All chemicals were purchased from Sigma Aldrich Company (Darmstadt, Germany) except for the others mentioned in the text.

For this study, specimens were collected alive from Iran (Zamani et al., 2021). The Agelena labyrinthica was photographed with a camera. The specimens were kept under suitable conditions, humidity (60%), and temperature (25°C) and fed by crickets and mealworms. The specimens were identified using the taxonomic keys (Nentwig et al., 2017). Epigyne of the female specimen was visualized with a stereoscope.

Female spiders were separated to extract the venom. Specimens were anesthetized with CO₂ in a small chamber, and the opisthosoma and carapace were removed under the stereoscope. The fangs and venom glands of the female specimen were visualized by a loop. Venom glands were collected into 4°C Phosphate buffered saline (PBS) were prepared in the laboratory with this recipe (137 mM NaCl, 3 mM KCl, 10 mM Na₂PO₄, 2 mM KH₂PO₄, and pH 7.4) and gently crushed with a glass stirrer for 30 min. Then, pieces of the venom gland were removed from the solution by centrifugation at 13,000 rpm for 30 min at 4°C, and the supernatant was lyophilized and stored at −70°C. Protein concentration was measured by Bradford assay with bovine serum albumin as standard protein.

To determine the LD₅₀, the albino mice (average weight 18–20 g) were intravenously (IV) injected with crude venom. After the injection, the animals were followed up for 1 day. The Spearman-Karber method was used to calculate the LD₅₀ dose for the crude venom and omega-agatoxin-Aa2a protein (Hamilton et al., 1977).

1 mg of the crude venom was dissolved in 1 ml PBS buffer and incubated at 4°C overnight. The supernatant was then centrifuged at 13,000 rpm for 4 min at 4°C. Then, 15 μl of dissolved crude venom was mixed with 5 μl loading buffer (1x) and heated for 10 min at 100°C. An amount of 20 μl of this solution was loaded into each well of the gel [15% Sodium Dodecyl Sulfate-Polyacrylamide Gel-Electrophoresis (SDS-PAGE)] which stained with coomassie blue dye for 45 min at 25°C. Hot water and a shaker were used to decolorize the gel. Finally, the gels were scanned by Bio-5000 Gel scanner device (Seyfi et al., 2019).

The lyophilized crude venom (10 mg) was resuspended in 1.5 mL of PBS buffer. The DNase (0.14 mg/ml) and RNase (0.14 mg/ml) enzymes were added to the sample and incubated for 2 h at 4°C. The clear solution was injected into a gel-filtration column (GE Healthcare HiLoad 16/600 Superdex^® 75 pg prep grade) and run over it using FPLC (Fast Protein Liquid Chromatography) system (Sykam, Germany). The column was washed with PBS Buffer. The injection volume was 1,200 μL and the flow rate was 0.7 mL/min. The fractions were observed with absorbance at 280 nm and collected in a 0.75 ml fraction. The chosen fractions (marked on the graph) were collected and injected into capillary electrophoresis and gel-electrophoresis (Mofid et al., 2021).

Capillary electrophoresis test was performed by Agilent 7100 equipped with a UV-Vis detector using a 50 μm uncoated silica column with a total length of 40 cm and a detector distance of 8.5 cm from the outlet. PBS buffer with pH 4.7 was used for both the sample, and running buffer. The capillary temperature was 25°C and the sample was injected at 100 mBar for 5 s. Electrophoresis was performed for 5 min at 25 kV normal polarity. The marked peaks were collected, and protein concentration was determined by Bradford assay. The desired peak was re-injected into the device to ensure purity, and the result was presented the collected fraction was subjected to 12% SDS-PAGE. The selected peak obtained from this method project into HPLC-ESI-MS (Keimasi et al., 2022).

