Introduction
Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders in the nervous system, which is characterized by difficulties in social interactions, as well as verbal and nonverbal communication, and repetitive behaviors (Llaneza et al., 2010 ). ASD is the most expensive medical condition in the USA, costing $61 billion annually, which is more than the costs of heart disease, stroke, and cancer combined (Buescher et al., 2014 ). According to the Centers for Disease Control and Prevention, one in 68 American children suffers from ASD (Christensen et al., 2018 ), a 10-fold increase over the last 40 years. Based on an epidemiological study, 26.4% of the rising prevalence of autism can be attributed to changes in diagnostic practices (King and Bearman, 2009 ).
ASD behavioral symptoms typically appear between the ages of 2 and 3 years old and persist throughout life (Elder et al., 2017 ; Benger et al., 2018 ). ASD is primarily diagnosed based on behavioral evaluation and parent questionnaires (Hennel et al. 2016 ; Elder et al., 2017 ), as reliable biomarkers are still in the research phase (Varcin and Nelson, 2016 ). It appears that genetic (De Rubeis et al., 2014 ; Hansen et al., 2015 ) and environmental (Chiarotti and Venerosi, 2020 ) factors influence the pathogenesis of ASD. Researchers have found that the maternal use of valproic acid (VPA) during pregnancy increases the risk of autism in humans (Ornoy, 2009 ) and rodents (Sabzalizadeh et al. 2022 ; Haratizadeh et al. 2023 ).
Animal studies have also reported social interaction deficits (Sabzalizadeh et al., 2022 ; Taheri et al., 2022 ) and increased repetitive behaviors in VPA-exposed animals (Baronio et al., 2015 ; Eissa et al., 2018b ). Our previous study showed social deficits in adult VPA-exposed rats (Taheri et al., 2022 ). Additionally, prenatal VPA caused increased anxiety in mice, as indicated by a significant decrease in the number of rearing and the frequency of entries into the center field during an open-field task (Yamaguchi et al., 2017 ; Gao et al., 2019 ).
According to clinical observations, about 75% of individuals with ASD experience learning and memory problems (Meador et al., 2009 ). The ability to recognize faces and executive functions are particularly impaired in children with ASD, which suggests recognition memory impairment (Golshan et al., 2019 ; Semino et al., 2021 ). Moreover, mice prenatally exposed to VPA exhibited impairments in recognition memory (Hara et al., 2017 ; Sungur et al., 2017 ). The hippocampus plays an important role in learning and memory formation (Miguez et al., 2015 ). ASD patients (Meador et al., 2009 ) and the VPA-induced animal model of autism (Gao et al., 2016 ) showed remarkable changes in the structure of the hippocampus, including apoptosis, pyramidal cell loss, and spine density of pyramidal cells (Takuma et al., 2014 ; Yang et al., 2016 ).
Several studies have focused on the various brain neurotransmitters such as histamine and their influence on behavior in brain disorders (Alhusaini et al., 2022 ; Taheri et al., 2022 ). Histamine plays a significant role in basic physiological functions such as sleep-wake cycles, sensory and motor functions, cognition, and attention, all of which are severely affected in neuropsychiatric disorders (Sadek et al., 2016a ).
The H1 receptors (H1Rs), H2 receptors (H2Rs), H3 receptors (H3Rs), and H4 receptors (H4Rs) are responsible for histamine’s functions. Among these receptors, H3Rs are presynaptic autoreceptors that block the synthesis and release of histamine from histaminergic neurons and heteroreceptors that control the release of other neurotransmitters (Panula and Nuutinen, 2013 ; Panula et al., 2015 ). By inhibiting H3Rs, histamine release, as well as the release of other neurotransmitters (Panula and Nuutinen, 2013 ), is enhanced in the regions of the brain necessary for alertness and information storage (Sadek et al., 2016a , 2016b ; Zlomuzica et al., 2016 ).
For disorders such as Alzheimer’s disease and narcolepsy, antagonists targeting H3Rs are considered promising alternative therapies (Sadek et al., 2016a ). Various cognitive disorders have been improved by increasing histamine in the synaptic space (Zlomuzica et al., 2016 ). Additionally, histamine has been shown to control several aspects of behavior and arousal in animal studies (Ferreira et al., 2012 ; Sadek et al., 2016b ; Eissa et al., 2018c ). H1Rs, H2Rs, and H3Rs are involved in the consolidation of object recognition memory in the histaminergic system (da Silveira et al., 2013 ). Several diseases such as Alzheimer’s disease, sleep disorders, and autism appear to target the histaminergic system of the brain (Haas and Panula, 2003 ).
To the best of our knowledge, no documented study has yet investigated the effects of H3R and H2R antagonists and their combination on the histological changes of neurons in the CA1 area of the hippocampus and cognitive performance in the offspring of rats exposed to VPA. As a result, the goal of the current investigation was to determine the impact of the H3R antagonist on hippocampal neuronal alterations and assess cognitive functions in the offspring of rats exposed to VPA. To ascertain the function of H2R in brain histaminergic neurotransmission and its interaction with H3R antagonist ciproxifan (CPX) in cognitive activities, the effects of the H2R antagonist famotidine (FAM) on behavioral and neuronal alterations in the hippocampus were also evaluated.
Methods
Subjects
Wistar rats were used in this experiment. The animals were housed in standard plastic cages in a room maintained at 23 ± 2°C under 12/12-h light-dark cycle. Animals had ad libitum access to food and water. The experimental procedures were conducted in accordance with guidelines relevant to the care of experimental animals, as approved by the Institutional Animal Research Ethics Committee of Kerman University of Medical Sciences (Ethics code: IR.KMU.REC.1400.059).
