Alfred Heller

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Age-dependent effects of 6-hydroxydopamine on locomotor activity in the rat

This experiment examined the effects on locomotor activity of intraventricular 6-hydroxydopamine (6-OHDA) administered to developing and adult rats. 6-OHDA was administered subsequent to pargyline treatment at 3 and 6 days of age; or 6-OHDA was administered subsequent to desmethylimipramine (DMI) treatment (6-OHDA/DMI) at 3 and 6 days of age, 11 and 14 days of age, 20 and 23 days of age, or 46 and 48 days of age. Locomotor activity of vehicle-treated rats assessed in stabilimeter cages peaked between 14 and 16 days of age and subsequently declined to levels characteristic of the adult. Treatment with pargyline and 6-OHDA at 3 days of age, or 6-OHDA/DMI at 3 and 6 or 11 and 14 days of age, did not alter the early rise in locomotor activity but prevented the decline in activity normally seen during the third and fourth weeks of life. When tested as adults, locomotor activity was greater in rats that had been treated with 6-OHDA/DMI at 3 and 6 and at 11 and 14 days of age than in those that had been treated at 20 and 23 days of age. Treatment with 6-OHDA/DMI at 46 and 48 days of age was without significant effect on locomotor activity. 6-OHDA (with pargyline pretreatment) produced large decreases in NE content in telencephalon and diencephalon and in dopamine (DA) content in striatum. 6-OHDA-DMI also produced large decreases in DA content in striatum and, in some of the treatment groups, only small decreases in norepinephrine (NE) content in telencephalon, diencephalon, and brain stem. These data suggest that the maturation of neuronal systems utilizing dopamine as a neurotransmitter is essential for the suppression of locomotor activity normally seen during development. The data further suggest that dopamine depletion per se does not lead to increased locomotor activity, but rather it is the destruction of dopamine-containing fibers prior to the normal period of locomotor suppression that increases locomotor activity.

Visual Pathway Mediating Pineal Response to Environmental Light

Activity of the melatoninforming enzyme, hydroxyindole-O-methyltransferase, in rat pineal is increased when the animal is exposed to continuous darkness, and it is decreased by exposure to continuous light. Response to environmental light is initiated in the retina and transmitted to the pineal by way of the central nervous system and the cervical sympathetics. The central visual pathway essential for mediation of this response is the inferior accessory optic tract. Visual pathways to thalamus and tectum do not participate in this response.

REGIONAL DEVELOPMENT OF CATECHOLAMINE BIBSYNTHESIS IN RAT BRAIN1

Abstract— The ontogenetic development of norepinephrine and dopamine and their associated biosynthetic and degradative enzymes was investigated in five anatomical regions of the rat brain. Clear regional differences were found in the development of both norepinephrine and tyrosine hydroxylase (EC 1.14.3.1). In the case of both norepinephrine and tyrosine hydroxylase, brainstem structures achieved adult levels well before forebrain structures. The development of DOPA decarboxylase (EC 4.1.1.26), monoamine oxidase (EC 1.4.3.4) and catechol‐0‐methyl transferase (EC 2.1.1.6) did not appear to differmarkedly from area to area. Further analysis of the data revealed that in forebrain structures both the amines and the biosynthetic enzymes developed concurrently. By contrast, in the brainstem structures, there was a dissociation of amine and enzyme development with development of tyrosine hydroxylase, in particular, markedly preceding that of norepinephrine and of DOPA decarboxylase. The bases for both the lower amine levels in the infant brain and the regional developmental differences are discussed in relation to the anatomical organization of the central catecholamine‐containing neurons.

Development of left ventricular hypertrophy in young spontaneously hypertensive rats after peripheral sympathectomy.

The effects of peripheral sympathectomy with nerve growth factor antisemm (NGFAS) on blood pressure, systemic hemodynamics, myocardial function, myocardial hypertrophy, and renin were studied in male spontaneously hypertensive (SH) rats of the Okamoto strain and normotensive control Kyoto-Wistar (WKY) rats. NGFAS prevented the development of hypertension in the SH rats but did not alter blood pressure in the WKY rats. The NGFAS-treated SH rats developed the same hemodynamic abnormalities as the sham-treated rats, including increased peripheral vascular resistance and depressed cardiac output. Indices of left ventricular performance, including peak flow velocity, stroke power, stroke work, dP/dtmax and flow acceleration (dF/dt), were diminished in the SH rats compared to the WKY rats. NGFAS treatment further depressed ventricular function in the SH rats, but had little effect on the WKY rats. Plasma renin activity in both the SH and WKY rats was unaffected by NGFAS treatment. Although NGFAS treatment effectively prevented the development of hypertension in the SH rats, it did not influence the development of left ventricular hypertrophy as reflected by increases in left ventricular mass, RNA, DNA, and hydroxyproline content. The data suggest that the development of myocardial hypertrophy and myocardial dysfunction in the SH rat is in part independent of hypertension and plasma renin activity.

