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Our objective was to evaluate the effect of a multispecies fungal extract (MFE) on growth performance, apparent total tract digestibility (ATTD), fermentation characteristics, and rumen microbiome composition of beef cattle fed forage-based diets. For experiment 1, ruminally cannulated Angus × SimAngus cows (n = 4; body weight [BW] = 569 ± 21 kg) were used in a randomized crossover design with two 21-d study periods and a 23-d washout period to evaluate the effect of dietary inclusion of an MFE on in situ digestion, ruminal fermentation, and the composition of the rumen microbiome. Treatments consisted of a forage-based diet with or without the inclusion of a MFE. Rumen samples were collected on days 5, 10, and 20. Experiment 2 evaluated different inclusion rates of the MFE in a randomized complete block design using Angus × SimAngus-crossbred steers (n = 80; BW = 370 ± 44 kg). Steers were blocked by BW and randomly assigned to one of four treatments (2 pens/treatment): diet with no MFE, 0.02%, 0.04%, and 0.08% of the MFE (dry matter [DM] basis). Steers were fed a forage-based diet for 122 d. Subsets of 10 steers/treatment were randomly selected for the determination of ATTD on d 20, 40, and 60. All data were analyzed using the MIXED procedure of SAS. In exp 1, adding the MFE to the diet tended to increase the ruminal disappearance rate of the DM on day 10 (P = 0.06). No interactions or treatment effects were observed for the short-chain fatty acid profile of the rumen fluid (P ≥ 0.13). Metagenomic analysis of the rumen microbiome showed an MFE × d interaction for the Fibrobacter genus (P = 0.01), which on day 20 was less abundant in the rumen of cows fed the MFE. In exp 2, steers supplemented with 0.04% of MFE had a lower average daily gain and were lighter at the end of the experiment (cubic, P ≤ 0.04) compared to steers supplemented with 0.02% MFE. Steers fed the diet with 0.02% of MFE had the greatest gain-to-feed ratio among the MFE-supplemented groups (cubic, P < 0.01). Dietary inclusion of the MFE increased neutral detergent fiber digestibility (linear, P = 0.05). Steers supplemented with 0.04% of MFE had the greatest acid detergent fiber digestibility among treatments (quadratic, P = 0.03). Collectively, results showed that ruminal disappearance rate and digestibility of forage-based diets increased due to MFE supplementation, but did not translate into growth performance improvements or beneficially alter rumen fermentation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Ongoing global research actions seek to comprehensively understand the adverse impact of stress and anxiety on the physical and mental health of both human beings and animals. Niaprazine (NIA) is a chemical compound that belongs to the class of piperazine derivatives. This compound has recently gained renewed attention due to its potential therapeutic properties for treating certain conditions such as anxiety. Despite its potential benefits, the behavioral effects of NIA have not been thoroughly investigated. This study aimed to examine NIA's potential as an anti-anxiety and anti-stress agent. After administering either vehicle or NIA in their drinking water to mice for 14 days, we conducted behavioral analyses using the Marble Burying Test and the Elevated Plus Maze test. NIA-treated mice spend more time in the open arms and bury fewer marbles. Moreover, a stability study confirmed the linear relationship between NIA concentration and its response across concentrations encompassing the NIA mother solution and the NIA solutions administered to mice. Also, a preliminary synaptic toxicity analysis showed no direct damage to cortical nerve endings. Here, we show that NIA can modulate anxiety-related behaviors without significantly impacting exploratory activity or adverse effects. Our work describes new findings that contribute to the research on safer and more tolerable anxiety management options.