Pregnancy determination
Male and female rats were mated overnight. Pregnancy was identified by the existence of the spermatozoa in vaginal smear or the presence of vaginal plugs (Taheri et al., 2019 ) the next morning, and that day was defined as embryonic day 0 (Taheri et al., 2018 ). Pregnant rats were randomly divided into two groups. VPA: Pregnant rats received an intraperitoneal injection of 600 mg/kg VPA which was diluted with normal saline to a concentration of 200 mg/mL at embryonic day 12.5 (E12.5) (Taheri et al., 2022 ). Saline: Pregnant rats received an intraperitoneal injection of saline at E12.5.
Drugs
Drugs used in the present study were H3R antagonist (CPX), sodium VPA, and urethane purchased from Sigma-Aldrich, USA. The pharmaceutical company, SMS Life Sciences India Limited, prepared FAM. The drugs were dissolved in normal saline, with the exception of FAM, which was dissolved in one drop of 1 M hydrochloric acid and then diluted in normal saline (Taheri et al., 2022 ). The solutions were freshly prepared daily. H3R antagonist CPX (1 and 3 mg/kg), H2R antagonist FAM (10, 20, and 40 mg/kg), or vehicle (saline) were administered 30 min before behavioral tests (Taheri et al., 2022 ).
Experimental groups
Offspring of both groups, VPA and Saline, were divided into seven subgroups according to treatment with Saline, CPX (1 and 3 mg/kg), FAM (10, 20, and 40 mg/kg), and a combination of effective doses of CPX and FAM (CPX3+ FAM20) (Fig. 1 ). One pup from each litter was used in each treatment group and totally, 98 male rats were tested during adolescence [postnatal day (PND) 48–50], and the sample size was n = 7 per condition. The timeline is shown in Fig. 2 . The experiments of the current study [marble burying task (MBT), open field, novel object recognition (NOR), and Passive avoidance tests] were carried out between 8:00 a.m. and 1 p.m. in 1 day.
Fig. 1: Animal groups arrangement. CPX, ciproxifan ; FAM, famotidine; VPA, valproic acid.
Fig. 2: Timeline diagram. VPA, valproic acid.
Behavioral assessments
Marble-burying test
MBT is an accurate reflection of repetitive digging behavior (Thomas et al., 2009 ). Briefly, cages (26 cm × 48 cm × 20 cm) were filled with fresh, unscented rat bedding material to a depth of 5 cm, and the bedding surface was leveled by placing another cage of the same size onto the surface of the bedding. For habituation, each rat was added individually. The rat was removed after 10 min, and 20 glass marbles (15 mm diameter) were arranged equidistantly in a 4 × 5 arrangement on the bedding surface. For 30 min, each rat was allowed to explore its designated test cage. We recorded the number of marbles buried (more than 50% of marble was covered by the bedding) (Kim et al., 2014 ). During this test, the number of marbles buried serves as a proxy measure of rat digging behavior.
Open-field test
The open-field test is used to evaluate exploratory, locomotor activity, and anxiety-like behaviors as well as repetitive behaviors in rodents (Servadio et al., 2015 ). The used apparatus was made up of Plexiglas (90 × 90 × 50 cm) with high walls and a dark floor. The box was divided into 16 small squares by imaginary lines in the Noldus Ethovision system; therefore, the mice could spend time in both peripheral and central areas. The animal was gently placed in the center of the apparatus and its locomotor and anxiety-like behaviors were recorded for 5 min using an automatic video camera fixed above the apparatus. Total movement distance and the velocity of the movement were measured to evaluate locomotor activity. In order to evaluate anxiety-like behavior, the time spent in the inner zone (central area) was measured (Fig. 3 ). Furthermore, the open-field test can be used to determine the amount of repetitive and monotonous activities (Servadio et al., 2015 ). Animal movements, such as wiping and licking the face and body, were recorded continuously and consecutively (Malkova et al., 2012 ). This level of movement was considered repetitive and monotonous in an animal model of autism (McFarlane et al., 2008 ). It has been shown that many animal models of autism exhibit stereotypical and repetitive movement parameters as well as anxiety-like behaviors in an open field (Moy et al., 2008 ). To avoid an odor signature, the apparatus was cleaned with 70% ethanol, and the test was performed in a light-controlled environment. A video camera on top of the field recorded these elements, and the animal’s performance was recorded by the Noldus Ethovision system, version 7.1, Noldus Company, Netherland. The number of grooming behavior was recorded by the experimenter (Taheri et al., 2020 ).
Fig. 3: Open-field task.
Novel object recognition test
The NOR task evaluates the rodents’ ability to recognize a novel object in the environment (Fig. 4 ). The task procedure consists of three phases: habituation, familiarization (training), and test phases. In the habituation phase, each rat is allowed to freely explore an empty wooden (60 × 60 × 40 cm) arena for 10 min. Twenty-four hours after the habituation, animals were allowed to explore two identical objects, at the same location inside the box for 5 min (training phase) (Fig. 4a ). After a retention interval of 15 min, during 3-min test phase, rat is returned to arena with two objects, one is familiar and other is novel (Fig. 4b ).
Fig. 4: Novel object recognition task.
The location of novel versus familiar objects is counterbalanced between trials. The rats spend more time exploring the novel object during the first few minutes of the test phase, and when this bias is observed, the rats can recall the sample object (Sivakumaran et al., 2018 ). In the training and test phases, animals that did not explore any of the objects were excluded from the analysis (Languille et al., 2015 ).
