Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression.

Elevation of the proinflammatory cytokine IL-6 has been implicated in depression; however, the mechanisms remain elusive. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression post-transcriptionally. The lethal-7 (let-7) miRNA family was suggested to be involved in the inflammation process and IL-6 was shown to be one of its targets. In the present study, we report elevation of Il6 in the prefrontal cortex (PFC) of a genetic rat model of depression, the Flinders Sensitive Line (FSL) compared to the control Flinders Resistant Line. This elevation was associated with an overexpression of LIN28B and downregulation of let-7 miRNAs, the former an RNA-binding protein that selectively represses let-7 synthesis. Also DROSHA, a key enzyme in miRNA biogenesis was downregulated in FSL. Running was previously shown to have an antidepressant-like effect in the FSL rat. We found that running reduced Il6 levels and selectively increased let-7i and miR-98 expression in the PFC of FSL, although there were no differences in LIN28B and DROSHA expression. Pri-let-7i was upregulated in the running FSL group, which associated with increased histone H4 acetylation. In conclusion, the disturbance of let-7 family biogenesis may underlie increased proinflammatory markers in the depressed FSL rats while physical activity could reduce their expression, possibly through regulating primary miRNA expression via epigenetic mechanisms.

Allele-specific programming of Npy and epigenetic effects of physical activity in a genetic model of depression.

Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

BDNF in schizophrenia, depression and corresponding animal models.

Understanding the etiology and pathogenesis schizophrenia and depression is a major challenge facing psychiatry. One hypothesis is that these disorders are secondary to a malfunction of neurotrophic factors. Inappropriate neurotrophic support during brain development could lead to structural disorganisation in which neuronal networks are established in a nonoptimal manner. Inadequate neurotrophic support in adult individuals could ultimately be an underlying mechanism leading to decreased capacity of brain to adaptive changes and increased vulnerability to neurotoxic damage. Brain-derived neurotrophic factor (BDNF) is a mediator involved in neuronal survival and plasticity of dopaminergic, cholinergic, and serotonergic neurons in the central nervous system (CNS). In this review, we summarize findings regarding altered BDNF in schizophrenia and depression and animal models, as well as the effects of antipsychotic and antidepressive treatments on the expression of BDNF.

Neurotrophic factors and receptors in the immature and adult spinal cord after mechanical injury or kainic acid.

Delivery of neurotrophic factors to the injured spinal cord has been shown to stimulate neuronal survival and regeneration. This indicates that a lack of sufficient trophic support is one factor contributing to the absence of spontaneous regeneration in the mammalian spinal cord. Regulation of the expression of neurotrophic factors and receptors after spinal cord injury has not been studied in detail. We investigated levels of mRNA-encoding neurotrophins, glial cell line-derived neurotrophic factor (GDNF) family members and related receptors, ciliary neurotrophic factor (CNTF), and c-fos in normal and injured spinal cord. Injuries in adult rats included weight-drop, transection, and excitotoxic kainic acid delivery; in newborn rats, partial transection was performed. The regulation of expression patterns in the adult spinal cord was compared with that in the PNS and the neonate spinal cord. After mechanical injury of the adult rat spinal cord, upregulations of NGF and GDNF mRNA occurred in meningeal cells adjacent to the lesion. BDNF and p75 mRNA increased in neurons, GDNF mRNA increased in astrocytes close to the lesion, and GFRalpha-1 and truncated TrkB mRNA increased in astrocytes of degenerating white matter. The relatively limited upregulation of neurotrophic factors in the spinal cord contrasted with the response of affected nerve roots, in which marked increases of NGF and GDNF mRNA levels were observed in Schwann cells. The difference between the ability of the PNS and CNS to provide trophic support correlates with their different abilities to regenerate. Kainic acid delivery led to only weak upregulations of BDNF and CNTF mRNA. Compared with several brain regions, the overall response of the spinal cord tissue to kainic acid was weak. The relative sparseness of upregulations of endogenous neurotrophic factors after injury strengthens the hypothesis that lack of regeneration in the spinal cord is attributable at least partly to lack of trophic support.

The selective kappa-opioid receptor agonist U50,488H attenuates voluntary ethanol intake in the rat.

