# 5-HT6 Receptor Inverse Agonists Modulate Diabetic Neuropathic Pain in Female Rats: Evidence for 5-HT6 Receptor Constitutive Activity

Nazarine Mokhtar, Marcin Drop, Lauriane Delay, Lusliany Josefina Rondón, Lorine Costerousse, Eric Chapuy, Laetitia Prival, Frédéric Lamaty, Xavier Bantreil, Vittorio Canale, et al.

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## To cite this version:

Nazarine Mokhtar, Marcin Drop, Lauriane Delay, Lusliany Josefina Rondón, Lorine Costerousse, et al. 5-HT6 Receptor Inverse Agonists Modulate Diabetic Neuropathic Pain in Female Rats: Evidence for 5-HT6 Receptor Constitutive Activity. Journal of Neurochemistry, 2025, 169 (8), pp.e70179. 10.1111/jnc.70179. hal-05390188

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Distributed under a Creative Commons Attribution 4.0 International License
5-HT₆ Receptor Inverse Agonists Modulate Diabetic Neuropathic Pain in Female Rats: Evidence for 5-HT₆ Receptor Constitutive Activity

Nazarine Mokhtar<fnref n="1" />, Marcin Drop<fnref n="2" />,<fnref n="3" />, Lauriane Delay<fnref n="1" />, Lusliany Josefina Rondón<fnref n="1" />,<fnref n="4" />,<fnref n="5" />, Lorine Costerousse<fnref n="1" />, Eric Chapuy<fnref n="1" />, Laetitia Prival<fnref n="1" />, Frédéric Lamaty<fnref n="2" />, Xavier Bantreil<fnref n="2" />, Vittorio Canale<fnref n="3" />, Philippe Marin<fnref n="6" />, Pawel Zajdel<fnref n="3" />, Stéphane Doly<fnref n="1" />, Christine Courteix<fnref n="1" />*

*Correspondence : Christine Courteix, Université Clermont Auvergne, INSERM, NEURO-DOL, F-63000 Clermont-Ferrand, France. Email : christine.courteix@uca.fr

<footnote n="1">¹Université Clermont Auvergne, INSERM, NEURO-DOL, F-63000 Clermont-Ferrand, France</footnote>

<footnote n="2">²Institut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, F-34095 Montpellier, France</footnote>

<footnote n="3">³Faculty of Pharmacy, Jagiellonian University Medical College, 30-688 Kraków, Poland</footnote>

<footnote n="4">⁴Molecular Physiology Laboratory, Center for Biophysics and Biochemistry, Venezuelan Institute of Scientific Research, Caracas, Venezuela</footnote>

<footnote n="5">⁵Biochemistry Department, School of Nutrition and Dietetics, Central University of Venezuela, Caracas, Venezuela</footnote>

<footnote n="6">⁶Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, F-34094 Montpellier, France</footnote>
Graphical abstract

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Painful neuropathy is a common complication of diabetes. We investigated the role of 5-HT₆R (serotonin type 6 receptor)/mTOR (mechanistic Target Of Rapamycin) signaling in neuropathic pain and cognitive comorbidity in streptozocin (STZ)-induced type 1 diabetes (D) in female rats. Mechanical hyperalgesia was attenuated by systemic injection of 5-HT₆R inverse agonists. Further, full but not partial 5-HT₆R inverse agonists alleviated cognitive deficits. Intrathecal administration of the mTOR inhibitor rapamycin or a cell-penetrating peptide that disrupts the interaction between the 5-HT₆R and mTOR also reduced pain and cognitive comorbidity. Together with previous data obtained in STZ-D male rats and in traumatic and toxic models of neuropathic pain, these results suggest that the analgesic and pro-cognitive effects of 5-HT₆R inverse agonists are sex-specific and dependent on the etiology of neuropathic pain, underscoring the importance of personalized treatment in neuropathic pain.
**Abstract**

Painful neuropathy is one of the most common complications of diabetes. First-line therapeutic agents such as tricyclic antidepressants, dual serotonin/noradrenaline reuptake inhibitors and alpha2-delta ligands of calcium channels (i.e., gabapentinoids), are poorly effective. New strategies targeting the serotonin type 6 receptor (5-HT<sub>6</sub>R) and mechanistic Target Of Rapamycin (mTOR) signaling have recently emerged. Until a few years ago, preclinical studies of pain in rodents were more often carried out in males than in females, despite compelling evidence of sex-specific mechanisms in pain. Here, we investigated the role of 5-HT<sub>6</sub>R/mTOR signaling in neuropathic pain in streptozocin (STZ)-induced type 1 diabetes (T1D) in female rats. Mechanical hyperalgesia was attenuated in female diabetic (STZ-D) rats by systemic injection of 5-HT<sub>6</sub>R inverse agonists. Further, administration of full (PZ-1386, SB258585), but not partial (PZ-1179) 5-HT<sub>6</sub>R inverse agonists alleviated cognitive deficits in female STZ-D rats. Intrathecal administration of the mTOR inhibitor rapamycin or a cell-penetrating peptide that disrupts the physical interaction between the 5-HT<sub>6</sub>R and mTOR also reduced pain and cognitive comorbidity in females. Together with previous data obtained in STZ-D male rats and in spinal nerve ligation (SNL) and oxaliplatin (OXA) models of neuropathic pain, these results suggest that the analgesic and pro-cognitive effects of 5-HT<sub>6</sub>R inverse agonists are sex-specific and dependent on the etiology of neuropathic pain, highlighting the importance of personalizing treatment that considers the patient's sex, etiology of neuropathy and the presence or absence of comorbid cognitive symptoms.

**Keywords:** neuropathic pain; diabetes; 5-HT<sub>6</sub>R; constitutive activity; inverse agonist; mTOR; Streptozocin; sex dimorphism.

**Abbreviations:** 5-HT, serotonin; A.U.C., area under the curve; a.u., arbitrary unit; CIPN, chemotherapy-induced peripheral neuropathy; DRG, dorsal root ganglia; GFP, green fluorescent protein; I.U., international unit; i.p., intraperitoneal; i.t., intrathecal; KI, knock-in; mTOR, mechanistic Target Of Rapamycin; NOR, novel object recognition; OXA, oxaliplatin; RRID, Research Resource Identifier; s.c., subcutaneous; SNL, spinal nerve ligation; STZ-D, streptozocin induced diabetic; T1D, type 1 Diabetes.

**1 Introduction**

Neuropathic pain is one of the most recalcitrant types of chronic pain affecting 6.9-10% of the general population (Chenaf et al. 2018; Bouhassira et al. 2008). It arises from a lesion or damage of the nervous somatosensory system (Jensen et al. 2011) caused by trauma or surgery, nerve compression or infiltrating tumors, infection, diabetes, chemotherapy, toxics, stroke or neurodegenerative disease.
A significant proportion of chronic pain patients also suffer from depression, anxiety, or cognitive comorbidities (Radat et al. 2013), yielding major economic and social burden and affecting the patients' quality of life (Schaefer et al. 2014).

Painful diabetic neuropathy affects 6-34% of diabetic patients in Europe (Alleman et al. 2015). Painful manifestations include spontaneous pain such as burning, pins, electric shock-like pains and evoked pain as well as sensory loss. Diabetic neuropathy first affects the most distal extremities (feet and legs) then ascends to the trunk and occasionally to upper limbs (hands and arms), thus corresponding to a « socks and gloves » distribution. Intensive blood glucose control is the first intervention known to prevent or reduce the progression of neuropathy in diabetic patients. Tricyclic antidepressants, serotonin noradrenaline reuptake inhibitors and gabapentin are recommended as the first-line therapeutic agents in the management of neuropathic pain (Moisset et al. 2021) but with only a partial clinical benefit (Finnerup et al. 2015). Recently, combination therapies such as amitriptyline in conjunction with pregabalin, pregabalin with amitriptyline and duloxetine with pregabalin have shown unequivocal efficiency, opening new opportunities to replace inadequate pain management with combined treatments (Tesfaye et al. 2022). Selective serotonin reuptake inhibitors (SSRIs) have no place in this range of treatments despite the major role played by serotonin (5-HT) in pain modulation, notably through descending projections originating from the rostral ventromedial medulla in the brain stem and projecting onto the dorsal horn of the spinal cord. In the spinal cord, 5-HT exerts both pronociceptive and antinociceptive effects depending on the 5-HT receptor subtype engaged, including 5-HT₁A, 5-HT₂A, 5-HT₃, 5-HT₆ and 5-HT₇ receptors (Mokhtar et al. 2023a; Dupuis et al. 2017; El Khamlichi et al. 2022; Liu et al. 2020).

