Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways.

Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.

Retroviral vectors derived from lentiviruses such as HIV-1 are promising tools for human gene therapy because they mediate the in vivo delivery and long-term expression of transgenes in nondividing tissues. We describe an HIV vector system in which the virulence genes env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. These data demonstrate the potential of lentiviral vectors in human gene therapy.

Regulation of gene expression in vivo following transduction by two separate rAAV vectors.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

LRRK2 mutations in a clinic-based cohort of Parkinson's disease.

In the last decade, major breakthroughs in the understanding of genetic contributions to Parkinson's disease (PD) have been achieved. Recently, mutations in LRRK2, encoding dardarin, have been found to be responsible for an autosomal dominant parkinsonism (OMIM 607060). We screened 311 subjects (cases: n = 202, controls: n = 109) for the three previously reported LRRK2 mutations. Our investigation revealed a sporadic case of PD with a heterozygous mutation G2019S (c.6055G>A). Here, we present the clinical phenotype of this patient and discuss the implications of genetic testing for the G2019S mutation in patients with sporadic PD.

Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra.

Guanosine triphosphate cyclohydrolase I (GTPCHI) is a critical enzyme in catecholamine function and is rate limiting for the synthesis of the catecholamine co-factor tetrahydrobiopterin. The present study assessed the distribution of GTPCHI immunoreactivity (-ir) within the monkey and human ventral midbrain and determined whether its expression is altered as a function of age. Light and confocal microscopic analyses revealed that young monkeys and humans displayed GTPCHI-ir within melanin-containing and tyrosine-hydroxylase-ir neurons in primate substantia nigra. Stereological counts revealed that there was a 67.4% reduction in GTPCHI-ir neuronal number, a 63.5% reduction in GTPCHI-ir neuronal density, and a 37.6% reduction in neuronal volume in aged monkeys relative to young cohorts. Similar age-related changes were seen in humans, in whom there were significant reductions in the number of GTPCHI-ir nigral neurons in middle age (58.4%) and aged (81.5%) cases relative to young cohorts. The density of GTPCHI-ir neurons within the nigra was similarly reduced in middle-aged (63.0%) and aged (81.8%) cases. In contrast to monkeys, aged humans did not display shrinkage in the volume of GTPCHI-ir nigral neurons. The presence of numerous melanin-positive, but GTPCHI-ir immunonegative, neurons in the aged monkey and human nigra indicates that these decreases represent an age-related phenotypic downregulation of this enzyme and not a loss of neurons per se. These data indicate that there is a dramatic decrease in GTPCHI-ir in nonhuman primates and humans as a function of age and that loss of this enzyme may be partly responsible for the age-related decrease in dopaminergic tone within nigrostriatal systems.

Characterization of GABA release from intrastriatal striatal transplants: dependence on host-derived afferents.

Extracellular levels of GABA, derived from cell suspension transplants of embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum, were measured using intracerebral microdialysis in halothane-anaesthetized rats. GABA overflow was monitored using loop type dialysis probes implanted into grafted, age-matched ibotenic acid-lesioned and intact striata, under baseline conditions and after different pharmacological manipulations. Basal and evoked GABA release, which was reduced by 58 and 96%, respectively, in the excitotoxin-lesioned striatum, was restored by the striatal grafts to levels close to or above those observed in normal striata. The graft-derived release of GABA was most likely of neuronal origin, since the K(+)-evoked (100 mM) GABA overflow was reduced by almost 80% when Ca++ was replaced by 20 mM Mg++ in the perfusion medium, and blockade of GABA uptake by nipecotic acid (0.5 mM), induced a greater than six-fold increase in GABA overflow. However, perfusion of the graft with 1 microM tetrodotoxin in combination with K+ (100 mM) resulted in little if any reduction in the K(+)-evoked overflow. Histological analysis demonstrated a dense tyrosine hydroxylase-positive fibre network in the grafts, which was removed after a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal pathway. The dopamine denervating lesion resulted in an increased K(+)-evoked GABA overflow both in the intact (+76%) and the grafted striata (+181%), suggesting that the tonic dopaminergic inhibitory control of GABA release, seen in the intact striatum, is also present in the grafted striata. The glutamate analogue, kainic acid (1 mM added to the perfusion fluid), evoked a 60-74% increase in GABA overflow both in intact striata (with or without dopaminergic denervation) and in the striatal grafts. This effect seemed to be dependent on an intact corticostriatal projection, since knife-cut transections of the frontal cortex at the level of the forceps minor, abolished the response in both the intact and grafted striata. These results demonstrate that grafts of fetal striatal tissue implanted into the excitotoxically lesioned striatum restore striatal GABA overflow in a neuron-dependent manner, close to or above that seen in the normal intact striatum. Furthermore, the graft-derived GABA release appears to be under normal regulatory control from the host dopaminergic and glutamatergic systems. Since the GABAergic striatal output system is critical for the expression of striatum-related behaviours, it is proposed that the graft-induced behavioural recovery in the striatal lesion model, at least in part, may depend on the restoration of striatal GABAergic neurotransmission.

Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum.

Dopamine receptor-mediated Fos protein expression in the striatum has been used to monitor dopamine receptor activation at the cellular level after dopaminergic denervation and reinnervation by fetal nigral transplants. The pattern of striatal Fos expression after systemic administration of either the dopamine receptor agonist, apomorphine, or the dopamine-releasing agent, amphetamine, was studied in rats which had received cell suspension grafts of fetal ventral mesencephalic neurons into the striatum after a complete 6-hydroxydopamine lesion of mesostriatal dopaminergic projection. Grafted animals, and normal and lesioned controls were killed 2 h after administration of either D-amphetamine (5 mg/kg, i.p.) or apomorphine (0.25 mg/kg, s.c.). Fos protein was detected immunohistochemically, and the density of Fos-immunoreactive cell nuclei was measured in 12 selected areas of caudate-putamen, nucleus accumbens and globus pallidus by computerized image analysis. Consistent with previous studies, amphetamine induced high Fos expression in the medial and dorsal parts of the intact caudate-putamen and significantly lower expression in the denervated caudate-putamen. A significant difference between lesioned and intact striata was present also in globus pallidus, but not in nucleus accumbens. In grafted rats, amphetamine-induced Fos activation was restored to normal or supranormal levels in the anterior and central caudate-putamen (i.e. close to the graft deposits), whereas in the tail of caudate-putamen Fos expression was significantly lower than normal. The side-to-side difference in globus pallidus seen in lesioned rats was no longer present in the grafted animals. Apomorphine led to high Fos activation throughout the dopamine-depleted caudate-putamen, whereas only very few immunopositive cells were observed in the intact caudate-putamen. Also in globus pallidus and nucleus accumbens, a significantly higher number of Fos-immunoreactive cells was detected on the denervated side. In the grafted rats, apomorphine-induced Fos activation was similar to normal in all striatal areas sampled, as well as in the globus pallidus. The graft-induced effect extended over a considerably larger area than that covered by the graft-derived tyrosine hydroxylase-immunoreactive innervation. These findings indicate that fetal ventral mesencephalic transplants normalize dopamine receptor-mediated function in the 6-hydroxydopamine-lesioned caudate-putamen and nucleus accumbens, as well as in a primary target of the striatal output neurons, the globus pallidus. The results support the idea that dopamine released from the grafted neurons, both under baseline conditions and after amphetamine administration, exerts functional effects over a larger volume of the striatum than that reached by the graft-derived fibers.

Long-term restoration of striatal L-aromatic amino acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a rodent model of Parkinson's disease.

As a potential treatment for Parkinson's disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinson's disease patients may be an attractive gene therapy strategy.

Independent effects of age and nucleus basalis magnocellularis lesion: maze learning, cortical neurochemistry, and morphometry.

The effects of age and lesion of the cholinergic nucleus basalis magnocellularis (NBm) were assessed behaviorally, morphologically, and biochemically. Groups consisted of rats lesioned 1 month before testing, rats lesioned 13 months before testing, and their respective age-matched controls. Both age and lesion independently induced behavioral deficits in performance on two water maze tasks. The combined effect of these two factors produced behavioral deficits equal to the sum of the individual impairments. NBm lesion produced a 28% decrease in anterior cortical choline acetyltransferase activity and a 20% decrease in synaptophysin immunoreactivity in the neocortex that was stable over a 12-month period. Neither neuritic plaque nor neurofibrillary-tanglelike structures were found in the brains of 18-month-old control rats, nor were they found in NBm-lesioned rats examined 15 months postlesion. There was an age-related decrease in homovanillic acid levels in both control and NBm groups, which suggests a decrease in dopamine turnover. These results show a lack of biochemical and behavioral recovery after NBm lesion and suggest that the effects of age on behavior are independent of NBm-cortical dysfunction.

