Rostral elements of sympatho-motor circuitry: a virally mediated transsynaptic tracing study

Abstract

Numerous physiological and emotionally motivated behaviors, including locomotion, exercise, escape, and attack behaviors as well as passive coping responses, require concomitant activation of motor and sympathetic efferents. Such functional heterogeneity suggests the existence of dual function neurons that can simultaneously coordinate motor and sympathetic output. Because previous physiological investigations have implicated a number of mesencephalic and telencephalic regions in mediating these behaviors, we hypothesized the presence of dual function sympatho-motor neurons in these neural structures. To test this hypothesis, we used recombinant strains of the pseudorabies virus (PRV) for transsynaptic tract-tracing. PRV-152, a strain that expresses enhanced green fluorescent protein, was injected into sympathectomized gastrocnemius muscle, whereas PRV-BaBlu, which expresses beta-galactosidase, was injected into the adrenal gland in the same animals. Although coinfected neurons were detected in a number of mesencephalic and telencephalic regions, >50% of such neurons were located within specific subdivisions of two general areas: the hypothalamus and periaqueductal gray. These subdivisions included the ventrolateral periaqueductal gray, dorsomedial hypothalamus, dorsolateral lateral hypothalamus, and ventral portion of the medial parvocellular subdivision of the paraventricular nucleus of the hypothalamus (PVN). A subset of the sympatho-motor neurons within the PVN also contained either arginine vasopressin or oxytocin. This sympatho-motor circuitry likely plays an important role in mediating different aspects of stress responses and emotionally motivated behaviors.

Figure 1.

Examples of sympatho-motor neurons in the midbrain. Transsynaptically labeled neurons infected with PRV-152, which was injected into the gastrocnemius muscle, appear green, whereas those infected with PRV-BaBlu were pseudocolored with red; double-infected neurons appear yellow or orange. Drawings in the left column are from Swanson's rat atlas (Swanson, 2004) and indicate locations of anatomical regions shown to the right. Areas shown in red indicate boundaries that were used for cell quantifications, whereas boxes drawn with dashed lines indicate approximate locations of images on the right. Images in the middle column were taken from an animal that survived 132 and 108 h after injections with PRV-152 and PRV-BaBlu, respectively. Images in the right column are from an animal that survived 144 and 120 h after injections with PRV-152 and PRV-BaBlu, respectively. Numbers on the left indicate approximate distances from bregma in millimeters. dmPAG, Dorsomedial periaqueductal gray. Scale bars, 100 μm.

Figure 2.

Examples of sympatho-motor neurons in the hypothalamus. The drawing at the top is from Swanson's (2004) rat atlas and indicates locations of anatomical regions shown below. Areas shown in red indicate boundaries that were used for cell quantifications, whereas boxes drawn with dashed lines indicate approximate locations of images below. The number on the left indicates approximate distance from bregma in millimeters. Images in the left column were taken from animals that survived 132 and 108 h after injections with PRV-152 and PRV-BaBlu, respectively. Images in the right column are from animals that survived 144 and 120 h after injections with PRV-152 and PRV-BaBlu, respectively. Dashed lines show the border of the third ventricle. Scale bars, 100 μm.

Figure 3.

Sympatho-motor neurons in the paraventricular nucleus of the hypothalamus. Images illustrate labeling at two different levels of the nucleus: A, caudal; B, rostral. Drawings at the top of each panel indicate different subdivisions of the nucleus; numbers to the left of the drawings indicate approximate distances from bregma in millimeters. Images A1 and B1 were taken from an animal that survived 132 and 108 h after injections with PRV-152 and PRV-BaBlu, respectively. Images in A2 and B2 are from animals that survived 144 and 120 h after injections with PRV-152 and PRV-BaBlu, respectively. Note the distinct pattern of labeling as the majority of double-infected cells are found within the lateral parvocellular, dorsal parvocellular, and medial parvocellular ventral subdivisions. Dashed lines outline the edge of the third ventricle. dp, Dorsal parvocellular; lp, lateral parvocellular; mpd, medial parvocellular dorsal division; mpv, medial parvocellular ventral division; pm, posterior magnocellular. Scale bars, 50 μm.

Figure 4.

