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.

Bortezomib treatment produces nocifensive behavior and changes in the expression of TRPV1, CGRP, and substance P in the rat DRG, spinal cord, and sciatic nerve.

To investigate neurochemical changes associated with bortezomib-induced painful peripheral neuropathy (PN), we examined the effects of a single-dose intravenous administration of bortezomib and a well-established "chronic" schedule in a rat model of bortezomib-induced PN. The TRPV1 channel and sensory neuropeptides CGRP and substance P (SP) were studied in L4-L5 dorsal root ganglia (DRGs), spinal cord, and sciatic nerve. Behavioral measures, performed at the end of the chronic bortezomib treatment, confirmed a reduction of mechanical nociceptive threshold, whereas no difference occurred in thermal withdrawal latency. Western blot analysis showed a relative increase of TRPV1 in DRG and spinal cord after both acute and chronic bortezomib administration. Reverse transcriptase-polymerase chain reaction revealed a decrease of TRPV1 and CGRP mRNA relative levels after chronic treatment. Immunohistochemistry showed that in the DRGs, TRPV1-, CGRP-, and SP-immunoreactive neurons were mostly small- and medium-sized and the proportion of TRPV1- and CGRP-labeled neurons increased after treatment. A bortezomib-induced increase in density of TRPV1- and CGRP-immunoreactive innervation in the dorsal horn was also observed. Our findings show that bortezomib-treatment selectively affects subsets of DRG neurons likely involved in the processing of nociceptive stimuli and that neurochemical changes may contribute to development and persistence of pain in bortezomib-induced PN.

The characteristics of neuronal injury in a static compression model of spinal cord injury in adult rats.

Studies of spinal cord injury using contusion (impact) injury paradigms have shown that neuronal death is an acute event that is largely over within 24 h. However, much less is known about cell death following compression injury, despite compression being a key component of natural spinal injuries. We have therefore used neuronal nuclei (NeuN) immunostaining to examine the spatiotemporal pattern of neuronal loss after static compression injury in adult rats. 3D reconstruction was used to reveal the full effect of the injury. Neuronal loss at the injury epicentre, assessed by NeuN immunostaining, amounted to 44% at 1 day but increased to 73% at 3 days and 81% at 1 month. Neuronal loss was also seen 5 mm rostral and caudal to the epicentre, but was not significant until 3 days. NeuN loss was greatest in the ventral horns and in the intermediate grey matter, with the lateral dorsal horns relatively spared. Cystic cavities formed after injury, but were not evident until 4 weeks and were small in size. In contrast to the slow profile of neuronal loss, the compression injury also evoked a transient expression of activating transcription factor-3 (ATF3) and activated c-Jun in neurons. ATF3 expression peaked at 3 days and declined at 7 days. Our spatiotemporal analysis of compression injury shows that neuronal loss is much more protracted than in contusion injury, and highlights the potential for neuroprotective strategies. This study is also the first indication of ATF3 involvement in spinal cord injury.

Somatostatin- and Substance P-ergic neurons and Glial cell line-derived neurotrophic factor in the human archicortex.

By means of immunohistochemistry, the localization of Somatostatin (SOM)- and Substance P (SP)-ergic neuronal populations was compared to the occurrence of Glial cell line-derived neurotrophic factor (GDNF) in the human hippocampus from prenatal to adult life stages. The results obtained i) confirm previous reports on the distribution of SOM and SP; ii) show that GDNF-like immunoreactivity occurs in an ample population of hippocampal neurons, with a main location in the pyramidal cells; iii) identify regions of codistribution of either neuropeptide with GDNF-positive elements. Although coexistence of GDNF with SOM or SP was not detected, the possibility that the trophic factor may act on the neuropeptide-containing neurons can be envisaged and is worth further analysis.

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.

Neurotrophin-like immunoreactivity in the human hippocampal formation.

The immunohistochemical occurrence and localisation of the neurotrophins nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-4, and neurotrophin-3 is described in the human post-mortem hippocampal formation from subjects aged 23 weeks of gestation to 68 years. Labeled neuronal cell bodies and processes were detectable for each neurotrophin at all examined ages with age-related changes in their distribution pattern. As a general rule, a higher number of immunoreactive perikarya was found in subjects at pre- and perinatal ages than in adults. At variance with the other neurotrophins, the BDNF antiserum labelled also extensive nerve fibre systems, whose occurrence and distribution widened with age. The results obtained provide a morphological ground in support to the concept that the neurotrophins play a functional role in the human hippocampal circuitry throughout life.

Haloperidol does not produce dopamine cell depolarization-block in paralyzed, unanesthetized rats.

