Gunnar Skagerberg

Neuropeptide Y (NPY) in the area of the hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat

Immunocytochemical studies have documented the presence of neuropeptide Y (NPY) in the hypothalamic paraventricular nucleus (PVN) which harbours a large number of neurones that contain corticotrophin-releasing factor (CRF). In this study the close morphological association between NPY fibres and CRF cell bodies in the PVN was confirmed. The localization of NPY terminals in the vicinity of CRF neurones forms a morphological basis for an action of NPY in the hypothalamic control of the pituitary-adrenocortical axis. We therefore microinjected NPY into the area of the PVN of both conscious, freely moving and anaesthetized rats and noted a powerful stimulatory effect on adrenocorticotropic hormone (ACTH) and corticosterone release as measured by radioimmunoassay. In experiments with conscious, freely moving rats, higher ACTH and corticosterone levels were detected following injection of NPY into the area of the PVN than following control injection (desamidated NPY). Intracerebroventricular injection of NPY produced a small, albeit significant, increase in circulating corticosterone levels as compared to control (saline-injected) rats. Anaesthetized rats responded to NPY (but not to saline) injected into the area of the PVN with elevated ACTH and corticosterone levels, while injection of NPY into the neocortex failed to affect the blood concentration of either ACTH or corticosterone. In conclusion, we have demonstrated an activating effect of NPY on the pituitary-adrenocortical axis both in conscious and anaesthetized rats which may reflect the anatomical relationship between NPY fibres and CRF neurones in the PVN.

Origin and termination of the diencephalo-spinal dopamine system in the rat

Using a combination of neonatal 6-hydroxydopamine and adult 5,7-dihydroxytryptamine treatment we have been able to achieve a 94-99% depletion of noradrenaline in the spinal cord. In such animals the dopamine levels are only marginally affected in the dorsal horn (at all levels) and in the intermediate zone at thoraco-lumbar levels. This combined treatment thus offers new possibilities for selective studies of the spinal dopamine projection. In agreement with the biochemical data the fluorescence histochemistry shows that the spinal dopamine innervation is mainly confined to the dorsal horn, the intermediolateral cell column and associated parts of the intermediate and central gray. Injections of fluorescent retrograde tracer combined with monoamine fluorescence histochemistry reveal that the diencephalic A11 cell group is the principal, and perhaps exclusive, source of this innervation. The area of termination, as well as the organizational similarities with certain diencephalic peptide-containing projections to the spinal cord, suggest that the diencephalo-spinal dopamine system may be importantly involved in autonomic regulatory processes.

Organization of diencephalic dopamine neurones projecting to the spinal cord in the rat

Using the aluminium-formaldehyde method for visualization of catecholamines in combination with injections of the fluorescent retrograde tracer True Blue we have studied those diencephalic dopamine (DA)-containing cell groups which have been proposed to give rise to the DA innervation of the spinal cord and investigated the organization of the diencephalospinal DA system in detail. The A13 cell group was found to contain 370, and the A11 cell group 140, DA-producing cells on each side, whereas only very few such cells were found in the paraventricular hypothalamic nucleus. Tracer injections into the spinal cord labelled only DA cells within the A11 group. The overall majority of labelled cells were found ipsilaterally but some cells were also found contralaterally indicating the existence of a minor crossed dopaminergic projection to the spinal cord. Large tracer injections which covered the hemicord at different levels generally resulted in very similar distributions and numbers of retrogradely-labelled DA cells. The labelled DA-containing cells constituted 30-50% of the total number of labelled neurones in the ipsilateral A11 area and about 20-40% of the total number of DA containing cells in this area were labelled. Small injections that did not extend into the nucleus reticularis or the adjacent part of the lateral funiculus failed to label any diencephalic DA cells but usually labelled some non-DA cells in the A11 area. It is concluded that the diencephalospinal DA neurones have long axons that extend over several segments and possibly traverse the entire length of the spinal cord, giving off collateral branches at various levels. From the anatomical data of the present study and previous pharmacological and electrophysiological findings it seems possible that diencephalospinal DA neurones could modulate both sympathetic activity and nociception.