A challenge of pain research is to develop preclinically relevant models of neuropathic pain that reproduce comorbidities, such as depressive-like behaviors, anxiety or cognition. While pain and comorbidities exhibit sex dimorphism (Mogil 2020), the inclusion of females in preclinical pain studies has long been lacking (Ghazisaeidi et al. 2023; Sadler et al. 2022). However, the use of both sexes in pain research has recently tended to increase (Plumb et al. 2023) given the importance of better characterizing the pathophysiological mechanisms and demonstrating the efficiency of treatments in both sexes. This provided the impetus for the present study that specifically examines the mechanism underlying neuropathic pain and comorbidities caused by type 1 diabetes (T1D) in female rats.

Diabetic neuropathy induced in rats by a single injection of streptozocin (STZ), an antibiotic cytotoxic for pancreatic beta-cells, results in drastic hyperglycemia and all classical signs of T1D, including polydipsia, polyuria, polyphagia and cessation of weight gain (Mokhtar et al. 2023a; Courteix et al. 1993). STZ-induced diabetes is also known to induce spinal sensitization (Deng et al. 2021) and
morphological (axonal demyelination) and functional alterations affecting the nerve conduction velocity of peripheral nerve fibers (Deng *et al.* 2021) that appear after the onset of painful symptoms (Zhang *et al.* 2021).

In the dorsal horn of the spinal cord, where painful information reaches the central nervous system to be modulated and relayed to higher centers, we have previously shown that mechanistic Target Of Rapamycin (mTOR) signaling under the control of constitutively active 5-HT<sub>6</sub>R plays a major role in neuropathic pain and cognitive comorbidities in male rats showing diabetic (Mokhtar *et al.* 2023b), traumatic (spinal nerve ligation, SNL) and chemically (oxaliplatin, OXA)-induced neuropathy (Martin *et al.* 2020). This suggests that 5-HT<sub>6</sub>R inverse agonists, already under clinical evaluation for other indications (cognitive decline, schizophrenia) (Nirogi *et al.* 2023) could be promising therapies to alleviate neuropathic pain. Studies have revealed the existence of sex-related differences in pain or memory processes involving 5-HT. For instance, the stronger functional connectivity between the periaqueductal gray and brain regions involved in pain processing might contribute to the higher vulnerability of females to migraine (Gecse *et al.* 2021). In light of these observations, we examined the role of 5-HT<sub>6</sub>R/mTOR signaling in neuropathic pain of different etiologies and the associated comorbid symptoms in female rats.

## 2 Materials and Methods

### 2.1 Ethics

All the experiments described here on rodents were conducted in accordance with the ARRIVE guidelines (Kilkenny *et al.* 2010) and were reviewed and approved by the local Animal Experiment Ethics Committee of Auvergne (CEMEA) and by the French Ministry of Higher Education, Research and Innovation (authorizations n° 5175 (STZ rats), n° 17573 (SNL rats), n° 24498 (OXA rats), n° 22133 (5-HT<sub>6</sub>-GFP-KI mice)).

### 2.2 Animals and Models of Neuropathic Pain

Sprague-Dawley female and male rats (RRID: IMSR_CRL:400) (initial weight 150-200 g) were purchased from Janvier Labs (Le Genest-Saint-Isle, France) 1 week before starting the experiment. Rats were housed three (STZ-D rats) or four (SNL and OXA rats) per cage. 5-HT<sub>6</sub>-GFP knock-in mice (males and females) were described elsewhere (Deraredj Nadim *et al.* 2016).

Animals were housed under standard laboratory conditions with a 12/12 h light/dark cycle, stable temperature (22°C ± 1°C), controlled humidity (55% ± 10%) and were given food and water *ad libitum*. At the end of the experiments, animals not collected for biochemical studies were euthanized by progressive carbon dioxide (CO<sub>2</sub>) inhalation (10%-20%/min).
The STZ-induced diabetic neuropathic pain model (Courteix et al. 1993) was used as previously described (Mokhtar et al. 2023b). Rats received a single intraperitoneal (i.p.) injection of STZ (75mg·kg⁻¹, Cat. no. S0130, Sigma-Aldrich, France) dissolved in water for injections. Age-matched rats received an equal volume of water for injections (i.p.) and served as control animals. One week after STZ injection, diabetes was confirmed by blood glucose levels measured with the Accu-Chek Performa glucometer (maximal detection: 30mM; Roche, France). At this stage, only animals with blood glucose >14mM, were considered diabetics and received a subcutaneous (s.c.) injection of insulin (2 I.U./injection every other day, LANTUS, Sanofi-Aventis, France) to ensure a good clinical condition during the entire experiment (Handwerker and Arendt-Nielsen 2013).

The spinal nerve ligation (SNL) model consists of tightly ligating the left spinal nerve L5 with a 4-0 silk suture (Mersilk, Ethicon LLC Johnson & Johnson, Norderstedt, Germany) in rats under xylazine 2%/ ketamine (80mg/kg i.p., Virbac, Carros, France) anesthesia, as previously described (Kim and Chung 1992; Mokhtar et al. 2023b). The same surgery without ligation was performed in healthy rats to serve as control (sham) animals. Immediately after the surgery, animals were treated with a non-steroidal anti-inflammatory drug (Meloxicam 2 mg/kg s.c., one injection per day for 2 days, Boehringer Ingelheim, France). During post-operative monitoring, none of the animals showed any signs of disability, distress, sensory loss or motor deficit, and all appeared to be in good health.

The chemotherapy-induced peripheral neuropathy (CIPN) model was induced by a single i.p. injection of oxaliplatin (OXA, 6 mg/kg, Leancare, Ltd, UK) dissolved in a 5% glucose solution (Ling et al. 2007; Mokhtar et al. 2023b).

## 2.3 Behavioral Tests

### 2.3.1 Evaluation of Mechanical Hyperalgesia and Tactile Allodynia

While tactile allodynia, characterized by hypersensitivity to the application of mild mechanical stimulation, is developed by the large majority of SNL and OXA rats, it is present in only 50% of diabetic neuropathic rats (personal data). In STZ-induced diabetic rats, for reasons of reproducibility and to reduce the number of animals used, mechanical hyperalgesia induced by the application of increasing mechanical stimulation exerting important force, was used to assess neuropathic pain.

Thus, in STZ-D rats, mechanical hyperalgesia to paw pressure was measured using the Ugo Basile analgesia meter (Bioseb, Vitrolles, France). The test consists of applying an increasing mechanical force exerting a strong pressure on the dorsum of the rat's left hind paw until vocalization or a struggling response occurs (vocalization/struggle threshold expressed in arbitrary units, a.u.) (Randall and Selitto 1957). The maximum force applied is limited to 450a.u. to avoid tissue damage.
In SNL and OXA rats, tactile hypersensitivity was measured using the von Frey hair test according to the Up-Down method (Chaplan *et al.* 1994) and previously described (Mokhtar *et al.* 2023b; Martin *et al.* 2020). Each rat was individually placed in transparent Plexiglas boxes (25 x 20 x 25cm) positioned on a raised metal floor and allowed to accommodate for approximately 15min. Beginning with the 6 g hair, von Frey filaments (1.4-26g) were applied perpendicularly to the central plantar surface of the hind paw to cause a slight buckling against the paw and held for 6s. Withdrawal or licking of the paw was considered as a positive response; in this case, the next weakest filament was applied. In case of a negative response (no withdrawal or licking of the paw), the next stronger filament was applied. This paradigm was continued until four measurements had been obtained after an initial change of behavior, or until four consecutive negative responses or five consecutive positive responses had been obtained. The 50% threshold (expressed in grams, g) was calculated using Dixon's formulae (Dixon 1980). The maximum 50% threshold was set at 34g.

The percentage of pain reduction was calculated according to the following formula: [(Post-drug threshold-Pre-drug threshold) / (Pre-STZ threshold-Pre-drug threshold)]×100. To investigate the global effects of the drugs, the areas under the time-course curves (A.U.C.) of the changes in vocalization (paw pressure test) or withdrawal (von Frey hair test) threshold between 0 and 120 min were calculated using the trapezoidal rule (expressed in a.u. x min).

### 2.3.2 Evaluation of Cognition

The novel object recognition (NOR) test was used to evaluate episodic memory in SNL and STZ-D rats, as previously described (Ennaceur and Delacour 1988; Mokhtar *et al.* 2023b). One day before the test, each rat was individually habituated to the arena (1-m long, 1-m wide). On the day of testing, the rat had a 5-min individual familiarization session with two identical objects in low-light conditions (30lx). The rat was transferred to its original cage for a 5-min inter-trial interval. The rat was then reintroduced into the arena for a training session, with one of the original objects replaced by a new one. Exploratory behavior defined as licking or touching the object while sniffing with active vibrissae was recorded for 5 min. The objects were plastic or wooden toys (10-cm high, 7-cm long, 7-cm wide) and were cleaned with 70% ethanol between sessions. Presentation sequence and object location were randomly assigned to each rat. The delay in onset of neuropathy in OXA rats (3 days) was too short to induce cognitive deficits in this animal model. For this reason, the NOR test was not performed in OXA rats.

