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(1) Adult male rats were injected with 50 μg of 5,6-dihydroxytryptamine (5,6-HT) into the left lateral ventricle. The process of de- and regeneration of the bulbospinal 5-hydroxytryptamine (5-HT) neuron systems was followed with monoamine fluorescence histochemistry, [3H]5-HT uptake measurements and estimations of the hindlimb extensor reflex. (2) Fluorescence histochemical estimations revealed a complete or almost complete disappearance of 5-HT nerve terminals in the gray matter of the spinal cord in all segments studied 8, 12 and 14 days after treatment as compared to controls. A gradual increase in a cranio-caudal direction was observed after 1 and 2 months and after 3 months there was a prominent increase in 5-HT nerve terminal density, to about one-half to two-thirds of original levels. (3) [3H]5-HT uptake showed a decrease to about 25% of control values 14 days after 5,6-HT and a recovery in a cranio-caudal direction as seen with fluorescence histochemistry 1, 2 and 3 months after treatment, reaching 75–85% of control values 3 months after 5,6-HT injection. (4) 5-HT receptor sensitivity was tested with the help of the hindlimb extensor reflex on acutely spinalized animals treated with nialamide 500 mg/kg i.p. for 1 h and l -tryptophan 50 mg/kg i.v. Two days after 5,6-HT injection no difference between controls and treated animals could be seen. A clearcut supersensitivity was present 8 and 14 days and 1 month after treatment. Parallel to the reappearance of 5-HT nerve terminals seen with fluorescence histochemistry and [3H]5-HT uptake a normalization of the reflex appeared, after 2 and 3 months. (5) 5-HT receptor supersensitivity was also demonstrated with 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a postsynaptic 5-HT receptor stimulating agent, 4 and 12 days after 5,6-HT injection. (6) We conclude that the 5,6-HT induced disappearance of spinal 5-HT nerve terminals causes 5-HT receptor supersensitivity and that (with the present dose of 5,6-HT) new terminals eventually grow out to normalize function of the 5-HT synapses as indicated by the disappearance of receptor supersensitivity.

The intraocular transplantation technique was used to study maturation of cerebellar homografts with electrophysiological and cytological methods. Taking of the transplants was optimal using donors with a gestational age of 15–17 days, whereafter it declined markedly. The time-table for the cytological maturation mimicked the in situ maturation surprisingly well, being retarded at the most by 5 days. External granule cells appeared 5 days postoperatively, and had completely disappeared about 5 weeks later. Internal granule cells appeared from about day 12. Purkinje cells were identified in clusters from day 8. They slowly fanned out to form a monolayer after about 3 weeks in oculo, situated between an internal granule cell layer and a molecular layer. The molecular layer was clearly recognized from the third week and often contained apical dendrites from the Purkinje cells. The overall cytological organization of the mature transplants was very similar to the normal cerebellum, also including some foliation and always including typical trialaminar areas. The intraocular transplantation technique is thus very suitable for producing cytologically homotypical cerebellar transplants. The electrophysiological maturation of the transplants was also followed using extracellular recordings. A spontaneous discharge pattern strongly suggesting the presence of Purkinje cells mimicked the well known in situ activity both regarding time course and way of firing. Thus, few single spikes were recorded 12 days postoperatively having very few high frequency bursts. The interspike intervals and the inter- and intraburst intervals then gradually decreased to reach values very close to those of normal adult cerebellum about 4 weeks postoperatively. Interspike and interburst interval histograms are presented to show these similarities. Also, potentials evoked by stimulating the base or the surface of the transplants showed action potential responses similar to those of normal cerebellum. Thus, (1) stimulating the base of the transplant, a short latency all-or-none single spike was elicited. (2) When stimulating the surface of the transplant often 1–3 driven spikes over a 5–10 msec period could be elicited. This response was invariably graded. (3) Stimulation of the surface often produced a short latency inhibition of spontaneous discharge lasting 40–120 msec. This response was also graded. The inhibition was shown to be independent of adrenergic fiber influence as shown by superior cervical ganglionectomy or parenteral reserpine administration. The inhibition was antagonized by the GABA-blocker bicuculline when administered parenterally. These 3 responses closely resemble the antidromic spike response, the parallel fiber excitation response and the basket-stellate cell inhibition response respectively, as seen in the normal adult cerebellum in situ. It is concluded that the intraocular cerebellar homografts develop and mature in a very homotypical way as shown by means of electrophysiology and histology in spite of the complete lack of normal connections with the rest of the brain and underlines the importance of intrinsic regulatory mechanisms in CNS ontogeny.

