Cell Electrokinetic Fingerprint: A Novel Approach Based on Optically Induced Dielectrophoresis (ODEP) for In-Flow Identification of Single Cells.

A novel optically induced dielectrophoresis (ODEP) system that can operate under flow conditions is designed for automatic trapping of cells and subsequent induction of 2D multi-frequency cell trajectories. Like in a "ping-pong" match, two virtual electrode barriers operate in an alternate mode with varying frequencies of the input voltage. The so-derived cell motions are characterized via time-lapse microscopy, cell tracking, and state-of-the-art machine learning algorithms, like the wavelet scattering transform (WST). As a cell-electrokinetic fingerprint, the dynamic of variation of the cell displacements happening, over time, is quantified in response to different frequency values of the induced electric field. When tested on two biological scenarios in the cancer domain, the proposed approach discriminates cellular dielectric phenotypes obtained, respectively, at different early phases of drug-induced apoptosis in prostate cancer (PC3) cells and for differential expression of the lectine-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcript levels in human colorectal adenocarcinoma (DLD-1) cells. The results demonstrate increased discrimination of the proposed system and pose an additional basis for making ODEP-based assays addressing cancer heterogeneity for precision medicine and pharmacological research.

NeuriTES. Monitoring neurite changes through transfer entropy and semantic segmentation in bright-field time-lapse microscopy.

One of the most challenging frontiers in biological systems understanding is fluorescent label-free imaging. We present here the NeuriTES platform that revisits the standard paradigms of video analysis to detect unlabeled objects and adapt to the dynamic evolution of the phenomenon under observation. Object segmentation is reformulated using robust algorithms to assure regular cell detection and transfer entropy measures are used to study the inter-relationship among the parameters related to the evolving system. We applied the NeuriTES platform to the automatic analysis of neurites degeneration in presence of amyotrophic lateral sclerosis (ALS) and to the study of the effects of a chemotherapy drug on living prostate cancer cells (PC3) cultures. Control cells have been considered in both the two cases study. Accuracy values of 93% and of 92% are achieved, respectively. NeuriTES not only represents a tool for investigation in fluorescent label-free images but demonstrates to be adaptable to individual needs.

Lateral Acromioplasty has a Positive Impact on Rotator Cuff Repair in Patients with a Critical Shoulder Angle Greater than 35 Degrees.

BACKGROUND: A Critical Shoulder Angle (CSA), evaluated on plain radiographs, greater than 35° is considered predictive of rotator cuff tears. The present prospective comparative study aimed, firstly, to develop a formula to calculate the amount of acromion that should be resected performing a lateral acromioplasty and, secondly, verify whether lateral acromioplasty to reduce the CSA associated with arthroscopic cuff repair decreased the rate of recurrence of the tears, and impacted favorably on clinical postoperative outcomes. METHODS: Patients undergoing arthroscopic rotator cuff repair (RCR) for rotator cuff tears with a CSA greater than 35° were included in this study and divided into two groups, based on whether the CSA had been reduced by arthroscopic resection of the lateral portion of the acromion. A new mathematical formula was developed in order to quantify the amount of bone to be resected while performing the lateral acromioplasty. Patients with traumatic tears, previous surgery, osteoarthritis or plain radiographs, not classified as A1 according to Suter-Henninger, were excluded. Clinical and radiographic outcomes were assessed at a minimum of 2 years of follow-up considering the tear size. RESULTS: 289 patients were included in this study. Thirty-seven were lost to follow-up. Group A (Lateral acromioplasty) patients included: 38 small tears, 30 medium tears, 28 large tears and 22 massive tears; Group B (control group) was composed of 40 small tears, 30 medium tears, 30 large tears and 23 massive tears. The Constants Score value and retear Rate were, respectively, significant higher (p = 0.007 and p = 0.004) and lower (p = 0.029 and p = 0.028) in Group A, both in the Small-and Medium-size subgroups. No complications were outlined. The mediolateral width of the acromion was reduced, according to the preoperatively calculated measure. CONCLUSION: Arthroscopic lateral acromioplasty decreased the CSA within the favorable range (30°-35°) in all patients treated, resecting the amount of bone predicted by the mathematical formula. Lateral acromioplasty is a safe and reproducible technique which may prevent recurrence of rotator cuff tears in patients with small and medium lesions. LEVEL OF EVIDENCE: II.

