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Biomedical subjects

C Pasqualini

Publications and source records attributed to C Pasqualini.

At least 19 recordsLinked to original sources

Impact of immigration and HIV infection on tuberculosis incidence in an area of low tuberculosis prevalence.

The aim of this study is to assess the effects of immigration from countries with a high prevalence of tuberculosis (HPCs), of HIV/AIDS prevalence, and the ageing of the indigenous population, on tuberculosis distribution in a low-prevalence area (LPCs), the Piedmont Region of Italy. Tuberculosis incidence and HIV cases were identified by linking records from the surveillance systems. Overall, 640 tuberculosis cases were identified and crude annual incidence was found to be 17.3/100000. The incidence rate ratio for HIV infection as a risk factor for tuberculosis (11.4 and 51.9 among individuals from HPCs and LPCs respectively) was greater than that for immigration from HPCs (6.7 and 30.9 among HIV+ and HIV- individuals). Immigration accounted for a larger number of incident cases [population attributable risk % (PAR %): 31.8 and 52.8% among HIV+ and HIV- individuals] than did HIV infection (PAR %: 5.4 and 11.1% among individuals from HPCs and LPCs). Efforts should be made to identify and treat young immigrants from HPCs.

AIDS-Related Opportunistic Infections↗

Dopaminergic inhibition of reproduction in teleost fishes: ecophysiological and evolutionary implications.

In many teleosts, dopamine (DA) exerts direct inhibitory control on gonadotropes, counteracting the stimulatory effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release. This dual control by GnRH and DA has been demonstrated in various adult teleosts and has major implications for aquaculture. Because of its unique life cycle, the European eel has provided a powerful model for demonstrating the key role of DA in the control of puberty. Data from tetrapods suggest that the inhibitory role of DA on reproduction is not restricted to the teleosts. Thus, DA inhibitory control could represent an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been differentially maintained throughout vertebrate evolution. The intensity of DA inhibition, its main site of action, and its involvement in the control of puberty, seasonal reproduction, ovulation, spermiation, or even sex change may differ among classes of vertebrates, as well as within smaller phylogenetic units such as teleosts or mammals. An inhibitory role for DA has been reported also in some invertebrates, indicating that neuronal DA pathways may have been recruited in various groups of metazoa to participate in the control of reproduction. In addition to the incontestable GnRH neurons, the recruitment of DA neurons for the neuroendocrine control of reproduction provides an additional brain pathway for the integration of various species-specific, internal, and environmental cues. In teleosts, the plasticity of the DA neuroendocrine role may have contributed to their large diversity of biological cycles and to their successful adaptation to various environments.

Animals↗

Brain expression of tyrosine hydroxylase and its regulation by steroid hormones in the European eel quantified by real-time PCR.

In the eel, dopamine inhibits pubertal development. To investigate the regulatory mechanisms involved, we developed a quantitative real-time RT-PCR assay for measurement of brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine. TH expression was highest in the olfactory bulb, followed by the di-/mesencephalic areas and the telencephalon/preoptic area. TH expression in the optic lobes and hindbrain was low or below the detection limit. In vivo treatment with testosterone, but not estradiol, resulted in increased TH expression in the forebrain, except the optic tectum, but not in the hindbrain. The results were confirmed by in situ hybridization.

Anguilla↗

Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.

Alternative Splicing↗

Computer-guided pericardiocentesis: experimental results and clinical perspectives.

Percutaneous pericardial puncture is a relatively safe and effective technique in case of large pericardial effusions when practiced under echographic or radiological control. The goal of our project is to improve the performance of this technique, mainly in case of smaller and loculated effusions using an accurate guidance towards a preplanned target, based on a model of the pericardial effusion. This paper presents preclinical results of this new computer-assisted technique used to reach the pericardial cavity. The procedure is divided into 3 steps: 1. acquisition of ultrasound data, using an echocardiographic device connected to a 3-D localizer and to a computer, 2. modeling procedure to define the optimal strategy taking into account the mobility of organs on a digital model, 3. guided puncture with a localized needle to reach the predefined target using a passive guidance system. After validation on a dynamic phantom and a feasibility study on dogs, an accuracy and reliability analysis protocol was realized on pigs with experimental pericardial effusion. Feasibility of the technique is demonstrated on animal study with an accuracy of at least 2.5 mm. Further clinical investigation is in progress using a more ergonomic and less cumbersome system. This study demonstrates the feasibility of computer-assisted pericardiocentesis. Beyond the simple improvement of the current technique, this could be a new way to reach the heart or a new tool for percutaneous access and image-guided puncture of soft tissues.

