Dampened neural activity and abolition of epileptic-like activity in cortical slices by active ingredients of spices

Abstract

Active ingredients of spices (AIS) modulate neural response in the peripheral nervous system, mainly through interaction with TRP channel/receptors. The present study explores how different AIS modulate neural response in layer 5 pyramidal neurons of S1 neocortex. The AIS tested are agonists of TRPV1/3, TRPM8 or TRPA1. Our results demonstrate that capsaicin, eugenol, menthol, icilin and cinnamaldehyde, but not AITC dampen the generation of APs in a voltage- and time-dependent manner. This effect was further tested for the TRPM8 ligands in the presence of a TRPM8 blocker (BCTC) and on TRPM8 KO mice. The observable effect was still present. Finally, the influence of the selected AIS was tested on in vitro gabazine-induced seizures. Results coincide with the above observations: except for cinnamaldehyde, the same AIS were able to reduce the number, duration of the AP bursts and increase the concentration of gabazine needed to elicit them. In conclusion, our data suggests that some of these AIS can modulate glutamatergic neurons in the brain through a TRP-independent pathway, regardless of whether the neurons are stimulated intracellularly or by hyperactive microcircuitry.

Figure 1. Voltage recordings of single layer 5 pyramidal neurons of the primary somatosensory cortex.

Compared voltage traces of different single cell recordings at high depolarization, in control situation (black) and in the presence of a particular AIS (red). Traces are grouped depending of the corresponding AIS receptors affinity: - TRPV1/3 ligands: 1. - Capsaicin 25 μM; 2. - Eugenol 200 μM. - TRPM8 ligands: 1. - Menthol 250 μM; 2. - Icilin 50 μM. - TRPA1 ligands: 1. - AITC 500 μM; 2. - Cinnamaldehyde 300 μM. Except for AITC all AIS tested tend to affect the generation the APs at high depolarization levels.

Figure 2. Recorded traces of stimuli eliciting AP affected by extracellular menthol 250 μM and corresponding analysis.

Single cell voltage recordings in control conditions (black) and in the presence of menthol 250 μM (red) from S1 somatosensory cortex layer 5 pyramidal neurons. The features extracted were affected by the presence of the AIS. Voltage recordings (A) under pulse stimuli, a set of 11 successive trials of increasing two-second long square current steps. Traces show action potential firing in the control situation (top, black) and under the effects of menthol 250 μM (bottom, red). The action potential amplitude drop rate (B) was obtained from each corresponding current step for both conditions (1) the amplitude drop rates for each condition were averaged and plotted (2) (n = 9 average ± S.E.; p < 0.005 assessed by Student's t-test). Further analysis was performed on the last action potential of each of the 11 trials per condition (C1). These features were plotted against their corresponding trials, for both conditions (C 2) (average ± S.E.; n = 9). Ramp stimuli (D1) was performed providing a measure for the action potential threshold (D2, Top) (p < 0.005; assessed by Student's t-test) and the action potential amplitude drop rate (D2, Bottom) (p < 0.05; assessed by Student's t-test) plotted for both conditions (average ± S.E.; n = 9).

Figure 3. Analysis of menthol 250 μM effects on neural activity through current stimulation.

The recordings of I/V analysis set of stimuli (A) provided voltage values (top traces) at peak and at steady state for control (black) and in the presence of menthol 250 μM (red; circle for peak voltage values and square for steady state.) at each level of current injected (bottom traces). Data was plotted for each cell and input resistance was obtained from both voltage levels for both conditions (B) by linear fit. When compared, (C) the input resistance at peak voltage and at steady state show no statistical difference between control (black) and in the presence of menthol 250 μM (red) (n = 9 average ± S.E.; assessed by Student's t-test). Action potential waveform analysis features, amplitude and width, were obtained from high sampling (20 kHz) voltage traces (D top traces) from control (black) and menthol 250 μM (red) conditions while applying square current pulses (bottom traces). Values were plotted and compared (E) presenting no statistical difference (n = 9 average ± S.E.; assessed by Student's t-test).

Figure 4. Plotted features obtained from S1 L5 pyramidal neurons through voltage recordings in naïve conditions () or under the presence of the TRPM8 channel blocker BCTC 15 μM ().

