Properties of GABAergic Neurons Containing Calcium-Permeable Kainate and AMPA-Receptors

Abstract

Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs), as well as NMDARs, play a pivotal role in plasticity and in regulating neurotransmitter release. Here we visualized in the mature hippocampal neuroglial cultures the neurons expressing CP-AMPARs and CP-KARs. These neurons were visualized by a characteristic fast sustained [Ca²⁺]_i increase in response to the agonist of these receptors, domoic acid (DoA), and a selective agonist of GluK1-containing KARs, ATPA. Neurons from both subpopulations are GABAergic. The subpopulation of neurons expressing CP-AMPARs includes a larger percentage of calbindin-positive neurons (39.4 ± 6.0%) than the subpopulation of neurons expressing CP-KARs (14.2 ± 7.5% of CB⁺ neurons). In addition, we have shown for the first time that NH₄Cl-induced depolarization faster induces an [Ca²⁺]_i elevation in GABAergic neurons expressing CP-KARs and CP-AMPARs than in most glutamatergic neurons. CP-AMPARs antagonist, NASPM, increased the amplitude of the DoA-induced Ca²⁺ response in GABAergic neurons expressing CP-KARs, indicating that neurons expressing CP-AMPARs innervate GABAergic neurons expressing CP-KARs. We assume that CP-KARs in inhibitory neurons are involved in the mechanism of outstripping GABA release upon hyperexcitation.

Keywords: Ca2+-permeable AMPA receptors; Ca2+-permeable kainate receptors; GABAergic neurons; GluA2 subunit; GluK1 subunit; spontaneous synchronous activity.

Figure 1

Identification and characteristics of neurons expressing GluK1-containing KARs. (A) Selective agonist of GluK1-containing receptors, ATPA (300 nM), induced insensitive to NASPM (30 µM) Ca²⁺ response. DoA (300 nM) evokes a rapid Ca²⁺ response in ATPA-sensitive neurons (black curves). N = 100, n = 5. (B,C) The effects of AMPARs, NMDARs (NBQX, 2 µM; D-AP5, 10 µM) on the ATPA- and DoA-induced [Ca²⁺]_i increase. N = 100, n = 5. (D) Diagram showing the influence of different antagonists on Ca²⁺ response to ATPA application. The ratio of responses to two repeated ATPA applications was used as a control. Kruskal-Wallis test; ns—p > 0.9999. (E) NH₄Cl (8 mM) induces an [Ca²⁺]_i increase without synchronous oscillations in 16 ± 1% of neurons (black and red curves) earlier than in other neurons (blue and purple curves). N = 100, n = 4. (F) ATPA (30 and 50 nM) increases [Ca²⁺]_i in neurons that faster responded to NH₄Cl application with an [Ca²⁺]_i increase (black curves) N = 100, n = 4. (C′) Fura-2 340/387 ratio images and (C″) and immunostaining of cells in this field with antibodies against NSE and GABA. White arrows indicate ATPA-sensitive neurons. The images in panels C′ and C″ correspond to the experiment presented in Panel C. The values in Panels A–C demonstrate the mean percentage of ATPA-responding neurons from a total number of neurons in a view field.

Figure 2

Identification of neurons expressing CP-AMPARs and CP-KARs. (A) [Ca²⁺]_i changes in 125 neurons in response to 300 nM DoA in the presence of bicuculline (10 µM) in control and in the presence of NASPM (50 µM). (B) The percentage of neurons responding and not responding to DoA with a sustained increase of basal [Ca²⁺]_i. Mean ± SD, n = 4. (C) The percentage of NASPM-sensitive and NASPM-insensitive neurons among those which respond to DoA with a sustained [Ca²⁺]_i increase, and the percentage of NASPM-insensitive neurons responding to ATPA. (D,E) Magnified calcium signals (from Panel A) during the first seconds after DoA application in control (D) and in the presence of NASPM (E). On all presented graphs, the red color corresponds to NASPM-sensitive neurons expressing CP-AMPARs, while the black color corresponds to ATPA-sensitive neurons expressing CP-KARs; the blue color corresponds to glutamatergic neurons. N = 125, n = 4.

Figure 3

Vital Ca²⁺ imaging and further immunostaining of neurons expressing CP-KARs and CP-AMPARs. (A) Calcium responses of representative neurons from each of the four subpopulations to DoA application (300 nM) in the presence of bicuculline (10 µM). The black curve—neurons expressing CP-KARs (NASPM-insensitive neurons); the red curve—neurons expressing CP-AMPARs (NASPM-sensitive neurons); the green and blue curves correspond to glutamatergic neurons. The vertical lines in Figure 3A show the moments corresponding to the images shown on the right panel. (B) Fluorescent images of cells stained with Fura-2. The upper image shows cells without any exposures. The middle image corresponds to the maximum of the calcium pulse (bright cells). Dark cells – astrocytes. The bottom image shows neurons (single bright cells) expressing CP-KARs and CP-AMPARs (neurons that respond faster to DoA). The numbers in the pictures show the time since the experiment beginning. (C) Immunostaining of neurons with antibodies against GAD 65/67 and NeuN. The presented images correspond to the area bordered with a red dotted square frame at the bottom of Figure 3B. The red arrow indicates neurons expressing CP-AMPARs; the black arrow indicates neurons expressing CP-KARs. (D) The percentage of GAD65/67-positive and GAD 65/67-negative neurons. (E,F). The percentage of neurons responding and not responding to DoA and ATPA in the groups of GAD 65/67-positive and GAD 65/67-negative neurons. Results are presented as the mean ± SD. N = 200, n = 6.

Figure 4

The presence of the calcium-binding proteins in neurons expressing CP-AMPARs and CP-KARs. (A) Immunocytochemical staining of neurons with antibodies against parvalbumin (PV), calbindin (CB), calretinin (CR), and GAD 65/67. The left column shows images of neurons loaded with Fura-2. The second column shows cells stained with antibodies against CBPs; the third column shows cells stained with antibodies against GAD 65/67; the fourth column merges Columns 2 and 3. The red arrow indicates a neuron expressing CP-AMPARs; the black arrow indicates neurons expressing CP-KARs. (B,C) The percentage of PV⁺, CB⁺, and CR⁺ neurons among GAD 65/67-positive neurons expressing CP-KARs (B) and CP-AMPARs (C). The results are presented as the mean ± SD, n = 4.

Figure 5

(A–D) The effect of NASPM on the activity of neurons from four subpopulations. [Ca²⁺]_i changes in neurons of different subpopulations from Fig. 2A. Calcium response to DoA and [Ca²⁺]_i oscillations in control (left parts) and in the presence of NASPM (right parts) in four subpopulations of neurons: GABAergic neurons are marked as CP-KAR (7 cells) and CP-AMPAR (10 cells); glutamatergic neurons are marked as Glut-1 (31 cells) and Glut-2 (19 cells). (E) The effects of NASPM on the amplitude of DoA-induced Ca²⁺ responses. Green and red markers—the cells responded to DoA; black markers—neurons that do not respond to DoA application. Bars show the 10% error. (F) The percentage of neurons from each subpopulation. (N = 125, n = 4). (G) Diagrams showing the amplitudes of [Ca²⁺]_i oscillations (left) and the amplitude of DoA-induced calcium responses (right) in control and in the presence of NASPM for each neuronal subpopulation. Paired t-test. CP-KAR: ns, p = 0.3677, * p = 0.0170; CP-AMPAR: ** p = 0.0062, * p = 0.0278; Glut-1: ** p = 0.0041, ** p = 0,0029; Glut-2: ns, p = 0.6238, ns, p = 0.2152.