Excitatory amino acid receptors in the dorsomedial hypothalamus mediate prostaglandin-evoked thermogenesis in brown adipose tissue

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Exp Physiol 96.2 pp 73–84 73

Research PaperResearch Paper

Excitatory amino acid receptors in the dorsomedialhypothalamus are involved in the cardiovascularand behavioural chemoreflex responses

Edson Alexandre Queiroz, Marcos Noboru Okada, Ubirajara Fumega, Marco Antonio Peliky Fontes,Marcio Flavio Dutra Moraes and Andrea Siqueira Haibara

Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil

The present study investigated the role of the dorsomedial hypothalamus (DMH) oncardiovascular and behavioural responses of chemoreflex activation in conscious rats. Thearterial chemoreflex was activated by potassium cyanide (KCN, 40 μg, i.v.) before and afterbilateral microinjection of lidocaine (2%) or kynurenic acid (2.7 nmol) into the DMH.Locomotor activity was measured to assess the chemoreflex behavioural response. Bilateralmicroinjection of lidocaine into the DMH produced a significant reduction in the pressorresponse induced by chemoreflex activation (+51 ± 4 versus +34 ± 5 mmHg, n = 5, P < 0.05).A similar reduction in the pressor chemoreflex response was also observed after microinjectionof kynurenic acid into the DMH (+50 ± 3 versus +22 ± 5 mmHg, n = 6, P < 0.05). Strikingly,the behaviour/locomotor activity induced by chemoreflex activation was virtually abolishedafter blockade of excitatory amino acid receptors in the DMH with kynurenic acid (44 ± 6versus 5 ± 4 cm, n = 6, P < 0.05). There was no correlation between the reduction in pressorand behavioural chemoreflex responses (r = −0.186, P > 0.05). The bradycardic response of thechemoreflex was not altered by lidocaine or kynurenic acid microinjected into the DMH. Theseresults strongly suggest that the excitatory amino acid receptors in the DMH are essential forfull expression of the behavioural response of the chemoreflex and participate, at least in part,in the integration of the pressor response of the chemoreflex in conscious rats.

(Received 12 July 2010; accepted after revision 24 September 2010; first published online 1 October 2010)Corresponding author A. S. Haibara: Department of Physiology and Biophysics, Institute of Biological Sciences, FederalUniversity of Minas Gerais, 31270-910, Belo Horizonte, MG, Brazil. Email: [email protected]

The activation of the peripheral chemoreceptorsin conscious rats produces a complex pattern ofcardiovascular, respiratory and behavioural responses,which are almost abolished by anaesthesia (Franchini& Krieger, 1993; Haibara et al. 1995; Barros et al.2002). The cardiovascular responses to the chemoreflexinclude increased blood pressure and bradycardia,which are mediated through activation of independentmechanisms, i.e. sympathetic and parasympatheticexcitation, respectively (Haibara et al. 1995). Thechemoreflex behavioural response is characterized bya sudden startle reaction followed by a rapid runningbehaviour, similar to the escape reaction of thedefence response (Franchini & Krieger, 1993). Moreover,stimulation of arterial chemoreceptors also increasesplasma corticosterone and adrenocorticotrophic hormone(ACTH) levels (Raff et al. 1982; Raff & Fagin, 1984; Honig

et al. 1996), hallmarks of the neuroendocrine response ofthe defence reaction to stress.

The defence reaction is a set of coordinated responsesinvolving behavioural and autonomic functions elicitedby the hypothalamus. Activation of hypothalamic defenceareas (HDAs) by peripheral chemoreceptors has beenconsidered an integral part of the response to systemichypoxia (Marshall & Metcalfe, 1988). Therefore, it isreasonable to assume that activation of the peripheralchemoreceptors can induce autonomic and behaviouralresponses associated with the alerting stage of the defencereaction (Hilton, 1982; Marshall, 1994). In addition,stimulation of the HDA can facilitate the cardiovascularand respiratory responses evoked by carotid chemoreflex(Hilton & Joels, 1965; Silva-Carvalho et al. 1993, 1995).

The dorsomedial hypothalamus (DMH) is consideredpart of the so-called HDAs (Yardley & Hilton, 1986).

C© 2010 The Authors. Journal compilation C© 2011 The Physiological Society DOI: 10.1113/expphysiol.2010.054080

74 E. A. Queiroz and others Exp Physiol 96.2 pp 73–84

Stimulation or disinhibition of the DMH neurons evokes apattern of autonomic, somatomotor and neuroendocrineresponses that closely mimics the classic defence reaction,including a marked increase in sympathetic activity, bloodpressure, heart rate, respiratory rate, locomotor activityand plasma ACTH (Keim & Shekhar, 1996; Bernardis &Bellinger, 1998; Fontes et al. 2001; DiMicco et al. 2002;Cao et al. 2004; McDowall et al. 2007). It has been shownthat excitatory amino acid (EAA) receptors in the DMHparticipate in the modulation of the defence reaction. Infact, all major EAA receptor subtypes are present at highlevels in the DMH (Meeker et al. 1994). Activation ofDMH neurons by EAA receptor agonists evokes a markedtachycardia, increases in blood pressure and behaviouralresponse (Silveira & Graeff, 1992; Soltis & DiMicco, 1992;De Novellis et al. 1995). Moreover, microinjection of EAAreceptor antagonists into the DMH inhibited experimentalstress-induced elevation of heart rate and blood pressure(Soltis & DiMicco, 1992; DiMicco et al. 1996). Thesefindings support a role for EAA receptors in the DMHin mediating cardiovascular and behavioural responses tostress.

Although the involvement of DMH neurons in thechemoreflex pathway has been previously suggested inan anatomical study that evaluated the Fos expression inhypothalamic areas during moderate hypoxia (Berquinet al. 2000), the functional role of the DMH in thereflex responses of chemoreceptor activation remains tobe understood. In the present study, we investigated apossible contribution of the DMH in the generation ofthe cardiovascular and behavioural components of thechemoreflex in conscious rats. The contribution of EAAreceptors in mediating these responses was also evaluated.

