Tonic GABA‐activated synaptic and extrasynaptic currents in dentate gyrus granule cells and CA3 pyramidal neurons along the mouse hippocampal dorsoventral axis

The hippocampus is a medial temporal lobe structure in the brain and is widely studied for its role in memory and learning, in particular, spacial memory and emotional responses. It was thought to be a homogenous structure but emerging evidence shows differentiation along the dorsoventral axis and even microdomains for functional and cellular markers. We have examined in two cell‐types of the hippocampal projection neurons, the dentate gyrus (DG) granule cells and CA3 pyramidal neurons, if the GABA‐activated tonic current density varied between the dorsal (septal) and the ventral (temporal) poles of the male mouse hippocampus. Tonic synaptic currents, arising from spontaneous and miniature inhibitory postsynaptic currents (sIPSC, mIPSC), and extrasynaptic tonic currents were evaluated. The results revealed different levels of sIPSC but not mIPSC density between the dorsal and ventral hippocampal neurons for both the DG granule cells and the CA3 pyramidal neurons. The extrasynaptic tonic current density was larger in the DG granule cells as compared to the CA3 pyramidal neurons but did not vary between the dorsal and ventral regions. IPSC bursting was observed in both cell‐types in the ventral hippocampus but was more common in the CA3 pyramidal neurons. Only in the dorsal DG granule cells was the level of the sIPSC and mIPSC density similar. The results indicate that the tonic GABAergic inhibition is particularly strong in the ventral hippocampal DG granule cells and enhanced in the dorsal as compared to the ventral hippocampal CA3 pyramidal neurons.

and spatial information from the sensory cortices whereas the ventral hippocampus has more connectivity with the prefrontal cortex, hypothalamus, and amygdala (Canteras & Swanson, 1992;Moser, Moser, & Andersen, 1993;Preston & Eichenbaum, 2013;Risold & Swanson, 1996;Strange et al., 2014). At the macroscopic level, the hippocampus appears to be a homogenous structure with a characteristic neuronal network module that is repeated in a parallel lamellar fashion along the longitudinal axis (Andersen, Bliss, & Skrede, 1971;Papatheodoropoulos, 2018). However, superimposed on the anatomical lamellar organization are molecular and functional gradients that run along the dorsoventral axis with no apparent "hard" boundaries (Fanselow & Dong, 2010;Strange et al., 2014). The hierarchies of the multiple domains that may result is currently not known. A common simplification of the endogenous diversification of the hippocampus is when the structure is divided along the dorsoventral axis into dorsal, intermediate, and ventral domains (Papatheodoropoulos, 2018;Strange et al., 2014).
Dorsal and ventral DG were defined in coronal slices according to Paxinos and Watson (1986). Hippocampal slices including the dorsal CA3 region were obtained by cutting the hemisphere in the coronal plane (the coronal sections) and the first three hippocampal slices were collected from the dorsal pole. For the ventral hippocampal slice preparation, the hemisphere was mounted on the flat dorsal surface.
The first four horizontal hippocampal slices were collected from the ventral region of the brain. The slices were then placed into chamber filled with the NMDG-based solution and incubated for 12-15 min at 32 C. After that, the slices were transferred to the incubation chamber filled with the HEPES-based holding solution (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH 2 PO 4 , 30 NaHCO 3 , 20 HEPES, 25 D-glucose, 2 MgSO 4 , 2 CaCl 2 , 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, pH 7.3-7.4 adjusted with NaOH when saturated with 95% O 2 and 5% CO 2 ; osmolarity 300-305 mOsm adjusted with sucrose. The slices were kept in the holding solution at room temperature (20-22 C) for at least 1 hr before use.