Selective histochemical demonstration of dopamine terminal systems in rat di- and telecephalon: New evidence for dopaminergic innervation of hypothalamic neurosecretory nuclei

The distribution of dopamine (DA)-containing fibers in the virtual absence of noradrenaline (NA)-containing ones has been mapped by aldehyde fluorescence histochemistry in rats subjected to a combined neurotoxin treatment (intracerebral 6-hydroxydopamine injections plus systemic injections of the selective NA neurotoxin DSP-4). This pretreatment left di- and telencephalic DA levels largely unaffected, but reduced the NA levels by at least 86-96%. The resulting DA:NA ratios suggested that the catecholamine-containing structures, demonstrable by fluorescence histochemistry in the di- and telencephalic regions, were predominantly the DA-containing ones. While the distribution of DA terminal systems in the neo- and allocortical regions conformed well to previous results, the combined neurotoxin treatment revealed new features of the distribution of DA fibers in the diencephalon. In addition to the previously described innervations of the tubero-hypophyseal system, the incerto-hypothalamic system, and the mesohabenular pathway, previously unknown innervations were revealed in the supraoptic, paraventricular and dorsomedial nuclei of the hypothalamus, and in the paraventricular nucleus of the thalamus. Apart from some scattered fibers in the periventricular and lateral hypothalamic areas and medical zona incerta, other diencephalic nuclei seemed to be devoid of any significant DA terminal networks. The dopaminergic nature of these innervations is supported by DA uptake experiments (evaluated by fluorescence histochemistry) as well as by independent biochemical and immunohistochemical evidence. It is suggested that the DA innervations of the hypothalamic neurosecretory nuclei originate in cell bodies of the diencephalic A11-A14 cell groups and that such intradiencephalic DA projections participate in the regulation of oxytocin and vasopressin release from the pituitary.

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.

Projections from the ventral tegmental area and mesencephalic raphe to the dorsal raphe nucleus in the rat

SummaryThe origins of the dopaminergic innervation of the rat dorsal raphe nucleus (NRD) have been investigated using a combination of fluorescent retrograde tracing and fluorescence histochemistry. Stereotaxic microinjections of True Blue were placed in the central, caudal and lateral portions of the NRD, and after 6–12 days survival the brains were processed for fluorescence histochemical detection of catecholamines. Retrogradely labeled neurons were searched for in the diencephalic A11 and A13 dopaminergic cell groups, substantia nigra, ventral tegmental area (VTA) and the linear, central superior and dorsal raphe nuclei. The various NRD injections consistently resulted in retrograde labeling of a small number of catecholamine-containing, presumed dopaminergic cell bodies, confined mainly to three regions: the VTA, the linear and central superior raphe nuclei and the NRD itself. The present findings indicate that not only dopaminergic neurons in the VTA but also the system of catecholamine-containing cells, extending dorsally and caudally from the VTA within the midline raphe area, project to the NRD. Although often similar in size, shape and distribution to the catecholaminergic neurons the majority of retrogradely labeled cells in these regions were, however, found to be non-catecholaminergic.

Further studies on the use of the fluorescent retrograde tracer true blue in combination with monoamine histochemistry

Some basic methodological aspects on the use of the retrograde fluorescent tracer True Blue (TB) was studied in freeze-dried material in the rat CNS. On the basis of fluorescence morphology and intensity at different filter settings it was possible to objectively delineate 3 distinct zones of the TB injection site, and define degrees of retrograde labelling from the different zones. Correlative studies of injection sites in subportions of the spinal cord and retrograde labelling in cell bodies of defined nuclei in the brainstem and cortex showed that effective uptake occurred only from the central area of the injection (zone I), while the uptake from the less intensely fluorescent zone II was variable and could only be documented for systems terminating within this zone. Time-course studies revealed that the size of the injection site and the resulting retrograde labelling is stable up to at least two months after injection and that relatively long survival times are often needed for optimal labelling. For practical purposes a bulk transport velocity of 20 mm/day can be used for estimating the survival time required for reasonable retrograde labelling. The accumulation of TB did not interfere with the visualization of monoamines in the same neurones, and the tracer was never seen to be anterogradely or transcellularly transported.

