Ultrastructural evidence for direct excitatory retroambiguus projections to cutaneous trunci and abdominal external oblique muscle motoneurons in the cat.

The nucleus retroambiguus (NRA) is a group of neurons, located laterally in the caudal medulla oblongata. The NRA is thought to modulate abdominal pressure in the framework of respiration, vomiting, vocalization, probably parturition, and, in all likelihood mating behavior. The NRA exerts this control through its projections to motoneurons to the nucleus ambiguus in the lateral medulla (innervating pharynx, larynx), and spinal cord (innervating cutaneous trunci, intercostal, abdominal, pelvic floor, and lower limb muscles). The nature of these NRA-motoneuronal projections is unknown. In this study we have determined the ultrastructure of the NRA-motoneuronal projections, and especially those to the abdominal external oblique and cutaneous trunci muscles. In four cats 0.1% cholera toxin subunit b was injected in the external oblique and cutaneous trunci muscles to retrogradely label their motoneurons in the spinal cord. Wheat germ agglutinin-conjugated horseradish peroxidase was injected into the NRA to anterogradely label its contralaterally descending fibers to the motoneurons of both muscles. In order to prevent anterograde labeling of ipsilaterally descending systems not originating from the NRA, a hemisection was made at the level of C2 prior to the NRA injection. The ultrastructural results indicate that the majority (60-74%) of the anterogradely labeled NRA-terminals made monosynaptic contacts with retrogradely labeled dendrites of the external oblique and the cutaneous trunci muscle motoneurons. The majority (86-95%) of the NRA terminals made asymmetric synaptic contacts and 79-84% contained round vesicles. These results demonstrate the existence of direct, presumably excitatory, projections from NRA to external oblique and cutaneous trunci muscle motoneurons.

Functional heterogeneity among neurons in the nucleus retroambiguus with lumbosacral projections in female cats.

Nucleus retroambiguus (NRA), in the caudal medulla, projects to all spinal levels. One physiological role is abdominal pressure control, evidenced by projections to intercostal and abdominal motoneurons from expiratory bulbospinal neurons (EBSNs) within NRA. The roles of NRA projections to the lumbosacral cord are less certain, although those to limb motoneurons may relate to mating behavior and those to Onuf's nucleus (ON) to maintaining continence. To clarify this we physiologically characterized NRA projections to the lumbosacral cord. Extracellular recordings were made in NRA under anesthesia and paralysis in estrus cats. Administered CO(2) gave a strong respiratory drive. Antidromic unit responses were recorded to stimulation of the contralateral ventrolateral funiculus of L(6), L(7), or sacral segments and to microstimulation in the region of semimembranosus motor nucleus or ON. All units were found at sites showing expiratory discharges. Units that showed collisions between antidromic and spontaneous spikes (all in late expiration) were identified as EBSNs. These were common from the ventrolateral funiculus (VLF) of L(6) (42.5%) or L(7) (32.9%), but rare from the sacral VLF or the motor nuclei. Antidromic latencies revealed a subthreshold respiratory drive in some non-EBSNs. This group had lower conduction velocities than the EBSNs. The remainder, with a negligible respiratory drive, had even lower conduction velocities. A new population of NRA neurons has thus been defined. They are not active even with a strong respiratory drive, but may provide most of the synaptic input from NRA to lower lumbar and sacral segments and could subserve functions related to mating behavior.

In cat four times as many lamina I neurons project to the parabrachial nuclei and twice as many to the periaqueductal gray as to the thalamus.

The spinothalamic tract, and especially its fibers originating in lamina I, is the best known pathway for transmission of nociceptive information. On the other hand, different studies have suggested that more lamina I cells project to the parabrachial nuclei (PBN) and periaqueductal gray (PAG) than to the thalamus. The exact ratio of the number of lamina I projections to PBN, PAG and thalamus is not known, because comprehensive studies examining these three projections from all spinal segments, using the same tracers and counting methods, do not exist. In the present study, the differences in number and distribution of retrogradely labeled lamina I cells in each segment of the cat spinal cord (C1-Coc2) were determined after large wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) injections in either PBN, PAG or thalamus. We estimate that approximately 6000 lamina I cells project to PBN, 3000 to PAG and less than 1500 to the thalamus. Of the lamina I cells projecting to thalamus or PAG more than 80%, and of the lamina I-PBN cells approximately 60%, were located on the contralateral side. In all cases, most labeled lamina I cells were found in the upper two cervical segments and in the cervical and lumbar enlargements.