Physostigmine improves water maze performance following nucleus basalis magnocellularis lesions in rats.

Bilateral excitotoxic lesions of the nucleus basalis magnocellularis in rats were used along with testing in the water maze task to assess whether inhibition of acetylcholinesterase with physostigmine would reverse the lesion-induced impairment. Rats were lesioned bilaterally in stages using ibotenic acid and then behaviorally tested 3 weeks after surgery. Lesioned animals were administered one of three doses of physostigmine (0.06, 0.19, or 0.32 mg/kg) or vehicle solution 15 min prior to water maze testing. Sham lesioned animals injected with vehicle solution served as an untreated control group. Animals were tested for 5 consecutive days followed by 2 days off and then tested for 5 additional days. The rats were then sacrificed and their frontal cortex was assayed for choline acetyltransferase. The nucleus basalis magnocellularis lesion caused approximately a 27% depletion of choline acetyltransferase in the frontal cortex of these animals. The lesion also impaired the performance of the rats given vehicle solution as compared to untreated controls. Two doses (0.06 and 0.19 mg/kg) of physostigmine improved performance relative to lesioned controls. The lower dose, 0.06 mg/kg, improved performance more than the 0.19 mg/kg dose of physostigmine. The highest dose of physostigmine impaired water maze performance relative to lesioned controls. These data are discussed in relation to the cholinergic hypothesis of Alzheimer's disease and the potential therapeutic use of physostigmine.

The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS.

A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Multisite recording of brain field potentials and unit activity in freely moving rats.

A technique has been developed to record from 16 different brain sites of the freely moving rat using subminiature MOSFET preamplifiers. The high input impedance, small size, durability and light weight of the amplifiers and connecting cable allows high quality multisite recording of field potentials and unit activity. In addition, a movable headstage for positioning multiple microelectrodes is described. The compact recording system permits one to construct neocortical EEG maps, instant depth profiles of evoked and spontaneous field data, and to study neuronal synchrony of distant cell populations.

Diencephalic lesions, learning impairments, and intact retrograde memory following acute thiamine deficiency in the rat.

Previous studies have shown that following an acute bout of pyrithiamine-induced thiamine deficiency (PTD) rats are impaired in learning appetitively and aversively motivated T-maze tasks. The present study examined if PTD-treated rats exhibit both anterograde and retrograde memory loss of an aversively motivated spatial navigation task. Histological examination revealed two consistent lesions in the PTD treated rats: a bilateral, symmetrical destruction of medial thalamus centered on the internal medullary lamina (IML), and a lesion of the medial nucleus of the mammillary body. In Experiment 1, control and recovered PTD rats were trained to find a hidden platform in a Morris water maze. PTD rats with the IML lesion were impaired in learning the water maze task but were eventually able to perform as well as controls and PTD animals without the IML lesion. In Experiment 2, half of the pretrained CT animals underwent thiamine deficiency (PTD2), were recovered, and subsequently were tested for retention of the platform location. The remaining CT animals and the PTD1 group were also tested for retention. No significant group differences were observed on any of the four postretention trials. When compared to their performance on the last four preretention trials, the performance of PTD1 and PTD2 animals with IML lesions were similar to those of the controls. These results demonstrate that acute thiamine deficiency in rats produces damage of medial thalamic and mammillary body nuclei, a mild anterograde learning deficit, but no loss of retrograde memory of the Morris water maze task.

Enhanced detection of nucleus basalis magnocellularis lesion-induced spatial learning deficit in rats by modification of training regimen.

