Enkephalin systems in diencephalon and brainstem of the rat

The immunocytochemical distribution of [Leu]enkephalin and an adrenal enkephalin precursor fragment (BAM-22P) immunoreactivity was investigated in the diencephalon and brainstem of rats pretreated with relatively high doses of colchicine (300–400 Μg/10 Μl intracerebroventricularly). The higher ranges of colchicine pretreatment allowed the visualization of extensive enkephalin-containing systems in these brain regions, some of which are reported for the first time. Immunoreactive perikarya were found in many hypothalamic and thalamic nuclei, interpeduncular nucleus, substan-tia nigra, the colliculi, periaqueductal gray, parabrachial nuclei, trigeminal motor and spinal nuclei, nucleus raphe magnus and other raphe nuclei, nucleus reticularis paragigantocellularis, vestibular nuclei, several nor-adrenergic cell groups, nucleus tractus solitarius, as well as in the spinal cord dorsal horn. In addition to the above regions, immunoreactive fibers were also noted in the habenular nuclei, trigeminal sensory nuclei, locus coeruleus, motor facial nucleus, cochlear nuclei, dorsal motor nucleus of the vagus, and hypoglossal nucleus. When adjacent sections to those stained for [Leu]enkephalin were processed for BAM-22P immunoreactivity, it was found that these two immunoreactivities were distributed identically at almost all anatomical locations. B AM-22P immunoreactivity was generally less pronounced and was preferentially localized to neuronal perikarya. The results of the present as well as the preceding studies (Khachaturian et ai., '83) strongly suggest substantial structural similarity between the adrenal proenkephalin precursor and that which occurs in the brain. Also discussed are some differences and parallels between the distribution of [Leu]enkeph-alin and dynorphin immunoreactivities.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50020/1/902200305_ftp.pd

Some perspectives on monoamine-opioid peptide interaction in rat central nervous system

Light microscopic immunocytochemistry was employed to investigate possible sites of interaction between the endogenous opioid peptides and monoamines in the rat central nervous system. The opioid and related peptides examined included beta-endorphin ([beta]-END), alpha-MSH ([alpha]-MSH) and leucine-enkephalin (Leu-ENK). The monoamines were examined using antisera generated against tyrosine hydroxylase, dopamine-[beta]-hydroxylase as well as serotonin. Due to the long-tract nature of the central monoamine projections as well as [beta]-END/[alpha]-MSH fiber systems, serial section analyses were performed utilizing parasagittal brain sections. Many areas rich in both the monoamines as well as opioid peptides were investigated. These included several thalamic and hypothalamic nuclei, several limbic structures, mesencephalic periaqueductal gray, brain stem noradrenergic cell groups and their rostral projections, the dopaminergic nigrostriatal system, and the serotonergic raphe nuclei and their projections. The results suggest a more intimate linkage between the monoamines and the opioid peptides than previously realized. Some of the intricacies of monoamine-opioid peptide interaction, in particular those pertaining to their possible role in pain and analgesia, catalepsy, and neuroendocrine effects are also discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23945/1/0000192.pd

Time of origin of opioid peptide-containing neurons in the rat hypothalamus

By using a combined technique of immunocytochemistry and [ 3 H]thymidine autoradiography, we have determined the “birth date” of opioid pep-tide-containing neurons in several hypothalamic nuclei and regions. These include proopiomelanocortin (POMC) neurons (represented by ACTH immunoreactivity) in the arcuate nucleus; dynorphin A neurons in the supraoptic and paraventricular nuclei and the lateral hypothalamic area; and leuenkephalin neurons in the periventricular, ventromedial, and medial mammillary nuclei, as well as in preoptic and perifornical areas. Arcuate POMC neurons were born very early in embryonic development, with peak heavy [ 3 H]thymidine nuclear labelling occurring on embryonic day E12. Supraoptic and paraventricular dynorphin A neurons were also labelled relatively early (peak at E13). The lateral hypothalamic dynorphin A neurons showed peak heavy labelling also on day E12, By contrast, leu-enkephalin neurons in the periventricular nucleus and medial preoptic area exhibited peak heavy nuclear labelling on day E14. Furthermore, perifornical and ventromedial leu-enkephalin neurons were also born relatively early (peak on days E12 and E13, respectively). However, the leu-enkephalin neurons in the medial mammillary nucleus were born the latest of all cell groups studied (i.e., peak at E15). The results indicate a differential genesis of these opioid peptide-containing neuronal groups in different hypothalamic nuclei and regions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50027/1/902360409_ftp.pd

MSG effects on beta-endorphin and alpha-MSH in the hypothalamus and caudal medulla

Monosodium glutamate (MSG) was given to neonatal male rats to determine its effects on neurons containing beta-endorphin ([beta]-END) and alpha-melanocyte stimulating hormone ([alpha]-MSH) within the basal hypothalamus (arcuate nucleus) and caudal medulla [nucleus tractus solitarius (NTS)] and on the levels of [beta]-END and [alpha]-MSH within these areas. Immunocytochemical studies demonstrated a reduction in the number of cells within the medial hypothalamic area (arcuate nucleus) amon MSG-treated animals versus saline controls. MSG did not reduce the number of cell bodies within the caudal medulla (NTS). MSG significantly reduced [beta]-END and [alpha]-MSH immunoreactive levels in the basal hypothalamus as determined by radioimmunoassay. Whereas a significant reduction in the level of [beta]-END occurred in the ventral caudal medulla (VCM), none occurred in the dorsal caudal medulla (DCM). In contrast, levels of [alpha]-MSH increased significantly in the DCM among animals receiving MSG compared to control animals. This study documents the contribution of beta-endorphin containing neurons of the basal hypothalamus to areas of the caudal medulla. The effect of MSG on beta-endorphin and [alpha]-MSH neurons in these areas and their differential effects on levels in the caudal medulla areas raises questions about the sites of origin of these peptides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27254/1/0000263.pd

Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain

Using adjacent section autoradiography-immunocytochemistry, the distribution of [3H]naloxone binding sites was studied in relation to neuronal systems containing [Leu]enkephalin, dynorphin A, or [beta]-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of [3H]naloxone, the pharmacological ([mu]-like) properties of which appear unaltered. In contrast, specific binding of the [delta] ligand [3H]D-Ala2,D-Leu5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, we have noted associations between [3H]naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex; for example, in caudate-putamen, patches of opioid receptors did not correspond to the distribution of enkephalin immunoreactivity, but there was a correspondence between subcallosal streaks of binding sites and enkephalin. The complexity of the association between [3H]naloxone binding sites and the multiple opioid systems, and previous reports of colocalization of [mu] and [kappa] receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post-translational processing in determining the physiological consequences of opioid neurotransmission.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25804/1/0000367.pd

Visualization of opiate receptors and opioid peptides in sequential brain sections

Autoradiographic and immunocytochemical studies were carried out on adjacent sections from formaldehyde-perfused rat brains in order to directly correlate the distribution of opiate receptors and opioid peptides. Perfusion fixation of the brains resulted in a partial loss of specific [3H]naloxone binding with essentially no change in the pharmacological properties of the remaining sites. When the distribution of sites was compared to that of enkaphalin immunoreactivity in adjacent sections, striking correlations were observed in a number of areas throughout the neuraxis. Adjacent section autoradiography-immunocytochemistry should provide a useful tool for relating the anatomical distribution of opiate receptor subtypes to different opioid peptide neuronal systems.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23888/1/0000127.pd

Immunocytochemical localization of pro-opiomelanocortin-derived peptides in the adult rat spinal cord

A dispersed descending pro-opiomelanocortin (POMC) fiber system has been demonstrated by peroxidase-antiperoxidase (PAP) immunocytochemistry in the adult rat spinal cord. [beta]-endorphin, adrenocorticotrophic hormone (ACTH), [alpha]-melanocyte-stimulating hormone ([alpha]-MSH) and 16K immunoreactive fibers exist in the spinal cord from cervical down to sacral level. Descending fibers running parallel in the dorsolateral and lateral funiculus send collaterals ventromedially or medially to terminate in the gray matter surrounding the central canal, where nociceptive neurons have recently been located, in addition to those nociceptive cells in the dorsal horn. After spinal transection at lower thoracic level, POMC peptide immunoreactivities disappeared below the lesion. Moreover, no POMC cell bodies were found in the spinal cord. Therefore, the descending fibers are most likely of supraspinal origin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26098/1/0000174.pd

Anatomical relationship between opioid peptides and receptors in rhesus monkey brain

To determine whether opioid peptide-receptor pharmacological associations found in vitro (e.g., enkephalin-[delta], dynorphin-[kappa]) predict anatomical relationships in situ, immunocytochemical and receptor autoradiographic studies were carried out on adjacent sections from the same brains of formaldehyde-perfused rhesus monkeys. Apparent [mu] and [kappa] opioid receptors (labeled, respectively, by [3H] naloxone and [3H]bremazocine under different incubation conditions), but not [delta] opioid receptors (labeled by [3H]D-Ala2, D-Leu5-enkephalin), survived the fixation procedure, and were found to be colocalized throughout the brain. We have observed complex associations between these binding sites and one, two, or all three opioid peptide systems (i.e., proopiomelanocortin, proenkephalin, and prodynorphin) in different brain regions. These multiple opioid peptide-receptor subtype associations are apparent, for example, in neural systems involved in the processing of pain stimuli, and may be important for mediating different types of analgesia. Since differential processing of proenkephalin and prodynorphin can give rise to opioids of varying receptor selectivities, the colocalization of opioid receptor subtypes may signify that such processing is a key regulatory event in determining which receptor subtype is activated and, thus, the physiological consequences of opioid neurotransmission.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24630/1/0000041.pd

Development of the catecholamine innervation of the supraoptic nucleus in the Brattleboro rat

The ontogenetic development of the noradrenergic innervation of the supraoptic nucleus was studied in the Brattleboro rat at late postcoital and early postnatal ages. This genetic mutant offers a useful model for analysis of neuronal development because of the absence of a specific peptide component of identifiable target neurons and has been used presently to eliminate the possibility that such substances are essential for the establishment of normal connectivity during postnatal development. In this model, catecholamine varicosities were seen in juxtaposition to vasopressin-deficient perikarya during the initial phases of postnatal development, but these varicosities gradually decreased in number suggesting the possibility that the target neuron peptide, or some functional aspect of the neuron, may be necessary for the normal maintenance of this neuronal interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24975/1/0000402.pd

Pro-opiomelanocortin mRNA and peptide co-expression in the developing rat pituitary

Pro-opiomelanocortin (POMC) is synthesized in both the pituitary gland and the brain. Various peptide products of this precursor, namely beta-endorphin, ACTH and alpha-MSH are co-localized in the anterior lobe corticotrophs, all intermediate lobe cells and in hypothalamic neurons. Messenger RNA (mRNA) for POMC has further been shown to exist in these tissues. In this study, we have shown that POMC mRNA, and peptide accumulation as detected by in situ hybridization and immunocytochemistry, respectively, occur simultaneously within the rat pituitary gland during ontogeny and that their maturation occurs in parallel during prenatal and early postnatal development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29476/1/0000562.pd