Targeting the annexin 1-formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS-induced pathologic changes in the murine adrenal cortex.

Hypothalamo-pituitary-adrenocortical dysfunction contributes to morbidity and mortality in a high proportion of patients with sepsis. Here, we provide new insights into the underlying adrenal pathology. Using a murine model of endotoxemia (LPS injection), we demonstrate that adrenal insufficiency is triggered early in the disease. LPS induced a local inflammatory response in the adrenal gland within 4 hours of administration, coupled with increased expression of mRNAs for annexin A1 (AnxA1) and the formyl peptide receptors [(Fprs) 1, 2, and 3], a loss of lipid droplets in cortical cells (index of availability of cholesterol, the substrate for steroidogenesis), and a failure to mount a steroidogenic response to ACTH. Deletion of AnxA1 or Fpr2/3 in mice prevented lipid droplet loss, but not leukocyte infiltration. LPS increased adrenal myeloid differentiation primary response gene 88 and TLR2 mRNA expression, but not lymphocyte antigen 96 or TLR4. By contrast, neutrophil depletion prevented leukocyte infiltration and increased AnxA1, Fpr1, and Fpr3 mRNAs but had no impact on lipid droplet loss. Our novel data demonstrate that AnxA1 and Fpr2 have a critical role in the manifestation of adrenal insufficiency in this model, through regulation of cholesterol ester storage, suggesting that pharmacologic interventions targeting the AnxA1/FPR/ALX pathway may provide a new approach for the maintenance of adrenal steroidogenesis in sepsis.

2-Chloroacetamidine, a novel immunomodulator, suppresses antigen-induced mouse airway inflammation.

BACKGROUND: Citrullination is a presently under-recognized posttranslational protein modification catalyzed by PAD enzymes. Immune responses to citrullinated neo-epitopes are identified in a growing number of inflammatory and autoimmune diseases. However, the involvement of hypercitrullination in the pathogenesis of bronchial asthma is still unknown. METHODS: As main experimental tool, we examined the effect of 2-chloroacetamidine (2CA), a PAD enzyme inhibitor, on OVA-immunized and airway-challenged BALB/c mice; a commonly used model of allergic airway inflammation. We also measured the effect of 2CA on ex vivo lymphocytes and cell lines. RESULTS: In vivo, 2CA dramatically suppressed lung tissue hypercitrullination, inflammatory cell recruitment, and airway-Th2 cytokine secretion. 2CA also suppressed systemic OVA-specific and total IgE production dramatically, effectively preventing de novo and diminishing established disease without measurably impacting general immunocompetence. In vitro, 2CA markedly inhibited the proliferation of mouse and human T cells with cell cycle block and apoptosis during a limited, postactivation phase. CONCLUSIONS: 2CA acts as narrow-spectrum immunosuppressant that selectively targets lymphocyte populations involved in active inflammatory tissue lesions. If hypercitrullination is generated in patients with asthma, 2CA may represent a novel disease modulator for human asthmatics/allergic diseases.

Leukocyte recruitment in the brain in sepsis: involvement of the annexin 1-FPR2/ALX anti-inflammatory system.

Unregulated inflammation underlies many diseases, including sepsis. Much interest lies in targeting anti-inflammatory mechanisms to develop new treatments. One such target is the anti-inflammatory protein annexin A1 (AnxA1) and its receptor, FPR2/ALX. Using intravital videomicroscopy, we investigated the role of AnxA1 and FPR2/ALX in a murine model of endotoxin-induced cerebral inflammation [intraperitoneal injection of lipopolysaccharide (LPS)]. An inflammatory response was confirmed by elevations in proinflammatory serum cytokines, increased cerebrovascular permeability, elevation in brain myeloperoxidase, and increased leukocyte rolling and adhesion in cerebral venules of wild-type (WT) mice, which were further exacerbated in AnxA1-null mice. mRNA expression of TLR2, TLR4, MyD-88, and Ly96 was also assessed. The AnxA1-mimetic peptide, AnxA1(Ac2-26) (100 µg/mouse, ∼33 µmol) mitigated LPS-induced leukocyte adhesion in WT and AnxA1-null animals without affecting leukocyte rolling, in comparison to saline control. AnxA1(Ac2-26) effects were attenuated by Boc2 (pan-FPR antagonist, 10 µg/mouse, ∼12 nmol), and by minocycline (2.25 mg/mouse, ∼6.3 nmol). The nonselective Fpr agonists, fMLP (6 µg/mouse, ∼17 nmol) and AnxA1(Ac2-26), and the Fpr2-selective agonist ATLa (5 µg/mouse, ∼11 nmol) were without effect in Fpr2/3(-/-) mice. In summary, our novel results demonstrate that the AnxA1/FPR2 system has an important role in effecting the resolution of cerebral inflammation in sepsis and may, therefore, provide a novel therapeutic target.

