A complete BAC-based physical map of the Arabidopsis thaliana genome.

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.

Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.

Linkage disequilibrium (LD) mapping provides a powerful method for fine-structure localization of rare disease genes, but has not yet been widely applied to common disease. We sought to design a systematic approach for LD mapping and apply it to the localization of a gene (IBD5) conferring susceptibility to Crohn disease. The key issues are: (i) to detect a significant LD signal (ii) to rigorously bound the critical region and (iii) to identify the causal genetic variant within this region. We previously mapped the IBD5 locus to a large region spanning 18 cM of chromosome 5q31 (P<10(-4)). Using dense genetic maps of microsatellite markers and single-nucleotide polymorphisms (SNPs) across the entire region, we found strong evidence of LD. We bound the region to a common haplotype spanning 250 kb that shows strong association with the disease (P< 2 x 10(-7)) and contains the cytokine gene cluster. This finding provides overwhelming evidence that a specific common haplotype of the cytokine region in 5q31 confers susceptibility to Crohn disease. However, genetic evidence alone is not sufficient to identify the causal mutation within this region, as strong LD across the region results in multiple SNPs having equivalent genetic evidence-each consistent with the expected properties of the IBD5 locus. These results have important implications for Crohn disease in particular and LD mapping in general.

Sonographic assessment of normal and abnormal patterns of fetal cerebral lamination.

OBJECTIVES: Prenatal development of the brain is characterized by gestational age-specific changes in the laminar structure of the brain parenchyma before 30 gestational weeks. Cerebral lamination patterns of normal fetal brain development have been described histologically, by postmortem in-vitro magnetic resonance imaging (MRI) and by in-vivo fetal MRI. The purpose of this study was to evaluate the sonographic appearance of laminar organization of the cerebral wall in normal and abnormal brain development. METHODS: This was a retrospective study of ultrasound findings in 92 normal fetuses and 68 fetuses with abnormal cerebral lamination patterns for gestational age, at 17-38 weeks' gestation. We investigated the visibility of the subplate zone relative to the intermediate zone and correlated characteristic sonographic findings of cerebral lamination with gestational age in order to evaluate transient structures. RESULTS: In the normal cohort, the subplate zone-intermediate zone interface was identified as early as 17 weeks, and in all 57 fetuses examined up to 28 weeks. In all of these fetuses, the subplate zone appeared anechoic and the intermediate zone appeared homogeneously more echogenic than did the subplate zone. In the 22 fetuses between 28 and 34 weeks, there was a transition period when lamination disappeared in a variable fashion. The subplate zone-intermediate zone interface was not identified in any fetus after 34 weeks (n=13). There were three patterns of abnormal cerebral lamination: (1) no normal laminar pattern before 28 weeks (n=32), in association with severe ventriculomegaly, diffuse ischemia, microcephaly, teratogen exposure or lissencephaly; (2) focal disruption of lamination before 28 weeks (n=24), associated with hemorrhage, porencephaly, stroke, migrational abnormalities, thanatophoric dysplasia, meningomyelocele or encephalocele; (3) increased prominence and echogenicity of the intermediate zone before 28 weeks and/or persistence of a laminar pattern beyond 33 weeks (n=10), associated with Type 1 lissencephaly or CMV infection. There was a mixed focal/diffuse pattern in two fetuses. In CMV infection, the earliest indication of the infection was focal heterogeneity and increased echogenicity of the intermediate zone, which predated the development of microcephaly, ventriculomegaly and intracranial calcification. CONCLUSIONS: The fetal subplate and intermediate zones can be demonstrated reliably on routine sonography before 28 weeks and disappear after 34 weeks. These findings represent normal gestational age-dependent transient laminar patterns of cerebral development and are consistent with histological studies. Abnormal fetal cerebral lamination patterns for gestational age are also visible on sonography, and may indicate abnormal brain development.

Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.

Pain prevalence in patients with chronic kidney disease.

