Leda Menescal-de-Oliveira

The cholinergic stimulation of the central amygdala modifying the tonic immobility response and antinociception in guinea pigs depends on the ventrolateral periaqueductal gray

Tonic immobility (TI), also known as death feigning or animal hypnosis, is a reversible state of motor inhibition that is triggered by postural inversion and/or movement restraining maneuvers but also by repetitive stimulation and pressure on body parts. Our previous studies demonstrated that cholinergic stimulation of the central amygdala (CEA) decreases the duration of TI in guinea pigs. Some reports have demonstrated that electrical or chemical stimulation of the CEA promotes antinociception. Evidence suggests that the CEA performs part of its functions by means of a connection with the ventrolateral periaqueductal gray (vlPAG). In the current study, we investigated the participation of a possible functional and anatomical CEA-vlPAG connection in guinea pigs in the regulation of the TI response and antinociception. Our results showed that the functional CEA-vlPAG connection is essential for the participation of the CEA in the modulation of TI and of antinociception. The reversible exclusion of the vlPAG by means of microinjection of 2% lidocaine blocked the inhibitory effect on TI duration and the antinociceptive effect, as determined by a decrease of the vocalization index (VI) obtained with the administration of carbachol (2.7 nmol/0.2 microl) into the CEA. On the other hand, the exclusion of the CEA by lidocaine did not block the antinociception or the increase in TI induced by microinjection of CCh into the vlPAG. Finally, microinjection of the retrograde neurotracer Fast Blue into the CEA or into the vlPAG demonstrated the existence of a reciprocal anatomical connection between the CEA and vlPAG.

Ventrolateral periaqueductal gray matter and the control of tonic immobility

Tonic immobility is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. The periaqueductal gray matter (PAG) contains neural circuits involved in descending pain modulation, as well as in the modulation of TI. We have reported previously that the cholinergic stimulation of the ventrolateral PAG increases the duration of TI in guinea pigs. In the present study, we attempted to characterize further the modulation of TI by pharmacological alteration of the neurochemistry of the ventrolateral PAG circuitry. We observed that both cholinergic (carbachol, 5.4 nmol/0.2 microl) and opioidergic stimulations (morphine, 4.48 nmol/0.2 microl) of the ventrolateral PAG increase the duration of TI and that these effects can be reversed by pre-treatment with naloxone (2.74 nmol/0.2 microl). Our results also showed that microinjection of the GABAergic agonist muscimol (1, 0.5, and 0.26 nmol/0.2 microl) decreased the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol/microl) increased it. Moreover, we observed that preadministration of muscimol (0.13 nmol/0.2 microl) at a dose that had no effect per se at this site antagonized the potentiating effect of morphine. Our results suggest that this modulation of TI from the ventrolateral PAG circuitry is accomplished by a complex interaction of cholinergic, opioidergic, and GABAergic mechanisms, similar to that proposed for descending antinociceptive circuits.

Involvement of the Cholinergic System and Periaqueductal Gray Matter in the Modulation of Tonic Immobility in the Guinea Pig

Unilateral microinjection of carbachol (CCh, 1.0 microg/0.2 microl) into the ventrolateral periaqueductal gray matter (vPAG) increased the duration of tonic immobility (TI) episodes induced by postural inversion and by movement restriction maneuvers in adult male guinea pig (Cavia porcellus), while stimulation with the same drug at the same concentration into the dorsolateral and dorsomedial periaqueductal gray matter (dl/dmPAG) decreased the duration of TI. Pretreatment with atropine (7.6 microg/0.4 microl) showed that the action of CCh is mediated by muscarinic receptors in the ventrolateral PAG but not in the dorsomedial and dorsolateral regions. These data suggest that the PAG and the cholinergic system are involved in the modulation of TI episodes and that different regions of the guinea pig PAG play distinct roles in the organization of this behavior.

The parabrachial region as a possible region modulating simultaneously pain and tonic immobility.