One hundred and twenty-one Angus × SimAngus-crossbred steers (body weight ( BW ) = 159 ± 22 kg) were used to evaluate the effects of different grain inclusion ( GI ) rates in diets provided to early-weaned calves and steroidal implants ( SI ) utilization on growth performance and carcass characteristics, particularly intramuscular fat deposition, of beef steers. The experiment was conducted as a randomized complete block design with a 2 × 2 factorial arrangement of treatments, consisting of two GI rates (35% vs. 58%, dry matter ( DM ) basis), each one associated or not to steroidal implant utilization (no implants vs. 80 mg trenbolone acetate ( TA ) + 16 mg estradiol followed by 120 mg TA + 24 mg of estradiol). After being early-weaned (124 ± 14 d of age), steers were offered an average of 4.5 kg/d (DM basis) of a concentrate-based diet with a greater or lesser GI rate for 60 d. After being fed a concentrate-based diet with different GI rates for 60 d, steers were fed a common backgrounding diet for 56 d and subsequently fed a common high-grain diet until harvested at a constant final BW (620 kg). Steers were not implanted until the beginning of the backgrounding phase and then re-implanted when initiating the finishing phase. Data were analyzed using PROC MIXED in SAS. There were no GI × SI interactions ( P  ≥ 0.62) for any of the growth performance parameters throughout the experimental period. Implanted steers tended to have a greater average daily gain ( P  = 0.10) during the finishing phase than nonimplanted steers. For the 12th rib fat thickness and yield grade ( YG ), a GI × SI interaction ( P  = 0.03) and a tendency for a GI × SI interaction ( P  = 0.10) was detected, respectively. Nonimplanted steers fed diets with greater GI rates presented the greatest 12th rib fat thickness and tended to have the greatest YG among treatments. No other interactions ( P  ≥ 0.33) were observed for the hot carcass weight, Longissimus muscle ( LM ) area, quality grade, marbling score, and kidney-pelvic-heart fat content. Steers fed diets with lesser GI rates tended to have a greater LM area than steers fed diets with greater GI rates ( P  = 0.10). Results from this experiment indicate that varying GI rates in diets provided to early-weaned calves and subsequent implantation with steroidal hormones did not affect marbling deposition., Competing Interests: The authors declare no real or perceived conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science.)

To evaluate the effect of supplementing beef cattle with a ruminal probiotic consisting of native rumen microbes (NRM; Chordicoccus furentiruminis, Prevotella albensis, and Succinivibrio dextrinosolvens) on methane (CH4) emissions, growth performance, carcass characteristics, and plasma metabolites, Angus × SimAngus-crossbred steers (n = 32; 8 per pen) and heifers (n = 48; 12 per pen) with an initial body weight (BW) of 353 ± 64 kg were used in randomized complete block design. Cattle were blocked by sex and BW and randomly assigned to 1 of 2 treatments (2 pens per treatment). Treatments consisted of diets offered for ad libitum intake with (NRM) or without (CON) the inclusion of the ruminal probiotic. Cattle were fed a growing diet for 49 d followed by a ground corn-based diet for 124 ± 27 d until reaching the targeted final BW (635 kg for steers and 590 kg for heifers). Methane emissions were estimated using the GreenFeed system (n = 12 per treatment) prior to trial commencement (baseline; period 1), and on three (2, 3, and 4), and two (5 and 6) different sampling periods throughout the growing and finishing stage, respectively. All data were analyzed using the PROC MIXED procedure of SAS. For CH4 production (g/d), there was a tendency for an NRM supplementation × period interaction (P = 0.07) where cattle-fed diets with NRM had lower production of methane in periods 3 and 4. Including NRM in the diet decreased CH4 yield (g/kg of dry matter intake (DMI)) by 20%. For CH4 emission intensity (g/kg of average daily gain (ADG)), an interaction (P < 0.01) of NRM supplementation × period occurred. In periods 2 and 3, cattle-fed diets with NRM inclusion had lower CH4 emission intensity than CON cattle. During the 84-d period when all cattle were still on the finishing diet, feeding NRM increased (P = 0.02) ADG and tended to increase (P = 0.10) DMI. At the end of the 84-d period, cattle-fed NRM tended to be heavier (P = 0.06) than CON cattle. Cattle supplemented with NRM required less (P = 0.04) days on feed to reach the targeted final BW. No differences (P ≤ 0.11) were detected for gain-to-feed ratio and carcass characteristics. Cattle-fed NRM had greater abundance of uncultured rumen bacteria that may improve rumen digestion when fed a high grain diet and potentially promote the reduction of enteric CH4 production. Results from this study suggest that daily administration of NRM may be a strategy to mitigate methanogenesis and improve