Enkephalin-like Immunoreactive Neurons Research Articles

Reciprocal synaptic relationships between angiotensin II-containing neurons and enkephalinergic neurons in the rat area postrema.

A preembedding double immunostaining technique was used to study synaptic relationships between angiotensin-II-like immunoreactive and enkephalin-like immunoreactive neurons in the rat area postrema. The angiotensin-II-like immunoreactive neurons were detected by silver-gold intensification of the DAB reaction results while the enkephalin-like immunoreactive neurons were detected by simple ABC-DAB reaction. The synaptic relationships were reciprocal between the two neurons. Most of the synapses found between these two neurons were the presynaptic enkephalin-like immunoreactive axon terminals that made synapses on the angiotensin-II-like immunoreactive perikarya and dendrites. Both the axo-somatic and axo-dendritic synapses were symmetrical. However, although angiotensin-II-like immunoreactive axon terminals also made synapses on enkephalin-like perikarya and dendrites, the axo-somatic synapses were symmetrical, while the axo-dendritic synapses were asymmetrical. The present results confirm the presence of angiotensin-II-like immunoreactive neurons in the area postrema and suggest that these angiotensinergic neurons in the area postrema may play a role in the regulation of blood pressure via coordinated synaptic interactions with enkephalinergic neurons.

Post-embedding double-gold labeling immunoelectron microscopic co-localization of neurotransmitters in the rat brain.

In this report, we describe a new quantitative electron microscopic protocol based on the use of double colloidal-gold post-embedding immunostaining procedure as markers to analyze the subcellular distribution of enkephalin (ENK) and GABA neurotransmitters simultaneously in the same ultrathin tissue sections in the periaqueductal gray area (PAG) of the rat brain. Double-gold particle signals were significantly improved using a number of technical adjustments for the immunogold electron microscopic co-localization technique of ENK and GABA. The GABA-like neuronal elements were immuno-reacted with 20 nm gold particles and the enkephalin-like immunoreactive neurons were labeled with 10 nm gold particles. This double labeling was more apparent in tissue sections that were deactivated for the gold staining of the first antibody. Excellent double labeling was obtained when we blocked antigenicity of the first antiserum with hot (80 degrees C) paraformaldehyde fumes. To minimize the clumping of the second gold particles around the first gold particles used against the first antibody, we tried different staining order for the neurotransmitters tested in this study. It was necessary to use a detergent (Triton X-100) at very low concentration (0.1%) instead of etching to expose the antigenic determinants of the neurotransmitters and at the same time to reduce the deleterious effects on the morphology of the tissue sections. Furthermore, the high-glutaraldehyde fixation and the decrease in the interval between cutting and labeling of the ultrathin sections significantly improved the results obtained in this study. Double labeling of sections with ENK and GABA produced co-localization in 23.1% and 1.2% of the immunoreactive axonal terminals and dendrites, respectively. Most of the double-labeled terminals contained more GABA-like than ENK-like immunolabeling. Half of the axon terminals [51%] and dendrites [56%] in the ventrolateral PAG were not labeled with either of GABA or ENK immunoreactivity. This procedure was found to be completely compatible with good double immunolabeling and ultrastructural preservation.

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 imrnunoreactive 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 μm 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 μm 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.

Identification and localization of a [Met 5]-enkephalin-like peptide in the mollusc, Lymnaea stagnalis

The goal of this study was to determine whether [Met 5]-enkephalin, or an analog, is present in identified neurons in the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis. High performance liquid chromatography and radioimmunoassay of CNS tissue homogenates revealed both [Met 5]-enkephalin and oxidized [Met 5]-enkephalin. No [Leu 5]-enkephalin, [Met 5]-enkephalin-Arg 6-Phe 7 or [Met 5]-enkephalin-Arg 6-Gly 7-Leu 8 were detected. Quantification of [Met 5]-enkephalin, by radioimmunoassay, revealed that the Lymnaea CNS contains approximately 2.2 fmol/CNS (undigested tissue) and 4.5 fmol/CNS (tissue enzymatically digested with trypsin and carboxypeptidase B). The increased amount of [Met 5]-enkephalin following tissue digestion indicates the presence of as yet unidentified extended forms of [Met 5]-enkephalin in Lymnaea. Using indirect immunocytochemistry, a [Met 5]-enkephalin-like peptide was localized to individual cells and cell clusters within the CNS, as well as to fibers in the atrium of the heart. A neuronal map depicting [Met 5]-enkephalin-like immunoreactive cells was produced. Among the immunoreactive neurons were four identified, well-characterized, giant cells: VD1, RPD2, LB1 and RB1. Identifiable [Met 5]-enkephalin-like immunoreactive neurons were characterized electrophysiologically and morphologically. Additionally, neurons VD1 and RPD2 were confirmed to be immunoreactive to Lymnaea α-peptide. The lack of both cross reactivity and sequence homology between α-peptide and [Met 5]-enkephalin suggests that a [Met 5]-enkephalin-like peptide and α-peptide are co-localized within these neurons.

