Uptake and retention of [3H]adrenaline by central monoaminergic neurons: a light- and electron-microscope radioautographic study after intraventricular administration in the rat

Paraventricular and paracisternal regions of adult rat central nervous system were investigated by light- and electron-microscope radioautography after intraventricular administration of tritiated adrenaline. In tissue primarily fixed by glutaraldehyde perfusion and post-fixed by immersion in osmium tetroxide, there were no aggregates of silver grains indicative of intraneuronal accumulation of the tracer, except over perivascular nerve terminals at the base of the brain. In contrast, when both fixation and postfixation were carried out by rapid vascular perfusion, preferentially labeled nerve cell bodies and axonal varicosities (i.e. terminals) were detected in various anatomical areas known to contain dopaminergic and/or noradrenergic neurons. Serotoninergic axonal varicosities in the supraependymal plexus and subcommissural organ, as well as a small group of nerve cell bodies of undetermined chemical identity in the n. paraventricularis thalami were also found to be labeled. Addition of a ten-fold higher concentration of non-radioactive serotonin to the solution of [3H]adrenaline suppressed the reactivity in the subcommissural organ and the supraependymal plexus but had no such effect elsewhere in brain. Lesioning of the nigrostriatal dopaminergic system with 6-hydroxydopamine prior to [3H]adrenaline injection eradicated axon terminal labeling in the ipsilateral neostriatum. Electron-microscopic examination of [3H]adrenaline-labeled varicosities in the neostriatum, lateral septum, arcuate nucleus and median eminence extended earlier observations on the ultrastructure of the catecholaminergic innervation of these regions. It was concluded that both dopaminergic and noradrenergic neurons as well as certain serotonin-containing axon terminals can take up and retain [3H]adrenaline, although they probably have lesser affinity for this amine than for their own transmitter. Due to the fact that presumptive adrenergic neurons are intermingled with dopaminergic and noradrenergic elements, further work will be needed to determine to which extent they also contributed to [3H]adrenaline uptake in the present experimental conditions.     

Possible resolution of a paradox concerning the use of p-chlorophenylalanine and 5-hydroxytryptophan: evidence for a mode of action involving adrenaline in manipulating the surge of luteinizing hormone in rats

Various functions involving the central nervous system can be manipulated by the sequential administration of p-chlorophenylalanine and 5-hydroxytryptophan, compounds which respectively inhibit and restore the synthesis of 5-hydroxytryptamine in the brain. An involvement of 5-hydroxytryptamine in the control of a particular function has been considered established when the effect of p-chlorophenylalanine on that function can be overcome by treatment with 5-hydroxytryptophan. This assumption is not, however, invariably substantiated when the functional consequences of other methods of depleting 5-hydroxytryptamine are considered; studies on the control of the daily surge of luteinizing hormone in oestrogen-treated ovariectomized rats present such a paradox. The surge can be prevented by p-chlorophenylalanine and restored by 5-hydroxytryptophan. Nevertheless, neurotoxin-induced lesions of the 5-hydroxytryptamine projections from the raphe nuclei are compatible with a normal occurrence of the surge. We have therefore examined the effects of p-chlorophenylalanine and 5-hydroxytryptophan on hypothalamic monoamines in oestrogen-treated ovariectomized rats and find that the drugs respectively suppress and elevate the concentration of adrenaline in addition to that of 5-hydroxytryptamine. Phenylethanolamine N-methyltransferase, the enzyme responsible for converting noradrenaline to adrenaline, is shown to be inhibited in vivo by p-chlorophenylalanine and in vitro by its metabolite, p-chlorophenylethylamine. The reciprocal effects of p-chlorophenylalanine and 5-hydroxytryptophan on the concentration of adrenaline are of particular interest since drugs which inhibit adrenaline synthesis can block the luteinizing hormone surge. It is proposed that when the 5-hydroxytryptophan-reversible effects of treatment with p-chlorophenylalanine are not reproduced by other procedures which deplete 5-hydroxytryptamine, the significant action of these compounds may involve adrenaline.     

