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Fetal spinal cord tissue was grafted to the anterior chamber of the eye of adult rats in order to evaluate survival and distribution of substance P- and enkephalin-immunoreactive neurons. Capsaicin treatment was used to evaluate any possible contribution of host iris-derived substance P fibres to the innervation of the grafts and to check for capsaicin sensitivity of graft substance P-positive systems. Substance P- and enkephalin-immunoreactive nerve fibres were present in grafts of half-transverse segments of the spinal cord and were clearly co-distributed throughout the grafts. Areas with a high density of substance P- and enkephalin-positive fibres resembling substantia gelatinosa were seen. Grafts of the dorsal horn alone had a moderate to high density of substance P- and enkephalin-positive fibres, while ventral horn grafts contained a low amount of such fibres. Capsaicin eliminated the substance P innervation of the host iris and the dorsal root ganglion-derived substance P innervation of the host spinal cord, while sparing the intrinsic substance P innervation of both host spinal cord and spinal cord grafts. These experiments show that intra-ocular grafts of defined spinal cord areas express relatively organotypic amounts of substance P- and enkephalin-positive nerve fibres, and thus emphasize the importance of intrinsic genetic determinants for spinal cord development.

By means of sensitive and specific methods for histochemical and biochemical determination of dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT) we have succeeded in demonstrating and mapping out a number of ascending monoamine neuron systems from the lower brain stem by studying the anterograde and retrograde changes that occur in these neurons after various types of brain lesions. In this way it has been possible to discover: 1) a large, uncrossed nigro-neostriatal DA neuron system; 2) a DA neuron system, arising from cell-bodies in the mesen-cephalon, ascending uncrossed in the medial forebrain bundle close to the nigro-neostriatal DA fibres, and innervating e.g. the tuberculum olfactorium and nuc. accumbens; 3) ascending NA neuron systems with cell-bodies situated mainly in the medulla oblongata and pons (locus coeruleus, formatio reticularis), and axons running uncrossed mainly in the medial forebrain bundle, innervating e.g. the limbic forebrain structures, the neocortex and the hypothalamus; 4) ascending 5-HT' neurons with cell-bodies situated mainly in the raphe nuclei of the mesencephalon (nuc. raphe dorsalis, nuc. raphe medianus), and axons running uncrossed mainly in the medial forebrain bundle, innervating e.g. the limbic forebrain structures and the hypothalamus. The effects observed on the amine levels of the neurons represent intraneuronal and not transsynaptic changes.

In vivo and in vitro studies using the histochemical fluorescence technique of Falck and Hillarp were made on the submaxillary and sublingual glands of the rat after the administration of catecholamines or dopa, or the incubation of slices of the glands with the same drugs. Catecholamines and in all probability also L-dopa were found to be bound extraneuronally in the salivary gland parenchyma under the conditions of the experiments. It is probable that the present experiments visualize the same type of extraneuronal binding as has been found with biochemical techniques. It appears that a certain activity of the parenchyma is necessary for the binding to occur. Apart from being a source of error in the evaluation of biochemical measurements, extraneuronal binding may be of importance related to transmitter mechanisms.

The early development of the peripheral adrenergic neurons of the rat was followed with the Falck-Hillarp fluorescence technique. In the 13 day embryo small fluorescent sympathicoblasts with short processes were found in segmentally arranged groups along the dorsal aspect of the embryo. The larger sympathetic ganglia and the strongly fluorescent adrenal medullary cells were easily identifiable at this stage. In the 18 day embryo, fluorescent axon bundles were found in most larger nerve trunks. The outgrowing fluorescent axons had a stronger fluorescence intensity than non-terminal axons of adult animals until at least 2 weeks after birth. The postnatal development of the plexuse of adrenergic nerves was followed in the iris, auricles of the heart, salivary glands, vas deferens and small intestine. The adrenergic innervation of the gut seemed to develop earlier than that of the other organs. Thus, the duodenal muscle layer had a well developed adrenergic innervation of the plexus of Auerbach at birth while the other organs investigated demonstrated well developed networks of adrenergic nerves from about the fourth day on. It was concluded from some pharmacological experiments that, under optimal conditions, the fluorescence technique can reveal all or almost all adrenergic nerves also during early development.

