Divergent Acute and Enduring Changes in 50-kHz Ultrasonic Vocalizations in Rats Repeatedly Treated With Amphetamine and Dopaminergic Antagonists: New Insights on the Role of Dopamine in Calling Behavior.

BACKGROUND: Rats emit 50-kHz ultrasonic vocalizations (USVs) in response to nonpharmacological and pharmacological stimuli, with addictive psychostimulants being the most effective drugs that elicit calling behavior in rats. Earlier investigations found that dopamine D1-like and D2-like receptors modulate the emission of 50-kHz USVs stimulated in rats by the acute administration of addictive psychostimulants. Conversely, information is lacking on how dopamine D1-like and D2-like receptors modulate calling behavior in rats that are repeatedly treated with addictive psychostimulants. METHODS: We evaluated the emission of 50-kHz USVs in rats repeatedly treated (×5 on alternate days) with amphetamine (1 mg/kg, i.p.) either alone or together with (1) SCH 23390 (0.1-1 mg/kg, s.c.), a dopamine D1 receptor antagonist; (2) raclopride (0.3-1 mg/kg, s.c.), a selective dopamine D2 receptor antagonist; or (3) a combination of SCH 23390 and raclopride (0.1 + 0.3 mg/kg, s.c.). Calling behavior of rats was recorded following pharmacological treatment, as well as in response to the presentation of amphetamine-paired cues and to amphetamine challenge (both performed 7 days after treatment discontinuation). RESULTS: Amphetamine-treated rats displayed a sensitized 50-kHz USV emission during repeated treatment, as well as marked calling behavior in response to amphetamine-paired cues and to amphetamine challenge. Antagonism of D1 or D2 receptors either significantly suppressed or attenuated the emission of 50-kHz USVs in amphetamine-treated rats, with a maximal effect after synergistic antagonism of both receptors. CONCLUSIONS: These results shed further light on how dopamine transmission modulates the emission of 50-kHz USVs in rats treated with psychoactive drugs.

Perturbation of serine enantiomers homeostasis in the striatum of MPTP-lesioned monkeys and mice reflects the extent of dopaminergic midbrain degeneration.

Loss of dopaminergic midbrain neurons perturbs l-serine and d-serine homeostasis in the post-mortem caudate putamen (CPu) of Parkinson's disease (PD) patients. However, it is unclear whether the severity of dopaminergic nigrostriatal degeneration plays a role in deregulating serine enantiomers' metabolism. Here, through high-performance liquid chromatography (HPLC), we measured the levels of these amino acids in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and MPTP-plus-probenecid (MPTPp)-treated mice to determine whether and how dopaminergic midbrain degeneration affects the levels of serine enantiomers in various basal ganglia subregions. In addition, in the same brain regions, we measured the levels of key neuroactive amino acids modulating glutamatergic neurotransmission, including l-glutamate, glycine, l-aspartate, d-aspartate, and their precursors l-glutamine, l-asparagine. In monkeys, MPTP treatment produced severe denervation of nigrostriatal dopaminergic fibers (⁓75%) and increased the levels of serine enantiomers in the rostral putamen (rPut), but not in the subthalamic nucleus, and the lateral and medial portion of the globus pallidus. Moreover, this neurotoxin significantly reduced the protein expression of the astrocytic serine transporter ASCT1 and the glycolytic enzyme GAPDH in the rPut of monkeys. Conversely, concentrations of d-serine and l-serine, as well as ASCT1 and GAPDH expression were unaffected in the striatum of MPTPp-treated mice, which showed only mild dopaminergic degeneration (⁓30%). These findings unveil a link between the severity of dopaminergic nigrostriatal degeneration and striatal serine enantiomers concentration, ASCT1 and GAPDH expression. We hypothesize that the up-regulation of d-serine and l-serine levels occurs as a secondary response within a homeostatic loop to support the metabolic and neurotransmission demands imposed by the degeneration of dopaminergic neurons.

Homeostasis of serine enantiomers is disrupted in the post-mortem caudate putamen and cerebrospinal fluid of living Parkinson's disease patients.

