5-HT1A and 5-HT2A receptor control of a panic-like defensive response in the rat dorsomedial hypothalamic nucleus

Abstract

The dorsomedial nucleus of the hypothalamus (DMH) has long been implicated in the genesis/regulation of escape, a panic-related defensive behavior. In the dorsal periaqueductal gray matter (dPAG), another key panic-associated area, serotonin, through the activation of 5-HT1A and 5-HT2A receptors, exerts an inhibitory role on escape expression. This panicolytic-like effect is facilitated by chronic treatment with clinically effective antipanic drugs such as fluoxetine and imipramine. It is still unclear whether serotonin within the DMH plays a similar regulatory action. The results showed that intra-DMH injection of the 5-HT1A receptor agonist 8-OH-DPAT, the preferential 5-HT2A receptor agonist DOI, but not the 5-HT2C agonist MK-212, inhibited the escape reaction of male Wistar rats evoked by electrical stimulation of the DMH. Local microinjection of the 5-HT1A antagonist WAY-100635 or the preferential 5-HT2A antagonist ketanserin was ineffective. Whereas chronic (21 days) systemic treatment with imipramine potentiated the anti-escape effect of both 8-OH-DPAT and DOI, repeated administration of fluoxetine enhanced the effect of the latter agonist. The results indicate that 5-HT1A and 5-HT2A receptors within the DMH play a phasic inhibitory role upon escape expression, as previously reported in the dPAG. Facilitation of 5-HT-mediated neurotransmission in the DMH may be implicated in the mode of action of antipanic drugs.

Keywords

Panic serotonin dorsomedial hypothalamus antidepressants imipramine and fluoxetine escape

Introduction

The importance of serotonin in the pathophysiology and treatment of anxiety disorders such as generalized anxiety and panic has been widely acknowledged (for a review see Deakin and Graeff, 1991; Hale et al., 2012; Maron et al., 2012). For instance, antidepressant drugs that selectively inhibit the reuptake of this indolamine (SSRIs), such as fluoxetine and escitalopram, are the first choice pharmacological treatment of these pathologies (Andrisano et al., 2013; Koen and Stein, 2011). Although the primary mechanism of action for these drugs is well established, the observation that SSRIs, as other antidepressants, are clinically effective only after chronic treatment (Millan, 2005; Nierenberg et al., 2000; Schneier et al., 1990) indicates that adaptive neuronal changes are required in order to attain their full beneficial effects. The nature of these brain substrate(s) and where the platic modifications take place are not yet fully understood and have been increasingly investigated in recent years (Blier and de Montigny, 1994; Castro et al., 2003; Li et al., 1996).

In a seminal paper, Deakin and Graeff (1991) proposed that 5-HT plays a different role in the mediation of defensive responses that have been related to anxiety and panic. According to the authors, the excessive release of 5-HT in brain areas involved in the genesis/regulation of defensive behaviors evoked by potential threats, such as the amygdala and frontal cortex, would lead to generalized anxiety-related symptoms. On the other hand, failure of the inhibitory control exerted by 5-HT upon areas implicated in proximal defense (e.g. escape), particularly the dorsal area of the periaqueductal gray matter (dPAG), would underlie susceptibility to panic attacks.

In regard to panic, one corollary of this hypothesis is that antipanic drugs, such as antidepressants, should, therefore, improve 5-HT-mediated neurotransmission in the dPAG and this effect should be evident after three or four weeks of continuous treatment, the average time for positive clinical responses (Nierenberg et al., 2000; Schneier et al., 1990). A wealth of evidence gathered in recent years supports this idea (for a review see Graeff and Zangrossi, 2010). For instance, long-term (21 days), but not short-term administration of fluoxetine (de Bortoli et al., 2006), sertraline (Zanoveli et al., 2007) or imipramine (Jacob et al., 2002; Mongeau and Marsden, 1997) facilitates the inhibitory effect on escape caused by the intra-dPAG injection of both 5-HT1A and 5-HT2A receptor agonists. This effect was not observed after long-term treatment with buspirone (de Bortoli et al., 2006; Zanoveli et al., 2005), a partial 5-HT1A receptor agonist with clinically relevant anxiolytic properties, but ineffective in panic disorder (Connor and Davidson, 1998; den Boer and Slaap, 1998). Besides changes in the reactivity of these 5HT receptors, a microdialysis study also revealed that chronic treatment with fluoxetine, but not buspirone, increases 5-HT release in the dPAG (Zanoveli et al., 2010). Altogether, the findings suggest that facilitation of 5-HT1A- and 5-HT2A-receptor mediated neurotransmission in the dPAG is implicated in the antipanic effects of antidepressants.

