Glucose metabolism in pancreatic α- and β-cells is believed to regulate secretion of glucagon and insulin, respectively. In β-cells, ATP links glucose metabolism to electrical activity and insulin secretion. In α-cells, ATP has been attributed various roles in glucose-regulated glucagon release, but the underlying mechanisms are poorly understood. Despite its importance in insulin and glucagon secretion little is known about ATP kinetics in α- and β-cells. In this thesis, the novel fluorescent ATP biosensor Perceval was used to monitor physiologically relevant ATP concentrations with little influence of ADP. Glucose stimulation of β-cells within mouse and human pancreatic islets induced pronounced rise of ATP with superimposed oscillations. Simultaneous measurements of the sub-plasma membrane ATP and Ca2+ concentrations revealed glucose-induced oscillations in opposite phase. ATP increased further and the oscillations ceased when voltage-dependent Ca2+ influx was prevented. In contrast, ATP promptly decreased in response to K+-depolarization-induced elevation of Ca2+. Also mobilization of Ca2+ from intracellular stores lowered ATP, but the negative effect was not due to increased ATP consumption by the sarco/endoplasmic reticulum Ca2+-ATPase. Store-operated Ca2+ entry alone had little effect but markedly elevated ATP when combined with muscarinic receptor activation. When comparing ATP and Ca2+ responses in α- and β-cells within the same islet, glucose-induced ATP generation was much less pronounced and the dose-response relationship left-shifted in the α-cells. At basal glucose, individual α-cells showed Ca2+ and concomitant ATP oscillations in opposite-phase with variable frequency. These oscillations largely cancelled out when averaging data from several α-cells. At high glucose, the Ca2+ and ATP oscillations in α-cells tended to synchronize with the corresponding β-cell oscillations. Since β-cell Ca2+ oscillations drive pulsatile insulin secretion, which is antiparallel to pulsatile glucagon secretion, there seems to be an inverse relationship between changes in α-cell Ca2+ and glucagon release. This paradox is attributed to paracrine inhibition overriding Ca2+ stimulation, since somatostatin receptor blockade potently stimulated glucagon release with little effect on α-cell Ca2+ signalling. The data indicate that complex ATP-Ca2+ interactions in α- and β-cells underlie cell-intrinsic regulation of glucagon and insulin secretion and that paracrine inhibition of glucagon release becomes important in hyperglycaemia.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...

Neonatal destruction of rat nigrostriatal dopaminergic fibers results in an enhanced oral activity response to both dopamine (DA) D1 and serotonin (5-HT) agonists. Because cholinergic systems represent another one of the neural circuits involved in oral behavior, it was of interest to determine whether muscarinic receptors might also be sensitized in the lesioned rats. At 3 days after birth, rats were pretreated with desipramine HCl (20 mg/kg, IP) 1 h before 6-hydroxydopamine (6-OHDA) HBr (100 μg in each lateral ventricle) or saline-ascorbic acid (0.1%) vehicle. Between 2 and 4 months, behavioral supersensitivity to a D1 agonist (SKandF 38393) and 5-HT agonist (m-chlorophenylpiperazine; m-CPP) was established before rats were challenged with the muscarinic receptor agonist, pilocarpine HCl (0.125 to 10.0 mg/kg, IP). The pilocarpine dose-effect curve was shifted to the left, with a maximal effect of 63.7 ± 8.6 oral movements being produced by a 1.0 mg/kg pilocarpine HCl dose in the 6-OHDA lesioned rats, versus 15.0 ± 2.4 oral movements in the control group (p < 0.001). The enhanced response to pilocarpine was attenuated by the muscarinic receptor antagonist, scopolamine HCl (0.1 mg/kg IP). These findings indicate that neonatal 6-OHDA treatment produces supersensitization of muscarinic receptors in rats.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...