Demonstration of serotoninergic axons terminating on luteinizing hormone-releasing hormone neurons in the preoptic area of the rat using a combination of immunocytochemistry and high resolution autoradiography

The synaptic relationship between serotoninergic terminals and luteinizing hormone-releasing hormone-containing neurons was investigated in the medial preoptic area using a combined technique. Axon terminals selectively taking up 5-[3H]hydroxytryptamine were labelled autoradiographically and luteinizing hormone-releasing hormone-containing neuronal elements were identified by means of immunocytochemistry. Synaptic contacts were observed between tritiated 5-hydroxytryptamine-labelled boutons and luteinizing hormone-releasing hormone-immunoreactive dendrites. About 5% of the boutons which formed synapses with luteinizing hormone-releasing hormone-immunoreactive dendrites were found to be labelled by the tritiated indolamine. Luteinizing hormone-releasing hormone-immunoreactive axon terminals occurred as presynaptic elements in contact with unidentified dendritic spines, shafts or perikarya. These observations provide morphological basis for the idea that 5-hydroxytryptamine-containing neurons can act directly on luteinizing hormone-releasing hormone release. Further, they support the assumption that luteinizing hormone-releasing hormone is not only a neurohormone but may also function as a neurotransmitter or neuromodulator.     

Morphological evidence for a dopaminergic terminal field in the hippocampal formation of young and adult rat

We have visualized the dopaminergic innervation of the hippocampal formation of the rat using two morphological methods: (1) tyrosine hydroxylase immunocytochemistry on noradrenaline-depleted animals and (2) fluorescence histochemistry after the uptake and storage of dopamine on hippocampal slices in vitro. The noradrenergic hippocampal terminal fields were destroyed by neonatal neurotoxin pretreatment and the validity of the lesion checked by the absence of dopamine beta-hydroxylase immunoreactivity. As observed on early postnatal ages, dopaminergic axons reached the hippocampal formation through the fimbria and the alveus, but also through the supracallosal bundle and the ventral amygdaloid area-entorhinal cortex. The temporal (ventral and caudal) part of the hippocampal formation received the bulk of the dopaminergic innervation whereas no fibers were observed in the septal pole. Very few positive axons were visualized in the hilus of the gyrus dentatus and CA3 field, only near the temporal pole. CA1 field (stratum oriens) was innervated throughout its ventral part. The most innervated area was the ventral part--especially the deep layers--of the subiculum, in particular the prosubiculum. The dorsal part of the subiculum displayed some positive axons, although to a lesser extent. The pre- and parasubiculum contained a few positive axons. In addition, some immunoreactive axons were observed in the anterior hippocampal continuation and the indusium griseum. The ventral junction prosubiculum-CA1 field appears to be the main target area for the hippocampal dopaminergic innervation. It is interesting that the same areas are characterized by their projections to the nucleus accumbens which receives dopaminergic afferents. Thus, the hippocampostriatal projections, that represent a link between the limbic and central motor mechanisms, could be under dopaminergic influence.     

Alteration of central alpha 2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin

A peripheral injection of DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] produced a marked, selective, and lasting depletion of norepinephrine in certain regions of the rat central nervous system. This depletion at 10 days after injection was associated with regional alterations in some, but not all, adrenergic binding sites (receptors) as determined by in vitro [3H]prazosin (alpha 1), [3H]p-aminoclonidine (alpha 2), and [3H]dihydroalprenolol (beta) binding. The neocortical alpha 1-receptor was not changed. The alpha 2-receptor in several regions was altered as indicated by an increase in ligand affinity; additionally, the density of this receptor was slightly decreased in some regions. Depending on the region, the beta-receptor either increased in density or was unchanged. The increased density of this receptor in neocortex corresponded to an increased activity of isoproterenol-sensitive adenylate cyclase. These two changes were not affected by subchronic treatment with desipramine, a norepinephrine uptake inhibitor. The changes were, however, partially or completely reversed by subchronic administration of clenbuterol, a centrally-acting beta-receptor agonist. The dopaminergic receptor in various regions was unaltered as assessed by in vivo and/or in vitro binding of [3H]spiperone. The in vivo binding of this ligand also indicated that the serotoninergic receptor in frontal neocortex was unchanged. Assessment of adrenergic receptors in neocortex at 50 days after injection indicated only the above affinity change of the (presumably postsynaptic) alpha 2-receptor. The alpha 1-receptor remained unaltered. The density of the beta-receptor had normalized, as had the activity of isoproterenol-sensitive adenylate cyclase. Implicit in these findings is the following rank order of receptor sensitivity to chronic norepinephrine depletion: alpha 2 greater than beta greater than alpha 1. The use of DSP-4 has clear advantages over other methods of depleting central norepinephrine. This neurotoxin can be administered by intraperitoneal injection, the depletion of norepinephrine can be readily checked by absence of the post-decapitation reflex, and the changes in other neurotransmitter concentrations are relatively minor or nonexistent. The alteration of alpha 2- and beta-receptors, as a consequence of DSP-4 treatment, may form the basis of a new animal model of adrenergic receptor supersensitivity. Such a model may clarify the importance of these central receptors to physiological and behavioral processes.     

