S. Bööj

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

The anterograde axonal transport of norepinephrine (NE), dopamine-beta-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 degrees 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.(ABSTRACT TRUNCATED AT 400 WORDS)

The contribution of cholinergic enzymes and acetylcholine from the lumbar sympathetic chain to the rat sciatic nerve

This study was performed to investigate how much of the acetylcholine (ACh), cholineacetyltransferase (ChAT) and ACh-esterase (AChE) in the rat sciatic nerve originate from the somatic motor input and from the automatic sympathetic input, respectively. The somatic motor axons to the sciatic nerve were eliminated by surgical transsection of the spinal roots, (rhizotomy) and the autonomie component was removed by surgical resection of the lumber sympathetic chain bilateraly (sympathectomy). Also combined operations were performed. In intact (non-crushed) sciatic nerve rhizotomy caused a reduction in ACh content by 70%, in ChAT-activity by 55%, and in AChE-activity by 41%. Sympathectomy alone had very little influence on ACh and ChAT, but reduced AChE by 20%. After crushing the nerve 13 hours before sacrifice, all three substances accumulated proximal to the crush region as described previously. When compared to the control group, sympathectomy alone caused a reduction in accumulated amounts of AChE only, while ACh and ChAT accumulations were essentially unchanged. Rhizotomy alone caused a substantial reduction in accumulated amounts of all three substances, but most prominently in ACh and ChAT-amounts. After symphathectomy in combination with rhizotomy ACh-accumulations were very low, and enzyme activities were reduced more than in the group with rhizotomy alone. A certain amount of residual ChAT and AChE was present in the nerve, and the location of this is discussed. The fact that combined sympathectomy and rhizotomy lowered ACh accumulations significantly more than would be expected from the results after either operation alone is commented upon. The results thus indicate that the major part of ACh enzymes in rat sciatic nerve is located in somatic motor axons. Very little ACh and ChAT, but about 20% of the ACh E is confined to the sympathetic axons. Some extraneuronal enzyme appears to be present.

The effect of chronic nicotine and withdrawal on intra-axonal transport of acetylcholine and related enzymes in sciatic nerve of the rat

The content of acetylcholine (ACh) and activities of the cholinergic enzymes choline acetyltransferase (CAT) and ACh-esterase (AChE) were studied in intact and crushed rat sciatic nerve afterchronic nicotine administration andwithdrawal 2 days before the final experiment. Nicotine was given in the drinking water during 8–10 weeks and the final dose reached was about 8 mg/kg/day, i.e. equivalent to that of the heavy cigarette smoker. In thechronic nicotine group, ACh levels and AChE activity of uncrushed nerve were significantly decreased as compared to the controls. The accumulation of ACh and AChE proximal to a single crush was also somewhat decreased, but significant only for AChE at 18 hours postoperatively. Afterwithdrawal of nicotine for 2 days the ACh content of both uncrushed and 12 hours crushed nerves were further decreased, while AChE was instead increased to control (uncrushed) or even supranormal (18-hour crush) levels.

The synthesis of NPY and DBH is independently regulated in adrenergic nerves after reserpine

A newly developed cytofluorimetric scanning technique was applied in a pharmacological study to investigate the influence of reserpine (10 mg/kg) on the axonal transport of norepinephrine (NE), dopamine-β-hydroxylase (DBH), tyrosine hydroxylase (TH), and neuropeptide Y (NPY)-like immunoreactivities (LI) in the adrenergic axons of the sciatic nerve of rat. Early after reserpine (18 hr and 24 hr after the reserpine injection) the amounts of NE accumulated proximal to a 12-hr crush werenil or very low, as observed in earlier studies. DBH-LI, TH-LI, and NPY-LI accumulations were also depressed but only to about 50% of control accumulations. This decrease in amounts of transported substances was probably caused by a decrease in protein synthesis and also a lowered velocity of fast axonal transport initially after reserpine, when body temperature is low. The amounts of accumulated NE, DBH-LI, TH-LI, and NPY-LI were normalized around day 2 after reserpine, but on day 4 NE, DBH-LI, and in some rats also TH-LI accumulated in supranormal amounts. However, NPY-LI accumulations were normal, indicating that DBH, butrot NPY, was trans- synaptically induced in rat sympathetic neurons, and that the biochemical composition of axonally transported organelles is altered for some days after reserpine.

Influence of descending bulbospinal monoamine neurons on axonal transport of acetylcholine and cholinergic enzymes

The influence of descending bulbospinal monoamine (MA) neurons on the intra-axonal transport of acetylcholine (ACh) and related enzymes (cholineacetyltransferase, CAT, and ACh-esterase, AChE) in rat sciatic nerve was studied in crush experiments following intracisternal injections of specific neurotoxins. The injection of 6-hydroxydopamine (6-OH-DA) and 5, 6-dihydroxytryptamine (5, 6-diOH-TA) (50μg×2) caused a degeneration of catecholamine (CA) and 5-hydroxytryptamine (5-HT) nerve terminals, respectively, and a combination of the two neurotoxins caused a loss of virtually all MA terminals in the lumbar spinal cord. The results of the neurotoxin injections were controlled by the Falck-Hillarp fluorescence method. The effect of neurotoxin treatment on the enzyme activities in the sciatic nerve was very small. The ACh levels of uncrushed nerves and in nerves proximal to a crush performed 12 hours before dissection decreased following either 6-OH-DA or 5, 6-diOH-TA. However, the combined treatment with both 6-OH-DA and 5, 6-diOH-TA had no influence on ACh accumulation and transport, as compared to the control group. In a previous study we have shown that mid-thoracic spinal cord transection increased AChE-transport while ACh-transport was decreased. The results of this study indicate that the bulbospinal MA neurons may be involved (perhaps indirectly) in the regulation of ACh levels and transport in motor neurons, but less important for the modulation of the cholinergic enzymes.