Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites

Abstract

Increases in cytosolic Ca2+ concentration ([Ca2+]i) mediated by NMDA-sensitive glutamate receptors (NMDARs) are important for synaptic plasticity. We studied a wide variety of dendritic spines on rat CA1 pyramidal neurons in acute hippocampal slices. Two-photon uncaging and Ca2+ imaging revealed that NMDAR-mediated currents increased with spine-head volume and that even the smallest spines contained a significant number of NMDARs. The fate of Ca2+ that entered spine heads through NMDARs was governed by the shape (length and radius) of the spine neck. Larger spines had necks that permitted greater efflux of Ca2+ into the dendritic shaft, whereas smaller spines manifested a larger increase in [Ca2+]i within the spine compartment as a result of a smaller Ca2+ flux through the neck. Spine-neck geometry is thus an important determinant of spine Ca2+ signaling, allowing small spines to be the preferential sites for isolated induction of long-term potentiation.

Figure 1. NMDAR-Mediated Ca²⁺ Signaling in Single Spines of CA1 Pyramidal Neurons in Response to Two-Photon Uncaging of MNI-Glutamate

(A) A three-dimensionally stacked two-photon fluorescence image of a region of a dendrite labeled with Alexa Fluor 594. Arrowheads indicate the line of laser scanning at 830 nm for fluorescence imaging, and the red dot represents the point of uncaging of MNI-glutamate. (B) Line-scan images of the spine shown in (A) for Alexa Fluor 594 (top), OGB-5N (middle), and Δ[Ca²⁺]_i obtained from the ratio of OGB-5N to Alexa Fluor 594 (bottom). The time of uncaging of MNI-glutamate is indicated by the arrowhead. The magnitude of Δ[Ca²⁺]_i is pseudocolor coded as indicated. (C) Δ[Ca²⁺]_i in the spine head (“H”) and at the base of the spine (“D”) shown in (A) as well as NMDAR-mediated current (I_NMDA) recorded in the whole-cell patch-clamp mode. Regions of averaging of fluorescence to estimate Δ[Ca²⁺]_i are indicated as “H” and “D” in (B). (D) Elimination by APV (100 μM) of the [Ca²⁺]_i and I_NMDA responses to uncaging of MNI-glutamate in the neuron shown in (A). (E and F) Stability of the maximal increase in the fluorescence ratio (ΔR) between OGB-5N and Alexa Fluor 594(E) and of the maximal I_NMDA (F) in a spine head subjected to uncaging of MNI-glutamate for >20 times. Horizontal lines indicate averages of three consecutive amplitudes. (G) Dependence of Δ[Ca²⁺]_i and NMDAR-mediated current on the laser power for uncaging in a spine head with a volume of 0.12 μm³.

Figure 2. Spatial Spread of NMDAR Activation and of Δ[Ca²⁺]_i in CA1 Pyramidal Neurons

(A) A stacked Alexa Fluor 594 image of a dendritic region. The red scale indicates the line along which uncaging of MNI-glutamate was sequentially induced, and the white line represents the scanning line for acquisition of fluorescence images. (B) NMDAR-mediated current as well as Δ[Ca²⁺]_i in the left (green circles) and right (orange circles) spines elicited by uncaging of MNI-glutamate at points along the red line in (A). (C) Stacked Alexa Fluor 594 image of a spine for which line scanning was applied at the line indicated by the arrowheads and uncaging of MNI-glutamate was induced at the red spot. (D and E) Line-scan images for Δ[Ca²⁺]_i (D) and averaged traces of Δ[Ca²⁺]_i (E) obtained from the spine shown in (C) during uncaging of MNI-glutamate at the red spot. Regions of averaging of fluorescence for Δ[Ca²⁺]_i are indicated as “H” and “D” in (D). (F–H) Panels corresponding to (C)–(E), respectively, for uncaging of MNI-glutamate at the position on the dendritic shaft indicated by the red dot in (F).

