Abstract

The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

Introduction

The medial hypothalamus (MH) is a cluster of nuclei located in the medial portion of the diencephalon. Specifically, the dorsomedial (DMH) and ventromedial (VMH) hypothalamus have the function of organizing defensive responses to aversive stimuli and the elaboration of adaptive physiologic reactions that accompany fear-induced behavior in the presence of environmental threats. In fact, it has been suggested that emotional behavior is at least partially organized by hypothalamic nuclei, and that dysfunctions in these nuclei in both humans and animals produce behavioral responses that are proposed to be putative models of psychiatric disorders (Schmitt et al. 1986; Ducrocq et al. 2001; Freitas et al. 2009; Wilent et al. 2010).

It has been suggested that γ-aminobutyric acid (GABA) exerts a tonic inhibitory control on neurons that is related to the elaboration of defensive emotional responses in the DMH (Shekhar and DiMicco 1987, Shekhar et al. 1993) and more caudal encephalic divisions (Coimbra and Brandão 1993; Eichenberger et al. 2002; Coimbra et al. 2006). In fact, in laboratory animals, a panic-prone state that is induced by chronic disruption of GABAergic inhibition in the DMH causes increased anxiety, heart rate, blood pressure, and respiration rate (DiMicco et al. 1996; Shekhar et al. 1996; Shekhar and Keim 1997). Additionally, it has been shown that dysfunction of the GABA-mediated neuronal system in the MH causes panic-like responses in laboratory animals, and that the elaborated escape behavior organized in both the DMH and VMH nuclei is followed by significant innate-fear-induced antinociception (Freitas et al. 2009; Biagioni et al. 2013).

The medial prefrontal cortex (MPFC) consists of a limbic system-related region subdivided into anterior cingulate, precentral, prelimbic, infralimbic, and medial orbital cortices. Moreover, the MPFC has functional differences that can result in distinct patterns of behaviors (Verberne and Owens 1998; Heidbreder and Groenewegen 2003; Resstel and Corrêa 2006). For example, the MPFC is involved in the elaboration of cardiovascular, neuroendocrine, and behavioral defensive responses (Frysztak and Neafsey 1994; Schulkin et al. 2005; Sierra-Mercado et al. 2006; Resstel et al. 2008; Radley et al. 2009; Tavares et al. 2009). It has also been shown that the MPFC is involved in fear-related endocrine and autonomic responses. Furthermore, lesions of the rodent MPFC have been shown to affect susceptibility to stress-induced ulceration (Sullivan and Gratton 1999).

Moreover, the MPFC has direct and/or indirect connections to subcortical structures believed to be involved in fear and anxiety, such as the amygdaloid complex, the hippocampus, the nucleus accumbens, the nucleus caudatus, the hypothalamus, and the periaqueductal gray matter (Kita and Oomura 1981; Swanson 1981; Sesack et al. 1989; Hurley et al. 1991; McDonald et al. 1996). Additionally, the Fos-like immunoreactivity, a functional indicator of neuronal activation that is present in the MPFC after exposure to a variety of anxiety-provoking challenges (Morrow et al. 1999) can be suppressed by benzodiazepines (Morrow et al. 2000). Finally, human imaging studies have shown that activation of the MPFC is altered in normal subjects during exposure to fearful stimuli, and abnormal MPFC activity has been associated with a number of anxiety disorders (Malizia 1999).

Glutamate is an important neurotransmitter of the mammalian central nervous system (Fleck et al. 1993; Khodorov 2004). There are 2 families of glutamatergic receptors, metabotropic and ionotropic, but the present work focuses on the activity of drugs on both the N-methyl-d-aspartate (NMDA) glutamate and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate (non-NMDA) receptors (Hollmann and Heinemann 1994; Kemp and McKernan 2002; Jingami et al. 2003). It has been shown that stress increases the extracellular concentration of l-glutamate in the MPFC of rodents (Moghaddam 1993). This finding indicates that stress increases the neural release of excitatory amino acids in the MPFC.

The MPFC also has a role in the elaboration of antinociception processes, because the microinjection of NMDA-glutamatergic receptor agonists into the MPFC elevates the nociceptive thresholds of rodents as measured by tail-flick and paw-withdrawal tests (Calejesan et al. 2000; Zhang et al. 2005).

To show the participation of the PL division of the MPFC in the organization of both panic-like responses organized by the MH and fear-induced antinociceptive processes, the present study sought to investigate whether the inputs to NMDA and AMPA/kainate glutamatergic receptors of the PL neurons modulate not only the responses to noxious stimulus, but also fear-induced behavioral responses. We investigated the effects of microinjections of 200 nL of LY235959 (an NMDA antagonist) and NBQX (an AMPA/kainate antagonist) at 2.0, 4.0, and 8.0 nmol in independent groups of Wistar rats on both the panic-like responses and innate fear-induced antinociception induced by GABAA receptor antagonism in the MH through the local administration of bicuculline at 40 ng/200 nL.

