RESUMEN
The use of methoxypoly(ethylene glycol) (mPEG) in PEG conjugates of proteins and non-protein therapeutic agents has led to the recognition that the polymer components of such conjugates can induce anti-PEG antibodies (anti-PEGs) that may accelerate the clearance and reduce the efficacy of the conjugates. Others have classified anti-PEGs as "methoxy-specific" or "backbone-specific". The results of our previous research on anti-PEGs in the sera of rabbits immunized with mPEG or hydroxyPEG (HO-PEG) conjugates of three unrelated proteins were consistent with that classification (Sherman, M.R., et al., 2012. Bioconjug. Chem. 23, 485-499). Enzyme-linked immunosorbent assays (ELISAs) were performed on rabbit antisera and rabbit monoclonal anti-PEGs with competitors including 10 kDa mPEG, 10 kDa PEG diol and six linear or cyclic oligomers of oxyethylene (CH2CH2O), with molecular weights of ca. 150-264 Da. Our results demonstrate that (1) the binding affinities of anti-mPEGs depend more on the backbone lengths of the polymers and the hydrophobicities of their end-groups than on their resemblance to the methoxy terminus of the immunogenic polymer; (2) anti-PEGs raised against HO-PEG-proteins are not directed against the terminal hydroxy group, but against the backbone; (3) rabbit anti-PEGs bind to and distinguish among PEG-like oligomers with as few as three oxyethylene groups; and (4) none of the monoclonal or polyclonal anti-PEGs was absolutely "methoxy-specific" or "backbone-specific", but displayed distinct relative selectivities. If these results are relevant to human immune responses, the clinical use of stable conjugates of HO-PEG with proteins and non-protein therapeutic agents would be expected to produce fewer and less intense immune responses than those induced by conjugates with mPEG or PEGs with larger alkoxy groups.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Afinidad de Anticuerpos/inmunología , Polietilenglicoles/metabolismo , Proteínas/inmunología , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Humanos , Polietilenglicoles/química , ConejosRESUMEN
Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.
Asunto(s)
Polietilenglicoles/química , Proteínas/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón-alfa/química , Proteínas/química , ConejosRESUMEN
The cholinergic input from the lateral dorsal tegmental area (LDTg) modulates the dopamine cells of the ventral tegmental area (VTA) and plays an important role in cocaine taking. Specific pharmacological agents that block or stimulate muscarinic receptors in the LDTg change acetylcholine (ACh) levels in the VTA. Furthermore, manipulations of cholinergic input in the VTA can change cocaine taking. In the current study, the ACh output from the LDTg was attenuated by treatment with the selective muscarinic type 2 (M2) autoreceptor agonist oxotremorine.sesquifumarate (OxoSQ). We hypothesized that OxoSQ would reduce the motivation of rats to self-administer both natural and drug rewards. Animals were tested on progressive ratio (PR) schedules of reinforcement for food pellets and cocaine. On test days, animals on food and on cocaine schedules were bilaterally microinjected prior to the test. Rats received either LDTg OxoSQ infusions or LDTg artificial cerebrospinal fluid (aCSF) infusions in a within-subjects design. In addition, infusions were delivered into a dorsal brain area above the LDTg as an anatomical control region. OxoSQ microinjection in the LDTg, compared to aCSF, significantly reduced both the number of self-administered pellets and cocaine infusions during the initial half of the session; this reduction was dose-dependent. OxoSQ microinjections into the area just dorsal to the LDTg had no significant effect on self-administration of food pellets or cocaine. Animals were also tested in locomotor activity chambers for motor effects following the above microinjections. Locomotor activity was mildly increased by OxoSQ microinjection into the LDTg during the initial half of the session. Overall, these data suggest that LDTg cholinergic neurons play an important role in modifying the reinforcing value of natural and drug rewards. These effects cannot be attributed to significant alterations of locomotor behavior and are likely accomplished through LDTg muscarinic autoreceptors.
