Introduction

Among many essential processes, learning and memory are the main functions of the brain (Amin et al., 2019). Learning and memory are cognitive functions that involve different stages, including acquisition, consolidation, and retrieval (More et al., 2016). The hippocampus, a limbic system structure, is located in the medial temporal lobe of the brain (Mu and Gage, 2011). Behavioral studies have suggested that the hippocampus plays a critical role in learning and memory (Kramer et al., 2004; Shahidi et al., 2017; Tahmasebi et al., 2015). Several neurotransmitters affect learning and memory through actions in different regions of the brain (Barzegar et al., 2015).

5-Hydroxytryptamine (5-HT, serotonin), a well-known monoamine, is an important neurotransmitter and neuromodulator in the central nervous system (CNS) (Osredkar and Kržan, 2009). In the CNS, serotonin is produced by serotonergic neurons located in the brain stem raphe nuclei (Butzlaff and Ponimaskin, 2016). Investigations on mammalian species have indicated high levels of 5-HT binding in the basal ganglia, substantia nigra, hippocampus, and related structures (Adham et al., 1993; Muchimapura et al. 2003; Waeber et al., 1989). Serotonin is also involved in regulating learning and memory and cognitive function (Butzlaff and Ponimaskin, 2016; Seyedabadi et al., 2014). The effects of 5-HT are due to its interaction with their receptors (Osredkar and Kržan, 2009). 5-HT receptors are currently classified into seven classes based on their structure, transduction mechanism, and pharmacological profile (Stasi et al., 2014). All serotonergic receptors, except the 5-HT3 receptor, are G-protein-coupled receptors (GPCRs) (Upadhyay, 2003). The class of 5-HT1 receptors includes several subtypes, such as 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-HT1E, and 5-HT1F (Filip and Bader, 2009; Stasi et al., 2014). The 5-HT1D and 5-HT1F receptors are attached to Gi/Go proteins to inhibit adenylyl cyclase and reduce cAMP levels (Berumen et al., 2012). According to the evidence from animal models, 5-HT1-7 receptors have numerous and complex effects on cognitive functions (Afshar et al., 2019; Hashemi-Firouzi et al., 2018; Liy-Salmeron and Meneses, 2007; Shahidi, S. et al., 2018).

The 5-HT1D receptors are expressed in various regions of the brain, especially in the raphe nuclei and cortex (Barnes and Sharp, 1999; Upadhyay, 2003). They negatively modulate the somatodendritic release of serotonin (Piñeyro et al., 1995). Also, the 5-HT1D receptor agonists exert their effects by reducing neurogenic inflammation and inhibiting neurotransmitter release (Neeb et al., 2010), leading to relieving migraine attacks (Wang et al., 2014) and attenuating hyperalgesia (Araldi et al., 2017). PNU142633, the 5-HT1D receptor agonist, and BRL15572 hydrochloride, the 5-HT1D receptor antagonist, bind with a high affinity and selectivity to the 5-HT1D receptor (Gomez-Mancilla et al., 2001; Price et al., 1997). PNU142633 and BRL15572 hydrochloride are effective in the acute treatment of migraine attacks (Barbanti et al., 2017).

On the other hand, the 5-HT1F receptor is prominently expressed in the neocortex, hippocampus, and dorsal raphe nucleus (Agosti, 2007; Muchimapura et al. 2003). Potent agonists for 5-HT1F receptors can be effective in the treatment of migraine (Neeb et al., 2010; Tfelt-Hansen and Olesen, 2012). However, the effect of 5-HT1D and 5-HT1F receptors on learning and memory has not been reported. LY 344864 is the selective 5-HT1F receptor agonist with a high affinity in humans and rabbits (Phebus et al., 1997). Activation of the 5-HT1F receptor by LY was effective in the treatment of migraine in animal models (Phebus et al., 1997), through the modulation of (anti) nociceptive impulses (Rubio-Beltrán et al., 2018) and attenuation of methamphetamine relapse (Shahidi, Siamak et al., 2018b).