Connectomic Basis for Tremor Control in Stereotactic Radiosurgical Thalamotomy.

BACKGROUND AND PURPOSE: Given the increased use of stereotactic radiosurgical thalamotomy and other ablative therapies for tremor, new biomarkers are needed to improve outcomes. Using resting-state fMRI and MR tractography, we hypothesized that a "connectome fingerprint" can predict tremor outcomes and potentially serve as a targeting biomarker for stereotactic radiosurgical thalamotomy. MATERIALS AND METHODS: We evaluated 27 patients who underwent unilateral stereotactic radiosurgical thalamotomy for essential tremor or tremor-predominant Parkinson disease. Percentage postoperative improvement in the contralateral limb Fahn-Tolosa-Marin Clinical Tremor Rating Scale (TRS) was the primary end point. Connectome-style resting-state fMRI and MR tractography were performed before stereotactic radiosurgery. Using the final lesion volume as a seed, "connectivity fingerprints" representing ideal connectivity maps were generated as whole-brain R-maps using a voxelwise nonparametric Spearman correlation. A leave-one-out cross-validation was performed using the generated R-maps. RESULTS: The mean improvement in the contralateral tremor score was 55.1% (SD, 38.9%) at a mean follow-up of 10.0 (SD, 5.0) months. Structural connectivity correlated with contralateral TRS improvement (r = 0.52; P = .006) and explained 27.0% of the variance in outcome. Functional connectivity correlated with contralateral TRS improvement (r = 0.50; P = .008) and explained 25.0% of the variance in outcome. Nodes most correlated with tremor improvement corresponded to areas of known network dysfunction in tremor, including the cerebello-thalamo-cortical pathway and the primary and extrastriate visual cortices. CONCLUSIONS: Stereotactic radiosurgical targets with a distinct connectivity profile predict improvement in tremor after treatment. Such connectomic fingerprints show promise for developing patient-specific biomarkers to guide therapy with stereotactic radiosurgical thalamotomy.

MEDI6012: Recombinant Human Lecithin Cholesterol Acyltransferase, High-Density Lipoprotein, and Low-Density Lipoprotein Receptor-Mediated Reverse Cholesterol Transport.

Background MEDI6012 is recombinant human lecithin cholesterol acyltransferase, the rate-limiting enzyme in reverse cholesterol transport. Infusions of lecithin cholesterol acyltransferase have the potential to enhance reverse cholesterol transport and benefit patients with coronary heart disease. The purpose of this study was to test the safety, pharmacokinetic, and pharmacodynamic profile of MEDI6012. Methods and Results This phase 2a double-blind study randomized 48 subjects with stable coronary heart disease on a statin to a single dose of MEDI6012 or placebo (6:2) (NCT02601560) with ascending doses administered intravenously (24, 80, 240, and 800 mg) and subcutaneously (80 and 600 mg). MEDI6012 demonstrated rates of treatment-emergent adverse events that were similar to those of placebo. Dose-dependent increases in high-density lipoprotein cholesterol were observed with area under the concentration-time curves from 0 to 96 hours of 728, 1640, 3035, and 5318 should be: mg·h/mL in the intravenous dose groups and 422 and 2845 mg·h/mL in the subcutaneous dose groups. Peak mean high-density lipoprotein cholesterol percent change was 31.4%, 71.4%, 125%, and 177.8% in the intravenous dose groups and 18.3% and 111.2% in the subcutaneous dose groups, and was accompanied by increases in endogenous apoA1 (apolipoprotein A1) and non-ATP-binding cassette transporter A1 cholesterol efflux capacity. Decreases in apoB (apolipoprotein B) were observed across all dose levels and decreases in atherogenic small low-density lipoprotein particles by 41%, 88%, and 79% at the 80-, 240-, and 800-mg IV doses, respectively. Conclusions MEDI6012 demonstrated an acceptable safety profile and increased high-density lipoprotein cholesterol, endogenous apoA1, and non-ATP-binding cassette transporter A1 cholesterol efflux capacity while reducing the number of atherogenic low-density lipoprotein particles. These findings are supportive of enhanced reverse cholesterol transport and a functional high-density lipoprotein phenotype. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02601560.

