Spontaneous and evoked synaptic rewiring in the neonatal neocortex

Abstract

The local microcircuitry of the neocortex is structurally a tabula rasa, with the axon of each pyramidal neuron having numerous submicrometer appositions with the dendrites of all neighboring pyramidal neurons, but is functionally highly selective, with synapses formed onto only a small proportion of these targets. This design leaves a vast potential for the microcircuit to rewire without extensive axonal or dendritic growth. To examine whether rewiring does take place, we used multineuron patch-clamp recordings on 12- to 14-day-old rat neocortical slices and studied long-term changes in synaptic connectivity within clusters of neurons. We found pyramidal neurons spontaneously connecting and disconnecting from each other and that exciting the slice with glutamate greatly increases the number of new connections established. Evoked emergence of new synaptic connections requires action potential activity and activation of metabotropic glutamate receptor 5, but not NMDA receptor or group II or group III metabotropic glutamate receptor activation. We also found that it is the weaker connections that are selectively eliminated. These results provide direct evidence for spontaneous and evoked rewiring of the neocortical microcircuitry involving entire functional multisynaptic connections. We speculate that this form of microcircuit plasticity enables an evolution of the microcircuit connectivity by natural selection as a function of experience.

Fig. 1.

Visualization and stimulation of TPC clusters. (A) IR differential interference contrast image of a seven-cell cluster patched. (Right) A neuron at first patch (“Before”) and repatched after 12 h (“After”). (B) Biocytin-stained cluster shows that only seven cells were patched despite the two separated sessions. (C) Whole-cell recordings of three of the six cells recorded while glutamate was puffed from the seventh patch pipette located ≈100 μm above the cluster (bottom trace). (D) Stability of the response to glutamate puffing over time. (E) Response of a cell to bath application of glutamate. “Control” shows the same time and voltage scale trace of a cell in normal ACSF.

Fig. 2.

Spontaneous and evoked emergences and disappearances of connections within 12 h. (A) A six-cell cluster showing the EPSPs elicited in response to a train of eight action potentials at 30 Hz and a recovery action potential 500 ms later (average of 30 trials). Each line and column represents a cell. Gray and red traces were recorded at first patch (“before”), and black and blue traces were recorded 12 h later (“after”) under Evoked 1 condition. The red trace indicates a disappearance, and blue traces indicate emergences. (B) Ei (no. of pairs newly connected “after”)/(no. of unconnected pairs “before”) for spontaneous (n = 182), Evoked 1 (Ev1) (n = 135), and Evoked 2 (Ev2) (n = 154) conditions (∗∗, P < 0.001). (C) Di (no. of pairs connected “before” that were not connected anymore “after”)/(no. of connected pairs “before”) for spontaneous (n = 24), Ev1 (n = 29), and Ev2 (n = 36) conditions. (D) Cumulative distributions of absolute synaptic efficacy (A) for recordings at first patch (gray) and emergences (blue) (n = 322 and 122, respectively; ∗∗∗, P < 0.001). (Insets) Bar graphs of A and Pr for the same data. (E) Same as in D but for disappearances (red) (n = 322 and 24, respectively; ∗∗∗, P < 0.001).

Fig. 3.

Emergences depend on mGluR5 and cell action potential activity. (A) Six cells cluster EPSPs during continuous MPEP perfusion. (B) Bar graphs of the antagonist effects on Ei (∗, P < 0.05). TTX, n = 209; CNQX, n = 166; AP5, n = 159; MPEP, n = 162; AP3, n = 42; EGLU, n = 88; CPPG, n = 126. (C) Bar graph of the antagonist effects on Di. TTX, n = 19; CNQX, n = 28; AP5, n = 28; MPEP, n = 14; AP3, n = 10; EGLU, n = 14; CPPG, n = 18.

Fig. 4.

Spontaneous and induced changes in existing synapses. (A) Same EPSP response as in Fig. 2A. (Left) Comparison of the raw traces at first recording (red) and 12 h later (blue). (Right) The same traces but with the first EPSP of the train scaled. The change is only in strength and not in dynamics. (Calibration: 10 ms and 0.5 mV.) (B) Bar graphs of the paired-pulse ratio (ppr) (Left) and A (Right) showing that the dynamics is constant and the strength varies over 12 h (n = 34 and 40 respectively; ∗, P < 0.05). (C) Variation of A in 12 h. Spontaneous, n = 41; Evoked 1 (Ev1), n = 63; Evoked 2 (Ev2), n = 47. (D) Antagonist effects on A variation when glutamate is puffed. TTX, n = 19; CNQX, n = 39; AP5, n = 44; MPEP, n = 19; AP3, n = 14; EGLU, n = 21; CPPG, n = 37.