Non-selective opioid receptor antagonists are increasingly used in the treatment of alcohol dependence. The clinical effects are significant but the effect size is rather small and unpleasant side effects may limit the benefits of the compounds. Ligands acting at mu- and/or delta- receptors can alter the voluntary intake of ethanol in various animal models. Therefore, the attenuating effects of selective opioid receptor ligands on ethanol intake may be of clinical interest in the treatment of alcoholism. The objective of this study was to examine the effects of a selective kappa-receptor agonist, U50,488H on voluntary ethanol intake in the rat. We used a restricted access model with a free choice between an ethanol solution (10% v/v) and water. During the 3-days baseline period, the rats received a daily saline injection (1 ml/kg, i.p.) 15 min before the 2 h access to ethanol. The animals had free access to water at all times. The control group received a daily saline injection during the 4-days treatment-period, whereas the treatment groups received a daily dose of U50,488H (2.5, 5.0 or 10 mg/kg per day). Animals treated with U50,488H dose-dependently decreased their ethanol intake. The effect of the highest dose of U50,488H was reduced by pre-treatment with the selective kappa-antagonist nor-binaltorphimine (nor-BNI). These results demonstrate that activation of kappa-opioid receptors can attenuate voluntary ethanol intake in the rat, and the data suggest that the brain dynorphin/kappa-receptor systems may represent a novel target for pharmacotherapy in the treatment of alcohol dependence.

Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen.

Physical activities such as long-distance running can be habit forming and associated with a sense of well-being to a degree that justifies comparison with drug-induced addictive behaviours. To understand molecular similarities and dissimilarities controlling these behaviours in humans we compared the effects of running in running wheels to the effects of chronic cocaine or morphine administration on mRNA levels in brain reward pathways in the inbred Fischer and Lewis rat strains. These strains are both inbred from the Sprague-Dawley strain; Lewis rats display a higher preference towards addictive drugs and running than do Fischer rats. After chronic cocaine or running a similar increase of dynorphin mRNA in medial caudate putamen was found in the Lewis rat, suggesting common neuronal adaptations in this brain region to both cocaine and running. Fischer and Lewis rats both responded to cocaine with increased dynorphin mRNA levels in medial caudate putamen. However, only Lewis rats increased dynorphin mRNA after running, possibly reflecting the much higher degree of running by the Lewis strain as compared to the Fischer strain. Moreover, the running-induced upregulation of dynorphin mRNA was blocked by the opioid receptor antagonist naloxone. We suggest that running increases dynorphin mRNA by a mechanism that involves endogenous opioids. The voluntary wheel-running model in rats might be used to study natural reward and compulsive behaviours and possibly also to screen candidate drugs for treatment of compulsive disorders.

Regulation of GluR2 promoter activity by neurotrophic factors via a neuron-restrictive silencer element.

The AMPA glutamate receptor subunit GluR2, which plays a critical role in regulation of AMPA channel function, shows altered levels of expression in vivo after several chronic perturbations. To evaluate the possibility that transcriptional mechanisms are involved, we studied a 1254-nucleotide fragment of the 5'-promoter region of the mouse GluR2 gene in neural-derived cell lines. We focused on regulation of GluR2 promoter activity by two neurotrophic factors, which are known to be altered in vivo in some of the same systems that show GluR2 regulation. Glial-cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) both induced GluR2 promoter activity. This was associated with increased expression of endogenous GluR2 immunoreactivity in the cells as measured by Western blotting. The effect of GDNF and BDNF appeared to be mediated via a NRSE (neuron-restrictive silencer element) present within the GluR2 promoter. The response to these neurotrophic factors was lost upon mutating or deleting this site, but not several other putative response elements present within the promoter. Moreover, overexpression of REST (restrictive element silencer transcription factor; also referred to as NRSF or neuron restrictive silencer factor), which is known to act on NRSEs in other genes to repress gene expression, blocked the ability of GDNF to induce GluR2 promoter activity. However, GDNF did not alter endogenous levels of REST in the cells. Together, these findings suggest that GluR2 expression can be regulated by neurotrophic factors via an apparently novel mechanism involving the NRSE present within the GluR2 gene promoter.

Differential patterns of induction of NGFI-B, Nor1 and c-fos mRNAs in striatal subregions by haloperidol and clozapine.

Disturbances of retinoid activated transcription mechanisms have recently been implicated as risk factors for schizophrenia. In this study we have compared the regulation of mRNAs for the nuclear orphan receptor NGFI-B, which forms a functional heterodimer with the retinoid x receptor and the related orphan nuclear receptor Nor1 with c-fos mRNA after acute and chronic treatments with haloperidol and clozapine. The antipsychotic drugs haloperidol and clozapine have different clinical profiles. Haloperidol is a typical neuroleptic giving extrapyramidal side effects (EPS), whereas the atypical compound clozapine does not. Acute haloperidol treatment increased NGFI-B, Nor1 and c-fos mRNAs in nucleus accumbens shell and core as well as medial and lateral caudate putamen. In contrast, clozapine lead to an increase of NGFI-B, Nor1 and c-fos only in the accumbens shell. No haloperidol or clozapine effect on these mRNAs was detected in cingulate, sensory or motor cortex. Chronic haloperidol lead to an increase of NGFI-B mRNA in the accumbens core. Acutely, it is possible that the increased levels of NGFI-B, Nor1 and c-fos mRNA levels in striatum and accumbens might indicate a neural activation which possibly can be used when screening for drugs that do not produce EPS. Also, the increased levels of NGFI-B, which is an important component in retinoid signaling, both after acute and chronic treatments of haloperidol suggests altered sensitivity to retinoids which could be an important component for the beneficial antipsychotic effect.

