HBK-15, a multimodal compound, induces procognitive effects through modulating hippocampal LTP and enhancing theta-gamma coupling in mice

doi:10.21203/rs.3.rs-3126208/v1

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HBK-15, a multimodal compound, induces procognitive effects through modulating hippocampal LTP and enhancing theta-gamma coupling in mice

https://doi.org/10.21203/rs.3.rs-3126208/v1

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Neuropsychiatric disorders present with an array of emotional and behavioral symptoms, as well as cognitive deficits. Likely rooted in a complex pathophysiology involving glutamatergic neurotransmission imbalance, cognitive deficits frequently elude treatment by current pharmacotherapies. This highlights the pressing need for innovative drugs specifically targeting and ameliorating cognitive deficits in neuropsychiatric disorders. Here we show that HBK-15, a multimodal compound, exhibits robust antiamnesic properties across several MK-801-induced mouse models of memory deficits, likely through counteracting LTP decline and enhancing theta-gamma coupling in the hippocampus. HBK-15 has shown efficacy in mitigating MK-801-induced cognitive deficits across recognition, emotional, and spatial memory domains without impacting motor skill learning. Its protective effects spanned the encoding, consolidation, and retrieval phases of memory processing. Furthermore, the test compound counteracted the decrease in the hippocampal LTP magnitude caused by MK-801, probably via influencing the L-type voltage-gated calcium channels (Cav1.2). Interestingly, HBK-15 and MK-801 exhibited opposing effects on the coupling between theta and gamma oscillations in the hippocampus. The promotion of theta-gamma coupling by HBK-15 suggests that the compound holds promise for enhancing learning and memory processes. Overall, our research underscores the potential of HBK-15 and compounds of a similar receptor profile in developing effective therapeutic strategies for cognitive deficits in neuropsychiatric conditions such as depression or schizophrenia.

Health sciences/Diseases/Psychiatric disorders/Depression

Health sciences/Diseases/Psychiatric disorders/Schizophrenia

Biological sciences/Neuroscience

Biological sciences/Drug discovery

Health sciences/Diseases/Psychiatric disorders/Bipolar disorder

memory deficits

MK-801

multimodal compound

recognition memory

emotional memory

spatial memory

2-methoxyphenylpiperazine derivative

LTP

Memory impairments are increasingly a significant concern as the human life span expands. These deficiencies in memory not only manifest as isolated disorders but often accompany a range of neuropsychiatric diseases, from diminished attention and working memory deficits to disrupted long-term memory and problem-solving abilities [1]. Unfortunately, cognitive impairments in neuropsychiatric disorders, such as depression, bipolar disorder, anxiety, or schizophrenia, have been underemphasized in drug discovery, leading to current pharmacotherapy being ineffective in mitigating memory decline. This oversight significantly contributes to an elevated risk of relapse and the persistence of residual symptoms in patients afflicted by neuropsychiatric disorders.

A critical aspect of developing novel drug candidates to alleviate both affective and cognitive symptoms involve identifying the cellular and molecular targets essential for the pathogenesis of neuropsychiatric disorders. One proposed mechanism pertains to glutamatergic imbalance [2]. Glutamate, the most abundant excitatory neurotransmitter in the central nervous system (CNS), modulates numerous brain functions and synaptic plasticity, including long-term potentiation (LTP). LTP, considered the neural basis of memory formation, can be triggered by activating NMDA receptors or NMDA-independent mechanisms (reviewed in [3]). Although the predominant type of LTP in the CNS is NMDA receptor-dependent, more intriguing mechanisms underlie the NMDA receptor-independent LTP. These may involve the activation of ionotropic receptors, such as nicotinic acetylcholine and serotonin 5-HT₃ receptors, or metabotropic receptors, including glutamate, serotonin 5-HT_1A, or 5-HT_2A receptors [3]. Moreover, NMDA receptor activity can be regulated by other receptors [4]. Consequently, revealing the complexity of potential therapeutic targets for treating memory deficits associated with neuropsychiatric disorders may facilitate the design of innovative drug candidates, ultimately leading to improved treatment efficacy.

HBK-15 has emerged as a promising compound for addressing depression-related cognitive deficits [5]. Previous research revealed a multimodal pharmacological profile of this methoxyphenyl piperazine derivative, exhibiting affinity for serotonin (5-HT_1A, 5-HT_2A, 5-HT₇), dopamine (D₂), and adrenergic (𝛼₁) receptors, serotonin transporter, and voltage-gated sodium channels, with antagonistic/inhibitory effects on these targets [6–10]. Various behavioral tasks demonstrated the antidepressant- and anxiolytic-like properties of HBK-15 following both single and repeated administration in naïve rodents [5–7] and mice subjected to depression models (corticosterone injections or unpredictable chronic mild stress procedure) [9, 10]. Additionally, a single injection of the compound normalized brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in the prefrontal cortex and hippocampus of depressed mice [9, 10]. Notably, HBK-15 is distinguished not only by its remarkable efficacy but also by its favorable safety profile [6, 8, 10–12]. While previous experiments have demonstrated its effectiveness against emotional memory impairments induced by a cholinergic system-disrupting drug [5], its impact on cognitive deficits caused by glutamatergic system imbalance remains unexplored.

Given the compelling evidence of glutamatergic system dysregulation in various neuropsychiatric disorders and its crucial role in learning and memory processes, our objective was to investigate the effects of HBK-15 on different types of memory impaired by MK-801, an NMDA receptor antagonist, using behavioral tasks. We also aimed to elucidate the underlying mechanisms by assessing the effects of the compound on hippocampal synaptic plasticity and neural oscillations.

Detailed methods can be found in Supplementary materials.

Animals

Depending on the experiment, we used naïve adult male BALB/c (Mossakowski Medical Research Institute, Polish Academy of Sciences or Charles River Laboratories (Barcelona, Spain or Edinburgh, UK)) or C57BL/6J (Mossakowski Medical Research Institute, Polish Academy of Sciences) weighing 24 ± 2 g (8–10 weeks old). The total number of animals used was 1188. All experiments were performed following the European (86/609/EEC; 2010/63/EU; 2012/707/EU) and Polish, Portuguese, or the United Kingdom Animals (Scientific Procedures) Act of 1986 Home Office regulations (approved by either the Local Ethics Committee for Experiments on Animals in Kraków: 485/2021, 547/2021, 548/2021, 549/2021, 550/2021, 591/2022, 604/2022 or by the Home Office in Strathclyde: PPL0688994).

