Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat.

Using the retrograde transport of horseradish peroxidase (HRP), a study has been made of projections to the ventral tegmental area of Tsai (VTA) and related dopaminergic cell groups (A 10). In order to minimise the possibility of damage to fibres of passage, a technique was evolved for the microiontophoresis of HRP such that minimal current strengths and durations were applied. In addition to a sham injection, control injections were also made to the medial lemnisuc, red nucleus, deep tegmental decussations, mesencephalic reticular formation and brachium conjunctivum. Following HRP injections confined to the areas of the VTA containing the dopamine cell groups, labelled neurons appeared in prefrontal cortex, dorsal bank of rhinal sulcus, nucleus accumbens, bed nucleus of stria terminalis, amygdala, diagonal band of Broca, substantis innominata, magnocellular preoptic area, medial and lateral preoptic areas, anterior, lateral and postero-dorsal hypothalamus, lateral habenular, nucleus parafascicular nucleus of thalamus, superior colliculus, nucleus raphe dorsalis, nucleus raphe nagnus and pontis, dorsal and ventral parabrachial nuclei, locus coeruleus and deep cerebellar nuclei. Regions containing catecholamine groups A 1, A 5, A 6, A 7, A 9, A 13 and the serotonin group B 7 corresponded to the topography of labeled cell groups. Injections of HRP to the interfascicular nucleus resulted in labeling predominantly confined to the medial habenular and median raphe nuclei. The results are discussed in relation to the known connections of these regions. Other regions of the brain labelled by VTA injections are assessed in relation to control injections and the limitations of the HRP technique. A review of the organisation of some of these afferents in relation to the known cortical-subcortical-mesencephalic projection systems, suggests that the VTA is in a position to recieve information from a massively convergent system derived ultimately from the entire archi-, paleo-, and neo-cerebral cortices. In addition A 10 dopaminergic neurons are known to project to restricted regions of both pre-frontal and entorhinal cortices, which themselves also recieve massively convergent association cortico-cortical connections. It would appear reasonable to propose that these neurons perform a correspondingly important integrative function.

The cytoarchitecture of the interfascicular nucleus and ventral tegmental area of Tsai in the rat.

The cytoarchitecture of the ventral tegmental area of Tsai (VTA) has been studied in detail in the rat with the acid of both conventional techniques and glyoxylic acid-fluorescence histochemistry. Three main dopamine containing cells groups can be distinguished: nucleus paranigralis, nucleus parabrachialis pigmentosus, nucleus linearis raphe caudalis. Nucleus parabrachialis pigmentosus lies dorsally, and nucleus paranigaralis ventrally in the VTA, while nucleus linearis continues posteriorly and medially from VTA and extends dorsally in the midline up towards the dorsal raphe nucleus. Fluorescent neurons in these three groups correspond to the A10 group. In addition to these previously described findings, the present study shows evidence for further small but cytoarchitecturally distinct dopaminergic group called the interfascicular necleus. This lies anteriorly and ventrally in the ventral tegmentum in the midline, dorsal to the rostral portion of the interpeduncular nucleus and interpeduncular fossa. The significance of these cytoarchitectural findings is discussed in relation to the known connections of the region.

A Golgi study of the ventral tegmental area of Tsai and interfascicular nucleus in the rat.