Asunto(s)
Cocaína/antagonistas & inhibidores , Receptor Muscarínico M2/fisiología , Tegmento Mesencefálico/fisiología , Animales , Cocaína/administración & dosificación , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Operante/fisiología , Relación Dosis-Respuesta a Droga , Alimentos , Masculino , Microinyecciones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Oxotremorina/administración & dosificación , Oxotremorina/análogos & derivados , Oxotremorina/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M2/agonistas , Esquema de Refuerzo , Recompensa , Autoadministración , Tegmento Mesencefálico/efectos de los fármacosRESUMEN
The perioculomotor urocortin-containing population of neurons (pIIIu: otherwise known as the non-preganglionic Edinger-Westphal nucleus) is sensitive to alcohol and is involved in the regulation of alcohol intake. A recent study indicated that this brain region is also sensitive to psychostimulants. Since pIIIu has been shown to respond to stress, we investigated how psychostimulant-induced pIIIu activation compares to stress- and ethanol-induced activation, and whether it is independent from a generalized stress response. Several experiments were performed to test how the pIIIu responds to psychostimulants by quantifying the number of Fos immunoreactive nuclei after acute i.p. injections of saline, 10-30 mg/kg cocaine, 5 mg/kg methamphetamine, 5 mg/kg amphetamine, 2.5 g/kg ethanol, 2 h of restraint stress, 10 min of swim stress, or six applications of mild foot shock in male C57BL/6 J mice. We also compared Fos immunoreactivity in pIIIu after acute (20 mg/kg cocaine) and repeated cocaine exposure (7 days of 20 mg/kg cocaine) injections in male C57BL/6 J mice in order to investigate the potential habituation of this response. Finally, we quantified the number of Fos immunoreactive nuclei in pIIIu after administration of saline, 2.5 g/kg ethanol, 20 mg/kg cocaine, or 2 h of restraint stress in male Sprague-Dawley rats. We found that exposure to psychostimulants and ethanol induced significantly higher Fos levels in pIIIu compared to stress in mice. Furthermore, repeated cocaine injections did not decrease Fos immunoreactivity as would be expected if this response were due to stress. In rats, exposure to ethanol, psychostimulant and restraint stress all induced pIIIu Fos immunoreactivity compared to saline-injected controls. In both mice and rats, ethanol- and cocaine-induced Fos immunoreactivity occurred exclusively in urocortin 1-positive, but not in tyrosine hydroxylase-positive, cells. These results provide evidence that the pIIIu Fos-response to psychostimulants is independent of a generalized stress in mice, but not rats. They additionally show that the pIIIu response to stress differs significantly between species.
Asunto(s)
Mesencéfalo/efectos de los fármacos , Mesencéfalo/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Urocortinas/metabolismo , Anfetamina/administración & dosificación , Animales , Depresores del Sistema Nervioso Central/administración & dosificación , Estimulantes del Sistema Nervioso Central/administración & dosificación , Cocaína/administración & dosificación , Etanol/administración & dosificación , Masculino , Metanfetamina/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-fos/metabolismo , Psicotrópicos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Estrés Psicológico/fisiopatología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Acetylcholine (ACh) is an important mediator of dopamine (DA) release and the behavioral reinforcing characteristics of drugs of abuse in the mesocorticolimbic pathway. Within the ventral tegmental area (VTA), the interaction of DA with ACh appears to be integral in mediating motivated behaviors. However, the effects of methamphetamine on VTA ACh and DA release remain poorly characterized. The current investigation performed microdialysis to evaluate the effects of methamphetamine on extracellular levels of ACh and DA. Male C57BL/6J mice received an i.p. injection (saline, 2 mg/kg, or 5 mg/kg) and an intra-VTA infusion (vehicle, 100 microM or 1 mM) of methamphetamine. Locally perfused methamphetamine resulted in no change in extracellular ACh compared with vehicle, but caused a strong, immediate and dose-dependent increase in extrasynaptic DA levels (1240% and 2473% of baseline, respectively) during the 20-min pulse perfusion. An i.p. injection of methamphetamine increased extrasynaptic DA to 275% and 941% of baseline (2 mg/kg and 5 mg/kg, respectively). Systemic methamphetamine significantly increased ACh levels up to 275% of baseline for 40-60 min (2 mg/kg) and 397% of baseline for 40-160 min (5 mg/kg) after injection. ACh remained elevated above baseline for 2-3 h post injection, depending on the methamphetamine dose. Methamphetamine-induced locomotor activity was dose-dependently correlated with extrasynaptic VTA ACh, but not DA levels. These data suggest that methamphetamine acts in the VTA to induce a robust and short-lived increase in extracellular DA release but acts in an area upstream from the VTA to produce a prolonged increase in ACh release in the VTA. We conclude that methamphetamine may activate a recurrent loop in the mesocorticolimbic DA system to stimulate pontine cholinergic nuclei and produce a prolonged ACh release in the VTA.