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10(-6) or 10(-5) M alpha-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic beta-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the beta-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.

Publisher Summary This chapter discusses the monoamine neuron systems in the normal and schizophrenic human brain. Fluorescence microscopy of the adult human brain is complicated by the heavy accumulations of lipofuscin in neurons and glia having a strong yellow autofluorescence. In vitro incubations of thin slices of tissue with α-methyl-NA sometimes following preincubation in 6-OH-Dopamine or DMI were carried out. Nerve densities and fluorescence intensities were estimated semiquantitatively. The basic cytoarchitecture of the monoamine neurons present in the rat is present also in man. It is found that there are descending bulbospinal noradrenaline and 5-HT systems, as well as ascending NA and 5-HT systems reaching almost all areas of the brain, having their cell bodies in the lower brain stem.

The effects of chlordiazepoxide, diazepam and nitrazepam on central DA and NA neurons of non-stressed and stressed rats were studied using histochemical and biochemical analysis of DA and NA. Estimations of DA and NA turnover in various regions of the brain were obtained by studying the decrease in stored DA and NA following treatment with a tyrosine hydroxylase inhibitor (H44/68) or a DA-β-hydroxylase inhibitor (FLA63). Stress was induced by immobilization. (1) Stress increased NA turnover in all parts of the brain and the spinal cord. By biochemical methods it was shown that DA turnover in the telencephalon was significantly reduced. (2) By histochemical methods it was shown that chlordiazepoxide and diazepam decreased NA turnover in the cortex cerebri and cortex cerebelli and the hippocampal formation but did not do so to any significant extent in the hypothalamus or the lower brain stem. These results show that the benzodiazepine derivatives decrease the impulse activity in the NA neurons of the locus coeruleus which innervate all cortices of the brain. (3) Chlordiazepoxide and diazepam caused a small but significant decrease in DA turnover in ascending DA neurons to the neostriatum and the limbic forebrain indicating reduced impulse flow in these neurons. (4) Chlordiazepoxide, diazepam and nitrazepam caused a blockade of the stress-induced activation of NA neurons by decreasing nervous activity in these neurons. (5) Chlordiazepoxide and diazepam potentiated the stress-induced decrease in nervous activity in ascending DA neurons. Stress combined with these drugs therefore decreased DA turnover in the telencephalon in comparison with normal controls. In view of the role which the central NA and DA neurons probably play in behavioral and EEG arousal it seems that the decrease in nervous activity in DA and NA neurons produced by chlordiazepoxide and diazepam may be responsible for part of the pharmacological effects of these tranquillizers such as sedation and EEG slowing.

The distribution of monoamine neurons in the human fetal brain was studied by Falck-Hillarp fluorescence histochemistry. Catecholamine (CA)- and 5-hydroxytryptamine (5-HT)-neuron systems were found in the smallest brain studied, obtained from an embryo having a total length of 2.1 cm and a gestational age of 7 weeks. A marked proliferation and differentiation of the monoamine neuron systems took place between the 7th and 23rd week of gestation (the range covered in the present investigation) permitting a mapping of major cell groups, as well as several axon pathways and terminal innervation patterns.

