KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner

Abstract

The molecular mechanisms controlling the termination of cortical interneuron migration are unknown. Here, we demonstrate that, prior to synaptogenesis, migrating interneurons change their responsiveness to ambient GABA from a motogenic to a stop signal. We found that, during migration into the cortex, ambient GABA and glutamate initially stimulate the motility of interneurons through both GABA(A) and AMPA/NMDA receptor activation. Once in the cortex, upregulation of the potassium-chloride cotransporter KCC2 is both necessary and sufficient to reduce interneuron motility through its ability to reduce membrane potential upon GABA(A) receptor activation, which decreases the frequency of spontaneous intracellular calcium transients initiated by L-type voltage-sensitive calcium channel (VSCC) activation. Our results suggest a mechanism whereby migrating interneurons determine the relative density of surrounding interneurons and principal cells through their ability to sense the combined extracellular levels of ambient glutamate and GABA once GABA(A) receptor activation becomes hyperpolarizing.

Figure 1. Characterizing the termination of cortical interneuron migration

(A) Illustration of our time-lapse set-up. See Suppl. Methods for details. (B–D) Merged images showing the decrease in motility of Lhx6-EGFP+ interneurons in cortical slices at embryonic day (E)14.5 (B), post-natal day (P)1 and P7 after ex vivo culture for 1–2 days. Frame corresponding to the start of imaging (t=0) is pseudo-colored in red merged with a frame captured six hours later is pseudo-colored in green. Yellow cell bodies indicate interneurons that did not move during these 6 hours. Arrowheads indicate migrating interneurons while arrows point to sedentary interneurons. (E) Movements of interneurons were quantified in the CP: by P7 the majority of interneurons show no detectable translocation. (F–G) Box plots indicate developmental decline in interneuron motility over time. Individual box indicates mid-50^th percentile range of individual neuron measurements taken. Whiskers extend from 10^th to 90^th percentiles. Horizontal bold line indicates the mean. The average moving speed showed an initial decrease between E15 and P1, but showed no significant decline between P1 and P7 (F). See Movies S1–3 for the corresponding time-lapse animations.

Figure 2. GABA decreases motility of interneurons expressing high levels of KCC2 via GABA_A receptor activation

(A) E14.5 wild-type neocortex was dissociated providing a 2-D substrate for the migration of MGE-derived EGFP-expressing interneurons. (B–F) Time-series of migrating interneurons pseudo-colored at equally spaced time frames before extracellular application of 20μM GABA (B, white; 180 minutes after start of imaging i.e. 180 minutes before application of GABA; C, blue 360 minutes after start of imaging corresponding to time of GABA application) and after application of GABA (D, red, 540 minutes after start of imaging i.e. 180 minutes after GABA application; E, green 720 minutes after start of imaging i.e. 360 minutes after GABA application). See Movie S7 for the animated version of this panel. (G–J) Interneurons cultures that were time-lapsed where fixed immediately after the last frame, and immunostained for MAP2, EGFP and KCC2. The same cells that were time-lapsed were then reimaged to assess their level of KCC2 expression. (K) This technique allows the matching of individual interneuron responses to pharmacological treatments with KCC2 expression in individual interneurons. Interneuron responses to GABA application were binned into low and high KCC2 populations (green and red, respectively) which demonstrate a significant shift in pausing frequency for the high KCC2-expressing subpopulation of interneurons but not for the low KCC2-expressing interneurons. (L–M) Box-whisker plots representation of interneuron responses to drug application binned according to KCC2 expression levels. Throughout the paper box plots in red represents putative responses to `hyperpolarizing' GABA<sub>A</sub> receptor activation (high KCC2 expressing interneurons) whereas green box plots indicate response to putative `depolarizing' GABA_A receptor activation. No significant changes in moving speed were detected in either sub-population following 20μM GABA (L) or GABA with concurrent application of GABA_A receptor antagonist bicuculline methiodide (BMI; 10μM; data not shown). (M) Low KCC2 interneurons showed no significant increase in pause time upon GABA application, while high KCC2 interneurons showed a significant (p=0.0004) increase in pausing after GABA application. Co-application of BMI with GABA abolished the effect of GABA alone (p=0.0308), leading to no significant difference between pre and post-drug pausing frequency. Conversely BMI co-application did cause any significant increase in pausing frequency for interneurons expressing low level of KCC2.

