The histamine H3R antagonist DL77 attenuates autistic behaviors in a prenatal valproic acid-induced mouse model of autism

Abstract

Autistic spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in social communication and restricted/repetitive behavior patterns or interests. Antagonists targeting histamine H3 receptor (H3R) are considered potential therapeutic agents for the therapeutic management of different brain disorders, e.g., cognitive impairments. Therefore, the effects of subchronic treatment with the potent and selective H3R antagonist DL77 (5, 10, or 15 mg/kg, i.p.) on sociability, social novelty, anxiety, and aggressive/repetitive behavior in male Tuck-Ordinary (TO) mice with ASD-like behaviors induced by prenatal exposure to valproic acid (VPA, 500 mg/kg, i.p.) were evaluated using the three-chamber test (TCT), marble burying test (MBT), nestlet shredding test (NST), and elevated plus maze (EPM) test. The results showed that VPA-exposed mice exhibited significantly lower sociability and social novelty preference compared to VPA-exposed mice that were pretreated with DL77 (10 or 15 mg/kg, i.p.). VPA-exposed mice presented a significantly higher percentage of buried marbles in MBT and shredded nestlet significantly more in NST compared to the control groups. However, VPA-exposed animals pretreated with DL77 (10 or 15 mg/kg, i.p.) buried a reduced percentage of marbles in MBT and presented a significantly lower percentage of shredding behavior in NST. On the other hand, pretreatment with DL77 (5, 10, or 15 mg/kg, i.p.) failed to restore the disturbed anxiety levels and hyperactivity observed in VPA-exposed animals in EPM, whereas the reference drug donepezil (DOZ, 1 mg/kg, i.p.) significantly palliated the anxiety and reduced the hyperactivity measures of VPA-exposed mice. Furthermore, pretreatment with DL77 (10 or 15 mg/kg, i.p.) modulated oxidative stress status by increasing GSH and decreasing MDA, and it attenuated the proinflammatory cytokines IL-1β, IL-6 and TNF-α exacerbated by lipopolysaccharide (LPS) challenge, in VPA-exposed mouse brain tissue. Taken together, these results provide evidence that modulation of brain histaminergic neurotransmission, such as by subchronic administration of the H3R antagonist DL77, may serve as an effective pharmacological therapeutic target to rescue ASD-like behaviors in VPA-exposed animals, although further investigations are necessary to corroborate and expand these initial data.

Figure 1

Schematic diagram of drug treatments, behavioral studies, and biochemical assessments with VPA mice. Pregnant mice were injected with VPA (500 mg/kg, i.p.) at embryonic day 12.5 (E12.5). Treatments started from postnatal day (P44). Injections (once daily) continued for 21 days until P64. Behavioral studies were carried starting from P51. All mice were then sacrificed at P64 for biochemical analyses.

Figure 2

Effects of DL77 and DOZ on sociability assessed in TCT paradigm. After 10 minutes of acclimatization, male subjects were allowed to explore all chambers for 10 min. The results obtained were (A) time spent in the chamber of the novel object (NO), the novel mouse (NM), or the central chamber (CC); (B) time spent exploring the novel object (NO) or novel mouse (NM); (C) abrogative effects of subchronic (21 days) systemic co-administration of RAMH (10 mg/kg, i.p. for IX), PYR (10 mg/kg, i.p. for X), or ZOL (10 mg/kg, i.p., for XI) on the DL77 (10 mg)-provided enhancement of sociability. Saline-exposed mice were injected with saline in group I, DL77 (10 mg/kg, i.p.) in group VII, or DOZ (1 mg/kg, i.p.) in group VIII (A,B). DL77 (at a dose of 5, 10, or 15 mg/kg in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in groups VI and VIII) was administered subchronically for 21 days. Figure shows mean ± SEM (n = 6–7). ^#P < 0.05 vs. time spent in the chamber of NO (A–C) or vs. SI of VPA-exposed mice (group II) (D). (D) Sociability index (SI). *P < 0.05 vs. time spent exploring NM of DL77(10 mg)-treated VPA-exposed mice (group IV) (C) or vs. SI of (saline)-treated saline-exposed mice (group I) (D). ^$P < 0.05 vs. DL77(10 mg)-treated VPA-exposed mice (group IV) (D).

Figure 3

Effects of DL77 and DOZ on social novelty assessed in TCT paradigm. After 10 minutes of acclimatization, male subjects were allowed to explore all chambers for 10 min. The results obtained were (A) time spent in the chamber of the familiar mouse (FM), novel mouse (NM), or central chamber (CC); (B) time spent exploring the familiar mouse (FM) or novel mouse (NM); (C) effects of subchronic (21 days) systemic co-administration of RAMH (10 mg/kg, i.p. for IX), PYR (10 mg/kg, i.p. for X), or ZOL (10 mg/kg, i.p., for XI) on the DL77 (10 mg)-provided enhancement of social novelty. Saline-exposed mice were injected with saline in group I, DL77 (10 mg/kg, i.p.) in group VII, or DOZ (1 mg/kg, i.p.) in group VIII (A,B). DL77 (at a dose of 5, 10, or 15 mg/kg in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in groups VI and VIII) was administered subchronically for 21 days. Figure shows mean ± SEM (n = 5–7). ^#P < 0.05 vs. time spent in the chamber FM (A–C) or vs. SNI of VPA-exposed mice (group II) (D). (D) Social novelty index (SNI). *P < 0.05 vs. time spent exploring NM of DL77(10 mg)-treated VPA-exposed mice (group IV) (C) or vs. SNI of (saline)-treated saline-exposed mice (group I) (D). ^$P < 0.05 vs. DL77(10 mg)-treated VPA-exposed mice (group IV) (D).