Methods

Ethical approval

All procedures and experimental protocols used in thisstudy were performed in accordance with the Guide forthe Care and Use of Laboratory Animals published by theUS National Institutes of Health (NIH publication no.85-23, revised 1996) and Ethical Principles for AnimalExperimentation established by the Brazilian Committeefor Animal Experimentation (COBEA) and approved bythe Ethical Committee for Animal Experimentation of theFederal University of Minas Gerais (protocol 0032/2007).

Animals

The experiments were performed on nineteen male Wistarrats weighing 270–300 g, obtained from the Animal Carefacility of the Federal University of Minas Gerais. Rats werekept on a 14 h–10 h light–dark circadian cycle, with foodand water ad libitum.

Placement of guide cannulae

Five days before the experiments, the rats were deeplyanaesthetized with tribromoethanol (250 mg kg−1 I.P.;Aldrich Chemical Company, Milwaukee, WI, USA) andplaced in a stereotaxic frame (Stoelting, Wood Dale, IL,USA) to allow implantation of bilateral guide cannulaedirected towards the DMH. Frequent toe pinching wasused to assess the depth of anaesthesia. If flexor reflex wasevident, anaesthesia was supplemented (tribromoethanol,250 mg kg−1 I.P.). A longitudinal incision in the skin of thehead was made, and bregma and lambda were maintainedat the same level. A small window was opened in theskull, through which 15-mm-long stainless-steel guidecannulae (22 gauge; Small Parts, Miami Lakes, FL, USA)were introduced in a perpendicular direction. Accordingto the atlas of Paxinos & Watson (2005), the followingcoordinates were used: 3.0 mm posterior to bregma,±0.6 mm lateral to the mid-line and 7.0 mm below theskull surface at bregma. The tips of the guide cannulae werepositioned approximately 1.7 mm above the DMH. Guidecannulae were fixed to the skull with methacrylate andscrews, and closed with an occluder until the experimentswere carried out. Intramuscular antibiotics were given toassist postoperative recovery.

Arterial and venous catheterization

One day before the experiments, under tribromoethanol(250 mg kg−1 I.P.) anaesthesia, a small incision was madein the inguinal region, and the femoral artery and veinwere exposed. Polyethylene catheters (PE-10 connected toPE-50; Clay Adams, Parsippany, NJ, USA) filled with saline(NaCl 0.9%) were inserted into the abdominal aorta via thefemoral artery and into the femoral vein. Both catheterswere tunnelled subcutaneously and exteriorized throughthe back of the neck. The catheter inserted in the femoralartery was used for recording of cardiovascular parametersand the catheter in the femoral vein was used forintravenous injection. Rats were placed in individual cagesand kept in the experimental room for environmentaladaptation.

Chemoreflex stimulation

The peripheral chemoreflex was activated by intravenousinjection of 100 μl potassium cyanide (KCN; 40 μg perrat; Merck, Darmstadt, Germany) in accordance withthe procedures described by Franchini & Krieger (1993)and previously validated for our experimental conditions(Haibara et al. 1995, 1999).

Recording of cardiovascular parameters

The catheter in the femoral artery was flushed withheparinized saline (0.9% NaCl) to prevent clotting andthen connected to the pressure transducer (model CDX

C© 2010 The Authors. Journal compilation C© 2011 The Physiological Society

Exp Physiol 96.2 pp 73–84 Dorsomedial hypothalamus and chemoreflex in conscious rats 75

III; Cobe Laboratories, Lakewood, CO, USA). Pulsatilearterial pressure (PAP) was continuously recorded by anA/D data acquisition system (MP100; Biopac Systems, Inc.,Santa Barbara, CA, USA). Mean arterial pressure (MAP)and heart rate (HR) were simultaneously derived fromarterial pulse waves by software (AcqKnowledge5; BiopacSystems).

Behavioural response measurement

To assess the behavioural chemoreflex response, thelocomotor activity induced by peripheral chemoreceptoractivation was measured in some animals in an exploratorybox by the open-field method, simultaneously with therecording of cardiovascular parameters. Measurements oflocomotor activity can provide information about animalbehaviour and can be used to index the behavioural effectsof various experimental manipulations (Hsieh & Yang,2008). The open-field box was 50 cm long, 30 cm wideand 20 cm high, with walls of opaque, black acrylic. Thechemoreflex behavioural response was recorded througha webcam mounted 70 cm above the central position ofthe cage. The sensitivity of the camera was high enoughto create a clear image of the rat, and the recording wassaved as an AVI format movie, at a rate of 3 frames s−1 and320 × 240 pixels per frame. The digital image processingwas done offline using MATLAB (The MathWorks, Inc.,Natick, MA, USA). The original RGB colour patternof each AVI frame was processed through a histogramshape-based threshold algorithm (Otsu, 1979) in order toproduce a black and white mask of the rat. The geometriccentre of the rat was determined, for each frame, bycalculating the arithmetic average of all overthresholdpixels in both x and y coordinates. The known dimensionof the open field was used to calibrate the pixel percentimetre resolution and, thus, determine the actualdistance of the geometric centre of the rat, sampled at3 Hz (3 frames s−1).

Drug solutions

The drugs used in experiments were lidocaine (MerrellLepetit, Sao Paulo, SP, Brazil) and kynurenic acid (EAAreceptor antagonist; RBI, Natick, MA, USA). The dosesof lidocaine and kynurenic acid used in the present studywere based on previous studies (Soltis & DiMicco, 1991,1992; Haibara et al. 2002; Reddy et al. 2005; Da Silvaet al. 2006). Kynurenic acid was dissolved in 0.9% salinesolution (NaCl), and sodium bicarbonate was added to thesolutions in order to adjust the pH to the 7.0–7.4 range.All the drugs used were freshly dissolved.