The uptake and accumulation of 3 H-NA was studied in vitro in iris, atrium, submaxillary and sublingual glands, and the muscular and mucosal layers of the duodenum from rats at different ages (0–60 days). Most of the 3 H-NA found in the tissues was located in the adrenergic nerves. The uptake of 3 H-NA increased progressively with age in all the organs examined, when calculated per tissue piece. When the radioactivity was expressed per gram tissue, the concentration in the iris and atrium increased progressively as the animal grew older, but the concentration in the salivary glands and in the gut decreased. The variation in the uptake of 3 H-NA per gram wet weight is probably due to differences in growth rates between neuronal and extraneuronal components of the different organs, rather than to differences in the uptake ability of the adrenergic nerves, during the time of 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.

Small pieces of rat adrenal medulla were homologously transplanted to the anterior chamber of the eye. The eyes were adrenergically denervated. Transplants became attached to and vascularized by the iris of the host eye. Transplants and irides were examined at various times postoperatively with the histochemical fluorescence method of Falck and Hillarp. It was shown that the adrenal medullary transplants were able to produce catecholamine-containing nerves that partly reinnervated the denervated host iris. The nerves derived at least partly from groups of highly fluorescent cells, similar to the adrenaline and/or noradrenaline cells of the normal adrenal medulla. The cells were thus not similar to sympathetic adrenergic nerve cells. Intravasal secretion of fluorescent material was observed in one case, indicating that the transplanted medullary tissue was also able to fullfill its normal endocrinological role of releasing hormones to the blood stream.

A method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA1 content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.

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.

Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.

Summary The effects of barbiturates and meprobamate on the central DA and NA neurones of non-stressed and stressed rats were studied using histochemical and biochemical analysis of DA and NA. Changes induced by the different drug and/or stress treatments in the rate of depletion of the CA stores 4 h after tyrosine hydroxylase inhibition were regarded as changes in turnover. Barbiturates, but not meprobamate, clearly decreased the turnover of NA in the cortical NA nerve terminals. No clear effects were found in the hypothalamus with the two drugs. Stress increased NA turnover in all parts of the brain studied, and these increases could be counteracted by both barbiturates and meprobamate. The net effect on DA turnover in the nerve terminals of the neostriatum and the median eminence was a reduction after 4 h of immobilization stress. The stress-induced decrease in turnover of the DA terminals in the neostriatum was further decreased by pretreatment with barbiturates or meprobamate. In the median eminence, on the other hand, these drugs accelerated DA turnover in the external layer as demonstrated histochemically. The present results indicate that the effects observed on CA turnover are related to the sedative and anti-stress actions of barbiturates and meprobamate. It is unlikely that the specific differences in turnover changes observed between different areas of the brain are the result of a general depressant action on the central nervous system. The effects of barbiturates and meprobamate on CA turnover found in the present study are essentially similar to the effects of the minor tranquillizers of the benzodiazepine group as found in a previous study.

The cerebral and cerebellar cortices of man are richly provided with varicose noradrenaline nerve terminals, which are visualized by fluorescence histochemistry of brain smears obtained by a new technique. The density of such nerves in human cortices equals that of the rat. The method permits simple and rapid analysis of noradrenergic nerves of the human cortex during routine neurosurgical operations.

Sympathische Ganglien, wenn mit Fasern desN. splenicus reinnerviert, bilden 2 pharmakologisch differente Synapsengruppen: normale, vom cholinergischen Typ und ca. 20% exitatorische vom «monoaminergischen» Typ, wenn sie von adrenergen Fasern reinnerviert wurden.

Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 3 1/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescent microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.

