Involvement of the periaqueductal gray in the hypotensive response evoked by l -glutamate microinjection in the lateral hypothalamus of unanesthetized rats

Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93 www.elsevier.com/locate/autneu

Involvement of the periaqueductal gray in the hypotensive response evoked by l-glutamate microinjection in the lateral hypothalamus of unanesthetized rats Gisela Pugliesi Pajolla, Rodrigo Fiacadori Tavares, Gislaine Garcia Pelosi, Fernando Morgan Aguiar Correˆa * Department of Pharmacology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes 3900, Ribeira˜o Preto, Sa˜o Paulo, 14040-900, Brazil Received 7 March 2005; received in revised form 1 September 2005; accepted 2 September 2005

Abstract The lateral hypothalamus (LH) is involved in cardiovascular control. l-glutamate (l-glu) stimulation of the LH of unanesthetized rats evoked hypotensive responses without significant heart rate changes. The neuronal pathway that mediates this response is unknown. There is evidence that the periaqueductal gray (PAG) is involved in the mediation of hypotensive responses evoked by electrical stimulation of the LH. In the present study, we attempted to verify the effect of an acute and reversible pharmacological ablation of the PAG with lidocaine or CoCl2 on the hypotensive response caused by l-glu injection in the LH of unanesthetized rats. Microinjection of the local anesthetic lidocaine or the unspecific synaptic blocker CoCl2 in the PAG significantly attenuated the hypotensive effects of l-glu stimulation of the LH, indicating the involvement of local synapses within the PAG in the hypotensive pathway activated by LH glutamatergic receptors. Microinjection of the neuronal tracer biotinylated dextran amine (BDA) in the PAG labeled neuronal cell bodies in the LH, indicating the existence of direct connections between these areas. In conclusion, the present results indicate that the hypotensive response evoked by l-glu stimulation of LH may involve a synaptic relay in the dorsal PAG. D 2005 Elsevier B.V. All rights reserved. Keywords: Lateral hypothalamus; Periaqueductal gray; l-glutamate; Blood pressure; Hypotension; Biotinylated dextran amine; BDA

1. Introduction The lateral hypothalamus (LH) is a large diencephalic region extending from the lateral preoptic area to the ventral tegmental area, including the medial forebrain bundle (Saper et al., 1979; Berk and Finkelstein, 1983). This complex region has been subject of many physiological studies, with a major focus on its role in cardiovascular regulation. Despite of the large amount of information implicating the LH in central autonomic regulation, there is little evidence on the efferent pathways involved in this control. * Corresponding author. Tel.: +55 16 6023206; fax: +55 16 6332301. E-mail address: [email protected] (F.M.A. Correˆa). 1566-0702/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2005.09.001

Neuroanatomic studies have shown that the LH projects to regions that can affect cardiovascular function either directly or indirectly, such as the periaqueductal gray (PAG), the parabrachial nucleus, the nucleus of the solitary tract, the intermediolateral cell column and the vagal nuclei (Berk and Finkelstein, 1983; Holstege, 1987; Hosoya and Matsushita, 1981; ter Horst et al., 1984). Arterial pressure and heart rate responses can be elicited by both electrical and chemical stimulation of the LH (Cechetto and Chen, 1992; Kabat et al., 1935; Van der Plas et al., 1995; Allen and Cechetto, 1992; Gelsema et al., 1989; Spencer et al., 1989). In previous studies we verified that the microinjection of l-glutamate (l-glu) in the LH of unanesthetized rats caused hypotensive responses without significant heart rate changes (Pajolla and Correˆa, 2004).

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

However, the neural pathway involved in the mediation of this hypotensive response is unknown. Van der Plas et al. (1995) reported that low-intensity electrical stimulation of the LH evoked hypotensive effects, which were similar to those caused by local microinjection of excitatory amino acids (Spencer et al., 1989; Pajolla and Correˆa, 2004). They also reported that electrical stimulation of the LH inhibited the activity of pressor-related PAG neurons, suggesting the involvement of the PAG in the hypotensive pathway activated by the electrical stimulation of hypothalamic areas. Considering that, it would be opportune to investigate if the PAG is also a relay in the hypotensive pathway activated by the injection of l-glu in the LH. A common approach to investigate possible involvement of specific brain areas in a functional neural pathway is based on information obtained by means of its reversible functional ablation. The technique is based on the administration of circumscribed microinjections of compounds that reversible block neuronal activity over a given period of time. Microinjections of lidocaine or CoCl2 in discrete brain areas have been used for reversible functional inactivation (Kretz, 1984; Sandku¨ler et al., 1987). Additionally, neuronal connections among areas of the brain may be studied by histological analysis of the distribution of neuronal tracers after their microinjection in selected brain areas. A particularly useful tracer is the low-molecular-weight biotynilated dextran amine (BDA), which is bidirectionally transported, evidencing both neural afferents from projecting neurons and efferent projections of a brain region under study (Vercelli et al., 2000). Considering the above, we studied the involvement of the PAG in the hypotensive pathway activated by the microinjection of l-glu in the LH of unanesthetized rats. We evaluated the effects of injections of lidocaine or CoCl2 in the PAG on the hypotensive response to l-glu injection in the LH. Additionally, neuronal connections between the injection sites in the LH and the PAG were traced after microinjection of BDA in the PAG.

