Effect of the neuropeptides vasoactive intestinal peptide, peptide histidine methionine and substance P on human major salivary gland secretion.

OBJECTIVE: The parasympathetic transmitters vasoactive intestinal peptide (VIP) and substance P (SP) are secretagogues in salivary glands of animals. Currently, we hypothesise that in human salivary glands, these neuropeptides and the VIP-related peptide histidine methionine (PHM) also exert secretory actions, reflected morphologically by exocytosis of acinar protein/glycoprotein-storing granules. MATERIALS AND METHODS: Submandibular and parotid gland tissues, exposed in vitro to VIP and PHM, and SP, respectively, were examined by light and transmission electron microscopy. For comparison, the response to in vitro stimulation of isoproterenol, phenylephrine and carbachol was examined. Moreover, the peptidergic innervation of the glands was examined by immunohistochemistry. RESULTS: Vasoactive intestinal peptide- and PHM-immunoreactive nerves were in close proximity to acini and ducts in the two glands, while these elements lacked a SP-positive innervation. While no morphological changes occurred in response to SP (parotid glands), VIP and PHM administration (submandibular glands) caused conspicuous acinar degranulation accompanied by luminal space broadening. In the two glands, both α1 - and ß-adrenergic receptor stimulation and muscarinic receptor stimulation caused similar changes as to VIP/PHM, although to varying extent. CONCLUSIONS: Vasoactive intestinal peptide and PHM, but not SP, are likely transmitters in the parasympathetic control of salivary (protein) secretion in humans.

The human hippocampal formation and parahippocampal gyrus: localization of substance P-like immunoreactivity in newborn and adult post-mortem tissue.

The localization of substance P-like immunoreactive nervous structures in the human post-mortem hippocampal formation and parahippocampal gyrus was studied by the indirect immunofluorescence technique of Coons and Kaplan (1950), J. exp. Med. 91, 1-9. Specimens were obtained from brains of both newborn and adult subjects. Substance P-positive perikarya were easily detectable in these regions, being particularly abundant in the stratum oriens of the hippocampus and in the hilus of the fascia dentata. The nerve fibres and terminals were also widely and unevenly distributed. Comparison of the newborn and adult tissue revealed differences in the distribution of substance P-like immunoreactivity in ontogenetic stages. Our results, together with data available in the literature, further suggest that substance P is differently distributed in the various cortical areas of the human brain, and that the distribution of substance P may vary among species.

Topographical localization of substance P in the human post-mortem brainstem. An immunohistochemical study in the newborn and adult tissue.

The localization of substance P-immunoreactive nervous structures in the newborn and adult human brainstem is studied by the indirect immunofluorescence technique of Coons and collaborators. Substance P-containing perikarya, nerve fibres and terminals are widely and unevenly distributed in several areas of the human brainstem, with a location similar to that described in other mammals; however, some remarkable differences are detectable, which speak in favour of the existence of species differences. For example, in the human brain there is a dense plexus of substance P-immunoreactive fibres in the cuneate fasciculus. The use of the newborn tissue is very helpful in the detection of the immunoreactive neuronal cell bodies. The comparative study of the newborn and the adult tissue reveals that the distribution of the substance P-like immunoreactivity changes in man during ontogeny and results in a more complete map of the substance P-containing neuron system.

Lack of copulatory behaviour in male castrated rats after p-chlorophenylalanine.

1 The effect of p-chlorophenylalanine (PCPA) on the copulatory behaviour of normal and castrated male rats with females in oestrus was studied.2 Castration 2 months before the experiment completely prevented the increased copulatory behaviour produced by PCPA in normal rats.3 The administration of testosterone restored the copulatory behaviour in the castrated rats indicating that testosterone is essential for this behaviour.

Somatostatin, galanin and peptide histidine isoleucine in the newborn and adult human trigeminal ganglion and spinal nucleus: immunohistochemistry, neuronal morphometry and colocalization with substance P.