Publisher Summary This chapter discusses the post-mortem fluorescence histochemistry of monoamine neuron systems in the human brain. Several factors such as cause of death, prior drug treatment, and post-mortem time can be supposed to influence the amount of amines detectable by fluorescence histochemistry after death. Post-mortem time is perhaps the most important single factor. The large amount of lipofuscin in both neural and non-neuronal elements of the adult human brain is a special problem in fluorescence microscopy because of its strong yellow autofluorescence. While this fluorescence complicates interpretation of black and white microphotographs, the differentiation between specific para formaldehyde induced monoamine fluorescence and lipofuscin fluorescence can mostly be safely made out in the microscope on the basis of the colours, fading characteristics, morphologies, and sharpness of borders of the fluorescent structures,. There are no qualitative differences between chronic schizophrenic and non-schizophrenic brains using Falck–Hillarp fluorescence.

Fetal parietal cerebral cortex was homologously transplanted to the anterior chambers of the eyes of adult rats. The transplants got vascularized, proliferated, as measured by in vivo stereoscopic inspections, and differentiated into brain tissue similar to cortex cerebri in situ and survived for long times, greater than 41/2 months. Fibers from the intact sympathetic adrenergic ground plexus of the iris were able to innervate the transplants in an organotypic way regarding fluorescence morphology, pattern of distribution of the nerve terminals and, to a certain extent, density of innervation, the only variable parameter being density of innervation. Thus, in unpretreated or MAO inhibited transplants only rather few to scattered terminals could be found, while after preincubation in 10(-5)M alpha-methyl-noradrenaline the number of visible terminals was normal or slightly less than normal, as compared to cortex cerebri in situ. When superior cervical ganglia (SCG) were transplanted together with fetal cortex tissue to sympathetically denervated eyes the ingrowth in the cortex tissue was similar to that after single cortex transplantation combined with 5 day old SCG, while a marked hyperinnervation was encountered when combined with adult SCG. It is concluded that the developing cortex cerebri, deprived of its normal CNS source of adrenergic nerves, is able to receive sympathetic adrenergic nerves from the iris in an organotypic way upon transplantation to the anterior chamber of the eye.

The present study provides morphological, biochemical, and functional evidence that after a lesion of descending bulbospinal 5-hydroxytryptamine (5-HT) neurons induced by 5,6-HT, there occurs an almost complete degeneration followed by a relatively complete reinnervation at all levels of the spinal cord. The reinnervation is probably accomplished at least partly by downgrowth of new axons from the surviving proximal ends close to the apparently undamaged cell bodies. These down-growing axons in turn give rise to the new terminal networks with a seemingly normal distribution pattern probably restoring functional integrity to the descending 5-HT pathway. Thus the supersensitivity of the 5-HT receptor that has developed in response to the degeneration disappears as the new 5-HT networks are being formed, suggesting that the 5-HT boutons are localized at the proper sites to restore 5-HT neurotransmission.

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.

The distribution of monoamine-containing neuron systems in adult human brains (including a few cases of mental disease) was studied post mortem by Falck-Hillarp fluorescence histochemistry. Endogenous intracellular monoamine stores decreased slowly with time after death, but varicose catecholamine-containing nerve terminals could be demonstrated in many regions several hours after death. A large number of catecholamine (CA), and probably 5-hydroxytryptamine (5-HT) nerve terminals retained an active concentration-dependent uptake mechanism over the terminals retained an active concentration-dependent uptake mechanism over the cell membrane even when the endogenous amines could no longer be visualized. Thus, e.g. the dopamine (DA) nerve terminals of the nucleus caudatus could be clearly visualized by in vitro incubations of caudate slices in Krebs-Ringer buffer containing 10 −6 Mor10 −5 M α-methyl-noradrenaline up to 7 h after death (longest post mortem time studied). A weak CA fluorescence was observed in some perikarya of the large heavily pigmented neurons of the locus coeruleus and more caudally in the pons, as well as in the substantia nigra. More strongly fluorescent CA nerve cell bodies were found in the hypothalamus close to the third ventricle. CA nerve terminals, probably of the NA type were found in the spinal cord and filum terminale, throughout the brain stem, and in other subcortical and cortical areas. 5-HT nerve terminals were less frequently seen but had a similar distribution. Diffuse CA fluorescence, probably of the DA type was clearly observed in sections of the nucleus caudatus and putamen, and in the nucleus amygdaloideus centralis, but not in the globus pallidus. The possibility of applying fluorescence histochemistry to post mortem brain material, especially following in vitro amine incubations means access to an inexhaustible source of material permitting a more detailed mapping of the monoamine neuron systems in adult man and insight into the development of these neuron systems in different human diseases.