The high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS) analysis was performed by Waters Alliance 2695 HPLC-Micromass Quattro micro API Mass Spectrometer. Liquid chromatography separation was performed on Atlantis T3-C18 column (3 μ, 2.1 × 100 mm) at 35°C. Mobile phases were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in H₂O. The gradient profile was 5% A held for 0.2 min and linearly increased to 90% in 10 min. Then, it was held for 5 min, which decreased to 5% over 3 min and finally held for 4 min. The flow rate was 0.2 mL/min, and the injection volume was 5 μL. The mass spectrometry method was included in positive mode with the capillary voltage, which was adjusted to 0.3 kV, and the source and dissolving temperatures were set at 120°C and 300°C respectively, with flow gas 200 L/h. The result was presented for the purified bio-active small protein peak (Jafari et al., 2014).

54 adult male Wistar rats, weighing 230–250 g were taken from the animal’s nest of the Faculty of Biological Sciences and Technology, University of Isfahan. They were kept in standard cages with controlled temperature (∼25°C) and humidity (∼40%), 12 h light; 12 h dark cycle, and free access to enough food and water. The ethics committee of the University of Isfahan approved the study.

The work was performed on male Wistar rats, which were spat into three groups (18 rats in each group). A small area on each rat’s skull was shaved while the head was fixed using a stereotaxic instrument (Stoelting Co., USA) to prepare for injection into the hippocampus. The PBS buffer was used as a vehicle for omega-agatoxin-Aa2a and N-Methyl d-Aspartate (NMDA). Rats were assigned into the following groups:

Control Group: Received 1 μl of PBS in the CA3 sub-region of the hippocampus twice and with an interval of 30 min.

NMDA-treated group: received a single dose of NMDA (1 μl, 10 μg/μl) in the CA3 sub-region of the hippocampus followed by 1 μl of PBS 30 min later (Jarrard, 2002).

Omega-agatoxin-Aa2a -treated group: received a single dose of NMDA (1 μl, 10 μg/μl) in the CA3 sub-region of the hippocampus followed by 1 μl of omega-agatoxin-Aa2a (2 μg/μl) 30 min later (Sousa et al., 2013).

One week before the start of the behavioral tests, all the animals were deeply anesthetized with an intraperitoneal (i.p.) injection of phenobarbital (40 mg/kg). The phenobarbital was purchased from Martindale Pharma Company (Buckinghamshire, England). The animals were wrapped in towels and the eyes were covered with Vaseline during the operation. Then, the CA₃ sub-region of the hippocampus (AP:−3.3 mm from bregma; ML: ± 3 mm from midline; DV: 3.5 mm from the skull surface) was found using the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2006). The injections were performed bilaterally on the hippocampi by stereotaxic apparatus.

The agents were bilaterally administered into the hippocampi using an injection needle (21 gauge) connected to a 5 μl Hamilton syringe through a polyethylene tube. The agents were slowly injected into the hippocampus area for 6 min. To avoid backflow of the fluid, the needle was slowly removed 2 min after the injection.

The behavioral evaluations including the Morris water maze and Passive avoidance tasks were performed a week after the stereotaxic surgery. These tasks have assessed spatial, long-term memory, and learning. Before the behavioral tests, animals were acclimatized over 2 days in the laboratory area to be adjusted to the experimental conditions and to minimize stress.

The Morris water maze test is one of the most common procedures to assess spatial memory and learning performance in rodents. The walls of the water maze room must have signs and symbols that rats can use their spatial memory to find the hidden platform in the target zone. Morris water maze task has three parts including habituation, training, and testing (it has been completely described in previous work) (Hosseini-Sharifabad et al., 2021). In this task, time spent in the target quadrant, distance moved in the target quadrant, the entry into the target quadrant, the velocity of rats, and swimming paths of rats in the last time of training were recorded to evaluate spatial memory and learning performance (ten rats in each group). All behavioral parameters of the rats were monitored by a video camera, fixed to the ceiling above the center of the pool and connected to a computerized tracking system (Auto vision Software, Designed by BorjSanat Company, Tehran, Iran).