Materials and Methods

Animals

Male Wistar rats (Rattus norvegicus, Rodentia, Muridae) weighing 220–250 g (N = 6–8 per group) from the animal facility of the School of Medicine of Ribeirão Preto of the University of São Paulo (FMRP-USP) were studied. The rats were kept (4 in a cage) in the experimental room (for at least 48 h prior to the experiments) with free access to water and food. The enclosure was kept under a light/dark cycle of 12/12 h (lights on from 7 AM to 7 PM) and maintained at a constant room temperature of 25 ± 1°C. All experiments were performed in accordance with the recommendations of the Commission of Ethics in Animal Experimentation of the FMRP-USP (proc. 192/2009), which is in agreement with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Commission of Ethics in Animal Research (CETEA) in 15 December 2008. All efforts were made to minimize pain or discomfort during each experiment.

Antinociceptive Procedure

The nociceptive thresholds of independent groups of rats (N = 6–8) were compared using the tail-flick test. Each animal was placed in a restraining apparatus (Insight, Brazil) with acrylic walls, and its tail was placed on a heated sensor (tail-flick Analgesia Instrument; Insight, Brazil). The temperature was progressively elevated and automatically interrupted at the moment when the animal removed its tail from the apparatus. Electrical current raised the temperature of the coil (Ni/Cr alloy; 26.04 cm in length × 0.02 cm in diameter) at the rate of 9°C/s (Prado and Roberts 1985), starting at room temperature (∼20°C). Small current intensity adjustments were performed, when necessary, for the beginning of the experiment (baseline records) to obtain 3 consecutive tail-flick latencies (TFLs) between 2.5 and 3.5 s. If the animal did not remove its tail from the heater within 6 s, the apparatus was turned off to prevent damage to the skin. Three baseline measurements of control TFLs were taken at 5-min intervals. TFLs were also measured at 60 min, immediately after the elaborated escape behavior.

Surgical Procedure

Animals were anaesthetized with ketamine at 92 mg/kg (Ketamine®) and xylazine at 9.2 mg/kg (Dopaser®) and fixed in a stereotaxic frame (David Kopf, USA). Stainless steel, guide cannulae (outer diameter 0.6 mm and inner diameter 0.4 mm) were implanted in the diencephalon targeting the MH and the PL division of the MPFC. The upper incisor bar was set at 3.3 mm below the interaural line, such that the skull was horizontal between bregma and lambda. The guide cannulae targeted at the MH were vertically introduced using the following coordinates with bregma serving as the reference: Anteroposterior, −2.80 mm; mediolateral, 0.5 mm; and dorsoventral, 7.8 mm. The stereotaxic coordinates for the implantation of the cannulae into the PL were selected from the rat brain atlas of Paxinos and Watson (1997): Anteroposterior, + 1.2 mm; anterolateral 0.5 mm; and dorsoventral, 2.6 mm with a lateral inclination of 29°. The guide cannulae were fixed to the skull using an acrylic resin and 2 stainless steel screws. At the end of the surgery, each guide cannulae was sealed with a stainless steel wire to protect it from obstruction.

Experimental Procedure

Microinjection of the NMDA and AMPA/Kainate Antagonists Into the PL cortex

Animals (N = 6–8) were surgically implanted with guide cannulae targeting the MH and PL cortex. Five days after surgery, baseline nociceptive thresholds, as measured by the tail-flick test, were recorded for each rodent. Immediately after this procedure, the PL cortex of independent groups of animals was pretreated with physiologic saline (NaCl 0.9%; 200 nL), 200 nL of LY235959 (NMDA receptor antagonist), or NBQX (AMPA/kainate receptor antagonist) at 2.0, 4.0, or 8.0 nmol. The injection needle was linked to a 5.0-μL hand-driven syringe (Hamilton) with polyethylene tubing. The injection was made using a thin dental needle (Mizzy, o.d. 0.3 mm) introduced through the guide cannulae until its lower end was 1 mm below the tip of the cannulae.

Ten minutes after the pretreatment of the PL cortex with either NMDA or AMPA/kainate antagonists or physiologic saline, the MH of independent groups of rodents was microinjected with the GABAA receptor antagonist bicuculline (40 ng/200 nL) or physiologic saline as a control. After these treatments, the following responses were recorded over 10 min in an open-field test arena (a circular enclosure 50 cm in diameter and 50 cm high) with the floor divided into 12 sections using a Sony Handycam camera: Exploratory behavior, expressed by the number of crossings (4 paws in a given division of the circular arena floor); the frequency and duration of rearing (upright posture); behavioral defensive reactions, expressed by the frequency and duration of rapid defensive backward movements; the frequency and duration of elaborated forward escape behaviors (running intercalated with exploratory behavior and jumps oriented to the upper side of the arena); the frequency and duration of defensive attention (alertness, a response operationally defined as the interruption of on-going behavior when the rodents orient themselves toward the stimulus that evokes attentive posture with small head movements, rearing and smelling of the surrounding air); and the frequency and duration of defensive immobility (“freezing,” operationally defined as body immobility accompanied by at least 2 of the following autonomic reactions: Urination, defecation, piloerection, or exophthalmos). The nociceptive responses (recorded as TFLs) were measured immediately after elaborated escape behavior and at 10, 20, 30, 40, and 60 min after the fear-related reactions. An investigator blind to the nature of the treatment groups performed the behavioral analysis.