L-serine generated in astrocytes plays a pivotal role in modulating essential neurometabolic processes, while its enantiomer, D-serine, specifically regulates NMDA receptor (NMDAR) signalling. Despite their physiological relevance in modulating cerebral activity, serine enantiomers metabolism in Parkinson's disease (PD) remains elusive. Using High-Performance Liquid Chromatography (HPLC), we measured D- and L-serine levels along with other amino acids known to modulate NMDAR function, such as L-glutamate, L-aspartate, D-aspartate, and glycine, in the post-mortem caudate putamen (CPu) and superior frontal gyrus (SFG) of PD patients. Moreover, we examined these amino acids in the cerebrospinal fluid (CSF) of de novo living PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) patients versus subjects with other neurological disorders (OND), used as control. We found higher D-serine and L-serine levels in the CPu of PD patients but not in the SFG, a cerebral region that, in contrast to the CPu, is not innervated by nigral dopaminergic terminals. We also highlighted a significant elevation of both serine enantiomers in the CSF samples from PD but not in those of AD and ALS patients, compared with control subjects. By contrast, none or only minor changes were found in the amount of other NMDAR modulating amino acids. Our findings identify D-serine and L-serine level upregulation as a biochemical signature associated with nigrostriatal dopaminergic degeneration in PD.

Involvement of the Protein Ras Homolog Enriched in the Striatum, Rhes, in Dopaminergic Neurons' Degeneration: Link to Parkinson's Disease.

Rhes is one of the most interesting genes regulated by thyroid hormones that, through the inhibition of the striatal cAMP/PKA pathway, acts as a modulator of dopamine neurotransmission. Rhes mRNA is expressed at high levels in the dorsal striatum, with a medial-to-lateral expression gradient reflecting that of both dopamine D2 and adenosine A2A receptors. Rhes transcript is also present in the hippocampus, cerebral cortex, olfactory tubercle and bulb, substantia nigra pars compacta (SNc) and ventral tegmental area of the rodent brain. In line with Rhes-dependent regulation of dopaminergic transmission, data showed that lack of Rhes enhanced cocaine- and amphetamine-induced motor stimulation in mice. Previous studies showed that pharmacological depletion of dopamine significantly reduces Rhes mRNA levels in rodents, non-human primates and Parkinson's disease (PD) patients, suggesting a link between dopaminergic innervation and physiological Rhes mRNA expression. Rhes protein binds to and activates striatal mTORC1, and modulates L-DOPA-induced dyskinesia in PD rodent models. Finally, Rhes is involved in the survival of mouse midbrain dopaminergic neurons of SNc, thus pointing towards a Rhes-dependent modulation of autophagy and mitophagy processes, and encouraging further investigations about mechanisms underlying dysfunctions of the nigrostriatal system.

Lack of Rhes Increases MDMA-Induced Neuroinflammation and Dopamine Neuron Degeneration: Role of Gender and Age.

Ras homolog enriched in striatum (Rhes) is a protein that exerts important physiological functions and modulates psychostimulant drug effects. On this basis, the object of this study was to assess 3,4-methylenedioxymethamphetamine (MDMA) effects on microglial (CD11b) and astroglial (GFAP) activation and on dopamine neuron degeneration (TH) in wild-type (WT) and Rhes knockout (KO) male and female mice of different ages. Motor activity was also evaluated. Adult (3 months) MDMA-treated mice displayed an increase in GFAP-positive cells in striatum (STR), whereas the substantia nigra pars compacta (SNc) was affected only in male mice. In these mice, the increase of CD11b was more extensive including STR, SNc, motor cortex (CTX), ventral tegmental area (VTA), and nucleus accumbens (NAc). MDMA administration also affected TH immunoreactivity in both STR and SNc of male but not female WT and Rhes KO mice. In middle-aged mice (12 months), MDMA administration further increased GFAP and CD11b and decreased TH immunoreactivity in STR and SNc of all mice. Finally, MDMA induced a higher increase of motor activity in adult Rhes KO male, but not female mice. The results show that Rhes protein plays an important role on MDMA-mediated neuroinflammation and neurodegeneration dependent on gender and age, and confirm the important role of Rhes protein in neuroinflammatory and neurodegenerative processes.

Modulation of Rat 50-kHz Ultrasonic Vocalizations by Glucocorticoid Signaling: Possible Relevance to Reward and Motivation.