Asunto(s)
Acetilcolina/metabolismo , Estimulantes del Sistema Nervioso Central/administración & dosificación , Dopamina/metabolismo , Metanfetamina/administración & dosificación , Área Tegmental Ventral/efectos de los fármacos , Análisis de Varianza , Animales , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Masculino , Ratones , Ratones Endogámicos C57BL , Microdiálisis/métodos , Actividad Motora/efectos de los fármacosRESUMEN
Previous studies using genetic and lesion approaches have shown that the neuropeptide urocortin 1 (Ucn1) is involved in regulating alcohol consumption. Ucn1 is a corticotropin releasing factor (CRF) -like peptide that binds CRF1 and CRF2 receptors. Perioculomotor urocortin-containing neurons (pIIIu), also known as the non-preganglionic Edinger-Westphal nucleus, are the major source of Ucn1 in the brain and are known to innervate the lateral septum. Thus, the present study tested whether Ucn1 could regulate alcohol consumption through the lateral septum. In a series of experiments Ucn1 or CRF was bilaterally injected at various doses into the lateral septum of male C57BL/6J mice. Consumption of 20% volume/volume ethanol or water was tested immediately after the injections using a modification of a 2-h limited access sweetener-free "drinking-in-the-dark" procedure. Ucn1 significantly suppressed ethanol consumption when administered prior to the third ethanol drinking session (the expression phase of ethanol drinking) at doses as low as 6 pmol. Ethanol intake was differentially sensitive to Ucn1, as equivalent doses of this peptide did not suppress water consumption. In contrast, CRF suppressed both ethanol and water intake at 40 and 60 pmol, but not at lower doses. Repeated administration of Ucn1 during the acquisition of alcohol consumption showed that 40 pmol (but not 2 or 0.1 pmol) significantly attenuated ethanol intake. Repeated administration of Ucn1 also resulted in a decrease of ethanol intake in sham-injected animals, a finding suggesting that the suppressive effect of Ucn1 on ethanol intake can be conditioned. Taken together, these studies confirm the importance of lateral septum innervation by Ucn1 in the regulation of alcohol consumption.
Asunto(s)
Consumo de Bebidas Alcohólicas , Conducta de Ingestión de Líquido/efectos de los fármacos , Núcleos Septales/efectos de los fármacos , Urocortinas/farmacología , Consumo de Bebidas Alcohólicas/fisiopatología , Animales , Conducta Animal , Depresores del Sistema Nervioso Central/administración & dosificación , Hormona Liberadora de Corticotropina/farmacología , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Etanol/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Microinyecciones/métodos , Núcleos Septales/fisiología , Factores de Tiempo , Urocortinas/efectos adversosRESUMEN
Hyperuricemia results from an imbalance between the rates of production and excretion of uric acid. Longstanding hyperuricemia can lead to gout, which is characterized by the deposition of monosodium urate monohydrate crystals in the joints and periarticular structures. Because such deposits are resolved very slowly by lowering plasma urate with available drugs or other measures, the symptoms of gout may become chronic. Persistent hyperuricemia may also increase the risk of renal and cardiovascular diseases. Unlike most mammals, humans lack the enzyme uricase (urate oxidase) that catalyzes the oxidation of uric acid to a more soluble product. This review describes the development of a poly(ethylene glycol) (PEG) conjugate of recombinant porcine-like uricase with which a substantial and persistent reduction of plasma urate concentrations has been demonstrated in a Phase 2 clinical trial. Two ongoing Phase 3 clinical trials include systematic assessments of gout symptoms, tophus resolution and quality of life, in addition to the primary endpoint of reduced plasma urate concentration.
