01; 8h: F_3,12 = 9.565, p < 0.01), but there were no statistically significant effects in the SHAM group, at any time points. In AAV5 animals, post-hoc Tukey HSD analyses revealed the largest differences at the 30-min time point, while subsequently these differences alleviated, but overall the significant food intake effect of DCZ remained until the end of the 8h recording period (Fig. 3/B). In the case of the AAV9 serotype transfection, the effects were even more robust (Fig. 3/C), all the post-hoc Tukey tests showed p < 0.001-level significance at 30 minutes, moreover the effects of all doses of DCZ remained significant until the end of the 8h recording period.

3.4. Effects of oral DCZ stimulation on the food intake of LH-hM4D(Gi) rats

Orally (per os, p.o.) applied DCZ resulted in much weaker effects compared to s.c. application (Fig. 3/D-F), even against the fact that we applied one logarithmic step higher oral doses of DCZ (oral: 100-300-1000 µg/kg vs. s.c. 30-100-300 µg/kg). In detail, factorial ANOVA on DOSE and SEROTYPE×DOSE were significant (DOSE: F_27,62 = 2.804, p < 0.001; SEROTYPE×DOSE: F_54,112 = 1.692, p < 0.05). One-way ANOVA tests showed a significant food intake decrease in the case of AAV9, at the first 5 food intake measurement time points (30 min: F_3,10 = 10.241, p < 0.01; 1h: F_3,10 = 10.768, p < 0.01; 2h: F_3,10 = 4.525, p < 0.05; 3h: F_3,10 = 4.116, p < 0.05; 4h: F_3,10 = 3.945, p < 0.05), but DCZ was not effective in either the AAV5 or the SHAM groups at any time points. In AAV9 animals, post-hoc Tukey HSD analyses revealed the largest differences at the 30-min time point, while subsequently these differences disappeared from the 5h measurement time point and remained non-significant until the end of the 8h recording period.

3.5. The timespan of the effect of DCZ

To further extend the functionality of our current rat food intake paradigm model, we tested the ability of the model to assess the timespan of effects of a selected actuator molecule. We chose DCZ with s.c. application in AAV5 LH-hM4D(Gi) rats, to test how long the food intake decreasing effect of DCZ last. We used the same food intake paradigm with a little modification: we applied different DCZ pre-treatment times (30 min, 1h, 3h, 16h before the reinstatement of food) and measured the 30-min food intake as the primary endpoint. We found that the pre-treatment time had a significant effect on the 30-min food intake (ANOVA: F_3,12 = 9.078, p < 0.01), and consequently, post-hoc Tukey HSD analyses revealed that the 0.5h and the 1h pre-treatment times resulted in significantly reduced food intake compared to the effect caused by 16h DCZ pre-treatment (30min.: 1.9 ± 0.4 g; 1h: 2.3 ± 0.4 g; vs. 16h: 6.1 ± 0.4 g; p < 0.01 in both comparisons). Food intake at the 3h pre-treatment point did not significantly differ from any other time points (food intake at 3h: 3.9 ± 1.0 g, N.S.), as it was numerically between the effect of the shorter (1-2h) and the longer (16h) pre-treatment times.

3.6. Histology

In the brains of all AAV-injected animals, an intensive expression of mCherry reporter protein was detected using confocal fluorescent microscopy (Fig. 4). The LH unambiguously expressed the reporter protein and therefore, confirmed the successful targeting and the sufficient DREADD receptor expression, regardless of the viral serotype. However, a somewhat larger area tended to be infected by AAV9 than AAV5. Furthermore, in both groups, the mCherry-labeled area appeared to confine adjacent brain regions outside the hypothalamus. Thus, in both groups, the zona incerta also showed the expression of the transgene, while in rats injected with AAV9, smaller parts of the ventral thalamus were also labeled with mCherry.

