Electrophysiological and Theoretical Analysis of Deltamethrin, a Type II Pyrethroid Insecticide, on the Gating Properties of Voltage-Gated Na⁺ Currents

1. Introduction

Deltamethrin (DLT, also known as decamethrin, see Figure 1) is a cyclopropanecarboxylate ester resulting from formal condensation of 3-(2,2-dibromovinyl)-2,2-dimethylcyclopanecarboxylic acid with cyano (3-phenoxyphenyl)methanol. It serves as the active insecticidal component of the proinsecticide tralomethrin [1]. Pyrethroids, such as DLT and tefluthrin, have been shown to alter the gating properties of voltage-gated Na⁺ (Na_V) channels [2,3,4,5]. Recent studies indicate that DLT and other structurally related pyrethroids can exert significant toxic effects across various species, including fish, laying hens, and rats [3,5,6,7,8,9,10,11,12,13]. Notably, DLT—a neurotoxic type II pyrethroid ester insecticide—has been associated with a reversible sequence of motor symptoms in rats, characterized by hind limb rigidity and choreoathetosis. Additionally, it has been implicated as a potential contributor to Cockayne syndrome [6,13,14]. Cockayne syndrome is a rare, inherited genetic disorder characterized by growth failure, neurological degeneration, photosensitivity, and premature aging [13,14].

Voltage-gated Na⁺ (Na_V) channels are essential for the excitability of mammalian tissues, including the central and peripheral nervous systems as well as endocrine and neuroendocrine cells, To date, nine isoforms (Na_V1.1 to 1.9), encoded by the SCN1A-SCN5A and SCN8A-SCN11A genes, have been identified across these systems [15]. Structurally, eukaryotic Na_V channels consist of a single α-subunit organized into four homologous domains (I-IV). Each domain contains six-transmembrane segments (S1-S6), which together form the functional architecture responsible for channel gating and ion conduction [15].

Following rapid membrane depolarization, Na_V channels undergo a sequence of conformational changes, transitioning from the closed (resting) state to an activated open state and subsequently to an inactivated state. These transitions generate the macroscopic Na⁺ current (I_Na) [15]. Repeated short-duration depolarizing stimuli can progressively enhance current inactivation, leading to a cumulative reduction in I_Na and thereby producing use-dependent changes of Na_V channel availability [16,17,18,19]. In addition to transient I_Na, increasing attention has been directed toward the persistent or late component of I_Na (I_Na(L)), whose magnitude appears to be dynamically regulated and may represent an important pharmacological target [20,21,22,23]. Activation of Na_V channels increases inward Na⁺ influx, further depolarizing the membrane and establishing a positive feedback loop that accelerates the rapid upstroke of action potentials (APs). Through this mechanism, Na_V channel activity is therefore a critical determinant of AP amplitude, firing frequency, and discharge patterns, and thereby contributing to the regulation of neuronal signal propagation and hormonal secretion across a variety of excitable cell types [20].

The Neuro-2a (N2a) cell line, derived from murine neuroblastoma, exhibits characteristics reminiscent of motor neurons and is widely utilized in Na_V 1.7 neuroscience research. Under specific conditions—such as treatment with retinoic acid—N2a can undergo differentiation, extending neuritis and acquiring neuron-like properties. This makes them a valuable in vitro model for investigating neuronal differentiation, neurotoxicity, and neurodegenerative disorders [18,21,22,23,24]. Additionally, N2a cells have been reported to express several Na_V channel isoforms, including Na_V1.2, 1.3, and 1.7 channels [23]. Na_V1.2 and 1.3 channels are predominantly expressed in the central nervous system and are implicated in the pathophysiology of epilepsy, while Na_V1.7 channel is primarily found in the peripheral nervous system and plays a key role in mediating neuropathic pain.

In this study, we aimed to explore the effects of DLT, a type II pyrethroid, and functionally related compounds such as dapagliflozin (Dapa), on transmembrane ionic currents, with a particular emphasis on the current density of I_Na. To assess how DLT influences I_Na gating behavior, we employed a theoretical approach to simulate I_Na dynamics. Furthermore, using a computational model of respiratory neurons located in the pre-Bötzinger complex (pre-BötC) of the mammalian brainstem [25], we investigated how exposure to DLT may alter the bursting firing patterns of these neurons.