Transplantations were made of fetal pineal glands (crown-rump length, CRL, 19-30 mm) or pineal glands from adult male rats to the anterior chamber of the eye of the rat. Studies were performed with regard to the importance of the age of the donor animal (and thereby the degree of maturation and innervation of the gland to be transplanted) for the possible development of denervation supersensitivity. The transplants were cultured in a medium containing 14C-serotonin. Increased production of 14C-N-acetylserotonin (NAcS) was used as the main criterion for beta-adrenergic stimulation. 4 experimental groups were obtained by transplanting fetal or adult pineals to intact or sympathetically denervated eyes. In all 4 groups beta1-stimulation (KWD 2033 10(-6) M) increased 14C-NAcS formation. The response to beta-stimulation was significantly higher in denervated fetal pineal transplants than in innervated fetal transplants and thus demonstrating beta-receptor supersensitivity. It was concluded that a) the ability to respond to beta-adrenoreceptor stimulation with increased 14C-NAcS formation develops between the 18th and 20th day of gestation, b) transplants derived from fetal as well as from adult rats can respond to beta-adrenergic stimulation, c) this sensitivity also develops in oculo in transplants that at the time of transplantation lacked the capacity to increase their 14C-NAcS formation in response to treatment with beta-agonist, d) denervation supersensitivity occurs in fetal transplants that became mature in sympathetically denervated eyes.

Immature rat pineal grafts were transplanted bilaterally to the anterior eye chamber of adult male rats. The transplants matured and became sympathetically innervated from the host iris and synthesized 5-hydroxytryptamine as revealed by fluorescence histochemistry. Transplants were used to investigate whether the newly formed nerve terminals were functional and thus could induce a rhythm in pineal N-acetyltransferase (NAT) enzyme activity and hydroxyindole-O-methyl transferase (HIOMT) enzyme activity. The in situ pineal glands from the recipient animals exhibited NAT and HIOMT levels and diurnal variations of enzyme activities no different from control rats without transplants. Sympathetically innervated transplants showed lower NAT enzyme activity than the in situ pineals but did yield a highly significant increase in darkness NAT activity as compared to light NAT activity (20:1). Sympathetically denervated transplants showed a much smaller rise in darkness NAT enzyme activity as compared to light values (1.8:1) but the rhythm was still statistically significant (p less then 0.01). Sympathetically innervated transplants had a higher HIOMT activity in darkness than in light (2.7:1)9 The HIOMT activity of the sympathetically denervated grafts was invariably low. Protein contents of transplants were found to be significantly lower than in situ pineal protein content but failed to show a diurnal variation.

After ligation of the main excretory ducts the rat submaxillary and sublingual glands atrophied by about two thirds due to a reduction of the cytoplasm of the acinar cells. This atrophy was not accompanied by a corresponding loss of noradrenaline, the level of which increased 2–3 times as a consequence of an increased density of the adrenergic ground plexus enclosing the acini in the submaxillary gland. A salivary gland with atrophied acinar cells has been found to be valuable in studies on postsynaptic events of adrenergic transmission.

After ligation of the excretory ducts and subsequent atrophy of the parenchymal cells of the rat sub-maxillary and sublingual glands, a severe reduction of the MAO activity was demonstrated both biochemically (more than 60 per cent decrease expressed as activity per gland) and histochemically. Chronic postganglionic sympathectomy produced a much smaller diminution of the MAO activity in these glands. It is concluded that the major part of the MAO in rat salivary glands occurs in the parenchymal cells.

Adrenergic receptor function in the human ductus arteriosus has been demonstrated by in vitro perfusion and spiral strip experiments and correlated with the finding of specific adrenergic nerve fibers in the smooth muscle layer of the media by histochemical fluorescence technique. The results support the possible role of an adrenergic mechanism involved in the closure of the ductus in neonates.

Reappearance of noradrenaline (NA) in the denervated sheep heart as a result of ingrowth of regenerating adrenergic nerves was first shown by Goodall (1951). Growth of the sympathetic adrenergic neurons has also been studied with the Falck—Hillarp fluorescence technique for histochemical visualization of monoamines under various experimental conditions. Thus, ingrowth of noradrenaline nerve terminals into pineal gland transplants was observed by Owman (1964). Little attention, however, has been given to the organization of newly formed sympathetic innervation apparatuses of transplanted tissues and organs. An investigation is therefore now being carried out to elucidate the development and appearance of such nerves in different grafted tissues. Small pieces of various organs were aseptically transplanted to the anterior chamber of the eye of adult Sprague-Dawley rats. Autologous, homologous and heterologous grafts (from N.M.R.I.-mice) were used. Host eyes and grafts were observed daily in vivo with a dissecting microscope and the animals sacrificed after different time intervals. Transplants and irides of the host eyes were then prepared for fluorescence microscopy as described earlier (Norberg and Hamberger 1964, Malmfors 1965, see also Corrodi and Jonsson 1967).