The passive avoidance task was applied to assess non-conceptual communication memory and learning, as this method has already been used in neurological disorders models in small laboratory animals (Salehifard et al., 2023). The term passive avoidance is commonly used to describe experiments in which animals learn to avoid a painful stimulus. A passive avoidance behavior study was performed on two consecutive days (training and test), as explained by Ebrahimpour et al. (2018). The latency time before entering the dark section, and the time spent in the dark section on the test day, were recorded to evaluate passive avoidance learning and memory (10 rats in each group).

The mRNA extraction was performed as formerly has been described by Gheysarzadeh et al. (2019) and mRNA expression measurement was done for the three groups (six rats in each group) (Gheysarzadeh et al., 2019). Total RNA was extracted from the hippocampus samples using RNX-PLUS reagent (SinaClon, Iran). cDNA was synthesized according to the manufacturer’s protocol with a cDNA synthesis kit (Takara, Japan). Real-time PCR assay was performed by StepOnePlus™ Real-Time PCR System. The sequences of Real-time PCR primers were as follows: 5′-GAGGAAGGTCTGAACCGCTCAT-3′, and 5′- CGTTCTCGGTAGTCTGACTGAG-3′ for mice syntaxin1A, 5′- AGGGCCTATGATGGACTTTCTG-3′, and 5′-TCCGTGGCCA TCTTCACATC-3′ for mice Synaptophysin, 5′-CGGCAAA CTGACTGTCATTC-3′ and 5′-GCC CCA GTG CTG TTG TAA CCA-3′ for mice Synaptotagmin 1, and 5′-CAGGG CTGCCTTCTCTTGTG-3′, and 5′-GATGGTGATGGGTTTC CCGT-3′ for mice Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative genomic expression was calculated by the 2^{–Δ Δ Ct} method. The mRNA levels were normalized to that of GAPDH (Gheysarzadeh et al., 2019).

The rats (six in each group) were anesthetized with urethane (1.5 g/kg; intraperitoneal injection) dissolved in 0.9% normal saline solution. The surgery and LTP recording procedures from the hippocampal CA₃ area were executed as formerly has been described by Keimasi et al. (2022).

The brains were washed with saline and sequentially fixed in 10% formaldehyde solution and then post-fixed in 15% and 25% sucrose solutions. Two-micrometer sections were cut by using a microtome instrument. Antigen retrieval was performed enzymatically for 20 min. To reduce non-specific antibody binding, the samples were blocked with the blocking agent [10% normal goat serum (Sigma, G9023) and 0.3% Triton X-100 in PBS] for 30 min at 37°C. After washing, tissue sections were incubated with mouse anti−SNAP25 monoclonal (Abcam, ab66066) as the primary antibody at 4°C. FITC−conjugated anti−mouse IgG (Sigma, F9137) were used as secondary antibody for 2 h at room temperature. The nuclei were counterstained with DAPI (Sigma, D9542). The slides were visualized using an AX70 Olympus fluorescence microscope (three in each group).

Tissue coronal slides were deparaffinized two times in xylene, each for 10 min. Rehydration was performed using gradient alcohol (100, 95, 70, and 50% alcohol) each time for 5 min. The brain slides were stained in cresyl violet solution (0.25% cresyl violet, 0.8% glacial acetic acid, 0.6 mM sodium acetate) for 20 min. The dehydration was done using gradient alcohol (70, 95, and 100% alcohol) each time for 3 min. Tissue slides were visualized using a BX40 Olympus light microscopy (three in each group).

Statistical analysis was performed using Graph Pad Prism statistics software (version 8.4.3). Statistical Data were evaluated by the D’Agostino-Pearson omnibus test to examine the normal distribution. The data collected were analyzed by one-way, and one-way repeated measures analysis of variance (ANOVA). For multiple comparisons, Tukey’s test was used. All data were shown as the mean ± standard errors of the means (P < 0.05 was considered a significant difference).

