The development of neuropeptide Y-immunoreactive neurons in a model of pure cortical culture.

The development of neuropeptide Y-immunoreactive neurons in the rat brain cerebral cortex was studied in a model of a pure cortical culture. In this model, development of neurons devoid of any afferents from other brain structures could be observed. Since mutual interactions between neuropeptide Y and catecholamines have been postulated, such a pure cortical culture offers a possibility of studying the development of neuropeptide Y neurons devoid of any brainstem monoaminergic afferents. A tissue dissected from 16-day-old rat fetuses and cultivated in a dissociated culture for 14 days was examined immunohistochemically for the presence of neuropeptide Y-immunoreactive neurons. Three main types of neuropeptide Y-immunoreactive neurons were found: unipolar, bipolar and multipolar. Cell processes and terminal varicose fibres were also observed. The results obtained indicate that neuropeptide Y-immunoreactive neurons and fibres may develop in a pure culture of the rat cerebral cortex without the influence of any other structures.     

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3'-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3'-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product; (ii) choline acetyltransferase detection by 3,3'-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyltransferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.     

Vasoconstrictor, vasodilator and pilomotor pathways in sympathetic ganglia of guinea-pigs.

Triple-labelling immunofluorescence and retrograde axonal tracing with fluorescent dyes have been combined to identify and characterize the neuropeptide content of vasoconstrictor, vasodilator and pilomotor neurons in the lumbar sympathetic ganglia of guinea-pigs. Postganglionic noradrenergic pilomotor neurons lacked immunoreactivity to neuropeptide Y and comprised up to about 30% of postganglionic neurons. Most post-ganglionic noradrenergic neurons that contained neuropeptide Y immunoreactivity were likely to be vasoconstrictor neurons, although some noradrenergic neurons containing neuropeptide Y projected to pelvic viscera. Vasoconstrictor neurons comprised up to about 60% of postganglionic neurons. About 15% of postganglionic neurons were non-noradrenergic and contained immunoreactivity to vasoactive intestinal peptide, neuropeptide Y and dynorphin. They mostly innervated blood vessels supplying skeletal muscles and were likely to be vasodilator neurons. Endings of presumed preganglionic neurons containing immunoreactivity to substance P were exclusively associated with vasodilator neurons. Conversely, presumed preganglionic endings containing immunoreactivity to calcitonin gene-related peptide were exclusively associated with vasoconstrictor neurons, although not all vasoconstrictor neurons had such endings associated with them. Presumed preganglionic terminals containing immunoreactivity to enkephalin were associated with some postganglionic neurons in each functional class. These results show that preganglionic and postganglionic sympathetic neurons lying in different functional pathways can be distinguished by their neuropeptide content as well as their projections. The identification of neurochemically distinct functional pathways begins to explain how the sympathetic nervous system is organized to allow the precise control of discrete target tissues.     

Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympathectomized rats.

We have examined the expression of tyrosine hydroxylase and neuropeptide Y in ciliary ganglia of normal adult rats and of adult rats in which the environment of these neurons was altered by sympathectomy at birth. Following neonatal 6-hydroxydopamine treatment, the proportion of tyrosine hydroxylase-immunoreactive and neuropeptide Y-immunoreactive neurons in ciliary ganglia was significantly increased. In ciliary neurons of both control and sympathectomized rats, neuropeptide Y immunoreactivity was preferentially co-localized with tyrosine hydroxylase. Immunoblot analysis confirmed the presence of tyrosine hydroxylase and its increase following sympathectomy. In situ hybridization studies revealed that many ciliary neurons contain mRNA for tyrosine hydroxylase and for neuropeptide Y. Like tyrosine hydroxylase immunoreactivity, the number of ciliary neurons containing tyrosine hydroxylase mRNA and the amount of mRNA per cell were increased in 6-hydroxydopamine-treated rats. In contrast, neuropeptide Y mRNA levels were the same in control and 6-hydroxydopamine-treated rats. Nerve growth factor is a candidate for mediating the effects of sympathectomy and most ciliary neurons in control and sympathectomized rats expressed immunoreactivity for the low-affinity nerve growth factor receptor. In addition, ciliary neurons from 6-hydroxydopamine-treated animals possessed increased nerve growth factor receptor immunoreactivity. These studies indicate that both tyrosine hydroxylase and neuropeptide Y in the ciliary ganglion are regulated by alterations in their environment. Their expression was enhanced by chemical sympathectomy which does not affect ciliary neurons directly but, rather, removes sympathetic innervation of shared targets, including the iris. In situ hybridization analysis suggests that the increased tyrosine hydroxylase and neuropeptide Y levels result from different mechanisms and provides evidence that neuropeptide levels can be regulated without changes in mRNA levels.     

