Distribution and colocalization of delta sleep-inducing peptide and luteinizing hormone-releasing hormone in the aged human brain: an immunohistochemical study

The distributions of delta sleep-inducing peptide (DSIP)- and luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons were investigated in the human brain with special emphasis on the basal forebrain (from the septum to the hypothalamus), using indirect immunofluorescence. With a modified elution technique, sequential stainings on the same section showed that DSIP- and LHRH-immunoreactivities were often colocalized. Small numbers of LHRH/DSIP-immunoreactive cells were essentially detected in the diagonal band of Broca, the medial septum and the ventral hypothalamus. The richest areas displaying fibres and terminal-like structures were the preoptic area, the ventromedial and ventrolateral hypothalamic areas, the periventricular region and certain circumventricular organs (i.e. median eminence, vascular organ of the lamina terminalis). Few isolated fibres were observed in the subfornical organ. The topographical relationships between DSIP- and LHRH-immunoreactivities in the neurosecretory systems suggest that DSIP may play a role as important as that of LHRH.     

Localization of neurons containing immunoreactive delta sleep-inducing peptide in the rat brain: An immunocytochemical study

Delta sleep-inducing peptide has been found in the peripheral circulation of animals entering slow-wave, or delta, sleep. An antiserum to this peptide was used to localize immunoreactive-like delta sleep-inducing peptide in the rat brain. The peptide was shown to have a rather widespread distribution. In the forebrain, the majority of these neurons were found to extend in a continuous rostral-caudal band in the ventral one-third of the brain from the primary olfactory cortex to the lateral hypothalamus. Neurons were also present in the basal ganglia, amygdala, septum, and thalamus. In the brainstem, the neurons were widespread and associated with the reticular formation, raphe nuclei, nuclei of the trigeminal complex, several auditory nuclei—nuclei of the lateral lemniscus, cochlear nuclei, and inferior colliculus—, cerebellum, locus ceruleus, periventricular gray, and vagal and hypoglossal nuclei. Immunoreactive fibers were, in general, difficult to demonstrate; they were seen mainly in the vicinity of the third ventricle and near blood vessels.The function of delta sleep-inducing peptide is unknown and its role in sleep is still under investigation. The distribution of delta sleep-inducing peptide in the present study suggests that the peptide is a component of several systems—arousal, locomotion, auditory, visual and sensory—both somatic and vestibular. The widespread distribution of the peptide, the lack of demonstrable immunoreactive fiber tracts, and the presence of these neurons in areas known to contain aminergic and peptidergic neurons, raises the possibility that neurons containing delta sleep-inducing peptide may exert their effect by projecting directly into blood vessels and/or interacting with neurons in their immediate vicinity.     

Modulatory actions of luteinizing hormone-releasing hormone on electrical activity of preoptic neurons in brain slices

Single unit activity was recorded from 378 neurons, in two preoptic nuclei rich in luteinizing hormone-releasing hormone neurons, using in vitro brain tissue slices which were prepared form either ovariectomized or ovariectomized plus estradiol-treated rats. To test possible transmitter-like actions, agents were injected into the perfusion medium. Luteinizing hormone-releasing hormone excited 46%, inhibited 7%, and evoked biphasic responses in 2% of the 250 units tested. By comparison, two other peptides, thyrotropin-releasing hormone and cholecystokinin-octapeptide sulfated were exclusively excitatory, acting on 55 and 67% of the neurons, respectively. The response to thyrotropin-releasing hormone, cholecystokinin-octapeptide sulfated, and neurotransmitters were prompt, large, and consistent from trial to trial. In contrast, responses to luteinizing hormone-releasing hormone were usually delayed, small, and variable. Responses to the agents tested were not affected by in vivo estradiol treatment. Possible modulatory actions of luteinizing hormone-releasing hormone were tested by comparing the responses of single neurons to norepinephrine and serotonin before and after an application of luteinizing hormone-releasing hormone. In 39 and 20% of the 119 neurons tested, the norepinephrine responses were potentiated and attenuated, respectively, by luteinizing hormone-releasing hormone. In 46 serotonin-responsive neurons, 28% were potentiated and 22% attenuated. These neuromodulatory actions of luteinizing hormone-releasing hormone were specific in affecting only certain responses of certain neurons, and they were not duplicated on the same neurons by thyrotropin-releasing hormone. It appears that luteinizing hormone-releasing hormone may be a neuromodulator in the preoptic area.     