Drugs

1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15) was synthesized in the Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College according to the methodology described earlier [6]. HBK-15 and MK-801 (Sigma, Germany) were dissolved in saline and administered intraperitoneally (ip). Control groups received saline. The doses of the studied compound and MK-801 for experiments were based on earlier studies [7].

For electrophysiological experiments, HBK-15 was supplied in a 1.0 mg/ml stock solution in saline and added to the slices in 1 µg/ml diluted in the artificial cerebral spinal fluid (aCSF). MK-801 was dissolved in a 10 mM stock solution in saline and added to the slices in a 10 µM diluted in aCSF.

Experimental procedures

In vitro binding assays

Binding studies were performed commercially in Eurofins Laboratories using testing procedures described elsewhere: kainate (KA), AMPA [13], NMDA (Glu) [14], NMDA (Gly) [15], NMDA (MK-801) [16], NMDA (PA) [17], 5-HT₃ [18], N-type calcium channels Cav 2.2.; (𝜔-conotoxin) [19], N-type calcium channels (Cav 2.2.; gabapentin) [20], L-type calcium channels (Cav 1.2.; phenylalkylamine) [21], mGluR1 [22], mGluR2 [23] and mGluR5 [22]. The results are presented as the inhibition of control-specific binding in the presence of HBK-15.

Behavioral tests

Behavioral tests were carried out as previously described (step-through passive avoidance task [24]; hot plate test [25]; object recognition test [26, 27]; Morris water maze [28, 29]; Barnes maze [30]; rotarod [31, 32] and detailed in the Supplementary material.

Step-through passive avoidance task

Acquisition phase: mice were placed individually in an illuminated white compartment of the apparatus with a closed door to a smaller dark compartment equipped with a grid floor (LE872, Bioseb, France). After 30 s, the door opened, and once the mice entered the dark compartment, the door closed, and an electric foot shock was delivered (0.2 mA for 2 s). The mice which did not enter the dark compartment within 60 s were excluded from the study. Test phase: the animals were placed again into the illuminated compartment and observed for up to 300 s (retention session), however, no electric foot shock was delivered when the mice entered the dark compartment.

HBK-15 was administered ip 30 min before or immediately after the familiarization session or 30 min before the test phase to evaluate whether the tested compound affected the learning phases (Fig. 1a, e, and h). MK-801 was injected ip 15 min after the administration of the tested compound. Moreover, we investigated how HBK-15 affected the animals' performance in various time intervals – the gap between acquisition and test phase was 15 min, 4 h, and 24 h (encoding process) or 4 h and 24 h (consolidation and retrieval processes).

Hot plate test

Mice were placed in the apparatus (Hot/Cold Plate, Bioseb, France) with electrically heated surface (55°C). Only mice with baseline latencies ≤ 30 s were selected for further analysis. 30 min post-injection of HBK-15, the latency to nocifensive reactions (licking hind paws or jumping) was measured. The animals that did not respond within 60 s were removed from the apparatus and assigned a score of 60 s.

Object recognition test

Familiarization session

mice were placed individually in the open field with two identical objects and left there until they reached the 20-s criterion of total exploration, but no longer than 10 min.

Test phase

mice were placed again in the open field, but this time one of the objects was replaced with the new one. Mice were again left until they reached the 20-s criterion of total exploration, but no longer than 10 min. To assess animals' performance in the object recognition test, the means of the novel object exploration time were compared with the chance level (10 s, equal exploration of the objects).

The administration schedule was the same as described in the previous paragraph regarding the passive avoidance task (Fig. 2a, e and h).

Morris water maze

Acquisition phase (days 1–6): Animals were placed in a circular pool (Panlab-Harvard Apparatus, Spain) filled with opaque water at a temperature of 24 ± 1°C and were learned to find a submerged platform using the visual cues around the tank. Each day mouse needed to complete four trials separated by 15-min intervals. If the animal did not find the platform within 60 s, it was gently guided to it and left for 15 s. HBK-15 was administered ip 30 min before the first trial for six consecutive days, and MK-801 was injected ip 15 min later (Fig. 3a). The following parameters were collected: the latency to enter the platform (i.e., escape latency), the total distance, and the swimming speed.

Probe test: 24 h after the last training session, no compounds were administered, and the platform was removed from the pool. Each mouse swam during a 60-s trial, and the following parameters were collected and analyzed: the latency and the covered distance to the target zone (a place where the platform was previously located), the percentage of time spent in the target quarter, number of target entries, and the swimming speed.

Barnes maze

The Barnes maze apparatus is a circular platform with 18 equally spaced holes (Panlab-Harvard Apparatus, Spain) and an escape box placed under one of the holes. The visual cues (boards with colored geometric figures) were placed around the maze.

Habituation phase (day 0)

the mouse was placed in a tube in the middle of the maze. 10 s later, the mouse was released, the aversive light (600 lux) and noise (80 dB) were turned on, and the animal could explore the maze for 1 min. Afterwards the animal was gently guided to the escape box, where it remained for the next 1 min. This phase was not preceded by compounds' injection.

Acquisition phase (days 1–4)

each mouse was placed individually in a plastic tube in the center of the platform, released after 10 s, and the aversive stimuli were turned on. The trial finished when the mouse entered the escape box or after 3 min have elapsed. Each day animals needed to complete four trials with a 15-minute interval between trials. Mice were injected ip with HBK-15 30 min before the first trial for four consecutive days, and MK-801 was administered ip 15 min later (Fig. 4a). During this phase, the number of primary errors (i.e., the number of errors before the mouse reached the escape box), total errors (i.e., the number of all errors made during 3 min of the test), primary and total latencies to find the escape box (i.e., the time required to find and time required to enter the escape box, respectively) were measured.

Test phase (days 5 and 12)

after the last training session, no compounds were administered, and the escape box was removed. Each mouse was allowed to explore the maze for the 90 s. Primary errors and latency to reach the former target (i.e., the escape box) location were measured.

Rotarod test

Acquisition phase (days 1–4)

each mouse was placed on the rotating rod with increasing speed (maximum speed 40 rpm) for 5 min or until they fell off (May Commat RR0711, Turkey). Within a day, each mouse performed four trials. 30 min before the first trial, animals were injected ip with HBK-15 and 15 min later, with MK-801 (Fig. S1a). The measured parameter was the latency to fall off the rotating rod.

Probe test

On the fifth day, the animals were again placed individually on a rotating rod with increasing speed. The duration of the experiment was again 5 min; however, no compounds were administered.

