Preprotachykinin Messenger RNA Research Articles

Differential effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNA expression in unique regions of the striatum

This study employed in situ hybridization to directly compare the effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNAs in distinct striatal regions. Male, Sprague–Dawley rats received a single administration of 15 mg/kg methamphetamine (s.c.) or 30 mg/kg cocaine (i.p.) and were killed 30 min or 3 h later. Methamphetamine and cocaine produced significant increases in preprotachykinin messenger RNA in the striatum after 3 h, but often in different subregions. Both drugs produced similar effects on preprotachykinin messenger RNA in the rostral striatum. However, methamphetamine produced significant increases in all regions of the caudal striatum, whereas cocaine-induced preprotachykinin messenger RNA expression was limited to dorsal regions of this striatal area. Methamphetamine also produced a significant increase in preprotachykinin messenger RNA in the caudal striatum after 30 min, whereas cocaine had no significant effect on preprotachykinin messenger RNA at this early time-point. The pattern of changes in neurotensin/neuromedin N messenger RNA caused by methamphetamine and cocaine after 3 h was even more distinct. Cocaine produced significant increases in neurotensin/neuromedin N messenger RNA in all regions of the rostral striatum, whereas methamphetamine had no effect in these areas. Furthermore, in more caudal sections, cocaine predominantly affected neurotensin/neuromedin N expression in dorsal aspects of the striatum, whereas methamphetamine significantly increased neurotensin/neuromedin N messenger RNA in all regions. There was much less effect of either drug on neuropeptide expression in the nucleus accumbens. The only significant effect was an increase in neurotensin/neuromedin N messenger RNA in the core region 3 h after methamphetamine administration. These results indicate that methamphetamine and cocaine increase preprotachykinin and neurotensin/neuromedin N messenger RNAs in distinct regions of the striatum. The ability of methamphetamine and cocaine to alter neuropeptide messenger RNA expression in unique regions of the striatum may be important for the long-term effects of these drugs, such as sensitization, since the striatum is not homogeneous in its connections and function.

Toluene diisocyanate enhances substance P in sensory neurons innervating the nasal mucosa.

Inhalation of irritants, such as toluene diisocyanate (TDI), stimulates substance P (SP) release from peripheral processes of sensory neurons innervating the airways. The purpose of this study was to determine if TDI inhalation affects intraneuronal levels of SP and preprotachykinin (PPT) messenger RNA (mRNA) in the sensory neurons of the trigeminal ganglion (TG) which innervate the nasal epithelium. The nasal cavity of Fisher-344 rats was instilled with rhodamine-labeled latex microspheres. Ten days later, the rats were exposed to 60 ppb of 2,4-2,6-TDI vapor for 2 h. The TG were removed 1, 12, 24, 48, 72, and 96 h after TDI treatment and prepared for SP immunocytochemistry and PPT in situ hybridization. SP nerve fiber density in nasal epithelium was significantly increased 12, 24, and 48 h after TDI exposure. The proportion of microsphere-labeled cell bodies expressing high levels of SP immunoreactivity was decreased at 24 h but was increased above controls at 48 and 72 h. The proportion of microsphere-labeled cell bodies expressing high levels of PPT mRNA was increased above control levels at 24 and 48 h. The percentage of leukocytes observed in nasal lavage fluid was significantly increased 12, 24, 48, and 72 h after inhalation. These studies indicate that SP production in TG neurons projecting to the nasal epithelium is transiently increased after TDI exposure, suggesting that TDI inhalation not only causes SP release but also increased intraneuronal neuropeptide levels. Increased neuronal SP levels may be involved in maintaining neurogenic inflammation or the development of airway hyperresponsiveness.