Immunohistochemical mapping of delta sleep-inducing peptide in the cat brain and hypophysis. Relationships with the LHRH system and corticotropes

Using the indirect immunofluorescence method, the distribution of the delta sleep-inducing peptide was studied in the cat brain and hypophysis. Delta sleep-inducing peptide-like-immunoreactive cell bodies mostly visualized in colchicine-pretreated animals were mainly found scattered throughout the diagonal band of Broca, the ventral septum and the anterior hypothalamic areas. A few immunoreactive cell somata were also seen in the ventrolateral hypothalamic area and more occasionally in the triangular septal nucleus. The heaviest concentrations of delta sleep-inducing peptide-like-immunoreactive varicose fibres and terminal-like structures were observed in the septo-preoptic region, in the median eminence and pituitary stalk. Some other brain regions supplied with few delta sleep-inducing peptide-immunoreactive fibres included the fimbria-fornix, the dorsal part of the subfornical organ, the medial habenular nucleus and more caudally, the periaqueductal gray. Elution-restaining experiments revealed that delta sleep-inducing peptide-like immunoreactivity frequently occurred in luteinizing hormone-releasing hormone-immunoreactive neurons and vice versa. At the pituitary level, delta sleep-inducing peptide-like immunoreactivity was detected in most, if not all, melanocorticotropes of the pars intermedia and further in a large subpopulation of corticotropes mainly located in the zona tuberalis of the pars distalis. Taken together these anatomical findings support the view that delta sleep-inducing peptide (or a closely related molecular form) could play a modulatory role at various levels of the hypothalamo-pituitary system.     

Comparison of the effects of two 'sleep' peptides, delta sleep-inducing peptide and arginine-vasotocin, on single neurons in the rat and rabbit brain stem

The effects of delta sleep-inducing peptide and arginine-vasotocin were assessed on single neurons in the nucleus reticularis gigantocellularis of the brain stem in rats and rabbits. Both peptides showed predominantly excitatory actions in both species when applied by microiontophoresis. A small proportion of cells was inhibited by delta sleep-inducing peptide in the rat. Responses to delta sleep-inducing peptide were short-lasting, dose-dependent and showed no significant desensitization to repeated applications. Responses to arginine-vasotocin were of very long time course and showed profound desensitization. No statistically significant correlation was seen between cells responsive to delta sleep-inducing peptide and those responsive to arginine-vasotocin. We conclude that both 'sleep' peptides have similar actions on central neurons and that they are active in both rats and rabbits. However, no evidence was found to suggest a common mechanism of action for both substances.     

Cascade afteraction of delta sleep-inducing peptide in rats

Delta sleep-inducing peptide is found to alter markedly the levels of substance P, β-endorphin, and corticosterone in the hypothalamus and blood plasma of rats, suggesting that the long-lasting stress-mitigating effects of this peptide are due to the considerable changes it causes in the content of other oligopeptides and hormones, involving them in various processes. Thus, DSIP itself appears to act only as a trigger, initiating a cascade of interdependent molecular reactions that correlate with the degree of resistance to stress. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 1, pp. 6–9, January, 1995     

Acetylcholinesterase and luteinizing hormone-releasing hormone distinguish separate populations of terminal nerve neurons

The terminal nerve is composed of a morphologically heterogeneous population of unipolar, bipolar and multipolar neurons located in the nasal and intracranial cavities of vertebrates. The question has arisen as to whether these neurons are neurochemically heterogeneous and therefore possibly functionally different as well. Among the substances localized in the terminal nerve are acetylcholinesterase and luteinizing hormone-releasing hormone-like immunoreactive material. We have developed a double-label procedure, combining immunocytochemistry and enzyme histochemistry to determine whether these two substances are localized within different populations of terminal nerve neurons. Compatibility of the two procedures was accomplished by modifications of the fixative and primary antibody solutions. In the immunocytochemical step, the avidin-biotin-peroxidase complex coupled to a new chromogen, Chromo-red, produced a bright red reaction product in neurons containing luteinizing hormone-releasing hormone-like material. This reaction product was easily differentiated from the black silver-intensified acetylcholinesterase label. In both neonatal and adult preparations, a large population of terminal neurons contained the acetylcholinesterase label only, whereas a smaller population contained both acetylcholinesterase and luteinizing hormone-releasing hormone-like material. The acetylcholinesterase-containing population of neurons was concentrated peripherally and included multipolar neurons. In contrast neurons with the two substances co-localized were unipolar or bipolar and were concentrated centrally. The simultaneous visualization of acetylcholinesterase and luteinizing hormone-releasing hormone-like material in the same tissue section enable the differentiation of two separate neurochemically defined populations of terminal neurons. The distribution of these two neuronal types was the same in neonatal and adult animals. These data provide support for a functional diversity of terminal neurons.     