Figure 4

Effects of DL77 and DOZ on elevated stereotyped repetitive behavior in VPA mice exposed to MBT. (A) Repetitive marble-burying behavior was measured after a 30-minute testing session. VPA-exposed mice (II) demonstrated elevated stereotyped, repetitive behaviors that were significantly increased compared to saline-exposed mice (I). DL77 (at a dose of 5, 10, or 15 mg/kg in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in groups VI and VIII) was administered subchronically for 21 days. (B) Effects of sub-chronic (21 days) systemic co-administration of RAMH (10 mg/kg, i.p. for IX), PYR (10 mg/kg, i.p. for X), or ZOL (10 mg/kg, i.p., for XI) on the DL77 (10 mg)-provided attenuation of stereotyped repetitive behavior of VPA-exposed mice in MBT. Saline-exposed mice were injected with saline in group I, DL77 (10 mg/kg, i.p.) in group VII, or DOZ (1 mg/kg, i.p.) in group VIII (A). Figures show mean ± SEM (n = 7). *P < 0.05 vs. saline-exposed mice. ^#P < 0.05 vs. VPA-exposed mice.

Figure 5

Effects of DL77 and DOZ on elevated stereotyped repetitive behavior in VPA mice exposed to NST. (A) Repetitive nestlet-shredding behavior was measured after a 30-minute testing session. VPA-exposed mice (II) demonstrated elevated stereotyped repetitive behaviors that were significantly increased compared to saline-exposed mice (I). DL77 (at a dose of 5, 10, or 15 mg/kg in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in groups VI and VIII) was administered sub-chronically for 21 days. (B) Effects of subchronic (21 days) systemic co-administration of RAMH (10 mg/kg, i.p. for IX), PYR (10 mg/kg, i.p. for X), or ZOL (10 mg/kg, i.p., for XI) on DL77 (10 mg)-provided attenuation of stereotyped repetitive behavior of VPA-exposed mice in NST. Saline-exposed mice were injected with saline in group I, DL77 (10 mg/kg, i.p.) in group VII, or DOZ (1 mg/kg, i.p.) in group VIII (A). Figures show mean ± SEM (n = 7). *P < 0.05 vs. saline-exposed mice. ^#P < 0.05 vs. VPA-exposed mice.

Figure 6

Effects of DL77 and DOZ pretreatment on exploratory behavior in the EPM test. VPA-exposed mice (II) demonstrated elevated impulsive attitude and deficits in cognition behaviors that were significantly increased compared to saline-exposed mice (I). DL77 (5, 10, or 15 mg/kg, i.p. in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p., in group VI) was administered subchronically for 21 days. DOZ (1 mg/kg, i.p. in groups VI and VIII) attenuated the increased percentage of time spent on the open arms of the EPM (A) the increased number of entries into the open arms (B) and the increased number of entries into the closed arms (C) in VPA mice. However, pretreatment with the H3R antagonist DL77 (5, 10, or 15 mg/kg, i.p.) did not affect any of the three parameters (A–C). Saline-exposed mice were injected with saline in group I and DOZ (1 mg/kg, i.p.) in group VIII (A–C). Data are expressed as the mean ± SEM (n = 5–7). *P < 0.05 vs. saline-exposed mice. ^#P < 0.05 vs. VPA-exposed mice.

Figure 7

Effects of DL77 and DOZ pretreatment on levels of malondialdehyde (MDA) and glutathione (GSH) in the cerebellum. VPA-exposed mice (II) showed a significant increase in MDA (A) and significant decrease in GSH (B) compared to saline-exposed mice (I). DL77 (5, 10, or 15 mg/kg, i.p. in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in group VI) was administered subchronically for 21 days. DL77 (10 or 15 mg/kg, i.p. in group IV or V, respectively) or DOZ (1 mg/kg, i.p. in group VI) significantly decreased MDA and significantly increased GSH. RAMH (10 mg/kg, i.p., co-administered in group IX) abrogated the modulating DL77 (10 mg)-provided effects on oxidative stress levels (A,B). Saline-exposed mice were injected with saline in group I (A,B). Data are expressed as the mean ± SEM (n = 5–7). *P < 0.05 vs. saline-exposed mice. ^#P < 0.05 vs. VPA-exposed mice. ^$P < 0.05 vs. DL77 (10 mg)-treated VPA-exposed mice.

Figure 8

Effects of DL77 and DOZ pretreatment on levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in the cerebellum. VPA-exposed mice (II) showed significantly increased IL-1β (A) IL-6 (B) and TNF-α (C) compared to saline-exposed mice (I) exacerbated by LPS challenge. DL77 (5, 10, or 15 mg/kg, i.p. in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in group VI) was administered subchronically for 21 days. DL77 (5, 10, or 15 mg/kg, i.p. in group III, IV, or V, respectively) or DOZ (1 mg/kg, i.p. in group VI) significantly decreased IL-1β (A), IL-6 (B) and TNF-α (C). RAMH (10 mg/kg, i.p., co-administered in group IX) reversed the modulating DL77 (10 mg)-provided effects on proinflammatory cytokines (A–C). Saline-exposed mice were injected with saline in group I (A–C). Data are expressed as the mean ± SEM (n = 5–7). *P < 0.05 vs. saline-exposed mice. ^#P < 0.05 vs. VPA-exposed mice. ^$P < 0.05 vs. DL77 (10 mg)-treated VPA-exposed mice.