Intrinsic properties of pyramidal neurons in the anterior and posterior IC of both sexes

In the rostro-caudal axis, the IC is divided into two regions: the anterior insula (aIC) and the posterior insula (pIC). While the distinct reciprocal connections of these regions with cortical and subcortical brain areas have been well-characterized, less is known about their intrinsic properties. Thus, electrophysiological characterizations of pyramidal neurons in both aIC and pIC were conducted in male and female subjects: layer V pyramidal neurons were whole-cell patch clamped in acute coronal slices from mouse aged between 90 to 120 postnatal days (PNDs) (Fig. 1A, B).

Fig. 1

Subregional intrinsic properties of anterior and posterior insular cortex pyramidal neurons in males and females. A, B Representative coronal sections of the brain from the anterior (Bregma + 1.98) and posterior (Bregma − 1.06) regions of the insular cortex (IC) showing recording sites of layer V pyramidal neurons. C, D Quantitative analysis of passive and active membrane properties revealed that anterior IC (aIC) pyramidal neurons are significantly larger (i.e., larger capacitance) and hyperpolarized compared to posterior IC (pIC) neurons, and that this difference is present in both male and female mouse. E, F Subregional differences in the current–voltage relationship between aIC and pIC pyramidal neurons were found in response to current injection steps of 50 pA, ranging from − 400 pA to + 50 pA, in adult females (E), but not in males (F). G An example of an action potential (AP) evoked by increasing current steps is illustrated, with the rheobase indicated. H The results indicated that the minimal current required to trigger an AP (rheobase) was significantly higher in aIC compared to pIC pyramidal neurons. Data are presented as box-and-whisker plots (minimum, maximum, median) for (C-D-H), and as mean ± SEM in XY plots for (E–F). Two-way ANOVA followed by Šídák's multiple comparison test was performed for (C-D-H), and Mann–Whitney U test was applied for (E–F). P-values < 0.05 are depicted in the graphs. The sample sizes for aIC male and female were 14/10 and 13/6, respectively, and for pIC male and female were 28/15 and 20/12, respectively

There were no significant differences in passive or active membrane properties when comparing males and females within each IC region. However, substantial differences were observed between the anterior and posterior IC. Specifically, pyramidal neurons in the aIC were notably bigger and hyperpolarized compared to those in the pIC (Fig. 1C, D, Table 1). Furthermore, aIC exhibited a higher rheobase compared to pIC neurons in both males and females, consistent with their hyperpolarized resting potential (Fig. 1H, Table 1).

Table 1 Active and passive membrane properties of aIC and pIC pyramidal neurons in both sexes

When comparing the membrane voltage response to hyperpolarizing current steps, we observed that in males, there were no significant differences between aIC and pIC (Fig. 1E, Table 2). However, in females, the voltage response of aIC was significantly lower compared to pIC (Fig. 1F, Table 2). Finally, no sex differences were observed when comparing the voltage membrane responses in both cortexes (Additional file 1: Fig. S1A, B, Additional file 4: Table S1). Principal Component Analysis (PCA, Fig. 3, Table 3) with membrane capacitance, rheobase, resting membrane potentials, neuronal excitabilities, and voltage membrane response to a different injected current steps as quantitative variables, indicated that subregion if the principal contributor to the dataset variance when all groups are considered (Fig. 3B).

Table 2 Voltage membrane response per current steps in aIC and pIC pyramidal neurons in both sexesTable 3 Principal component analysis (PCA) of IC pyramidal neurons intrinsic propertiesExcitability differences in anterior and posterior insular cortex pyramidal neurons by sex and subregion

The observed differences in passive and active membrane properties between the aIC and pIC suggest distinct levels of excitability of the principal projecting neurons in these two subregions.

To assess the intrinsic excitability of IC principal neurons, we recorded their membrane response profiles in reaction to a series of somatic current steps. Both male and female mouse exhibited significant differences in membrane profiles between the aIC and pIC, indicating a higher excitability of pyramidal neurons in the posterior IC (Fig. 2A, B, Table 4). However, when comparing sexes, pyramidal neurons showed similar excitability (Additional file 1: Fig. S1C, D, Additional file 4: Table S1). Furthermore, within the “physiological range”, neurons in the aIC fired less frequently than those in the pIC, independently of sex. Notably, among males, pyramidal neurons in the posterior IC fired more frequently than in females (Fig. 2D, Table 1). Consistent with these intrinsic passive properties, PCA revealed that the two distinct subregions were the primary contributors to the variance in this dataset (see Fig. 3B).