A total of 150 spiders were collected from Iran (Zamani et al., 2021), and separated by species and gender. One hundred female Agelena labyrinthica specimens were selected for the next phase of the study. The specimens on the net web and epigyne of this species are shown in Figure 1.

1.03 g of the lyophilized crude venom was extracted from Agelena labyrinthica specimens. Then, 10 mg of the crude venom was dissolved in PBS buffer. The protein concentration of the Agelena labyrinthica lyophilized crude venom was 7.2 mg/ml, which was determined by the Bradford method. The fangs, venom glands, and lyophilized crude venom of Agelena labyrinthica are presented in Figure 2.

Based on the results of IV injections of the crude venom and omega-agatoxin-Aa2a protein into the albino mice, the LD₅₀ was determined as 32.68 mg/kg and not toxic.

The gel electrophoresis of the crude venom of Agelena labyrinthica showed a lot of protein bands separated based on their molecular weights. The strongest bands were the 158 and the band range of 3–10 k Da. The 3–10 k Da band was related to the agatoxin family, which has a molecular weight in this range (Herzig et al., 2010; Consortium, 2019; Figure 3).

As shown in Figure 4A, the gel-filtration chromatography of the Agelena labyrinthica venom had five peaks. The fourth fraction of Agelena Labyrinthica venom was collected from 160 to 190 min. 1.57 mg of the lyophilized fraction was obtained from 21 ml of the solution, taken from the instrument. The protein concentration of this fraction was 1.03 mg/ml.

The gel electrophoresis of the 4, 5, and 6 fractions are displayed in Figure 4B. This figure is completely consistent with the gel-filtration chromatography pattern, shown in Figure 4A.

Figure 5, part A displays the capillary electrophoresis pattern, obtained from the fourth fraction of Agelena labyrinthica venom. Four peaks were observed at 1.71, 1.77, 3.95, and 4.78 min. The fourth peak was collected and re-injected to confirm the purity of this small bioactive protein and then injected into HPLC-ESI-MS for mass determination.

The gel electrophoresis of the selected peak revealed a single band of less than 13 kDa (Figure 5B).

The molecular mass of omega-agatoxin-Aa2a is 10,982 Da, according to the Sousa et al. (2013) study. The spectrum results demonstrated the existence of omega-agatoxin-Aa2a in the selected peak (Figure 6A). The mass-to-charge ratio of this ligand was consistent with omega-agatoxin-Aa2a. The charge-to-mass ratio of omega-agatoxin-Aa2a is as follows: (M + Na + 5H)⁶⁺ = 1835, (M + Na + 6H)⁷⁺ = 1573, (M + Na + 7H)⁸⁺ = 1,376.5. The Mass/Mass spectrum of Quadro charge ion at 1,376.5 was selected for N-terminal partial sequencing as follows: b₃ = 274, b₄ = 403, b₅ = 516, b₆ = 573, b₇ = 630, b₈ = 745, b₉ = 848, b₁₀ = 963, b₁₁ = 1,020, b₁₂ = 1,183, b₁₃ = 1,311, b₁₄ = 1,440, and b₁₅ = 1,568. The results of the spectrum indicated a good consistency with the omega-agatoxin-Aa2a (Accession number: P15971) sequence (Figure 6B).

The intra-hippocampal administration of NMDA, significantly decreased the time spent in the target area (P < 0.0001), and distance moved in the target zone (P < 0.0001), when compared to the control group. NMDA treatment with administration of omega-agatoxin-Aa2a increased the time spent in the target zone (P < 0.01), and distance moved in the target zone (P < 0.01) of the maze, compared to the NMDA-treated group (P < 0.01). Also, significant differences in the time spent (P < 0.01), and distance moved (P < 0.01) in the target zone of the maze were observed between the control, and NMDA + Aga groups (Figure 7A).