Deciphering Cancer Cell Behavior From Motility and Shape Features: Peer Prediction and Dynamic Selection to Support Cancer Diagnosis and Therapy.

Cell motility varies according to intrinsic features and microenvironmental stimuli, being a signature of underlying biological phenomena. The heterogeneity in cell response, due to multilevel cell diversity especially relevant in cancer, poses a challenge in identifying the biological scenario from cell trajectories. We propose here a novel peer prediction strategy among cell trajectories, deciphering cell state (tumor vs. nontumor), tumor stage, and response to the anticancer drug etoposide, based on morphology and motility features, solving the strong heterogeneity of individual cell properties. The proposed approach first barcodes cell trajectories, then automatically selects the good ones for optimal model construction (good teacher and test sample selection), and finally extracts a collective response from the heterogeneous populations via cooperative learning approaches, discriminating with high accuracy prostate noncancer vs. cancer cells of high vs. low malignancy. Comparison with standard classification methods validates our approach, which therefore represents a promising tool for addressing clinically relevant issues in cancer diagnosis and therapy, e.g., detection of potentially metastatic cells and anticancer drug screening.

A Camera Sensors-Based System to Study Drug Effects On In Vitro Motility: The Case of PC-3 Prostate Cancer Cells.

Cell motility is the brilliant result of cell status and its interaction with close environments. Its detection is now possible, thanks to the synergy of high-resolution camera sensors, time-lapse microscopy devices, and dedicated software tools for video and data analysis. In this scenario, we formulated a novel paradigm in which we considered the individual cells as a sort of sensitive element of a sensor, which exploits the camera as a transducer returning the movement of the cell as an output signal. In this way, cell movement allows us to retrieve information about the chemical composition of the close environment. To optimally exploit this information, in this work, we introduce a new setting, in which a cell trajectory is divided into sub-tracks, each one characterized by a specific motion kind. Hence, we considered all the sub-tracks of the single-cell trajectory as the signals of a virtual array of cell motility-based sensors. The kinematics of each sub-track is quantified and used for a classification task. To investigate the potential of the proposed approach, we have compared the achieved performances with those obtained by using a single-trajectory paradigm with the scope to evaluate the chemotherapy treatment effects on prostate cancer cells. Novel pattern recognition algorithms have been applied to the descriptors extracted at a sub-track level by implementing features, as well as samples selection (a good teacher learning approach) for model construction. The experimental results have put in evidence that the performances are higher when a further cluster majority role has been considered, by emulating a sort of sensor fusion procedure. All of these results highlighted the high strength of the proposed approach, and straightforwardly prefigure its use in lab-on-chip or organ-on-chip applications, where the cell motility analysis can be massively applied using time-lapse microscopy images.

Learning Cancer-Related Drug Efficacy Exploiting Consensus in Coordinated Motility Within Cell Clusters.

OBJECTIVE: The ability of cells to collectively move is essential in various biological contexts including cancer metastasis. In this paper, we propose an automatic video analysis tool to correlate the cell movement inhibition with replication block induced by dose-dependent chemotherapy administration. METHODS: The novel approach combines individual and collective cell kinematic analysis performed over time-lapse microscopy video frames. Cells are first localized and tracked, and then kinematic descriptors are extracted for each track. Selective track identification is performed assuming diversified cell roles within the same cluster (spontaneously forming groups of cells), and finally individual results are grouped exploiting consensus of coordinated motility within cell clusters. RESULTS: Recognition performance of three different experimental conditions (no drug, 0.5-5 µM merged in the same condition, and 50 µM) reached an average accuracy value of 88% over 958 different tracks collected in 36 clusters of diverse dimensions in eight independent experiments. CONCLUSION: An extensive application of this methodology could give a different point of view of the cancer mechanisms.

Cooperative strategy for a dynamic ensemble of classification models in clinical applications: the case of MRI vertebral compression fractures.

