Anders Bjo¨rklund

Plastic changes in the adrenergic innervation of the rat septal area in response to denervation

Abstract The adrenergic innervation of the rat septal area was studied using the Falck-Hillarp histofluorescence method in normal animals and in ones in which the septum had been denervated by a unilateral hippocampal lesion. In the normal septum adrenergic terminals are found in all of the principal septal nuclei with the heaviest innervation apparent in the lateral septal nucleus, the nucleus accumbens and the interstitial nucleus of the stria terminalis. A smaller but significant innervation is also present in the medial septal nucleus, nucleus of the diagonal band, posterior septal nucleus and the hippocampal rudiment. Following a total, unilateral transection of the hippocampal fimbria, there is a marked increase in the adrenergic innervation of those septal nuclei that receive a significant projection from the hippocampus. This first appears between 8 and 15 days after operation, becomes marked by 30–60 days, and persists for at least 100 days after the septal denervation. The increase in the number of adrenergic terminals is initially found in the caudal part of the lateral septal nucleus and it subsequently is evident through most of the lateral septal nucleus, the medial septal nucleus and the posterior septal nucleus. No changes were noted in the other nuclei of the septal area. Concomitant with the histochemically demonstrable increase in adrenergic innervation, there is a biochemically detectable increase in the norepinephrine content of the septum. The source of the adrenergic innervation in both the normal and denervated septum appears to be axons traversing the medial forebrain bundle since section of this tract produces a substantial reduction in the adrenergic innervation of the septal nuclei. It is concluded that the increase in norepinephrine-containing terminals produced by removal of the non-adrenergic hippocampal afferents is due to the formation of new terminals which have sprouted from intact norepinephrine-containing axons normally innervating the septal nuclei.

Evidence of an incerto-hypothalamic dopamine neurone system in the rat

With the recently introduced glyoxylic acid histochemical fluorescence method, a previously unknown catecholamine-containing fibre system has been revealed in the zona incerta, hypothalamus and the caudal septum. These fibres, designated the incerto-hypothalamic system, have a characteristic, very delicate, finely varicose appearance, and they have a weak fluorescence, indicating an unusually low intra-neuronal amine content. On the basis of their distribution a caudal and a rostral part can be discriminated: the caudal part extends from the area of the dopamine-containing cell bodies in the caudal thalamus, the posterior hypothalamic area and the medial zona incerta (the A11 and A13 cell groups) into the dorsal part of the dorso-medial nucleus and the dorsal and anterior hypothalamic areas; the rostral part extends from the area of the rostral periventricular dopaminergic cell system (the A14 cell group) into the medial preoptic area and the periventricular and suprachiasmatic preoptic nuclei. The system probably extends also into the most caudal portion of the lateral septal nucleus. From a series of lesions and in vitro uptake studies, evidence has been obtained that the incerto-hypothalamic fibres are the projections of short, intradiencephalic dopaminergic neurones whose cell bodies are located in the A11, A13 and A14 cell groups. The projection areas of these neurones signify an involvement of the system in the control of secretion of pituitary hormones.

Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival

Neuronal cell suspensions, prepared by trypsination and mechanical disruption from embryonic CNS tissue, are currently used for intracerebral neuronal grafting to deep brain sites. In the present study the viability of suspended neurons from different brain regions has been monitored with a fluorescent vital stain, and studied as a function of time after dissociation and age of the donor rat embryos. Subsequently, the validity of the in vitro viability rates as a criterion for the in vivo survivability of each individual suspension was tested for suspensions prepared from the developing mesencephalon, rich in dopamine-containing neurons. The results indicate that mechanically dissociated embryonic CNS neurons remain viable for several hours in a simple glucose-saline solution at room temperature. The in vitro viability scores declined faster in suspensions prepared from mesencephalon than in those prepared from telencephalon (striatum and basal forebrain), and they declined faster in suspensions prepared from older embryos. The fetal cells were sensitive to the mechanical trauma caused by excessive pipetting, and tissue from older embryos seemed generally more vulnerable in the trypsination-dissociation procedure. The grafting experiments showed a good correlation between the in vitro cell viability counts and in vivo neuronal survival after grafting, indicating that the vital stain, at least under certain conditions, can be used as a simple and practical routine test to check and standardize cell suspensions to be used in intracerebral grafting experiments.