How many spinothalamic tract cells are there? A retrograde tracing study in cat.

The spinothalamic tract, well known for its role in nociception, is the most frequently studied ascending pathway originating from the spinal cord. It is known that spinothalamic neurons are located in all segments of the spinal cord, but in most mammals the total number of spinothalamic neurons is not known. In three cats, after large wheat germ agglutinin-conjugated horseradish peroxidase injections involving all parts (one case) or almost all parts of the thalamus (two cases), the number of retrogradely labeled profiles was counted in a 1:4 series of sections of all spinal segments from C1 to Coc2. After applying the correction factor of Abercrombie (Anat. Rec. 94 (1946) 239), it appears that a total of 12,000 cells in the spinal cord project to the thalamus.

Less than 15% of the spinothalamic fibers originate from neurons in lamina I in cat.

Lamina I neurons sending their axons into the spinothalamic tract are thought to play a crucial role in nociception, but many spinothalamic fibers do not originate from lamina I neurons. In cat, no consensus exists about what percentage of the spinothalamic tract cells are located in lamina I. After wheat germ agglutinin-conjugated horseradish peroxidase injections that covered large parts of the thalamus, retrogradely labeled cells were plotted and counted in all segments of the spinal cord. Results show that, averaged over all spinal segments, the percentage of labeled lamina I neurons was 4.9-14.2%. These results demonstrate that, in contrast to what is concluded in several previous studies, lamina I in the cat provides only a limited part of the total spinal input to the thalamus.

Direct projections from the nucleus retroambiguus to cricothyroid motoneurons in the cat.

Vocalization can be elicited by stimulation in the periaqueductal gray (PAG). Light-microscopical tracing and physiological studies have revealed that the PAG uses the nucleus retroambiguus (NRA) as a relay to excite the vocalization muscle motoneurons. Direct NRA projections have been demonstrated to pharyngeal and abdominal wall muscle motoneurons, but not to laryngeal motoneurons. In two cats 0.1% cholera toxin subunit b was injected in the cricothyroid muscle of the larynx to retrogradely label its motoneurons, and 2.5% wheat germ agglutinin-horseradish peroxidase was injected into the NRA to anterogradely label its fibers. The electronmicroscopical results indicate that the NRA fibers make monosynaptic contacts with cricothyroid motoneuronal dendrites. Almost all NRA terminal profiles had asymmetrical synapses and contained mostly round or pleiomorphic vesicles, which strongly suggests that the NRA-cricothyroid motoneuronal projection is an excitatory pathway.

Lamina I-periaqueductal gray (PAG) projections represent only a limited part of the total spinal and caudal medullary input to the PAG in the cat.

The periaqueductal gray is well known for its involvement in nociception control, but it also plays an important role in the emotional motor system. To accomplish these functions the periaqueductal gray receives input from the limbic system and from the caudal brainstem and spinal cord. Earlier studies gave the impression that the majority of the periaqueductal gray projecting cells in caudal brainstem and spinal cord are located in the contralateral lamina I, which is involved in nociception. The present study in the cat, however, demonstrates that of all periaqueductal gray projecting neurons in the contralateral caudal medulla less than 7% was located in lamina I. Of the spinal periaqueductal gray projecting neurons less than 29% was located in lamina I. However, within the spinal cord large segmental differences exist: in few segments of the enlargements the lamina I-periaqueductal gray projecting neurons represent a majority. In conclusion, although the lamina I-periaqueductal gray projection is a very important nociceptive pathway, it constitutes only a limited part of the total projection from the caudal medulla and spinal cord to the periaqueductal gray. These results suggest that a large portion of the medullo- and spino-periaqueductal gray pathways conveys information other than nociception.

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.

Ultrastructural evidence for direct projections from the pontine micturition center to glycine-immunoreactive neurons in the sacral dorsal gray commissure in the cat.

