3H]DPDPE binding to delta opioid receptors in the rat mesocorticolimbic and nigrostriatal pathways is transiently increased by acute ethanol administration

Dopaminergic transmission in the mesolimbic and nigrostriatal pathways plays a key role in the reinforcement mechanisms and brain sensitivity to ethanol, respectively. Ethanol reinforcement and high alcohol drinking behaviour have been postulated to be partially mediated by a neurobiological mechanism involving the ethanol-induced activation of the endogenous opioid system. Activation of opioid neural pathways by ethanol may include alterations in the processing, release and/or the receptor binding of opioid peptides. The aim of this work was to investigate the effects of acute ethanol administration on delta opioid receptors in the rat mesocortical, meso-accumbens and nigrostriatal pathways by quantitative receptor autoradiography, using [(3)H] (2-D-penicillamine, 5-D-penicillamine)-enkephalin as radioligand. A significant increase in [(3)H] (2-D-penicillamine, 5-D-penicillamine)-enkephalin binding was observed in the substantia nigra pars reticulata 1 h after ethanol treatment. Two hours after drug exposure, ligand binding was significantly increased in the frontal and prefrontal cortices, the core and shell regions of the nucleus accumbens, and in the anterior-medial and medial-posterior regions of the caudate-putamen. In contrast, ligand binding was significantly decreased in the posterior region of the caudate-putamen 30 min after ethanol administration. The observed effects may reflect ethanol-induced changes in ligand binding affinity and/or in receptor density. Our results suggest that transitory changes in delta opioid receptors with different kinetic patterns may be involved in ethanol reinforcement and brain sensitivity to the drug. Ethanol-induced delta receptor up- and down-regulation mechanisms may participate in modulation of dopaminergic transmission in the mesocorticolimbic and nigrostriatal pathways.     

Exofocal alterations in opioidergic receptor densities following focal cerebral ischemia in the mouse

In previous studies of our group, we have reported differential alterations in opioidergic receptor subtypes densities in infarcted and periinfarcted brain tissue following middle cerebral artery occlusion (MCAO) in mice. Other studies have also described subcortical alterations consecutive to focal cortical ischemia. For a better understanding of ischemic processes in exofocal areas, we have investigated the evolution of opioidergic receptors following focal cortical ischemia through the quantification of relative binding densities, B(max) and K(d) values for the mu, delta, and kappa subtypes. Our results demonstrate that opioid receptor subtypes exhibit adaptations at distance from the ischemic core, mainly in the striatum, the thalamus, and the substantia nigra. Indeed, mu and delta B(max) values were increased in ventral thalamic nuclei, while kappa relative binding densities were transiently increased in nucleus medialis dorsalis and nucleus lateralis, pars posterior. Moreover, the B(max) of mu and delta receptors were transiently decreased at 6 h post-MCAO in ipsi- and contralateral patches and matrices of the striatum. Conversely, the mu B(max) values were increased in ipsi- and contralateral substantia nigra, pars compacta, and pars reticulata, 24 h following MCAO. In contralateral substantia nigra, pars compacta, kappa B(max) was found to be decreased at 24 h post-MCAO. These alterations could reflect neuronal dysfunction in exofocal brain structures, consecutively to the degeneration of defined neuroanatomical pathways. Our study indicates that opioidergic receptors could be used as markers of the neuronal reorganization that take place in subcortical areas following an ischemic insult of the brain cortex.     

Long-term alterations in μ, δ and κ opioidergic receptors following middle cerebral artery occlusion in mice

Alterations in the opioidergic system may play a role in the molecular mechanisms underlying neurochemical responses to cerebral ischaemia. The present study aimed to determine the delayed expression of μ, δ and κ opioid receptors, following 1, 2, 7, and 30 days of middle cerebral artery occlusion (MCAO) in mice. Using quantitative autoradiography, we highlighted significant decreases in μ, δ and κ opioid receptor expression in ipsilateral cortices from day 1 post-MCAO. Moreover, in contralateral nucleus lateralis thalami pars posterior, ipsi- and contralateral nucleus medialis dorsalis thalami, and ipsilateral substantia nigra, pars reticulata (SNr), κ receptors were increased; μ receptor densities were decreased in nucleus ventralis thalami, pars posterior (VThP), and SNr. δ-Binding sites were increased in the striatum on day 30 post-MCAO. The alterations in opioid receptors in cortical infarcts were correlated with strong histological damage. Further reductions in opioid receptor densities in cortical infarcts were observed at later time points. In subcortical brain regions, opioid receptor densities were also altered but no histological damage was seen, except in the VThP, in which cell density was increased on day 30. Delayed reductions in opioid receptor densities in the infarct appeared as the continuation of the early processes previously demonstrated. However, changes in subcortical opioid receptor expression may correlate with neuronal alterations in remote brain regions. Changes in opioidergic receptor expression in these regions may be involved in the long-term consequences of stroke and could be used as biomarker of neuronal alteration through the use of imaging techniques in the clinic. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kindling does not cause persistent changes in firing rates or transmitter sensitivities of substantia nigra pars reticulata neurons