Asunto(s)
Resistencia a Medicamentos , Gota/tratamiento farmacológico , Hiperuricemia/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Urato Oxidasa/uso terapéutico , Animales , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , HumanosRESUMEN
The role of autocrine growth factors in the stimulation of lung cancer growth is well established. Nicotine is an agonist for acetylcholine receptors and stimulates lung cancer growth. This suggests that if lung cancers synthesize acetylcholine (ACh), then ACh may be an autocrine growth factor for lung cancer. Analysis of normal lung demonstrated that the cells of origin of lung cancers express the proteins necessary for non-neuronal ACh storage and synthesis. Analysis of mRNA from squamous cell lung carcinoma, small cell lung carcinoma (SCLC) and adenocarcinoma showed synthesis of choline acetyltransferase (ChAT) and nicotinic receptors. Immunohistochemical analysis of a retrospective series of SCLC and adenocarcinomas showed that more than 50% of the lung cancers screened expressed ChAT and nicotinic receptors. To study the effect of endogenous ACh synthesis on growth, SCLC cell lines were studied. SCLC cell lines were found to express ChAT mRNA and to secrete ACh into the medium as measured by HPLC separation and enzymatically-coupled electrochemical detection. The SCLC cell line NCI-H82 synthesized highest levels of ACh. Showing that the endogenously synthesized ACh interacted with its receptors to stimulate cell growth, addition of muscarinic and nicotinic antagonists slowed H82 cell proliferation. These findings demonstrate that lung cancer cell lines synthesize and secrete ACh to act as an autocrine growth factor. The existence of a cholinergic autocrine loop in lung cancer provides a basis for understanding the effects of nicotine in cigarette smoke on lung cancer growth and provides a new pathway to investigate for potential therapeutic approaches to lung cancer.
Asunto(s)
Acetilcolina/biosíntesis , Comunicación Autocrina/fisiología , Carcinoma de Células Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Transporte Vesicular , Acetilcolina/fisiología , Animales , Atropina/farmacología , Carcinoma de Células Pequeñas/patología , Proteínas Portadoras/metabolismo , División Celular/fisiología , Colina O-Acetiltransferasa/metabolismo , Haplorrinos , Inmunohistoquímica , Pulmón/patología , Neoplasias Pulmonares/patología , Mecamilamina/farmacología , Proteínas de Transporte de Membrana/metabolismo , Antagonistas Muscarínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Colinérgicos/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
Uricase-deficient mice develop uric acid nephropathy, with high mortality rates before weaning. Urate excretion was quantitated and renal function was better defined in this study, to facilitate the use of these mice as a model for evaluating poly(ethylene glycol)-modified recombinant mammalian uricases (PEG-uricase) as a potential therapy for gout and uric acid nephropathy. The uric acid/creatinine ratio in the urine of uricase-deficient mice ranges from 10 to >30; on a weight basis, these mice excrete 20- to 40-fold more urate than do human subjects. These mice consistently develop a severe defect in renal concentrating ability, resulting in an approximately sixfold greater urine volume and a fivefold greater fluid requirement, compared with normal mice. This nephrogenic diabetes insipidus leads to dehydration and death of nursing mice but, with adequate water replacement, high urine flow protects adults from progressive renal damage. Treatment of uricase-deficient mice with PEG-uricase markedly reduced urate levels and, when initiated before weaning, preserved the renal architecture (as evaluated by magnetic resonance micros-copy) and prevented the loss of renal concentrating function. PEG-uricase was far more effective and less immunogenic than unmodified uricase. Retention of uricase in most mammals and its loss in humans and some other primates may reflect the evolution of renal function under different environmental conditions. PEG-uricase could provide an effective therapy for uric acid nephropathy and refractory gout in human patients.