Background: Rats emit 50-kHz ultrasonic vocalizations (USVs) to communicate positive emotional states, and these USVs are increasingly being investigated in preclinical studies on reward and motivation. Although it is the activation of dopamine receptors that initiates the emission of 50-kHz USVs, non-dopaminergic mechanisms may modulate calling in the 50 kHz frequency band. To further elucidate these mechanisms, the present study investigated whether the pharmacological manipulation of glucocorticoid signaling influenced calling. Methods: Rats were administered corticosterone (1-5 mg/kg, s.c.), the glucocorticoid receptor antagonist mifepristone (40 or 100 mg/kg, s.c.), or the corticosterone synthesis inhibitor metyrapone (50 or 100 mg/kg, i.p.). The effects of these drugs on calling initiation and on calling recorded during nonaggressive social contacts or after the administration of amphetamine (0.25 or 1 mg/kg, i.p.) were then evaluated. Results: Corticosterone failed to initiate the emission of 50-kHz USVs and did not influence pro-social and amphetamine-stimulated calling. Similarly, mifepristone and metyrapone did not initiate calling. However, metyrapone suppressed pro-social calling and calling stimulated by a moderate dose (1 mg/kg, i.p.) of amphetamine. Conversely, mifepristone attenuated calling stimulated by a low (0.25 mg/kg, i.p.), but not moderate (1 mg/kg, i.p.), dose of amphetamine and had no influence on pro-social calling. Conclusions: The present results demonstrate that glucocorticoid signaling modulates calling in the 50 kHz frequency band only in certain conditions and suggest that mechanisms different from the inhibition of corticosterone synthesis may participate in the suppression of calling by metyrapone.

Role of adenosine A2A receptors in motor control: relevance to Parkinson's disease and dyskinesia.

Adenosine is an endogenous purine nucleoside that regulates several physiological functions, at the central and peripheral levels. Besides, adenosine has emerged as a major player in the regulation of motor behavior. In fact, adenosine receptors of the A2A subtype are highly enriched in the caudate-putamen, which is richly innervated by dopamine. Moreover, several studies in experimental animals have consistently demonstrated that the pharmacological antagonism of A2A receptors has a facilitatory influence on motor behavior. Taken together, these findings have envisaged A2A receptors as a promising target for symptomatic therapies aimed at ameliorating motor deficits. Accordingly, A2A receptor antagonists have been extensively studied as new agents for the treatment of Parkinson's disease (PD), the epitome of motor disorders. In this review, we provide an overview of the effects that adenosine A2A receptor antagonists elicit in rodent and primate experimental models of PD, with regard to the counteraction of motor deficits as well as to manifestation of dyskinesia and motor fluctuations. Moreover, we briefly present the results of clinical trials of A2A receptor antagonists in PD patients experiencing motor fluctuations, with particular regard to dyskinesia. Finally, we discuss the interaction between A2A receptor antagonists and serotonin receptor agonists, since combined administration of these drugs has recently emerged as a new potential therapeutic strategy in the treatment of dyskinesia.

Brain dysfunctions and neurotoxicity induced by psychostimulants in experimental models and humans: an overview of recent findings.

Preclinical and clinical studies indicate that psychostimulants, in addition to having abuse potential, may elicit brain dysfunctions and/or neurotoxic effects. Central toxicity induced by psychostimulants may pose serious health risks since the recreational use of these substances is on the rise among young people and adults. The present review provides an overview of recent research, conducted between 2018 and 2023, focusing on brain dysfunctions and neurotoxic effects elicited in experimental models and humans by amphetamine, cocaine, methamphetamine, 3,4-methylenedioxymethamphetamine, methylphenidate, caffeine, and nicotine. Detailed elucidation of factors and mechanisms that underlie psychostimulant-induced brain dysfunction and neurotoxicity is crucial for understanding the acute and enduring noxious brain effects that may occur in individuals who use psychostimulants for recreational and/or therapeutic purposes.

Positive relation between dopamine neuron degeneration and metabolic connectivity disruption in the MPTP plus probenecid mouse model of Parkinson's disease.