Figure 3. Dependence of NMDAR-Mediated Current and Ca²⁺ Signaling on Spine-Head Volume

(A) Stacked Alexa Fluor 594 image of a dendritic region in which uncaging of MNI-glutamate was induced at the numbered spines and line-scan imaging was performed along the axis of each spine. Original xy images are shown in Figure S5. (B–D) V_H dependence of NMDAR-mediated current (I_NMDA) (B) as well as of Δ[Ca²⁺]_i in the spine head (c_H) (C) and in the dendritic shaft at the base of the spine (c_D) (D). Straight lines in (B) are linear regression lines. Uncaging was induced at the spines (red circles) indicated in (A) and at spines in three other dendrites (yellow, blue, and white circles). The amplitude of currents was measured at their peak, and Δ[Ca²⁺]_i was measured at the time of the maximal value in spine heads. (E) V_H dependence of the coupling ratio (c_R = c_D/c_H) for each spine. (F and G) V_H dependence of Ca²⁺ conductance of the spine neck (g_N) and its double logarithmic plot, respectively. Smooth curves in (C)–(G) were drawn according to (24), (25), (26), (27) and (28) with four different sets of dendritic parameters as described in “Spine Ca²⁺ Model.”

Figure 4. Spread of Ca²⁺ along the Axis of a Dendritic Shaft

(A) Stacked Alexa Fluor 594 fluorescence image of a dendritic region subjected to line scanning along both the axis of a spine (S-scan) and that of the parent dendritic shaft (D-scan). Uncaging of MNI-glutamate was performed at the position indicated by the red dot. (B) Averaged Δ[Ca²⁺]_i in the spine head (H) and at the base of the spine (D). (C) Spatial gradient of Δ[Ca²⁺]_i at three different time periods (boxes 1, 2, 3) depicted in (B). Smooth curves are exponential functions with length constants of 1.6 μm, 1.9 μm, and 2.4 μm for the periods 1, 2, and 3, respectively. (D) Time course of Δ[Ca²⁺]_i in a dendritic shaft whose soma was depolarized to −10 mV for 40 ms. The decay of Δ[Ca²⁺]_i was fitted with an exponential curve with a time constant (τ_D) of 219 ms.

Figure 5. Spine-Neck Geometries

(A) Stacked Alexa Fluor 594 fluorescence images of dendritic spines with different c_R values. Necks appear to be shorter and thicker for spines with a larger c_R. Two values of spine-neck Ca²⁺ conductance, g_N and g_N*, were estimated from c_R and from fluorescence analysis as described below, respectively. (B) Stacked Alexa Fluor 594 fluorescence image of a spine subjected to analysis of neck geometry. (C) Fluorescence profile along the dashed line for the spine shown in (B). Four regions were selected for fluorescence measurement. The neck region (1) is flanked by the edges of the spine head and dendritic shaft, which are determined by curve fitting (see Experimental Procedures). For those stubby spines whose neck structures were not well resolved, we assumed l_N as 0.05 μm and obtained r_N at the base of the spine. Regions of background fluorescence for the spine head (2) and the dendritic shaft (3) are placed at the predicted edge of a spherical spine and cylindrical shaft. A region of nonspecific background fluorescence (4) was positioned between 2 and 5 μm from the shaft. (D) Relation between g_N* and g_N. Data obtained from the same dendrites are represented by the same colors as in Figure 3. We estimated g_N* from neck fluorescence according to Equation 4 and assuming D_app = 12 μm² s⁻¹ (see Experimental Procedures). (E) Double logarithmic plot of the V_H dependence of g_N*. Straight lines in (D) and (E) are linear regression lines. Triangles in (D) and (E) indicate those stubby spines whose values of g_N* were obtained by assuming l_N = 0.05 μm.