Microinjection procedures

For microinjections into the DMH, a 33 gauge needle(Small Parts) 1.7 mm longer than the guide cannulae wasconnected by PE-10 tubing to a 1 μl syringe (Hamilton,

Reno, NV, USA). After removal of the occluder, themicroinjection needle was carefully inserted into the guidecannula, and manual injection was started 30 s later. Theneedle was repositioned on the contralateral side andthe second injection performed. The volume for eachmicroinjection was 200 nl in all experimental protocols.

Experimental protocols

All studies were performed in conscious, freely movinganimals after their recovery from instrumentation. Onlyanimals that presented no evidence of pain or distress wereused in the experiments. The experimental protocol forthe evaluation of the DMH in the chemoreflex neuronalpathways consisted of activation of the chemoreflex beforeand after bilateral microinjection of lidocaine, a localanaesthetic, into the DMH (n = 5), in order to produce areversible blockade of neuronal activity of this region. Thepeak changes in MAP and HR in response to intravenousinjection of KCN (40 μg) were evaluated before and 2, 5and 15 min after the bilateral microinjection of lidocaine(2%) into the DMH. In order to evaluate the role of EAAreceptors in the DMH on cardiovascular and behaviouralchemoreflex responses, the peak changes in MAP, HRand locomotor activity in response to KCN (40 μg) wereevaluated before and 10, 20, 30 and 45 min after thebilateral kynurenic acid (2.7 nmol) microinjection intothe DMH (n = 6). As a control, in another group of rats,the cardiovascular and behavioural chemoreflex responseswere evaluated before and 10, 20, 30 and 45 min afterbilateral microinjection of the vehicle (0.9% NaCl) intothe DMH (n = 4).

Histological examination

At the end of each experiment, Alcian Blue dye solution(2.5%) was microinjected bilaterally to mark the sitesof injection. Animals were killed with an overdose ofthiopental sodium (90 mg kg−1, I.V., Cristalia, Itapira, SP,Brazil) and perfused via the intracardiac route with saline(0.9% NaCl) followed by 10% buffered formalin. Brainswere removed and stored in buffered formalin for 2 daysand then serial coronal sections (50 μm thickness) wereobtained and stained with Neutral Red (1%) using theNissl method. The histology was considered positive whenthe centre of microinjection reached the DMH bilaterally.The microinjections performed in areas outside theDMH (i.e. peri-DMH) were used as a control misplacedmicroinjections group. The locations of the microinjectionsites were plotted on representative drawings from a ratstereotaxic atlas (Paxinos & Watson, 2005).

Data analysis

All data are expressed as means ± S.E.M. The results wereanalysed by one-way ANOVA followed by Dunnett’s post



hoc test. The differences between individual means werecompared by Student’s paired t test. Correlation betweenthe alterations in pressor and behavioural responsesinduced by chemoreflex activation after kynurenic acidmicroinjection into the DMH was assessed using Pearson’scorrelation coefficient. In all statistical analysis, the levelof significance was P < 0.05.

Results

Effect of bilateral microinjection of lidocaineinto the DMH on the cardiovascular responsesto chemoreflex activation

Figure 1 shows typical representative traces of pulsatilearterial pressure, mean arterial pressure and heart rateresponses to chemoreflex activation by KCN (40 μg) in onerat, before and after bilateral microinjection of lidocaine(2%) into the DMH. As expected, the chemoreflexactivation evoked an increase in blood pressure andbradycardia. Lidocaine microinjected bilaterally into the

DMH attenuated the pressor response, but not thebradycardic response of the chemoreflex.

Figure 2 shows grouped data summarizing the peakchanges in mean arterial pressure and heart rate inresponse to chemoreflex activation before and after themicroinjection of lidocaine (n = 5) into the DMH. Thesedata show that 2 and 5 min after lidocaine microinjection,the chemoreflex pressor response was significantlyreduced (+51 ± 4 versus +34 ± 5 and +41 ± 3 mmHg,respectively, P < 0.05). Although locomotor activity hasnot been quantified in this experimental group, we mightnote that the behavioural response of the chemoreflexwas also reduced after lidocaine microinjection into theDMH. However, during the period studied, lidocaineproduced no changes in the bradycardic response inducedby chemoreflex activation (−151 ± 45 versus −155 ± 47,−109 ± 25 and −138 ± 47 beats min−1 at 2, 5 and 15 min,respectively).

The effect of lidocaine microinjection into theDMH was reversible, considering that 15 min after themicroinjection, the pressor response of chemoreflex

Figure 1. Representative traces of cardiovascular responses to chemoreflex activation before and aftermicroinjections of lidocaine into the DMHTypical recordings of pulsatile arterial pressure (PAP), mean arterial pressure (MAP) and heart rate (HR), illustratingthe pressor and bradycardic responses to an intravenous bolus injection of KCN (40 μg, I.V.) before (KCN control)and 2, 5 and 15 min after bilateral microinjection of 2% lidocaine into the DMH of the conscious animal. Thearrows indicate the time of KCN injection.



Table 1. Baseline mean arterial pressure (MAP) and heart rate (HR) before and after bilateral microinjection of lidocaine (2%,n = 5), kynurenic acid (2.7 nmol, n = 6) or saline (n = 4) into the DMH of conscious rats

Before microinjection After microinjection

A. Lidocaine 2 min 5 min 15 minMAP (mmHg) 96.2 ± 4.4 98.0 ± 4.4 96.8 ± 3.9 100.4 ± 3.3HR (beats min−1) 357.2 ± 16.6 360.4 ± 12.7 350.0 ± 16.2 396.2 ± 9.9

B. Kynurenic acid 10 min 20 min 30 min 45 minMAP (mmHg) 118.5 ± 2.3 110.2 ± 3.8∗ 111.2 ± 3.6∗ 111.7 ± 2.7∗ 113.0 ± 1.4HR (beats min−1) 398.3 ± 17.6 358.2 ± 19.1∗ 353.2 ± 22.7∗ 357.5 ± 24.4∗ 354.7 ± 21.9

C. Saline 10 min 20 min 30 min 45 minMAP (mmHg) 113.0 ± 5.4 114.8 ± 3.9 113.8 ± 5.9 111.0 ± 4.4 111.3 ± 3.6HR (beats min−1) 386.5 ± 44.3 394.3 ± 31.1 376.8 ± 30.4 374.8 ± 34.7 365.5 ± 33.6

Values are means ± S.E.M. ∗ Statistical difference was observed when values before and after bilateral microinjection werecompared (P < 0.05).

activation was similar to that observed in the controlperiod (+51 ± 4 versus +46 ± 4 mmHg, control versus15 min, respectively). Lidocaine microinjected into theDMH did not elicit significant changes in baseline meanarterial pressure or baseline heart rate (Table 1).