Annexin A1 and the formyl peptide receptor family: neuroendocrine and metabolic aspects.

Annexin A1 (ANXA1, formerly termed lipocortin 1 or macrocortin) is an important protein mediator of the feedback actions of glucocorticoids within the hypothalamo-pituitary-adrenocortical (HPA) axis. Here we consider the mechanisms by which ANXA1 exerts these actions, with particular reference to the potential role of the formyl peptide receptors (FPRs), a family of G-protein-coupled receptors which has only very recently been implicated in the regulation of neuroendocrine function. In addition, we discuss evidence that ANXA1 contributes to the regulation of other aspects of endocrine and metabolic function and to the aetiology of sexual dimorphisms.

Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells.

OBJECTIVES: A gene-environment interaction between HLA-DR shared epitope genes and smoking in anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis (RA) has been reported. Identification of citrullinated proteins in bronchoalveolar lavage (BAL) cells from smokers has led to the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA. OBJECTIVE: To confirm and extend these findings. METHODS: Immunohistochemistry was performed on BAL cells and bronchial mucosal biopsy sections obtained through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. Two antibodies recognising citrullinated proteins, two antibodies recognising peptidylarginine deiminase (PAD)2 enzyme and one recognising PAD4 enzyme were used. RESULTS: Citrullinated proteins are upregulated in BAL cells of healthy smokers compared with healthy non-smokers. This was associated with higher expression of the PAD2 enzyme. The same level of citrullinated proteins was present in bronchial mucosal biopsy specimens of healthy smokers and non-smokers, despite higher expression of PAD2 in smokers. CONCLUSION: This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

The distribution and significance of CNS adrenoceptors examined with in situ hybridization.

Several of the established alpha 1-, alpha 2- and beta-adrenoceptors have now been isolated and cloned. The in situ hybridization method has been used to map the distribution of many of these adrenoceptors within cells of the CNS. These studies add complementary and new information to our knowledge of adrenoceptor localization provided previously by radioligand-mediated autoradiography. Neuronal cell groups containing one or more mRNAs for seven adrenoceptor subtypes throughout the rat CNS have been mapped. In the present review Anthony Nicholas, Tomas Hökfelt and Vincent Pieribone will examine these localizations and discuss the additional information these maps supply, as well as some implications for understanding central noradrenaline and adrenaline systems.

Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study.

Selective 35S-labeled oligonucleotide probes were designed to sequences of the rat alpha-2A (RG20), alpha-2B (RNG), and alpha-2C (RG10) adrenoreceptor mRNAs for use in in situ hybridization experiments on sections of unfixed rat brain, spinal cord and kidney. After hybridized sections were exposed to film or dipped in autoradiographic emulsion, specific and selective labeling patterns characteristic for each probe and region of the central nervous system were observed. Alpha-2A mRNA labeling was most pronounced in neurons in layer six of the cerebral cortex, hypothalamic paraventricular nucleus, reticular thalamic nucleus, pontine nuclei, locus coeruleus, vestibular nuclei, trapezoid nuclei, deep cerebellar nuclei, nucleus tractus solitarii, ventrolateral medullary reticular formation, and the intermediolateral cell column of the thoracic spinal cord. In some of these locations, the receptor mRNA, in all probability, is present in noradrenaline and perhaps adrenaline neurons. The alpha-2B probe, which primarily labels the kidney, gave only a very light signal in the thalamus in the central nervous system after extended exposure times. Alpha-2C mRNA labeling was primarily observed in the olfactory bulb, cerebral cortex, islands of Calleja, striatum, hippocampal formation, cerebellar cortex, and dorsal root ganglia. Labeling patterns disappeared when excess unlabeled probes were added to their respective radiolabeled probes, or when sense probes were employed. When a hybrid antisense probe homologous to all three alpha-2 probes was used, labeling patterns also disappeared. The present study therefore justifies the pharmacological subclassification of alpha-2 receptors by providing anatomical evidence for specific and selective cell groups in the rat central nervous system containing mRNA for three alpha-2 receptor subtypes.

