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Cet article revisite l’histoire du concept de contre-transfert en mettant en exergue une evolution vers la reconnaissance de differentes modalites de manifestations contre-transferentielles comprenant de nos jours les manifestations corporo-somatiques. Alors que Freud opere une mise a distance du corps physique de l’analyste dans la pratique analytique, celui-ci devient aujourd’hui lieu d’ecoute contre-transferentielle, clef d’acces a l’inconscient du patient.

Methodological differences may explain discrepancies in adult mammalian neurogenesis in the brain outside the widely accepted neurogenic regions, i.e. hippocampus and olfactory bulb/subventricular zone. Here, we describe a method to dissolve and administer bromodeoxyuridine (BrdU) at high concentrations (150mg/mL) into the adult mouse brain to demonstrate neuronal incorporation of this thymidine analogue in CNS regions with a low rate of neurogenesis. The dosage and duration of BrdU appear critical, since exposure to low doses did not result in a robust label using this marker for proliferation [Zhao et al., 2003. Proc Natl Acad Sci USA 100; 7925]. Mice (five per cage in an enriched environment) received BrdU in the right cerebral ventricle from a subcutaneous osmotic pump (0.9mg/day, 3 weeks, infused at 0.25microL/h). To avoid labelling of cells with a fast turnover, the mice were allowed to survive 3 weeks after ending the BrdU delivery. Midbrain sections were processed for tyrosine hydroxylase (TH) immunohistochemistry, post-fixed with 4% paraformaldehyde, denaturated with 2N HCl and 0.025% pepsin, followed by immunolabelling of nuclear BrdU in single-stranded DNA. Double-labelled cells were analysed in a confocal laser microscope and showed segmented nuclear BrdU-label surrounded by TH-immunoreactive cytoplasm, never displaying apoptotic morphological features. Nigral neuronal proliferation was confirmed using another marker [(3)H]thymidine, delivered via an intraperitoneal osmotic pump. The protocol was well tolerated by the mice and not found to be toxic for the region studied, i.e. did not alter the total number of nigral neurons identified with TH/cresyl violet immunohistochemistry.

Object Intracranial lesions affecting the facial nerve are usually associated with significant morbidity and poor functional restitution, despite the fact that a peripheral nerve injury normally recovers well. Mechanistic explanations are needed to direct future therapies. Although neonatal motor neurons are known to die as a result of apoptosis after axotomy, this cell death mechanism has not been explicitly demonstrated after peripheral cranial nerve transection in adult mammals. Methods The authors induced substantial retrograde neuronal death in the adult rodent by transecting the facial nerve during its intracranial course. Neuronal apoptosis was demonstrated as shrunken facial motor neurons, retrogradely labeled with fluorogold and with nuclei positively labeled by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick–end labeling (TUNEL). Glial apoptosis was demonstrated by double labeling with respect to cell type. On postinjury Days 7 and 14, the intracranial axotomy led to neuronal apoptosis, corresponding to a neuronal loss that was observed quantitatively in cresyl violet–stained tissue sections obtained using a stereological method. In contrast, no neuronal apoptosis was observed after creating a distal lesion of the facial nerve, which causes less neuronal loss. In addition, glial apoptosis was seen in the facial nucleus after both distal and proximal axotomy. Whereas the proximal intracranial axotomy led to TUNEL-positive nuclei in cells showing markers for oligodendrocytes and microglia, only the latter glial cell population was double labeled with TUNEL-positive nuclei after distal lesioning. Conclusions These findings may ultimately lead to new therapeutic strategies in patients suffering from facial nerve palsy due to an intracranial lesion.

New neurons are generated from stem cells in a few regions of the adult mammalian brain. Here we provide evidence for the generation of dopaminergic projection neurons of the type that are lost in Parkinson's disease from stem cells in the adult rodent brain and show that the rate of neurogenesis is increased after a lesion. The number of new neurons generated under physiological conditions in substantia nigra pars compacta was found to be several orders of magnitude smaller than in the granular cell layer of the dentate gyrus of the hippocampus. However, if the rate of neuronal turnover is constant, the entire population of dopaminergic neurons in substantia nigra could be replaced during the lifespan of a mouse. These data indicate that neurogenesis in the adult brain is more widespread than previously thought and may have implications for our understanding of the pathogenesis and treatment of neurodegenerative disorders such as Parkinson's disease.

Striatal 6-[ 18 F]fluoro-L-DOPA (FDOPA) kinetic rate constants were measured by positron emission tomography (PET) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys. After scanning, stereological counts of dopaminergic neurons were done in substantia nigra, and dopamine (DA) and metabolite concentrations were determined in the caudate, putamen, and substantia nigra. Graded doses of MPTP produced animals with mild to moderate reductions (10-35%) in dopaminergic neurons, where the percent of cell loss was proportional to the amount of MPTP given. Striatal DA and metabolite concentrations were relatively unchanged in animals given 1.0 and 1.5 mg/kg of MPTP, but were significantly reduced after 2.0 mg/kg of MPTP. All animals injected with a single dose of MPTP showed no overt signs of parkinsonism. In contrast, DA and metabolite concentrations in the substantia nigra were significantly reduced for all MPTP-treated animals. Reduction of dopaminergic indices in the substantia nigra did not parallel reductions in the striatum, indicating differential sensitivity of the nigrostriatal pathway to the neurotoxic effects of MPTP. The percent change in FDOPA uptake (Ki) and decarboyxlation (k 3 ) after MPTP showed significant positive correlations to striatal DA levels, but not to the number of dopaminergic neurons. This suggests that FDOPA is a good index of striatal DA levels.

Presynaptic denervation is likely to play an important role in the pathophysiology of dyskinesias that develop after levodopa administration to patients with Parkinson's disease. In this study, the thresholds of nigrostriatal damage necessary for the occurrence of parkinsonism and levodopa-induced involuntary movements were compared in squirrel monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animals treated with a regimen of MPTP that caused parkinsonism displayed > or =95% striatal dopamine depletion, 90% reduction of striatal dopamine uptake sites, and 70% nigral neuronal loss. Levodopa administration ameliorated the parkinsonian signs of these monkeys but also induced dyskinesias. A separate group of animals was treated with a milder MPTP regimen that caused 60%-70% striatal dopamine depletion, a 50% decrease in dopamine transporter, and 40% loss of dopaminergic nigral neurons. While these monkeys displayed no behavioral signs of parkinsonism, they all became dyskinetic after levodopa administration. The priming effect of levodopa, that is, the recurrence of dyskinesias in animals previously exposed to the drug, was compared in severely versus mildly lesioned monkeys. When severely injured parkinsonian animals underwent a second cycle of levodopa treatment, they immediately and consistently developed involuntary movements. In contrast, the recurrence of dyskinesias in primed monkeys with a partial nigrostriatal lesion required several levodopa administrations and remained relatively sporadic. The data indicate that moderate nigrostriatal damage which does not induce clinical parkinsonism predisposes to levodopa-induced dyskinesias. Once dyskinesias have been induced, the severity of denervation may enhance the sensitivity to subsequent levodopa exposures.

