Shortly, HEK293TN cells were grown to 70–80% confluence in Dulbecco modified eagle medium supplemented with 5% fetal bovine serum. Cells were cotransfected with pD2 (serotype 2) + pAAV-CAG-NGF-IRES-GFP (vector carrying human NGF cDNA) or pD2 + pAAV-CAG-IRES-GFP (control viruses) using branched PEI (Sigma). Three days after transfection, the cells were collected and lysed in a cell lysis buffer (3 × freezing/thaw at −80°C); cell debris were removed by centrifugation (3,900 g, 5 min, 4°C). Virus suspension was treated with Benzonase nuclease and incubated in water bath at 37°C for 30 min; then, the viral suspension was centrifuged (3,900 g, 5 min, 4°C), and supernatant was filtered (PES 0.45 μm). AAVs were purified using HiTrap HEPARIN HP columns and concentrated on Amicon 100 k filters. Then the viruses were sterilized using an Acrodisk filter (0.22 μm) and stored at −80°C. Viral titer was 10¹¹–10¹² viral particles/mL.

Drug administration was performed using standard stereotaxic methods. Animals were anesthetized with isoflurane (Baxter, United States) and mounted in a Kopf stereotaxic frame. 192IgG-saporin (1.5 mg) or PBS was injected into the medial septum area (0.4 mm anterior, 1.5 lateral to bregma, angle 14°). This dose of the toxin was chosen based on our previous data that the immunotoxin dose of 1.5 mg induced loss of choline acetyltransferase (ChAT)–positive neurons in the medial septum area (Dobryakova et al., 2020b, a). AAV suspension was bilaterally injected into the CA1 area of the hippocampus (2.9 mm posterior, 1.7 lateral to bregma, 3.3 mm depth; 1 μL/side). All injections were performed through the Hamilton syringe (Hamilton company, United States) using a microinfusion pump (Stoelting Co., United States) at a rate of 0.5 μL/min. After each injection, the needle was left in situ for 10 min. Rats were allowed to recover for 21 days after the surgery.

Electrophysiological experiments were performed as previously described (Dobryakova et al., 2020a). Rats were anaesthetized with urethane (1.75 g/kg, intraperitoneally) and mounted in a stereotaxic frame for surgical preparation for the recording session. Stimulating nickel-chrome electrode (diameter 80 μm) was implanted into the ventral hippocampal commissure (VHC) (1.3 mm posterior, 1.0 mm lateral to bregma, approximately 3.5 mm ventral to dura). Recording electrode was placed into the CA1 area (2.7 mm posterior, 1.5 lateral to bregma, approximately 2.2 mm ventral to dura) (Paxinos and Watson, 2007). One electrode under the skin served as a ground and as a reference electrode.

To study whether NGF overexpression can prevent disturbances caused by the immunotoxin, the fEPSP amplitude in the CA1 field evoked by paired VHC stimulation (interstimulus interval 30 ms; intertrain time 20 s at intensity of 100–300 μA; 10 paired stimulations) was recorded every 10 min. The intensity of testing paired pulse stimulation was set to evoke 40–50% of the maximum fEPSP amplitude. The magnitude of stimulation current for paired pulse stimulation did not differ between the groups [138 ± 14, 145 ± 6, and 133 ± 11 μA for PBS-GFP, SAP-GFP, and SAP-NGF, respectively; F(2, 24) = 3.95, p = 0.68]. The amplitude of baseline fEPSP also did not differ between the groups [0.99 ± 0.09, 1.0 ± 0.13, and 1.0 ± 0.1 mV for PBS-GFP, SAP-GFP, and SAP-NGF, respectively; F(2, 24) = 0.02, p = 0.98]. Long-term potentiation (LTP) in the CA1 was induced by high-frequency stimulation of VHC (theta burst stimulation, five series, four trains of five stimuli with frequency of 100 Hz, intertrain interval was 200 ms; interseries interval was 30 s) after 30-min baseline recording. In our experiments for long-term recordings, we applied urethane anesthesia, which is used for non-recovery procedures of exceptionally long duration where preservation of autonomic reflexes is essential and thus does not need any additional euthanasia procedure.

All rats were anesthetized with urethane (1.75 g/kg) and were perfused transcardially with ice-cold 0.9% NaCl. The brains were removed, and the frontal part of the brain, which contains septum, was dissected and postfixed in 4% formalin (Panreac, Spain) solution in PBS (Biolot, Russia) for at least 2 days. The rest of the brain was cut along the midline. The hippocampus was isolated from the left hemisphere and used for assessment of NGF content and AChE activity. The right hemisphere was postfixed in the 4% formalin for control of transduction efficiency and immunohistochemistry.