These studies were undertaken to determine whether the kindling process induces persistent alterations in the functional status of neurons of the substantia nigra pars reticulata, a brain area identified previously as a site important in regulating the expression of generalized motor seizures. Extracellular, single-unit recordings of pars reticulata neurons were made in chloral hydrate-anesthetized, fully kindled rats (2–3 weeks after the last seizure), or unkindled control rats of the same age and weight. Kindling caused no alterations in several electrophysiological parameters examined. For instance, neither the number of active pars reticulata cells encountered, nor their firing rates, were significantly different between kindled and control groups. In addition, kindling failed to alter the sensitivities of pars reticulata neurons to iontophoretic application of two inhibitory transmitters, γ-aminobutyric acid and glycine, and two transmitters that excite these cells, glutamate and acetylcholine. These results suggest that while kindling produces enduring increases in seizure susceptibility, it causes no persistent interictal changes in either basal activity or several measures of transmitter sensitivity of substantia nigra pars reticulata neurons.     

Matrix metalloproteinase-9 is elevated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice

Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of degrading components of the extracellular matrix. Recent evidence has implicated MMPs in the pathogenesis of neurodegenerative diseases as Alzheimer’s disease and amyotrophic lateral sclerosis. In this study, we investigated the involvement of MMP-9 (gelatinase B) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease using zymography, immunohistochemistry, and Western blot analysis. The activity of MMP-9 was upregulated at 3 h after MPTP injection in the striatum and after 24 h in the substantia nigra. Although MMP-9 expression decreased in the striatum by 72 h, it remained elevated in the substantia nigra compared to controls up to 7 d after MPTP administration. Immunohistochemistry showed that neurons and microglia are the source of MMP-9 expression after MPTP administration to mice. Treatment with a hydroxamate-based MMP inhibitor, Ro 28-2653 significantly reduced dopamine depletion and loss of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta. MMP-9 expression as measured via zymography in the substantia nigra was reduced by the MMP inhibitor. These results indicate that MMP-9 is induced after MPTP application in mice and that pharmacologic inhibition of MMPs protects against MPTP neurotoxicity.     

Expression of ErbB4 in substantia nigra dopamine neurons of monkeys and humans

Abnormal neuregulin-1 signaling through its receptor (ErbB4) might be associated with schizophrenia, although their neuropathological contribution remains controversial. To assess the role of neuregulin-1 in the dopamine hypothesis of schizophrenia, we used in situ hybridization and immunoblotting to investigate the cellular distribution of ErbB4 mRNA in the substantia nigra of Japanese monkeys (Macaca fuscata) and human postmortem brains. In both monkeys and humans, significant signal for ErbB4 mRNA was detected in substantia nigra dopamine neurons, which were identified by melanin deposits. The expression of ErbB4 mRNA in nigral dopamine neurons was confirmed with an independent RNA probe, as well as with combined tyrosine hydroxylase immunostaining. Immunoblotting appeared to support the observation of in situ hybridization. Immunoreactivity for ErbB4 protein was much more enriched in substantia nigra pars compacta containing dopamine neurons than in neighboring substantia nigra pars reticulata. These observations suggest that ErbB4 is expressed in the dopaminergic neurons of primate substantia nigra and ErbB4 abnormality might contribute to the dopaminergic pathology associated with schizophrenia or other brain diseases.     