The effect of 5-HT1F receptor activation or LY 344864 is not known in episodic-like memory. The aim of the present study was to investigate the role of 5-HT1D and 5-HT1F receptors in the retrieval stage of learning and memory processes by selected agonists or antagonists in the hippocampus area.

Materials and methods

Animals

Fifty adult male Wistar rats (200–250 g) were obtained from the laboratory animal center of Hamadan University of Medical Sciences, Hamadan, Iran. The rats were kept under standard laboratory conditions (temperature: 22 ± 2 °C and a 12:12-h light:dark cycle). All animals had free access to water and food. All experimental procedures were approved by the Ethics Committee of the Hamadan University of Medical Sciences (IR.UMSHA.REC.1394.358) and were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985).

Drugs

PNU142633 (5-HT1D receptor agonist), BRL15572 hydrochloride (5-HT1D receptor antagonist), and LY344864 (5-HT1F receptor agonist) were purchased from Tocris Bioscience Company (Bristol, UK). All compounds were dissolved in saline dimethyl sulfoxide (DMSO). These chemicals were stored at −20 °C before use.

Experimental design

The animals were divided into five groups randomly, each consisting of ten rats: (1) Intact control with no surgical or injection procedure; (2) sham-operated group, which received bilateral intrahippocampal (IHP) injection of normal saline (1μl); (3) PNU group, which received a bilateral IHP injection of PNU-142633 (1 μg/μl); (4) BRL group, which received a bilateral IHP injection of BRL-15572 (1 μg/μl); and (5) LY group, which received a bilateral IHP injection of LY 344864 (1 μg/μl).

Surgical procedures

The animals were anesthetized intraperitoneally by a mixture of ketamine (100 mg/kg, BehbodDarou, Tehran, Iran) and xylazine (10 mg/kg, Alfasan, Woerden, and The Netherlands). After being fixed in the stereotaxic apparatus (Stoelting Co., Chicago, IL), cannulas (10 mm, 23- gauge) were implanted bilaterally into the hippocampus using the stereotaxic coordinates, AP: −3.8 mm from bregma; ML: ±2.3 mm from the midline, and DV: −2.8 mm from the skull surface (Beigi et al., 2018; Paxinos and Watson, 1998). Each rat was then returned to an individual cage (40×28×15 cm) and allowed to recover for 7 days. After recovery, the cages were replaced with other cages with diameters of 55 cm × 37 cm × 25 cm for a group of up to four rats.

Microinjection procedure

For the microinjection procedure, the cannula stylet was removed and replaced by an injection needle (30-gauge) connected with a short piece of polyethylene tubing to a Hamilton syringe. In order to prevent mechanical damage to the hippocampus, the needle was inserted 1 mm below the tip of the cannula so that it was just above or slightly within the target brain region. Then, the rat received infusions of either saline or drugs (1 μl per infusion) into the hippocampus over a 2-min period (Lashgari et al., 2008). The needle was left in place for another 60 s before it was slowly withdrawn. Then, the microinjection was performed on the other side. The doses of chemical substances were chosen according to previous studies (Centurión et al., 2001; Muñoz-Islas et al., 2006). After treatment, rats were subjected to the open field test, novel object recognition test, and the passive avoidance learning test. There was an interval of 5 days between behavioral tests (Fig. 1).

Fig. 1
figure 1

A schematic of the experimental design and timeline

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Open field test

The open field test was used to assess the rats’ locomotor activity (Afshar et al., 2018; Shahidi et al., 2019). The foursquare apparatus comprised a black wooden floor (48 cm×41.5 cm×36 cm) with low ambient light and a video recording system. The animal was placed in the middle of an open field and allowed to explore the whole field. The behavior of the animal was recorded using video recording for 10 min. The total distance traveled and mean velocity were analyzed using the Ethovision tracking program (Borj Sanat Azma, Iran).