The NOR test was performed in healthy rats under ketamine-induced memory deficit as previously described (Białon *et al.* 2020). The day following the arena habituation, healthy rats were
injected with ketamine 30 min before the familiarization session. Then, the NOR test was performed
in the same conditions as in neuropathic rats.

NOR memory was expressed as a recognition index (D2 score), defined as the ratio of time spent exploring the novel object to time spent exploring the two objects.

## 2.4 Intrathecal Injection

Rats were anesthetized with isoflurane (4% induction, 2% maintenance) and held at the pelvic girdle in order to inject the drugs between lumbar vertebrae L5 and L6. A 50 µl Hamilton syringe with a 25-gage x 1-in. needle was used to deliver the drugs into the subarachnoid space. The success of the injection was confirmed by a rapid tail flick. The syringe was held in position for a few seconds after the injection to prevent solution reflux.

## 2.5 Chemicals

SB258585 hydrochloride (MW=523.8g.mol⁻¹, Bio-Techne Cat. no. 1961) previously described as a full 5-HT₆R inverse agonist and Rapamycin an mTOR inhibitor (MW=914.2g.mol⁻¹, Bio-technne Cat. no. 1292) were purchased from Bio-technne (Tocris Bioscience). Ketamine hydrochloride (Ketamine 1000) was purchased from Virbac (Carros, France).

The full 5-HT₆R inverse agonists PZ-1388 hydrochloride, 2-(3-fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]-N-(piperidin-4-yl)-1H-pyrrole-3-carboxamide hydrochloride (MW = 534.86 g.mol⁻¹) and PZ-1386 hydrochloride, 2-(4-fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]- N-(piperidin-4-yl)-1H-pyrrole-3-carboxamide (MW=534.86g.mol⁻¹) and the partial 5-HT₆R inverse agonist PZ-1179 hydrochloride, (R)-2-(4-fluorophenyl)-1-[(3-chlorophenyl)sulfonyl]-N-(pyrrolidine-3-yl)-1Hpyrrole-3-carboxamide hydrochloride (MW=521.33g.mol⁻¹) were synthesized as previously described (Martin *et al.* 2020; Drop *et al.* 2021b; Drop *et al.* 2021a).

Tat-VEPE ([NH₂]YGRKKRRQRRR-FFVTDSVEPVE[COOH], purity > 98%, MW=2711.1g.mol⁻¹) and Tat-SCA ([NH₂]YGRKKRRQRRR-TVNEK-VSCA[COOH], purity >98%, MW=2491.2g.mol⁻¹) peptides were synthesized by Thermo Fisher Scientific (France) as previously designed (Martin *et al.* 2020).

## 2.6 Western Blot Analysis

Spinal cord lysates were solubilized in lysis buffer RIPA (Santa Cruz Biotechnology, France), protease and phosphatase inhibitor mixture EDTA free (Roche cOmplete Cat. no. 4693132001) for 2 h at 4 °C. Samples were centrifuged at 11000rpm for 20min at 4°C. Solubilized proteins were mixed with Laemmli buffer (Bio-Rad Cat. no. 1610747), heated at 95°C for 5min. Equal amounts of protein (25µg) from each sample were resolved onto 10% acrylamide SDS-PAGE gels (BioRad Cat. no. 4568034, TGX
Stain-Free FastCastAcrylamide Solutions, 1.5h at 90V) in Tris-Glycine-SDS buffer. Proteins were transferred to a nitrocellulose membrane (BioRad Cat. no. 1704270, Trans-Blot Turbo RTA Mini Nitrocellulose Kit). Membranes were blocked for 1h in Tris-buffered saline containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) (Sigma-Aldrich Cat. no. A3912) at room temperature and immunoblotted with primary antibodies overnight at 4°C: anti-total mTOR (1:1000, Cell Signaling Technology Cat. no. 2983, RRID:AB_2105622), antiphospho-Ser<sup>2448</sup>-mTOR (1:1000, Cell Signaling Technology Cat. no. 2976, RRID:AB_490932), antitotal S6 (1:1000, Cell Signaling Technology Cat. no. 2217, RRID:AB_331355), antiphospho-Ser<sup>240/244</sup>-S6 (1:1000, Cell Signaling Technology Cat. no. 2215, RRID:AB_331682). After three washes, membranes were incubated with anti-rabbit HRP-conjugated secondary antibody (1:5000, Thermo Fisher Scientific Cat. no. 32460, RRID:AB_1185567) for 1.5h at room temperature. Immunoreactivity was detected with an enhanced chemiluminescence method using the ChemiDocMP Imaging System (Bio Rad, RRID:SCR_019037). Immunoreactive bands were quantified by densitometry using Image Lab 6.1 (RRID:SCR_014210).

## 2.7 Immunohistochemistry

Immunodetection of 5-HT<sub>6</sub>R was performed in healthy 5-HT<sub>6</sub>-GFP KI mice of both sexes. Animals were deeply anesthetized by i.p. injection of 0.1mL pentobarbital sodium (1/10 Euthasol vet., Dechra Veterinary Products, Montigny-Le-Bretonneux, France) and transcardially perfused with 4% paraformaldehyde (Sigma-Aldrich Cat. no. 47608) in 0.1M phosphate-buffered saline (PBS). Lumbar spinal cord enlargement was harvested and post-fixed for 24h in the same solution at 4°C, before being cryoprotected with 30% sucrose solution (Sigma-Aldrich Cat. no. 57903) in 0.1M PBS (Gibco PBS Tablets, Thermo Fisher Scientific Cat. no. 18912014) for 3 days at 4°C. All tissues were embedded in tissue freezing medium (General Data Healthcare, Thermo Fisher Scientific Cat. no. 15-183-13) and 30µm-thick sections were cut at -22°C using a cryotome (Microm HM450, RRID:SCR_015959). The sections were permeabilized and saturated with 0.2% Triton X-100 (VWR Chemicals Cat. no. 28817-295), 5% BSA (Sigma-Aldrich Cat. no. A3912) in 0.1M PBS for 1h and incubated overnight at room temperature and under agitation with primary antibody: anti-GFP (1:500, Thermo Fisher Scientific Cat. no. A10262, RRID:AB_2534023) diluted in the same solution. After washing, the sections were incubated for 1h with Alexa Fluor 488 conjugated antibody (1:1000, Thermo Fisher Scientific Cat. no. A32723, RRID:AB_2633275) in the same solution. Immunofluorescence staining was observed using a Zeiss LSM 880 with Airyscan Confocal Laser Scanning Microscope (RRID:SCR_020925).

## 2.8 Experimental Design

Baseline mechanical thresholds were measured before and 21 days after STZ injection. Similarly, baseline tactile thresholds were determined before SNL surgery or OXA injection, then 14
days or 3 days after, respectively. At those times, STZ-D, SNL, and OXA rats were considered hyperalgesic (STZ-D rats) or allodynic (SNL- and OXA-rats) if their thresholds were reduced to at least 25%, compared with thresholds measured before the induction of the neuropathy. Animals that do not develop diabetes or hyperalgesia (STZ-D rats) or allodynia (SNL and OXA rats) were excluded from the study and euthanized by progressive CO₂ inhalation (10%-20% /min).

A total of 472 rats were used: 251 rats were injected with STZ, 34 rats served as healthy controls; 100 rats were submitted to spinal nerve ligation and 27 rats received oxaliplatin. Among the 251 rats injected with STZ, two died, 227 developed hyperglycemia, 203/227 developed mechanical hyperalgesia and five were excluded. Among the 100 rats submitted to surgery (SNL), 12 died during anesthesia, 67 among the 82 still alive developed tactile allodynia and seven rats were excluded. Among the 24 rats receiving oxaliplatin, 21 developed tactile allodynia. Sixty healthy rats were used in the NOR test under ketamine-induced memory deficit.

Among the animals excluded, three STZ-rats were removed from the analysis due to incorrect intrathecal injections, as confirmed by post-procedural evaluation. One SNL-rat was excluded from the von Frey hair procedure due to abnormal position (slight eversion) of the ligated paw and an additional eight rats (2 STZ and 6 SNL) were excluded for not meeting the predefined response criteria for the novel object recognition test. Specifically, animals were excluded if their total time exploring both objects during the familiarization phase was less than 15s, indicating insufficient engagement with the task. Thus, no statistical tests for outliers were performed.

