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. 2006 Apr 20;26(4-6):633–641. doi: 10.1007/s10571-006-9015-2

Rostrocaudal Somatotopy in the Neural Connections Between the Lateral Hypothalamus and the Dorsal Periaqueductal Gray of the Rat Brain

Gislaine G Pelosi 1, Rodrigo F Tavares 1, Fernando M A Corrêa 1,✉
PMCID: PMC11520621  PMID: 16625431

Abstract

1. The lateral hypothalamus (LH) and the dorsal periaqueductal gray area (dPAG) are two important brain structures involved in central cardiovascular control.

2. In the present study, we searched for possible rostrocaudal somatotopy in the neural connections from the three subdivisions of the LH (anterior—LHa; tuberal—LHt and posterior—LHp) to the different rostrocaudal portions of the dPAG.

3. The bidirectional neuronal tracer biotinylated-dextran-amine (BDA) was microinjected into different rostrocaudal coordinates of the dPAG (AP 3.4–2.7 mm) of male Wistar rats. One week later, animals were sacrificed and brain slices were processed and analyzed to detect neuronal efferent projections from the LH to the dPAG.

4. Neuronal cell body staining was observed along all the rostrocaudal axis of the LH when BDA was microinjected in more rostral dPAG coordinates. When the BDA was microinjected into more caudal dPAG regions, labeled neurons were observed only in the caudal portion of the LH.

5. Efferent projections from the LHa were directed only to the rostral portion of the dPAG. Projections from the rostral and medial portions of the LHt were also directed to the rostral dPAG, whereas both rostral and caudal dPAG received projections from the caudal portion of the LHt. Efferent projections from the anterior portion of the LHp were directed to both rostral and caudal dPAG, whereas projections from the caudal LHp were only directed to the rostral portion of the dPAG.

6. The results suggest a somatotopic correlation in LH projections to the dPAG with main connections to the rostral dPAG, which are efferent from the three divisions of the LH. More caudal regions of the dPAG received afferents only from posterior sites in the LH.

7. Moreover, the results point out to extensive and complex neural somatotopic projections from all LH subdivisions to different rostrocaudal portions of the dPAG, reinforcing the idea of significant functional interactions between the brain structures.

KEY WORDS: dorsal periaqueductal gray, lateral hypothalamus, biotinylated-dextran-amine, neural connections

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). The LH is divided in anterior (LHa), tuberal (LHt) and posterior (LHp) subareas (Saper et al., 1979). This complex region has been subject of many physiological studies, with a major focus on its role in cardiovascular control. Neuroanatomic studies have shown LH projections to regions involved in cardiovascular control, such as the periaqueductal gray area, the parabrachial nucleus, the nucleus of the solitary tract, the vagal nuclei and the intermediolateral cell column (Hosoya and Matsushita, 1981; Berk and Finkelstein, 1983; ter Horst et al., 1984; Holstege, 1987).

The periaqueductal gray area (PAG) is a mesencephalic region surrounding the aqueduct and extending from the posterior commissure to rostral locus coeruleus (Beitz, 1995). Many studies have described this structure as involved with behavioral responses associated with cardiovascular changes (Nashold et al., 1969; Jenck et al., 1989; Huang et al., 2000). The PAG is functionally subdivided in four longitudinal columns along its rostrocaudal axis: the dorsomedial, the dorsolateral, the lateral and the ventrolateral column (Bandler et al., 1991).

The PAG receives projections from the LH among other brain regions involved with autonomic regulation, such as the amygdala, the zona incerta, the superior and inferior colliculus and the nucleus of the solitary tract (Mantyh, 1982; Bandler and Tork, 1987; Semenenko and Lumb, 1992; Beitz, 1995; Paredes et al., 2000). Both the LH and the PAG are important modifiers of autonomic nervous system responses and fear behavior (Lyon, 1964; Liebman et al., 1970; Yardley and Hilton, 1986; Allen and Cechetto, 1992). Studies by Iwata et al. (1986) and LeDoux et al. (1988) on conditioned fear responses suggested that lesions of the LH or the PAG disrupt the cardiovascular and behavioral responses that accompany conditioned fear responses, suggesting orchestration between these areas. Chemical ablation of the dorsal region of the PAG (dPAG) evoked decreased depressor responses to L-glutamate microinjection into the LH, suggesting a dPAG relay in this hypotensive pathway (Pajolla et al., 2005).

Although neuroanatomic connections have been reported between the LH and the dPAG, projections from these regions as well as the possible rostrocaudal somatotopic correlation between the different LH and dPAG regions was not yet studied. Connections among brain areas may be studied by histological analysis of neuronal tracers’ distribution after microinjection in selected brain areas. A useful tracer is the low-molecular-weight biotinylated-dextran-amine (BDA) with bidirectional transport evidencing both neural afferents from projecting neurons and efferent projections of a brain region under study (Vercelli et al., 2000).