Fig. 2

Sex and subregional differences in the excitability of insular cortex pyramidal neurons. A, B As indicated by the response of action potentials to progressive depolarizing current injection, pyramidal neurons exhibit a notably higher level of excitability in the pIC compared to the aIC, and this trend is consistent across both sexes. (500 ms, ranging from 0 to 600 pA in 50 pA steps). C Representative voltage traces evoked by the injection of hyper- and depolarizing current steps (500 ms, ranging from − 150 to 250 pA in 50 pA steps) in both anterior and posterior IC pyramidal neurons of both sexes. D During physiological depolarizing current stages, neurons from the aIC displayed less frequent firing compared to those from the pIC. Additionally, male pIC neurons showed a higher level of excitability than those of their female counterparts. E An example of firing patterns triggered by the injection of + 150 pA depolarizing currents for each group is provided. Data are presented as mean ± SEM in XY plots for (A, B), and as box-and-whisker plots (minimum, maximum, median) for (C). Mann-Whitney U test was applied for (A, B), and two-way ANOVA followed by Šídák's multiple comparison test was performed for (C). P-values < 0.05 are depicted in the graphs. The sample sizes for aIC male and female were 14/10 and 14/6, respectively, and for pIC male and female were 20/15 and 16/12, respectively

Table 4 Intrinsic excitability of aIC and pIC pyramidal neurons in both sexesFig. 3

Principal component analysis (PCA) of intrinsic properties indicating that subregion is the main factor contributing to the variance in the dataset. This analysis was carried out using membrane capacitance, rheobase, resting membrane potentials, neuronal excitabilities, and the voltage membrane's response to varying injected current steps as quantitative variables. A Plotting the percentage of explained variance by each PC (histogram) reveals that most of the data set’s variance is explained by PC1 (65.90%), followed by PC2 (12.15%) and PC3 (5.33%). Black dots represent the cumulative percentage of explained variance. B, C PCA graphs of individuals were built with PC1 and PC2 which together explained more then 80% of the variance (see A). Small dots represent individuals colored according to their belonging to one the following qualitative supplementary variables: subregion (top), sex (below). Ellipses represent the barycenter of individuals (i.e., mean) for each category. B PCA show that the subregion is the major contributor in the overall variance. C In contrast PCA showed that in both insular cortices male and female largely overlap

The excitatory/ inhibitory balance of across sex and IC subregions

Alterations in excitatory (glutamatergic)/ inhibitory (GABAergic) neurotransmission in the insular cortex have been proposed to play a causal role in the development of chronic pain state and perception of painful stimuli [14]. However, the E/I balance of IC subregions has not been characterized yet. First, we quantified the total charge transferred from whole-cell recorded spontaneous AMPA-mediated EPSCs (sEPSCs; Fig. 4A) and GABA-mediated IPSCs (sIPSCs; Fig. 4B) a parameter which accounts for both frequency and amplitude of spontaneous events. The total charge transfer of sEPSCs in aIC was greater than that of the pIC in male only (Fig. 4C, Table 5). The total charge transfer of sIPSCs was similar across sexes and subregions (Fig. 4D, Table 5). Subsequently, we compared the relative distribution of sEPSCs and sIPSCs total charge transfer [15, 16]. Interestingly, in both sexes and subregions, sIPSCs were predominant (Fig. 4E–H) as show by the right-shift in the sIPSCs cumulative distribution, indicating that the E/I balance is shifted towards inhibition (Fig. 4E–H, Dot plots). Although the E/I balance in the IC is mainly influenced by inhibition, it is important to note that in both male and female the percentage of inhibition is larger in the pIC than in the aIC (Fig. 4E–H, Pie chart).