A widely accepted theory postulates that chronic treatment with neuroleptics causes, in rats, the depolarization block of the majority of midbrain dopamine (DA) neurons. However, we reported that such treatment fails to reduce the number of spontaneously active DA neurons when the neuronal sampling is performed in the d-tubocurarine-paralyzed instead of chloral-hydrate anesthetized preparation. The present experiments were aimed at verifying whether the negative results might be due to the use of d-tubocurarine as paralyzing agent. Rats were chronically treated with haloperidol (0.5 mg kg-1 i.p., daily) for 3 to 4 weeks. Two to three hours after the last injection, the number of spontaneously active DA neurons in the ventral tegmental area (VTA) were sampled, and their discharging characteristics analyzed, both in animals under chloral hydrate anesthesia and in rats immobilized either with d-tubocurarine, gallamine or succinylcholine. The results indicate that chronic treatment with haloperidol reduced the number of spontaneously active VTA-DA neurons by about 65% in animals under chloral hydrate anesthesia, but failed to modify the number of spontaneously firing DA neurons in rats immobilized with d-tubocurarine, gallamine or succinylcholine. The results indicate that the depolarization block of DA neurons does not occur in the paralyzed preparation and raise doubts about the presence of this phenomenon in the intact non- anesthetized unrestrained animal.

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.

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.

Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy.

Dorsal root ganglia (DRG) neuronopathy was induced in rats by chronic treatment (2 mg/kg twice a week for nine injections) with the antineoplastic drug cisplatin. Morphological alterations and changes in peptide [calcitonin gene-related peptide (CGRP), substance P, galanin (Gal), and somatostatin] concentration were studied in the DRG, the spinal cord, and the sciatic nerve. Peptide concentration was increased in DRG neurons, with CGRP and Gal showing the highest increase. Conversely, in the sciatic nerve there was a general decrease in peptide content. In DRG a reduction in the nuclear, cytoplasmic, and nucleolar areas of primary sensory neurons was evident and was accompanied by clear-cut aspects of nucleolar structural damage. In peripheral nerves only extensive morphometric determinations could evidence a reduction in nerve conduction velocities and impairment in pain detection and coordination. Some of the nerve fibers presented axonal and adaxonal accumulations, suggesting the presence of an axonopathy. These results confirm that DRG cells are the primary target of cisplatin-induced neurotoxicity. Milder alterations can be detected in peripheral nerves. The increase in peptide concentration in DRG is probably due to cisplatin-related damage to the axonal transport system rather than to an increased synthesis.

GAP-43 in the spinal trigeminal and dorsal column nuclei of the newborn and adult man: immunohistochemical distribution and comparison with that of the neuropeptides SP and CGRP.

By means of immunohistochemistry the presence of the growth-associated protein GAP-43 and its codistribution with substance P (SP) and calcitonin gene-related peptide (CGRP) are studied in the human spinal trigeminal, gracile, and cuneate nuclei at perinatal and adult life stages. The results obtained show that the distribution pattern of GAP-43 in the areas examined varies with age and that the immunohistochemical detectability of the protein persists in discrete subregions of the trigeminal and cuneate nuclei of the adult, where its localization closely matches that of SP and CGRP. It is suggested that neuronal plasticity may be pronounced throughout life in areas of the human nervous system involved in the neurotransmission of protopathic stimuli at the first synaptic level. Discrete subregions of the cuneate nucleus, bearing neurochemical characteristics strikingly similar to those of the substantia gelatinosa of the trigeminal subnucleus caudalis are pointed out.

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.

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.

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.

Selective MPP+ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity.

1. In the present study we provide evidence for a saturable, Mg2+/ATP- and temperature-dependent, tetrabenazine-, dopamine-, and amphetamine-sensitive uptake of 1-methyl-4-phenylpyridinium ion (MPP+) in synaptic vesicles from mouse striatum. 2. Similarity in the properties of the vesicular uptake suggests that in the striatum dopamine and MPP+ share the vesicular carrier. 3. The presence of MPP+ vesicular uptake in dopamine-rich regions such as striatum, olfactory, tubercles and hypothalamus, as well as its absence in cerebellum, cortex and pons-medulla, suggest that monoamine vesicular carriers differ between highly and poorly dopamine-innervated regions. 4. The restriction of active MPP+ uptake to the dopaminergic regions, which reflects the previously shown distribution of [3H]-MPP+ binding sites in mouse brain membranes, indicates MPP+ as a marker of the vesicular carrier for dopamine in dopaminergic neurones. 5. A role in MPP+ neurotoxicity is suggested for this region-specific, vesicular storage of the toxin.

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.