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In this study co-localization of galanin- with calcitonin gene-related peptide (CGRP)-like immunoreactivity was examined in dorsal root ganglion neurons 14 days after sciatic nerve cut using a laser scanning confocal microscope. CGRP- and galanin-like immunoreactivities were also analysed in the dorsal horn of the spinal cord of these animals with immunofluorescence microscopy. The ultrastructural changes in galanin-immunoreactive, presumably primary afferent terminals in the superficial dorsal horn, were studied as well as the relationship between galanin-, substance P- and CGRP-like immunoreactivities in primary afferent terminals. Local galanin-positive neurons in lamina II were also analysed after peripheral axotomy. Under the confocal microscope, CGRP-like immunoreactivity was located in the perinuclear region, probably the Golgi complex, and in dot-like structures, probably representing large dense-core vesicles, in normal dorsal root ganglion neurons. However, after peripheral axotomy CGRP was mainly detected in dot-like structures. Only a slight decrease in percentage of CGRP neurons in dorsal root ganglion was seen after axotomy, and about 84% of the galanin-positive neurons contained CGRP. The field of galanin-positive nerve fibres in the superficial lumbar (L)4 and L5 dorsal horn expanded and the intensity of staining for CGRP was reduced in these regions 14 days after sciatic nerve cut. Using pre-embedding immunoelectron microscopy, several morphological changes were observed in galanin-positive terminals in laminae I and II ipsilateral to the lesion. Most importantly, the most frequently occurring type of galanin-positive terminals (type 1) showed distinct changes with a granular matrix, many immunoreactive, peripherally located large dense-core vesicles, empty large vesicles and synaptic vesicles which were displaced from the presynaptic zone. Other galanin-positive terminals underwent even more pronounced morphological changes, including extensive vesiculolysis, also of large dense-core vesicles, filamentous degeneration or formation of axonal labyrinths. An increased number of galanin-positive nerve terminals was observed in lamina III of the ipsilateral dorsal horn after axotomy. They did not form glomeruli and contained few large dense-core vesicles. Post-embedding immunocytochemistry combined with quantitative analysis revealed that significant changes occurred in a proportion of terminals also with regard to peptide content in large dense-core vesicles after axotomy. Thus, the percentage of galanin-positive large dense-core vesicles increased in several cases and that of substance P- and CGRP-immunoreactive ones decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Using immunofluorescence histochemistry and pre- and post-embedding immunoelectron microscopy the rat lumbar dorsal horn was analysed in normal rats and 14 days after unilateral transection of the sciatic nerve. A marked increase in neuropeptide Y-like immunoreactivity was observed in the ipsilateral, superficial dorsal horn, especially in laminae III and IV, of the lumbar 4-5 spinal cord segments after peripheral axotomy. In the ipsilateral lamina II two types of neuropeptide Y-immunoreactive, presumably primary afferent terminals could be identified at the ultrastructural level. The first type contained many large dense-core vesicles (100-155 nm in diameter), whereas a second, more common type had only a few and smaller large dense-core vesicles (80-100 nm in diameter), plus synaptic vesicles of varying diameter (50-85 nm), large empty vesicles and tubular structures. Only occasionally were neuropeptide Y-positive terminals in lamina II involved in the formation of axonal labyrinths. In the ipsilateral lamina III, the number of neuropeptide Y-positive nerve terminals markedly increased after axotomy, with a moderate increase in lamina IV. These neuropeptide Y-positive terminals were morphologically similar to the second type of neuropeptide Y-positive terminal in lamina II, i.e. contained many synaptic vesicles (45-50 nm in diameter), a few small large dense-core vesicles (80-100 nm in diameter), electron-dense granular matrix and a few tubular structures. Fusion of synaptic vesicles with the plasma membrane was often observed at these synapses. These terminals frequently formed glomeruli but were not involved in axonal labyrinths. With regard to local neurons, neuropeptide Y-like immunoreactivity was observed in many dendrite-like profiles mostly making synaptic contacts with neuropeptide Y-negative dendrites and only rarely contacting the central terminal of the glomeruli. Neuropeptide Y-positive nerve endings were mainly seen in lamina I and the outer third of lamina II. After peripheral axotomy the number of vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive terminals was increased in laminae I and II. They contained many large dense-core vesicles (100-120 nm in diameter), and some of them were positive for vasoactive intestinal polypeptide/peptide histidine isoleucine. Morphologically, the terminals were characterized by a granular matrix, tubular structures, empty vesicles, reduction in synaptic vesicles and absence of postsynaptic densities. Vasoactive intestinal polypeptide/peptide histidine isoleucine-like immunoreactivities were often found in association with labyrinth formation.(ABSTRACT TRUNCATED AT 400 WORDS)