PURPOSE: In clinical practice, the constructive consultation among experts improves the reliability of the diagnosis and leads to the definition of the treatment plan for the patient. Aggregation of the different opinions collected by many experts can be performed at the level of patient information, abnormality delineation, or final assessment. METHODS: In this study, we present a novel cooperative strategy that exploits the dynamic contribution of the classification models composing the ensemble to make the final class assignment. As a proof of concept, we applied the proposed approach to the assessment of malignant infiltration in 103 vertebral compression fractures in magnetic resonance images. RESULTS: The results obtained with repeated random subsampling and receiver operating characteristic analysis indicate that the cooperative system statistically improved ([Formula: see text]) the classification accuracy of individual modules as well as of that based on the manual segmentation of the fractures provided by the experts. CONCLUSIONS: The performances have been also compared with those obtained with those of standard ensemble classification algorithms showing superior results.

Computer-aided diagnosis of plus disease via measurement of vessel thickness in retinal fundus images of preterm infants.

Changes in the characteristics of retinal vessels such as width and tortuosity can be signs of the presence of several diseases such retinopathy of prematurity (ROP) and diabetic retinopathy. Plus disease is an indicator of ROP which requires treatment and is signified by an increase in posterior venular width. In this work, we present image processing techniques for the detection, segmentation, tracking, and measurement of the width of the major temporal arcade (MTA), which is the thickest venular branch in the retina. Several image processing techniques have been employed, including the use of Gabor filters to detect the MTA, morphological image processing to obtain its skeleton, Canny's method to detect and select MTA vessel-edge candidates, least-squares fitting to interpolate the MTA edges, and geometrical procedures to measure the width of the MTA. The results, obtained using 110 retinal fundus images of preterm infants, indicate a statistically highly significant difference in MTA width of normal cases as compared to cases with plus disease (p<0.01). The results provide good accuracy in computer-aided diagnosis (CAD) of plus disease with an area under the receiver operating characteristic curve of 0.76. The proposed methods may be used in CAD of plus disease and timely treatment of ROP in a clinical or teleophthalmological setting.

Analysis of structural similarity in mammograms for detection of bilateral asymmetry.

We hypothesize that quantification of structural similarity or dissimilarity between paired mammographic regions can be effective in detecting asymmetric signs of breast cancer. Bilateral masking procedures are applied for this purpose by using automatically detected anatomical landmarks. Changes in structural information of the extracted regions are investigated using spherical semivariogram descriptors and correlation-based structural similarity indices in the spatial and complex wavelet domains. The spatial distribution of grayscale values as well as of the magnitude and phase responses of multidirectional Gabor filters are used to represent the structure of mammographic density and of the directional components of breast tissue patterns, respectively. A total of 188 mammograms from the DDSM and mini-MIAS databases, consisting of 47 asymmetric cases and 47 normal cases, were analyzed. For the combined dataset of mammograms, areas under the receiver operating characteristic curves of 0.83, 0.77, and 0.87 were obtained, respectively, with linear discriminant analysis, the Bayesian classifier, and an artificial neural network with radial basis functions, using the features selected by stepwise logistic regression and leave-one-patient-out cross-validation. Two-view analysis provided accuracy up to 0.94, with sensitivity and specificity of 1 and 0.88, respectively.

Δ9-tetrahydrocannabinol prevents methamphetamine-induced neurotoxicity.