Access of luteinizing hormone-releasing hormone neurons to the vasculature in the rat

Luteinizing hormone-releasing hormone is secreted into the hypophysial portal vasculature through which it controls the release of the gonadotropins. The peptide also acts as a neurotransmitter in various loci within the central nervous system. It is not known whether these roles are performed by separate populations of luteinizing hormone-releasing hormone neurons. Some recent tracing experiments suggest that this is the case (Silverman et al., J. Neurosci. 7, 2312, 1987; Jennes and Stumpf, Neuroscience 18, 403, 1986). One aspect of this question was addressed in the current study by intraperitoneal introduction of Fluoro-Gold (a retrograde tracer) into male and female rats under various age and hormonal conditions. Brain sections from the anterior olfactory nuclei to the median eminence were treated for the immunocytochemical demonstration of luteinizing hormone-releasing hormone. In all cases, regardless of the age, sex or hormonal condition of the animal, the Fluoro-Gold tracer was found in more than 90% of the luteinizing hormone-releasing hormone neurons. We conclude that virtually all luteinizing hormone-releasing hormone neurons in the rat secrete outside the blood-brain barrier, including those which project to sites within the central nervous system.     

Effect of delta sleep-inducing peptide on macromolecule biosynthesis in brain tissue of stressed rodents

For evaluation of the nature of adaptogenic properties of delta sleep-inducing peptide we studied the effect of this substance on macromolecule biosynthesis in the brain of rats and mice exposed to burn injury and psychoemotional stress, respectively. Anabolic activity of delta sleep-inducing peptide depended on the purpose of adaptation corresponding to the type of stress. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 400–403, April, 2006     

Chicken II luteinizing hormone-releasing hormone inhibits the M-current of bullfrog sympathetic neurons

The M-current of dissociated bullfrog sympathetic neurons, measured by the whole-cell patch clamp technique, is powerfully inhibited by chicken II LH-RH (luteinizing hormone-releasing hormone), with 50% inhibition near 1 nM. Chicken II LH-RH is approximately 100 times more potent than salmon LH-RH, and at least 1000 times more potent than other known naturally occurring LH-RH analogs (chicken I LH-RH, mammalian LH-RH, and lamprey LH-RH). This high potency makes chicken II LH-RH a candidate for the endogenous transmitter mediating the late, slow excitatory postsynaptic potential (EPSP) in bullfrog sympathetic ganglia.     

Delta sleep-inducing peptide blocks excitatory effects of glutamate on rat brain neurons

Microiontophoresis of delta sleep-inducing peptide primarily activated neurons in the dorsal hippocampus, anteroventral thalamic nucleus, lateral hypothalamus, and sensorimotor cortex. Microiontophoretic administration of glutamate markedly enhanced neuronal activity, while preliminary microionophoresis of delta sleep-inducing peptide blocked the excitatory effect of glutamate on neurons in these brain structures.     

Aging changes in synaptology of luteinizing hormone-releasing hormone neurons in male rat preoptic area

This study was undertaken to examine some aspects of the anatomical substrate for reproductive senescence. Immunocytochemically identified luteinizing hormone-releasing hormone neurons and their processes in the male rat brain preoptic area were compared in young adult (2-4 months), middle-aged (12-14 months) and old (20-23 months) animals. At the light microscopic level there were no age-dependent differences in total numbers or sizes of LHRH neurons nor in their distribution in the brain. Examination of these neurons at the electron microscopic level did reveal significant differences in certain organelles and in the degree and kind of synaptic input. Random sections of middle-aged luteinizing hormone-releasing hormone neurons more frequently passed through the nucleolus and the incidence of nematosomes was higher than in luteinizing hormone-releasing hormone neurons from the young and old animals. Quantitative measures of synaptic input to luteinizing hormone-releasing hormone soma and dendrites as well as to unidentified neurons in the same thin section were made. These are reported as percent of membrane that showed synaptic structure. Dendrites of both luteinizing hormone-releasing hormone and nonidentified neurons were more densely innervated than perikarya. The density of synaptic input to luteinizing hormone-releasing hormone neurons was significantly greater than that to nonidentified neurons in young and middle-aged animals, but was equal to that of nonidentified neurons by old age. Age-related changes were noted in synaptic organization with the most significant change being an increased input to luteinizing hormone-releasing hormone perikarya. Indeed, synaptic input to luteinizing hormone-releasing hormone perikaryal membrane was increased three-fold by middle age and ten-fold by old age. Density of synaptic input to luteinizing hormone-releasing hormone dendritic membrane did not change with age. There were no aging changes in percentage of membrane with synaptic structure in nonidentified elements. Synapses were also classified on the basis of their synaptic vesicle content. There were proportionately more synaptic boutons containing round clear than pleomorphic vesicles in the young sample. The proportion of synapses with pleomorphic vesicles increased with age onto both luteinizing hormone-releasing hormone perikarya and their dendrites. The proportion of boutons containing some electron dense-core vesicles along with clear vesicles decreased with age onto both luteinizing hormone-releasing hormone and nonidentified neurons and their processes.     

