Norberto Cysne Coimbra

Neural substrate of defensive behavior in the midbrain tectum

It has been shown that the gradual increase in the intensity of electrical stimulation of the dorsal periaqueductal gray (DPAG), deep layers of the superior colliculus (DLSC) and inferior colliculus of rats induces, in a progressive manner, characteristic aversive responses such as arousal, freezing, and escape behavior. The DPAG-DLSC together with the periventricular gray substance of the diencephalon, amygdala and the inferior colliculus, constitute the neural substrate of aversion in the brain. In general, the behavioral responses induced by midbrain tectum stimulation are accompanied by increases in the mean arterial blood pressure, heart rate, and respiration. Both the behavioral and autonomic consequences of electrical stimulation of the mesencephalic tectum have been shown to be attenuated by minor tranquilizers, probably through enhancement of GABAergic neurotransmission. Besides GABAergic mechanisms several lines of evidence have clearly implicated opioid, serotonergic, and excitatory amino acids-mediated mechanisms in the control of the neural substrates commanding defensive behavior in the brain aversive system.

GABAergic nigro-collicular pathways modulate the defensive behaviour elicited by midbrain tectum stimulation.

Midbrain tectum (MT) structures such as the dorsal periaqueductal gray matter and deep layers of superior colliculus are well-known for the organization and generation of defensive behaviour. Electrical stimulation or microinjection of GABA antagonists into these structures produce aversive behaviour. In order to determine whether the nigrocollicular GABAergic fibers exert some control over this behaviour, rats bearing neurochemical lesions with kainic acid in the substantia nigra, pars reticulata (SNpr) and compacta (SNpc), were submitted to MT microinjections of bicuculline or electrical stimulation at aversive thresholds. The same procedure was carried out after enhancement or inhibition of GABAergic transmission in SNpr through microinjections of muscimol or bicuculline, respectively. Animals with SNpr neurochemical lesion exhibited a significant decrease in the aversive thresholds and an increase in the responsiveness to bicuculline microinjections. An opposite effect was observed following microinjections of bicuculline into the SNpr. The enhancement of the GABAergic transmission into the SNpr following microinjection of muscimol mimicked the effects produced by the lesion with kainic acid. These results suggest an inhibitory control of GABAergic fibers from the substantia nigra, pars reticulata, on aversive behaviour induced by midbrain stimulation.

Effects of lesions of amygdaloid nuclei and substantia nigra on aversive responses induced by electrical stimulation of the inferior colliculus

Stimulation of the central nucleus of the inferior colliculus causes defensive behavior. In this work we examined the influence of lesions of brain structures involved in the expression of fear, such as periaqueductal gray matter, amygdala, and substantia nigra pars reticulata (SNpr), on these aversive responses. Thus, rats were implanted with an electrode in the central nucleus of the inferior colliculus, for the determination of the thresholds of alertness, freezing, and escape responses. Each rat also bore a cannula implanted in the periaqueductal, amygdala or Snpr for injection of the neurotoxin N-methyl-D-aspartate (8 micrograms/0.8 microliters). The data obtained show that lesion of the central nucleus of the amygdala increases the thresholds of aversive responses whereas lesion of the basolateral complex decreases the threshold of these responses. Lesion of the Snpr increased the aversive consequences of the electrical stimulation of the inferior colliculus whereas periaqueductal gray lesions, either dorsal or ventral regions, did not change these responses. From the evidences obtained in this work, it is suggested that the expression of the defensive behavior induced by activation of the neural substrates of the inferior colliculus does not seem to depend on the integrity of the periaqueductal gray. On the contrary, the basolateral complex inhibits and the central nucleus amplifies the aversive responses integrated in the inferior colliculus. Furthermore, SNpr seems also to be an important motor output for the defensive behavior induced by stimulation of the inferior colliculus, in agreement with what has been suggested for other brain structures implicated in the expression of fear.