Ultrastructural studies on peptides in the dorsal horn of the spinal cord--I. Co-existence of galanin with other peptides in primary afferents in normal rats.

The aim of the present study was to investigate galanin-like immunoreactivity in primary afferent terminals and its relationship to other neuropeptides in laminae I and II of the fourth and fifth lumbar segments of normal rat spinal cord using immunofluorescence and pre- and post-embedding electron-microscopic immunocytochemistry. Triple-immunofluorescence staining showed that galanin-like immunoreactivity co-localized with substance P- and calcitonin gene-related peptide-like immunoreactivities in many nerve fibres and terminals in laminae I and II of the dorsal horn. At the ultrastructural level, using pre-embedding immunocytochemistry, galanin-like immunoreactivity was found in type I glomeruli with an electron-dense central terminal containing many densely packed synaptic vesicles and several large dense-core vesicles. Both the cytoplasm and the core of the large vesicles were immunoreactive. In type II glomeruli with an electron-lucent central terminal and loosely packed synaptic vesicles the large dense-core vesicles and the cytoplasm were only weakly galanin-positive. Post-embedding immunocytochemistry revealed that galanin-like immunoreactivity co-existed with substance P- and calcitonin gene-related peptide-like immunoreactivities in many terminals and in individual large dense-core vesicles in lamina II. These terminals were considered to represent primary afferents, since there is evidence that calcitonin gene-related peptide in the dorsal horn only occurs in nerve endings originating in dorsal root ganglia. Evidence was also unexpectedly obtained for the occurrence of several other peptides in calcitonin gene-related peptide-positive terminals, i.e. in presumably primary afferents. Thus galanin-like immunoreactivity sometimes also co-localized with cholecystokinin- and neuropeptide tyrosine-like immunoreactivities in calcitonin gene-related peptide-immunoreactive terminals and in some large dense-core vesicles in such terminals. A small number of calcitonin gene-related peptide immunoreactive, presumably primary afferent terminals contained enkephalin-, neurotensin- (and galanin-)like immunoreactivities. These results indicated that galanin can be co-stored with several other neuropeptides in large dense-core vesicles in primary afferent terminals and may presumably be released together with them in the superficial layer of the dorsal horn. Since various combinations of peptides, presumably at varying concentrations, occur in the large dense-core vesicles in a given nerve ending, it is likely that the individual large dense-core vesicles produced in a neuron are heterogenous with regard to peptide content and thus to the message that they transmit upon release.

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord--II. Co-existence of galanin with other peptides in local neurons.

Using light microscopic immunoperoxidase and immunofluorescence histochemistry, double-staining methodology, and electron microscopic pre-embedding and post-embedding immunocytochemistry, we studied galanin-immunoreactive neurons in the superficial dorsal horn of the rat spinal cord. Co-existence of galanin with other neuropeptides was also analysed. The lumbar 4 and 5 segments of normal rats and after rhizotomy or spinal cord transection were studied. Galanin-positive local neurons in lamina II were often islet cells and could be classified as type A, which had abundant electron-dense cytoplasm containing many large dense-core vesicles, and type B, which had electron-lucent cytoplasm with only a few large dense-core vesicles. Galanin-positive and -negative peripheral afferent terminals made synaptic contact mostly with galanin-negative dendrites and cell bodies, but also with type B galanin cell bodies and with galanin-positive dendrites of unidentified type. Galanin-immunoreactive terminals from local neurons could also be classified into two types. Type alpha terminals were most common; they contained densely packed synaptic vesicles and many large dense-core vesicles, were strongly immunostained and most frequently made synaptic contact with galanin-negative dendrites. Type beta terminals contained loosely packed synaptic vesicles and a few large dense-core vesicles, and were weakly immunostained. Axosomatic synaptic contact were sometimes found between type beta terminals and type B galanin-positive cell bodies, but were most often associated with galanin-negative dendrites. Double immunostaining showed that galanin-like immunoreactivity co-localized mainly with enkephalin-like, but sometimes also with neuropeptide Y-like immunoreactivity in some local neurons in lamina II. Galanin-like and substance P-like immunoreactivities were identified in the same neurons in deeper layers of the dorsal horn. Coexistence of these neuropeptides and neurotensin with galanin was demonstrated not only in terminals in lamina II but also in large dense-core vesicles, as revealed by post-embedding immunocytochemistry. These results show that galanin-immunoreactive neurons in lamina II receive inputs directly from primary afferents and frequently make synaptic contacts with other intrinsic neurons. Galanin in the superficial dorsal horn may be released both from primary afferents and local neurons to modulate sensory processing in many different ways, including interacting with enkephalin, neuropeptide Y, neurotensin and substance P released from the same and/or other local neurons.