the growth performance of beef cattle., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We evaluate the effect of the weaning strategy (WS; early, 130 ± 21 d vs. normal, 187 ± 20 d) and backgrounding management (BGM) on growth, carcass characteristics, and relative mRNA expression in the longissimus muscle (LM) of beef steers. One hundred and twenty Angus × SimAngus-crossbred steers (body weight (BW) = 130 ± 11.2 kg) were used in a randomized complete block design. Steers, blocked by age and BW, were randomly assigned to one treatment (2 × 2 factorial). Treatments consisted of early weaned (EW) or normal weaned (NW) steers subsequently backgrounded (BG) on either a forage-based (FB) or concentrates-based (CB) diet. The EW steers (d 0) were ad libitum-fed a grain-based diet for 49 d until nursing calves were NW. Steers were ad libitum-fed either a FB diet for 214 d or a CB diet for 95 d afterward. Steers were finished on a high-grain diet until harvested at an estimated constant 12th-rib fat thickness (1.5 cm). Expression of mRNA in the LM was measured over time. Data were analyzed using PROC MIXED in SAS. The EW steers were heavier (P ˂ 0.01) at the beginning of the backgrounding and finishing period. When the finishing phase began, steers FB were heavier (P ˂ 0.01) than CB steers. There was a tendency for a WS × BGM interaction (P = 0.08) for final BW, where NW-FB steers were heavier than the steers on the other three treatments, which do not differ between them. In the finishing phase, steers BG on a FB diet had greater dry matter intake and average daily gain, but lower gain-to-feed ratio (P ˂ 0.01). There was a WS × BGM interaction (P = 0.03) for days on feed (DOF) in the finishing diet, where backgrounding steers in a FB diet decreased DOF required to reach the harvesting target among EW steers, but not within NW steers. No interactions or treatment effects (P ≥ 0.17) were detected for marbling score (MS). For ZFP423, EW steers showed a greater mRNA expression on d 112 and a lower expression on d 255 than NW steers (P ˂ 0.01). In d 57, steers BG on a CB diet presented a greater delta-like homolog 1 mRNA expression than steers BG on a FB diet, whereas in d 255, this was inverted (P ˂ 0.01). For CCAAT/enhancer binding protein D (C/EBPD) mRNA expression, a tendency for a WS × BGM interaction was observed (P = 0.06), where a greater expression of C/EBPD was observed in steers BG on a FB diet among EW steers, but not within NW steers. In this study, early grain feeding followed by different BGM does not support MS improvements of beef carcasses., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sixty Angus × SimAngus-crossbred steers (body weight [BW] 279 ± 16 kg) were used to evaluate the effect of calcium salts of palm oil inclusion (CPO) and the amount of feed offered (AFO) on plasma glucose-dependent insulinotropic polypeptide (GIP) concentration and its association with energy metabolism and marbling score (MS) in feedlot steers. Steers were blocked by BW and gain to feed (G:F) and randomly assigned to individual feedlot pens. Treatments (2 × 2 factorial) consisted of ad libitum-fed steers without (ANF) or with (AWF) the inclusion of CPO or restricted-fed steers (85% of the ad libitum intake of ANF) without (RNF) or with the inclusion of CPO (RWF). After weaning, steers were adapted to individual pens and fed a corn silage-based diet for 30 d and subsequently placed in a ground corn (GC)-based diet. Diets were given ad libitum or at 85% of the ANF intake and with or without CPO. After 59 d on the finishing diet, all steers had ad libitum access to the finishing diet until harvest. Measurements of CO2 emission and O2 consumption to estimate respiratory quotient (RQ) were taken (n = 9/treatment). Correlations between plasma GIP and insulin concentrations and RQ were analyzed. A linear regression was performed to evaluate the association of plasma GIP and MS. All data were analyzed using the PROC MIXED procedure of SAS. During the first 103 d of the trial, there were AFO × CPO interactions (P ≤ 0.01) for BW, dry matter intake (DMI), average daily gain (ADG), and net energy for maintenance (NEm) intake. Ad libitum-fed steers without CPO presented the greatest DMI among dietary treatments and had greater BW and ADG compared with steers in the RWF and RNF treatments. After all steers had ad libitum access to dietary treatments, steers that were previously restricted showed a 30% and 19% increase (P ≤ 0.01) in ADG and G:F, respectively. There was a three-way interaction time × CPO × AFO (P = 0.04) for plasma GIP concentration. There was no correlation (P = 0.96) of GIP with RQ, whereas insulin demonstrated marginal significance for a positive (P = 0.07) and negative (P = 0.08) correlation with plasma GIP and RQ, respectively. There was no association (P = 0.30) between GIP and MS. These data indicate that GIP secretion results from an interaction between CPO and energy intake depending on the time relative to feed intake that GIP might indirectly regulate energy metabolism through insulin secretion, and that GIP does not appear to be associated with MS., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