2. Materials and Methods

2.1. Chemicals, Compounds, Drugs, Reagents, and Solutions

Deltamethrin (DLT, also referred to as decamethrin; molecular formula C₂₂H₁₉Br₂NO₃; IUPAC name: (S)-α-cyano-3-phenoxybenzyl (1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylate, Figure 1) was purchased from MedChemExpress through Asia Biomed Inc., (Taipei, Taiwan). Dapagliflozin (Dapa, marketed under the trade names Farxiga^® and Forxiga^®) was retrieved from Cayman Chemical (Ann Arbor, MI). Atropine, tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were acquired from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan). Because deltamethrin is susceptible to both photodegradation and microbial decomposition [1,26,27], a 10 mM stock solution was freshly prepared in dimethyl sulfoxide (DMSO). The stock solution was protected from light by wrapping the container with aluminum foil and stored at −20 °C until used to preserve its stability.

Cell culture preparations, including fetal bovine serum, L-glutamine, trypsin–EDTA, culture media, were purchased from HyClone^TM (Thermo Fisher Scientific, Tainan, Taiwan). Additional chemicals, such as CsCl, CsOH, CdCl₂, and other reagents and solvents used throughout the experiments, were of analytical grade and obtained from Sigma-Aldrich through Genechain (Kaohsiung, Taiwan).

Whole-cell recordings were performed using a standard HEPES-buffered Tyrode’s solution as the extracellular bathing medium. The solution contained (in mM): 136.5 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.53 MgCl₂, 5.5 glucose, and 5.5 HEPES, with the pH adjusted to 7.4 using NaOH. For the measurement of K⁺ currents, recording pipettes were digitally subtracted were filled with an intracellular solution composed of (mM): 130 K-aspartate, 20 KCl, 1 MgCl₂, 1 KH₂PO₄, 3 Na₂ATP, 0.1 Na₂GTP, 0.1 EGTA, and 5 HEPES, with the pH adjusted to 7.2 using KOH. To isolate voltage-gated Na⁺ current (I_Na), K⁺ in the intracellular solution were completely substituted with an equimolar concentration of Cs⁺ to suppress endogenous K⁺ conductances. The pH of the Cs⁺-based internal solution was adjusted to 7.2 with CsOH before use.

2.2. Cell Preparation

Neuro-2a (N2a) cells, a mouse neuroblastoma-derived clonal cell line originating from a spontaneous tumor in strain A albino mice (Mus musculus), were obtained from the Bioresources Collection and Research Center (BCRC-60026, Hsinchu, Taiwan). The cell line was originally established by the American Type Culture Collection (ATCC^® CCL-131^TM; Manassas, VA) and has been extensively employed as an electrically excitable neuronal model for electrophysiological and pharmacological investigations [18,22].

N2a cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate. Cell cultures were maintained at 37 °C in a humidified incubator with an atmosphere of 5% CO₂ and 95% air [18]. Routine passaging was performed using 0.025% trypsin–EDTA (HyClone^TM) containing 0.01% sodium N,N-diethyldithiocarbamate. Electrophysiological experiments were conducted on cultures that had reached approximately 60–70% confluence, generally within 5–7 days after plating [18,19].

2.3. Electrophysiological Recordings Using the Patch-Clamp Technique

Following enzymatic dissociation, several drops of the Neuro-2a cell suspension were placed into a custom-designed recording chamber positioned on the stage of an inverted microscope. The cells were superfused with normal Tyrode’s solution of the ionic composition described above and maintained at room temperature (20-25 °C). Before each measurement were initiated, sufficient time was allowed for the cells to settle and firmly attach to the bottom surface of the chamber.