Objects were randomly changed to avoid the natural preference of animals and both objects and boxes were cleaned with 70% ethanol before the trial. During the test phase, sniffing or touching the object at a distance of less than 2 cm was quantified by the camera. Finally, a discrimination ratio (recognition index) was defined as the ratio of time spent exploring each object to the total time spent exploring both objects multiplied by 100 in the training and test phases (Taheri et al., 2020 ).
Passive avoidance test
The Passive avoidance task is a fear-aggravated test used to evaluate associative learning and memory in rodents. The animal learns to avoid an environment in which a prior aversive stimulus has been delivered. Shuttle-box device with dimensions of 100 [L] × 25[W] × 25[H] (cm) consisting of two compartments (light and dark) separated by a door was used. Each animal was first habituated to the test equipment by placing it in the light compartment for 5 min, the animal was allowed to move to the dark chapter, and then it was returned to the home cage. This process was repeated once and if an animal failed to move into the dark compartment, it was removed from the study. Finally, 2 h later, the animal was placed into the light compartment, the door was opened after 10 s and, on entering the dark compartment, it was given an electric shock (50 Hz, 0.5 mA, 2 s; via wires embedded in the dark chamber floor). This final part of the process was repeated at 2 min intervals until the animal learned to avoid the dark compartment (remained in the light compartment for at least 120 s) and the number of shocks required for learning was recorded. The assessment phase of the test was undertaken 24 h after the learning phase. The animal was placed in the light chamber (door closed) and, after 10 s, the door was opened, and the time taken by the animal to enter the dark chamber was recorded as the step-through latency (STL). The total time spent in the dark compartment during a period of 5 min after door opening was also recorded (Rehman et al., 2020 ).
The behavioral tests were performed 30 min after the injection of saline or different doses of CPX and FAM.
Histology : hematoxylin and eosin
After the end of the behavioral tests, all of the groups were anesthetized with carbon dioxide and then sacrificed. The brain was immediately fixed in 10% buffered formalin and processed according to a histological method. Paraffinized brain tissues were cut into 8-μm sections using a semi-automated microtome, and the sections were stained with hematoxylin and eosin (Sigma, Missouri, USA). For assessment of neuronal counting, stained sections of the hippocampal CA1 area were evaluated. Neurons were counted in three microscopic fields at 400X magnification, and neurons with visible round nucleus, prominent nucleolus, and intact cytoplasm were counted as intact neurons.
Statistical analysis
To evaluate the effect of H3 antagonist on measured parameters, two-way analysis of variance (ANOVA) was used to compare the mean differences between groups in behavioral tests (MBT, open field, NOR, and Passive avoidance). To analyze the histological results, one-way ANOVA was used. If there was statistical significance between the groups, Turkey’s multiple-comparison was performed as a post-hoc. The data were presented as mean ± SEM and differences among groups were considered significant at a P < 0.05. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, California, USA).
Results
Effect of valproic acid on crooked-tail phenotype
Genetic mutations, malnutrition, and teratogen exposure during gestation can cause neural tube defects (NTD) (Kaufman, 2004 ). Previous studies determined that VPA-induced crooked tail phenotypes, a mild form of NTD, and autistic-like behavior impairments in mice and rats.
(Ornoy, 2009 ; Kim et al., 2011 ). VPA induced the crooked-tail phenotype in all rats in the current study (Fig. 5 ).
Fig. 5: Neural tube defects by VPA exposure. Malformations of tail structure occurred in the VPA-exposed group. VPA, valproic acid.
Stereotyped repetitive behavior test
Effects of H3 antagonist (ciproxifan ) on stereotyped repetitive behavior in valproic acid-exposed rats in marble-burying test
The MBT test was used to evaluate repetitive behavior . MBT behavioral is an accurate reflection of repetitive digging behavior. Two-way ANOVA showed a significant difference among groups in repetitive behavior [F (2, 36) = 4.372]. In the VPA + Saline group, repetitive behaviors were significantly higher than in the Saline + Saline group. Indeed, the VPA + Saline group buried significantly more marbles than the Saline + Saline group (P < 0.01). In the VPA + CPX3 group, CPX3 treatment significantly decreased the elevated percentage of marbles buried compared to the VPA + Saline group (P < 0.01) (Fig. 6a ).
Fig. 6: Effects of CPX, FAM, and combination of (CPX and FAM) on stereotyped repetitive behavior in the VPA-exposed rats in MBT. (a) VPA-exposed rats buried significantly more marbles compared to the saline-exposed rats. After treatment with CPX, the elevated percentage of marbles buried significantly decreased in the VPA + CPX3 group compared to the VPA + Saline group. (b) FAM had no effect on the percentage of buried marbles. (c) Combination of CPX and FAM had no effect on the percentage of buried marbles. Two-way ANOVA was used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05,**P < 0.01 vs. Saline + Saline, ##P < 0.01 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
Effects of H2 antagonist (famotidine) on stereotyped repetitive behavior in valproic acid-exposed rats in marble-burying test
We found no significant difference between the VPA + Saline group and the VPA + FAM10, VPA + FAM20, and VPA + FAM40 groups in the MBT test. FAM did not affect the percentage of marbles buried (Fig. 6b ).
Effects of combination of H2 H3 antagonists (ciproxifan and famotidine) on stereotyped repetitive behavior in valproic acid-exposed rats in marble burying task
ANOVA analysis showed no significant difference among groups in the percentage of marbles buried after treatment with CPX and FAM (Fig. 6c ).