Following the pioneering studies of Hess and Brugger (1943) with electrical stimulation of specific brain areas, Fernandez de Molina and Hunsperger (1959, 1962) reported that activation of gray areas surrounding either the aqueduct, where the dPAG lies, or the third ventricle, at the level of the hypothalamus, induces defensive reactions in cats, such as escape or fight, similar to those observed when these animals are confronted with predators. Since then, the involvement of the hypothalamus, particularly its dorsomedial nucleus (DMH), in the genesis/regulation of proximal defense in different animal species has been confirmed by several studies (Duan et al., 1996; Freitas et al., 2009; Markgraf et al., 1991; Silveira et al., 1995). It was also shown that antipanic drugs such as imipramine and clonazepam block the behavioral and cardiovascular responses evoked by disinhibition of the DMH by local injection of the GABAA receptor antagonist bicuculline (Shekhar, 1994). Results such as these have led to the proposal that either electrical or chemical stimulation of the DMH can be used to model some aspects of panic disorder, mainly its core symptom: panic attacks (Freitas et al., 2009; Graeff and Zangrossi, 2002; Johnson and Shekhar, 2012).

In the present study, we investigated whether change in 5-HT-mediated neurotransmission within the DMH, as observed in the dPAG (for a review see Graeff and Zangrossi, 2010), interferes with the defensive reaction caused by electrical stimulation of this brain area. More specifically, we evaluated whether activation of 5-HT1A, 5-HT2A or 5-HT2C receptors, which are found in the DMH (Clemett et al., 2000; Li et al., 1997b), inhibits the escape response observed during electrical current delivery. It was also of interest to investigate whether chronic administration of imipramine and fluoxetine may affect the anti-escape effect that was found after the activation of 5-HT1A and 5-HT2A receptors within the DMH.

Materials and methods

Animals

Male Wistar rats (University of São Paulo, Campus Ribeirão Preto), weighing 250–300 g on the day of surgery, were housed in groups of four to five per cage under a 12-h light/dark cycle (lights on 07:00 hours) at 22±1°C, with free access to food and water, except during tests. All experiments were performed in accordance with the Brazilian Society of Neuroscience and Behaviour (SBNeC) for the care and use of laboratory animals and were approved by the Experimental Animal Ethical Committee of the School of Medicine of Ribeirão Preto, University of São Paulo. All efforts were made to minimize animal suffering.

Apparatus

The escape behavior induced by DMH electrical stimulation was evaluated in a bowl-shaped cage (round bottom bowl, model MD1500 - Bioanalytical Systems, USA; height 35 cm, top and base diameter 40 and 25 cm, respectively). Brain stimulation was generated by a sine-wave stimulator (Marseillan, 1977). The stimulation current (peak to peak) was monitored on the screen of an oscilloscope (Minipa, Brazil). The brain electrode was connected to the stimulator by means of an electromechanical swivel and a flexible cable, allowing ample movement of the animal inside the experimental cage.

Drugs

The following drugs were used: 5-hydroxytryptamine creatinine sulfate (5-HT; Sigma, USA), (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT; Sigma, USA), (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI; Sigma, USA), 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK-212; Sigma, USA), N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl cyclohexanecarboxamide maleate (WAY-100635; Sigma, USA), ketanserin tartrate (Sigma, USA), imipramine hydrochloride (Sigma, USA) and fluoxetine hydrochloride (Lily, Brazil). The drugs were dissolved in sterile saline (0.9%), except fluoxetine and ketanserin, which were dissolved in a solution containing sterile saline with 2% Tween 80. Doses are expressed in terms of the salt weights.

Surgery

Rats were anesthetized with 2,2,2 tribromoethanol (250 mg/kg, i.p.) associated with local anesthesia (2% lidocaine with a vasoconstrictor; Harvey, Brazil) and fixed in a stereotaxic frame (David Kopf, USA) for the implant of a chemitrode in the DMH. The chemitrode was made of a stainless-steel guide cannula (outside diameter 0.6 mm, 16 mm long) glued to a brain electrode made of stainless-steel wire (diameter 250 μm), enamel insulated, except at the cross-section of the tip, reaching 1.5 mm below the lower end of the cannula. The electrode wire was connected to a male pin, parallel to the outer end of the cannula that could be plugged into a socket at the end of a flexible electrical cable and used for brain stimulation. The chemitrode was implanted using coordinates according to the atlas of Paxinos and Watson (2007). Holding the incisor bar 3.5 mm below the horizontal plane, the chemitrode was vertically introduced using the following coordinates, with bregma serving as the reference: anteroposterior −2.8 mm from bregma; mediolateral 0.5 mm, and dorsoventral −8.7 mm. In experiment 3, besides a chemitrode in the DMH, a guide cannula made of stainless steel (0.6 mm outer diameter, 0.4 mm inner diameter) was also implanted in the midbrain, aimed at the dPAG. Briefly, holding the incisor bar 2.5 mm below the interaural line, the cannula was introduced 1.9 mm lateral to lambda at an angle of 22^o with the sagittal plane, until it was 3.2 mm below the surface of the skull.

The guide-cannulae and chemitrodes were attached to the skull by means of acrylic resin and two stainless steel screws. A stylet with the same length as the guide cannula was introduced inside it to prevent obstruction.

