Immunohistochemical evidence for a neurotensin striatonigral pathway in the rat brain.

The distribution and origin of neurotensin-like immunoreactivity in the substantia nigra pars reticulata of the rat have been analysed using immunohistochemistry combined with different drug treatments and lesioning techniques. In normal rats, a distinct but weakly fluorescent network of neurotensin-immunoreactive fibers was found in the central part of the substantia nigra pars reticulata. When the animals were treated with reserpine, which suppresses dopamine transmission, a similar pattern of immunoreactivity was found, though the intensity of staining was slightly enhanced. However, when rats were treated with methamphetamine, a potent dopamine releaser, the intensity of immunoreactivity was dramatically increased. In particular, densely packed neurotensin-immunoreactive fibers were found at the dorsal border and at the ventral periphery of the substantia nigra pars reticulata. This pattern of immunoreactivity was found to be similar to that displayed by dynorphin. In the nucleus caudatus, several neurotensin-immunoreactive cell bodies were seen after reserpine treatment. Morphologically similar perikarya were observed in methamphetamine-treated rats, but they were less numerous, whereas no cell bodies were detectable in untreated animals. When a unilateral mechanical transection or an ibotenic acid injection was performed in the striatum, the patterns of neurotensin as well as dynorphin and substance P immunoreactivities in the substantia nigra pars reticulata were strongly affected. Both types of lesion caused a marked, parallel depletion of all three immunoreactive substances on the side ipsilateral to the lesion, where a restricted area was virtually devoid of immunoreactive elements. Thus the present study provides evidence for the existence of a unilateral neurotensin striatonigral pathway, terminating in the pars reticulata. The origin of the neurotensin fibers in the pars compacta has not been established but does not appear to be the caudate nucleus. These results together with evidence from the literature suggest that methamphetamine induced a massive release of dopamine from nigral dendrites acting on presynaptic D1 dopamine receptors located on neurotensinergic terminals leading to a marked increase in neurotensin-like immunoreactivity in the pars reticulata.

Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat.

Although the existence of receptor transport has been clearly demonstrated in peripheral nerves, there is no clear cut evidence in the brain of such a process for neuropeptide receptors. Because of the localization of neurotensin receptors on dopaminergic terminals, the dopaminergic nigrostriatal pathway appears to be the system of choice for studying the axonal transport of neuropeptide receptors in the brain. When labelled neurotensin was injected into the rat striatum, a delayed accumulation of radioactivity in the ipsilateral substantia nigra was observed about 2 h after injection. An essential requirement to clearly observe this phenomenon was the pretreatment of animals with kelatorphan in order to prevent the labelled neurotensin degradation. The appearance of this labelling was prevented by injection of an excess of unlabelled neurotensin or of neurotensin 8-13, an active neurotensin fragment, but not by neurotensin 1-8, which had no affinity for neurotensin receptors. This process was saturable, microtubule-dependent and occurred only in mesostriatal and nigrostriatal dopaminergic neurons as identified after 6-hydroxydopamine lesion and by autoradiography. These results demonstrate that neurotensin was retrogradely transported by a process involving neurotensin receptors. The retrograde transport of receptor-bound neuropeptide may represent an important dynamic process which conveys information molecules from the synapse towards the cell body.

Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostriatal dopaminergic neurons.