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.

Publisher Summary This chapter discusses the growth characteristics of the sympathetic adrenergic nerves particularly, the problem of morphological and functional reinnervation of denervated tissues in adult animals. The sympathetic innervation apparatus constitutes a widespread 3-dimensional plexus of nerve terminals in most organs. It is involved in a multitude of important regulatory autonomic functions. Almost any kind of injury to the body will inevitably include axotomy of a number of postganglionic sympathetic axons and consequently at least partial sympathetic denervation of a certain region. Reinnervation of such areas in the adult animal then becomes one of the necessities for re-establishment of proper function of the denervated tissues. Reinnervation of transplanted tissues is a specific and rapid process leading to a morphologically normal pattern of nerves that restores adrenergic neurotransmission in the transplants.

Publisher Summary This chapter discusses the post-mortem fluorescence histochemistry of monoamine neuron systems in the human brain. Several factors such as cause of death, prior drug treatment, and post-mortem time can be supposed to influence the amount of amines detectable by fluorescence histochemistry after death. Post-mortem time is perhaps the most important single factor. The large amount of lipofuscin in both neural and non-neuronal elements of the adult human brain is a special problem in fluorescence microscopy because of its strong yellow autofluorescence. While this fluorescence complicates interpretation of black and white microphotographs, the differentiation between specific para formaldehyde induced monoamine fluorescence and lipofuscin fluorescence can mostly be safely made out in the microscope on the basis of the colours, fading characteristics, morphologies, and sharpness of borders of the fluorescent structures,. There are no qualitative differences between chronic schizophrenic and non-schizophrenic brains using Falck–Hillarp fluorescence.

Intermittent electrical stimulation of central monoamine pathways has been carried out in anesthetized rats pretreated with inhibitors of amine synthesis. Depletion of catecholamines, as assessed by the histochemical fluorescence technique, was observed in DA and NA nerve terminal systems in various areas of the brain according to the area of stimulation. Without inhibition of amine synthesis no amine depletion was observed. Stimulation of the dorsal bundle (a collection of NA-containing fibere probably mainly arising from the locus coeruleus (group A6)) resulted in amine depletion mainly of NA terminals in the cortex cerebri and the hippocampal formation. Stimulation of the ventral NA bundle in the mesencephalon resulted in amine depletion mainly of NA terminals in the ventral diencephalon (e.g. hypothalamus) and the ventral parts of the limbic system. Similarly, stimulation of the DA axons arising from the zona compacta of the substantia nigra (group A9) led to depletion of amine from DA nerve terminals in the neostriatum. Stimulation in the A10 cell body area resulted in depletion of amines from NA (hypothalamus + ventral parts of the limbic system) and/or DA (limbic forebrain and/or neostriatum) nerve terminals in the tel- and diencephalon. It is suggested that these effects are the results of stimulation of DA cell bodies and fibers in this area and NA fibers of passage originating from the ventral NA bundle. As the stimulus parameters used in this study were determined in separate experiments on intracerebral self-stimulation, it seems likely that, with certain electrode placements, the levels of stimulation used in such behavioral experiments may be effective in releasing central catecholamines.

