BDNF-TrkB signaling in striatopallidal neurons controls inhibition of locomotor behavior.

The physiology of brain-derived neurotrophic factor signaling in enkephalinergic striatopallidal neurons is poorly understood. Changes in cortical Bdnf expression levels, and/or impairment in brain-derived neurotrophic factor anterograde transport induced by mutant huntingtin (mHdh) are believed to cause striatopallidal neuron vulnerability in early-stage Huntington's disease. Although several studies have confirmed a link between altered cortical brain-derived neurotrophic factor signaling and striatal vulnerability, it is not known whether the effects are mediated via the brain-derived neurotrophic factor receptor TrkB, and whether they are direct or indirect. Using a novel genetic mouse model, here, we show that selective removal of brain-derived neurotrophic factor-TrkB signaling from enkephalinergic striatal targets unexpectedly leads to spontaneous and drug-induced hyperlocomotion. This is associated with dopamine D2 receptor-dependent increased striatal protein kinase C and MAP kinase activation, resulting in altered intrinsic activation of striatal enkephalinergic neurons. Therefore, brain-derived neurotrophic factor/TrkB signaling in striatopallidal neurons controls inhibition of locomotor behavior by modulating neuronal activity in response to excitatory input through the protein kinase C/MAP kinase pathway.

Effects of pallidotomy on motor symptoms in an animal model of Parkinson's disease.

The present study was designed to evaluate the motor effects of lesioning the internal globus pallidus in an animal model of Parkinson's disease. Fourty rats were divided into four groups (each of 10 rats) which received either unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle (mfb) plus sham surgery to the pallidum, sham surgery of mfb plus N-methyl-D-aspartate (NMDA) induced pallidal lesions, combined 6-OHDA mfb + NMDA pallidal lesions or sham surgery to both structures. Animals with 6-OHDA lesions developed significant ipsilateral biases in head position, body axis and circling after amphetamine challenge (all P < 0.05). Prominent contralateral deficits were present in sensorimotor response latency and contralateral circling was induced by apomorphine challenge (both P < 0.05). The addition of an NMDA pallidal lesion, improved the head position and body axis biases, as well as dopamine-agonist induced rotation and contralateral reaction time in a sensorimotor task (all P < 0.05). There was, however, a slight worsening of sensorimotor response on the ipsilateral side (P < 0.05). Pallidal lesions in the absence of 6-OHDA lesions produced contralateral head position and body axis biases (both P < 0.05). These data indicate that pallidotomy improves some, but not all aspects of parkinsonian motor dysfunction in an animal model of Parkinson's disease (PD).

Regional and subcellular compartmentation of the dopamine transporter and tyrosine hydroxylase in the rat ventral pallidum.

The ventral pallidum (VP) is a major intermediary in the prefrontal cortical circuitry regulating sensorimotor gating and locomotor behavior, both of which are potently modulated by catecholamines. The VP catecholaminergic innervation is derived from midbrain dopaminergic neurons that differ in expression levels of the dopamine transporter (DAT) and from brainstem noradrenergic neurons without DAT. The preferentially low level of DAT in dopaminergic terminals in the prefrontal cortex and in striatal regions projecting more extensively to the VP medial (VPm) compared with VP lateral (VPl) compartment suggests possible region-specific differences in VP axonal distribution of DAT. To test this hypothesis, we examined the electron microscopic localization of DAT and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the VPm and VPl of rat brain. In both regions, DAT and TH were localized primarily in small unmyelinated axons and morphologically heterogeneous axon terminals. DAT-immunogold particles were few in number, but mostly located on the plasma membrane. In contrast, TH immunoreactivity was distributed in the cytoplasm of individual profiles, many of which were without detectable DAT. In comparison with TH, the mean area density of DAT-labeled axons was low throughout the VP. The mean area density of DAT-immunogold axon terminals, however, was significantly higher in VPl than in VPm, whereas that of TH-labeled axons was higher in VPm than in VPl. This dissociation suggests that, compared to the VPl, the VPm receives the greatest input from catecholaminergic afferents that are either nondopaminergic or characterized by having low levels or less terminal distributions of DAT.

Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats.

Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn/kg/day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson's disease.

Changes in the mitochondrial enzyme activity in striatal projection areas after unilateral excitotoxic striatal lesions: partial restoration by embryonic striatal transplants.