Open-field test
Effects of H3 antagonist (ciproxifan ) on locomotor activity and anxiety-like behavior of valproic acid-exposed rats in open field
To evaluate mood-related behavior, an open-field test was conducted. After CPX treatment, two-way ANOVA showed no significant difference among groups in total distance (Fig. 7a ) and velocity (Fig. 7b ). Two-way ANOVA showed significant differences among groups in time spent in inner zone [F (2, 36) = 10.47]. Saline + CPX1 and VPA + Saline groups spent less time in the inner zone than the Saline + Saline group (Saline + CPX1: P < 0.05, and VPA + Saline: P < 0.01). As well as, the VPA + CPX3 group spent more time in the inner zone than the VPA + Saline group (P < 0.05) (Fig. 7c ).
Fig. 7: Effects of H3 antagonist (CPX) on locomotor activity and anxiety-like behavior in open field. (a) There was no significant difference among groups in total distance and (b) velocity among groups. (c) Time spent in the inner zone was decreased in Saline + CPX1 and VPA + Saline groups compared to the Saline + Saline group. (d) Grooming behavior was higher in the VPA + Saline group compared to the Saline + Saline group. VPA + CPX3 had lower grooming behavior compared to the VPA + Saline group. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Saline + Saline, #P < 0.05, ##P < 0.01 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble burying test; VPA, valproic acid.
Two-way ANOVA showed that there was significant difference among groups in grooming behavior [F (2, 36) = 9.055]. VPA increased grooming behavior in the VPA + Saline group compared to the Saline + Saline group (P < 0.001). In addition, CPX3 decreased grooming behavior in the VPA + CPX3 group compared to the VPA + Saline group (P < 0.01) (Fig. 7d ).
Effects of H2 antagonist (famotidine) on locomotor activity and anxiety-like behavior of valproic acid-exposed rats in open field
After FAM treatment, two-way ANOVA showed that there was no significant difference among groups in total distance (Fig. 8a ) and velocity (Fig. 8b ). Two-way ANOVA showed that time spent in inner zone was significantly different among groups [F (3, 48) = 15.08]. So the Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline groups spent less time in the inner zone than the Saline + Saline group (Saline + FAM10: P < 0.01, Saline + FAM20 and Saline + FAM40: P < 0.001, VPA + Saline: P < 0.05) (Fig. 8c ). As well as, the VPA + FAM20, VPA + FAM40 groups spent less time in the inner zone than the VPA + Saline group (P < 0.05) (Fig. 8c ).
Fig. 8: Effects of H2 antagonist (famotidine) on locomotor activity and anxiety-like behavior in open field. (a) There was no significant difference among groups in total distance and (b) velocity among groups. (c) Time spent in the inner zone was decreased in VPA + Saline, Saline + FAM10, Saline + FAM20, and Saline + FAM 40 groups compared to the Saline + Saline group. In addition, time spent in the inner zone was decreased in VPA + FAM20 and VPA + FAM40 groups compared to the VPA + Saline group. (d) Grooming behavior was higher in Saline + FAM20, Saline + FAM40, and VPA + Saline compared to the Saline + Saline group. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05, **P < 0.01, ***P < 0.001 vs Saline + Saline, # P < 0.05 vs VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
There was a significant difference among groups in grooming behavior [F (3, 48) = 1.161]. FAM increased grooming behavior in Saline + FAM20 and Saline + FAM40 groups compared to the Saline + Saline group (Saline + FAM20: P < 0.05 and Saline + FAM40: P < 0. 01, VPA + Saline: P < 0.001) (Fig. 8d ).
Effects of the combination of H3 and H2 antagonists (ciproxifan and famotidine) on locomotor activity and anxiety-like behavior of valproic acid-exposed rats in open field
Two-way ANOVA showed that there was no significant difference among groups in total distance (Fig. 9a ) and velocity (Fig. 8b ). A two-way ANOVA showed that time spent in inner zone was significantly different among groups [F (1, 24) = 25.99]. So that, the Saline + CPX3 + FAM20 group spent less time in the inner zone than the Saline + CPX3 group (P < 0.05) (Fig. 9c ). Also, the VPA + CPX3 + FAM20 group spent less time in the inner zone than the VPA + CPX3 group (P < 0.01) (Fig. 9c ). There was no significant difference among groups in grooming behavior (Fig. 9d ).
Fig. 9: Effects of the combination of H3 and H2 antagonists (CPX and FAM) on locomotor activity and anxiety-like behavior in open field. (a) There was no significant difference among groups in total distance and (b) velocity among groups. (c) Time spent in the inner zone was decreased in the Saline + CPX3 + FAM20 group compared to the Saline + CPX3 group. In addition, time spent in the inner zone was decreased in the VPA + CPX3 + FAM20 group compared to the VPA + CPX3. (d) There was no significant difference among groups in grooming behavior. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05 vs. Saline + CPX3, ## P < 0.01 vs VPA + CPX3. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble burying test; VPA, valproic acid.