At the end of the surgery, all animals were injected intramuscularly with 0.2 mL of antibiotic preparation (benzylpenicillin and streptomycin, Pentabiotico Veterinário Pequeno Porte; Forte Dodge, Brazil) to prevent possible infections. In addition, flunixin meglumine (Scheringe Plough, Brazil; 2.5 mg/kg), a drug with analgesic, antipyretic and anti-inflammatory properties, was administered subcutaneously for post-surgery analgesia. In experiments 1–3, the animals were left undisturbed for 5–7 days after the surgery, except for normal handling for cage cleaning.

Procedure

Intracerebral injections

For drug injection into the DMH or dPAG, a needle (outside diameter, 0.3 mm) was introduced through the guide cannula until its tip was 1.5 mm below the cannula end. A volume of 0.2 μL was injected for 120 s using a 10 μL microsyringe (Hamilton 701-RN, USA) attached to a microinfusion pump (KD Scientific, USA). The displacement of an air bubble inside the polyethylene catheter connecting the syringe needle to the intracerebral needle was used to monitor the microinjection. The needle was removed 60 s after the injection was finished.

Escape threshold determination

One day before the test, the animals were gently handled by the experimenter for 5 min in the morning and afternoon, except in experiments 4 and 5.

On the test day, the animals were placed into the experimental cage, and the escape threshold was determined through an electrical stimulus (AC, 60 Hz, 10 s) presented through the implanted chemitrode. The interstimulus interval was 10 s. The current intensity started at a level of 20 μA and was increased by steps of 4 μA until the rat presented running or jumping reactions, characterizing the escape behavior. When these behaviors were observed, application of electrical stimulation to the DMH was interrupted by the experimenter. The basal escape threshold was defined as the lowest current intensity that evoked escape in three successive trials of electrical stimulation. Animals with basal thresholds above 150 μA were excluded from the study. Drug effects were determined as the difference between post- and pre-treatment escape thresholds. An increase in this value was taken as a panicolytic-like effect.

In experiment 1, after basal threshold determination and according to previous randomization, each rat received four microinjections in counterbalanced order. An interval of 24 h was allowed between injections. In experiment 1A, the animals were microinjected with the 5-HT1A receptor antagonist WAY-100635 (0.18, 0.37 or 0.74 nmol) or saline (n=7), in experiment 1B with the preferential 5-HT2A receptor antagonist ketanserin (5, 10 or 20 nmol) or saline (n=6) and in experiment 1C with the 5-HT2C receptor agonist MK-212 (0.1, 1 or 10 nmol) or saline (n=13) and the escape threshold was reanalyzed 10 min later.

In experiment 2, the same experimental design adopted in experiment 1 was followed, except that the animals (n=13) were injected into the DMH with either the endogenous agonist 5-HT (20 nmol), the 5-HT1A receptor agonist 8-OH-DPAT (8 nmol), the 5-HT2A receptor agonist DOI (16 nmol) or saline. The escape threshold was reanalyzed 10 min (8-OH-DPAT, 5-HT and saline) or 20 min (DOI) later according to previous studies using these drugs in the dPAG (de Bortoli et al., 2006, 2008; Nogueira and Graeff, 1995).

In experiment 3, we investigated whether the anti-escape effect of 8-OH-DPAT or DOI observed in experiment 2 was due to the diffusion of these drugs from the DMH to the dPAG. For this, after basal threshold determination (by electrically stimulating the DMH), each rat (n= 9) was injected into the dPAG with 8-OH-DPAT (8 nmol), DOI (16 nmol) or saline in counterbalanced order and respecting an interval of 24 h between injections. The escape threshold was reanalyzed 10 min (8-OH-DPAT and saline) or 20 min (DOI) later.

In experiment 4, rats were daily intraperitoneally injected with fluoxetine (10 mg/kg, n=10) or vehicle solution (n=8) for 23 days. The surgery for chemitrode implantation was performed on the 16th day after the beginning of the systemic injections and basal escape determination was first performed five days later. After that, and according to previous randomization, each rat received three DMH microinjections in counterbalanced order, following an interval of 24 h between injections. 8-OH-DPAT (8 nmol), DOI (16 nmol) or saline were administered 3 h after injection of fluoxetine or vehicle solution. Therefore, in each of the systemically injected groups, animals served as their own controls for the central effects of the drugs. The escape threshold was reanalyzed 10 min (8-OH-DPAT and saline) or 20 min (DOI) later, as in experiment 2.

In experiment 5, rats were daily intraperitoneally injected with imipramine (15 mg/kg, n=9) or saline (n=8) for 23 days; the same test protocol for drug microinjection in the DMH (i.e. drugs, doses and time intervals) described in experiment 4 was followed.

The doses of the drugs used in all these experiments were selected on the basis of previous results with the dPAG (Audi and Graeff, 1984; de Bortoli et al., 2006, 2008; de Paula Soares and Zangrossi, 2004; Jacob et al., 2002; Jenck et al., 1998; Nogueira and Graeff, 1995; Schütz et al., 1985; Yamashita et al., 2011; Zanoveli et al., 2005, 2007). Information on the selectivity of these drugs to different 5-HT receptors subtypes was taken from The International Union of Basic and Clinical Pharmacology (IUPHAR) database (http://www.iuphar-db.org).