2. Materials and methods 2.1. Subjects Experimental procedures were carried out following protocols approved by the ethical review committee of the School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo. Male Wistar rats weighing 220 –280 g (n = 21) were used in the present experiment. Animals were housed individually in plastic cages in a temperature-controlled room (25 -C) in the Animal Care Unit of the Department of Pharmacology of the School of Medicine of Ribeira˜o Preto under a 12 : 12 h light – dark cycle. Animals had free access to water and standard laboratory chow, except during the experimental period.

85

2.2. Surgical preparation For implantation of stainless steel guide cannulas in the LH and PAG, the animals were anesthetized with tribromoethanol (250 mg/kg, i.p). After local anesthesia with 2% xylocaine, the skull was surgically exposed and stainless steel guide cannulas (24 G) were implanted 1 mm above the injection sites using a stereotaxic apparatus (Stoelting, USA). Stereotaxic coordinates for cannula implantation in the LH were selected from the brain atlas of Paxinos and Watson (1997): AP=+ 6.2 mm from the interaural line; L=+ 1.8 mm from the medial suture and V= 7.6 mm from the skull. Stereotaxic coordinates for cannula implantation in the PAG were AP=+2.7 mm from the interaural line, L=+2.5 mm from the medial suture and V= 4.7 mm from the skull with a lateral inclination of 26-. Cannulas were fixed to the skull with dental cement and one metal screw. A tight-fitting trocar was kept inside the guide cannula to avoid its occlusion. After surgery, animals were treated with 100,000 units of benzyl penicillin. Two days later, a polyethylene catheter was implanted in the femoral artery of the rats. The rats were anesthetized with tribromoethanol and a polyethylene catheter was implanted in the femoral artery for chronic blood pressure recording. The arterial catheter consisted of a segment of PE-10 tubing (4.0 cm) heat-bonded to a longer segment of PE-50 tubing (10 – 12 cm). The catheter was filled with 0.3% heparin (5000 UI/ml) in sterile saline (0.9% NaCl). The PE-10 segment was introduced in the femoral artery until the tip reached the aorta. The catheter was secured in position with thread and the PE-50 segment was passed under the skin to be extruded at the dorsum of the animals. After surgery, the animals were allowed to recover for 24 h. Animals were kept in individual cages during the postsurgery period. 2.3. Measurement of cardiovascular responses After surgery, the animals were kept in individual cages in the Animal Care Unit, which were transported to the experimental room. Animals were allowed a period of 15 min to adapt to the conditions of the experimental room, such as sound and illumination, prior to start of the blood pressure and heart rate recording. The experimental room was acoustically isolated and had constant background noise generated by an air exhauster. At least another period of 15 min was allowed before the experiments were initiated and care was taken to start injection whenever stable blood pressure and, specially, heart rate were observed. The injection needle was slowly introduced in the guide cannula without touching or restraining the animals. The mean arterial pressure of freely moving animals was recorded using an HP-7754A preamplifier (Hewlett Packard, USA) and an acquisition board (MP100A, Biopac Systems Inc, USA) connected to a computer.

86

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

Heart rate values were derived from the blood pressure recordings and processed online. Blood pressure baseline values were calculated as the average of the 3-min recording prior to the injection whenever blood pressure was considered to be stable. Mean arterial pressure and heart rate responses were measured at the peak of the hypotensive effect, which was observed approximately 15 s after the lglu injection. 2.4. Drug microinjection in the LH or the PAG l-glu (Sigma, USA) and CoCl2 (Sigma, USA) were dissolved in sterile 0.9% NaCl. Sodium bicarbonate was added to adjust the pH to 7.0 – 7.4. On the first day of the experiment, unanesthetized rats received 100 nL microinjections of 0.1 M l-glu in the LH. On the second day, the same rats received 100 nL microinjections of 0.1 M l-glu in the LH 10 min after the pretreatment of the PAG with 100 nL of saline, 2% lidocaine or 1 mM CoCl2. A 1.0 AL syringe (KH7001, Hamilton, USA) connected to a 33 G injection needle by a segment of PE-10 tubing was used to microinject l-glu in the LH or lidocaine or CoCl2 in the PAG. The injection needles were 1.0 mm longer than the guide cannulas. Drugs were microinjected in 5 s and the needle remained in place for at least 1 min before being removed from the guide cannula.

experiment. The rats were anesthetized with tribromoethanol (250 mg/kg, i.p.). After local anesthesia with 2% lidocaine, the skull was surgically exposed. 3000 MW BDA (Molecular Probes Inc., USA) was microinjected under pressure in the PAG (AP=+2.7 mm, L=+ 2.5 mm from the medial suture and V= 4.8 mm from the skull with a lateral inclination of 26-) according to brain atlas of Paxinos and Watson (1997), using a stereotaxic apparatus (Stoelting, USA). The injection needle (33 G) was connected to a 1.0 Al syringe (7001 KH, Hamilton, USA) through a segment of PE-10 polyethylene (Intramedic, Clay-Adams, USA). The skull was surgically exposed and the needle was introduced in the brain. A 10% BDA solution in 0.01 M phosphate buffer (pH = 7.4) was injected in a volume of 50 nL. After the injection, the needle was removed, the skull was closed with dental cement and animals were treated with 100,000 units of benzyl penicillin.