Patch pipettes were fabricated from Kimax^®-51 borosilicate glass capillaries (catalog no. DWK34500-99; Kimble^®, Merck, Tainan, Taiwan) and fire-polished to obtain a final tip resistance of 2–4 MΩ when filled with the internal solution. Each pipette was secured in a sealed electrode holder fitted with a lateral suction port to facilitate seal formation. Electrical continuity between the amplifier and the pipette solution was established using an Ag/AgCl wire. Membrane currents were measured in the whole-cell configuration of the patch-clamp technique with an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France), operated under a modified recording protocol, as described previously [2,28].

A liquid junction potential arose from the difference in ionic composition between the extracellular bathing solution and the pipette internal solution. This potential was compensated immediately before establishing the gigaohm (GΩ) seal and was further corrected during subsequent analysis of whole-cell recordings. To reduce capacitive currents associated with the activation of I_Na during depolarizing voltage steps, the currents elicited by an isoelectric hyperpolarizing pulse were provided previously digitally subtracted from the recorded traces.

2.4. Statistical Analyses

Results are presented as the mean ± standard error of the mean (SEM), and n denotes the number of individual cells included in each analysis. Linear and nonlinear models were fitted by minimizing the least-squares error using either Microsoft^® Excel^® for Microsoft 365 MSO (64-bit; version 16.017628.20006) with the Solver add-in (Microsoft Corporation, Redmond, WA) or OriginPro^® 2024b (OriginLab Corporation, Northampton, MA). Differences between two groups were evaluated using paired or unpaired two-tailed Student’s t-tests, as appropriate. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test was applied. Unless otherwise specified, statistical analyses were conducted with SPSS statistics, version 23.0 (IBM Corporation, Armonk, NY). A P-value below 0.05 was considered indicative of statistical significance.

2.5. Computer Simulations

To simulate the DLT-induced alterations in the amplitude and gating kinetics of I_Na, a modified model, originally developed by Abdulla et al., 2022 [25], was mathematically constructed for this study. Below is a description of the kinetic scheme of I_Na that accounts for the experimentally observed results.

$$I_{Na\left(T\right)}=G_{Na\left(T\right)}\times m_{Na\left(T\right)}^3\times h_{Na\left(T\right)}\times \left(V-E_{Na}\right)$$

$$I_{Na\left(L\right)}=G_{Na\left(L\right)}\times m_{Na\left(L\right)}\times h_{Na\left(L\right)}\times h_{Na\left(L\right)}\times \left(V-E_{Na}\right)$$

$$I_{Na\left(Tail\right)}=G_{Na\left(Tail\right)}\times {(1-m_{Na\left(L\right)})}^2\times \left(1-h_{Na\left(L\right)}\right)\times (V-E_{Na})$$

$$I_{Na\left(Tot\right)}=I_{Na\left(T\right)}+I_{Na\left(L\right)}+I_{Na\left(Tail\right)}$$

The expressions of I_Na(T), I_Na(L), and I_Na(Tail) are derived from the voltage- and time-dependent characteristics of the Na_V channels. The I_Na(Tail) is given by:

The currents I_Na(T) and I_Na(L) are expressed as the product of their maximal conductances, voltage- and time-dependent gating variables, and the term (V - E_Na), which represents the difference between the membrane potential and the Na⁺ reversal potential. Each of the Na⁺ gating variables x ϵ {m_Na, h_Na, m_Na(L), h_Na(L)} satisfies the following equation

$${\tau }_x\left(V\right)·{\displaystyle \frac{dx}{dt}}=x_{\infty }\left(V\right)-x,$$

where

$$x_{\infty }\left(V\right)={\left[1+\exp \left({\displaystyle \frac{V_x-V}{k_x}}\right)\right]}^{-1},$$

$${\tau }_x\left(V\right)={\tau }_x/[\cosh \left({\displaystyle \frac{V-V_{{\tau }_x}}{k_{{\tau }_x}}}\right).$$

The parameter values used for these equations, including the equilibrium potential for Na⁺ ions (E_Na), were provided previously [28].

