Marcelo J. Villar

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin.

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy

The L4 and L5 dorsal root ganglia were studied in untreated rats and rats subjected to unilateral transection of the sciatic nerve, using the indirect immunofluorescence technique and antibodies to the peptide galanin (GAL). In control rats only low numbers of small ganglion cells contained GAL-like immunoreactivity (LI). After axotomy a marked increase in the number and intensity of GAL-immunoreactive ganglion cell bodies was seen on the lesion side. Thus, some primary sensory neurons react to transection of their peripheral branches by expressing increased GAL levels. A similar reaction has been described by other groups for vasoactive intestinal polypeptide.

Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels.

SummaryIn situ hybridization histochemistry and indirect immunofluorescence histochemistry were used to study changes in the expression of vasopressin (VP), oxytocin (OXY), tyrosine hydroxylase (TH), galanin (GAL), dynorphin (DYN) and cholecystokinin (CCK) in hypothalamic magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of rats. After prolonged administration of 2% sodium chloride as drinking water (salt-loading), the treatment increased the levels of VP, OXY, TH, GAL, DYN and CCK mRNA in the PVN and SON. The increase in CCK mRNA was, however, proportionally higher in the PVN than in the SON. Within cell bodies of the PVN and SON of salt-loaded rats, a depletion of VP- and OXY-like immunoreactivity (LI) and an increase in TH-LI were seen. In salt-loaded/colchicine-treated rats, a marked decrease in GAL- and DYN-LI, but no specific changes in CCK-LI were observed. Within nerve fibers of the posterior pituitary of salt-loaded rats, a marked depletion of VP-, GAL- and DYN-LI was found. Less pronounced depletion was observed in OXY- and CCK-LI, and no specific changes in TH-LI were seen. The results show that high plasma osmolality induces increased mRNA levels for VP, OXY, TH, GAL, DYN and CCK, presumably indicating increased synthesis, an increased export from cell somata of VP, OXY, GAL and DYN, and a decrease in levels of these peptides in the posterior pituitary, suggesting increased release. The catecholamine-synthesizing enzyme TH, however, which has a cytoplasmic localization and is not released from nerve endings, remains high in the cell bodies and nerve endings during this state of increased activity.

Further studies on galanin-, substance P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: Effects of dorsal rhizotomies and sciatic nerve lesions

The peptides galanin (GAL), substance P (SP), and calcitonin gene-related peptide (CGRP) were analyzed with immunohistochemistry and radioimmunoassay in the spinal cord, dorsal root ganglia, dorsal roots, and sciatic nerve of normal rats and rats subjected to several experimental procedures, including ligation, crush, and/or sectioning of nerves. The results show that peripheral nerve transection induces a dramatic increase in GAL content both in dorsal roots and sciatic nerve, demonstrating that this lesion causes an increased out-transport of the newly synthesized peptide both into the central and peripheral branches of the primary sensory neurons. In contrast evidence was obtained for decreased out-transport of SP and CGRP. The functional significance of these findings remains to be analyzed.

Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: An immunohistochemical study using experimental manipulations