肝性脑病大鼠γ氨基丁酸转运体mRNA的表达

目的 研究肝性脑病模型大鼠γ氨基丁酸转运体(GAT)mRNA在各脑区的表达.方法 雄性SD大鼠12只按随机数字表法分为模型组和对照组,每组6只,采用硫代乙酰胺腹腔注射制作大鼠急性肝性脑病模型.对照组注射等量生理盐水.采用经地高辛标记的针对GAT-1的寡核苷酸探针,应用原位杂交方法 检测大鼠基底核、皮层、黑质网状部和致密部及海马各区GAT-1mRNA的表达. 结果 与对照组比较,模型组大鼠基底核、黑质网状部和致密部、海马各区GAT-1 mRNA表达均降低,差异有统计学意义(P<0.05),而大脑皮层其表达无明显变化. 结论 肝性脑病的发生可能与GAT mRNA的表达改变有关.     

Prenatal ethanol exposure modifies locomotor activity and induces selective changes in Met-enk expression in adolescent rats

Several studies suggest that prenatal ethanol exposure (PEE) facilitates ethanol intake. Opioid peptides play a main role in ethanol reinforcement during infancy and adulthood. However, PEE effects upon motor responsiveness elicited by an ethanol challenge and the participation of opioids in these actions remain to be understood. This work assessed the susceptibility of adolescent rats to prenatal and/or postnatal ethanol exposure in terms of behavioral responses, as well as alcohol effects on Met-enk expression in brain areas related to drug reinforcement. Motor parameters (horizontal locomotion, rearings and stereotyped behaviors) in pre- and postnatally ethanol-challenged adolescents were evaluated. Pregnant rats received ethanol (2 g/kg) or water during gestational days 17–20. Adolescents at postnatal day 30 (PD30) were tested in a three-trial activity paradigm (habituation, vehicle and drug sessions). Met-enk content was quantitated by radioimmunoassay in several regions: ventral tegmental area [VTA], nucleus accumbens [NAcc], prefrontal cortex [PFC], substantia nigra [SN], caudate-putamen [CP], amygdala, hypothalamus and hippocampus. PEE significantly reduced rearing responses. Ethanol challenge at PD30 decreased horizontal locomotion and showed a tendency to reduce rearings and stereotyped behaviors. PEE increased Met-enk content in the PFC, CP, hypothalamus and hippocampus, but did not alter peptide levels in the amygdala, VTA and NAcc. These findings suggest that PEE selectively modifies behavioral parameters at PD30 and induces specific changes in Met-enk content in regions of the mesocortical and nigrostriatal pathways, the hypothalamus and hippocampus. Prenatal and postnatal ethanol actions on motor activity in adolescents could involve activation of specific neural enkephalinergic pathways.     

Differential expression of kainate receptors in the basal ganglia of the developing and adult rat brain

Glutamate is the principal excitatory transmitter of the mammalian brain and plays a particularly important role in the physiology of the basal ganglia structures responsible for movement regulation. Using in situ hybridization with oligonucleotide probes, we examined the expression patterns of the five known kainate type glutamate receptor subunit genes, KA1, KA2 and GluR5–7, in the basal ganglia of adult and developing rat brain. In the adult rat, a highly organized and selective pattern of expression of the kainate subunits was observed in the basal ganglia and associated structures as well as in other regions of the brain. KA2 mRNA was abundant in the striatum, nucleus accumbens, subthalamic nucleus and substantia nigra pars compacta, and was present at lower levels in the globus pallidus and substantia nigra pars reticulata. Neither KA1 nor GluR5 expression was observed in the basal ganglia of adult rats, although these messages were present in other regions. GluR6 was highly expressed in the striatum and subthalamic nucleus and to a lesser extent in the substantia nigra pars reticulata, while no hybridization signal was detectable in the large, presumably dopaminergic neurons of the substantia nigra pars compacta. In contrast, GluR7 was strongly expressed in the substantia nigra pars compacta, was present at lower levels in the striatum, globus pallidus and substantia nigra pars reticulata, and was not detectable in the subthalamic nucleus. During postnatal development, expression of the kainate receptor subunits was characteristically highest on postnatal day 1 and declined to adult levels by day 20; however, in the globus pallidus we did observe the transient expression of KA1 and GluR5 between day 1 and day 10. These results demonstrate that the neuronal structures comprising the basal ganglia express a distinct combination of kainate receptor subunit genes, suggesting that the pharmacological properties of the resultant glutamate receptors are likely to be regionally specific. The organization of expression of these genes is established early in life, which is consistent with the important role they may play in establishing the functions of the motor system.     

