Elsevier

Experimental Neurology

Volume 197, Issue 1, January 2006, Pages 93-112
Experimental Neurology

Regular Article
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

https://doi.org/10.1016/j.expneurol.2005.08.022Get rights and content

Abstract

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 GABAA 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 GABAA 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 rat's 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.

Introduction

Studies on the neural bases of the defensive responses evoked by activation of the prosencephalic, diencephalic, and mesencephalic structures have been performed with the aim of contributing for the understanding of the neuromorphological basis of fear-induced reactions and, by extension, of the neurobiology of some psychiatric diseases, such as panic disorders (Coimbra and Brandão, 1993, Grove et al., 1997, Gorman et al., 2000, de Oliveira et al., 2000, Coimbra et al., 2000, Eichenberger et al., 2002, Vianna et al., 2003, Borelli et al., 2004, De Oliveira et al., 2005). Among them, histochemical methods and immunohistochemical techniques for identification of protooncogenes activity have been very useful (Fischer and Zeman, 1959, Fischer et al., 1961, Ceccatelli et al., 1989, Brudzynski and Wang, 1996, de Oliveira et al., 2000, Vianna et al., 2003, Borelli et al., 2005). Another interesting approach showed that changes resembling those of reversible denaturation enhanced dye–protein interaction in protein extracts from electrically stimulated brain tissue (Fischer and Zeman, 1959, Fischer et al., 1961), and this method consists in another interesting histochemical approach for the study of the neural activity.

The dorsal periaqueductal gray matter (dPAG), deep layers of the superior colliculus (dlSC), and the inferior colliculus play important role in the integration of defensive behaviors and analgesia (Bandler, 1988, Graeff, 1990, Bandler et al., 1991, Carrive, 1993, Coimbra et al., 1992, Coimbra and Brandão, 1993), and these structures are connected to monoaminergic nuclei of the reticular formation, such as the dorsal raphe nucleus and the locus coeruleus (Freitas et al., 2005). It has been proposed that the ascending dorsal raphe serotonergic pathway facilitates conditioned avoidance responses to potential or distal threat, and 5,7-dihydroxytryptamine-induced lesions of the dorsal raphe nucleus neural networks impair inhibitory avoidance while facilitate one-way escape from the open arms of the elevated T-maze (Sena et al., 2003), but these monoaminergic inputs to the dorsal midbrain are not sufficient for explain the fear-induced analgesia.

Serotonin (5-HT), endogenous opioid, and other neuropeptides in the nervous tissue of these brainstem regions have been detected using electrochemical biosensors associated with cyclic voltammetry (Cespuglio, 1982, Eichenberger et al., 1995), immunofluorescence histochemistry (Dahlstrom and Fuxe, 1964, Dahlstrom and Fuxe, 1965, Fuxe and Jonsson, 1967, Glazer and Basbaum, 1981, Shipley et al., 1991), and autoradiograph (Mansour et al., 1988, Basbaum et al., 1982). These neurotransmitters are involved in defense reaction and anti-nociception (Basbaum et al., 1983, Mathiasen and Vaught, 1987, Fang et al., 1986, Porreca et al., 1987, Dauge et al., 1987, Herz and Millan, 1988, McGowan and Hammond, 1993a, McGowan and Hammond, 1993b, Holmes and Fujimoto, 1994, Shekhar et al., 1994, Rosa et al., 1998, Coimbra et al., 2000, Osaki et al., 2003, Rebouças et al., 2005).

Basbaum and Fields described a neuroanatomical circuitry involving critical structures that contribute to control of pain transmission (Basbaum and Fields, 1978, Basbaum and Fields, 1984, Mulcahy and Basbaum, 1984, Ho and Takemori, 1989, Fields and Basbaum, 1989). They demonstrated connections from midbrain periaqueductal gray to dorsal raphe nucleus and, via the dorsolateral funiculus, to the dorsal horn of the spinal cord. In the meantime, it was found that the spinal projections potentially relevant to analgesia, vocalizations, and motor activities arise from ventral aspects of the periaqueductal gray matter (PAG) (Carstens et al., 1979, Holstege, 1989, Morgan et al., 1991, Keay and Bandler, 1992, Mouton and Holstege, 1994). However, the precise functional relevance of the involvement of opioid peptides and serotonin in defensive behavior and anti-nociception elicited by dorsal midbrain stimulation (Bennett and Mayer, 1979, Nichols et al., 1989, Coimbra et al., 1992, Coimbra et al., 1996, Borelli et al., 2004) is still unclear. This is probably due to the complexity of both opioid and serotonergic pathways in the central nervous system (Palkovits et al., 1974, Steinbusch, 1981, Mansour et al., 1988, Eichenberger et al., 2002, Osaki et al., 2003). In addition, it is not clear if the anti-nociception that followed defensive reactions elicited by electrical and chemical stimulation of the intermediate and deep layers of the superior colliculus is only modulated by serotonin-containing inputs to the dorsal midbrain or if this phenomenon may be organized through a possible direct projection from the deep layers of the superior colliculus to the reticular formation monoaminergic nuclei.

