Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat.

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus.

Autoradiographic localization of neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei of the rat.

The hypoglossal and motor trigeminal nuclei contain somatic motoneurons innervating the tongue, jaw, and palate. These two cranial motor nuclei are myotopically organized and contain neurotransmitter binding sites for thyrotropin-releasing hormone, substance P, and serotonin. Quantitative autoradiography was used to localize thyrotropin-releasing hormone, substance P, and serotonin-1A and serotonin-1B binding sites in the hypoglossal and motor trigeminal nuclei and to relate the relative distributions of these binding sites to the myotopic organizations of the two nuclei. In the hypoglossal nucleus, high-to-moderate concentrations of all four binding sites were present in the dorsal and ventromedial subnuclei, whereas low concentrations were noted in the ventrolateral subnucleus. In the motor trigeminal nucleus, high concentrations of serotonin-1B, moderate densities of thyrotropin-releasing hormone, and low levels of substance P and serotonin-1A binding sites were present in both the ventromedial and dorsolateral subnuclei. These observations demonstrate that neurotransmitter binding sites in the hypoglossal and motor trigeminal nuclei are heterogeneously localized and that their distributions correspond to the previously described myotopic organizations of each nucleus.

Natural history and prognosis of advanced lung disease.

Large gaps exist in our knowledge of the natural history of advanced lung disease and of the impact of various therapies upon prognosis and survival. Applying the results of population-based epidemiologic studies or limited clinical trials to a specific patient is hazardous because of marked individual variation in survival, even with the most grim of prognoses. Obtaining such prognostic information is essential, however, in addressing current key issues in advanced lung disease-the efficacy of various therapies, timing lung transplantation, referring to hospice care, providing palliative therapy, and determining medical futility.

Raphespinal and reticulospinal neurons project to the dorsal vagal complex in the rat.

Stimulation of the caudal raphe nuclei alters visceral functions. The caudal raphe nuclei project to the nucleus of the solitary tract, which receives the central terminations of vagal afferents and plays an important role in the central integration of autonomic activities. The caudal raphe nuclei also project to the somatic and preganglionic autonomic motoneurons of the spinal cord. Diamidino yellow was injected into the nucleus of the solitary tract, and fast blue was injected into either the cervical, thoracic, or lumbar spinal cord. Large numbers of double-labeled neurons were present within the caudal raphe nuclei and the adjacent reticular formation of the medial tegmental field. This observation documents that individual raphespinal and reticulospinal neurons project an axon collateral to the nucleus of the solitary tract. These data demonstrate the anatomic substrate for global modulation of the autonomic motoneuron pool by the caudal raphe nuclei.

Origin of serotoninergic afferents to the hypoglossal nucleus in the rat.

The hypoglossal nucleus contains serotonin and several different serotonin receptors, and serotonin is present in fibers and terminals contacting hypoglossal motoneurons. Serotonin alters the excitability of hypoglossal motoneurons, and may influence hypoglossal motoneuron activity in a variety of physiological processes. Since the hypoglossal nucleus contains no serotoninergic somata, the present study sought to identify the sources of serotoninergic afferents to the hypoglossal nucleus. Fluorogold was injected into the hypoglossal nucleus and serotoninergic immunofluorescence was utilized in a dual-fluorescence technique to identify the sources of serotoninergic afferents to the hypoglossal nucleus. The results demonstrate that most serotoninergic afferents to the hypoglossal nucleus originate from the nuclei raphe pallidus and obscurus, while fewer originate from the nucleus raphe magnus and the parapyramidal region. Other regions of the medial tegmental field and the pons that contain both serotoninergic neurons and neuronal afferents to the hypoglossal nucleus contain no double-labeled neurons.

Effects of vagotomy on neurotransmitter receptors in the rat dorsal vagal complex.