Methamphetamine (METH) is a potent psychostimulant with neurotoxic properties. Heavy use increases the activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrites, microglia stimulation, and induces hyperthermia and anorectic effects. Most METH recreational users also consume cannabis. Preclinical studies have shown that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 and CB2 receptor agonists exert neuroprotective effects on different models of cerebral damage. Here, we investigated the neuroprotective effect of Δ9-THC on METH-induced neurotoxicity by examining its ability to reduce astrocyte activation and nNOS overexpression in selected brain areas. Rats exposed to a METH neurotoxic regimen (4 × 10 mg/kg, 2 hours apart) were pre- or post-treated with Δ9-THC (1 or 3 mg/kg) and sacrificed 3 days after the last METH administration. Semi-quantitative immunohistochemistry was performed using antibodies against nNOS and Glial Fibrillary Acidic Protein (GFAP). Results showed that, as compared to corresponding controls (i) METH-induced nNOS overexpression in the caudate-putamen (CPu) was significantly attenuated by pre- and post-treatment with both doses of Δ9-THC (-19% and -28% for 1 mg/kg pre- and post-treated animals; -25% and -21% for 3 mg/kg pre- and post-treated animals); (ii) METH-induced GFAP-immunoreactivity (IR) was significantly reduced in the CPu by post-treatment with 1 mg/kg Δ9-THC1 (-50%) and by pre-treatment with 3 mg/kg Δ9-THC (-53%); (iii) METH-induced GFAP-IR was significantly decreased in the prefrontal cortex (PFC) by pre- and post-treatment with both doses of Δ9-THC (-34% and -47% for 1 mg/kg pre- and post-treated animals; -37% and -29% for 3 mg/kg pre- and post-treated animals). The cannabinoid CB1 receptor antagonist SR141716A attenuated METH-induced nNOS overexpression in the CPu, but failed to counteract the Δ9-THC-mediated reduction of METH-induced GFAP-IR both in the PFC and CPu. Our results indicate that Δ9-THC reduces METH-induced brain damage via inhibition of nNOS expression and astrocyte activation through CB1-dependent and independent mechanisms, respectively.

Development and validation of a fully automated system for detection and diagnosis of mammographic lesions.

We present a comprehensive and fully automated system for computer-aided detection and diagnosis of masses in mammograms. Novel methods for detection include: selection of suspicious focal areas based on analysis of the gradient vector field, rejection of oriented components of breast tissue using multidirectional Gabor filtering, and use of differential features for rejection of false positives (FPs) via clustering of the surrounding fibroglandular tissue. The diagnosis step is based on extraction of contour-independent features for characterization of lesions as benign or malignant from automatically detected circular and annular regions. A new unified 3D free-response receiver operating characteristic framework is introduced for global analysis of two binary categorization problems in cascade. In total, 3,080 suspicious focal areas were extracted from a set of 156 full-field digital mammograms, including 26 malignant tumors, 120 benign lesions, and 18 normal mammograms. The proposed system detected and diagnosed malignant tumors with a sensitivity of 0.96, 0.92, and 0.88 at, respectively, 1.83, 0.46, and 0.45 FPs/image, with two stages of stepwise logistic regression for selection of features, a cascade of Fisher linear discriminant analysis and an artificial neural network with radial basis functions, and leave-one-patient-out cross-validation.

Measures of radial correlation and trend for classification of breast masses in mammograms.

In this paper, a novel approach for classification of breast masses is presented that quantifies the texture of masses without relying on accurate extraction of their contours. Two novel feature descriptors based on 2D extensions of the reverse arrangement (RA) and Mantel's tests were designed for this purpose. Measures of radial correlation and radial trend were extracted from the original gray-scale values as well as from the Gabor magnitude response of 146 regions of interest, including 120 benign masses and 26 malignant tumors. Four classifiers, Fisher-linear discriminant analysis, Bayesian, support vector machine, and an artificial neural network based on radial basis functions (ANN-RBF), were employed to predict the diagnosis, using stepwise logistic regression for feature selection and the leave-one-patient-out method for cross-validation. The ANN-RBF resulted in an area under the receiver operating characteristic curve of 0.93. The experimental results show the effectiveness of the proposed approach.

Automatic detection of the nipple in screen-film and full-field digital mammograms using a novel Hessian-based method.