Stereotaxic electrode placement in the neonatal rat

A head holder for stereotaxic electrode placement in the neonatal rat is described. The neonatal head is held in a fixed and reproducible position by means of a mouth bar and a recurved needle hooked into the foramen magnum. With the aid of this instrument, a stereotaxic atlas for the 3-day-old rat has been prepared. The head holder has been used for rats up to 10 days of age and permits electrode placement in the neonatal rat in a manner essentially analogous in ease and precision to that of stereotaxic methods for the adult animal.

Enhanced dopamine cell survival in reaggregates containing telencephalic target cells.

Dissociated, 14-day-old embryonic cells of the rostral mesencephalic tegmentum (RMT), including the dopamine neurons of this region, were allowed to reaggregate and develop in rotatory culture for 7 days in the presence of dissociated embryonic cells from the target areas of the dopaminergic neurons, corpus striatum (CS) or frontal cortex (FCx). Alternatively, RMT cells were allowed to reaggregate by themselves or in the presence of dissociated cells from a telencephalic area, occipital cortex (OCx), or mesencephalic area, tectum (T), which are not target areas for the dopamine neurons. Histofluorescence analysis revealed the number of dopamine neurons contained within reaggregates of any given type. Approximately 4 times as many dopamine neurons were found in RMT-CS coaggregates and 1.5 times as many in RMT-FCx coaggregates than in aggregates constituted from cells of the RMT either alone, or in coaggregates from RMT-OCx or RMT-T. Since axonal process formation and maintenance can only be observed in RMT-CS and RMT-FCx coaggregates, the enhanced dopamine neuron survival is probably due to an interaction of dopaminergic axonal processes with target cells within the reaggregates.

Control of brain serotonin and norepinephrine by specific neural systems.

Publisher Summary This chapter discusses the experiment technique of selective destruction of discrete groups of central neurons employed to study the monoamines, 5-HT, and NE. The experiments are initiated to identify neuronal systems in brain responsible for the presence and metabolism of these substances as one step in the elucidation of their functional role in this organ. Each area whose destruction is demonstrated to lower brain levels of either 5-HT or NE, involves neural elements within or directly related to the neurons of the medial forebrain bundle. Septa lesions reduce hippocampal 5-HT and result in some minimal reduction in cortical 5-HT and NE, which may be either polysynaptic in character or a function of loss of monoamine neurons innervating these areas. Each central neuron receives input over much of the surface of its soma and dendrites and may be influenced by vast numbers of presynaptic elements. The neurochemical integrity of any given cell is interpreted as a function of this afferent pool of innervation. Stimulation of the tegmentum causes release of 5-HT within the telencephalon. Similarly, the syndrome of adipsia and aphagia resulting from lateral hypothalamic lesions represents distant changes in the functional activity of widespread areas on the basis of transsynaptic changes. The lesion technique has proved a useful approach to the study of neuronal systems controlling brain monoamines. The maintenance of 5-HT and NE in brain appears to be a function of the integrity of a specific system of hypothalamic neural elements, the medial forebrain bundle.

Analysis of huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons

Huntingtin, the protein product of the Huntingtons disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin‐associated protein 1 (HAP‐1), a brain‐enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP‐1 is unknown, but recent evidence supports a role in microtubule‐dependent organelle transport. We examined the subcellular localization of HAP‐1 with an antibody made against the NH2‐terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP‐1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP‐1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP‐1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP‐1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP‐1 localized to membrane‐bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP‐1 was also strongly associated with an unusual large “dense” organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP‐1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP‐1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons. J. Comp. Neurol. 403:421–430, 1999.

Methamphetamine concentrations in fetal and maternal brain following prenatal exposure

Levels of methamphetamine in maternal striatum and whole fetal mouse brain were assessed at 0.5, 1, 2, and 4 h postinjection on gestational day 14 (GD14) following a single, subcutaneous injection of 40 mg/kg (+)-methamphetamine hydrochloride to pregnant mice. In the dams, striatal concentrations of methamphetamine peaked at 1 h postinjection, reaching levels of approximately 510 ng/mg protein. Amphetamine, the primary metabolite of methamphetamine, increased to 77 ng/mg protein at 2 h and remained elevated by 4 h postinjection. In the fetal brain, peak methamphetamine concentrations of approximately 122 ng/mg protein were attained at 1 h. Amphetamine was only detectable in fetal brain at 2 and 4 h postinjection. Regional analysis of methamphetamine levels in fetal striatum, cortex, and brainstem revealed that the drug was not uniformly distributed. Maternal administration of methamphetamine results in fetal brain drug concentrations, which approximate those reported in human infants whose mother abused methamphetamine. This dosage regimen, therefore, serves as an appropriate animal model for assessing the potential risks to human offspring exposed to methamphetamine in utero.