SUMMARYThe activity of the pineal enzyme arylamine: N-acetyltransferase (NAT) was determined following direct stimulation ofthe preganglionic or post-ganglionic nerves of the superior cervical ganglia.1. Stimulation of the preganglionic trunks at 10 c/sec during the day or night was sufficient to increase NAT activity approximately 50-fold, to levels comparable to those observed at night in the intact animal. The time course of this effect of nerve stimulation differed between day and night.2. The responses of pineal NAT to certain frequencies of stimulation were similar for preganglionic and post-ganglionic stimulation. In both cases the responses to stimulation at 5 c/sec appeared to be maximal, 10 c/sec causing no further increase. However, at 1-0 c/sec, stimulation was more effective post-ganglionically than preganglionically.3. Various patterns of preganglionic stimulation, having the same average frequency, differed in their ability to increase the activity of NAT. Some, though not all, of these differences between patterns were observed during post-ganglionic stimulation.4. Unilateral stimulation of the preganglionic nerves produced an increase in NAT activity that was less than half the increase produced by bilateral stimulation, suggesting that the innervation from the two ganglia interact within the pineal gland.5. These data indicate that changes in the firing rates of sympathetic nerves innervating the pineal gland, within the range of frequencies typically observed for sympathetic neurones, would be sufficient to account for the circadian rhythm in NAT activity observed in the intact rat. Changes in the over-all pattern of sympathetic activity, in addition to changes in the total number of stimuli, could play a significant role in the pineal response.

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the two superior cervical ganglia and whose axons reach the gland via the two internal carotid nerves (ICNs). Bilateral decentralization of the superior cervical ganglia, produced by lesioning both cervical sympathetic trunks (CSTs), abolishes the circadian rhythm in the activity of the pineal enzyme serotonin N-acetyltransferase (NAT). We have examined the effects on NAT activity of unilaterally cutting the ICN or the CST. During the first night after either operation, nocturnal NAT activity was reduced by 75% compared to controls. However, during the second night after unilaterally cutting the ICN, NAT activity was restored to control values, and normal enzyme activity was seen in these lesioned animals for up to 1 month after this operation. On the other hand, following unilateral decentralization of one superior cervical ganglion, enzyme activity was reduced for at least 5 months. The high enzyme activity in animals with one ICN cut was abolished by cutting the contralateral CST, indicating that the recovery of NAT activity depended on the remaining intact sympathetic neurons. Electrical stimulation of the intact ICN during the daytime in animals in which the contralateral ICN was cut produced an increase in pineal NAT activity which was greater than the increase seen when similar stimulation was performed in sham-operated animals or in animals in which the contralateral superior cervical ganglion had been decentralized. The time course of the recovery of nocturnal NAT activity after unilateral denervation of the pineal gland was similar to the time course of the decrease in norepinephrine uptake sites in the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of the enzyme serotonin N-acetyltransferase (NAT) in the rat pineal gland exhibits a large circadian rhythm, with peak activity occurring at night. This rhythm is dependent on stimulation of the pineal gland by neurons whose cell bodies are in the superior cervical ganglia and whose axons reach the gland via the internal carotid nerves (ICNs). Two days after both ICN were cut, crushed, or frozen, nighttime NAT activity was decreased by 90%. The remaining low level of enzyme activity was not affected by decentralization of the superior cervical ganglia. Thus, this enzyme activity did not depend on the activity of neurons in these ganglia. Bilateraliy lesioning the ICN also abolished the neuronal uptake of norepinephrine in the pineal, further indicating that the sympathetic innervation of the gland had been destroyed. Three months after crushing both ICNs, nighttime NAT activity was only 20% of control values. However, in these animals, bilateral decentralization of the superior cervical ganglion reduced this low level of NAT activity by 90%. Thus, NAT activity, although low, was again dependent on sympathetic nerve stimulation. In contrast to this rather small recovery of nocturnal NAT activity, the norepinephrine uptake capacity of the gland recovered to 60% of control values. A similar discrepancy between the extent of recovery of NAT activity and of norepinephrine uptake was observed when the ICNs were frozen rather than crushed. To determine to what extent the sympathetic nerves that had reinnervated the pineal gland in these lesioned animals were capable of regulating NAT activity, their cervical sympathetic trunks were stimulated electrically at 5 Hz for 3 hr during the daytime. NAT activity increased in these animals, as it did in sham-operated animals, from low daytime values to near peak nighttime values. Thus, the sympathetic nerves reinnervating the pineal gland are capable of increasing NAT activity to high levels when electrically stimulated, and yet these animals do not recover a normal NAT rhythm. We hypothesize that, following bilateral lesioning of the ICN, the pineal gland is reinnervated by different sympathetic neurons than those that had previously innervated this tissue and that these neurons do not receive the type of neural information from the central nervous system that is necessary for regulating a normal circadian rhythm in NAT activity.

The activity of serotonin N-acetyltransferase (NATase) in the rat pineal gland exhibits a large (approximately 100-fold) circadian variation, with peak activity occurring in the dark part ofthe light/dark cycle. Surgical removal ofboth superior cervical ganglia abolishes this rhythm in enzyme activity. Unilateral ganglionectomy caused a 75% decrease in NATase activity during the dark period immediately following the operation; however, by the subsequent dark period (32 hr after operation) the rhythm in NATase activity had returned to normal. Similar results were found after the internal carotid nerve was cut, and data are presented indicating that this is the postganglionic trunk by which sympathetic neurons reach the pineal gland. Denervation of one superior cervical ganglion (unilateral "decentralization") also produced a 75% decrease in NATase activity during the dark period immediately following the operation; however, after decentralization, enzyme activity did not return to normal in subsequent cycles. It is hypothesized that this recovery is due to loss of norepinephrine uptake sites in the degenerating sympathetic nerve terminals. As a result of decreased norepinephrine uptake, the effectiveness of the norepinephrine released by surviving neurons may be enhanced. This hypothesis is supported by experiments in which pharmacological blockade of norepinephrine uptake in unilaterally decentralized animals increased NATase activity to control levels. We propose that neural systems which use transmitter uptake as the mechanism of transmitter inactivation have a built-in "reserve stimulatory capacity."Although a great deal of interest has been focused in recent years on the ability of the nervous system to recover after subtotal neural damage, there are only a few systems available in which functional recovery has been studied quantitatively on a cellular level. A useful system for such an investigation should have two characteristics: (i) permit the making of a lesion of a reproducible size in a specific part of the nervous system, and (ii) permit quantitative measurement ofthe degree offunctional recovery after the lesion is made. The innervation of the rat pineal gland offers such a model system.The pineal gland is a midline structure innervated by adrenergic sympathetic neurons whose cell bodies are located in the right and left superior cervical ganglia (SCGs) (1-4). In the rat these ganglia are the primary source ofneurons innervating this tissue (3)(4)(5). Ganglionic neurons are important in regulating a number of aspects of the biochemistry of the pineal parenchymal cells, particularly the synthesis of the. hormone melatonin. Pinealocytes synthesize melatonin from serotonin in two steps catalyzed by the enzymes serotonin N-acetyltransferase (arylamine N-acetyltransferase, EC 2.3.1.5) (NATase) and hydroxyindole O-methyltransferase (acetylserotonin methyltransferase, EC 2.1.1.4).The sympathetic neurons innervating the pineal gland appear to constitute the final pathway by which changes in environ...

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.