Novel object recognition test
Effects of H3 antagonist (ciproxifan ) on novel object recognition in valproic acid-exposed rats
In the first trial (Training phase) with two familiar objects, all the groups spend a similar amount of time exploring the objects. There was no significant difference in discrimination ratio among groups in the training phase (Fig. 10a ). In the second trial (Test phase), the object recognition memory was assessed by replacing the object of the first trial with a copy of the original object (familiar object) and a novel object (unfamiliar object). NOR memory was reflected in performance for exploring the novel object. There was a significant difference in discrimination ratio among groups [F (2, 36) = 6.384]. The new objective recognition memory was significantly disrupted in the VPA + Saline group rats. VPA + Saline group rats failed to spend a greater amount of time investigating the novel object than the familiar object, and they were not significantly biased toward the novel object. In these animals, the discrimination ratio was significantly lower than that in the Saline + Saline group (P < 0.001) (Fig. 10b ). While administration of the CPX3, 30 min prior to the test, improved the recognition memory impairment so that the discrimination ratio was significantly increased in the VPA + CPX3 group compared to the VPA + Saline group (P < 0.05) (Fig. 10b ). Two-way ANOVA test was used.
Fig. 10: The effects of H3 antagonist (CPX) on Novel object recognition test. (a) There was no significant difference in exploring time in the training session between all groups. (b) There was a significant decreased in discrimination ratio in the VPA + Saline group compared to the Saline + Saline group in the test phase. The discrimination ratio increased in the VPA + CPX3 group compared to the VPA + Saline group in the test phase. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. ***P < 0.001 vs. Saline + Saline, #P < 0.05 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
Effects of H2 antagonist (famotidine) on novel object recognition in valproic acid-exposed rats
In the first trial (Training phase) was no significant difference in the discrimination ratio among groups in the training phase (Fig. 11a ). In the second trial (Test phase), there was a significant difference in discrimination ratio among groups [F (3, 48) = 39.18]. The new objective recognition memory was significantly disrupted in Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline group rats. These groups failed to spend a greater amount of time investigating the novel object than the familiar object, and they were not significantly biased toward the novel object. In these animals, the discrimination ratio was significantly lower than that in the Saline + Saline group (Saline + FAM10: P < 0.05, Saline + FAM20, Saline + FAM40, and VPA + Saline: P < 0.001). In addition, the discrimination ratio was significantly lower in the VPA + FAM40 group compared to the VPA + Saline group (P < 0.01) (Fig. 11b ). Two-way ANOVA followed test was used.
Fig. 11: Effects of H2 antagonist (FAM) on Novel object recognition test. (a) There was no significant difference in exploring time in the training phase between all groups. (b) There was a significant decreased in discrimination ratio in Saline + FAM20, Saline + FAM40, and VPA + Saline groups compared to the Saline + Saline group in the test phase. The discrimination ratio decreased in the VPA + FAM40 group compared to the VPA + Saline group in the test phase. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Saline + Saline, ##P < 0.01 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble burying test; VPA, valproic acid.
Effects of the combination of H2 and H3 antagonists (ciproxifan and famotidine) on novel object recognition in valproic acid-exposed rats
There was no significant difference in discrimination ratio among groups in the training phase (Fig. 12a ). In the test phase, there was a significant difference in discrimination ratio among groups [F (1, 24) = 64.95]. The new objective recognition memory was significantly disrupted in Saline + CPX3 + FAM20 and VPA + CPX3 + FAM20 groups. These animals failed to spend a greater amount of time investigating the novel object than the familiar object, and they were not significantly biased toward the novel object. So, the discrimination ratio was significantly lower in the Saline + CPX3 + FAM20 compared to the Saline + CPX3 group (P < 0.001) (Fig. 12b ). In addition, the discrimination ratio was lower in the VPA + CPX3 + FAM20 compared to the VPA + CPX3 group (P < 0.001) (Fig. 12b ). Two-way ANOVA followed test was used.
Fig. 12: Effects of the combination of H3 and H2 antagonists (CPX and FAM) on Novel object recognition test. (a) There was no significant difference in exploring time in the training phase between all groups. (b) There was a significant decreased in discrimination ratio in Saline + CPX3 + FAM20 and VPA + CPX3 + FAM20 groups in the test phase. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. ***P < 0.001 vs. Saline + CPX3, ### P < 0.001 vs. VPA + CPX3. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
Passive avoidance test
Effects of H3 antagonist (ciproxifan ) on Passive avoidance learning and memory in valproic acid-exposed rats
The influence of CPX3 on Passive avoidance learning is shown in Fig. 13 . There was no significant difference in shock number between groups. Figure 13b and c ) shows the effect of CPX3 on Passive avoidance memory. Two-way ANOVA showed a significant difference in time spent in the dark compartment among groups [F (2, 36) = 12.54]. So that time spent in the dark compartment increased in the VPA + Saline group compared to the Saline + Saline group (P < 0.001) (Fig. 13b ). In addition, the VPA + CPX3 group showed a significant decrease in time spent in the dark compartment compared to the VPA + Saline group (P < 0.001) (Fig. 13b ).
Fig. 13: The effects of H3 antagonist (CPX) on learning trial (shock number) (a) and retention test including time spent in dark compartment (b) and step-through latency (c). (a) There was no significant difference in shock number between VPA + Saline and Saline +Saline groups. (b) Time spent in the dark compartment significantly increased in the VPA + Saline group compared to the Saline +Saline. Also, step-through latency significantly decreased in the VPA + Saline group compared to the Saline +Saline. CPX3 decreased time spent in the dark compartment (b) in the VPA+CPX3 compared to the VPA + Saline and also increased step-through latency in the VPA+CPX3 compared to the VPA + Saline. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05, ***P < 0.001 vs. Saline + Saline, #P < 0.05, ###P < 0.001 vs. VPA + Saline. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble burying test; VPA, valproic acid.