The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment approaches. Biomarkers to identify the early stages of PD are therefore of paramount importance for the advancement of the therapy of PD. In the present study, by using a mouse model of PD obtained by subchronic treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the clearance inhibitor probenecid (MPTPp), we identified prodromal markers of PD by combining in vivo positron emission tomography (PET) imaging and ex vivo immunohistochemistry. Longitudinal PET imaging of the dopamine transporter (DAT) by [18F]-N-(3-fluoropropyl)-2ß-carboxymethoxy-3ß-(4-iodophenyl) nortropane ([18F]-FP-CIT), and brain glucose metabolism by 2-deoxy-2-[18F]-fluoroglucose ([18F]-FDG) were performed before MPTPp treatment and after 1, 3, and 10 MPTPp administrations, in order to assess relation between dopamine neuron integrity and brain connectivity. The results show that in vivo [18F]-FP-CIT in the dorsal striatum was not modified after the first administration of MPTPp, tended to decrease after 3 administrations, and significantly decreased after 10 MPTPp administrations. Post-mortem immunohistochemical analyses of DAT and tyrosine hydroxylase (TH) in the striatum showed a positive correlation with [18F]-FP-CIT, confirming the validity of repeated MPTPp-treated mice as a model that can reproduce the progressive pathological changes in the early phases of PD. Analysis of [18F]-FDG uptake in several brain areas connected to the striatum showed that metabolic connectivity was progressively disrupted, starting from the first MPTPp administration, and that significant connections between cortical and subcortical regions were lost after 10 MPTPp administrations, suggesting an association between dopamine neuron degeneration and connectivity disruption in this PD model. The results of this study provide a relevant model, where new drugs that can alleviate neurodegeneration in PD could be evaluated preclinically.

Withania somnifera influences MDMA-induced hyperthermic, cognitive, neurotoxic and neuroinflammatory effects in mice.

Withania somnifera (WS) is utilized in Ayurvedic medicine owing to its central and peripheral beneficial properties. Several studies have accrued indicating that the recreational amphetamine-related drug (+/-)- 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) targets the nigrostriatal dopaminergic system in mice, inducing neurodegeneration and gliosis, causing acute hyperthermia and cognitive impairment. This study aimed to investigate the effect of a standardized extract of W. somnifera (WSE) on MDMA-induced neurotoxicity, neuroinflammation, memory impairment and hyperthermia. Mice received a 3-day pretreatment with vehicle or WSE. Thereafter, vehicle- and WSE-pretreated mice were randomly divided into four groups: saline, WSE, MDMA alone, WSE plus MDMA. Body temperature was recorded throughout treatment, and memory performance was assessed by a novel object recognition (NOR) task at the end of treatment. Thereafter, immunohistochemistry was performed to evaluate in the substantia nigra pars compacta (SNc) and striatum the levels of tyrosine hydroxylase (TH), as marker of dopaminergic degeneration, and of glial fibrillary acidic protein (GFAP) and TMEM119, as markers of astrogliosis or microgliosis, respectively. MDMA-treated mice showed a decrease in TH-positive neurons and fibers in the SNc and striatum respectively, an increase in gliosis and body temperature, and a decrease in NOR performance, irrespective of vehicle or WSE pretreatment. Acute WSE plus MDMA counteracted the modifications in TH-positive cells in SNc, GFAP-positive cells in striatum, TMEM in both areas and NOR performance, as compared to MDMA alone, while no differences were observed as compared to saline. Results indicate that WSE acutely administered in combination with MDMA, but not as pretreatment, protects mice against the noxious central effects of MDMA.

Stereotactic body radiotherapy with CyberKnife® System for low- and intermediate-risk prostate cancer: clinical outcomes and toxicities of CyPro Trial.