BACKGROUND: The prevalence of pain in patients with chronic kidney diseases (CKD) is not known. In the current study, we aim to determine the prevalence of pain in CKD patients and its associations with various medical and psychosocial factors. METHODS: Consecutive CKD patients who were presented to the renal clinic at Olive View-UCLA Medical Center, a Los Angeles County tertiary referral center, over a 3-month period were interviewed on their medical and psychosocial histories and a history of pain including duration, severity and source. Chi2-testing for independence and binary logistic regression performed for the presence of pain and CKD stages as well as other medical and psychosocial factors were determined. A separate survey for pain was also done for 100 consecutive non-CKD patients who were presented to our ambulatory medicine clinic for routine care. RESULTS: 54.6% of 130 patients with known CKD interviewed were women. Any type of pain of at least a 2 week duration was reported in 72.9%. The most common source of pain was musculoskeletal. The presence of pain of less than a 2 week duration was associated with worse CKD stages (3 - 5 versus 1 - 2) and non-exercisers. Higher body mass index was associated with having pain lasting longer than a 2 week duration. Among patients who had pain, 33.8% used acetaminophen, 15.4% nonsteroidal anti-inflammatory drugs and 7.8% combination analgesics. In contrast to CKD patients, only 9% of non-CKD patients reported to have any type or duration of pain. CONCLUSIONS: Pain was much more prevalent among our CKD compared with non-CKD patients.

Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor.

BACKGROUND: Inbred mouse strains exhibit striking differences in the susceptibility of their macrophages to the effects of anthrax lethal toxin (LeTx). Previous data has shown that this difference in susceptibility lies downstream of toxin entry into macrophages. A locus controlling this phenotype, called Ltxs1, has been mapped to chromosome 11, but the responsible gene has not been identified. RESULTS: Here, we report the identification of the Ltxs1 gene as Kif1C, which encodes a kinesin-like motor protein of the UNC104 subfamily. Kif1C is the only gene in the Ltxs1 interval exhibiting polymorphisms between susceptible and resistant strains. Multiple alleles of Kif1C determine the susceptibility or resistance of cultured mouse macrophages to LeTx. Treatment of resistant macrophages with brefeldin-A (which alters the cellular localization of Kif1C) induces susceptibility to LeTx, while ectopic expression of a resistance allele of Kif1C in susceptible macrophages causes a 4-fold increase in the number of cells surviving LeTx treatment. We also show that cleavage of map kinase kinase 3, a target of LeTx proteolysis, occurs in resistant cells. CONCLUSIONS: We conclude that mutations in Kif1C are responsible for the differences in the susceptibility of inbred mouse macrophages to LeTx and that proper Kif1C function is required for LeTx resistance. Since the LeTx-mediated proteolysis of map kinase kinase 3 occurs even in resistant cells, Kif1C does not affect cellular entry or processing of LeTx and likely influences events occurring later in the intoxication pathway.

Ethnic differences in catechol O-methyltransferase pharmacogenetics: frequency of the codon 108/158 low activity allele is lower in Kenyan than Caucasian or South-west Asian individuals.

Catechol O-methyltransferase (COMT) inactivates neurotransmitters, hormones and drugs such as levodopa. COMT activity is inherited in an autosomal recessive manner and individuals with low activity have thermolabile COMT protein. A low activity allele has been demonstrated at codon 108/158 of the soluble and membrane bound COMT protein, respectively, whereby a G to A transition results in a valine to methionine substitution, rendering the protein more thermolabile. As ethnic differences in erythrocyte COMT activity have been previously demonstrated, the frequency of low activity alleles were investigated in 265 British Caucasian, 99 British South-west Asian and 102 Kenyan individuals. Genotyping of COMT codon 108/158 was performed using a minisequencing method. Erythrocyte COMT activity was measured in 60 British Caucasian individuals by radiochemical assay. The frequency of low activity alleles was 0.54 in Caucasians, 0.49 in South-west Asians, and 0.32 in Kenyans. There was a much lower frequency of individuals with homozygous low activity allele in the Kenyan population (9%) than in Caucasians (31%) or South-west Asians (27%). Erythrocyte COMT activity was lower and less thermostable in individuals with homozygous low activity alleles. The data provide molecular evidence that low COMT is less common in African individuals than the Caucasian population.