One-hundred twenty crossbreed steers (initial body weight (BW) 566 ± 42 kg) were used to evaluate the interaction of protein source (PS) and nonroughage NDF content (NRFC) in finishing diets of feedlot cattle fed free-choice hay on performance and carcass characteristics. Steers were stratified by BW and randomly assigned to 8 pens (2 × 2 factorial) and fed for 104 ± 10 d. Four dietary treatments were investigated: (1) distillers' dry grains with solubles (DDGS) and a low NRFC (DLF), (2) DDGS and a high NRFC (DHF), (3) soybean meal (SBM) and a low NRFC (SLF), (4) SBM and a high NRFC (SHF). Free-choice grass hay and concentrates were offered in a different bunk. Data were analyzed as a randomized complete block design. Do to the confounded effect of PS and protein intake, a linear regression was used to evaluate the effect of protein intake in growth performance. For gain to feed ratio (G:F) an interaction tended to occur ( P = 0.10) between PS and NRFC. Steers on the DHF treatment had a lower G:F compared with SLF and SHF. Feeding SBM increased ( P = 0.05) final BW, tended to increase ( P = 0.06) average daily gain (ADG), and decreased ( P = 0.05) hay intake (HI) compared with steers fed DDGS. There was a positive association ( P ˂ 0.01) of crude protein intake with ADG and FBW. Dietary NRFC did not change ( P ≥ 0.3) final BW, ADG, DMI, and HI. Protein source did not affect ( P ≥ 0.16) hot carcass weight (HCW), longissimus muscle (LM) area, dressing, 12th rib fat thickness, or marbling score (MS). No differences were detected between NRFC for dressing, HCW, LM area, or MS ( P ≥ 0.18); but diets with greater NRFC decreased ( P = 0.03) the 12th rib fat thickness. Steers in the SHF treatment presented the lesser kidney-pelvic-heart fat compared with the remaining treatments (PS × NRFC interaction, P = 0.04). Soybean meal inclusion/increase in protein intake improved growth performance of feedlot steers compared with DDGS, despite protein intake meet the protein requirement. Increasing the NRFC did not affect growth or HI but decreased feed efficiency of steers fed DDGS., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The objective was to evaluate the effect of adding an exogenous glucoamylase (GA) enzyme from the fungus Trichoderma reesei on in vitro fermentation, growth performance, and carcass characteristics of feedlot steers fed a dry-rolled corn (DRC)-based diet. Experiment 1 evaluated three levels of added enzyme (0, 0.24, and 0.72 GA enzyme units) and two corn particle sizes (CPS; 2 and 4 mm) in a factorial arrangement using a 7 h in vitro batch culture fermentation. Addition of GA increased ( P < 0.01) in vitro dry matter disappearance by 13% and decreased final pH ( P < 0.01). Molar proportion of propionate increased with GA inclusion ( P < 0.01). A smaller CPS increased ( P < 0.01) in vitro dry matter disappearance and total volatile fatty acid and decreased final pH ( P < 0.01). A smaller CPS also decreased ( P < 0.01) the molar proportion of acetate and increased ( P < 0.01) the molar proportion of butyrate. In experiment 2, Angus × Simmental steers ( n = 105; initial body weight [BW] = 329 ± 38 kg) were used to evaluate the inclusion of an exogenous GA on growth performance and carcass characteristics. Steers were fed a basal diet consisting of 60% DRC, 17.5% modified distillers grains with solubles, 12.5% corn silage, and 10% dry supplement on a dry matter basis for 136 d. Steers were blocked by weight and allotted to pens. Pens were randomly assigned to one of three treatments (5 pens/treatment): diet with no GA (CON), low inclusion of GA (122 enzyme units/kg dry matter [DM]; LGA), or high inclusion of GA (183 enzyme units/kg DM; HGA). Inclusion of GA did not affect ( P ≥ 0.23) final BW, dry matter intake (DMI), or average daily gain (ADG) for the 136-d feeding period. Feed conversion was affected ( P = 0.02) by treatment with steers fed HGA having ~8% greater G:F compared with LGA and CON. Treatment did not affect ( P = 0.32) fecal starch. Inclusion of GA did not affect ( P ≥ 0.19) carcass traits including hot carcass weight, 12th rib fat thickness, yield grade, longissimus muscle area, or marbling score. Overall, results suggest inclusion of exogenous GA enzyme increased in vitro dry matter disappearance in batch culture and improved feed conversion in steers fed 183 enzyme units/kg DM during the finishing phase., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [ 3 H]D-aspartate ([ 3 H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH 2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [ 3 H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cisani, Olivero, Usai, Van Camp, Maccari, Morley-Fletcher and Pittaluga.)