It is well established that the activity of cytochrome oxidase (CO), a mitochondrial enzyme, reflects the long-term, steady-state levels of neuronal activity. The present study investigated the long-term effects of unilateral striatal lesions induced by quinolinic acid on CO activity in primary striatal targets, including the globus pallidus (GP), entopeduncular nucleus (EP), and substantia nigra pars reticulata (SNR) and a secondary striatal projection area, such as subthalamic nucleus (STN), in rats. The activity of CO was determined by measuring staining intensity on brain sections processed for CO histochemistry. We also examined whether intrastriatal transplants of embryonic striatal tissue could affect the lesion-induced changes in the CO activity of those brain structures. Unilateral striatal lesions were found to lead to increases in the CO activity of the GP, EP, and SNR ipsilateral to the lesions. By contrast, the activity of the ipsilateral STN was decreased following striatal lesions, probably due to the increased inhibitory effect of the GP on the STN. Intrastriatal implantation of the lateral ganglionic eminence (LGN), but not the medial ganglionic eminence (MGE), reversed the lesion-induced changes in the CO activity of the GP and STN with concomitant attenuation of apomorphine-induced rotational asymmetry. The grafts failed to affect the activity of either the EP or SNR. The present results indicate that striatal lesions induce changes in the functional activity of basal ganglia nuclei and that the LGE grafts placed in the damaged striatum partly reverse the alterations in the functional state of the basal ganglia circuitry.

Alteration of pallidal cholinergic activity in MPTP-treated monkeys: effect of dihydro-alpha-ergocryptine (DEK).

Monkeys, intravenously administered with MPTP at the dose of 0.3 mg/kg for 5 consecutive days, develop a severe Parkinson-like syndrome. Cholinergic enzyme activities are increased in the internal segment of the globus pallidus (GPi) and into a lesser extent in the external globus pallidus (GPe). Cholinergic activities are not significantly affected in the caudate and putamen nor in the frontal, parietotemporal, occipital cortices and in the cerebellum. The treatment of the animals twice daily for 2 weeks with dihydro-alpha-ergocryptine (DEK) starting 5 days before the first MPTP administration counteracts the neurotoxin-induced alteration in the internal pallidum and ameliorates some motor related parkinsonian symptoms.

Direct in vivo gene transfer to ependymal cells in the central nervous system using recombinant adenovirus vectors.

To evaluate the potential for adenovirus-mediated central nervous system (CNS) gene transfer, the replication deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for beta-galactosidase) and Ad-alpha 1AT (coding for human alpha 1-antitrypsin) were administered to the lateral ventricle of rats. Ad.RSV beta gal transferred beta-galactosidase to ependymal cells lining the ventricles whereas Ad-alpha 1AT mediated alpha 1-antitrypsin secretion into the cerebral spinal fluid for 1 week. These observations, together with beta-galactosidase activity in the globus pallidus and substantia nigra following stereotactic administration of Ad.RSV beta gal to the globus pallidus, suggest that adenovirus vectors will be useful for CNS gene therapy.

Transsynaptic induction of c-fos in basal forebrain, diencephalic and midbrain neurons following AMPA-induced activation of the dorsal and ventral striatum.

In these experiments, induction of the immediate early gene c-fos following excitation of striatal neurons has been used to investigate the organization of the ventral and dorsal striatopallidal systems and the relationship between striatal neurons and cholinergic neurons of the nucleus basalis magnocellularis (of Meynert, nbM). The results demonstrate that FOS immunoreactivity (ir) can be detected in ventral and dorsal striatal neurons following infusions of the non-N-methyl-D-aspartic acid (NMDA) glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). This activation and increased expression of FOS in striatal neurons was itself associated with the sustained appearance of FOS-ir in neurons of the ipsilateral ventral and dorsal pallidum, subthalamic nucleus and some thalamic nuclei. Infusions of AMPA into the ventral striatum (VS), but not the dorsal striatum (DS), also resulted in the appearance of FOS-ir in a proportion (17%) of the cholinergic neurons of the nbM. By combining the retrograde transport of Fluoro-Gold with FOS immunocytochemistry, it was also possible to demonstrate that approximately 46% and 58% of the pallidal neurons containing FOS-ir after infusions of AMPA into the VS or DS, respectively, directly project to the subthalamic nucleus. Taken together, these observations suggest that visualizing the protein product of transsynaptic c-fos induction provides an effective way to study the topographic and transsynaptic, within-system consequences of striatal activation.

Drug discrimination learning in rats with excitotoxic lesions of nucleus basalis and ventral globus pallidus.