In this model, the membrane potential (V) is governed by the current balance equations:

$$C·{\displaystyle \raisebox{1ex}{$dV$}\!\left/ \!\raisebox{-1ex}{$dt$}\right.}=-\left(I_{Na\left(T\right)}+I_{Na\left(L\right)}+I_{Na\left(Tail\right)}+I_{K\left(DR\right)}+I_L+I_{Syn}\right).$$

The membrane currents in (I) include the transient Na⁺ current (I_Na(T)), the late (or persistent) Na⁺ current (_Na(L)), the delayed rectifier K⁺ current (I_K(DR)), the leakage current (I_L), and the synaptic current (I_Syn) [28]. This respiratory model neuron exhibits bursting behavior by coupling membrane potential dynamics with the intrinsic mechanisms of Na⁺ and K⁺ currents.

The primary platform for these in silico experiments was XPP, where simulations were developed using the XPP simulation package. Additionally, parts of the numerical simulations were cross-validated using either the web-based version of Microsoft Excel^® or Python implementations [29,30].

3. Results

3.1. Effect of DLT on Voltage-Gated Na⁺ Current (I_Na) Present in Neuro-2a Neuronal Cells

In the initial experiment, we investigated the potential effects of DLT on the I_Na elicited by step depolarization in these cells. To isolate I_Na, the cells were bathed in a Ca²⁺-free Tyrode’s solution supplemented with 0.5 mM CdCl₂ and 10 mM tetraethylammonium chloride (TEA). CdCl₂ was used to block nonspecific Ca²⁺ currents, while TEA was included to suppress the majority of nonspecific K⁺ currents. The ionic composition of the Tyrode’s solution is described in detail in the Materials and Methods section. Whole-cell voltage-clamp recordings of I_Na were performed using a Cs⁺-containing pipette solution.

As illustrated in Figure 2A, Neuro-2a cells were initially held at a membrane potential of −80 mV. To ensure complete inactivation of I_Na, the cells were hyperpolarized to −100 mV for 40 msec. This was followed by a depolarizing step to −10 mV for 40 msec to activate I_Na, which produced a downward deflection in the current trace. To assess I_Na(Tail), the membrane potential was then repolarized to −50 mV for another 40 msec. Finally, the voltage was returned to the initial holding potential of −80 mV. The corresponding voltage protocol is shown in the upper panel of Figure 2A.

In these recordings, the cell was depolarized from −100 to −10 mV for 40 msec, which reliably evoked a robust, transient inward I_Na (I_Na(T))—a rapidly activating component known to be sensitive to blockade by 1 μM tetrodotoxin (TTX). Upon exposure to 10 μM DLT, both the peak density of I_Na(T) and the late or persistent component (I_Na(L)) of I_Na density were enhanced. Furthermore, as the membrane potential was repolarized to more hyperpolarized levels following the depolarizing step, the current density of I_Na(Tail) progressively increased. Despite these changes, the kinetics of fast and slow inactivation course of I_Na evoked by the short depolarizing pulse remained unaffected by 10 μM DLT. Notably, the DLT-induced increase in I_Na(L) density was significantly greater than that observed for I_Na(T). In addition, the density of tail component (I_Na(Tail)) measured at the level of −50 mV or −80 mV was markedly elevated in the presence of 10 μM DLT.

Figure 2B and 2C summarize the effects of DLT alone and in combination with dapagliflozin (Dapa) on the densities of I_Na(T) and I_Na(L) in Neuro-2a neuronal cells. Dapa, a well-characterized inhibitor of the Na⁺-glucose co-transporter, has recently been reported to selectively suppress I_Na(L) [2,24,31]. In this study, treatment with DLT significantly increased overall I_Na density in Neuro-2a cells, despite having no observable effect on the fast or slow inactivation kinetics of the current. Interestingly, the DLT-induced enhancement appeared to be more prominent in the I_Na(L) and I_Na(Tail) components than in I_Na(T) component, suggesting a degree of selectivity in its modulatory action. Furthermore, the addition of Dapa in the continued presence of DLT partially reversed the DLT-mediated increase in I_Na density, particularly affecting the I_Na(L) and I_Na(Tail) components. These findings highlight a potential interaction between DLT and Dapa in modulating I_Na dynamics in neuronal cells.