2.7. Biotinylated dextran amine (BDA) injection in the PAG

2.7.1. Tissue processing Five days after the surgery, the animal was anesthetized with 1.2 g/kg i.p. urethane (Sigma, USA) and perfused transcardiacally, at a rate of 3.5 mL/min, with 20 mL of saline, followed by 100– 200 mL of 4% paraformaldehyde (Sigma, USA) in 0.1 M phosphate buffer, pH = 7.4. Brains were removed and kept for 2 h in this solution and then stored in a 30% sucrose solution at 4 -C until further processing. A cryostat (CM1900, Leica, Germany) was used to cut two consecutive sets of 60 Am frozen sections in the frontal plane, which were collected in 0.1 M phosphate buffer, pH = 7.4. The set of sections processed for neuronal tracing was rinsed in 50%, 70% and 50% (v / v) ethanol –water solution for 15 min in order to block endogenous peroxidase activity. The sections were rinsed three times in 0.1 M phosphate buffer, pH = 7.4. For BDA visualization, an avidin –biotin kit (Vectastain ABC Elite; Vector Laboratories, USA) was used. The sections were rinsed three times in 0.1 M phosphate buffer (pH = 7.4) and then incubated in the ABC solution for 60 min at room temperature under slow-motion agitation. Subsequently, the sections were rinsed three times in 0.1 M phosphate buffer, pH = 7.4. The tissue was then processed for peroxidase visualization using 3-3V diaminobenzidine dihydrochloride (DAB tablets, Sigma, USA). The sections were incubated at 4 -C on a shaker for 10 min in 100 mL phosphate buffer (pH = 7.4) containing 50 mg DAB, 2.5 mL of 1% CoCl2 and 2 mL of 1% nickel ammonium sulfate (Fisher, USA). Hydrogen peroxide was then added to a final concentration of 0.003% and the tissue was incubated for an additional period of 50 min. The sections were rinsed and mounted onto gelatin-coated slices, air-dried, dehydrated and counterstained with 1% neutral red (Sigma, USA). The consecutive set of sections was stained with Cresyl violet for structure identification.

The anterograde and retrograde tracer BDA was used to determine the connections between LH and PAG. Male Wistar rats (240 – 250 g, n = 5) were used in the present

2.7.2. Microscopic analysis of BDA-labeled sections Light microscopy analysis was performed using an Eclipse E-600 microscope (Nikon, Japan) and magnification

2.5. Histological determination of the microinjection sites At the end of the experiments, the animals were anesthetized with urethane (1.25 g/kg, i.p.) and 100 nL of 1% Evan’s blue dye was microinjected in the brain as a marker of the injection site. The injection needle remained in place for 1 min before being removed. The chest was surgically opened, the descending aorta occluded, the right atrium severed and the brain perfused with 0.9% NaCl followed by 10% formalin through the left ventricle. The brains were removed and post-fixed for 24 h at 4 -C and serial 40 Am thick sections were cut with a cryostat (CM 1900, Leica, Germany). Brain sections were stained with 1% neutral red for light microscopy analysis. The actual placement of the microinjection needles was determined considering the serial sections and according to the rat brain atlas of Paxinos and Watson (1997). 2.6. Statistical analysis Statistical analysis was performed using two-way ANOVA (Prism, Graphpad, USA). The level of significance was set at p < 0.05. Data are presented as mean T SEM.

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

87

neurons. The existence of labeled fibers with varicosity indicates efferent projections from the injection site to the labeled area.

3. Results 3.1. Effects of the pretreatment with saline, 2% lidocaine or 1 mM CoCl2 in the PAG on the hypotensive responses caused by l-glu microinjection in the LH of unanesthetized rats

Fig. 1. Changes in mean arterial pressure (DMAP) and heart rate (DHR) caused by the injection of 100 nL of 0.1 M l-glu in the LH before (open columns) and after (filled columns) the injection of 100 nL of vehicle (n = 3); 2% lidocaine (n = 7) or 1 mM CoCl2 (n = 4) in the PAG of unanesthetized rats. Bars represent the SEM. *p < 0.05, two-way ANOVA followed by Bonferroni’s post hoc test.

