O.T. Phillipson

A Semiautomated Analysis Method for Catecholamines, Indoleamines, and Some Prominent Metabolites in Microdissected Regions of the Nervous System: An Isocratic HPLC Technique Employing Coulometric Detection and Minimal Sample Preparation

The application of a commercially available coulometric electrochemical detector to the automated HPLC analysis of some monoamines and their metabolites in microdissected areas of the rat nervous system is described. Apart from the stability and high sensitivity of the system, other appealing features of the technique are the facile sample preparation and long‐term sample storage characteristics which show minimal analyte degradation. Basal values of some regional monoamine and metabolite concentration are listed together with a brief appendix that serves as a users guide to the operation and maintenance of the detection system.

Afferent projections to the dorsal thalamus of the rat as shown by retrograde lectin transport. II. The midline nuclei.

Afferent projections to midline thalamic cell groups which innervate nucleus accumbens, were identified by the retrograde transport of unconjugated wheat germ agglutinin followed by the identification of labelled cell groups with immunocytochemistry. Large numbers of neurones were labelled in a variety of hypothalamic nuclei; principally in the medial preoptic area, anterior hypothalamic area, ventromedial, periventricular, arcuate and posterior hypothalamic nuclei, and in the supramammillary and lateral hypothalamic areas. Following rostral thalamic injections labelled cells were also found in the lateral septum, bed nucleus of the stria terminalis, and zona incerta. Prominent and localised label was found in the nucleus reticularis of thalamus at its most rostral medial tip. Cortical label was found in the ventral subiculum following rostral injections, and in the perirhinal cortex following mid-thalamic injections. In the brainstem label was found in central grey, laterodorsal tegmental nucleus, raphe, dorsal and ventral parabranchial nuclei and nucleus of the tractus solitarius. The results are discussed in the context of striatal function, particularly the nucleus accumbens, which is a component of the ventral striatum. Thus, the midline thalamic nuclei may provide an interface where a variety of inputs from many limbic regions and hypothalamic nuclei can influence nucleus accumbens function. Comparison of afferents to several thalamic nuclei directly related to striatal function and the prefrontal cortex show, that forebrain thalamic afferents from pallidal and hypothalamic sites, are organised with a clear topography. Some afferents suggest specific routes which may allow the reticular activating system to participate in the regulation of basal ganglia function.

Afferent projections to the dorsal thalamus of the rat as shown by retrograde lectin transport—I. The mediodorsal nucleus

The topography of afferent projections to the mediodorsal thalamic nucleus of the rat has been studied using the retrograde transport of unconjugated wheat germ agglutinin as identified by immunocytochemistry. Inputs were defined according to the lateral, central or medial segments of the nucleus injected, and controlled by additional injections into the habenula, central medial and paraventricular nuclei of the thalamus. Cortical afferents to the lateral segment arose from anterior cingulate and prelimbic areas on the medial surface of the hemisphere, those to the central segment arose mainly from ventral orbital area, whilst those to the medial segment arose from the infra-limbic and agranular insular areas. This strict cortical topography was matched by the organization of afferents from the reticular thalamic nucleus; i.e. lateral, intermediate and medial reticular neurons from the rostral nucleus projected to lateral, central and medial segments of the mediodorsal thalamus respectively. In the basal forebrain ventral pallidum projected only to the medial segment, whilst magnocellular preoptic region projected only to the central segment. Lateral preoptic area projected to lateral and central segments and the diagonal band mainly to central segment. Projections from substantia innominata were found regardless of the area of mediodorsal nucleus injected. The paraventricular nucleus of thalamus, lateral habenula and substantia nigra reticulata projected to the lateral segment only, whilst central gray projected only to the medial segment. Projections from amygdala (mainly basolateral and central nucleus) were found only following central and medial segment injections. All regions of the mediodorsal nucleus injected received input from the lateral hypothalamus, the ventral tegmental area and the dorsal tegmental gray. The results are discussed and particular emphasis is placed on the possible functions of the thalamocortical connections and the role of the reticular thalamic nucleus as a potential regulator of thalamocortical activity.

