Estrogen receptor-alpha immunoreactive neurons in the brainstem and spinal cord of the female rhesus monkey: species-specific characteristics.

The distribution pattern of estrogen receptors in the rodent CNS has been reported extensively, but mapping of estrogen receptors in primates is incomplete. In this study we describe the distribution of estrogen receptor alpha immunoreactive (ER-alpha IR) neurons in the brainstem and spinal cord of the rhesus monkey. In the midbrain, ER-alpha IR neurons were located in the periaqueductal gray, especially the caudal ventrolateral part, the adjacent tegmentum, peripeduncular nucleus, and pretectal nucleus. A few ER-alpha IR neurons were found in the lateral parabrachial nucleus, lateral pontine tegmentum, and pontine gray medial to the locus coeruleus. At caudal medullary levels, ER-alpha IR neurons were present in the commissural nucleus of the solitary complex and the caudal spinal trigeminal nucleus. The remaining regions of the brainstem were devoid of ER-alpha IR neurons. Spinal ER-alpha IR neurons were found in laminae I-V, and area X, and were most numerous in lower lumbar and sacral segments. The lateral collateral pathway and dorsal commissural nuclei of the sacral cord and the thoracic intermediolateral cell column also contained ER-alpha IR neurons. Estrogen treatment did not result in any differences in the distribution pattern of ER-alpha IR neurons. The results indicate that ER-alpha IR neurons in the primate brainstem and spinal cord are concentrated mainly in regions involved in sensory and autonomic processing. Compared with rodent species, the regional distribution of ER-alpha IR neurons is less widespread, and ER-alpha IR neurons in regions such as the spinal dorsal horn and caudal spinal trigeminal nucleus appear to be less abundant. These distinctions suggest a modest role of ER-alpha in estrogen-mediated actions on primate brainstem and spinal systems. These differences may contribute to variations in behavioral effects of estrogen between primate and rodent species.

Projections from estrogen receptor-alpha immunoreactive neurons in the periaqueductal gray to the lateral medulla oblongata in the rhesus monkey.

The periaqueductal gray (PAG) contains numerous estrogen receptor-alpha immunoreactive (ER-alpha IR) neurons that are distributed in a species-specific way. These neurons might modulate different types of behavior that are mediated by the PAG such as active and passive coping responses, analgesia, and reproductive behavior. In primates, it is not known whether ER-alpha IR PAG neurons represent local interneurons and/or neurons that project to brainstem areas that control these behaviors. In this double labeling study, we asked whether ER-alpha IR neurons in the PAG of the rhesus monkey project to the nucleus retroambiguus (NRA), an area in the ventrolateral caudal medulla oblongata that is involved in expiration, vocalization, and reproductive behavior. Tracer was injected into the caudal lateral medulla oblongata to retrogradely label PAG neurons, and ER-alpha was visualized immunohistochemically. Although ER-alpha IR neurons and NRA-projection neurons were present at similar levels of the PAG, their distributions hardly overlapped. ER-alpha IR PAG neurons that project to the lateral caudal medulla represented less than 2% of ER-alpha IR PAG neurons. These double-labeled neurons were mainly located in the ipsilateral caudal PAG. The cluster of neurons in the medial part of the lateral PAG that projects specifically to the NRA-region did not contain double-labeled cells. The results indicate that only a few ER-alpha IR PAG neurons project to the NRA-region. This might be related to the modest effects of estrogen on mating-related behavior in primates compared most other mammalian species. Remaining ER-alpha IR PAG neurons might act locally on other PAG neurons, or they might represent neurons that project to other areas. Furthermore, the finding that the distributions of ER-alpha IR neurons and neurons that project to premotor neurons in the NRA-region scarcely overlap illustrates that the PAG in primates is very highly organized into anatomically distinct regions compared with other species.

Monosynaptic projections from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey.