Comparative biochemical pharmacology of central nervous system dopamine D1 and D2 receptors.

The biochemical properties of central nervous system (CNS) dopamine (DA) D1 and D2 receptors were examined using the specific antagonists [3H]SCH23390 and [3H]raclopride, respectively. There is a different participation of sulfhydryl (-SH) and disulfide (-SS-) groups in the binding site and/or coupling to second messenger systems of D1 and D2 receptors. The ionic studies with [3H]SCH23390 showed slight agonist and antagonist affinity shifts for the D1 receptor. On the other hand, the D2 receptor is very sensitive to cations; even if lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions that cannot be explained by surface charge mechanisms. The distribution of D1 and D2 receptors was heterogenous in both species, with the greatest densities in the neostriatum, where the highest concentrations of DA and metabolites were measured. Regions with low endogenous DA content (cerebral cortex and hippocampus) had lower densities of DA receptors. Furthermore, these binding sites were differentially localized within the various regions, and there were substantially more D1 than D2 receptors. The functional significance and heterogeneities in the distribution of D1 and D2 receptors can be related to dopaminergic innervation and turnover.

Prediction of level of serotonin 2A receptor binding by serotonin receptor 2A genetic variation in postmortem brain samples from subjects who did or did not commit suicide.

OBJECTIVE: Postmortem studies have indicated that suicide victims have greater serotonin receptor 2A (5-HTR2A) binding in prefrontal brain regions. However, there remains some controversy regarding the biological specificity of these findings. The authors hypothesized that the variance observed in brain 5-HTR2A binding is genetically mediated, at least in part. METHOD: Postmortem data from 56 subjects who had committed suicide and 126 normal comparison subjects were studied; brain tissue was available from 11 subjects who committed suicide and 11 comparison subjects. Homogenate binding assays were carried out with [3H]ketanserin. Variation at the 5-HTR2A gene (HTR2A) was investigated by means of two polymorphisms: T102C and A-1438G. RESULTS: 5-HTR2A binding was greater in the prefrontal cortex of the subjects who committed suicide. In addition, the findings suggest that HTR2A variation significantly affects 5-HTR2A binding. However, no interaction between suicidal behavior and this locus was observed. CONCLUSIONS: These results confirm previous reports of greater 5-HTR2A binding in subjects who committed suicide; they also provide preliminary evidence suggesting that the number of 5-HTR2A receptors is genetically mediated.

Changes of D1 and D2 receptors in adult rat neostriatum after neonatal dopamine denervation: quantitative data from ligand binding, in situ hybridization and iontophoresis.

The specific binding of [3H]SCH23390 to D1 and of [3H]raclopride to D2 dopamine receptors was measured by autoradiography in the rostral and caudal halves of neostriatum and in the substantia nigra of adult rats subjected to near total destruction of nigrostriatal dopamine neurons by intraventricular 6-hydroxydopamine soon after birth. Three months after this lesion, [3H]SCH23390 binding (D1 receptors) was slightly but significantly decreased in the rostral neostriatum (22%), but unchanged in its caudal half and in the substantia nigra. In contrast, [3H]raclopride binding (D2 receptors) was considerably increased throughout the neostriatum (10-40%), while markedly decreased in the substantia nigra (80%). In the rostral neostriatum, there were no parallel changes in D2 receptor messenger RNA levels, as measured by in situ hybridization on adjacent sections. Caudally, however, slight but significant increases in D2 messenger RNA could be observed (10-20%). As assessed by quantitative iontophoresis, there was a marked enhancement (63%) of the inhibitory responsiveness of spontaneously firing units in the rostral neostriatum to dopamine and the D1 agonist, SKF38393, in neonatally lesioned compared to control rats. On the other hand, responsiveness to PPHT, a potent D2 agonist, appeared to be unchanged. Such opposite changes in the number of D1 and D2 binding sites, dissociated from the expression of D2 receptor messenger RNA and from the sensitivity to dopamine and D1 and D2 agonists, suggested independent adaptations of these various parameters following the neonatal dopamine denervation of neostriatum. They also provided further evidence for mechanisms other than the dopamine innervation in the control of the expression of neostriatal D2 receptor messenger RNA during ontogenesis, and emphasized that the effects of dopamine and its D1 and D2 agonists in neostriatum do not depend strictly on the number of D1 and D2 primary ligand recognition sites.