Expression and regulation of CNTF receptor-alpha in the in situ and in oculo grafted adult rat adrenal medulla.

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.

NGFI-B and nor1 mRNAs are upregulated in brain reward pathways by drugs of abuse: different effects in Fischer and Lewis rats.

The two inbred Fischer and Lewis rat strains display differences in acquisition of drug self-administration, suggesting genetic factors controlling the vulnerability to drugs of abuse. In this study, we analyzed the effects of acute and chronic cocaine and morphine on mRNAs encoding the NGFI-B/Nur77 family of nuclear orphan receptors in reward pathways in Fischer and Lewis rats. After a single injection of cocaine, a similar upregulation of NGFI-B mRNA in striatal subregions and cortex cinguli was seen in both Fischer and Lewis rats. In contrast, Nor1 mRNA was only significantly upregulated by cocaine in the Fischer rats. Morphine increased NGFI-B mRNA in medial caudate putamen and cortex cinguli in Lewis rats and Nor1 mRNA in medial caudate putamen in Fischer rats. Chronic cocaine upregulated NGFI-B mRNA in nucleus accumbens core, lateral caudate putamen and cingulate cortex in Fischer rats, whereas no effect was seen in Lewis rats. In contrast, Nor1 mRNA levels were upregulated in Lewis rats in medial caudate putamen and cingulate cortex after chronic cocaine and in cingulate cortex after chronic morphine. No effect on Nor1 mRNA levels was seen in Fischer rats after chronic treatments. Our results demonstrate different responses in addiction-prone Lewis rats as compared to the less addiction-prone Fischer rats with respect to NGFI-B and Nor1 mRNA regulation after acute and repeated administration of cocaine and morphine. Thus, we suggest that the transcription factors NGFI-B and Nor1 might be involved in the control of behaviors such as sensitized locomotor response, craving and aversion that appears after repeated administration of abused drugs.

Addiction-prone Lewis but not Fischer rats develop compulsive running that coincides with downregulation of nerve growth factor inducible-B and neuron-derived orphan receptor 1.

We have examined the effects of chronic voluntary running for 30 d on the levels of nerve growth factor inducilble-B (NGFI-B) and neuron-derived orphan receptor 1 (NOR1) mRNAs in Fischer and Lewis rats. The aim was to compare the addiction-prone Lewis rat strain to the Fischer strain in a plausible model for natural reward. The Lewis strain ran markedly more than the Fischer strain, as indicated by the length of running per day when given free access to running wheels. Both strains progressively increased their amount of daily running. By day 14, Lewis rats had reached a maximal level corresponding to 10 km/d, which slowly decreased to approximately 8 km/d. Fischer rats ran considerably less, averaging approximately 1. 5 km/d by day 30. After 30 d of running, levels of mRNA encoding NGFI-B and Nor1 were decreased in cerebral cortex in Lewis but not Fischer rats. The downregulation of NGFI-B mRNA in Lewis rats could not be attenuated by the opioid receptor antagonist naloxone. Instead, naloxone by itself downregulated NGFI-B in striatum and cerebral cortex in both strains. In contrast, naloxone had no effect on Nor1 mRNA levels, although the running-induced downregulation of Nor1 was, in most cases, attenuated by naloxone. Data from the present study suggest that the same genetic factors contributing to the drug addiction-prone behavior of Lewis rats also control the excessive running behavior and that this coincides with downregulation of transcription factors of the NGFI-B family.

A tachykinin NK1 receptor antagonist, CP-122,721-1, attenuates kainic acid-induced seizure activity.