Effect of bilateral microinjection of kynurenic acidinto the DMH on the cardiovascular and behaviouralresponses to chemoreflex activation

Figure 3 is a typical trace from one rat representativeof the group, showing the cardiovascular responses tochemoreflex activation by KCN (40 μg) before and afterbilateral microinjection of kynurenic acid (2.7 nmol) intothe DMH. Bilateral microinjection of kynurenic acid intothe DMH produced a clear reduction in the magnitude ofthe pressor response but not in the bradycardic responseto chemoreflex activation.

The data summarized in Fig. 4A show that kynurenicacid (n = 6) microinjected into the DMH produced asignificant reduction in the pressor response 10, 20, 30and 45 min after microinjection (+50 ± 3 versus +22 ± 5,+31 ± 8, +31 ± 6 and +29 ± 7 mmHg, respectively,P < 0.05), but produced no changes in bradycardicresponse of the chemoreflex during the period studied(−229 ± 21 versus −178 ± 37, −174 ± 33, −181 ± 19and −208 ± 18 beats min−1). Figure 4A also shows thatkynurenic acid microinjected into the DMH reducedthe locomotor activity of the behavioural response tochemoreflex stimulation (44 ± 6 versus 5 ± 4, 6 ± 4,13 ± 7 and 21 ± 7 cm, P < 0.05) at 10, 20, 30 and 45 minafter the microinjection, respectively (Supplementaryvideos 1 and 2). Interestingly, there was no correlationbetween the reduction in pressor and behaviouralresponses of the chemoreflex (r = −0.186, P > 0.05;Fig. 5).

Misplaced microinjection of kynurenic acid in thevicinity of the DMH (i.e. peri-DMH, n = 4) producedno significant changes in pressor (+63 ± 2 versus

+56 ± 2, +55 ± 4, +58 ± 2 and +60 ± 2 mmHg) orin bradycardic responses (−189 ± 32 versus −161 ± 35,−192 ± 27, −198 ± 43 and −161 ± 42 beats min−1) tochemoreflex activation (Fig. 6). In the same way,bilateral microinjection of saline (n = 4) into the DMHproduced no changes in pressor (+35 ± 3 versus +33 ± 7,+34 ± 5, +37 ± 8 and +38 ± 8 mmHg), bradycardic(−152 ± 20 versus −191 ± 44, −160 ± 30, −197 ± 51and −181 ± 35 beats min−1) or behavioural responses(39 ± 9 versus 44 ± 10, 32 ± 8, 35 ± 14 and 46 ± 7 cm) ofchemoreflex activation at 10, 20, 30 and 45 min after the

Figure 2. Changes in the pressor and bradycardic responses tochemoreflex activation before and after microinjection oflidocaine into the DMHChanges in mean arterial pressure (�MAP) and heart rate (�HR) inresponse to chemoreflex activation with KCN (40 μg, I.V.) before (KCNcontrol) and 2, 5 and 15 min after bilateral microinjection of lidocaine(2%; n = 5) into the DMH of conscious animals. ∗ Statistically differentin relation to the control response (P < 0.05).



microinjection, respectively, in relation to control values(Fig. 4B).

Table 1 shows the absolute values of MAP and HRimmediately before and 10, 20, 30 and 45 min afterbilateral microinjection of kynurenic acid into the DMH.The data indicate that kynurenic acid microinjected intothe DMH elicited a small but significant reduction in base-line cardiovascular parameters. Saline microinjected intothe DMH did not elicit significant changes in baselinemean arterial pressure or baseline heart rate (Table 1).

Histological analysis of microinjection sites

Figure 7 summarizes the microinjection sites from allexperimental protocols of this study. Figure 7B is aphotomicrograph of a transverse section of the brain ofone rat representative of the group, showing the sites ofbilateral microinjections into the DMH, whose locationis shown in Fig. 7A in a schematic representation adaptedfrom the stereotaxic atlas of Paxinos & Watson (2005).Schematic drawings of coronal sections of the rat brainto illustrate the relative location of microinjection centresalong the rostrocaudal extension of the DMH are shown

in Fig. 7C. The majority of the injection sites were locatedin, or in the immediate vicinity of, the compact portionof the DMH, approximately −3.24 mm caudal to bregma.In four other rats, we failed to achieve the desired cannulaplacement in the DMH, and the microinjection centreswere localized in the peri-DMH region.