Afferent projections to the parafascicular thalamic nucleus of the rat, as shown by the retrograde transport of wheat germ agglutinin

Afferent projections to the parafascicular nucleus of the rat have been mapped using the retrograde transport of unconjugated wheat germ agglutinin and immunohistochemistry using very short survival times. Retrogradely labelled neurones were found in laminae V and V1 of primary motor cortex, lamina V1 of primary somatosensory cortex, and deep laminae of gustatory cortex; in the reticular thalamic nucleus and zona incerta; and in the caudate-putamen, entopeduncular nucleus, mesencephalic reticular formation and pretectum. Additional label was found in the laterodorsal tegmental nucleus, nucleus tegmenti pedunculopontinus, dorsal and ventral parabrachial nuclei, vestibular nuclei and the lateral cervical, medial and interpositus nuclei of the cerebellum. These results are discussed in the context of the connections of parafascicular nucleus with the motor system, particularly the basal ganglia. Of particular interest are inputs from laterodorsal tegmental nucleus, nucleus reticularis of thalamus, mesencephalic reticular formation, nucleus tegmenti pedunculopontinus, primary motor cortex and deep cerebellar nuclei. These indicate that the parafascicular nucleus lies at an interface between the reticular activating system on the one hand, and the motor system on the other. This result thus enlarges on present concepts of the parafascicular nucleus. Comparison of afferent projections to a variety of non-specific thalamic nuclei, the parafascicular, paraventricular and mediodorsal thalamic nuclei, indicate a remarkable set of topographic parallels from cortical, reticular thalamic, hypothalamic and brainstem sites. These comparisons provide clues as to the organisational principles of these non-specific thalamic nuclei, particularly in the context of the reticular activating system.

Demonstration of synaptic input from prefrontal cortex to the habenula in the rat

Using lesion-degeneration techniques at the EM level, it is confirmed that a pathway from prefrontal cortex projects to the lateral habenula, and further that it makes synaptic contacts predominantly with dendrites of neurons in the medial sector of the lateral nucleus. The available neuroanatomical evidence points to a role for habenula as a regulator of the activity of the meso-cortical dopamine pathway by the interaction of this cortico-habenular pathway with a wide variety of limbic inputs in the medial sector of lateral habenula.

Dopaminergic innervation of the primary visual cortex in the rat, and some correlations with human cortex

Dopaminergic terminals have been identified in the primary visual cortex with three techniques; immunocytochemistry with an anti-dopamine antiserum, retrograde axonal transport techniques using unconjugated wheat germ agglutinin and HPLC determination of catecholamines and metabolites in microdissected sub-regions of occipital cortex in the rat. The results demonstrate a specific dopaminergic innervation, arising from the ventral tegmental area, which is found mainly in laminae VI and V, but with minor innervation also in lamina I. Dopaminergic innervation to adjacent cortical regions is also described. Neurochemical data from post-mortem human material suggests that a similar innervation exists in man. An analysis of the distribution of dopaminergic fibres in relation to the known connections and possible functions of the deep laminae of visual cortex suggests that dopaminergic axons may participate in the corticofugal control of visual afferent pathways.

Regulation of dopamine function in the nucleus accumbens of the rat by the thalamic paraventricular nucleus and adjacent midline nuclei.

SummaryThe effects of unilateral treatments applied to non-dopamine containing output neurones of the thalamic paraventricular nucleus and adjacent midline nuclei (PV-MLT) were observed on dopamine (DA) utilisation of the nucleus accumbens (NAc). The ratios of [metabolite]: [parent amine] were used as indices of DA utilisation. In general, these indices were observed to increase in NAc in a bilaterally symmetrical fashion immediately after infusion of low doses (5 μM) of a cell-selective chemical excitant (quisqualic acid, QUIS) into either rostral or caudal PV-MLT. Moreover, the increases appeared to be entirely due to changes in the tissue content of metabolite. Electrical stimulation of caudal PV-MLT also enhanced DA utilisation ratios in NAc but appeared to do so by decreasing the tissue content of DA itself. Attempts to lesion caudal PV-MLT neurones by infusion of a higher dose of QUIS (50 mM) followed by long-term recovery (7 days) produced ratios of DA utilisation in NAc that were no different from those of controls. DA utilisation ratios in NAc were no different from control values immediately after infusion into caudal PV-MLT of an ‘intermediate’ dose (10 mM) of another chemical excitant (N-methyl-d-aspartic acid, NMDA). Since DA utilisation ratios in this area were also unaffected by histologically verifiable lesions of caudal PV-MLT neurones produced 7 days after infusion of high doses (100 mM) of NMDA it is argued that the former treatment may lead to an acute firing inactivation of PV-MLT neurones. In conclusion, experimental treatments that attempt to enhance the activity of PV-MLT efferent neurones produce increased DA utilisation ratios in NAc, whereas those treatments designed to reduce the activity of PV-MLT neurones appear to have no detectable effect on DA function in this terminal region.