This thesis is comprised of two parts. In the first part, we investigated a) the pharmacology of GNTI, a selective kappa opioid receptor antagonist, as a scratch-inducing compound in mice and b) possible mediators and receptors that may be involved in GNTI-induced scratching (itch). We studied if GNTI induces scratching through opioid, histamine, gastrin-releasing peptide (GRP) and/or muscarinic M1 receptors. In the second part, we established similarities and differences between pain and itch using GNTI-induced scratching and formalin-induced nociception models in mice. We found that GNTI (0.03-3 mg/kg, s.c., behind the neck) induces compulsive and vigorous scratching behavior in a dose-dependent manner. A standard submaximal dose (0.3 mg/kg) of GNTI caused animals to scratch 500-600 times in a 30 min observation period. Intrathecal (i.t.) or intraperitoneal (i.p.) administration of GNTI did not elicit scratching behavior. Duration of action of GNTI was 60-70 min and tolerance to the repetitive behavior did not develop. C-fos expressing neurons, in response to GNTI injection, were localized on the lateral side of the superficial layers of the dorsal horn of the cervical spinal cord. Compound 48/80, a chemically different pruritogen, evoked c-fos expression in neurons which are located on the lateral side of the superficial layer of the dorsal horn. These data suggest that both GNTI and compound 48/80 activate a group of sensory neurons located on the lateral side of lamina I and II. Pretreating (at -20 min) and posttreating (at +5 min) mice with the kappa opioid receptor agonist, nalfurafine (0.001-0.03 mg/kg, s.c.), significantly attenuated scratching induced by GNTI (0.3 mg/kg). These effects were not a consequence of behavioral depression. Tolerance did not develop to the anti-scratch activity of nalfurafine. Pretreating mice with nalfurafine (0.02 mg/kg) prevented both GNTI- and compound 48/80-provoked c-fos expression. Our c-fos results suggest that the preclinical antipruritic activity of nalfurafine occurs at the spinal level. Moreover, our results reinforce the need to evaluate nalfurafine as a potentially useful antipruritic in human conditions involving itch. GNTI still elicited excessive scratching in mice lacking mu, delta or kappa opioid receptors, respectively, as well as in mice pretreated with either naloxone or norbinaltorphimine. The H1 receptor antagonist, fexofenadine, or the H4 receptor antagonist, JNJ 10191584, did not attenuate GNTI-induced scratching. Also, pretreating mice with the peptide GRPR antagonist, [D-Phe6]bombesin(6-13) methyl ester, or the non-peptide GRPR antagonist, RC-3095, did not antagonize scratching induced by GNTI. Furthermore, GRPR mRNA levels did not change in response to GNTI injection. Telenzepine, a standard M1 receptor antagonist, had no marked effect against GNTI-elicited scratching, however (unexpectedly) McN-A-343, an M1 receptor agonist, attenuated this behavior in a dose-dependent manner. In the second part of our studies, we found that pretreating mice with lidocaine (i.d., behind the neck) inhibits GNTI-induced scratching and prevents GNTI-provoked c-fos expression in the dorsal horn of the spinal cord. Similarly, lidocaine (i.d., hind leg) inhibits formalin-induced nociception as well as formalin-provoked c-fos expression. While injection (s.c.) of formalin to the face of mice induced only wiping (indicating pain) by forepaws of the injection side, injection (s.c.) of GNTI to the face elicited grooming and scratching (indicating itch). In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. In short, our main results indicate that a) the scratch-inducing activity of GNTI is not mediated by opioid, histamine or GRP receptors; b) kappa opioid receptors are involved, at least in part, in the inhibition of itch sensation and thus, on the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch; and c) agonism at M1 receptors inhibits GNTI-induced scratching therefore the M1 receptor may be a key target for antipruritic drug development. / Pharmacology

Purpose: To establish a tool, termed as antimuscarinic activity (AMA), to predict the incidence of antimuscarinic adverse events (AMAEs).Methods: A literature review, focused on drugs having off-target interaction with muscarinic receptors, was performed. Prototypical drugs olanzapine, diphenhydramine, paroxetine were selected for the analysis. Scopolamine and darifenacin were included as positive and negative controls, respectively. Physiochemical properties, pharmacokinetic data, and clinical incidence of AMAEs for the selected drugs were collected from reported literature. Extrapolation of literature data was carried-out to obtain exposure data. To determine the drugs muscarinic affinity (Ki values), experiments were performed using 3H-QNB and membrane suspensions of M1, M2, and M3. Cmax, values were combined with Ki values to generate the relevant AMA. Validation of the AMA biomarker was carried-out against the reported AMAEs incidence. Results: With the exclusion of scopolamine and olanzapine for CNS and peripheral AMAEs, respectively, AMA ranking was related to the drugs AMAEs.

