Adenosinergic modulation of rat basal forebrain neurons during sleep and waking: neuronal recording with microdialysis

Abstract

1. The cholinergic system of the basal forebrain (BF) is hypothesized to play an important role in behavioural and electrocortical arousal. Adenosine has been proposed as a sleep-promoting substance that induces sleep by inhibiting cholinergic neurons of the BF and brainstem. However, adenosinergic influences on the activity of BF neurons in naturally awake and sleeping animals have not been demonstrated. 2. We recorded the sleep-wake discharge profile of BF neurons and simultaneously assessed adenosinergic influences on wake- and sleep-related activity of these neurons by delivering adenosinergic agents adjacent to the recorded neurons with a microdialysis probe. Discharge rates of BF neurons were recorded through two to three sleep-wake episodes during baseline (artificial cerebrospinal fluid perfusion), and after delivering an adenosine transport inhibitor (s-(p-nitrobenzyl)-6-thioinosine; NBTI), or exogenous adenosine, or a selective adenosine A1 receptor antagonist (8-cyclopentyl-1, 3-dimethylxanthine; CPDX). 3. NBTI and adenosine decreased the discharge rate of BF neurons during both waking and non-rapid eye movement (NREM) sleep. In contrast, CPDX increased the discharge rate of BF neurons during both waking and NREM sleep. These results suggest that in naturally awake and sleeping animals, adenosine exerts tonic inhibitory influences on BF neurons, supporting the hypothesized role of adenosine in sleep regulation. 4. However, in the presence of exogenous adenosine, NBTI or CPDX, BF neurons retained their wake- and sleep-related discharge patterns, i.e. still exhibited changes in discharge rate during transitions between waking and NREM sleep. This suggests that other neurotransmitters/neuromodulators also contribute to the sleep-wake discharge modulation of BF neurons.

Figure 1. Microdialysis with adjacent unit recording

A, a schematic representation of the microdrive and microdialysis probe arrangements. The microwires were carried in a screw-driven microdrive, which was adjacent to the fixed microdialysis probe. The recorded neurons were within the perfusion area of the microdialysis probe (0.5-1.0 mm). B, photomicrograph of a brain section showing microwire tracts (small arrows) and the tract of the microdialysis probe (large arrows). ac, anterior commissure; 3V, third ventricle; oc, optic chiasm.

Figure 8. Line drawings of two representative brain sections showing the areas of neuronal recording at two rostral-caudal planes

Left side of the sections shows the distribution of the neurons studied for the effects of 10 μm NBTI, 300 μm adenosine and 5 μm CPDX, according to their sleep-wake discharge patterns. ChAT-positive neurons are plotted on the right side of the section (•). Most of the recorded neurons were within or close to areas containing ChAT-positive neurons. ac, anterior commissure; oc, optic chiasm; 3V, third ventricle.

Figure 2. Effects of microdialysis perfusion of 10 μm NBTI on the neuronal activity of an individual wake-related neuron during waking and NREM sleep

A, a 48 min continuous recording session showing the discharge rate (spikes s⁻¹) of a neuron during baseline, NBTI perfusion and recovery. The discharge rate of this neuron increased during each arousal state indicated by elevation of EMG activity and desynchronized EEG. This neuron also exhibited an increased discharge during REM sleep, as is typical of BF neurons. NBTI was perfused for about 12 min. A strong suppression of the neuronal activity after NBTI delivery, and recovery following wash out can be seen. Lower panels are 5 min expanded tracings of the areas marked in A showing wake- and sleep-related neuronal discharge rate during baseline (B), NBTI perfusion (C) and recovery (D). In response to NBTI, this neuron exhibited a strong suppression of discharge during waking (C). During NREM sleep, this neuron had a low baseline discharge rate but exhibited further suppression of discharge due to NBTI perfusion. The action potentials to the right of B–D represent the average waveforms of all the action potentials recorded during the representative section in each condition. There was no change in spike shape or amplitude as a result of microdialysis perfusion of ACSF or NBTI. EEG, electroencephalogram; EMG, electromyogram; Unit, neuronal activity.

Figure 3. Neuronal responses to NBTI

The effects of 10 μm NBTI on discharge rate (spikes s⁻¹; means and s.e.m.) of wake-related neurons (A), state-indifferent neurons (C) and sleep-related neurons (E) during waking and NREM sleep. The NREM/wake discharge ratios of wake-related (B), state-indifferent (D) and sleep-related (F) neurons during baseline and after NBTI perfusion are also shown. NBTI suppressed the discharge rate of wake-related and state-indifferent neurons during both waking and NREM sleep without producing any significant change in the NREM/wake discharge ratio. Sleep-related neurons showed suppression only during waking resulting in an increased NREM/wake discharge ratio. , control (ACSF perfusion); ▪, 10 μm NBTI perfusion; * P < 0.05, ** P < 0.01 (Wilcoxon matched-pair signed rank test).

Figure 4. Effects of microdialysis perfusion of 300 μm adenosine on the discharge rate of simultaneously recorded wake-related (Unit 1) and sleep-related (Unit 2) neurons during waking and NREM sleep

Neuronal discharge is shown during baseline ACSF (A) and adenosine perfusion (B). The action potentials to the right of A and B represent the average waveforms of all the action potentials recorded for each neuron during the representative sections. Although the action potentials from the two neurons were similar in shape, they differed in terms of their spike amplitude and sleep-wake discharge patterns. Adenosine suppressed the mean discharge rate of this wake-related neuron during waking and NREM sleep. In contrast, adenosine increased the discharge rate of this sleep-related neuron during waking and NREM sleep. Abbreviations as in Fig. 2.

Figure 5. Neuronal responses to adenosine

The effects of 300 μm adenosine on discharge rate (spikes s⁻¹; means and s.e.m.) of wake-related neurons (A), state-indifferent neurons (C) and sleep-related neurons (E) during waking and NREM sleep. The NREM/wake discharge ratios of wake-related (B), state-indifferent (D) and sleep-related (F) neurons during baseline and after adenosine perfusion are also shown. Note that adenosine produced effects similar to those produced by NBTI. , control (ACSF perfusion); ▪, 300 μm adenosine perfusion; * P < 0.05 (Wilcoxon matched-pair signed rank test).

Figure 6. Effects of 5 μm CPDX on the neuronal activity of an individual wake-related neuron (A and B) and a sleep-related neuron (C and D) during waking and NREM sleep

The action potentials to the right of A–D represent the average waveforms of all the action potentials recorded during the representative sections in each condition. CPDX increased the discharge rate of this wake-related neuron during both waking and NREM sleep (B). CPDX increased the discharge rate of this sleep-related neuron during waking with a smaller effect during NREM sleep (D). Abbreviations as in Fig. 2.

Figure 7. Neuronal responses to CPDX

The effects of 5 μm CPDX on discharge rate (spikes s⁻¹; means and s.e.m.) of wake-related neurons (A), state-indifferent neurons (C) and sleep-related neurons (E) during waking and NREM sleep. The NREM/wake discharge ratios of wake-related (B), state-indifferent (D) and sleep-related (F) neurons during baseline and after CPDX perfusion are also shown. In response to CPDX, all neuronal types exhibited increased discharge during both waking and NREM sleep. However, sleep-related neurons exhibited a smaller increase in discharge during NREM sleep resulting in a significant decrease in their NREM/wake discharge ratio. , control (ACSF perfusion); ▪, 5 μm CPDX perfusion; * P < 0.05; ** P < 0.01 (Wilcoxon matched-pair signed rank test).