The ventral tegmental area of Tsai (VTA) and interfascicular nucleus of the adult rat brain has been studied with two variants of the Golgi method in three planes of section. Neurons were studied in relation to the cytoarchitectural groupings of the VTA. Dendritic organisation and dendritic fields were mapped out for each cytoarchitectural subgroup and cell types within each subgroup were classified on the basis of cell size and dendritic morphology. In each subnucleus of VTA, neurons had distinct characteristics. In nucleus paranigralis neurons were small to medium in size and their dendritic fields organised in an approximately horizontal plane orientated in an anteromedial direction and slanting dorsally over the interpeduncular nucleus and fossa. Neurons of the parabrachial group were small to medium sized with no preferential orientation. In nucleus linearis raphe caudalis small neurons were strongly orientated in the plane of the nucleus in a dorso-ventral direction slanting forwards. Neurons in the interfascicular group were small to very small and their maximum dendritic extents were seen in the horizontal plane. In frontal section they formed a compact ball of cells in the midline and were separated on either side from the larger neurons in the medial edge of nucleus paranigralis. In general VTA neurons tended to fall into one of two morphological categories. Type 1 were small to medium, and had two to four primary dendrites which divided into varicose secondary dendrites. Type 2 were medium sized, with two to five primary dendrites. Both primary and secondary dendrites and the cell soma of Type 2 neurons were moderately spiny. Secondary dendrites were not varicose. Forms also occurred which were intermediate between Types 1 and 2. In the nucleus paranigralis, Type 1 was more common medially, while Type 2 was more common laterally, particularly in the ventrolateral paranigral region. Only neurons of Type 1 were seen in nucleus linearis raphe and interfascicular nuclei. Local axon circuits were observed to arise from the primary dendrites of Type 1 neurons and to ramify close to neighbouring neurons. Axon swellings from such circuits were observed to make apparent contact with primary dendrites of nearby neurons and clusters of axon swellings were observed near cell somas of neighbouring impregnated neurons of similar type. The results are discussed and particular attention is paid to the similarities and differences between VTA and the substantia nigra pars compacts (SNC). The major difference appears to be that, whereas in SNC dendrites are organised in vertical as well as horizontal planes, in the VTA no long ventrally directed dendrites were observed. Combining these results with known cytoarchitecture and connections of VTA and SNC, it appears that fundamental differences occur between VTA and some neurons of the SNC, both in the nature of their morphology and intrinsic organisation, and in the organisation of their efferent and afferent connections.

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.

Thalamic control of dopaminergic functions in the caudate-putamen of the rat--I. The influence of electrical stimulation of the parafascicular nucleus on dopamine utilization.

A neurochemical response of four dopamine-rich brain regions to unilateral electrical stimulation of the parafascicular thalamic nucleus was examined in the halothane-anaesthetized rat. Tissue concentrations of dopamine and its two major metabolites, 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid, were assayed by a high performance liquid chromatographic technique in samples of caudate-putamen complex, nucleus accumbens, prefrontal cortex and substantia nigra. The ratios of metabolite to parent amine concentrations were taken as indices of dopamine utilization. Halothane anaesthesia alone evoked significant bilateral increases of dopamine utilization in every brain region studied. Electrical stimulation of one parafascicular nucleus produced further bilateral elevations of dopamine utilization in the caudate-putamen complex without altering these parameters in the substantia nigra. In the prefrontal cortex, however, thalamic stimulation resulted in significant bilateral decreases of dopamine utilization. Electrical stimulation of cortical or other thalamic areas did not evoke this regional pattern of dopamine utilization. It is argued that these indices of dopamine utilization together serve as reliable indicators of synaptic dopamine release and it is concluded that the parafascicular thalamus is capable of facilitating dopaminergic neurotransmission in the caudate-putamen by a mechanism that is probably independent of changes in dopamine cell firing rate. An anatomical analysis suggests that a thalamo-cortical-striatal route is most likely to mediate this function.

The topographic order of inputs to nucleus accumbens in the rat.

