Author response: Functional specification of CCK+ interneurons by alternative isoforms of Kv4.3 auxiliary subunits

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract CCK-expressing interneurons (CCK+INs) are crucial for controlling hippocampal activity. We found two firing phenotypes of CCK+INs in rat hippocampal CA3 area; either possessing a previously undetected membrane potential-dependent firing or regular firing phenotype, due to different low-voltage-activated potassium currents. These different excitability properties destine the two types for distinct functions, because the former is essentially silenced during realistic 8–15 Hz oscillations. By contrast, the general intrinsic excitability, morphology and gene-profiles of the two types were surprisingly similar. Even the expression of Kv4.3 channels were comparable, despite evidences showing that Kv4.3-mediated currents underlie the distinct firing properties. Instead, the firing phenotypes were correlated with the presence of distinct isoforms of Kv4 auxiliary subunits (KChIP1 vs. KChIP4e and DPP6S). Our results reveal the underlying mechanisms of two previously unknown types of CCK+INs and demonstrate that alternative splicing of few genes, which may be viewed as a minor change in the cells’ whole transcriptome, can determine cell-type identity. Introduction The biophysical and morphological properties of complementary GABAergic cell classes are specifically tuned for regulating the activity of the much more populous principal cells in broad temporal (from second to sub-millisecond; Hu et al., 2014; Overstreet-Wadiche and McBain, 2015) and spatial (from axons to distal dendrites Freund and Buzsáki, 1996) domains. Thus, specialized features of GABAergic neurons help the hippocampus to comply with the vast computational demand related to various behaviors (Klausberger and Somogyi, 2008). However, the functional diversity of currently known GABAergic cell types cannot match the vast amount of hippocampal behavioral tasks. Therefore, a more complete understanding of GABAergic cells is one of the major goals of current research. Emerging evidence using single-cell transcriptomics suggests that the number of GABAergic types may be higher than previously recognized (Földy et al., 2016; Fuzik et al., 2016; Harris et al., 2018; Que et al., 2019; Tasic, 2018; Zeisel et al., 2015). While these studies accelerated cell classification efforts by identifying a large number of genes that appear to define further subtypes in neuronal taxonomy, the functional relevance of most transcriptomic marker genes remain poorly understood. To better understand the relationship between gene expression and physiological function, and how these properties define neuronal types, we employed a multidisciplinary approach to study a previously unrecognized level of functional diversity among CCK-expressing hippocampal interneurons (CCK+IN) in rat CA3 area. Several properties distinguish CCK+INs from other major GABAergic cell classes. Unlike in other GABAergic cells, the axons of CCK+INs are highly enriched with CB1 receptors, which mediate activity-dependent regulation of GABA release (Freund and Katona, 2007). As a result of this delicate control, CCK+INs are ideally suited for dynamic inhibition of a subset of principal cells based on the context of ongoing activity. The in vitro firing of CCK+INs is believed to be homogeneous typically displaying intermediate AP widths (between that of pyramidal cells and classical fast-spiking interneurons) and clear spike frequency accommodation (Cea-del Rio et al., 2011; Glickfeld and Scanziani, 2006; Szabadics and Soltesz, 2009; Szabó et al., 2014). These excitability properties are crucial for the generation of characteristic CCK+INs firing in vivo, which is observed during exploration-associated with theta and gamma oscillations (Klausberger et al., 2005; Lasztóczi et al., 2011). In contrast to their apparent biophysical homogeneity, the morphology and molecular content of CCK+INs are diverse. Based on their axonal morphology basket-, mossy fiber-associated, Schaffer collateral-associated and perforant path-associated types (Cope et al., 2002; Vida and Frotscher, 2000; Vida et al., 1998) can be distinguished. These distinct morphological types selectively control excitation from different afferents (hence their names). At the molecular level, complimentary expression of marker genes characterize subsets of CCK+INs, such as VGluT3 and VIP (Somogyi et al., 2004), and single-cell RNA-sequencing also uncovered several additional molecular varieties (Fuzik et al., 2016). Due to their distinction from other major classes and large interclass variability, CCK+INs are ideal for examining relationships between genes and cellular identity and boundaries between GABAergic cell classes. In this study, we investigated hippocampal CCK+INs from a broader perspective that could reveal