Asunto(s)
Diabetes Insípida/tratamiento farmacológico , Diabetes Insípida/enzimología , Polietilenglicoles/uso terapéutico , Urato Oxidasa/deficiencia , Urato Oxidasa/uso terapéutico , Animales , Agua Corporal/metabolismo , Diabetes Insípida/patología , Diabetes Insípida/fisiopatología , Modelos Animales de Enfermedad , Gota/tratamiento farmacológico , Humanos , Capacidad de Concentración Renal , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/uso terapéutico , Urato Oxidasa/genética , Ácido Úrico/orinaRESUMEN
Identifying the neurocircuitry involved in behavioral responses to drugs of abuse is an important step towards understanding the mechanisms of drug addiction. The present study sought to distinguish brain regions involved in pharmacological effects of cocaine and ethanol from secondary effects by administering these drugs in the presence or absence of pentobarbital anesthesia. Changes in neuronal activity were assessed by immunohistochemical analysis of expression of an inducible transcription factor (ITF), c-Fos, in the brain of rats habituated to repeated pentobarbital anesthesia or saline administration. Cocaine administration (15 mg/kg, i.v.) in non-anesthetized animals produced a strong induction of c-Fos in the striatum and large number of other brain areas. Ethanol administration (2 g/kg, i.p.) induced c-Fos in a smaller number of characteristic brain areas, including the central nucleus of amygdala and paraventricular nucleus of hypothalamus. However, neither of these drugs was able to induce c-Fos in pentobarbital-anesthetized rats (50 mg/kg, i.v.). The suppressive effects of pentobarbital were not specific to c-Fos, such that pentobarbital also suppressed expression of ITFs FosB and Egr1 in the striatum of cocaine-treated rats. On the other hand, pentobarbital by itself strongly induced c-Fos expression in the lateral habenula of saline-, cocaine-, and ethanol-injected rats. It is not clear whether the suppressive effects of anesthesia on ITF expression in other areas are mediated by activation of lateral habenula, or are independent of this event. Our data suggest that in the absence of conscious awareness of drug-associated cues, cocaine and alcohol activate only a fraction of the neural elements engaged in the unanesthetized state.
Asunto(s)
Encéfalo/efectos de los fármacos , Cocaína/farmacología , Interacciones Farmacológicas/fisiología , Etanol/farmacología , Neuronas/efectos de los fármacos , Pentobarbital/farmacología , Factores de Transcripción/efectos de los fármacos , Anestésicos/farmacología , Animales , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Encéfalo/citología , Encéfalo/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Masculino , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Vías Nerviosas/citología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Estrés Fisiológico/inducido químicamente , Estrés Fisiológico/tratamiento farmacológico , Estrés Fisiológico/fisiopatología , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Trastornos Relacionados con Sustancias/metabolismo , Trastornos Relacionados con Sustancias/fisiopatología , Factores de Transcripción/metabolismoRESUMEN
Evidence is presented for an acetylcholine (ACh) input to the midbrain ventral tegmental area (VTA) as part of a system for self-stimulation and ingestive behavior. Male rats were prepared with an electrode in the perifornical lateral hypothalamus and an ipsilateral guideshaft for microdialysis in the VTA. Extracellular ACh increased in the VTA during self-stimulation, auto-stimulation, eating, or drinking. Infusion of atropine into the VTA via the microdialysis probe was sufficient to stop self-stimulation and reduce intake of food. It is concluded that ACh acts at muscarinic receptors in the VTA as part of a circuit that modulates hypothalamic self-stimulation and ingestive behavior.