Enkephalin-like immunoreactive neurons in the central nervous system of gastropods (Helix pomatia, Lymnaea stagnalis, Aplysia californica): a comparative immunocytochemical study

The distribution and morphology of met-enkephalin-like immunoreactive (MetLI) and leu-enkephalin-like immunoreactive (LeuLI) neurons were investigated in the central nervous system of three gastropod species, Helix pomatia, Lymnaea stagnalis and Aplysia californica. Differences between the three species were observed in (1) the characteristics of immunostaining with antibodies to met-enkephalin and leu-enkephalin antibodies; (2) the number of immunostained neurons; (3) the projection areas of imunostained elements; (4) the specificity of immunostaining. Differences in the appearance of MetLI and LeuLI neurons were apparent: in Aplysia, both MetLI and LeuLI neurons could be observed, whereas in Lymnaea LeuLI was only found in fibers; only MetLI neurons occurred in Helix. According to an absorbtion control specificity test, a part of the LeuLI seen in the neuropil of Aplysia ganglia did not represent authentic leu-enkephalin. In Helix pomatia, significantly more MetLI neurons were present than in the CNS of Lymnaea and Aplysia; the majority of these neurons were concentrated in the cerebral ganglia and were small-size (12–25 μm) interneurons. In addition to central and peripheral projections observed in the three species, the connective tissue sheath around the ganglia and peripheral nerves contained MetLI varicose axons only in Helix, where a possible neurohormonal role could be attributed to these substance. The mapping and detailed chemical-neuroanatomical description of enkephalin-immunoreactive elements may furnish a chemical-neuroanatomical background to facilitate further neurophysiologic and pharmacologic analysis of enkephalinergic mechanisms in the gastropod CNS.

Immunohistochemical evidence for coexistence of methionine-enkephalin and tyrosine hydroxylase in neurons of the locus coeruleus complex projecting to the spinal cord of the cat

Previous studies have revealed the presence of pontospinal neurons with either methionine-enkephalin-or tyrosine hydroxylase-like immunoreactivity in the dorsolateral pontine tegmentum of the cat. Using a combined fast blue retrograde transport technique and simultaneous immunofluorescence histochemistry, the present study was designed to reveal the coexistence of enkephalin and tyrosine hydroxylase in cat coerulospinal neurons and to determine if and to what extent the coerulospinal pathway is heterogeneous. Fast blue-labelled neurons with tyrosine hydroxylase- and enkephalin-like immunoreactivities were found in the nucleus locus coeruleus, nucleus subcoeruleus, Kölliker-Fuse nucleus, and the medial and lateral parabrachial nuclei. Approximately 87% of tyrosine hydroxylase-like immunoreactive neurons had enkephalin-like immunoreactivity, whereas about 76% of the enkephalin-like immunoreactive neurons had tyrosine hydroxylase-like immunoreactivity. About 71% of all coerulospinal neurons exhibited both tyrosine hydroxylase- and enkephalin-like immunoreactivities. These findings indicate that coerulospinal activity may lead to spinal cord effects reflecting both norepinephrine and enkephalin activity in most cases but do not rule out each transmitter's isolated functions.

Enkephalin-like immunoreactive neurons appear in superficial layers of superior colliculus after enucleation

Enkephalin-like immunoreactive neurons appear in superficial layers of superior colliculus after enucleation

Chapter 1 Relation of enkephalin-like immunoreactive neurons to other neuropeptide and monoamine-containing neurons in the ventrolateral medulla

Publisher Summary This chapter presents a study in which a series of experiments was done to map the presence of neuronal perikarya containing leucine-enkephalin (Leu-ENK) and methionine (Met)-enkephalin (Met-ENK) in the cat ventrolateral medulla (VLM). ENK immunoreactive neurons were identified in animals after the administration of colchicine into the cisterna magna and in animals that did not receive colchicine. Colchicine is known to disrupt axonal transport and result in the accumulation of peptides in neuronal perikarya. There is now a considerable amount of experimental evidence suggesting that neurons in the VLM are involved in the maintenance and reflex regulation of systemic arterial pressure. Recently it has been shown in the rat that the microinjections of an enkephalin analogue into the rostra1 VLM elicit decreases in arterial pressure, whereas microinjections into the caudal VLM elicit increases in arterial pressure. These data suggested that an enkephalinergic system in the VLM might be involved in controlling the circulation. In support of this suggestion, the rat VLM has been shown to contain neurons that are immunoreactive to Met-ENK and β -endorphin.