Simulation studies on I_Na(T), I_Na(L), and I_Na(Tail)

The activity of I_Na, including its persistent component I_Na(L), has been implicated in the generation of respiratory rhythms within the pre-BötC [32]. To explore the potential effects of DLT on I_Na, we developed a computational model that incorporates I_Na(T), I_Na(L), and I_Na(Tail) components. The simulated I_Na model was adapted from the work of Abdulla et al. [25], with a detailed description of the kinetic scheme provided in the Materials and Methods section. As shown in Figure 3, following appropriate parameter adjustments, we successfully reproduced the I_Na in Neuro-2a cells, clearly distinguishing the three components: I_Na(T), I_Na(L), and I_Na(Tail). The total I_Na (I_Na(Tot)) represents the sum of these three components. The voltage-clamp protocol applied in these simulations is depicted at the top of Figure 3 and Figure 4. To ensure comparability across experiments, current densities were normalized to membrane capacitance.

3.2. Simulation of Increases in I_Na(T), I_Na(L), and I_Na(Tail) to Mimic DLT Effects on I_Na

To simulate the effects of DLT on I_Na, we adjusted specific parameters (G_Na(T), G_Na(L), and G_Na(Tai)) to generate simulated I_Na with increased I_Na(T), I_Na(L), and I_Na(Tail) components. The simulation results, shown in Figure 4, illustrate the different forms of I_Na, namely I_Na(T), I_Na(L), and I_Na(Tail). Notably, the simulated densities of I_Na(L) and I_Na(Tail) exhibited a greater increase compared to I_Na(T), closely mirroring the experimental observations (I_Na) shown in Figure 2. Although the densities of I_Na(L) and I_Na(Tail) were significantly elevated relative to I_Na(T), the inactivation time course of I_Na elicited by short depolarizing pulse remained unchanged.

Neuronal burst firing of action potentials (APs) as simulated I_Na was incorporated to the modeled respiratory neuron

We next incorporated the simulated I_Na into the bursting pattern of a modeled respiratory neuron. The upper part of Figure 5 illustrates this neuron’s distinct bursting pattern, which aligns with earlier observations [25]. The interburst rate was approximately 0.33 Hz, and a progressive shortening in intraburst intervals from 92 to 38 msec was observed. The AP amplitude within the bursts progressively decreased, along with a gradual shortening of the intraburst AP intervals. The lower part of Figure 5 depicts the corresponding I_Na(Tot), showing a progressive decline in its density. This decline reflects the frequency dependence of I_Na, consistent with previous studies [16,17,18,19].

Figure 6 illustrates the AP bursting patterns resulting from the modification of I_Na to mimic the effects of DLT on I_Na(T), I_Na(L), and I_Na(Tail). Figure 7 provides an enlarged view of the dashed box in the uppermost part of Figure 6. Notably, two distinct patterns of AP bursting, categorized as slow and fast types, were clearly observed, as indicated with asterisk (*) and double asterisks (**), in the potential trace at the top of Figure 6.

The slow bursting pattern exhibited an intraburst rate that progressively shortened from 271 to 128 msec across an individual burst, while the fast bursting pattern demonstrated a quicker intraburst rate, with intervals decreasing from 90 to 39 msec. One bursting pattern exhibited a slow intraburst rate with a progressive shortening of intraburst AP intervals from 271 to 128 msec, while the other was noted to display fast intraburst rate with a progressive shortening of intraburst intervals from 90 to 39 msec. Both patterns showed reverse spike-frequency adaptation during their respective slow or fast bursting firing [33]. The simulated interburst rate during both fast and slow bursts was approximately 0.24 Hz.

During slow-type bursts, the resting membrane potential depolarized from −54.8 to −52.9 mV. This depolarization was more pronounced during fast-type bursts, where the resting potential shifted from −53,4 to −50.2 mV. Additionally, the amplitude of APs during fast-type bursts progressively decreased from 26.7 to 15.6 mV, while that in slow-type bursts, the amplitude declined from 28.7 to 23.6 mV. The post-burst hyperpolarizing potential was −55.1 mV during slow- versus fast-type bursts and −56.1 mV during fast-type bursts.