Adrenaline turnover rates in the medial preoptic area and mediobasal hypothalamus in relation to the release of luteinizing hormone in female rats

The turnover rates of adrenaline in the medial preoptic area and mediobasal hypothalamus, areas which, respectively, include the cell bodies and terminals of luteinizing hormone-releasing hormone neurons, have been measured in female rats on pro-oestrus, the day of the preovulatory surge of luteinizing hormone, and on dioestrus, the preceding day. A rise in the rate of turnover was found in the medial preoptic area coinciding with the surge of luteinizing hormone in the late afternoon of pro-oestrus; the rate of turnover at this time was higher than at the same time on dioestrus. No changes in turnover rate were found in the mediobasal hypothalamus within either of these days.The results indicate that the adrenaline-containing projections to the preoptic area may be actively involved in the production of the spontaneous preovulatory surge of luteinizing hormone in rats.     

Changes in body weight and food-related behaviour induced by destruction of the ventral or dorsal noradrenergic bundle in the rat

Three experiments contrasted the effects of 6-hydroxydopamine-induced lesions of the ventral noradrenergic and dorsal noradrenergic projections, predominantly to hypothalamus and cortex, respectively, upon body weight changes and food-related behaviour in the rat. In general, ventral noradrenergic bundle lesions enhanced weight gain and these effects were exaggerated by the provision of palatable cheese to the standard chow diet. In contrast, lesions of the dorsal noradrenergic bundle produced minor changes in body weight. Associated with the effects of ventral noradrenergic bundle lesions were hyperphagia, enhanced suppression of intake of food adulterated with quinine, (at high concentration), a small attenuation of food neophobia, and enhanced acquisition, but not performance, of the eating response to tail-pinch stimulation. These ventral noradrenergic bundle lesions failed to alter basal activity levels, amphetamine anorexia or the diurnal pattern of eating or activity. In contrast, lesions of the dorsal noradrenergic bundle did not produce either hyperphagia or enhanced rejection of food adulterated with quinine. However, there was a strong attenuation of food neophobia and a retarded acquisition (but unimpaired performance) of eating in response to tail-pinch stimulation.

The results are discussed in connection with previous studies of ventral and dorsal noradrenergic bundle lesions, with the effects of ventromedial hypothalamic lesions and with the underlying behavioural and physiological processes that mediate these contrasting effects of different neuroanatomical patterns of central noradrenaline depletion.     

Looking at neurotransmitters in the microscope

This review article covers the early period of my career. I first summarize research initiated by the late Nils-Åke Hillarp, after his appointment in 1962 as professor in the Department of Histology at Karolinska Institutet. He only lived for three more years, but during this short period he started up a group of ten students who explored various aspects of the three monoamine transmitters, dopamine, noradrenaline and 5-hydroxytryptamine, using the new formaldehyde fluorescence method developed by Bengt Falck and Hillarp in Lund. This method allowed visualization of the cellular localization in the microscope of these monoamines, which introduced a new discipline in neurobiology—chemical neuroanatomy. I then deal with work aiming at localizing the monoamines at the ultrastructural level, as well as attempts to use radioactively labeled aminoacids, especially γ-aminobutyric acid (GABA), and autoradiography, to identify, in the microscope, neurons using such transmitters. Finally, our immunohistochemical work together with Kjell Fuxe and the late Menek Goldstein, using antibodies to four monoamine-synthesizing enzymes is summarized, including some aspects on the adrenaline neurons, which had escaped detection with the Falck–Hillarp technique.