We previously demonstrated the existence of a retrograde axonal transport of radioactivity to the substantia nigra pars compacta following injection of mono-iodinated neurotensin in rat neostriatum. In the present study, the topographical and cellular distribution of this retrogradely transported material was examined by light and electron microscopic autoradiography. Four and a half hours after unilateral injection of [125I]neurotensin in the caudoputamen, retrogradely labelled neuronal cell bodies were detected by light microscopic autoradiography throughout the ipsilateral substantia nigra pars compacta as well as within the ventral tegmental area and retrorubral field. In semithin sections, silver grains were prevalent over the perinuclear cytoplasm of neuronal cell bodies but were also detected over neuronal nuclei. Analysis of electron microscopic autoradiographs revealed that the vast majority (greater than 85%) were associated with neuronal perikarya, unmyelinated and myelinated axons, dendrites and terminals. Within the soma, a significant proportion of silver grains (16% of somatic grains) was detected over the nucleus. However, the majority were identified over the cytoplasm where they often encompassed cytoplasmic organelles, including rough endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, and multi-vesicular bodies. In dendrites and axons, a substantial percentage of silver grains (63-89%) was localized over the plasma membrane. A minor proportion (13% of total) of the autoradiographic labelling was detected over myelin sheaths, astrocytes, and oligodendrocytes. The present results are consistent with previous light-microscopic evidence for internalization and retrograde transport of intrastriatal neurotensin within nigrostriatal dopaminergic neurons. They further suggest that retrogradely transported neurotensin may be processed along a variety of intracellular pathways including those mediating degradation in lysosomes and recycling in rough endoplasmic reticulum. The detection of specific autoradiographic labelling in the nucleus supports the concept that neurotensin alone, or complexed to its receptor, might be involved in the regulation of gene expression through direct or indirect interactions with nuclear DNA. Consequently, the retrograde transport of neurotensin in nigrostriatal dopaminergic neurons might provide a vehicle through which events occurring at the level of the axon terminal may initiate long-term biological responses.

Neurotensin increases tyrosine hydroxylase messenger RNA-positive neurons in substantia nigra after retrograde axonal transport.

In previous studies we have shown that labelled neurotensin injected into the rat striatum was found to be transported retrogradely in dopaminergic neurons through a process which was receptor and microtubule dependent. Now, we show, by in situ hybridization, the consequences of the striatal injection of neurotensin on the gene expression of tyrosine hydroxylase in the substantia nigra. Rats were injected with neurotensin or its fragments in the striatum of one side and with saline or the inactive fragment on the other. The number of nigral cells expressing tyrosine hydroxylase mRNA was found to increase by 40% after injection of neurotensin or its active fragment (neurotensin 8-13). In the same experimental conditions, the inactive fragment (neurotensin 1-8) was without effect. Time-course experiments revealed that the tyrosine hydroxylase mRNA was increased 4 h after neurotensin injection but not at 1 or 16 h. The fact that the increase of mRNA parallels the appearance of labelled neurotensin in the substantia nigra indicates that the changes in the gene expression of tyrosine hydroxylase might be the consequence of the retrograde axonal transport of neurotensin. These results represent the first evidence for the existence of a long-distance retrograde signalling process in which the neuropeptide and presumably its receptor may serve as information molecule between synapses and the cell body.

Marked increase in cholecystokinin B receptor messenger RNA levels in rat dorsal root ganglia after peripheral axotomy.

It is now well established that the expression of peptides in rat primary sensory neurons is dramatically changed in response to peripheral nerve injury. Thus, as first shown by Jessell et al. peripheral axotomy causes a decrease in substance P levels in the dorsal horn of the corresponding spinal cord segments, and this is due to down-regulation of peptide synthesis in dorsal root ganglion neurons. In contrast, other peptides such as vasoactive intestinal polypeptide and peptide histidine isoleucine, galanin and neuropeptide Y are all markedly upregulated in the rat L4 and L5 dorsal root ganglia after sciatic nerve sectioning. The levels of another peptide, cholecystokinin and its messenger RNA are normally very low or undectable in rat primary sensory neurons, but after peripheral axotomy approximately 30% of the ganglion neurons express cholecystokinin messenger RNA. During the last few years a number of peptide receptors have been cloned, and they all belong to the family of G-protein coupled receptors with seven membrane spanning segments, among them the two cholecystokinin receptors cholecystokininA and cholecystokininB. Ghilardi et al. have recently described presence of cholecystokininB binding sites in rat dorsal root ganglia neurons. In the present study we report that the messenger RNA for the cholecystokininB receptor is present at very low levels in normal dorsal root ganglia of the rat, but axotomy causes a very marked increase in the number of sensory neurons of all sizes expressing cholecystokininB receptor messenger RNA, suggesting an increased sensitivity to cholecystokinin for many primary sensory neurons of different modalities after lesion.

Retrograde axonal transport of neurotensin in rat nigrostriatal dopaminergic neurons. Modulation during ageing and possible physiological role.