Histology

Upon completion of the experiments, the animals were anaesthetized with ketamine at 92 mg/kg (Ketamina®) and xylazine at 9.2 mg/kg (Dopaser®) and perfused through the left cardiac ventricle. The blood was washed out with Tyrod's buffer (40 mL at 4°C) followed by 200 mL ice-cold 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 15 min at a pressure of 50 mmHg. The brains were quickly removed and soaked for 4 h in fresh fixative at 4°C. After fixation, the brains were sectioned, and the diencephalon was rinsed in 10% and 20% sucrose dissolved in 0.1 M sodium phosphate buffer (pH 7.3) at 4°C for at least 12 h in each solution. Tissue pieces were immersed in 2-methylbutane (Sigma), frozen on dry ice (30 min), embedded in Tissue Tek, and cut on a cryostat (Leica CM 1950). The slices were then mounted on glass slides coated with chrome alum gelatin to prevent detachment and stained in a robotic autostainer (CV 5030 Leica Autostainer) with hematoxylin–eosin to localize the positions of the guide cannulae tips according to the Paxinos and Watson's stereotaxic atlas (Paxinos and Watson 1997) under a motorized photomicroscope (AxioImager Z1, Zeiss). Data from rats in which the guide cannulae tips were located outside the MH (dorsomedial and ventromedial nuclei of the hypothalamus) or PL cortex were not included in the statistical analysis.

Drugs

Bicuculline methiodide (Tocris) at 40 ng/200 nL was microinjected into the MH. PL pretreatment was performed with a volume of 200 nL of the following drugs at 2.0, 4.0, and 8.0 nmol: LY235959 (Sigma) or NBQX (Sigma) dissolved in physiologic saline shortly before use. A peristaltic pump (Stoelting Co.®) was used to infuse drugs at a rate of 100 nL per minute.

Statistical Analysis

Data from independent groups of animals in the neurophysiological and neuropharmacological studies that received GABAA receptor blockade in the MH were submitted to a one-way analysis of variance (ANOVA) followed by Newman–Keuls' post hoc test. Kolmogorov–Smirnov tests for normal distributions of data showed Gaussian distributions inside the intervals studied in all cases. Data from experiments to determine nociceptive thresholds during the organization of innate fear-related responses evoked by chemical stimulation of the MH were submitted to a repeated-measures ANOVA followed by Duncan's post hoc test. All values are reported as the mean ± standard error of the mean. A P-value <0.05 was considered statistically significant.

Results

Effect of PL cortex Pretreatment with an NMDA Antagonist on Panic-Like Responses

The blockade of GABAergic receptors in the MH by intradiencephalic microinjection of bicuculline at 40 ng/200 nL produced defensive alertness, defensive immobility, and escape behaviors with running and vertical jumps toward the upper side of the arena and expressive exploratory behaviors that were as if the animals were searching for a way to escape from the environment in which they were stimulated (Fig. 1); these behaviors are the so-called oriented or elaborated escape behaviors (Freitas et al. 2009; Biagioni et al. 2012, 2013). The Newman–Keuls post hoc test showed that treatment of the PL cortex with physiologic saline followed by microinjection of bicuculline into the MH caused a significant increase in the frequency (F5,34 = 15.41; P < 0.01) and duration (F5,34 = 15.56; P < 0.001) of defensive attention, in the frequency (F5,34 = 7.77; P < 0.001) and duration (F5,34 = 5.90; P < 0.001) of defensive immobility, in the frequency (F5,34 = 10.97; P < 0.001) and duration (F5,34 = 9.70; P < 0.001) of defensive backward movements, in the frequency (F5,34 = 5.43; P < 0.001) and duration (F5,34 = 9.02; P < 0.001) of forward escape responses (Fig. 1A–H), in the frequency of crossings (F5,34 = 14.93; P < 0.001), and in the frequency (F5,34 = 4.59; P < 0.001) and duration (F5,34 = 3.49; P < 0.001) of rearing when compared with the PL physiologic saline + intra-MH physiologic saline-treated group (Fig. 2A–C).

Figure 1.

Effect of the microinjection of 200 nL of LY235959 (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%/200 nL) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (40 ng/200 nL) into the medial hypothalamus (MH) on the frequency and duration of defensive attention (A and B), defensive immobility (C and D), defensive backward movements (E and F), and forward escape behaviors (G and H) (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the saline (MPFC) + bicuculline (MH)-treated group; ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA, followed by Newman–Keuls' post hoc tests.

Figure 1.

