Neonatal rearing conditions distinctly shape locus coeruleus neuronal activity, dendritic arborization, and sensitivity to corticotrophin-releasing factor

Abstract

Early life events influence vulnerability to psychiatric illness. This has been modelled in rats and it has been demonstrated that different durations of maternal separation shape adult endocrine and behavioural stress reactivity. One system through which maternal separation may act is the locus coeruleus (LC)-norepinephrine system that regulates emotional arousal. Here we demonstrate that different durations of maternal separation have distinct effects on LC physiology and dendritic morphology. Rat pups were separated from the dam for 15 min/d (HMS-15) or 180 min/d (HMS-180) from post-natal days 2-14. Others were either undisturbed (HMS-0) or were vendor-purchased controls. LC characteristics were compared at age 22-35 d using whole-cell recordings in vitro. Cells were filled with biocytin for morphological analysis. LC neurons of HMS-180 rats were tonically activated compared to HMS-15 and control rats, with firing rates that were 2-fold higher than these groups. Corticotrophin-releasing factor (CRF) application did not further activate LC neurons of HMS-180 rats but increased LC firing rate in HMS-0 and control rats. LC neurons of HMS-15 rats were resistant to excitation by CRF. Maternal separation also affected LC dendritic morphology. LC dendrites of HMS-15 rats exhibited less branching and decreased total dendritic length, an effect that could decrease the probability of contacting limbic afferents that terminate in the pericoerulear region. This effect may provide a structural basis for an attenuated magnitude of emotional arousal. Together, these results demonstrate long-term consequences of early life events on the LC-norepinephrine system that may shape adult behaviour.

Fig. 1

Early rearing environment affects locus coeruleus (LC) spontaneous firing rate. (a–d) Representative traces of whole-cell recordings from LC neurons in current clamp, showing the different baseline firing rates of spontaneously active LC neurons from (a) control, (b) HMS-0, (c) HMS-15 and (d) HMS-180 rats. On average cells from HMS-180 had increased spontaneous firing rates compared to LC neurons from controls and HMS-15 rats.

Fig. 2

Early rearing environment differentially affects the sensitivity of locus coereleus (LC) neurons to the excitatory effects of corticotrophin-releasing factor (CRF) via CRF₁. (a) Graphical representation of the differential effects of CRF on the mean firing rate of LC neurons. Bath application of 11 nm CRF significantly increased the firing rate of cells from control and HMS-0 animals (paired t test). In contrast, CRF did not elevate the mean firing rate of cells from HMS-15 or HMS-180 rats. For control, HMS-0, HMS-15 and HMS-180 groups the number of rats used was 7, 15, 10, and 7, respectively. (b) The selective CRF₁ antagonist, antalarmin (ANT), blocked the excitatory effects of CRF. The number of cells analysed in each group is indicated in parentheses (* p<0.05).

Fig. 3

Photomicrographs of filled cells from a HMS-15 rat and HMS-180 rat. The top panels show biocytin-labelled cells visualized with a rhodamine filter. Middle panels show tyrosine hydroxylase (TH) immunoreactivity in same section visualized using a fluorescein filter. Bottom panels show the merged image indicating that the biocytin-labelled cells are TH expressing and within the nucleus locus coereleus. Top is dorsal and medial is to the left. Scale bar, 60 μm.

Fig. 4

Quantification of different morphological parameters of recorded cells. The mean number of primary dendrites per cell (a) and the length of the longest dendrite (c) were not significantly different across animal groups. In contrast, there was a significant difference in dendritic branching (b) between the four groups [F(3, 90)=4.3, p=0.007]. Post-hoc comparison between groups revealed significant differences in branch number between the control and HMS-15 groups as well as the HMS-0 and HMS-15 groups (* p<0.01, ** p<0.005). In addition, the total dendritic length (d) differed significantly [F(3, 90)=4.6, p<0.005] between the HMS-0 and HMS-15 groups as well as the HMS-180 and HMS-15 groups (** p<0.005). The number of cells analysed in each group are indicated in parentheses in panel (a). For control, HMS-0, HMS-15 and HMS-180 groups the number of rats used was 7, 15, 10 and 7, respectively.