There was a significant difference in STL among groups [F (2, 36) = 9.396]. STL significantly decreased in the VPA + Saline group compared to the Saline + Saline group (P < 0.05) (Fig. 13c ). STL significantly increased in the VPA + CPX3 group compared to the VPA + Saline group (P < 0.05) (Fig. 13c ).
Effects of H2 antagonist (famotidine) on Passive avoidance learning and memory in valproic acid-exposed rats
The results of Passive avoidance test showed that shock numbers were no significant difference among groups (Fig. 14a ). Two-way ANOVA showed a significant difference in time spent in the dark compartment among groups [F (1, 24) = 2.191]. So that after treatment with FAM, time spent in the dark compartment increased in Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline groups compared to the Saline + Saline group (Saline + FAM10: P < 0.05, Saline + FAM20 and Saline + FAM40: P < 0.001, VPA + Saline: P < 0.05). In addition, the VPA+FAM20 group showed significant increase in time spent in the dark compartment compared to the VPA + Saline group (Fig. 14b . P < 0.05).
Fig. 14: The effects of H2 antagonist (FAM) on learning trial (shock number) (a) and retention test including time spent in dark compartment (b) and step-through latency (c). (a) There was no significant difference in shock number among groups. (b) Time spent in the dark compartment significantly increased in Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline groups compared to the saline. Also, step-through latency significantly decreased in Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline groups compared to the Saline + Saline group. FAM increased time spent in the dark compartment (b) in the VPA + FAM20 compared to the VPA + Saline also increased step-through latency in VPA + FAM10, VPA + FAM20, and VPA + FAM40 compared to the VPA + Saline. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. *P < 0.05, **P < 0.01, ***P < 0.001, vs. Saline + Saline, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. VPA + Saline. CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
There was a significant difference in STL among groups [F (1, 24) = 6.331]. STL significantly decreased in the Saline + FAM10, Saline + FAM20, Saline + FAM40, and VPA + Saline groups compared to the Saline + Saline group (Saline + FAM10, Saline + FAM20, and Saline + FAM40: P < 0.001, VPA + Saline: P < 0.01) (Fig. 14b and c ). In addition, STL increased in VPA+FAM10, VPA+FAM20, and VPA+FAM40 groups compared to the VPA + Saline group (VPA+FAM10, and VPA+FAM40: P < 0.001, VPA+FAM20: P < 0.01) (Fig. 14c ).
Effects of the combination of H2 and H3 antagonists (ciproxifan and famotidine) on Passive avoidance learning and memory in valproic acid-exposed rats
The influence of the combination of H3 and H2 antagonists (CPX + FAM) on Passive avoidance learning has shown in (Fig. 15 ). Our findings showed that there was no significant difference in shock number among groups (Fig. 15a ). Figure 15b and c ) shows the effect of the combination of H2 and H3 antagonists (CPX + FAM) on Passive avoidance memory. Two-way ANOVA showed significant difference in time spent in the dark compartment among groups [F (1, 24) = 2.191].
Fig. 15: The effects of the combination of H3 and H2 antagonists (CPX and FAM) on learning trial (shock number) (a) and retention test including time spent in the dark compartment (b) and step-through latency (c). (a) There was no significant difference in shock number among groups. (b) FAM increased time spent in the dark compartment. Time spent in the dark compartment significantly increased in the Saline + CPX3 + FAM20 group compared to the Saline + CPX3 group. Also, step-through latency significantly decreased in the VPA + CPX3 + FAM20 compared to the VPA + CPX3 group. Two-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. **P < 0.01, ***P < 0.001 vs. Saline + CPX3, ###P < 0.001 vs. VPA + CPX3. CPX, ciproxifan ; FAM, famotidine; MBT, marble burying test; VPA, valproic acid.
The Saline + CPX3 + FAM20 group showed a significant increase in time spent in the dark compartment compared to the Saline + CPX3 group (P < 0.01) (Fig. 15b ). In addition, time spent in the dark compartment increased in the VPA + CPX3 + FAM20 group compared to the VPA + CPX3 group (P < 0.001) (Fig. 15b ).
There was a significant difference in STL among groups [F (1, 24) = 6.331]. STL decreased in the Saline + CPX3 + FAM20 group compared to the Saline + CPX3 group (Fig. 14c ; P < 0.001). In addition, STL decreased in the VPA + CPX3 + FAM20 group compared to the VPA + CPX3 group (Fig. 15c ; P < 0.001).
Histological evaluations
After obtaining effective doses of CPX and FAM (CPX 3 mg/kg and FAM 20 mg/kg, respectively), a histological study was performed.
Histological evaluation showed that VPA decreased the number of CA1 neurons compared to the saline group, but this decrease was not significant (P = 0.43). On the other hand, our morphological investigation showed that VPA treatment did not lead to neuronal degeneration in the CA1 subfield of the hippocampus in rats’ pups. CPX3 increased the number of CA1 neurons in VPA + CPX3 group compared to the VPA + Saline group, but this increase was not significant (P = 0.99). The number of CA1 neurons decreased in VPA + FAM20 and VPA + CPX3 + FAM20 groups, respectively, compared to the VPA + Saline and VPA + CPX3 group, but these decreased was not significant (P = 0.85, P = 0.97, respectively).