Beta-endorphin-, adrenocorticotrophic hormone- and neuropeptide y-containing projection fibers from the arcuate hypothalamic nucleus make synaptic contacts on to nucleus preopticus medianus neurons projecting to the paraventricular hypothalamic nucleus in the rat

The nucleus preopticus medianus is known to be situated in a key site in pathways regulating the paraventricular hypothalamic nucleus. To investigate the innervation pattern to nucleus preopticus medianus neurons by afferent fibers containing beta-endorphin, adrenocorticotrophic hormone and neuropeptide Y, a retrograde tracing method was combined with immunohistochemistry for these peptides in the rat. In the first experiment with injection of a retrograde tracer in the nucleus preopticus medianus, retrogradely labeled neurons were found in many regions throughout the brain. Among these, the arcuate hypothalamic nucleus contained a number of retrogradely labeled neurons showing immunoreactivity to the neuropeptides examined. About 20%, 20% and 40% of retrogradely labeled arcuate hypothalamic nucleus neurons showed beta-endorphin, adrenocorticotrophic hormone and neuropeptide Y immunoreactivity, respectively. About 18% and 57% of retrogradely labeled neurons in the nucleus tractus solitarius and ventrolateral medulla, respectively, were immunoreactive to neuropeptide Y. There were many more neuropeptide Y-immunoreactive projections to the nucleus preopticus medianus from the arcuate hypothalamic nucleus than those from the medulla. None of the retrogradely labeled neurons in the medulla showed immunoreactivity to beta-endorphin or adrenocorticotrophic hormone. In the second experiment with injection of a retrograde tracer in the paraventricular hypothalamic nucleus, electron microscopic observation revealed that retrogradely labeled neurons in the nucleus preopticus medianus were in synaptic contact with beta-endorphin-, adrenocorticotrophic hormone- and neuropeptide Y-immunoreactive axon terminals.The present finding indicates that nucleus preopticus medianus neurons projecting to the paraventricular hypothalamic nucleus are innervated by beta-endorphin-, adrenocorticotrophic hormone- and neuropeptide Y-containing arcuate hypothalamic nucleus neurons in addition to being innervated by neuropeptide Y-containing catecholaminergic medullary neurons which have been reported in our previous study.     

Neuropeptide Y reduces epileptiform discharges and excitatory synaptic transmission in rat frontal cortex in vitro

Neuropeptide Y reduced spontaneous and stimulation-evoked epileptiform discharges in rat frontal cortex slices perfused with a magnesium-free solution and with the GABA(A) receptor antagonist picrotoxin. To investigate the mechanism of that action, effects of neuropeptide Y on intrinsic membrane properties and synaptic responses of layer II/III cortical neurons were studied using intracellular recording. Neuropeptide Y (1 microM) had no detectable effect on the membrane properties of neurons. The evoked synaptic potentials were attenuated by neuropeptide Y. Moreover, the pharmacologically isolated excitatory postsynaptic potentials, mediated by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, were reversibly depressed by neuropeptide Y. The most pronounced inhibitory effect of neuropeptide Y was observed on late polysynaptic excitatory postsynaptic potentials. To assess a putative postsynaptic action of neuropeptide Y, N-methyl-D-aspartate was locally applied in the presence of tetrodotoxin. The N-methyl-D-aspartate-evoked depolarizations were unaffected by neuropeptide Y, which suggests that the depression of excitatory postsynaptic potentials was due to an action at sites presynaptic to the recorded neurons.These data show that neuropeptide Y attenuates epileptiform discharges and the glutamate receptor-mediated synaptic transmission in the rat frontal cortex. The above results indicate that neuropeptide Y may regulate neuronal excitability within the cortex, and that neuropeptide Y receptors are potential targets for an anticonvulsant therapy.     

