Neurotoxicity of 1,2,3,4-tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ) and effects on catecholamine homeostasis in SH-SY5Y cells.

We have investigated the potential neurotoxicity of the catecholamine depleting agent 1,2,3,4-tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ) in SH-SY5Y neuroblastoma cells. TMIQ induced a time and dose related inhibition of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction and an increase in lactate dehydrogenase release. After 72 h TMIQ (30 µM) significantly (P < 0.05) inhibited MTT reduction, and significantly increased LDH release. TMIQ cytotoxicity was not prevented by the inclusion of monoamine oxidase inhibitors (clorgyline or deprenyl), antioxidants (α-tocopherol or Trolox C) or the uptake(1) inhibitor imipramine. TMIQ also induced a dose dependent stimulation of [(3)H]noradrenaline (NA) uptake, with maximum at 100 µM and EC(50) of 8 µM. This stimulation of [(3)H]NA uptake was not prevented by the inhibition of protein kinase C, or activation of adenylate or guanylate cyclases. In addition, TMIQ significantly (P < 0.05) displaced [(3)H]nisoxetine binding from the uptake(1) recognition site with a K(i) of 71 ± 8 µM. However, as this interaction occurs at concentrations of TMIQ well above the EC(50) for [(3)H]NA uptake, it is unlikely to explain TMIQ stimulated NA uptake. Furthermore, TMIQ inhibited potassium evoked [(3)H]NA release from SH-SY5Y cells, with an IC(50) of 490 µM. Thus, TMIQ is cytotoxic to SH-SY5Y cells. However, the exact mechanism of toxicity requires further investigation, since it appears not to involve monoamine oxidase bioactivation, and is not mediated through membrane based free radical damage. Furthermore, although TMIQ inhibits mitochondrial Complex I (IC(50) = 1.5 mM) with potency apparently greater than MPTP (2.7 mM), mitochondrial respiration was unaffected. The present studies suggest that the mechanism of toxicity differs from that causing depletion of catecholamines and inhibition of tyrosine hydroxylase by TMIQ described in previous studies.

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.

C1-3 adrenergic medullary neurones project to the paraventricular thalamic nucleus in the rat.

Adrenergic afferents to the thalamus are almost entirely concentrated in the paraventricular thalamic nucleus (PV). The origins of this projection from medullary C1-3 neurones were quantified, using a combination of retrograde fluorescent markers and immunocytochemical localisation for phenylethanolamine N-methyltransferase (PNMT) in the rat. C1 neurones contributed 51%, C2 29% and C3 20% of the total adrenergic input. Many apparently non-adrenergic retrogradely labelled neurones were also found amongst the C1-3 neurones. The C3 region contained the largest adrenergic population (67%) of retrogradely labelled neurones. The neuronal networks associated with PV suggest a role for these adrenergic projections in regulating specific autonomic, locomotor and behavioural events.

1,2,3,4-Tetrahydro-2-methyl-4,6,7-isoquinolinetriol inhibits tyrosine hydroxylase activity in rat striatal synaptosomes.

1,2,3,4-tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ), a tetrahydroisoquinoline derivative of adrenaline, was tested for potency as an analog of the dopamine depleting agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in assays of tyrosine hydroxylase (TH) activity in the striatal synaptosome preparation. TMIQ inhibited TH activity with an IC50 (4 x 10(-6)M) similar to that found for MPTP (IC50 1 x 10(-6)M). TH inhibitions produced by IC50 concentrations of TMIQ were reversed by monoamine oxidase (MAO)-A or MAO-B inhibitors (clorgyline or deprenyl), or the dopamine reuptake blocker nomifensine, or excess cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin. TMIQ did not appear to act at the presynaptic D2 sulpiride sensitive autoreceptor for dopamine synthesis modulation. These in vitro data are consistent with earlier findings that TMIQ acts as a dopamine depleting agent, and with the possibility that TMIQ may have a degree of MPTP-like activity in vivo.

1,2,3,4-Tetrahydro-2-methyl-4,6,7-isoquinolinetriol depletes catecholamines in rat brain.