Vocalization and straining-related activities require the activation of laryngeal muscles. The control of laryngeal muscles during these activities is thought to be mediated by a pathway from the periaqueductal gray via premotor neurons in the nucleus retroambiguus to laryngeal motoneurons in the nucleus ambiguus. However, direct contacts between the nucleus retroambiguus and laryngeal motoneurons have never been demonstrated anatomically. Moreover, data in primates about the nucleus retroambiguus-nucleus ambiguus pathway are lacking. Therefore, the present study examines the projection from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey at the light and electron microscopic levels. Injections with wheat germ agglutinin-horseradish peroxidase were made into the nucleus retroambiguus in five rhesus monkeys to anterogradely label fibers in the nucleus ambiguus. In two of these animals, the cricothyroid muscle was injected with cholera toxin subunit b to identify the motoneurons that supply it. The results show that the nucleus retroambiguus region most densely projects to the compact formation of the nucleus ambiguus, whereas cricothyroid motoneurons, which surround the compact formation, receive a moderate projection. The projections are bilateral, with a contralateral predominance. Ultrastructurally, anterogradely labeled terminal profiles from the nucleus retroambiguus contact cholera toxin subunit b-labeled dendrites of cricothyroid motoneurons. The terminal profiles contain primarily spherical vesicles and form asymmetrical contacts with cricothyroid motoneurons. This study demonstrates that the nucleus retroambiguus region projects to the nucleus ambiguus in the primate. Some of these projections include monosynaptic connections to laryngeal motoneurons. This pathway is important for the control of the vocal folds during vocalization and straining-related activities.

Estrogen receptor-alpha immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat.

Estrogen affects autonomic functions such as micturition. The sacral cord is important in the control of micturition and contains numerous estrogen receptor-alpha immnoreactive (ER-alpha IR) neurons. Therefore, the present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are immunoreactive for ER-alpha. In the sacral cord of seven female ovariectomized cats, the distribution of ER-alpha IR neurons was studied using the H222 and 1D5 antibodies. Choleratoxin subunit b (CTb) was injected into the bladder wall to visualize its preganglionic neurons. ER-alpha IR was present in the nuclei of cells in laminae I, II, V, VII, and X, and in nuclei and cytoplasm of neurons in the sacral parasympathetic nucleus. The vast majority of CTb labeled neurons contained ER-alpha IR nuclei, indicating that preganglionic neurons innervating the bladder express ER-alpha. The results suggest that estrogen modulates micturition in the cat via ER-alpha in bladder preganglionic neurons.

Monosynaptic projections from the nucleus retroambiguus to motoneurons supplying the abdominal wall, axial, hindlimb, and pelvic floor muscles in the female rhesus monkey.

The nucleus retroambiguus (NRA) consists of premotor neurons in the caudal medulla. It is involved in expiration, vomiting, vocalization, and probably reproductive behavior by means of projections to distinct motoneuronal cell groups. Because no information is available about the NRA and its efferent pathways in primates, the present study examines NRA projections to the lumbosacral spinal cord in female rhesus monkeys. To identify the NRA, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was injected into the lumbosacral cord in three monkeys. To study the distribution of NRA axons in the lumbosacral cord, WGA-HRP injections were made into the NRA in seven monkeys. To identify motoneuronal cell groups receiving input from the NRA, the same seven monkeys also received cholera toxin subunit b (CTb) injections into different hindlimb, axial, and pelvic floor muscles. The results show that NRA neurons projecting to the lumbosacral cord are mainly located between 1 to 4 mm caudal to the obex. They send numerous axons to external oblique and pelvic floor motoneurons, whereas projections to iliopsoas and axial motoneurons are less numerous. The projections are bilateral, but show a clear contralateral predominance in the iliopsoas, axial, and pelvic floor motoneuronal cell groups. At the ultrastructural level, NRA-terminal profiles make asymmetrical contacts with labeled and unlabeled dendrites in these motoneuronal cell groups and contain large amounts of spherical and a few dense core vesicles. It is concluded that the NRA is well developed in the monkey and that there exists a direct pathway from the NRA to lumbosacral motoneurons in this species. The finding that the NRA projects to a somewhat different set of motoneuronal cell groups compared with other species fits the concept that it is not only involved in expiration-related activities but also in species specific receptive and submissive behavior.

Monosynaptic projections from the lateral periaqueductal gray to the nucleus retroambiguus in the rhesus monkey: implications for vocalization and reproductive behavior.

The periaqueductal gray (PAG) is known to be essential for vocalization and reproductive behavior. The PAG controls components of these behaviors by means of projections to the nucleus retroambiguus (NRA), a group of premotor neurons in the caudal medulla oblongata. In the accompanying study (VanderHorst et al., 2000 [accompanying study]), the NRA and its lumbosacral projections have been identified in the rhesus monkey. The present light and electron microscopical tracing study describes the PAG-NRA pathway in primates. To locate midbrain neurons projecting to the NRA, wheat germ agglutinin horseradish peroxidase (WGA-HRP) was injected into the NRA in six monkeys. To determine the distribution pattern of PAG axons in the medulla oblongata, WGA-HRP was injected into the PAG and adjacent tegmentum in three additional monkeys. In one of these three monkeys, biotinylated dextran amine and cholera toxin subunit b were injected into the lumbosacral cord to retrogradely identify NRA neurons. The results show that a compact group of neurons in the medial part of the lateral PAG at the intercollicular level sends a dense projection to the NRA. The projection is bilateral with a clear ipsilateral predominance. At the ultrastructural level, there are monosynaptic contacts between PAG fibers and NRA neurons, including NRA neurons that project to the lumbosacral cord. The synaptic contacts were primarily asymmetrical and the labeled terminal profiles contained spherical and dense core vesicles. It is concluded that there exists a strong and direct PAG-NRA pathway in the rhesus monkey. Because NRA neurons projecting to the lower lumbar cord are included, the PAG-NRA projection is likely to be involved not only in vocalization but also in other behaviors, such as receptive posture.