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is dependent upon the MAO-B (monoamine oxidase type B)-catalyzed production of 1-methyl-4-phenylpyridinium ion (MPP(+)) and is likely to involve a perturbation of energy metabolism. Protection against MPTP neurotoxicity has been shown by treating mice with 7-nitroindazole (7-NI), a reversible inhibitor of both MAO-B and neuronal nitric oxide synthase (nNOS) activity. The objective of the present study was to evaluate (i) the relationship between the neuroprotective effect of 7-NI and MPTP-induced energy deficiency, and (ii) the role of nitric oxide production as a potential mechanism for energy perturbation after MPTP exposure. Maximum protection against striatal dopamine depletion and nigral neuronal loss was achieved when 7-NI (50 mg/kg, i.p.) was administered to C57BL/6 mice immediately before and after MPTP (50 mg/kg, s.c.). This short-term regimen of 7-NI administration parallels the time when MPTP exposure causes energy failure. 7-NI also completely prevented the loss of striatal ATP that occurs in mice during the initial hours after MPTP administration. In contrast, N(G)-nitro-L-arginine (two injections of 50 mg/kg each, given i.p. 20 and 4 h prior to MPTP), another NOS inhibitor, failed to affect MPTP-induced ATP depletion. Taken together, data indicate that (i) a temporal and causal relationship exists between the neuroprotective effect of 7-NI and its ability to counteract ATP reduction, and (ii) MAO-B rather than NOS inhibition is the mechanism by which 7-NI counteracts MPTP-induced ATP depletion.

Parkinson's disease is characterized by motor deficits caused by loss of midbrain dopaminergic neurons. Neurotrophic factors and cell transplantation have partially restored function in models of Parkinson's disease, but have had limited effects in humans. Here we show that intracerebroventricular administration of platelet-derived growth factor-BB can offer an alternative strategy to restore function in Parkinson's disease; In animal models of nigrostriatal injury, a two weeks treatment with platelet-derived growth factor-BB resulted in long-lasting restoration of striatal dopamine transporter binding sites and expression of nigral tyrosine hydroxylase. It also normalized amphetamine-induced rotational behavior in 6-hydroxydopamine lesioned rats. Platelet-derived growth factor-BB promoted proliferation of neural progenitor cells in the subventricular zone. The effects on dopaminergic neurons and functional recovery could be blocked by co-infusion with a proliferation inhibitor, indicating a link between the proliferative and anti-parkinsonian effects. Based on the current data, we consider platelet-derived growth factor-BB a clinical candidate drug for treatment of Parkinson's disease.

Methylmercury (MeHg) is an environmental contaminant with recognized neurotoxic effects, particularly to the developing nervous system. In the present study, we show that nanomolar concentrations of MeHg can induce long-lasting effects in neural stem cells (NSCs). We investigated short-term direct and long-term inherited effects of exposure to MeHg (2.5 or 5.0 nM) using primary cultures of rat embryonic cortical NSCs. We found that MeHg had no adverse effect on cell viability but reduced NSC proliferation and altered the expression of cell cycle regulators (p16 and p21) and senescence-associated markers. In addition, we demonstrated a decrease in global DNA methylation in the exposed cells, indicating that epigenetic changes may be involved in the mechanisms underlying the MeHg-induced effects. These changes were observed in cells directly exposed to MeHg (parent cells) and in their daughter cells cultured under MeHg-free conditions. In agreement with our in vitro data, a trend was found for decreased cell proliferation in the subgranular zone in the hippocampi of adult mice exposed to low doses of MeHg during the perinatal period. Interestingly, this impaired proliferation had a measurable impact on the total number of neurons in the hippocampal dentate gyrus. Importantly, this effect could be reversed by chronic antidepressant treatment. Our study provides novel evidence for programming effects induced by MeHg in NSCs and supports the idea that developmental exposure to low levels of MeHg may result in long-term consequences predisposing to neurodevelopmental disorders and/or neurodegeneration.

Loss of genetic information from chromosome 22 has been implicated in the development of neurofibromatosis type 2, meningioma, and several other neoplasia. Molecular studies indicate that genes within chromosomal band 22q12 may be involved in tumorigenesis. We have mapped 29 loci into 16 groups in this region, using pulsed-field gel electrophoresis, fluorescence in situ suppression hybridization, and somatic cell hybrid mapping. The region spans more than 5 Mb of genomic DNA and contains the genes for neurofibromatosis type 2 and meningioma. The order of loci presented here provides the framework for the fine mapping of this region using cosmids and yeast artificial chromosomes, and it facilitates the speedy cloning of novel genes from 22q12.

In extracellular fluids the insulin-like growth factors (IGFs) are bound to specific binding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBP1 gene to human chromosomal region 7q14–p12 and the IGBP2 gene to region 2q33–q34. In this study, somatic cell hybrid analysis indicated that IGBP3 is also located on chromosome 7. Pulsed-field gel electrophoresis was used to demonstrate the close physical linkage between IGBP1 and IGBP3. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBP1 DNA sequence disclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for Apa LI and Taq I in the IGBP1 locus.

We have constructed a physical map of the region q12-13 on chromosome 11 by combining data generated from a panel of radiation-reduced somatic cell hybrids and pulsed-field gel electrophoresis (PFGE). Twenty different genetic markers have been sublocalized and ordered within this region and a total of 8.0 megabases has been mapped in detail using rare-cutting restriction endonucleases and PFGE. In two instances, the long-range restriction PFGE map spans the total distance between pairs of loci that have been previously mapped by genetic linkage in reference families. Comparison of this physical map with the available linkage map indicates a great variation in the recombination frequency over the region. The recombination rate is higher than expected, particularly for markers flanking the MEN1 region. Thus, for the closest pair of linked markers on the centromeric side, one centimorgan corresponds to approximately 300 kilobases, and for markers on the telomeric side, one centimorgan corresponds to approximately 350-600 kilobases.

Stereology is a valuable tool for neuroscientists, allowing them to obtain three-dimensional information from two-dimensional measurements made on appropriately sampled sections (usually obtained from histological sections or MRI/CT/PET scans). This 3-D information is invaluable in correlating structural/functional relationships in the pursuit of far greater understanding of the function of the central nervous system. However, in carrying out such measurements, often based on limited data sets, there is a risk of experimenter bias. An important feature of modern design based stereology is to be aware of potential sources of bias and eliminate them during the data collection. With many of the major neuroscience journals now insisting that quantitative data be presented, there is a greater need than ever for neuroscientists to understand the theory and practice behind quantitative methods, such as those offered by stereology. This book is a cookbook of stereological methods. Throughout the book, the emphasis is on practical guidance, rather than discussions and formulae.