1,2,3,4-Tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ) was synthesised and tested for activity as a dopamine-depleting agent in rat brain. After intracerebroventricular infusion, TMIQ caused reductions in dopamine concentrations in substantia nigra, striatum, hypothalamus, and dorsal raphe, and reduction in noradrenaline concentrations in locus coeruleus. TMIQ also reduced 5-hydroxytryptamine concentrations in dorsal raphe and substantia nigra, although with a lower potency. Comparisons between TMIQ and MPTP showed that they were approximately equipotent in depleting dopamine in the substantia nigra, hypothalamus, and dorsal raphe. Pretreatment of animals with a combination of monoamine oxidase A and B inhibitors completely prevented the TMIQ-induced reductions in dopamine concentrations in substantia nigra and hypothalamus. Direct unilateral intrastriatal injections of TMIQ produced marked ipsilateral reductions in striatal dopamine, correlating with a behavioural response consisting of turning towards the side of injection. The results suggest that TMIQ should be evaluated further as a possible MPTP-like compound, which may derive from endogenous beta-hydroxylated catecholamines.

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.

Processing of spatial contrast in peripheral vision in Parkinson's disease.

Two experiments were carried out to test the hypothesis, based on anatomical evidence, that contrast gain might be reduced in the retinal periphery in Parkinson's disease. In the first experiment, subjects set contrast thresholds before and after adaptation to a vertical grating of 2 cycles per degree (c/deg), either stationary or oscillating sideways through its spatial period at 8 Hz, presented either in central vision or 7 degrees peripherally. Threshold elevations were similar for central viewing in both patients and controls. However, for peripheral viewing, elevations were greater in the controls, but smaller in the patients, than for central viewing. In the second experiment, a staircase procedure was used to find the contrast of a peripherally viewed grating of either 4 or 1.2 c/deg which apparently matched that of a centrally viewed grating of the same spatial frequency. Patients needed more contrast (about 1.6 times, at both spatial frequencies) than controls for a match. These results suggest that contrast gain may be lowered in the peripheral retina in Parkinson's disease, perhaps because of an abnormality of dopamine amacrine cells, whose density peaks in the peripheral retina.

Tilt aftereffect reveals early visual processing deficits in Parkinson's disease and in chronic schizophrenic patients on depot neuroleptic.

Previous studies suggest that the tilt aftereffect (TAE) may be used to indicate the site of dopaminergic abnormalities in the human visual system. In this study we investigated the TAE in patients with Parkinson's disease, and in chronic schizophrenic patients receiving depot injections of neuroleptic. The results suggest that the retina may be abnormal in both groups of patients, and that schizophrenic patients may also have cortical changes. The results are discussed in terms of the clinical implications of these visual changes.

Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat.

The connections of the laterodorsal tegmental nucleus (LDTg) have been investigated using anterograde and retrograde lectin tracers with immunocytochemical detection. Inputs to LDTg were found from frontal cortex, diagonal band, preoptic areas, lateral hypothalamus, lateral mamillary nucleus, lateral habenula; the interpeduncular nucleus, ventral tegmental area, substantia nigra and retrorubral fields; the medial terminal nucleus, interstitial nucleus, supraoculomotor central grey, medial pretectum, nucleus of the posterior commissure, paramedian pontine reticular formation, paraabducens and paratrochlear region; the parabrachial nuclei and nucleus of the tractus solitarius. Terminal labelling from PHA-L injections of LDTg was found in infralimbic, cingulate and hippocampal cortex, lateral septum, septofimbrial and triangular nuclei, horizontal limb of diagonal band and preoptic areas; in the anterior, mediodorsal, reuniens, centrolateral, parafascicular, paraventricular and laterodorsal thalamic nuclei, rostral reticular thalamic nucleus, and zona incerta; the lateral habenula and the lateral hypothalamus. A number of brainstem structures apparently associated with visual functions were also innervated, mainly the superior colliculus, medial pretectum, medial terminal nucleus, paramedian pontine reticular formation, inferior olive, supraoculomotor, paraabducens and supragenual regions, prepositus hypoglossi and nucleus of the posterior commissure. Also innervated were substantia nigra compacta, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, dorsal and medial raphe, pedunculopontine tegmental region, parabrachial nuclei, and nucleus of the tractus solitarius. These findings suggest the LDTg to be a highly differentiated part of the ascending "reticular activating" system, concerned not only with specific cortical and thalamic regions, especially those associated with the limbic system, but also with the basal ganglia, and visual (particularly oculomotor) mechanisms. Additional links with the habenula-interpeduncular system are discussed in this context.

