Atomoxetine modulates spontaneous and sensory-evoked discharge of locus coeruleus noradrenergic neurons

Abstract

Atomoxetine (ATM) is a potent norepinephrine (NE) uptake inhibitor and increases both NE and dopamine synaptic levels in prefrontal cortex, where it is thought to exert its beneficial effects on attention and impulsivity. At the behavioral level, ATM has been shown to cause improvements on the measures of executive functions, such as response inhibition, working memory and attentional set shifting across different species. However, the exact mechanism of action for ATM's effects on cognition is still not clear. One possible target for the cognitive enhancing effects of ATM is the noradrenergic locus coeruleus (LC), the only source of NE to key forebrain areas such as cerebral cortex and hippocampus. Although it is known that ATM increases NE availability overall by blocking reuptake of NE, the effects of this agent on impulse activity of LC neurons have not been reported. Here, the effect of ATM (0.1-1 mg/kg, ip) on NE-LC neurons was investigated by recording extracellular activity of LC neurons in isoflurane-anesthetized rats. ATM caused a significant decrease of the tonic activity of LC single-units, although leaving intact the sensory-evoked excitatory component of LC phasic response. Moreover, the magnitude of the inhibitory component of LC response to paw stimulation was increased after 1 mg/kg of ATM and its duration was prolonged at 0.3 mg/kg. Together, these effects of ATM produced an increase in the phasic-to-tonic ratio of LC phasic response to sensory stimulation. ATM also modulated the average sensory-evoked local field potential (LFP) and spike-field coherence in LC depending on the dose tested. The lower dose (0.1 mg/kg) significantly decreased early positive and negative components of the sensory-evoked LFP response. Higher doses (0.3-1 mg/kg) initially increased and then decreased the amplitude of components of the evoked fields, whereas the spike-field coherence was enhanced by 1 mg/kg ATM across frequency bands. Finally, coherence between LC fields and EEG signals was generally increased by 1 mg/kg ATM, whereas 0.1 and 0.3 mg/kg respectively decreased and increased coherence values in specific frequency bands. Taken together these results suggest that ATM effects on LC neuronal activity are dose-dependent, with different doses affecting different aspects of LC firing. This modulation of activity of LC-NE neurons may play a role in the cognitive effects of ATM. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Fig. 1

EEG traces were continuously monitored to ensure a stable level of anesthesia. EEG activity during extracellular recordings was characterized by slow, high amplitude oscillations and punctuated by regular spindle-like activity. a) The level of the anesthetic was regulated throughout the experiment so as to achieve reliable EEG responses to acoustic (finger snaps) sensory stimulation. At this level of anesthesia, LC tonic activity is comparable to that of awake animals (Aston-Jones and Bloom, 1981a, b) Comparison of the ‘spike’ trace (100–3K Hz bandpass) and LFP trace before (1–1K Hz bandpass; ‘raw’ LFP) and after digital filtering (1–30 Hz bandpass; ‘spike-free’ LFP). The digital filter applied off-line successfully removed spike contamination from the LFP trace. c) Representative coronal section through the LC from one of the experimental subjects. Pontamine blue deposit in ventral LC confirmed that all electrode tracks were in the intended area. Abbreviations: EEG, electroencephalogram; LFP, local field potential; ME5, mesencephalic nucleus 5; ventr, ventricle.

Fig. 2

Spontaneous firing activity of LC neurons recorded from 0 to 30 min after ATM injection. a) Upper panels depicts representative rate histograms (10s bins) from 3 different LC neurons recorded in animals that received 0.1, 0.3 or 1 mg/kg ATM. It can be seen that the two higher doses, but not 0.1 mg/kg, attenuated LC spontaneous activity and that 1 mg/kg caused a much faster onset of this effect. The lower panels depict the inter-spike intervals (ISI) of the above recordings (5ms bins). b) ATM at the 0.3 and 1 mg/kg doses decreased spontaneous LC firing activity and this variable was different from the pre-drug condition during the first two time windows considered (30–90 min and 90–150 min). Firing rates recovered between 150–210 min for both effective doses. c) Representative sampling of waveforms from one LC neuron during a 30 min period. (* = p < .05, for time).

Fig. 3

a) Schematic representation of the method used to separate the different components of the LC sensory-evoked response in single-unit recordings (see text). b) Effects of ATM on LC evoked discharge. The single-unit PSTHs depict two representative neurons per dose. For most neurons, tonic activity was more sensitive to the inhibitory effects of ATM, while largely sparing the excitatory response with a consequent increase in the phasic-to-tonic ratio.

Fig. 4

Effects of different doses of ATM on LC single-unit sensory-evoked response expressed as percentage change from the pre-drug condition. a) Baseline activity between trials was significantly decreased at 0.3 and 1 mg/kg of ATM (both p < .005) and between 90–150 min after ATM administration. The excitatory component (b) was not affected by ATM administration compared to pre-drug levels, whereas the magnitude of the inhibitory component (c) was increased during the second time-period (90–150 min) and at 1mg/kg (p < .05). The duration of the inhibitory component (d) was increased by 3 mg/kg (p < .05) only, whereas 1mg/kg of ATM increased the phasic-to-tonic ratio (e) of LC excitatory response to paw stimulation (p < .05). f) Both 0.3 and 1 mg/kg of ATM increased the phasic-to-tonic (P:T) ratio of the inhibitory response compared to pre-drug condition (p < .05 and p < .005, respectively) and the second time window (90–150 min) was different from all the other epoch considered. Increased variability was observed on some of the measures, but only during specific time-windows. This indicates that this variability is not due to noise in the data, but to the time course of ATM effects (* = p < .05 for time).

Fig. 5

Normalized average evoked LFPs in LC following paw stimulation before and after different doses of ATM. During the first time-period (a and d; 30–90 min), although having no effect on single-unit activity in the LC, the lower dose (0.1 mg/kg) tended to flatten the LFP response to paw stimulation and significantly decreased both the Neg1 and Pos3 components. Higher doses increased negative components only. In the second time window considered (b and e; 90–150 min), 1 mg/kg of ATM significantly decreased the early negative and the positive components, whereas 0.1 mg/kg decreased only the Pos3. c) Example of the average LFP response before normalization and relative components. (* = p < .05, compared to pre-drug condition).

Fig. 6

Effects of ATM on spike-field and EEG-field coherence during the first 30–90 min post-administration for the four frequency bands analyzed: delta (2–4 Hz), theta (4–8 Hz), alpha (8–12 Hz) and beta (12–18 Hz). a) ATM generally increased spike-field coherence at the high dose (1 mg/kg) across all frequency bands considered, with the exception of the delta frequency range. b) EEG-field coherence was affected by ATM across frequencies, with 1 mg/kg generally increasing coherence, whereas 0.3 mg/kg increased coherence only in the alpha and beta range. The low dose of ATM (0.1 mg/kg) decreased coherence across delta, theta and alpha frequency bands. (* = p < .05, compared to pre-drug condition).