Serotonin is unevenly distributed in caudal aspects of brainstem areas, with high concentrations found in the raphe nuclei (Palkovits et al., 1974). Electrophysiological and behavioral studies have shown that 5-HT1 and 5-HT2 receptors play a significant role in the modulation of aversive states at the level of the dorsal PAG and deep layers of the superior colliculus (Graeff, 1990, Brandão et al., 1991). Considerable evidence suggests that this analgesia may be part of the defense reaction (Fardin et al., 1984, Fanselow, 1991, Coimbra, 1995). The analgesia induced by electrical stimulation of these regions seems to be of serotonergic rather than of opioid nature (Cannon et al., 1982, Basbaum and Fields, 1984, Nichols et al., 1989, Coimbra et al., 1992, Coimbra, 1995, Coimbra and Brandão, 1997).

Immunocytochemical techniques associated to neuropharmacological studies may provide important clues to the understanding of the anatomy and physiology of the neural systems that modulate the neural substrates of aversion and fear-induced analgesia (Coimbra, 1995, Eichenberger et al., 2002, Osaki et al., 2003). The involvement of the opioid system of the dorsal mesencephalon in defensive behaviors has been recently substantially studied (Bach and Yaksh, 1995, 2000; Coimbra et al., 1996, Coimbra et al., 2000, Kishimoto et al., 2001, Eichenberger et al., 2002, Osaki et al., 2003). However, there is no study on the morphology of serotonin-mediated system associated with the neural substrates of the aversion and anti-nociception in the midbrain tectum, which have been substantially associated with the emergence of panic attacks (Schenberg et al., 1990, Vargas et al., 2000, Vianna et al., 2001a, Vianna et al., 2001b, Borelli et al., 2004, Borelli et al., 2005).

The present work examines neurons containing serotonin in the midbrain tectum, in sites in which the chemical stimulation produces fear-related responses and defensive motor behavior, as well as the neural hodology connecting this neural network with monoaminergic nuclei of the pain endogenous inhibitory system.

Section snippets

Materials and methods

Aiming the elucidation of the structural and functional interface between the dorsal midbrain neurons involved with fear- and panic-related responses and the reticular formation, the present work examines in more detailed morphological and neuropsychobiological approaches (a) the effect of the GABA-A blockade in the dorsal midbrain on the enhanced dye–protein interaction associated to the increased activity of the mesencephalic neurons involved with the generation and elaboration of the

Results

The present results showed that microinjections of the GABAA receptor blocker bicuculline into the midbrain tectum elicited defensive immobility followed by an explosive reaction of escape, which lasted approximately 20–30 min (Table 1).

Fear-like responses induced by GABAA receptor blockade in the dorsal mesencephalon, in its cranial aspects, were followed by activation of neurons in the deep layers of the superior colliculus and dlPAG, as identified by the neuronal-dye sorption technique, that

Discussion

In addition to the excellent description of the serotonergic immunoreactive perikarya and terminals in the brainstem reported by Steinbusch (1981) and Takeuchi et al. (1982), our results show the presence of serotonin-containing cell groups in the dorsal aspects of the PAG and in the intermediate and deep strata of the superior colliculus, mesencephalic structures which have been associated with the neural substrates involved with the organization and elaboration of defense reactions. In fact,

Acknowledgments

This work was supported by FAPESP (Proc. 95/8418-4, 96/5645-2, 97/11097-0, 98/11187-2, 02/03705-0, and 02/01496-5 to N.C. Coimbra and M.L. Brandão; 03/02186-2 and 04/11732-3 to L.L. Melo), CNPq (proc. 470119/2004-9 to N.C. Coimbra), and FAEPA (proc. 1291/97, 68/2001, 15/2003, and 06/2004 to N.C. Coimbra) grants. L.A. da Silva was the recipient of a post-doctoral fellowship (JP-3) from FAPESP (proc. 96/06946-6). K.G. Borelli was the recipient of Master (MS) and Doctoral (PhD) fellowships from

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