Effects of monovalent cations on neostriatal dopamine D2 receptors labeled with [3H]raclopride.

Specific [3H]raclopride binding to dopamine D2 receptors in the rabbit neostriatum was investigated in the presence of the monovalent cations sodium, lithium and potassium. NaCl and LiCl produced concentration-dependent elevations in specific [3H]raclopride binding with sodium inducing approximately 50% more binding than lithium. Inhibition of [3H]raclopride binding by the antagonist (+)-butaclamol was unaffected by the presence of sodium or lithium in the incubation medium. In contrast, the potency of dopamine to compete with [3H]raclopride was decreased by these two ions. This effect was more pronounced in the presence of sodium than lithium and was observed for both the high- and low-affinity states of the D2 receptor. The guanine nucleotide derivative 5'-guanylylimidodiphosphate (Gpp(NH)p) reduced the potency of dopamine to compete with [3H]raclopride binding in both the presence and absence of cations; however, this effect of Gpp(NH)p was a shift of the D2 receptors from a high to a lower affinity state. Saturation binding curves in the presence of sodium or lithium were compared with experiments carried out in the absence of monovalent cations (sucrose) and demonstrated that these ions increased the affinity (judged by the equilibrium dissociation constant Kd) of the neostriatal [3H]raclopride binding sites. While NaCl produced a significantly greater change in the Kd of [3H]raclopride binding as compared to LiCl, no differences were apparent in the maximum binding capacity (Bmax) values determined in the presence of these two cations. In conclusion, the results indicate that [3H]raclopride binding to rabbit neostriatal membranes exhibits a sensitivity to monovalent cations that is consistent with the ionic regulatory properties of the D2 receptor. Moreover, although lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions.

Potentiation of the behavioural effects of the antidepressant phenelzine by deuterium substitution.

Phenelzine in the rat induced biphasic behavioural stimulation, which was profoundly potentiated by deuterium substitution. Doses of 12.5 or 25.0 mg/kg phenelzine had little or no effect on spontaneous activity, whereas the same doses of deuterated phenelzine produced hyperactivity, wet-dog shakes, forepaw padding, splayed hind limbs, backward walking, sniffing and stereotyped grooming 2-12 h after injection. Similarly, the behavioural response induced by 50.0 mg/kg phenelzine was strongly potentiated by deuterium substitution. It appears likely that the increased behavioural response induced by deuterated phenelzine may be due to its greater potency as a monoamine oxidase inhibitor compared to undeuterated phenelzine. Since phenelzine is an antidepressant that is particularly efficacious in the treatment of severe anxiety, a deuterated analogue of the drug seems likely to be clinically useful.

Effects of denervation and hyperinnervation on dopamine and serotonin systems in the rat neostriatum: implications for human Parkinson's disease.

The research on central synaptic neurotransmission has greatly benefited from the use of the neurotoxin 2,4,5-trihydroxyphenylethylamine, or 6-hydroxydopamine (6-OHDA), that destroys catecholamine-containing neuronal cell bodies and nerve terminals. Refinements in the use of this neurotoxin led to the use of dopamine-denervated animals as models of human Parkinson's disease, in which the loss of dopaminergic neurons is a prominent feature. Here we review structural, pharmacological, and biochemical studies carried out in the adult and neonatal 6-OHDA lesioned animals. These models have become useful and interesting paradigms to examine alterations in the expression of receptors and in their sensitivity to agonist drugs; some of these modifications may underlie the altered responsiveness of the dopamine-lesioned animals to dopamine, but also to other compounds, including serotoninergic drugs. We have also reviewed studies of amino acids as well as of monoamine metabolism and of uptake mechanisms that may underlie some of the behavioural alterations in these models that have become relevant for our understanding of the sprouting and plastic properties of spared neurons, and of the alternate neuronal projections that replace lesioned terminals, enabling compensatory adaptations. Although 6-OHDA-lesioned animals, that display some biochemical characteristics of Parkinson's disease in humans, do not express all of the neurological features exhibited by patients, the increasing knowledge that can be obtained from studies in simplified experimental models will undoubtedly lead to the development of innovative drugs and other replacement therapies for degenerative brain diseases.