Novel object recognition test

The novel object recognition test has been extensively used to measure memory in rodents. The apparatus used in this study consisted of a black square cage with a floor arena covered with wood tips. The test had the following three sessions: habituation, training, and retention. In the habituation session, the rats were individually placed in the box for 10 min without any objects to explore (day 1). The training session was performed 24 h after the habituation session, in which two identical objects were placed on one side of the box, and the rats were allowed to freely explore the two objects for 10 min. The retention session was performed 24 h after the training session, and 5 min prior to the retention session, animals received a single dose of saline or drugs. In the retention trial, one of the two familiar objects was replaced by a novel object, and the time taken to explore each of them was again recorded. Object exploration was defined as follows: sniffing, touching, and directing attention to the object. Animal behaviors were recorded with a video camera system and the results were manually analyzed. The recognition index (%) was calculated as the time spent exploring the novel object/total exploration time × 100 (Afshar et al., 2018).

Passive avoidance learning test

Apparatus

Passive avoidance learning was tested in an apparatus with two (light and dark) compartments of the same size (20×20×30 cm each) separated by a guillotine door. The light chamber was made of transparent plastic, and the dark chamber walls were made of dark opaque plastic. The floor of the dark chamber was equipped with stainless-steel rods, and electric shocks (50 Hz, 0.4 mA, 1.5 s) were delivered to the grid floor of the dark compartment by a stimulator (Hasanein and Shahidi, 2012).

Training

All rats were habituated to the experiment room for 30 min before the training and testing experiments. First, all groups were given two trials to habituate and acclimatize them to the apparatus. We habituated the rats to the apparatus as follows: the rats were placed in the lighted compartment of the apparatus for 30 s, and then, the guillotine door was opened to allow access to the dark compartment. The rat has a natural preference for the dark environment. Once the rat entered the dark compartment, the door was closed, and after 30 s, the rat was removed from the dark compartment and placed in its home cage. This habituation trial was repeated 30 min later and followed by the first acquisition trial after the same interval.

For the training of the animals, when they had spontaneously entered the dark compartment, the door was closed and after 30 s, a mild electrical shock (0.4 mA, 1.5 s) was applied. The entrance latency to the dark compartment (step-through latency in acquisition, STLa) was recorded when the animal had all four paws in the dark compartment. After 20 s, the rat was removed from the apparatus and placed in the home cage. Two minutes later, the procedure was repeated. If the rat did not enter the dark compartment in 120 s, successful acquisition of inhibitory avoidance response was recorded. Otherwise, when the rat entered the dark compartment (before 120 s), the door was closed, and the animal received the same shock. The number of trials (entries into the dark chamber) was recorded.

Retention

The retention test was performed 24 h after the passive avoidance training trial to evaluate long-term memory. Five minutes prior to the retention session, animals received a single dose of saline or drugs. The rat was placed in the light compartment as in the passive avoidance training session, the door was opened after 10 s, and the step-through latency during the retention trial and the time spent in the dark compartment were recorded up to 300 s. The test session was terminated when the rat either entered the dark compartment or remained in the light compartment for 300 s. During the retention test, the electric shocks were not applied to the grid floor (Shahidi et al., 2019).

Statistical analysis

Statistical analyses were performed using SPSS 16.0 statistical software. The Kolmogorov–Smirnov test was used to check the data normality. The data were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test. A P < 0.05 was considered statistically significant. The results were expressed as mean ± SD.

Results

Open field test

One-way ANOVA results showed no significant difference in the total distance moved (F (4,49) = 0.47, 0.75, P > 0.05) and the velocity [F (4, 49) =0.37, P=0.82 > 0.05] between the groups (data are not shown).

Novel object recognition test

One-way ANOVA results indicated significant differences between the recognition indices of objects between groups [F (4, 49) = 9.735, P< 0.001] (Fig. 2). Tukey’s test results showed that the recognition index of the BRL group was significantly higher than that of the control (P<0.05) and sham (P<0.05) groups. The recognition index in rats that received LY was not significantly different compared to the control and sham groups. The rats receiving PNU had a significantly lower recognition index than the BRL and LY groups (P < 0.001).