Reduced density of neuropeptide Y neurons in the somatosensory cortex of old male and female rats: relation to cholinergic depletion and recovery after nerve growth factor treatment

Synthesis of neuropeptide Y in the neocortex and activity of the basalocortical cholinergic system are both reduced in the aging brain. We hypothesized that, by stimulating the activity of the basal forebrain cholinergic neurons, nerve growth factor might also be capable of restoring the synthesis of neuropeptide Y in cortical neurons. Old male and female rats were intraventricularly infused with nerve growth factor for 14 days and their brains were analyzed in order to quantify the densities of neuropeptide Y-immunoreactive neurons and of fiber varicosities stained for vesicular acetylcholine transporter protein in layers II/III, V and VI of the primary somatosensory barrel-field cortex. The areal densities of neuropeptide Y neurons and of vesicular acetylcholine transporter protein varicosities in all cortical laminae were found to be dramatically decreased in old rats when compared with young rats. However, infusions of nerve growth factor, known to exert a powerful trophic effect upon cortically projecting cholinergic neurons, have led to considerable recovery of vesicular acetylcholine transporter protein-positive terminal fields, which was paralleled by complete restoration of function in neuropeptide Y-producing neurons. With respect to the gender differences, although the density of cortical neuropeptide Y neurons was found to be significantly higher in young females than in young males and the opposite was true for vesicular acetylcholine transporter protein-positive varicosities, the general pattern of age- and treatment-related changes in these neurochemical markers was similar in both sexes. Overall, the age- and treatment-related variations in the density of cortical neuropeptide Y cells were found to correlate with those observed in the density of vesicular acetylcholine transporter protein varicosities. These results lend support to the idea that there is a causal relationship between age-related changes in cortical cholinergic and neuropeptide Y-ergic neurotransmitter systems.     

Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: an immunocytochemical and in situ hybridization study.

The neuronal distributions of somatostatin and neuropeptide Y and their respective mRNAs in hippocampal slice cultures were examined by immunohistochemical staining and in situ hybridization. For the in situ hybridization we used an alkaline phosphatase-labelled oligodeoxynucleotide probe for somatostatin mRNA and an 35S-labelled oligodeoxynucleotide probe for neuropeptide Y mRNA. For both neuropeptides the immunostained and hybridized neurons displayed a comparable, organotypic distribution. Most labelled neurons were located in the dentate hilus and stratum oriens of CA3 and CA1. Additional neurons were found in stratum radiatum and pyramidale of CA3, but very few in the corresponding layers of CA1. In all locations the density of somatostatin- and neuropeptide Y-reactive cells exceeded that observed in vivo. Also, the hybridization signal of the individual neurons appeared enhanced in the slice cultures. Methodologically it was noted that the non-radioactive alkaline phosphatase-labelled oligodeoxynucleotide probe gave excellent in situ hybridization results with detailed cellular resolution and no apparent problems of tissue penetration, even when used on whole-mount explants. These results demonstrate that somatostatin and neuropeptide Y-immunoreactive and mRNA containing neurons retain their organotypic distribution and basic morphological characteristics in the slice cultures. The supernormal density of these neurons and their hybridization signals indicate that a transient developmental increase in neuropeptide expression may persist in vitro.     

The neuropeptide tyrosine Y1R is expressed in interneurons and projection neurons in the dorsal horn and area X of the rat spinal cord

