Decrease in NMDA receptor-signalling activity in the anterior cingulate cortex diminishes defensive behaviour and unconditioned fear-induced antinociception elicited by GABAergic tonic inhibition impairment in the posterior hypothalamus

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Abstract

Acute γ-aminobutyric acid (GABA) disinhibition in the posterior hypothalamus (PH) elicits defensive reactions that are considered anxiety- and panic attack-like behaviour, and these defensive reactions are followed by antinociception. Evidence indicates that the PH connects with the medial prefrontal cortex, particularly the anterior cingulate cortex (ACC), which seems to regulate these unconditioned fear-induced defensive responses. However, few studies have shown the participation of cortical regions in the control of behavioural and antinociceptive responses organised by diencephalic structures. It has been suggested that the glutamatergic system can mediate this cortical influence, as excitatory imbalance is believed to play a role in both defensive mechanisms. Thus, the aim of the present study was to investigate the involvement of ACC glutamatergic connections via blockade of local N-methyl-D-aspartate (NMDA) receptors to elaborate panic-like defensive behaviours and unconditioned fear-induced antinociception organised by PH neurons. Wistar rats were treated with microinjections of 0.9% NaCl or LY235959 (a selective NMDA receptor antagonist) in the ACC at different concentrations (2, 4 and 8 nmol/0.2 μL), followed by GABAA receptor blockade in the PH. Defensive reactions were analysed for 20 min, and the nociceptive threshold was then measured at 10-min intervals for 60 min. Pretreatment of the ACC with LY235959 reduced both panic-like defensive behaviour and fear-induced antinociception evoked by PH GABAergic disinhibition. Our findings suggest that ACC NMDA receptor-signalled glutamatergic inputs play a relevant role in the organisation of anxiety- and panic attack-like behaviours and in fear-induced antinociception.

Introduction

Fear-related emotional reactions are mediated by complex neural circuitry in part comprising the medial prefrontal cortex (mPFC), e.g., prelimbic (de Freitas et al., 2014), infralimbic (Lemos et al., 2010) and anterior cingulate cortex (ACC) (Falconi-Sobrinho et al., 2017) and diencephalic regions, such the hypothalamus (Biagioni et al., 2012, Shekhar et al., 1990). In addition, there is some evidence showing that these regions of the limbic cortex are connected with the diencephalon (Azuma and Chiba, 1996, Falconi-Sobrinho et al., 2017, Reppucci and Petrovich, 2016) and can modulate anxiety- and fear-induced hypothalamic defensive reactions (de Freitas et al., 2013, de Freitas et al., 2014, Falconi-Sobrinho et al., 2017).

The hypothalamus has been considered a putative neural substrate for studying the mechanisms that induce panic attacks (Shekhar, 1994). Studies have proposed that there is a distinct circuit in the medial hypothalamic zone (MHZ), which in part consists of neural hypothalamic networks, that participates of the elaboration of fear-related defensive responses elicited in rodents submitted to threatening situations (Canteras, 2002). Rodents exposed to their natural predator showed an increase in the expression of Fos protein in certain nuclei of the hypothalamus that composes part of the MHZ, such as the anterior nucleus of the hypothalamus (AHN), the dorsomedial subdivision of the ventromedial nucleus of the hypothalamus (VMHdm) and the dorsal pre-mammillary nucleus (dPM) (Canteras et al., 1997). In addition, there is evidence that the posterior hypothalamic (PH) nucleus connects with these hypothalamic nuclei (Abrahamson and Moore, 2001), and shows also an increase in expression of Fos protein in rodents that are confronted by their predator (Canteras et al., 1997). This justifies the importance of further research on the specific role of the PH in defensive behaviour related to panic attacks. Furthermore, studies have shown that this hypothalamic nucleus when chemically stimulated also induces defensive behavioural responses. For example, GABAergic disinhibition by microinjections of bicuculline (a GABAA receptor antagonist) in the PH of laboratory rats elicits defensive reactions that are considered anxiety- and panic attack-like behaviour (Falconi-Sobrinho et al., 2017, Shekhar et al., 1990). Additionally, these defensive reactions elaborated in the hypothalamus are followed by antinociception (Biagioni et al., 2012). The antinociceptive response that follows defensive behaviours may be elicited by neurons that connect the hypothalamus with other nuclei of the endogenous pain modulation system (Biagioni et al., 2013). Descending pathways derived from the hypothalamus recruit midbrain (e.g., periaqueductal grey matter; PAG) and/or medulla oblongata (e.g., raphe nuclei) structures (Aimone et al., 1988, Falconi-Sobrinho et al., 2017) before reaching the dorsal horn of the spinal cord (DHSC) (Basbaum and Fields, 1984, da Silva et al., 2013). In addition, the hypothalamus is connected to different regions of the cerebral cortex that are also involved in the control of emotion-related pain (Buchanan et al., 1994, Falconi-Sobrinho et al., 2017). In fact, previous evidence demonstrated descending projections to the PH from the limbic forebrain, specifically from the ACC (Abrahamson and Moore, 2001). Corroborating these neuroanatomical findings, through morphological studies, Falconi-Sobrinho et al. (2017) showed retrograde-labelled neuronal bodies in the Cg1 region of the ACC after microinjection of a bidirectional neurotracer in the PH. Furthermore, positive fibres and terminal boutons containing glutamatergic synaptic vesicles were also identified surrounding PH neuronal perikarya after depositing an anterograde neurotracer into the Cg1, providing evidence that this region of the ACC sends glutamatergic projections to the PH.