A human genomic clone encompassing exons 1-3 of the neurotensin/neuromedin N gene was identified using a canine neurotensin complementary DNA probe. Sequence comparisons revealed that the 120-amino acid portion of the precursor sequence encoded by exons 1-3 is 89% identical to previously determined cow and dog sequences and that the proximal 250 bp of 5' flanking sequences are strikingly conserved between rat and human. The 5' flanking sequence contains cis-regulatory sites required for the induction of neurotensin/neuromedin N gene expression in PC12 cells, including AP1 sites and two cyclic adenosine-5'-monophosphate response elements. Oligonucleotide probes based on the human sequence were used to examine the distribution of neurotensin/neuromedin N messenger RNA in the ventral mesencephalon of schizophrenics and age- and sex-matched controls. Neurotensin/neuromedin N messenger RNA was observed in ventral mesencephalic cells some of which also contained melanin pigment or tyrosine hydroxylase messenger RNA. Neurons expressing neurotensin/neuromedin N messenger RNA were observed in the ventral mesencephalon of both schizophrenic and non-schizophrenic humans.

Two compounds, [D-Arg]kyotorphin and thiorphan, unique in their mechanisms for producing an opioid-like analgesic response, were infused for six days into the lateral cerebral ventricle of rats. [D-Arg]kyotorphin (62.5 micrograms/microliter per h) and thiorphan (75 micrograms/5 microliter per h), but not vehicle infused animals, displayed certain behaviors characteristic of precipitated morphine withdrawal upon naloxone (10 mg/kg i.p.) challenge. Acute intracerebroventricular (i.c.v.) administration of [D-Arg]kyotorphin (62.5 micrograms/5 microliter) or thiorphan (75 micrograms/5 microliter) and subsequent challenge with naloxone resulted in no abnormal behavior. These data indicate that compounds which produce their analgesic effect through the modulation of endogenous opioids can cause physical dependence.

The cholecystokinin (CCK) antagonist, proglumide, administered chronically (41.0-53.5 mg/kg per day, 14 days) to rats via osmotic mini-pumps, produced a significant 13% increase in the number of [3H]spiperone labeled binding sites (Bmax) in the striatum. There was no associated change in the affinity (Kd) of [3H]spiperone for the striatal binding sites. Given chronically at lower dose levels (10.4-13.6 or 21.8-29.8 mg/kg per day), or acutely in doses of 10, 20 or 40 mg/kg s.c., proglumide failed to alter the binding of [3H]spiperone to rat striatal tissue. These data indicate long-term proglumide administration increases the number of binding sites for [3H]spiperone, thought to be a ligand for dopamine D-2 receptors.

The effects of depletion of somatostatin in the region of the retrorubral field on in vivo tyrosine hydroxylation in the A8 cell group were assessed. Local injections of cysteamine into the central amygdaloid nucleus, source of a somatostatin input to the A8 cell group region, or directly into the retrorubral field resulted in an increase in in vivo tyrosine hydroxylation in the A8 cell group. Levels of somatostatin-like immunoreactivity in the retrorubral field were decreased by approximately 50% by the cysteamine treatment. These data suggest that somatostatin may hold A8 dopamine neurons under tonic inhibition.