The localization of the neuropeptide tyrosine Y1 receptor was studied with immunohistochemistry in parasagittal and transverse, free-floating sections of the rat lumbar spinal cord. At least seven distinct Y1 receptor-positive populations could tentatively be recognized: Type 1) abundant small, fusiform Y1 receptor-positive neurons in laminae I-II, producing a profuse neuropil; Type 2) Y1 receptor-positive projection neurons in lamina I; Type 3) small Y1 receptor-positive neurons in lamina III, similar to Type 1 neurons, but less densely packed; Type 4) a number of large, multipolar Y1 receptor-positive neurons in the border area between laminae III-IV, with dendrites projecting toward laminae I-II; Type 5) a considerable number of large, multipolar Y1 receptor-positive neurons in laminae V-VI; Type 6) many large Y1 receptor-positive neurons around the central canal (area X); and Type 7) a small number of large Y1 receptor-positive neurons in the medial aspect of the ventral horns (lamina VIII). Many of the neurons present in laminae V-VI and area X produce craniocaudal processes extending for several hundred micrometers. Retrograde tracing using cholera toxin B subunit injected at the 9th thoracic spinal cord level shows that several Type 5 neurons in laminae V-VI, and at least a few Type 2 in lamina I and Type 6 in area X have projections extending to the lower segments of the thoracic spinal cord (and perhaps to supraspinal levels). The present results define distinct subpopulations of neuropeptide tyrosine-sensitive neurons, localized in superficial and deep layers of the dorsal, in the ventral horns and in area X. The lamina II neurons express somatostatin [The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons. Eur J Neurosci 11:2211-2225] and are presumably glutamatergic [Todd AJ, Hughes DI, Polgar E, Nagy GG, Mackie M, Ottersen OP, Maxwell DJ (2003) The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn. Eur J Neurosci 17:13-27], that is they are excitatory interneurons under a Y1 receptor-mediated inhibitory influence. The remaining Y1 receptor-positive spinal neurons need to be phenotyped, for example if the large Y1 receptor-positive laminae III-IV neurons (Type 5) are identical to the neurokinin (NK)1R-positive neurons previously shown to receive neuropeptide tyrosine positive dendritic contacts [Polgár E, Shehab SA, Watt C, Todd AJ (1999) GABAergic neurons that contain neuropeptide Y selectively target cells with the NK1 receptor in laminae III and IV of the rat spinal cord. J Neurosci 19:2637-2646]. If so, neuropeptide tyrosine could have an antinociceptive action not only via Y1 receptor-positive interneurons (Type 1) but also projection neurons. The present results show neuropeptide tyrosine-sensitive neuron populations virtually in all parts of the lumbar spinal cord, suggesting a role for neuropeptide tyrosine signaling in many spinal functions, including pain.     

Transient expression of neuropeptide Y and its C-flanking peptide immunoreactivities in the spinal cord and ganglia of human embryos and fetuses.

An immunohistochemical study of spinal cord, dorsal root and sympathetic ganglia of human embryos and fetuses demonstrated that neuropeptide Y and its C-flanking peptide could be detected in seven-week-old embryos but were absent or difficult to demonstrate after the 17th week of gestation. The peptides were found in several structures of the spinal cord, e.g. fibres in the dorsal portion of the lateral funiculus, cell bodies and fibres in the dorsal horn, and motoneurons, and also in numerous primary sensory neurons of dorsal root ganglia. They were also present in sympathetic neurons and since these are the only structures expressing neuropeptide Y and its C-flanking peptide in the adult, it must be concluded that their presence in other neurons is a transient developmental feature. To assist in understanding the relationship of these transient structures with other spinal and sensory neurons, a comparison was made with other neuronal structures showing immunoreactivity for two general neuronal markers, neurofilaments and protein gene product 9.5, and two neuropeptides present in primary sensory afferents, somatostatin and substance P. In the dorsal root ganglia, numerous neuropeptide Y- and C-flanking peptide-immunoreactive neurons were observed before substance P- or somatostatin-immunoreactive cells could be detected. Therefore, neuropeptide Y and its C-flanking peptide could represent a primitive peptidergic system appearing before primary sensory neurons express their characteristic adult phenotype. The fibres of the lateral funiculus showing immunoreactivity for neuropeptide Y and its C-flanking peptide were longitudinally orientated and could be detected at all cephalocaudal levels of the spinal cord. Comparison with the other immunohistochemical markers indicated that they were not primary sensory afferents. At least some of them probably originated from neuropeptide Y- and C-flanking peptide-immunoreactive neurons of the dorsal horn, that may be considered to be a subset of early-appearing interneurons.     

Influence of reserpine administration on neuropeptide Y immunoreactivity in the locus coeruleus and caudate-putamen nucleus of the rat brain.

The effects of treatment with reserpine (10 mg/kg, i.p.) a monoamine depleting agent, on neuropeptide Y immunoreactivity were studied immunohistochemically in neurons of two rat brain structures: locus coeruleus and caudate-putamen nucleus. It was found that reserpine after 24 h increased neuropeptide Y immunoreactivity level but no significant changes were observed 4 and 72 h or 5 days after the injection. The results indicate that despite the known co-existence of neuropeptide Y and noradrenaline in some neurons of the locus coeruleus no concomitant decrease in neuropeptide Y immunoreactivity level was found after reserpine when noradrenaline was depleted from nerve cell bodies and terminals. The increase in neuropeptide Y immunoreactivity observed 24 h after reserpine injection may suggest that the neuropeptide Y-containing neuronal systems of the locus coeruleus and caudate-putamen nucleus are controlled by monoaminergic afferents.     