Rats can readily acquire conditional discriminations in which mixtures of drugs serve as compound internal discriminative stimuli. Excitotoxic lesions in the region of the nucleus basalis have been shown to impair the acquisition of conditional discriminations based upon external visual stimuli, but nothing was known about their effects on discrimination of internal stimuli. A baseline of undiscriminated bar-pressing for food reinforcers was established prior to surgery. Lesions were made by infusing either ibotenic or quisqualic acid bilaterally into the basal forebrain (the ibotenate-induced lesions had been shown previously to impair cortical cholinergic function and to produce non-specific damage). After surgery, rats were trained to discriminate effects of drug mixtures using a standard, two-bar operant conditioning procedure. The ibotenate, but not the quisqualate, lesion impaired the acquisition of a discrimination of a mixture of (+)-amphetamine plus pentobarbitone, while neither lesion impaired acquisition with a mixture of (-)-nicotine plus midazolam. The ibotenate lesions also reduced overall rates of responding in both experiments. Thus, the deficit in the acquisition of drug discrimination in rats with ibotenate lesions had some pharmacological specificity, but could not be related easily to disturbances in neocortical cholinergic function. In comparisons with other published data, the results suggest a possible dichotomy in the processing of interoceptive and external information in the basal forebrain, a major target of ventral striatal overflow.

Membrane localization of endopeptidase-24.11 and peptidyl dipeptidase A (angiotensin converting enzyme) in the pig brain: a study using subcellular fractionation and electron microscopic immunocytochemistry.

Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand-binding assays using (-)-[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase-24.11 exhibited a biphasic distribution, consistent with its presence on both pre- and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity-purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity-purified antibody to endopeptidase-24.11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre- and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase-24.11 is present on both pre- and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.

Distribution of endopeptidase-24.15 in rat brain nuclei using a novel fluorogenic substrate: comparison with endopeptidase-24.11.

A novel fluorogenic substrate for the neutral metalloendopeptidase-24.15 (E.C.3.4.24.15; EP-24.15) was synthesized which allowed continuous assay of the enzyme. The substrate, Glutaryl-Phe-Ala-Ala-Phe-4-methoxynaphthylamide (G-FAAF-4MN) is cleaved at the Phe-Ala bond by EP-24.15 (Km = 0.026 mM). The product, AAF-4MN is subsequently hydrolyzed to its constituent amino acids and the potent fluorophore 4MN by aminopeptidase M. This method has allowed the measurement of the specific activity EP-24.15 within microdissected nuclei of rat brain. The enzyme was found to have a relatively broad distribution within brain nuclei, and the activity ranged from 15-80 nmol 4MN/mg prot/h in all areas examined. The activity of EP-24.15 was relatively high in the medial and lateral pre-optic nuclei, where potential substrates include the dynorphin-like peptides and LHRH. The activity of EP-24.15 was compared with that of endopeptidase-24.11 (E.C.3.4.24.11, 'enkephalinase', EP-24.11), another peptide-cleaving metalloenzyme. EP-24.11 appeared to have a much more narrow distribution, with very high specific activity in basal ganglia as well as in the supraoptic and suprachiasmatic nuclei.

Histochemical visualization of neutral endopeptidase-24.11 (enkephalinase) activity in rat brain: cellular localization and codistribution with enkephalins in the globus pallidus.

We have developed a novel fluorescent histochemical method to localize the enzyme neutral endopeptidase-24.11 (NEP, E.C. 3.4.24.11, enkephalinase) in the rat brain in order to directly compare the relative distributions of the enzyme and its putative peptide substrate, the enkephalins. The method is based on the sequential cleavage of the synthetic peptide substrate, glutaryl-alanyl-alanyl-phenylanyl-4-methoxy-2-naphthylamide, by NEP and exogenous aminopeptidase M to yield free 4-methoxy-2-naphthylamine (MNA). In the presence of nitrosalicylaldehyde, free MNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of NEP activity. The specificity of the method was demonstrated using the selective NEP inhibitors thiorphan, phosphoramidon, and JHF26. All NEP staining throughout the brain was abolished using a 50-nM concentration of these inhibitors. The enzyme was richly localized to many regions, including the cerebral cortex, caudate putamen, globus pallidus, hippocampus, substantia nigra, periaqueductal gray, several cranial nerve nuclei, nuclei of the reticular formation of the medulla. In most regions, reaction product was associated with cell bodies of varying size and morphology. In a number of regions, colchicine increased the amount of NEP staining, particularly in cell processes. The regional distribution pattern of the enzyme, however, did not change in response to colchicine and was similar to that of untreated animals. The histochemical localization of NEP was combined with fluorescent immunocytochemical visualization of the enkephalins in order to localize both in the same tissue section. In the globus pallidus, this combined fluorescent technique revealed numerous NEP-positive cell bodies surrounded by fiber pathways displaying intense enkephalin-like immunoreactivity. The source of the NEP in the globus pallidus was studied using the neurotoxic agent, N-methyl-D-aspartate (NMDA). A pronounced decrease in NEP cellular staining was observed within 7 d in response to NMDA, persisted for at least 16 weeks, and correlated with injury of pallidal neurons. There was no apparent change in enkephalin-like immunoreactivity in the globus pallidus in response to NMDA. These data provide evidence that NEP and enkephalin in the globus pallidus derive from different sources. This study supports the hypothesis that NEP localizes to enkephalin-rich regions of the rat brain, and that the enzyme may be involved in the inactivation of synaptically released enkephalins.