Histology

After the experiments, animals were sacrificed under deep anesthesia with chloral hydrate. The brain was perfused through the heart with saline solution followed by 10% formalin solution, before being removed and fixed in 10% formalin. Frozen sections of 55 μm were cut using a microtome to localize the positions of the chemitrode tips according to the atlas of Paxinos and Watson (2007). Only data from rats having chemitrode tips inside the DMH were included in the statistical analysis. Misplacement of the chemitrodes in the DMH was found in 37% of all animals tested. In experiment 3, incorrect localization of injection sites in the dPAG (considered as the area comprising the dorsolateral and dorsomedial PAG) was found in 35% of all animals tested.

Statistical analysis

Repeated-measures analysis of variance was used to analyze data from experiments 1–3, with treatment (5-HT antagonists or agonists) as the independent factor and the escape threshold as the dependent variable. Trials (days of testing) were considered as the repeated measure. In experiments 4 and 5, besides the repeated measure, two independent factors were considered: the intraperitoneal treatment with the antidepressants and the intra-DMH treatment with the 5-HT agonists. When appropriate, post hoc comparisons were performed by Duncan’s test.

Results

Figure 1 depicts the sites of chemitrode placement in the DMH and of drug injections into the dPAG of animals tested in the current study.

In all experiments performed in this study, no significant change in the basal escape threshold was found between two consecutive days of testing, indicating that the effect of a given pharmacological treatment washed out after 24h.

Experiment 1 – As can be seen in Table 1, microinjection of WAY-100635, ketanserin or MK-212 did not significantly change the intensity of current required to induce escape reactions.

Experiment 2 – Figure 2 shows that administration of either 5-HT, 8-OH-DPAT or DOI into the DMH significantly raised the escape threshold (treatment effect, F(3,36)=11.90, p<0.01). There was no significant effect of trials or an interaction between the two factors analyzed.

Experiment 3 – As can be seen in Table 1, microinjection of 8-OH-DPAT or DOI in the dPAG did not significantly change the intensity of current required to induce escape reactions from the DMH.

Experiment 4 – As shown in Figure 3 (upper panel), intra-DMH administration of 8-OH-DPAT and DOI raised the escape threshold (central treatment effect, F(2,32)=27.46, p<0.01). There was also a significant effect of systemic treatment with fluoxetine (F(1,16)=7.07, p<0.05) and an interaction between central and systemic treatments (F(2,32)=3.98; p<0.05). The post hoc analysis revealed that the panicolytic-like effect of DOI, but not of 8-OH-DPAT, was higher (p<0.05) in rats chronically treated with fluoxetine than in those systemically injected with vehicle.

Experiment 5 – The lower panel of Figure 3 shows that, as in the previous studies, microinjection of 8-OH-DPAT and DOI raised the escape threshold (central treatment effect, F(2,30)=61.84, p<0.01). There was also a significant effect of systemic treatment with imipramine (F(1,15)=12.60, p<0.01) and an interaction between central and systemic treatments (F(2,30)=8.14, p<0.01). Duncan’s test revealed that the panicolytic-like effect of both DOI and 8-OH-DPAT, was higher (p<0.05) in rats chronically treated with imipramine than in those systemically injected with vehicle.

Table 1.

Effect (mean ± SEM) of intra-DMH or intra-dPAG injection of drugs on escape threshold (µA) evoked by electrical stimulation of the DMH.

Drug (nmol)	n	Basal escape threshold	Post-drug escape threshold
Intra-DMH injections
Saline	7	102.86 ± 5.70	114.29 ± 5.51
WAY-100635 (0.185)	7	98.86 ± 8.45	110.86 ± 9.47
WAY-100635 (0.37)	7	105.14 ± 10.24	113.71 ± 12.22
WAY-100635 (0.74)	7	105.14 ± 9.47	116.57 ± 10.42
Saline	6	90.67 ± 6.67	105.33 ± 8.24
Ketanserin (5)	6	89.33 ± 11.11	102.00 ± 11.94
Ketanserin (10)	6	80.67 ± 11.10	92.00 ± 13.15
Ketanserin (20)	6	82.67 ± 12.12	98.00 ± 12.30
Saline	13	72.00 ± 6.32	78.46 ± 6.45
MK-212 (0.1)	13	70.46 ± 5.56	74.46 ± 6.35
MK-212 (1)	13	71.08 ± 5.30	77.85 ± 6.57
MK-212 (10)	13	68.62 ± 5.24	77.23 ± 5.98
Intra-dPAG injections
Saline	9	70.67 ± 8.41	71.11 ± 7.74
8-OH-DPAT (8)	9	69.78 ± 7.92	72.89 ± 8.56
DOI (16)	9	76.89 ± 9.21	78.22 ± 7.60

DMH: dorsomedial nucleus of the hypothalamus; dPAG: dorsal area of the periaqueductal gray matter

Discussion

The results of the present study show that stimulation of 5-HT1A and 5-HT2A receptors within the DMH inhibits the escape response evoked by electrical stimulation of this diencephalic region. The blockade of these receptors with the 5-HT1A receptor antagonist WAY-100635 or the preferential 5-HT2A receptor antagonist ketanserin did not interfere with this defensive response, indicating that in the DMH, similarly as previously reported in the dPAG (de Oliveira Sergio et al., 2011; de Paula Soares and Zangrossi, 2004, de Paula Soares et al., 2010), 5-HT exerts a phasic regulatory control on neurons that are engaged with this task. This contrasts with the tonic inhibitory role played by GABA in both periventricular structures as indicated by studies showing that microinjection of the GABAA receptor antagonist bicuculline in the DMH or dPAG evokes marked escape reactions (Brandão et al., 2005; Freitas et al., 2009; Jenck et al., 1988).