Indirect immunofluorescence histochemistry was used to investigate the distribution and extent of co-localization of chemical messengers in magnocellular neurons of the supraoptic and paraventricular nuclei. In order to increase the number of neurons immunoreactive to the antisera used, experimental manipulations were employed. The homozygous Brattleboro (diabetes insipidus) rat was also investigated. In untreated rats, only vasopressin- and oxytocin-like immunoreactivities could be observed. Colchicine treatment alone resulted in appearance of galanin-, dynorphin-, cholecystokinin-, [Leu]enkephalin- and thyrotropin-releasing hormone-positive cells. In hypophysectomized rats, all these markers, except tyrosine hydroxylase, showed substantial further increases. In addition, peptide histidine-isoleucine-immunoreactive cell bodies could now be seen. After salt-loading alone, tyrosine hydroxylase-like immunoreactivity was markedly increased, whereas vasopressin- and oxytocin-like immunoreactivity were very weak or undetectable. When salt-loaded rats received colchicine, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity in addition increased, whereas galanin- and dynorphin-like immunoreactivity markedly decreased. The Brattleboro rats resembled untreated rats, except their lack of vasopressin-like immunoreactivity, the marked increase in tyrosine hydroxylase-like immunoreactivity, and smaller increase in galanin- and dynorphin-like immunoreactivity. Addition of colchicine to Brattleboro rats resulted in some distinct further changes in that dynorphin-like immunoreactivity decreased in some neurons and that [Leu]enkephalin-, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity increased substantially. Several similarities could be observed between the salt-loaded and Brattleboro rats, with or without colchicine. However, a marked difference in immunoreactive [Leu]enkephalin levels was observed with no difference in dynorphin-like immunoreactivity, and opposite changes in galanin-like immunoreactivity. The results confirm the traditional view that hypothalamic magnocellular neurons in the supraoptic and paraventricular nuclei contain two separate cell populations, characterized by vasopressin and oxytocin, respectively, and that they contain additional messenger molecules in specific patterns. Vasopressin-containing neurons primarily express tyrosine hydroxylase, galanin, dynorphin, [Leu]enkephalin and peptide histidine-isoleucine, and to a minor extent cholecystokinin and thyrotropin-releasing hormone. Oxytocin-containing neurons mainly have cholecystokinin and corticotropin-releasing factor, and to a minor extent galanin, dynorphin, [Leu]enkephalin and thyrotropin-releasing hormone. Furthermore, our results detail individual co-existence situations among these putative messenger molecules. Thus, magnocellular neurons respond in a differential way to various stimuli and they store multiple bioactive substances in specific combinations.

The effects of intrathecal galanin and C-fiber stimulation on the flexor reflex in the rat

Galanin (GAL) was applied intrathecally (i.t.) at the lumbar level in decerebrate, spinalized, unanesthetized rats. GAL had no effect on the amplitude of the monosynaptic reflex over a wide concentration range, but at low concentrations if briefly facilitated the flexor reflex and at higher concentrations the facilitation was sometimes followed by a depression. GAL decreased the facilitatory effect of a conditioning stimulus train to C-fibers in the sural nerve. The depressive effect of GAL could be prevented by the i.t. coadministration of calcitonin gene-related peptide (CGRP), but not substance P (SP) and was not reversed by i.t. naloxone or bicuculline. The results illustrate the complex effect of GAL on the spinal cord, possibly exhibiting a biphasic effect. The observed effects on the flexor reflex are probably not due to changes in the excitability of motoneurons. Descending inhibitory pathways or local inhibitory non-GAL interneurons probably are not involved in the depressive effect of GAL. The possibility that the observed effects are related to primary sensory afferents containing not only GAL but also CGRP, and/or to local GAL neurons in the dorsal horn is discussed.

On the Role of Galanin, Substance P and Other Neuropeptides in Primary Sensory Neurons of the Rat: Studies on Spinal Reflex Excitability and Peripheral Axotomy.

The interaction of intrathecally (i.t.) applied galanin (GAL) with substance P (SP), calcitonin gene‐related peptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) and C‐fibre conditioning stimulation (CS) with regard to their effects on the spinal nociceptive flexor reflex was studied in decerebrate, spinalized, unanaesthetized rats with intact or sectioned sciatic nerves. SP, CGRP, VIP and SOM applied onto the surface of lumbar spinal cord or a brief CS train (1 Hz, 20 s) to the sural nerve facilitated the flexor reflex for several minutes in animals with intact or sectioned nerves. Pretreatment with GAL, which by itself had a biphasic effect on the flexor reflex in a dose‐dependent manner, antagonized the reflex facilitation induced by sural CS before and after sciatic nerve section. SP‐induced facilitation of the flexor reflex was antagonized by GAL in rats with intact sciatic nerves, but not after nerve section. In contrast, VIP‐induced reflex facilitation was antagonized by GAL only after sectioning of the sciatic nerve. GAL was effective in antagonizing the facilitatory effect of CGRP under both situations, but had no effect on SOM‐induced facilitation. A parallel immunohistochemical study revealed that after sciatic nerve section GAL‐like immunoreactivity (LI) and VIP‐LI are increased in the dorsal root ganglia and that these two peptides coexist in many cells. The present results indicate that GAL antagonizes the excitatory effect of some neuropeptides which exist in the spinal cord. This antagonism could explain the inhibitory effect of GAL on C‐fibre CS‐induced facilitation of the flexor reflex, which is presumably due to the release of some of these neuropeptides from the terminals of primary afferents. Furthermore, the interaction between GAL and other neuropeptides is altered by sciatic nerve section, paralleling changes in the levels of these neuropeptides in primary afferents and their pattern of coexistence after nerve section. It is proposed that SP and CGRP are important mediators of the spinal flexor reflex in intact rats. However, after axotomy VIP may replace SP in this capacity, paralleling the decrease in SP and marked increase in VIP levels. In general the study provides further support for involvement of peptides in sensory function.