Effect of the microinjection of 200 nL of LY235959 (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%/200 nL) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (40 ng/200 nL) into the medial hypothalamus (MH) on the frequency and duration of defensive attention (A and B), defensive immobility (C and D), defensive backward movements (E and F), and forward escape behaviors (G and H) (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the saline (MPFC) + bicuculline (MH)-treated group; ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA, followed by Newman–Keuls' post hoc tests.

Figure 2.

(A–C) Effect of the microinjection of 200 nL of LY235959 (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on the frequency of crossing (A) and the frequency and duration of rearing (B and C) during elaborated escape behavior (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05 and ***P < 0.001 compared with the saline (MPFC) + bicuculline (MH)-treated group; #P < 0.05; ##P < 0.01, and ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to one-way ANOVA followed by Newman–Keuls' post hoc tests. (D) Effect of the microinjection of LY235959 (at 3 different concentrations) or saline (NaCl 0.9%) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on nociceptive thresholds measured with the tail-flick test (TFL) (N = 6–8). *P < 0.05 compared with the saline (MPFC) + bicuculline (MH) group; #P < 0.05 compared with the saline (MPFC) + saline (MH) group; −, P < 0.05 compared with the LY235959 2 nmol (MPFC) + bicuculline (MH) group; +, P < 0.05 compared with the LY235959 4 nmol (MPFC) + bicuculline (MH) group according to a repeated-measures ANOVA followed by Duncan's post hoc tests.

Figure 2.

(A–C) Effect of the microinjection of 200 nL of LY235959 (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on the frequency of crossing (A) and the frequency and duration of rearing (B and C) during elaborated escape behavior (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05 and ***P < 0.001 compared with the saline (MPFC) + bicuculline (MH)-treated group; #P < 0.05; ##P < 0.01, and ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to one-way ANOVA followed by Newman–Keuls' post hoc tests. (D) Effect of the microinjection of LY235959 (at 3 different concentrations) or saline (NaCl 0.9%) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on nociceptive thresholds measured with the tail-flick test (TFL) (N = 6–8). *P < 0.05 compared with the saline (MPFC) + bicuculline (MH) group; #P < 0.05 compared with the saline (MPFC) + saline (MH) group; −, P < 0.05 compared with the LY235959 2 nmol (MPFC) + bicuculline (MH) group; +, P < 0.05 compared with the LY235959 4 nmol (MPFC) + bicuculline (MH) group according to a repeated-measures ANOVA followed by Duncan's post hoc tests.

The Newman–Keuls post hoc test also showed that the pretreatment of the PL cortex with LY235959 at any concentration significantly decreased the frequency (F5,34 = 15.41; P < 0.01) and duration (F5,34 = 15.56; P < 0.001) of defensive attention (Fig. 1A,B). In addition, LY235959 at any concentration significantly decreased the frequency (F5,34 = 7.77; P < 0.01) and duration (F5,34 = 5.90; P < 0.001) of defensive immobility (Fig. 1C,D). The pretreatment of the PL cortex with the NMDA antagonist at any dose followed by MH treatment with bicuculline also caused significant decreases in the frequency (F5,34 = 10.97; P < 0.001) and duration (F5,34 = 9.70; P < 0.01) of defensive backward movements (Fig. 1E,F) and in the frequency (F5,34 = 5.43; P < 0.01) and duration (F5,34 = 9.02; P < 0.01) of forward escape responses when compared with the PL physiologic saline + intra-MH bicuculline-treated group (Fig. 1G,H).

The post hoc analysis showed that, at all concentrations (2.0, 4.0, and 8.0 nmol), LY235959 administered into the PL cortex caused significant decreases in the frequencies of crossing (F5,34 = 14.93; P < 0.001) and rearing (F3,34 = 3.61; P < 0.05) compared with control animals (intra-PL physiologic saline + intra-MH bicuculline-treated animals) during the elaborated escape reactions (Fig. 2A–C). In addition, the lower and intermediate concentrations of LY235959 (2.0 and 4.0 nmol) were also able to significantly decrease the duration of rearing (F5,34 = 3.49; P < 0.05; Fig. 2C).

Effect of the Pretreatment of PL cortex with NMDA Receptor Antagonist on Elaborated Fear-Induced Antinociception

There was a clear increase in pain thresholds after innate fear-induced behavioral responses that characterized innate fear-induced antinociception. The repeated-measures ANOVA showed statistically significant effects of treatment (F5,34 = 20.90; P < 0.001), time (F9,27 = 7.14; P < 0.001), and a significant interaction of treatment and time (F9,45 = 30.14; P < 0.001). One-way ANOVA followed by Duncan's post hoc test showed a significant increase in the nociceptive threshold induced by elaborated escape behavior of animals pretreated with PL cortex microinjections of physiologic saline + treatment of the MH with bicuculline when compared with the PL cortex physiologic saline + intra-MH physiologic saline-treated group (P < 0.001). Post hoc analysis showed a significant decrease in fear-induced antinociception after the pretreatment of the PL cortex with LY235959 at the higher concentration (8.0 nmol) from 0 to 30 min after the elaborated escape behavioral response. The pretreatment of the PL cortex with LY235959 at the intermediate concentration (4.0 nmol) decreased fear-induced antinociception from 10 to 30 min after the escape behavior. The pretreatment of the PL cortex with LY235959 at the lowest concentration decreased fear-induced antinociception 10, 20, and 30 min after the elaborated escape behavior induced by GABAA receptor antagonism into the MH compared with the control group (intra-PL cortex physiologic saline + intra-MH bicuculline-treated animals) (P < 0.05). These findings are shown in Figure 2D.