Defensive reactions evoked by activation of NMDA receptors in distinct sites of the inferior colliculus

The inferior colliculus (IC) is primarily involved in conveying auditory information to higher cortical structures. Recently we have shown that this structure may also be part of a brain system commanding defensive behaviour. There is evidence that the neural substrates responsible for defensive behaviour in the inferior colliculus are regulated by GABAergic, serotonergic and opioid mechanisms and that these substrates may also be depressed by benzodiazepines as part of their anxiolytic action. Here we present evidence for the involvement of excitatory amino acids in the IC in the expression of defensive reactions. Microinjections of NMDA (5-40 nmol)--an excitatory amino acid--into the ventrolateral division of the central nucleus of the IC of rats placed inside a circular arena induced aversive reactions, characterized by running, rearing, and jumping. This hyperactivity was interspersed by immobility states which often progressed to convulsive seizures. These reactions were inhibited by the NMDA specific antagonist AP7 previously microinjected into the IC. It is suggested that NMDA receptor mediated mechanisms are called into play during the display of the defensive behaviour.

Neuroanatomical approaches of the tectum-reticular pathways and immunohistochemical evidence for serotonin-positive perikarya on neuronal substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear-induced analgesia.

Deep layers of the superior colliculus, the dorsal periaqueductal gray matter and the inferior colliculus are midbrain structures involved in the generation of defensive behavior and fear-induced anti-nociception. Local injections of the GABA(A) antagonist bicuculline into these structures have been used to produce this defense reaction. Serotonin is thought to be the main neurotransmitter to modulate such defense reaction in mammals. This study is the first attempt to employ immunohistochemical techniques to locate serotonergic cells in the same midbrain sites from where defense reaction is evoked by chemical stimulation with bicuculline. The blockade of GABA(A) receptors in the neural substrates of the dorsal mesencephalon was followed by vigorous defensive reactions and increased nociceptive thresholds. Light microscopy immunocytochemistry with streptavidin method was used for the localization of the putative cells of defensive behavior with antibodies to serotonin in the rats midbrain. Neurons positive to serotonin were found in the midbrain sites where defensive reactions were evoked by microinjection of bicuculline. Serotonin was localized to somata and projections of the neural networks of the mesencephalic tectum. Immunohistochemical studies showed that the sites in which neuronal perikarya positive to serotonin were identified in intermediate and deep layers of the superior colliculus, and in the dorsal and ventral columns of the periaqueductal gray matter are the same which were activated during the generation of defense behaviors, such as alertness, freezing, and escape reactions, induced by bicuculline. These findings support the contention that serotonin and GABAergic neurons may act in concert in the modulation of defense reaction in the midbrain tectum. Our neuroanatomical findings indicate a direct neural pathway connecting the dorsal midbrain and monoaminergic nuclei of the descending pain inhibitory system, with profuse synaptic terminals mainly in the pontine reticular formation, gigantocellularis nucleus, and nucleus raphe magnus. The midbrain tectum-gigantocellularis complex and midbrain tectum-nucleus raphe magnus neural pathways may provide an alternative output allowing the organization of the fear-induced anti-nociception by mesencephalic networks.

Neuroanatomical and psychopharmacological evidence for interaction between opioid and GABAergic neural pathways in the modulation of fear and defense elicited by electrical and chemical stimulation of the deep layers of the superior colliculus and dorsal periaqueductal gray matter

The effects of central administration of opioid antagonists on the aversive responses elicited by electrical (at the freezing and escape thresholds) or chemical stimulation (crossings, rearings, turnings and jumps, induced by microinjections of bicuculline) of the midbrain tectum were determined. Central microinjections of naloxone and naltrexone in the mesencephalic tectum caused a significant increase in the freezing and escape thresholds elicited by electrical midbrain tectum stimulation. Furthermore, both opioid antagonists caused a significant decrease in the mean incidence of aversive behavioral responses induced by microinjections of bicuculline in the deep layers of the superior colliculus (DLSC) and in dorsal aspects of the periaqueductal gray matter (DPAG), as compared with controls. These findings suggest an opioid modulation of the GABAergic inhibitory inputs controlling the aversive behavior elicited by midbrain tectum stimulation. In fact, immunohistochemical evidence suggests that the dorsal mesencephalon is rich in beta-endorphin-containing neurons and fibers with varicosities. Iontophoretical microinjections of the neurotracer biodextran in the substantia nigra, pars reticulata (SNpr), show nigro-tectal pathways connecting SNpr with the same neural substrate of the DPAG rich in neuronal cells immunoreactive for opioid peptides. Labeled neurons of the DLSC and periaqueductal gray matter send inputs with varsicosities to ipsi- and contralateral DPAG and ipsilateral SNpr. These findings, in addition to the psychopharmacological evidence for the interaction between opioid and GABAergic mechanisms, offer a neuroanatomical basis of a possible presynaptic opioid inhibition of GABAergic nigro-tectal neurons modulating the fear in aversive structures of the cranial mesencephalon, in a short link, and maybe through a major neural circuit, also in GABA-containing perikarya of nigro-tectal neurons.