Estrogen receptor-alpha-immunoreactive neurons in the periaqueductal gray of the adult ovariectomized female cat.

Anatomical and physiological studies in rodent and cat have shown that distinct parts of the midbrain periaqueductal gray (PAG) are important for the estrogen dependent, female reproductive behavior. The present study gives a detailed overview of the estrogen receptor-alpha-immunoreactive (ER-IR) neurons in the PAG in the cat. ER-IR neurons were found throughout the rostrocaudal extent of the PAG and laterally adjacent tegmentum, but were most numerous at caudal levels. The lateral and dorsal PAG contained most ER-IR neurons, whereas moderate numbers were found dorsolaterally. In these areas, only very few ER-IR neurons were found near the border of the ependymal layer. Except for the rostral dorsal raphe nucleus, the ventral PAG contained only few ER-IR neurons.

Estrogen receptor-immunoreactive neurons in the lumbosacral cord projecting to the periaqueductal gray in the ovariectomized female cat.

The periaqueductal gray (PAG) plays a crucial role in reproductive behavior. The present study investigates whether lumbosacral PAG-projecting neurons contain estrogen receptors. In four ovariectomized adult female cats, injections with cholera toxin subunit (CTb) were made into the PAG to retrogradely label PAG projecting neurons in the lumbosacral cord. Estrogen receptor immunoreactive ER-IR neurons in the lumbosacral cord were identified immunohistochemically using the antibody H222. PAG-projecting neurons that were immunoreactive for the estrogen receptor were very scarce, and predominantly present in the medial part of the ventral horn. The results indicate that only very few of the neurons relaying information from the urogenital organs to the PAG contain estrogen receptors.

Evidence for a periaqueductal gray-nucleus retroambiguus-spinal cord pathway in the rat.

The nucleus retroambiguus in the cat has been shown to receive strong projections from the periaqueductal gray and to send fibres to distinct motoneuronal cell groups in brainstem and spinal cord. The nucleus retroambiguus plays a role in the production of vocalization and possibly copulatory (lordosis and mounting) behaviour. The question arises of whether a periaqueductal gray nucleus retroambiguus-spinal cord projection also exists in the rat. In the present study, using the retrograde wheatgerm agglutinin-horseradish peroxidase tracing technique, the nucleus retroambiguus was defined as the area in the caudal medulla oblongata (1.0-2.0 mm caudal to the obex) which sends its fibres mainly through the contralateral spinal cord. Further retrograde tracing experiments demonstrated that a relatively large number of neurons in the lateral and ventral periaqueductal gray and immediately adjacent tegmentum projects to the caudal medullary lateral tegmentum. Anterograde wheatgerm agglutinin-horseradish peroxidase tracing studies finally showed that neurons in the lateral periaqueductal gray and immediately adjoining tegmentum project specifically to the nucleus retroambiguus and not to the lateral tegmentum in general, which seems to be the case for the neurons in the ventral periaqueductal gray. The results indicate that in the rat a periaqueductal gray nucleus retroambiguus spinal cord projection also exists, which may be of crucial importance for the study of the anatomical and physiological framework of respiration, vocalization, and female and male reproductive behaviour in this animal.

Organization of lumbosacral motoneuronal cell groups innervating hindlimb, pelvic floor, and axial muscles in the cat.