Cellular localization of messenger RNA for beta-1 and beta-2 adrenergic receptors in rat brain: an in situ hybridization study.

Selective, 35S-labeled, oligonucleotide probes were designed from sequences of the rat beta-1 and beta-2 adrenoceptor messenger RNAs for use in situ hybridization experiments on sections of unfixed rat brain and spinal cord. After hybridized sections were exposed to film or dipped in autoradiographic emulsion, specific and selective labeling patterns characteristic for each receptor messenger RNA and region of the central nervous system were observed. For example, labeling for beta-1 messenger RNA was found in the anterior olfactory nucleus, cerebral cortex, lateral intermediate septal nucleus, reticular thalamic nucleus, oculomotor complex, vestibular nuclei, deep cerebellar nuclei, trapezoid nucleus, abducens nucleus, ventrolateral pontine and medullary reticular formations, the intermediate gray matter of the spinal cord and in the pineal gland, while beta-2 messenger RNA labeling was strongest in the olfactory bulb, piriform cortex, hippocampal formation, thalamic intralaminar nuclei and cerebellar cortex. In some of these regions the beta-1 labeling seemed mainly confined to the cell nucleus. Whether or not this apparently nuclear labeling is specific, i.e. indicates synthesis of beta-1 receptor, remains to be established. However, all labeling patterns described disappeared when excess unlabeled probes were added to their respective radiolabeled probes or when sense probes were employed. Since the in situ method labels only cell bodies that produce the messenger RNA for these two beta receptor subtypes, a comparison between these maps and those of past autoradiographic studies mapping the location of central beta receptors using drugs as radioligands may produce further insights regarding the pre- and postsynaptic localization of these receptors in the various parts of the central nervous system circuitry.

Serotonin-, substance P- and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate.

Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaca fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple messengers in descending serotonin neurons: localization and functional implications.

In the present review article we summarize mainly histochemical work dealing with descending bulbospinal serotonin neurons which also express a number of neuropeptides, in particular substance P and thyrotropin releasing hormone. Such neurons have been observed both in rat, cat and monkey, and may preferentially innervate the ventral horns of the spinal cord, whereas the serotonin projections to the dorsal horn seem to lack these coexisting peptides. More recent studies indicate that a small population of medullary raphe serotonin neurons, especially at rostral levels, also synthesize the inhibitory neurotransmitter gamma-amino butyric acid (GABA). Many serotonin neurons contain the glutamate synthesizing enzyme glutaminase and can be labelled with antibodies raised against glutamate, suggesting that one and the same neuron may release several signalling substances, causing a wide spectrum of post- (and pre-) synaptic actions.

Glutamate-like immunoreactivity in medulla oblongata catecholamine/substance P neurons.

Immunofluorescence histochemistry was performed on sections of the rat medulla oblongata with a well characterized antibody to the amino acid glutamate (GLU) combined with antisera to catecholamine synthesizing enzymes and substance P. GLU-like immunoreactive (LIR) neurons were seen in many areas of the medulla, and were particularly intensely stained in the rostral ventrolateral medulla. In this area, nearly all adrenaline neurons were GLU-LIR. This immunoreactivity was also seen in catecholamine neurons of the C2, C3, A1 and A2 cell groups. Many adrenaline neurons, especially of the C1 group, contained substance P-LI in addition to GLU-like immunoreactivity (LI).