Animals↗

Stage- and region-specific expression of estrogen receptor alpha isoforms during ontogeny of the pituitary gland.

The expression time course of estrogen receptor alpha (ER alpha) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ER alpha messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ER alpha mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ER alpha mRNA. On Western blots from rat pituitaries of different ages, an ER alpha-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ER alpha mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ER alpha to be present first in the cytosol of intermediate lobe cells (around embryonic day 16). Only 5 days later, nuclear staining became detectable in the anterior lobe. We argue that the observed cytosolic staining will be essentially due to short ER alpha isoforms, which are indeed more abundantly expressed in the intermediate lobe. These data suggest that during pituitary development, the activity of the ER alpha might be specifically regulated by differential splicing of its primary transcript, resulting in a differential subcellular localization of the isoforms.

Age Factors↗

Phosphorylated and non-phosphorylated neurofilament epitopes are co-distributed in the fish Mauthner axon.

The neurofilament (NF) polypeptides of the fish giant Mauthner cell axons (MAs) and their degree of phosphorylation were investigated in the adult by means of immunoblot and immunohistochemical staining. Fasciculus longitudinalis medialis (FLM) axons of much smaller caliber, among which MAs run in the ventral spinal cord, were also analyzed in two teleost fish belonging to continuously growing species. To detect NF polypeptide subunits, commercially available monoclonal antibodies against mammalian NF-L (68 kDa), NF-M (160 kDa), phosphorylated (P) and non-phosphorylated (nP) NF-H (200 kDa) epitopes were used. These antibodies labelled bands of the identical molecular weight in fish cervical spinal cord total protein immunoblots. A peculiar NF composition of the MAs was observed, following immunohistochemical staining i.e. a low NF-M expression and a codistribution of P and (nP)-NF-H epitopes. Moreover, on the basis of immunoperoxidase staining in ultrathin longitudinal MA sections, we suggest that P NF-H are arranged in bundles, whilst (nP)-NF-H are likely to be free in the axoplasm. By contrast, FLM axons were found reactive with antibodies only against P NF-H. These results confirm that in carp and trout, NF have epitopes cross-reacting with monoclonal antibodies directed against the mammalian NF subunits. Furthermore, as regards the co-distribution of phosphorylated and non-phosphorylated NF-H epitopes in the M-cell axons, these might be considered as not yet completely mature axons taking into account that carp and trout belong to continuously growing species.

Animals↗

Acute stimulatory effect of estradiol on striatal dopamine synthesis.

The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17 alpha or 17 beta E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17 beta (but not 17 alpha) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10(-9) M 17 beta (but not 17 alpha) E2 to the superfusing fluid immediately evoked an approximately 50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki(DA)) was calculated. Incubation of striatal slices in the presence of 10(-9) M 17 beta (but not 17 alpha) E2 indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

Evidence for protein kinase C involvement in the short-term activation by prolactin of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons.

The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a Ki(DA) value of 29.92 +/- 0.49 microM, the other being approximately 15-fold more sensitive to DA inhibition with a Ki(DA) of 1.96 +/- 0.09 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low Ki(DA) remained unaffected, whereas the Ki(DA) of the purported active form of TH increased to 62.6 +/- 0.8 microM, suggesting an increase in the enzyme phosphorylation. This increase in the Ki(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12-O-tetradecanoylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.

Animals↗

Short-term inhibitory effect of estradiol on tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons in vitro.

The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30-40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a Ki(DA) of 26.4 +/- 2 microM and the other being approximately 10-fold more sensitive to DA inhibition, with a Ki(DA) of 2.56 +/- 0.17 microM, likely corresponding to a phosphorylated and active form and to a nonphosphorylated and poorly active form, respectively. Conversely, after estradiol treatment all TH molecules exhibited the same Ki(DA) of 2.5 +/- 0.3 microM. This effect was stereospecific, because 17 alpha-estradiol could not promote it, whereas with 17 beta-estradiol, it could be observed at only 10(-11) M and after a short delay (30 min). Finally, this decrease in the Ki(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.

Animals↗

[Decrease of pineal AMPc and TOH activity in the superior cervical ganglia after ablation of submaxillary glands in rats].

In order to investigate a possible functional relationship between the submandibular salivary gland (SSG) and the central nervous system (CNS), we have extirpated the salivary organs from thirty male rats. Twenty days after ablation both the pineal glands and the cervical superior ganglions (CSG) were dissected, homogenized and frozen until AMPc and TOH were assayed respectively. We observed a significant decrease in pineal AMPc (53.9 +/- 6.2 vs 76.1 +/- 7.6% of maximum value; p less than 0.02) which seems to be linked with a significant drop in TOH activity measured at CSG level (1.5 +/- 0.6 vs 3.7 +/- 0.9 nmoles of DOPA/h/pair GCS; p less than 0.03). Our results suggest that both findings might be due to the lack of NGF normally reaching the CSG from SSG. This data reinforces the idea of a functional link between SSG and CNS via the pineal gland.