Simple summary: Stereotactic body radiotherapy (SBRT) of 35-36.25 Gy in five fractions with the CyberKnife System yields excellent control with low toxicity in low-intermediate-risk prostate cancer patients. We found no differences in biochemical control and overall survival in relation to dose. There were no significant differences in toxicity or quality of life between the two groups. Aims: Stereotactic body radiotherapy (SBRT) is an emerging therapeutic approach for low- and intermediate-risk prostate cancer. We present retrospective data on biochemical control, toxicity, and quality of life of CyPro Trial. Materials and methods: A total of 122 patients with low- and intermediate-risk prostate cancer were treated with the CyberKnife System at a dose of 35 Gy or 36.25 Gy in five fractions. Biochemical failure (BF)/biochemical disease-free survival (bDFS) was defined using the Phoenix method (nadir + 2 ng/ml). Acute/late rectal and urinary toxicities were assessed by the Radiation Therapy Oncology Group (RTOG) toxicity scale. Quality of life (QoL) was assessed by the European Organisation for Research and Treatment of Cancer (EORTC) QLQ C30 and PR25. International Erectile Function Index-5 (IIEF5) and International Prostate Symptom Score (IPSS) questionnaires were administered at baseline, every 3 months after treatment during the first years, and then at 24 months and 36 months. Results: The 1-, 2-, and 5-year DFS rates were 92.9%, 92.9%, and 92.3%, respectively, while the 1-, 2-, and 5-year bDFS rates were 100%, 100%, and 95.7%, respectively. With regard to risk groups or doses, no statistically significant differences were found in terms of DFS or bDFS. Grade 2 urinary toxicity was acute in 10% and delayed in 2% of patients. No Grade 3 acute and late urinary toxicity was observed. Grade 2 rectal toxicity was acute in 8% and late in 1% of patients. No Grade 3-4 acute and late rectal toxicity was observed. Grade 2 acute toxicity appeared higher in the high-dose group (20% in the 36.25-Gy group versus 3% in the 35-Gy group) but was not statistically significant. Conclusion: Our study confirms that SBRT of 35-36.25 Gy in five fractions with the CyberKnife System produces excellent control with low toxicity in patients with low-intermediate-risk prostate cancer. We found no dose-related differences in biochemical control and overall survival. Further confirmation of these results is awaited through the prospective phase of this study, which is still ongoing.

Dose-Tracking Software: A Retrospective Analysis of Dosimetric Data in CT Procedures.

ABSTRACT: The increasing use of ionizing radiation in healthcare is causing growing alarm about radiation protection of patients and the doses they receive during procedures. Radiation dose assessment for patients in radiodiagnostic procedures is the subject of interest in view of the recent Italian D.Lgs 31 July 2020, n. 101 (Decreto Legislativo 31 luglio 2020, n. 101) and one of its most important focuses is the prescription to provide patient exposure information as an integral part of the examination report. Dose monitoring systems are therefore essential for the collection of the dosimetric data. In order to analyse potential and critical issues of these software, different systems, adopted at the Antonio Cardarelli Hospital in Naples, were employed. Data extracted from the DoseWatch software (GE Healthcare) and Gray Detector (EL.CO. S.r.l. Healthcare Solutions, Italy) and relating to several protocols adopted for computed tomography (CT), were retrospectively analysed for the purpose of identifying critical issues in the data acquisition and recording phase, comparing with Italian nationwide diagnostic reference levels (DRLs), as provided for in regulatory provisions for radiation safety. Multiphase examinations were also included in this study. Once the distributions of volumetric CT Dose Index (CTDIvol) and dose-length product (DLP) were determined for each acquisition phase and total DLP (DLPtot) for each examination, the 25th, 50th and 75th percentiles were calculated for each distribution and then compared with the relevant Italian nationwide DRLs. In addition, to improve protocol optimization and dose reduction the magnitude of the CT acquisition settings chosen in each procedure was evaluated. In conclusion, these systems allow accurate analysis of radiation dose according to equipment and protocol over time. For the application of optimization measures, a constant use of the dose tracking software is required, which can be translated into actions on scan parameters and prospective data analysis.

D2R signaling in striatal spiny neurons modulates L-DOPA induced dyskinesia.

Degeneration of dopaminergic neurons leads to Parkinson's disease (PD), characterized by reduced levels of striatal dopamine (DA) and impaired voluntary movements. DA replacement is achieved by levodopa treatment which in long-term causes involuntary movements or dyskinesia. Dyskinesia is linked to the pulsatile activation of D1 receptors of the striatal medium spiny neurons (MSNs) forming the direct output pathway (dMSNs). The contribution of DA stimulation of D2R in MSNs of the indirect pathway (iMSNs) is less clear. Using the 6-hydroxydopamine model of PD, here we show that loss of DA-mediated inhibition of these neurons intensifies levodopa-induced dyskinesia (LID) leading to reprogramming of striatal gene expression. We propose that the motor impairments characteristic of PD and of its therapy are critically dependent on D2R-mediated iMSNs activity. D2R signaling not only filters inputs to the striatum but also indirectly regulates dMSNs mediated responses.