Increased alpha 1B-adrenoreceptor mRNA levels in the rat kidney after thyroidectomy.

Oligonucleotide probes were designed to sequences of the rat alpha 1B- and alpha 2B-adrenergic receptor mRNA and used for in situ hybridization histochemistry on tissue sections of kidneys from control and thyroidectomized rats. Both alpha 1B- and alpha 2B-receptor mRNA labelling was demonstrated in proximal tubule cells in the outer stripe of the outer medulla, with tubular rays radiating into the cortex. Thyroidectomy induced a more than 4-fold increase in mRNA for the alpha 1B-receptor in the kidney, whereas no change in alpha 2B-receptor mRNA levels could be demonstrated in thyroidectomized rats as compared to control animals. The results suggest that thyroid hormone plays an important role in regulating expression of alpha 1B-receptors in renal tubule cells.

Evidence for projections from the rostral medullary raphe onto medullary catecholamine neurons in the rat.

Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral medullary raphe, which included portions of the caudal nucleus raphe magnus, rostral nucleus raphe pallidus, rostral nucleus raphe obscurus and rostral nucleus reticularis paragigantocellularis, two-color immunoperoxidase staining was employed to demonstrate contiguity between PHA-L-immunoreactive (PHA-LI) varicose fibers and boutons and medullary catecholamine (CA) cells. Raphe projections were contiguous with phenylethanolamine N-methyltransferase-immunoreactive (PNMTI) neurons in the C1, C2 and C3 cell groups and with tyrosine hydroxylase-immunoreactive (THI) neurons in the A1 and A2 cell groups. Contiguity between PHA-LI processes and medullary CA cells was observed most frequently in the C1 cell group. Preliminary findings of this study have been presented previously.

Projections from the rostral ventrolateral medulla to brainstem monoamine neurons in the rat.

Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral ventrolateral medulla (RVL), two-color immunoperoxidase staining was employed to demonstrate contiguity between PHA-L-immunoreactive (PHA-LI) varicose fibers and boutons and brainstem monoaminergic cells. Black-stained PHA-LI cells in the deposition site were found to be located among amber-stained phenylethanolamine N-methyl transferase-immunoreactive (PNMT-I) neurons of the C1 cell group. RVL projections were contiguous with PNMT-I neurons of the C1, C2 and C3 cell groups, with tyrosine hydroxylase-immunoreactive (TH-I) neurons of the A1, A2 and A5 cell groups, and with serotonin-immunoreactive (5-HT-I) neurons of the B1, B2 and B3 cell groups. Preliminary findings of this study have been presented previously (Soc. Neurosci. Abstr., 15 (1989) 451).

An immunohistochemical investigation of the opioid cell column in lamina X of the male rat lumbosacral spinal cord.

Tri-color immunohistochemistry was employed to examine enkephalin-like immunoreactive neurons in lamina X of the rat lumbosacral spinal cord. Serial coronal sections from levels L1 to S3 were examined. A rostral group of large (40-50 microm diameter), pyramidal-shaped enkephalin-like immunoreactive neurons were shown from levels L1 to L4-5. Essentially all of these neurons were also immunoreactive for galanin and cholecystokinin. A second enkephalin-like immunoreactive cell group, extending from L5 to approximately the S2-3 level, contained smaller (20-30 microm diameter), ovoid-shaped perikaryia. Approximately 75% of these enkephalin-like immunoreactive neurons were also immunoreactive for neuropeptide Y. Neurotensin-immunoreactivity was also present in this area, having varying amounts of co-localization with these other two peptides. These results demonstrate that the lumbosacral opioid cell column in lamina X is not a neurochemically homogenous structure.

Oxytocin-immunoreactive projections onto medullary adrenaline neurons.

With the use of two-color immunoperoxidase staining, oxytocin-like immunoreactive (OLI) processes have been observed along the surface of phenylethanolamine N-methyl transferase-like immunoreactive (PNMTLI) cells in the ventrolateral and dorsomedial medulla of the rat. OLI boutons were also observed on PNMTLI cells retrogradely labeled with HRP injected into the rostral thoracic spinal cord. The close spatial relationships between OLI processes and PNMTLI cells argues for a functional connection which could provide for direct hypothalamic control of medullary adrenaline cells involved in cardiovascular regulation.