By means of indirect immunofluorescence the neuropeptides somatostatin, galanin and peptide histidine isoleucine were localized in cell bodies, nerve fibres and terminal-like elements in the ganglion and spinal nucleus of the human trigeminal nerve in perinatal and adult ages. No immunoreactivity to vasoactive intestinal polypeptide was observed. In the gasserian ganglion somatostatin-, galanin- and peptide histidine isoleucine-containing neurons and nerve fibres occurred frequently in pre- and full-term newborns, but were scarce to absent in adults. Somatostatin- and galanin-positive pericellular basket-like structures around non-immunoreactive perikarya were observed in newborn specimens. Immunoreactivity to somatostatin, galanin and peptide histidine isoleucine labelled nerve fibers and punctate and felt-like nerve terminals in the pars interpolaris and subnucleus caudalis of the spinal trigeminal nucleus, with immunostaining and distribution patterns characteristic for each peptide. In addition, somatostatin-containing neuronal cell bodies frequently were detected. At variance with those containing somatostatin, the number of galanin- and peptide histidine isoleucine-like immunoreactive elements were dramatically reduced in the adult tissue compared to the newborn one. Double immunostaining revealed that each of the three peptides partially colocalizes with substance P, the degree of coexistence being very low for somatostatin/substance P and high for galanin/substance P and peptide histidine isoleucine/substance P both in the gasserian ganglion and in the spinal nucleus. The results obtained suggest that somatostatin, galanin and peptide histidine isoleucine may play functional roles in primary sensory neurons and at the first synaptic level of the human trigeminal sensory system.

Neuropeptides in the human celiac/superior mesenteric ganglionic complex: an immunohistochemical study.

The occurrence of vasoactive intestinal polypeptide (VIP), peptide histidine-isoleucine (PHI), calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), galanin (GAL) and enkephalins (ENK) is studied in the human celiac/superior mesenteric ganglionic complex of pre- and full-term newborns, and adult subjects by means of immunohistochemistry. The antisera used labelled nerve fibres and terminal-like networks for each examined peptide, as well as VIP- and SOM-positive postganglionic neurons. Differences in the relative amount and density of the structures immunoreactive to the various peptides were observed. Moreover, variations in the amount and type of labelled elements were appreciable for each peptide when specimens from subjects at perinatal and adult ages were compared. Double-labelling immunofluorescence for SP and each other peptide showed that co-localization with SP is very frequent for CGRP, moderate to scarce for GAL and SOM, and rare to absent for PHI, VIP and ENK. VIP-, ENK- and CGRP-immunolabeled perikarya bearing the morphological features of the small intensely fluorescent (SIF) cells occurred in the organ. The presence of a paraganglion in one of the specimens examined allowed the detection of VIP- and ENK-positive cell bodies and VIP-, ENK-, SP- and GAL-like immunoreactive varicose nerve fibres in it. The results obtained provide substantial morphological data in support of the involvement of the examined peptides in the chemical interneuronal signalling in the human celiac/superior mesenteric ganglia.

Calcitonin gene-related peptide in the human trigeminal sensory system at developmental and adult life stages: immunohistochemistry, neuronal morphometry and coexistence with substance P.

The distribution of calcitonin gene-related peptide (CGRP) has been examined by the indirect immunofluorescence technique in the Gasserian ganglion and spinal nucleus of the human trigeminal nerve. In the ganglion CGRP is present in almost 50% of primary sensory neurons, in varicose and non-varicose nerve fibres and in pericellular basket-like plexuses around non-immunoreactive ganglionic perikarya. Morphometric analysis reveals that the CGRP-positive neuronal population is heterogeneous in cell size. Observation of specimens from subjects at fetal, perinatal and adult life stages reveals that the percentage of CGRP-immunoreactive cells reaches a maximum at perinatal stages and then remains constant, declining only in old age. Pericellular basket-like nerve fibres are detectable only in fetal and pre-term and full-term newborn tissue. Coexistence between CGRP and substance P (SP) occurs, SP being present in about one quarter of the CGRP-immunoreactive neurons and CGRP being localized in a little more than half of the SP-immunoreactive neurons. However, perikarya, nerve fibres and pericellular fibres containing only one or other peptide are also present. Bundles of immunoreactive fibres and dot-like nerve terminals occur in the spinal tract and superficial and deep regions of the spinal trigeminal nucleus. A particularly dense plexus is present in the peripheral nuclear layers. Double immunostaining shows a similar regional distribution for SP. However, in inner substantia gelatinosa the density of CGRP-immunoreactive fibres is much higher than that of SP-immunoreactive ones. The results obtained add information to our knowledge of the organization of neurochemically identified neurons in the human trigeminal sensory system.