## 2.9 Drug Treatment

For each experimental series, hyperalgesic/allodynic rats were randomly assigned to treatment groups. Pain thresholds were measured before and after drug injection every 30min for 180min to follow the effects of the treatments. SB258585, PZ-1388, PZ-1386, and PZ-1179 were injected intraperitoneally (i.p., 5mL/kg). Rapamycin and Tat-VEPE peptide were administered intrathecally (i.t., 10µL/rat).

For experiments exploring the effect of drugs on cognition in neuropathic animals, drugs were administered with a delay coinciding with the maximal anti-hyperalgesic or anti-allodynic effect of the drug.

For the NOR test under ketamine-induced memory deficit, ketamine (20 mg/kg i.p.) was administered 30 min before the familiarization trial, and PZ-1386 or SB258585 was delivered 30min before ketamine injection.
All in vivo experiments were performed blind using a parallel group design. Treatments were anonymized and arbitrarily allocated to rats. Block randomization has been created depending on the experiment and the number of treatments to create homogeneous groups and eliminate selection bias. Different animals were used in each experiment. The number of animals used for the study was determined on previous studies using similar procedures.

## 2.10 Statistical Analysis

GraphPad Prism software (version 10) was used to analyse the data. The Gaussian distribution of the values was assessed using the Shapiro-Wilk test. Biochemical and behavioral data were analysed either by one-way ANOVA followed by Tukey's post-hoc test or by two-way ANOVA followed by Dunnett's post-hoc test, or by an unpaired t-test. Results were expressed as mean ± standard error of the mean (SEM); statistical significance was set at 5% (*p*<0.05).

## 3. Results

### 3.1 Diabetes, SNL and OXA Injection Induced Mechanical Hyperalgesia and Tactile Allodynia in Female Rats

Ninety-one percent (229/251) of STZ-injected rats developed severe hyperglycemia (> 30 mM). STZ-D rats also presented disturbances of clinical signs such as polydipsia, polyuria, polyphagia, and a slowing of weight gain (194.3±0.99g before STZ and 224.1±2.4g, 3 weeks after-STZ). Insulin injections (every other day) maintained diabetic rats in a good health status, even though two diabetic rats died. Eighty-nine percent (203/227) of hyperglycemic rats showed a significant reduction in vocalization threshold to paw pressure 21 days after the induction of diabetes (198.6±18.5a.u. 3 weeks after STZ vs. 309.2±17.2a.u. before STZ), suggesting mechanical hyperalgesia. None of the compounds tested (SB258585, PZ-1388, PZ-1386, PZ-1179, Rapamycin, Tat-VEPE) reduced hyperglycemia.

Eighty-one percent (67/82) of SNL rats developed tactile allodynia to plantar stimulation with von Frey monofilaments 14 days after surgery (30.43 ± 5.2 vs. 12.59 ± 7.12 g after surgery). Anesthesia during surgery caused 18% (18/100) of deaths.

A single OXA injection induced tactile allodynia in 24/27 (88%) of injected rats.

### 3.2 5-HT<sub>6</sub>R Inverse Agonists Attenuate Mechanical Hyperalgesia in STZ-Induced Diabetic Female Rats

The effect of 5-HT<sub>6</sub>R inverse agonists (SB258585, PZ-1388, PZ-1386 and PZ-1179) has been previously characterized in male (Mokhtar *et al.* 2023b; Martin *et al.* 2020) but not in female rats. Injection of each drug (25µmol/kg, i.p., each) significantly attenuated diabetes-induced mechanical
hyperalgesia, 60-90 (SB258585), 30-90 (PZ-1388 and PZ-1386) and 30-60 (PZ-1179) min after injection (Figure 1a-d, Table 1). The time course of the anti-hyperalgesic effect of the drugs showed a maximal effect at 90min for SB258585, 60min for PZ-1388, PZ-1386 and PZ-1179 and corresponded to a pain reduction of 39%±7%, 59%±11%, 83%±21% and 50%±16%, respectively. The overall effect of the drugs, assessed by calculating the area under the time curve (A.U.C., 0-120min) showed that the vocalization thresholds of all compounds were significantly higher than that of the STZ-D vehicle-treated group (Figure 1e, Table 1).

We then investigated whether these analgesic effects measured in STZ-D female rats could be extended to toxic and traumatic neuropathic pain models. PZ-1386 and PZ-1179 administration (25µmol/kg i.p. each) reduced tactile hypersensitivity evoked by von Frey hair stimulation in OXA and SNL female rats with a maximal effect observed at 60 min post-injection. Pain reduction varied from 51%±14% to 69%±14% for PZ-1386 in OXA and SNL rats respectively and from 48%±23% to 61%±14% for PZ-1179 in OXA and SNL rats respectively (Figure S1a,b, Table 1).

### 3.3 Rapamycin and Tat-VEPE Attenuate Hyperalgesia-Induced by Diabetes in Female Rats

Pharmacological inhibition of mTOR activity or disrupting the physical interaction between the C-terminal domain of the 5-HT<sub>6</sub>R and mTOR, which prevents receptor-operated mTOR activation (Meffre et al. 2012), was shown to reduce mechanical hyperalgesia/tactile allodynia caused by diabetic, traumatic and toxic neuropathic pain in male rats (Mokhtar et al. 2023b; Martin et al. 2020; Drop et al. 2021b). Intrathecal administration of rapamycin to female STZ-D rats (0.3, 3 and 10nmol/rat) increased the vocalization threshold at 30-120min after injection (Figure 2a, Table 1). The maximal effect observed at 90min (266±16.0a.u.) in animals injected with 10nmol rapamycin corresponded to a complete reversal of mechanical hyperalgesia (80%±14% of pain reduction). No dose-dependent effect of rapamycin was found with the A.U.C., but a significant increase in vocalization thresholds measured between 0 and 120min was only found for the 10nmol-treated group (Figure 2b, Table 1).

Administration of Tat-VEPE (300ng/rat i.t.) but not a control mutant peptide not capable of preventing 5-HT<sub>6</sub>R-mTOR interaction (Tat-SCA, 300ng/rat i.t.), significantly reduced mechanical hyperalgesia 30-90min after injection in painful STZ-D female rats (Figure 3a, Table 1). The maximal anti-hyperalgesic effect (308±18.8a.u.) was measured 60 min after injection, resulting in a 123%± 2% reversal of mechanical hyperalgesia. The overall anti-hyperalgesic effect of Tat-VEPE, assessed by the A.U.C. of threshold variations, confirmed its analgesic effect, compared with the Tat-SCA peptide or with the vehicle-treated groups (Figure 3b, Table 1).
### 3.4 Effect of 5-HT<sub>6</sub>R Ligands, Rapamycin and Tat-VEPE on Comorbid Cognitive Symptoms

Impaired cognitive functions and poor cognitive performance were consistently reported in patients with T1D mellitus (T1DM) (Li et al. 2017). A cross-sectional, observational, case-control study also showed reduced cognitive scores in T1DM patients with painful and painless diabetic peripheral neuropathy (Croosu et al. 2023). Likewise, working memory deficits and impaired hippocampal synaptic plasticity have been reported in STZ-D rats (Sasaki-Hamada et al. 2021). In models of traumatic neuropathic pain, cognitive impairment has been shown using different paradigms such as the NOR and social interaction tests. Furthermore, 5-HT<sub>6</sub>R inverse agonists reduce cognitive impairment in SNL male rats (Martin et al. 2020). We therefore investigated whether these compounds, rapamycin and Tat-VEPE peptide would be effective in improving cognitive deficits associated with chronic pain in hyperalgesic STZ-D female rats in the NOR task. STZ-D female rats treated with vehicle failed to distinguish a novel object from a familiar one (Figure 4a,b, Table 1). Systemic (i.p.) administration of PZ-1386 (25µmol/kg) and SB258585 (25µmol/kg) but not PZ-1179 (25µmol/kg) restored episodic memory deficit in STZ-D female rats (Figure 4a, Table 1). Similarly, intrathecal injection of rapamycin (10nmol/rat) or Tat-VEPE (300ng/rat) increased novelty discrimination in STZ-D female rats (Figure 4b, Table 1).

We next examined whether the rescue of episodic memory deficit induced by PZ-1386 or SB258585 administration was due to the improvement of neuropathic pain or to its own pro-cognitive action. To address this issue, we tested the pro-cognitive effect of the PZ-1386 and SB258585 two compounds in healthy, pain-free rats exhibiting episodic memory deficit induced by administration of ketamine (20mg/kg, i.p.). Intraperitoneal administration of PZ-1386 (25µmol/kg) did not restore novelty discrimination in the NOR test (Figure S2, Table 1) whereas SB258585 (5 µmol/kg) abolished it in ketamine-treated female rats. These results indicate that the pro-cognitive effect of PZ-1386 in diabetic neuropathic rats depends on its anti-hyperalgesic effect mediated by suppression of constitutive activity of spinal 5-HT<sub>6</sub>R.