During micturition, according to the concept of Blok, Holstege, and colleagues ([1997] Neurosci. Lett. 233:109-112), the pontine micturition center (PMC) elicits bladder contraction by way of direct excitation of the parasympathetic bladder motoneurons. At the same time, the PMC elicits relaxation of the external urethral sphincter (EUS) by excitation of gamma-aminobutyric acid (GABA)-ergic interneurons in the sacral dorsal gray commissure (DGC), which, in turn, inhibit EUS motoneurons. The question is whether the inhibitory neurotransmitter glycine is also involved in this system. The present study investigated, first, whether there are glycine immunoreactive interneurons in the sacral DGC and, second, whether they receive direct PMC afferents. Finally, it was determined whether glycine and GABA are colocalized in DGC interneurons. In two adult male cats, the PMC was identified by electrical stimulation. Subsequently, the identified region was injected with the anterograde tracer WGA-HRP. Sections of sacral cord segments were processed for light and electron microscopic detection of anterograde labeling, as well as for glycine and GABA, using postembedding immunogold labeling with antibodies. In total 128 labeled PMC terminals were found in the DGC, which contained many round vesicles and asymmetric synapses. About 31.3% (40 of 128) made contact with glycine-immunoreactive dendrites. Eleven of them were selected for serial sectioning, which showed that 54.6% (6 of 11) of the glycine-immunoreactive dendrites were also immunoreactive for GABA. The results demonstrate that the PMC projects directly to dendrites of interneurons in the sacral DGC, which are immunoreactive for both glycine and GABA. These interneurons are thought to inhibit the EUS motoneurons during micturition.

Segmental and laminar organization of the spinal neurons projecting to the periaqueductal gray (PAG) in the cat suggests the existence of at least five separate clusters of spino-PAG neurons.

The present retrograde tracing study in the cat describes the spinal cord projections to the periaqueductal gray (PAG), taking into account different regions of the PAG and all spinal segments. Results show that injecting different parts of the PAG leads to different laminar and segmental distributions of labeled spinal neurons. The impression was gained that at least five separate clusters of spinal neurons exist. Cluster I neurons are found in laminae I and V throughout the length of the cord and are probably involved in relaying nociceptive information to the PAG. Cluster II neurons lie in the ventrolateral part of laminae VI-VII of the C1-C4 spinal cord and were labeled by injecting the ventrolateral or lateral part of the rostrocaudal PAG or the deep tectum. Cluster III neurons are located in lamina X of the thoracic and upper lumbar cord and seem to target the PAG and the deep tectum. Cluster IV neurons are located in the medial part of laminae VI-VII of the lumbosacral cord and seem to project predominantly to the lateral and ventrolateral caudal PAG. These neurons may play a role in conveying tactile stimuli to the PAG during mating behavior. Neurons of cluster V are located in the lateral part of lamina I of L6-S2 and in laminae V-VII and X of S1-S3. They are labeled only after injections into the central portion of the lateral and ventrolateral caudal PAG and probably relay information concerning micturition and mating behavior.

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.

Retroambiguus projections to the cutaneus trunci motoneurons may form a pathway in the central control of mating.

Our laboratory has proposed that the nucleus retroambiguus (NRA) generates the specific motor performance displayed by female cats during mating and that it uses direct pathways to the motoneurons of the lower limb muscles involved in this activity. In the hamster a similar NRA-projection system could generate the typical female mating posture, which is characterized by lordosis of the back as well as elevation of the tail. The present study attempted to determine whether this elevation of the tail is also part of the NRA-mating control system. The basic assumption was that elevation of the tail is a function of the cutaneous trunci muscle (CTM), which was verified by bilateral tetanic stimulation of the lateral thoracic nerves innervating the CTM. It resulted in upward movement of the tail to a position similar to the tail-up position during the lordosis posture. Retrograde tracing results showed that CTM motoneurons are located in the ventral and ventrolateral part of the C(7)-C(8) ventral horn, those innervating the tail region ventrolateral to those innervating the axillary region. Anterograde tracing studies showed that NRA fibers terminate bilaterally in both parts of the CTM motoneuronal cell groups. Electron microscopical studies revealed that labeled NRA terminals make monosynaptic contacts with retrogradely labeled dendrites of CTM motoneurons. Almost all of these terminal profiles had asymmetric synapses and contained spherical vesicles, which suggests an excitatory function. The observation that 15% of the labeled NRA terminals make more than one synaptic contact with a retrogradely labeled CTM motoneuronal dendrite within the same section indicates how powerful the NRA-CTM projection is. The results indicate that during mating the NRA not only could generate the lordosis posture but also the elevation of the tail.

The pontine micturition center in rat receives direct lumbosacral input. An ultrastructural study.

The act of micturition differs strongly among species. For example, adult cats and humans urinate primarily in a safe environment ('guarded urination'), while rats urinate more reflexively ('reflex urination'). This study in adult rats investigates the existence of direct lumbosacral cord projections to spinally projecting neurons in the pontine micturition center (PMC). Bilateral injections of wheat germ agglutinin horseradish peroxidase in the caudal lumbar and rostral sacral cord resulted in labeled profiles, including retrogradely labeled neurons in the PMC. At the ultrastructural level, anterogradely labeled terminals in the PMC were found, which were filled with many round and some pleiomorphic and flat vesicles. About eleven percent of the terminals contacted retrogradely labeled dendrites. Of the labeled terminals 80% contained asymmetric synaptic clefts, and 20% symmetric synaptic clefts. The results provide evidence that in the rat, unlike the cat, a direct lumbosacral pathway to the PMC exists, which might explain the differences in micturition behavior between rats and cats.