Figure 6. Determinants of Spine Ca²⁺ Signaling

(A and B) V_H dependence of κ_T and g_H, respectively, among spines. (C) V_H dependence of the g_N/g_H ratio. (D) Predicted values of NMDAR-mediated current (I_NMDA*) from Equation 8 plotted against actual I_NMDA for each spine. Values of I_NMDA were obtained at the peak of spine [Ca²⁺]_i as shown in Figure 8E. (E and F) I_NMDA dependence of c_H and c_D, respectively, among spines. Straight lines in (A)–(D) and (F) are linear regression lines. (G and H) g_N dependence of c_H and c_D, respectively, among spines. Data obtained from the same dendrites are represented by the same colors as those in Figure 3. The smooth lines in (G) and (H) were obtained from (21) and (22), respectively, assuming g_H = 1 μm³ s⁻¹, g_D = 7 μm³ s⁻¹, I_NMDA = 5 pA, κT = 102, and g_N = 250 V_H².

Figure 7. Functional Expression of AMPARs and NMDARs in the Same Spines

(A and B) Fluorescence images (left) of spines and their maps of AMPAR-mediated current (right). Current amplitude is pseudocolor coded as indicated. (C and D) Maximal AMPAR-mediated currents in the spines shown in (A) and (B), respectively. (E and F) NMDAR-mediated currents evoked by uncaging of MNI-glutamate at the tips of the spines show in (A) and (B), respectively. (G) V_H dependence of the maximal AMPAR-mediated currents for 31 spines on four dendrites represented by different colors. (H) Relation between the maximal amplitudes of NMDAR-mediated and AMPAR-mediated currents recorded from the same spines. Data in (G) and (H) were obtained from preparations different from those shown in Figure 3.

Figure 8. Spine Ca²⁺ Signals

(A) NMDAR-mediated influx of Ca²⁺ (dots) in small and large spines. The narrow neck of small spines results in larger and more confined increases in [Ca²⁺]_i in the spine head, allowing induction of LTP at the level of the single spine. The thick neck of large spines gives rise to smaller increases in spine [Ca²⁺]_i and greater outflow of Ca²⁺ into the dendritic shaft. (B) Diagram showing the relative effects of spine-head volume (V_H) on spine-neck Ca²⁺ conductance (g_N), spine-head Ca²⁺ conductance (g_H), and I_NMDA, as well as of the latter parameters on spine-head [Ca²⁺]_i (c_H) and [Ca²⁺]_i in the dendritic shaft adjacent to the spine (c_D). (C) Stationary model of Ca²⁺ signaling in a spine, where g_D, j_H, and κ _T represent Ca²⁺ conductance of the dendritic shaft, Ca²⁺ influx, and the total Ca²⁺ binding ratio, respectively. (D) Time-dependent model, where τ_H and τ_D represent the time constants of [Ca²⁺]_i in the spine head and dendritic shaft, respectively.Ĵ_H(s), Ĉ_H(s), and Ĉ_D(s) are the Laplace transforms of J_H (t), C_H (t), and C_D (t), respectively, which represent the time courses of Ca²⁺ influx and of [Ca²⁺]_i in the spine head and in the dendritic shaft adjacent to the spine, respectively. (E and F) Simulation of C_H(t) and C_D(t) based on the model in (D) and (29) and (30), where J_H(t) is either EPSC-like (E) or impulsive (F), and g_N, g_D , and g_H are all set to 7.0 μm³ s⁻¹ . As indicated in (E), c_H, c_D, and j_H are obtained at the time when C_H(t) is maximal, and c_IH, c_IH′, c_ID, and j_I are obtained at the time when J_H(t) is maximal. (G) V_H dependence of c_R (equal to c_D/c_H) in the time-dependent model in (D), where c_D and c_H are obtained as indicated in (E). The value of g_N is either fixed (thin lines) or V_H dependent (thick line). The values of g_D, τ_D, τ_H are set at 7 μm³ s⁻¹ , 0.2 s, 0.05 s, respectively, and g_H is altered according to V_H/τ_H.