Posttraumatic stress disorder (PTSD) is an anxiety disorder that may follow exposure to severe emotional trauma and presents with various symptoms of anxiety, hyperarousal and cognitive anomalies. Interestingly, only 10-30% of an exposed population will go on to develop full-blown PTSD. Cholinergic neurotransmission is implicated in anxiety as well as other typical manifestations of PTSD, particularly cognitive changes. The frontal cortex and hippocampus regulate and in turn are affected by stress, and have also been implicated in the underlying neuropathology of PTSD. These areas are densely innervated by cholinergic neurons originating from the basal forebrain. In this study, the time dependent sensitization (TDS) model was used to induce symptoms of PTSD in animals. The study was designed to determine the long-term effects of an intense, prolonged aversive procedure on central muscarinic acetylcholine receptor (mAChR) characteristics and the correlation if any of those findings to cognitive aspects and general arousal as characteristics associated with PTSD. In order to achieve this goal, male Sprague-Dawley rats were exposed to the TDS stress paradigm with behavioral/neuro-receptor assessments performed on day 7 post re-stress (duration of each experiment in whole is 14 days). Acoustic startle reflex (ASR) was used to determine emotional state (hyperarousal), while the conditioned taste aversion (CTA) paradigm was implemented in order to assess aversive memory. Muscarinic receptor binding studies were performed in the frontal cortex and hippocampus. Moreover, both the stress-exposed and control animals were pre-tested in the acoustic startle chamber in order to attempt to separate stress sensitive from stress-resilient animals based on predetermined ASR criteria. The ASR niodel was previously validated in our laboratory, while the CTA model was validated in this project before application. In the CTA model, an i.p. injection with lithium chloride (LiCl) (associated with digestive malaise), was used as unconditioned stimulus (US) and was paired with a saccharinlcyclamate drinking solution as conditioned stimulus (CS) to induce aversion to the novel taste (CS) when presented in the absence of the US. Population data of animals tested in the ASR experiment indicated no statistical significant difference between stressed and control animals. However, when each animal was assessed individually, 22.5 % of the exposed population displayed all increase above the predetermined criteria of 35 % in startle response, indicating a state of heightened arousal. In contrast, only 4.2 O h of control animals (no stress) displayed an increase in arousal based on the above mentioned criteria. Muscarinic receptor densities (Bm,) in the total population of animals exposed to stress showed a statistical significant increase in both the hippocampus and frontal cortex when compared to controls, with no changes in & values observed in either one of the areas. In the CTA experiment, TDS stress was implemented as US paired with a saccharinlcyclamate drinking solution as CS. An acute session of prolonged stress (as used in the TDS model) effectively induced aversion to a novel taste and a subsequent reminder of the stress (restress) paired with the CS sustained the acquire adversive memory. Furthermore, LiCl was reintroduced as US in order to assess the effect of prior exposure to two types of stress (acute and TDS) on subsequently acquired CTA memory. Prior exposure to acute stress had no significant effect on subsequently acquired aversive memory when measured either 3- or 7 days post-conditioning (CS-US). Stress-restress (TDS) exposure, however, indicated a significant decrease in aversive memory from 3- to 7 days post-conditioning (CS-US) as well as a significant decrease in aversive memory between the control- and the TDS group 7 days post-conditioning. The mAChR density (B,,) in the frontal cortex; but not in the hippocampus, was elevated at the same point in time (7 days post CS-US pairing) that CTA memory was impaired following TDS stress (stress-restress). Ultimately, these data support an association between altered cholinergic receptors and hyperarousallanxiety in an animal model of PTSD. The data also support the phenomenon of individual susceptibility to stress in animals that parallels that observed in humans exposed to severe trauma. Impaired aversive memory (CTA) is a consequence of prior exposure to TDS stress, but not acute stress, and is likewise mediated by an altered central cholinergic transmission displayed as an increase in mAChRs in the frontal cortex. The lack of studies regarding the influence of the cholinergic system in PTSD related behavior earns ,this project value as inimitable PTSD research. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Posttraumatic stress disorder (PTSD) is an anxiety disorder that may follow exposure to severe emotional trauma and presents with various symptoms of anxiety, hyperarousal and cognitive anomalies. Interestingly, only 10-30% of an exposed population will go on to develop full-blown PTSD. Cholinergic neurotransmission is implicated in anxiety as well as other typical manifestations of PTSD, particularly cognitive changes. The frontal cortex and hippocampus regulate and in turn are affected by stress, and have also been implicated in the underlying neuropathology of PTSD. These areas are densely innervated by cholinergic neurons originating from the basal forebrain. In this study, the time dependent sensitization (TDS) model was used to induce symptoms of PTSD in animals. The study was designed to determine the long-term effects of an intense, prolonged aversive procedure on central muscarinic acetylcholine receptor (mAChR) characteristics and the correlation if any of those findings to cognitive aspects and general arousal as characteristics associated with PTSD. In order to achieve this goal, male Sprague-Dawley rats were exposed to the TDS stress paradigm with behavioral/neuro-receptor assessments performed on day 7 post re-stress (duration of each experiment in whole is 14 days). Acoustic startle reflex (ASR) was used to determine emotional state (hyperarousal), while the conditioned taste aversion (CTA) paradigm was implemented in order to assess aversive memory. Muscarinic receptor binding studies were performed in the frontal cortex and hippocampus. Moreover, both the stress-exposed and control animals were pre-tested in the acoustic startle chamber in order to attempt to separate stress sensitive from stress-resilient animals based on predetermined ASR criteria. The ASR niodel was previously validated in our laboratory, while the CTA model was validated in this project before application. In the CTA model, an i.p. injection with lithium chloride (LiCl) (associated with digestive malaise), was used as unconditioned stimulus (US) and was paired with a saccharinlcyclamate drinking solution as conditioned stimulus (CS) to induce aversion to the novel taste (CS) when presented in the absence of the US. Population data of animals tested in the ASR experiment indicated no statistical significant difference between stressed and control animals. However, when each animal was assessed individually, 22.5 % of the exposed population displayed all increase above the predetermined criteria of 35 % in startle response, indicating a state of heightened arousal. In contrast, only 4.2 O h of control animals (no stress) displayed an increase in arousal based on the above mentioned criteria. Muscarinic receptor densities (Bm,) in the total population of animals exposed to stress showed a statistical significant increase in both the hippocampus and frontal cortex when compared to controls, with no changes in & values observed in either one of the areas. In the CTA experiment, TDS stress was implemented as US paired with a saccharinlcyclamate drinking solution as CS. An acute session of prolonged stress (as used in the TDS model) effectively induced aversion to a novel taste and a subsequent reminder of the stress (restress) paired with the CS sustained the acquire adversive memory. Furthermore, LiCl was reintroduced as US in order to assess the effect of prior exposure to two types of stress (acute and TDS) on subsequently acquired CTA memory. Prior exposure to acute