Sympatho-motor neurons in the motor cortex. The red box in the top drawing indicates the approximate location from which the images were digitized. Images A–C were taken from an animal that survived 120 and 96 h after injections of PRV-152 and PRV-BaBlu, respectively. Images D–F are from animals that survived 144 and 120 h after injections of PRV-152 and PRV-BaBlu, respectively. A and D show neurons infected with PRV-BaBlu, which send transsynaptic projections to the adrenal gland; B and E show neurons infected with PRV-152, which send transsynaptic projections to the gastrocnemius muscle; C and F show overlaid images of A plus B and D plus E, respectively. Arrows indicate double-labeled neurons. Scale bars, 100 μm.

Figure 5.

Effect of increasing survival times on the number of double-infected neurons. There was a significant correlation between the length of the postinjection survival period for both PRV-152 (r² = 0.48; p < 0.05; A) and PRV-BaBlu (r² = 0.48; p < 0.05; B). DL, Double labeled.

Figure 6.

Distribution of sympatho-motor neurons among different regions of the midbrain and hypothalamus. Black bars indicate areas that contained >50% of all of the double-infected cells that were detected in each animal. *p < 0.05, significantly >19 other regions; ^#p < 0.05, significantly >15 other regions. cDLPAG, Caudal dorsolateral periaqueductal gray; cDMPAG, caudal dorsomedial periaqueductal gray; cPH, caudal posterior hypothalamus; iDLPAG, intermediate dorsolateral periaqueductal gray; iDMPAG, intermediate dorsomedial periaqueductal gray; irDLPAG, intermediate rostral dorsolateral periaqueductal gray; irDMPAG, intermediate rostral dorsomedial periaqueductal gray; irVLPAG, intermediate rostral ventrolateral periaqueductal gray; rDLPAG, rostral dorsolateral periaqueductal gray; rDMPAG, rostral dorsomedial periaqueductal gray; pfLH, perifornical lateral hypothalamus; PVNdp, paraventricular nucleus, dorsal parvocellular subdivision; PVNmpd, paraventricular nucleus, medial parvocellular dorsal subdivision; rPH, rostral posterior hypothalamus; rVLPAG, rostral ventrolateral periaqueductal gray.

Figure 7.

Relationship of sympatho-motor neurons to AVP- and OT-containing neurons in the PVN. Sections were processed for simultaneous visualization of viral reporter proteins (β-gal and eGFP) as well as AVP (A1, B1) or OT (A2, B2). β-Gal and eGFP were tagged with red and green fluorophores, whereas AVP and OT were tagged with a blue fluorophore. Note the extensive interdigitation of AVP-positive (A1, B1) and OT-positive (A2, B2) neurons with virally infected cells. Also note the differences in the distribution of AVP and OT within the PVN. Although AVP-positive neurons are found within all of the different subdivisions of the PVN, these cells are enriched in the posterior magnocellular (pm) subdivision (B1). In contrast, OT-containing cells show only light clustering within the pm subdivision but instead are scattered throughout the PVN (A2, B2). Abbreviations are as in Figure 3. Scale bars, 50 μm.

Figure 8.

Neurons expressing viral reporter proteins are colocalized with AVP (A, B) or OT (C, D) within the PVN. AVP- and OT-expressing neurons were tagged with a blue fluorophore (A1, B1, C1, D1), those infected with PRV-BaBlu were tagged with a red fluorophore (A2, B2, C2, D2), and cells infected with PRV-152 were tagged with a green fluorophore (A3, B3, C3, D3). Colocalization of these antigens was determined by overlaying the three images (A4, B4, C4, D4). Arrowheads show examples of triple-labeled neurons in the posterior magnocellular (A), lateral parvocellular (B), dorsal parvocellular (C), and medial parvocellular ventral (D) subdivisions on the PVN. Arrows show examples of neurons coinfected with both viral strains but not colocalized with either peptide. Scale bar, 20 μm.

Figure 9.

Regional differences in colocalization of AVP with sympatho-motor neurons and that of OT with sympatho-motor neurons. In the case of AVP, nearly one-half (49%) of the cells that colocalized with AVP and both viral reporter proteins were found within the posterior magnocellular (pm) subdivision (A). In contrast, only a small proportion (10%) of sympatho-motor neurons that colocalized with OT was located within the pm subdivision (B). Instead, such neurons were found predominantly (79%) within the lateral parvocellular (lp), dorsal parvocellular (dp), and medial parvocellular ventral (mpv) subdivisions (B). These regions of the PVN also contained a considerable fraction (35%) of AVP-positive sympatho-motor neurons (A).