The full extent of septum and right hemisphere of the hippocampus were sectioned at a 50-μm-thick coronal brain slices using a vibrating blade microtome (VT1200 S; Leica, Germany). Two slices from the septum area of each rat were selected and stained for ChAT to confirm cholinergic depletion in the medial septum area. Two slices from the hippocampal area from each rat were selected and stained for GFP to confirm the virus expression. To study the distribution of p75/NGFR receptors in the hippocampus and medial septal area, several slices from these structures from each experimental group were double-stained for p75/NGFR and NeuN. Slices from the hippocampus and medial septum area were also stained for TrkA to determine the localization of the receptor after NGF overexpression.

For ChAT immunostaining, the sections were preincubated in 0.3% triton X-100 (SERVA, Germany) in 0.01 M PBS (PBS-T) three times for 5 min at room temperature. Endogenous peroxidases were quenched using 15-min incubation in 3% H₂O₂, and the sections were washed thrice with PBS-T. Then, the sections were incubated for 1 h in blocking solution [5% normal goat serum or normal rabbit serum (Sigma–Aldrich, United States) in PBS-T] and after that in blocking solution with primary rabbit anti-choactase antibodies diluted as 1:500 (Santa Cruz Biotechnology, United States) or goat anti-TrkA antibodies diluted as 1:20 (R&D Systems, United States) at 4°C overnight. The next day, the sections were washed in PBS-T and incubated with secondary antibodies [1:800, goat anti-rabbit immunoglobulin G (IgG)–biotin, Sigma–Aldrich or 1:500 rabbit anti-goat IgG-biotin, Jackson Laboratories, both United States] in blocking solution at room temperature for 1 h. After additional washing in PBS, the sections were incubated with avidin–biotin–horseradish peroxidase (HRP) complex (ABC Elite kit, Vector Labs, United States) for 1 h, and 3,3-diaminobenzidine (Sigma–Aldrich) was used as a chromogen for development of staining.

GFP fluorescence was enhanced using immunostaining. In brief, after the preincubation step as above, the sections were placed for 1 h in blocking solution (5% normal goat serum in PBS, Sigma–Aldrich) and after that in blocking solution with primary anti-GFP antibodies diluted as 1:500 (Invitrogen, United States) at 4°C overnight. The next day, the sections were washed in PBS-T and incubated with secondary goat anti-rabbit Alexa 488 conjugated antibodies (diluted as 1:500 in the same blocking solution at room temperature for 1 h).

Two additional sections from the hippocampus and medial septal area were double-stained for p75 and NeuN. In brief, the sections were washed in PBS three times for 5 min at room temperature. Endogenous peroxidases were quenched using 15-min incubation in 3% H₂O₂, and the sections were additionally washed thrice with PBS. Then, they were incubated for 1 h in blocking solution [0.3% triton X-100 (SERVA, Germany)] in 0.01 M PBS (PBS-T) and 5% normal goat serum (Sigma–Aldrich) and, after that, in blocking solution with primary mouse monoclonal anti-p75 NGF receptor antibodies diluted as 1:500 (Abcam, United Kingdom) at 4°C overnight. The next day, sections were washed in PBS and incubated with goat anti-mouse IgG-biotin, antibodies 1:500 (Jackson Laboratories, United States) in blocking solution at room temperature for 2 h. After additional washing in PBS, the sections were incubated with avidin–biotin–HRP complex (ABC Elite kit, Vector Labs, United States) for 1 h, and 3,3-diaminobenzidine with nickel enhancement (Vector Labs, United States) was used as a chromogen for development of staining. Then, the brain sections were washed thrice with PBS for 5 min and then placed in blocking solution with rabbit anti-NeuN antibodies diluted as 1:500 (Chemicon, United States) at 4°C overnight. The next day, the sections were washed and incubated with avidin–biotin–HRP complex (ABC Elite kit, Vector Labs, United States) for 1 h, and chromogenic detection was performed using Vector NovaRed Substrate Kit (Vector Labs, United States).

All images were acquired with an epifluorescent Leica DM6000B microscope (Leica). Imaging parameters were set to avoid signal saturations. Two sections from the medial septal area were selected in accordance to rat brain atlas (Paxinos and Watson, 2007) with a distance of 300 μm between them. All ChAT-positive cells within the medial septum area were counted. The mean number of cells per section was counted and was considered as a representative value for one animal. The density of cells, expressing p75 and TrkA receptors, was calculated in the adjacent sections, using eye peace grid and a × 40 lens. Number of cells is presented as percent of the value counted in the PBS-GFP group.