Neurotransmitter levels

Tissue homogenates

Tissue and homogenates preparation: 30 min after acute administration of HBK-15 or saline, naïve mice were sacrificed, and their brains were rapidly removed and chilled in an ice-cold saline solution. The hippocampi were dissected, frozen, and stored at − 80°C until the assay. On the day of experiments, tissues were thawed on ice and homogenized (1:10 w/v) in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 0.32 M sucrose using bead homogenizer (Bead Rupture elite, Omni International, USA).

Analytical method

Concentrations of dopamine (DA), serotonin (5-HT), acetylcholine (ACh), glutamic acid (Glu), histamine (HIS), and norepinephrine (NE) in the hippocampi were measured by liquid chromatography-tandem mass spectrometry method (LC-MS/MS). After deproteinization and centrifugation of homogenate samples supernatants were transferred into the autosampler vials. Analytes were separated on the XBridge HILIC (2.1 x 150 mm, 3.5 µm, Waters, USA) analytical column using an Exion LC AC HPLC system (Sciex, USA). The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile mixed at the ratio of 30/70 (v/v). A QTRAP 4500 (Sciex, USA) tandem mass spectrometer equipped with an electrospray ionization (ESI) was operated at unit resolution monitoring the transitions presented in Table S1. Corresponding deuterated internal standards were used for analyte quantification. Calibration curves were linear in the range from 1 to 1000 ng/ml for ACh, 5-HT, and DA; from 2.5 to 1000 ng/ml for HIS; from 10 to 1000 ng/ml for Glu and from 25 to 1000 ng/ml for NE. Then, concentrations of neurotransmitters in the analyzed samples were calculated per g of brain tissue by applying appropriate dilution factors.

Microdialysates

Surgery

Animals were anesthetized with 2.5% isoflurane (5% for induction) and guide cannulas (MAB 10.8.IC, AgnTho’s AB, Sweden) were implanted over the hippocampus (AP – 1.93 mm; ML + 1.5 mm; DV – 1.8 mm from the bregma; Fig. S2), according to the stereotaxis atlas of Franklin and Paxinos [33]. After surgery, mice were housed in a high-roofed cage with ad libitum access to water and food and monitored for recovery (normal eating, drinking, and defecation).

Microdialysis: Following a 3-day recovery period, microdialysis probes (MAB 10.8.1.PES with cut off: 6 kD, AgnTho’s AB, Sweden) were connected to the syringe pump (Univentor 864 Syringe Pump, AgnTho’s AB, Sweden) that delivered aCSF composed of (mM) 147 NaCl, 4 KCl, 2.2 CaCl₂ and 1.0 MgCl₂ at a flow rate 1 µl/min. The monitoring of extracellular levels of neurotransmitters has been performed in freely moving animals. After a 2-h stabilization period, a baseline sample was collected for 30 min. Thereafter, HBK-15 at the dose of 2.5 mg/kg was injected ip and three samples were further collected every 30 min. Placement of dialysis probes was verified post-mortem in coronal sections (Fig. S2b).

Analytical method

Concentrations of ACh and Glu in microdialysis samples from mouse hippocampus were measured by LC-MS/MS method similar to the one described above with some minor modifications. The sample preparation procedure involved the addition of 3 µL of an internal standard (IS) mixture to 30 µL of dialysate. Then, samples were vortex mixed, transferred to autosampler vials and injected into the LC-MS/MS system. Analytes were separated using gradient elution with the mobile phase consisting of 25 mM ammonium formate (pH = 3.5) in water and acetonitrile. Calibration curves were prepared in the aCSF and were linear in the range from 1 to 1000 ng/ml for ACh and Glu.

Electrophysiology ex vivo

Hippocampal slices

The hippocampus was dissected in ice-cold aCSF and slices (400 µm thick) were cut with a McIlwain tissue chopper and allowed to recover functionally and energetically for 1 h in a resting chamber as previously described [34].

Field postsynaptic potentials (fEPSPs) recording

fEPSP were recorded through an extracellular microelectrode placed in the stratum radiatum of the CA1 area (Fig. S3a). Stimulation was delivered through an electrode placed on the Schaffer collateral–commissural fibers, in the stratum radiatum near CA3–CA1 border (Fig. S3a) as previously described [34].

Long-term potentiation (LTP) induction and quantification

Stimulation was delivered alternatively to two independent pathways through bipolar concentric electrodes placed on Shaffer collateral/commissural fibers in stratum radiatum (Fig. S3b). A θ-burst protocol was induced consisting of four trains of 100 Hz, and four stimuli, separated by 200 ms as previously described (Fig. S3c) [34]. To evaluate the effects of the tested compounds upon LTP, each individual drug was added to the superfusing bath before induction of LTP and remained in the bath up to the end of the experiment. In the experiments where both compounds were tested, HBK-15 was added 15 min before MK-801 and remained until the end of the experiments.

Electrophysiology in vivo

10 male BALB/c mice (8–10 weeks old) were used. Briefly, two bone screws were implanted in the frontal region (AP + 1.5 mm, ML ± 1 mm) for cortical electroencephalograms (EEGs). Two twisted wires were inserted into the neck muscles for electromyography (EMG). A bipolar electrode was inserted into the hippocampal CA1 (AP -2 mm, ML + 1.5 mm, -1.5 mm from the cortical surface; Fig. S4). Electrophysiological signals were monitored at 1 kHz using a headstage (C3334, Intan Technologies) and an interface board (C3100, Intan Technologies). In each recording session, the animals received one of the following four pharmacological treatments: 1) saline and saline, 2) saline and MK-801 (0.125 mg/kg), 3) HBK-15 (2.5 mg/kg) and saline, and 4) HBK-15 and MK-801. All data analysis was performed offline using custom-written scripts (MATLAB R2022a, MathWorks). The power of theta (4–10 Hz) and gamma (30–45 Hz) was calculated and normalized by the total power at ≤ 40 Hz. The effect of treatments was assessed by calculating the average Z-scored power of theta and gamma oscillations between 30 and 60 min after the second injection. To estimate the phase-amplitude coupling (PAC) between theta and gamma oscillations, LFPs were bandpass-filtered at theta (4–10 Hz) and gamma (30–45 Hz) bands. The instantaneous phase of theta oscillations and the amplitude envelope of gamma oscillations were estimated by the Hilbert transformation. The mean amplitude of gamma oscillations at a certain phase of theta oscillations was computed in every 30-s window and 45° phase bin. The modulation index was defined as the ratio between the maximum and minimum mean amplitudes in each time window.