Effect of the Pretreatment of the PL cortex with an AMPA/Kainate Receptor Antagonist on Panic-Like Responses

GABAergic antagonism in the MH through intradiencephalic microinjection of bicuculline at 40 ng/200 nL was followed by exploratory behavior and oriented/elaborated escape responses. The Newman–Keuls post hoc test showed that the pretreatment of the PL cortex with physiologic saline followed by microinjection of bicuculline into the MH caused a significant increase in the frequency (F5,34 = 16.70; P < 0.01) and duration (F5,34 = 15.56; P < 0.001) of defensive attention, in the frequency (F5,34 = 5.34; P < 0.05) and duration (F5,34 = 6.24; P < 0.01) of defensive immobility, in the frequency (F5,34 = 7.26; P < 0.001) and duration (F5,34 = 9.59; P < 0.001) of defensive backward movements, in the frequency (F5,34 = 7.42; P < 0.001) and duration (F5,34 = 8.07; P < 0.001) of forward escape responses (Fig. 3A–H), in the frequency of crossings (F5,34 = 7.89; P < 0.001), and in the frequency (F5,34 = 9.10; P < 0.01) and duration (F5,34 = 12.43; P < 0.001) of rearing when compared with the PL cortex physiologic saline + intra-MH physiologic saline-treated group (Fig. 4A–C).

Figure 3.

Effect of the microinjection of 200 nL of NBQX (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (40 ng/200 nL) into the MH on the frequency and duration of defensive attention (A and B), defensive immobility (C and D), defensive backward movement (E and F), and forward escape behavior (G and H) (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05 and **P < 0.01 compared with the saline (MPFC) + bicuculline (MH)-treated group; #P < 0.05; ##P < 0.01, and ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA followed by Newman–Keuls post hoc tests.

Figure 3.

Effect of the microinjection of 200 nL of NBQX (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) division of the medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (40 ng/200 nL) into the MH on the frequency and duration of defensive attention (A and B), defensive immobility (C and D), defensive backward movement (E and F), and forward escape behavior (G and H) (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. *P < 0.05 and **P < 0.01 compared with the saline (MPFC) + bicuculline (MH)-treated group; #P < 0.05; ##P < 0.01, and ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA followed by Newman–Keuls post hoc tests.

Figure 4.

(A–C) Lack of the effect of the microinjection of 200 nL of NBQX (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on the frequency of crossing (A) and the frequency and duration of rearing (B and C) during elaborated escape behavior (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA followed by Newman–Keuls post hoc tests. (D) The effects of the microinjection of NBQX (at 3 different concentrations) or saline (NaCl 0.9%/200 nL) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on nociceptive thresholds as measured by the tail-flick test (TFL) (N = 6–8). *P < 0.05 compared with the saline (MPFC) + bicuculline (MH) group; #P < 0.05 compared with the saline (MPFC) + saline (MH) group according to a repeated-measures ANOVA followed by Duncan's post hoc tests.

Figure 4.

(A–C) Lack of the effect of the microinjection of 200 nL of NBQX (at 2.0, 4.0, and 8.0 nmol) or saline (NaCl 0.9%) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on the frequency of crossing (A) and the frequency and duration of rearing (B and C) during elaborated escape behavior (N = 6–8). The columns represent the means, and the bars represent the standard errors of the means. ###P < 0.001 compared with the saline (MPFC) + saline (MH)-treated group according to a one-way ANOVA followed by Newman–Keuls post hoc tests. (D) The effects of the microinjection of NBQX (at 3 different concentrations) or saline (NaCl 0.9%/200 nL) into the prelimbic (PL) medial prefrontal cortex (MPFC) followed by the microinjection of bicuculline (at 40 ng/200 nL) into the MH on nociceptive thresholds as measured by the tail-flick test (TFL) (N = 6–8). *P < 0.05 compared with the saline (MPFC) + bicuculline (MH) group; #P < 0.05 compared with the saline (MPFC) + saline (MH) group according to a repeated-measures ANOVA followed by Duncan's post hoc tests.