Functional reinnervation of rat hippocampus by locus coeruleus implants

Transplants of the embryonic locus coeruleus (LC) region were implanted into the circuity of the hippocampal formation in adult rats in which the normal adrenergic afferents to the hippocampus had been removed. The growth of new adrenergic axons from the implant in the denervated hippocampus was followed for 1-14 months after surgery by means of fluorescence histochemistry, and the function of the implant-hippocampal connections was tested electrophysiologically after 2-3 months survival. In the successful cases the entire hippocampal formation was reinnervated from the LC implant within 3-6 months after operation, and the newly formed innervation still persisted unchanged by 14 months. The reinnervation was equally effective irrespective of the route by which the axons entered the hippocampus, i.e. along the lesioned fornix-fimbria or along a retrosplenial route. The pattern formed by the ingrowing LC axons mimicked to a large extent that of the normal LC afferents. Little growth was seen into denervated terminal fields of the commissural, septal or entorhinal afferents, pointing to a preference of the ingrowing LC fibers for the areas normally innervated by adrenergic afferents. In the electrophysiological experiments, stimulation of the LC implants caused (in 20 out of 29 cells monitored) an inhibition of the spontaneous activity of neurons in the host hippocampus. This inhibition had a relatively long latency and a long duration, similar to that observed after stimulation of the innate LC in the intact rat. As in the normal rat, the inhibitory responses were blocked by systemic or local application of the beta-adrenergic receptor blockers propranolol or sotalol. It is concluded that the adult rat brain is capable of carrying out all steps involved in correct functional reinnervation of a denervated region. Moreover, the implant-hippocampal preparation should be a highly suitable model system for functional studies of a central noradrenergic connection.

In vivo evidence for a hippocampal adrenergic neuronotrophic factor specifically released on septal deafferentation

Denervation of the hippocampal formation in adult rats through lesions of the septohippocampal pathway was found to induce a trophic growth response in intracortical grafts of sympathetic superior cervical ganglia, and to stimulate regeneration of the intrinsic locus coeruleus adrenergic neurons following chemically induced axotomy. The grafted sympathetic adrenergic neurons grew very poorly into the adjacent hippocampus in animals with the septohippocampal pathways intact. A lesion of the ipsilateral fimbria-fornix or of the medial septum-diagonal band area caused a massive stimulation of axonal growth from the transplanted ganglionic neurons into the denervated hippocampus. This increase was more than 100-fold by 1 month after lesion and about 10-fold by 3 months after lesion. Fluorescence histochemistry revealed that the lesion-induced ingrowth occurred primarily into those areas of the dentate gyrus and hippocampus which were denervated of their septal cholinergic afferents. In addition, the septal and fimbria-fornix lesions induced a marked increase in size and noradrenaline fluorescence of the grafted sympathetic neurons, without any clear-cut effects on the numbers of surviving neurons in the graft. This lesion-induced trophic growth response (increases in axonal outgrowth, cell body size and noradrenaline content) was specific for lesions of the septal (probably primarily cholinergic) innervation of the hippocampus. Thus, extensive denervations induced by lesions of the commissural or perforant path afferents, as well as selective lesions of the intrinsic adrenergic afferents from the locus coeruleus, had no clear-cut effects. The intrinsic central adrenergic neurons were also found to be responsive to the lesion-induced growth-stimulating mechanism. Thus, lesions of the fimbria-fornix or the medial septum-diagonal band area had a marked stimulatory effect on the regeneration of the locus coeruleus noradrenergic neurons after selective chemical axotomy (induced by 5,7-dihydroxytryptamine; 5,7-DHT). Thus, the adrenergic reinnervation of the initially denervated hippocampus was significantly accelerated by 3 weeks after the fimbria-fornix or septal lesions, and the increase persisted for at least 8-10 months after transplantation. These results provide evidence for an adrenergic neuronotrophic factor whose production in the hippocampus normally is under the control of non-adrenergic (probably cholinergic) afferents originating in the septal-diagonal band area. The actions of this putative factor on sympathetic adrenergic neurons resemble those induced by nerve growth factor (NGF). Interestingly, however, the results obtained after 5,7-DHT-induced axotomy indicate that central and peripheral adrenergic neurons are equally responsive, and thus that the putative central adrenergic neuronotrophic factor may play a normal physiological role in the regulation of axonal growth and regeneration within the central nervous system.

