The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum.

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures--the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 microl)--a selective mu-receptor agonist--or nor-binaltorphimine (BNI; 2.5 and 5 microg/0.2 microl)--a selective kappa-receptor antagonist with tardive action--on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region.

Previous spatial learning is required for the development of place preference in rats following chronic administration of and withdrawal from morphine

The positive reinforcing properties of addictive drugs have a primary role in the development of drug dependence. In the field of opiates, great attention has been given to this phenomenon, as well as to the negative properties eliciting craving and inducing relapse during withdrawal. This study was designed to evaluate whether elevated plus-maze (EPM) experienced rats withdrawn from low doses of subcutaneous (s.c.) morphine (10 mg/kg), in which a high anxiety level is the most prominent withdrawal symptom, acquire place preference when submitted to a conflict paradigm in which drug effects are paired with an aversive context: the distal part of the open arms of an EPM. Both the anxiety test and place preference conditioning were measured in the same apparatus, a biased version of the plus-maze. In order to verify the influence of previous EPM spatial learning on the performance of morphine-withdrawn rats, half the animals in this study experienced the EPM prior to treatment. Additional groups were also tested under the influence of morphine effects. The effects of the treatments were quantified through the analysis of three types of measures: anxiety was inferred from the use of the conventional measures (percentage of entries and time spent in the open-arms) and risk-assessment behaviours (frequency of stretched-attending postures - SAP, and time spent at the centre of the maze). Place preference conditioning was evaluated through analysis of the number of entries, total time spent and distance run in the open-arm extremities, which is where the animals were conditioned. The number of closed-arm entries was taken as an index of locomotor activity. Our results showed that (i) EPM naïve rats pre-treated with morphine did not develop place preference, behaving like control rats; (ii) rats that had previous experience in the EPM showed no changes in open-arm avoidance on the second exposure, when compared with rats naïve for this condition; ...(AU)

Previous spatial learning is required for the development of place preference in rats following chronic administration of and withdrawal from morphine

The positive reinforcing properties of addictive drugs have a primary role in the development of drug dependence. In the field of opiates, great attention has been given to this phenomenon, as well as to the negative properties eliciting craving and inducing relapse during withdrawal. This study was designed to evaluate whether elevated plus-maze (EPM) experienced rats withdrawn from low doses of subcutaneous (s.c.) morphine (10 mg/kg), in which a high anxiety level is the most prominent withdrawal symptom, acquire place preference when submitted to a conflict paradigm in which drug effects are paired with an aversive context: the distal part of the open arms of an EPM. Both the anxiety test and place preference conditioning were measured in the same apparatus, a biased version of the plus-maze. In order to verify the influence of previous EPM spatial learning on the performance of morphine-withdrawn rats, half the animals in this study experienced the EPM prior to treatment. Additional groups were also tested under the influence of morphine effects. The effects of the treatments were quantified through the analysis of three types of measures: anxiety was inferred from the use of the conventional measures (percentage of entries and time spent in the open-arms) and risk-assessment behaviours (frequency of stretched-attending postures - SAP, and time spent at the centre of the maze). Place preference conditioning was evaluated through analysis of the number of entries, total time spent and distance run in the open-arm extremities, which is where the animals were conditioned. The number of closed-arm entries was taken as an index of locomotor activity. Our results showed that (i) EPM naïve rats pre-treated with morphine did not develop place preference, behaving like control rats; (ii) rats that had previous experience in the EPM showed no changes in open-arm avoidance on the second exposure, when compared with rats naïve for this condition; (iii) previous spatial learning of the EPM contextual cues was, in fact, a requirement for anxiety-inducing place preference for the open-arms in morphine-withdrawn rats and, (iiii) conditioned place preference was achieved both in rats under the effects of morphine and in withdrawal, probably through the influence of the positive or negative reinforcing effects promoted by the presence or absence of the drug in the central nervous system.

GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine.

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol-a GABA(A) receptors agonist-and DAMGO-a mu-opioid receptors agonist-on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence model. Besides, mechanisms mediated by mu- and GABA(A)-receptors in the CeA appear to exert an inhibitory influence on the anxiety-like behavior induced by withdrawal from acute morphine treatment.

Involvement of the midbrain tectum in the unconditioned fear promoted by morphine withdrawal.

The midbrain tectum structures, dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), are involved in the organization of fear and anxiety states during the exposure to dangerous stimuli. Since opiate withdrawal is associated with increased anxiety in both humans and animals, this study aimed to investigate the possible sensitization of the neural substrates of fear in the midbrain tectum and its influence on the morphine withdrawal-induced anxiety. For the production of drug withdrawal, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of the chronic treatment, independent groups were probed in the elevated plus-maze and open-field tests. Additional groups of animals were implanted with a bipolar electrode into the dPAG or the IC and submitted to the electrical stimulation of these structures for the determination of the freezing and escape thresholds after 48 h of withdrawal. Our results showed that the morphine withdrawal promoted clear-cut levels of anxiety without the somatic signs of opiate withdrawal. Moreover, morphine-withdrawn rats had an increase in the reactivity to the electrical stimulation of the dPAG and the IC. These findings suggest that the increased anxiety induced by morphine withdrawal is associated with the sensitization of the neural substrates of fear in the dPAG and the IC. So, the present results give support to the hypothesis that withdrawal from chronic treatment with morphine leads to fear states possibly engendered by activation of the dPAG and IC, regardless of the production of somatic symptoms.