Effects of docosanyl ferulate, a constituent of Withania somnifera, on ethanol- and morphine-elicited conditioned place preference and ERK phosphorylation in the accumbens shell of CD1 mice.

BACKGROUND: Docosanyl ferulate (DF) is a behaviourally active GABAA receptor complex (GABAAR) agonist, recently isolated from the standardized methanolic extract of Withania somnifera Dunal (WSE) root. Previous studies have shown that WSE prevents both ethanol- and morphine-dependent acquisition and expression of conditioned place preference (CPP) and stimulation of dopamine release in the nucleus accumbens shell (AcbSh). AIMS: The study aimed at determining (a) whether DF contributes to WSE's ability to affect the acquisition and expression of ethanol- and morphine-elicited CPP and, given that phosphorylation of extracellular signal-regulated kinase (pERK) in the AcbSh is involved in associative learning and motivated behaviours, (b) whether WSE and DF may affect ethanol- and morphine-induced ERKs phosphorylation in the AcbSh. METHODS: In adult male CD1 mice, DF's effects on the acquisition and expression of ethanol- and morphine-elicited CPP were evaluated by a classical place conditioning paradigm, whereas the effects of WSE and DF on ethanol- and morphine-elicited pERK in the AcbSh were evaluated by immunohistochemistry. RESULTS AND CONCLUSIONS: The study shows that DF, differently from WSE, affects only the acquisition but not the expression of ethanol- and morphine-induced CPP. Moreover, the study shows that both WSE and DF can prevent ethanol- and morphine-elicited pERK expression in the AcbSh. Overall, these results highlight subtle but critical differences for the role of GABAARs in the mechanism by which WSE affects these ethanol- and morphine-dependent behavioural and molecular/cellular responses and support the suggestion of WSE and DF for the control of different components of drug addiction.

SBRT for Localized Prostate Cancer: CyberKnife vs. VMAT-FFF, a Dosimetric Study.

In recent years, stereotactic body radiation therapy (SBRT) has gained popularity among clinical methods for the treatment of medium and low risk prostate cancer (PCa), mainly as an alternative to surgery. The hypo-fractionated regimen allows the administration of high doses of radiation in a small number of fractions; such a fractionation is possible by exploiting the different intrinsic prostate radiosensitivity compared with the surrounding healthy tissues. In addition, SBRT treatment guaranteed a better quality of life compared with surgery, avoiding risks, aftermaths, and possible complications. At present, most stereotactic prostate treatments are performed with the CyberKnife (CK) system, which is an accelerator exclusively dedicated for stereotaxis and it is not widely spread in every radiotherapy centre like a classic linear accelerator (LINAC). To be fair, a stereotactic treatment is achievable also by using a LINAC through Volumetric Modulated Arc Therapy (VMAT), but some precautions must be taken. The aim of this work is to carry out a dosimetric comparison between these two methodologies. In order to pursue such a goal, two groups of patients were selected at Instituto Nazionale Tumori-IRCCS Fondazione G. Pascale: the first group consisting of ten patients previously treated with a SBRT performed with CK; the second one was composed of ten patients who received a hypo-fractionated VMAT treatment and replanned in VMAT-SBRT flattening filter free mode (FFF). The two SBRT techniques were rescaled at the same target coverage and compared by normal tissue sparing, dose distribution parameters and delivery time. All organs at risk (OAR) constraints were achieved by both platforms. CK exhibits higher performances in terms of dose delivery; nevertheless, the general satisfying dosimetric results and the significantly shorter delivery time make VMAT-FFF an attractive and reasonable alternative SBRT technique for the treatment of localized prostate cancer.

Reirradiation of Locally Recurrent Prostate Cancer with Cyberknife® System or Volumetric Modulated Arc Therapy (VMAT) and IGRT-Clarity®: Outcomes, Toxicities and Dosimetric Evaluation.