Alterations in the turnover rate of dopamine D1 but not D2 receptors in the adult rat neostriatum after a neonatal dopamine denervation.

Adult rats that were treated with intracerebral ventricular injection of 6-hydroxydopamine (6-OHDA) as neonates exhibit a profound loss of nigrostriatal dopamine innervation in addition to a variety of other neurochemical and anatomical changes, including alterations in the number of neostriatal D1 and D2 receptor binding sites. In the present study, the turnover of neostriatal dopamine D1 and D2 receptors was measured in rats previously treated with 6-OHDA or ascorbic acid vehicle as neonates at various time intervals after peripheral N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ, 10 mg/kg) administration. Dopamine receptors were labelled with [3H]SCH23390 (D1) and [3H]raclopride (D2), while the degree of dopamine denervation was assessed by the measurement of neostriatal dopamine, homovanillic acid and dihydroxyphenylacetic acid content. Two days after acute EEDQ treatment, the maximal binding capacity (Bmax) of [3H]SCH23390 and [3H]raclopride binding was significantly decreased to 58 and 32% of control values, respectively, without any significant alteration in their equilibrium dissociation constants (Kd). A time-dependent increase in the density of [3H]SCH23390 and [3H]raclopride binding was observed in both treatment groups following a single dose of EEDQ. The rate of recovery of D1 receptors was significantly slower in the 6-OHDA-lesioned animals as compared to controls with a half-life of 103 compared to 53 h, respectively. No differences were observed in the rate of recovery of D2 receptors in these two treatment groups. These data are consistent with the findings of decreased expression of D1 receptors in neonatal 6-OHDA-lesioned rats owing to decreased receptor synthesis, and further suggest that in this model the up-regulation of D2 receptors is a result of a post-transcriptional mechanism, such as an increased rate of post-synthetic maturation.

Dopamine receptor alterations correlate with increased GABA levels in adult rat neostriatum: effects of a neonatal 6-hydroxydopamine denervation.

The effects of neonatal intracerebroventricular 6-hydroxydopamine (6-OHDA) injection on the densities of dopamine (DA) receptors and GABA levels were determined in the rostral neostriatum of adult rats. Measurement of GABA turnover indicated that increased tissue GABA in the DA-lesioned neostriatum is a consequence of higher GABA synthesis rate (205%). Binding experiments with [3H]SCH23390 (D1 receptors) and [3H]raclopride (D2 receptors) point to a correlation between tissue GABA content and altered DA receptors. Three months after the lesion there was a 27% decrease in D1 receptors and a 22% increase in D2 receptors. In control neostriatum, GABA levels were inversely related to D2 receptors and this relationship was reversed after 6-OHDA treatment. In contrast, the positive correlation between GABA and D1 receptors remained unchanged after the lesion. Irreversible blockade of DA receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) decreased both D1 and D2 sites (73-87%) in both control and lesioned neostriatum, but increased GABA levels by 25% only in animals which have received 6-OHDA just after birth. Following acute inhibition of DA synthesis or of DA catabolism, GABA levels remained unchanged. The present results indicated that DA depletion by itself is not the cause for the increase in GABA levels. The augmented GABAergic activity following neonatal 6-OHDA is seemingly influenced primarily by DA receptor status; presumably, changes in D2 receptor properties during maturation may be a principal cause for an increase in neostriatal GABA content.

Effects of chronic lithium treatments on central dopaminergic receptor systems: G proteins as possible targets.