The lack of effect of MK-212 on escape threshold indicates that 5-HT2C receptors of the DMH are not involved in the regulation of this defensive behavior. This agonist, which has a ten-fold higher affinity for 5-HT2C as for 5-HT2A receptors (Knight et al., 2004), when administered in the dPAG was equally ineffective in changing the escape reaction generated by electrical stimulation of this midbrain area or by the elevated T-maze (Yamashita et al., 2011).

Other studies in the literature support an inhibitory role of 5-HT on the aversive consequences generated by electrical stimulation of the hypothalamus. For instance, Leroux and Myers (1975) reported that in different sites within this brain area where electrical stimulation induces aversion, local microinjection of serotonin, but not of dopamine, acetylcholine or noradrenaline, attenuates this effect. Schmitt and coworkers (1983) showed that electrical stimulation of the medial area of the dorsal raphe nucleus (DRN), a source of 5-HT innervation to the DMH (Commons et al., 2003; Phelix et al., 1998), inhibits the escape response evoked by electrical stimulation of the DMH.

An important finding of this study is that the changes in escape response observed after manipulation of 5-HT neurotransmission in the DMH are similar to those previously reported in the dPAG. Thus, intra-dPAG injection of 5-HT1A and 5-HT2A receptor agonists inhibits escape expression either after electrical stimulation of the dPAG or in the elevated T-maze (de Bortoli et al., 2006, 2008; Jacob et al., 2002; Nogueira and Graeff, 1995; Yamashita et al., 2011; Zanoveli et al., 2005, 2007). Besides, the results of experiment 3 showed that the anti-escape effect generated by 8-OH-DPAT or DOI in the DMH was not due to the diffusion of these agonists to the dPAG. Therefore, stimulation of 5-HT1A and 5-HT2A receptors seems to cause a pervasive inhibitory effect on periventricular structures controlling escape behavior. It is noteworthy, however, that neither injection of 8-OH-DPAT or DOI into the ventromedial hypothalamus (VMH) affected escape expression in the elevated T-maze (da Silva et al., 2011). The VMH, as the DMH, has also been implicated in the genesis/regulation of panic-related defensive responses (Freitas et al., 2009; Wilent et al., 2010). To determine the generality of this finding, we are currently investigating the effects of intra-VMH injection of 5-HT1A- and 5-HT2-related drugs on the escape reaction evoked by electrical stimulation of the VMH.

Interestingly, although the activation of both 5-HT1A and 5-HT2A receptors in the DMH resulted in the same behavioral effect, at the cellular level, stimulation of these receptor subtypes induces opposite responses. Thus, while agonist interaction with 5-HT1A leads to hyperpolarization of the cell membrane (Aghajanian, 1995), activation of 5-HT2A receptors promotes depolarization, by increasing the levels of intracellular calcium (Hoyer et al., 2002). In the case of the dPAG, it has been reported that the majority of 5-HT2A receptor-labeled cells present in this midbrain area also show immunoreactivity to GABA (Brandão et al., 1991; Griffiths and Lovick, 2002). We recently observed that previous intra-dPAG injection of the GABAA receptor antagonist bicuculline blocked the anti-escape effect of local injection of DOI (de Oliveira Sergio et al., 2011). Altogether, these results indicate that the antipanic-like effect resulting from DOI interaction with 5-HT2A receptors in the dPAG is indirectly mediated by the activation of inhibitory GABAergic interneurons. It is therefore conceivable that a similar mechanism may have accounted for DOI effects in the DMH.

Our results also showed that, as previously reported in the dPAG (Jacob et al., 2002; Zanoveli et al., 2005), chronic systemic treatment with imipramine potentiated the anti-escape effect of 8-OH-DPAT and DOI, suggesting that in the DMH, as in the dPAG, the reactivity of both 5-HT1A and 5-HT2A receptors was enhanced by this tricyclic antidepressant. On the other hand, chronic administration of fluoxetine only increased the responsiveness of the latter receptors, contrasting with the facilitatory effect of this SSRI on both ligand sites in the dPAG (de Bortoli et al., 2006; Zanoveli et al., 2007).

Electrophysiological and neurochemical evidence indicates that chronic treatment with fluoxetine may induce regionally specific adaptations in 5-HT1A sensitivity as found in our studies with the dPAG (de Bortoli et al., 2006; Zanoveli et al., 2007) and DMH (experiment 4 of the current study). Whereas enhanced sensitivity has been reported in the hippocampus (Beck et al. 1997; Castro et al., 2003; Shen et al. 2002), desensitization has been observed in other anxiety-associated areas such as the lateral septum (Shen et al. 2002) and the DRN (Blier and de Montigny 1994; Castro et al. 2003).