The management of prostate cancer recurrence following external beam radiotherapy is not defined yet. Stereotaxic body reirradiation therapy showed encouraging results for local and biochemical control. From April 2017 to December 2020, 29 patients with prostate cancer recurrence were collected, joining the retrospective studies CyPro (prot. 46/19 OSS) and CLARO (Prot. 19/20 OSS) trials. Patients received Cyberknife® treatment (17 pts) or alternatively VMAT (Volumetric Modulated Arc Technique) therapy by IGRT (Image-Guided Radiation Therapy)/Clarity® (12 pts). By comparing the reirradiation of two groups, urinary (GU), rectal (GI) toxicities, and biochemical control were investigated. Further, the two techniques were dosimetrically compared by rival plans. The VMAT-IGRT Clarity® treatments were replanned with an optimized template developed for prostate VMAT-SBRT in FFF mode keeping the same dose and fractionation scheduled for Cyberknife Group (30 Gy in 5 fx, at 80% isodose). In the CK group, 23% of patients experienced grade 2 acute GU, while 6% grade 2 acute GI. In the VMAT-Clarity® group, acute GU toxicity was recorded in 17%, while for 8% grade 2 late toxicity was recorded. The dosimetric analysis shows that the VMAT-FFF allows to deliver a biological equivalent dose to CK, with the advantage of reducing the likelihood of toxicities arising.

Characterization of Nasco grape pomace-loaded nutriosomes and their neuroprotective effects in the MPTP mouse model of Parkinson's disease.

Grape pomaces have recently received great attention for their richness in polyphenols, compounds known to exert anti-inflammatory and antioxidant effects. These pomaces, however, have low brain bioavailability when administered orally due to their extensive degradation in the gastrointestinal tract. To overcome this problem, Nasco pomace extract was incorporated into a novel nanovesicle system called nutriosomes, composed of phospholipids (S75) and water-soluble maltodextrin (Nutriose® FM06). Nutriosomes were small, homogeneously dispersed, had negative zeta potential, and were biocompatible with intestinal epithelial cells (Caco-2). Nasco pomace extract resulted rich in antioxidant polyphenols (gallic acid, catechin, epicatechin, procyanidin B2, and quercetin). To investigate the neuroprotective effect of Nasco pomace in the subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), Nasco nutriosomes or Nasco suspension was administered intragastrically and their neuroprotective effects were evaluated. Degeneration of nigro-striatal dopaminergic neurons induced by subacute MPTP treatment, the pathological hallmark of PD, was assessed through immunohistochemical evaluation of tyrosine hydroxylase (TH) in the caudate-putamen (CPu) and substantia nigra pars compacta (SNc), and the dopamine transporter (DAT) in CPu. Immunohistochemical analysis revealed that Nasco nutriosomes significantly prevented the reduction in TH- and DAT-positive fibres in CPu, and the number of TH-positive cells in SNc following subacute MPTP treatment, while Nasco suspension counteracted MPTP toxicity exclusively in SNc. Overall, these results highlight the therapeutic effects of Nasco pomace extract when administered in a nutriosome formulation in the subacute MPTP mouse model of PD and validate the effectiveness of the nutriosome preparation over suspension as an innovative nano-drug delivery system for in vivo administration.

Association between Novel Object Recognition/Spontaneous Alternation Behavior and Emission of Ultrasonic Vocalizations in Rats: Possible Relevance to the Study of Memory.

Rats emit ultrasonic vocalizations (USVs) in situations with emotional valence, and USVs have also been proposed as a marker for memories conditioned to those situations. This study investigated whether USV emissions can predict and/or be associated with the behavior of rats in tests that evaluate unconditioned memory. To this end, rats were subjected to "tickling", a procedure of heterospecific play that has emotional valence and elicits the emission of USVs, and afterwards evaluated in the novel object recognition test (NOR) and in the single trial continuous spontaneous alternation behavior (SAB) test in a Y maze. The number of 22-kHz USVs (aversive) and 50-kHz USVs (appetitive) emitted in response to tickling and during NOR and SAB tests were scored, and the correlations among them and with rats' behavior evaluated. Rats emitted 50-kHz USVs, but not 22-kHz USVs, during the NOR and SAB tests, and such calling behavior was not linked with the behavioral readouts indicative of memory function in either test. However, rats that prevalently emitted 22-kHz USVs in response to tickling displayed an impaired NOR performance. These findings suggest that measuring the emission of USVs could be of interest in studies of unconditioned memory, at least with regard to 22-kHz USVs.