Serotonin-2 receptor stimulation normalizes striatal preprotachykinin messenger RNA in an animal model of Parkinson's disease

Dopamine and serotonin neurotransmission regulate striatal preprotachykinin messenger RNA levels. In the present study, we investigated serotonin 2A/2C receptor-mediated regulation of preprotachykinin messenger RNA expression in the rat striatum after adult dopamine depletion produced with 6-hydroxydopamine. Significant reductions (46–61% of control values) in preprotachykinin messenger RNA levels were detected by in situ hybridization in rostral, central and caudal regions of the striatum after >85% dopamine depletion. Repeated administration of the specific serotonin 2A/2C receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine hydrobromide, to dopamine-depleted rats completely reversed the reduction in preprotachykinin messenger RNA levels in rostral, central and dorsal-caudal striatal regions. In unlesioned (vehicle-injected) control animals, repeated administration of (±)-2,5-dimethoxy-4-iodoamphetamine hydrobromide did not affect preprotachykinin messenger RNA expression in rostral, central and ventral-caudal striatal regions, but decreased preprotachykinin messenger RNA levels in the dorsal-caudal striatal subregion. In addition, serotonin turnover in the dopamine-depleted rostral striatum was significantly increased by 35–45% which is consistent with serotonin hyperinnervation after 6-hydroxydopamine lesions. These data show that the decrease in striatal preprotachykinin messenger RNA after dopamine depletion can be normalized with repeated serotonin 2A/2C receptor stimulation. We hypothesize that this serotonin 2A/2C receptor regulation of preprotachykinin messenger RNA expression after 6-hydroxydopamine is a consequence of serotonin hyperinnervation, which may include increased striatal serotonin 2A/2C receptors, induced by dopamine depletion. We also propose that the serotonin system could be pharmacologically targeted to restore the direct striatal tachykinin pathway in Parkinson's disease.

Opiate receptors modulate estrogen-induced cholecystokinin and tachykinin but not enkephalin messenger RNA levels in the limbic system and hypothalamus

Cholecystokinin, substance P and methionine enkephalin all regulate the display of reproductive behaviour. Their expression is exquisitely regulated by estrogen in the limbic–hypothalamic circuit, a circuit that regulates the display of estrogen-sensitive female reproductive behaviour. Relatively little is known, however, about the interaction of endogenous opioid peptides with cholecystokinin and substance P in the limbic–hypothalamic circuit. Opiates antagonize the release of cholecystokinin and substance P in the hypothalamus and periaqueductal gray and stimulate cholecystokinin messenger RNA levels in the amygdala. To determine the effect of endogenous opioid input on estrogen-induced cholecystokinin, enkephalin and substance P expression, in situ hybridization histochemistry was used to examine estrogen-induced messenger RNA levels of these neuropeptides in specific nuclei of the limbic system and hypothalamus in the presence of opiate receptor antagonists. Estrogen treatment of ovariectomized rats significantly elevated cholecystokinin messenger RNA levels in the central portion of the medial preoptic nucleus, the encapsulated portion of the bed nucleus of the stria terminalis and the posterodorsal medial amygdala, as well as increased preproenkephalin and preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus and the posterodorsal medial amygdala. The universal opiate receptor antagonist naltrexone and the δ-opiate receptor antagonist naltrindole each potentiated the estrogen-induced increase and elevated cholecystokinin messenger RNA levels an additional 1.9- to 2.8-fold depending on the nucleus examined, but had no effect on the estrogen-induced expression of either preproenkephalin or preprotachykinin messenger RNA. β-Funaltrexamine, a μ-opiate receptor antagonist, had no effect on the medial preoptic or medial amygdaloid cholecystokinin messenger RNA levels or on the estrogen-induced expression of preproenkephalin messenger RNA but did cause a decrease in estrogen-induced cholecystokinin messenger RNA levels in the bed nucleus of the stria terminalis and a decrease in the preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus. These results indicate that endogenous opioids, acting on the δ-opiate receptor within nuclei of the limbic–hypothalamic circuit, restrain the estrogen-induced increase of cholecystokinin messenger RNA expression. Activation of the μ-opiate receptor, however, may facilitate cholecystokinin messenger RNA expression in the bed nucleus of the stria terminalis and preprotachykinin messenger RNA expression in the ventromedial hypothalamic nucleus. Thus, endogenous opioid peptides may act in a site- and receptor-specific manner to modulate estrogen-induced neuropeptide levels in the limbic system and hypothalamus.