In a study on descending pathways from the nucleus retroambiguus (NRA) to hindlimb motoneurons (see accompanying paper), it appeared impossible, using data from the literature, to precisely determine which muscles were innervated by the motoneurons receiving the NRA fibers. This lack of data made it necessary to produce a detailed map of the lumbosacral motoneuronal cell groups in the cat. Therefore, 50 different muscles or muscle compartments of hindlimb, pelvic floor and lower back were injected with horseradish peroxidase (HRP) in 135 cases. The respective muscles were divided into ten groups: I, sartorius and iliopsoas; II, quadriceps; III, adductors; IV, hamstrings; V, gluteal and other proximal muscles of the hip; VI, posterior compartment of the distal hindlimb; VII, anterior compartment of the distal hindlimb; VIII, long flexors and intrinsic muscles of the foot; IX, pelvic floor muscles; and X, extensors of the lower back and tail. The L4-S2 segments were cut and incubated, and labeled motoneurons were counted and plotted. A new method was developed that made it possible, despite variations in size and segmental organization between the different cases, to compare the results of different cases. The results show that the spatial interrelationship between the hindlimb and pelvic floor lumbosacral motoneuronal cell groups remains constant. This finding enabled the authors to compose an accurate overall map of the location of lumbosacral motoneuronal cell groups. The general distribution of the motoneuronal cell groups is also discussed in respect to their dorsoventral, mediolateral, and rostrocaudal position within the lumbosacral ventral horn.

Nucleus retroambiguus projections to lumbosacral motoneuronal cell groups in the male cat.

Recently, in the female cat, nucleus retroambiguus (NRA) projections have been described as distinct motoneuronal cell groups in the lumbar enlargement, possibly involved in lordosis behavior. The present study deals with the NRA-lumbosacral pathway in the male cat. Lumbosacral injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were made to localize and quantify retrogradely labeled neurons in the caudal medulla. These injections were preceded by spinal hemisections to distinguish between neurons with ipsi- and contralaterally descending axons. The NRA-lumbosacral fibers descended almost exclusively contralaterally, but neurons in areas surrounding the NRA projected mainly ipsilaterally. Injections of WGA-HRP were made in the region of the NRA to determine its targets in the lumbosacral cord. To distinguish between the contralateral NRA pathways and the ipsilateral projections from neurons in the adjoining lateral tegmentum, the injections were preceded by ipsilateral hemisections in C2. A new scaling method was used to compare the results of the different cases, despite variations in size and segmental organization. The results show that the distribution pattern of anterogradely labeled fibers in the lumbosacral cord matched precisely the location of certain motoneuronal cell groups. The NRA projected densely to the abdominal wall and pelvic floor motoneurons in Onuf's nucleus, moderately to adductor longus, semimembranosus, and biceps femoris anterior motoneuronal cell groups, and only sparsely to iliopsoas and semitendinosus motoneuronal cell groups. Compared with the findings in the female, the NRA in the male cat projects more heavily to the biceps anterior and adductor longus and only sparsely to the iliopsoas and semitendinosus motoneuronal cell groups. These male-female differences are discussed.

Evidence for monosynaptic projections from the nucleus retroambiguus to hindlimb motoneurons in the cat.

The nucleus retroambiguus (NRA) is a group of premotor neurons at the transition between brainstem and spinal cord. It projects to certain motoneuronal cell groups, among which is a distinct set of motoneurons in the lumbar enlargement innervating muscles including iliopsoas, adductor longus, and hamstrings. To find out whether these NRA-motoneuronal projections are monosynaptic, injections of wheat germ-agglutinin horseradish peroxidase (WGA-HRP) into the NRA were combined with injections of cholera toxin subunit b (CTb) into the hamstring muscles. Electron microscopical examination revealed that the NRA terminal profiles make monosynaptic contacts with dendrites of motoneurons innervating these muscles. The NRA terminal profiles formed asymmetrical synapses, and contained spherical and a few dense core vesicles. These findings provide evidence of monosynaptic NRA-hindlimb motoneuronal projections which are likely to be excitatory.

Estrogen induces axonal outgrowth in the nucleus retroambiguus-lumbosacral motoneuronal pathway in the adult female cat.

In 1995, we discovered a new pathway in the cat, which originates from the nucleus retroambiguus (NRA) and terminates in a distinct set of lumbosacral hindlimb, axial, and pelvic floor motoneuronal cell groups [VanderHorst VGJM, Holstege G (1995) Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis. J Comp Neurol 359:457-475]. The NRA is a compact group of interneurons located laterally in the caudal medulla oblongata. Its projection to lumbosacral motoneurons is thought to represent the final common pathway for male mounting and for female receptive or lordosis behavior. However, females only display lordosis behavior. However, females only display lordosis behavior when they are in estrus, which suggests that the NRA-lumbosacral pathway is only active during estrus. This raised the question of whether estrogen affects this pathway. The effect of estrogen on the NRA-lumbosacral projection was studied light microscopically, using wheat-germ agglutinin horseradish peroxidase (WGA-HRP) as a tracer. The rubrospinal pathway served as control. The density of labeled NRA fibers in their target hindlimb motoneuronal cell groups appeared abundant in estrous and very weak in nonestrous cats. Such differences were not found in the rubrospinal pathway. For electron microscopical study, the NRA projection to the semi-membranosus motoneuronal cell group was selected. In this cell group, an almost ninefold increase of labeled profiles was found in estrous versus nonestrous cats. Moreover, the semimembranous motoneuronal cell group contained labeled growth cones in estrous, but not in nonestrous, cats. The present study is the first to show that estrogen induces axonal outgrowth of a precisely identified pathway in the adult mammalian central nervous system. The possible mechanisms underlying this outgrowth are discussed.