Distribution of D4 dopamine receptor in rat brain with sequence-specific antibodies

The distribution of the dopaminergic D4 receptor in rat brain was studied employing site directed polyclonal antibodies. Antisera were raised in rabbits to two oligopeptides corresponding to amino acids 160-172 of the second extracellular loop (P1) and amino acids 260-273 of the third intracellular loop (P2) of the D4 receptor sequence. Affinity-purified antibodies (anti-P1 and anti-P2) specifically recognized two major bands of 42-45 and 95 kDa in Western blots of denatured preparations of various rat brain areas. Immunocyto-chemistry studies showed that D4 receptor is widely distributed in rat central nervous system (CNS) showing higher labelling in the hippocampus (CA1, CA2, CA3 and dentate gyrus) frontal cortex, entorhinal cortex, caudate putamen, nucleus accumbens, olfactory tubercle, cerebellum, supraoptic nucleus and sustancia nigra pars compacta. In addition, anti-P1 decreased the binding of the antagonist [3H]YM-09151-2 selective for D2, D3 and D4 receptors but did not modify the binding of [3H]raclopride an antagonist selective for D2 and D3, in striatal synaptosomes. Anti-P2 did not modify the binding of these ligands. These results confirm the selectivity of the antibodies towards the D4 receptor and suggest that the binding site for the antagonists might be located at or close to the second extracellular loop of the protein sequence. D4 receptor protein is mainly expressed in plasma membranes and in the peripheral cytoplasm of neurons and is more widely distributed than was originally proposed based on mRNA localization, since it is present both in limbic, diencephalic and motor areas of rat brain.

Nitric oxide synthase increases in hypothalamic magnocellular neurons after salt loading in the rat. An immunohistochemical and in situ hybridization study

Magnocellular hypothalamic neurons of the paraventricular (PVN) and supraoptic (SON) nuclei have been shown to contain a wide variety of messenger molecules in addition to vasopressin and oxytocin, including the nitric oxide (NO)-synthesizing enzyme (NOS). In this paper we have investigated the effects of salt loading on the expression of NOS by means of immunohistochemistry and in-situ hybridization. The results show an increase in the number of NOS-immunoreactive (IR) neurons both in the PVN and the SON after 5 and 14 days of salt loading. Several of these neurons were double labelled with vasopressin antiserum. In situ hybridization showed a marked increase in the number of neurons expressing NOS mRNA and a stronger signal in individual neurons. The present results suggest a role for NO in the magnocellular hypothalamic system after salt loading.

Intrathecal galanin potentiates the spinal analgesic effect of morphine: Electrophysiological and behavioural studies

The interaction between intrathecally (i.t.) applied galanin (GAL) and morphine was examined in electrophysiological and behavioural experiments. The physiological experiments were performed on decerebrate, spinalized, unanesthetized rats where the effects of i.t. GAL and morphine on the hamstring flexor reflex were studied. In the behavioural experiments sensitivity to noxious thermal stimulation was assessed on the hot plate test in rats injected with GAL and morphine via chronically implanted i.t. catheters. GAL at 100 ng in 10 microliters, which by itself has no depressive effect, potentiated the depressive effect of morphine on the flexor reflex. In the behavioral study the same dose of GAL potentiated the antinociceptive effect of morphine on the hot plate test without having an analgesic effect by itself. It is suggested that GAL may enhance the analgesic effect of opiates in the spinal cord.