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [35S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [35S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells, demonstrating clearly that these dual-labelled cells expressed both messenger RNAs. By contrast, the hybridization signals for proneurotensin and enkephalin, and proneurotensin and dopamine and adenylate cyclase phosphoprotein-32 were generally coincident, at least within the neostriatum; most proneurotensin messenger RNA-positive cells expressed enkephalin messenger RNA and were also positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA. However, occasional proneurotensin messenger RNA-positive striatal cells were identified that were single-labelled and did not express enkephalin messenger RNA. Within the septal nucleus, enkephalin messenger RNA and substance P messenger RNA were expressed essentially within segregated cell populations.These studies illustrate further the utility of co-expression techniques for investigating the chemical phenotype of cells within the CNS and demonstrate that the distribution of neuropeptide co-expressing cells is different within different brain regions. That several populations of proneurotensin messenger RNA-positive striatal cells may exist, of which one population is sensitive to haloperidol, co-expresses enkephalin messenger RNA and is positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA may be of some significance in neuropsychiatric/neurological disorders given that the translated peptide, neurotensin, is known to influence and interact closely with the dopamine systems.

Effects of l-DOPA on preproenkephalin and preprotachykinin gene expression in the MPTP-treated monkey striatum

The cellular expression of the genes encoding the neuropeptides enkephalin and substance P were examined in the caudate nucleus and putamen of parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus monkeys by in situ hybridization using radioactive antisense oligonucleotides coupled with computer-assisted image analysis. Behavioural evaluation of the animals revealed two levels of motor impairment; one group moderately impaired and the other severely disabled. A marked increase in the cellular content of preproenkephalin A messenger RNA was observed in medium-sized (106 +/- 9 microns2) cells in the caudate-putamen of all MPTP animals when compared with controls, the increase being greatest in the most severely impaired animals. By contrast, a marked reduction in the cellular abundance of preprotachykinin gene expression was detected in striatal cells (101 +/- 16 microns2) of these same MPTP animals. These changes in neuropeptide gene expression were not associated with a change in the density (approximately 10 cells per mm2) of messenger RNA-expressing cells. L-DOPA treatment of two of the severely-impaired MPTP monkeys resulted in a dissociation of expression of these two genes: the cellular abundance of preproenkephalin A remained elevated whilst preprotachykinin levels were normalized and comparable with controls. No change in the cellular abundance of preprotachykinin messenger RNA was observed in cells of the insular cortex or a small discrete population of large cells (208 +/- 27 microns2) in the ventral putamen. These results demonstrate that MPTP treatment of primates results in a marked potentiation in preproenkephalin messenger RNA coupled with a attenuation in preprotachykinin messenger RNA in the dopamine-denervated caudate-putamen. L-DOPA therapy given on an intermittent schedule reverses the decrease in preprotachykinin messenger RNA, but fails to reverse the increase in preproenkephalin messenger RNA in the same animal. These observations suggest that a dissociation of the activity of these two neuropeptide systems may underlie the improvement in motor skill that accompanies dopamine replacement therapy and that this dissociation may be instrumental in the long-term complications associated with L-DOPA therapy.

Chronic L-DOPA treatment differentially regulates gene expression of glutamate decarboxylase, preproenkephalin and preprotachykinin in the striatum of 6-hydroxydopamine-lesioned rat.