Animals↗

Estrogen-like effects of 7,12-dimethylbenz(a)anthracene on the female rat hypothalamo-pituitary axis.

We have recently demonstrated that 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors in rodents, can in vitro decrease the number of membrane dopamine D2 receptors and stimulate prolactin (PRL) release, by direct estrogen-like actions on anterior pituitary. In the present study, we tested the ability of DMBA to mimic the in vivo estradiol (17 beta E2) effects on pituitary D2 receptors and on PRL as well as LH release. We have found that DMBA, like 17 beta E2, when injected to ovariectomized rats, induced a decrease in the number of anterior pituitary D2 receptors, a release of PRL and exerted a biphasic (acute negative and longer term positive) action on LH secretion. We thus examined the ability of DMBA to interact with 17 beta E2 receptors in the hypothalamo-pituitary axis: DMBA binds to the pituitary cytosolic estrogen receptors with an affinity 0.001% that of 17 beta E2. Finally [3H]DMBA binds to hypothalamus-containing brain sections. This binding was displaced partially by RU 2858 a pure estrogen agonist and totally by tamoxifen, a purported estrogen antagonist. No competition for [3H]DMBA binding was observed with an androgen (RU 1881) or a glucocorticoid (RU 26988) agonist. From these data, it may be concluded that DMBA can act as a partial estrogen in pituitary and hypothalamic tissues.

9,10-Dimethyl-1,2-benzanthracene↗

Median eminence dopamine and serotonin neuronal activity. Temporal relationship to preovulatory prolactin and luteinizing hormone surges.

Using a high-performance liquid chromatography (HPLC) system coupled with an electrochemical detector, the concentrations of dopamine (DA) and 5-hydroxytryptamine (5-HT) and their major specific metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA), respectively, were measured in the median eminence (ME) throughout the rat estrous cycle. The ME DA content remained fairly constant throughout the estrous cycle except on estrus when 17.00 h values were significantly lower than 10.00 h values (40% decrease, p less than 0.05). The ME 5-HT content determined at 10.00 h was higher on proestrus than on any other day of the cycle. The ME DOPAC concentrations did not differ between 10.00 and 17.00 h on diestrus I, diestrus II or estrus. On the contrary, there was an almost linear decline between 10.00 and 17.00 h on proestrus (36% decrease, p less than 0.05). The ME 5-HIAA content did not differ between 10.00 and 17.00 h on any day of the estrous cycle. Significant changes were recorded for the DOPAC/DA and 5-HIAA/5-HT ratio in the ME on proestrus. There was a progressive decrease, starting from 10.00 h in the DOPAC/DA ratio with minimal values (42% decrease, p less than 0.05) at 16.00 h followed by an increase from 16.00 to 19.00 h. On the other hand, the 5-HIAA/5-HT ratio increased between 10.00 and 17.00 h (97% increase, p less than 0.05) and subsequently declined until 19.00 h (67% decrease vs. 17.00 h, p less than 0.05).2+hese data show that a concomitant

3,4-Dihydroxyphenylacetic Acid↗

In vitro estrogen-like effects of 7,12-dimethylbenz(a)anthracene on anterior pituitary dopamine receptors of rats.

The ability of 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors, to mimic short term effects of estradiol (17 beta-E2) on the anterior pituitary, was tested in vitro. Incubation of anterior pituitaries from ovariectomized rats with DMBA resulted in a marked depletion of membrane dopamine receptors (labeled with [3H] spiperone) and a parallel stimulation of prolactin (PRL) release. Maximal receptor depletion and PRL release were obtained after 15-30 min of incubation with 10(-8) M DMBA. These effects were reversible and already significant after a 5-min incubation. Their magnitude, dose dependency, and time course were identical to those reported for 17 beta-E2. A structurally related noncarcinogenic polycyclic aromatic hydrocarbon, phenanthrene, had no effect on [3H]spiperone binding or PRL release. When DMBA and 17 beta-E2, at suboptimal concentrations, were simultaneously added to the culture medium, no synergistic effect could be observed. When 10(-8) M of both compounds were introduced simultaneously, the decrease in dopamine receptors and the increase in PRL release were not greater than those observed in the presence of 10(-8) M of only one compound, indicating that the same mechanism(s) can be involved. These data suggest that DMBA in desensitizing lactotrophs to dopamine and in releasing PRL, by direct estrogen-like actions on anterior pituitary, may provide a hormonal state conducive to tumor development.