Fig. 4

Sex and subregional profiles of the Excitatory/Inhibitory (E/I) balance of the Insular Cortex. A, B Schematic illustration of synaptic charge transferred by each sEPSCs (A) and sIPSCs (B) calculated as the area inside each event as indicated by the arrows. C An analysis of the total charge of AMPA-sEPSCs measured over a 6-min period across sexes revealed similar total charges in both male and female in both the aIC and pIC. However, there was a higher total charge transfer observed in the aIC compared with pIC, but this was only observed in males. D Similarly, the total charge of GABA-sIPSCs, when measured over a 6-min period considering both area and sex, showed a comparable amount of charge transferred. This was analogous to the observations made in 'C' for AMPA-sEPSCs. E–H Cumulative frequency distribution of sEPSCs (excitation) and sIPSCs (inhibition) total charge transfer obtain from each Insular cortex within male and female animals. Insets: dot plots and pie graph showing the proportion of E versus I extrapolated at P = 0.5 from the corresponding cumulative frequency. C, D Data are presented as box-and-whisker plots (minimum, maximum, median) and analyzed via two-way ANOVA followed by Šídák's multiple comparison. P-values < 0.05 are depicted in the graphs. C The sample sizes for aIC male were 12/10 for pIC male 22/15, for aIC female 13/10 and for pIC female 12/10. D The sample sizes for aIC male were 19/11 for pIC male 13/9, for aIC female 13/10 and for pIC female 16/10

Table 5 Total charge transferred by AMPA- and GABA-mediated events in aIC and pIC of both sexesSubregional differences of excitatory synaptic transmission

To explore the hypothesis that synaptic connectivity is responsible for the observed subregional differences in E/I balance, we conducted recordings and compared spontaneous AMPA-mediated post-synaptic currents in layer V pyramidal neurons within both sexes and subregions (Figs. 5, 6). In males, it became evident that the mean amplitude of post-synaptic currents in the pIC increased, while the mean frequency was lower compared to the anterior aIC (Fig. 5C–E, Table 6. Further analysis of the frequency distribution confirmed a higher proportion of larger but less frequent excitatory events in pIC compared to aIC (Fig. 5D–F).

Fig. 5

Region-specific spontaneous excitatory synaptic activity in adult male IC pyramidal neurons. A, B Representative spontaneous excitatory postsynaptic currents (sEPSCs) recorded at − 70 mV in male aIC and pIC. C, D On average, excitatory events are larger and less frequent in the pIC than the aIC. E, F Log-normal curve fittings with confidence intervals (± CI) reveal that the amplitude and frequency distribution are skewed to the right in the pIC as compared to the aIC. Data are presented as box-and-whisker plots (minimum, maximum, median) and analyzed via Mann–Whitney U test. P-values < 0.05 are depicted in the graphs. The sample size for aIC male was 14/9, and for pIC male was 27/16

Fig. 6

Differences in excitatory synaptic activity in female insular pyramidal neurons by region. A, B Representative spontaneous excitatory postsynaptic currents (sEPSCs) recorded at − 70 mV in female aIC and pIC. C, D On average, excitatory events are comparable in amplitude and frequency in both aIC and pIC. E, F Log-normal curve fittings with confidence intervals (± CI) reveal higher proportion of smaller and more frequent events in the aIC as compared to the pIC. Data are presented as box-and-whisker plots (minimum, maximum, median) and analyzed via Mann–Whitney U test. P-values < 0.05 are depicted in the graphs. The sample size for aIC female was 13/6, and for pIC female was 17/12

Table 6 sE/IPSCs within the IC in both sexes

In contrast, for females, the mean amplitude and frequency of spontaneous excitatory post-synaptic currents (sEPSCs) remained similar in both aIC and pIC (Fig. 6C–E, Table 6). A closer examination of the frequency distribution of individual sEPSCs, however, revealed a higher proportion of smaller and more frequent synaptic currents in aIC compared to pIC in females (Fig. 6D–F, Table 6). Notably, when comparing across sexes and subregions, a sex-specific difference was observed solely in pIC. Specifically, the primary frequency of sEPSCs was higher in females than in males (Additional file 2: Fig. S2B, Additional file 5: Table S2). Finally, no significant differences were found in the kinetics of excitatory events across both subregions and sexes (Additional file 2: Fig. S2C-D, Additional file 5: Table S2).

Differential GABAergic transmission in aIC and pIC of adult males

Spontaneous GABAA-mediated inhibitory post-synaptic currents were recorded in IC principal neurons in whole-cell configuration (Figs. 7, 8). As shown in Fig. 7C, in adult male the mean amplitude of sIPSCs remained similar among the two cortexes. In contrast the frequency was lower in pIC when compared to those in aIC (Fig. 7E, Table 6). Indeed, the frequency distribution confirmed that inhibitory events were more frequent in aIC (Fig. 7F). In contrast, in female group both mean amplitude and frequency were comparable (Fig. 7C–E, Table 6). When compared across sex and subregion no further differences were found in the mean amplitude, frequency or kinetics of sIPSCs (Additional file 3: Fig. S3A–D, Additional file 5: Table S2).