Nitric oxide synthase in the hypothalamic paraventricular nucleus of the female rat; organization of spinal projections and coexistence with oxytocin or vasopressin

We investigated the distributions and interrelations of neuronal nitric oxide (NO) synthase- (nNOS), oxytocin- (OT), and 8-arginine vasopressin- (AVP) immunoreactive (IR) neurons in the paraventricular nucleus (PVN), and the occurrence and distribution of nNOS spinally projecting neurons in the PVN of the female rat. Using double labelling immunohistochemistry, we mapped the distribution of nNOS-, OT- and AVP-immunoreactive (IR) neuronal cell bodies in the different parts of the PVN. About 80% of nNOS-IR cell bodies were magnocellular. About 30% of the nNOS-IR cell bodies were OT-IR, colocalization being most frequent in the rostral parts. In comparison, only approximately 3% of all nNOS-IR cell bodies were AVP-IR, evenly distributed throughout the PVN. True Blue (TB), administered unilaterally into the spinal cord, disclosed that most spinally projecting cell bodies in the PVN were localized in caudal parts. Combined TB tracing and nNOS immunohistochemistry showed that approximately 30% of spinally projecting neurons in the PVN were nNOS-IR, and that approximately 40% of these were magnocellular. Ipsilateral nNOS spinal projections were about eight times more frequent than the contralateral nNOS projections. The study describes the detailed neuroanatomical organization of nNOS neurons coexpressing OT or AVP, and of nNOS spinally projecting neurons within defined parts of the PVN. In contrast to the paraventriculo-spinal system in general, we show that the nNOS paraventriculo-spinal pathway to a large extent originates in magnocellular cell bodies. The results suggest that NO is an important messenger in the paraventriculo-spinal pathway that may in part act in concert with OT.

Spinal projections of hypothalamic histidine decarboxylaseimmunoreactive neurones

The existence of a histidine decarboxylase (HDC)-immunoreactive diencephalo-spinal pathway in the rat was demonstrated using an antiserum raised against HDC from fetal rat liver. HDC-immunoreactive nerve cell bodies were numerous in the ventral and lateral caudal hypothalamus. More caudally, in the mesencephalon, no cell bodies were observed but fairly many, transversely cut nerve fibres, were found in association with the fasiculus longitudinalis medialis bilaterally. At the most caudal medullary level, these longitudinally passing fibres became displaced ventrally to a position just laterally to the pyramidal decussation. In the spinal cord the fibres were more dispersed and rather sparse in most areas. The existence of a diencephalo-spinal HDC-immunoreactive pathway was verified by analyzing material from rats which had received injections of the retrograde fluorescent tracer True Blue into the cervical spinal cord. True Blue fluorescence and HDC immunofluorescence were found to coexist in a subpopulation of the HDC-immunoreactive neurones in the hypothalamus.

Cerebral Physiological and Biochemical Changes During Vasogenic Brain Oedema Induced by Intrathecal Injection of Bacterial Lipopolysaccharides in Piglets

Summary.Background: The objective of the study was to evaluate biochemical and physiological changes in an experimental model of vasogenic brain oedema utilising techniques also used in routine neuro-intensive care.Method: 32 piglets were randomised to control or experimental group. The latter received an intrathecal injection of lipopolysaccharide (LPS) from E.coli (LPS group). Intracranial pressure (ICP) and mean arterial pressure (MAP) were measured continuously. Intracerebral microdialysis was used for analysing interstitial levels of glucose, pyruvate, lactate, glutamate, glycerol and urea every 30 min. Repeated calculations of mean hemispheric CBF were performed utilising an extra-cranial scintillation detector and intracarotid injection of 133Xe. Cerebral specific gravity was measured and the brains were fixed for histological examinations.Findings: After LPS injection ICP increased reaching a plateau phase after 4–7 hours and CBF increased by 46%. Histological examination showed inflammation with pronounced extravasation of granulocytes. A significant decrease in brain specific gravity (p= 0.022) was obtained. LPS caused a significant decrease in cerebral interstitial concentration of glucose (p=0.0035), and significant increases in lactate concentration (p=0.002) and lactate/pyruvate ratio (p=0.0017). A small but significant increase in glutamate was obtained (p=0.0219). Glycerol did not change significantly.Interpretation: Intrathecal LPS caused an inflammatory reaction with extravasation of granulocytes, increased blood-brain barrier permeability and cerebral oedema. Biochemical analyses indicate increased glycolysis but no signs of cell membrane degradation.