Unilateral microinjection of carbachol (1 microgram) into the dorsal parabrachial region (PBR) of conscious guinea pigs produced a 100% increase in the duration of restraint-induced tonic immobility (TI) episodes. In another group of animals with a subcutaneous electrode introduced into the thigh region, microinjections of equivalent doses of the same drug in similar sites also significantly reduced the motor defense and vocalization responses elicited by the application of a noxious electrical stimulus to the skin. Both effects were blocked by pretreatment with atropine. The possible simultaneous activation of mechanisms modulating TI and the response to a noxious stimulus may be of adaptive importance since analgesia may reinforce immobility to permit the use of other defense mechanisms in a situation of prey/predator confrontation.

Central nucleus of the amygdala and the control of tonic immobility in guinea pigs.

Tonic immobility (TI) is a temporary state of profound motor inhibition induced by situations that supposedly generate intense fear, with the objective to protect the animal from attacks by predators. A previous study by our group demonstrated that cholinergic stimulation of the central, basolateral, and lateral posterior nuclei of the amygdala decreases the duration of TI in guinea pigs. In the current study, we attempted to investigate the effects of cholinergic, opioidergic, and gamma-aminobutyric acid (GABA)ergic stimulation of the central amygdala (CEA) on TI modulation. We observed that both cholinergic (carbachol, 2.7 nmol/0.2 microl) and opioidergic (morphine, 2.2 and 4.4 nmol/0.2 microl) stimulation of the CEA decreased the duration of TI and that these effects could be reversed by pretreatment with naloxone (1.3 and 2.7 nmol/0.2 microl). Our results also showed that microinjection of the GABAergic agonist muscimol (0.26 nmol/0.2 microl) reduced the duration of TI episodes, whereas microinjection of the GABAergic antagonist bicuculline (BIC, 1 nmol/0.2 microl) increased it. Thus, the present experiments demonstrated that cholinergic, opioidergic, and GABAergic systems of the CEA have an inhibitory action on the duration of TI in guinea pigs. Furthermore, the current study suggests an interaction of cholinergic and opioidergic mechanisms. In addition, the GABAergic circuit of the CEA has a tonic inhibitory influence on the duration of TI and is mediated by GABA(A) receptors.

Role of cholinergic, opioidergic and GABAergic neurotransmission of the dorsal hippocampus in the modulation of nociception in guinea pigs

AIMS Several physiological, pharmacological and behavioral lines of evidence suggest that the hippocampal formation is involved in nociception. The hippocampus is also believed to play an important role in the affective and motivational components of pain perception. Thus, our aim was to investigate the participation of cholinergic, opioidergic and GABAergic systems of the dorsal hippocampus (DH) in the modulation of nociception in guinea pigs. MAIN METHODS The test used consisted of the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal. KEY FINDINGS Our results showed that, in guinea pigs, microinjection of carbachol, morphine and bicuculline into the DH promoted antinociception, while muscimol promoted pronociception. These results were verified by a decrease and an increase, respectively, in the vocalization index in the vocalization test. This antinociceptive effect of carbachol (2.7 nmol) was blocked by previous administration of atropine (0.7 nmol) or naloxone (1.3 nmol) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol) into the DH was prevented by pretreatment with naloxone (1.3 nmol) or muscimol (0.5 nmol). At doses of 1.0 nmol, muscimol microinjection caused pronociception, while bicuculline promoted antinociception. SIGNIFICANCE These results indicate the involvement of the cholinergic, opioidergic and GABAergic systems of the DH in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalin from interneurons of the DH, which would inhibit GABAergic neurons, resulting in antinociception.

Role of the amygdaloid nuclei in the modulation of tonic immobility in guinea pigs.

Tonic immobility (TI) is thought to represent the terminal reaction in the chain of antipredatory responses involved in maintaining survival. TI is an inhibitory behavioral response in which the animal presents a significant decrease in body activity and responsiveness to the environment induced by some form of physical restraint. This response is induced in the laboratory by inversion of the animal and brief postural contention of its movements. In nature, the TI response may be triggered by some threatening or predatory stimulus, indicating the physical contact between response occurs when there is physical contact between prey and predator. In this case, the physical inactivity of the prey may prevent the continuation of the attack. The neural substrate of this response is not well known, and the objective of the present study was to investigate the effect of cholinergic stimulation of amygdala regions on TI modulation in guinea pigs. Microinjection of carbachol (0.5 microg/0.2 microL) into the central (CEA), basolateral (BLA), and lateral posterior (LPA) nuclei of the amygdala promoted a reduction in the duration of TI episodes. Pretreatment with atropine (0.5 microg/0.2 microL) showed that the action of carbachol is mediated by muscarinic receptors.

