Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Apr 2.
Published in final edited form as: Neurosci Res. 2025 Jan 31;213:156–160. doi: 10.1016/j.neures.2025.01.008

Positive allosteric modulator of GLT-1 reduces methamphetamine hyperlocomotion, sensitization and conditioned place preference in mice

Prateek Mokkarala a, Sonita Wiah a, Ellen M Unterwald a,b, Xiangdang Shi a, Danielle R Stern a, Andreia CK Fontana c, Joseph M Salvino d, Scott M Rawls a,b,*
PMCID: PMC11963643  NIHMSID: NIHMS2068712  PMID: 39894415

Abstract

NA-014 is a positive allosteric modulator (PAM) of glutamate transporter subtype 1 (GLT-1) that increases glutamate reuptake. Since enhanced glutamate transmission facilitates methamphetamine (METH) hyperlocomotion, behavioral sensitization, and conditioned place preference (CPP), we hypothesized that NA-014 (60 mg/kg) would reduce these effects in male mice. NA-014 reduced acute ambulation induced by METH and behavioral sensitization during repeated METH. NA-014 given after METH conditioning reduced expression of METH CPP. NA-014 injected during METH conditioning did not affect development of METH CPP, and NA-014 did not affect spatial memory. These results suggest that GLT-1 PAMs reduce METH-induced behavioral effects in mice.

Keywords: NA-014, Methamphetamine, GLT-1, Glutamate, PAM, CPP, Locomotor

NA-014 is a selective positive allosteric modulator (PAM) that activates glutamate transporter subtype 1 (GLT-1), a protein that clears extracellular glutamate in the forebrain (Reeb et al., 2024; Fontana et al., 2024). NA-014 stimulates GLT-1 activity in glial cultures and transfected COS-7 cells without affecting related glutamate transporters (e.g., GLAST or EAAT3) or NMDA and AMPA receptors (Fontana et al., 2024). The unique mechanistic feature of NA-014 is that it acts as a PAM of GLT-1 by increasing Vmax by 2–3-fold without changing substrate affinity (KM). NA-014 shows 52 % brain penetrability in rats (Reeb et al., 2024) and 83 % in mice (Fontana et al., 2024). Unlike β-lactam compounds such as ceftriaxone (CTX) and clavulanic acid (CLAV) that rely on upregulation of GLT-1 protein levels for CNS efficacy, NA-014 does not increase cortical GLT-1 levels in rats (Reeb et al., 2024). Early NA-014 analogs (e.g., GT949) also have in vitro neuroprotective efficacy (Falcucci et al., 2019), suggesting therapeutic promise for neurological and psychiatric disorders (Fontana et al., 2024).

GLT-1 is an important molecular target for psychostimulant use disorders, as the glutamatergic system facilitates psychotropic effects of psychostimulants like methamphetamine (METH). Here, we tested the hypothesis that NA-014 reduces acute and sensitized locomotor responses to METH and reduces METH conditioned place preference (CPP) in male mice. The hypothesis is based on evidence that β-lactam GLT-1 enhancers reduce hyperlocomotion induced by acute METH or amphetamine injection; behavioral sensitization caused by repeated METH or amphetamine exposure, and METH seeking in CPP assays (Rasmussen et al., 2011; Abulseoud et al., 2012; Walters et al., 2024).

Adult male C57Bl/6 mice (2 months old) from Envigo Laboratories were used. Animal use procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Temple University Institutional Animal Care and Use Committee. Mice were housed on a 12 h light/dark cycle and provided food and water ad libitum.

Methamphetamine hydrochloride (METH) (Sigma-Aldrich) was dissolved in physiological saline (0.9 %). NA-014 was synthesized as described (Fontana et al., 2024) and dissolved in 10 % Kolliphor/10 % DMSO/ 80 % physiological saline. Both drugs were injected intraperitoneally (IP).

Locomotor activity was measured using a Digiscan DMicro system (Potula et al., 2023). Following a 60-min habituation in activity chambers, mice were injected with NA-014 (10, 30, 60 mg/kg) or vehicle. Fifteen min later, all mice were injected with METH (3 mg/kg), and ambulatory and stereotypical activities were measured for 105 min. The selected NA-014 dose range and pretreatment time (15 min) are effective against cocaine-induced behaviors in rats (Reeb et al., 2024). For locomotor sensitization experiments, mice were injected with METH (3 mg/kg) every other day for 15 d. Fifteen min before each METH (3 mg/kg) injection, mice were injected with NA-014 (60 mg/kg) or vehicle. Ambulatory and stereotypical activities were measured on days 1, 5 and 15 for 105 min after METH injection.

