Pro-inflammatory cytokines, IL-1ß and TNF-α, produce persistent compromise in tonic immobility defensive behaviour in endotoxemia guinea-pigs.

AIM: Sepsis has been associated with acute behavioural changes in humans and rodents, which consists of a motivational state and an adaptive response that improve survival. However, the involvement of peripheral cytokines synthesized during systemic inflammation as modulators of the tonic immobility (TI) defensive behaviour remains a literature gap. Our purposes were to characterize the TI defensive behaviour in endotoxemia guinea-pigs at acute phase and after recovery from the initial inflammatory challenge. Furthermore, we investigated whether peri-aqueductal grey matter (PAG) exists as a brain structure related to this behaviour and also pro-inflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, act at this mesencephalic nucleus. METHODS: Endotoxemia was induced by lipopolysaccharide (LPS) administration in guinea-pigs. The parameters evaluated included TI defensive behaviour, survival, cytokines production, as well as neuronal activation and apoptosis in the PAG. RESULTS: Endotoxemia guinea-pigs exhibited a reduction in the duration of TI episodes, starting at 2 h after LPS administration and persisting throughout the experimental period evaluated over 7 days. Moreover, endotoxemia increased the c-FOS immunoreactivity of neurones in the ventrolateral PAG (vlPAG), as well as the caspase-3 expression. The LPS microinjection into vlPAG reproduces the same compromise, that is a decrease in the duration of TI defensive behaviour, observed after the peripheral administration. The immunoneutralization against IL-1ß and TNF-α into vlPAG reverts all the effects produced by peripheral LPS administration. CONCLUSION: Our findings confirm that vlPAG is an important brain structure involved in the behavioural alterations induced by endotoxemia, possibly changing the neuronal activity caused by pro-inflammatory cytokines produced peripherally.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Cholinergic-opioidergic interaction in the central amygdala induces antinociception in the guinea pig.

Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs.

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. 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. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 +/- 6 and 2 +/- 1 in phases 1 and 2) and lickings (6 +/- 2 and 1 +/- 1 in phases 1 and 2) in the formalin test when compared with control (75 +/- 13 and 22 +/- 6 flinches in phases 1 and 2; 28 +/- 7 and 17 +/- 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 +/- 0.1 and 0.53 +/- 0.13, respectively) when compared with control (-0.10 +/- 0.08 at 30 min and -0.09 +/- 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. 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. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 + or - 6 and 2 + or - 1 in phases 1 and 2) and lickings (6 + or - 2 and 1 + or - 1 in phases 1 and 2) in the formalin test when compared with control (75 + or - 13 and 22 + or - 6 flinches in phases 1 and 2; 28 + or - 7 and 17 + or - 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 0.1 and 0.53 + or - 0.13, respectively) when compared with control (-0.10 + or - 0.08 at 30 min and -0.09 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses

Baroreceptor control of heart rate in the awake toad: peripheral autonomic effectors and arterial baroreceptor areas.

Systemic injection of sodium nitroprusside (30 microg/kg, i.v.) in the awake Bufo paracnemis toad induced a fall in arterial blood pressure and tachycardia. This tachycardia, but not the hypotension, was significantly reduced in toads with bilateral electrolytic lesion of the caudal and commissural regions of the solitary tract nucleus and in animals with transection of the spinal cord, 2 mm below the obex. This indicates that the tachycardia is reflex, depends on the integrity of the solitary tract nucleus and is due to descending spinal autonomic activation. Pretreatment with propranolol (4 mg/kg, i.v.) significantly reduced the tachycardia but did not block it completely, showing the importance of beta-adrenoceptors in its genesis. The reflex increase in heart rate induced by nitroprusside was not statistically significant in animals with bilateral section of the laryngeal nerve, whose baroreceptor fibers originate from the pulmocutaneous artery or in animals in which the bilateral section of the laryngeal nerve was performed together with section of the glossopharyngeal nerves, which incorporate fibers originating from the carotid labyrinth. The reduction of the reflex tachycardia was significant in toads with aortic arch denervation alone or combined with section of the laryngeal nerves or in animals with complete denervation of the three baroreceptors areas. These results suggest that the region of the aortic arch, when submitted to unloading, is the most important baroreceptor zone for cardiac compensation in toads.