previously undetected excitability parameters suitable for specific physiological functions. A hint that such diverse physiological parameters exist came from previous in vivo recordings that showed that individual CCK+INs are differentially active during various oscillatory states (Klausberger et al., 2005; Lasztóczi et al., 2011), where many of them appeared to prefer either lower or higher frequency ranges. We found two types of hippocampal CCK+INs in rats based on their different excitability, with potentially different contributions to network events, particularly in the range of theta oscillations. Detailed realistic simulations showed that switching only the properties of Kv4.3-mediated currents can sufficiently convert one functional cell type to the other. Combined analyses of the complete mRNA content and protein expression of single cells revealed that the pronounced functional distinction between these two CCK+IN type is defined by differential isoform usage of three auxiliary subunits of the Kv4.3 channels. Results A large portion of CCK+INs show state-dependent firing in the CA3 area To explore potential differences in the excitability of individual CCK+INs in the CA3 region of the rat hippocampus, first we characterized their firing properties in two different conditions. Specifically, we recorded their spiking in response to current steps from two, physiologically plausible membrane potential ranges of post hoc identified CCK+INs. We focused mostly on the CA3 region because here the diversity of CCK+INs is the largest within the hippocampus. When CCK+INs (n = 557 cells) were stimulated from slightly depolarized membrane potentials (MP, range: −60 – −65 mV) relative to rest (−64.7 ± 0.4 mV), action potential (AP) firing always showed spike-frequency accommodation, which is one of the most characteristic features of this cell class (Cea-del Rio et al., 2011; Glickfeld and Scanziani, 2006; Szabadics and Soltesz, 2009; Szabó et al., 2014). However, we noticed that numerous CCK+INs (n = 290 cells) showed MP-dependent firing: their initial spiking was strongly inhibited and its onset was delayed when it was evoked from hyperpolarized MPs (between −75 to −85 mV, Figure 1A–B). On average, these cells started firing after a 252 ± 15 ms silent period from hyperpolarized MP (measured from the start of the current injection). We named these cells as Transient Outward Rectifying cells or TOR cells (a term that was used to describe cells with similar firing patterns in other brain regions: Stern and Armstrong, 1996). The rest of CCK+INs (n = 267 cells) were characterized as regular spiking or RS cells, as they fired regularly irrespective of their MP and they started firing with a short delay (33 ± 2 ms) when stimulated from hyperpolarized MP. At depolarized MP (−55 to −65 mV), the first APs of both TOR and RS cells occurred with similar short delays (48 ± 3 ms and 26 ± 1 ms, respectively, Student t-test, p=0.09, t(160) = −1.706). Figure 1 Download asset Open asset Two distinct firing patterns within CA3 CCK+ cells. (A) Firing properties of two representative CCK+INs in the CA3 hippocampal region. Firing was elicited with square pulse current injection of identical amplitude, but from depolarized (grey traces), or hyperpolarized MPs (blue traces). Several trials are superimposed to show the stability of the timing of the first action potential. Insets show the immunolabelling of the biocytin filled (BIO) recorded cells for CCK. (B) Average time course of AP occurrence in TOR and RS cells from two MP ranges (n = 120 and 113 representative cells, respectively). (C) Timing of the first AP and probability of APs during the first 150 ms of the square pulse stimulus shows steep MP-dependence in TOR cells, whereas the initial spikes are stable in the RS cells. The amplitude of stimulating current steps was standardized for each cell and only the preceding holding current (3 s) was varied in individual trials. Traces show a representative recording from a TOR cell. The average data derived from 85 TOR and 81 RS cells. Figure 1—source data 1 Electrophysiological parameters of TOR and RS CCK+INs. Note that only those cells were included in this table from which all parameters could be retrieved. Thus, the number of analyzed cells may differ from specific analysis of the main text. https://cdn.elifesciences.org/articles/58515/elife-58515-fig1-data1-v2.docx Download elife-58515-fig1-data1-v2.docx Next, we applied a protocol allowing the detailed quantification of the MP-dependence of firing in individual cells (n = 81 RS and 85 TOR cells). Specifically, firing was evoked by a current step that was calibrated for each cell to elicit a similar mean firing frequency (10–20 Hz) from slightly depolarized MP. The holding current preceding the current step was systematically varied to reach a wide range of steady-state MPs (3 s, in the range of −50 and −90 mV, above