After the electrophysiological recording, animals were submitted to transcardial perfusion with ice-cold 0.9% NaCl. The isolated left hippocampus was used for NGF assessment. To extract proteins, the hippocampal samples were homogenized in PBS containing inhibitor cocktail (Roche, Switzerland) and 0.1% NP-40 (Helicon, Russia). Then, protein lysates were kept on ice for 15 min to allow complete tissue disintegration and cell lysis. This step was followed by sample centrifugation at 15,000 g for 30 min at 4°C. The supernatants were used to detect rat NGF concentration in the hippocampus using a rat β-NGF enzyme-linked immunosorbent assay (ELISA) kit RAB0883 (Sigma–Aldrich). Briefly, to perform the assay, 100-μL volumes of samples (duplicate) or standards were added to the wells, followed by 100-μL volume of the detection antibody. After 1-h incubation at room temperature on a plate shaker set to 300 revolutions/min (rpm), the wells were washed 3 × 300 μL 1X wash buffer to remove unbound material. Then, 100 μL of Streptavidin solution was added to each well and incubated for 45 min at room temperature. Then, TMB One-Step Substrate Reagent was added to each well followed by incubation for 30 min in the dark (avoiding standard signal saturation) on a plate shaker set to 300 rpm. This reaction was stopped by addition of 100 μL of Stop Solution to each well, completing any color change from blue to yellow. Signal was generated proportionally to the amount of bound analyte, and the intensity was measured at 450 nm with a plate reader.

The activity of AChE was assessed using a standard method for continuous monitoring of AChE activity in a fluorescence microplate using the Amplex Red Assay Kit (Thermo Fisher Scientific). In the kit, AChE activity is monitored indirectly using Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), a sensitive fluorogenic probe for H₂O₂. Isolated hippocampi were homogenized in ice-cold homogenization buffer consisting of 0.9% NaCl, 0.1% NP-40 (Helicon), leupeptin (Sigma–Aldrich), and aprotenin (Sigma–Aldrich). Then, protein lysates were kept on ice for 15 min to allow complete tissue disintegration and cell lyses. Homogenates were centrifuged at 15,000 g for 30 min at 4°C. The supernatants were used to detect acetylcholinesterase. All samples were diluted in 1X reaction buffer from the kit. A volume of 100 μL was used for each reaction. The samples and positive controls were duplicated. Then, 100 μL of the Amplex Red working solution was added to each well. Working solution contained 400 μM Amplex Red reagent, 2 U/mL HRP, 0.2 U/mL choline oxidase, and 100 μM acetylcholine. The fluorescence was measured at multiple time points for 30 min (6-min intervals) at 20°C. The excitation was measured in the range of 530–560 nm and emission detection at 590 nm. The results were expressed as U/g tissue weight. One unit of AChE is the enzyme that generates 1 μmol of TNB per min.

All data are presented as mean ± SEM. The distribution of variables in the samples was normal according to Shapiro-Wilk test. One-way analysis of variance (ANOVA) with a between-factor “Group,” followed by Tukey honestly significant difference post hoc test, was used to reveal the differences between the groups. Mixed-design ANOVA with “Group” and “Time after stimulation” as between and within factors, respectively, followed by Tukey HSD post hoc test, was used to analyze electrophysiological data. The differences were considered as significant at p < 0.05.

Administration of AAV-based vectors in the hippocampus resulted in the thorough expression of carried genes in the CA1 and DG regions of the hippocampus (Figures 1A–C). Furthermore, in the NGF-SAP group, the level of NGF detected by ELISA in the hippocampal extracts was approximately twice higher as compared to the samples from PBS-GFP or SAP-GFP animals [Figure 1D; F(2, 26) = 13.5, p < 0.0001]. These data support the efficiency of neuronal transduction using our vectors.

In order to estimate a possible effect of 192IgG-saporin and a possible protective effect of NGF after the simultaneous administration of the toxin and Ngf-containing or control vectors into the medial septum or hippocampus, respectively, we conducted a series of behavioral experiments. The series included several tests that allowed analyzing different aspects of animal behavior related to memory. Spontaneous alternation was assessed in the T-maze and Y-maze tests. These tests allow studying exploratory activity and short-term memory, although in a primitive form. In PBS-GFP rats, the overall number of errors over the first 3 days was lower compared to the SAP-GFP group in the T-maze test. NGF tended to decrease the number of errors. However, these changes were not significant (Figure 2A) (p = 0.05). The overall numbers of errors over all testing periods were not significantly different due to high number of errors over the last 2 testing days in all groups. In contrast, ANOVA revealed a significant group effect for the percentage of alternations [F(2, 26) = 14.5, p < 0.0001] in the Y-maze. 192IgG-saporin significantly decreased a percentage of correct choices and NGF overexpression induced a significant improvement of the alternation back to the control level (Figure 2B).

In order to conclude that the observed behavioral disturbances in the Y-maze were associated with effects on the mechanisms of short-term memory, and not on the general activity, rat locomotion and exploration were studied in the open field test. We did not reveal any significant differences in these indices among the groups injected with 192IgG-saporin, NGF or a combination of both (Figures 2C–G). We observed only a weak trend to a decrease in the number of center crosses in 192IgG-saporin–treated group (Figure 2E).