Estrogen receptor-alpha-immunoreactive neurons in the mesencephalon, pons and medulla oblongata of the female golden hamster.

Recent studies have revealed brainstem-spinal pathways involved in the generation of receptive behavior in hamster and cat, and the enormous influence of estrogen on these pathways. The present study gives an overview of the location of estrogen receptor-alpha-immunoreactive neurons (ER-alpha-IR) in the brainstem of the female hamster. In the mesencephalon, ER-alpha-IR cells were found in the arcuate and peripeduncular nuclei as well as throughout the rostrocaudal extent of the periaqueductal gray (PAG), and the laterally adjoining tegmentum. In the caudal brainstem, groups of ER-alpha-IR cells were present in the ventrolateral parabrachial nucleus, the solitary nucleus, and in contrast to the cat, in the nucleus retroambiguus. No ER-alpha-IR cells were found in any other part of the brainstem. The functional implications of these findings are discussed.

Two pontine micturition centers in the cat are not interconnected directly: implications for the central organization of micturition.

The urinary bladder muscle and its external urethral sphincter are innervated, respectively, by the parasympathetic preganglionic motoneurons in the sacral intermediolateral cell column and somatic motoneurons in Onuf's nucleus. Neurons coordinating the activity of these muscles during micturition and urinary continence are not located in the sacral cord but in two pontine regions, the medial (M)-region (or pontine micturition center) and the lateral (L)-region (or pontine storage center). The M-region excites the bladder muscle through projections to its motoneurons and inhibits the urethral sphincter through excitatory projections to sacral cord gamma-amino butyric acid (GABA)-immunoreactive interneurons, which, in turn, inhibit urethral sphincter motoneurons. The L-region, through direct projections, excites urethral sphincter motoneurons. The present study investigated whether there are interconnections between the M- and L-regions. Anterograde tracing injections in the M-region resulted in labeled fibers to the intermediolateral cell column containing bladder motoneurons but not to Onuf's nucleus. No specific projections were found to the L-regions or to the contralateral M-region. L-region injections resulted in distinct projections to the Onuf's nucleus but not to the sacral intermediolateral cell column. No specific projections were observed either to the M-region or to the contralateral L-region. In conclusion, the M- and L-regions have direct long fiber projections, respectively, to the motoneurons of the bladder muscle and the external urethral sphincter, but they do not influence one another through direct pathways. The results strongly suggest that the M- and L-regions represent separate functional systems that act independently.

Descending projections from the nucleus retroambiguus to the iliopsoas motoneuronal cell groups in the female golden hamster: possible role in reproductive behavior.

In the cat, the nucleus retroambiguus (NRA) projects to expiratory motoneurons in the brainstem and spinal cord. Recently, it has been demonstrated that the NRA sends fibers to a specific set of motoneurons in the lumbosacral cord, which pathway is thought to play a crucial role in mating behavior. The question is whether such projections exist in the hamster, because the female of this species displays a very distinctive receptive behavior. In the hamster, lumbosacral cord injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) combined with hemisection 1 or 2 segments rostral to injection sites in three of the five cases demonstrated retrogradely labeled neurons in the NRA at levels 1.0-2.25 mm caudal to the obex, contralateral to the injection sites. Injections of WGA-HRP into the NRA and adjoining reticular formation revealed that NRA fibers crossed the midline in the caudal medulla and descended in the contralateral lateral and ventrolateral funiculi to terminate bilaterally, but mainly contralaterally, in the motoneuronal cell groups of the abdominal wall and iliopsoas muscles. NRA projections to levels caudal to lumbar segment 5 were virtually absent. Electron microscopic examination revealed that, of the 162 labeled NRA terminal profiles found in the ultrathin sections, 144 (89%) made monosynaptic contacts with retrogradely labeled dendrites of iliopsoas motoneurons. These NRA terminals formed asymmetrical synapses and contained spherical vesicles indicative of an excitatory function. The results indicate that, in the hamster, direct contralateral NRA projections exist to iliopsoas motoneurons. A concept is discussed in which this pathway plays a crucial role in mating behavior.