In the present study, we microinjected BDA into different rostrocaudal dPAG coordinates to identify neuronal projections from the three LH subareas to the extent of the dPAG axis and evaluate possible somatotopic correlation.

METHODS

Biotinylated-Dextran-Amine (BDA) injection into the dPAG

The anterograde and retrograde tracer BDA was used to detect afferent projections from the different subareas of the LH to the dPAG rostrocaudal axis. Male Wistar rats (240–250 g) were used in the present experiment (n=8). The rats were anesthetized with tribromoethanol (250 mg/kg, i.p.). After local anesthesia with 2% xylocaine, the head was immobilized on a stereotaxic apparatus (Stoelting, USA) and the skull surgically exposed. BDA (3000 MW, Molecular Probes Inc., USA) was microinjected into different rostrocaudal coordinate sites of the dPAG (AP=+3.4 mm; +3.27 mm; +2.96 mm and +2.70 mm from the interaural line) and L=+2.5 mm from the medial suture and V=−5.8 mm from the skull, with a lateral inclination of 26°. Coordinates were obtained from the rat brain atlas of Paxinos and Watson (1997). The injection needle (33G) was connected to a 1 μL syringe (7001KH, Hamilton, USA) through a PE-10 polyethylene (Clay-Adams, USA) segment. BDA (10% solution) solution in 0.01 M phosphate buffer (pH 7.4) was injected in a volume of 50 nL in the dPAG. After the injection, the needle was removed, the skull closed with dental cement and animals were treated with 100,000 units of benzyl penicillin.

Tissue Processing

One week after the surgery, animals were anesthetized with 1.2 g/kg i.p. urethane (Sigma, USA) and perfused through the heart, at a rate of 3.5 mL/min, with 20 mL of saline solution 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 (CM 1900, Leica, Germany) was used to cut 60 μm frozen sections in the frontal plane that were collected in 0.1 M phosphate buffer, pH 7.4.

Sections were rinsed in ethanol at different concentrations: 50, 70 and 50% (v/v) 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. An avidin-biotin kit (Vectastain ABC Elite, Vector Laboratories, USA) was used for BDA visualization. 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 3-3′-diaminobenzidine dihydrochloride (DAB tablets, Sigma, USA). 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 CoCl2 and 2 mL nickel ammonium sulphate (Fisher, USA). Hydrogen peroxide was then added to a final concentration of 0.003% and the tissue incubated for 50 min. Sections were then rinsed and mounted on to gelatin-coated slices, air-dried, dehydrated and counterstained with 1% neutral red (Sigma, USA).

Microscopic Analysis of BDA Labeled Sections

BDA is a neuronal anterograde and retrograde tracer. Labeled neuronal cell bodies indicate inputs from that area to the injection site. The existence of labeled fibers with varicosity indicates existence of efferent projections from the injection site to this area. Photomicrographs were taken using a digital camera (CoolSnap, Media Cybernetics, USA) mounted on a microscope (Nikon E600, Japan) and computer processed using an image capture software (ImagePro Plus, Media Cybernetics, USA).

RESULTS

BDA Injection into the dPAG

BDA (50 nL) was microinjected in different sites along the rostrocaudal axis of the dPAG (AP=3.4, 3.2, 2.96 and 2.7 mm). Two animals were analyzed for each site of injection (n=8). Retrograde labeling was observed throughout the three subareas of the LH (Fig. 1A). BDA microinjection sites in the dPAG are represented in Fig. 1B.

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(A) Diagrammatic representation of rat brain frontal sections modified from Paxinos and Watson (1997) showing the (A) distribution of retrogradely labeled neurons in the LH (dots symbolize neuronal cell bodies) (B) dispersion of the injection sites of the neuronal tracer BDA in different rostrocaudal coordinates of the dPAG of the rats used in the present work (filled circles). Numbers represent the stereotaxic coordinates (mm from the interaural line). LHa: anterior lateral hypothalamus; LHt: tuberal lateral hypothalamus; LHp: posterior lateral hypothalamus; dPAG: dorsal periaqueductal gray area.

Neurons were observed along the rostrocaudal axis composing the LHa, LHt, and LHp, when BDA was microinjected in rostral coordinate of the dPAG of rats (AP=3.40 and 3.20 mm). This observation suggests that these dPAG portions receive efferent projections from all LH subareas (Fig. 1). Photomicrographs showing an injection site of BDA in the rostral portion of the dPAG and labeled neurons in the LH are represented in Fig. 2.

Fig. 2.

Fig. 2.

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Photomicrographs of brain sections showing the injection site of BDA within the rostral coordinate of the dPAG of the rats and labeled neurons in the LH (4× magnification). Plate A, the arrow indicates the center of BDA injection in the dPAG. Labeled neurons were observed in all extension of the LH (arrows, plates B and C). The neurons indicated with white arrows in plates B and C are magnified (20×) within the square. IA: interaural (mm); dPAG: dorsal periaqueductal gray area; LH: lateral hypothalamus.