Afferents to the nucleus accumbens have been studied with the retrograde transport of unconjugated wheatgerm agglutinin as detected by immunohistochemistry using the peroxidase-antiperoxidase method, in order to define precisely afferent topography from the cortex, thalamus, midbrain and amygdala. Cortical afferent topography was extremely precise. The largest number of cells was found following injections to the anterior accumbens. Anteromedial injections labelled a very large extent of the subiculum and part of the entorhinal cortex. Anterolateral injections produced less subicular and entorhinal label but also labelled the posterior perirhinal cortex. Posteromedial injections labelled only the ventral subiculum and a few cells in the adjacent medial entorhinal cortex. Posterolateral injections labelled few lateral entorhinal neurones but did label a long anteroposterior strip of perirhinal cortex. Prefrontal cortex label was found only after anterior accumbens injections. In the amygdala labelled neurones were found in cortical, central, lateral posterior, anteromedial and basolateral nuclei. Basolateral amygdala projected chiefly to the anteromedial accumbens and central nucleus to anterolateral accumbens. Only a weak amygdala label was found after posterior accumbens injections. In the ventral tegmental area, the midline interfascicular nucleus projected only to medial accumbens. The paranigral ventral tegmentum projected chiefly to the medial accumbens and the parabrachial area chiefly to the lateral accumbens. In the thalamus, heaviest label was found after anterior accumbens injections. Most cells were found in the paraventricular, reuniens and rhomboid nuclei and at posterior thalamic levels lying medial to the fasciculus retroflexus. There was only restricted topography found from thalamic sites. Retrograde label was also found in the ventral pallidum and lateral hypothalamus. Single small injection sites within accumbens received input from the whole anteroposterior extent of the thalamus and ventral tegmentum. The medial accumbens was found to have a close relationship to habenula, globus pallidus and interfascicular nucleus. It appeared that the heaviest volume of inputs projected to anteromedial accumbens, where output from hippocampus (CAI), subiculum, entorhinal and prefrontal cortices converged with output from amygdala, midline thalamus and ventral tegmentum.

Thalamic control of dopaminergic functions in the caudate-putamen of the rat--II. Studies using ibotenic acid injection of the parafascicular-intralaminar nuclei.

We have investigated the role of the parafascicular-intralaminar thalamus in the regulation of dopaminergic function in the caudate-putamen by making unilateral injections of the excitotoxin, ibotenic acid, into the thalamus of the halothane-anaesthetized rat. Dopamine utilization was measured at 4 h, 18 h, and 7 days after operation in microdissected tissue from caudate-putamen, substantia nigra, and nucleus accumbens. High performance liquid chromatography with electrochemical detection was used to simultaneously determine dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Dopamine utilization was recorded as a ratio of metabolite to its parent amine. At 4 h following injection large bilateral increases in dopamine utilization were recorded in both medial and lateral sectors of caudate-putamen. The percentage increases found for homovanillic acid-based ratios were larger than those found for 3,4-dihydroxyphenylacetic acid-based ratios. These results probably reflect increased dopamine release resulting from the acute effects of ibotenic acid. These changes were independent of dopamine utilization ratios recorded in the substantia nigra, which showed no change either ipsilateral or contralateral to the injection. In contrast to these findings, at 7 days following injection, dopamine utilization ratios were reduced both ipsilateral and contralateral to the injection, although only the ipsilateral reductions were significant. Again, no change was found for this survival time in the substantia nigra. At 18 h survival an intermediate pattern between the 4 h and 7 day result was found. In the nucleus accumbens, ibotenic acid injection produced similar results to those found in caudate-putamen, i.e. a bilateral increase in dopamine utilization at early time intervals and a unilateral ipsilateral decrease at long intervals following injection. These results show that dopamine release in the caudate-putamen is sensitive to experimentally induced changes in neural activity and lesions of its thalamic input. Since the effect of presumed stimulation is markedly greater than lesion, it would appear that, under the conditions employed in these experiments, the thalamus is relatively silent; a suggestion consistent with other evidence. Furthermore, since the changes found occurred in the absence of changes in utilization ratios in the substantia nigra, the mechanisms whereby thalamus regulates dopamine release may be exerted via a local circuit and/or a presynaptic mechanism in the region of dopamine terminals. The anatomical routes responsible for these effects are discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Collateral axons of cholinergic pontine neurones projecting to midline, mediodorsal and parafascicular thalamic nuclei in the rat.

The organization of collateral axons projecting from neurones in the pontine laterodorsal tegmental nucleus (LDTg) has been examined using combinations of retrograde neuronal tracers with immunocytochemical markers for the acetylcholine-synthesizing enzyme choline acetyltransferase (CHAT), focussing on projections to the midline, mediodorsal and parafascicular thalamic nuclei and the ventral tegmental area. 25-59% of LDTg neurones projecting to the mediodorsal nucleus provided collaterals to the midline nuclei. Virtually all (87-96%) of these double retrogradely labelled neurones appeared cholinergic. 9-18% of LDTg neurones projecting to the parafascicular nuclei also provided a collateral to the midline nuclei and 50-78% of these double retrogradely labelled neurones stained for CHAT. 26-29% of the single LDTg neurones which projected collaterals to both the mediodorsal and midline nuclei, were found to project a third collateral to the ventral tegmental area. These anatomical findings, taken together with functional evidence, suggest that cholinergic terminals arising from LDTg are involved in coordinating thalamic mechanisms of brain state control; and in regulating dopaminergic pathways, both directly and via the thalamus.