It is well-known that the health properties attributed to several fruits, herbs, seeds and their processed foods/beverages are due to an important group of natural polyphenols classified as hydrolysable tannins (HT) named ellagitannins (ETs), that encompass both one or more gallic acid (GA) units and one or more hexahydroxydiphenoic acid (HHDP) units, ester-connected with a sugar residue. In vivo, ETs are rather not absorbed and in gastrointestinal tract (GIT), they are hydrolysed providing mainly ellagic acid (EA). Due to its trivial water-solubility, first pass effect, metabolism in GIT, or irreversible binding to cellular DNA and proteins, EA has a very low bioavailability. Some authors are studying methods to increase EA water-solubility and thus to improve its bioavailability. At the same, EA metabolism to urolithins (UROs), whose concentration and activity is inter-individual and intra-individual dependent, is still under study and not completely elucidate. Numerous in vitro and in vivo studies have been carried out to define the molecular and cellular events underlying the beneficial effects that this compound and its metabolites exert in pathological conditions. The anti-inflammatory and the antioxidant properties of EA attracted the interest of researchers for its potential health benefits in humans, including anti-cancer, anti-diabetes activities and cardio-protection. Nevertheless, lately the attention paid to EA is focusing on its potential protective action towards several neurodegenerative disorders. Thus, EA is investigated as a potential "lead compound" endowed with multi-target pharmacological properties on CNS. Since the identification of the pharmacophore(s) responsible for both health benefits and collateral effects of this compound is crucial in drug discovery, this review aims to provide an all-round updated analysis of the literature concerning EA involvement in several CNS disorders, hoping that such information will be useful to researchers involved in multi-target drug design for CNS., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

This research takes place in the context of an Alcotra Italy-France trans-frontier project called FINNOVER, which includes among its objectives the "green" innovation of agro-industrial chains. Bud-derivatives are a category of natural products produced macerating meristematic tissues of trees and plants. They are quite expensive compared to other botanicals, since the collection period of their raw materials is extremely limited over the time. Pulsed Ultrasound-Assisted Extraction has been employed to extract further valuable material from the buds by-products remaining after the production of Castanea sativa Glyceric Macerates. UV-Visible spectra coupled with chemometrics were employed, as untargeted phytochemical fingerprints, to quickly screen the best experimental conditions of extraction: a duty cycle of 80%, an extraction time of 15 min and a solvent/ratio of 1/10. Targeted phytochemical fingerprints by HPLC have been used to identify and quantify the main bioactive compounds of the most promising marcs extract comparing it with the corresponding commercial Castanea sativa Glyceric Macerate. An innovative extraction and re-use strategy to obtain value-added products from botanicals by-products was developed in alternative to incineration or composting. It was applied to Castanea sativa buds production as case study, but it could be analogously applied for other herbal preparations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This review focuses on a specific interaction occurring between the nicotinic cholinergic receptors (nAChRs) and the glutamatergic receptors (GluRs) at the nerve endings level. We have employed synaptosomes in superfusion and supplemented and integrated our findings with data obtained using techniques from molecular biology and immuno-cytochemistry, and the assessment of receptor trafficking. In particular, we characterize the following: (1) the direct and unequivocal localization of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) glutamatergic receptors on specific nerve terminals, (2) their pharmacological characterization and functional co-localization with nAChRs on the same nerve endings, and (3) the existence of synergistic or antagonistic interactions among them. Indeed, in the rat nucleus accumbens (NAc), the function of some AMPA and NMDA receptors present on the dopaminergic and glutamatergic nerve terminals can be regulated negatively or positively in response to a brief activation of nAChRs. This effect occurs rapidly and involves the trafficking of AMPA and NMDA receptors. The event takes place also at very low concentrations of nicotine and involves the activation of several nAChRs subtypes. This dynamic control by cholinergic nicotinic system of glutamatergic NMDA and AMPA receptors might therefore represent an important neuronal presynaptic adaptation associated with nicotine administration. The understanding of the role of these nicotine-induced functional changes might open new and interesting perspectives both in terms of explaining the mechanisms that underlie some of the effects of nicotine addiction and in the development of new drugs for smoking cessation.

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.