Mixed electrical-chemical transmission between hippocampal mossy fibers and pyramidal cells

Abstract

Morphological and electrophysiological studies have shown that granule cell axons, the mossy fibers (MFs), establish gap junctions and therefore electrical communication among them. That granule cells express gap junctional proteins in their axons suggests the possibility that their terminals also express them. If this were to be the case, mixed electrical-chemical communication could be supported, as MF terminals normally use glutamate for fast communication with their target cells. Here we present electrophysiological studies in the rat and modeling studies consistent with this hypothesis. We show that MF activation produced fast spikelets followed by excitatory postsynaptic potentials in pyramidal cells (PCs), which, unlike the spikelets, underwent frequency potentiation and were strongly depressed by activation of metabotropic glutamate receptors, as expected from transmission of MF origin. The spikelets, which persisted during blockade of chemical transmission, were potentiated by dopamine and suppressed by the gap junction blocker carbenoxolone. The various waveforms evoked by MF stimulation were replicated in a multi-compartment model of a PC by brief current-pulse injections into the proximal apical dendritic compartment, where MFs are known to contact PCs. Mixed electrical and glutamatergic communication between granule cells and some PCs in CA3 may ensure the activation of sets of PCs, bypassing the strong action of concurrent feed-forward inhibition that granule cells activate. Importantly, MF-to-PC electrical coupling may allow bidirectional, possibly graded, communication that can be faster than chemical synapses and subject to different forms of modulation.

Figure 1

Mossy fiber activation evokes spikelets in PCs of CA3 at resting membrane potential in the presence of glutamate, GABA_A and ACh-M₁ receptor antagonists at physiological calcium concentration. (A₁) Spikelets are evoked in an all-or-none fashion once threshold is reached. The variability in the threshold current intensity for eliciting fast spikelets is due to the probability with which the electrode location and the stimulation intensity are capable of activating a MF that actually establishes gap junctions with the recorded PC. When stimulation is given at a depolarized membrane potential (A₂ and displaced trace in B₁), the PC fires an action potential, which is preceded by a notch (arrow) corresponding to an underlying spikelet. By contrast, hyperpolarization of the membrane potential by ≥ 15 mV suppresses the spikelet. (B₂) Stimulation 300 μm away from the first stimulation site does not evoke any response in the same cell, indicating that the spikelet originated by stimulation of the first site is synapse-specific and not due to electrical artifacts. Similar results were obtained when stimulating farther away from the initial effective site (C1) and when placing the stimulus electrode in the pyramidal cell layer of CA3 (C₂), which evoked an IPSP, only in the presence of glutamatergic antagonists.

Figure 2

(A) Spontaneous spikelets with similar rise-time as the evoked ones were recorded in most of the responsive PCs. (B) Spikelets (arrow) occurred either preceding action potentials (single asterisk) or isolated (double asterisk). (C) Spontaneous events depicted in B with asterisks, at an expanded time scale. (D) Evoked spikelet followed by an action potential. (E) The waveforms of the experimentally obtained synaptic events in C and D were reproduced by a compartmental model. We simulated gap junctional inputs to PCs, from MF action potentials, by injecting brief depolarizing current pulses (3 nA, 0.4 ms) into the proximal apical dendritic compartment. (E₁) The current pulses evoked isolated spikelets with relatively larger afterhyperpolarization conductance values. (E2) The pulses evoked spikelets leading into bursts at smaller afterhyperpolarization values. (F1, F2) A MF-evoked spikelet at resting membrane potential is enhanced when preceded by a hyperpolarizing pulse. This is consistent with removal of inactivation from fast Na⁺ channels. (G) Carbenoxolone (100 μM), a gap junction blocker, strongly depressed the MF-evoked spikelets, while DA (10 μM) strongly enhanced them (H).

Figure 3

Mossy fiber activation evokes mixed electrical and chemical synaptic responses in PCs under normal neurotransmission conditions. (A) Increasing stimulation intensities enhanced the chemical component of the synaptic response, while the spikelet was evoked upon reaching threshold intensity and not further modified by increasing the stimulus intensity. (B) Input-output curve of the chemical (EPSP) and electrical (spikelet) synaptic components. Note that the spikelet is evoked in an all-or-none fashion. (C, D) The chemical component following the spikelet was better observed after increasing the stimulation frequency from 0.05 to 1 Hz, which eventually produced action potentials riding on the EPSP (arrow in avg 1 Hz). (D) Plot of the behavior of the EPSP (area under the curve; left y axis) and of the spikelet (amplitude; right y axis) at 0.01 and 1 Hz and during the perfusion of DCG-IV. APth on the left hand axis signals action potential threshold. Because the effect of high frequency stimulation and of DCG-IV was estimated by measuring the area under the curve of the EPSP, no complete depression could be observed when perfusing DCG-IV, which isolates the area under the curve of the depolarization following the spikelet. The spikelet itself was not affected (E).

Figure 4

Integration of electrical communication at high frequencies. In 2 (out of 4) experiments, stimulation at 10 (A₁) and 50 Hz (A₂) produced spikelets for each stimulus of the train in the presence of ionotropic glutamatergic and GABAergic antagonists. Asterisks signal stimulus artifacts; arrows signal spikelets. (B₁) Under normal neurotransmission conditions (ACSF), stimulation of the MF at 50 Hz produces hyperpolarization of the cell membrane due to feed-forward inhibition. (B₂) Perfusion of ionotropic glutamatergic and GABAergic receptors antagonists (NBQX+APV+BICU) blocks all synaptic responses. (B₃) Under these conditions, stimulation at an intensity that recruits an electrically-coupled MF produces spikelets that give rise to action potentials over a slow depolarization produced by temporal summation of the responses (in 2 out of 4 experiments). (C₁) Using the same model as in Figure 2, brief current pulses (0.4 ms, 5 nA) were injected into the proximal apical dendrite, at 50 Hz, corresponding to putative synchronized presynaptic terminal spikes in structures electrically coupled to the simulated cell. Note the brief bursts of spikes (often with a small spikelet on the rising phase of action potentials), alternating with larger spikelets. (C₂) First 4 responses of the 50 Hz train, depicted in B₃. The first and third responses in the train are spikelets (arrows), while the second and fourth stimuli (short, thin arrows) originated action potentials.