The effect of a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle in rats and subsequent L-DOPA treatment for eight weeks on preproenkephalin, preprotachykinin and glutamate decarboxylase (M(r) 67,000) gene expression in the striatum was investigated by in situ hybridization. A 6-hydroxydopamine lesion of the medial forebrain bundle markedly increased the level of preproenkephalin messenger RNA (+66%) and modestly elevated the level of glutamate decarboxylase (M(r) 67,000) messenger RNA (+36%) in the denervated striatum, but caused a decrease in the level of preprotachykinin messenger RNA (-54%) relative to the intact striatum and to sham-lesioned control animals. Treatment with L-DOPA (200 mg/kg/24 h) for eight weeks reduced but did not abolish the 6-hydroxydopamine lesion-induced elevation of preproenkephalin messenger RNA and slightly reduced the elevation of glutamate decarboxylase (M(r) 67,000) messenger RNA in denervated striatum relative to intact side and control groups. However, L-DOPA treatment almost completely reversed the decrease in preprotachykinin messenger RNA caused by 6-hydroxydopamine lesioning when compared to intact side and control groups. The effect of L-DOPA on the gene expression of preproenkephalin and glutamate decarboxylase (M(r) 67,000) differs from the increase in striatal enkephalin content and glutamate decarboxylase activity previously found following L-DOPA treatment. In contrast, L-DOPA reversed the changes in preprotachykinin messenger RNA, reflecting previously reported increases in substance P content. The findings provide new evidence that chronic L-DOPA treatment differentially affects direct striatonigral and indirect striatopallidal pathways at the molecular level.

Chronic blockade of muscarinic cholinergic receptors by systemic trihexyphenidyl (Artane) administration modulates but does not mediate the dopaminergic regulation of striatal prepropeptide messenger RNA expression.

A striatal dopaminergic denervation leads to changes in the expression of messenger RNA encoding prepropeptides contained in striatal efferent neurons. Such a dopaminergic lesion also abolishes a functional equilibrium between dopaminergic and cholinergic transmissions, generally believed to operate within the neostriatum, which constitutes the theoretical basis for the clinical use of antimuscarinic drugs in extrapyramidal diseases. It is possible, therefore, that changes in prepropeptide messenger RNA expression are mediated by an alteration in cholinergic transmission. To test this hypothesis, we have examined in rats whether trihexyphenidyl, an antimuscarinic drug of wide clinical use, can counteract the changes in preproenkephalin, preprotachykinin and preprodynorphin messenger RNA expression produced by a unilateral 6-hydroxydopamine lesion of substantia nigra dopaminergic neurons. Two weeks after the lesion, trihexyphenidyl was continuously administered through an osmotic minipump (5 mg/day for 15 days) to half of the lesioned and sham-operated rats, the other half receiving the vehicle. Using quantitative in situ hybridization histochemistry, messenger RNAs were analysed at two rostrocaudal levels (anterior and central) of the neostriatum. In parallel, M1 muscarinic receptors were measured by autoradiography of [3H]pirenzepine binding sites. In sham-operated rats, trihexyphenidyl administration produced a significant increase (17-27%) in M1 binding sites. In addition, preproenkephalin messenger RNA levels were decreased (-38%) in the central part, while preprodynorphin messenger RNA levels were significantly increased (+22%) at both striatal levels. In 6-hydroxydopamine-lesioned rats, the expected changes in messenger RNAs were observed when ipsi- versus contralateral side values were compared, but changes were not always detected when comparison was established between values from the dopamine-denervated neostriatum and those from sham-operated rats. The trihexyphenidyl administration in 6-hydroxydopamine-lesioned animals was unable to reproduce the up-regulation of M1 receptors, even in the intact neostriatum. This antimuscarinic treatment further increased preproenkephalin messenger RNA levels in the denervated anterior neostriatum, amplifying the ipsi- versus contralateral difference. It also potentiated the imbalance in preprotachykinin messenger RNA expression, mainly as a result of an increase of preprotachykinin messenger RNA levels in the intact neostriatum. In contrast, trihexyphenidyl treatment by increasing preprodynorphin messenger RNA in both neostriata abolished the ipsi- versus contralateral difference observed in lesioned rats. In conclusion, with the exception of preprodynorphin messenger RNA, trihexyphenidyl treatment was unable to counteract the imbalance in prepropeptide messenger RNA expression produced by a unilateral striatal dopaminergic denervation and even amplified this effect. These results question the neostriatum as the site of action of antimuscarinic drugs in producing their therapeutic effect in extrapyramidal syndromes.