Regulation of striatal serotonin release by the lateral habenula-dorsal raphe pathway in the rat as demonstrated by in vivo microdialysis : role of excitatory amino acids and GABA

Striatal extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored with the microdialysis technique during electrical stimulation of the lateral habenula-dorsal raphe (LHb-NRD) pathway in halothane anaesthetized rats. A new double-loop probe, with an improved recovery factor, was implanted into the head of the caudate-putamen and perfused with Ringer solution containing 1 microM of the 5-HT uptake blocker indalpine. Samples were collected every 15 min and analyzed with HPLC coupled to fluorimetric detection. Low frequency stimulation of the LHb (1.5 and 3 Hz, 0.5 mA) produced no detectable changes in striatal indole levels, whereas 15 Hz stimulation induced a 70% increase in 5-HT release. This effect was most likely mediated by a direct LHb-NRD link, since it persisted after ibotenic acid lesions of the interpeduncular nucleus (which is the major projection area for the medial habenular nucleus), but was completely abolished after transection of the fasciculus retroflexus, which carries the axons of the LHb-NRD pathway. The possible identity of the transmitter operating in the LHb-NRD pathway was investigated by NRD injections of kynurenic acid, a potent blocker of excitatory amino acid transmission, and by NRD injections of the GABA antagonist bicuculline. Kynurenic acid (300 nl, 50 mM) did not by itself induce any detectable changes in spontaneous indole output, but completely blocked the effect of LHb stimulation. Injection of bicuculline (300 nl, 2 mM) increased the striatal 5-HT output by about 70%, and potentiated the effect of LHb stimulation by a further 50%. In none of the experiments performed in this study were there any significant changes in striatal 5-HIAA output. These data are compatible with the idea that excitatory amino acids in the LHb-NRD pathway are involved in the regulation of striatal 5-HT release, and that this influence is modulated by GABAergic synaptic activity at the level of the NRD.

Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis

The role of monoamines in the functional regulation of the septo-hippocampal cholinergic system was studied using in vivo microdialysis of acetylcholine (ACh) release in the hippocampus of awake unrestrained rats. Systemic administration of the dopamine receptor agonist apomorphine (2.0 mg/kg) resulted in a 170% increase in hippocampal ACh overflow. Similarly the catecholamine-releasing agent amphetamine (2.5 mg/kg) produced a 400% increase in ACh overflow. The effect induced by amphetamine, but not that of apomorphine, was blocked in animals pretreated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). The effect of amphetamine on ACh release was reduced by 75% after a 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA) but was not affected by 6-OHDA lesions of the noradrenergic dorsal and ventral bundles. However, baseline ACh overflow was increased by 130% by the dorsal and ventral bundle lesions. The serotonin-releasing agent p-chloroamphetamine (2.5 mg/kg) produced a 160% increase in hippocampal ACh release, and this effect was enhanced after a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the serotonin projection system. The results show that surgical or pharmacological manipulations of the ascending brainstem monoaminergic systems, which innervate wide areas of the forebrain, including the septum and the hippocampal formation, have pronounced effects on septo-hippocampal cholinergic activity. Thus, the present data provide support for the view that information regarding behavioral state and arousal is conveyed to the septo-hippocampal system via ascending monoaminergic systems.