Serotoninergic activation of the basolateral amygdala and modulation of tonic immobility in guinea pig

Tonic immobility (TI), also known as death feigning or animal hypnosis, is a reversible state of motor inhibition that is not only triggered by postural inversion and/or movement restraining maneuvers but also by repetitive stimulation and pressure on body parts. Evidence has demonstrated that the basolateral nucleus of the amygdala (BLA) is particularly associated with defensive behavior that involves the emotional states of fear and anxiety. In addition, some reports have demonstrated that serotonin (5-HT) released in the amygdala is increased during states of stress and anxiety, principally in the BLA. In the present study, we investigated the effects of serotonergic activation of the BLA on the duration of TI. The results showed that the microinjection of 5-HT (3.0 microg) into the BLA decreased the duration of TI. Similarly, the administration of a 5-HT1A agonist (0.1 microg of 8-hydroxy-dipropylaminotretalin) or 5-HT2 agonist (0.1 microg of alpha-methyl-5-HT) into the BLA reduced the TI duration. The effect of 5-HT2 agonist was reversed by pretreatment with a dose that had no effect per se (0.01 microg) of ketanserin (5-HT2 receptor antagonists) into the BLA. Moreover, the activation of 5-HT1A and 5-HT2 receptors in the BLA did not alter the spontaneous motor activity in the open field test. The results of the present study indicate that the serotonergic system of the BLA possibly produces a reduction in fear and/or anxiety that reduces the TI duration in guinea pigs, but this is not due to increased spontaneous motor activity induced by serotonergic activation, which might affect TI duration non-specifically.

Role of opioidergic and GABAergic neurotransmission of the nucleus raphe magnus in the modulation of tonic immobility in guinea pigs

Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. Previous results from our laboratory have demonstrated that nucleus raphe magnus (NRM) is also a structure involved in the modulation of TI behavior, as chemical stimulation through carbachol decreases the duration of TI in guinea pigs. In view of the fact that GABAergic and opioidergic circuits participate in the regulation of neuronal activity in the NRM and since these neurotransmitters are also involved in the modulation of TI, the objective of the present study was to evaluate the role of these circuits of the NRM in the modulation of the behavioral TI response. Microinjection of morphine (4.4 nmol/0.2 microl) or bicuculline (0.4 nmol/0.2 microl) into the NRM increased the duration of TI episodes while muscimol (0.5 nmol/0.2 microl) decreased it. The effect of morphine injection into the NRM was blocked by previous microinjection of naloxone (2.7 nmol/0.2 microl). Muscimol at 0.25 nmol did not produce any change in TI duration; however, it blocked the increased response induced by morphine. Our results indicate a facilitatory role of opioidergic neurotransmission in the modulation of the TI response within the NRM, whereas GABAergic activity plays an inhibitory role. In addition, in the present study the modulation of TI in the NRM possibly occurred via an interaction between opioidergic and GABAergic systems, where the opioidergic effect might be due to inhibition of tonically active GABAergic interneurons.

The lateral hypothalamus in the modulation of tonic immobility in guinea pigs.

THE lateral hypothalamus has been reported to be involved in the organization of aggression and predatory attack but not in behavioral inhibition responses such as tonic immobility (TI). TI may be defined as an inborn behavioral inhibition characterized by profound physical inactivity and relative lack of responsiveness to the environment, triggered by an intense sensation of fear generated during prey—predator confrontation. Our study indicates that cholinergic stimulation of anterior regions of the lateral hypothalamus of guinea pigs potentiates the duration of TI episodes, while stimulation of medial and posterior regions of this structure promotes a decrease in TI duration, suggesting that the lateral hypothalamus modulates the duration of TI episodes in a differentiated manner.