Increasing incidence of Parkinson's disease with advancing age suggests that age-related processes predispose the nigrostriatal dopaminergic system to neurodegeneration. Several hypotheses concerning the effects of aging on nigrostriatal neurons were assessed in this study using a non-human primate model. First, we examined the possibility that the total number of dopaminergic neurons decline in the substantia nigra as a function of age. Stereological counting based on both tyrosine hydroxylase immunoreactivity (TH-ir) and neuromelanin (NM) content revealed no difference in cell number between young, middle-aged and old squirrel monkeys. We then determined whether advancing age changed the relative proportion of neurons characterized by 1) TH-ir in the absence of NM, 2) the presence of both TH-ir and NM, or 3) NM without TH-ir. Indeed, a progressive age-related depletion of TH only cells was paralleled by an increase in NM only neurons. The possibility that these changes could underlie a functional impairment of the nigrostriatal system was supported by striatal dopamine measurements showing a decrease in older monkeys. Finally, we tested the hypotheses that aging may enhance cell vulnerability to injury and that different dopaminergic subpopulations display varying degrees of susceptibility. When monkeys were exposed to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, cell loss was markedly more pronounced in older animals, and the ranking of vulnerability was TH only < TH/NM < NM only cells. The data indicate that, even in the absence of an overall neuronal loss, changes in the characteristics of dopaminergic cells reflect functional deficits and increased vulnerability to injury with age. NM content appears to be an important marker of these age-related effects. J. Comp. Neurol. 471:387–395, 2004. © 2004 Wiley-Liss, Inc.

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.

Although nontoxic when administered alone, diethyldithiocarbamate (DDC) is known to enhance the dopamine-depleting effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse striatum. The purpose of the present study was twofold: (i) to carefully characterize the effects of DDC on MPTP-induced degeneration of dopaminergic neurons in substantia nigra pars compacta using unbiased, stereological cell counting techniques and (ii) to determine whether or not DDC can convert a nontoxic dose of MPTP into one which is clearly toxic on dopaminergic neurons in the substantia nigra. A single low dose of MPTP (15 mg/kg intraperitoneally (ip)) was used for these studies, which failed to induce any neurochemical or histological effects on the nigrostriatal system of C57BL/6 mice when administered alone. However, when animals were pretreated with DDC (400 mg/kg ip), the same dose of MPTP resulted in a 50% loss of neurons in the substantia nigra pars compacta, as well as a 70% reduction in striatal dopamine (DA). A 31% reduction of DA in the ventral mesencephalon was also seen. This combined regimen of DDC and MPTP was not significantly different from a maximally tolerated "toxic" dose of MPTP alone (15 mg/kg x 4, 1 h apart, ip). As expected, animals receiving DDC alone did not show any dopamine depletion nor nigral neuronal loss. The present study confirms previous work suggesting that DDC enhances MPTP-induced nigral cell loss and shows for the first time that DDC can "unmask" MPTP toxicity. These observations could have implications for theories on the cause of Parkinson's disease.

Spontaneous reoccurrence of neurofilament (NF)-positive fibers has been described after spinal cord lesions in rats. However, previously introduced methods to evaluate the lesion and the regenerative fiber outgrowth suffer from several biases, why a new concept of quantitative, morphological analysis after spinal cord injury is needed. Length quantification of the putatively spontaneously regenerating fibers has been difficult until recently, when two length estimators based on sampling with isotropic virtual planes within thick physical sections were introduced. The applicability of these techniques to estimate the total length of NF-positive fibers was evaluated in photochemically induced ischemic lesions of thoracic spinal cords in young rats 6 weeks postlesion. Fiber length was found to be the most consistent measure with a mean of 3.71 m (coefficient of variation, CV = 0.16) in the 0.90 mm3 (CV = 0.26) large lesions. Whether or not the NF-positive fibers observed inside the lesion represent spontaneously regenerating axons needs to be confirmed in longitudinal, functional, and ultrastructural studies. J. Comp. Neurol. 400:441–448, 1998. © 1998 Wiley-Liss, Inc.

The present study was carried out with a variety of neuroanatomical techniques to investigate the consequences of chronic continuous nicotine treatment (0.125 mg · kg−1 · h−1, sc, 14 days) on the lesion-induced effects of a partial meso-diencephalic hemitransection. Both the striatonigral substance P (SP) and the nigrostriatal dopamine (DA) pathways were studied. The lesion-induced degenerative changes were most pronounced in the lateral parts of the ipsilateral substantia nigra and striatum. We have previously demonstrated that chronic nicotine infusion counteracts the lesion-induced loss of nigral tyrosine hydroxylase (TH) immunoreactive/Nissl stained DA neurons. The main finding of this study is that this phenomenon also involves changes in the striatonigral pathways. Thus, nicotine induced a disappearance of SP immunoreactive nerve terminals in substantia nigra pars compacta on the lesioned side, while it was again shown to counteract the lesion-induced disappearance of nigral TH immunoreactivity in the same animals. These data are interpreted on the basis of previous electrophysiological findings, where nicotine under similar experimental conditions counteracted the lesion-induced increase in burst firingin vivoin nigral dopamine neurons. Taken together these results indicate that nicotine may act by a reduced SP excitatory input to the nigral DA cells, which rescues them from dying. It is likely that the surviving cells are functional, since increased extracellular striatal DA levels have been observed after nicotine treatment ipsilateral to the lesion in a previous microdialysis experimentin vivo.These findings might contribute to the development of new neuroprotective therapies for patients suffering from neurodegenerative disorders such as Parkinson's disease.

The effect of chronic continuous (-)nicotine treatment in lesioned nigrostriatal dopamine (DA) systems was studied in rats with a partial unilateral mesodiencephalic lesion. (-)Nicotine significantly counteracted both the lesion-induced reduction in total number of nigral tyrosine-hydroxylase-like (TH-li) immunoreactive neurons and the lesion-induced upregulation of the high-affinity binding sites of the striatal DA D2 receptors analyzed with [3H]N-propylnorapomorphine. The suggested mechanism for the neuroprotection is a functional desensitization of the nicotinic cholinoceptors located on DA nerve cells. In a different lesion model, (-)nicotine either attenuated or enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice depending on the dose-schedule. Here, we suggest an action of MPTP on the nicotinic cholinoceptors preventing functional desensitization from taking place.

To further elucidate the previously demonstrated protective actions of nicotine on lesioned nigrostriatal dopamine (DA) systems (Janson and Moller, Neuroscience, 57 (1993) 931–941), the present receptor binding experiments were carried out. Rats were partially hemitransected at the meso-diencephalic junction and the effects of chronic continuous (-)nicotine treatment (osmotic pumps s.c., 0.125 mg/kg/h, 14 days) on [ 3 H] N- propylnorapomorphine ([ 3 H]NPA) and [ 3 H]methylcarbamylcholine ([ 3 H]MCC) binding were investigated in striatal coronal sections to study the agonist binding sites of DA D 2 receptors and nicotinic cholinoceptors, respectively. In saline-treated but not in nicotine-treated rats, the lesion led to an increased B max value of [ 3 H]NPA binding. The B max value of [ 3 H]MCC binding was increased by nicotine treatment and decreased by the partial hemitransection. These results indicate that chronic nicotine treatment counteracts the lesion-induced upregulation of the high-affinity agonist binding site of the DA D 2 receptor, which may be explained by an increased presence of DA via a protective effect of nicotine on neostriatal DA terminals. This action of nicotine may be of interest in the treatment of neurodegenerative diseases such as Parkinson's disease.