Statistical analysis

Results are presented as means ± standard deviation (SD) or standard error of mean (SEM) for parametric analysis, or medians and interquartile ranges (IQR) for non-parametric analysis. Data normality and homogeneity were verified via Shapiro-Wilk and Brown-Forsythe tests. Various ANOVAs, with appropriate post hoc tests (Dunnett's, Tukey’s, Bonferroni), were used for group comparisons. Non-parametric Kruskal-Wallis analysis with Dunn's post hoc test and Welch ANOVA test with Dunnett's T3 test were used when assumptions were not met. A one-sample t-test compared novel object exploration time to chance level in an object recognition test. A p-value < 0.05 signified significance. Analyses were done with GraphPad 9.5.0 and MATLAB (R2022a).

HBK-15 displayed a significant affinity for the L-type voltage-gated calcium channel (Cav1.2)

Previously, we found that HBK-15 interacted strongly with serotonin 5-HT_1A, 5-HT₇, dopamine D₂, α₁-adrenoceptors, and voltage-gated sodium channels [6–10]. Yet, we had not probed its potential affinity for glutamatergic receptors and additional ion channels, known for their roles in learning and memory. Addressing this gap, the current study evaluated HBK-15's interaction with a range of these cognitively important biological targets. Our radioligand binding studies demonstrated that HBK-15 showed a substantial affinity for the L-type voltage-gated calcium channel (Cav1.2), but not to other chosen biological targets, including kainate, AMPA, NMDA (at glutamate, glycine, MK-801, and polyamine (PA) binding sites), 5-HT₃ receptors, and N-type calcium channels (Cav2.2.) (Table 1).

Table 1

*In vitro* binding assays for HBK-15
Molecular target	Source	% inhibition of control-specific binding
KA	Rat cerebral cortex	-8
AMPA	Wistar rat cerebral cortex	-9
NMDA (Glu)	Wistar rat cerebral cortex	7
NMDA (Gly)	Wistar rat cerebral cortex	-4
NMDA (MK-801)	Wistar rat brain (minus cerebellum)	-6
NMDA (PA)	Wistar rat cerebral cortex	1
mGluR1	Rat cerebellum	-6
mGluR2	Human recombinant Chem-1 cells	-22
mGluR5	Human recombinant CHO-K1 cells	-9
5-HT3	Human recombinant CHO cells	-1
Cav1.2	Wistar rat cerebral cortex	57
Cav2.2 (𝜔-conotoxin)	Human recombinant CHO cells	2
Cav2.2 (gabapentin)	Human recombinant CHO cells	-6
HBK-15 was tested at concentrations 10^− 6 M. The results are presented as % inhibition of control-specific binding in the presence of HBK-15. Results showing an activity > 50% were considered to represent significant effects of the test compound; results showing an inhibition between 25% and 50% indicate moderate to weak effect; results showing an inhibition < 25% are not considered significant and mostly attributable to the variability of the signal around the control level. Binding studies were performed commercially in Eurofins Laboratories (Poitiers, France).
The binding sites for NMDA receptors are presented in brackets. Glu – glutamate, Gly – glycine, PA – polyamines

HBK-15 countered MK-801-induced emotional memory deficits at all learning stages

In our prior work, we demonstrated the rapid antidepressant- and anxiolytic-like effects of HBK-15 in rodents, alongside its procognitive potential in a mouse model of scopolamine-induced amnesia [5–7, 9, 10]. Recognizing that cognitive deficits in neuropsychiatric diseases often stem from glutamatergic system imbalance [35, 36], we investigated the potential of HBK-15 in mitigating MK-801-induced emotional memory deficits throughout the learning process using step-through passive avoidance task in mice (Fig. 1a, e, and h). Our findings revealed that HBK-15 possessed antiamnesic properties, evidenced by increased latency to enter the dark chamber, compared to the MK-801-treated control group (Fig. 1b-d). In addition, depending on the time interval between the acquisition and test phase (15 min, 4 h, or 24 h), HBK-15 was active at two (0.625 and 5 mg/kg), three (0.625, 2.5 and 5 mg/kg) or four doses (0.3, 0.625, 2.5 and 5 mg/kg), respectively, when investigating the encoding process (15 min: W(6,22.87) = 4.088, p < 0.01); 4 h: (H(7,66) = 22.05, p < 0.01); 24 h: (F(7,65) = 6.976, p < 0.0001; n = 8–10; Figs. 1b-d).

Given the observed antiamnesic properties of HBK-15 during the encoding of memory engrams and the distinct cellular processes involved in different learning phases, we further investigated the compound's potential to alleviate MK-801-induced impairments in emotional memory during the consolidation and retrieval phases. While the tested compound exhibited antiamnesic effects during all learning phases, the effect was more pronounced with a longer interval following the acquisition session. A single administration of the tested compound after the acquisition phase significantly reversed MK-801's reduction in latency at three doses (0.625, 2.5, and 5 mg/kg) for 4 h intervals and at five doses (0.3, 0.625, 1.25, 2.5 and 5 mg/kg) for 24 h interval (4 h: F(6,52) = 4.229, p < 0.01; 24 h: F(7,58) = 5.166, p < 0.001; Figs. 1f-g). We observed a similar pattern for the retrieval phase, with only two doses of HBK-15 active (0.625 and 5 mg/kg) after a 4h-delay, while a 24-h delay increased the number of effective doses (0.625, 1.25, 2.5, and 5 mg/kg) (4 h: H(7,61) = 23.09, p < 0.001); 24 h: (F(6,53) = 7.11, p < 0.0001; Figs. 1i-j).

Furthermore, we also conducted the hot plate test to exclude the possibility of false-negative results in the passive avoidance task due to the tested compound's analgesic properties. As a result, we demonstrated that none of the tested doses of HBK-15 affected the nociceptive threshold in naïve animals (F(6,54) = 2.915, p < 0.05; ), n = 7–9, Fig. S5). Overall, our study suggests that HBK-15 shows promise in addressing emotional memory deficits linked to glutamatergic system imbalance.