Projections to the rostral reticular thalamic nucleus in the rat.

Afferent pathways to the rostral reticular thalamic nucleus (Rt) in the rat were studied using anterograde and retrograde lectin tracing techniques, with sensitive immunocytochemical methods. The analysis was carried out to further investigate previously described subregions of the reticular thalamic nucleus, which are related to subdivisions of the dorsal thalamus, in the paraventricular and midline nuclei and three segments of the mediodorsal thalamic nucleus. Cortical inputs to the rostral reticular nucleus were found from lamina VI of cingulate, orbital and infralimbic cortex. These projected with a clear topography to lateral, intermediate and medial reticular nucleus respectively. Thalamic inputs were found from lateral and central segments of the mediodorsal nucleus to the lateral and intermediate rostral reticular nucleus respectively and heavy paraventricular thalamic inputs were found to the medial reticular nucleus. In the basal forebrain, afferents were found from the vertical and horizontal limbs of the diagonal band, substantia innominata, ventral pallidum and medial globus pallidus. Brainstem projections were identified from ventrolateral periaqueductal grey and adjacent sites in the mesencephalic reticular formation, laterodorsal tegmental nucleus, pedunculopontine nucleus, medial pretectum and ventral tegmental area. The results suggest a general similarity in the organisation of some brainstem Rt afferents in rat and cat, but also show previously unsuspected inputs. Furthermore, there appear to be at least two functional subdivisions of rostral Rt which is reflected by their connections with cortex and thalamus. The studies also extend recent findings that the ventral striatum, via inputs from the paraventricular thalamic nucleus, is included in the circuitry of the rostral Rt, providing further evidence that basal ganglia may function in concert with Rt. Evidence is also outlined with regard to the possibility that rostral Rt plays a significant role in visuomotor functions.

The influence of dopamine on spatial vision.

Contrast thresholds for, and contrast matches between, stationary gratings of three spatial frequencies (0.5, 2, and 8 c/deg) were measured on eight subjects with a history of schizophrenia, just before, and again two to three days after, a therapeutic injection of depot neuroleptic. The drug enhanced sensitivity at the low, and reduced it at the medium and high spatial frequency. After injection, subjects required more contrast to match the apparent contrast of the high, and less contrast to match that of the low, to that of the medium spatial frequency. Pupillary measurements suggested that these effects were not due to drug-induced changes in pupil size. The results are discussed in terms of the functional role of dopamine in the retina, and a possible application in therapy for amblyopia.

Object-name selection in psychiatric patients before and after neuroleptic therapy.

An object-name selection test was administered to 16 newly admitted schizo phrenic, non-schizophrenic psychotic and non-psychotic patients on admission when they were drug-free, and again 4-6 weeks later, following treatment with neuroleptics. Sixteen healthy, drug-free age-matched and sex-matched controls were similarly tested on two occasions 4-6 weeks apart. Schizophrenic and non-schizophrenic psychotic subjects made significantly more association-type errors than non-psychotic subjects and controls. A signifi cant reduction in association-type errors which was observed in both psychotic patient groups on the second testing occasion may be attributed to medication. The data suggest that overinclusive thinking, as measured by the object-name selection test, is a feature of both schizophrenic and non-schizophrenic psychosis. It is concluded that tests of overinclusion are therefore of limited diagnostic utility although they may be of value in monitoring response to treatment.

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.

Excitatory amino acid treatment of the ventromedial globus pallidus enhances dopamine utilization in the prefrontal cortex of the rat via the thalamic mediodorsal nucleus.