Publisher Summary This chapter describes the new morphological and functional features of fibroblast growth factor-2 (FGF-2) and gangliosides in the basal ganglia, especially in relation to the nigrostriatal dopamine system, which is degenerated in Parkinson's disease. The mapping of FGF-2 IR demonstrates an astroglial and neuronal localization within the substantia nigra and the globus pallidus, while in the neostriatum, the FGF-2 IR is confined to the astroglial populations. In the postnatal period, all areas of the basal ganglia including substantia nigra contained neuronal but not glial FGF-2 IR. In contrast to the adult rat, also a nuclear localization of FGF-2 IR can be observed within the nerve cells. In adulthood, the nuclear localization of FGF-2 IR is not seen in most neurons with the present antiserum but mainly in the astroglial cell populations. The FGF-2 IR DA nerve cells demonstrates within the zona compacta of the substantia nigra also a codistribution with the ganglioside positive nerve terminals, with high and low affinity FGF receptors and with FGF-2 mRNA level.

The vigilance promoting drug Modafinil has been demonstrated to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity (40 mg/kg sc) in the nigrostriatal dopamine (DA) system of the black mouse. 16, 29 A single dose of Modafinil (30 mg/kg ip, at different time intervals before and after MPTP) significantly counteracted the MPTP-induced depletion of striatal dopamine (DA) as analyzed 14 days later. A similar treatment protocol showed that Modafinil significantly counteracted the paradoxical MPTP-induced increase in locomotion and motility. Chronic administration of Modafinil (30 mg/kg ip daily for 14 days) led to a more pronounced counteraction of the MPTP-induced decrease in striatal DA and also attenuated the MPTP-induced increase in DA turnover. After a similar chronic treatment with Modafinil (10–100 mg/kg) the disappearance of tyrosine hydroxylase immunoreactive neurons in substantia nigra appeared to be dose-dependently counteracted. In summary, biochemical, behavioral and morphological evidence indicates a potential neuroprotective role of Modafinil, which could eventually lead to new pharmacological strategies in Parkinson's disease, once the mechanism of action has been established.

We studied the ability of the vigilance-promoting drug modafinil to modulate the anterograde and retrograde changes in tyrosine hydroxylase (TH) immunoreactivity and in dopamine (DA) stores in the nigro-neostriatal DA neurons, following a partial hemitransection of this ascending DA system, using a combined morphometrical, biochemical and behavioural analysis. Modafinil was given daily i.p. in doses of 10-100 mg/kg, starting 15 min after the lesion, and the partially hemitransected rats were killed 2 weeks later. Changes in TH-immunoreactive nerve cell bodies and nerve terminals induced by the partial hemitransection were studied in the substantia nigra and neostriatum in combination with image analysis. The substantia nigra and neostriatum were also subjected to biochemical analysis of DA, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels. Modafinil treatment dose-dependently (10-100 mg/kg) counteracted the hemitransection-induced disappearance of nigral TH-immunoreactive nerve cell body profiles and neostriatal TH-immunoreactive nerve terminal profiles. A 2-week treatment with 100 mg/kg of modafinil also counteracted the hemitransection-induced depletion of DA stores in the neostriatum and the ventral midbrain. Moreover, the repeated daily treatment with modafinil (100 mg/kg) protected against the hemitransection-induced disappearance of striatal 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and noradrenaline levels. Striatal DA function was analysed by studying apomorphine-induced (1 mg/kg, s.c.) ipsilateral rotational behaviour 4 and 11 days after the operation. A marked dose-dependent reduction of ipsilateral rotational behaviour was demonstrated after the daily modafinil treatment in the partially hemitransected rats. In another model involving unilateral nigral microinjections of 6-hydroxydopamine, acute (one single dose) modafinil (100 mg/kg) did not affect the contralateral rotational behaviour induced by apomorphine (0.05 mg/kg s.c.), when given 30 min before the apomorphine. Taken together, morphological, neurochemical and behavioural evidence has been obtained that anterograde and retrograde changes induced in the DA stores and TH immunoreactivity of the nigro-neostriatal DA neurons by a partial hemistransection are counteracted by modafinil in a dose dependent way with 100 mg/kg producing a significant protective action against impairment of DA transmission. The results of this study open up the possibility that modafinil may protect against the anterograde and retrograde degeneration of nigrostriatal DA neurons seen after mechanically induced injury.

Basic fibroblast growth factor (bFGF, FGF-2) is a trophic factor for neurons and astrocytes and has recently been demonstrated in the vast majority of dopamine (DA) neurons of the ventral midbrain of the rat. Potential neuroprotective actions of FGF-2 in the l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) model have also been reported. The actions of the FGF-2 have now been further analyzed in a combined morphological and behavioural analysis in the MPTP model of the adult black mouse, using a continuous human recombinant FGF-2 (hrFGF-2) intraventricular (i.v.t.) administration in a heparin-containing (10 IU heparin/ml) mock cerebrospinal fluid (CSF) solution. Tyrosine hydroxylase (TH) immunocytochemistry in combination with computer assisted microdensitometry demonstrated a counteraction of the MPTP-induced disappearance of neostriatal TH-immunoreactive (ir) nerve terminals following the FGF-2 treatment. Unbiased estimates of the total number of nigral TH ir neurons, using stereological methods involving the optical disector (Olympus), showed that the MPTP-induced reduction in the number of nigral TH ir nerve cell bodies counterstained with cresyl violet (CV; by 56%) was partially counteracted by the FGF-2 treatment (by 26%). The behavioral analysis demonstrated an almost full recovery of the MPTP-induced reduction of the locomotor activity after FGF-2 treatment. This action was maintained also 1 week after cessation of treatment. The hrFGF-2 produced an astroglial reaction as determined in the lateral neostriatum and in the substantia nigra (SN) far from the site of the infusion, indicating that the growth factor may have reached these regions by diffusion to activate the astroglia. Immunocytochemistry revealed FGF-2 immunoreactivity (IR) in the nuclei of the astroglia cell population in the dorsomedial striatum and the microdensitometric and morphometric evaluation demonstrated an increase in the number, but not in the intensity, of these profiles on the cannulated side, suggesting the possibility that hrFGF-2 stimulates FGF-2 synthesis in astroglial cells with low endogenous FGF-2 IR. These results indicate that hrFGF-2, directly and/or indirectly via astroglia, upon i.v.t. infusion exerts trophic effects on the nigrostriatal DA system and may increase survival of nigrostriatal DA nerve cells exposed to the MPTP neurotoxin.