Moreover, during initial generation of I_Na(T) became progressively declined; however, the density of I_Na(L) and I_Na(Tail) was gradually and evidently increased. Such simulations show that DLT exerts a significant impact on burst patterns of this modeled respiratory neuron through its stimulation of marked under the trade name channels. Consequently, this could lead to substantial disturbances in the respiratory movement of organisms [34,35].

3.3. Molecular Docking on Interaction Between Na_V Channel and DLT

Since our results have shown that DLT has stimulatory effects on the current density and gating kinetics of I_Na, we also conducted further investigations to examine the direct binding of DLT on the Na_V protein. The docking of DLT with the Na_V1.8-channel protein was performed using PyRx software, and the resulting predicted docking sites of the compound are depicted in Figure 8. Of note, DLT can form hydrophobic contacts with eleven amino-acid residues Cys10(A), Ile11(A), Val12(A), Pro13(A), Phe17(A), Tyr19(A), Cys20(A), Pro22(A), Leu24(A), Ile25(A), and Cys26(A). The docking site appears to be located near the Ile-Phe-Met motif and the N-terminal helix, as noted by Chen et al., 2024 [34]. Based on the results from in-silico docking studies, it suggests that the DLT molecule has the potential to bind to the specific region of the Na_V1.8 channel with a binding affinity of −6.3 kcal/mol.

4. Discussion

This study reports six key findings: (1) In Neuro-2a neuroblastoma cells, deltamethrin (DLT), a known gating modifier of voltage-gated Na⁺ current (I_Na), significantly enhanced the densities of I_Na(T), I_Na(L), and I_Na(Tail); (2) Among these, the increase in the density of transient I_Na (I_Na(T)) induced by DLT was less pronounced compared to the more substantial enhancements observed in late I_Na (I_Na(L)) and tail I_Na (I_Na(Tail)); (3) Co-application of dapagliflozin (Dapa) effectively reversed the DLT-induced increase in all three forms of I_Na (I_Na(T), I_Na(L), and I_Na(Tail)), even in the continued presence of DLT; (4) A computational model was constructed to replicate DLT’s stimulatory effects on these distinct components of I_Na; (5) Numerical simulations were performed using this model to assess how DLT might influence the bursting pattern of respiratory neurons; and (6) Molecular docking analysis predicted potential docking sites for DLT on specific amino acid residues within the Na_V1.8 channel. Together, these findings demonstrate that DLT-induced alterations on I_Na amplitude and gating properties—particularly in I_Na(T), I_Na(L), and I_Na(Tail)—can significantly disrupt neuronal bursting patterns [32].

Previous studies have suggested that the neurotoxic effects of DLT might involve its interaction with muscarinic receptors [36,37,38]. Neuro-2a cells are known to express muscarinic receptors on their membranes [21]. However, in our study, treatment with atropine—a muscarinic receptor antagonist—did not reverse the DLT-induced alterations in I_Na. This finding indicates that DLT likely exerts a direct effect on Na_V channels, rather than acting indirectly through muscarinic receptor pathways. Additionally, it appears unlikely that the DLT-induced changes in I_Na are primarily mediated by effects on voltage-gated anion channels or Ca²⁺-activated Cl^- channels, as recently proposed in other studies [24,39].

Dapagliflozin (Dapa), a known inhibitor of Na⁺-glucose co-transporters (SGLT), is primarily used to manage type 2 diabetes by lowering blood glucose levels [40]. In the present study, however, Dapa was found to inhibit DLT-induced alterations in the amplitude and gating properties of the I_Na. Although SGLT activity has been observed in some endocrine cells [43,44], it has not been reported in Neuro-2a cells. This suggests that Dapa may exert a direct effect on Na_V channels in Neuro-2a cells, consistent with previous findings [2,31,41,42,43].