CPP experiments were conducted as described (Wiah et al., 2023). In an initial 30-min pre-test, the compartment in which a mouse spent less time was determined and designated as the METH-paired side. A 4-day conditioning paradigm, with morning and afternoon sessions, was then initiated. Mice were injected in the morning with METH (3 mg/kg) and confined to the drug-paired compartment for 30 min. In the afternoon, mice were injected with saline and confined to the opposite compartment for 30 min. One day after the conditioning phase, mice were injected once with NA-014 (60 mg/kg) or vehicle, placed into chambers and allowed access to both compartments for 30 min. Experiments investigating effects of NA-014 on development of METH CPP were conducted similarly with two exceptions: NA-014 (or vehicle) was injected 15 min before METH (3 mg/kg) on each day of METH conditioning, and NA-0–14 was not injected on the day of the post-test. To control for aversive or rewarding effects of NA-014, otherwise drug--naïve mice were conditioned with NA-014 (60 mg/kg) for 4 d and time spent in the light for pre- and post-conditioning was determined.

For Y-maze testing of spatial memory and exploratory behavior (Lalonde, 2002), mice were injected with NA-014 (60 mg/kg) or vehicle once daily for 3 d. Fifteen min after the last injection, mice were placed in the central zone of a Y-maze and allowed to explore the maze for 5 min. Behaviors were captured using a webcam positioned above the maze and later analyzed with EthoVision video tracking software. Number of entries and alternations between arms were measured. Alternations were defined as entry into all three arms on consecutive choices in overlapping triplet sets. Percent spontaneous alternations were calculated as the ratio of actual to possible (total number of arm entries minus two) alternations X 100.

Locomotor data were analyzed by two-way ANOVA followed by a Bonferroni test. CPP and Y-maze data were analyzed by a Student’s t-test. Statistical significance was set at P < 0.05.

For acute ambulation (Fig. 1A), two-way ANOVA revealed effects of NA-014 [F (3, 594) = 7.967, P < 0.001] and time [F (21, 594) = 28.90, P < 0.001] and an interaction [F (63, 594) = 1.396, P < 0.05]. In METH-exposed mice, NA-014 (60 mg/kg) reduced ambulation from 15 to 30 min after METH exposure. A lower NA-014 dose (30 mg/kg) reduced ambulation in METH-exposed mice 30 min after METH injection (P < 0.05). For acute stereotypies (Fig. 1B), two-way ANOVA revealed effects of NA-014 [F (3, 616) = 16.89, P < 0.001] and time [F (21, 616) = 6.991, P < 0.001]. NA-014 (60 mg/kg) increased stereotypical activity in METH-exposed mice from 80 to 95 min post-METH exposure. NA-014 itself (60 mg/kg) did not affect spontaneous ambulatory or stereotypical activity (P > 0.05) (Fig. 1C–D).

Fig. 1. NA-014 reduced METH-evoked ambulation and enhances METH-evoked stereotypy but does not affect spontaneous motor activities.

Fig. 1.

Open in a new tab

(A, B) NA-014 + METH: Mice injected with NA-014 (10, 30, 60 mg/kg) or vehicle were injected with METH (3 mg/kg) 15 min later, and ambulatory and stereotypical activities were measured for 105 min.*P < 0.05, **P < 0.01, or ***P < 0.001 compared to VEH + METH. N = 7–8 mice/group. (C, D) NA-014 by itself: Mice were injected with NA-014 (60 mg/kg) or vehicle, and ambulatory and stereotypical activities were measured for 105 min. N = 7–8 mice/group.

For sensitization results, two-way ANOVA revealed effects of 60 mg/kg NA-014 [F (1, 42) = 15.58, P < 0.001] and day [F (2, 42) = 7.369, P < 0.05] on ambulatory responding (Fig. 2A). For mice exposed only to METH, ambulatory activity was greater on days 5 (P < 0.05) and 15 (P < 0.05) than on day 1. For mice exposed to both NA-014 and METH, ambulatory activity was greater on day 15 (P < 0.05) than on day 1. For day 5, mice treated repeatedly with METH and NA-014 displayed less ambulation than mice treated repeatedly with METH alone (P < 0.05). NA-014 (60 mg/kg) effects on stereotypical sensitization to METH were similar (Fig. 2B). Two-way ANOVA revealed an interaction (NA-014 × day) [F (2, 42) = 5.120, P < 0.05]. For mice exposed to METH by itself, cumulative stereotypical activity was greater on days 5 (P < 0.01) and 15 (P < 0.01) than on day 1. For mice exposed to both NA-014 and METH, cumulative ambulatory activity was not different across days (P > 0.05). For day 5, mice treated repeatedly with METH and NA-014 displayed less stereotypical activity than mice treated repeatedly with METH alone (P < 0.05). A lower dose of 30 mg/kg NA-014 did not affect METH behavioral sensitization (data not shown).

