Nicotine activates immature "silent" connections in the developing hippocampus

Abstract

In the hippocampus at birth, most glutamatergic synapses are immature and functionally "silent" either because the neurotransmitter is released in insufficient amount to activate low-affinity alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors or because the appropriate receptor system is missing or nonfunctional. Here we show that, in the newborn rat, a brief application of nicotine at immature Schaffer collateral-CA1 connections strongly enhances neurotransmitter release and converts presynaptically silent synapses into conductive ones. This effect is persistent and can be mimicked by endogenous acetylcholine released from cholinergic fibers. Thus, during a critical period of postnatal development, activation of nicotinic acetylcholine receptors contributes to the maturation of functional synaptic contacts and the wiring of adult hippocampal circuitry.

Figure 1

Nicotine induces the appearance of mEPSCs in silent neurons and enhances their frequency in active ones. (a) Representative traces from a P3 silent neuron before (Left) and after (Right) the application of nicotine (1 μM). (b) The amplitude of individual events (●, cell shown in a is plotted against time). (c) The amplitude of miniature events before (●), during (filled bar), and after nicotine application is plotted against time. Note complete suppression of miniature events with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (open bar). (d and e) Cumulative distributions of interevent interval (d) and amplitude (e) of mEPSCs shown in c before (thick line) and after (thin line) nicotine application. (f and g) Summary plot of mEPSCs frequency versus time for cells recorded at 24°C (n = 7; f) and 33°C (n = 7; g). Each column represents the number of miniatures per min. Dotted lines represent the mean mEPSC frequency in control conditions (also including silent cells). (Insets) mEPSCs amplitude (mean ± SEM of three cells in f and five in g) recorded before (open columns) and after (filled columns) nicotine application.

Figure 2

Nicotine switches on presynaptically silent synapses. (a) The amplitudes of the first EPSC (Left, ●) and second EPSC (Right, ○) evoked by pair of stimuli before, during (bar), and after nicotine application are plotted against time. (Upper) Averages of 100 individual responses (successes and failures) obtained before (control) and after nicotine application. (Lower) Ten individual responses evoked in control and 20 min after washing out nicotine. (b) Mean percentage of successes to the first and second stimulus for all P1–P5 CA1 pyramidal neurons examined (n = 13) before (control) and 20 min after nicotine application. (c) PPR calculated in seven low probability cells before (control) and after nicotine application. (d and e) Summary plot of mean number of successes versus time in response to the first and second stimulus for 10 cells (including those presynaptically silent). Each column represents the number of successes per min. *, P < 0.05; **, P < 0.01.

Figure 3

Nicotine effect is mediated by α7 receptors. (a) The amplitudes of the first EPSC (Left, ●) and second EPSC (Right, ○) evoked by a pair of stimuli before, during, and after nicotine application in the presence of DHβE are plotted against time. (Upper) Averages of 100 individual responses (successes and failures) obtained before (control) and 20 min after nicotine application. (Lower) Ten individual responses evoked in control and after washing out nicotine. Note the lack of potentiation when nicotine was applied in the presence of the antagonist. This cell is the same shown in Fig. 2 (after washing out DHβE). (b) Mean percentage of successes (n = 13; Left) and PPR (n = 7; Right) obtained before and after nicotine application in the presence of DHβE. (c) Number of successes to the first (open column) and second (filled column) stimulus observed 20 min after application of nicotine in the presence of different nAChR antagonists. Successes are normalized to those present in control (dotted line). Note that DHβE (1 μM) did not block nicotine-induced potentiation (*, P < 0.05). Number of cells for α-BGT, MLA, and DHβE are five, six, and six, respectively.

Figure 4

Activation of nicotinic receptors by endogenously released ACh converts presynaptically silent synapses into conductive ones and strongly enhances synaptic efficacy. (a) Schematic diagram showing localization of stimulating and recording electrodes. Cholinergic fibers were stimulated in the presence of atropine (1 μM). Sch, Schaffer collaterals; mf, mossy fiber; pp, perforant path; DG, dendate gyrus; EC, entorhinal cortex. (b) Amplitudes of the first response obtained before and after stimulation of cholinergic fibers (arrows) are plotted against time. Note the lack of EPSC potentiation when cholinergic fibers were stimulated in the presence of DHβE. (Insets) Ten superimposed individual responses evoked by pair of stimuli delivered to the Schaffer collateral 15 min after repetitive stimulation of the cholinergic pathway in the presence (Left) or absence (Right) of DHβE. (c) Summary plot of the mean number of successes per min versus time for six cells (silent and nonsilent). Repetitive stimulation of the cholinergic fibers is marked by an arrow. (d) Mean percentage of successes to the first and second stimulus for all P3-P5 CA1 pyramidal neurons examined (n = 7) before and 20 min after stimulation of cholinergic fibers. *, P < 0.05.