Action potential-triggered somatic exocytosis in mesencephalic trigeminal nucleus neurons in rat brain slices

Abstract

The neurons in the mesencephalic trigeminal nucleus (MeV) play essential roles in proprioceptive sensation of the face and oral cavity. The somata of MeV neurons are generally assumed to carry out neuronal functions but not to play a direct role in synaptic transmission. Using whole-cell recording and membrane capacitance (C(m)) measurements, we found that the somata of MeV neurons underwent robust exocytosis (C(m) jumps) upon depolarization and with the normal firing of action potentials in brain slices. Both removing [Ca(2+)](o) and buffering [Ca(2+)](i) with BAPTA blocked this exocytosis, indicating that it was completely Ca(2+) dependent. In addition, an electron microscopic study showed synaptic-like vesicles approximated to the plasma membrane in somata. There was a single Ca(2+)-dependent releasable vesicle pool with a peak release rate of 1912 fF s(-1). Importantly, following depolarization-induced somatic exocytosis, GABA-mediated postsynaptic currents were transiently reduced by 31%, suggesting that the somatic vesicular release had a retrograde effect on afferent GABAergic transmission. These results provide strong evidence that the somata of MeV neurons undergo robust somatic secretion and may play a crucial role in bidirectional communication between somata and their synaptic inputs in the central nervous system.

Figure 1. Ca²⁺ dependence of exocytosis in somata of MeV neurons

A, left upper: photomicrograph of MeV neurons in a rat brain slice. Right: C_m jump evoked by a depolarizing pulse (from −60 mV to +10 mV, 100 ms). The Ca²⁺ currents (I_Ca) and {Ca²⁺}_i with Ca²⁺ imaging (F₃₄₀/F₃₈₀) recorded simultaneously. The baseline C_m of the neuron was 53.02 pF (dotted line). The decay of the C_m response was fitted by a single exponential function with τ= 2.1 s (continuous line). B, pre-puffing 0 mm Ca²⁺ attenuated the C_m response (triggered by a depolarizing pulse to +10 mV, 100 ms). Both Ca²⁺ currents and {Ca²⁺}_i decreased. C, statistical bar graph showing pulse stimulation-triggered C_m jumps as in B, that were abolished by 10 mm BAPTA in the pipette. D, C_m jump and Ca²⁺ current as a function of membrane potential. The C_m response was observable at membrane potentials positive to −30 mV, and peaked round 0 mV (n = 8). The Ca²⁺ current was recorded at different depolarizing potentials (100 ms, from −60 mV to 60 mV, +10 mV steps) in different cells (n = 8).

Figure 2. Depolarization-induced C_m jump in somata of isolated MeV neurons

A, photomicrograph of two isolated MeV neurons under bright field DIC, one with a typical monopolar axon (upper) and the other being whole-cell patch-clamped (lower). B, a C_m jump occurred abruptly in response to 500 ms depolarization (to 0 mV). Similar results were obtained in four other neurons.

Figure 3. Amperometric spikes in DA-preloaded MeV neurons in brain slices

High potassium (KCl, 80 mm)-induced amperometric spikes in a MeV soma. The slowly descending background current is an artifact caused by puffing KCl (Chen et al. 2005). Inset: a single expanded amperometric spike. Similar amperometric signals were obtained from 3 slices.

Figure 4. Vesicular release rate in somata of MeV neurons

A, C_m responses induced by depolarization (to +10 mV) with pulse durations from 20 to 500 ms. Lower traces: Ca²⁺ currents induced by pulse stimulation. B, C_m as a function of depolarization duration. The C_m increased as a single exponential function (f_max= 749, τ= 321 ms) against pulse duration (n = 8).

Figure 5. Natural firing patterns modulate C_m responses of MeV neurons

A, representative firing patterns, phasic and tonic, in MeV neurons in response to depolarization pulses (1 s, 200 pA current injection) under current clamp. B, using the phasic and tonic firing patterns recorded by current clamp in A as stimulation templates, we recorded C_m responses with voltage clamp in a MeV neuron soma. C, statistics of averaged C_m responses in B.

Figure 6. Depolarization evoked exocytosis and modulated GABAergic inputs

A, field stimulation-induced currents recorded from the soma of a MeV neuron. Bicuculline (10 μm) abolished the stimulation-induced inward current, demonstrating that the inward current was mediated by GABAergic inputs. B, representative experiment showing postsynaptic currents (PSC) from the soma of an MeV neuron following 0.2 Hz field stimulation. The whole-cell soma depolarization (from −60 mV to 0 mV, 200 ms), which triggers somatic release (267 fF in the example trace, similar to Figs 1 and 4), was delivered in the interval between two PSCs (at the time marked by the arrow during field stimulation). The PSCs shown here were averaged from two sequential recordings during 10 s both before and after soma depolarization. C, field stimulation-evoked PSCs at 0, 10, 20 and 30 s following no somatic depolarization (open columns, n = 5), somatic depolarization (cis-stripe, n = 6), and somatic depolarization plus 10 mm{EGTA}_i dialysis (trans-stripe, n = 5).