| Slice electrophysiology
Whole-cell patch-clamp recordings  were per- sIPSCs were recorded for at least 5 min after baseline stabilization. For mIPSCs recordings, tetrodotoxin (TTX, 1 μM, bath application for 10-12 min) was added to the ACSF to block voltage-activated sodium channels and, therefore, action potential-dependent GABA release (Edwards et al., 1990). To reveal the extrasynaptic GABA A receptors mediated tonic currents, 100 μM picrotoxin or bicuculline methiodide was applied.
Picrotoxin was dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO in the ACSF was 0.1% (v/v).

| Data analysis
The sIPSCs and mIPSCs were analyzed using the MiniAnalysis software 6.0 (Synaptosoft, Decatur). IPSC events were first detected if larger than a threshold value, which was set as 5xRMS (root-mean-square of the baseline noise), and then visually inspected to remove false events. As the frequency of IPSCs differs between DG granule cells and CA3 pyramidal neurons, we used time (3-5 min) and number of events (200-300 events), respectively, for the analysis. Only IPSC events with a single peak were used for analysis of amplitude and kinetics of the currents (10-90% rising time ≤5 ms, current decay (ms), charge transfer Q (fC), Figure S1a). The total synaptic current (sIPSC T or mIPSC T ) was defined as frequency (s −1 ) × Q (fC) for the individual neuron. To account for variable cell size, the IPSC T was normalized to the cell membrane capacitance (Cm (pF)) and expressed as total synaptic current density, sIPSC T density or mIPSC T density (pA/pF), Figure  IPSCs. The bursts were separated by basal sIPSC activity. In order to characterize recurrent bursting activity, we determined burst parameters, that is, burst frequency, interburst interval, and burst charge transfer (burst Q). The total bursts current was determined according to burst T (pA) = burst frequency (s −1 ) × burst Q (pC) and the density, burst T density (pA/pF) = burst T (pA)/Cm (pF). In total, we analyzed 14 individual bursts from 5 DG granule cells and 81 bursts from 19 CA3 neurons.
The values from each burst were averaged per cell to obtain the mean value for the individual neuron. The extrasynaptic tonic current amplitude was measured as the shift of baseline at the holding current after application of a GABA A receptors inhibitor, picrotoxin, or bicuculline (Bai et al., 2001;pCLAMP 10.5 software, Axon Instruments, Molecular Devices). To account for size variation among the cells, the extrasynaptic tonic current density was expressed as the extrasynaptic tonic current amplitude normalized to the cell membrane capacitance (Stell, Brickley, Tang, Farrant, & Mody, 2003), Figure S1b.

| Statistics
The data were analyzed using the GraphPad Prism 8 (GraphPad Software La Jolla, CA). Values are presented as mean ± SEM, n refers to the number of analyzed cells. All data were included in the analysis unless statistically defined as outliers by the Tukey method. Each group of the data sets was tested for normality with the D'Agostino test and histogram distribution. To compare the normally distributed data, the Student's two-tailed t-test was used to determine statistical differences between two experimental groups. Data sets that are not normally distributed were compared using a nonparametric Mann-Whitney U-test. Correlations between variables were assessed using a nonparametric Spearman rank correlation. A p-value less than .05 was statistically significant.