Effect of delta sleep-inducing peptide on catalytic properties of mitochondrial malate dehydrogenase from rat brain

Delta sleep-inducing peptide is shown to alter properties of malate dehydrogenase in brain mitochondria. The regulatory activity of the peptide is manifested in stabilization of catalytic properties of the enzyme at a higher level, which prevents their change during hypoxic stress. Regulation of malate dehydrogenase is presumed to occur through direct action of the peptide on mitochondrial membranes. Translated fromByulleten Eksperimental'noi Biologii i Meditsiny, Vol. 119, No. 2, pp. 141–143, February, 1995 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Distribution of labeled amino acids and delta sleep-inducing peptide in the body after instillation into the rabbit conjunctiva

P. K. Anokhin Research Institute of Normal Physiology, Academy of Medical Sciences of the USSR. Institute of Molecular Genetics, Academy of Sciences of the USSR. I. M. Sechenov First Moscow Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 9, pp. 236–237, September, 1990.     

LHRH类似物在乳猪体内的药代动力学研究

目的 :研究黄体激素释放激素类似物 (luteinizinghormone releasinghormoneanalogue ,LHRH -A)在健康乳猪体内的药代动力学规律。方法 :采用氯胺T法 ,用12 5I标记LHRH A ,放化纯度 96.2 %。按 1mg·kg 1体重于乳猪颈背部两侧分别肌注 ,药后 2 1个不同时点分别取血 0 .2 0～ 0 .2 5ml做血药浓度测定 ,用大型药理学计算软件DAS统计其药代动力学参数。结果 :LHRH A在体内的分布、消除以二室模型方式进行。Tmax :1 .1 667± 0 .40 82h ,Cmax :98.82 0 0± 69.5 5 86μg·L 1,T1 /2 β :5 5 8.3 71 4± 3 76.93 2 8h ,AUC :5 5 65 .1 2 41± 3 5 60 .1 962 μg·L 1·h 1,Vd/F :0 .0 0 2 2± 0 .0 0 1 6L。结论 :LHRH A在乳猪体内的分布、消除方式为二室模型 ,且消除较慢     

Mechanism of action of delta sleep-inducing peptide injected after L-dopa

Laboratory of Cytochemistry, Brain Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. S. Adrianov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 4, pp. 440–442, April, 1989.     

Production and immunohistochemical application of monoclonal antibodies against delta sleep-inducing peptide

Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunola belling systems Furthermore, it was demonstrated that DSIP5 9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.     

Localization of luteinizing hormone-releasing hormone in the mammalian hypothalamus

Utilizing immunohistochemistry with rabbit antiserum to synthetic luteinizing hormone-releasing hormone (LRH), LRH was localized in the peripheral region of the median eminence in the mouse and rat, and more generally in the median eminence of the guinea pig.     

Immunocytochemical localization of luteinizing hormone-releasing hormone in neurons in the medial basal hypothalamus of the female rat

Summary Luteinizing hormone-releasing hormone (LHRH)-neurons were localized with a LHRH antiserum (WP-1) in the medial basal hypothalamus (MBH) of the female rat using the sagittal hypothalamic slice preparation in combination with the peroxidase-antiperoxidase immunocytochemical technique of Sternberger (1979). Numerous darklystained perikarya were visualized in the cell-poor zone, lateral arcuate nucleus and the median eminence. The processes of these neurons contributed to the intensely-stained fiber bundle above the tubero-infundibular sulcus. The fibers in this tract run in a rostrocaudal plane in the lateral external zone of the median eminence. Also, numerous fibers course into the internal zone of the median eminence, perpendicular to the rostrocaudal plane. Several LHRH-immunoreactive perikarya also were identified in the periventricular area caudal to the optic chiasm (retrochiasmatic area). The LHRH neurons were small (10 m) bipolar neurons with fibers (dendrites) exiting from either pole which showed very little branching. It appears that the sagittal slice in combination with the appropriate fixation procedure and LHRH antiserum enabled us to demonstrate the presence of LHRH-immunoreactive perikarya in the MBH of the female rat.Supported by Health Research Services Foundation Grant W-22 and NIH Grant NS16419     

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus

Expression of P2X₁, P2X₂, P2X₃, P2X₄, P2X₅ and P2X₆ receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X₂, P2X₄, P2X₅ and P2X₆ receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X₂ and P2X₆ on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.