Effect of 6-hydroxydopamine on neuropeptide Y and corticotropin-releasing factor expression in rat amygdala

The influence of dopaminergic denervation on neuropeptide Y and corticotropin-releasing factor-containing neurons in the amygdala was investigated in rats by examining the effects of a selective, unilateral 6-hydroxydopamine lesion of mesencephalic dopaminergic neurons in both the substantia nigra and the ventral tegmental area on these peptides and their messenger RNA expression, observed eight to 10 days after the lesion. The studies were conducted by immunocytochemical and in situ hybridization methods. Neuropeptide Y or corticotropin-releasing factor-immunoreactive neurons were counted in sections of the amygdala under a microscope, and the messenger RNA expression was measured as optical density units in autoradiograms. A significant increase in both neuropeptide Y and corticotropin-releasing factor messenger RNA expression was found in the amygdala on the lesioned side in comparison with the contralateral one, as well as with the ipsilateral side of vehicle-injected controls. Immunohistochemical studies showed that the number of neuropeptide Y-immunoreactive neurons increased in the whole amygdala on the lesioned side. At the same time, the number of corticotropin-releasing factor-immunoreactive neurons grouped in the central amygdaloid nucleus declined, and so did the staining intensity. The obtained results indicate that dopaminergic denervation stimulates the synthesis of neuropeptide Y and corticotropin-releasing factor in rat amygdala, but the peptide levels are differently regulated, which points to a diverse release of these peptides.     

Locus coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or galanin and their efferent projections to the spinal cord, cerebral cortex and hypothalamus

The efferent projections of locus coeruleus neurons which contain neuropeptide Y-, tyrosine hydroxylase- or galanin-like immunoreactivity were investigated using the indirect immunofluorescence technique combined with the retrograde transport of the fluorescent substance Fast Blue. Four groups of rats received injections of Fast Blue: (1) bilaterally into the mid-thoracic spinal cord (T6-T7); (2) unilaterally into the low cervical spinal cord (C4-C5); (3) unilaterally into the paraventricular, periventricular and dorsomedial hypothalamic nuclei; and (4) unilaterally into five sites in the cerebral cortex (frontal, cingulate and striate cortex). Efferent projections to the spinal cord, hypothalamus and cerebral cortex from neuropeptide Y-, tyrosine hydroxylase- and galanin-containing locus coeruleus cells were observed. A higher percentage of the peptidergic locus coeruleus neurons projected to the hypothalamus than to the spinal cord or cerebral cortex. The distribution and morphology of the neuropeptide Y- and galanin-containing neurons in the locus coeruleus were also investigated. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were found in small, medium and large multipolar neurons, as well as in fusiform locus coeruleus cells. The neuropeptide Y- and galanin-immunoreactive neurons were found throughout the locus coeruleus. In the caudal locus coeruleus, they were primarily located in the dorsal portion. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were only seen in a few tyrosine hydroxylase-positive neurons of the subcoeruleus group. The data show that the peptide-containing locus coeruleus neurons have efferent projections to the spinal cord, hypothalamus and cerebral cortex. The locus coeruleus may be divided into functional subdivisions dependent on the region of the locus coeruleus, the neurotransmitter/neuropeptide(s) contained within the neurons and their efferent projections.     

Neuropeptide Y modulates excitatory synaptic transmission in the olfactory bulb

Although the olfactory bulb contains one of the highest concentrations of neuropeptide Y in the CNS, its function in the bulb remains unclear. In this study, we used whole-cell electrophysiological, molecular, and primary culture techniques to investigate neuropeptide Y gene expression and neuromodulatory actions of neuropeptide Y on rat olfactory bulb neurons. Northern analysis showed that neuropeptide Y mRNA increases with animal age or time in culture, in a parallel manner. In electrophysiology experiments, agonists that activate neuropeptide Y receptors (whole neuropeptide Y) and the Y2 receptor subtype (neuropeptide Y 13-36) reduced spontaneous excitatory activity in bulb interneurons. In investigating potential presynaptic effects, both agonists reduced the amplitude of calcium channel currents in the presynaptic (mitral/tufted) cell. Also consistent with a presynaptic effect, both agonists reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (or "minis") in interneurons. In examining potential postsynaptic effects, both agonists slightly increased membrane resistance but had no effect on currents evoked by glutamate. Together, these data suggest that neuropeptide Y inhibits excitatory neurotransmission between olfactory bulb neurons via a presynaptic effect on transmitter (glutamate) release.     