Origin, course and termination of the mesohabenular dopamine pathway in the rat

This study describes the organization of the mesohabenular dopamine (DA) system in the rat as revealed by fluorescence histochemistry in combination with lesions, DA uptake experiments and injections of a retrograde tracer. The DA axons were found to be aggregated in a dense terminal field located in the caudal two thirds of the medial part of the lateral habenular nucleus. Microknife lesions of the stria medullaris left this DA innervation unaffected while cuts through the fasciculus retroflexus resulted in the virtual disappearance of the DA innervation. Injections of the fluorescent retrograde tracer True Blue (TB) into the lateral habenula produced labeling of both DA and non-DA-containing cells in the ventral mesencephalon, mainly in the interfascicular nucleus ipsilateral to the injection. This study thus documents the existence of a mesohabenular DA pathway whose cell bodies are located in the ventral mesencephalon and whose axons ascend with the fasciculus retroflexus to terminate in the caudomedial part of the lateral habenular nucleus. This information, taken together with insights gained from other studies, suggests a role for the mesohabenular DA system in modulating telencephalic feedback onto the mesencephalic DA-neurons and also in regulating the output from the dorsal raphe nucleus.

Basic characteristics of noradrenaline release in the hippocampus of intact and 6-hydroxydopamine-lesioned rats as studied by in vivo microdialysis

Extracellular levels of noradrenaline (NA) were measured in the hippocampus of awake and halothane-anaesthetized rats, using intracerebral dialysis coupled to a highly sensitive radioenzymatic assay. In awake animals, steady state NA output after 1 h of perfusion was 69 +/- 21 fmol/30 microliter (15 min). Non-noxious sensory stimulation increased the steady state NA output by 35%, while halothane anaesthesia reduced the levels by 63-72%. During halothane anaesthesia KCl (100 mM) or desipramine (5 microM) added to the perfusion medium increased the NA levels 27-fold and 8-fold, respectively. Tetrodotoxin added in the presence of desipramine reduced the extracellular NA levels by 85%. In 6-OHDA lesioned animals the NA levels were below the limit of detection in virtually all samples (i.e. less than 3 fmol/30 microliter) at 10-16 days after lesion, but there was a partial recovery at longer postoperative time points. These results support the conclusion that the extracellular NA levels recovered in the dialysis perfusates are neuronally derived and can be used as an index of noradrenergic synaptic activity in the brain.

The indolaminergic innervation of the inferior olive. 2. Relation to harmaline induced tremor

The possible involvement of serotoninergic mechanisms in the induction of harmaline generated tremor in the inferior olive has been investigated electrophysiologically in the cat and rat. Mass recordings of Purkinje cell activity in the cat showed that harmaline induces strong, synchronous and rhythmic activity in those parts of the climbing fibre system originating in the caudal part of the medial accessory olive and the caudolateral parts of the dorsal accessory nucleus. These are the areas of the cat olive shown to receive a dense serotoninergic innervation. In the rat, the selective removal of the serotoninergic innervation--produced by an intraventricular injection of 5,6-dihydroxytryptamine, or 5,7-dihydroxytryptamine in combination with desipramine--caused a significant attenuation of both the tremor and the climbing fibre activity induced by an intravenous harmaline injection. In the 5,6-dihydroxytryptamine-treated animals the reappearance of the harmaline tremor seemed to parallel the regrowth of new serotoninergic axon sprouts in the inferior olive. On the basis of the present results it is proposed that the serotoninergic afferents to the accessory olivary nuclei are of critical importance for the tremor induction of harmaline in the inferior olive. It is suggested that harmaline, rather than acting directly on the olivary neurones, exerts its effect through an interference with a serotoninergic (possibly inhibitory) innervation of these cells.