The dorsal vagal complex contains many different neurotransmitter receptors. The cyto-architectural localizations of some of these receptors remain largely unknown. In rats, vagotomy was performed to destroy vagal afferents terminating in the nucleus of the solitary tract and to produce chromatolysis of preganglionic motoneurons in the dorsal motor nucleus of the vagus. Quantitative receptor autoradiography was then employed to determine the effect of vagotomy upon the distribution of receptors for thyrotropin-releasing hormone, substance P, and serotonin within individual regions and subnuclei of the entire dorsal vagal complex. Vagotomy reduced the concentrations of thyrotropin-releasing hormone and substance P, but not serotonin1A, or serotonin1B, receptors in the dorsal motor nucleus of the vagus. Within the nucleus of the solitary tract, substance P receptors were reduced in only the medial and central subnuclei after vagotomy. In contrast, no effect was observed upon the concentrations of thyrotropin-releasing hormone, serotonin1A, or serotonin1B receptors in any subnuclei of the solitary tract following vagotomy. These results suggest that in the dorsal motor nucleus of the vagus, thyrotropin-releasing hormone and substance P receptors are localized upon vagal preganglionic motoneurons, while serotonin1A and serotonin1B receptors are present upon interneurons or other neuronal elements. These results also suggest that thyrotropin-releasing hormone, substance P, serotonin1A, and serotonin1B receptors in the nucleus of the solitary tract are localized upon internuncial neurons in the nucleus of the solitary tract.

Raphespinal and reticulospinal axon collaterals to the hypoglossal nucleus in the rat.

Neurons in the medial tegmental field project directly to spinal somatic motoneurons and to cranial motoneuron pools such as the hypoglossal nucleus. The axons of these neurons may be highly collateralized, projecting to multiple levels of the spinal cord and to many diverse regions at different levels of the neuraxis. We employed a double fluorescent retrograde tracer technique to examine whether medial tegmental neurons that project to the spinal cord also project to the hypoglossal nucleus. Injections of Diamidino Yellow into the hypoglossal nucleus and Fast Blue into the spinal cord produced large numbers of double labeled neurons in the medial tegmental field, particularly in the caudal raphe nuclei and adjacent ventromedial reticular formation. In these structures the number of neurons projecting to both the hypoglossal nucleus and the spinal cord was equivalent to the number of neurons projecting to multiple levels of the spinal cord observed in control animals. Fewer neurons projecting to both the hypoglossal nucleus and the spinal cord were observed in several other nuclei and subregions of the medial tegmental field, while almost no such neurons were observed in the lateral tegmental field or other pontomedullary structures. These results demonstrate that neurons of the caudal raphe nuclei and adjacent ventromedial reticular formation project to both the spinal cord and the hypoglossal nucleus, and support the concept that the diffuse projections to motoneuron pools from the medial tegmental field globally modulate both spinal and cranial somatic motoneuron excitability.

Neurons of the motor trigeminal nucleus project to the hypoglossal nucleus in the rat.

The hypoglossal nucleus (Mo12) contains motoneurons that innervate the tongue, while the motor trigeminal nucleus (Mo5) contains motoneurons that elevate or depress the mandible. Previous studies have revealed lateral and medial tegmental field neuronal afferents to the Mo12 adjacent to, but not within, the motor trigeminal nucleus (Mo5). The current studies demonstrate the presence of retrogradely labeled neuronal afferents to the Mo12 within the Mo5 produced by as little as 10 nl of Fast Blue (FB) injected into the Mo12. Retrograde labeling of Mo5 afferents to the Mo12 with injections of Diamidino Yellow (DY) combined with injections of FB into the lumbar spinal cord showed these neuronal afferents to the Mo12 are not part of the diffuse projections to motoneurons from the nucleus subcoeruleus. Retrograde labeling of Mo5 afferents to the Mo12 with DY combined with injections of FB into the masseter revealed these neuronal afferents to the Mo12 are not trigeminal motoneurons. These results indicate that Mo5 interneurons are part of the lateral tegmental field projections to the Mo12, and are likely to comprise part of the neural substrate coordinating the motor activity of the jaw and tongue.

Organization of serotonin 1A and 1B receptors in the nucleus of the solitary tract.