| IPSCs differ between dorsal and ventral hippocampal neurons
The dorsal and ventral parts of the local hippocampal neuronal networks participate in different extended circuitries (Papatheodoropoulos, 2018;Strange et al., 2014) and hence, the inhibitory input of the intrinsic pri- This raised the question whether the current density varied, or not, between the cells in the dorsal and ventral hippocampus. In order to estimate the neuronal size which may vary along the dorsoventral axis, we recorded the cell membrane capacitance. The DG granule cells membrane capacitance was larger, on the average, in the ventral as compared to the dorsal hippocampus ( Figure 1h). The current density for cells from the two hippocampal regions is shown in Figure 1i. It revealed that the sIPSC T density in the ventral DG granule cells was 2.3 fold larger than that of the dorsal DG granule cells. Interestingly, the sIPSC T density and the mIPSC T density were similar for the dorsal neurons. . Summary graphs for the mean frequency (d), the median amplitude (e), the median charge transfer (f), and the total synaptic current (g, IPSC T ) of sIPSCs (−TTX) and mIPSCs (+TTX) recorded from DG granule cells of the DH and the VH. (h) The membrane capacitance of DG granule cells in the VH was significantly higher compared with the DH (DH vs VH: 58.5 ± 3.2 n = 54 vs 89.9 ± 3.5 n = 61, p < .001). (i) The sIPSCs (−TTX) and mIPSCs (+TTX) total current density recorded from the DG granule cells of the dorsal and ventral regions. Data is presented as a scatter dot plot for cell values and a box and whiskers plot with a median value plotted as a line and a mean value shown as "+." Outliers are marked as dot plot (filled black circles) and defined by the Tukey method. Statistical analysis was performed by excluding outliers and only statistically significant differences are marked on the graph. In total, records from 46/41 granule cells in the DH/VH, respectively, were analyzed for sIPSCs (−TTX) and 17/15 granule cells in the DH/VH, respectively, were analyzed for mIPSCs (+TTX). Unpaired Students t-test/ nonparametric Mann-Whitney U-test, *p < .05, **p < .01, ***p < .001 [Color figure can be viewed at wileyonlinelibrary.com] 3.2 | Extrasynaptic tonic-current density is celltype dependent Baseline excitability of neuronal networks is regulated by a number of mechanisms including the extrasynaptic tonic GABA-activated currents (Bai et al., 2001;Birnir et al., 1994Birnir et al., , 2000Brickley et al., 1996;Pavlov, Savtchenko, Kullmann, Semyanov, & Walker, 2009;Rossi & Hamann, 1998;Wlodarczyk et al., 2013). We examined if the extrasynaptic toniccurrent density differed for cells along the longitudinal hippocampal axis and between the DG granule cells and the CA3 pyramidal neurons.
F I G U R E 2 Legend on next page.

| DG granule cells
Extrasynaptic tonic current identified as a shift of the baseline currents with picrotoxin or bicuculline is shown in Figure 3a for a dorsal and a ventral hippocampal DG granule cell. As the neurons vary in membrane capacitance ( Figure S5a), the current density was calculated by dividing the tonic current amplitude by the membrane capacitance of the cell. No difference was observed between the dorsal and ventral DG granule cells extrasynaptic tonic-current density but addition of TTX (1 μM) significantly decreased the tonic current by 31% in the ventral, but not the dorsal, DG granule cells (Figure 3c).