Effects of acute and chronic fluoxetine and diazepam on freezing behavior induced by electrical stimulation of dorsolateral and lateral columns of the periaqueductal gray matter

The defensive responses induced by electrical stimulation of the dorsal periaqueductal gray matter (dPAG) of the rat have been proposed as a model of panic attacks in humans. In the present study we investigated the acute and chronic effects of fluoxetine and diazepam on freezing and escape reactions elicited by electrical stimulation of the dorsolateral (dlPAG) and lateral (lPAG) columns of the periaqueductal gray matter (PAG). The frequencies of crossing, rearing, bouts of micturition and fecal boli were also recorded. Electrodes were unilaterally implanted in the brainstem aimed at the PAG. Drug treatments were given daily for 2 weeks with fluoxetine (5, 10 and 20 mg/kg ip), a selective inhibitor of serotonin reuptake, diazepam (1, 2 and 4 mg/kg ip), or saline. Drug effects were assessed acutely (15 min after the first injection) and chronically (15 min after the 14th injection). Chronic, but not acute, administration of fluoxetine caused a significant increase in the threshold of freezing without affecting the escape response elicited by dlPAG/lPAG stimulation. This characteristic pattern of effects could not be attributed to motor deficit, since this drug did not change the number of crossings and rearings. In contrast, no significant threshold changes were observed following acute and chronic treatment with diazepam. These data give further evidence for (a) an antiaversive effect of chronic treatment with fluoxetine, which caused a selective reduction in freezing behavior and neurovegetative responses associated with fearlike reaction elicited by dlPAG/lPAG electrical stimulation; (b) the involvement of the dlPAG and lPAG in the generation and organization of defensive responses and that freezing may probably be associated with panic attacks; and (c) the lack of effect of diazepam in this model is in line with its inefficacy as a panicolytic drug. The study of the unconditioned freezing behavior evoked by dlPAG/lPAG stimulation may constitute a new and interesting model for the study of panic disorder.

Evidence for the involvement of serotonin in the antinociception induced by electrical or chemical stimulation of the mesencephalic tectum.

A great deal of evidence has shown that electrical stimulation or microinjections of GABAA blockers, such as bicuculline, into the midbrain tectum (MT) produce escape behavior, which has been associated to fear. This study was aimed to examine the characteristics of the analgesia that follows the escape behavior induced by electrical (freezing and escape thresholds) and chemical (bicuculline microinjections) stimulation of the midbrain tectum. Immediately after the expression of the aversive responses the rats were submitted to the tail-flick test. The obtained results show that analgesia always follows aversive responses integrated at the MT level regardless of the kind of stimulation applied. The antinociceptive effects induced by either electrical or chemical stimulation of the MT were not antagonized by central microinjections of naloxone. On the other hand, the non-specific serotonin antagonist methysergide microinjected into the MT was effective in antagonizing the analgesia induced by any of the aversive stimulations. Based on these results we suggest that serotonin, but not opioid mechanisms, may be involved in the integration of antinociceptive responses to stimulation of the midbrain tectum.

Pharmacological and Biochemical Aspects of GABAergic Neurotransmission: Pathological and Neuropsychobiological Relationships

Abstract1. The GABAergic neurotransmission has been implicated in the modulation of many neural networks in forebrain, midbrain and hindbrain, as well as, in several neurological disorders.2. The complete comprehension of GABA system neurochemical properties and the search for approaches in identifying new targets for the treatment of neural diseases related to GABAergic pathway are of the extreme relevance.3. The present review will be focused on the pharmacology and biochemistry of the GABA metabolism, GABA receptors and transporters. In addition, the pathological and psychobiological implications related to GABAergic neurotransmission will be considered.

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.