Figure 3. Manipulating KCC2 expression is sufficient to reproduce effects observed in binning interneurons high and low KCC2 expressing populations

(A–D) KCC2 was over-expressed in E14.5 wild-type MGE explants by electroporation of EGFP-IRES human KCC2 construct (KCC2*). (B–D) Expression was verified by high immunofluorescence after 4.5div. (E–J) Short hairpin RNAi targeted against mouse KCC2 (shKCC2) is very effective to knockdown endogenous KCC2 expression in interneurons. (E–G) Interneuron electroporated with a construct encoding EGFP at E14.5 was immunostained for KCC2 (red) at 7div. (H–J) Interneuron electroporated with a control EGFP and a construct encoding shKCC2 at E14.5 shows no KCC2 expression at 7div. (K) Quantification shows a significant reduction in KCC2 with use of shKCC2. Measurements of background KCC2 immuno-reactivity show KCC2 is approaching undetectable background levels with introduction of shKCC2. Fluorescence was measured in 12 bits (value range of 0–4095). (L) Similar to interneurons expressing low levels of KCC2, interneurons expressing shKCC2 (KCC2 knockdown) respond to BMI application (10 μM) by pausing significantly more frequently (p<0.0001). KCC2* rescues the knockdown by pausing them more with GABA_A receptor activation (10 μM muscimol; p<0.0001) and less with blocking the receptors (p<0.0001). Box plots are color-coded to represent putative depolarized (green), hyperpolarized (red) or mixed (yellow) interneuron population depending on the treatment. Light-grey shading shows the 25^th–75^th percentile range of appropriate control.

Figure 4. KCC2 is strongly correlated with the termination of interneuron migration

(A) E14.5 EGFP-MGE explants were placed on E14.5 wild-type dissociations and cultured for 7div. These interneurons were then time-lapsed for 6 hours (B–D), fixed and immunostained for KCC2 (F–H). (E) Box plots show 10^th, 25^th, 50^th, 75^th and 90^th percentiles KCC2 fluorescence in moving versus sedentary interneurons. Light-grey shading shows the 25^th–75^th percentile range of KCC2 expression in the motile population of interneurons. Binning interneurons into moving and non-moving populations reveals significantly higher KCC2 expression in the sedentary population (p<0.0001). Note yellow `co-labeling' in time-lapse representation (D) corresponds with yellow co-labeling of KCC2 with interneurons (H). Suppl. Movie S8 provides an example of the raw time-lapse data.

Figure 5. KCC2 expression is necessary and sufficient for the inhibition of cortical interneuron migration

(A) Wild-type embryonic cortical slices (300 microns thick) were injected in the MGE with either control EGFP constructs or EGFP-IRES-KCC2* expressing constructs and subsequently electroporated. Inset shows early EGFP expression restricted to the MGE after 1div. (B–C) While EGFP controls (B) show robust migration of interneurons from the striatum into the dorsal telencephalon, precocious expression of KCC2* in slices reduces migration to the cortex by approximately 2-fold. (D) Significant decrease (p<0.0001) in the percentage of MGE-derived interneurons migrating into the cortex in KCC2-expressing interneurons compared to control. (E–G) MGEs electroporated with either (E) control plasmid, (F) shKCC2 or (G) shKCC2+KCC2* were explanted on wild-type cortical dissociated cultures for 7div and time-lapsed for 6 hours. Pictures show initial frame (t=0) in red and frame captured 3 hours later in green. Yellow labeled cells indicates sedentary interneurons. (H) Box plots show change in pausing frequency of cortical interneuron populations expressing the indicated constructs, before and after the indicated drug treatment Light-grey shading shows middle 50^th percent range of appropriate control population. Quantification shows a significant decrease in the pausing frequency of interneurons expressing shKCC2 (p<0.0001) and a significant rescue with KCC2* (p<0.0001). See also Movies S9–11 for representative examples.