We then investigated whether the lack of effect of PZ-1179 on cognitive deficit in STZ-D females, that was previously reported in STZ-D male rats (Mokhtar et al. 2023b) is specific of neuropathic pain of metabolic etiology. To address this issue, the pro-cognitive effect of PZ-1179 was evaluated in traumatic neuropathic pain induced by SNL in female and male rats. In this experiment, PZ-1386 served as a positive control since it has previously been shown to ameliorate cognitive comorbidity in STZ-D male rats (Mokhtar et al. 2023b) and in the current study, in STZ-D female rats. Systemic administration of PZ-1179 (25µmol/kg) and PZ-1386 (25µmol/kg) to SNL female rats
improved novelty discrimination (Figure S3a, Table 1), whereas only PZ-1386, but not PZ-1179, induced
a procognitive effect in the NOR test in male rats (Figure S3b, Table 1).

## 3.5 Lack of Effect of STZ and of 5-HT<sub>6</sub>R Inverse Agonists, Rapamycin and Tat-VEPE on mTOR Signaling in the Dorsal Spinal Cord

No increase in mTOR activity has been observed in the dorsal horn spinal cord of diabetic female rats (Figure 5, Table 1) as well as diabetic male rats. Consequently, the mTOR activity level in STZ-D female rats, as assessed by mTOR phosphorylation on Ser<sup>2448</sup> and S6 phosphorylation on Ser<sup>240/244</sup> was not affected by peripheral administration of PZ-1386 or PZ-1179 nor by intrathecal administration of Rapamycin or Tat-VEPE (Figure 5).

## 3.6 Absence of Sex-Related Dimorphism in the Number of 5-HT<sub>6</sub>R-Positive Cilia in the Dorsal Spinal Cord of 5-HT<sub>6</sub>-GFP KI Mice

To exclude that the differential analgesic effect of 5-HT<sub>6</sub>R inverse agonists in female and male rats (i.e., greater maximal effect of SB258585, PZ-1388 and PZ-1179 in male rats) is due to a differential expression of 5-HT<sub>6</sub>R in primary cilia of dorsal spinal cord neurons in males and females, we quantified 5-HT<sub>6</sub>R-positive cilia (visualized with a GFP antibody) in KI mice expressing a GFP-tagged version of the 5-HT<sub>6</sub>R (5-HT<sub>6</sub>-GFP KI mice). No significant difference in neurons encompassing 5-HT<sub>6</sub>R-positive cilia was found between female and male rats (16.35±0.74 and 16.89±0.73 positive cilia/0.125mm², respectively) (Figure 6, Table 1).

## 4 Discussion

In the present study, we investigated the anti-hyperalgesic effect of full and partial 5-HT<sub>6</sub>R inverse agonists in painful neuropathic diabetic female rats. The STZ-D rat is a preclinical model commonly used for diabetic neuropathic pain in both males and females (Mokhtar et al. 2023a). For comparison purposes, we applied exactly the same housing and health care conditions as those previously published in males (Mokhtar et al. 2023b) but it cannot be ruled out experimental differences between the present study and the previous one and any comparison must be treated with caution. In these experimental conditions, 89% of STZ-D rats developed mechanical hyperalgesia. The baseline thresholds and severity of mechanical hyperalgesia observed in female rats 21 days after diabetes induction was similar to those previously reported in male rats (Mokhtar et al. 2023b). Cognitive alteration, currently observed in diabetic patients (Croosu et al. 2023) and already reported in diabetic male rats (Mokhtar et al. 2023b; Sasaki-Hamada et al. 2021) was also observed in the present study in female diabetic rats. The D2 score observed in healthy female rats was similar to that previously reported in healthy male rats (Mokhtar et al. 2023b). Likewise, the magnitude of cognitive
deficits 21 days after STZ injection were similar in STZ-D females and males (Mokhtar *et al.* 2023b). Reminiscent of the analgesic effect of 5-HT<sub>6</sub>R inverse agonists previously observed in male rats in STZ-D, SNL and OXA models (Mokhtar *et al.* 2023b; Martin *et al.* 2020), these compounds attenuated mechanical hyperalgesia and tactile hypersensitivity induced by neuropathic pain in female STZ-D, SNL and OXA rats. Additionally, the full (SB258585, PZ-1386) but not partial (PZ-1179) 5-HT<sub>6</sub>R inverse agonists also alleviated cognitive deficits in STZ-D female rats.

Almost exclusively located within the central nervous system and negligibly in the peripheral nervous system (Delay *et al.* 2022), the 5-HT<sub>6</sub>R was shown to display constitutive activity in neuropathic pain conditions of traumatic (SNL), diabetic (STZ) or toxic (OXA) etiology (Mokhtar *et al.* 2023b; Martin *et al.* 2020; Drop *et al.* 2021b). In the SNL model, the constitutive activity of spinal 5-HT<sub>6</sub>R is responsible for an increase in mTOR activity, which underlies the associated neuropathic pain. Correspondingly, administration of SB258585 or PZ-1388 and its two derivatives, PZ-1386 and PZ-1179, which behave as full and partial 5-HT<sub>6</sub>R inverse agonists (Martin *et al.* 2020; Drop *et al.* 2021b; Drop *et al.* 2021a), respectively, decreases neuropathic pain in male rats, including STZ-D male rats. Likewise, in STZ-D female rats, all the inverse agonists tested alleviated mechanical hyperalgesia, but with different efficacy and duration of effect. PZ-1388 and PZ-1386 increased vocalization thresholds with a similar magnitude, leading to an 59%-83% improvement in pain and had a similar duration of effect, of 90 min, whereas SB258585 and PZ-1179 were a bit less efficient, with an effect corresponding to a 39%-50% pain reduction (Table S1). Furthermore, PZ-1179 had a shorter analgesic effect (60 min). Despite the different potency to reduce AMPc production of the carboxamide-derived 5-HT<sub>6</sub>R inverse agonists (i.e., IC<sub>50</sub> values of 164nM for PZ-1188 and PZ-1386 and of 265nM for PZ-1179) (Mokhtar *et al.* 2023b), the global effect of PZ-1388 and PZ-1386, measured by the A.U.C., was not significantly different from that of PZ-1179. A previous study reported a complete suppression of mechanical hyperalgesia in STZ-D male rats treated with SB258585, PZ-1388 and its derivative PZ-1179 with a long duration of effect (>180min) (Mokhtar *et al.* 2023b). With the limitation that experiments were not performed side by side, this suggests that female-specific pharmacokinetic or pharmacodynamic parameters may modify the analgesic activity of 5-HT<sub>6</sub>R ligands, and underlines the importance of sex-specific adjustment of treatment, consistent with a recent literature review (Merlin *et al.* 2024). To determine whether this different efficacy in males and females is specific to neuropathic pain of metabolic etiology or to a general sex-related dimorphism, we also examined the effect of PZ-1386 and PZ-1179 in OXA and SNL female rats and found that both inverse agonists partially reversed (by 69% and 61% in SNL rats, respectively and by 51% and 48% in OXA rats, respectively) tactile hypersensitivity to the application of von Frey hair. This suggests that the modest efficacy of PZ-1179 is sex-dependent, whereas the analgesic effect of PZ-1386 rather depends on the etiology of the neuropathic pain.
Very few studies have reported sex differences in the efficacy of drugs acting on the 5-HT system, and more specifically in experimental models of painful diabetic neuropathy. Peripheral 5-HT induces thermal hyperalgesia in males and females, which is exacerbated in females in proestrus and estrus, and mechanical allodynia only in females in proestrus and estrus (Kaur *et al.* 2018), suggesting female-specific mechanisms in 5-HT-mediated pain neurotransmission. Similarly, sex-dependent effects of 5-HT<sub>6</sub>R on hippocampal synaptic activity (at the CA3/CA1 connections) through changes in excitation/inhibition balance, have been recently reported (Lahogue *et al.* 2023).