The swimming path of experimental animals during the last time of training is displayed in Figure 7B. The swimming path of the control group was short and the rats found the hidden platform, easily. But, the swimming path of the NMDA group was long and the rats failed to locate the hidden platform. The majority of the movements of these rats were limited to the area close to the wall of the pool. However, the swimming path of the NMDA-treated with a single dose of omega-agatoxin-Aa2a group was almost short and the evaluated rats were able to find the hidden platform after a quick exploration in the water pool.

According to Figure 8A, our data revealed that administration of a single dose of NMDA into the hippocampus, significantly decreases the latency time in the NMDA group, compared to the control group (P < 0.0001), in the passive avoidance task. NMDA treatment with a single dose of omega-agatoxin-Aa2a increased the latency time in the NMDA + Aga group compared to the NMDA-treated group (P < 0.05). The Control group was significantly different from the NMDA + Aga group (P < 0.0001).

As shown in Figure 8B, our results demonstrated that the intra-hippocampal injection of NMDA significantly increases the time spent in the dark chamber in the NMDA-treated group when compared to the control group (P < 0.0001). Accordingly, NMDA treatment with omega-agatoxin-Aa2a decreases the time spent in the dark chamber in the NMDA + Aga group compared to the NMDA-treated group (P < 0.0001), in the passive avoidance task. There is a significant difference between the NMDA + Aga and the control groups (P < 0.0001).

Figure 9 displays the effect of NMDA-treated and NMDA-treated + Aga groups on, SY1A, SYT1, and SYN genes expression in the rat hippocampus. This figure reveals that administration of NMDA in the rat hippocampus distinctly has decreased SY1A (P < 0.0001), SYT1 (P < 0.0001), and SYN (P < 0.0001) mRNAs expression when compared to the control group. The NMDA-treated group with a single dosage injection of omega-agatoxin-Aa2a increased SY1A (P < 0.05), SYT1 (P < 0.01), and SYN (P < 0.05) mRNAs expression with respect to the NMDA-treated group. Significant differences were observed between the two examined groups (mRNAs expression: NMDA-treated, and NMDA-treated with a single dose of omega-agatoxin-Aa2a) for SY1A (P < 0.001), SYT1 (P < 0.001), and SYN (P < 0.0001).

According to the Figure 10A, the injection of NMDA significantly decreased the field excitatory postsynaptic potentials (fEPSP) amplitude in the NMDA-treated group after LTP induction in the CA₃ area of the hippocampus when compared to the control group (P < 0.001). Administration of a single dose of omega-agatoxin-Aa2a after the injection of NMDA in the NMDA-treated + Aga group remarkably increased the fEPSP amplitude after LTP induction with respect to the NMDA-treated group (P < 0.01). The fEPSP amplitude in the NMDA-treated + Aga group represented a significant difference compared to the control group (P < 0.01).

Figure 10B, demonstrates the traces of the recorded fEPSPs in the hippocampal CA₃ neurons, before and after LTP induction using the high-frequency stimulation (HFS) technique in all the compared groups.

Our data showed that the single injection of NMDA significantly decreased the expression of SNAP-25 protein in the CA₃ area of the hippocampus compared to the control group (P < 0.0001). The single administration of omega-agatoxin-Aa2a after injection of NMDA distinctly enhanced the expression of SNAP-25 in the CA₃ area of rat hippocampus with respect to the NMDA group (P < 0.01). The NMDA + Aga group showed a significant differences in the CA₃ sub-region of the rat hippocampus compared to the control group (P < 0.01; Figures 11A–C.

Our data showed that the single injection of NMDA significantly decreased the expression of living pyramidal neurons in the CA₃ sub-region of the hippocampus compared to the control group (P < 0.001). The single administration of omega-agatoxin-Aa2a after injection of NMDA, distinctly enhanced the living pyramidal neurons in the CA₃ sub-region of the hippocampus with respect to the NMDA-treated group (P < 0.01). The NMDA-treated + ligand group showed a significant difference in the CA₃ sub-region of the hippocampus compared to the control group (P < 0.01; Figures 12A, B).