Numerous biochemical and electrophysiological studies have proposed a role for dopamine (DA) in the therapeutic efficacy of lithium (Li+) salts. The effects of ex vivo chronic Li+ treatments on neostriatal DA receptors, as well as on the G protein adenylyl cyclase complex and on tissue cAMP levels were investigated in adult rats. The animals were administered LiCl in their drinking water (1 g/l) for varying periods of time, i.e. 1, 15 and 28 days. After sacrifice by decapitation, their brains were removed and the neostriatum dissected out to assay DA receptors and adenylyl cyclase activity. The antagonists [3H]SCH23390 and [3H]raclopride were employed to label D1 and D2 receptors, respectively. Chronic Li+ treatments did not modify the saturation binding of either ligand. However, competition studies of the same antagonists by DA revealed biphasic curves, and the inhibition constant of the high-affinity site was significatively increased after chronic Li+. The data suggest an alteration in the coupling efficacy between G proteins and DA receptors. Moreover, chronic (28 day) Li+ treatment, but not a 1 day Li+ administration, lead to a reduction of the GTP-induced and DA-sensitive adenylyl cyclase activity, without changes in the basal activity or in forskolin-induced cAMP production. The results demonstrate that chronic Li+ treatments diminish neostriatal dopaminergic activity, probably through a direct action on the G protein itself. The underlying mechanisms do not appear to involve modifications in either the D1 or the D2 receptor primary ligand recognition sites, but may represent alterations in both the coupling process and the capacity of the G proteins, once activated, to stimulate adenylyl cyclase.

Serotonin 5-HT1 and 5-HT2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study.

Neonatal destruction of nigrostriatal dopamine neurons by cerebroventricular injection of 6-hydroxydopamine (6-OHDA) results in a serotonin (5-HT) hyperinnervation of the rostral neostriatum in adult rat. Quantitative ligand-binding autoradiography was used to compare the density of various 5-HT receptor subtypes in the adult brain of control and neonatally 6-OHDA-lesioned rats. 5-HT1A, 5-HT1B, 5HT1nonAB and 5-HT2 sites were labeled with [3H]8-OH-DPAT, [125I]cyanopindolol, [3H]5-HT and [125I]DOI, respectively, and measured in the rostral and caudal halves of neostriatum and selected forebrain or midbrain regions. 5-HT1A binding, measured after 6 months, was unchanged in all regions examined including the dorsal raphe nucleus. Three months after the lesion, 5-HT1B binding was increased throughout the neostriatum (30%), but also in the substantia nigra (50%) and globus pallidus (30%), suggesting an up-regulation and an increased axonal transport of these receptors in neostriatal projection neurons. 5-HT1nonAB binding was also increased throughout the neostriatum (40%) and in the substantia nigra (50%), but unchanged in the globus pallidus, as if this up-regulation preferentially involved striatonigral as opposed to striatopallidal neurons. 5-HT2 binding showed an even greater increase (60%), which was restricted to the rostral half of neostriatum and also seemed imputable to an up-regulation as heteroreceptors. Even though the exact cause(s) of these receptor increases could not be determined, their anatomical distribution suggested that they were somehow related to the initial dopamine denervation in the case of the 5-HT1B and 5-HT1nonAB receptors, and more tightly linked to the 5-HT hyperinnervation in the case of the 5-HT2 receptors. Such receptor changes could participate in adaptive mechanisms implicating other transmitters and behavioral disturbances observed in this particular experimental model. Interestingly, they could also account for an enhancement of neostriatal 5-HT function even in a condition where extracellular levels of 5-HT apparently remain normal because of increased uptake.

Adenosine deaminase-'like' immunoreactivity in cerebellar Purkinje cells of rat.