| Bursts of IPSCs in the dorsal and ventral hippocampal neurons
In 10% of the DG granule cells from the ventral hippocampus  (Freund & Buzsaki, 1996;Papatheodoropoulos, 2018). It is well-known that neurogenesis in adult mice also has a dorso-ventral gradient with more neurogenesis in dorsal DG region (Overstreet-Wadiche & Westbrook, 2006;Pedroni et al., 2014) but immature neurons have distinct electrophysiological properties and the F I G U R E 2 GABA A receptor-mediated synaptic currents in CA3 pyramidal neurons in the mouse dorsal and ventral hippocampus. Microphotographs of a dorsal (a, DH) and a ventral (b, VH) hippocampal slices and corresponding drawings depict schematic recording pipette (Rec) positioning. Voltage-clamp recordings of sIPSCs and mIPSCs (1 μM TTX) in the CA3 pyramidal neurons of the DH (aA) and VH (bA) hippocampus at a holding potential of −60 mV. Regions marked with filled circles are shown on an expanded scale in (B). (c) Cumulative probability distribution for the interevent interval (IEI) and the median amplitude of (A) sIPSCs (left, IEI: 378/275 events analyzed for the DH/VH; Amplitude: 315/242 events analyzed for the DH/VH) and (B) mIPSCs (right, IEI: 333/300 events analyzed for the DH/VH; Amplitude: 277/252 events analyzed for the DH/VH) recorded from CA3 principal neuron of the DH and VH, from the representative traces (a) and (b). Summary plots for the mean frequency (d), the median amplitude (e), the median charge transfer (f), and the total synaptic current (g, IPSC T ) of sIPSCs (−TTX) and mIPSCs (+TTX) recorded from CA3 pyramidal neurons of the DH and the VH. (h) The membrane capacitance of CA3 neurons was significantly higher in the VH compared with the DH (DH vs. VH: 201.6 ± 6.4 n = 63 vs. 248.1 ± 11.1 n = 63, p < .001). (i). The total current density of the sIPSCs (−TTX) and mIPSCs (+TTX) recorded from the CA3 pyramidal neurons of the DH and the VH. Data is presented as a scatter dot plot for neuron values and a box and whiskers plot with a median value plotted as a line and a mean value shown as "+." Outliers, defined by the Tukey method, are marked as dot plot (filled black circles). Statistical analysis was performed by excluding outliers and only statistically significant differences are marked on the graph. In total, the records from 38/40 CA3 neurons in the DH/VH, respectively, were analyzed for sIPSCs (−TTX) and 22/22 CA3 neurons in the DH/VH, respectively, were analyzed for mIPSCs (+TTX). Unpaired students t-test/nonparametric Mann-Whitney U-test, *p < .05, **p < .01, ***p < .001 [Color figure can be viewed at wileyonlinelibrary.com] four, dorsal DG immature granule cells we identified were not included in the current analysis. Nevertheless, because subpopulations of DG granule cells in the DH can be at different stages of maturation, this may contribute to the lower synaptic current density in dorsal compared with ventral DG. The similar mIPSC T density in a given cell-type along the dorsoventral axis despite the increased mIPSC frequency in the ventral hippocampal neurons may potentially be related to more inhibitory synapses on larger neurons in the ventral hippocampus (Freund & Buzsaki, 1996;Neddens & Buonanno, 2010).