Bilateral excitotoxic lesions of the nucleus basalis magnocellularis (NBM) in the rat cause deficits in the water maze, a spatial memory paradigm. Previous investigations aimed at reversing the water maze performance deficit with anticholinesterase treatments have been unable to demonstrate a consistent drug effect due to the relatively good acquisition of the task seen following NBM lesions. The present investigation tested three different water maze training regimens designed to separate the learning curves. F-344 rats received bilateral NBM injections of ibotenic acid; sham-operated rats served as controls. The animals were tested in three groups in the water maze as follows: (1) four trials per day with no intertrial interval (standard paradigm), (2) four trials per day with a 10-minute intertrial interval, and (3) two trials per day with no intertrial interval. Each group was tested in the water maze for five consecutive days, followed by two days of rest, and then tested for an additional five days. The two-trial per day paradigm was more difficult than the standard paradigm for both lesions and controls and yielded the most difference between lesions and controls as compared to the other two testing regimens. The 10-min intertrial interval schedule was more difficult than the standard paradigm for lesioned animals but acquisition was not affected in control rats. These data demonstrate that the nucleus basalis lesions cause a deficit in the water maze task regardless of training parameters. Further, while all rats showed some acquisition of the water maze task, training schedule affected the level of learning of both lesioned and control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative estimate of the role of striatal D-2 receptor proliferation in dopaminergic behavioral supersensitivity: the contribution of mesolimbic dopamine to the magnitude of 6-OHDA lesion-induced agonist sensitivity in the rat.

Rats with unilateral depletions of neostriatal dopamine display increased sensitivity to dopamine agonists estimated to be 30 to 100 x in the 6-hydroxydopamine (6-OHDA) rotational model. Given that mild striatal dopamine D-2 receptor proliferation occurs (20-40%), it is difficult to explain the extent of behavioral supersensitivity by a simple increase in receptor density. This study was designed to investigate the quantitative aspects of the rotational behavior model utilizing constrained non-linear curve fitting routines. A dose-response curve for the rotational response arising from apomorphine stimulation of the normosensitive striatum was obtained in animals bearing unilateral lesions of striatal efferents (predominantly the striato-nigral pathway as previously described). After the control dose-response experiment, rats received a dopamine- (DA) depleting lesion in the contralateral hemisphere. In one group, 6-OHDA was infused into the medial forebrain bundle (MFB), a placement which is common in the literature and is known to deplete DA in both the striatum and nucleus accumbens. In a second group of rats, 6-OHDA was infused into the globus pallidus at a site which depletes caudate DA, but leaves n. accumbens DA relatively intact. The two experimental groups were tested in identical apomorphine-induced rotation dose-response experiments. The ED50's of the MFB- and caudate-lesioned rats were reduced by 36 and 5.8 fold, respectively, as compared to the control dose-response curve. The MFB and caudate lesions depleted striatal DA and produced a 30 and 36% increase in striatal D-2 binding sites, respectively. Modeling the behavioral and biochemical data with the null model for receptor occlusion indicated that increased striatal D-2 receptor density could account for the magnitude of behavioral supersensitivity in neither the MFB-lesioned group, nor even in the caudate-lesioned group. Thus simple up-regulation or D-2 receptors is unlikely to account for supersensitization as measured in the rotational model. Further, we suggest that quantitative modeling of such hypotheses is a valuable experimental technique for assessing relationships between biochemical and behavioral variables.

Quantification of lesion-induced dopaminergic supersensitivity using the rotational model in the mouse.

A dual lesion technique was used to determine the degree of supersensitivity resulting from nigrostriatal lesions in C57BL/6J mice. Internal capsule lesions encroaching on globus pallidus resulted in reliable ipsilateral rotation both to apomorphine and amphetamine. Dose-response curves to apomorphine were determined before and 21 days after 6-hydroxydopamine lesion of the contralateral nigrostriatal pathway. A 31.5-fold shift to the left was observed following the nigrostriatal lesion, with no change in slope. The extent and placement of the internal capsule lesion, as well as the magnitude of supersensitization correspond closely to those previously reported in the rat.

Amphetamine induces excess release of striatal acetylcholine in vivo that is independent of nigrostriatal dopamine.