Discussion

Several studies have suggested the involvement ofhypothalamic areas in the chemoreflex pathways (Thomas& Calaresu, 1972; Marshall, 1994; Silva-Carvalho et al.1995; Olivan et al. 2001; Cruz et al. 2008). A previousstudy using Fos immunoreactivity supports the hypothesisthat the neuronal population of the DMH is involved inchemoreflex networks (Berquin et al. 2000). However,the effective role of this nucleus in the reflex responsesof chemoreflex activation in conscious rats has notbeen previously evaluated. The present study showedthat bilateral microinjection of lidocaine into the DMHproduced a significant reduction of the pressor responseinduced by chemoreflex activation. Thus, this is the firstfunctional study to provide clear evidence that the DMH

Figure 3. Representative traces of cardiovascular responses to chemoreflex activation before and aftermicroinjections of kynurenic acid into the DMHTypical recordings of PAP, MAP and HR, illustrating the pressor and bradycardic responses to an intravenous bolusinjection of KCN (40 μg, I.V.) before (KCN control) and 10, 20 and 30 min after bilateral microinjection of kynurenicacid (2.7 nmol) into the DMH of the conscious animal. The arrows indicate the time of KCN injection.



is part of the neural pathway involved in the generation ofthe sympathoexcitatory component of the chemoreflex.However, considering that lidocaine inactivates bothneuronal soma and fibres of passage, it is possible that thiseffect was due to inactivation of axonal signals receivedfrom structures entirely outside the DMH. The findingthat bilateral microinjection of kynurenic acid into theDMH also produced an important reduction in pressorand behavioural chemoreflex responses confirms andprovides additional evidence that activation of neuronsin the DMH, through EAA receptors, is essential for thecomplete expression of the cardiovascular and behaviouralresponses of the chemoreflex.

There is considerable evidence that EAA receptorsin the DMH play a critical role in the integration ofcardiovascular responses induced in emotional stressmodels. Microinjection of EAA agonists in the DMHevokes a pattern of cardiovascular and neuroendocrineresponses identified as the defence reaction, involving, forinstance, tachycardia, a modest increase in arterial pressureand increased plasma levels of ACTH (Soltis & DiMicco,

1992; Bailey & DiMicco, 2001). Moreover, blockade of EAAreceptors in the DMH elicited an important reduction inthe cardiovascular responses induced by air jet stress orchemical disinhibition of the DMH (Soltis & DiMicco,1991, 1992; DiMicco et al. 1996). The participationof EAA receptors in the DMH in the modulation ofbehaviour/locomotor responses has also been previouslyreported. Microinjection of EAA agonists into the DMHof conscious rats produces a pattern of behaviouralresponses closely related to the defence reaction (Silveira& Graeff, 1992; Bailey & DiMicco, 2001). In addition,studies in anaesthetized rats have demonstrated thatmicroinjection of L-glutamate into the DMH initiateslocomotor stepping responses (Marciello & Sinnamon,1990). Antagonists of EAA receptors also modulate thecardiovascular and behavioural lactate-induced panic-likeresponses in rats with GABA dysfunction in the DMH(Johnson & Shekhar, 2006). Accordingly, the attenuationof pressor and behavioural chemoreflex responses inducedby kynurenic acid in our study strongly supports thehypothesis that EAA receptors in the DMH are involved

Figure 4. Changes in the pressor, bradycardic and behavioural responses to chemoreflex activationbefore and after microinjection of kynurenic acid or saline into the DMHChanges in mean arterial pressure (� MAP), heart rate (� HR) and locomotor activity in response to chemoreflexactivation with KCN (40 μg, I.V.), before (KCN control) and 10, 20, 30 and 45 min after bilateral microinjection ofkynurenic acid (2.7 nmol; n = 6; A) or saline (0.9% NaCl; n = 4; B) into the DMH of conscious animals. ∗ Statisticallydifferent in relation to the control response (P < 0.05).



in the modulation of autonomic and somatomotorresponses.

In the present study, we observed that bilateral blockadeof the EAA receptors in the DMH did not abolish thepressor response of the chemoreflex, but only reduced it.As only one dose of the kynurenic acid antagonist wasused, it is possible to consider that the pressor responseof the chemoreflex was not abolished because the EAAreceptors were only partly blocked. However, in the sameanimals this dose of kynurenic acid virtually abolished thebehavioural response of the chemoreflex, which indirectlysuggests that the dose of 2.7 nmol effectively blocked theEAA receptors. In addition, bilateral microinjection oflidocaine into the DMH was also only able to attenuate thepressor response of the chemoreflex. These results suggestthat the DMH is essential to integrate the behaviouralresponse of the chemoreflex, whereas the pressor responseis partly dependent on integration in the DMH.

In addition to eliciting changes in the reflex responsesto activation of chemoreceptors, blockade of the EAAreceptors in the DMH elicited a small, but significant,reduction in the baseline MAP and HR. These dataare consistent with a previous study on anaesthetizedrats (Soltis & DiMicco, 1992) and suggest that theEAA receptors are tonically activated, contributing tothe maintenance of baseline cardiovascular parameters.Therefore, our data confirm and extend the results ofprevious studies, suggesting that EAA receptors in theDMH are significantly involved in tonic and reflex controlof blood pressure.

It is important to note that the reduction in thebehavioural response of the chemoreflex induced byblockade of the EAA receptors in the DMH does not

Figure 5. Correlation between reduction in pressor andbehavioural chemoreflex responses after kynurenic acidmicroinjection into the DMHValues are expressed as a percentage of control responses (beforekynurenic acid).

appear to be related to the impairment of motor functionor to a possible anxiolytic effect. Studies by Jardim &Guimaraes (2001) showed that microinjection of EAAantagonists into the DMH did not produce an anxiolyticeffect in the elevated plus maze model, but did reduce theexploratory activity. This reduction in locomotor responsewas not due to impairment of the motor function,evaluated by the rota-rod test (Jardim & Guimaraes,2004). In fact, Chou et al. (2003) reported that chemicallesion of the DMH reduced the circadian rhythms oflocomotor activity, suggesting that the level of behaviouralresponse is dependent on this region. Taken together, theseresults suggest that EAA neurotransmission in the DMHinfluences the locomotor behaviour. This hypothesis isconsistent with the effects observed in our study, in whichkynurenic acid microinjected into the DMH abolished thebehavioural response of the chemoreflex.

