Vertigoheel promotes rodent cognitive performance in multiple memory tests

Abstract

Introduction: Cognitive impairment associated with old age or various brain disorders may be very disabling for affected individuals, placing their carers and public health services under considerable stress. The standard-of-care drugs produce only transient improvement of cognitive impairment in older people, so the search for novel, safe and effective therapeutics that would help to reverse or delay cognitive impairment is warranted. Repurposing pharmacological therapies with well-established safety record for additional indications is a promising recent trend in drug development. Vertigoheel (VH-04), a multicomponent drug made of Ambra grisea, Anamirta cocculus L., Conium maculatum, and Petroleum rectificatum, has been successfully used for several decades in the treatment of vertigo. Here, we investigated effects of VH-04 on cognitive performance in standard behavioral tests assessing different types of memory and explored cellular and molecular underpinnings of VH-04's biological activity.

Methods: In the majority of behavioral experiments, namely in the spontaneous and rewarded alternation tests, passive avoidance test, contextual/cued fear conditioning, and social transmission of food preference, we examined the ability of single and repeated intraperitoneal administrations of VH-04 to improve cognitive parameters of mice and rats disrupted by the application of the muscarinic antagonist scopolamine. In addition, we also assessed how VH-04 affected novel object recognition and influenced performance of aged animals in Morris water maze. Furthermore, we also studied the effects of VH-04 on primary hippocampal neurons in vitro and mRNA expression of synaptophysin in the hippocampus.

Results: Administration of VH-04 positively influenced visual recognition memory in the novel object recognition test and alleviated the impairments in spatial working memory and olfactory memory caused by the muscarinic antagonist scopolamine in the spontaneous alternation and social transmission of food preference tests. In addition, VH-04 improved retention of the spatial orientation memory of old rats in the Morris water maze. In contrast, VH-04 did not have significant effects on scopolamine-induced impairments in tests of fear-aggravated memory or rewarded alternation. Experiments in vitro showed that VH-04 stimulated neurite growth and possibly reversed the age-dependent decrease in hippocampal synaptophysin mRNA expression, which implies that VH-04 may preserve synaptic integrity in the aging brain.

Discussion: Our findings allow a cautious conclusion that in addition to its ability to alleviate manifestations of vertigo, VH-04 may be also used as a cognitive enhancer.

Keywords: Vertigoheel; forgetfulness; multicomponent drug; neurite length; olfactory memory; spatial orientation memory; spatial working memory; visual recognition memory.

Figure 1

Schematic illustration of the experiments. Behavioral tests or in vitro assays are described in individual boxes. Arrows connect experiments where the same set of animals was used following breaks of several weeks to allow wash-out of the drugs from the previous experiment. Upper row of boxes illustrates experiments in rats or involving rat tissue. Lower row of boxes illustrates experiments in mice or involving mouse tissue. The initial number of animals allocated per group is indicated under each box. Occasionally, data from some animals were excluded (see section 2.3 Behavioral tests for details and justification of such exclusions). Headings above the boxes indicate the cognitive domain or other experimental parameter tested.

Figure 2

VH-04 positively modulates visual recognition memory. (A,B) Discrimination indices (DIs) based on the time (A) and number of contacts (B) with the novel object during the retention trial in 30 min or 24 h after the initial acquisition trial in rats that received saline, VH-04, or donepezil. (C) Distances ran by rats during the retention trial. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 10–12). Statistical significance of differences was determined by one-way ANOVA followed by the Dunnett’s multiple comparisons test (vs. “Saline, 24 h) and is illustrated as follows: *p < 0.05; **p < 0.01; ****p < 0.0001 (see Supplementary Table 1 for the full breakdown of statistical results).

Figure 3

Modulation of the spatial working memory by VH-04. Drug effects were determined in the T-maze spontaneous (A) and rewarded (B) alternation tests in CD1 mice and Wistar rats, respectively. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values). For experiments in (A), N = 10 mice per each group. For experiments in (B), N = 9–15 rats in each group. Statistical significance of differences was determined by one-way ANOVA followed by the Dunnett’s multiple comparisons test [vs. “SCO (1 mg/kg)”]: **p < 0.01; ****p < 0.0001 (see Supplementary Table 2 for the full breakdown of statistical results). b.i.d., twice a day; q.d., daily.