Our findings do not allow conclusions on the nature of the changes induced by imipramine on 5-HT1A and 5-HT2A receptors or by fluoxetine on 5-HT2A binding sites. However, a previous study in the literature (Li et al., 1993a) reveals that long-term treatment (21 days) with fluoxetine, at the dose used here, potentiates DOI-induced elevation in ACTH and oxytocin secretion, indicating, as in our study, a functional supersensitivity of hypothalamic 5-HT2A receptors. Later, it was shown that this effect was correlated with a gradual increase in the coupling of hypothalamic 5-HT2A receptors to their G proteins (Li et al., 1997a).

Regarding 5-HT1A receptors, Li and coworkers (Li et al., 1993b) reported that 8-OH-DPAT, as observed with DOI (Li et al., 1993a), enhanced ACTH and oxytocin secretion, but this effect, differently from that observed with the preferential 5-HT2A agonist, was inhibited by chronic administration of fluoxetine, suggesting desensitization of hypothalamic 5-HT1A receptors. Interestingly, the same research group failed to find any significant effect of fluoxetine on the density or the degree of coupling of 5-HT1A receptors in whole hypothalamic homogenates (Li et al., 1994) or in different hypothalamic subnuclei, including the DMH (Li et al., 1997b). Since 8-OH-DPAT was administered systemically, which allows its interaction with both pre- and post-synaptic 5-HT1A receptors, the authors suggested that changes in the former sites might be indirectly related to the inhibitory effect of fluoxetine on hormonal responses (Li et al., 1994). This idea is compatible with the lack of effect of this antidepressant on the responsiveness of 5-HT1A receptors in the DMH found in the current study. However, given the scarcity of evidence, further analyses are still required in order to determine the nature of the changes induced in DMH by both imipramine and fluoxetine.

In conclusion, our results indicate that 5-HT1A and 5-HT2A receptors within the DMH play a phasic inhibitory role upon escape expression, as previously reported in the dPAG. Facilitation of 5-HT-mediated neurotransmission in the DMH, through different cellular mechanisms, may be implicated in the mode of action of antipanic drugs such as imipramine and fluoxetine.

Footnotes

Acknowledgements

The authors thank Afonso Paulo Padovan for expert technical assistance.

Conflict of interest

The authors declare no conflict of interest.

Funding

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP and CNPq, Brazil (grant numbers: 2007/03685-3 and 2008/03123-8).

References

Aghajanian

(1995) Electrophysiology of serotonin receptor subtypes and signal transduction pathways. In: Bloom

Kupfer

(eds) Psychopharmacology: The fourth generation of progress. New York: Raven, pp.1451–1459.

Andrisano

Chiesa

Serretti

(2013) Newer antidepressants and panic disorder: A meta-analysis. Int Clin Psychopharmacol 28: 33–45.

Audi

Graeff

(1984) Benzodiazepine receptors in the periaqueductal grey mediate anti-aversive drug action. Eur J Pharmacol 103: 279–285.

Beck

Birnstiel

Choi

. (1997) Fluoxetine selectively alters 5-hydroxytryptamine1A and gamma-aminobutyric acid B receptor-mediated hyperpolarization in area CA1, but not area CA3, hippocampal pyramidal cells. J Pharmacol Exp Ther 281: 115–122.

Blier

de Montigny

(1994) Current advances and trends in the treatment of depression. Trends Pharmacol Sci 15: 220–226.

Brandão

Borelli

Nobre

. (2005) Gabaergic regulation of the neural organization of fear in the midbrain tectum. Neurosci Biobehav Rev 29: 1299–1311.

Brandão

Lopez-Garcia

Graeff

. (1991) Electrophysiological evidence for excitatory 5-HT2 and depressant 5-HT1A receptors on neurones of the rat midbrain tectum. Brain Res 556: 259–266.

Castro

Diaz

del Olmo

. (2003) Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT1A receptors in rat brain. Neuropharmacology 44: 93–101.

Clemett

Punhani

Duxon

. (2000) Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 39: 123–132.

10.

Commons

Connolley

Valentino

(2003) A neurochemically distinct dorsal raphe-limbic circuit with a potential role in affective disorders. Neuropsychopharmacology 28: 206–215.

11.

Connor

Davidson

JRT

(1998) Generalized anxiety disorder: Neurobiological and pharmacotherapeutic perspectives. Biol Psychiatry 44: 1286–1294.

12.

da Silva

Poltronieri

Nascimento

. (2011) Facilitation of 5-HT(2A/2C)-mediated neurotransmission in the ventromedial hypothalamic nucleus decreases anxiety in the elevated T-maze. Behav Brain Res 216: 692–698.

13.

Deakin

JFW

Graeff

(1991) 5-HT and mechanisms of defense. J Psychopharmacol 5: 305–315.