The noradrenaline (NA)-axons of the rat sciatic nerve were studied with the histochemical fluorescence technique of Falck and Hillarp. Using freeze-dried and spreadprepared specimens, it was possible to visualize most of the normally present NA-axons in spite of their low content of noradrenaline. The degenerative and regenerative patterns of these fibres were examined after cutting or compressing the nerve. The superior cervical ganglion was transplanted to the sciatic nerve in order to study the outgrowth of NA-axons from the ganglion into the nerve trunk. The findings were as follows: 1. The normally present NA-axons run singly or in small groups in the nerve trunk and have a lower fluorescence intensity than adrenergic nerve terminals. They are relatively smooth in their most proximal parts, but become more beaded distally. 2. After interruption of the axons by cutting or compression, the fluorescent fibres in the distal stump loose their fluorescence and thus the noradrenaline content within 45 hrs probably in a proximo-distal fashion. 3. It was confirmed that a heavy accumulation of noradrenaline rapidly develops above a compression. The accumulated parts closest to the compression desintegrate into degeneration spheroids. At this level the regeneration then starts. 4. Regenerating fibres show great morphological variability. Thin sprouting fibres as well as larger typical growth cones are described. The thinnest fibres were found to grow initially as fast as 9.2 mm/24 hrs. The minimum mean growth rate for most of the fluorescent fibres in the nerve was 1.4 mm/24 hrs during the first week, and 2.9 mm/24 hrs during the second week. 5. NA-axons from the superior cervical ganglion transplanted to the proximally cut sciatic nerve grow vigorously in both directions into the nerve. The growth rate for these fibres was at least equal to that mentioned above. When transplanted to the intact sciatic nerve, however, the adrenergic neurons of the ganglion seemed to give rise to fluorescent fibres to a much lesser extent.

Various tissues from mice treated with a nerve-growth factor (NGF) were studied with the histochemical technique ofFalck andHillarp, which visualizes the adrenergic transmitter in the sympathetic postganglionic neurons. Growth stimulation was detectable in all parts of the sympathetic adrenergic neurons. An increased density of the adrenergic ground plexus was observed in e.g. the iris, submaxillary and parotid glands, blood vessels and intramural ganglionic plexuses of the intestinal tract. Normally non-innervated tissues were also found to contain a considerable number of adrenergic terminals. Of special interest is the striking increase in number of adrenergic terminals in various types of autonomic ganglia, in all probability with an inhibitory effect on ganglionic transmission.

The adrenergic innervation of the major salivary glands in the rat has been studied by a specific histochemical method for the visualization of the adrenergic transmitter. Adrenergic varicose nerve fibres were found, located in a typical adrenergic ground plexus closely surrounding the serous acini of the submaxillary and parotid glands, but not the acini of the mainly mucous sublingual gland. The ducts were found to be completely devoid of adrenergic innervation. Arterioles and venules in the stroma of all three glands and certain very small vessels, possibly the sphincters of arterio-venous anastomoses, were also richly innervated by adrenergic vasomotor fibres. The relationship of the adrenergic nerve fibres to the different functional units of the gland parenchyma is discussed.

The early ontogeny of the monoamine neuron systems in the rat brain has been analysed using Falck-Hillarp fluorescence histochemistry. Serial sagittal sections of embryos with a crown rump length between 7 and 13 mm, approximately corresponding to gestational days 12 to 15 were obtained from mothers treated with a monoamine oxidase inhibitor given in order to increase the monoamine levels of the embryos. 5-hydroxytryptamine (5-HT)-neurons made their first appearance in the 8 mm embryo, dopamine (DA)-neurons in the 9 mm embryo, and noradrenaline (NA)-neurons in the 11 mm embryo. Small, rounded, weakly fluorescent cell bodies forming sparse aggregations appeared first. Fluorescent processes of two types soon appeared. Short processes from the cell bodies were running perpendicular to the long axis of the brain stem within the cell groups, while long slender axon bundles could be traced ascending through the met- and mesencephalon and into the prosencephalon as well as descending in the myelencephalon andspinal cord. In the 12 mm embryo the primordial DA cell formation of the substantia nigra with its striatal projections, the 5-HT neuron formations of the caudal mesencephalon, met- and myelencephalon as well as the NA neurons of the met- and myelencephalon are relatively well developed. It is concluded that the monoamine-neurons develop mechanisms for synthesis and storage of amines at a very early stage during ontogeny, thus recapitulating the phylogeny of these old systems. Likewise, monoamine oxidase is present early. The presence of neurotransmitters specifying the different developing neurons long before development of their nerve terminal areas and therefore before the establishment of normal synaptic function may indicate a role of these substances during ontogeny other than transmission of nerve impulses.