The overarching goal of our study was to develop a high translational value rodent behavioral assay, that supports the development of new chemogenetic systems in various CNS indications, offering a simple but robust in vivo efficacy readout to straightforwardly prove the effectiveness of any new chemogenetic constructs. Additionally, we were aiming to utilize the same assay to provide a simple way to estimate the basic pharmacokinetic properties of systemically applied actuator drugs. Finally, we also aimed to provide the first human translatable in vivo POC validating the therapeutic potential of chemogenetic treatments in obesity indication.

We utilized exenatide as a food-intake-reducing reference drug to validate the human relevance of our model and to prove the translational power and predictability of our results. Exenatide is presumed to primarily promote weight loss in humans by reducing food consumption [21, 22], via acting as a mimetic of glucagon-like peptide 1 (GLP-1). Accordingly, the reported exenatide-induced robust food-intake-reducing effects (Fig. 1) validated the current preclinical model and proved its high translational value and human relevance.

We hereby demonstrated that several methodological parameters could affect the overall biological effectiveness of chemogenetic methods, including the serotype of the applied AAV viruses, the characteristics of the actuator drug, including its brain penetration, pharmacokinetic/pharmacodynamic properties, dose, and application route (i.e., oral or parenteral). From the currently investigated several parameters, we identified the AAV9 serotype, and DCZ with s.c. application, as the most effective combination of factors, producing a robust food intake decrease in rats comparable to the effects of exenatide (Fig. 3/C). Nevertheless, the AAV5 serotype, DCZ with oral application, and CNO were also shown to be significantly effective, but with a slightly weaker efficacy (Fig. 2, Fig. 3). We also noticed that with all applied combinations of experimental parameters, the first 30-min food intake (after re-feeding) provided the greatest effect, therefore this seems to be the most ideal experimental endpoint. We also demonstrated that with different actuator pre-treatment times the timespan of the effect of any actuator molecule can be conveniently assessed, and the acquired data can be used as an indirect pharmacokinetic estimation, that could be valuable when optimizing the effectiveness of newly developed actuator-receptor combinations, without the need of separate pharmacokinetic/pharmacodynamic studies. Finally, in terms of AAV serotype effectiveness, AAV9 seemed to provide a more robust hypothalamic (LH) and possibly some scattered extra-hypothalamic expression (Fig. 4) that seemed to correlate with its somewhat higher food intake decreasing potential. The AAV5 serotype, on the other hand, showed a slightly smaller, more focused LH infection, which, in turn, can possibly explain its marginally less efficacy. If one would like to further explore the food intake or obesity-related effects of selective LH chemogenetic inhibition, it would be a logical next step to try to investigate the effectiveness of other serotypes (e.g., AAV2 or AAV8), and perhaps to try to reduce the affected brain area size by decreasing the applied virus volume (< 1000 nL) and/or the virus titer (< 10¹² vg/ml).

Taken these methodological considerations together, we have successfully developed, optimized, and validated an in vivo preclinical experimental model that is suitable for supporting various chemogenetic drug discovery projects, by providing a robust and reliable, but, at the same time, inexpensive and simple tool that can serve as a “first in vivo” model for characterizing the effectiveness of any newly developed CNS-targeted chemogenetic treatments. Secondarily, we are presenting hereby the first human translatable evidence that CNS-targeted neuron-specific, however, subtype general chemogenetic stimulation can be similarly effective in reducing food intake than the currently most effective GLP-1 agonist pharmacological therapy available on the market in obesity indication [23]. Since food intake is a primary etiological biomarker for obesity and weight loss, the current study can be considered as the first indirect POC, that verifies the feasibility of the chemogenetic approach inhibiting LH neurons, as a potential new human treatment opportunity in obesity. Subsequent studies are warranted to provide a direct POC via using diet-induced obesity models, and weight loss, as the primary endpoint, and possibly investigating other feasible target areas of the hypothalamus that could similarly induce food intake decrease and weight loss, like the arcuate nucleus.