Brain-derived neurotrophic factor and modify neurotransmitter-related gene expression in the 6-hydroxydopamine-lesioned rat striatum

Disruption of dopaminergic neurotransmission in the striatum by neurotoxic lesions of the substantia nigra leads to increases in glutamic acid decarboxylase and proenkephalin messenger RNA expression, and to decreases in preprotackykinin (the precursor molecule for substance P) messenger RNA expression in the two populations of striatal medium-sized spiny projection neurons. These cells also express TrkB, the neurotrophin receptor for brain-derived neurotrophic factor and neurotrophin 4/5, and TrkC, the receptor for neurotrophin-3. Since there is some indication that exogenous brain-derived neurotrophic factor can exert neuromodulatory effects in the basal ganglia, we studied the effects of repeated intrastriatal injections of the four members of the neurotrophin family of neural growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 on the expression of striatal neurotransmitter-related genes in the unilaterally 6-hydroxydopamine-lesioned rat using in situ hybridization histochemistry. We found that 4 micrograms/day of brain-derived neurotrophic factor or neurotrophin-4/5 when injected intrastriatally for eight consecutive days led to a normalization of the denervation-induced decrease of preprotachykinin messenger RNA when compared to animals injected with equivalent doses of nerve growth factor, neurotrophin-3, or vehicle. Neurotrophin-4/5 alone also normalized expression of messenger RNA encoding the 67 x 10(3) mol. wt isoform of glutamate decarboxylase, while none of the neurotrophins had a significant effect on preproenkephalin messenger RNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in nerve growth factor and preprotachykinin messenger RNA levels in the iris and trigeminal ganglion in diabetic rats; effects of treatment with insulin or nerve growth factor

This study was designed to explore effects of experimental diabetes mellitus on expression of substance P in the trigeminal ganglion and of nerve growth factor (NGF) in the iris. Rats with streptozotocin-diabetes showed an increase in NGF mRNA in the iris (P < 0.01) which was corrected by insulin treatment. There was also an increase in the levels of mRNA for the substance P precursor, preprotachykinin (PPT), in the trigeminal ganglion of these animals, despite there being no change in GAP-43 mRNA. Since expression of both of these peptides is sensitive to NGF in vitro, we examined the effect of treatment of diabetic rats with NGF at three different doses (0.2, 0.5 and 1.0 mg/kg body weight). There was a dose-dependent increase in both gamma-PPT and GAP-43 mRNA in the trigeminal ganglia of NGF-treated diabetic rats. The findings indicate that increased NGF may be responsible for raised substance P levels in the iris.

The human neostriatum shows compartmentalization of neuropeptide gene expression in dorsal and ventral regions: an in situ hybridization histochemical analysis.

Expression of neuropeptide messenger RNAs in striatal neurons was studied in post mortem human brain tissue by the use of in situ hybridization histochemistry. Clusters of cells expressing high levels of prodynorphin messenger RNA, and less strikingly, preprotachykinin messenger RNA, were prominent in the caudate nucleus and were present but less pronounced in the putamen. Proenkephalin and prosomatostatin messenger RNA-containing cells were more homogeneously distributed throughout the striatum, though the latter were much sparser. The four neuropeptide messenger RNA patterns in the nucleus accumbens were rather homogeneous compared with the dorsal striatum. Of these, prodynorphin messenger RNA showed a higher level of expression per cell in the nucleus accumbens relative to the dorsal striatum. The relationship of neuropeptide-containing cell clusters to the striosomal organization was characterized by looking at the register of these markers with patterns of low acetylcholinesterase activity and dense mu opiate receptor binding. In the caudate and putamen, clusters of cells expressing high levels of dynorphin and preprotachykinin messenger RNAs were clearly in register with the striosomes. The accumbens was defined by high prodynorphin messenger RNA levels, both low and high levels of acetylcholinesterase staining, and very low to absent mu opiate receptor binding. The distribution of high-expressing prodynorphin messenger RNA-containing cells--to the patch compartment and throughout the entire ventral striatum/nucleus accumbens region--defines the limbic domain of the neostriatum and suggests particular relevance to human striatal organization and function, because the distribution of this opioid neuropeptide is considerably more compartmentalized in human than in non-human species.