The blockade of AMPA-kainate and NMDA receptors in the dorsal periaqueductal gray reduces the effects of diazepam withdrawal in rats.

It is well established that the most persistent sign of withdrawal from chronic benzodiazepine use in humans is anxiety. In contrast to other types of drugs of abuse, the emergence of this anxiety does not seem to be linked directly to alterations in the levels of dopamine in the mesolimbic system. Some studies have proposed that fear-like behaviors elicited by benzodiazepine withdrawal could be the result either of alterations in the sensitivity of GABAA receptors or in the neuronal hyperexcitability that results from neuroadaptative responses to chronic treatment, probably mediated by glutamate. The increased fear-like behaviors induced by benzodiazepine withdrawal are similar to the defense reaction displayed by animals exposed to dangerous situations or submitted to electrical or chemical stimulation of the dorsal periaqueductal gray (dPAG), a key structure of the brain aversive system. However, the involvement of the dPAG in drug abuse has been investigated only in the context of the physical effects of drug dependence. Thus, in this study we investigated the effects of injections into the dPAG of the glutamic acid diethyl ester (GDEE) and 2-amino-7-phosphonoheptanoate (AP-7) (AMPA-kainate and NMDA receptors antagonists, respectively) on fear-like behaviors promoted by benzodiazepine withdrawal in rats submitted to aversive events (foot-shocks) immediately before chronic diazepam administration in a conditioning place-preference paradigm, using a light-dark box. Our results showed that inhibition of the glutamatergic neurotransmission in the dPAG reduces the consequence of the diazepam withdrawal in rats, implicating the excitatory amino acids of the dPAG in the modulation of the aversive state induced by benzodiazepine drugs withdrawal.

The distribution of fos immunoreactivity in rat brain following freezing and escape responses elicited by electrical stimulation of the inferior colliculus.

Several sources of evidence indicate that the inferior colliculus also integrates acoustic information of an aversive nature besides its well-known role as a relay station for auditory pathways. Gradual increases of the electrical stimulation of this structure cause in a hierarchical manner alertness, freezing and escape behaviors. Independent groups of animals implanted with bipolar electrodes into the inferior colliculus received electrical stimulation at one of these aversive thresholds. Control animals were submitted to the same procedure but no current was applied. Next, analysis of Fos protein expression was used to map brain areas activated by the inferior colliculus stimulation at each aversive threshold and in the controls. Whereas alertness elicited by stimulation of the inferior colliculus did not cause any significant labeling in any structure studied in relation to the respective control, electrical stimulation applied at the freezing threshold increased Fos-like immunoreactivity in the central amygdaloid nucleus and entorhinal cortex. In contrast, escape response enhanced Fos-like immunoreactivity in the nucleus cuneiform and the dorsal periaqueductal gray matter of the mesencephalon. This evidence supports the notion that freezing and escape behaviors induced by electrical stimulation of the inferior colliculus activate different neural circuitries in the brain. Both defensive behaviors caused significant expression of c-fos in the frontal cortex, hippocampus and basolateral amygdaloid nucleus. This indistinct pattern of c-fos distribution may indicate a more general role for these structures in the modulation of fear-related behaviors. Therefore, the present data bring support to the notion that amygdala, dorsal hippocampus, entorhinal cortex, frontal cortex, dorsal periaqueductal gray matter and cuneiform nucleus altogether play a role in the integration of aversive states generated at the level of the inferior colliculus.

Produçäo e caracterizaçäo de alfa-amilase produzida por Rhizopus sp / Production and characterization of alfa-amilase from Rhizopus sp

A strain of Rhizopus sp. screened among more than 800 filamentous fungi showed great ability to produce a thermostable alfa-amylase by solid state fermentation. The best production was obtained with a bran moisture content of 40 per cent when the enzyme activity reached 60 EU/g. of medium. During the purification procedures, a column of DEAE- Sephadex A-50 separeted the enzyme in two fractions and the larger (85 per cent of the total activity) showed optimum pH in a range from 4.0 to 5.6. Optimum temperature was found at 60-65§ C and in this range no loss of activity was observed after 60 min. of treatment in pH 5,0. Its Km and Vm are, respectively, of 5.0 mg/ml of starch and 10,01 uMol of reducing sugar/min./mg. of protein. Its molecular weight was calculated in 64.000 by gel filtration in Sephadex G-200. The dextrinization power of the enzyme was observed preferentialy on substrates compound by chain with higher ramifications, traht is: amylopectin > starch> amylose. Other aspects of the enzyme pattern action are also discussed