Newman–Keuls post hoc tests also showed that the pretreatment of the PL cortex with NBQX did not modify the frequency (P > 0.05) or duration (P > 0.05) of defensive attention (Fig. 3A,B) or the frequency (P > 0.05) or duration (P > 0.05) of defensive immobility (Fig. 3C,D). The pretreatment of the PL cortex with the AMPA/kainate receptor antagonist NBQX at the higher dose (8.0 nmol) followed by GABAA receptor antagonism in the MH was able to decrease the frequency (F5,34 = 7.26; P < 0.05) and duration (F5,34 = 9.59; P < 0.05) of backward escape behavior, as shown in Figure 3E,F. Finally, the pretreatment of the PL cortex with the AMPA/kainate receptor antagonist NBQX modified neither the frequency (P > 0.05) nor the duration (P > 0.05) of forward escape behaviors (Fig. 3G,H) compared with the control group (physiologic saline intra-PL cortex + bicuculline intra-MH). In addition, a Newman–Keuls post hoc test showed that, at all concentrations used in the present work (2.0, 4.0, and 8.0 nmol), the AMPA/kainate receptor antagonist administered into the PL cortex was not able to significantly modify either the frequency of crossing (P > 0.05) or the frequency (P > 0.05) or duration (P > 0.05) of rearing compared with the physiologic saline intra-PL cortex + bicuculline intra-MH-treated group after the elaboration of oriented escape behavior (Fig. 4A–C).

Effect of the Pretreatment of the PL cortex with an AMPA/Kainate Receptor Antagonist on Elaborated Fear-Induced Antinociception

The analyses of nociceptive thresholds after elaborated/oriented escape behaviors using a repeated-measures ANOVA revealed statistically significant effects of treatment (F5,34 = 10.80; P < 0,001), time (F9.25 = 20.92; P < 0,001), and treatment by time interaction (F9,45 = 6.00; P < 0,001). One-way ANOVA followed by Duncan's post hoc tests showed a significant increase of fear-induced antinociception recorded immediately and 10 min after fear-related responses of PL cortex physiologic saline-pretreated + MH bicuculline-treated animals when compared with the PL cortex physiologic saline-pretreated + intra-MH physiologic saline-treated groups (P < 0.001). Post hoc analysis showed a significant decrease in innate fear-induced antinociception after the pretreatment of the PL cortex with NBQX at all doses followed by MH treatment with bicuculline recorded immediately after the elaborated escape behavior and at 10 min after this defensive response (P < 0.001; Fig. 4D).

Diagrams of the microinjection sites for physiologic saline or LY235959 within the PL cortex and MH treatments with physiologic saline or bicuculline are represented in Figure 5A,B, respectively. Diagrams of the microinjection sites for physiologic saline or NBQX within the PL and MH treatments with physiologic saline or bicuculline are represented in Figure 5C,D, respectively.

Figure 5.

(A–B) Schematic coronal section of the brain of R. norvegicus showing the sites of central administrations in the prelimbic (PL) division of the medial prefrontal cortex (A) and medial hypothalamus (MH) (B) of the (open triangles) saline intra-PL cortex + saline intra-MH, (filled squares) saline intra-PL cortex + bicuculline intra-MH, (open circles) LY235959 (2.0 nmol) intra-PL cortex + bicuculline intra-MH, (closed circles) LY235959 (4.0 nmol) intra-PL cortex + bicuculline intra-MH, (open squares) LY235959 (8.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled triangles) LY235959 (8.0 nmol) intra-PL cortex + saline intra-MH groups depicted on illustrations from Paxinos and Watson's stereotaxic atlas (1997). (C and D) Schematic coronal section of the brain of R. norvegicus showing the sites of central administrations in the PL cortex (A) and MH (B) of the (open triangles) saline intra-PL cortex + saline intra-MH, (filled squares) saline intra-PL cortex + bicuculline intra-MH, (open circles) NBQX (2.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled circles) NBQX (4.0 nmol) intra-PL cortex + bicuculline intra-MH, (open squares) NBQX (8.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled triangles) NBQX (8.0 nmol) intra-PL cortex + saline intra-MH groups depicted on illustrations from Paxinos and Watson's stereotaxic atlas (1997).

Figure 5.

(A–B) Schematic coronal section of the brain of R. norvegicus showing the sites of central administrations in the prelimbic (PL) division of the medial prefrontal cortex (A) and medial hypothalamus (MH) (B) of the (open triangles) saline intra-PL cortex + saline intra-MH, (filled squares) saline intra-PL cortex + bicuculline intra-MH, (open circles) LY235959 (2.0 nmol) intra-PL cortex + bicuculline intra-MH, (closed circles) LY235959 (4.0 nmol) intra-PL cortex + bicuculline intra-MH, (open squares) LY235959 (8.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled triangles) LY235959 (8.0 nmol) intra-PL cortex + saline intra-MH groups depicted on illustrations from Paxinos and Watson's stereotaxic atlas (1997). (C and D) Schematic coronal section of the brain of R. norvegicus showing the sites of central administrations in the PL cortex (A) and MH (B) of the (open triangles) saline intra-PL cortex + saline intra-MH, (filled squares) saline intra-PL cortex + bicuculline intra-MH, (open circles) NBQX (2.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled circles) NBQX (4.0 nmol) intra-PL cortex + bicuculline intra-MH, (open squares) NBQX (8.0 nmol) intra-PL cortex + bicuculline intra-MH, (filled triangles) NBQX (8.0 nmol) intra-PL cortex + saline intra-MH groups depicted on illustrations from Paxinos and Watson's stereotaxic atlas (1997).

