Cholinergic responses and intrinsic membrane properties of developing thalamic parafascicular neurons

Abstract

Parafascicular (Pf) neurons receive cholinergic input from the pedunculopontine nucleus (PPN), which is active during waking and REM sleep. There is a developmental decrease in REM sleep in humans between birth and puberty and 10-30 days in rat. Previous studies have established an increase in muscarinic and 5-HT1 serotonergic receptor-mediated inhibition and a transition from excitatory to inhibitory GABA(A) responses in the PPN during the developmental decrease in REM sleep. However, no studies have been conducted on the responses of Pf cells to the cholinergic input from the PPN during development, which is a major target of ascending cholinergic projections and may be an important mechanism for the generation of rhythmic oscillations in the cortex. Whole cell patch-clamp recordings were performed in 9- to 20-day-old rat Pf neurons in parasagittal slices, and responses to the cholinergic agonist carbachol (CAR) were determined. Three types of responses were identified: inhibitory (55.3%), excitatory (31.1%), and biphasic (fast inhibitory followed by slow excitatory, 6.8%), whereas 6.8% of cells showed no response. The proportion of CAR-inhibited Pf neurons increased with development. Experiments using cholinergic antagonists showed that M2 receptors mediated the inhibitory response, whereas excitatory modulation involved M1, nicotinic, and probably M3 or M5 receptors, and the biphasic response was caused by the activation of multiple types of muscarinic receptors. Compared with CAR-inhibited cells, CAR-excited Pf cells showed 1) a decreased membrane time constant, 2) higher density of hyperpolarization-activated channels (I(h)), 3) lower input resistance (R(in)), 4) lower action potential threshold, and 5) shorter half-width duration of action potentials. Some Pf cells exhibited spikelets, and all were excited by CAR. During development, we observed decreases in I(h) density, R(in), time constant, and action potential half-width. These results suggest that cholinergic modulation of Pf differentially affects separate populations, perhaps including electrically coupled cells. Pf cells tend to show decreased excitability and cholinergic activation during the developmental decrease in REM sleep.

FIG. 1.

Measurement of intrinsic membrane properties. A: representative I-V steps under current-clamp mode. Time constant (tau) was determined using the following equation: V = −ΔV × e^−t/tau. B: single I-V step under voltage-clamp mode. R_in = 50 mV/I(Ins). Membrane capacitance (C) = tau/R_in. Normalized rebound current (or I_h current) = I(Rebound) [or I_h]/C. AP, action potential; Amp, amplitude; Thr, threshold; I(ins), instantaneous current.

FIG. 2.

Localization and morphology of parafascicular (Pf) neurons. A: location of some of the recorded Pf cells immediately posterior or anterior to the fasciculus retroflexus reconstructed on a 400-μm parasagittal thalamic section. B: 2-dimensional confocal image of 2 17-day Pf neurons identified by intracellular neurobiotin conjugated to Cy2 labeling from a single frame. The cell posterior to the fr was inhibited by carbachol (CAR) application and had a capacitance of 106 pF. The neuron anterior to the fr showed no response to CAR and had a capacitance of 101 pF. Note the long sparsely branching processes of both neurons. The morphology of CAR-excited and -inhibited and nonresponsive cells was similar.

FIG. 3.

Responses of Pf neurons to CAR or cholinergic antagonists were via postsynaptic receptors. A: repetitive application of CAR produced a slight rundown of the inward current induced by CAR. The 1st application of CAR induced a 60-pA inward current, with as much as 40 pA induced by the 4th application. B: outward current triggered by CAR decreased slightly with repetitive application. C: summary graph showing the average rundown ratio of the inward current produced by repetitive application of CAR. Baseline was the amplitude of the 1st response. D: summary graph showing the average rundown ratio of the outward current caused by repetitive application of CAR. E: TTX and excitatory synaptic blockers (ESBs) (ESB: CNQX and APV) failed to block the inward current induced by CAR. Top recording shows the effect of CAR alone, with a 50-pA inward current being induced. In the presence of TTX, the CAR-induced inward current decreased to 35 pA. ESBs did not affect the inward current, but excitatory postsynaptic currents (EPSCs) were blocked. F: the outward current produced by CAR was not blocked by TTX or by inhibitory synaptic blockers (ISB: GBZ + CGP + STR + YOH + WAY + KET). Black bars indicate the period when neuroactive agents were applied. Horizontal scale bars in E and F are 500 ms. Vertical scales in E are 40 pA and in F are 15 pA.

FIG. 4.