HBK-15 prevented MK-801-induced recognition memory impairments throughout the encoding, consolidation, and recall stages

Recognition memory impairment is a common cognitive deficit observed in psychiatric disorders like schizophrenia or depression [37, 38]. Therefore, we investigated the potential of HBK-15 to alleviate MK-801-induced impairments in recognition memory using the object recognition test. As previously, we examined the effects of HBK-15 on different memory stages (short-, intermediate-, and long-term) as well as learning processes (encoding, consolidation, and retrieval) (Figs. 2a,e,h). HBK-15 effectively mitigated memory deficits observed at 15 min, 4 h, and 24 h following the familiarization session when examining the encoding phase of learning (Figs. 2b-d). Moreover, a single administration of the tested compound resulted in statistically significant increase in the novel object exploration time compared to the chance level of 10 s for three out of the five tested doses (i.e., 0.625, 2,5, and 5 mg/kg) (15 min – 0.625 mg/kg: t(8) = 5.253, p < 0.001; 2.5 mg/kg: t(8) = 6.766, p < 0.001; 5 mg/kg: t(7) = 5.955, p < 0.001; 4 h – 0.625 mg/kg: t(9) = 4.345, p < 0.01; 2.5 mg/kg: t(9) = 2.561, p < 0.05; 5 mg/kg: t(9) = 2.275, p < 0.05; 24 h – 0.625 mg/kg: t(7) = 3.251, p < 0.05; 2.5 mg/kg: t(8) = 2.954, p < 0.05; 5 mg/kg: t(7) = 4.548, p < 0.01; n = 7–10; Figs. 2b-d).

Given that HBK-15 exhibited an antiamnesic effect throughout all learning phases of emotional memory, we were curious to determine whether a similar pattern could be observed in recognition memory. A single injection of HBK-15 effectively reversed MK-801-induced memory consolidation impairments at one (0.625 mg/kg) or four (0.625, 1.25, 2.5, and 5 mg/kg) doses after 4 and 24 h post-familiarization phase, respectively (4 h – 0.625 mg/kg: t(8) = 3.555, p < 0.01); 24 h – 0.625 mg/kg: t(7) = 2.454, p < 0.05; 1.25 mg/kg: t(8) = 3.17, p < 0.05; 2.5 mg/kg: t(7) = 2.515, p < 0.05; 5 mg/kg: t(7) = 2.79, p < 0.05; Figs. 2f-g). Additionally, during the retrieval phase, HBK-15 attenuated memory deficits, increasing novel object exploration time at four doses (0.625, 1.25, 2.5, and 5 mg/kg) when examining effects after 4 h from the familiarization phase and at three tested doses (0.625, 1.25 and 2.5 mg/kg) in the case when a 24-h break occurred between familiarization and test phases (4 h – 0.625 mg/kg: t(7) = 4.714, p < 0.01; 1.25 mg/kg: t(7) = 3.128, p < 0.05; 2.5 mg/kg: t(8) = 3.014, p < 0.05; 5 mg/kg: t(7) = 3.883, p < 0.01; 24 h – 0.625 mg/kg: t(8) = 3.3, p < 0.05; 1.25 mg/kg: t(7) = 3.351, p < 0.05; 2.5 mg/kg: t(7) = 4.692, p < 0.01; Figs. 2i-j). Altogether, HBK-15 demonstrated significant efficacy in alleviating MK-801-induced recognition memory impairments across all learning phases.

HBK-15 mitigated MK-801 negative effect on spatial learning in the Morris water maze

Spatial memory is another memory type often impaired in neuropsychiatric disorders [39]. Therefore, the subsequent phase of our study was designed to examine the influence of HBK-15 on spatial memory, utilizing the Morris water maze as our experimental paradigm (Fig. 3a). We observed that MK-801 administration led to spatial memory impairments, as gauged by metrics such as latency to find a hidden platform and the distance covered. HBK-15 demonstrated the capability to counteract MK-801-induced memory deficits. However, not all doses were effective from the first day, and efficacious doses varied depending on the parameters measured. For latency to find a platform, only a dose of 2.5 mg/kg proved effective on the third and fourth days of spatial learning. On the fifth day, the 0.3 and 1.25 mg/kg also reduced the latency, and by the sixth day, all doses of HBK-15 and vortioxetine were effective (time: F(3.634,261.6) = 112.2, p < 0.0001; treatment: F(7,72) = 9.814, p < 0.0001; interaction: F(35,360) = 3.572, p < 0.0001; n = 8–10; Fig. 3b).

Interestingly, examining another parameter, such as the distance required to reach the hidden platform, the antiamnesic effect of HBK-15 was discernible only on the sixth day of the acquisition phase at two doses (0.625 and 1.25 mg/kg) (time: F(4.085,294.1) = 182.9, p < 0.0001; treatment: F(7,72) = 6.316, p < 0.0001; interaction: F(35,360) = 1.644, p < 0.05; Fig. 3c). This divergence may stem from variations in animal swimming behavior as we noted that MK-801 reduced swim speed, a change that HBK-15 reversed (time: F(2.842,204.6) = 8.128, p < 0.0001; treatment: F(7,72) = 4.202, p < 0.001; interaction: F(35,360) = 1.711, p < 0.01; Fig. S6b). Given that literature suggests MK-801 triggers motor disturbances, particularly in aquatic conditions, it is challenging to ascertain if HBK-15 enhanced spatial learning or motor functions. However, considering that HBK-15 did not counteract motor impairments in the rotarod test, it is plausible to infer that the observed effect in the Morris water maze was related to its antiamnesic properties.

In the subsequent probe test, where no compounds were administered, no group speed differences were observed (H(8,76) = 10.29, p = 0.1728; Fig. S6c). However, the other measured parameters (latency and covered distance to find the target zone, the percentage of time spent in the target quadrant, but not number of target entries) highlighted the spatial memory deficits in animals previously treated with MK-801 (latency: H(8,76) = 29.6, p < 0.001; distance: H(8,76) = 29.14, p < 0.001; percentage of time: F(7,72) = 3.464, p < 0.01; number of target entries: H(8,78) = 12.09, p = 0.0975; Figs. 3d-g). Further, HBK-15 at doses 0.3, 0.625, and 2.5 mg/kg altered these parameters (Figs. 3d-g), confirming that the tested compound's effect arises from its antiamnesic activity. Altogether, HBK-15 prevented MK-801-induced spatial learning and memory impairments.