Discussion

In the present study, antagonism of GABAA receptors in the MH after microinjections of physiologic saline in the prelimbic medial prefrontal cortex induced panic-like behavioral responses characterized by defensive attention, defensive immobility, risk assessment, defensive backward movements (backward escape behavior), forward elaborated escape behaviors (running and oriented jumps toward the arena's upper open area), and exploratory behavior. Additionally, we demonstrated that an antinociceptive process is evoked by innate fear-related behaviors induced by GABAergic blockade in the MH. However, after the pretreatment of the PL cortex with the NMDA receptor antagonist LY235959, the panic-like responses and innate fear-induced antinociception that appeared after GABAA receptor blockade in the MH were decreased. The pretreatment of the PL cortex with NBQX, an AMPA/kainate receptor antagonist, only decreased innate fear-induced antinociception without consistently decreasing the elaborated defensive responses induced by GABAA receptors antagonism in the MH, except by the decreasing of backward defensive behavior at the highest dose.

In fact, GABAA-mediated neurotransmission has been suggested to be involved in the anxiety and panic-like defensive behaviors that are elaborated by the DMH (Shekhar 1993; Shekhar et al. 1996). Furthermore, the GABAergic system of the dorsomedial division of the ventromedial hypothalamic nucleus has also been implicated in the modulation of behavioral escape responses (Bueno et al. 2007).

There is anatomic evidence showing reciprocal connections between the prefrontal cortex and the hypothalamus. For example, the MPFC sends inputs to the tuberal lateral hypothalamus (Gabbott, et al. 2005; Morshedi and Meredith 2008), a structure that is related to panic-like responses (Salgado-Rohner et al. 2011), in a region that is roughly coextensive with the ventromedial hypothalamic nucleus. In addition, although the projections from the DMH nucleus are mainly distributed into the MH (Thompson et al. 1996), some connections reach the medial division of the lateral hypothalamic area. The involvement of the MPFC in emotion (Frysztak and Neafsey 1994; Morgan and LeDoux 1995) may utilize pathways connecting the prefrontal cortex and the lateral/ventromedial hypothalamic nuclei. However, it has been shown that the MPFC does not have a primary role in mediating basic fear and anxiety responses; rather, the MPFC acts as an inhibitory center of control through which neocortical regions modulate fear responses based on complex temporal and/or contextual information processed by the cortex (Ledoux 1993).

There is evidence showing that MPFC lesions in rats cause anxiolytic-like effects in the elevated plus maze test (Lacroix et al. 2000), and it has also been reported that the MPFC modulates conditioned fear via the amygdaloid complex (Quirk et al. 2003; Malin et al. 2007). Excitotoxic lesions of the rat medial prefrontal cortex attenuate fear-related responses in the elevated plus maze, during social interaction, and in the shock probe burying test (Shah and Treit 2003).

However, given the complex temporal and/or contextual cues processed by neocortical neurons, it is possible that the prefrontal cortex does not play a primary role in mediating innate fear- and anxiety-related responses, but rather exerts an inhibitory influence on more caudal structures, such as the hypothalamus and periaqueductal gray matter. There is additional evidence suggesting that the prefrontal region has a secondary role in modulating neural systems directly involved with the elaboration of fear-related behavior (Leonard et al. 2001).

However, we (de Freitas et al. 2011; Freitas et al. 2011) have shown additional evidence revealing a consistent attenuation of fear reactivity in a wide range of innate fear-induced behaviors caused by a transitory blockade of synapses by microinjection into the PL of cobalt chloride, suggesting that this cortical region may have a more direct role in mediating innate fear-related behavior. In fact, the breadth of evidence suggests that the PL cortex has a general role in aversive stimuli processing and may be critical for the coordination or modulation of the more directional and oriented behavioral responses induced by behavioral reaction related to innate fear and anxiety (e.g., the behavioral responses organized by the hypothalamic nuclei). The present work offers additional evidence supporting this notion.

We also found that microinjections of the NMDA receptor antagonist LY235959 into the PL cortex decreased defensive behaviors and fear-induced antinociception recorded after the elicitation of elaborated escape reactions. However, the pretreatment of the PL cortex with the AMPA/kainate receptor antagonist NBQX changed the intensity of at least part of defensive behavioral responses (only defensive backward movement) and also significantly decreased the fear-induced antinociception. In agreement with these findings, the blockade of GABA synthesis in the dorsomedial hypothalamic nucleus causes increases in both panic-like responses and NMDA receptor expression in this structure, indicating that the glutamatergic system is involved in the defensive behavior organized by the MH (Johnson and Shekhar 2006).