Figure 4

Effect of VH-04 administration on spatial orientation learning and memory retention in the Morris water maze. (A–D) Latency (A) and distance (B) to reach the platform, speed (C), and the extent of thigmotaxis (D) during acquisition trials in vehicle-treated young rats (3 months), and in vehicle- or VH-04-treated aged rats (25 months) trained in the Morris water maze. Data are presented as the mean ± S.E.M. (N = 8–10 rats in each group). Two-way repeated measures ANOVA and subsequent Holm-Šídák multiple comparisons did not reveal significant differences in any of the four parameters between aged rats treated with vehicle or VH-04 at any of the acquisition trials. (E–G) Time spent in different quadrants (E), number of passes through the platform counter (F), and time spent within the platform counter area (G) during the probe trial. Data are presented as the mean ± S.E.M. (N = 8–10 rats in each group). Data in (E–G) were analyzed by two-way ANOVA followed by the Holm-Šídák multiple comparisons test to compare values in each quadrant within the same experimental group. Statistical significance of differences from the values in target quadrant 3 is illustrated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (see Supplementary Table 3 for the full breakdown of statistical results).

Figure 5

Administration of VH-04 does not improve passive avoidance performance impaired by scopolamine. VH-04 was given either simultaneously with scopolamine or for 3 days q.d. before the treatment with scopolamine. Latencies to enter the dark chamber on the training day (Day 1) and next day (Day 2) for different experimental groups of rats are illustrated. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 15 or 16 rats in each group). Statistical significance of differences was determined by one-way ANOVA followed by the Dunnett’s multiple comparison test (vs. “SCO (1 mg/kg)”): ****p < 0.0001 (see Supplementary Table 4 for the full breakdown of statistical results). q.d., daily.

Figure 6

VH-04 administration does not ameliorate the amnesic effect of scopolamine in contextual/cued fear conditioning. Fractions of time spent freezing to context (A), altered context (B), and cue in altered context (C) are shown. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 13–15 mice in each group). Statistical significance of differences was determined by one-way ANOVA followed by the Dunnett’s multiple comparisons test (vs. “SCO (1 mg/kg)”): **p < 0.01 (see Supplementary Table 5 for the full breakdown of statistical results). q.d., daily.

Figure 7

Restoration of the preference for cued food impaired by scopolamine following treatment with VH-04 or donepezil in the social transmission of food preference test. Absolute amounts of cued and non-cued food consumed by “observer” mice are illustrated. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 13–15 mice in each group). Ordinary two-way ANOVA with food and treatment type as factors showed a significant effect of factor interaction [F_{(5, 156)} = 2.818, p = 0.0182]. Asterisks indicate groups in which the difference in the amount of cued and non-cued food was significant according to the Holm-Šídák multiple comparisons test: *p < 0.05; ***p < 0.001; ****p < 0.0001 (see Supplementary Table 6 for the full breakdown of statistical results). q.d., daily.

Figure 8

Age-related changes in the concentrations of various metabolites in the hippocampal region of rats are not restored by VH-04 administration. Measurements were performed in animals used in the Morris water maze test (Figure 3) in 1–4 days following the probe trial. Data are presented as the mean ± S.D. (N = 8 or 10 mice in each group). Data for each metabolite were analyzed by one-way ANOVA followed by the Holm-Šídák multiple comparisons test. Statistical significance of differences from the values in vehicle-treated young rats is illustrated as follows: *p < 0.05; ***p < 0.001; ****p < 0.0001 (see Supplementary Table 7 for the full breakdown of statistical results). CHO, choline; CR, creatine; GLN, glutamine; GLU, glutamate; INS, myo-inositol; NAA, N-acetyl-aspartate; NAAG, N-acetyl-aspartyl-glutamate; PCR, phosphocreatine; TAU, taurine.

Figure 9

Positive effect of incubation with VH-04 on neurite length in mouse primary hippocampal neurons. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 19–70 neurites in each group). Statistical significance of differences was determined by the unpaired Student’s or Welch’s t-test, depending on the equality of variances: *p < 0.05; ****p < 0.0001 (see Supplementary Table 8 for the full breakdown of statistical results).

Figure 10

Effect of treatment with VH-04 on synaptophysin mRNA expression in hippocampal samples of aged rats. Expression levels were normalized by Gapdh mRNA levels. Data are presented as box-whisker plots (middle line: median; box: 25th and 75th percentiles; cross: mean value; whiskers: smallest and largest values; N = 5–9 in each group). Group factor significantly affected the relative Syp mRNA expression level [F_{(2, 5.964)} = 6.37; p = 0.0331, Brown-Forsythe ANOVA; see Supplementary Table 9 for the full breakdown of statistical results].