We utilized 3H-8-hydroxy-N,N-dipropyl-2-aminotetralin (3H-DPAT) and 125I-iodocyanopindolol (125I-CYP) to label serotonin (5HT) 1A and 5HT1B receptors, respectively, in sections of the rat brain after characterizing the pharmacologic specificity of these agents. We then used quantitative autoradiography to measure the concentrations of 5HT1A and 5HT1B receptors in individual subnuclei of the nucleus of the solitary tract (NTS) and adjacent structures of the dorsal vagal complex. The highest 5HT1A receptor concentrations were observed within the central and intermediate subnuclei of the NTS, with low quantities of 3H-DPAT binding sites observed in the hypoglossal nucleus and dorsal motor nucleus of the vagus. In contrast, the density of 5HT1B receptors was relatively homogeneous through all NTS subnuclei, with the highest concentrations localized within the ventrolateral subnucleus. The hypoglossal and dorsal motor nuclei had slightly higher 5HT1B receptor densities than the NTS subnuclei, whereas the area postrema had a very low density. These data suggest that 5HT1A receptors are organized in a manner consistent with the cytoarchitectural and hodological parcellation of the NTS into individual subnuclei. The high concentrations of 5HT1A receptors in the central and intermediate subnuclei suggest a role for these receptors in medullary reflex pathways subserving deglutition. The relatively high density of 5HT1B receptors in the ventrolateral subnucleus suggests that these receptors modulate respiratory neurons, whereas the diffuse organization of 5HT1B receptors in the remaining subnuclei suggests that they are associated with central 5HT afferent pathways to the NTS. Further studies will be required to understand the physiologic role of 5HT1 receptors within the NTS.

Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex.

We utilized quantitative autoradiography to localize receptors for thyrotropin-releasing hormone (TRH) and substance P in individual subnuclei of the rat nucleus tractus solitarii (NTS) and the dorsal vagal complex. Within the NTS, TRH receptor concentrations were highest within the gelatinosus and centralis subnuclei and the medial subnucleus rostral to the area postrema, moderate within the intermediate subnucleus and the medial subnucleus adjacent to the area postrema, and low within the ventrolateral and commissural subnuclei and the medial subnucleus caudal to the area postrema. In contrast, substance P receptor concentrations were high throughout the medial subnucleus, moderate in all other subnuclei medial to the tractus solitarius, and relatively low in subnuclei lateral to the tractus solitarius. The dorsal motor nucleus of the vagus contained high concentrations of both TRH and substance P receptors, whereas we observed low TRH and moderate substance P receptors in the area postrema. High TRH and moderate substance P receptors were observed in the adjacent hypoglossal nucleus. In addition, we compared the concentrations of TRH receptors between chloroform-defatted and nondefatted tissue sections, and noted little effect of white matter tritium quench upon the observed TRH receptor concentrations. These results suggest that neurotransmitter receptors within the rat dorsal vagal complex are organized in a manner consistent with previous cytoarchitectural and hodological partitioning of the NTS and that the distribution of an individual neurotransmitter receptor in the NTS may correspond to the role of that transmitter in modulating autonomic function.

Autoradiographic distribution of thyrotropin-releasing hormone receptors in the African lungfish Protopterus annectens.

We used quantitative autoradiography to examine the distribution of thyrotropin-releasing hormone (TRH) receptors in the central nervous system (CNS) of the African lungfish Protopterus annectens. We found that the distribution of TRH receptors throughout the CNS of the lungfish was heterogeneous with the highest concentrations (500-800 fmol/mg protein) in the olfactory bulb and telencephalon, moderately high concentrations (200-500 fmol/mg protein) in the diencephalon, and moderate (50-200 fmol/mg protein) to low (less than 50 fmol/mg protein) concentrations in the brainstem and spinal cord. Except for the motor nuclei of the cranial nerves and spinal cord, TRH receptors were concentrated in the acellular regions. In the telencephalon and diencephalon, the receptor density was inversely related to cellular density. These results provide a neuroanatomic and neuropharmacologic basis for further investigations of TRH in the African lungfish.

TRH and TRH receptors in the spinal cord.