Automatic detection of the nipple in mammograms is an important step in computerized systems that combine multiview information for accurate detection and diagnosis of breast cancer. Locating the nipple is a difficult task owing to variations in image quality, presence of noise, and distortion and displacement of the breast tissue due to compression. In this work, we propose a novel Hessian-based method to locate automatically the nipple in screen-film and full-field digital mammograms (FFDMs). The method includes detection of a plausible nipple/retroareolar area in a mammogram using geometrical constraints, analysis of the gradient vector field by mean and Gaussian curvature measurements, and local shape-based conditions. The proposed procedure was tested on 566 mammographic images consisting of 372 randomly selected scanned films from two public databases (mini-MIAS and DDSM), and 194 digital mammograms acquired with a GE Senographe 2000D FFDM system. A radiologist independently marked the centers of the nipples for evaluation of the results. The average error obtained was 6.7 mm (22 pixels) with reference to the center of the nipple as identified by the radiologist. Only two out of the 566 detected nipples (0.35 %) had an error larger than 50 mm. The method was also directly compared with two other techniques for the detection of the nipple. The results indicate that the proposed method outperforms other algorithms presented in the literature and can be used to identify accurately the nipple on various types of mammographic images.

Characterization of COR627 and COR628, two novel positive allosteric modulators of the GABA(B) receptor.

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.

Evaluation of amphetamine-induced hyperlocomotion and catalepsy following long-acting risperidone administration in rats.

It has been proposed that long-acting risperidone could provide a constant antipsychotic efficacy associated with a reduced liability to induce extra-pyramidal symptoms. To ascertain this hypothesis, antagonism of amphetamine-induced hyperlocomotion and catalepsy were analyzed in rats for a period of 1-6 weeks following long-acting risperidone (20-60 mg/kg) injection. Long-acting risperidone reduced amphetamine-induced hyperlocomotion after 2-5 weeks from drug injection, without producing significant extra-pyramidal symptoms. Following the administration of long-acting risperidone a constant ability to antagonize amphetamine-induced hyperlocomotion was observed during the day, but not when the antipsychotic was chronically administered using a short-acting formulation. The pre-clinical results confirmed that long-acting risperidone may represent an advance in antipsychotic therapy.

Delta-9-tetrahydrocannabinol differently affects striatal c-Fos expression following haloperidol or clozapine administration.

It was previously shown that haloperidol, but not clozapine, induced intense rat catalepsy when co-administered with delta-9-tetrahydrocannabinol. The present study investigated whether similar alterations could be observed on striatal c-Fos immunoreactivity after administration of the same drug combinations. Western Blot and immunocytochemistry stereological analyses indicated that delta-9-tetrahydrocannabinol (0.5 mg/kg) increased striatal c-Fos immunoreactivity induced by haloperidol (0.1 mg/kg). Conversely, no significant alterations of striatal c-Fos immunoreactivity were observed after injections of clozapine (10 mg/kg)+vehicle, clozapine+delta-9-tetrahydrocannabinol or vehicle+delta-9-tetrahydrocannabinol. The present results indicate that the behavioral effects induced by delta-9-tetrahydrocannabinol in haloperidol- and clozapine-treated rats are associated with different striatal c-Fos expressions.

Blockade of neurotensin receptors affects differently hypo-locomotion and catalepsy induced by haloperidol in mice.

Antipsychotic drug treatment increases neurotensin (NT) neurotransmission, and the exogenous administration of NT produces antipsychotic-like effects in rodents. In order to investigate whether "endogenous" NT may act as a natural occurring antipsychotic or may mediate antipsychotic drug activity, the effects of the selective NT receptor antagonists SR 48692 and SR 142948A were analyzed in different behavioural tests of locomotor activity using vehicle, amphetamine, or haloperidol in mice. SR 48692 (0.1-1 mg/kg, i.p.) and SR 142948A (0.03-0.1 mg/kg, i.p.) failed to affect mouse spontaneous locomotor activity and amphetamine-induced (2.5 mg/kg, i.p.) hyper-locomotion. However, SR 48692 (0.1 and 0.3 mg/kg, i.p.) and SR 142948A (0.03 and 0.05 mg/kg, i.p.) significantly alleviated the reduction of locomotor activity elicited by haloperidol (0.01 and 0.04 mg/kg, s.c.) in vehicle- or amphetamine-treated mice. Finally, SR 48692 (0.3 mg/kg, i.p.) and SR 142948A (0.05 and 0.1 mg/kg, i.p.) increased mouse catalepsy produced by haloperidol (0.3 mg/kg, s.c.). The present results indicate that while endogenous NT is not involved in the modulation of either mouse spontaneous locomotor activity or amphetamine-induced hyper-locomotion, it might act by enhancing the therapeutic effects of haloperidol and by attenuating the extrapyramidal side effects elicited by this antipsychotic.