stress had no significant effect on subsequently acquired aversive memory when measured either 3- or 7 days post-conditioning (CS-US). Stress-restress (TDS) exposure, however, indicated a significant decrease in aversive memory from 3- to 7 days post-conditioning (CS-US) as well as a significant decrease in aversive memory between the control- and the TDS group 7 days post-conditioning. The mAChR density (B,,) in the frontal cortex; but not in the hippocampus, was elevated at the same point in time (7 days post CS-US pairing) that CTA memory was impaired following TDS stress (stress-restress). Ultimately, these data support an association between altered cholinergic receptors and hyperarousallanxiety in an animal model of PTSD. The data also support the phenomenon of individual susceptibility to stress in animals that parallels that observed in humans exposed to severe trauma. Impaired aversive memory (CTA) is a consequence of prior exposure to TDS stress, but not acute stress, and is likewise mediated by an altered central cholinergic transmission displayed as an increase in mAChRs in the frontal cortex. The lack of studies regarding the influence of the cholinergic system in PTSD related behavior earns ,this project value as inimitable PTSD research. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

A sinalização muscarínica exerce função modulatória sobre diferentes aspectos da cognição e emoções. Todos os cinco subtipos de receptores muscarínicos (mAChR), M1 a M5, são expressos no hipocampo de mamíferos e são ativados de forma sobreposta pela maioria dos fármacos, dificultando avanços significativos na compreensão da contribuição de cada componente desse sistema. A toxina muscarínica 3 (MT3) é um antagonista seletivo para o subtipo M4, permitindo a investigação das ações modulatórias deste receptor no aprendizado, memória e plasticidade sináptica. Os M4 são receptores acoplados à proteína G (GPCRs) que atuam via Gi/o desencadeando efeitos inibitórios sobre as células em que estão presentes. Estudos comportamentais anteriores indicam que a administração de MT3 imediatamente após o treino em uma tarefa aversiva produz efeito amnéstico, enquanto que a administração antes da evocação, causa facilitação. Uma explicação para estes resultados é que os circuitos locais envolvidos na consolidação e na evocação da memória diferem em sua natureza. Nesse contexto, sugere-se que o efeito amnéstico da MT3 sobre a consolidação seja consequência da supressão da inibição de interneurônios GABAérgicos; enquanto que na evocação, esse efeito se daria sobre as sinapses glutamatérgicas. Assim, no presente trabalho, com o objetivo de investigar como o receptor M4 modula a plasticidade sináptica de longa duração e interage com uma dessas sinalizações, no caso a GABAérgica, utilizou-se a técnica de eletrofisiologia in vivo de hipocampo de ratos anestesiados. Para tanto, foram realizados registros extracelulares do potencial excitatório pós-sináptico de campo (fEPSP) de CA1 evocados por estimulação contralateral da via Colateral de Schaffer com infusão dos fármacos 15 min antes ou depois da estimulação elétrica de alta ou baixa frequência (HFS: 10 trens 0,5 Hz, 20 pulsos 100 Hz; ou LFS: 600 pulsos 1 Hz, respectivamente). MT3 (4,0 μg/μl), bicuculina (0,06 μg/μl), baclofen (0,2 μg/μl) e veículo, isoladamente ou combinados, não alteraram a amplitude da resposta evocada basal ou a facilitação por pulso pareado (FPP) 15 min após a infusão. MT3 aparentemente atenuou, mas não de forma significativa, a potenciação de longa duração (LTP) em relação ao controle (potenciação 60 min após a HFS de 31,8% e 66,0%, respectivamente). Além disso, não houve diferença significativa entre a amplitude do fEPSP no período basal e 60 min após a HFS sob ação da MT3. Bicuculina, embora não tenha abolido a LTP e nem causado alteração na FPP, produziu uma potenciação de apenas 36,4%. Baclofen promoveu uma potenciação semelhante à dos controles. A administração de baclofen também reduziu significativamente a FPP em relação ao basal. A administração conjunta de MT3 com bicuculina ou baclofen promoveu uma potenciação semelhante ao controle. MT3 não