Leif Wiklund

Afferent and efferent connections of striatal grafts implanted into the ibotenic acid lesioned neostriatum in adult rats

SummaryThe afferent and efferent connections of grafts of fetal caudate-putamen, implanted into the ibotenic acid (IA)-lesioned striatum of adult rats, have been studied with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) as a combined retrograde and anterograde tracer, and with aldehyde fluorescence histochemistry for the visualisation of dopamine-containing nigrostriatal afferents from the host. The WGA-HRP was deposited in crystalline form (within a capillary tip) either into the depth of the graft tissue, or into the IA lesioned host striatum as a control. Labelling was only evaluated in specimens where the WGA-HRP deposit was entirely confined within the graft. Retrogradely labelled neurons were most consistently found in the ipsilateral host substantia nigra and the spared portions of the host CP, and in one case also in the midline and intralaminar thalamic nuclei normally projecting to the striatum. Some neurons, although weakly labelled, occurred in the deep layers of the frontal cortex in all grafted rats. Signs of anterograde WGA-HRP labelling in the host were found in one of the five animals in the ipsilateral globus pallidus and substantia nigra, pars reticulata. Fluorescence histochemistry revealed extensive ingrowth of dopamine-containing fibres from the host striatum into the grafted striatal tissue. The ingrowing fibres formed distinct and partly interconnected patches, most prominently in the peripheral regions of the grafts. The results provide evidence that intrastriatal grafts of fetal striatal tissue receive extensive dopaminergic afferents from the host substantia nigra, and that they may be capable of establishing connections also with thalamus, neocortex and globus pallidus of the host, as well as with the spared portions of the host caudate-putamen. The afferent connections from the thalamus and neocortex were notably more variable and sparse. However, since the control WGA-HRP deposits (into the lesioned host striatum) labelled the cortical and thalamic afferent neurons only poorly, it appears that the cortico-striatal and thalamo-striatal afferents (in contrast to the nigro-striatal ones) had undergone substantial degenerative changes (atrophy and/or cell death) in the long-term (6–11 months) IA-lesioned rats. The sparse thalamic and cortical afferent connections to the grafts may thus reflect an inability of the grafted striatal tissue to prevent the course of degenerative changes in these striatal input systems.

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and d-[3H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benzidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in the lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surrounding the perihypoglossal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminalis, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of D-[3H]aspartate (D[3H]Asp) and autoradiography consistently revealed retrograde labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of D-[3H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an excitatory amino acid as transmitter. Significant numbers of D-[3H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large D-[3H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to surrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents with relatively lower affinity for D-[3H]Asp. Several afferents to NRD failed to label with D-[3H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.

Description of an indolaminergic cell component in the cat locus coeruleus: A fluorescence histochemical and radioautographic study

Using Falck-Hillarp fluorescence histochemical and radioautographic techniques, it has been found that, in addition to the well-known catecholaminergic cells, the locus coeruleus (LC) of the cat contains a sizeable component of indolaminergic neurons. Indolaminergic cell bodies occur in all subdivisions of the LC complex. They are most numerous in the LC proper and subcoeruleus area, but are also present in the medial and lateral parabrachial, and Kölliker-Fuse nuclei. In all, the indolaminergic cells are estimated to make up 7-10% of the monoaminergic neuronal population of the LC complex. With the exception of the Kölliker-Fuse nucleus, where somewhat larger cells occur, the indolaminergic cell bodies in different parts of the LC complex share a common fluorescence histochemical appearance. They display round to fusiform shapes and measure 30 x 18 micron on the average, which makes them cytoarchitectonically similar to the small type of noradrenergic cells in the LC. The formaldehyde-induced fluorescence of the indolaminergic cells in the LC complex was analyzed microspectrofluorometrically and the recorded excitation and emission spectra (maxima at 370 and 530 nm, respectively) were found to be identical with those recorded from midline raphe neurons. No evidence of noradrenaline content was found in the indolaminergic cells of the LC. Radioautographic experiments after intratissular injections of tritiated serotonin showed that the indolaminergic cells of the LC complex possess uptake mechanisms for serotonin. Taken together these results provide strong evidence for serotonin being the transmitter of the indolaminergic neurons discovered in the LC of the cat.

Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations

Abstract The degenerative and regenerative processes following 75 μg 5,6-dihydroxytryptamine (5,6-DHT) have been investigated in the bulbospinal serotoninergic system with a fluorescence histochemical technique. Acutely, 1–3 weeks after 5,6-DHT an extensive disappearance of serotoninergic terminals was observed in the spinal gray, inferior olive, and the facial and hypoglossal nuclei, while the spinal trigeminal nucleus and regions of the reticular formation were partially denervated. Concomitantly, large numbers of swollen, highly fluorescent profiles, interpreted as the proximal stumps of lesioned serotoninergic axons, appeared in the medulla oblongata and most rostral cervical spinal cord. The first signs of regenerative sprouting from the swollen proximal stumps appeared one week after 5,6-DHT, and was prominent during subsequent weeks. The rate of reinnervation of different target areas was dependent on the distance to the regrowing axons; it was most rapid in brain stem nuclei. About one month after 5,6-DHT a terminal plexus of normal density had reappeared in both inferior olive and the facial nucleus. The axonal proliferation continued, however, to produce increasingly abnormal hyperinnervations. Certain abnormalities in terminal fiber morphology occurred: the fibers were coarser and exhibited higher fluorescence than normal, and in the facial and motor trigeminal nuclei rather thick, smooth fibers with pericellular arrangement appeared after long survival times (7–19 months). The reinnervation of spinal cord followed a slower time course. A normal amount of innervation was only reformed in the rostral 3–4 mm of the cervical spinal cord, while thoracic and lumbar levels only recovered a small part of the original terminal density. The regenerative growth demonstrated a high degree of specificity, which indicates precise mechanisms of guidance and recognition. These are presumably similar, or identical, to those regulating ontogenetic development of the serotoninergic system. Limitations of these regulatory mechanisms were, however, demonstrated both by the abnormal proximo-distal distribution of regenerated terminals, and in the inferior olivary complex, where the regrowing fibers invaded areas normally devoid of serotoninergic innervation. It is suggested that the drive of axonal growth is derived from an inherent program of the serotoninergic neurons to establish a predetermined amount of axonal arborizations. The role of the target tissue may be a mere provision of cues for recognition. The regulatory neuronal program seems to lack a mechanism to terminate further proliferation when an innervation of normal density has been reformed.

A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with the antiserum to nerve growth factor

In the adult mouse, the antiserum to nerve growth factor (NGF) induced marked atrophic changes of the ganglionic cell bodies in the superior cervical ganglion (SCG) and a disappearance of adrenergic nerve terminals in several peripheral tissues. By fluorescence histochemistry a lower-than-normal content of the noradrenaline (NA) transmitter was observed within the entire adrenergic neurone only 1 day after a single injection of NGF-antiserum (0.1 ml/g body weight). An atrophy of adrenergic nerve cell bodies and a disappearance of adrenergic nerve terminals were observed after 3 days, but the antiserum-induced effects did not appear maximally developed until 7 days after treatment. These fluorescence histochemical findings were paralleled by a gradual decrease of the endogenous NA levels in peripheral tissues and also of the weight of the SCG. A gradually proceeding restoration towards normal of the adrenergic innervation apparatus was observed fluorescence histochemically following a 5-day treatment with NGF-antiserum (0.1 ml/g body weight each dose), and after 6 weeks to 3 months a normal or close to normal fluorescence microscopical appearance was regained in the peripheral tissues and also in the SCG. These findings were parelleled by the results of the determinations of endogenous NA in peripheral tissues and by the results of weighing the SCG. We discuss some important differences between NGF-antiserum and 6-hydroxydopamine with respect to their mode of action on the mature sympathetic nervous system. Finally, we suggest that a decreased availability of NGF in a terminal area, due to an interference with endogenous NGF by NGF-antibodies, may temporarily result in an impaired function of the supplying adrenergic neurone, including a degeneration of nerve terminals.

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.

Autoradiographic and electrophysiological evidence for excitatory amino acid transmission in the periaqueductal gray projection to nucleus raphe magnus in the rat

Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25-50 nl 10(-2) or 10(-3) M D-[3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2-12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and gamma-D-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N-methyl-D-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.

Mechanisms of regrowth of the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. I. Biochemical correlates