Enkephalins occur and colocalize with substance P in human trigeminal ganglion neurones.

Immunohistochemical evidence is provided for (i) the occurrence of a primary sensory neuronal population immunoreactive to methionine- and leucine-enkephalin (EK) in the human trigeminal ganglion; (ii) colocalization of EK and substance P (SP) in a subpopulation of ganglion neurones and in nerve fibres and terminal-like structures in the human trigeminal spinal nucleus. The results obtained indicate that part of the EK-positive innervation of the spinal nucleus may be of ganglionic origin and raise the possibility that EK and SP are co-stored in and co-released from primary afferent terminals, thus adding to the complexity of the sites and ways of interaction between these neuropeptides in the processing of sensory information.

trk-like immunoreactivity in the human trigeminal ganglion and subnucleus caudalis.

The immunohistochemical occurrence of trkA, trkB and trkC receptors was examined in the human trigeminal ganglion and spinal nucleus of subjects at all ages and compared with that of substance P (SP) and calcitonin gene-related peptide (CGRP), trk-like immunoreactive (LI) material was detectable in discrete subpopulations of primary sensory neurones from 25 weeks of gestation to adult life. Each subpopulation overlapped partially with those immunoreactive to SP and CGRP, trkA- and trkC-positive filamentous and punctate elements occurred in the trigeminal subnucleus caudalis. While immunostaining for trkC was restricted to rare isolated elements, that for trkA outlined the superficial laminae of the nucleus and was more intense early in life than in adults.

Neurotrophin-like immunoreactivity in the human trigeminal ganglion.

The localization of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) was demonstrated immunohistochemically in discrete neuronal subsets of the human trigeminal ganglion at ages ranging from 23 weeks of gestation to adulthood. Neurotrophin-containing subpopulations partially overlapped with each other and with those immunoreactive for the relevant trk receptor. Glial elements could also be immunostained, labelled satellite cells being particularly abundant in NT-3 stained sections. These results suggest that the neurotrophins are of functional significance for the human trigeminal primary sensory neurones throughout life. Their localization in the ganglion cellular components supports their function as target-derived trophic factors and as molecules effective in autocrine/paracrine interactions.

GAP-43 persists in adulthood and coexists with SP and CGRP in human trigeminal sensory neurones.

Immunohistochemistry was used to examine the occurrence and colocalization of the growth-associated protein GAP-43 with substance P (SP) and calcitonin gene-related peptide (CGRP) in the human trigeminal ganglion and sensory nucleus at perinatal and adult life stages. The results obtained show that: GAP-43-like immunoreactive (LI) material persists in trigeminal primary sensory neurones of the normal adult; the GAP-43-LI ganglionic population partially overlaps with those immunoreactive to SP and CGRP; the distribution pattern of the protein in the spinal nucleus varies with age; in the adult subnucleus caudalis GAP-43 is co-distributed with SP and CGRP. It is suggested that the trigeminal GAP-43-LI neuronal system may retain the capacity for structural and functional plasticity in adult life.

Neostriatal enkephalin-immunoreactive neurones project to the globus pallidus.