To elucidate possible actions of nicotine on dopamine D2 receptor binding, the effect of chronic continuous (-)nicotine treatment (osmotic pumps s.c., 0.125 mg kg h-2, 14 days) was studied in the binding of [3H]N-propylnorapomorphine ([3H]NPA) and [125I]sulpride in coronal cryostat sections in the rat. Quantitative autoradiography showed that nicotine decreased the binding of [3H]NPA in the basal ganglia, preferentially in the nucleus accumbens and olfactory tubercle. In contrast, [125I]sulpride binding was not affected. Nicotine decreased the KD value of [3H]NPA by 27% and decreased the Bmax value by 17%, using filter-wiped sections. These results indicate that chronic continuous nicotine treatment affects the D2 receptor and that this effect may be involved in the development of nicotine dependence.

Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.

The effects of modafinil on acetylcholine and GABA outflow from the cerebral cortex of awake freely moving guinea pigs provided with an epidural cup were studied. In the dose range of 3-30 mg/kg s.c. modafinil produced a dose dependent significant inhibition of GABA outflow without influencing cortical acetylcholine release. Methysergide (2 mg/kg, i.p.) and ketanserin (0.5 mg/kg, i.p.) but not prazosin (0.14 mg/kg, i.p.) counteracted the inhibitory action of modafinil on cortical GABA outflow. Modafinil both acutely and chronically in the same dose range increased striatal 5-HIAA levels and 5-HT utilization in the rat (acute) and mouse (chronic). The action on cortical GABA release may be dependent on activity at 5-HT2 receptors, since the action of modafinil in this respect is blocked by the non-selective 5-HT antagonist methysergide and the 5-HT2 antagonist ketanserin. The involvement of 5-HT mechanisms in the inhibitory action of modafinil on cortical GABA release is also suggested by the findings that 5-HT metabolism may become increased by modafinil at least in the striatum. The reduction of cortical GABA outflow via 5-HT2 receptors by modafinil is probably related to some of its actions on the central nervous system including behavioural effects.

The effect of chronic treatment with (−)-nicotine on the decrease in extracellular dopamine (DA) levels in neostriatum induced by a unilateral transection at the meso-diencephalic junction in rats was studied. At the lesion time, Alzet minipumps filled with (−)-nicotine were implanted subcutaneously. Two weeks later, microdialysis probes were implanted bilaterally into the neostriatum. Perfusates were assayed for DA, acetylcholine (ACh) and metabolites in HPLC systems under basal and KCl stimulated conditions. The unilateral hemitransection produced an ipsilateral decrease in neostriatal extracellular DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), but not in ACh levels. Chronic nicotine treatment counteracted the lesion-induced decrease in DA, but had no effect on extracellular DA levels in the contralateral neostriatum or in normal rats. The results support the idea that chronic treatment may protect against degeneration of central DA neurons.

Chronic continuous administration of nicotine (0.125 mg/kg/h, 14 days) to male Sprague-Dawley rats with a partial hemitransection at the meso-diencephalic junction caused a significant reduction in burst firing of remaining dopamine (DA) neurons in the zona compacta, substantia nigra, whereas neither the firing rate nor the number of spontaneously active DA cells per track were altered in comparison with saline-treated, hemitransected controls. The reduced functional activity of the remaining DA cells subjected to nicotine treatment provides a physiological correlate to the previously observed, reduced DA utilization in these neurons. It may also help to explain the increased nigral DA cell survival found after chronic nicotine treatment in similar lesion experiments.

Morphological changes in ventral mesencephalic dopamine (DA) neurons of a monkey sustaining a unilateral electrolytic lesion of the ventromedial mesencephalic tegmentum four years earlier were examined. Substantia nigra (A9) DA neurons lateral to the lesion underwent hypertrophic changes. The mean area of these neurons was enlarged by approximately 30% relative to corresponding neurons in the contralateral substantia nigra. Semi-quantitative immunohistochemical measurements of the intensity of tyrosine hydroxylase-like immunoreactivity (TH-li) indicated an increase in the amount of TH-li protein per cell in the hypertrophied neurons. Hypertrophic changes were also observed in ipsilateral A11 DA neurons of the caudal hypothalamus, suggesting that the increase in size was related to transection of the axons of DA neurons as they pass through the midbrain in their projections to target sites. The lesion did not overtly change the density or pattern of the substance P innervation of the substantia nigra, indicating that the striato- and pallido-nigral projections were spared by the lesion. These data suggest that hypertrophy may be a compensatory mechanism of dopaminergic neurons in response to partial lesions of the nigrostriatal system, and thus represent a morphological counterpart to the compensatory biochemical processes effected in response to partial lesions of the striatal dopaminergic innervation.

In a series of studies on the effects of chronic nicotine treatment via minipumps on retrograde and anterograde degenerative processes in nigrostriatal dopamine (DA) neurons following a partial hemitransection, morphological, biochemical, functional and neurophysiological evidence has been obtained of protective actions of chronic nicotine treatment against the degeneration of nigrostriatal DA neurons in the male rat. 1. By means of tyrosine hydroxylase (TH) immunocytochemistry in combination with image analysis it was demonstrated that chronic nicotine treatment via minipumps ((-)nicotine hydrogen (+)tartrate: 0.125 mg/kg/h for 2 weeks) significantly counteracted the disappearance of TH immunoreactive (IR) nerve cell body, dendrite and terminal profiles of the nigrostriatal DA neurons produced by a partial di-mesencephalic hemitransection. The most marked protection was observed against the degeneration of the TH dendritic profiles in the substantia nigra. 2. By means of DA fluorescence histochemistry in combination with quantitative histofluorimetry as well as of biochemical analysis of DA stores it was demonstrated that this type of chronic nicotine treatment also protected against the disappearance of DA stores within the substantia nigra and within nucleus caudatus putamen and the nucleus accumbens induced by a partial di-mesencephalic hemitransection. By the use of the TH inhibition method using a-methyl-(±)-p-tyrosine methyl ester (α-MT) evidence was also obtained that chronic nicotine treatment preerentially and substantially reduces forebrain DA utilization on the lesioned side. 3. By means of analysis of local cerebral blood flow and glucose utilization within the neostriatum by computer assisted autoradiography using the radioligands 14C-deoxyglucose and 14C-iodoantipyrin it was demonstrated that the partial hemitransec- tion at the di-mesencephalic level produced a substantial reduction in both glucose utilization and in blood flow in the neostriatum of the lesioned side. It was demonstrated that this reduction of both glucose utilization and of cerebral blood flow in the neostriatum was counteracted by the chronic nicotine treatment using minipumps (same dose treatment schedule as above). 4. By means of the standard single cell recording method the extracellular activity of nigral DA neurons was analyzed two weeks following partial hemitransection at the meso-diencephalic level. It was found that the chronic nicotine treament as described above produced a significantly lower burst firing in the nicotine treated hemitransected animals compared with the hemitransected group treated with saline. No differences in firing rate or regularity of firing was observed between the two groups. It is suggested that the mechanism underlying the protective action of nicotine in this mechanical lesion model is represented a desensitization of the excitatory nicotinic cholino- ceptors located on the nigral DA nerve cells and on the DA fore- brain terminals. Desensitization of the nicotinic cholinoceptors leads to the demonstrated reduction of burst firing, and thus to a preferential and marked reduction of DA utilization in the surviving DA nerve terminals. In this way reduced energy demands develop in the surviving nigrostriatal DA neurons as well as a reduced calcium ion influx, which is known to have neurotoxic activity. Desensitization of the nicotinic cholinergic receptors per se will also lead to a reduced calcium influx in view of the closure of the ligand gated kation channel in the nicotinic cholinoceptors. In conclusion, chronic nicotine treatment, via desensitization of the excitatory nicotinic cholinoceptors, may enhance repair mechanisms in central neuronal systems, possessing large numbers of nicotinic receptors, such as the monoaminergic and cholinergic neurons. There is an urgent need to further evaluate protective actions of chronic nicotine treatment in animal models of Parkinson’s disease and Alzheimer’s disease in view of its potential therapeutic role (see also Janson et al., this symposium).