HBK-15 prevented MK-801-induced spatial memory deficits in the Barnes maze

To corroborate the findings from the Morris water maze concerning the antiamnesic qualities of HBK-15, we evaluated the impact of this compound on spatial memory using the Barnes maze (Fig. 4a). This model, although also employs aversive stimuli, exposes the subjects to milder adversities compared to the aquatic conditions of the Morris water maze, utilizing light and noise as stimuli. Given its land-based setup, the results are less susceptible to the motor disruptions triggered by MK-801. On the fourth day of training, HBK-15 mitigated the memory impairments induced by MK-801, as evidenced by a reduction in primary latency across all tested doses and a decrease in primary errors at four doses (0.625, 1.25, 2.5 and 5 mg/kg) (primary latency: time: F(2.061,148.4) = 51.61, p < 0.0001; treatment: F(7,72) = 1.074, p = 0.389; interaction: F(21,216) = 1.375, p = 0.1327; primary errors: time: F(2.266,163.2) = 30.7, p < 0.0001; treatment: F(7,72) = 0.4708, p = 0.8526; interaction: F(21,216) = 1.655, p < 0.05; n = 8–10; Figs. 4b-c). In terms of other parameters, HBK-15 at doses 0.625, 1.25, and 2.5 mg/kg reduced the total latency and decreased the number of total errors at doses 0.625 and 1.25 mg/kg (total latency: time: F(1.953,140.6) = 10.8, p < 0.0001; treatment: F(7,72) = 2.609, p < 0.05; interaction: F(21,216) = 1.163, p = 0.2868; total errors: time: F(2.182,157.1) = 45.26, p < 0.0001; treatment: F(7,72) = 1.405, p = 0.2168; interaction: F(21,216) = 1.692, p < 0.05; Figs. S7b-c).

Intriguingly, during the probe test on the fifth day of the experiment, mice that had previously received HBK-15 at doses 2.5 and 5 mg/kg committed fewer errors and required less time to locate the target zone (errors: H(8,78) = 22.21, p < 0.01; latency: W(7.000,28.57) = 4.638, p < 0.01; Figs. 4d-e). However, by the twelfth day, the antiamnesic activity of HBK-15 had largely diminished, except for the highest dose of HBK-15, which manifested its effectiveness in terms of latency to find a target zone (errors: H(8,80) = 7.257, p = 0.4026; latency: H(8,79) = 10.53, p = 0.1607; Figs. S7d-e). Overall, these results confirmed that HBK-15 prevented the spatial memory disturbances caused by MK-801 regardless of the type of test and environmental conditions.

HBK-15 did not protect against motor skill learning impairments caused by MK-801

Distinct brain regions are involved in various memory types. Emotional, recognition, and spatial memory rely on the prefrontal cortex, hippocampus, and/or amygdala [40–42], while the dorsal striatum is crucial for motor skill learning [43]. Inspired by the positive effects of HBK-15 on different memory types, we next explored its impact on motor skill learning impairments induced by MK-801 using the rotarod test (Fig. S1a). Mice administered with MK-801 exhibited poorer performance compared to the saline-treated control group on the third and fourth days of training (time: F(2.455,154.7) = 1.288, p = 0.2805; treatment: F(6,63) = 4.699, p < 0.001; interaction: F(18,189) = 2.588, p < 0.001; n = 9–10; Fig. S1b). Notably, none of the tested doses of HBK-15 alleviated motor disturbances. Additionally, during the probe test, there were no statistically significant differences between the HBK-15- and MK-801-treated groups (H(7,65) = 22.13, p < 0.01; Fig. S1c). In conclusion, HBK-15 did not mitigate motor learning and memory impairments.

A single administration of HBK-15 had no impact on the levels of various neurotransmitters

Neurotransmitters such as monoamines, glutamate, or acetylcholine are widely recognized as modulators of memory. To explore the potential mechanism of underpinning the antiamnesic activity of HBK-15, we analyzed the impact of a single administration of the compound on the levels of selected neurotransmitters in the hippocampus of naïve animals. We selected a 2.5 mg/kg dose of HBK-15 for this and further experiments due to its consistent antiamnesic efficacy in the above behavioral tests. The hippocampus was chosen for investigation due to its crucial role in memory and learning processes. Our study revealed no significant alterations in the levels of serotonin (F(5,54) = 1.798, p = 0.8842), noradrenaline (H(6,59) = 2.902, p = 0.715), histamine (F(5,54) = 2.165, p = 0.0716), dopamine (concentration below LOQ), glutamate (W(5,23.18) = 2.502, p = 0.0596), or acetylcholine (H(6,60) = 2.797, p = 0.7312) neither in the homogenates (n = 8–10, Table S2) nor in the microdialysates (glutamate: F(2.346,18.77) = 0.1948, p = 0.8561; acetylcholine: F(1.495,13.45) = 0.6435, p = 0.4973; n = 9–10, Table S3). These results suggest that HBK-15 did not induce changes in neurotransmitter levels within the hippocampus at any dosage tested.

HBK-15 countervailed the decrease in the LTP magnitude caused by MK-801

To ascertain whether HBK-15 alters LTP that could be linked to the observed memory impairments incited by MK-801, we conducted a series of electrophysiological experiments.

As depicted in Fig. 5a and b, both MK-801 (10µM) and HBK-15 (1µg/mL), when individually administered to the hippocampal slices, did not substantially impact the slope of fEPSP (n = 4 and 5, respectively). A similar result was obtained when both drugs were co-applied to the hippocampal slices (Fig. 5c, n = 3).

LTP, characterized by the sustained augmentation of synaptic connections, is regarded as an electrophysiological model for the fundamental mechanisms involved in learning and memory formation [44, 45]. Moreover, abnormal performance in memory tasks, specifically hippocampal-dependent spatial memory tasks such as the Morris water maze and Barnes maze, has been linked to deficiencies in LTP [30, 46, 47]. Consistent with this, akin to the impairments seen in vivo in the Morris water maze and in the Barnes maze in MK-801 treated animals, LTP was similarly compromised in hippocampal slices exposed to MK-801. Correspondingly, in the presence of MK-801, there was a significant decrease in LTP magnitude compared to the control condition (F(3,15) = 15.24, p < 0.0001; control: 53.38 ± 5.78, n = 8 vs MK-801: -8.40 ± 6.08, n = 3, p < 0.0001, Figs. 5d,g,j).

Remarkably, in the presence of HBK-15, the LTP magnitude was restored to levels comparable to the control (control: 53.38 ± 5.78, n = 8 vs HBK-15 + MK-801: 28.88 ± 9.61, n = 3, p = 0.0756, Figs. 5f,i,j) and distinct from the condition with MK-801 alone (p < 0.05). Independently, HBK-15 did not significantly alter LTP magnitude when compared to the control condition (control: 53.38 ± 5.78, n = 8 vs HBK-15: 36.27 ± 2.63, n = 5; p = 0.1666, Figs. 5e,h,j). Our results indicate that HBK-15 prevented the disruption of hippocampal LTP by MK-801.

