Lesioning noradrenergic neurons of the locus coeruleus in C57Bl/6 mice with unilateral 6-hydroxydopamine injection, to assess molecular, electrophysiological and biochemical changes in noradrenergic signaling

Abstract

The locus coeruleus (LC) is the major loci of noradrenergic innervation to the forebrain. Due to the extensive central nervous system innervation of the LC noradrenergic system, a reduction in the number of LC neurons could result in significant changes in noradrenergic function in many forebrain regions. LC noradrenergic neurons were lesioned in adult male C57Bl/6 mice with the unilateral administration of 6-hydroxydopamine (6OHDA) (vehicle on the alternate side). Noradrenergic markers were measured 3 weeks later to determine the consequence of LC loss in the forebrain. Direct administration of 6OHDA into the LC results in the specific reduction of noradrenergic neurons in the LC (as measured by electrophysiology, immunoreactivity and in situ hybridization), the lateral tegmental neurons and dopaminergic neurons in the substantia nigra (SN) and ventral tegmental region were unaffected. The loss of LC noradrenergic neurons did not result in compensatory changes in the expression of mRNA for norepinephrine (NE) synthesizing enzymes. The loss of LC noradrenergic neurons is associated with reduced NE tissue concentration and NE transporter (NET) binding sites in the frontal cortex and hippocampus, as well as other forebrain regions such as the amygdala and SN. Adrenoreceptor (AR) binding sites (α1- and α2-AR) were not significantly affected on the 6OHDA-treated side compared to the vehicle-treated side, although there is a reduction of AR binding sites on both the vehicle- and 6OHDA-treated side in specific forebrain regions. These studies indicate that unilateral stereotaxic injection of 6OHDA into mice reduces noradrenergic LC neurons and reduces noradrenergic innervation to many forebrain regions, including the contralateral side.

Depressive-like behavior observed with a minimal loss of locus coeruleus (LC) neurons following administration of 6-hydroxydopamine is associated with electrophysiological changes and reversed with precursors of norepinephrine

“…Animals were sutured, removed from the stereotaxic apparatus and allowed to recover. Previous work indicated injection of 6-OHDA into the LC specifically affected the LC noradrenergic neurons (Szot et al, 2012b, c). …”

“…The minimum number of animals was used for these studies and care was taken to minimize any suffering. Injection of LC 6-OHDA or vehicle (0.2% ascorbic acid/saline) bilaterally is described in detail in previously published work (Szot et al, 2012b, c). Briefly, animals were anesthetized with isofluorane and mounted into a stereotaxic apparatus.…”

“…Catecholamine concentrations were measured by HPLC using a slightly modified version previously described (Szot et al, 2012b, c) to include analysis of serotonin (5-HT) and its metabolite 5-hydroxy indole acetic acid (5-HIAA) along with NE, dopamine (DA), the NE/DA precursor DOPA; and metabolites of NE and DA: 3-methoxy-4-hydroxy-phenethyleneglycol (MHPG), dihydroxyphenylglycol (DHPG), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). The catecholamines from the L-DOPA treated group were analyzed together with the animals that received DOPS.…”

“…TH-IH was performed in the LC to assess the number of noradrenergic neurons in vehicle- and 6-OHDA-treated animals as previously described (Szot et al, 2012b, c). The number of TH-immunoreactive (IR) cell bodies in the LC were counted in three consecutive atlas-matched sections bilaterally in all animals and expressed as the average ± SEM for each group.…”

Apathy and noradrenaline

“…Dopamine and noradrenaline are both tyrosine-derived catecholamines; their interaction may be of particular interest [83], given that the LC-NE system has widespread cortical projections (Fig. 2) [84]; noradrenaline may protect against dopaminergic deficiency [85]; and noradrenaline modulates dopaminergic activation [86]. Indeed, recent research in learning and decision-making has already moved beyond the bounds of the basal ganglia to scrutinize LC-NE's contribution to these functions [87,88].…”