Protective activity of (-) nicotine on l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine-induced (MPTP, 50 mg/kg sc) neurotoxicity is demonstrated in the mesostriatal dopamine (DA) system in the male black mouse with an acute intermittent (-)nicotine treatment (0.5 mg/kg x 4, starting immediately following the MPTP injection with a 30-minute time interval) together with a two-week continuous administration of (-) nicotine via Alzet minipumps implanted subcutaneously the day before MPTP is given. In contrast, when omitting the acute (-) nicotine treatment a dose-dependent increase in the MPTP-induced neurotoxicity is seen.

Patients with the heritable form of retinoblastoma carry a constitutional mutation in the retinoblastoma locus in heterozygous form. The majority of such cases are the result of new mutations, which may be inherited by their offspring. We have identified such constitutional mutations within the retinoblastoma locus in 3 out of 66 investigated unrelated gene carriers, using Southern blot analysis and Rb-gene cDNA-probes. The identified mutations were found to be located in different regions of the gene. These analyses may be used to identify or exclude close relatives at risk for the disease. In 2 of the 3 cases, the identified aberrations were used for informed genetic counselling of relatives.

In order to further study the previously demonstrated protective action of chronic nicotine treatment on lesioned meso-striatal dopamine (DA) pathways, the following study was carried out on DA utilization in these lesioned neurons. Male Sprague-Dawley rats were partially hemitransected at the meso-diencephalic junction and treated with nicotine (0.125 mg · kg−1 · h−1) by means of Alzet minipumps implanted subcutaneously for 2 weeks. The overall serum nicotine level obtained was 64.6 ± 2.7 ng · ml−1. The results demonstrated that partial di-mesencephalic hemitransections produced a marked reduction of DA fluorescence (quantitative histofluorimetry) on the lesioned side in the nucleus caudatus putamen, anterior nucleus accumbens and posterior lateral tuberculum olfactorium. No significant effects were observed on the intact side. Furthermore, studies on changes in DA utilization as evaluated 2 h after tyrosine hydroxylase inhibition showed an augmentation in the α-methyl-(±)-p-tyrosine methyl ester (α-MT)-induced depletion of the DA stores on the hemitransected side in comparison with the operated side of the sham-operated animals. On the hemitransected side chronic nicotine treatment increased DA stores in the DA nerve terminals of the nucleus caudatus putamen and the posterior lateral tuberculum olfactorium. No significant effects were observed on the intact side. Following chronic nicotine treatment a marked and preferential attenuation of the α-MT-induced depletion of DA stores was seen in the various DA nerve terminal systems of the forebrain on the hemitransected side. In the substantia nigra reduced DA levels (HPLC) were demonstrated on the hemitransected side, while no effects on the non-operated side were observed. Also an increase of the α-MT-induced depletion of the DA stores was seen on the hemitransected side in comparison with the operated side of the sham-operated animals. In contrast, on the non-operated side an attenuation of the a MT-induced depletion of the DA stores was found. Following chronic nicotine treatment the lesion induced reduction of the nigral DA stores on the hemitransected side was counteracted, as was the lesion induced increase in the α-MT-induced depletion of DA stores, which was replaced by a reduction of the α-MT-induced depletion of the nigral DA stores. However, on the non-operated side an increased DA depletion was observed after α-MT treatment in rats treated chronically with nicotine. Chronic nicotine treatment under the present conditions did not significantly alter serum levels of corticosterone and reduced prolactin serum levels in sham-operated rats. The present results indicate that a partial hemitransection produces marked increases in DA utilization of the forebrain and of the substantia nigra on the lesioned side; whereas on the non-operated side a reduction in nigral DA utilization was found. On the hemitransected side chronic nicotine treatment increases DA stores of the nucleus caudatus putamen, tuberculum olfactorium and substantia nigra, suggesting a protective action of nicotine. Chronic nicotine treatment preferentially and substantially reduces striatal, accumbens and nigral DA utilization on the lesioned side. On the non-operated side chronic nicotine treatment abolished the lesion-induced reduction of DA utilization in the substantia nigra. These results are in support of the hypothesis that a protective action of chronic nicotine treatment on ascending DA systems may be produced via a desensitization of excitatory nicotine cholinoceptors regulating the ascending DA pathways, leading to reduced firing rates and thus to reduced energy demands. The endocrine system does not seem to be involved in these effects.

(1) In the present study the occlusion method was employed to evaluate the overall coexistence of neuropeptide Y and phenylethanolamine-N-methyl transferase, neuropeptide Y and tyrosine hydroxylase as well as cholecystokinin and phenylethanolamine-N-methyl transferase immunoreactivity in nerve cell bodies of the dorsal subnuclei of the nucleus tractus solitarius of the male rat. A high degree of coexistence was established for neuropeptide Y/phenylethanolamine-N-methyl transferase, cholecystokinin/phenylethanolamine-N-methyl transferase and for tyrosine hydroxylase/neuropeptide Y immunoreactivity. (2) Sulfated [ 12 I]cholecystokinin-8 was used as radioligand to study the densities of cholecystokinin-8 binding sites in the dorsal medulla oblongata by means of quantitative receptor autoradiography. High densities of binding sites were observed in parts of the nucleus tractus solitarius and in the area postrema. Labeling was also observed in the dorsal motor nucleus of the vagus. (3) In the physiological studies adrenaline (0.15–1.0 nmol), neuropeptide Y (0.075–0.75 nmol) and sulfated cholecystokinin-8 (0.3–3.0 nmol) were administered alone or in combination with neuropeptide Y or adrenaline intracisternally into α-chloralose anaesthetized male rats. Especially the hypotensive and bradycardic responses of adrenaline were counteracted in the adrenaline/cholecystokinin co-treated animals, whereas the cardiovascular effects of neuropeptide Y when co-administered with cholecystokinin-8 (0.3 nmol) appeared to be more resistant to the antagonistic effect of cholecystokinin 8. In addition, cholecystokinin-8 further enhanced the neuropeptide Y-induced bradynpnea and increase in the tidal volume. The present results indicate the existence of neuropeptide Y, adrenaline and cholecystokinin-8 immunoreactivity in the same neurons of the dorsal subnuclei of the nucleus tractus solitarius. Furthermore, binding sites for cholecystokinin-8 seem to at least partly co-distribute with α-2 adrenergic and neuropeptide Y binding sites in the nucleus tractus solitarius. In the functional analysis, an antagonistic interaction between cholecystokinin-8 and adrenaline as well as between cholecystokinin and neuropeptide Y is demonstrated opening up the possibility that cholecystokinin peptides act as intrinsic modulators in the putative cholecystokinin/neuropeptide Y/adrenaline synapses in the nucleus tractus solitarius.