Additionally, the sensorimotor coordination was evaluated in the beam-walking test. Noteworthy, cholinergic deficit resulted in a significant reduction of the beam-walking score in SAP-GFP group (SAP-treated group vs. control group) whereas NGF overexpression in the hippocampus improved the sensorimotor coordination and, thus, increased the score in the SAP-NGF group compared to SAP-GFP rats. ANOVA also showed a significant group effect [F(2, 26) = 6.96, p < 0.01] for beam-walking scores (Figure 2H).

Finally, the behavior in a step-through passive avoidance task was used to evaluate the mechanism of long-term memory formation and recall. Twenty-four hours after the training session, all the animals spent most of testing time in the light part of the experimental box exhibiting normal memory capacities. Thus, neither induction of cholinergic deficit nor NGF overexpression influenced animal behavior in this test (Figure 2I). Altogether, our data indicate that intraseptal administration of 192IgG-saporin induced mild impairment of short-term memory and motor coordination without any significant effect on general locomotor and exploratory activity and long-term memory formation and/or recall.

Could the mild effects of NGF overexpression on animal behavior be associated not with an improvement in cholinergic transmission but with an effect on neuroplasticity in the hippocampus? To tackle this question, we examined whether NGF overexpression under conditions of cholinergic deficit would have protective effect on the LTP induction at VHC-CA1 synapses in vivo. LTP-inducing tetanic stimulation was applied in animals when the baseline fEPSP was stable for at least 30 min. Analysis of fEPSP slope using mixed-design ANOVA revealed no significant group effect [F(2, 23) = 1.1, p < 0.3] and interaction between group × time [F(22, 253) = 1.0, p < 0.4]. However, significant time effect [F(11, 253) = 30.1, p < 0.00001] was found for this parameter reflecting LTP induction. In contrast, analysis of fEPSP amplitude revealed a trend to a group effect [F(2, 23) = 2.7, p < 0.09], a significant time effect [F(11, 253) = 34.8, p < 0.001], and strong time × group interaction [F(22, 253) = 2.3, p < 0.0001]. Data in Figure 5A demonstrate that although an increase in fEPSP amplitude was found in all groups of rats, the magnitude of this fEPSP increase in the SAP-GFP group was significantly lower as compared with the control or SAP-NGF rats. Thus, NGF overexpression prevented a negative effect of cholinergic deficit on the LTP magnitude. We also estimated paired-pulse facilitation (PPF) during baseline and after LTP induction (30-ms interstimulus interval) and did not observe any group effect on PPF whereas a strong effect of time [F(11, 253) = 31.94, p < 0.00001] and time × group interaction [F(22, 253) = 4.14, p < 0.0001] were found. The post hoc test revealed that LTP induction led to a significant decrease in the PPF under the control conditions (Figure 5B), which was absent in the SAP-GFP rats. The development of PPF after the LTP induction was normalized by NGF overexpression.

As field response is multicomponent and has contribution of different factors, we also evaluated changes that were produced by cholinergic deficit and NGF in the characteristics of second fEPSP. Unlike the characteristics of the first response, the second response was not affected by any of the treatments. Mixed-design ANOVA revealed no significant group effect [F(2, 21) = 0.18, p < 0.8] and interaction between group × time [F(20, 210) = 0.14, p < 1.0]. However, LTP was induced in all groups which, is supported by significant time effect [F(10, 210) = 30.1, p < 0.00001] for the amplitude of the second response. Similarly, the group effect was absent for the slope of the second response [F(2, 22) = 1.8, p < 0.19], and no interaction group × time interactions were observed [F(20, 220) = 1.3, p < 0.2]. However, significant time effect [F(10, 220) = 14.055, p < 0.0001] was also observed for the fEPSP slope reflecting LTP induction in all experimental groups. The data of electrophysiological experiments suggest that induction of cholinergic deficit leads to suppression of LTP magnitude and NGF restores it; however, this effect is observed only for the first fEPSP response.

We also studied whether the above effects were mediated by local overexpression of NGF receptors in the hippocampus. For this purpose, we detected expression of TrkA and p75 receptors in the hippocampus using specific antibodies. In the control rats, there were no neurons stained with anti-TrkA antibodies except of some cell profiles that were observed along the needle track (Figure 6A). Similar pattern of immunostaining was observed in two other groups of animals (Figures 6B,C). Single NGFR-positive cell profiles were scattered in the hippocampus, mainly in the region of injection, but the pattern of staining did not differ among groups (Figures 6D–F). Thus, overexpression of NGF produced in the hippocampus by AAV-mediated transduction did not substantially affect the expression of NGF receptors in this brain region.