HBK-15 enhanced theta-gamma coupling in the hippocampus in vivo

To examine how pharmacological treatment of HBK-15 and MK-801 affect hippocampal activity in vivo, we conducted electrophysiological recordings from the dorsal hippocampal CA1 region in a freely behaving condition while the animal received one of the four treatment options: 1) saline + saline, 2) saline + MK-801, 3) HBK-15 + saline and 4) HBK-15 + MK-801 (Fig. 6a). The electrode position was confirmed in the CA1 region based on post-mortem histological analysis (Figs. 6b and S4).

Given that theta and gamma oscillations are prominent neural oscillations in the hippocampus [48–50], we began by computing the power of theta (4–10 Hz) and gamma (30–45 Hz) oscillations from hippocampal local field potentials (LFPs) (Figs. 6c-f). 30–60 min after the second injection, we did not observe any significant effect of treatment on theta power (F(3,36) = 1.75, p = 0.17, n = 10) (Fig. 6d). On the other hand, we observed a significant increase in gamma power in the administration of both HBK-15 and MK-801 (F(3,36) = 7.59, p < 0.01 n = 10) (Fig. 6f) while MK-801 administration alone tended to increase gamma power too.

To further elucidate the effect of HBK-15 on hippocampal oscillations, we computed the phase-amplitude coupling (PAC) between theta and gamma oscillations (Figs. 6g,h), a phenomenon implicated in hippocampal memory function [51–53]. Intriguingly, we observed opposing trends in the PAC between HBK-15 and MK-801 administrations (Fig. 6g): HBK-15 tended to facilitate the PAC, whereas MK-801 tended to disorganize the coupling. We confirmed this by comparing the normalized modulation index (MI) between HBK-15- and MK-801-treated conditions (F(3,36) = 3.40, p < 0.05, n = 10) (Fig. 6h). When both drugs were administered, the negative impact of MK-801 tended to be improved. Overall, HBK-15 can modulate the coordination of hippocampal oscillations in vivo.

Our study revealed the previously uncharted potential of a multimodal compound, HBK-15, in mitigating MK-801-induced cognitive deficits across multiple domains, specifically recognition, emotional, and spatial memory. We identified a lack of impact of the compound on motor memory, suggesting its nuanced brain region selectivity. HBK-15’s effects spanned all stages of memory processing, potentially linked to its ability to counteract the MK-801-induced decline in LTP, a fundamental process in memory formation. Additionally, we uncovered HBK-15's capability to modulate hippocampal oscillations, specifically enhancing theta-gamma coupling. This work marks a significant stride in the field of neuropsychiatric disorders, providing new insights into therapeutic strategies for memory impairments.

Available pharmacotherapeutic strategies face challenges in addressing memory impairments of diverse etiological and pathophysiological origins. In some disorders, cognitive deficits remain insufficiently treated and overlooked, despite their significant influence on disease progression. This malpractice is particularly noticeable in neuropsychiatric diseases, such as depression or schizophrenia [54]. Even with a plethora of drugs exhibiting various mechanisms of action, the cognitive symptoms are still inadequately alleviated [55]. During our past investigations, we identified a methoxyphenylpiperazine derivative, HBK-15, that demonstrated fast antidepressant-and anxiolytic-like effects, as well as a potential to reverse memory impairments [5–7, 9, 10].

Given that memory disorders in neuropsychiatric diseases likely originate from dysfunctions within the glutamatergic system [56], we tested if HBK-15 could rectify cognitive impairments induced by MK-801 administration. Our findings indicate that HBK-15 successfully counteracted short-, intermediate-, and long-term deficits in recognition and emotional memory. It is important to emphasize that different mechanisms underpin these memory types. Short-term memory depends on changes in the synaptic strength of pre-existing neuronal connections, driven by modifications of pre-existing proteins [57]. Conversely, long-term memory formation relies on the synthesis of new proteins and morphological modifications [57]. Differentiation between short-, intermediate-, and long-term memory also hinges on the involvement of various signaling proteins [58, 59], distinct stages of gene expression [60], neuronal activity [61] and synaptic plasticity (early and late LTP) [62].

Additionally, the compound alleviated deficits across various stages of memory formation, including acquisition, consolidation, and retrieval. Each learning phase engages separate signaling proteins, kinases, receptors, or brain circuits [63–65]. The involvement of such diverse cellular and molecular mechanisms in each stage of memory formation implies that the mechanism underpinning the antiamnesic effect of HBK-15 may be more intricate than initially suspected, potentially engaging a variety of brain processes.

Another intriguing aspect of our study is the non-linear dose-response relationship of HBK-15 across various types of memory and different learning phases, exhibiting binary, biphasic, or inverted U-shaped patterns. We suspect several explanations of this discovery. First, due to HBK-15's affinity for various receptors and, consequently, its involvement with different signaling pathways, it may exhibit antiamnesic activity at diverse doses based on the investigated memory type, stage, and learning phase. Alternatively, HBK-15 may dose-dependently saturate receptors involved in distinct memory types, leading to observable effects at certain doses and no effects at others. Nevertheless, further research is necessary to explain this phenomenon more comprehensively.

The finding that HBK-15 demonstrated antiamnesic activity in tasks assessing recognition, emotional, and spatial memory, but not motor memory, may suggest a potential regional selectivity of the compound. Different types of memory engage distinct brain structures. For instance, recognition memory draws upon the hippocampus, entorhinal cortex, and medial prefrontal cortex [40], while emotional memory formation involves the amygdala and hippocampus [41]. Motor memory engages the primary motor cortex, basal nuclei, and cerebellum [66], and the hippocampus and entorhinal cortex play crucial roles in spatial memory [42]. Given the varied receptor expression observed in these brain regions, HBK-15 as a multimodal compound, could potentially modulate the activity of these brain areas in a differentiated manner.

Neurotransmitters exert control over learning and memory processes, prompting us to explore if a single dose of HBK-15 alters the concentration of selected neuromodulators in hippocampal homogenates. Our investigations, however, did not identify significant alterations in neurotransmitter levels from any of the administered doses. This outcome might imply that the influence of HBK-15 resides either within other brain structures or within particular regions of the hippocampus.

With these implications in mind, we narrowed our investigation to the CA1 region of the hippocampus. Rodent hippocampal CA1 neurons crucially contribute to the processing of hippocampus-dependent memory [67, 68], warranting our focus. We concentrated on glutamate and acetylcholine, the two vital neurotransmitters for proper memory function, and employed the microdialysis method in our research. Our results revealed an absence of alterations in the extracellular concentration of the chosen neurotransmitters. This discovery suggests that the fluctuation in neurotransmitter levels within the CA1 region of the hippocampus may not significantly contribute to the antiamnesic effects of HBK-15. Thus, it is possible that other hippocampal regions are key to HBK-15's antiamnesic effects, or that these effects result from different mechanisms.