Dopamine function in the prefrontal cortex of the rat is sensitive to a reduction of tonic GABA-mediated inhibition in the thalamic mediodorsal nucleus

SummaryDopamine (DA) utilisation has been determined in the medial bank of the prefrontal cortex (FCx) and the agranular insular cortex (AgCx) of the rat in response to a unilateral reduction of γ-aminobutyric acid (GABA)-mediated inhibition in the thalamic mediodorsal nucleus (MD). The ratios of 3,4-dihydroxyphenylacetic acid (DOPAC) ∶ DA and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA): DA were used as indices of DA utilisation. A bilateral increase in both ratios was found in FCx and AgCx following unilateral infusion of GABA antagonists (1 mM) into MDc. When this concentration was infused into one MDL no change was detected in DA utilisation of FCx, although a bilateral increase was observed in AgCx. However, a correspondence with the known anatomical connections was attained following infusion of lower concentrations (0.5 mM) into MDL in that a significant bilateral elevation of DA utilisation was shown in FCx. The changes induced in these ratios by the above treatments were, in general, due to increases in the concentration of metabolite and slight decreases in that of DA. However, unilateral lesions to the presumed GABA-containing neurones of the rostrodorsal thalamic reticular nucleus (TRNd), which topographically innervate MDL, produced increases in both metabolite and DA concentrations in FCx of both hemispheres, whilst those in AgCx were unaffected. Despite the slightly different results obtained using these two experimental approaches, it is argued that a reduction of tonic GABA-mediated inhibition in MD may tend to activate the DA system in cortical target regions.

The agranular insular cortex: a site of unusually high dopamine utilisation

Dopamine (DA) utilisation (expressed as homovanillic acid:DA) was compared in the medial prefrontal cortex (FCx), the agranular insular cortex (AgCx), the caudate-putamen (medial, CPM and lateral, CPL divisions) and the nucleus accumbens (NAc). DA utilisation in these regions decreased in the order AgCx greater than FCx greater than CPM, CPL, NAc, whilst the concentration of DA decreased in the reverse order. Thus, although fewer DA neurones appear to innervate AgCx compared with FCx, the rate of DA utilisation/release is much greater in AgCx. It is suggested that this apparently more marked activity in DA neurones innervating AgCx may reflect a relative lack of autoreceptor control.

Thalamic control of dopaminergic functions in the caudate-putamen of the rat—III. The effects of lesions in the parafascicular-intralaminar nuclei on D2 dopamine receptors and high affinity dopamine uptake

Dopamine receptor binding in the caudate-putamen was studied following bilateral lesions of the thalamostriatal pathway. Receptor binding was assayed using [3H]spiperone and defined with both (+)-butaclamol and S(-)-sulpiride. Radiofrequency lesions resulted in an increase in the Bmax of [3H]spiperone binding defined with both (+)-butaclamol and S(-)-sulpiride between 7 and 14 days following surgery. At longer survival times a fluctuating response was seen in which a decrease in receptor binding was observed at 28 days following lesion and a further rise again at 70 days. At no time point was significant change in Kd recorded. Further experiments were carried out to control for the possible effects of damage to fibres of passage and for inadvertent damage to habenula, as well as to define the receptor subtype involved. Ibotenic acid lesions resulted in similar effects to those reported with the radiofrequency method. Thus, 7 days following lesion, Bmax for (+)-butaclamol-defined [3H]spiperone binding increased by approximately 14-20% over that recorded in sham-lesioned animals. Using S(-)-sulpiride to define binding, Bmax was found to increase 13-17% in the same membrane preparations. Similar results were obtained in experiments at 14 days following ibotenic acid induced lesions. Again, no change in Kd was recorded. When radiofrequency lesions were made, which were largely restricted to habenula and associated fibres of passage, only small [(+)-butaclamol defined] or insignificant [S(-)-sulpiride defined] changes in Bmax were recorded. Combined radiofrequency lesions of habenula and ibotenic acid lesions of the thalamus resulted in a larger increase in Bmax for (+)-butaclamol defined binding than with S(-)-sulpiride defined binding. Our interpretation of these findings, in the light of the histology of the lesions, is that the predominant effect of removing thalamic input to the caudate-putamen is an increase in the number of D2 receptors, but without any change of affinity. A small component of the change in Bmax defined with (+)-butaclamol found with radiofrequency lesions may be due to a response at non-dopamine sites (possibly a 5-hydroxytryptamine receptor subtype) following damage to other caudate-putamen afferents which pass near the habenula or fasciculus retroflexus. Following unilateral ibotenic acid lesions of the thalamus, the number of high affinity uptake sites for dopamine was increased at long survival times.(ABSTRACT TRUNCATED AT 400 WORDS)