Development of the ultrastructural features of neuropeptide Y-immunoreactive neurons in the rat visual cortex

Summary Immunohistochemical studies have localized neuropeptide Y into a small population of non-pyramidal neurons in the mammalian cerebral cortex. In the rat, these cells are distributed in layers II–VI and are characterized at the ultrastructural level by an abundance of cytoplasm containing a plethora of organelles, most conspicuous of which are cisternae of granular endoplasmic reticulum stacked in parallel arrays. In the present study, we used electron microscopic immunocytochemistry to examine the ultrastructural development of neuropeptide Y-labelled neurons in the rat visual cortex from birth, when they first appear in this cortical area, until postnatal day 32. At birth and in the subsequent few days, neuropeptide Y neurons, found exclusively in layers V and VI, often show a deeply infolded nucleus and little cytoplasm containing few organelles. At the end of the first postnatal week, labelled cells are still restricted to layers V and VI and display immature features. However, at this stage, cells often show irregularly enlarged proximal dendrites filled with organelles. During the second postnatal week, neuropeptide Y-immunoreactive cell bodies appear for the first time in layers II and III, and at the end of this week they have a distribution similar to that observed in the adult. Labelled cells are overall more differentiated than at earlier ages showing some of the ultrastructural features which distinguish them in the adult. No differences in maturation are evident between immunoreactive neurons located in the superficial layers and those in the deep layers, suggesting that the neuropeptide Y neurons in the more superficial layers express the peptide after having completed their migration and have acquired their characteristic ultrastructural features. Maturation proceeds during the third postnatal week. At the end of this stage, neuropeptide Y-containing cells acquire their mature nuclear and cytoplasmic features and an adult complement of synapses.     

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the “late” time intervals investigated (30–90 days after kainic acid).

These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at “late” time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide.

It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.     

Dendritic morphology of presumptive vasoconstrictor and pilomotor neurons and their relations with neuropeptide-containing preganglionic fibres in lumbar sympathetic ganglia of guinea-pigs

We have used intracellular dye-filling combined with multiple-labelling immunofluorescence to examine the dendritic morphology of neurons and their relations with neuropeptide-containing preganglionic terminals in the lumbar sympathetic chain of guinea-pigs. Presumptive vasoconstrictor neurons with immunoreactivity for both tyrosine hydroxylase and neuropeptide Y dendritic fields that were significantly smaller, on average, than those of presumptive pilomotor neurons containing immunoreactivity to tyrosine hydroxylase but not to neuropeptide Y. However, there was considerable variation in the sizes of the dendritic fields of the vasoconstrictor neurons. Preganglionic nerve terminals containing immunoreactivity to calcitonin gene-related peptide, but not to substance P, only surrounded cell bodies of vasoconstrictor neurons containing immunoreactivity to tyrosine hydroxylase and neuropeptide Y. In most cases, the neuropeptide-containing preganglionic terminals were not associated closely with the distal dendrites of these neurons. Few neuropeptide-containing terminals were associated closely with either the cell bodies or dendrites of the pilomotor neurons.

These results show that there is a considerable range in the size of dendritic trees of sympathetic final motor neurons. Some of this variation is related to the pathways within which the neurons lie, so that presumptive pilomotor neurons generally are larger than presumptive vasoconstrictor neurons. The marked variation in size of vasoconstrictor neurons raises the possibility that there may be a size dependent recruitment of these neurons, similar to that seen in pools of spinal motor neurons. The distribution of the peptide-containing preganglionic endings suggests that they would act predominantly at the cell body and proximal dendrites of the final motor neurons.     

A comparative analysis of neurons containing catecholamine-synthesizing enzymes and neuropeptide Y in the ventrolateral medulla of rats, guinea-pigs and cats.