Infusion of a low dose (5 microM) of the cell-selective chemical excitant quisqualic acid (QUIS) into rostral ventromedial globus pallidus (GP) had no immediate effect on DA utilization (assessed as [DOPAC]:[DA] and [HVA]:[DA] ratios) in either the medial bank of the prefrontal cortex (FCx) or the agranular insular cortex (AgCx). In contrast, a larger dose (630 microM) of another excitant sodium ibotenate (IBO) produced an immediate bilaterally symmetrical increase in both indices of DA utilization in FCx. There was also a marked trend towards a bilateral increase in these indices of DA utilization in AgCx. In order to determine whether these effects on cortical DA utilization are mediated by a direct cortical route or via the thalamic mediodorsal nucleus (lateral division, MDL), infusions of IBO into GP were repeated in animals with a 1-week-old N-methyl-D-aspartate lesion of MDL. The increase in DA utilization of FCx following infusion of IBO into GP was abolished, although the trend towards increased DA utilization in AgCx was still maintained. Since MDL innervates FCx but not AgCx and since we have previously shown that MDL lesions alone have no effect on DA utilization in either cortical region, the present results suggest that the changes in cortical DA utilization are probably mediated via MD. Thus in addition to the well-documented control exerted by the thalamus over brain DA function, this has now been extended in the present study to include GP, which projects both directly and indirectly to the thalamus.

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

The 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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Thalamic control of subcortical dopamine function in the rat and the effects of lesions applied to the medial prefrontal cortex.

Dopamine (DA) utilisation has been assessed in medial and lateral segments of the caudate-putamen complex (CPM and CPL, respectively) in response to unilateral manipulations aimed at the thalamic mediodorsal nucleus, lateral division (MDL). The ratios of 3,4-dihydroxyphenylacetic acid (DOPAC):DA and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA):DA are used as indices of DA utilisation and, in the case of HVA:DA, may also reflect DA release. Neither electrical stimulation nor ibotenate (IBO) treatment followed by long recovery periods (2 days or 1 week) had any significant effect on DA utilisation in CPM or CPL. Cell-specific activation of neurones produced by short-term (1 h recovery) infusions of IBO aimed unilaterally at MDL (right side) resulted in bilateral increases of DA utilisation in both CP sectors. These changes tended to be slightly more marked in the hemisphere ipsilateral to the side of IBO infusion. Unilateral infusions of IBO were then aimed at MDL of either (1) the left or right hemisphere of animals which had already received a 1-week-old unilateral (right side) prefrontal cortex (FCx) lesion or (2) the right hemisphere of animals which had previously received a 1 week-old bilateral FCx lesion. The pattern of changes, when expressed relative to the 'sham-operated' animals which received the FCx lesion alone, were similar to those described above following intra-MDL infusions of IBO into animals with an intact cortex. The FCx lesions themselves were shown to have no significant effect on DA utilisation in any CP sector. In view of the known neuroanatomical connections, it is likely that the effects observed in CP are not due to activation of MDL neurones themselves but are more likely the result of activation of neurones in the intralaminar nuclei which border MDL. Nevertheless, these findings support the concept that activation of thalamic nuclei will enhance DA function in a variety of forebrain areas in the rat.

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.

Mediodorsal and reticular thalamic nuclei receive collateral axons from prefrontal cortex and laterodorsal tegmental nucleus in the rat.

Quantitative analysis of the branching pattern of output projections from the prefrontal cortex and the laterodorsal tegmental nucleus of the brainstem, has been carried out using the newly developed method for double retrograde labelling of neurons with fluorescent labelled latex microspheres. At least 34% of cortical mediodorsal projection neurones in lamina VI send collateral axons to the reticular thalamic nucleus, and at least 40% of cortical reticular projection neurones and collateral axons to mediodorsal nucleus. In the brainstem, laterodorsal neurones are also highly collateralized; at least 44% of mediodorsal projection neurones also innervating nucleus reticularis, and at least 47% of reticularis projection neurones innervating the mediodorsal nucleus.

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.