from 40 to 400. Images were acquired using a CoolSnap-Pro digital camera (Roper Scientific, USA) and the ImagePro-Plus 4.5 acquisition program (MediaCybernetics, USA). The areas surveyed comprised the injection site of BDA in the PAG in brain sections corresponding to the coordinates + 2.96 to 3.4 mm from the interaural line from the rat brain atlas of Paxinos and Watson (1997) and sections corresponding to the coordinates comprising the injection sites of l-glu in the LH, which were located in the posterior and tuberal portions of the LH as well as the anterior portion of the LH (coordinates from +4.84 to +7.2 mm from the interaural line). Labeled neuronal cell bodies indicate that the injection site receives inputs from the area containing the labeled

The injection of 100 nL of 0.1 M l-glu in the LH of normotensive unanesthetized rats (MAP baseline = 99.0 T 2.5 mmHg, HR baseline = 337.0 T 5.0 bpm, n = 14) caused hypotensive responses without significant HR changes (Fig. 1, open columns and Fig. 2). The pretreatment of the PAG with 100 nL of 2% lidocaine (n = 7) or 1 mM CoCl2 (n = 4) did not affect basal MAP (before lidocaine: 91.0 T 5.7 and after: 94.0 T 6.0 mmHg; before CoCl2: 93.0 T 17.7 and after: 91.0 T 17.5 mmHg) or HR (before lidocaine: 319 T 9 and after: 307 T 13 bpm; before CoCl2: 317 T 14 and after: 321 T11 bpm). Both pretreatments significantly reduced the hypotensive response caused for the injection of 0.1 M lglu in the LH (Fig. 1, filled columns). The pretreatment of the PAG with 100 nL of vehicle (n = 3) did not affect basal MAP (before vehicle: 91.0 T 5.7 and after: 94.0 T 6.0 mmHg) or HR (before vehicle: 301 T 23 and after: 292 T 24 bpm) or alter the hypotensive response to the l-glu in the LH (Fig. 1, filled column). Statistical analysis of the MAP data corresponding to the treatment with saline, lidocaine and CoCl2 was performed using two-way ANOVA. The overall analysis indicated that a significant difference among drugs ( F = 8.88, p < 0.001, df = 2) and between injection prior and after treatments ( F = 23.93, p < 0.0001, df = 1). Breaking the analysis into two groups (saline vs lidocaine and saline vs CoCl2), the two-way ANOVA followed by the Bonferroni’s post hoc test indicated no significant differences between control injection previous to saline and lidocaine (t = 0.9923, ns) or

Fig. 2. Recordings showing the effects of the injection of 100 nL of 0.1 M l-glu (Glu) in the LH before (A) and 10 min after (C) the pretreatment of the PAG with 100 nL of 1 mM CoCl2 (B) on mean arterial pressure (MAP), pulsatile arterial pressure (PAP) and heart rate (HR) of one unanesthetized rat.

88

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

saline and CoCl2 (t = 1.665, ns) and significant reduction after the treatment with lidocaine (t = 3.774, p < 0.01) or CoCl2 (t = 5.469, p < 0.001). No significant differences were observed among HR data from the different treatments. The overall analysis of HR data indicated no significant difference among drugs ( F = 0.3011, ns, df = 2) and between injection prior and after treatments ( F = 2.308, ns, df = 1). Blood pressure and HR recordings showing the pattern of the hypotensive responses to LH injection of 100 nL of 0.1 M l-glu are presented in Fig. 2. In Fig. 2, we also observe that the pretreatment with the nonselective synapse blocker CoCl2 markedly reduced the hypotensive response to l-glu. The dispersion of the injection sites within the sub regions of the LH and PAG is represented in Fig. 3. A photomicrograph showing injection sites in the LH and PAG is presented in Fig. 4. Injection of CoCl2 in the superior colliculus immediately above the dorsal PAG did not affect the hypotensive response to the injection of l-glu in the LH. The MAP

and HR responses to the injection of l-glu in the LH were 13.6 T 2.3 mmHg and 5 T 4.4 bpm (n = 3) before and 16.0 T 4.0 mmHg and 8 T 7.0 bpm (n = 3) after the injection of CoCl2 in the superior colliculus, respectively. The injection sites in the superior colliculus are represented in Fig. 3. 3.2. BDA injection in the PAG Both anterograde and retrograde labeling were observed after BDA injection in the dorsal PAG region (Figs. 5 and 6). A diagrammatic representation of the injection sites of BDA in the PAG of five rats used in the present protocol is presented in Fig. 5A. A schematic illustration of the distribution of retrogradely labeled neurons observed in the LH and other hypothalamic areas is presented in Fig. 5B. A photomicrography showing the injection site within the PAG of one animal is presented in Fig. 6A. An expressive number of labeled cell bodies was observed in the dorsal

Fig. 3. Diagrammatic representation of rat brain frontal sections modified from Paxinos and Watson (1997) showing the distribution of Evan’s blue stain corresponding to the injection sites of (A) exclusively l-glu in the LH and (B) vehicle, lidocaine or CoCl2 in the PAG or CoCl2 in the superior colliculus (SC) of unanesthetized rats (n = 17). The symbol key only applies to part B of the figure. In some cases there was overlapping. Numbers represent the stereotaxic coordinates (in millimeters from the interaural line).