14.

de Bortoli

Nogueira

Zangrossi

Jr (2006) Effects of fluoxetine and buspirone on the panicolytic-like response induced by the activation of 5-HT1A and 5-HT2A receptors in the rat dorsal periaqueductal gray. Psychopharmacology (Berl) 183: 422–428.

15.

de Bortoli

Nogueira

Zangrossi

Jr (2008) Alprazolam potentiates the antiaversive effect induced by the activation of 5-HT(1A) and 5-HT (2A) receptors in the rat dorsal periaqueductal gray. Psychopharmacology (Berl) 198: 341–349.

16.

den Boer

Slaap

(1998) Review of current treatment in panic disorder. Int Clin Psychopharmacol 13(Suppl. 4): S25–S30.

17.

de Oliveira Sergio

de Bortoli

Zangrossi

Jr (2011) Serotonin-2A receptor regulation of panic-like behavior in the rat dorsal periaqueductal gray matter: The role of GABA. Psychopharmacology (Berl) 218: 725–732.

18.

de Paula Soares

Zangrossi

Jr (2004) Involvement of 5-HT1A and 5-HT2 receptors of the dorsal periaqueductal gray in the regulation of the defensive behaviors generated by the elevated T-maze. Brain Res Bull 64: 181–188.

19.

de Paula Soares

Campos

Bortoli

. (2010) Intra-dorsal periaqueductal gray administration of cannabidiol blocks panic-like response by activating 5-HT1A receptors. Behav Brain Res 213: 225–229.

20.

Duan

Winters

McCabe

. (1996) Behavioral characteristics of defense and vigilance reactions elicited by electrical stimulation of the hypothalamus in rabbits. Behav Brain Res 81: 33–41.

21.

Fernandez

Molina

Hunsperger

(1959) Central representation of affective reactions in forebrain and brain stem: Electrical stimulation of amygdala, stria terminalis, and adjacent structures. J Physiol 145: 251–265.

22.

Fernandez

Molina

Hunsperger

(1962) Organization of the subcortical system governing defence and flight reactions in the cat. J Physiol 160: 200–213.

23.

Freitas

Uribe-Mariño

Castiblanco-Urbina

. (2009) GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception. Brain Res 1305: 118–131.

24.

Graeff

Zangrossi

Jr (2002) Animal models of anxiety disorders. In: D’Haenen

den Boer

Westenberg

(eds) Textbook of biological psychiatry. London: Wiley, pp.879–893.

25.

Graeff

Zangrossi

Jr (2010) The dual role of serotonin in defense and the mode of action of antidepressants on generalized anxiety and panic disorders. Cent Nerv Syst Agents Med Chem 10: 207–217.

26.

Griffiths

Lovick

(2002) Co-localization of 5-HT 2A -receptor- and GABA-immunoreactivity in neurones in the periaqueductal grey matter of the rat. Neurosci Lett 326: 151–154.

27.

Hale

Shekhar

Lowry

(2012) Stress-related serotonergic systems: Implications for symptomatology of anxiety and affective disorders. Cell Mol Neurobiol 32: 695–708.

28.

Hess

Brugger

(1943) Das subkortikale zentrum der affectiven adwehrreaktion. Helv Physiol Pharmacol Acta 1: 33–52.

29.

Hoyer

Hannon

Martin

(2002) Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 71: 533–554.

30.

Jacob

Cabral

Almeida

. (2002) Chronic imipramine enhances 5-HT(1A) and 5-HT(2) receptors-mediated inhibition of panic-like behavior in the rat dorsal periaqueductal gray. Pharmacol Biochem Behav 72: 761–766.

31.

Jenck

Moreau

Berendsen

. (1998) Antiaversive effects of 5HT2C receptor agonists and fluoxetine in a model of panic-like anxiety in rats. Eur Neuropsychopharmacol 8: 161–168.

32.

Jenck

Moreau

Karli

(1988) Modulation by morphine of aversive-like behavior induced by GABAergic blockade in periaqueductal gray or medial hypothalamus. Pharmacol Biochem Behav 31: 193–200.

33.

Johnson

Shekhar

(2012) An animal model of panic vulnerability with chronic disinhibition of the dorsomedial/perifornical hypothalamus. Physiol Behav 107: 686–698.

34.

Knight

Misra

Quirk

. (2004) Pharmacological characterisation of the agonist radioligand binding site of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors. Naunyn Schmiedebergs Arch Pharmacol 370: 114–123.

35.

Koen

Stein

(2011) Pharmacotherapy of anxiety disorders: A critical review. Dialogues Clin Neurosci 13: 423–437.

36.

Leroux

Myers

(1975) Action of serotonin microinjected into hypothalamic sites at which electrical stimulation produced aversive responses in the rat. Physiol Behav 14: 501–505.

37.

Battaglia

van

Kar

(1997b) Autoradiographic evidence for differential G-protein coupling of 5-HT1A receptors in rat brain: lack of effect of repeated injections of fluoxetine. Brain Res 769: 141–151.

38.

Brownfield

Battaglia

. (1993a) Long-term treatment with the antidepressants fluoxetine and desipramine potentiates endocrine responses to the serotonin agonists 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) and (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI). J Pharmacol Exp Ther 266: 836–844.