Outward current induced by CAR was caused by the activation of M2 receptors mainly via opening the inwardly rectifying potassium channels in the Pf. A: outward current induced by CAR in the presence of TTX (recording I) was blocked by pretreatment with methoctramine (MTO) (recording II) in this Pf neuron. B: input resistance (R_in) change during recordings in A. CAR decreased the R_in from ∼700 to ∼300 MΩ in the presence of TTX, which recovered to ∼600 MΩ after wash with artificial cerebrospinal fluid (ACSF) (recording I). In the presence of MTO, CAR failed to decrease R_in (recording II). C: I-V relationship obtained in recordings (A). Subtraction of current ramp at the peak CAR effect (b) from that in control condition (a) showed that the CAR-induced outward current reversed at approximately −72 mV (b − a); however, in the presence of MTO, subtraction of control from CAR (d − c) showed no deviation from the voltage axis at 0 pA, suggesting that no current was activated. D: the CAR-induced outward current in the presence of TTX (recording I) was significantly reduced by Ba²⁺ (recording II). E: R_in change during recordings in D. Note that Ba²⁺ slightly increased R_in and significantly reduced the R_in decrease induced by CAR. F: subtractions of current ramps in different conditions indicated that the CAR-induced outward current reversed at approximately −72 mV in the presence of TTX (b − a), Ba²⁺ induced a small inward current reversing at approximately −87 mV (d − c), and almost completely blocked the current induced by CAR (e − d). Black bars show the period when drugs were applied. Horizontal scale bars are 100 s, and vertical bars are 10 pA.

FIG. 5.

Excitatory inward currents induced by CAR involved the activation of M1, nicotinic cholinergic receptor and probably M3 or M5 receptors in the Pf. A: CAR-induced inward currents were not blocked by mecamylamine (MEC) alone but were blocked during co-pretreatment with pirenzepine (PRZ) and MEC. Note the 2 phases of inward current in the top recording, whereas only 1 phase was present in the 2nd recording. B: inward currents induced by CAR were partially blocked by PRZ or co-application of PRZ and MEC. Note the 2 phases in the 2nd recording. Black bars show the period during which neuroagents were applied, and scale bars are 100 s and 20 pA for the horizontal and vertical axes, respectively.

FIG. 6.

Biphasic cholinergic response of the Pf was induced by the activation of muscarinic cholinergic receptors (mAChRs). A: a fast outward current followed by a slow inward current was produced by the administration of CAR with or without TTX, which was blocked by atropine (ATR). B: I-V relationship obtained from subtraction of current ramps at the peak CAR effect from that in control conditions. Recording I: I-V curve of (b − a) showed that the reversal potential of the CAR-activated fast outward current was approximately −62 mV and that of (c − a) suggested a reversal potential higher than −45 mV for the CAR-induced inward current. Recording (II): in the presence of TTX, the reversal potential did not show remarkable changes compared with that in recording I. Recording III: I-V relationship of (b − a) showed a parallel line to the voltage-axis at ∼0 pA, indicating no current was activated. Black bars show the period when drugs were applied. Vertical scale bars are 20 pA and horizontal bars are 100 s.

FIG. 7.

Membrane property records of Pf neurons with differential cholinergic responses. A: CAR induced an inward current in this Pf neuron, as well as rhythmic spikelets. a and b are shorter time scale representative records in controls (in ACSF) and during CAR application, showing the occurrence of EPSCs and spikelets, respectively. B: autocorrelograms of records shown in a and b indicated that spikelets occurred rhythmically at a frequency ∼5 Hz. C: representative I-V steps under voltage-clamp mode from 2 12-day-old Pf cells, 1 of them was excited by CAR, and the other was inhibited. D: relationship between voltage and instantaneous current [I(ins)] of 2 representative cells in C. Although a small rectifying current was present, the excited cell still showed a higher slope, indicating a lower R_in, whereas the inhibited cell exhibited a lower slope, indicating a higher R_in. E: relationship between voltage and I_h for the 2 cells shown in C. F: action potentials of 2 12-day-old representative Pf cells with different cholinergic responses. The excited cell showed a lower action potential threshold and a shorter half-width.

FIG. 8.

Development of cholinergic responses and intrinsic membrane properties of Pf neurons. A: proportion of Pf cells with different cholinergic responses changed with development. A decrease in the proportion of CAR-excited neurons and an increase in inhibited cells were found. Biphasic cholinergic response appeared in the 12- to 14-day group. B and C: developmental changes in the proportion of Pf neurons with Ih and spontaneous or CAR-induced spikelets. Numbers above each column indicate the cell number of cells. D–F: scatter plots of the developmental changes of I_h, R_in, and AP half-width. Straight lines represent the corresponding linear regression fits for each group of cells.