Given the role of LTP as a cellular mechanism fundamental to learning and memory, we sought to determine if the antiamnesic properties of HBK-15 might be linked to concurrent changes in hippocampal synaptic plasticity. Prior research demonstrated that MK-801 impaired LTP in the hippocampus [69, 70], which could potentially correlate with the observed behavioral performance decline in animals during various memory assessment tests. It is important to note that HBK-15 administration counteracted MK-801-induced LTP reduction, offering a plausible rationale for the compound's antiamnesic effects. Intriguingly, a review of HBK-15's receptor profile indicates that its LTP induction could be NMDA-independent since HBK-15 did not bind to any of this receptor's sites. In addition, HBK-15 exhibited no affinity for AMPA, kainate, metabotropic glutamate, 5-HT3, and nicotinic receptors [71] or N-type voltage-gated calcium channels (Cav2.2). However, HBK-15 showed a substantial affinity for the L-type voltage-gated calcium channel (Cav1.2), a significant contributor to NMDA-independent LTP formation in the hippocampus (reviewed in [3]). Therefore, we suspect that this channel might be involved in HBK-15's mitigation of the MK-801-induced LTP decrease. Alternatively, HBK-15 may be indirectly influencing glutamate receptors integral to LTP modulation.

Notably, our previous research suggested that HBK-15 acted as an antagonist at the 5-HT₃ receptor in a biofunctional assay [9]. Yet, our current study using radioligand binding assays found no evidence of HBK-15 binding to the 5-HT₃ receptor. This seeming contradiction could be attributed to the different methodologies employed to investigate HBK-15's effect on the 5-HT₃ receptor i.e., in vitro radioligand binding assays in the current study versus ex vivo isolated guinea pig ileum in the prior research. The former study yielded a false-positive outcome, construed as HBK-15's antagonistic effect on the 5-HT₃ receptor, likely due to the compound's interaction with voltage-gated sodium [8] and/or calcium channels. Existing research affirms that blocking voltage-gated sodium and/or calcium channels can inhibit 5-HT₃ receptor activity [72, 73], which may account for the previous misinterpretation of the experimental results.

Additionally, alternations in the hippocampal oscillatory activity correspond to impaired memory functions. Studies indicated that MK-801 could augment gamma oscillations and disrupt theta/gamma coupling within the hippocampus during spatial working memory tasks [74]. Our findings resonate with these studies, demonstrating that a single administration of the NMDA receptor antagonist, MK-801, can disrupt this coupling. Interestingly, the co-administration of HBK-15 and MK-801 appeared to improve the theta-gamma coupling, although the differences were not statistically significant. Since we used only one dose for testing, which was selected for its optimal performance in behavioral tests, it is possible that we missed more significant improvements in theta-gamma coupling that could have been observed with a different dosage. On the other hand, HBK-15's antiamnesic effects might not solely depend on hippocampal oscillation coupling, and other mechanisms could be at play. Interestingly, when administered independently, HBK-15 promoted the theta-gamma coupling, suggesting the compound’s potential to improve learning and memory processes. Several studies associated increased theta-gamma coupling in the hippocampus with enhancements in spatial reference memory [75] or associative memory [76]. However, further research is warranted to explore this hypothesis.

In conclusion, our study demonstrates that HBK-15 exhibited antiamnesic properties, effectively counteracting the MK-801-induced deficits in recognition, emotional, and spatial memory. However, it did not show the same mitigating effects on motor memory deficits. The memory-enhancing capacities of HBK-15 extended across all stages of recognition and emotional memory processing - encoding, consolidation, and retrieval. Our findings suggest that the observed pharmacological effects of HBK-15 most likely hinge on its ability to counteract the MK-801-induced decline in the LTP, potentially involving the L-type voltage-gated calcium channel (Cav1.2). However, the mechanism underlying the antiamnesic effect of HBK-15 is likely more intricate than initially suspected, potentially engaging a variety of brain processes. Moreover, HBK-15 promoted the theta-gamma coupling in the hippocampus, indicating the compound's potential to improve learning and memory processes.

Overall, our findings shine a light on HBK-15 as a promising novel candidate for the treatment of memory impairments, particularly those related to neuropsychiatric disorders, extending its therapeutic potential beyond its previously known antidepressant- and anxiolytic-like effects. Our work not only provides significant insights into the unique properties of HBK-15 but also catalyzes new directions for further research in unraveling its complex mode of action and understanding memory processes at a molecular level.

Acknowledgments

We would like to thank Dr Małgorzata Więcek for the resynthesis of HBK-15.

Funding

This study was financially supported by the National Science Centre, Poland (grant number 2019/34/E/NZ7/00454) to KP, Santa Casa da Misericórdia de Lisboa (MB35-2021) to MJD and Medical Research Council (MR/V033964/1) and Horizon2020-ICT (DEEPER, 101016787) to SS.

Author contributions

Conceptualization: KP, SS, SX, MJD, KS; Formal analysis: KS, SI-O, SS; Investigation: KS, KL, MS, SI-O, PS, LM, EM, JF; Writing – Original Draft: KS, MS, SI-O, MJD, SS, KP; Writing – Review & Editing: KS, KL, SX, SS, LM, KP; Funding acquisition: KP, SS, MJD; Supervision: KP, SS, MJD, SX

Conflict of Interest

none

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The authors have declared there is NO conflict of interest to disclose

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HBK-15, a multimodal compound, induces procognitive effects through modulating hippocampal LTP and enhancing theta-gamma coupling in mice

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Abstract

Figures

Introduction

Materials and methods

Animals

Drugs

Experimental procedures

Behavioral tests

Step-through passive avoidance task

Hot plate test

Object recognition test

Morris water maze

Barnes maze

Rotarod test

Neurotransmitter levels

Tissue homogenates

Microdialysates

Statistical analysis

Results

HBK-15 displayed a significant affinity for the L-type voltage-gated calcium channel (Cav1.2)

HBK-15 countered MK-801-induced emotional memory deficits at all learning stages

HBK-15 prevented MK-801-induced recognition memory impairments throughout the encoding, consolidation, and recall stages

HBK-15 mitigated MK-801 negative effect on spatial learning in the Morris water maze

HBK-15 prevented MK-801-induced spatial memory deficits in the Barnes maze

HBK-15 did not protect against motor skill learning impairments caused by MK-801

A single administration of HBK-15 had no impact on the levels of various neurotransmitters

HBK-15 countervailed the decrease in the LTP magnitude caused by MK-801

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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