Partial hemitransection was performed through a knife lesion at the meso-diencephalic level in rats to sever the mesostriatal dopamine system. During the subsequent 2 weeks the animals received 0.125 mg/kg/h of nicotine continuously via an osmotic minipump implanted s.c. To achieve prompt high nicotine levels, 4 i.p. injections of 0.5 mg/kg nicotine were, in addition, given during the first 2 h following the lesion. The total treatment corresponded to a mean plasma level of 50 ng/ml nicotine, measured at the end of the experiment. Control animals received corresponding volumes of 0.9% saline. Quantitative autoradiographic analysis of the glucose utilization in the caudate nucleus using Sokoloff's [14C]2-deoxyglucose method demonstrated a 16% side-to-side difference in the lesioned control animals, whereas the asymmetry was counteracted by the nicotine treatment. Although there was an overall tendency to a lower rate of glucose utilization (by 6%) in the nicotine-treated animals compared to the controls receiving saline only, the difference was not statistically significant. The eliminated asymmetry probably reflects an increased survival of the dopamine neurons and/or of striatal nerve cells on the lesioned side due to protective effects of nicotine resulting from desensitization of nicotinic-type cholinergic receptors following continuous administration of the drug.

Somatostatin/catecholamine as well as growth hormone releasing factor/catecholamine interactions have been characterized in the hypothalamus and the preoptic area using morphometrical and quantitative histofluorimetrical analyses. 1. (1) The morphometrical analysis of adjacent coronal sections of the rat median eminence demonstrated a marked overlap of somatostatin and tyrosine hydroxylase immunoreactive nerve terminals as well as of growth hormone releasing factor and tyrosine hydroxylase immunoreactive nerve terminals in the medial and lateral palisade zones of the rostral and central parts. Furthermore, the studies on codistribution of growth hormone releasing factor and tyrosine hydroxylase immunoreactivity indicate that only a limited proportion of the growth hormone releasing factor and the dopamine nerve terminals may costore dopamine and growth hormone releasing factor respectively in the medial and lateral palisade zones (see Meister et al. , 1985). 2. (2) Intravenous injections of somatostatin 1–14 (100 μg/kg, 2 h) into the hypophysectomized male rat produced an increase in dopamine utilization in the medial and lateral palisade zones of the median eminence. 3. (3) Intravenous injections of rat hypothalamic growth hormone releasing factor (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence but increased noradrenaline utilization in the ventral zone of the hypothalamus and produced a depletion of noradrenaline stores in the paraventricular hypothalamic nucleus. 4. (4) Intravenous injections of human pancreatic growth hormone releasing factor 1–44 (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence, but reduced noradrenaline utilization in the subependymal layer and increased noradrenaline utilization in the suprachiasmatic preoptic nucleus. The combined results of the present and previous studies have led us to put forward the medianosome concept. The medianosome is defined as an integrative unit, which consists of well defined aggregates of transmitter identified nerve terminals interacting with one another in the external layer of the median eminence. Our present data indicate the existence of putative medianosomes consisting predominantly of growth hormone releasing factor nerve terminals costoring dopamine as well as of somatostatin and dopamine nerve terminals, which interact locally to control growth hormone secretion. A complementary control of growth hormone secretion may be exerted by noradrenaline mechanisms in the subependymal layer, in the ventral zone and/or in the suprachiasmatic preoptic nucleus. However, further analyses in view of the differential effects seen with the present doses of rat hypothalamic and human pancreatic growth hormone releasing factor have to be done. The results also indicate the possible existence of growth hormone releasing factor receptors in the median eminence which may participate in the feedback control of the growth hormone releasing factor immunoreactive neurons in the ventral zone of the hypothalamus.

The present results demonstrate that chronic nicotine treatment can in part protect against mechanically-induced and neurotoxin-induced degeneration of nigrostriatal DA neurons. These results indicate that in sufficient doses chronic treatment with nicotine may be considered in the pharmacological treatment of Parkinson's disease. It remains to be demonstrated whether these protective actions can be extended to include also other injured neurons such as the cholinergic neurons, known to be severely affected in Alzheimer's disease.

Publisher Summary The morphological studies in the past have been performed by means of computer-assisted morphometry and microdensitometry to give an objective representation of the central adrenaline (A) nerve cell groups in the medulla oblongata. The functional and biochemical studies of the A neurons within the medulla oblongata, especially within the dorsal cardiovascular region, have given evidence that the adrenergic neurons have an important vasodepressor function in the central nervous system (CNS). The existence of neuropeptide Y–like immunoreactivity in the A cell groups C1, C2, and C3 in the medulla oblongata has been demonstrated by studies in the past. Based on these observations, the effects of centrally administered neuropeptide Y (NPY) on cardiovascular and respiratory parameters and on pre- and postsynaptic mechanisms in central A nerve terminal networks in the dorsal cardiovascular region of the medulla oblongata of the rat have been analyzed. These studies have shown that NPY given intracutaneously in the α-chloralose anesthetized rat can reduce arterial blood pressure and heart rate. These actions have been observed in the presence of α-adrenergic receptor blockade.