Fig. 2
figure 2

The effect of treatment with PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist) on the recognition index. Data are shown as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (n=10 per group). *P < 0.05 compared to the control group; #P < 0.05 compared to the sham group, and !!!P < 0.001 compared to the PNU group

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Passive avoidance learning test

One-way ANOVA results showed no significant difference in step-through latency in acquisition between the experimental groups in the first acquisition trial (before the electrical shock) [F (4,49) = 1.021, P=0.407> 0.05; Fig. 3]. There was also no significant difference in the number of trials during the first acquisition between the experimental groups [F (4,49) = 1.164, P=0.339> 0.05; Fig. 4].

Fig. 3
figure 3

The effect of treatment with PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist) on the step-through latency in acquisition in the passive avoidance learning test. Data are expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (n=10 per group)

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Fig. 4
figure 4

The effect of treatment with PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist) on the number of trials in the passive avoidance learning test. Data are expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (n=10 per group)

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According to one-way ANOVA results, there was a significant difference in the step-through latency during the retention trial between the groups [F (4,49) = 12.369, P<0.001] (Fig. 5). Also, Tukey’s test results showed that STLr was significantly lower in the BRL group than in the control (P < 0.001), sham (P < 0.01), and PNU (P < 0.001) groups. In addition, STLr in rats that received LY was significantly lower than step-through latency during the retention trial in the control (P < 0.01), sham (P < 0.01), and PNU (P < 0.001) groups. There was no significant difference between the PNU, control, and sham groups in the memory test.

Fig. 5
figure 5

The effect of treatment with PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist) on step-through latency in the retention trial in the passive avoidance learning test. Data are expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (n=10 per group). **P < 0.01 and ***P < 0.001 compared to the control group; ##P < 0.01 compared to the sham group; and !!!P < 0.001 compared to the PNU group

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A statistically significant difference was detected in the time spent in the dark compartment between the experimental groups evidenced by one-way ANOVA [F (4,48) = 2.657, P=0.045<0.05]. As shown in Fig. 6, the time spent in the dark compartment of the BRL group was significantly greater than that of the control (P < 0.01) and PNU (P < 0.001) groups. The time spent in the dark compartment in the LY group was significantly higher than time spent in the dark compartment in the control (P<0.05) and PNU (P < 0.01) groups.

Fig. 6
figure 6

The effect of treatment with PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist) on the time spent in the dark compartment (TDC) in the passive avoidance learning test. Data are expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (n=10 per group). *P < 0.05 and **P < 0.01 compared to the control group and !!P < 0.01 and !!!P < 0.001 compared to the PNU group

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Discussion

The present study investigated the role of serotonin receptors in the retrieval stage of learning and memory by PNU (5-HT1D receptor agonist), BRL (5-HT1D receptor antagonist), and LY (5-HT1F receptor agonist). Briefly, the results indicated that IHP injection of BRL increased the recognition index in the novel object recognition test. BRL decreased the step through latency during the retention trial and increased the time spent in the dark compartment in the passive avoidance learning test. It can be concluded that acute inhibition of the 5-HT1D receptor potentiated the recognition memory but attenuated the avoidance memory. The acute activation of the 5-HT1D receptor by an IHP injection of PNU had no effects on memory. Also, the administration of LY decreased the step-through latency during the retention trial and increased the time spent in the dark compartment in the passive avoidance learning test.

These conflicting results could be due to the different brain structures involved in the memory processes. The recognition memory is a form of non-spatial memory in the object and passive avoidance. The novel object recognition test is a valuable measurement of cognition in rodents (Antunes and Biala, 2012). The prefrontal cortex and hippocampus are involved in object memory (Chao et al., 2020). The hippocampus plays a main role in the encoding, consolidation, and retrieval stages in object memory (Cohen and Stackman Jr, 2015). The hippocampus is responsible for long-term object recognition by receiving inputs from the perirhinal cortex (Reger et al., 2009). In addition, hippocampal formation has the main role in memory for contextual information (Antunes and Biala, 2012). On the other hand, the entorhinal cortex processes object information (Clemow et al., 2020). The hippocampus and prefrontal cortex contribute to forming memory (Rolls, 2022).