The existence of neural circuits that can simultaneouslyregulate the activity of somatomotor and sympatheticefferent systems has been demonstrated (Kerman, 2008).The presence of these sympathomotor neurons wasdetected in several hypothalamic nuclei, including those inthe DMH region (Kerman et al. 2006). Thus, consideringthat blockade of the EAA receptors in the DMH elicitedan important reduction in the pressor response andsimultaneously abolished the behavioural response ofthe chemoreflex, it could be argued that the reductionin the pressor response was a secondary effect of the

Figure 6. Changes in the pressor and bradycardic responses tochemoreflex activation before and after microinjection ofkynurenic acid in the vicinity of the DMHChanges in mean arterial pressure (� MAP) and heart rate (� HR) inresponse to chemoreflex activation with KCN (40 μg, I.V.) before (KCNcontrol) and 10, 20, 30 and 45 min after bilateral microinjection ofkynurenic acid (2.7 nmol; n = 4) in the peri-DMH region of consciousanimals.



abolition of the behavioural response of the chemoreflex.Although we cannot exclude this possibility, there is nocorrelation between the reduction in pressor response andreduction in locomotor activity induced by chemoreflexactivation. Therefore, considering this indirect evidence,it is reasonable to suggest that the decrease in chemoreflexpressor response does not appear to be associated withreduced behavioural response. This observation indicatesthat the EAA receptors in the DMH may play animportant role in the processing of the chemoreflexpressor response. However, the present study did not haveenough data to further define the appropriate mechanismsby which the DMH neurons coordinate the complexpattern of cardiovascular and behavioural responses ofthe chemoreflex. Such questions need to be addressed byfuture studies.

The effect of kynurenic acid on the chemoreflex seemsto be restricted to the DMH, because microinjection ofthis antagonist into the vicinity of the DMH did not elicitany change in the chemoreflex cardiovascular responses.

Moreover, the average of the anterior–posterior extensionof the area stained by Alcian Blue dye was 0.57 ± 0.04 mmin the animals in which the microinjection centre ofkynurenic acid was located in the DMH. This is a relevantpoint to take into account, because the paraventricularnucleus of the hypothalamus (PVN), located about1 mm from the DMH, has also been described as anintegrating centre for autonomic and endocrine responsesto stress (Swanson & Sawchenko, 1980). In addition,several studies reported involvement of the PVN in thechemoreflex pathways (Berquin et al. 2000; Cruz et al.2008). Electrolytic lesion of the PVN produced a markedreduction in the magnitude and duration of the pressorresponse to chemoreflex activation in conscious rats,indicating that this hypothalamic nucleus may play apermissive role in the sympathoexcitatory componentof the chemoreflex (Olivan et al. 2001). Microinjectionof an EAA antagonist (Kubo et al. 1997) or lidocaine(Reddy et al. 2005) into the PVN elicited an importantreduction in the pressor response and sympathoexcitatory

Figure 7. Photomicrograph and schematic drawings of the sites of microinjection into the DMH ofconscious ratsA, schematic drawing showing the location of the DMH. Abbreviations: DMC, compact portion of the dorsomedialhypothalamus; DMD, diffuse portion of the dorsomedial hypothalamus; VMH, ventromedial hypothalamus;f, fornix; and 3V, third ventricle. B, representative photomicrograph of a coronal section in which bilateralmicroinjections were made into the DMH. C, schematic drawings illustrating the microinjection centres located inthe DMH (•) and in the peri-DMH region (◦) of conscious rats. Distance from bregma is indicated. Redrawn fromPaxinos & Watson (2005).



component of the peripheral chemoreflex in anaesthetizedrats. However, the absence of dye in the PVN in ourstudy indicates that the responses observed after themicroinjection of the kynurenic acid into the DMH arerestricted to this region. Recently, Cruz & Machado (2009)have reported that the bilateral microinjection of a largerdose of kynurenic acid (7.2 nmol) into the PVN didnot affect the cardiovascular responses to chemoreflexactivation in conscious rats. In addition, studies by Chenet al. (2003) and Li et al. (2006) also showed that EAAantagonist in the PVN does not affect the baseline MAPor HR. Therefore, we can exclude the possibility that theeffect of kynurenic acid on the chemoreflex observed inour study is due to diffusion of this antagonist to neuronswithin the PVN.

Although the respiratory changes in response tochemoreflex activation after injection of lidocaine orkynurenic acid into the DMH were not measured inthe present study, we cannot rule out the possibleinvolvement of the DMH in the tachypnoeic responses tochemoreflex activation (Berquin et al. 2000). Stimulationor disinhibition of the DMH neurons in anaesthetizedrats elicited an increase in respiratory frequency (Shekhar,1993; Tanaka & McAllen, 2008). Thus, the activation ofrespiratory neurons in the DMH may induce an additionalincrease in the firing of inspiratory neurons on theventral surface of the brainstem, hence an increase inthe activity of sympathetic preganglionic neurons locatedin the rostral ventrolateral medulla (Guyenet & Koshiya,1995; Koshiya & Guyenet, 1996). We may suggest thatblockade of the neuronal activity of the DMH by lidocaineor kynurenic acid affects the ventilatory responses tochemoreflex activation and consequently the increase insympathetic activity and MAP. More recently, McDowallet al. (2007) showed that microinjection of bicucullineinto the DMH elicits an increase in the frequency andamplitude of phrenic nerve activity, as well as an increasein renal sympathetic nerve activity. However, this samestudy showed that there was no correlation betweenphrenic nerve activity and renal sympathetic nerve activity,indicating that the increase in sympathetic activity wasnot a consequence of the increase in respiratory activity.Despite this finding, further studies are needed to evaluatethe respiratory and cardiovascular components of thechemoreflex during the blockade of the EAA receptorsin the DMH to better understand their role in integratingthese responses.

In conclusion, the data of the present study showthat the DMH is part of the neural pathways involvedin the integration of the chemoreflex. In addition, thisstudy showed that the EAA receptors in the DMH playa key role in the processing of the behavioural responseof the chemoreflex and participate, at least in part, inthe neurotransmission of the pressor response of thechemoreflex in conscious rats.

References

Bailey TW & DiMicco JA (2001). Chemical stimulation of thedorsomedial hypothalamus elevates plasma ACTH inconscious rats. Am J Physiol Regul Integr Comp Physiol 280,R8–R15.