39.

Brownfield

Levy

. (1994) Attenuation of hormone responses to the 5-HT1A agonist ipsapirone by long-term treatment with fluoxetine, but not desipramine, in male rats. Biol Psychiatry 36: 300–308.

40.

Levy

Cabrera

. (1993b) Long-term fluoxetine, but not desipramine, inhibits the ACTH and oxytocin responses to the 5-HT1A agonist, 8-OH-DPAT, in male rats. Brain Res 630: 148–156.

41.

Muma

Battaglia

. (1997a) Fluoxetine gradually increases [125I]DOI-labelled 5-HT2A/2C receptors in the hypothalamus without changing the levels of Gq- and G11-proteins. Brain Res 775: 225–228.

42.

Muma

NA and

van

Kar

(1996) Chronic fluoxetine induces a gradual desensitization of 5-HT1A receptors: Reductions in hypothalamic and midbrain Gi and G(o) proteins and in neuroendocrine responses to a 5-HT1A agonist. J Pharmacol Exp Ther 279: 1035–1042.

43.

Markgraf

Winters

Liskowsky

. (1991) Hypothalamic, midbrain and bulbar areas involved in the defense reaction in rabbits. Physiol Behav 49: 493–500.

44.

Maron

Nutt

Shlik

(2012) Neuroimaging of serotonin system in anxiety disorders. Curr Pharm Des 18: 5699–5708.

45.

Marseillan

(1977) A solid state sine-wave stimulator. Physiol Behav 19: 339–340.

46.

Millan

(2005) Serotonin 5-HT2C receptors as a target for the treatment of depressive and anxious states: Focus on novel therapeutic strategies. Therapie 60: 441–460.

47.

Mongeau

Marsden

(1997) Effect of imipramine treatments on the 5-HT1A-receptor-mediated inhibition of panic-like behaviours in rats. Psychopharmacology 131: 321–328.

48.

Nierenberg

Farabaugh

Alpert

. (2000) Timing of onset of antidepressant response with fluoxetine treatment. Am J Psychiatry 157: 1423–1428.

49.

Nogueira

Graeff

(1995) Role of 5-HT receptor subtypes in the modulation of dorsal periaqueductal gray generated aversion. Pharmacol Biochem Behav 52: 1–6.

50.

Paxinos

Watson

(2007) The rat brain in stereotaxic coordinates, 6th ed. London: Academic Press.

51.

Phelix

Adai

Cantu

. (1998) Immunohistochemical demonstration of serotonin-containing axons in the hypothalamus of the white-footed mouse, Peromyscus leucopus. Brain Res 808: 197–219.

52.

Schmitt

Sandner

Colpaert

. (1983) Effects of dorsal raphe stimulation on escape induced by medial hypothalamic or central gray stimulation. Behav Brain Res 8: 289–307.

53.

Schneier

Liebowitz

Davies

. (1990) Fluoxetine in panic disorder. J Clin Psychopharmacol 10: 119–121.

54.

Schütz

de Aguiar

Graeff

(1985) Anti-aversive role of serotonin in the dorsal periaqueductal grey matter. Psychopharmacology (Berl) 85: 340–345.

55.

Shekhar

(1994) Effects of treatment with imipramine and clonazepam on an animal model of panic disorder. Biol Psychiatry 36: 748–758.

56.

Shen

Meller

(2002) Repeated treatment with antidepressants differentially alters 5-HT1A agonist-stimulated [35S]GTP gamma S binding in rat brain regions. Neuropharmacology 42: 1031–1038.

57.

Silveira

Sandner

Di Scala

. (1995) c-fos immunoreactivity in the brain following electrical or chemical stimulation of the medial hypothalamus of freely moving rats. Brain Res 674: 265–274.

58.

Wilent

Buetefisch

. (2010) Induction of panic attack by stimulation of the ventromedial hypothalamus. J Neurosurg 12: 1295–1298.

59.

Yamashita

de Bortoli

Zangrossi

Jr (2011) 5-HT2C receptor regulation of defensive responses in the rat dorsal periaqueductal gray. Neuropharmacology 60: 216–222.

60.

Zanoveli

Nogueira

Zangrossi

Jr (2005) Chronic imipramine treatment sensitizes 5-HT1A and 5-HT2A receptors in the dorsal periaqueductal gray matter: Evidence from the elevated T-maze test of anxiety. Behav Pharmacol 16: 543–552.

61.

Zanoveli

Nogueira

Zangrossi

Jr (2007) Enhanced reactivity of 5-HT1A receptors in the rat dorsal periaqueductal gray matter after chronic treatment with fluoxetine and sertraline: evidence from the elevated T-maze. Neuropharmacology 52: 1188–1195.

62.

Zanoveli

Pobbe

de Bortoli

. (2010) Facilitation of 5-HT1A-mediated neurotransmission in dorsal periaqueductal gray matter accounts for the panicolytic-like effect of chronic fluoxetine. Int J Neuropsychopharmacol 13: 1079–1088.