According to Shahidi et al., the intracerebroventricular administration of PNU decreased long-term potentiation components and synaptic transmission in methamphetamine-treated rats (Shahidi, Siamak et al., 2018a). Also, the administration of GR46611 (a 5-HT1B and 5-HT1D receptor agonist) impaired the consolidation of learning in the autoshaping learning task (Meneses, 2001; Meneses et al., 1997). Najar et al. reported that intra-septum post-training administration of CP94253 (5-HT1B and 5-HT1D receptor agonist) reduced the step-through latency during the retention trial in the passive avoidance learning test, while GR127935 (5-HT1B and 5-HT1D receptor antagonist) did not alter memory consolidation (Najar et al., 2015).

PNU142633 is a selective and high-affinity 5-HT1D receptor agonist, which can activate the receptor by the inhibition of adenylyl cyclase activity (Hensler, 2012). Central 5-HT1D receptors are found at low densities and low levels in the cortex and hippocampus (Blackburn, 2009; Hoyer, 2019). Our results showed that PNU142633 had no effects on memory tests. The activation of the brain’s 5-HT1D receptors is linked to a reduction in serotonin, glutamate, and acetylcholine release in many brain regions and can impair some aspects of learning and memory (Filip and Bader, 2009). The low concentration of PNU142633 (1 μg/μl) can be the reason for no effect of 5-HT1D receptor on memory in the present study.

Our finding show that 5-HT1D receptor antagonist enhanced recognition memory. The hippocampus contains a high density of 5-HT1D sites (Bruinvels et al., 1994). 5-HT1D receptors are negatively coupled to adenylate cyclase (Hoyer et al., 1990) and act as inhibitory autoreceptors in axon terminals of serotonergic neurons (Sari, 2004). 5-HT1D receptors are found at pre- and postsynaptic sites but presynaptic receptors are predominantly located on 5-HT hippocampal nerve terminals (Fink and Göthert, 2007; Muchimapura et al. 2003). Presynaptic 5-HT1D receptors participate in the release of 5-HT and other neurotransmitters such as, acetylcholine and glutamate (Bruinvels et al., 1994; De Deurwaerdere and Di Giovanni, 2021; Pauwels, 1997; Rutz et al., 2006). Selective blockade of 5-HT1D autoreceptors facilitates serotonin neurotransmission (Pauwels, 1997) and elevated extracellular 5-HT concentrations (Pullar et al., 2004). Inhibition of 5-HT1D receptor enhances release of neurotransmitter and improves memory impairments (Meneses et al., 1997).

On the other hand, the present finding shows that 5-HT1D receptor antagonist attenuates avoidance memory. The 5-HT1D receptors are located in postsynaptic neurons (Pauwels, 1997). 5-HT1D receptors negatively modulate the somatodendritic release of 5-HT (Piñeyro et al., 1995). BRL15572 blockade the postsynaptic sites of 5-HT1D receptor, and inhibition of 5-HT1D receptors induces a serotonergic activation on adrenergic neurotransmission (García-Pedraza et al., 2013). These events may modulate the avoidance memory.

The different effects of 5-HT1D receptor antagonist in object recognition and passive avoidance tests is related to the hippocampus and other brain area, which link to cognition or avoidance memory. The hippocampus plays an important role in memory, and in this process, performs via synapses, neuronal circuits, and pathways. Serotonin has positive role in memory. The cognitive deficits are associated with reduced serotonin levels (Cowen and Sherwood, 2013; Mendelsohn et al., 2009). Hippocampus, entorhinal system, and neocortex involved in cognition memory (Lisman et al., 2017).