Effects of lorazepam on human contrast sensitivity.

The anxiolytic lorazepam was studied for its effects on contrast sensitivity to gratings flickering in counterphase in normal volunteers. The drug significantly reduced contrast sensitivity at low spatial frequencies in a dose-related manner. The results are discussed with reference to possible GABA-mediated processes in the retina and lateral geniculate nucleus.

Effects of chlorpromazine and promazine on the visual aftereffects of tilt and movement.

The effects of chlorpromazine (CPZ) and promazine on the visual aftereffects of tilt and motion were measured. CPZ markedly reduced the strength of both aftereffects, while promazine produced a smaller and not always significant reduction. Control experiments suggested that the effects were produced in the central visual system rather than by several possible peripheral artefacts or by drowsiness. The effects are discussed with reference to the pharmacological activity of the drugs and their influence on the strength of inhibition in the visual cortex, both in normal subjects and in schizophrenic illness.

Effects of haloperidol and nomifensine on the visual aftereffects of tilt and movement.

An antipsychotic and two antidepressant drugs were studied for their effects on the visual aftereffects of tilt and movement in normal volunteers. Compared with placebo, haloperidol reduced the aftereffects. The antidepressant nomifensine enhanced the tilt but not the movement aftereffect, while maprotiline (another antidepressant) had no significant effect on either aftereffect. Control experiments showed that these changes were unlikely to have resulted from drug induced alterations in scanning eye movements during adaptation. The results are discussed with reference to possible dopaminergic influences on the visual system, and to some of the symptoms of schizophrenia.

Modification of visual orientation illusions by drugs which influence dopamine and GABA neurones: differential effects on simultaneous and successive illusions.

The effects of haloperidol, nomifensine and lorazepam on the visual tilt illusion were studied in normal volunteers. Haloperidol and nomifensine produced no significant changes in the illusion, although in previous work they had been found to reduce and enhance, respectively, a closely related illusion, the tilt aftereffect. By contrast, lorazepam produced a dose-related increment in the size of the tilt illusion, but had no effect on the tilt aftereffect. The results are discussed in relation to proposed mechanisms which may underlie the two kinds of illusion. The differential effects of individual drugs on the two illusions may reflect their differing actions on two processes: lateral inhibition and adaptation in visual channels.

Quantitative analysis of axonal branching using the retrograde transport of fluorescent latex microspheres.

A method is described for the quantitative analysis of double retrograde labelled neuronal cell bodies following labelling of branched axonal projections. This exploits the known ability of retrograde translocator proteins to transport latex microspheres following their uptake at nerve terminals. Conditions necessary for uptake and transport include small bead diameter (0.05-micron) and carboxylation of the latex particle. Using coumarin- and rhodamine-labelled microspheres a reliable, sensitive, rapid method has been developed, which results in double retrograde cell labelling in branched axonal pathways from the frontal cortex, basal forebrain, and brainstem. The technique has several advantages over currently available double retrograde labelling methods and yields repeatable quantitative estimates of populations of neurones bearing branched axons.

Demonstration of synaptic input from prefrontal cortex to the habenula i 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.

Anterograde and retrograde labelling of CNS pathways with unconjugated lectins using the unlabelled antibody enzyme method.