Discussion
This study investigated, for the first time, the effects of the H3R antagonist (CPX) and the co-administration of FAM and CPX on the cognitive performance and histology of neurons in the CA1 area of the hippocampus of VPA-exposed rats. We found that repetitive behaviors increase in VPA-exposed rats during the MBT. In the present study, repetitive behaviors were assessed using the digging behavior as a parameter (Eissa et al., 2018b ), and an increase in the number of marbles buried was observed in VPA-exposed rats in the MBT paradigm. Similarly, in various other studies, an increased preference for repetitive behavior in VPA-exposed rats has been reported compared to the control rats (Kim et al., 2014 ; Eissa et al. , 2018b , 2018c ). Repetitive behaviors in rodent models of autism include self-grooming, jumping, marble burying, and forelimb movements and involve several molecular and neural pathways (Gandhi and Lee, 2020 ). These behaviors might be the result of impaired function of the hippocampus, amygdala, and basal ganglia, the regions that are known to influence repetitive behaviors in animal models (Calderoni et al., 2014 ).
As shown in Fig. 6a , CPX at a concentration of 3 mg/kg significantly decreased repetitive behaviors in VPA-exposed rats, which is consistent with the previous findings indicating that blocking H3R decreases repetitive digging behavior in VPA-exposed rats (Baronio et al., 2015 ; Eissa et al., 2018b ). In this study, the administration of the H2R antagonist (FAM) and the co-administration of FAM and CPX had no effect on repetitive behaviors in VPA-exposed rats (Fig. 6b and c ). The mechanism by which repetitive behaviors are improved is not clear, but it might be explained by the ability of CPX, as a potent H3R antagonist, to mediate the release of different neurotransmitters in addition to histamine in the brain. Evidence has shown that signaling pathways involving different neurotransmitters and their receptors, such as glutamate, gamma-aminobutyric acid, serotonin, and dopamine, are also affected by the pathophysiology of stereotypical motor behaviors (Garner, 2005 ).
Anxiety in autistic individuals is common and the estimates of anxiety prevalence in those with ASD range from 22% to 84% (Russell et al., 2016 ; Nimmo-Smith et al., 2020 ). In the present study, time spent in the inner zone was used as a parameter in the open-field test to assess anxiety. A decrease in time spent in the inner zone was observed in VPA-exposed rats showing strong anxiogenic behavior. Moreover, grooming was used as a parameter to assess repetitive behaviors, and an increase in grooming was observed in VPA-exposed rats in the open field. Grooming behavior represents repetitive, stress-coping activities associated with anxiety in rodents (O’Leary et al., 2013 ). This behavior might be the result of impaired neurotransmitter function in the hippocampus, the region is known to influence anxiety-related behaviors in animal models (Engin and Treit, 2007 ). In agreement with our results, previous studies have shown that prenatal exposure to VPA may lead to cognitive deficits, such as increased anxiety-like behaviors (Yamaguchi et al., 2017 ; Gao et al., 2019 ; Lai et al., 2019 ). Moreover, recent studies have indicated that anxiety symptoms are associated with abnormal amygdala morphology in ASD (Fisler et al., 2013 ; Mueller et al., 2013 ).
According to previous studies, pretreatment with the H3R antagonist (E100) attenuates abnormal anxiety levels in the open-field (Eissa et al., 2019 ) and elevated plus maze (Eissa et al., 2020a ) tests in VPA-exposed mice. As shown in Fig. 7 , CPX at a concentration of 3 mg/kg significantly ameliorated anxiety-like behaviors in the open-field paradigm in VPA-exposed rats, which is consistent with the finding that blocking H3R enhances the potential anxiolytic effect of extracellular histamine (Eissa et al., 2019 ). According to the results of the present study, anxiety-like behaviors were increased by the H2R antagonist through pretreatment with FAM (20 mg/kg) (Fig. 8c ). FAM (20 mg/kg) completely reversed the CPX-induced improvement in anxiety-like behaviors in rats when co-administered with CPX (Fig. 9c ). In VPA-exposed rats, histamine appears to alter anxiety-like behavior processes by activating postsynaptically located H2Rs. However, pretreatment of VPA-exposed rats with CPX (1 and 3 mg/kg) or FAM (10, 20, and 40 mg/kg) did not change locomotor activity as measured by total distance and velocity in the open-field task.
Interventions in ASD, such as impairments in communication, stereotype, and anxiety, may be intensified by increased memory disorders (Llaneza et al., 2010 ; Eissa et al., 2018a ). VPA exposure affected the novel object paradigm in this study, and there was a deficit in recognition memory in VPA-exposed rats (Fig. 10 ). Previous reports have indicated that prenatal exposure to VPA leads to recognition memory impairments (Hara et al., 2017 ; Yamaguchi et al., 2017 ; Matsuo et al., 2020 ) in rodents. In the current study, CPX (3 mg/kg) improved object recognition memory impairments in VPA-exposed rats (Fig. 10 ). In addition, FAM treatment deteriorated object recognition memory in the novel object task (Fig. 11 ). CPX (3 mg/kg) improved the discrimination ratio in the NOR test, which was completely reversed after the co-administration of CPX with FAM (H2R antagonist) in VPA-exposed rats (Fig. 12 ). This finding suggests that histamine may modulate learning and memory processes, probably through the activation of H2R.
Another paradigm that has been used to examine the effect of VPA is fear conditioning, that is, the Passive avoidance learning task (Baarendse et al., 2008 ). In the present study, VPA exposure did not result in hippocampus-dependent fear learning (Fig. 13a ). In the memory phase, retention time in a dark chamber and STL was disrupted in the VPA-exposed group compared to the saline group (Fig. 13b and c ). Similar results were observed in (Varadinova et al., 2019 ) where VPA exposure disrupted fear conditioning in comparison with the saline group. The possible mechanism may involve VPA-induced morphological changes (pyramidal cell loss and apoptosis) in the hippocampus (Lind et al., 2013 ).