Fig. 2. NA-014 reduced METH behavioral sensitization and expression of METH CPP.

Fig. 2.

Open in a new tab

(A, B) METH sensitization: Mice were injected every other day for 15 d with 3 mg/kg METH. NA-014 (60 mg/kg) or vehicle was injected 15 min before each METH injection. On days 1, 5 and 15, data are presented as (A) cumulative ambulatory counts or (B) cumulative stereotypy counts for the 105-min interval after METH injection. *P < 0.05 or **P < 0.01 compared to day 1 VEH + METH or #P < 0.05 compared to day 1 NA-014 + METH. +P < 0.05 or ++P < 0.01 compared to day 5 of VEH + METH group. N = 8 mice/group. (C, D) METH CPP: Mice conditioned with METH (3 mg/kg) for 4 d were injected with NA-014 (60 mg/kg) or vehicle (C) once (after conditioning on CPP test day) or (D) on each day of conditioning 15 min before METH injection. For (C) Expression and (D) Development, data expressed as a difference score (difference in time spent on METH-paired side between post-test and pre-test). N = 10–14 mice/group. *P < 0.05 compared to VEH METH.

For expression of METH CPP (Fig. 2C), METH-conditioned mice treated with NA-014 (60 mg/kg) on post-test day displayed less place preference than METH-conditioned mice treated with vehicle (i.e., approximately 61 % reduction) (P < 0.05). A lower dose of NA-014 (30 mg/kg) reduced expression of METH CPP by approximately 37 %, but the effect did not reach statistical significance (P > 0.05). For development of METH CPP (Fig. 2D), mice conditioned with NA-014 (60 mg/kg) and METH displayed place preference that was not different from mice conditioned with METH (P > 0.05). For mice conditioned only with NA-014 (60 mg/kg), time spent in the light compartment before (584 ± 58 s) and after (662 ± 75 s) conditioning was not different (P > 0.05, Students t-test, n = 7).

For Y-maze experiments, there were no differences between mice injected with NA-014 (60 mg/kg) or vehicle in number of alternations (Fig. 3A) or arm entries (Fig. 3B) or percent alternations (Fig. 3C) (P > 0.05). These results indicate NA-014 did not affect activity, exploration or spatial memory.

Fig. 3. NA-014 did not affect exploratory activity or spatial memory in the Y-maze.

Fig. 3.

Open in a new tab

Mice were injected with NA-014 (60 mg/kg) or vehicle once daily for 3 d. Fifteen min after the last injection, mice were placed in the central zone of a Y-maze and allowed to freely explore the maze for 5 min while being video recorded. Number and sequence of arm entries were measured. Data are presented as (A) total alternations, (B) total arm entries, and (C) % alternations. There were no significant differences between groups on any measure (Student’s t-test). N = 8 mice/group.

The three main findings of the present study were that NA-014 reduced ambulation induced by acute METH, behavioral sensitization during repeated METH exposure, and METH CPP. Effects of NA-014 against an addictive drug have only been demonstrated in one prior study, which showed that NA-014 reduced cocaine hyperlocomotion and CPP in male and female rats (Reeb et al., 2024). Here, NA-014 reduced ambulation induced by acute METH in mice, which is consistent with established GLT-1 enhancers reducing ambulation caused by acute amphetamine exposure. For example, repeated CTX reduces amphetamine-evoked ambulation in rats (Rasmussen et al., 2011). FDA-approved riluzole, which enhances glutamate reuptake and inhibits glutamate release, reduces METH-evoked hyperlocomotion in mice (Itzhak and Martin, 2000; Lourenço Da Silva et al., 2003). Troriluzole, a pharmacokinetically enhanced riluzole prodrug, reduces METH-evoked hyperlocomotion in rats (Wiah et al., 2023). One difference between NA-014 and established GLT-1 enhancers was duration of inhibition of METH hyperlocomotion. Inhibition by NA-014 persisted for only 30 min after METH exposure while GLT-1 enhancers, especially CTX (Rasmussen et al., 2011), produce enduring inhibition. The shorter duration of action of NA-014 may be related to pharmacokinetic differences (e.g. NA-014 rapidly penetrates the brain, but brain levels decline within 30–45 min) or fewer effects on non-GLT-1 targets (Reeb et al., 2024; Fontana et al., 2024). CTX reduces cocaine-evoked extracellular dopamine in the nucleus accumbens of rats (Barr et al., 2015), which could contribute to more persistent inhibition of psychostimulant-induced hyperlocomotion. Another distinction between NA-014 and CTX is effects on stereotypical behaviors. NA-014 caused a delayed but significant increase in METH-evoked stereotypies (80–100 min after METH injection) whereas CTX reduces amphetamine-evoked stereotypical counts in rats (Rasmussen et al., 2011).