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

Fig. 4. Photomicrograph of injection site in the LH (A, MT=mammillothalamic tract, periaqueductal gray area, and

coronal rat brain sections indicating the arrow) and in the dorsal PAG region (B). F=fornix, LH=lateral hypothalamus, PAG= IA=interaural line (mm).

and lateral portion of the PAG, contralateral to the injection site of BDA (Fig. 6B). Reciprocal connections were observed between the PAG and the LH in all animals. Labeled neuronal cell bodies were observed throughout the LH extension (Figs. 5B, 6C and D) and only ipsilateral to the injection site of BDA in the PAG. The number of labeled neurons in the LH observed in the set of slides processed for neuronal tracing ranged from 41 to 54/animal (46.2 T 3, n = 5). Labeled neurons were also observed in the zona incerta, subincertal nucleus, anterior hypothalamus, dorsal hypothalamus, ventral medial hypothalamus and tuber cinereum (Fig. 5B).

4. Discussion In a previous study, it was reported that the microinjection of l-glu in sites located throughout the LH of unanesthetized rats caused hypotensive responses without

89

significant HR changes (Pajolla and Correˆa, 2004). In the present study, we report that the hypotensive pathway activated by l-glu microinjection in the LH involves a synaptic relay in the PAG. The PAG is an important mesencephalic region that is involved in several biological functions such as defense and reproductive behavior, pain, anxiety, cardiovascular and respiratory control (Bandler et al., 2000; Carrive et al., 1987; Graeff et al., 1993; Huang et al., 2000; Oliveira and Prado, 2001; Sakuma and Plaff, 1980). Bandler et al. (1991) reported that the PAG has a longitudinal columnar organization, based on the cardiovascular response observed after chemical stimulation of specific portions of that area in the cat. A similar organization was also observed in the rat (Lovick, 1985, 1992). Consequently, the PAG was subdivided in four longitudinal columns along its rostrocaudal axis: the dorsomedial, the dorsolateral, the lateral and the ventrolateral columns. Electrical stimulation of the dorsal, dorsolateral or the lateral PAG evoked hypertension, tachycardia and hindlimb vasodilatation in anesthetized rats (Hamalainen and Lovick, 1997; Lovick, 1992). Chemical stimulation of PAG with dl-homocysteic acid also caused region-dependent cardiovascular responses. Microinjection in the dorsolateral PAG of anesthetized rats resulted in hypertension and tachycardia, whereas depressor and bradycardiac responses were evoked after injections in the ventrolateral PAG (Carrive et al., 1989; Carrive and Bandler, 1991; Huang et al., 2000; Lovick, 1992; Rossi et al., 1994). In the present study, we injected the local anesthetic lidocaine or the nonselective synapse blocker CoCl2 in the PAG before the injection of l-glu in the LH to investigate if the PAG is involved in the cardiovascular pathway activated by glutamatergic stimulation of the LH. Microinjections were directed to the dorsolateral and dorsomedial regions of the PAG, which have been previously shown to be involved with cardiovascular modulation. Chemical ablation of this PAG area caused by local injection of lidocaine or CoCl2 significantly reduced the hypotensive response to the injection of l-glu in the LH of unanesthetized rats. Considering that the effects of CoCl2 are due to specific blockade of neuronal synaptic transmission (Kretz, 1984), the observation that lidocaine or CoCl2 microinjections had similar effects suggests that the hypotension evoked by stimulation of LH is mediated by synaptic neurotransmission within the PAG and is not related to fibers of passage. Additionally, microinjection of CoCl2 outside the PAG, in the superior colliculus, did not affect the hypotensive response to l-glu in the LH, favoring the idea of a synaptic relay in the PAG. Because the stimulation of the dorsal PAG is associated with pressor responses, one could hypothesize a tonic influence of that PAG area on the blood pressure control. However, the ablation of the dorsal PAG by local injection of lidocaine or CoCl2 did not affect basal blood pressure or heart rate. Fisk and Wyss (2000) reported similar results

90

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

Fig. 5. Diagrammatic representation of rat brain frontal sections modified from Paxinos and Watson (1997) showing: A) the dispersion of the injection sites of the neuronal tracer BDA in the PAG of the five rats used in the present experiment and B) the distribution of fibers and retrogradely labeled neurons in the lateral hypothalamus (filled circles) and other hypothalamic nuclei (open circles). Numbers represent the stereotaxic coordinates (in millimeters from the interaural line). LH=lateral hypothalamus; PAG=periaqueductal gray area; ZI=zona incerta; AHC=anterior hypothalamic area, central part; AHP=anterior hypothalamic area, posterior part; VMH=ventromedial hypothalamic nucleus; DA=dorsal hypothalamic area; DMD=dorsomedial hypothalamic nucleus, dorsal part; VMHDM=ventromedial hypothalamic nucleus, dorsomedial part; VMHVL=ventromedial hypothalamic nucleus, ventrolateral part; SPF=subparafascicular thalamic nucleus; PH=posterior hypothalamic nucleus; and PMD=premammillary nucleus, dorsal part.