The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration.

Tachykinin peptides such as substance P and neurokinin B have been widely studied as mediators of pain transmission. The expression of neurokinin-1 and neurokinin-3 receptor messenger RNAs in the spinal cord is increased following intense nociception. The opiate ligands morphine and naltrexone alter behavioral responses to formalin-induced pain and alter evoked substance P release. This study investigated whether these opiates similarly alter the expression of substance P-, neurokinin B-, neurokinin-1 receptor- and neurokinin-3 receptor-encoding messenger RNAs in spinal systems following formalin-induced nociception. Expression levels of various messenger RNAs were quantitated using solution hybridization-nuclease protection assays. Six hours after hindpaw treatment, the levels of substance P-encoding preprotachykinin messenger RNA in the lumbar dorsal root ganglia and neurokinin B, neurokinin-1 receptor and neurokinin-3 receptor messenger RNAs in the lumbar dorsal horn were increased by approximately two-fold as compared to sham-treated controls. Pretreatment with naltrexone resulted in a further increase in the nociception-induced substance P messenger RNA expression in the dorsal root ganglia; preprotachykinin messenger RNA expression was not affected by morphine. Nociception-induced neurokinin-1 receptor messenger RNA expression in the dorsal horn was blocked by morphine, but was not affected by naltrexone. Both morphine and naltrexone blocked the formalin-induced increases in neurokinin B and neurokinin-3 receptor messenger RNA levels. Increased neurokinin B messenger RNA expression may reflect increased neurokinin B turnover in spinal interneurons activated by nociception. Neurokinin-3 receptor messenger RNA expression levels varied closely with, and thus may be regulated by, the levels of neurokinin B messenger RNA in the same regions. The results of this study indicate that pretreatment with opiate ligands modulates the expression of tachykinin peptide and neurokinin receptor encoding mRNAs in spinal systems following a peripheral chemogenic inflammatory stimulus. Thus, endogenous opioid systems may be involved in activity-induced changes in the expression of spinal tachykinin peptides and neurokinin receptors.

Neurotransmitter-related gene expression in intrastriatal striatal transplants—II. Characterization of efferent projecting graft neurons

The phenotypic characteristics of identified graft neurons in intrastriatal striatal transplants which give rise to efferent projections innervating the host brain were examined using a combination of in situ hybridization histochemistry and fluorescent retrograde tracing. Cell suspension grafts of embryonic day 14-15 rat striatal primordia (including both the medial and lateral ganglionic eminences) were implanted into the previously excitotoxically lesioned striatum of adult rats, and after longer than one year the retrograde tracer Fluoro-Gold was injected bilaterally into either the globus pallidus or the substantia nigra. Injections into the globus pallidus resulted in significant retrograde labelling of graft neurons within most of the experimental animals, whereas very few graft cells were labelled after the nigral injections. The vast majority of the neurons retrogradely labelled from the globus pallidus occurred in clusters or patches in the caudal half of the transplants, which corresponded well with DARPP-32 messenger RNA expressing (i.e. striatal) regions of the grafts. Indeed, within these Fluoro-Gold-labelled graft patches, the proportion of retrogradely labelled cells found to contain DARPP-32 messenger RNA was identical to that observed in the intact striatum after similar pallidal injections (93%). In addition, some Fluoro-Gold-labelled cells were found scattered outside the DARPP-32-positive cell clusters; these cells were overall larger and rarely (c. 9%) DARPP-32 messenger RNA-positive. Messenger RNA encoding for glutamate decarboxylase (which was found in 95% of Fluoro-Gold-labelled neurons in the intact striatum) was detected in almost all retrogradely labelled graft neurons located in both the DARPP-32-positive patches of retrograde labelling (93%) and in the DARPP-32-negative regions (82%). In the intact striatum, neurons labelled after pallidal injections of Fluoro-Gold were observed to express preproenkephalin messenger RNA to a greater extent than preprotachykinin messenger RNA (81% vs 21%). Conversely, within the grafts, retrogradely labelled neurons in the patches of Fluoro-Gold-labelled cells were more often found to contain preprotachykinin messenger RNA (50%) than preproenkephalin messenger RNA (21%). The Fluoro-Gold-labelled cells scattered outside the patches of retrograde labelling rarely expressed either preproenkephalin or preprotachykinin messenger RNA. Fluoro-Gold injections into the host substantia nigra resulted in very few retrogradely labelled graft neurons; however, many (85%) of these cells were observed to express glutamate decarboxylase messenger RNA, while only rarely were they observed to contain either DARPP-32, preproenkephalin or preprotachykinin messenger RNAs (c. 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmitter-related gene expression in intrastriatal striatal transplants--I. Phenotypical characterization of striatal and non-striatal graft regions.