Asunto(s)
Ingestión de Líquidos/fisiología , Ingestión de Alimentos/fisiología , Hipotálamo/fisiología , Área Tegmental Ventral/metabolismo , Animales , Atropina/farmacología , Masculino , Microdiálisis , Ratas , Ratas Sprague-Dawley , AutoestimulaciónAsunto(s)
Acetilcolina/fisiología , Cocaína/administración & dosificación , Cocaína/farmacología , Núcleo Accumbens/fisiología , Refuerzo en Psicología , Recompensa , Animales , Alimentos , Masculino , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Autoadministración , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
RATIONALE: The neurochemical effects of psychostimulant exposure may depend on how these drugs are encountered. A useful method for examining this issue is to compare neurotransmitter release following response-dependent, or self-administered, drug exposure and response-independent exposure. OBJECTIVES: This experiment examined the effect of active and passive cocaine administration on acetylcholine (ACh) efflux in the shell region of the nucleus accumbens (NAc) in rats. METHODS: One group of rats (CSA: cocaine self-administration) was trained to lever-press for intravenous infusions of cocaine (0.42 mg/kg per infusion) on a fixed-ratio-1 schedule of reinforcement. Cocaine infusions were accompanied by the onset of a stimulus light that signaled a 20-s time-out period. Control rats received intravenous cocaine (cocaine non-contingent: CNC) or saline (SAL) in a manner that was not contingent upon their behavior. Drug infusions in these groups were determined by the lever-press behavior of the animals in the CSA group, i.e. they were yoked to rats in the self-administration group such that CNC animals received equal amounts of cocaine as CSA rats. Animals received cocaine or saline in 3-h sessions for 13 consecutive days before testing. On day 14, extracellular ACh was measured in 15-min intervals before, during and after a 3-h session of cocaine exposure using unilateral microdialysis probes located in the NAc shell coupled with HPLC. RESULTS: ACh efflux was significantly increased above baseline in both groups of rats that received cocaine but CSA rats had significantly higher ACh levels during the self-administration period compared to their yoked counterparts. In addition, ACh efflux remained elevated longer in CSA animals relative to CNC rats following cessation of cocaine exposure. CONCLUSIONS: These results demonstrate that ACh interneurons in the NAc shell are responsive to cocaine exposure. In addition, these findings suggest that the manner in which the drug is administered (i.e. either by active self-administration or passive exposure) may be relevant to the magnitude of the neural response.
Asunto(s)
Acetilcolina/metabolismo , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Núcleo Accumbens/metabolismo , Animales , Cocaína/administración & dosificación , Condicionamiento Operante/efectos de los fármacos , Inhibidores de Captación de Dopamina/administración & dosificación , Masculino , Microdiálisis , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , AutoadministraciónRESUMEN
Alterations in the density of GABA and glutamate immunolabeling within nerve terminals in the shell region of the nucleus accumbens were assessed in rats withdrawn from intravenous cocaine exposure. Four groups of rats were used: one group self-administered cocaine (0.42 mg/kg/infusion) in daily 3-h sessions for approximately 2 weeks, two additional groups received either saline or cocaine in a noncontingent fashion, and a fourth comprised a drug-naive, age-matched control group. Immunogold electron microscopy was used to quantify presynaptic terminal GABA and glutamate density within the vesicular and mitochondrial pools approximately 18 days following the last drug or saline exposure in the treatment groups. A significant 27.7% decrease in vesicular glutamate density within asymmetrical nerve terminals was observed in animals that self-administered cocaine as compared to controls. This group also showed an 18.6% decrease in vesicular nerve terminal glutamate immunolabeling as compared to animals that were administered a similar total dose of cocaine in a response-independent fashion. No significant changes in the density of nerve terminal GABA vesicular immunolabeling were observed in any groups. For both transmitters, no differences were detected in the density of immunolabeling within the presynaptic mitochondrial (i.e., metabolic) pool. These results demonstrate that glutamate density is suppressed in the shell region of the nucleus accumbens following withdrawal from 2 weeks of cocaine exposure. The findings also suggest that the motivational aspects that accompany self-administration may participate in this reduction.