Publisher Summary This chapter discusses the monoamine neuron systems in the normal and schizophrenic human brain. Fluorescence microscopy of the adult human brain is complicated by the heavy accumulations of lipofuscin in neurons and glia having a strong yellow autofluorescence. In vitro incubations of thin slices of tissue with α-methyl-NA sometimes following preincubation in 6-OH-Dopamine or DMI were carried out. Nerve densities and fluorescence intensities were estimated semiquantitatively. The basic cytoarchitecture of the monoamine neurons present in the rat is present also in man. It is found that there are descending bulbospinal noradrenaline and 5-HT systems, as well as ascending NA and 5-HT systems reaching almost all areas of the brain, having their cell bodies in the lower brain stem.

Publisher Summary This chapter describes the nerve growth specificity and regulation as revealed by the intraocular brain tissue transplants. The central monoamine neurons have a remarkable growth potential. They develop early, often innervate large areas, and in most cases respond to axotomy by pronounced regenerative growth also in the mature animal. Using the group-one type of monoamine neuroblast-containing transplants from the brain stem more or less homotypical innervation patterns were found. After a month or more in oculo, clusters of mature multipolar monoamine nerve cell bodies were found that gave rise to varicose nerve terminals innervating other areas of the transplant containing groups of nonfluorescent neurons. It is found that when NA and 5-HT neurons or DA and 5-HT neurons were present together, they tended to form separate groups of cell bodies while their terminal ramifications were closely intermingled, often seemingly innervating the same cells in the transplants. A most interesting feature of the central monoamine neurons was their pronounced ability to reinnervate the sympathetically denervated host iris. It is found that from the site of attachment of the transplants, heavy bundles of fluorescent axons radiated out over the surface of the iris just like adrenergic nerves fan out over the iris from a transplant of an adrenergic ganglion.

A new freeze-drier for histochemical use is described. It uses a refrigerated cooling bath for outer cooling and large amounts of phosphorous pentoxide as water vapor trap.

Publisher Summary This chapter describes the enhanced sensitivity to the noradrenaline (NA) receptor stimulating agent, clonidine, following degeneration of noradrenaline pathways. Systemic injections of 6-OH-DA to newborn rats led to a marked reduction of NA nerve terminals in the cortex cerebri and in the spinal cord, whereas the pons-medulla region became highly innervated by NA terminals. The resting values of arterial pressure, heart rate, and respiration rate were in the same order of magnitude in the treated animals and in the controls. The effect of clonidine on arterial pressure and heart rate did not differ among the groups, but there was a potentiation of the respiratory rate lowering effect in the 6-OHDA- treated group. This finding was interpreted as because a supersensitivity development of the NA receptors in areas lacking NA terminals, such as the cerebral cortex and spinal cord. 6-OH-DA was injected into the dorsal NA bundle. After 2 weeks, there was an almost complete denervation of the NA projections to the limbic cortex and neocortex. Clonidine was given to these rats and the effects were compared with controls. There was no significant difference in the resting levels of arterial pressure, heart rate, or respiration rate between the treated rats and the controls.

Nach einseitigen Lasionen im Rattenhirn an der Grenze zwischen Pons und Mesencephalon, konnte auf Grund von anterograden und retrograden neuronalen Veranderungen gezeigt werden, dass mindestens 2/3 der noradrenergenen Endigungen im Telencephalon und Diencephalon hauptsachlich zu den ungekreuzten, von Zellkorpern aus Pons und Medulla oblongata stammenden, Axonen gehoren.

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).