In the present study, we have re-examined the heterogeneous nature of intrastriatal striatal transplants derived from embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum of adult rats, using in situ hybridization histochemistry to localize neurotransmitter-related messenger RNAs. These grafts are characterized by discrete patches of DARPP-32 messenger RNA expression, which cover approximately one-third of the cross-sectional graft area. The messenger RNAs encoding for preproenkephalin (the enkephalin precursor), preprotachykinin (precursor to substance P), choline acetyltransferase, as well as the D1 and D2 dopamine receptors, which are abundant in the normal striatum, were all present in the striatal grafts and were expressed almost exclusively in the DARPP-32-positive graft regions. In these graft regions, the expression of the neurotransmitter-related messenger RNAs was generally similar to that seen in the intact striatum, although the level of expression of preproenkephalin and preprotachykinin messenger RNAs varied notably among the patches of expression. Cellular analysis performed on individual patches showed that the expression per cell of preproenkephalin and preprotachykinin messenger RNAs was inversely related, such that patches with higher than normal preproenkephalin messenger RNA levels displayed lower than normal preprotachykinin messenger RNA levels, and vice versa. Moreover, messenger RNA expression for the dopamine D2 receptor was overall lower than that for the dopamine D1 receptor, both with respect to the level per cell and the number of positive cells within the DARPP-32 patches. Glutamate decarboxylase messenger RNA was expressed throughout the grafts, in 98% of all neurons located in the DARPP-32-positive regions and in 75% of all neurons in the non-DARPP-32 regions of the graft. Interestingly, the cellular expression of glutamate decarboxylase messenger RNA was considerably higher in the non-DARPP-32 expressing regions than that in the DARPP-32 messenger RNA-rich areas, where it approximated that of the intact striatum. Furthermore, grafted neurons located outside the DARPP-32-expressing regions displayed similar levels of expression to those found in the overlying cortex and in the closely adjacent globus pallidus. To further characterize the DARPP and non-DARPP graft compartments, messenger RNAs encoding the alpha 1 and beta 2 subunits of the GABAA receptor were studied. These receptor subunits, which exhibit a high expression in the host cortex and pallidum but little in the intact striatum, were found in discrete patches situated outside, but often closely associated with, the DARPP-32-rich areas of the graft.(ABSTRACT TRUNCATED AT 400 WORDS)

Fourth Isoform of Preprotachykinin Messenger RNA Encoding for Substance P in the Rat Intestine

Three different isoforms of preprotachykinin mRNA (PPT mRNA) encode for substance P and related neuropeptides (1). Here we report a fourth isoform of PPT mRNA which is generated by alternative exclusion of exon-4 and exon-6 from the PPT mRNA. It was present mainly in ileal smooth muscle and mucosa, colon, heart and brain and low level of this mRNA was detected in the jejunal smooth muscle and mucosa. This was not detected in the kidney or uterus. The level of this PPT mRNA was enhanced significantly by 60% during colitis in rat induced by trinitro benzene sulphonic acid.

Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: Differential effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels

In situ hybridization histochemistry was used to analyse the expression of the messenger RNAs encoding for enkephalin, substance P and dynorphin in the striatum of normal rats, rats subjected to a unilateral 6-hydroxydopamine lesion of the mesostriatal dopamine pathway and lesioned rats bearing intrastriatal transplants of fetal nigral neurons. About half of the rats in each group received twice-daily subcutaneous injections of 5 mg/kg apomorphine and the other half received control injections of saline, for nine days. Three hours after the last injection, the rats were killed by decapitation. Cryostat sections through the striatum were incubated with, 35 S-labeled oligodeoxyribonucleotide probes hybridizing with preproenkephalin, preprotachykinin or prodynorphin messenger RNA. One additional series of sections was incubated with [ 3H]GBR 12935 in order to label dopamine uptake sites. Quantitative evaluation of the hybridization signal was performed both at the macroscopic level (autoradiographic film analysis) and at the cellular level (optical density of silver grains over identified cells). The grafted nigral neurons reversed the lesion-induced up-regulation of preproenkephalin messenger RNA in the whole striatal complex. By contrast, the graft-induced effect on the lesion-induced down-regulation of preprotachykinin messenger RNA was restricted to the region of the host striatum where the graft-derived dopamine fibers exhibited their densest distribution (up to 0.5 mm from the border of the grafts). However, following chronic treatment with apomorphine, preprotachykinin messenger RNA expression approached control levels in a wider portion of the grafted striata (up to 1 mm from the border of the grafts). Basal prodynorphin messenger RNA expression, which was also down-regulated in the lesioned striata, was only partially restored by the transplants. Repeated injections of apomorphine enhanced prodynorphin messenger RNA in the lesioned striata to levels several fold higher than normal. This massive increase in prodynorphin messenger RNA expression was completely prevented by the transplants over a large volume of the host striatum (> 1 mm from the graft-host border), but a trend towards an abnormally high prodynorphin messenger RNA expression was still present in peripheral striatal areas that were not reached by graft-derived dopamine fibers. The present results indicate that fetal nigral neurons transplanted to the 6-hydroxydopamine-lesioned striatum have differential effects on the activity of enkephalin-containing (i.e. mainly striatopallidal) and substance P- or dynorphin-containing (i.e. mainly striatonigral) neurons. An inhibitory control over the activity of striatopallidal neurons is completely restored by the grafts, even in non-reinnervated striatal regions, suggesting that neurohumoral mechanisms underlie this effect. A facilitatory control over the activity of striatonigral neurons is restored by the grafts only in the densely reinnervated portion of the host striatum, which indicates that synaptic transmission is likely to mediate this effect. The ability of the grafts to prevent an abnormal up-regulation of prodynorphin messenger RNA expression by apomorphine indicates, however, that graft-derived dopamine release is also sufficient to induce a complete and widespread normalization of dopamine receptor sensitivity on striatonigral neurons.

Preprotachykinin messenger RNA detected by in situ hybridization in striatal neurons of the human brain

Sections from postmortem human brain were processed for in situ hybridization histochemistry using a 35S-labelled RNA probe transcribed from a cDNA coding for the human preprotachykinin which contains both substance P and K. Labelled neurons were observed in the caudate nucleus and the putamen but not the cerebellum. The labelled cells were of medium size and their distribution and morphology were compatible with previous data on substance P-like immunoreactivity in the human brain. The results confirm the presence of preprotachykinin mRNA in a subpopulation of striatal neurons in the human and show that in situ hybridization can be used to detect specific neurotransmitter-related mRNAs in postmortem tissue from normal and diseased humans.