As a third major outcome of this study, we provided proof – in accordance with previous preclinical reports [7, 8, 24, 25] - showing that DCZ is a more ideal chemogenetic actuator molecule for stimulating muscarinic DREADDs, compared to CNO, therefore we also do recommend a transition from the still widespread use of CNO to DCZ in all CNS-targeted preclinical studies. We agree with the previous reports, that the higher efficacy and faster pharmacodynamic effect of DCZ can be explained by its better brain penetrance, better selectivity for the M3/M4 DREADD receptors, and its better metabolic stability, meaning that the effects of DCZ are not compromised by its metabolites with divergent pharmacological properties like in the case of CNO [5, 26].

Furthermore, the current results will strengthen the hypothesis that emerging innovative “genetic neuroengineering” techniques, like G-protein coupled DREADD chemogenetics, can be very promising potential future human therapeutic strategies, in general. This assumption is mostly based on the idea that chemogenetics can provide superior spatial targeting and selectivity, compared to conventional CNS pharmacotherapy methods. The only currently approved CNS therapeutic option, with similarly high spatial neuromodulatory ability is DBS, however, DBS needs chronically implanted devices that are permanently left in the brain, posing a considerable risk factor that might negatively influence the success of the otherwise highly effective method in the clinic [3].

To conclude our current report, the impact of the present research can be numerous. As it was described above, first and foremost, it provides a high translational value preclinical evidence for the feasibility of LH chemogenetic stimulation as a tool for CNS-targeted chemogenetic drug discovery, and simultaneously, it delivers the first translatable indirect in vivo POC for the therapeutic feasibility of chemogenetics in obesity indication. Based on our current results, we also support the hypothesis that future genetic neuroengineering treatments can offer highly effective alternative therapeutic options to both DBS and conventional pharmacotherapy in various CNS conditions, providing better spatial selectivity than pharmacotherapy, and offering a more tolerable approach for DBS-eligible patients. To aid this development, future studies must further explore the effectiveness (e.g., length of effect, viral serotype, titer, and dose dependence, etc.) and also the side-effect profile (e.g., potential neurotoxicity) of AAV-delivered CNS-targeted chemogenetic approaches in relevant preclinical models of obesity.

Acknowledgements

The authors would like to thank Nikoletta Balog for her assistance in experiments, and Mrs. Marika Antalné for animal care.

Statements and Declarations

Data availability statement: The authors declare that they make all experimental data available upon request to the corresponding author.

Funding: This study was supported by the National Research, Development, and Innovation Office of the Hungarian Government (grant no. ‘K 143816’, IH). The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Competing interests: The authors declare no conflict of interest.

Ethics approval statement: The experiments were approved by the Animal Welfare Committee of the University of Pécs, and the National Scientific Ethical Committee on Animal Experimentation (ÁTET) at the Ministry of Agriculture (ethical license no.: BA02/2000-40/2021). All procedures fully complied with Decree No. 40/2013. (II. 14.) of the Hungarian Government and the EU Directive 2010/63/EU on the Protection of animals used for scientific purposes.

Author Contributions: Conceptualization: PK, TK, ZKB, ZP, IH; Methodology: PK, ZKB, IH; Investigation: ZKB, LVN, AB; Data curation: PK, ZKB, AB; Formal analysis: PK; Project administration: PK, TK; Funding acquisition: ZP; Resources: ZP, IH; Supervision: IH; Writing – original draft: PK; Writing – review & editing: TK, ZKB, IH; Visualization: PK.

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published 17 May, 2024

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Chemogenetic inhibition of the lateral hypothalamus in rats: A simple but robust tool with high translational relevance to facilitate the development of central nervous system-targeted gene therapies

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Abstract

Aims

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Intracranial injection of AAVs

2.3. Experimental design

2.4. Food intake measurements

2.5. Histology

2.6. Data analysis

3. Results

3.1. Effect of exenatide on the food intake of control rats

3.2. Effects of LH-hM4D(Gi) activation by CNO on the food intake of rats

3.3. Effects of s.c. DCZ stimulation on the food intake of LH-hM4D(Gi) rats

3.4. Effects of oral DCZ stimulation on the food intake of LH-hM4D(Gi) rats

3.5. The timespan of the effect of DCZ

3.6. Histology

4. Discussion

Declarations

References

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