Somatic recordings of DG granule cells and pyramidal neurons have
shown that the majority of the IPSCs recorded originate from synapses close to or at the soma (Cossart et al., 2000;Miles et al., 1996;Soltesz et al., 1995;Williams, Buhl, & Mody, 1998), and are particularly effective in controlling the output of the cells. Indeed, it has been shown that removal of the bulk of the dendritic tree (>50%) does not change the characteristics of mIPSCs recorded at the soma (Soltesz et al., 1995). In contrast, IPSCs generated in the dendrites originate from firing of dendritic projecting interneurons and primarily serve to control local excitatory conductance's and their impact on somatic output as distance-dependent scaling of the IPSCs does not take place (Andrasfalvy & Mody, 2006;Cossart et al., 2000;Miles et al., 1996;Soltesz et al., 1995). Accordingly, the majority of the results in this study are expected to originate from synapses at or close to the soma and impact sodium-dependent action potential generation in the DG granule and CA3 pyramidal neurons. This study identified a significantly higher sIPSC T density as compared to the mIPSC T in both the dorsal and the ventral hippocampal CA3 pyramidal neurons whereas the sIPSC T density was only larger than the mIPSC T in the ventral hippocampal DG granule cells. For both cell-types, where the current density was amplified, then the sIPSCs frequency and median amplitude were also increased as compared to the mIPSCs. The finding that in the dorsal hippocampus, in the DG granule cells the spontaneous and the action-potential independent IPSC T density were similar, is consistent with a low level of spontaneous firing by the interneurons synapsing on the soma of the dorsal DG granule cell. The difference, or lack there-of, between the sIPSCs and mIPSCs observed may be related to the suggestion that GABA can regulate neuronal activity by evoking either hyperpolarization or depolarization in a dose-dependent manner that may be activity dependent (Freund & Buzsaki, 1996;Staley & Mody, 1992;Staley, Soldo, & Proctor, 1995).
Intense IPSCs activity may result in shift of the chloride reversal potential to more depolarized values due to increased chloride concentration in the postsynaptic cell. Where no difference between sIPSCs and mIPSCs was recorded that is, in the dorsal hippocampal F I G U R E 3 Extrasynaptic GABA A receptor-mediated tonic currents in hippocampal DG granule cells and CA3 pyramidal neurons in the mouse dorsal and ventral hippocampus. (a). GABA-evoked tonic currents recorded in DG granule cells of the DH (dorsal hippocampus, black trace) and VH (ventral hippocampus, red trace). (b). GABA-evoked tonic currents from CA3 pyramidal neurons of the DH (black trace) and VH (red trace) under basal physiological conditions. (c). GABA-evoked extrasynaptic tonic-current density in DG granule cells and CA3 neurons from the DH and VH in the absence (−TTX) or presence of TTX (+TTX 1 μM, acute application). Upward shift of the baseline with the application of an inhibitor (picrotoxin, PTX or bicuculline, BIC, 100 μM) reveals the extrasynaptic tonic-current amplitude (the difference between the dashed lines). Data is presented as a scatter dot plot for individual cells and a box and whiskers plot with median values plotted by Tukey's method. Mean values shown as "+." Only statistically significant differences are marked on the graph. Records from 5 to 13 neurons in the DH and VH were analyzed in the absence/ presence of TTX. Nonparametric Mann-Whitney U-test, *p < .05; **p < .01. V hold = −60 mV [Color figure can be viewed at wileyonlinelibrary.com] DG granule cells, GABA might be expected to only contribute to inhibition of the primary neurons.
Tonic conductance is not only evoked by sIPSC and mIPSC, but also by low concentrations of interstitial GABA activating extrasynaptically-located high-affinity GABA A receptors or spontaneously opening GABA A receptors resulting in long-lasting tonic inhibition (Bai et al., 2001;Birnir et al., 1994;Jin, Jin, Kumar-Mendu, et al., 2011;Kasugai et al., 2010;Otis et al., 1991;Semyanov et al., 2003;Soltesz et al., 1995;Wlodarczyk et al., 2013). The current density for the extrasynaptic tonic current did not vary along the longitudinal hippocampal axis for neither the DG granule cells nor the CA3 pyramidal neurons. In the DG granule cells the extrasynaptic tonic current was more prominent than in the CA3 pyramidal neurons and may be related to, at least in part, spill-over of GABA from synapses (Brickley et al., 1996;Rossi & Hamann, 1998) as the difference between the two cell-types disappeared in the presence of TTX. Moreover, extrasynaptic tonic current density positively correlated with total synaptic current density in the ventral DG granule cells. What remained of the current in TTX must have been generated either by spontaneously opening GABA A receptors channels (Birnir et al., 2000;Korol et al., 2018;Wlodarczyk et al., 2013) or interstitial GABA-activated high-affinity GABA A receptors (Bai et al., 2001;Birnir et al., 1994;Jin, Jin, Kumar-Mendu, et al., 2011;Kasugai et al., 2010;Semyanov et al., 2003). The distinct types of tonic conductances, the sIPSCs, mIPSCs, and extrasynaptic tonic currents, reflect the multimodal nature of the neuronal inhibition present in the hippocampal neurons.
