Selective lesion of the developing central noradrenergic system: short‐ and long‐term effects and reinnervation by noradrenergic‐rich tissue grafts

Abstract

J. Neurochem. (2010) 114, 761–771. Abstract The possibility to selectively remove noradrenergic neurons in the locus coeruleus/subcoeruleus (LC/SubC) complex by the immunotoxin anti‐dopamine‐β‐hydroxylase (DBH)‐saporin has offered a powerful tool to study the functional role of this projection system. In the present study, the anatomical consequences of selective lesions of the LC/SubC on descending noradrenergic projections during early postnatal development have been investigated following bilateral intraventricular injections of anti‐DBH‐saporin or 6‐hydroxydopamine to immature (4 day old) rats. Administration of increasing doses (0.25–1.0 μg) of the immunotoxin produced, about 5 weeks later, a dose‐dependent loss of DBH‐immunoreactive neurons in the LC/SubC complex (≈ 45–90%) paralleled by a similar reduction of noradrenergic innervation in the terminal territories in the lumbar spinal cord. Even at the highest dose used (1.0 μg) the immunotoxin did not produce any detectable effects on dopaminergic, adrenergic, serotonergic or cholinergic neuronal populations, which, by contrast, were markedly reduced after administration of 6‐hydroxydopamine. The ≈ 90% noradrenergic depletion induced by 0.5 and 1.0 μg of anti‐DBH‐saporin remained virtually unchanged at 40 weeks post‐lesion. Conversely, the ≈ 45% reduction of spinal innervation density estimated at 5 weeks in animals injected with the lowest dose (0.25 μg) of the immunotoxin was seen recovered up to near‐normal levels at 40 weeks, possibly as a result of the intrinsic plasticity of the developing noradrenergic system. A similar reinnervation in the lumbar spinal cord was also seen promoted by grafts of fetal LC tissue implanted at the postnatal day 8 (i.e. 4 days after the lesion with 0.5 μg of anti‐DBH‐saporin). In these animals, the number of surviving neurons in the grafts and the magnitude of the reinnervation, with fibers extending in both the grey and white matter for considerable distances, were seen higher than those reported in previous studies using adult recipients. This would suggest that the functional interactions between the grafted tissue and the host may recapitulate the events normally occurring during the ontogenesis of the coeruleo‐spinal projection system, and can therefore be developmentally regulated. Thus, the neonatal anti‐DBH‐saporin lesion model, with the possibility to produce graded noradrenergic depletions, holds promises as a most valuable tool to address issues of compensatory reinnervation and functional recovery in the severed CNS as well as to elucidate the mechanisms governing long‐distance axon growth from transplanted neural precursors.

Essential role of hippocampal noradrenaline in the regulation of spatial working memory and TDP‐43 tissue pathology

“…Selective lesions of the developing noradrenergic system were performed on 4‐day‐old (post‐natal day, PD, 4) pups under hypothermic anesthesia (Coradazzi, Gulino, Garozzo, & Leanza, ). The anti‐DBH‐saporin immunotoxin (Advanced Targeting Systems, San Diego, CA), was used at a dose of 0.50 µg dissolved in sterile phosphate‐buffered saline (PBS).…”

“…Four weeks later, a noradrenergic‐rich cell suspension was prepared as previously described (Coradazzi et al, ; Leanza, Cataudella, Dimauro, Monaco, & Stanzani, ) following a modified protocol based on the cell suspension technique (Björklund, Stenevi, Schmidt, Dunnett, & Gage, ). Briefly, the developing LC was bilaterally dissected from the dorsolateral pons of 13‐ to 14‐days‐old donor rat foetuses (crown‐to‐rump length, CRL, 9–13 mm) of the same strain as the graft recipients, and collected in cold Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Italy).…”

“…Using a freezing microtome (Leitz Wetzlar), 40 mm sections were cut from the level of the prefrontal cortex through the hippocampus, to the brainstem regions containing the LC and collected into six series. Immunohistochemistry was conducted on free‐floating sections, following a standard avidin‐biotin ABC procedure (Coradazzi et al, ). The following primary antisera were used: Mouse monoclonal antibody C4F10.2 against DBH (1:2,000; Merck Millipore, Darmstadt, Germany, Cat# MAB 308, RRID: AB_2245740); Rabbit polyclonal antibody against TDP‐43 (1:2,000; Proteintech, Rosemont, IL, Cat# 10782.2.AP, RRID: AB_615042).…”

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

“…Another neurotoxin, 6-hydroxydopamine (6OHDA), has been shown to reduce LC noradrenergic neurons when administered directly into the lateral ventricles, but dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are reduced as well (Descarries and Saucier, 1972; Coradazzi et al, 2010). Administration of 6OHDA directly into the LC also results in a significant loss of noradrenergic neurons (Harik, 1984; Biancardi et al, 2008), although a comprehensive analysis of the consequence of this loss on noradrenergic terminals has not been performed.…”

Noradrenaline neuron degeneration contributes to motor impairments and development of L-DOPA-induced dyskinesia in a rat model of Parkinson's disease

“…Anti-dopamine-β-hydroxylase (DBH)-Saporin (here called DBH-Saporin) is an interesting alternative. This 6 immunotoxin, which consists of a monoclonal antibody to DBH coupled by a disulfide bond to saporin (a ribosome inactivating protein), has been shown to be selectively toxic to NA neurons, and when injected into the ventricular space in rats it has been shown to be effective in killing the NA neurons in the LC, A5, and A7, eliminating cell bodies without any detectable effect on other monoaminergic neurons in the brain (Wrenn et al, 1996;Coradazzi et al, 2010). The high selectivity and efficiency of the DBH-Saporin lesion makes it an ideal tool for studies involving lesions of the NA system, but it has so far not been used to explore the role of NA neurons in the development of motor impairments and dyskinesia in the rat PD model.…”