Substance P (SP) can play an important role in neuronal survival. To analyze the role of SP in excitotoxicity, kainic acid (KA) was administered to rats and in situ hybridization was used to analyze the levels of the SP encoding preprotachykinin-A (PPT-A) mRNA in striatal and hippocampal subregions 1, 4, and 24 h and 7 days after KA. In striatum and piriform cortex, PPT-A mRNA peaked 4 h after KA while in hippocampus, levels peaked after 24 h. KA caused seizures and neuronal toxicity as indicated by a reduction of the number of neurons in the hippocampal CA1 subregion after 7 days. KA was later administered alone or following pretreatment with the tachykinin NK1 receptor antagonist CP-122,721-1 (0.3 mg/kg). The pretreatment decreased seizure activity and a negative correlation was found between seizure activity and survival of CA1 neurons. Conclusively, treatment with CP-122,721-1 has a seizure inhibiting property and may possibly counteract KA-induced nerve cell death in CA1.

Expression of messenger RNAs encoding ionotropic glutamate receptors in rat brain: regulation by haloperidol.

In situ hybridization was used to study the regional distribution of messenger RNAs encoding ionotropic glutamate receptor subtypes in the rat brain's dopaminergic cell body regions and their forebrain projection areas. Short oligonucleotide probes specific for the messenger RNAs encoding the flip or flop splice forms of the GluR1 and GluR2 AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor subunits, or for the messenger RNAs encoding the N-methyl-D-aspartate R1 subunit, were used. Significant differences were seen in the relative messenger RNA levels, and the distribution of the flip and flop splice forms, of GluR1 and GluR2. In the dopaminergic cell groups of the substantia nigra pars compacta and the ventral tegmental area, the flip form of both GluR1 and GluR2 dominated over the flop form. Similarly, in the core division of the nucleus accumbens, GluR1 and GluR2 flip forms dominated over the flop forms. In contrast, in the accumbens shell, the GluR1 and GluR2 flop forms dominated over the flip forms. As a comparison to the AMPA receptor subunits, N-methyl-D-aspartate R1 messenger RNA was relatively evenly distributed in all the regions analysed. The results demonstrate a heterogeneous distribution of the flip and flop splice forms of GluR1 and GluR2 in the brain's dopaminergic pathways, which could contribute to physiological differences in regulation of the pathways by glutamatergic neurotransmission. We also studied regulation of glutamate receptor subunit expression in these regions by antipsychotic drugs, based on previous reports of altered levels of subunit immunoreactivity after drug treatment. Chronic administration of the typical antipsychotic drug, haloperidol, caused a small but significant induction of GluR2 flip messenger RNA in the dorsolateral caudate putamen. This effect was not seen after chronic administration of the atypical antipsychotic drug, clozapine. Significant drug regulation of the other glutamate receptor subunits studied was not observed.

Schizophrenia and neurotrophin-3 alleles.

Studies of brain anatomy and premorbid functioning indicate that schizophrenia may be of neurodevelopmental origin. In the neurotrophic factor neurotrophin-3 (NT-3) gene, the A3/147-bp allele in a dinucleotide repeat polymorphism located in the promoter region was found to be associated with schizophrenia in a Japanese study. Another NT-3 polymorphism (Glu63Gly) indicated an association with schizophrenic patients with a putative neurodevelopmental form of the disease. We examined Swedish schizophrenic patients (n = 109) and control subjects (n = 78) for the same two NT-3 polymorphisms, as well as a third silent exonic polymorphism (at Pro55). No significant difference was found between the two groups. However, in a meta-analysis including the present and previous studies of Caucasian subjects, the A3/147-bp allele frequency was found to be significantly higher in the schizophrenic patients. In the present study, carriers of the A3/147 bp allele tended to have an earlier age of onset and to display more extrapyramidal symptoms. In the silent exonic polymorphism (at Pro55), female schizophrenic patients had higher adenine and lower guanine allele frequencies than control female subjects. Together with previous studies, the results provide some support for an association between the NT-3 gene and certain forms of schizophrenia. This warrants further investigation of NT-3 and other neurotrophic factors with additional polymorphisms and larger patient samples.

Lack of evidence for allelic association between personality traits and the dopamine D4 receptor gene polymorphisms.

OBJECTIVE: Personality traits in human subjects have shown considerable heritable components. Recently, two research groups reported associations between dopamine D4 receptor genotypes and the personality trait known as novelty seeking. This study was an attempt to replicate these findings. METHOD: Three different exonic dopamine D4 receptor polymorphisms were genotyped in 126 healthy Swedish subjects. Personality traits of the subjects were assessed with the Karolinska Scales of Personality. RESULTS: Although there was a tendency in the direction hypothesized, no significant association between genotype constellations and personality traits was found. CONCLUSIONS: The previously reported association between dopamine D4 receptor alleles and novelty seeking was not replicated. Possible reasons for this include differences in personality inventories, ethnicity, and type I or type II errors.

Tyrosine hydroxylase and dopamine D4 receptor allelic distribution in Scandinavian chronic alcoholics.