apresentou efeito sobre a manutenção da LTP quando aplicada 15 min após a HFS. Por fim, não foi possível induzir a depressão de longa duração (LTD) com o protocolo de LFS utilizado. Embora não tenha ocorrido diferença estatisticamente significativa entre os grupos devido ao baixo número de animais utilizados, os dados sugerem a possibilidade de uma amplitude reduzida da LTP quando da injeção de bicuculina. Baclofen alterou a FPP em relação ao fEPSP basal, o mesmo não tendo sido observado no grupo controle. Com a administração concomitante de MT3, tais alterações deixam de ser identificadas. Ainda que os achados experimentais sejam inconclusivos e preliminares, este trabalho permitiu a padronização da técnica de eletrofisiologia in vivo em animal anestesiado o que abre portas para futuras investigações. / The cholinergic muscarinic system exerts modulatory function over different aspects of cognition and emotion. All five muscarinic receptors subtypes (mAChR), M1 to M5, are expressed at mammals hippocampus and at least two of them are simultaneously activated by most of the drugs, hindering significant advances on the role of each component of this system. The muscarinic toxin 3 (MT3) is a selective antagonist for the M4 subtype, allowing the investigation of the modulatory actions of this receptor over learning, memory and synaptic plasticity. The M4 are G protein coupled receptors (GPCRs) that act through Gi/o triggering inhibitory effects on which cells they are occur. Previous behavioral studies have shown that administration of MT3 soon after aversive task training exerts amnestic effects over memory, while administration prior to recall, leads to facilitation. A possible explanation to these results could be that the local circuits involved on memory consolidation and recall are different in nature. On this perspective, the amnestic effect of MT3 over memory consolidation should be consequence of GABAergic interneurons inhibition suppression; while the effect on recall, should be over glutamatergic synapses modulation. Thereby, the present work, with the objective to investigate how the M4 receptor modulates long-term synaptic plasticity and interacts with the GABAergic system, in vivo electrophysiological approach of anesthetized rats’ hippocampus was applied. Hence, field excitatory postsynaptic potentials (fEPSP) from CA1 were recorded after stimulation of contralateral Schaffer Collateral pathway with drugs infusion 15 min before or after high or low frequency electric stimulation (HFS: 10 trains 0.5 Hz, 20 pulses 100 Hz; LFS: 600 pulses 1 Hz, respectively). Neither MT3 (4.00 μg/μl), bicuculline (0.06 μg/μl), baclofen (0.20 μg/μl) nor vehicle, isolated or combined, changed the baseline evoked response amplitude 15 min after infusion nor the paired-pulse facilitation ratio (PPF). MT3 apparently attenuated, but not significantly, the long-term potentiation (LTP) compared to control (31.8% and 66.0% potentiation 60 min after HFS, respectively). In addition, there was no significant difference between baseline and 60 min after HFS fEPSP amplitude at MT3 group. Bicuculline, although did not abolish LTP neither changed PPF, it did produce a potentiation of only 36.4%. Baclofen induced a potentiation similar to control group. Baclofen administration also significantly reduced PPF compared to baseline. The simultaneous administration of MT3 and bicuculline or baclofen led to a potentiation similar to the control group. MT3 did not show any effect over LTP maintenance when applied 15 min after HFS. Lastly, it was not possible to induce long-term depression (LTD) with the used LFS protocol. Although there was no statistical significance between groups due to the low animal numbers used, data suggest that bicuculline had reduced LTP amplitude. Baclofen did alter PPF and the same was not observed on control group. When bicuculline or baclofen were injected with MT3, those alterations were not observed. These are inconclusive and preliminary results, notwithstanding this work allowed to set up the in vivo electrophysiology technique in anesthetized animals what will provide new tools for future research.