Lack of drug- and cue-stimulated emissions of ultrasonic vocalizations in C57BL/6J mice repeatedly treated with amphetamine.

The emission of ultrasonic vocalizations (USVs) is thought to communicate the behavioral and emotional states elicited in rodents by social and non-social stimuli. On this basis, studies of psychopharmacology in rats are increasingly utilizing USVs as a behavioral marker to evaluate the effects of drugs on the emotional state. Conversely, very limited information is available as to whether psychoactive drugs influence USV emissions in mice. To provide new insights in this respect, we evaluated the emission of USVs in C57BL/6J mice subjected to repeated treatment with the dopaminergic psychostimulant of abuse amphetamine. Mice were first allowed to perform social contacts in dyads, and 2 days later they received amphetamine (1-4 mg/kg, i.p.) in a test cage (× 5 administrations) on alternate days. Seven days after treatment discontinuation, mice were re-exposed to the test cage to evaluate whether the presentation of drug-paired environmental cues elicited calling behavior, and thereafter received an amphetamine challenge. An additional group of animals received the dopamine receptor agonist apomorphine (1-4 mg/kg, i.p.), to further clarify the role of dopamine transmission in calling behavior of mice. C57BL/6J mice emitted USVs during social contacts, but did not significantly vocalize after amphetamine administration, in response to amphetamine-paired environmental cues, and after apomorphine administration. These results indicate that C57BL/6J mice may respond differently to social and pharmacological stimuli in terms of USV emissions, and may lay the foundation for future studies aimed at clarifying whether USVs may be a useful behavioral marker in studies of psychopharmacology in mice.

Neuroinflammation and L-dopa-induced abnormal involuntary movements in 6-hydroxydopamine-lesioned rat model of Parkinson's disease are counteracted by combined administration of a 5-HT1A/1B receptor agonist and A2A receptor antagonist.

Several lines of evidence have strongly implicated neuroinflammation in Parkinson's disease (PD) progression and l-dopa-induced dyskinesia. The present study investigated whether early subchronic pretreatment with the serotonin 5-HT1A/1B receptor agonist eltoprazine plus the adenosine A2A receptor antagonist preladenant counteracted l-dopa-induced abnormal involuntary movements (AIMs, index of dyskinesia), and neuroinflammation, in unilateral 6-hydroxydopamine(6-OHDA)-lesioned rat model of PD. The immunoreactivity of glial fibrillary acidic protein (GFAP), and the colocalization of ionized calcium binding adaptor molecule-1 (IBA-1), with interleukin (IL)-1ß, tumor-necrosis-factor-α (TNF-α) and IL-10 were evaluated in the denervated caudate-putamen (CPu) and substantia nigra pars-compacta (SNc). The combined subchronic pretreatment with l-dopa plus eltoprazine and preladenant reduced AIMs induced by acute l-dopa challenge in these rats and decreased GFAP and IBA-1 immunoreactivity induced by the drug in both CPu and SNc, with reduction in IL-1ß in IBA-1-positive cells in both CPu and SNc, and in TNF-α in IBA-1-positive cells in SNc. Moreover, a significant increase in IL-10 in IBA-1-positive cells was observed in SNc. Evaluation of immediate early-gene zif-268 (index of neuronal activation) after l-dopa challenge, showed an increase in its expression in denervated CPu of rats pretreated with l-dopa or l-dopa plus preladenant compared with vehicle, whereas rats pretreated with eltoprazine, with or without preladenant, had lower zif-268 expression. Finally, tyrosine hydroxylase and dopamine transporter examined to evaluate neurodegeneration, showed a significant equal decrease in all experimental groups. The present findings suggest that combination of l-dopa with eltoprazine and preladenant may be promising therapeutic strategy for delaying the onset of dyskinesia, preserving l-dopa efficacy and reducing neuroinflammation markers in nigrostriatal system of 6-OHDA-lesioned rats.