Associations of polymorphic genetic markers at the tyrosine hydroxylase (TH) and dopamine D4 receptor (DRD4) loci were examined in Scandinavian chronic alcoholics (n = 72) and control subjects (n = 67). Patients were divided into subgroups with regard to the presence of parental alcoholism and age of onset. Neither the TH nor the DRD4 allele distributions were significantly different when alcoholic samples were compared with control subjects. However, a tendency to high prevalence for 1 of the 5 TH alleles assayed (TH-K3) was observed in a subsample of 44 alcoholics characterized by late onset when compared with control subjects (27.3% vs. 10.6%, p = 0.041). Results suggest that no major influence on alcoholism is exerted through genes associated with the DRD4 or TH allelic markers examined.

Dopamine-related genes and their relationships to monoamine metabolites in CSF.

Monoamine metabolite (MM) levels in lumbar cerebrospinal fluid (CSF) are extensively used as indirect estimates of monoamine turnover in the brain. In this study we investigated genotypes for DNA polymorphisms in the D2 (DRD2), D3 (DRD3), and D4 (DRD4) dopamine receptor and tyrosine hydroxylase (TH) genes and their relationships to CSF MM in healthy volunteers (n = 66). Concentrations of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were corrected for back length, a confounding variable. Corrected MM levels were not related to age, gender, height, weight heredity, season or atmospheric pressure at sampling. Individuals with specific DRD2 and TH allele and genotype configurations significantly differed in HVA and MHPG concentrations. DRD3 homo- and heterozygotic genotypes had significantly different CSF 5-HIAA levels. DRD4 genotypes were not related to MM concentrations. The results suggest that specific DRD2, DRD3, and TH genotypes participate in the regulation of monoamine turnover in the central nervous system. Accordingly monoamine receptors and synthesizing enzyme genotypes appear to be variance factors influencing MM concentrations in CSF. The relationships found in this study support MM concentrations as markers for monoamine transmission in the human brain.

A search for association between schizophrenia and dopamine-related alleles.

Dopamine receptor dysfunction and altered tyrosine hydroxylase activity have both been implicated in the pathophysiology of schizophrenia. Schizophrenic patients and control subjects were examined for allele frequencies in the tyrosine hydroxylase and dopamine D2 and D4 receptor genes. No significant differences of allele or genotype frequencies were found between the two groups after adjustment for multiple comparisons. Neither were any significant relationships observed between allele frequencies and a number of clinical variables within the schizophrenic subsample. When no adjustment was made for multiple testing a few significant tendencies were obtained which warrant further research in extended patient and control materials. The results are compatible with the view that the tyrosine hydroxylase, dopamine receptor D2 and D4 gene polymorphisms examined are not of major importance in the aetiology or pathophysiology of schizophrenia.

Distribution of messenger RNAs for D1 dopamine receptors and DARPP-32 in striatum and cerebral cortex of the cynomolgus monkey: relationship to D1 dopamine receptors.

Messenger RNAs for the D1 dopamine receptor and dopamine- and cyclic AMP-regulated phosphoprotein of relative mass 32,000 (DARPP-32) were examined by in situ hybridization in the cynomolgus monkey brain. The messenger RNA distribution was compared to the distribution of D1 dopamine receptors using [3H]SCH 23390 autoradiography. In the caudate nucleus and putamen, D1 dopamine receptor messenger RNA-positive cells were unevenly distributed. Clusters of cells with an approximately three-fold higher intensity of labeling, as compared to surrounding regions, were found. Some of these D1 dopamine receptor messenger RNA intensive cell clusters in the caudate nucleus appeared to some extent to be matched to regions of higher intensity of [3H]SCH 23390 binding. The distribution of cells expressing DARPP-32 messenger RNA in the caudate nucleus and putamen was found to be non-clustered. In neocortical regions, cells of different sizes expressing D1 dopamine receptor messenger RNA were present in layers II-VI. D1 dopamine receptor messenger RNA-positive cells were most abundant in layer V. Unexpectedly, no DARPP-32 messenger RNA signal was detected in neocortex. Chronic SCH 23390 administration did not change the relative levels of messenger RNAs for the D1 dopamine receptor and DARPP-32 or [3H]SCH 23390 binding as measured by quantitative image analysis. The clustered distribution of D1 dopamine receptor messenger RNA is in contrast to that of DARPP-32 messenger RNA. This suggests that D1 dopamine receptors may play a more significant role in regulating DARPP-32 function in patch regions as compared to matrix regions. D1 dopamine receptor messenger RNA-expressing cells could also be visualized in several layers of the primate neocortex, implying that dopamine acts through D1 dopamine receptors within functionally different neuronal circuits of the neocortex.