The origin of enkephalin-immunoreactive nerve terminals in the globus pallidus was investigated by combining immunocytochemistry with stereotaxic injection of neurotoxic agents (colchicine and kainic acid) and microknife deafferentations. The intracerebral administration of colchicine, irrespective of whether in the caudate putamen or in globus pallidus, induces the appearance of enkephalin-immunoreactive cell bodies and fibres in the caudate putamen. No immunoreactive cell bodies were depicted in the globus pallidus after this treatment. Kainic acid injections in the caudate putamen produced topographic depletions of enkephalin-immunoreactive terminals in the globus pallidus. The more anterior injections produced medial-anterior depletions, while posterior injections gave latero-posterior depletions. Injections in the globus pallidus produced only a non-specific loss of fluorescence restricted to the tip of the cannula. Coronal microknife cuts produced a combination of build-up and depletion of enkephalin immunofluorescence according to the position of the cut. The build-up of immunoreactive materials was always observed in the caudate-putamen side of the cut while depletions observed in the globus pallidus were related to the extent of deafferentation of this nucleus from the caudate putamen. All these observations confirmed the neostriatal origin (caudate putamen) of the enkephalinergic fibres present in the paleostriatum (globus pallidus).

The central and peripheral ends of the substance P-containing sensory neurones in the rat trigeminal system.

The distribution of substance P (SP) immunoreactivity in the spinal nucleus of the rat trigeminal nerve and in the skin of the lower lip was examined following (a) unilateral electrolytic lesions of the trigeminal ganglion, (b) trigeminal rhizotomy, and (c) unilateral interruption of the mental nerve, the sensory branch of the trigeminal nerve innervating the lower lip. A marked depletion of SP immunoreactivity in the ipsilateral trigeminal spinal nucleus followed lesions of the trigeminal ganglion or rhizotomy. The reticular formation ventral and medial to the spinal nucleus showed a small decrease in SP immunofluorescence on the operated side. Some loss of SP immunoreactivity was observed in the skin of the lower lip following ganglionectomy or rhizotomy. After sectioning the mental branch SP-immunofluorescent fibres of the skin of the lower lip disappear completely on the denervated side. It was concluded that some trigeminal ganglion neurones store, and might release, SP at their axon terminals in the medulla oblongata and at their sensory terminals in the skin.

Spontaneous and amphetamine-induced behavior after bilateral injection of ethanolamine-O-sulfate into the substantia nigra.

Injections of ethanolamine-O-sulfate (EOS) (200 microgram/kg) through chronic indwelling cannulae into the zona reticulata of the substantia nigra in rats produced immediate contraversive turning (away from the injection side). Bilateral injections produced spontaneous stereotyped behaviors including sniffing and biting. Twenty-four hours after the first injection of EOS the rats showed a significantly decreased locomotor response to 1.5 mg/kg D-amphetamine. The spontaneous behaviors elicited following the initial injection of EOS increased both in nature and duration with the second and third injections of EOS, and persevered for 24 h. At this time 1.5 mg/kg D-amphetamine intensified this spontaneous stereotyped behavior. GABA levels were significantly higher in substantia nigra, hypothalamus, corpus striatum and cerebral cortex 24 h following single or repeated (3 x) injections of 200 microgram/kg of EOS into the zona reticulata of the SN. Dopamine levels in the corpus striatum were unchanged 24 h following the EOS treatment except by the injection procedure itself. Control injections into the thalamus produced no turning or stereotypy, nor an enhanced response to 1.5 mg/kg D-amphetamine. GABA levels were, however, significantly higher in the substantia nigra and thalamus 24 h following repeated injections (3 x) of EOS into the thalamus. Results are consistent with the hypothesis that GABA containing neurons comprise part of the efferent output of the corpus striatum and do not act exclusively through a dopaminergic substrate.

Substance P-like immunoreactivity in human sympathetic ganglia.

Substance P-like immunoreactive nerve fibres and terminals are found in the human sympathetic paravertebral ganglia in connection with postganglionic neurones. Substance P may act as neurotransmitter or neuromodulator in the autonomic transmission in man.

Substance P in the human brainstem. Preliminary results of its immunohistochemical localization.