In light of the role of mTOR signaling in neuropathic pain models of various etiologies, including STZ-D males, and the activation of mTOR signaling by constitutively active 5-HT<sub>6</sub>R (Mokhtar *et al.* 2023b; Martin *et al.* 2020), we hypothesized that the analgesic effects of 5-HT<sub>6</sub>R inverse agonists in STZ-D female rats might result from their ability to inhibit mTOR signaling. Consistent with this hypothesis, intrathecal delivery of the Tat-VEPE peptide which uncouples 5-HT<sub>6</sub>R from mTOR and prevents 5-HT<sub>6</sub>-R-operated mTOR activation and, thus, mimics the effects of 5-HT<sub>6</sub>R inverse agonists (Martin *et al.* 2020), reduced mechanical hyperalgesia in STZ-D female rats with an apparently similar global effect to that previously reported in STZ-D male rats (Mokhtar *et al.* 2023b)(Table S1). No increase in mTOR activity, as assessed by the phosphorylation of mTOR (at Ser<sup>2448</sup>) and its downstream substrate S6 (at Ser<sup>240/244</sup>), was measured in the dorsal spinal cord of STZ-D female rats. Consistently, there was no effect of inverse agonists on mTOR and S6 phosphorylation. This apparently contrasts with previous observations indicating an overactivation of the mTOR pathway in the spinal cord dorsal horn of db/db mice, a model of T2D (Wang *et al.* 2020) and in the STZ model of T1D in rats (He *et al.* 2019). On the other hand, fluctuations in insulin and glucose levels due to every-other-day insulin administration in the STZ-T1D model (present study) could mask an increase in pain-related mTOR signaling that has been observed in a model of traumatic neuropathic pain model (Martin *et al.* 2020) and in non-insulin-treated STZ-T1D rats (He *et al.* 2019). Indeed, insulin receptors are expressed in nociceptive neurons of the DRG and dorsal horn (Sugimoto *et al.* 2002). Insulin, via the insulin receptor substrate (IRS-1), increases phosphorylation of mediators of the PI3K-Akt-mTOR pathway, and in turn, activation of S6K1 by mTORC1 phosphorylates inhibitory serine residues of IRS-1 (Grote and Wright 2016). This insulin-dependent regulatory loop could occur in primary terminals and dorsal horn neurons and reduce mTOR signaling in the STZ-T1D model, while pain increases the pathway in interneurons expressing the 5-HT<sub>6</sub> receptor. This remains to be explored in non-insulin-treated STZ-T1D rats. Whatever, intrathecal administration of rapamycin, an mTOR inhibitor, consistently reversed STZ-induced neuropathic pain in female rats. The overall effect of rapamycin, measured by the A.U.C. appeared similar to that previously reported in STZ-D males (Mokhtar *et al.* 2023b) (Table S1) but with a shorter duration of effect (120min) to that previously reported in SNL male rats (24h)
(Martin *et al.* 2020). This behavioral data are also consistent with previous studies showing that mTOR signaling inhibition alleviates pain in experimental models of traumatic (Martin *et al.* 2020; Wang *et al.* 2016; Asante *et al.* 2010; Obara *et al.* 2011; Zhang *et al.* 2013), metabolic (He *et al.* 2019), and toxic (Martin *et al.* 2020; Duan *et al.* 2018) etiologies.

The full 5-HT<sub>6</sub>R inverse agonist SB258585 and PZ-1386 but not the partial 5-HT<sub>6</sub>R inverse agonist PZ-1179 restored normal episodic memory in STZ-D female rats. The D2 score measured in females treated with PZ-1386 and subjected to the NOR test was equivalent to that previously measured in STZ-D males (Mokhtar *et al.* 2023b). Reminiscent of its lack of pro-cognitive effect in STZ-D male rats, PZ-1179 did not alleviate cognitive deficit in STZ-D females. In the SNL model of traumatic neuropathy, PZ-1179 also failed to improve cognitive impairment in males, but restored novelty discrimination in females. These results suggest that the pro-cognitive effect of PZ-1179 depends on both the etiology of neuropathic pain (traumatic vs. metabolic) and the sex. These observations stress the need for personalized treatment, which takes into account the patient's sex, the etiology of neuropathic pain and comorbid cognitive symptoms. The present results also suggest that the pro-cognitive effect of PZ-1386 on comorbid cognitive symptoms might be related to its anti-hyperalgesic effect since it failed to restore normal episodic memory in ketamine-induced cognitive impairment in healthy rats.

Beyond a role in centrally regulating glucose metabolism, insulin is involved in memory, cognition, and mood regulation (Martin *et al.* 2021). Removal of insulin signaling in the hippocampus using viral delivery of insulin receptor antisense sequences, impaired hippocampal plasticity related to long-term potentiation and spatial learning of rats (Grillo *et al.* 2015). In STZ-D rats, the hippocampus was shown to be more responsive to insulin administration compared to nondiabetic rats and sustained insulin delivery to diabetic rats could normalize insulin receptor activity (King *et al.* 2015). This suggests that insulin treatment may provide benefits to cognitive deficits associated with diabetes. In the present study, STZ-D animals receiving insulin displayed cognitive impairments. Comparing cognitive performance in STZ-D rats treated or not with insulin would certainly help to solve this conundrum.

## 5 Conclusions

The present study is consistent with growing evidence implicating constitutively active 5-HT<sub>6</sub>R as important modulators of neuropathic pain and comorbid cognitive deficits. It provides new insights into the sex-and pain etiology-dependent therapeutic potential of 5-HT<sub>6</sub>R ligands, underscoring the importance of the need for personalized treatment in neuropathic pain.
**Funding:** This research was funded by the Agence Nationale de la Recherche (ANR) (ANR-19-CE18-0018 and ANR-22-CE92-0073-01) and program "Investissements d'Avenir" (16-IDEX-0001 CAP 20-25), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Auvergne-Rhône-Alpes Region (AURA, France), the Université Clermont Auvergne (UCA), the Université de Montpellier (UM). N.M. received a scholarship from the french Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI).

**Data Availability Statement**

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

**Acknowledgments**

The authors thank the technical support from personnel at the animal facility at the UCA.

**Conflicts of Interest**

The authors declare no conflict of interest.

**Peer Review**

The peer review history for this article is available at
https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/jnc.70179.

**References**

Alleman C. J. M., Westerhout K. Y., Hensen M., Chambers C., Stoker M., Long S., Van Nooten F. E. (2015) Humanistic and economic burden of painful diabetic peripheral neuropathy in Europe: A review of the literature. *Diabetes Research and Clinical Practice* **109**, 215-225.

Asante C. O., Wallace V. C., Dickenson A. H. (2010) Mammalian target of rapamycin signaling in the spinal cord is required for neuronal plasticity and behavioral hypersensitivity associated with neuropathy in the rat. *J Pain* **11**, 1356-67.

Białoń M., Żarnowska M., Antkiewicz-Michaluk L., Wąsik A. (2020) Pro-cognitive effect of 1MeTIQ on recognition memory in the ketamine model of schizophrenia in rats: the behavioural and neurochemical effects. *Psychopharmacology (Berl)* **237**, 1577-1593.

Bouhassira D., Lantéri-Minet M., Attal N., Laurent B., Touboul C. (2008) Prevalence of chronic pain with neuropathic characteristics in the general population. *Pain* **136**, 380-387.

Chaplan S. R., Bach F. W., Pogrel J. W., Chung J. M., Yaksh T. L. (1994) Quantitative assessment of tactile allodynia in the rat paw. *J. Neurosci. Methods* **53**, 55-63.

Chenaf C., Delorme J., Delage N., Ardid D., Eschalier A., Authier N. (2018) Prevalence of chronic pain with or without neuropathic characteristics in France using the capture-recapture method: a population-based study. *Pain* **159**, 2394-2402.
Courteix C., Eschalier A., Lavarenne J. (1993) Streptozocin-induced diabetic rats: behavioural evidence for a model of chronic pain. *Pain* **53**, 81–8.

Croosu S. S., Gjela M., Røikjer J., Hansen T. M., Mørch C. D., Frøkjaer J. B., Ejskjaer N. (2023) Cognitive function in individuals with and without painful and painless diabetic polyneuropathy-A cross-sectional study in type 1 diabetes. *Endocrinol Diabetes Metab* **6**, e420.

Delay L., Barbier J., Aissouni Y., Jurczak A., Boudieu L., Briat A., Auzeloux P., et al. (2022) Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis. *Pain* **163**, e837–e849.

Deng X., Ma P., Wu M., Liao H., Song X.-J. (2021) Role of Matrix Metalloproteinases in Myelin Abnormalities and Mechanical Allodynia in Rodents with Diabetic Neuropathy. *Aging and disease* **12**, 1808.

Deraredj Nadim W., Chaumont-Dubel S., Madouri F., Cobret L., De Tauzia M.-L., Zajdel P., Bénédetti H., Marin P., Morisset-Lopez S. (2016) Physical interaction between neurofibromin and serotonin 5-HT₆ receptor promotes receptor constitutive activity. *Proceedings of the National Academy of Sciences*, 201600914.

Dixon W. J. (1980) Efficient analysis of experimental observations. *Annu Rev Pharmacol Toxicol* **20**, 441–62.

Drop M., Canale V., Chaumont-Dubel S., Kurczab R., Satała G., Bantreil X., Walczak M., et al. (2021a) 2-Phenyl-1 *H* -pyrrole-3-carboxamide as a New Scaffold for Developing 5-HT<sub>6</sub> Receptor Inverse Agonists with Cognition-Enhancing Activity. *ACS Chem. Neurosci.* **12**, 1228–1240.