Abstract The regrowth of the bulbospinal serotonin neurons after 5,6-dihydroxytryptamine (5,6-DHT)-induced axotomy has been followed between 1–2 weeks and 8 months after neurotoxin injection, using determinations of endogenous serotonin (5-HT) and [ 3 H]5-HT uptake, and measurements of the conversion of [ 3 H]tryptophan ([ 3 H]TP) into [ 3 H]5-HT and [ 3 H]5-HIAA in vitro. In the spinal cord there was, at 2 weeks after the 5,6-DHT treatment (75 μg), a drastic reduction in all three parameters throughout the cord signifying that the 5,6-DHT-induced axotomy causes an almost complete denervation of the spinal cord from the lumbar up to the most cranial cervical segments. With time there was a gradual recovery in the [ 3 H]5-HT uptake, which appeared to progress in a cranio-caudal direction. By 2 months the recovery was noted mainly in the upper cervical segments; by 4 months it was evident also in the lower cervical segments, and by 8 months it had reached the thoracic and lumbar segments. By 7–8 months, when the [ 3 H]5-HT uptake had recovered to 60% of normal in the upper cervical segments and to 25% of normal in the lumbar cord, endogenous 5-HT had recovered to 34% of the age-matched vehicle-treated controls in the cervical cord and to 13% in the lumbar cord, while the [ 3 H]5-HT synthesis from [ 3 H]TP had recovered to about 40–50%. The [ 3 H]5-HIAA/[ 3 H]5-HT ratio (reflecting the rate of metabolism of the newly synthesized transmitter) and the medium/tissue ratio (reflecting the rate of spontaneous release under the in vitro conditions) had undergone a more than two-fold increase in the long-term 5,6-DHT-treated rats, indicating an increased turnover of the transmitter in the partially reinnervated spinal cord. In medulla oblongata, where the cell bodies of origin of the bulbospinal serotonin neurons, as well as proximal terminal areas are located, there was acutely a partial (40–50%) reduction in [ 3 H]5-HT uptake and [ 3 H]5-HT synthesis capacity, whereas the endogenous 5-HT content was increased by 30% (due to accumulation of the transmitter in the lesioned axon stumps). There was a rapid and extensive recovery, resulting in supranormal values in all three parameters by 7–8 months. This was accompanied by a marked retardation of the in vitro turnover of newly-synthesized [ 3 H]5-HT (reflected in marked reductions in the [ 3 H]5-HIAA/[ 3 H]5-HT and the medium/tissue ratios). The results demonstrate an extensive recovery of the neurotransmitter biosynthetic machinery during regeneration of the chemically axotomized serotonin neurons. Moreover, the observations of increased transmitter turnover in the incompletely reinnervated areas in the spinal cord and decreased turnover in the hyperinnervated brain stem areas suggest that regional compensatory mechanisms may alleviate the functional consequences of the abnormal distribution of the regenerated terminals. It is proposed that with a combination of axonal regeneration, increased utilization of the transmitter, and development of receptor supersensitivity also an incomplete regeneration, resulting in a partial reinnervation of an area, can become efficient in restoring normal function in the CNS.

The indolaminergic innervation of the inferior olive. 1. Convergence with the direct spinal afferents in the areas projecting to the cerebellar anterior lobe.

Abstract The distribution of indolaminergic innervation has been mapped in the inferior olivary complex using the Falck-Hillarp formaddehyde histofluorescence method. The results demonstrate that olivary neurones projecting to different sagittal zones of the cerebellar cortex receive widely different densities of indolamine-containing (probably serotoninergic) terminals. In the cat the densest innervation was found in the caudal part of the medial accessory nucleus and the caudal and caudolateral parts of the dorsal accessory nucleus, known to project to the medial A zone and the lateral B zone, respectively, of the anterior lobe vermis. Interestingly, these olivary areas seem to coincide with those previously shown to receive direct afferent connections from the spinal cord. In contrast, the rostromedial part of the dorsal accessory nucleus, projecting to the C 1 and C 3 zones of the cerebellar pars intermedia, showed only sparse innervation. In the rostral part of the medial accessory nucleus, projecting to the C 2 zone of pars intermedia, virtually no indolaminergic terminals were found. Indolaminergic innervation of medium density occurred in olivary areas that have been described to project further caudally in the cerebellar vermis, such as the dorsomedial cell column, the β-subnucleus and the dorsal cap of Kooy. In the rat inferior olive, the indolaminergic innervation was confined to the lateral part of the dorsal accessory nucleus, all other regions exhibiting no or only sparse terminal supplies. A comparison between the patterns of indolaminergic and catecholaminergic innervations has also been made, showing that the distributions of the two types of monoaminergic olivary afferents are very different in both species. A particularly interesting situation was observed in the dorsal accessory nucleus where the two innervations seemed to be complementary. Thus, the catecholaminergic innervation was confined to that part of the nucleus having only sparse indolaminergic fibre supply.

Distribution and numbers of indoleamine cell bodies in the cat brainstem determined with Falck-Hillarp fluorescence histochemistry

Using the Falck-Hillarp method, the cat brainstem was found to contain approximately 60,000 indoleamine (IA) cells. Most of these (46,000 or 77%) are located within the raphe nuclei. Nissl-stained material demonstrated both medium- and small-sized perikarya in the raphe nuclei, and quantitation revealed that the IA cells comprise only part of the medium-sized cells. Thus, the raphe dorsalis holds about 24,000 IA cells representing some 70% of its medium-sized cells. Corresponding values were for raphe pallidus 8,000 IA cells (55%), raphe centralis superior 7,400 (35%), raphe magnus 2,400 (15%), raphe obscurus 2,300 (33%), linearis intermedius 2,100 (23%), and raphe pontis 280 (9%). A considerable number of IA cells (13,600, representing 23% of the total) were found in locations outside the raphe nuclei: in ventral brainstem as lateral extensions from the raphe, among the bundles of fasciculus longitudinalis medialis, in periventricular gray and adjacent tegmentum, mixing with the noradrenergic cells of the locus coeruleus complex, among the mesencephalic dopamine cells, and in the nucleus interpeduncularis.