NA-014 reduced development of behavioral sensitization during repeated METH exposure. CTX also reduces development of behavioral sensitization to repeated amphetamine or cocaine exposure in rats (Rasmussen et al., 2011; Sondheimer and Knackstedt, 2011), and CLAV blocks development of behavioral sensitization to METH in mice and to morphine in rats (Walters et al., 2024; Schroeder et al., 2014). Mechanistically, increased glutamate transmission at NMDA and AMPA receptors during repeated METH exposure facilitates sensitization (Karler et al., 1991; Wolf and Khansa, 1991). For our experiments, during NA-014 and METH co-exposure, extracellular glutamate may have been cleared more effectively due to enhanced glutamate reuptake by NA-014, diminishing the normal increase in glutamate transmission during METH exposure and reducing the degree of behavioral sensitization.

NA-014 reduced expression of METH CPP (i.e., NA-014 given once after METH conditioning) but did not affect development of METH CPP (i.e., NA-014 given during METH conditioning). The CPP outcomes for NA-014 resemble the profile for CLAV, which blocks expression of cocaine CPP and disrupts maintenance of both METH and cocaine CPP but is ineffective against development of cocaine CPP (Althobaiti et al., 2019; Philogene-Khalid et al., 2022). Troriluzole displays broader efficacy against METH CPP in rats as it blocks development and expression and disrupts maintenance (Wiah et al., 2023). The dissimilar effects of NA-014, CLAV, and troriluzole on psychostimulant-induced CPP are likely due to mechanistic differences at GLT-1 or differences in effects at other glutamatergic and non-glutamatergic targets. For example, troriuzole’s broader efficacy may be related to a more diverse mechanism including enhanced glutamate reuptake, reduced glutamate release, and inhibition of protein kinase C (PKC) signaling (Cheah et al., 2010).

In summary, GLT-1 PAMs, compared to conventional GLT-1 protein expression enhancers, could offer a more rapid onset of action against CNS diseases without long term GLT-1upregulation that increases cognitive risks. For NA-014, future priorities include optimization of pharmacokinetic properties, determination of effects against psychostimulant seeking in self-administration assays, and characterization of potential sex differences.

Acknowledgements

NIH grants R01 DA051205 and P30 DA013429 supported this work.

Footnotes

CRediT authorship contribution statement

Shi Xiangdang: Formal analysis, Investigation, Methodology, Supervision, Writing – review & editing. Stern Danielle R: Investigation, Methodology, Writing – review & editing. Rawls Scott: Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Unterwald Ellen M.: Investigation, Methodology, Supervision, Writing – review & editing. Fontana Andreia C. K.: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization. Salvino Joseph M.: Writing – review & editing, Funding acquisition, Conceptualization. Mokkarala Prateek: Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. Wiah Sonita: Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization.

Declaration of Competing Interest

The authors have no conflicts of interest.