Previous studies have shown that type-I pyrethroids, such as tefluthrin, increase the amplitude of I_Na(T) and concurrently slow its inactivation time course [28,44]. In contrast, type II pyrethroids, including DLT, also enhance I_Na(T) amplitude but do not significantly affect the inactivation time constant of either the fast or slow components of the current. Notably, both the current study and earlier reports [2] have demonstrated that DLT additionally augments I_Na(L) and I_Na(Tail). Structurally, type II pyrethroids are distinguished from type I pyrethroids by the presence of an α-cyano group on the benzyl moiety of the compound (Figure 1 and Figure 8), which is absent in type-1 pyrethroids. These findings suggest that the α-cyano group in the pyrethroid molecule may play a role in the differential modulation of Na_V channel gating by various pyrethroids.

An important consideration from our experimental results is that while Neuro-2a cells can display motor neuron-like properties, they are not identical to primary motor neurons. As a transformed cell line with cancerous origins, Neuro-2a cells can exhibit significant differences in behavior and characteristics compared to primary neurons [22,23,24]. Researchers frequently use Neuro-2a cells due to their ease of culture and manipulation. However, it is crucial to validate findings obtained from Neuro-2a cells using primary neurons or in vivo models to ensure their relevance and accuracy.

In simulations where DLT was present, perturbations to I_Na resulted in two distinct burst patterns in the modeled respiratory neuron (Figure 6 and Figure 7). This model, originally developed to simulate pre-Bötzinger complex (pre-BötC) neuronal activity [25], revealed one pattern characterized by a slowing of intraburst AP firing, while the other exhibited a faster intraburst firing rate. Both patterns also demonstrated a progressive shortening of intraburst intervals over time. This phenomenon is indicative of reverse spike-frequency adaptation where a neuron exhibits in increase in its firing rate during intraburst activity, as previously described in subthalamic nucleus neurons [33]. This is in contrast to the more common spike-frequency adaptation, where the firing rate typically decreases over time during intraburst activity. These findings suggest that the activity of I_Na(L) and I_Na(Tail), particularly under conditions involving DLT exposure, may be responsible for the reverse spike-frequency adaptation observed in the intraburst firing of this modeled respiratory neuron.

In these simulations, the AP amplitude exhibited a significant and progressive decrease during fast-type bursts, while the reduction during slow-type bursts was less pronounced. This observation suggests that the cumulative inhibition of I_Na, as previously described [17,19,45], coupled with reverse spike-frequency adaptation, is more prominent during fast-type bursts compared to slow-type bursts. Additionally, the post-burst hyperpolarizing potential was notably larger during fast-type bursts, and the extent of resting membrane potential depolarization was also more pronounced during fast-type bursts compared to slow-type bursts.

Bursting patterns in different modeled neurons are significant because they play a crucial role in understanding the complex dynamics of neural activity, such as neural coding and information processing, synchronization and network dynamics, and pathophysiological insights [46]. Collectively, these findings suggest that exposure to DLT is capable of inducing changes in the density and gating properties of I_Na [2], significantly influencing the burst patterns of respiratory neurons and potentially impacting respiratory function, as previously reported [6,10,20,25,32,35].

In our docking prediction of the DLT molecule on Na_V channel (Figure 8), we found that DLT forms hydrophobic contacts with several amino-acid residues in the Na_V1.8 channel, indicating that these regions may have a specific role in modulating DLT’s binding affinity to Na_V1.8. Na_V1.8 channels, known as a Na_V channel α subunit 8 (SCN10A), are a type of Na_V channel primarily expressed in peripheral sensory neurons, particularly in dorsal root ganglia neurons [47,48]. If this region contains these amino-acid residues, performing knockdown or knockout maneuvers is likely to result in these treated cells showing increased resistance to DLT or other type II pyrethroids’ modifications of I_Na [1,4,37,52]. However, whether DLT produces similar docking sites for the amino acids in Na_V1.2, Na_V1.3, and Na_V1.7 channels in Neuro-2a cells [23] remains to be further investigated. Additionally, further investigation is needed to determine whether DLT or other type II pyrethroids interact with the auxiliary β subunits of Na_V channels.