after the injection of lidocaine in the PAG and local injection of muscimol into the dorsal PAG also did not affect basal blood pressure values (da Silva et al., 2003), arguing against the hypothesis of a sustained tonic influence of the dPAG on blood pressure control. Although the dPAG may not have a tonic influence on blood pressure, it is well known to be involved in the mediation of cardiovascular correlates of behavioral responses and predominantly with the pressor responses. The dPAG was implicated in the mediation of the pressor response elicited by acute disinhibition of the dorsomedial hypothalamus (da Silva et al., 2003). Additionally, disinhibition of the dorsal PAG by local injection of bicuculine was reported to elicit sustained pressor responses (Hayward et al., 2003), agreeing with the idea of massive inhibitory input to the dorsal PAG that would involve both forebrain

inputs as well as local GABAergic interneurons (Bandler et al., 2000; Brandao et al., 1999; Schmitt et al., 1986). Consequently, one could hypothesize that under normal conditions there is equilibrium between pressor and depressor influences inputting on the PAG, which consequently would minimize its positive drive upon medullar sympathetic premotor areas. Under this condition, there is a possibility that an acute further stimulation of these inhibitory inputs could result in moderated depressor responses such as those observed after the stimulation of the LH by l-glu. The recording of spontaneous activity of PAG neurons during the electrical stimulation of hypothalamic sites and the zona incerta resulted mainly in neuronal inhibition (Van der Plas et al., 1995), suggesting that an inhibitory action of the LH on PAG neurons could be responsible for the hypotensive responses observed after

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

Fig. 6. Photomicrography of a brain section showing the injection site of BDA within the dorsal PAG region of the rat (plate A, the arrow indicates the center of BDA injection). Labeled neurons and axons were observed in the dorsal and lateral portions of the PAG contralateral to the injection site (plate B) as well as in the LH ipsilateral to the injection site of BDA in the PAG (plates C and D). In B, C and D arrows indicate labeled fibers and neurons.

hypothalamic stimulation. Agarwal and Calaresu (1993) reported that the hypotensive response to l-glu stimulation of the LH in anesthetized rats was associated with inhibition of cardiovascular neurons in the ventrolateral medulla (VLM). Additionally, Cechetto and Chen (1992) reported that the hypotension caused by microinjection of dlhomocysteic acid in the LH of anesthetized rats was associated with a decrease in renal sympathetic nerve activity and that this response was reduced after microinjection of CoCl2 in the VLM. Considering the above information, it could be hypothesized that an acute further increase in the inhibitory output from the LH to the pressor neurons of the dorsal PAG could be responsible for the hypotensive response observed after the glutamatergic stimulation of the LH. Because the ventrolateral PAG has been associated with the generation of hypotensive responses (Carrive et al., 1989; Carrive and Bandler, 1991), an alternative possibility is that stimulation of the LH would lead to the activation of depressor neurons in the ventrolateral PAG, which were proposed to be involved in the intrinsic modulation between columns of the PAG (Jansen et al., 1998). However, further neurophysiologic experiments are necessary to clarify this issue.

91

The existence of neural connections between the injection site of l-glu in LH and the injection site of lidocaine or CoCl2 in the PAG was presently observed when the neuronal tracer BDA was microinjected in the PAG. Neuronal cell bodies were found ipsilaterally in the LH region corresponding to the injection site of l-glu, thus providing evidence of a direct neural connection between the LH and the PAG relay site of the depressor pathway. The fact that BDA-labeled neuronal cell bodies were only observed ipsilateral to the BDA injection site in the PAG suggests anatomical laterality. Nevertheless, lidocaine or CoCl2 caused similar blockade of the hypotensive response to the injection of l-glu in the LH, regardless of being injected ipsi- or contralerally suggesting a lack of laterality in terms of response. A possible explanation for this apparent contradiction would be the existence of intrinsic neuromodulation between opposite columns in the PAG, as proposed by Jansen et al. (1998). In agreement we observed labeled neurons in the contralateral dorsal PAG as well as in the ipsilateral and contralateral lateral columns that project to the ipsilateral dorsal column (BDA injection site). Another alternative would be the existence of intermediary synaptic relays between the LH and PAG that project bilaterally to the PAG. Consequently, further studies will be necessary to clarify if the direct LH –PAG neural connections are indeed associated with the LH-related hypotensive pathway and to exclude the possible existence of intermediary synaptic relays between the LH and the PAG. Concerning the possible functional significance of this hypotensive pathway, there is evidence indicating that both the LH and the PAG are important modifiers of autonomic nervous system responses and fear behavior (Allen and Cechetto, 1992; Liebman et al., 1970; Lyon, 1964; Yardley and Hilton, 1986). Studies by Iwata et al. (1986) and LeDoux et al. (1988) on conditioned fear responses indicated that lesions of the LH or the PAG disrupt the cardiovascular and behavioral responses that accompany conditioned fear responses, suggesting an orchestrated interaction between these areas. Consequently, the presently reported hypotensive pathway from the LH to the PAG involves the modulation of cardiovascular responses associated to behavioral responses such as fear and stress. In conclusion, our results suggest that the hypotensive pathway activated by glutamatergic stimulation of the LH involves a relay in the PAG before reaching the RVLM.

