Coordination of neuronal activity in developing visual cortex by gap junction-mediated biochemical communication

Abstract

During brain development, endogenously generated coordinated neuronal activity regulates the precision of developing synaptic circuits (Shatz and Stryker, 1988; Weliky and Katz, 1997). In the neonatal neocortex, a form of endogenous coordinated activity is present as locally restricted intercellular calcium waves that are mediated by gap junctions (Yuste et al., 1992). As in other neuronal and non-neuronal systems, these coordinated calcium fluctuations may form the basis of functional cell assemblies (for review, seeWarner, 1992; Peinado et al., 1993b). In the present study, we investigated the cellular mechanisms that mediate the activation of neuronal domains and the propagation of intercellular calcium waves in slices from neonatal rat neocortex. The occurrence of neuronal domains did not depend on intercellular propagation of regenerative electrical signals because domains persisted after blockade of sodium and calcium-dependent action potentials. Neuronal domains were elicited by intracellular infusion of inositol trisphosphate (IP3) but not of calcium, indicating the involvement of IP3-related second-messenger systems. Pharmacological stimulation of metabotropic glutamate receptors, which are linked to the production of IP3, elicited similarly coordinated calcium increases, whereas pharmacological blockade of metabotropic glutamate receptors dramatically reduced the number of neuronal domains. Therefore, the propagating cellular signal that causes the occurrence of neuronal domains seems to be inositol trisphosphate but not calcium. Because coordination of neuronal calcium changes by gap junctions is independent of electrical signals, the function of gap junctions between neocortical neurons is probably to synchronize biochemical rather than electrical activity.

Fig. 1.

Neuronal domains in fura-2-stained slices in neonatal rat visual cortex. Slices were illuminated with 385 nm light at which fura-2 emission decreases with increasing calcium concentration. Video images were taken before (left) and during (middle) the occurrence of neuronal domains. On the right, the changes in the fura-2 fluorescence are expressed in pseudocolor as ΔF/F (in percent) and overlaid onto the images shown in themiddle. Each individual image is the average of 16 background-subtracted single frames taken at video rate. The pial surface is indicated by the arrowheads; the white matter is identified by the dashed line. A, Neuronal domains elicited by temperature drop under control conditions (2 μm TTX). Coronal slice of a P3 rat. B, Neuronal domains elicited by temperature drop in the presence of 2 mm Ni²⁺ and 2 μm TTX. These neuronal domains are similar in size and shape to those observed under control conditions, indicating that neither sodium-dependent action potentials nor extracellular Ca²⁺ entry is required for the occurrence of neuronal domains. Coronal slice of a P2 rat.

Fig. 2.

Effects of nickel, thapsigargin, and t-ACPD on the number and size of neuronal domains. A, Average number of neuronal domains elicited by temperature drop under control conditions and in the presence of 2 mm nickel chloride (Ni) or 10 μm thapsigargin (thap). Blockade of voltage-gated calcium channels with 2 mm nickel had no effect on the number of domains. In contrast, depletion of intracellular calcium stores with 10 μm thapsigargin almost completely abolished neuronal domains (p < 0.01, Student’st test). B, Average size of neuronal domains elicited by temperature drop under control conditions and in the presence of 2 mm nickel (Ni) and elicited by bath application of t-ACPD (40–100 μm). Neuronal domains elicited by t-ACPD were smaller (p < 0.01) than were domains elicited by temperature drop. In all cases, the bath solution contained 2 μm TTX. Numbers abovebars indicate the number of slices (A) or number of neuronal domains (B). Asterisks indicate a significant difference (p ≤ 0.05; student’st test).

Fig. 3.

A, Activation of the metabotropic glutamate agonist t-ACPD elicits neuronal domains. Theleft and middle images were taken before and during the application of 40 μm t-ACPD. In theright image, changes in fura-2 emission are pseudocolor coded as ΔF/F (in percent) and overlaid onto the middle image. Each individual image is the average of 16 background-subtracted frames. Numerous neuronal domains are visible, as are individual cells, the [Ca²⁺]_i of which increased by activation of metabotropic glutamate receptors. B, t-ACPD-elicited neuronal domains depend on functional gap junctions. In the presence of the gap junction blocker octanol (1 mm), application of 200 μm t-ACPD elicits only single-cell responses but no neuronal domains. The pial surface is to theupper right in A and up inB. A, Coronal slice of a P3 rat.B, Coronal slice of a P4 rat.

Fig. 4.

Metabotropic glutamate receptors are required for initiation of neuronal domains. A, Bath application of 1 mm (+)-MCPG dramatically reduced the number of neuronal domains (p < 0.01, Student’st test). B, Bath application of 1 mm (+)-MCPG does not affect the size of the neuronal domains that persist, indicating that metabotropic glutamate receptors are only involved in the initiation of neuronal domains.Numbers above bars indicate the number of slices (A) or number of neuronal domains (B). An asterisk indicates a significant difference (p ≤ 0.01; student’st-test).

Fig. 5.

Neuronal domains are elicited by increasing [IP₃]_i but not [Ca²⁺]_i. A, Neuronal domains elicited by intracellular infusion of inositol 1,4,5-trisphosphate are shown. Changes in the fura-2 fluorescence signal are pseudocolor coded as ΔF/F(in percent) and superimposed on background-subtracted videoframes. The arrow points to the filled cell in the first frame that was taken 2 sec (−2) before rupture of the cell membrane (frame 0). Increasing [IP₃]_i triggered a concentric calcium wave with a diameter of ∼80 μm. Tangential slice of a P1 rat. B, Increasing [Ca²⁺]_i in a single cell (arrow) by depolarizing voltage steps failed to elicit intercellular calcium waves. The cell was depolarized from a holding potential of −70 mV to +10 mV by a 40 sec train consisting of 50-msec-long depolarizations delivered at 10 Hz. The neuron was also filled with 100 μm fluo-3 to visualize its basic structure (right). Coronal slice of a P3 rat.

Fig. 6.

Model for the initiation and propagation of interneuronal calcium waves underlying neuronal domains. Waves are initiated either by ambient or synaptically released glutamate (Glu; open triangles) that acts on metabotropic glutamate receptors (mGluR). This stimulates the G-protein (G)-phospholipase C (PLC) cascade that results in the production of inositol trisphosphate (IP₃; filled circles). IP₃ activatesIP₃ receptors (IP₃R; small filled ovals) and thereby causes calcium release (filled squares) from intracellular stores. In addition,IP₃ also diffuses through gap junctions (GJ) into neighboring neurons where it causes calcium release. In coupled cells, IP₃ could be regenerated by calcium-mediated positive feedback loops (dashed arrows), including the sensitization ofIP₃R and stimulation ofPLC.