The localization of adenosine deaminase (ADA) throughout the rat CNS was investigated immunohistochemically with 5 different affinity purified polyclonal antibody preparations against ADA. Except in the cerebellum, identical immunostaining patterns of ADA-immunoreactivity were observed with all 5 antibodies. In the cerebellum, Purkinje cells and presumptive swellings on their axons were immunostained by only one of the 5 antibodies. Preabsorption with purified ADA abolished immunostaining with all 5 antibodies. Biochemical tests showed that the Km for substrate, the specific activity of immunoprecipitable enzyme and the isozymic characteristics of cerebellar ADA was not different from ADA in whole brain or several other brain regions examined. The atypical immunohistochemical behavior of cerebellar ADA, despite its biochemical similarity to ADA elsewhere in the brain, suggests that the enzyme in the cerebellum has some unique features which must be taken into account when considering its possible role in regulating the neuromodulatory actions of adenosine in the cerebellar cortex.

Distinct adenosine deaminase-containing inputs to the substantia nigra from the striatum and tuberomammillary nucleus.

Immunohistochemical, neuroanatomical and lesion methods were used to investigate the projections of adenosine deaminase immunoreactive (ADA-IR) neurons in the striatum (caudate/putamen) and hypothalamus to the substantia nigra (SN). Striatal ADA-IR neurons were distributed within two zones; anteriorly in the medial and ventromedial extreme of the head and body of the striatum, and posteriorly in the tail of the striatum. The posterior hypothalamus contained ADA-positive neurons which were confined to the tuberomammillary nucleus (TM). The SN was devoid of ADA-positive neurons, but contained two distinct types of ADA-IR fiber terminations. One type was confined to bands located at the ventrolateral and dorsomedial borders of the pars reticulata and consisted of fine puncta. The other type was distributed throughout the SN and consisted of long, beaded fibers. Injections of the retrograde tracer Fluoro-gold (FG) into the SN gave rise to FG-labelling of significant numbers of ADA-IR neurons in both the striatum and TM. Medial SN injections preferentially labelled ADA-IR neurons in the anterior striatum and lateral SN injections labelled posterior ADA-IR striatal neurons. Kainic acid lesions of the anterior medial striatum selectively abolished the punctate ADA-IR band in the medial SN and left the long, ADA-IR nigral fibers in an apparently hypertrophied state. Despite depletion of ADA-IR neurons in the striatum by kainic acid, ADA activity increased significantly at striatal lesion sites. The results suggest that the SN receives two topographically segregated fine terminal fields from striatal ADA-IR neurons, and a substantial innervation from ADA-IR neurons in the TM as well. These findings add to the heterogeneous chemical composition of nigral afferents and are discussed in the context of adenosine neuromodulatory mechanisms in the striatonigral system.

Elevation of dopamine D2 but not D1 receptors in adult rat neostriatum after neonatal 6-hydroxydopamine denervation.

Monoamine levels and the binding properties of [3H]SCH23390, a D1-specific ligand, and [3H]raclopride, a D2-specific ligand, were measured in the rostal and caudal neostriatum to investigate the fate of dopamine receptors following bilateral cerebroventricular injection of 6-hydroxydopamine in 3-day-old rats. After survival times of 15, 30 or 90 days, measurement of monoamine levels and of [3H]SCH23390 binding were also obtained from the cerebral cortex. At all three survival times, dopamine content was reduced by more than 90% of control values in both the rostral and caudal neostriatum; in cerebral cortex, the dopamine depletion was less profound (80%) and noticeable only after 1 and 3 months. In the rostral but not the caudal neostriatum, serotonin and 5-hydroxyindoleacetic acid concentrations were markedly increased at 1 and 3 months; cortical serotonin also was augmented at 3 months. There were no changes in neostriatal [3H]SCH23390 binding at any of the survival times, but a transient elevation occurred in the cortex at 1 month. In the rostral but not the caudal neostriatum, [3H]raclopride binding showed a slight elevation at 1 month and a further, highly significant increase at 3 months. As measured in individual rats, this increase in [3H]raclopride binding was linearly correlated with the increase in serotonin turnover (ratio of 5-hydroxyindoleacetic acid/serotonin). Such an up-regulation of D2 receptors, restricted to the rostral neostriatum which was also the site of a serotonin hyperinnervation, was probably indicative of a serotonin control on the expression of D2 receptors after dopamine denervation.