Acknowledgments The authors would like to thank Ms Ida´lia I.B. Aguiar, Ivanilda A.C. Fortunato and Simone S. Guilhaume for technical support. Gisela P. Pajolla (Fapesp 00/00314-5) and Gislaine G. Pelosi (Fapesp 02/14147-9) were PhD students enrolled in the Graduation Program on Pharmacology of the School of Medicine of Ribeira˜o Preto. Rodrigo F. Tavares is

92

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93

a post-doc fellow (Fapesp 04/01270-2), supported by a grant from CNPq/PADCT- 505394/2003-0.

References Agarwal, S.K., Calaresu, F.R., 1993. Supramedullary inputs to cardiovascular neurons of rostral ventrolateral medulla in rats. Am. J. Physiol. 265, R111 – R116. Allen, G.V., Cechetto, D.F., 1992. Functional and anatomical organization of cardiovascular pressor and depressor sites in the lateral hypothalamic area: I. Descending projections. J. Comp. Neurol. 315, 313 – 332. Bandler, R., Carrive, P., Depaulis, A., 1991. Introduction: emerging principles of organization of the midbrain periaqueductal gray matter. In: Depaulis, A., Bandler, R. (Eds.), The Midbrain Periaqueductal Gray Matter: Functional, Anatomical, and Neurochemical Organization. Plenum Press, New York, pp. 1 – 8. Bandler, R., Keay, K.A., Floyd, N., Price, J., 2000. Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Res. Bull. 53, 95 – 104. Berk, M.L., Finkelstein, J.A., 1983. Long descending projections of the hypothalamus in the pigeon, Columba livia. J. Comp. Neurol. 220, 127 – 136. Brandao, M.L., Anseloni, V.Z., Pandossio, J.E., De Araujo, J.E., Castilho, V.M., 1999. Neurochemical mechanisms of the defensive behavior in the dorsal midbrain. Neurosci. Biobehav. Rev. 23, 863 – 875. Carrive, P., Bandler, R., 1991. Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study. Brain Res. 541, 206 – 215. Carrive, P., Dampney, R.A.L., Bandler, R., 1987. Excitation of neurones in a restricted portion of the midbrain periaqueductal grey elicits both behavioural and cardiovascular components of the defence reaction in the unanaesthetised decerebrate cat. Neurosci. Lett. 81, 273 – 278. Carrive, P., Bandler, R., Dampney, R.A., 1989. Somatic and autonomic integration in the midbrain of the ananesthetized decerebrate cat: a distinctive pattern evoked by excitation of neurons in the subtentorial portion of the midbrain periaqueductal grey. Brain Res. 483, 251 – 258. Cechetto, D.F., Chen, S.J., 1992. Hypothalamic and cortical sympathetic responses relay in the medulla of the rat. Am. J. Physiol. 263, R544 – R552. da Silva, L.G., de Menezes, R.C., dos Santos, R.A., Campagnole-Santos, M.J., Fontes, M.A., 2003. Role of periaqueductal gray on the cardiovascular response evoked by disinhibition of the dorsomedial hypothalamus. Brain Res. 984, 206 – 214. Fisk, G.D., Wyss, J.M., 2000. Descending projections of infralimbic cortex that mediate stimulation-evoked changes in arterial pressure. Brain Res. 859, 83 – 95. Gelsema, A.J., Roe, M.J., Calaresu, F.R., 1989. Neurally mediated cardiovascular responses to stimulation of cell bodies in the hypothalamus of the rat. Brain Res. 482, 67 – 77. Graeff, F.G., Silveira, M.C., Nogueira, R.L., Audi, E.A., Oliveira, R.M., 1993. Role of the amygdala and periaqueductal gray in anxiety and panic. Behav. Brain Res. 58, 123 – 131. Hamalainen, M.M., Lovick, T.A., 1997. Role of nitric oxide and serotonin in modulation of the cardiovascular defence response evoked by stimulation in the periaqueductal grey matter in rats. Neurosci. Lett. 229, 105 – 108. Hayward, L.F., Swartz, C.L., Davenport, P.W., 2003. Respiratory response to activation or disinhibition of the dorsal periaqueductal gray in rats. J. Appl. Physiol. 94, 913 – 922. Holstege, G., 1987. Some anatomical observations on the projections from the hypothalamus to brainstem and spinal cord: an HRP