Drop M., Jacquot F., Canale V., Chaumont-Dubel S., Walczak M., Satała G., Nosalska K., et al. (2021b) Neuropathic pain-alleviating activity of novel 5-HT6 receptor inverse agonists derived from 2-aryl-1H-pyrrole-3-carboxamide. *Bioorg Chem* **115**, 105218.

Duan Z., Su Z., Wang H., Pang X. (2018) Involvement of pro-inflammation signal pathway in inhibitory effects of rapamycin on oxaliplatin-induced neuropathic pain. *Mol Pain* **14**, 1744806918769426.

Dupuis A., Wattiez A.-S., Pinguet J., Richard D., Libert F., Chalus M., Aissouni Y., et al. (2017) Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release. *Pharmacological Research* **118**, 93–103.

El Khamlichi C., Reverchon F., Hervouet-Coste N., Robin E., Chopin N., Deau E., Madouri F., et al. (2022) Serodolin, a β-arrestin-biased ligand of 5-HT<sub>7</sub> receptor, attenuates pain-related behaviors. *Proc. Natl. Acad. Sci. U.S.A.* **119**, e2118847119.

Ennaceur A., Delacour J. (1988) A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. *Behav. Brain Res.* **31**, 47–59.

Finnerup N. B., Attal N., Haroutounian S., McNicol E., Baron R., Dworkin R. H., Gilron I., et al. (2015) Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. *Lancet Neurol* **14**, 162–73.

Gecse K., Baksa D., Dobos D., Aranyi C. S., Galambos A., Kocsel N., Szabó E., et al. (2021) Sex Differences of Periaqueductal Grey Matter Functional Connectivity in Migraine. *Front Pain Res (Lausanne)* **2**, 767162.

Ghazisaeidi S., Muley M. M., Salter M. W. (2023) Neuropathic Pain: Mechanisms, Sex Differences, and Potential Therapies for a Global Problem. *Annu. Rev. Pharmacol. Toxicol.* **63**, 565–583.

Grillo C. A., Piroli G. G., Lawrence R. C., Wrighten S. A., Green A. J., Wilson S. P., Sakai R. R., et al. (2015) Hippocampal Insulin Resistance Impairs Spatial Learning and Synaptic Plasticity. *Diabetes* **64**, 3927–3936.

Grote C. W., Wright D. E. (2016) A Role for Insulin in Diabetic Neuropathy. *Front Neurosci* **10**, 581.

Handwerker H. O., Arendt-Nielsen L., eds (2013) *Pain models: translational relevance and applications*. IASP Press, Washington, D.C.

He W.-Y., Zhang B., Zhao W.-C., He J., Wang Y., Zhang L., Xiong Q.-M., Wang H.-B. (2019) mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats. *Mol Pain* **15**, 1744806919880643.
Jensen T. S., Baron R., Haanpaa M., Kalso E., Loeser J. D., Rice A. S., Treede R. D. (2011) A new definition of neuropathic pain. *Pain* **152**, 2204–5.

Kaur S., Benton W. L., Tongkhuya S. A., Lopez C. M. C., Uphouse L., Averitt D. L. (2018) Sex Differences and Estrous Cycle Effects of Peripheral Serotonin-Evoked Rodent Pain Behaviors. *Neuroscience* **384**, 87–100.

Kilkenny C., Browne W., Cuthill I. C., Emerson M., Altman D. G. (2010) Animal research: reporting in vivo experiments: the ARRIVE guidelines. *Br J Pharmacol* **160**, 1577–9.

Kim S. H., Chung J. M. (1992) An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. *Pain* **50**, 355–63.

King M. R., Anderson N. J., Liu C., Law E., Cundiff M., Mixcoatl-Zecuatl T. M., Jolivalt C. G. (2015) Activation of the insulin-signaling pathway in sciatic nerve and hippocampus of type 1 diabetic rats. *Neuroscience* **303**, 220–228.

Lahogue C., Billard J.-M., Freret T., Bouet V. (2023) 5-HT6 Receptors Sex-Dependently Modulate Hippocampal Synaptic Activity through GABA Inhibition. *Biomolecules* **13**, 751.

Li W., Huang E., Gao S. (2017) Type 1 Diabetes Mellitus and Cognitive Impairments: A Systematic Review. *J Alzheimers Dis* **57**, 29–36.

Ling B., Coudoré-Civiale M.-A., Balayssac D., Eschalier A., Coudoré F., Authier N. (2007) Behavioral and immunohistological assessment of painful neuropathy induced by a single oxaliplatin injection in the rat. *Toxicology* **234**, 176–184.

Liu Q. Q., Yao X. X., Gao S. H., Li R., Li B. J., Yang W., Cui R. J. (2020) Role of 5-HT receptors in neuropathic pain: potential therapeutic implications. *Pharmacological Research* **159**, 1049–9.

Martin H., Bullich S., Guiard B. P., Fioramonti X. (2021) The impact of insulin on the serotonergic system and consequences on diabetes-associated mood disorders. *J Neuroendocrinology* **33**, e12928.

Martin P.-Y., Doly S., Hamieh A. M., Chapuy E., Canale V., Drop M., Chaumont-Dubel S., et al. (2020) mTOR activation by constitutively active serotonin6 receptors as new paradigm in neuropathic pain and its treatment. *Prog Neurobiol* **193**, 101846.

Meffre J., Chaumont-Dubel S., Mannoury la Cour C., Loiseau F., Watson D. J., Dekeyne A., Seveno M., et al. (2012) 5-HT(6) receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia. *EMBO Mol Med* **4**, 1043–56.

Merlin E., Salio C., Ferrini F. (2024) Painful Diabetic Neuropathy: Sex-Specific Mechanisms and Differences from Animal Models to Clinical Outcomes. *Cells* **13**, 2024.

Mogil J. S. (2020) Qualitative sex differences in pain processing: emerging evidence of a biased literature. *Nat Rev Neurosci* **21**, 353–365.

Moisset X., Bouhassira D., Attal N. (2021) French guidelines for neuropathic pain: An update and commentary. *Revue Neurologique* **177**, 834–837.

Mokhtar N., Doly S., Courteix C. (2023a) Diabetic Neuropathic Pain and Serotonin: What Is New in the Last 15 Years? *Biomedicines* **11**, 1924.

Mokhtar N., Drop M., Jacquot F., Lamoine S., Chapuy E., Prival L., Aissouni Y., et al. (2023b) The Constitutive Activity of Spinal 5-HT6 Receptors Contributes to Diabetic Neuropathic Pain in Rats. *Biomolecules* **13**, 364.

Nirogi R., Jayarajan P., Shinde A., Mohammed A. R., Grandhi V. R., Benade V., Goyal V. K., Abraham R., Jasti V., Cummings J. (2023) Progress in Investigational Agents Targeting Serotonin-6 Receptors for the Treatment of Brain Disorders. *Biomolecules* **13**, 309.

Obara I., Tochiki K. K., Géranton S. M., Carr F. B., Lumb B. M., Liu Q., Hunt S. P. (2011) Systemic inhibition of the mammalian target of rapamycin (mTOR) pathway reduces neuropathic pain in mice: *Pain* **152**, 2582–2595.

Plumb A. N., Lesnak J. B., Berardi G., Hayashi K., Janowski A. J., Smith A. F., Bailey D., et al. (2023) Standing on the shoulders of bias: lack of transparency and reporting of critical rigor characteristics in pain research. *Pain* **164**, 1775–1782.

Radat F., Margot-Duclot A., Attal N. (2013) Psychiatric co-morbidities in patients with chronic peripheral neuropathic pain: a multicentre cohort study. *Eur J Pain* **17**, 1547–1557.
Randall L. O., Selitto J. J. (1957) A method for measurement of analgesic activity on inflamed tissue. *Arch Int Pharmacodyn Ther* **111**, 409-19.

Sadler K. E., Mogil J. S., Stucky C. L. (2022) Innovations and advances in modelling and measuring pain in animals. *Nat Rev Neurosci* **23**, 70–85.

Sasaki-Hamada S., Fujiwara A., Satoh S., Iwai T., Oka J.-I. (2021) GLP-2 restores impairments in spatial working memory and hippocampal LTD via the MEK/ERK pathway in juvenile-onset diabetes rats. *Behav Brain Res* **406**, 113235.

Schaefer C., Sadosky A., Mann R., Daniel S., Parsons B., Tuchman M., Anschel A., Stacey B. R., Nalamachu S., Nieshoff E. (2014) Pain severity and the economic burden of neuropathic pain in the United States: BEAT Neuropathic Pain Observational Study. *Clinicoecon Outcomes Res* **6**, 483-496.