Electronmicroscopic immunocytochemistry of pig brain shows that endopeptidase-24.11 is localized in neuronal membranes.

An ultrastructural study of endopeptidase-24.11 in the globus pallidus from the brain of a newborn pig is reported. The antigen was localized by an immunoperoxidase method using an affinity-purified polyclonal antibody in which staining was performed on thick vibratome sections prior to osmication and flat embedding. When areas selected by light microscopy for re-embedding were examined in the electron microscope a minority of the axonal membranes in the fields examined were observed to be immunostained for endopeptidase-24.11. These were unmyelinated fibres and the membrane staining included not only the length of an axon but also some boutons synapsing with dendrites. Positively staining dendritic and glial membranes were not observed. These results support the view that endopeptidase-24.11 may play a role in inactivating some neuropeptides after their release at the synapse.

Neuronal localization of the neutral endopeptidase 'enkephalinase' in rat brain revealed by lesions and autoradiography.

The cellular localization of rat brain enkephalinase was studied after induction of selective unilateral lesions using in vitro quantitative autoradiography of the specific binding of the enzyme inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly). Twenty-one days following injection of kainic acid in the caudate-putamen (CP) [3H]HACBO-Gly binding was locally decreased by 52% with a concomitant reduction of 67 and 78% in the ipsilateral substantia nigra (SN) and globus pallidus (GP), respectively. Inhibition of axonal transport in the CP by unilateral stereotaxic injection of colchicine induced a large (30-60%) and progressive decrease in enkephalinase labelling within the ipsilateral GP and SN. Taken together these results strongly suggest that in the CP a large fraction of enkephalinase is localized on intrinsic striatal neurones, and that the enzyme present both in the GP and the SN is partly localized on nerve terminals originating from neurones in the CP. No change in [3H]HACBO-Gly binding was observed in the CP following injection of 6-hydroxydopamine in the nigrostriatal bundle, contrasting with the 30% depletion in opioid receptors. This would indicate that enkephalinase is present in only very low amounts, if at all, on striatal dopaminergic nerve terminals.

Hallervorden-Spatz disease: cysteine accumulation and cysteine dioxygenase deficiency in the globus pallidus.

We describe neurochemical abnormalities found in the brains of 2 patients with autopsy-confirmed Hallervorden-Spatz (HS) disease. In 1 patient, contents of cystine and of glutathione-cysteine mixed disulfide in the globus pallidus were markedly elevated above values for appropriate control subjects. Activity of cysteine dioxygenase, which converts cysteine to cysteine sulfinic acid, was reduced in the globus pallidus, but normal in the frontal cortex and putamen of both patients. gamma-Aminobutyric acid content was markedly decreased in the globus pallidus and substantia nigra of both patients. These results suggest that cysteine accumulates locally in the globus pallidus in Hallervorden-Spatz disease as a result of an enzymatic block in the metabolic pathway from cysteine to taurine. Accumulated cysteine may chelate iron, accounting for the local increase in iron content in Hallervorden-Spatz disease. The combined excess of cysteine and ferrous iron may generate free radicals that damage neuronal membranes to cause the typical morphological changes observed in this disorder.

Altered succinate dehydrogenase activity of basal ganglia following to mesotelencephalic dopaminergic projection.

Unilateral damage to the mesotelencephalic dopamine-containing projection of rats by intracerebral 6-hydroxydopamine injection, alters the staining of the tricarboxylic acid cycle enzyme succinate dehydrogenase in the basal ganglia. Relative to the intact hemisphere, the neostriatum ipsilateral to the damage shows diminished enzyme staining, while the ipsilateral globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata all show enhanced deposition of reaction product. These asymmetries are detectable in some animals by 3 days after surgery and increase in magnitude at longer postoperative survival times. The alterations in succinate dehydrogenase staining appear to be related to the severity of the sensorimotor impairments resulting from the injury.

Reduced cortical choline acetyltransferase activity in senile dementia of Alzheimer type is not accompanied by changes in vasoactive intestinal polypeptide.

Post-mortem brain tissue from 7 patients who died with a diagnosis of senile dementia of Alzheimer type (SDAT) was compared with tissue obtained from 7 control patients at routine post mortem. A significant fall in choline acetyltransferase (ChAT) activity was apparent in the cerebral cortex of the SDAT cases which was maximal in the temporal lobe. The fall in ChAT activity was not accompanied by changes in cortical vasoactive intestinal polypeptide (VIP) measured by radioimmunoassay.