Asunto(s)
Cocaína/efectos adversos , Ácido Glutámico/metabolismo , Núcleo Accumbens/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Animales , Cocaína/administración & dosificación , Inmunohistoquímica , Masculino , Microscopía Electrónica , Núcleo Accumbens/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Autoadministración , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoAsunto(s)
Acetilcolina/fisiología , Amígdala del Cerebelo/metabolismo , Dopamina/fisiología , Microdiálisis/métodos , Núcleo Accumbens/metabolismo , Acetilcolina/metabolismo , Amígdala del Cerebelo/química , Animales , Dopamina/metabolismo , Masculino , Núcleo Accumbens/química , Ratas , Ratas Sprague-DawleyRESUMEN
Rats were prepared with two implanted guide shafts, one for microdialysis to measure extracellular dopamine (DA) and acetylcholine (ACh) in the posterior, medial nucleus accumbens (NAc), and the other for microinjection of galanin, neuropeptide Y or saline in the hypothalamic paraventricular nucleus (PVN). There was an increase in DA release and a decrease in ACh in the NAc following microinjections of galanin into the PVN. The effect was observed only in rats for which identical galanin injections induced feeding in separate tests. Ringer injections had no effects. Unlike galanin, neuropeptide Y in the PVN induced eating without altering DA/ACh; whereas earlier results showed that norepinephrine in the PVN works like galanin. These results suggest that galanin initiates feeding, in part, by activating the mesolimbic DA system and suppressing intrinsic cholinergic activity in the NAc. This may prime instrumental behavior with DA while disinhibiting behavior by lowering ACh.
Asunto(s)
Acetilcolina/metabolismo , Dopamina/metabolismo , Conducta Alimentaria/efectos de los fármacos , Galanina/farmacología , Núcleo Accumbens/metabolismo , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Animales , Mapeo Encefálico , Conducta Alimentaria/fisiología , Masculino , Neuropéptido Y/farmacología , Núcleo Hipotalámico Paraventricular/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
It is known that lateral hypothalamic stimulation or self-stimulation can release dopamine in the nucleus accumbens (NAc). The present experiment illustrates that an aversively motivated behavior can also do this. Rats were prepared with microdialysis probes in the NAc and electrodes in the lateral hypothalamus (LH) or medial hypothalamus (MH). Automatic stimulation of the LH increased extracellular dopamine in the NAc 30% as reported earlier. The animals would perform both self-stimulation to turn the current on and stimulation-escape to turn it off, suggesting a combination of reward and aversion. Escape responding increased extracellular dopamine (DA) 100%, even though there was less total stimulation. Automatic stimulation of the MH did the opposite of the LH by decreasing accumbens dopamine (-20%), and the animals would only perform stimulation-escape, indicative of pure aversion. But again, extracellular DA in the NAc increased 100% during escape responding. Thus DA can be released during negative reinforcement when an animal's behavior is reinforced by escape from lateral or medial hypothalamic stimulation. This suggests that DA release was correlated with stimulation-escape behavior, rather than the aversiveness of automatic stimulation.
Asunto(s)
Dopamina/metabolismo , Reacción de Fuga/fisiología , Hipotálamo/fisiología , Núcleo Accumbens/metabolismo , Autoestimulación/fisiología , Animales , Mapeo Encefálico , Estimulación Eléctrica , Electrodos Implantados , Área Hipotalámica Lateral/fisiología , Hipotálamo Medio/fisiología , Masculino , Microdiálisis , Ratas , Ratas Sprague-DawleyRESUMEN
To assess the interaction of dopamine and acetylcholine systems in the rat nucleus accumbens in response to direct D-amphetamine administration, in vivo microdialysis measures of acetylcholine were used during reverse dialysis of amphetamine alone and in combination with D1 and D2 receptor antagonists SCH 23390 and sulpiride, respectively. During a 15-min exposure to amphetamine (50 microM) in the nucleus accumbens, acetylcholine increased to 33% above pre-infusion levels, became maximal at 15 min post-infusion (+41%) and gradually returned to baseline levels by 60 min post-amphetamine. Conversely, amphetamine (1 mM) administration caused a biphasic change in acetylcholine release with a trend toward a decrease (-14%) during exposure followed by a significant increase (+36%) at 30 min post-amphetamine that returned to baseline levels by 60 min after infusion. The increases observed during amphetamine (50 microM) exposure and during recovery from amphetamine (1 mM) were both blocked by co-administration with the D1 antagonist, SCH 23390 (10 microM), but not with the D2 antagonist, sulpiride (10 microM). Co-infusion of sulpiride eliminated the trend toward reduced acetylcholine release observed during 1 mM amphetamine whereas co-administration of SCH 23390 potentiated this decrease. A possible tonic D1 facilitation of nucleus accumbens acetylcholine release was indicated by the consistent reductions in acetylcholine release observed during infusion of SCH 23390. These results suggest that amphetamine administration in the nucleus accumbens induces a bidirectional change in acetylcholine release that is dependent on dose and opposing effects of nucleus accumbens D1 and D2 activation. In general, relatively low doses of amphetamine administered into the nucleus accumbens caused an increase in acetylcholine release that was dependent on dopamine D1 receptors whereas higher doses of amphetamine resulted in a D2-mediated decrease.