Memory of inhibitory avoidance is processed by the hippocampus, amygdala, entorhinal cortex, striatum, and parietal cortex (González-Salinas et al., 2018; Izquierdo and Medina, 1997). Memory of habituation to a novel environment is performed only by the hippocampus (Izquierdo and Medina, 1993). Retrieved inhibitory avoidance memory is performed through memory reconsolidation by interactions between the amygdala prefrontal cortex and hippocampus (Fukushima et al., 2021). The entorhinal cortex is the gate to control the flow of information into and out of the hippocampus (Lei, 2012).

Hippocampal injection of 5-HT1D receptors antagonist decreases the avoidance memory. Although, the hippocampus contains a high density of 5-HT1 sites (Waeber et al., 1989) and serotonin receptors express at high density in CA3, low density in dentate gyrus, and lowest density in CA1 (Hensler, 2006), but 5-HT1D binding sites in hippocampus have very low presence (Bruinvels et al., 1993). Distribution of 5-HT1D receptors is high concentrations in the nigro-striatal pathway, entorhinal cortex, and cortex (Hoyer et al., 1990). It seems that the low binding sites of 5-HT1D in hippocampus are responsible for the decrease on avoidance memory in shuttle box.

In the current study, the hippocampal injection of LY decreased avoidance memory in the passive avoidance learning task but had no effects on object recognition memory. These results may be due to the differences in cell signaling mechanisms and the neuronal circuits involved in each task. However, no studies have investigated the role of LY in memory function. LY attenuated the reinstatement of methamphetamine-seeking behavior (Shahidi, Siamak et al., 2018b). Moreover, 5-HT1 receptor subtypes improved cognitive function. Also, 5-HT1A receptor blockade by NAD-299 (Afshar et al., 2018; Lüttgen et al., 2005) and WAY-100635 (Misane and Ögren, 2003) improved memory in rats in the passive avoidance test.

LY344864 is a selective 5-HT1F receptor agonist. LY344864 administration leads to the inhibition of cyclic AMP accumulation in cells following in vitro electrical stimulation (Phebus et al., 1997). Also, the 5-HT1F agonist exerts its effect on memory by inhibiting neuropeptide and neurotransmitter release (Clemow et al., 2020). Although the protein and neurotransmitter were not measured in our study, BRL15572 and LY344864 had similar effects on the recognition and avoidance memory.

BRL and LY had similar effects on the performed tests. BRL is the 5-HT1D receptor antagonist and LY is the 5-HT1F receptor agonist. It means that the inhibition of the 5-HT1D receptor and stimulation of the 5-HT1F receptor can lead to similar effects on memory.

There were some limitations in this study that should be discussed. This study investigated the effect of drugs in the learning phase (before the learning test) and preliminary information was obtained. More studies should be done on different stages of learning and memory in impaired memory models. It is necessary to investigate the effect of the receptor on neuronal plasticity and changes in the expression of proteins, such as brain-derived neurotrophic factor. This study was further limited by the choice of behavioral test. Although the passive avoidance task and novel object recognition test evaluated memory and cognition, they did not test spatial memory, which can provide more information about memory. As mentioned in the methods section, acute treatment with the agonist and antagonist was done using microinjections into the hippocampus so that the stimulation or inhibition of the serotonin receptor was observed over a relatively short period of time. The effectiveness of the substance through systemic consumption was not studied. Examining the effect of long-term treatment with 5-HT1D receptor agonist and 5-HT1F receptor antagonist is essential in understanding the function of the serotonin receptors.

Conclusion

In conclusion, the present study showed that the inhibition of the hippocampal 5-HT1D receptor improved the recognition memory but impaired avoidance memory. Activation of the hippocampal 5-HT1F receptor had no effect on recognition memory but impaired aversive memory. However, more studies should be performed to confirm these results. Also, further investigations, such as molecular and behavioral experiments, are required to clarify the more detailed mechanism of our findings.