It is possible that some of the functional diversity we identified may be explained by differential expression of GABA A receptors subtypes in the postsynaptic neurons. Expression of GABA A receptors varies with developmental stage, brain region, and neuronal types  (Bhandage et al., 2014;Hortnagl et al., 2013;Jin, Bazov, et al., 2011;Wisden, Laurie, Monyer, & Seeburg, 1992). mRNA and protein expression in the dorsal and ventral mouse hippocampus of the various GABA A subunits has been studied by Hörtnagl et al. (Hortnagl et al., 2013). The α2, α5, and γ2 were prominently expressed in the DG granule and CA3 pyramidal layers in both the dorsal and ventral hippocampus (Hortnagl et al., 2013). The α1 and α4 subunits general expression was somewhat lower and considerably weaker in CA3 area as compared to the DG area (Hortnagl et al., 2013). The expression of the α3, α6, and γ1 was not detected in the DG or the CA3 area. The δ subunit expression was almost exclusively in the DG granule layer and no labelling was observed in the CA3 area (Hortnagl et al., 2013).
The beta subunits were present in both areas and form an integral part of GABA A receptors (Kasugai et al., 2010). All GABA A receptors subunits can be found outside of synapses (Kasugai et al., 2010) but some appear to form GABA A receptors located preferentially outside of synapses. The α4, α5, and the δ subunit are almost exclusively located outside of synapses and, therefore, mainly contribute to extrasynaptic GABA A receptors generating the extrasynaptic tonic currents, whereas the α1, α2, and γ2 subunits form most of the synaptic receptors and thus, generate IPSCs when activated by GABA. The similar expression pattern of the subunits may suggest that the larger dorsal sIPSCs and mIPSCs amplitudes in CA3 pyramidal neurons, as compared to the ventral CA3 hippocampal sIPSCs, can be related to a greater number of receptors at the dorsal postsynaptic sites rather than to an expression of a different subtype of GABA A receptors in the two regions. In the absence of action potentials, the extrasynaptic tonic current was similar in both cell-types at both the dorsal and the ventral hippocampal pole, despite the GABA A receptors probably being different, a mix of at least α4βδ, α4βγ2, and α5βγ2 in the DG granule cells but mainly α5βγ2 GABA A receptors in the CA3 pyramidal neurons. Contributions of α4 and α5 GABA A receptors to extrasynaptic tonic currents in the hippocampal primary neurons have been reported (Caraiscos et al., 2004;Glykys, Mann, & Mody, 2008;Jin, Jin, Kumar-Mendu, et al., 2011;Mtchedlishvili & Kapur, 2006;Scimemi, Semyanov, Sperk, Kullmann, & Walker, 2005). A recent study has further demonstrated that δ subunit-containing receptors contribute to sIPSCs in DG granule cells to a greater extent than has been previously reported (Sun et al., 2018). Compared to the CA3 pyramidal neurons, the DG granule cells have more subtypes of GABA A receptors extrasynaptically that may, importantly, vary in affinity for GABA (Jin, Jin, Kumar-Mendu, et al., 2011;Lindquist & Birnir, 2006) enabling greater effective ambient GABA-concentration range in the DG region as compared to the CA3 region.
The dorsal hippocampus is well known for its role in learning and spatial memory formation (Strange et al., 2014) while normal affective processing relies on well-functioning ventral hippocampus where disruption or malfunctioning of ventral hippocampal networks may promote anxiety phenotypes (Papatheodoropoulos, 2018;Zeidler et al., 2018). It is possible that the low tonic GABAergic inhibition we identified in the ventral CA3 neurons may, at least partly, underlie, for example, the higher susceptibility of the ventral hippocampus to generate epileptiform activity.
The findings in this study revealed differential dorsoventral and cell-type specific GABAergic inhibition in two types of the principal neurons in the mouse hippocampal lamellar circuit network. The results are consistent with cell-type specific variation in the inhibitory tone along the longitudinal axis of the mouse hippocampus and are expected to have a significant impact on the different processing of information in the two regions.

ACKNOWLEDGMENTS
The study was funded by Swedish Research Council grants 2018-02952 and 2015-02417 to Bryndis Birnir, the Swedish Brain Foundation grant,

Excellence of Diabetes Research in Sweden (EXODIAB) to Bryndis
Birnir. We thank professor Costas Papatheodoropoulos for critical comments on the manuscript and Ms. Chang Li for participation in some initial experiments.

CONFLICT OF INTEREST
The authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.