Reserpine-induced effects in the adrenergic neuron as studied with cytofluorimetric scanning

Abstract

The anterograde axonal transport of norepinephrine (NE), dopamine-β-hydroxylase (DBH), neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was studied in the rat sciatic nerve after reserpine (10 mg/kg). The histochemical method of Hillarp and Falck was used to study NE and the indirect immunofluorescence technique to study DBH-, NPY- and TH-immunoreactive material (IR), using antisera produced in rabbits. The rats were given reserpine 18 hr, or 1–7 days before sacrifice. Before perfusion fixation one nerve was dissected, frozen and freeze-dried for studies on NE, and the contralateral nerve was then processed for immunofluorescence. The amount of fluorescent material accumulated proximal to a 12 hr crush was quantified in longitudinal sections of the nerve using a cytofluorimetric scanning method. During the early phase after reserpine (18 and 25 hr) the amounts of accumulated NE were undetectable or very low, near control levels at 2 day, and overshooting to 160% of control at 4 d after reserpine. Accumulations of DBH-IR, NPY-IR and TH-IR were also depressed initially to 60–70% of control. DBH-IR and TH-IR thereafter increased to supranormal levels (140% of control) at day 4, while NPY-IR did not exhibit any overshooting but accumulated in the normal range at 2, 4, and 7 days. The results indicate that the amount of material transported distally early after reserpine is depressed. After local vinblastine-treatment of the lumbar sympathetic ganglia the amounts of DBH-IR and TH-IR in the perikarya were markedly lower in reserpine treated rats than in controls, probably due to a decreased perikaryal synthesis of these two enzymes. Rectal temperature in these rats decreased during the initial 10 hr after reserpine by up to 3°C. Thus, the decreased synthesis may be caused by the lowered body temperature, which also may slow down the rate of anterograde axonal transport. The later overshooting in accumulated amounts of NE, DBH-IR an TH-IR gives support to the hypothesis that amine granules containing DBH and NE are produced and transported in supranormal amounts around the 4th day after reserpine. Also, the results indicate that a considerable fraction of TH-IR is transported with organelles, probably amine storage granules, in adrenergic axons. NPY, shown to be localized in amine storage granules, did not overshoot at day 4 after reserpine, in contrast to DBH-IR, TH-IR and NE. We suggest that the induction in perikaryal synthesis, triggered by reserpine, is restricted to mRNA coding for DBH and TH, while, mRNA coding for NPY is not induced. This may imply an altered biochemical composition (less NPY/DBH) of the amine granules in transit distally around day 4 after reserpine in rat adrenergic axons.