A method for demonstrating the anterograde and retrograde transport of unconjugated lectins in the CNS is given using the labelled antibody enzyme technique. Successful labelling of a variety of pathways is demonstrated using wheat germ agglutinin (WGA) and concanavalin A (Con A). Exceptionally large numbers of neurones labelled by retrograde transport were detected in each area. Using this method afferents to the mediodorsal thalamus are reported from prefrontal, cingulate and perirhinal cortex, substantia innominata, magnocellular and lateral preoptic area, ventral pallidum and diagonal band of Broca. In addition it is shown that large numbers of neurones of the periventricular hypothalamus and periaqueductal central grey of the mesencephalon techniques and further applications of the method are outlined.

Single neurones of the basal forebrain and laterodorsal tegmental nucleus project by collateral axons to the olfactory bulb and the mediodorsal nucleus in the rat.

The branching pattern of axons arising from cells in the basal forebrain and laterodorsal tegmental nucleus of the pontine grey, was examined using the double retrograde transport of rhodamine- and coumarin-labelled latex microspheres injected into the olfactory bulb and the mediodorsal thalamic nucleus. About 30-50% of basal forebrain neurones were double-labelled, and approximately 30-40% of laterodorsal tegmental neurones were double-labelled ipsilateral to the injections. No contralateral single or double label was observed in the basal forebrain, but approximately 30% of the contralateral laterodorsal tegmental projection was also double-labelled.

The effect of hydrocortisone and adrenocorticotrophic hormone on monoamine oxidase and tyrosine hydroxylase in explant cultures of embryonic chick sympathetic ganglia.

Sympathetic ganglia from 13- to 15-day-old embryonic chicks were cultured for up to 2 days in Leighton tubes. The influence of hydrocortisone and ACTH added to the culture medium on the enzymes monoamine oxidase (MAO) and tyrosine hydroxylase was studied. Hydrocortisone (5 times 10(-5)M) had no effect on tyrosine hydroxylase but increased MAO activity by up to 46 percent over control values under conditions of low or zero nerve growth factor (NGF) concentration. ACTH also increased ganglionic MAO activity, the effect again depending on NGF concentration. This time the maximal response (an increase of 50 percent over controls) was seen at high NGF concentrations. This response was similar to the effect of 1 mM dibutyryl cyclic AMP, and was blocked by 1 times 10-5 M propranolol and 10 muM prostaglandin E(1). ACTH only slightly increased tyrosine hydroxylase activity and this effect was due to a small (18 percent) increase in sympathetic neurone number. Guanosine 5-diphosphate (0.5 mM) was found to increase tyrosine hydroxylase activity by 57 percent and this effect was blocked by the presence of ACTH.

The influence of nerve growth factor, potassium depolarization and dibutyryl (cyclic) adenosine 3',5'-monophosphate on explant cultures of chick embryo sympathetic ganglia.

Optimal nerve growth factor (NGF) and potassium concentrations for the culture of explants of single chick embryo sympathetic ganglia in Leighton tubes are described. NGF increases ganglionic monoamine oxidase (MAO; E.C.1.4.3.4) activity in a dose dependent fashion with maximum effects at a concentration of about 8 U/ml NGF. Peak activity compares closely with values seen in chick ganglia in vivo. Potassium-induced depolarization (45 mM K-+) also increases MAO activity; but the extent of the increase depends upon (NGF concentration, for there is little or no increase at high NGF concentrations. Dibutyryl adenosine 3',5'-cyclic monophosphoric acid (db cyclic AMP) can mimic the potassium-induced increase in MAO.

Distribution of axons showing neurophysin-like immunoreactivity in cortical and anterior basal forebrain sites.

The distribution of fibres reacting with neurophysin antisera has been studied in the frontal cortex and anterior basal forebrain of young and adult rats. Fibres reacting with antisera raised against neurophysin A (vasopressin-neurophysin) were observed in the medial bank of frontal cortex, the anterior hippocampal rudiment, the rostral extension of substantia innominata lying between nucleus accumbens and olfactory tubercle, bed nucleus of stria terminalis, ventral pallidum, substantia innominata, lateral preoptic and magnocellular preoptic area. Axons were also identified lying in the anterior tip of the lateral ventricle and amongst the cells of the proliferative subependymal layer lining the ventricle. The significance of these findings is discussed. Particular attention is paid to the known connections of these, and associated regions, with limbic dopaminergic cell groups.