The extracted features are obtained from different types of stimulation protocols performed on control (black) and under the effect of menthol 250 μM (red). Firing pattern features from successive incremental current square pulses are plotted and compared (A). Last AP analysis (A1) shows little or no difference on the effect produced by menthol 250 μM (red), whether if it is measured in the absence (; n = 9) or in the presence of BCTC 15 μM (; n = 8) (average ± S.E.). A similar situation can be observed in the AP drop rate slope measured (A2; average ± S.E.; *p < 0.05, ***p < 0.005, estimated by Student's t test). Parameters obtained from incremental current ramp stimulation are plotted (B). The effect produced by menthol 250 μM (red) on the threshold (B, left) was not blocked by BCTC 15 μM (; n = 8 Average ± S.E. * p < 0.05, estimated by Student's t test). On the action potential amplitude drop rate (B, right) the effect of menthol 250 μM (red) can be observed in naïve conditions (; n = 9) but stops being statistically significant in the presence of BCTC 15 μM (; n = 8) (average ± S.E.; *** p< 0.005, estimated by Student's t test). The parameters that describe the action potential wave-form are plotted (C) and compared. The lack of effect produced by menthol (red) on the AP amplitude (left) and on the width (right) persists in naïve conditions (; n = 9) and in the presence of BCTC (; n = 8; Average ± S.E.; estimated by Student's t test). However, the blocker seems to have some effect on the width. The input resistance values were extracted from I/V analysis and plotted (D) for voltage at peak (left) and at steady state (right). For each case the resistance appears to remain invariant for all conditions (estimated by Student's t test).

Figure 5. Voltage recordings from a single L5 pyramidal neuron of a C57BL6/J TRPM8 KO mouse primary somatosensory cortex.

The response corresponds to different current stimuli in control (left), menthol 250 μM (red, center) and washout (right) conditions. Voltage traces of suprathreshold square pulse stimuli (A) with low current (top row) and high current stimulation (bottom row) in all three conditions. Firing pattern is strongly affected by the presence of menthol 250 μM (A, red, center) especially at high depolarization levels. Firing behaviour is partly recovered after washout (A, right). Voltage response to an increasingly depolarizing ramp (B) from subthreshold to suprathreshold values show a difference between control (left) and menthol 250 μM (red, center) conditions. Firing pattern is partly recovered on the washout (right). Voltage traces corresponding the AP wave-form (C) and to I/V Analysis (D) show no difference between control (left) and menthol 250 μM (red, center) conditions.

Figure 6. Plotted features obtained from S1 L5 pyramidal neurons through voltage recordings in wild type animals () or in a TRPM8 KO strain ().

The extracted features are obtained from different types of stimulation protocols performed on control (black) and under the effect of menthol 250 μM (red).Firing pattern features from successive incremental current square pulses are plotted and compared (A). Last AP analysis (A1) shows little or no difference on the effect produced by menthol 250 μM (red), whether if it is measured in wild type (; n = 7) or in TRPM8 KO animals (; n = 7) (average ± S.E.). Similar situation can be observed in the AP drop rate slope measured (A2, average ± S.E.; *p < 0.05, ***p < 0.005, estimated by Student's t test). Parameters obtained from incremental current ramp stimulation are plotted (B). The effect produced by menthol 250 μM (red) on the threshold (B, left) and on the action potential amplitude drop rate (B, right) is present whether obtained from wt (; n = 7) or TRPM8 KO (; n = 7) animals (average±S.E.; *p < 0.05, ***p < 0.005, estimated by Student's t test). The parameters that describe the action potential wave-form are plotted (C) and compared. The lack of effect produced by menthol (red) on the AP amplitude (C, left) and on the width (C, right) persists in wt (; n = 7) and in TRPM8 KO animals (; n = 7) (average ± S.E.; estimated by Student's t test). The input resistance values were extracted from I/V analysis and plotted (D) for voltage at peak (D, left) and at steady state (D, right). For each case the resistance appears to remain invariant for all conditions (estimated by Student's t test).

Figure 7. Menthol prevents seizure-like bursts in the neocortex.

Voltage recordings of layer 5 pyramidal neurons of mouse primary somatosensory cortex while applying gabazine through increasing concentration steps (A). Elicited bursts increase in duration and the number of APs elicited as the concentration of gabazine becomes higher (C). The increasing concentrations steps of gabazine protocol are repeated while permanently applying menthol 250 μM (red). In order to obtain bursts higher gabazine is required (B) and the duration as well as the number of APs elicited decrease (D).

Figure 8. Voltage recordings in the absence (control) or in the presence of the different AIS were obtained while Gabazine was applied in an incremental manner through 5 min steps.

AP bursts were spontaneously generated (A). AP bursts were analyzed and quantified for two different concentrations of Gabazine (10 μM and 20 μM), by arbitrarily placing a depolarization limit at −40 mV (B). Data was plotted as total number of events (C), and total duration of the depolarized phase (D).