Figure 6. Calcium signals in tangentially migrating interneurons are reduced with KCC2 up-regulation

(A–D) Migrating cortical interneurons show spontaneous calcium transients. (A) An mRFP-electroporated E14.5 MGE interneuron was loaded with Oregon green BAPTA-AM and time-lapsed. Pseudo-colored images show calcium signal at low (B) and high (C) periods of activity in the outlined migrating interneuron. (D) Pseudo-colored strips show a kymograph obtained by an orthogonal re-section through the cell body during the course of the time-lapse. (E) Calcium signals observed in control conditions shows wide range of dynamics. (F–J) KCC2 expression reduces the calcium transients in the 0.003-0.03Hz range. Calcium signals from Oregon Green BAPTA-loaded interneurons electroporated with either mRFP and a plasmid encoding shKCC2 (F), mRFP-IRES-KCC2 (G) or shKCC2+10μM BMI (H) are shown. Neither KCC2 over-expressing nor shKCC2+BMI interneurons show these types of calcium transients. See Movies S12–15 for corresponding time-lapse. (I) A spectral analysis done on individual interneurons and averaged for each group reveals a significant decrease in calcium signaling in the 0.003–0.03Hz frequency range upon either KCC2 over-expression or GABA_A receptor blockage. (J) The relative power spectral densities were binned into 0.003–0.03Hz and >0.03Hz categories. (K–M) Time-lapsed Lhx6-EGFP interneurons at E15 are shown with initial frame (t=0) in blue, 3 hours frame in green, and 6 hours frame in red. Non-moving cells appear white. Note that chelation of intracellular calcium with 25μM BAPTA-AM significantly increases the number of stationary interneurons (L) relative to control (K). (M) Quantification shows a decrease (Chi-Square Analysis p<0.0001) in the proportion of migrating interneurons following intracellular calcium chelation. See Movies S1 and S16 for corresponding time-lapse.

Figure 7. Reduced activation of VSCCs by direct pharmacological antagonism or indirectly by blocking glutamate signaling further increases pause times in interneurons

(A–C) E14.5 interneurons electroporated with shKCC2 and plated on wild-type dissociated cortex increased in pausing with addition of (A) the N-type VSCC antagonist ω-conotoxin GVIA (5μM) at 4.5div (p<0.0001) and an even greater effect with the addition of (B) L-type VSCC antagonist, nifedipine (10μM; p<0.0001). (C) Box plots showing percentage of time pausing for migrating cortical interneurons in conditions shown in A–B. Light-grey shading indicates the 25^th–75^th range of percentiles of control values. See S17 and S18 for corresponding movies. (D–F) KCC2 knockdown interneurons show little effect on pausing when eliminating depolarization due to glutamate signaling (100μM APV and 10μM NBQX) as long as depolarization due to muscimol (10μM) are still present (D,F). Box plots are color coded to indicate whether drug treatment and KCC2 modification would be depolarizing (green) or hyperpolarizing (red) with respect to appropriate control. Note that when KCC2 is up-regulated and muscimol is hyperpolarizing, the effect of eliminating AMPA/NMDA-mediated depolarization is highly significant (E,F). See Suppl. Movies 19 and 20 for corresponding time-lapse.

Figure 8. Model – KCC2 expression facilitates the termination of cortical interneuron migration

(A) Initial migration state. Green `halo' represents motogenic effect of glutamate and GABA. Blue shade of interneurons and pyramidal neurons represents degree of KCC2 expression. Membrane magnification illustrates transporter and channel composition of interneurons throughout the figure. Initially, ambient GABA and glutamate stimulate the migration of interneurons by inducing membrane depolarization and calcium influx through activation of VSCCs. (B) KCC2 up-regulation act as a switch rendering ambient GABA hyperpolarizing, reducing Ca²⁺ influx through VSCCs which significantly reduces interneuron motility. (C) Developmental decrease of ambient glutamate, mostly due to astrocytic re-uptake and confinement to synaptic release, further reduces Ca²⁺ influx and contributes to the termination of interneuron migration by reducing global AMPA/NMDA receptor activation on interneurons.