The localization of substance P in the human post-mortem brainstem is studied by the indirect immunofluorescence technique. Positive neuronal cell bodies, nerve fibers and terminals are unevenly present over both sensory and effector areas and nuclei. The pattern of distribution of substance P in the human brainstem, similar to that seen in the laboratory animals, supports the hypothesis of a neurotransmitter role of this compound in man.

Glial cell line-derived neurotrophic factor-like immunoreactivity in human trigeminal ganglion and nucleus.

Glial cell line-derived neurotrophic factor (GDNF) is shown by immunohistochemistry in human trigeminal sensory system from 22 weeks of gestation to adulthood. In the trigeminal ganglion, a distinct subpopulation of GDNF-positive neurones is observed, which amounts to about 15% at early pre-term and adult ages and peaks to around 30% at perinatal ages. Labelled neurones are mostly small- and medium-sized. Occasionally, Schwann and satellite cells are stained. GDNF/substance P (SP) and GDNF/calcitonin gene-related peptide (CGRP) double stained neurones occur at all ages examined, whereas GDNF/trkA coexistence can be observed in pre- and full-term newborns only. Centrally, GDNF-immunostained fibers and terminal-like structures are mainly restricted to the spinal trigeminal nucleus, where they are codistributed with SP and CGRP. In the subnucleus caudalis, positive neurones can also be observed both in the superficial laminae and in the magnocellular part, with higher frequency in adults. These results suggest that GDNF may play a functional role in human trigeminal primary sensory neurones throughout life and provide indication for its possible involvement in the regulation of pain-related neuronal circuits in human trigeminal sensory system.

Effect of discrete kainic acid-induced lesions of corpus caudatus and globus pallidus on glutamic acid decarboxylase of rat substantia nigra.

Locally applied kainic acid was used in order to destroy pallidal perikarya without damaging axons en passage, in an effort to clarify the role of the globus pallidus as a source of nigral GABAergic terminals. Rats were microinjected unilaterally with kainic acid in the globus pallidus, head, body and tail of the caudate and were sacrificed 7 days later. The forebrain of each rat was examined histologically in order to establish the extent of the lesion and nigral glutamate decarboxylase (GAD) was assayed as a marker of GABAergic terminals. Kainic acid produced in the globus pallidus loss of neuronal perikarya and reactive gliosis. Large multipolar neurons of the globus pallidus were characteristically absent on the lesioned-side. Lesions of the pallidum resulted in a non-significant (5.5%) reduction of nigral GAD. Kainate lesions restricted to the head of the caudate resulted in a significant (19%) drop of nigral GAD, while lesions of the caudate body provided the largest reductions of nigral GAD (53%). Lesions of the caudate tail were without effect. The results indicate that nigral GAD arises mostly from the body and, in part, also from the head of the caudate but not from the globus pallidus or from the tail of the caudate.

Ipsiversive turning behaviour after discrete unilateral lesions of the dorsal mesencephalic reticular formation by kainic acid.

Discrete axon-sparing lesions were placed unilaterally in the mesencephalon by the local injection of kainic acid. Unilateral lesions of the dorsal reticular formation just beneath the superior colliculus and lateral to the periacqueductal grey resulted in consistent ipsiversive apomorphine-induced circling. Only weak motor asymmetries were observed after unilateral lesions restricted to the superior colliculus or to the periacqueductal grey. The results indicate that the dorsal mesencephalic reticular formation plays an essential role as an output station for striatal postural functions.

Evidence for the presence of substance P-like immunoreactivity in the human cerebellum.

Preliminary results on the localization of substance P-like immunoreactivity in the human cerebellum are presented. Cerebella from newborn and adult subjects were examined. While only sporadic positive fibres were detected in the adult tissue, the immunoreactive material appeared more abundant in the cerebella from newborn subjects. Varicose and non-varicose fibres and dot-like nerve terminals were present with different density in various regions. The paucity of immunoreactive perikarya suggests that most of the cerebellar substance P-like immunoreactive material has an extrinsic origin.