Sugimoto K., Murakawa Y., Sima A. A. F. (2002) Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord. *J Peripher Nerv Syst* **7**, 44-53.

Tesfaye S., Sloan G., Petrie J., White D., Bradburn M., Julious S., Rajbhandari S., et al. (2022) Comparison of amitriptyline supplemented with pregabalin, pregabalin supplemented with amitriptyline, and duloxetine supplemented with pregabalin for the treatment of diabetic peripheral neuropathic pain (OPTION-DM): a multicentre, double-blind, randomised crossover trial. *The Lancet* **400**, 680–690.

Wang R., Qiu Z., Wang G., Hu Q., Shi N., Zhang Z., Wu Y., Zhou C. (2020) Quercetin attenuates diabetic neuropathic pain by inhibiting mTOR/p70S6K pathway-mediated changes of synaptic morphology and synaptic protein levels in spinal dorsal horn of db/db mice. *Eur J Pharmacol* **882**, 173266.

Wang X., Li X., Huang B., Ma S. (2016) Blocking mammalian target of rapamycin (mTOR) improves neuropathic pain evoked by spinal cord injury. *Transl Neurosci* **7**, 50-55.

Zhang Q., Song W., Zhao B., Xie J., Sun Q., Shi X., Yan B., Tian G., Liang X. (2021) Quercetin Attenuates Diabetic Peripheral Neuropathy by Correcting Mitochondrial Abnormality via Activation of AMPK/PGC-1α Pathway in vivo and in vitro. *Front. Neurosci.* **15**, 636172.

Zhang W., Sun X. F., Bo J. H., Zhang J., Liu X. J., Wu L. P., Ma Z. L., Gu X. P. (2013) Activation of mTOR in the spinal cord is required for pain hypersensitivity induced by chronic constriction injury in mice. *Pharmacol Biochem Behav* **111**, 64-70.

**Supporting Information**

Additional supporting information can be found online in the Supporting Information section.

**Appendix S1: jnc70179-sup-0001-AppendixS1.pdf.**
<table><caption>TABLE 1. Detailed statistics for figures</caption><thead><tr><th>Figures</th><th>Analysis</th><th>Statistics</th><th>p</th></tr></thead><tbody><tr><td>Figure 1a-d</td><td>2-way RM ANOVA</td><td>F(30, 282)=4.715</td><td>p&lt;0.0001</td></tr><tr><td>Figure 1e</td><td>1-way ANOVA</td><td>F(5, 46)=9.155</td><td>p&lt;0.0001</td></tr><tr><td>Figure 2a</td><td>2-way RM ANOVA</td><td>F(18, 180)=3.417</td><td>p&lt;0.0001</td></tr><tr><td>Figure 2b</td><td>1-way ANOVA</td><td>F(3, 27)=6.696</td><td>p=0.0016</td></tr><tr><td>Figure 3a</td><td>2-way RM ANOVA</td><td>F(12, 114)=11.33</td><td>p&lt;0.0001</td></tr><tr><td>Figure 3b</td><td>1-way ANOVA</td><td>F(2, 17)=26.42</td><td>p&lt;0.0001</td></tr><tr><td>Figure 4a</td><td>1-way ANOVA</td><td>F(4, 44)=8.796</td><td>p&lt;0.0001</td></tr><tr><td>Figure 4b</td><td>1-way ANOVA</td><td>F(3, 52)=11.69</td><td>p&lt;0.0001</td></tr><tr><td>Figure 5a</td><td>1-way ANOVA</td><td>F(5, 6)=1.7</td><td>p=0.2677</td></tr><tr><td>Figure 5b</td><td>1-way ANOVA</td><td>F(5, 6)=2.331</td><td>p=0.1661</td></tr><tr><td>Figure 6</td><td>Student's t-test</td><td>t=0.6692; df= 22</td><td>p=0.5046</td></tr><tr><td>Figure S1a</td><td>2-way RM ANOVA</td><td>F(15, 125)=5.97</td><td>p&lt;0.0001</td></tr><tr><td>Figure S1b</td><td>2-way RM ANOVA</td><td>F(14, 105)=1.26</td><td>p=0.2452</td></tr><tr><td>Figure S2</td><td>1-way ANOVA</td><td>F(3, 57)= 32.46</td><td>p&lt;0.0001</td></tr><tr><td>Figure S3a</td><td>1-way ANOVA</td><td>F(3, 21)= 5.393</td><td>p=0.0065</td></tr><tr><td>Figure S3b</td><td>1-way ANOVA</td><td>F(3, 33)= 6.136</td><td>p=0.0020</td></tr></tbody></table>
<figure><img src="image_4.png" /></figure>

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<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**FIGURE 1** Systemic administration of 5-HT<sub>6</sub>R inverse agonists reduces STZ-induced mechanical hyperalgesia (paw pressure test) in female rats. (a) Intraperitoneal (i.p.) administration of SB258585 (b) PZ-1388, (c) PZ-1386 and (d) PZ-1179 (25µmol/kg each) but not vehicle (water for injections) improves mechanical hyperalgesia to paw pressure in STZ-induced diabetic (STZ-D) rats (n=4-14/group). Vocalization thresholds are expressed as arbitrary units (a.u.). ###P<0.001 versus values measured before STZ; \*p<0.05, \*\*p<0.01, \*\*\*p<0.001 vs. values measured before the drug/vehicle (water for injections) injection (D21 Post-STZ), two-way ANOVA. (e) Area under the curve (A.U.C.) of vocalization threshold variations (0-120min) in STZ-D and control rats calculated by the trapezoidal rule (in a.u. x min). Points represent individual data. \*P<0.05, \*\*\*P<0.001 vs. STZ-D Vehicle group, one-way ANOVA. Bars represent SEM.</figcaption></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**FIGURE 3** Tat-VEPE suppresses mechanical hyperalgesia in STZ diabetic (STZ-D) female rats. (a) Intrathecal (i.t.) administration of a cell-permeable interfering peptide fused to the transduction domain of the HIV Tat protein (Tat-VEPE, 300ng/rat), increases the vocalization threshold to paw pressure in STZ-D rats (n=5-8/group). ###p<0.001 versus values measured before STZ. ***p<0.001 versus values measured before the drug/vehicle (water for injections) injection (D21 Post-STZ), two-way ANOVA. (b) Area under the curve (A.U.C.) of vocalization threshold variations (0-120min) in STZ-D rats calculated by the trapezoidal rule (in a.u. x min). Points represent individual data. §§§p<0.001 versus STZ-D Vehicle group; **p<0.001 versus corresponding group, one-way ANOVA. Bars represent SEM.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**FIGURE 4** Effect of systemic administration of the 5-HT<sub>6</sub>R inverse agonists PZ-1386, PZ-1179 or SB258585, or of intrathecal (i.t.) injection of rapamycin or Tat-VEPE on cognitive deficits (novel object recognition test) induced by diabetic neuropathic pain in female rats. (a) Intraperitoneal (i.p.) injection of PZ-1386 (25µmol/kg) or SB258585 (25µmol/kg) but not PZ-1179 (25µmol/kg), or vehicle (water for injections) improves novelty discrimination in STZ-Diabetic (STZ-D) female rats (n=7-12/group). (b) Intrathecal (i.t.) injection of Tat-VEPE (300ng/rat) or rapamycin (Rapa, 10nmol/rat), but not vehicle (water for injections), improves novelty discrimination in STZ-D rats (n= 3-15/group). Points represent individual data. \*p<0.05, \*\*\*p<0.001 versus corresponding group, one-way ANOVA. Bars represent SEM.</figcaption></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**FIGURE 5** Effect of 5-HT<sub>6</sub> receptor inverse agonists, rapamycin and Tat-VEPE on STZ-induced mTOR signaling in the dorsal spinal cord of STZ-diabetic female rats. Diabetic neuropathy in rats does not induce significant changes in S6 phosphorylation (at Ser<sup>240/244</sup>) (a) or mTOR phosphorylation (at Ser<sup>2448</sup>) (b) (n=2/group). One-way ANOVA. Bars represent SEM.</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**FIGURE 6** Immunostaining of 5-HT<sub>6</sub>R in primary cilia of dorsal horn spinal cord neurons from 5-HT6-GFP KI male and female mice. (a, b) Immunolabeling of 5-HT6R (indicated by arrowheads) was detected in the dorsal horn of the spinal cord in female (a) and male (b) 5-HT6-GFP knock-in (KI) mice using a GFP antibody. (c) Quantification of GFP-positive primary cilia in dorsal horn neurons from 5-HT<sub>6</sub>-GFP KI mice (n=31 cryostat sections per mouse; n=2/group). Data points for individual mice are represented by distinct colors. ns, not significant (Student's t-test). Bars represent SEM.</figcaption></figure>