Fig. 7

Differences in inhibitory synaptic activity among male pyramidal neurons in the insular cortex, by region. A, B Representative spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at − 70 mV in male aIC and pIC. C, D Upon conducting a quantitative analysis of the mean amplitude and frequency across these areas, it was found that although the amplitudes were similar, there was a lower frequency of inhibitory events in the pIC when compared to the aIC. E, F Log-normal curve fittings with confidence intervals (± CI) reveal that the amplitude and frequency distribution are skewed to the right and to the left respectively in the aIC as compared to the pIC. Data are presented as box-and-whisker plots (minimum, maximum, median) and analyzed via Mann–Whitney U test. P-values < 0.05 are depicted in the graphs. The sample size for aIC male was 22/10, and for pIC male was 14/11

Fig. 8

Similarities in inhibitory synaptic activity among female pyramidal neurons across different subregions of the insular cortex. A, B Representative spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at − 70 mV in female aIC and pIC. C, D On average, ihibitory events are comparable in amplitude and frequency in both aIC and pIC. E, F Log-normal curve fittings with confidence intervals (± CI) reveal higher proportion of smaller events in pIC compared to aIC. Data are presented as box-and-whisker plots (minimum, maximum, median) and analyzed via Mann–Whitney U test. P-values < 0.05 are depicted in the graphs. The sample size for aIC female was 15/10, and for pIC female was 19/11

LTP at the glutamatergic synapses in a sex- and subregion-specific manner

The IC exhibits reciprocal connections with cortical and subcortical regions associated with sensory, cognitive, and memory functions, suggesting the involvement of synaptic plasticity mechanisms. While Long-Term Potentiation (LTP) has been studied in the aIC, our understanding of LTP in the pIC is limited. Additionally, there is a paucity of knowledge regarding potential sex differences in synaptic processes within the IC. Thus, excitatory post synaptic transmission in each IC were recorded in layer V pyramidal neuron in both male and female (Fig. 9A, B). We fist compared input–output (I/O) curves of fEPSP (Fig. 9C, D, Table 7). The I/O relationship did not differ between aIC and pIC in male (Fig. 9C), in contrast female showed a marked difference across subregions. Indeed, along the entire I/O curve the aIC showed a greater synaptic strength compared to the pIC (Fig. 9D). LTP could be elicited in both aIC and pIC in male (Fig. 9E, F) and in contrast, LTP could only be induced in the aIC of female (Fig. 9G, H).

Fig. 9

Long-term potentiation in the insular cortex varies based on sex and subregion. A, B A schematic illustration indicates the aIC and pIC, both delineated by red dashed lines. Stimulation and recordings were applied and gathered respectively from layer V pyramidal neurons in each insular cortex. C, D An examination of the input–output (I/O) relationship showed a comparable synaptic strength in both the aIC and pIC of adult males (C). Conversely, female aIC exhibited a greater synaptic strength in comparison to pIC. E–G The field excitatory postsynaptic potential (fEPSP), shown as a percentage of the baseline, was observed before and after the application of high-frequency stimulation (HFS). The point in time at which HFS was applied is shown by the arrow. E When HFS protocol was administered to layer V pyramidal neurons in adult males, it led to long-term potentiation (LTP) in both the anterior and posterior insular cortices. F The fEPSP magnitude at baseline (from 10 to 0 min) and during LTP (from 20 to 30 min after induction), corresponding to the normalized values in 'C', demonstrated a notable difference in the 10-min baseline period and the final 10 min of recording in both male insular cortices. G In contrast, the same protocol, when applied in adult females, only induced a strong LTP in the aIC. H The fEPSP magnitude at baseline (from 10 to 0 min) and during LTP (from 20 to 30 min post-induction), corresponding to the normalized values in 'F', showed a significant difference in the 10-min baseline period and the final 10 min of recording, but only in the aIC of adult females. C-D-E-G Data are presented as mean ± SEM in XY plots and analyzed via Mann–Whitney U test (C, D). P-values < 0.05 are depicted in the graphs. F–H Data are shown as pre-post individual experiments and analyzed via Wilcoxon P test. P-values < 0.05 are depicted in the graphs. C–H The sample sizes for aIC and pIC male and female were 8 and 7, respectively

Table 7 Input–output (I/O) Relationship of aIC and pIC of adult males and females