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.

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.

Participation of the medial and anterior hypothalamus in the modulation of tonic immobility in guinea pigs.

Tonic immobility (TI) is an inhibitory behavioral response during which the animal presents profound physical inactivity and a relative lack of response to the environment. This response is induced in the laboratory by postural inversion of the animal and brief postural contention of its movements. In nature, the 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 hypothalamic regions on TI modulation in guinea pigs (Cavia porcellus). Microinjection of carbachol (1.0 microg/0.2 microL) into the anterior hypothalamus promoted an increase in the duration of TI episodes. Microinjection of carbachol into the ventro- and dorsomedial hypothalamus, however, promoted a reduced duration of TI episodes. Pretreatment with atropine (0.5 microg/0.2 microL) showed that the action of carbachol is mediated by muscarinic receptors in the anterior and ventromedial hypothalamus but not in the dorsomedial hypothalamus. The results suggest that the hypothalamic regions may play different roles in the organization of defensive behavioral responses such as TI.

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 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.

Temporal modulation of antinociception by reciprocal connections between the dorsomedial medulla and parabrachial region.

Microinjection of carbachol into the dorsal parabrachial regio (PBRd) of guinea pigs induces analgesia from the 5th to the 15th min postinjection, as evaluated by the reduction of the vocalization in response to an electric shock applied to one paw. When reversible blockade of the dorsomedial medulla or specifically of the nucleus tractus solitarius (NTS) is performed with xylocaine 5 min after microinjection of carbachol into the PBRd, the analgesic effect continues up to the 45th and to the 60th min, respectively. Blockade of the dorsomedial medulla is achieved by topical application of xylocaine to the area postrema (AP) or microinjection of the drug into the NTS. A prolongation of the duration of the analgesic effect also occurs after the inverse procedure, i.e., after reversible blockade of the PBRd 5 min after topical application of carbachol (1 microgram/microliter)to the AP or microinjection of carbachol into the NTS. In this case, the analgesic action, which lasted up to 30 min when carbachol was applied to the AP and 60 min when microinjected into the NTS, was prolonged up to 60 min and to 80 min, respectively, after reversible blockade of PBR. The present data suggest that the reciprocal connections between the different regions of the dorsomedial medulla and the PBR play an important role in the modulation of the duration of the analgesic effect, and that this fact may be of adaptive importance in the defensive analgesia that occurs in the confrontation between prey and predator.

Participation of the periaqueductal gray matter in the modulation of tonic immobility in the guinea pig.

Unilateral microinjection of carbachol (CCh, 1 microgram/0.2 microliter) into the specific sites in the ventral and ventrolateral portions of the periaqueductal gray (PAG) matter, which is known to be involved in analgesia, increases the duration of restraint-induced tonic immobility (TI) episodes induced in 23 adult male guinea pigs (Cavia porcellus). Mean duration of TI episodes was 107 +/- 16.38 s in the control group and increased to 220.7 +/- 40.24 s in the group microinjected with CCh. The potentiating effect of carbachol on TI duration was blocked by pretreatment with atropine (7.6 micrograms/0.4 microliter). These data suggest that PAG and the cholinergic system are involved in the modulation of the motor inhibition characteristic of TI which may be activated by the same stimuli that induce defensive analgesia.

Participation of the periaqueductal gray matter in the modulation of tonic immobility in the guinea pig

Unilateral microinjection of carbachol (CCh, 1 microgram/0.2 microliter) into the specific sites in the ventral and ventrolateral portions of the periaqueductal gray (PAG) matter, which is known to be involved in analgesia, increases the duration of restraint-induced tonic immobility (TI) episodes induced in 23 adult male guinea pigs (Cavia porcellus). Mean duration of TI episodes was 107 +/- 16.38 s in the control group and increased to 220.7 +/- 40.24 s in the group microinjected with CCh. The potentiating effect of carbachol on TI duration was blocked by pretreatment with atropine (7.6 micrograms/0.4 microliter). These data suggest that PAG and the cholinergic system are involved in the modulation of the motor inhibition characteristic of TI which may be activated by the same stimuli that induce defensive analgesia.