References

  1. Abulseoud OA, Miller JD, Wu J, Choi DS, Holschneider DP, 2012. Ceftriaxone upregulates the glutamate transporter in medial prefrontal cortex and blocks reinstatement of methamphetamine seeking in a condition place preference paradigm. Brain Res 1456, 14–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Althobaiti YS, Alshehri FS, Hakami AY, Hammad AM, Sari Y, 2019. Effects of Clavulanic Acid Treatment on Reinstatement to Methamphetamine, Glial Glutamate Transporters, and mGluR 2/3 Expression in P Rats Exposed to Ethanol. J. Mol. Neurosci 67, 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barr JL, Rasmussen BA, Tallarida CS, Scholl JL, Forster GL, Unterwald EM, Rawls SM, 2015. Ceftriaxone attenuates acute cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens of the rat. Br. J. Pharmacol 172, 5414–5424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheah BC, Vucic S, Krishnan AV, Kiernan MC, 2010. Riluzole, neuroprotection and amyotrophic lateral sclerosis. Curr. Med. Chem 17, 1942–199. [DOI] [PubMed] [Google Scholar]
  5. Falcucci RM, Wertz R, Green JL, Meucci O, Salvino J, Fontana ACK, 2019. Novel Positive Allosteric Modulators of Glutamate Transport Have Neuroprotective Properties in an in Vitro Excitotoxic Model. ACS Chem. Neurosci 10, 3437–3453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fontana ACK, Poli ANR, Gou RJ, Srikanth YVV, Anastasi N, Ashok D, Khatiwada A, Reeb KL, Cheng MH, Bahar I, Rawls SM, Salvino JM, 2024. Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators. J. Med. Chem 67, 6119–6143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Itzhak Y, Martin JL, 2000. Effect of riluzole and gabapentin on cocaine- and methamphetamine-induced behavioral sensitization in mice. Psychopharmacology 151, 226–233. [DOI] [PubMed] [Google Scholar]
  8. Karler R, Calder LD, Turkanis SA, 1991. DNQX blockade of amphetamine behavioral sensitization. Brain Res 552, 295–300. [DOI] [PubMed] [Google Scholar]
  9. Lalonde R, 2002. The neurobiological basis of spontaneous alternation. Neurosci. Biobehav. Rev 26, 91–104. [DOI] [PubMed] [Google Scholar]
  10. Lourenço, Da Silva A, Hoffmann A, Dietrich MO, Dall’Igna OP, Souza DO, Lara DR, 2003. Effect of riluzole on MK-801 and amphetamine-induced hyperlocomotion. Neuropsychobiology 48, 27–30. [DOI] [PubMed] [Google Scholar]
  11. Philogene-Khalid HL, Morrison MF, Darbinian N, Selzer ME, Schroeder J, Rawls SM, 2022. The GLT-1 enhancer clavulanic acid suppresses cocaine place preference behavior and reduces GCPII activity and protein levels in the rat nucleus accumbens. Drug Alcohol Depend 232, 109306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Potula R, Gentile TA, Meissler JJ, Shekarabi A, Wiah S, Farkas DJ, Inan S, Eisenstein TK, Rawls SM, 2023. Purinergic P2X7 receptor antagonist inhibits methamphetamine-induced reward, hyperlocomotion, and cortical IL-7A levels in mice: A role for P2X7/IL-17A crosstalk in methamphetamine behaviors? Brain Behav. Immun 107, 47–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rasmussen B, Unterwald EM, Rawls SM, 2011. Glutamate transporter subtype 1 (GLT-1) activator ceftriaxone attenuates amphetamine-induced hyperactivity and behavioral sensitization in rats. Drug Alcohol Depend 118, 484–488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Reeb KL, Wiah S, Patel BP, Lewandowski SI, Mortensen OV, Salvino JM, Rawls SM, Fontana ACK, 2024. Positive allosteric modulation of glutamate transporter reduces cocaine-induced locomotion and expression of cocaine conditioned place preference in rats. Eur. J. Pharmacol 984, 177017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schroeder JA, Tolman NG, McKenna FF, Watkins KL, Passeri SM, Hsu AH, Shinn BR, Rawls SM, 2014. Clavulanic acid reduces rewarding, hyperthermic and locomotor-sensitizing effects of morphine in rats: a new indication for an old drug? Drug Alcohol Depend 142, 41–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sondheimer I, Knackstedt LA, 2011. Ceftriaxone prevents the induction of cocaine sensitization and produces enduring attenuation of cue- and cocaine-primed reinstatement of cocaine-seeking. Behav. Brain Res 225, 252–258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Walters TH, Wiah S, Shekarabi A, Milton M, Reddy S, Zhao P, Mokkarala PS, Potula R, Rawls SM, 2024. Clavulanic acid inhibits methamphetamine locomotor sensitization in mice and normalizes methamphetamine-induced changes in glutaminase mRNA levels in the nucleus accumbens. Physiol. Behav 284, 114646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wiah S, Roper A, Zhao P, Shekarabi A, Watson MN, Farkas DJ, Potula R, Reitz AB, Rawls SM, 2023. Troriluzole inhibits methamphetamine place preference in rats and normalizes methamphetamine-evoked glutamate carboxypeptidase II (GCPII) protein levels in the mesolimbic pathway. Drug Alcohol Depend 242, 109719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wolf ME, Khansa MR, 1991. Repeated administration of MK-801 produces sensitization to its own locomotor stimulant effects but blocks sensitization to amphetamine. Brain Res 562, 164–168. [DOI] [PubMed] [Google Scholar]

RESOURCES