The effect of amphetamine on striatal acetylcholine (ACh) release was studied by an in vivo intrastriatal microdialysis technique. Although we expected systemic amphetamine to inhibit baseline striatal ACh release, the opposite was found. In addition, we found that the amphetamine-induced striatal ACh release did not depend on nigrostriatal DA since 6-hydroxydopamine (6-OHDA) lesions had no effect on amphetamine-induced ACh release. Local intrastriatal injection of amphetamine via the microdialysis probe had no effect on striatal ACh release even when the probe was located more laterally in striatum to take advantage of the medial to lateral gradient of striatal ACh and D2 receptors. The hypothesis that amphetamine increased extracellular striatal ACh by increasing the release of biogenic amines besides dopamine was tested by pharmacological manipulations designed to specifically increase local striatal norepinephrine or serotonin levels. The serotonergic and noradrenergic manipulations had no effect on striatal ACh levels. These results indicate that amphetamine-induced release of ACh in striatum is mediated via distal brain regions that are functionally connected with the striatum.

Fos expression in intrastriatal striatal grafts: regulation by host dopaminergic afferents.

Previous studies have shown that transplants of fetal striatum, implanted into the ibotenic acid-lesioned striatum of adult rats, become innervated from the host nigrostriatal dopamine (DA) pathway. In the present study we have used DA-receptor-mediated expression of the Fos protein (i.e. the product of the immediate-early c-fos gene) as a cellular marker for functional dopaminergic host-graft interactions in the striatal grafts. Amphetamine (5 mg/kg; 2 h) induced Fos-like immunoreactivity in clusters of cells located mainly within the DARPP-32-positive areas within the transplants, i.e. within the striatum-like graft compartment which is preferentially innervated by the host DA afferents. As in the normal striatum, this effect was largely, although not completely, abolished by a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal DA pathway. Apomorphine (0.25 mg/kg; 2 h) had no detectable effect in grafts with an intact host DA system. Two to 3 weeks after a 6-OHDA lesion of the host DA pathway (i.e. a time sufficient for DA receptor supersensitivity to develop), apomorphine-induced extensive Fos-activation selectively within the DARPP-32-positive areas of the graft. The magnitude of the response was similar to that seen in the DA-denervated host striatum. Dual Fos/DARPP-32 immunostaining revealed that the activated graft neurons were, at least in part, DARPP-32-positive. In intrastriatal grafts of fetal neocortical tissue, which were studied for comparison, the amphetamine- and apomorphine-induced effects on Fos expression were much smaller and similar to that seen in the DARPP-32-negative, non-striatal compartment within the striatal grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: a microdialysis study in the rat.

Dopamine (DA) and noradrenaline (NA) extracellular levels have been measured by microdialysis in the medial frontal cortex (MFC), nucleus accumbens (NAc) and caudate-putamen (CP) under baseline conditions in awake and halothane-anaesthetized rats, and after application of three types of stimuli which are likely to activate the brainstem catecholaminergic systems: mild stressors (handling and tail pinch), rewarded behavior (eating palatable food without prior food deprivation) and electrical stimulation of the lateral habenular nucleus. Changes were studied with and without uptake blockade (10 microM nomifensine in the perfusion fluid). The influence of calcium concentration (1.2 or 2.3 mM in the perfusion fluid) on DA and NA overflow was tested in some cases. Handling and tail pinch stimulated both DA and NA overflow in MFC, and enhanced NA overflow in NAc. By contrast, these mildly stressful stimuli had only marginal effects on DA overflow in NAc and no effects on either DA or NA overflow in CP. Eating behavior was accompanied by increased DA and NA overflow in MFC but had no effect in NAc. These regional differences were similar also when the manipulations were applied under uptake blockade, which indicates that the more pronounced changes seen in MFC did not simply reflect a more sparse innervation (i.e. lower density of uptake sites) in the MFC compared to the more densely innervated NAc and CP areas. Stimulation of the lateral habenula induced a 2-3-fold increase in NA overflow in both MFC, NAc and CP but had no consistent effect on DA overflow in any region. The effect on NA release was abolished by a transection of the ipsilateral fasciculus retroflexus (which carries the efferent output of the lateral habenula). The results show that the forebrain DA and NA projections to cortical and striatal targets are differentially regulated during ongoing behavior, that the mesocortical and mesostriatal DA systems respond quite differently to stressful and rewarding stimuli; and that the NA projection to MFC (like the dopaminergic one) is more responsive to stressful and rewarding stimuli than the ones innervating the striatum (NAc and CP). The results support the view that environmental stimuli evoking emotional arousal (whether aversive or non-aversive) are accompanied by increased DA and NA release above all in the MFC and only to a minor extent in limbic and striatal areas.