the theoretical reversal potential of potassium current, Figure 1C). In TOR cells, the number of APs within the first 150 ms and the timing of the first AP showed steep voltage dependence (V1/2 value of the Boltzmann fits were −67.4 mV and −73 mV, respectively, R2 = 0.995 and 0.999, Figure 1C). In contrast, the delay and number of spikes did not show membrane potential-dependence in RS cells. Importantly, both RS and TOR firing was stable in prolonged patch-clamp recordings, which lasted for 45–64 min (average 58 min, n = 4 TOR and 3 RS cells). In contrast to the difference in initial spiking, other firing and membrane properties of TOR and RS cells were similar, including input resistance, AP threshold, half-width, rate of rise and after-hyperpolarization (Figure 1—source data 1). Furthermore, the frequency and amplitude of spontaneous synaptic events (including both IPSCs and EPSCs; 2.47 ± 0.57 Hz and 3.26 ± 0.86 Hz, Student t-test, p=0.45, t(15) = −0.78; −51.6 ± 3.3 pA and −51.5 ± 2.5 pA, Student t-test, p=0.98, t(15) = −0.02, n = 9 TOR and 8 RS cells) were similar. We found TOR cells in the CA1 region as well. However, here their prevalence was lower compared to CA3 (2 TOR out of 13 CA1 CCK+IN). Finally, both TOR (n = 15 cells) and RS (n = 12 cells) CCK+INs were detected in the CA3 region of adult rats (older than 70 days) suggesting that these biophysical features are not age-dependent. In summary, the firing of TOR cells shows a remarkable sensitivity to a physiologically plausible 20 mV shift in the MP, despite having no other distinctive passive electrical or spiking properties compared to RS cells. TOR and RS firing types do not correlate with previously known subtypes of CCK+ cells The CCK+IN class has been previously divided into several subtypes based on various functionally relevant features. Therefore, next we investigated whether the two MP-dependent firing phenotype could be linked to previously identified subtypes of CCK+INs. Three subtypes have been previously identified within CA3 CCK+INs based on the target zones of their axons: (i) basket cells (BCs) that innervate the soma and proximal dendrites of pyramidal cells (Hendry and Jones, 1985), (ii) mossy fiber-associated cells (MFA) with axons branching in stratum lucidum and hilus regions that are also occupied by the axons of dentate gyrus granule cells (Vida and Frotscher, 2000), and (iii) Schaffer-collateral associated cells (SCA), targeting the same regions as Schaffer collaterals (Cope et al., 2002). Altogether, 172 of the recorded CCK+INs were unequivocally identified either as BCs (n = 31), MFAs (n = 96) or SCAs (n = 45; Figure 2A). Both TOR and RS firing types occurred similarly among these morphological subtypes (Figure 2B) indicating that neither of the two firing phenotypes could be assigned to any of the morphology-based CCK+IN subtypes. This assessment was further supported by quantitative analyses of axonal and dendritic features (Figure 2—figure supplements 1 and 2). Figure 2 with 3 supplements see all Download asset Open asset Different excitability does not correlate with previously known diversity of CCK+ cells. (A) Axonal (black) and dendritic (blue) reconstructions of four CCK+INs in the CA3 region representing two major morphological types, the mossy-fiber associated cells and basket cells. The firing of cell#1 and #2 are shown in detail in Figure 1A. The firing from hyperpolarized MP is shown for each cell. Immuno-labelling for CCK and the nuclear protein Satb1 are shown next to each recorded cell. (B) Prevalence of TOR and RS cells within three major morphological types of CA3 CCK+. Numbers of identified cells are indicated on each bar (BC: CCK+ basket cells, MFA: mossy-fiber associated cells, SCA: Schaffer-collateral associated cells). (C) Single cell RNAseq characterization of recorded TOR (left, n = 8) and RS (right, n = 8) cells for known GABAergic cell markers. Each column corresponds to single identified CCK+INs. The bars on the right shows the logarithm of p values of the statistical comparison of TOR and RS cells for each genes. Significantly different mRNA content between TOR and RS cells was found for Satb1 and Lypd1 (p=0.0095 and p=0.0006, Mann-Whitney Test). (D) Comparison of immunohistochemical and single cell RNAseq data. Bar plots show the fraction of the recorded cells with detectable RNA content (threshold: 0.2) for the selected markers and immunopositivity for the same proteins (for examples of the immunolabelling see Figure 2—figure supplement 3). The number of tested cells are shown on each bar. In addition to the variable morphological features, CCK+INs heterogeneously express several molecular markers (Somogyi et al., 2004). To study their molecular heterogeneity, we performed single cell RNA sequencing (SC-RNAseq) (Földy et al., 2016) of individually recorded TOR cells (n = 8) and RS cells (n = 8). Only those cells were analyzed in