9,10-Dimethyl-1,2-benzanthracene↗

Changes in tuberoinfundibular dopaminergic neuron activity during the rat estrous cycle in relation to the prolactin surge: alteration by a mammary carcinogen.

An attempt was made to correlate the physiological or the dimethylbenz(a)anthracene (DMBA)-enhanced serum prolactin (PRL) surge, which occurs in the afternoon of proestrus in female Sprague-Dawley (SD) rats, with physiological or pathological changes in two biochemical estimates of the tuberoinfundibular dopaminergic (TIDA) neuron activity. Dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) concentrations as well as tyrosine hydroxylase (TH) activity were measured in the median eminence (ME) of control or DMBA-pretreated SD rats throughout the estrous cycle in relation to PRL secretion. In both groups of females, while the DA content was fairly constant, the DOPAC content and TH activity in the ME fluctuated markedly throughout the estrous cycle. Thus, in control animals, the DOPAC content, DOPAC/DA ratio and TH activity which were stable on the days of diestrus and morning of proestrus were markedly decreased at noon and early afternoon when serum PRL levels began to rise. Later in the afternoon of proestrus, when serum PRL levels were maximal, there was a marked but transient increase in the DOPAC content and DOPAC/DA ratio as well as a brief surge in TH activity. In the evening of the same day, when serum PRL returned to basal levels, the DOPAC content, DOPAC/DA ratio and TH activity were low. Finally on estrus morning, the DOPAC content, DOPAC/DA ratio and TH activity increased again to reach the diestrus levels. In DMBA-pretreated females, similar fluctuations in TIDA neuronal activity occurred during the estrous cycle, but the dynamics of these changes was altered: the DOPAC/DA ratio and TH activity first showed a marked increase in the morning of proestrus day, before decreasing dramatically.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

Direct effect of estradiol on the number of dopamine receptors in the anterior pituitary of ovariectomized rats.

The effect of 17 beta-estradiol (17 beta E2) on anterior pituitary dopaminergic receptor (D2) content was studied in vitro in relation to PRL secretion. Anterior pituitaries from ovariectomized rats were incubated for short periods in medium 199, with or without the steroid. Dopamine (DA) receptors in partially purified pituitary membranes were quantified by equilibrium binding using [3H]spiperone; the PRL released into the incubation medium was analyzed by RIA. Addition of 10(-10) to 10(-6) M 17 beta E2 to the incubation medium of anterior pituitaries rapidly and reversibly decreased the number of DA receptors (P less than 0.01 to 0.001), while increasing PRL release, in a dose-related fashion. The maximal effect on both receptor numbers and PRL secretion was obtained with 10(-8) M 17 beta E2. This effect involved no change in receptor affinity (Kd = 0.11 +/- 0.01 nM in presence or in absence of 17 beta E2). This estrogen-induced decrease in DA-binding capacity was apparently not the result of the occupation of spiperone binding sites by the steroid, since after a 30-min incubation with 10(-8) M [3H]17 beta E2, no radioactivity was detectable on the partially purified membranes. Moreover, the presence of 17 beta E2 at the same time as the labeled D2 ligand did not modify the kinetics of association or dissociation of spiperone with pituitary membranes. This decrease in anterior pituitary DA receptor content and the increase in PRL release were already significant after a 7-min incubation in the presence of 10(-8) M 17 beta E2. Finally, these effects of 17 beta E2 were not mimicked by its 17 alpha-stereoisomer, nor by progesterone, or testosterone. These results suggest that the stimulatory effect of 17 beta E2 on PRL secretion may be due, at least in part, to the desensitization of anterior pituitary cells to DA. The steroid may produce this desensitization directly by decreasing the number of D2. The short latency of this effect likely discards the possibility of a genomic action of 17 beta E2.

Animals↗

[Direct effect of estradiol on the number of dopaminergic receptors in the anterior pituitary, in ovariectomized rats].

The effect of estradiol on anterior pituitary dopaminergic receptor content was studied in vivo and in vitro, in relation with the serum PRL secretion. A progressive and significant decrease in the number of these receptors was observed, a few hours before the serum release of PRL induced in ovariectomized females by a sequential treatment with different doses of estradiol benzoate. This decrease in the number of dopaminergic membrane receptors can be obtained as well in vitro, when anterior pituitaries, from ovariectomized rats, are incubated with 17 beta-estradiol. These results suggest that the stimulatory effect of estradiol on PRL secretion may be due, at least in part, to the direct "desensitization" to DA of anterior pituitary cells, which is produced by the decrease of dopaminergic receptor level.

Animals↗