Enkephalin-like immunoreactive neurons and fibers in the ventromedial hypothalamic nucleus

Enkephalin-like immunoreacitve (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10–25 μm in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (60–230 nm with a predominance in the 60–80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from unlabeled axonal boutons containing many small, clear vesicles and occasional LCV. The ultrastructural features of the ENK-IR cells in the VMH seemed to correspond to the “common cells” described by Milhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synaptic contacts with the cell bodies and dendrites of unlabeled neurons. The findings suggest that opioid peptides directly influence VMH neurons through synaptic contacts.

Ultrastructural evidence of enkephalinergic input to glucoreceptor neurons in ventromedial hypothalamic nucleus

A combination of intracellular injection of horseradish peroxidase into the ventromedial hypothalamic nucleus glucoreceptor neurons and immunohistochemical staining for enkephalin allowed ultrastructural visualization of interrelations between these electrophysiologically identified neurons and enkephalin-like immunoreactive (ENK-IR) terminals. The results show that glucoreceptor neurons receive direct inputs from ENK-IR neurons. This is the first evidence that glucoreceptor neurons, identified electrophysiologically, can be affected monosynaptically by endogenous enkephalin, probably released from ENK-IR terminals.

An enkephalinergic projection from the hypothalamic paraventricular nucleus to the hypothalamic ventromedial nucleus of the rat: An experimental immunohistochemical study

The distribution and origins of enkephalin-like immunoreactive (ENK-IR) fibers in the nucleus ventromedialis hypothalami (vm) of the rat were examined using immunohistochemistry. A dense plexus was evenly distributed in the vm with no regional differences. A group of ENK-IR neurons was concentrated in the ventrolateral portion of the vm. The destruction of the hypothalamic paraventricular nucleus (pv) which contained numerous ENK-IR neurons, resulted in a marked decrease in ENK-IR fibers in the vm on the operated side. In addition, the destruction of the magnocellular portion of the pv, while leaving most of parvocellular portion intact, failed to substantially decrease the ENK-IR fibers in the vm, suggesting that these fibers originate from ENK-IR neurons located in the parvocellular portion of the pv. The present study further showed that the axons of these neurons first proceeded laterally to the perifornical area, next ran ventrolaterally to the ventrolateral portion of the anterior hypothalamic nucleus and finally turned medially to the vm.

Survival and growth of neurons with enkephalin-like immunoreactivity in fetal brain areas grafted to the anterior chamber of the eye

Areas of fetal rat brain and spinal cord known to contain enkephalin-like immunoreactive cell bodies and/or terminal fields were transplanted to the anterior chamber of the eye of adult rats. Enkephalin-like immunoreactive neurons survive and produce an enkephalin-like immunoreactive fiber network within grafts of spinal cord, ventral medulla oblongata, ventrolateral pons, tectum, locus coeruleus, substantia nigra and the areas containing columna fornicis and globus pallidus. Although single intraocular grafts of neocortex do not apparently contain enkephalin-like immunoreactive fibers, such grafts contain a variable amount of sparsely distributed enkephalin-like fibers when sequentially grafted in oculo with either locus coeruleus or spinal cord. Combinations of locus coeruleus and globus pallidus contained a rich enkephalin fiber network in the locus coeruleus part and a sparse innervation of the globus pallidus part. We conclude that enkephalin-like immunoreactive neurons in small areas of fetal rat brain can be successfully transplanted to the anterior chamber of the eye. They are able to survive and develop to maturity in complete isolation from the rest of the brain. In general, the enkephalin-like immunoreactive fiber density in the various single grafts approximated that of their brain counterparts in situ. Fiber formation can be reinitiated in mature enkephalin-like immunoreactive neurons by addition of new brain target areas. Thus, the technique permits establishment of isolated, defined enkephalin systems and pathways accessible to functional analysis.