detail, which had detectable mRNA for both CCK and cannabinoid-receptor type 1 (CB1R or Cnr1; Figure 2C). In accordance with previous observations (Földy et al., 2016), the included cells contained the transcripts of at least 3000 different genes (the range of detected genes in the included cells was between 4173 and 9629 with mean 6946 ± 430 genes). Most CCK+IN-relevant marker genes (calbindin/Calb1, vesicular glutamate transporter VGlut3/Slc17a8, vasoactive intestinal protein/VIP, serotonin receptor subtype 3a/5HT3a, Reelin, neuropeptide-Y/Npy) were expressed at comparable levels in both TOR and RS cells (Figure 2C). One of the exception was the activity-dependent transcription regulator, Satb1 (Close et al., 2012), whose mRNA was present in TOR cells (n = 8 out of 8), whereas only 4 of the eight tested RS cells had detectable levels (Mann-Whitney test, p=0.0095, z = 2.59, U = 57). Furthermore, we found that Lypd1, which is enriched in a subpopulation of CCK+INs in the CA1 (Harris et al., 2018) was expressed in all tested TOR cells (n = 8) and in only one of the RS cells (1 out of 8, Mann-Whitney test, p=0.0006, z = 3.45, U = 64). Despite these differences, hierarchical cluster analysis of the total transcriptome did not categorize these cells into two distinct groups that would correspond to their firing phenotype (Figure 2—figure supplement 2), suggesting that these cells have highly similar transcriptomic identities (Fuzik et al., 2016; Harris et al., 2018). Our complete SC-RNAseq dataset is available at NCBI GEO as GSE133951. To confirm these findings at the level of protein expression, we performed immunohistochemistry on identified CCK+ TOR and RS cells in separate experiments (Figure 2D and Figure 2—figure supplement 3). As expected, CB1R protein was detected in the axon of almost all tested cells (15 out of 18 cells). Furthermore, VGluT3 (23 out of 46 TOR cells, 16 out of 30 RS cells), Reelin (all 11 tested TOR and 7 out 10 RS) and Calb1 (10 out 30 TOR and 7 out of 29 RS) proteins occurred similarly in RS and TOR types. By contrast, while the majority of TOR cells were positive for Satb1 protein (109 out of 132, 82.6%), only few RS cells were Satb1 positive (14 out of 128 tested, 10.9%). Satb1 protein was also prevalent in non-recorded CCK+ cells within the CA3 region of the acute slices (126 Satb1+/CCK+ out of 425 CCK+), suggesting that the detection of this activity-dependent protein was not a consequence of patch-clamp recording. The somata of Satb1+/CCK+ cells were found throughout CA3 strata oriens, pyramidale, lucidum and radiatum and their proportion to all CCK+ cells were similar (34.1%, 21.4%, 33.6%, and 27.2%, respectively). In agreement with the lower occurrence of TOR cells in the CA1, non-recorded CCK+ cells in this region were less likely to be Satb1 positive of CCK+ cells). In the region of the dentate gyrus the proportion of Satb1+/CCK+ cells was also of CCK+ cells). We have also detected Satb1 and CCK in derived from Satb1+/CCK+ out of CCK+ cells in the CA3 To transcriptomic and immunohistochemical data that Satb1 is of TOR identity in hippocampal CCK+INs and that these TOR and RS firing phenotypes are not related to previously known subtypes within CCK+INs. in low-voltage-activated potassium currents underlie the of CCK+IN firing Next, we investigated that are for the two firing types in CCK+INs. We focused on potassium currents because they have been shown to firing similar in various brain regions et al., et al., et al., 2002; Stern and Armstrong, 1996). we characterized each cell as TOR or RS type recording conditions. Next, we recorded potassium currents between and mV, in the presence of and (3 and 10 hoc immunolabelling that all included cells were CCK+. TOR cells had a amount of potassium currents at firing (Figure ± pA at mV, n = By contrast, potassium currents in RS cells at more positive and were much at ± pA, n = p=0.0006, The low-voltage-activated potassium currents which are 30 to mV AP threshold, can underlie the inhibition of AP generation in TOR cells, whereas in RS cells is much at voltage Figure 3 with 2 supplements see all Download asset Open asset in the currents underlie the of CCK+IN (A) from two single cells and average voltage dependence of of in TOR (n = and RS (n = cells. The AP ± mV) the large amount of current in TOR cells at (B) and voltage dependence of of at mV from different holding potentials (n = and 10 for TOR and RS cells, respectively). and the voltage ranges from which state-dependent firing was tested Figure 1). (C) and average voltage dependence of in from TOR and RS cells (n = 7 and 8, respectively). The are shown both as the average of and data to the differences both in the amount and voltage dependence of the in the right currents in the presence of (1 (D) CCK+INs firing patterns recorded during and after response to identical current and the average AP in TOR (n = and RS cells (n = from hyperpolarized MP ranges to Average delay of the first AP and of individual in representative TOR and RS cells and the average voltage dependence (n = 7 for both TOR and RS cells). Next, we investigated the availability of at different these potassium currents were evoked by a voltage step to mV various potentials between to The of the average was ± mV R2 = 0.999, mean of from individual ± mV, n = 13 cells, Figure in TOR cells. Importantly, the majority of was available at slightly hyperpolarized MPs ± at mV), where the inhibition of firing was during the characterization of TOR cells Figure 1 at −60 mV, where the inhibition of spiking was not the majority of currents in TOR cells were only ± of the total current to the the of in RS cells was positive voltage ranges ± mV, n = 8 cells, mean of individual ± 2 mV, comparison with TOR cells, see p=0.0006, and a portion of this current was available at −60 mV ± Figure Thus, of the RS cells cannot a amount of In the time of was in RS cells compared to TOR cells ± ms, n = vs. ± ms, n = at mV, The from was in TOR compared to RS cells, and it showed steep voltage dependence time of the were ± ms, ± ms, ± 2.5 ms and ± ms at −85 and mV, whereas in RS ± ms, ± ms, ± ms and 3.3 ± 0.4 ms, respectively). Due to these currents in RS cells AP either from or −60 mV preceding In summary, the different properties of particularly the and may underlie the differences in TOR and RS firing In recordings the majority of the membrane which voltage at more depolarized MPs and the the voltage dependence of Therefore, we compared the in TOR and RS cells in The better voltage control to currents at more depolarized voltage ranges and the total of in the two types of CCK+INs. The of in TOR cells was compared to RS cells (Figure ± ± n = 7 and 8). However, with findings using at a more hyperpolarized voltage range in TOR cell (V1/2 ± mV, ± mV, with and 15 ± and ± average at mV, respectively, Mann-Whitney test, z = U = n = 7 and 8) and the current had time in TOR ± ms, n = compared to RS ± ms, n = 8, = cells. Thus, in of the in TOR cells than in RS cells during ms voltage steps to mV ± and ± = n = 7 and 8). and do not on the of we investigated currents by from the soma and dendrites of RS and TOR cells (Figure supplement 1). These recordings that the of was in RS cells ± n = compared to that of the TOR ± n = = cells. The of dendritic was similar in TOR and RS cells ± 3.3 n = vs. ± n = Furthermore, we did not a the dendrites to from R2 = n = R2 = n = On average, the of currents those of currents and in TOR cells. However, was a pronounced of between individual from but not cells (Figure supplement when were from the same cell. In many TOR cell the current the and currents of the RS cells. While in other TOR cell much currents were also This be due to the occurrence of potassium channels in GABAergic cells see et al., Kv4 channels are for both types of currents in CCK+INs Next, we investigated the identity of potassium subunits for the differences of in RS and TOR cells and for the MP-dependent firing in TOR cells. and 10 a of and the initial firing in TOR cells (Figure supplement 2A). Only a of was to the TOR with the above voltage the of Kv4 channels et al., of which Kv4.3 has been shown to be present in hippocampal CCK+INs et al., et al., To specifically the of Kv4 channels to the TOR we applied 1 which selectively and Kv4.3 channels by the voltage dependence of the et al., 2011; et al., In the presence of more APs were evoked in TOR cells during the first ms of the stimulus compared to control in the same cells (Figure from mV preceding MP, ± ± n = = Furthermore, the delay of the first APs was from ± ms to ± ms (Figure = t-test, n = 8 TOR cells). However, did not change the number and temporal of APs in TOR cells from −60 mV not and the firing of RS cells also both at and −60 mV (n = 8 cells). Furthermore, had only minor on the of the APs ± ms, ± ms, n = 8, = RS ± ms, ± ms, n = 8, = Finally, the was on the TOR which additional evidence for the stability of the TOR in individual cells during recording In summary, currents are crucial for the TOR Next, we analyzed the of currents to in TOR and RS cells. In both types had of currents. In TOR cells, the currents at more voltage (Figure and the time of was than in RS cells ± ms vs. ± ms, at The voltage dependence of of the current was also in TOR relative to RS cells (V1/2 ± mV, n = ± mV, n = In with the known of et al., 2011; et al., the voltage of was to more depolarized membrane potentials in both TOR and RS cells in ± 3.3 mV, n = ± mV, n = Figure which in a similar in the at mV ± n = ± n = To results that the majority of potassium currents are by Kv4 channels in both TOR and RS cells, in of the different properties of the currents. of TOR and RS firing In the previous we excitability properties in CCK+INs using steady-state current However, excitation of neurons in vivo is