Neuroprotective and possible trophic actions of nicotine were studied in two types of experimental models: (1) one in which the meso-striatal dopamine system was subjected to partial hemitransection, and regional glucose utilization (using 2-[3H]deoxyglucose) and blood flow (using [14C]iodoantipyrine) were measured by computer-assisted quantitative autoradiography based on a double-isotope technique; and (2) another where the sympathetic cranial nervous system supplying the brain vasculature was subjected to decentralization, axotomy, and partial or complete ganglionectomy, and the neuronal survival and fiber regeneration were elucidated by fluorescence histochemistry of noradrenaline, tyrosine hydroxylase, and neuropeptide Y. Continuous nicotine infusion for 4 weeks failed to significantly affect the neuronal response to the surgical interference of the sympathetic noradrenergic system. The same nicotine treatment for 2 weeks significantly improved glucose utilization and blood flow in caudate-putamen on the side in which the meso-striatal dopamine system had been transected, thus eliminating the 16% side-to-side asymmetry in the metabolism caused by the axotomy. The dopamine agonist, EMD 23,448, was without significant effect on this asymmetry. The hemitransection produced marked reduction in metabolism and flow also in the ventro-lateral thalamus. In substantia nigra, glucose utilization was markedly elevated--perhaps as a consequence of a regenerative increase in protein synthesis--opposite to a considerable reduction in nigral blood flow. Little or no effect of the hemitransection was seen in hippocampus or nucleus accumbens. In neither of these four regions did nicotine (or EMD 23,448) have any overt influence on glucose metabolism or blood flow. It is concluded that nicotine, mainly through its protective action on the meso-striatal dopaminergic system, is able to improve striatal glucose utilization and associated blood flow, probably reflecting a tendency to amelioration of neurotransmission function of surviving terminals belonging to the nigro-striatal dopamine neurons.

Glucocorticoid receptor (GR) immunoreactivity (IR) was analyzed semi-automatically in the forebrain and in the lower brain stem of male rats treated for two weeks with imipramine (10 mumol/kg). Serum corticosterone and aldosterone levels were determined by means of radioimmunoassay procedures. The microdensitometric analysis demonstrated a selective increase in the GR IR in the nerve cell nuclei of the locus coeruleus (A6), of the ventral part of the reticular gigantocellular nucleus (B3L) and of the nucleus raphae magnus (B 3 M), whereas a small reduction of GR IR was found in the nucleus raphe obscurus (B2). In the morphometric analysis significant increases in the mean profile area of nuclear GR IR, which may be secondary to the increase in GR IR, were observed in the B 3 M. The serum corticosterone and aldosterone levels were not found to be significantly altered. The selective changes of GR IR may reflect the presence of an altered number of GR in these nerve cell groups and/or an altered translocation of GR to the nuclei. It is of substantial interest that these changes were observed in the presence of unchanged serum levels of corticosterone and aldosterone. It seems possible that adaptive changes in monoamine synapses induced by the chronic imipramine treatment may be responsible for the changes in GR IR found in the noradrenaline (NA) and 5-hydroxytryptamine (5-HT) cell bodies, respectively. The present results open up the possibility that chronic imipramine treatment may help to maintain the glucocorticoid receptor function in the locus coeruleus and in the 5-HT cell groups of the rostral ventromedial medulla of depressed patients.

Male rats were exposed to severe 14 day immobilization stress. Body weight, body temperature, food and water intake, behavioral parameters, and serum corticosterone levels were measured during and after the stress period. On the 7th day after cessation of stress the experimental animals together with the control rats were taken to immunocytochemical analysis involving morphometry and microdensitometry of tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), various neuropeptide, and glucocorticoid receptor (GR) immunoreactivities (IRs) in a large number of regions of the central nervous system. In addition, adrenocorticotropic hormone (ACTH) IR was analyzed in the pituitary gland. Seven days following cessation of the chronic stress food intake, total locomotion and forward locomotion had been restored to normal. Serum corticosterone levels appeared to remain increased even 6 days following cessation of the chronic immobilization stress, probably caused by increased release of ACTH. Paraventricular corticotropin releasing hormone (CRF) IR was negatively correlated with the pituitary ACTH IR, indicating that the increase in ACTH release was produced by an increased release of CRF from the hypothalamus. The major immunocytochemical change observed 7 days after cessation of stress was a disappearance of 5-HT IR in the 5-HT cell groups B 1, B 2, B 3, and B 7. 5-HT IR in nerve terminals was only affected in the dorsal horn, where 5-HT IR was increased in the substantia gelatinosa. GR IR was found to be significantly increaed in monoaminergic cell groups: serotoninergic B 7, dopaminergic A 12, and noradrenergic A 1, A 2, and A 6. A trend for a reduction of TH IR was observed in nigral DA cells associated with significant reductions in TH IR in striatal DA nerve terminals. Finally, increases in 5-HT and substance P (SP) IR were found in the nerve terminals of the substantia gelatinosa of the cervical spinal cord in the stress group. In the present experimental model evidence has been obtained for a maintained activation of the hypothalamic-pituitary-adrenal axis as evaluated 7 days after cessation of severe chronic immobilization stress. The reduction of 5-HT IR in various 5-HT cell groups indicates a reduction of 5-HT synthesis, which may also be associated with reduced 5-HT release from the nerve terminals, since no depletion was observed in terminal regions and in one case an increase in 5-HT IR was noted (substantia gelatinosa). The increase in GR IR, demonstrated in the NA and 5-HT cell groups in the presence of a maintained hypersecretion of corticosterone may represent signs of an upregulation of GR synthesis and/or increased translocation, which take place in the presence of maintained hypersecretion of corticosterone. Thus, 5-HT and NA neurons may respond more effectively to circulating glucocorticoids after severe chronic stress. In this way glucocorticoids may protect against stress-induced exhaustion of neurons leading to impairment of transmission. Studies on TH IR suggest a deficit in the DA transmission line of the nigrostriatal DA neurons, but of no other CA neurons studied. Such effects may contribute to behavioral suppression. Finally, the stress-induced increases in 5-HT and SP IR in the substantia gelatinosa may in part underlie the phenomenon of stress-induced analgesia.

Using molecular cloning, we earlier isolated the "retinoblastoma gene"; mutations or deletions at this locus are associated with the hereditary predisposition to some human cancers, especially retinoblastoma and osteosarcoma. To develop diagnostic tests for such a predisposition, we identified restriction-fragment-length polymorphisms (RFLPs) within the retinoblastoma gene and tested their usefulness in predicting the risk of cancer in 20 families with members who had hereditary retinoblastoma. We were able to make predictions in 19 of the 20 kindreds. In 18 kindreds, we demonstrated a consistent association of marker RFLPs with the mutation predisposing to retinoblastoma. In the 19th kindred, there may be a lack of cosegregation of the DNA polymorphisms within the gene and the site of the mutation predisposing to retinoblastoma. However, there is uncertainty about the clinical diagnosis of the retinal lesion in a key member of this kindred; if the lesion is not a retinoblastoma, there is no discrepancy between the DNA polymorphisms and the retinoblastoma trait. We conclude that it is feasible and clinically useful to use these DNA polymorphisms to determine the risk of cancer.

Retinoblastoma may be caused by constitutional mutations in the retinoblastoma gene which segregates as an autosomal dominant inherited predisposition for developing retinoblastoma tumours. Since 75% of these cases are new mutations, there is a need for methods to identify carriers of such germ-line mutations, so that informed genetic counselling is available to patients and close relatives. We have used pulsed-field gel electrophoresis in screening 20 unrelated cases with bilateral retinoblastoma. One constitutional mutation could be detected, and was found to be caused by a balanced chromosome (4; 13) translocation with the breakpoint within intron 17 of the retinoblastoma gene.