Angiotensin II receptor binding associated with nigrostriatal dopaminergic neurons in human basal ganglia

In the human brain, receptor binding sites for angiotensin are found in the striatum and in the substantia nigra pars compacta overlying dopamine-containing cell bodies. In contrast, angiotensin-converting enzyme occurs in the substantia nigra pars reticulata and is enriched in the striosomes of the striatum. In this study, using quantitative in vitro autoradiography, we demonstrate decreased angiotensin receptor binding in the substantia nigra and striatum of postmortem brains from patients with Parkinson's disease. In the same brains the density of binding to angiotensin-converting enzyme shows no consistent change. We propose, from these results, that angiotensin receptors in the striatum are located presynaptically on dopaminergic terminals projecting from the substantia nigra. In contrast, the results support previous studies in rats demonstrating that angiotensin-converting enzyme is associated with striatal neurons projecting to the substantia nigra pars reticulata. These findings raise the possibility that newly emerging drugs that interact with the angiotensin system, particularly converting enzyme inhibitors and new nonpeptide angiotensin receptor blockers, may modulate the brain dopamine system.     

The novel antiepileptic drug levetiracetam (ucb L059) induces alterations in GABA metabolism and turnover in discrete areas of rat brain and reduces neuronal activity in substantia nigra pars reticulata

Levetiracetam ((S)-α-ethyl -2-oxo-pyrrolidine acetamide, ucb L059) is a novel anticonvulsant drug presently in clinical development. Its mechanism of action is unknown although a recently reported novel specific binding site for [³H]levetiracetam, unique to brain, may be involved. This binding site has not yet been characterized, but some evidence suggested a possibly indirect interaction with the GABA system. We therefore examined levetiracetam's effects on GABA metabolism and turnover in several rat brain regions after systemic administration of anticonvulsant doses. Furthermore, in order to study functional effects of levetiracetam on a well defined system of GABAergic neurons in a brain region that has been critically involved in anticonvulsant drug action, we examined levetiracetam's action on spontaneous firing of substantia nigra pars reticulata (SNR) neurons in anesthetized rats. Although levetiracetam did not alter the activity of the GABA synthesizing and degrading enzymes glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T) in vitro, systemic administration induced significant alterations in these enzymes in several brain regions, indicating that these enzyme alterations were no direct drug effects but a consequence of postsynaptic changes in either GABAergic or other neurotransmitter-related systems. In the striatum, levetiracetam, 170 mg/kg i.p., induced a significant increase in GABA-T activity while GAD activity markedly decreased. When GABA turnover was estimated after inhibition of GABA-T by aminooxyacetic acid (AOAA), treatment with levetiracetam (given 15 min prior to injection of AOAA) significantly reduced GABA turnover in the striatum. Since the substantia nigra pars reticulata (SNR) receives a strong GABAergic input from the striatum, we examined if the alterations in GABA metabolism and turnover in the striatum led to functional alterations in neuronal activity in the SNR by recording single unit activity of SNR neurons after i.p. injection of levetiracetam. While injection of vehicle did not affect SNR neuronal activity, a significant decrease in spontaneous neuronal firing was recorded after levetiracetam. Since a substantial body of evidence suggests that the SNR is a critical site at which decrease of neuronal firing results in protection against various seizure types, the suppressive effect of levetiracetam on SNR activity may contribute to the anticonvulsant action of this drug.     

Possible mechanisms involved in the stereotyped behavior elicited by amphetamine.

In a variety of animals, amphetamine administration produces an increase in locomotor behavior and an induction of repetitive, stereotyped behaviors. There is now considerable evidence to suggest that the induction of stereotyped behaviors is accomplished, in part, by alterations in catecholaminergic transmission in the central nervous system. By recording the spontaneous activity of neurons in the rat brain substantia nigra, reticular formation, basal ganglia, and elsewhere during systemic administration of amphetamine and related drugs, or during administration by means of microinfusions directly into these brain regions, relationships may be drawn between the biochemical and behavioral effects of these drugs and drug-induced changes in neuronal activity in the central nerovous system. Current evidence, for example, suggests that amphetamine produces an inhibition of neuronal activity in the neostriatum and pars compacta of the substantia nigra by means of dopamine released from dopaminergic terminals in the neostriatum and dopaminergic dendrites in the substantia nigra respectively. In addition, current evidence suggests the possibility of a GABA-mediated functional antagonism between excitatory cortical and/or thalamic input to the neostriatum and dopaminergic input from the substantia nigra which could be involved in the apparently mutually exclusive occurrence of amphetamine-induced locomotion and stereotyped behaviors that follow amphetamine administration. Such evidence may also have relevance to a variety of behavioral disorders involving the basal ganglia and catecholaminergic transmission in the central nervous system.     