Barros RC, Bonagamba LG, Okamoto-Canesin R, de OliveiraM, Branco LG & Machado BH (2002). Cardiovascularresponses to chemoreflex activation with potassium cyanideor hypoxic hypoxia in awake rats. Auton Neurosci 97,110–115.

Bernardis LL & Bellinger LL (1998). The dorsomedialhypothalamic nucleus revisited: 1998 update. Proc Soc ExpBiol Med 218, 284–306.

Berquin P, Bodineau L, Gros F & Larnicol N (2000). Brainstemand hypothalamic areas involved in respiratorychemoreflexes: a Fos study in adult rats. Brain Res 857,30–40.

Cao WH, Fan W & Morrison SF (2004). Medullary pathwaysmediating specific sympathetic responses to activation ofdorsomedial hypothalamus. Neuroscience 126, 229–240.

Chen QH, Haywood JR & Toney GM (2003).Sympathoexcitation by PVN-injected bicuculline requiresactivation of excitatory amino acid receptors. Hypertension42, 725–731.

Chou TC, Scammell TE, Gooley JJ, Gaus SE, Saper CB & Lu J(2003). Critical role of dorsomedial hypothalamic nucleus ina wide range of behavioral circadian rhythms. Neurosci 23,10691–10702.

Cruz JC, Bonagamba LG, Machado BH, Biancardi VC & SternJE (2008). Intermittent activation of peripheralchemoreceptors in awake rats induces Fos expression inrostral ventrolateral medulla-projecting neurons in theparaventricular nucleus of the hypothalamus. Neuroscience157, 463–472.

Cruz JC & Machado BH (2009). GABA and nitric oxide in thePVN are involved in arterial pressure control but not in thechemoreflex responses in rats. Auton Neurosci 146,47–55.

Da Silva LG, Menezes RCA, Villela DC & Fontes MAP (2006).Excitatory amino acid receptors in the periaqueductal graymediate the cardiovascular response evoked by activation ofdorsomedial hypothalamic neurons. Neuroscience 139,1129–1139.

De Novellis V, Stotz-Potter EH, Morin SM, Rossi F & DiMiccoJA (1995). Hypothalamic sites mediating cardiovasculareffects of microinjected bicuculline and EAAs in rats. Am JPhysiol Regul Integr Comp Physiol 269, R131–R140.

DiMicco JA, Samuels BC, Zaretskaia MV & Zaretsky DV(2002). The dorsomedial hypothalamus and the response tostress: part renaissance, part revolution. Pharmacol BiochemBehav 71, 469–480.

DiMicco JA, Stotz-Potter EH, Monroe AJ & Morin SM (1996).Role of the dorsomedial hypothalamus in the cardiovascularresponse to stress. Clin Exp Pharmacol Physiol 23,171–176.

Fontes MA, Tagawa T, Polson JW, Cavanagh SJ & Dampney RA(2001). Descending pathways mediating cardiovascularresponse from dorsomedial hypothalamic nucleus. Am JPhysiol Heart Circ Physiol 280, H2891–H2901.



Franchini KG & Krieger EM (1993). Cardiovascularresponses of conscious rats to carotid body chemoreceptorstimulation by intravenous KCN. J Auton Nerv Syst 42,63–70.

Guyenet PG & Koshiya N (1995). Working model of thesympathetic chemoreflex in rats. Clin Exp Hypertens 17,167–179.

Haibara AS, Bonagamba LGH & Machado BH (1999).Sympathoexcitatory neurotransmission of the chemoreflexin the NTS of awake rats. Am J Physiol Regul Integr CompPhysiol 276, R69–R80.

Haibara AS, Colombari E, Chianca DA Jr, Bonagamba LGH &Machado BH (1995). NMDA receptors in NTS areinvolved in bradycardic but not in pressor response ofchemoreflex. Am J Physiol Heart Circ Physiol 269,H1421–H1427.

Haibara AS, Tamashiro E, Olivan MV, Bonagamba LGH &Machado BH (2002). Involvement of the parabrachialnucleus in the pressor response to chemoreflex activation inawake rats. Auton Neurosci 101, 60–67.

Hilton SM (1982). The defence-arousal system and itsrelevance for circulatory and respiratory control. J Exp Biol100, 159–174.

Hilton SM & Joels N (1965). Facilitation of chemoreceptorreflexes during the defence reaction. J Physiol 176,20P.

Honig A, Wedler B, Oppermann H, Gruska S & Schmidt M(1996). Effect of arterial chemoreceptor stimulation withalmitrine bismesylate on plasma renin activity,aldosterone, ACTH and cortisol in anaesthetized,artificially ventilated cats. Clin Exp Pharmacol Physiol 23,106–110.

Hsieh YL & Yang CC (2008). Age-series characteristics oflocomotor activities in spontaneously hypertensive rats: acomparison with the Wistar–Kyoto strain. Physiol Behav 93,777–782.

Jardim MC & Guimaraes FS (2001). GABAergic andglutamatergic modulation of exploratory behavior in thedorsomedial hypothalamus. Pharmacol Biochem Behav 69,579–584.

Jardim MC & Guimaraes FS (2004). Role of glutamateionotropic receptors in the dorsomedial hypothalamicnucleus on anxiety and locomotor behavior. PharmacolBiochem Behav 79, 541–546.

Johnson PL & Shekhar A (2006). Panic-prone state induced inrats with GABA dysfunction in the dorsomedialhypothalamus is mediated by NMDA receptors. J Neurosci26, 7093–7104.

Keim SR & Shekhar A (1996). The effects of GABAA receptorblockade in the dorsomedial hypothalamic nucleus oncorticotrophin (ACTH) and corticosterone secretion in malerats. Brain Res 739, 46–51.

Kerman IA (2008). Organization of brain somatomotor-sympathetic circuits. Exp Brain Res 187, 1–16.