Large segmental differences in the spinal projections to the periaqueductal gray in the cat.

The periaqueductal gray (PAG) is involved in motor activities, such as movements of the neck, back and hind limbs, cardiovascular regulation, micturition, vocalization, and mating behavior, as well as in nociception control. To accomplish these functions the PAG uses information from other parts of the limbic system, from the lower brainstem, and from the spinal cord. To study the ascending projections from the spinal cord to the PAG, tracer was injected in different parts of the PAG, and the number of retrogradely labeled neurons were counted for each spinal segment. Results show that large segmental differences exist in the number of PAG projecting neurons throughout the length of the spinal cord and that different parts of the spinal cord project to specific areas in the PAG.

Distinct cell groups in the lumbosacral cord of the cat project to different areas in the periaqueductal gray.

The periaqueductal gray (PAG) is involved in aggressive and defensive behavior, micturition, and lordosis. Especially for the latter two functions, PAG afferents from the lumbosacral cord are of vital importance because, in addition to information regarding homeostasis and thermoregulation, they convey information from the pelvic viscera and sex organs. In the present retro- and antero-grade tracing study, the projection patterns of different lumbosacral cell groups in the PAG were determined. In the retrograde study, wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injections were made in the PAG and/or adjacent tegmentum, and in the anterograde study, WGA-HRP was injected in different lumbosacral segments. The results revealed that lumbosacral-PAG neurons could be divided into three groups. The first and largest group was present in lumbar 7-sacral 3 segments (L7-S3) and consisted of small, oval, and fusiform neurons. It extended from the dorsolateral part of lamina I in L7, along the lateral part of the dorsal horn in S1, and into lamina V of S2. In the lateral part of S2, some of its neurons formed clusters with intervals of +/- 230 microns. The location of the first group overlapped extensively with the termination area of pelvic and pudendal afferents. The main midbrain target of the first group was the medial part of the lateral PAG. The second group consisted of small to large multipolar neurons in laminae VIII and medial VII of caudal L6, L7, and rostral S1. This group projected strongly to a distinct region in the lateral part of the lateral PAG and the laterally adjacent tegmentum. About 10% of the labeled neurons did not fit in the two groups. They were evenly distributed throughout lumbar 4-coccygeal 3 segments (L4-Co3) and consisted of large multipolar lamina V neurons and small lamina I neurons that projected diffusely to the lateral and dorsal PAG. The large lamina V neurons also targeted the laterally adjacent tegmentum. The possible involvement of the lumbosacral-PAG projections in micturition, lordosis, and defensive and aggressive behavior is discussed.

Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis.

The nucleus retroambiguus (NRA) projects to distinct brainstem and cervical and thoracic cord motoneuronal cell groups. The present paper describes NRA projections to distinct motoneuronal cell groups in the lumbar enlargement. Lumbosacral injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were made to localize and quantify the retrogradely labeled neurons in the caudal medullary lateral tegmentum. These injections were combined with spinal hemisections to distinguish between neurons having ipsi-or contralaterally descending axons. The NRA-lumbosacral fibers descend almost exclusively contralaterally, but neurons in areas surrounding the NRA project mainly ipsilaterally. In an anterograde tracing study, injections of WGA-HRP or tritiated leucine were made in the region of the NRA to determine the NRA targets in the lumbosarcral cord. Hemisections in C2 made it possible to distinguish between NRA projections and projections from neurons in the adjoining lateral tegmentum. The results show delicate NRA projections to distinct lumbosacral motoneuronal cell groups innervating specific hindlimb muscles (iliopsoas, adductors, and hamstrings) as well as axial muscles (medial longissimus and proximal tail muscles). The projection is bilateral, with a contralateral predominance. Ipsilaterally terminating fibers are derived from NRA neurons whose axons cross the midline at the level of the obex, descend through the contralateral spinal white matter, and recross at the level of termination. A conceptual description is presented in which the periaqueductal gray-NRA-lumbosacral projections form the final common pathway for lordosis in the cat.