After the BDA microinjection into the caudal coordinates of the dPAG (interaural: 2.96 and 2.70 mm) labeled neurons were only visualized in the posterior LHt and in the anterior LHp sites (AP=5.4–4.46 mm). This suggests a specific efferent connection from those hypothalamic regions to the caudal portion of the dPAG (Fig. 1). Photomicrographs showing an injection site of BDA in the caudal portion of the dPAG and labeled neurons in the LH are represented in Fig. 3.

Fig. 3.

Fig. 3.

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Photomicrographs of brain sections showing the injection site of BDA within the caudal coordinate of the dPAG of the rats and labeled neurons in the LH (4× magnification). The neuron indicated with the white arrow in plate B is magnified (20×) within the square. The square in plate C is a magnification (20×) of the rostral region of the LH showing the absence of labeled neurons after BDA microinjection in the caudal site of the dPAG. IA: interaural (mm); dPAG: dorsal periaqueductal gray area; LH: lateral hypothalamus.

A somatotopic correlation was observed between the anterior portions of LH and dPAG. Neurons in the LHa and the rostral three-fourth of the LHt only projected to the rostral portion of the dPAG (AP=3.4–3.2 mm) (Fig. 4). Neurons in the most caudal quarter of the LHt projected to the whole extent of the dPAG from AP=3.4–2.7 mm (Fig. 4). Neurons in the anterior portion of the LHp projected to the whole extent of the dPAG, whereas neurons in the caudal portion of the LHp only projected to the rostral portion of the dPAG (AP=3.4–3.2 mm) (Fig. 4).

Fig. 4.

Fig. 4.

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Schematic representation of the neural connections between the anterior (LHa), tuberal (LHt), or posterior (LHp) regions of the lateral hypothalamus and the different rostrocaudal sites of the dPAG. Numbers represent the stereotaxic coordinates (mm from the interaural line). Arrows indicate efferent projections of the LH.

Fibers were observed throughout the LH rostrocaudal axis regardless of the microinjection site in the dPAG, suggesting an extensive connection between the LH subareas and the dPAG connects.

DISCUSSION

We report rostrocaudal somatotopic neuroanatomic connections between LH subareas and rostrocaudal dPAG regions.

Chemical stimulation of the dPAG with NA in rats evoked pressor responses with different intensities according to the rostrocaudal coordinate targeted, suggesting that NA-sensitive cardiovascular neurons are preferentially located in the rostral portion of the dPAG (Pelosi and Corrêa, 2005). Van der Plas et al. (1995) reported that LH stimulation inhibited pressor-related PAG neurons. L-glutamate microinjection throughout the extent of the LH evoked depressor response in conscious or anesthetized rats (Pajolla and Corrêa, 2004). The depressor response to L-glutamate in the LHt and anterior portion of the LHp was reduced, but not abolished, by reversible pharmacological ablation of the rostral portion of the dPAG with lidocaine or CoCl2 (Pajolla et al., 2005). This suggested possible projections from the LH also to the caudal portion of the dPAG.

The functional evidence of extensive connections between the LH subareas and the different dPAG rostrocaudal portions, prompted us to study the possible existence of somatotopic correlations between LH efferent projections and the different regions along the dPAG rostrocaudal axis. In order to evaluate that hypothesis, we microinjected BDA into four rostrocaudal portions along the dPAG area.

Labeled neurons were observed throughout the LH after the BDA microinjection in anterior coordinates of the dPAG. Neurons were evidenced only in the LHt and LHp when the tracer was microinjected in caudal levels of the dPAG.

The LHa, LHt, and LHp connect preferentially with rostral sites in the dPAG. The LHt and LHp regions project exclusively to caudal dPAG sites, suggesting the existence of somatotopic neural output from the LH to the dPAG among their rostrocaudal axes. The existence of projections from the LHt and LHp to the caudal dPAG may explain the partial blockade of the hypotensive responses to L-glutamate microinjected into those hypothalamic regions, reported by Pajolla et al. (2005). However, the actual functional meaning of these specific neural connections has yet to be clarified. The complex neural interaction between LH subareas and the dPAG column should be considered when studying PAG involvement in hypothalamus-evoked autonomic responses.

ACKNOWLEDGMENTS

The authors would like to thank Ms Ivanilda A.C. Fortunato, Simone S. Guilhaume and Idalia I.B. Aguiar for technical support. Gislaine G. Pelosi is a Ph.D. student (Fapesp-02/14147-9) enrolled in the Graduation Program on Pharmacology of the School of Medicine of Ribeirão Preto and Rodrigo F. Tavares is a post-doc fellow (Fapesp 04/01270-2). This study was supported by grant from CAPES and CNPq (306381/2003-6 and 505394/2003-0).

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