Treatment with CPX at a concentration of 3 mg/kg led to significant improvement in VPA-induced memory deficit in associative learning and memory (Fig. 13b and c ). However, there was no significant difference in the learning phase in the Passive avoidance task. Our results revealed that FAM attenuated memory deficits in rats (Fig. 14 ). Furthermore, the effects of CPX (3 mg/kg) on memory were completely reversed after its co-administration with FAM20 (H2R antagonist) in VPA-exposed rats (Fig. 15 ). Passive avoidance learning depends on N -methyl-D-aspartate (NMDA) receptors (Baarendse et al., 2008 ); therefore, it may be hypothesized that VPA-exposure downregulates glutamate receptors and histamine increases NMDA receptor current (Bekkers, 1993 ).
According to our morphological analysis, VPA treatment did not cause neuronal degeneration in the CA1 subfield of the hippocampus of VPA-exposed rats (Fig. 16 ). However, Yang et al. (2016 ) demonstrated that VPA causes hypocellularity in the CA1-subiculum (Yang et al., 2016 ). In the histological assessment, the number of CA1 neurons in the hippocampus was increased by CPX3, but this increase was not significant. However, FAM did not significantly decrease the number of CA1 neurons in VPA + FAM20 and VPA + CPX3 + FAM20 groups.
Fig. 16: The effects of H3 and H2 antagonists (CPX and FAM) and the combination of them on neural counting in the hippocampal CA1 region of male offspring exposed to VPA. There was no significant difference between groups. One-way ANOVA used for statistical analysis. The data are presented as mean ± SEM. n = 7. ANOVA, analysis of variance; CPX, ciproxifan ; FAM, famotidine; MBT, marble-burying test; VPA, valproic acid.
As histamine modulates a wide range of functions, including long-term social recognition memory , arousal, cognition, learning, memory, aggression, and emotions, many studies have investigated its effect on treating mental health disorders (Hu and Chen, 2017 ; Rani et al., 2021 ). The administration of an H3R antagonist alleviates autistic-like behaviors in the bar-biting behavior (BTBR) in mice (Eissa et al., 2020b ). Histamine injection into the ventral hippocampus improves scopolamine-induced working memory deficits (Xu et al., 2009 ). Studies have shown that H3R antagonists improve cognitive impairments in mice (Sadek et al., 2016b ; Eissa et al., 2018c ). CPX improved memory retrieval in mice with non-stress and stress conditions (Chauveau et al., 2019 ) and learning and memory in the Alzheimer’s disease model in the Passive avoidance test (Bardgett et al., 2009 ; Bardgett et al., 2011 ). The possible mechanism(s) of the involvement of CPX in memory improvement is not clear, but it might be explained by the ability of CPX, as a potent H3R antagonist, to mediate the release of different neurotransmitters in addition to histamine, such as acetylcholine, serotonin, and dopamine, in several specific brain areas (Sadek et al., 2016a ).
Moreover, the effects of the selective H2R antagonist suggest that histamine adjusts memory by acting on H2Rs (Huang et al., 2003 ). It has been shown that H2R antagonists cause cognitive deficits through their anticholinergic activities (Slugg et al., 1992 ). Histamine modulates recognition memory consolidation through H2Rs; therefore, intra-hippocampal treatment with ranitidine (an H2R antagonist) improved recognition memory consolidation (da Silveira et al., 2013 ).
By increasing the NMDA current in the brain, histamine improves Passive avoidance memory (Bekkers, 1993 ). Studies have shown that the cholinergic neurotransmitter system in the brain plays an important role in controlling ASD-related behavioral features including attention (Chen et al., 2017 ), cognitive flexibility (Hellmer and Nyström, 2017 ), social interaction (Arnold et al., 2002 ), and stereotyped behaviors (Wang et al., 2015 ; Karimi et al., 2017 ). Recent studies have indicated that acetylcholine is regulated by the activity of histamine H3R in the brain (Esbenshade et al., 2008 ). On the other hand, the ability of H3R antagonists to facilitate the histamine cycle in the brain may enhance cognitive pathways through the action of histamine on postsynaptic H1Rs and H2Rs (Zlomuzica et al., 2016 ). Therefore, H3Rs in the histaminergic system can be an important therapeutic target in cognitive deficits in many diseases, such as Alzheimer’s, schizophrenia, autism , sleep disorders, and attention deficit hyperactivity disorder (Haas and Panula, 2003 ). Given that changes in the gene expression of the enzyme responsible for the central metabolism of histamine, that is, histamine N -methyltransferase leads to a possible decrease in histamine levels in ASD patients, it is likely that increased histamine may improve some cognitive deficits caused by VPA (Taheri et al. , 2022). However, more studies are needed, especially at the cellular and molecular levels of ASD.
In summary, our results showed that prenatal VPA exposure was associated with anxiety-like behaviors, increased repetitive behaviors, and impaired memory in VPA-exposed rat offspring. CPX could improve VPA-induced impairments and it decreased repetitive and anxiety-like behaviors and ameliorated memory deficits in the VPA + CPX3 group. Moreover, the results revealed that the histaminergic system affects the modulation of cognition behaviors through H2R. The morphological investigation by histological assessment showed that VPA treatment did not lead to neuronal degeneration in the CA1 subfield of the hippocampus in rat pups.
Acknowledgements
This study was supported by the Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran.
Conflicts of interest
There are no conflicts of interest.
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