and autoradiographic tracing study in the cat. J. Comp. Neurol. 260, 98 – 126. Hosoya, Y., Matsushita, M., 1981. Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography. Neurosci. Lett. 24, 111 – 116. Huang, Z., Subramanian, S.H., Balnave, R.J., Turman, A.B., Chow, C.M., 2000. Roles of periaqueductal gray and nucleus tractus solitarius in cardiorespiratory function in the rat brainstem. Respir. Physiol. 120, 185 – 195. Iwata, J., LeDoux, J.E., Reis, D.J., 1986. Destruction of intrinsic neurons in the lateral hypothalamus disrupts the classical conditioning of autonomic but not behavioral emotional responses in the rat. Brain Res. 368, 161 – 166. Jansen, A.S.P., Farkas, E., Sams, J.M., Loewy, A.D., 1998. Local connections between the columns of the periaqueductal gray matter: a case for intrinsic neuromodulation. Brain Res. 784, 329 – 336. Kabat, H., Magoun, H.W., Ranson, S.W., 1935. Electrical stimulation of points in the forebrain and midbrain. The resultant alteration in blood pressure. Arch. Neurol. Psychiat. (Chicago) 34, 931 – 955. Kretz, R., 1984. Local cobalt injection: a method to discriminate presynaptic axonal from postsynaptic neuronal activity. J. Neurosci. Methods 11, 129 – 135. LeDoux, J.E., Iwata, J., Cicchetti, P., Reis, D.J., 1988. Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J. Neurosci. 8, 2517 – 2529. Liebman, J.M., Mayer, D.J., Liebeskind, J.C., 1970. Mesencephalic central gray lesions and fear-motivated behavior in rats. Brain Res. 23, 353 – 370. Lovick, T.A., 1985. Ventrolateral medullary lesions block the antinociceptive and cardiovascular responses elicited by stimulating the dorsal periaqueductal grey matter in rats. Pain 21, 241 – 252. Lovick, T.A., 1992. Inhibitory modulation of the cardiovascular defence response by the ventrolateral periaqueductal grey matter in rats. Exp. Brain Res. 89, 133 – 139. Lyon, M., 1964. The role of central midbrain structures in conditioned responding to aversive noise in the rat. J. Comp. Neurol. 122, 407 – 429. Oliveira, M.A., Prado, W.A., 2001. Role of PAG in the antinociception evoked from the medial or central amygdala in rats. Brain Res. Bull. 54, 55 – 63. Pajolla, G.P., Correˆa, F.M.A., 2004. Cardiovascular responses to the injection of l-glutamate in the lateral hypothalamus of unanesthetized or anesthetized rats. Auton. Neurosci. 116, 19 – 29. Paxinos, G., Watson, C., 1997. The rat brain in stereotaxic coordinates. Compact, 3rd edition. Academic Press, San Diego. Rossi, F., Maione, S., Berrino, L., 1994. Periaqueductal gray area and cardiovascular function. Pharmacol. Res. 29 (1), 27 – 36. Sakuma, Y., Plaff, D.W., 1980. LHRH in the mesencephalic central grey can potentiate lordosis reflex of female rats. Nature 283, 566 – 567. Sandku¨ler, J., Maisch, B., Zimmermann, M., 1987. The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block. Exp. Brain Res. 68, 168 – 178. Saper, C.B., Swanson, L.W., Cowan, W.M., 1979. An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat. J. Comp. Neurol. 183, 689 – 706. Schmitt, P., Carrive, P., Di Scala, G., Jenck, F., Branda˜o, M., Bagri, A., Moreau, J.L., Sandner, G., 1986. A neuropharmacological study of the periventricular neural substrate involved in flight. Behav. Brain Res. 22, 181 – 190. Spencer, S.E., Sawyer, W.B., Loewy, A.D., 1989. Cardiovascular effects produced by l-glutamate stimulation of the lateral hypothalamic area. Am. J. Physiol. 257, H540 – H552. ter Horst, G.J., Luiten, P.G.M., Kuipers, F., 1984. Descending pathways from hypothalamus to dorsal motor vagus and ambiguous nuclei of the rat. J. Auton. Nerv. Syst. 11, 59 – 75. Van der Plas, J., Wiersinga-Post, J.E.C., Maes, F.W., Bohus, B., 1995. Cardiovascular effects and changes in midbrain periaqueductal gray

G.P. Pajolla et al. / Autonomic Neuroscience: Basic and Clinical 122 (2005) 84 – 93 neuronal activity induced by electrical stimulation of the hypothalamus in the rat. Brain Res. Bull. 37, 645 – 656. Vercelli, A., Repici, M., Garbossa, D., Grimaldi, A., 2000. Recent techniques for tracing pathways in the central nervous system of developing and adult mammals. Brain Res. Bull. 51, 11 – 28.

93

Yardley, C.P., Hilton, S.M., 1986. The hypothalamic and brainstem areas from which the cardiovascular and behavioural components of the defense reaction are elicited in the rat. J. Auton. Nerv. Syst. 15, 227 – 244.