Neurons in the ventrolateral medulla oblongata of rats, guinea-pigs and cats that contain tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine-N-methyltransferase and neuropeptide Y have been demonstrated immunohistochemically in serial coronal sections of tissue taken from the level of the cervical spinal cord to the level of the facial nucleus. The anatomical distribution of these neurons has been described, quantified and reconstructed in three dimensions to compare the neuron populations between species. In all species, between 50 and 90% of immunoreactive neurons lay rostral to the level of the obex. There were no significant differences in the number and distribution of neurons containing catecholamine-synthesizing enzymes between control animals and those pretreated with colchicine, with two exceptions: all dopamine-beta-hydroxylase neurons were weakly immunoreactive without colchicine pretreatment in cats, and pretreatment with colchicine revealed a small rostral group of tyrosine hydroxylase-positive neurons in guinea-pigs. There were remarkable similarities in the rostrocaudal distributions of neurons containing tyrosine hydroxylase, dopamine-beta-hydroxylase and neuropeptide Y in relation to comparable anatomical landmarks across the species. However, the distributions of neurons containing tyrosine hydroxylase. Phenylethanolamine-N-methyltransferase-positive neurons, while densely stained in rats, were only faintly stained in cats and absent in guinea-pigs; the distribution of these neurons was similar to the distribution of neurons containing only tyrosine hydroxylase. The similarity of the distribution of neurons demonstrated using tyrosine hydroxylase, dopamine-beta-hydroxylase and neuropeptide Y immunohistochemistry implies that homologous catecholamine-containing neuron groups do exist in the ventrolateral medulla despite the variation in phenylethanolamine-N-methyltransferase between species. In contrast to the previous classification of neuron groups into A1 and C1 based on the presence or absence of this latter enzyme, the data suggest that a discrete group of tyrosine hydroxylase-immunoreactive neurons, which probably do not contain dopamine-beta-hydroxylase or neuropeptide Y, can be distinguished in the rostral ventrolateral medulla of all species. The absence of detectable dopamine-beta-hydroxylase in this group of neurons suggests that they may not synthesize either adrenaline or noradrenaline.     

Projections of peptide-containing neurons in rat colon

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, substance P, neuropeptide Y, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or enkephalin were studied in the midcolon of the rat by immunocytochemistry and immunochemistry. Most of these nerve fibers had an intramural origin as was established by extrinsic denervation (serving of mesenterial nerves). Extrinsic denervation eliminated neuropeptide Y-containing fibers of presumably sympathetic origin together with sensory nerve fibers containing both substance P and calcitonin gene-related peptide. Co-existence of two peptides in the same neuron was studied by double immunostaining. This revealed co-existence of neuropeptide Y and vasoactive intestinal peptide in one population of intramural neurons; an additional population of intramural neurons was found to contain vasoactive intestinal peptide but not neuropeptide Y. All somatostatin-containing neurons in the submucous ganglia were found to harbor calcitonin gene-related peptide. A much larger population of submucous neurons containing calcitonin gene-related but not somatostatin was also detected. Some perivascular calcitonin gene-related peptide-containing nerve fibers (of intrinsic origin) harbored vasoactive intestinal peptide while others (of extrinsic origin) harbored substance P. The polarities and projections of the various peptide-containing intramural neurons in the transverse colon were studied by analysing the loss of nerve fibers upon local disruption of enteric nervous pathways (myectomy or intestinal clamping). Myenteric neurons containing vasoactive intestinal peptide, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or vasoactive intestinal peptide/neuropeptide Y gave off 5-10-mm-long descending projections while those containing substance P or enkephalin issued approx. 5-mm-long ascending projections. Submucous neurons containing calcitonin gene-related peptide, somatostatin/calcitonin gene-related peptide or gastrin-releasing peptide issued both ascending (2-6 mm) and descending (2-6 mm) projections, those containing vasoactive intestinal peptide issued ascending (approx. 2 mm) projections, while those containing galanin or vasoactive intestinal peptide/neuropeptide Y lacked demonstrable oro-anal projections. Enkephalin-containing fibers could not be detected in the mucosa and the mucosal substance P-containing nerve fibers were too few to enable us to delineate their projections.     

Neuropeptide Y immunoreactivity in the locus coeruleus of the rat brain

The detailed distribution of neuropeptide Y immunoreactive neurons and fibres is given for the rat locus coeruleus. The studies were carried out using indirect immunofluorescence and avidin-biotin-peroxidase techniques. It was shown that in colchicine pretreated rats, about 15-20% of locus coeruleus neurons contain neuropeptide Y immunoreactivity. Neuropeptide Y immunoreactive neurons form two populations: (1) medium-sized or large neurons, poorly immunostained, situated mainly in the dorsal and central locus coeruleus nucleus, and (2) small, strongly immunostained neurons in ventromedial parts of the nucleus. Neuropeptide Y immunoreactive fibres and terminals are scattered throughout the locus coeruleus, but are more numerous in its ventromedial and ventrorostral parts.