Frontal cortical areas can influence the transmission of nociceptive information from the DHSC through activation or inhibition of the descending modulatory pain system (Zhang et a., 2005) via the brainstem (Ohara et a., 2005). On the other hand, less well established is the idea that cortical modulation of nociceptive processes can also occur through a diencephalic relay before reaching the DHSC. Recent studies have shown that the mPFC can modulate hypothalamus-mediated anxiety/fear-induced antinociception (de Freitas et al., 2014, Falconi-Sobrinho et al., 2017). Inactivation of efferent excitatory pathways from the Cg1 region of the ACC to the PH via intracortical administration of lidocaine attenuated defensive mechanisms, including an anxiety/fear-induced antinociception organised in the PH. In the same study, the blockade of glutamatergic terminals in the PH from Cg1 long projection neurons reduced both running and jump defensive behaviours and antinociception, suggesting that glutamate release in the PH is required for the organisation of hypothalamic defensive reactions (Falconi-Sobrinho et al., 2017). A similar effect on the hypothalamic defensive behaviours and the antinociception that follows these anxiety/fear emotions was also seen after blockade of ionotropic glutamatergic receptors in the prelimbic cortex (de Freitas et al., 2014). These findings demonstrated that the mPFC regions, including the Cg1 division of the ACC, can modulate the expression of hypothalamus-related fearful defensive behaviours, such as panic attack-like defensive reactions and antinociception, through reducing the cortico-hypothalamic glutamatergic pathway activity.

Glutamate activates two families of receptors, metabotropic and ionotropic receptors (Hollmann and Heinemann, 1994, Kemp and McKernan, 2002). Among ionotropic receptors, NMDA (Kemp and McKernan, 2002) receptors have been proposed to play a key role in fear-related defensive behaviours and nociceptive processes. Several studies demonstrated the involvement of these receptors in ACC on the expression of conditioned fear (Descalzi et al., 2012), and pain perception (Zugaib et al., 2014). However, few studies have shown the participation of glutamatergic neurotransmission in the limbic forebrain in descending modulation of defensive behaviour and unconditioned fear-induced antinociception (de Freitas et al., 2014, Falconi-Sobrinho et al., 2017).

To show the role of the ACC in defensive behaviours and unconditioned fear-induced antinociception organised by PH neurons, we investigated whether inputs to NMDA glutamatergic receptors of ACC neurons modulate both of these hypothalamic defensive mechanisms. The hypothesis of the present work is that pretreatment of the Cg1 region of the ACC with the selective NMDA receptor antagonist LY235959 at different concentrations may impair the hypothalamic defensive responses and fear-related hypoalgesia.

Section snippets

Animals

Male Wistar rats weighing 240–260 g (N = 64; n = 8 per group) from the animal facility of Ribeirão Preto Medical School of the University of São Paulo (FMRP-USP) were studied. They were housed 4 to a cage and were habituated in the experimental room for at least 48 h prior to the experiments with free access to water and food. The enclosure was maintained under a light/dark cycle of 12/12 h (lights on from 7 am to 7 pm) and at a constant room temperature of 24 °C ± 1 °C. All efforts were made to

Defensive behaviour

The blockade of GABAA receptors in the PH via intradiencephalic microinjection of bicuculline elicited alertness and escape behaviours (Figure 1A–F). There was no freezing behaviour. The injection of vehicle into the Cg1 did not affect any behavioural responses induced by PH-bicuculline treatment. On the other hand, the blockade of NMDA receptors with microinjection of LY235959 into the Cg1 caused one phase decay responses in all behaviours analysed (Figure 1A-F’).

According to two-way ANOVA,

Discussion

Defensive reactions were evoked by GABAA receptor blockade in the PH via local microinjection of bicuculline. Our results are in agreement with previous studies demonstrating that hypothalamic GABAergic disinhibition in rodents elicits defensive attention and escape reactions (Shekhar and DiMicco, 1987, Shekhar et al., 1990). It has been suggested that GABAergic neurotransmission mediates tonic inhibitory control of hypothalamic neurons (Milani and Graeff, 1987), including those of the PH that

Role of Funding Source

This research was supported by FAPESP (processes 2007/01174-1, 2012/03798-0, and 2017/11855-8) and CNPq (processes 483763/2010-1 and 474853/2013-6). Each organisation had no further role in the study design, the collection, analysis, or interpretation of the data, writing the report, or the decision to submit this paper for publication.

Author contributions

L. L. Falconi-Sobrinho performed the experiments, analysed and interpreted the data, and wrote the manuscript. R. de Oliveira performed the experiments and analysed and interpreted the data. T. dos Anjos-Garcia performed the statistical analysis and designed the figures. N. C. Coimbra designed the experiments, analysed and interpreted the data, wrote the manuscript, and approved the final manuscript. All authors have approved the final version of the manuscript.

Conflicts of interest

The authors declare that there are no conflicts of interest with respect to the presented work.

Acknowledgements

L.L. Falconi-Sobrinho was supported by FAPESP (M.Sc. process 2013/10984-8) and CNPq (M.Sc. fellowship process 134267/2013-3; Sc. D. fellowship process 145258/2015-7). T. dos Anjos-Garcia was supported by CNPq (M.Sc. fellowship process 130124/2012-5; Sc.D. fellowship process 141124/2014-8). R. de Oliveira was a post-doctoral researcher who was supported by FAPESP (process 2011/09850-1). N.C. Coimbra is a researcher (level 1 A) at CNPq (processes 301905/2010-0 and 301341/215-0). The authors thank

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