Ritanserin counteracts both rat vacuous chewing movements and nigro-striatal tyrosine hydroxylase-immunostaining alterations induced by haloperidol.

The effect of subchronic co-administration of ritanserin (1.5 mg/kg, i.p., twice a day) and haloperidol (1 mg/kg, i.p., twice a day) on rat vacuous chewing movements and on tyrosine hydroxylase-immunostaining was investigated. Ritanserin significantly reduced rat vacuous chewing movements observed following 2, 3 and 4 weeks of haloperidol administration and after 5 days of haloperidol withdrawal. Furthermore, ritanserin prevented the reduction of striatal tyrosine hydroxylase-immunostaining and the shrinkage of nigral dopaminergic cell bodies induced by haloperidol. The present results indicate that ritanserin may possess protective properties on both dopaminergic nigro-striatal neuron alterations and vacuous chewing movements induced by haloperidol, and provide further evidence indicating a possible association between these two haloperidol-induced effects.

Haloperidol versus risperidone on rat "early onset" vacuous chewing.

Similarly to acute rat catalepsy, "early onset" vacuous chewing movements (VCMs) induced by subchronic treatment with antipsychotic have recently been proposed as a model of human extrapyramidal symptoms. In the present study, the propensities of haloperidol and risperidone in inducing rat "early onset" VCMs were compared using doses of the two antipsychotics that acutely induce similar catalepsy. Comparable rat catalepsy states were observed when the effects produced by 0.1, 0.5, and 1mg/kg of haloperidol were compared with those induced by 1, 4, and 10mg/kg of risperidone, respectively. These doses of the two antipsychotics were then administered twice a day for 4 weeks and VCMs scored after 12h, 5 days, or 3 weeks of drug withdrawal. Among the haloperidol-treated groups, only those rats injected with 0.5 and 1mg/kg showed high levels of VCMs after 12h and 5 days of drug withdrawal when compared to vehicle-treated rats, while basal levels of VCMs were reached after 3 weeks from the last injection. High VCMs levels were observed in risperidone-treated rats only at the dose of 10mg/kg and after 12h of drug withdrawal, but not after 5 days or 3 weeks. The present results indicated that haloperidol possessed a much higher propensity to induce rat "early onset" VCMs than risperidone.

The cerebellar GABAA alpha6 subunit is differentially modulated by chronic ethanol exposure in normal (R100R) and mutated (Q100Q) sNP rats.

Sardinian alcohol non-preferring (sNP) rats carry a point mutation (R100Q) in the cerebellar expressed GABAA receptor alpha6 subunit gene, leading to a higher sensitivity to ethanol and diazepam. The role of the alpha6 subunit gene cluster in the ethanol non-preferring phenotype was here investigated by measuring the levels of alpha1, alpha6 and gamma2 peptide in the cerebellum of normal (RR) and mutated (QQ) sNP rats after 2 weeks of chronic ethanol administration. Western blot analysis revealed that the alpha6 subunit is increased in RR sNP rats after chronic ethanol exposure (25.44%+/-8.69 versus control), while it remained unchanged in mutated QQ sNP rats. Interestingly, chronic ethanol administration decreased alpha1 peptide levels in the cerebellum of both rat lines to a similar extent (30.99%+/-6.74 and 27.12%+/-9.83 in RR and QQ rats, respectively), while gamma2 peptide levels remained unchanged. To further correlate the genetic and biochemical difference of the normal and mutated sNP rats with their aversive phenotype, we exposed sNP rats to a protocol of acquisition and maintenance of ethanol drinking. QQ sNP rats drank less ethanol than RR rats during the acquisition phase, but such difference was lost during the maintenance phase. These data may contribute to elucidating the mechanisms of alcohol avoidance in rat lines selected for this behavior when exposed to ethanol solution.