Asunto(s)
Acetilcolina/metabolismo , Anfetamina/farmacología , Benzazepinas/farmacología , Núcleo Accumbens/fisiología , Receptores de Dopamina D1/fisiología , Receptores de Dopamina D2/fisiología , Sulpirida/farmacología , Anfetamina/administración & dosificación , Animales , Benzazepinas/administración & dosificación , Antagonistas de Dopamina/administración & dosificación , Antagonistas de Dopamina/farmacología , Infusiones Parenterales , Cinética , Masculino , Microdiálisis , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sulpirida/administración & dosificación , Factores de TiempoRESUMEN
Norepinephrine (NE) was microinjected into the paraventricular nucleus (PVN), while microdialysis was used to monitor extracellular dopamine (DA) and acetylcholine (ACh) in the nucleus accumbens (NAc). The PVN is a site where exogenously administered NE can act through alpha 2 receptors to elicit eating behavior and preference for carbohydrates. It was hypothesized that NE in the PVN acts on a behavior reinforcement system by altering the DA/ACh balance in the NAc. NE microinjections (80 nmol in 0.3 microliter), which effectively elicited feeding in satiated rats in a separate test, caused a significant increase in extracellular DA (109%) and decrease in ACh (-27%) when the same animals were tested in the absence of food. In contrast when the food was available and ingested, ACh increased (51%) instead of decreasing. These results support the hypothesis that a functional link exists between the PVN and the NAc in which DA helps initiate and ACh helps stop appetitive behavior involved in the reinforcement of eating.
Asunto(s)
Acetilcolina/metabolismo , Dopamina/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Norepinefrina/farmacología , Núcleo Accumbens/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Animales , Espacio Extracelular/metabolismo , Masculino , Microdiálisis , Microinyecciones , Núcleo Accumbens/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
A number of experimental results has pointed to a cholinergic involvement in the stress response. Recently, analytical techniques have become available to measure acetylcholine release in vivo during exposure to various stressors. In these experiments, microdialysis was used to monitor acetylcholine output every 15 min in the dorsal hippocampus, amygdala, nucleus accumbens and prefrontal cortex before, during and after 1 h of restraint, including a 15-min session of intermittent tail-shock (1/min, 1 mA, 1-s duration) in rats. In response to the stressful event, acetylcholine release was significantly increased in the prefrontal cortex (186%; p < 0.01) and hippocampus (168%; P < 0.01) but not in the amygdala or nucleus accumbens. The sole effects observed in the amygdala and nucleus accumbens occurred upon release from the restrainer, at which point acetylcholine levels were significantly elevated in both areas (amygdala: 150%; P < 0.05; nucleus accumbens: 13%; P < 0.05). An enhanced acetylcholine release was also evident during this sample period in the hippocampus and prefrontal cortex. These data demonstrate an enhancement of cholinergic activity in response to stress in two acetylcholine projection systems (hippocampus and prefrontal cortex) but not in the intrinsic acetylcholine system of the nucleus accumbens or the extrinsic innervation of the amygdala. Moreover, the data showed that relief from stress was accompanied by a more ubiquitous acetylcholine response that extended to each site tested.