Alterations of cerebellar mRNA specific for BDNF, p75NTR, and TrkB receptor isoforms occur within hours of ethanol administration to 4-day-old rat pups

Developing cerebellar Purkinje cells of the rat are extremely sensitive to ethanol during postnatal days (PN) 4-6, but not at later times during development. Ethanol exposure during this vulnerable window induces rapid apoptotic Purkinje cell death that is hypothesized to result from ethanol inhibition in brain-derived nerve growth factor (BDNF)-TrkB neurotrophic signaling that results in loss of apoptotic suppression. In this study, the effect that different concentrations of ethanol (1.5, 3.0, 4.5 and 6.0 g/kg) have on steady-state mRNA expression of BDNF and different TrkB receptor isoforms in the cerebellum on PN4 was determined at 1, 4, 6, and 8 h after treatment. Significant decreases in mRNA specific for BDNF and TrkB isoforms were detected within 1 h after ethanol administration. No significant alterations in expression of mRNA specific to the low affinity p75(NTR) receptor were identified. These alterations are concurrent with the PN4 vulnerable period for Purkinje cells since equivalent treatment of PN9 rat pups does not produce significant alterations in mRNA specific to BDNF or TrkB at 4 h after exposure. These results support the hypothesis that ethanol induces a disruption of BDNF-TrkB signaling that results in loss of apoptotic suppression in vulnerable Purkinje cells by growth factor withdrawal.     

Does cholecystokinin colocalize with dopamine in the human substantia nigra?

In situ hybridization was used to examine the distribution of neurons containing cholecystokinin (CCK) mRNA in human, monkey and rat brain. In rat and monkey brain CCK mRNA was visualized in the substantia nigra pars compacta and in the ventral tegmental area. The dopaminergic cell bodies in the human substantia nigra did not however show detectable amounts of CCK mRNA. Low levels of CCK mRNA were observed in the nucleus paranigralis, the human equivalent of the rodent ventral tegmental area. High levels of CCK mRNA were seen in other regions of the same brains including the cortex and the hippocampus. Thus, the adult human substantia nigra dopaminergic cells, in contrast to primate and rodent substantia nigra, do not express CCK. These results question the hypothesis of an involvement of CCK in the regulation of dopaminergic neurons and help to explain the absence of decreased CCK levels in the caudate and putamen of Parkinson's disease victims.     

Changes in vesicular monoamine transporter (VMAT2) and synaptophysin in rat Substantia nigra and prefrontal cortex induced by psychotropic drugs.

We investigated the regulatory effect of the dopaminergic agent L-dopa, the mood stabilizer lithium and the nonselective monoamine oxidase inhibitor phenelzine on brain vesicular monoamine transporter (VMAT2) expression. Rats were treated chronically (21 days) with the three psychoactive drugs. VMAT2 gene expression at the protein level was assessed in the prefrontal cortex and striatum by autoradiography with high-affinity [(3)H]dihydrotetrabenazine ([(3)H]TBZOH) binding and at the mRNA level in the substantia nigra pars compacta by in situ hybridization. In addition, the effect of various treatments on the synaptophysin mRNA level was determined in the substantia nigra by in situ hybridization. Chronic administration of L-dopa resulted in a significant decrease (28%, p < 0.05) in the density of [(3)H]TBZOH binding in the prefrontal cortex but had no effect on VMAT2 and synaptophysin mRNA levels in the substantia nigra. Lithium treatment increased [(3)H]TBZOH-specific binding in the prefrontal cortex (23%, p < 0.05) but had no effect on VMAT2 and synaptophysin mRNA levels. Phenelzine did not modulate VMAT2 gene expression but reduced the synaptophysin mRNA level (19%, p < 0.05). The modulatory activities of these drugs, although relatively weak, may be relevant to the drug-induced synaptic and neuronal plasticity as well as to the molecular and cellular pathophysiology of monoamine-related neuropsychiatric disorders.