Kerman IA, Akil H & Watson SJ (2006). Rostral elements ofsympatho-motor circuitry: a virally mediated transsynaptictracing study. J Neurosci 26, 3423–3433.

Koshiya N & Guyenet PG (1996). Tonic sympatheticchemoreflex after blockade of respiratory rhythmogenesis inthe rat. J Physiol 491, 859–869.

Kubo T, Yanagihara Y, Yamaguchi H & Fukumori R (1997).Excitatory amino acid receptors in the paraventricularhypothalamic nucleus mediate pressor response induced bycarotid body chemoreceptor stimulation in rats. Clin ExpHypertens 19, 1117–1134.

Li YF, Jackson KL, Stern JE, Rabeler B & Patel KP (2006).Interaction between glutamate and GABA systems in theintegration of sympathetic outflow by the paraventricularnucleus of the hypothalamus. Am J Physiol Heart Circ Physiol291, H2847–H2856.

McDowall LM, Horiuchi J & Dampney RA (2007). Effects ofdisinhibition of neurons in the dorsomedial hypothalamuson central respiratory drive. Am J Physiol Regul Integr CompPhysiol 293, R1728–R1735.

Marciello M & Sinnamon HM (1990). Locomotor steppinginitiated by glutamate injections into the hypothalamus ofthe anesthetized rat. Behav Neurosci 104, 980–990.

Marshall JM (1994). Peripheral chemoreceptors andcardiovascular regulation. J Physiol Rev 74, 543–594.

Marshall JM & Metcalfe JD (1988). Analysis of thecardiovascular changes induced in the rat by graded levels ofsystemic hypoxia. J Physiol 407, 385–403.

Meeker RB, Greenwood RS & Hayward JN (1994). Glutamatereceptors in the rat hypothalamus and pituitary.Endocrinology 134, 621–629.

Olivan MV, Bonagamba LG & Machado BH (2001).Involvement of the paraventricular nucleus of thehypothalamus in the pressor response to chemoreflexactivation in awake rats. Brain Res 895, 167–172.

Otsu N (1979). A threshold selection method from gray-levelhistograms. IEEE Trans Sys Man Cyber 9, 62–66.

Paxinos G & Watson C (2005). The Rat Brain in StereotaxicCoordinates. Elsevier Academic Press, San Diego.

Raff H & Fagin KD (1984). Measurement of hormones andblood gases during hypoxia in conscious cannulated rats. JAppl Physiol 56, 1426–1430.

Raff H, Tzankoff SP & Fitzgerald RS (1982). Chemoreceptorinvolvement in cortisol responses to hypoxia in ventilateddogs. J Appl Physiol 52, 1092–1096.

Reddy MK, Patel KP & Schultz HD (2005). Differential role ofthe paraventricular nucleus of the hypothalamus inmodulating the sympathoexcitatory component ofperipheral and central chemoreflexes. Am J Physiol RegulIntegr Comp Physiol 289, R789–R797.

Shekhar A (1993). GABA receptors in the region of thedorsomedial hypothalamus of rats regulate anxiety in theelevated plus-maze test. I. Behavioral measures. Brain Res627, 9–16.

Silva-Carvalho L, Dawid-Milner MS, Goldsmith GE & SpyerKM (1993). Hypothalamic-evoked effects in cat nucleustractus solitarius facilitating chemoreceptor reflexes. ExpPhysiol 78, 425–428.

Silva-Carvalho L, Dawid-Milner MS, Goldsmith GE & SpyerKM (1995). Hypothalamic modulation of the arterialchemoreceptor reflex in the anaesthetized cat: role of thenucleus tractus solitarii. J Physiol 487, 751–760.

Silveira MC & Graeff FG (1992). Defense reaction elicited bymicroinjection of kainic acid into the medial hypothalamusof the rat: antagonism by a GABAA receptor agonist. BehavNeural Biol 57, 226–232.



Soltis RP & DiMicco JA (1991). Interaction of hypothalamicGABAA and excitatory amino acid receptors controllingheart rate in rats. Am J Physiol Regul Integr Comp Physiol261, R427–R433.

Soltis RP & DiMicco JA (1992). Hypothalamic excitatoryamino acid receptors mediate stress-induced tachycardia inrats. Am J Physiol Regul Integr Comp Physiol 262, R689–R697.

Swanson LW & Sawchenko PE (1980). Paraventricular nucleus:a site for the integration of neuroendocrine and autonomicmechanisms. Neuroendocrinology 31, 410–417.

Tanaka M & McAllen RM (2008). Functional topography of thedorsomedial hypothalamus. Am J Physiol Regul Integr CompPhysiol 294, R477–R486.

Thomas MR & Calaresu FR (1972). Responses of single units inthe medial hypothalamus to electrical stimulation of thecarotid sinus nerve in the cat. Brain Res 44, 49–62.

Yardley CP & Hilton SM (1986). The hypothalamic andbrainstem areas from which the cardiovascular andbehavioural components of the defence reaction are elicitedin the rat. J Auton Nerv Syst 15, 227–244.

Acknowledgements

We are thankful to Jose Roberto da Silva for skilful technicalassistance. This study was supported by Fundacao de Amparo a

Pesquisa do Estado de Minas Gerais (FAPEMIG, grant CBB-APQ-3157-3.13/07). M. A. P. Fontes was supported by CNPqand FAPEMIG. E. A. Queiroz was a recipient of CAPES masterfellowship of the Post-graduation Program in Biological Science:Physiology and Pharmacology, ICB, UFMG. U. Fumega was arecipient of CAPES doctoral fellowship of the Post-graduationProgram in Bioinformatics, ICB, UFMG.

Supporting Information

Online supplementary material for this paper can be accessed atWiley Online Library:

Video 1. Representative video of cardiovascular andbehavioural responses to chemoreflex activation beforemicroinjection of kynurenic acid into the DMH.Video 2. Representative video of cardiovascular andbehavioural responses to chemoreflex activation aftermicroinjection of kynurenic acid into the DMH.

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