Over the past 12 years, substantial progress has been made in delineating the localization of TRH and TRH receptors in spinal cord. High concentrations of both the peptide and its receptor have been observed in the ventral horn in the region of the motoneurons and in the dorsal horn in the substantia gelatinosa. As noted, pharmacological effects of TRH administration on various parameters of spinal cord function have been reported in a number of studies. To date, however, substantial questions remain regarding the physiological role of TRH in the spinal cord. Nevertheless, it is hoped that the extensive information that has been obtained on localization of TRH and TRH receptors in spinal cord will provide a basis for answering these complex questions.

Characterization of thyrotropin-releasing hormone in the central nervous system of African lungfish.

Central administration of thyrotropin-releasing hormone (TRH) produces potent effects on various physiological parameters, such as arousal, respiration, and cardiovascular function, in several species. As part of an investigation into the evolution of this tripeptide as a central modulator of these parameters, we examined its distribution in the central nervous system of the African lungfish (Protopterus). Lungfish brains were dissected into three regions: telencephalon, diencephalon, and medulla. Each region was assayed for TRH by radioimmunoassay and for norepinephrine, dopamine, and serotonin by HPLC/electrochemical methods. TRH immunoreactivity (IR-TRH) was present in all regions of lungfish brain examined. The telencephalon contained the highest concentrations of TRH, the diencephalon also contained a high concentration of TRH, and the medulla contained a markedly lower concentration. Similar concentration gradients (telencephalon greater than diencephalon greater than medulla) were observed for norepinephrine, dopamine, and serotonin. The identity of IR-TRH as authentic TRH was confirmed by elution profiles on HPLC. The results of this investigation demonstrated that TRH and the monoamine neurotransmitters are present in high concentrations in various regions of lungfish brain. The lungfish may represent a promising model for further studies of the interactions of TRH with these neurotransmitter systems.

Alterations in receptors for thyrotropin-releasing hormone, serotonin, and acetylcholine in amyotrophic lateral sclerosis.

We utilized quantitative autoradiography to examine thyrotropin-releasing hormone (TRH) receptors, serotonin type 1A (5-HT1A) receptors, muscarinic cholinergic receptors, choline uptake sites, beta-adrenergic receptors, and norepinephrine uptake sites in discrete laminae of spinal cord from patients with amyotrophic lateral sclerosis (ALS) and non-neurologic controls. We found decreases of over 50% in the concentration of TRH receptors in lamina IX of cervical, thoracic, and lumbar spinal cord from ALS patients. Similar reductions were noted in concentrations of muscarinic cholinergic receptors in lamina IX of spinal cords from ALS patients. Significant increases of up to 140% in 5-HT1A receptor densities were noted in lamina IX of spinal cords from ALS patients. No differences were noted between the concentrations of beta-adrenergic receptors or norepinephrine uptake sites in patients with ALS and controls. These findings suggest that TRH and 5-HT may be involved in the pathophysiology of ALS, and act in a comodulatory role in the normal spinal cord.

Localization and quantification of beta-adrenergic receptors in human brain.

Little information is currently available on the localization of noradrenergic systems in the human CNS. We used quantitative autoradiography with [125I] iodopindolol to examine beta-adrenergic receptors in postmortem human brain. The concentration of beta-receptors was highest in all subfields of the hippocampus, followed by cerebellum, and then thalamic nuclei, basal ganglia, midbrain, and cerebral cortex. Low levels were found in white matter and hypothalamus. This distribution differed from the distribution of beta-receptors reported in membrane homogenates of human brain and also from the distribution of beta-receptors in rat brain determined by autoradiography. The similarities and differences between the distribution of beta-receptors in the human and rat brains may have implications regarding the role of norepinephrine in the CNS of these two species.

Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland.

Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3-125I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci.

Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures.

Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides. Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin. This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2). Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH. In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate. A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells. A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not. Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate. The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of thyrotropin releasing hormone receptors in human brain.

We used quantitative autoradiography to localize thyrotropin releasing hormone (TRH) receptors in human brain. Highest concentrations of TRH receptors were localized within the cortical, basal, and lateral nuclei of the amygdala and the molecular layer of the hippocampus. Low levels were found in the cortex, diencephalon, and basal ganglia. The radioligand bound with similar affinity and pharmacology to pituitary gland as to brain. These data suggest that authentic TRH receptors in the hippocampus and amygdala may mediate the putative effects of TRH on the human brain.