Alteration of the motor response to an acute painful stimulus during tonic immobility.

The motor responses caused by liminal (1.0 and 3.0 mA, applied for 3 s) and subliminal electrical stimuli (40% below the liminal value) applied to the dental pulp of the upper incisors of adult guinea pigs (N = 41) are more intense during tonic immobility (TI) than in control situations. Tonic immobility is a temporary state of profound motor inhibition elicited by some form of physical restraint in our experiment induced by maneuvers of lateral postural inversion and movement restraint. This suggests the occurrence of hyperalgesia in the trigeminal territory in situations of direct confrontation with a predator, as in the case for tonic immobility. This hyperalgesia may protect in a reflex manner vital regions of the head and neck from fatal bites inflicted by the predator, but without interrupting the state of immobility, since in our experiments the electric stimuli had no disruptive effect on TI episodes.

Alteration of the motor response to an acute painful stimulus during tonic immobility

The motor responses caused by liminal (1.0 and 3.0 mA, applied for 3 s) and subliminal electrical stimuli (40 below the liminal value) applied to the dental pulp of the upper incisors of adult guinea pigs (N = 41) are more intense during tonic immobility (TI) than in control situations. Tonic immobility is a temporary state of profound motor inhibition elicited by some form of physical restraint in our experiment induced by maneuvers of lateral postural inversion and movement restraint. This suggests the occurrence of hyperalgesia in the trigeminal territory in situations of direct confrontation with a predator, as in the case for tonic immobility. This hyperalgesia may protect in a reflex manner vital regions of the head and neck from fatal bites inflicted by the predator, but without interrupting the state of immobility, since in our experiments the electric stimuli had no disruptive effect on TI episodes.

The basal midbrain as a region modulating the level of alerting in the toad, Bufo paracnemis.

Reversible thiopental blockade of sites located in the basal midbrain of conscious toads bearing chronically implanted cannulae induced a response in which the animal withdrew to the bottom of the experimental box with flexed head and forelegs. Partial or total eyelid closure, myosis, and reduced ventilatory movements also occurred. No significant changes in arterial pressure levels or heart rate occurred and the reflex wiping response to the application of a painful stimulus (acid) to the skin was also unaffected. However, the somatic and visceral responses to nonpainful stimuli were reduced. There was a decrease, but not abolition, of forebrain waves in the 11 Hz range grouped in spindles, perhaps due to the reduced ventilatory drive. On the basis of these findings and of previous data from our laboratory and others on other nonmammalian groups, we postulate the existence of neurons involved in the modulation of behavioral and visceral alerting in the basal midbrain.

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.

Origin of the telencephalic spindles of the toad Bufo paracnemis

Spindles (8-11 Hz and up to 100 uV in amplitude) recorded on the surface of the surface of the telencephalic hemispheres and olfaactory bulbs of the conscious toad disappear after transection between these two structures, indicating that their presence depends onm the integrity of their interconnections. Spindles continue to be present, although in reduced numbers, after transection between the hemispheres and the diencephalon, indicating that caudal regions are not essential for generating spindles but modulate the neurons responsible for spindle genesis. In contrast, the olfactoryy nerves, in addition to their known phasis activity on the spindles, exert a tonic action since after their section there is a change in the duration and amplitude of component waves

Disruptive effect of the mesencephalic reticular formation on tonic immobility in guinea pigs.

Cholinergic stimulation of the mesencephalic reticular formation with carbachol impairs the induction of tonic immobility (TI) by restraining maneuvers and reduces the duration of immobility episodes in guinea pigs. This finding apparently disagrees with the hypothesis that environmental monitoring occurs during TI which permits the animal to evaluate the best time for escape. It is possible that this monitoring involves circuits and neurotransmitters other than the ascending cholinergic system originating in the mesencephalic reticular formation.