Glutamate is an important neurotransmitter in the ventral division of the MPFC (Gigg et al. 1994). Relative to AMPA/kainate receptors, NMDA receptors can be more critically involved in the elaboration of physiologic responses related to emotion. Binding studies have revealed that the MPFC has a large amount of ionotropic glutamatergic receptors, including the NMDA receptor (Nicolle and Baxter 2003).

In the present work, we showed that the NMDA receptors of PL cortex are critically involved in the organization of the innate fear- and panic attack-related behavioral responses induced by transitory GABAergic dysfunction in the MH. In agreement with the present findings, there is evidence that the social interaction-induced tachycardia response, tachypnea, and increases in anxiety levels that are evoked by chronic inhibition of GABA synthesis and the consequent increase in glutamatergic activation of MH neurons are modulated or reversed by intrahypothalamic microinjections of NMDA receptor antagonists (Johnson and Shekhar 2006; Johnson et al. 2008). However, based on the present findings, it is possible that both NMDA and AMPA/kainate receptors in the PL cortex are involved in the elaboration or modulation of antinociception following defensive behavioral responses organized by the MH.

Recent brain imaging studies have shown activation of cortical areas during pain processing; the cerebral cortex is not only the final target of the ascending nociceptive pathways, but also recruits descending projections involved in the modulation of pain threshold and the emotional aspects of pain (Jasmin et al. 2003; Petrovic et al. 2004). In fact, human pain tolerance levels are increased by transcranial magnetic stimulation of the dorsolateral prefrontal cortex (Graff-Guerrero et al. 2005).

Hypothalamic nuclei provide important descending projections to the periaqueductal gray matter (Beitz 1982). The nucleus raphe magnus and the periaqueductal gray matter are reciprocally connected to the lateral hypothalamus (Aimone et al. 1988; Cameron, Khan, Westlund, Cliffer, et al. 1995; Cameron, Khan, Westlund, Willis, et al. 1995). In fact, neuroanatomic approaches have demonstrated the existence of pathways connecting the MH with the midbrain tectum structures involved in the organization of panic attack-like responses and antinociceptive processes (Stamford 1995; Lumb 2004). Retrograde double-labeling neurotracing has shown neural connections between the hypothalamus and the dorsal raphe nucleus (DRN) as well as locus coeruleus (LC), both of which are important structures in the endogenous pain modulatory system (Lee et al. 2005), and fear induced-elaborated defensive responses elicited by GABAergic blockade in the MH recruit monoamine-containing pathways connecting both the DRN and LC (Biagioni et al. 2013).

Recent findings obtained in our laboratory (de Freitas et al. 2011; Freitas et al. 2011) suggest that the PL cortex is directly involved both in the organization of defensive responses and in the elaboration of the innate fear-induced antinociception evoked by the pretreatment of the MH with the GABAA receptor antagonist bicuculline, considering that the pretreatment of the PL cortex with cobalt chloride, a blocker of synapses, decreased the panic attack-related defensive behavior as well as the innate fear-induced antinociception. In the present work, we also demonstrated that NMDA receptors localized in the PL cortex are critically involved in the elaboration of panic attack-like behavioral responses and innate fear-induced antinociception. However, the AMPA/kainate receptors of the PL cortex seem to be more involved in the innate fear-induced antinociception than in the panic-like responses induced by GABAA receptor blockade in the MH.

Funding

This study was supported by CNPq (proc.483763/2010-1), FAEPA (proc. 345/2009), and FAPESP (proc. 07/01174-1, 2012/03798-0). R.L.d.F was the recipient of a Scientific Initiation Scholarship (proc. 01/03752-6), a Magister Scientiae (M.Sc.) fellowship (proc. 03/05256-1) sponsored by FAPESP, a Scientiae Doctor (Sc.D.) Fellowship sponsored CAPES, and a Post Doctorate Fellowship sponsored by FAPESP (proc. 2009/17258-5). C.J.S.-R. was supported by CNPq (proc. 135930/2009-0; M.Sc. fellowship). A.F.B. was supported by FAPESP (proc. 2008/08955-1 for M.Sc., and 2010/15140-4 for Sc.D.). P.M. was supported by FAPESP (2010/14446-2 for M.Sc.). N.C.C. was granted a research fellowship (level 1A) from CNPq (proc. 301905/2010-0) and was a CNPq postdoctoral fellow (proc. 200629/2005-0) in the Departments of Physiology, Anatomy, and Genetics and in the Department of Clinical Neurology (FMRIB Centre) of the University of Oxford, UK. J.A.S.C. (1C) and J.E.C.H. (2) were granted a research fellowship by CNPq.

Notes

The authors are grateful to D.H. Elias-Filho for expert technical assistance. D.H. Elias-Filho received a technician scholarship from FAPESP (TT-2, proc. 02/01497-1) and was the recipient of scholarships sponsored by CNPq (proc. 501858/2005-9, 500896/2008-9, and 505461/2010-2). Conflict of Interest: None declared.

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