Biochemical and anatomical data are reported which demonstrate for the first time the existence of a retrograde axonal transport process for a neuropeptide, neurotensin, in rat brain. Neurotensin receptors are mainly located in the striatum on nerve terminals of the nigrostriatal dopaminergic pathway. Thus, the association of specific neurotensin receptors on a well defined pathway provides an excellent model to investigate the existence of such a process. Two hours after the intrastriatal injection of iodinated neurotensin, radioactivity started to accumulate in the ipsilateral substantia nigra. The levels were maximal during the fourth hour. The appearance of this labelling was prevented by injection of a large excess of unlabelled neurotensin or of neurotensin 8-13, an active neurotensin fragment, but not by neurotensin 1-8 which had no affinity for neurotensin receptors. These results suggest that the appearance of radioactivity in the ipsilateral substantia nigra was dependent on the initial binding of this peptide to its receptors in the striatum. HPLC studies demonstrated that the radioactivity found in the substantia nigra corresponded to intact neurotensin and to degradation products of this peptide. Moreover, it has been shown that this retrograde transport was microtubule-dependent and occurred in dopaminergic nigrostriatal neurons. Light and electron microscopic data confirmed and extended the present results. Four and a half hours after intrastriatal injection of iodinated neurotensin, silver grains were mainly detected in dopaminergic perikarya of the substantia nigra pars compacta. The vast majority were associated with neuronal elements and their localization within cell bodies suggests that retrogradely transported neurotensin may be processed along a variety of intracellular pathways including those mediating recycling in the rough endoplasmic reticulum and degradation in lysosomes. However, the presence of silver grains over the nucleus, as well as the increase in tyrosine-hydroxylase mRNA expression in the ipsilateral substantia nigra 4 hr after intrastriatal injection of neurotensin support the concept that neurotensin alone, or associated with its receptor, might be involved in the regulation of gene expression. Finally, we have demonstrated that in old rats the quantity of retrogradely transported neurotensin was significantly decreased as compared to that observed in young adult rats. This retrograde axonal transport of a neuropeptide may represent, as already suggested for growth factors, an important dynamic process conveying information from nerve terminals to the cell body.

Modulation of the neurotensin striato-nigral pathway by D1 receptors.

It has been reported that methamphetamine (METH) upregulates striatal neurotensin (NT) mRNA levels. The present in situ hybridization study demonstrates, using the dopamine D1 antagonist SCH23390 that this effect is mediated through D1 receptors. Sulpiride, a selective D2 antagonist, induces an upregulation of NT expression, which in some striatal areas is additive to the METH effect. This suggests the existence of at least two NT striatal neuronal populations. One, sensitive to D1 receptors, corresponds to the recently described striatonigral NT pathway. The second one, modulated by D2 receptors, may project to the globus pallidus and/or represent interneurones.

Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease.

Direct intracerebral gene transfer to neural cells has been demonstrated with recombinant adenovirus encoding beta-galactosidase. To explore the potential of recombinant adenovirus for the therapy of neurological disease we constructed a recombinant adenovirus encoding tyrosine hydroxylase and optimized intracerebral injection to express the gene in the striatum of unilaterally denervated rats. These animals have dopamine depletion in their lesioned striatum, causing a rotation asymmetry induced by apomorphine. One and two weeks after intracerebral injection this sensorimotor asymmetry was decreased by the adenovirus encoding tyrosine hydroxylase and not by a control adenovirus encoding beta-galactosidase. Histological analysis showed that tyrosine hydroxylase was preferentially expressed in astrocytes.

Effects of ICV administration of neurotensin and analogs on EEG in rats.

The electroencephalographic (EEG) effects of the ICV administration of neurotensin (NT 1-13), NT 1-8 (an inactive neurotensin fragment) and D TYR-11 NT (a long-lasting analog of neurotensin) were studied in rats. In awake rats, NT 1-13 (30 micrograms) and D TYR-11 NT (10 micrograms) induced an increase of the power spectrum in the theta range activity (4-7 Hz). In rats recorded during the sleep-wakefulness cycles, NT 1-13 (10 and 30 micrograms) and D TYR-11 NT (10 micrograms) had an awakening effect and also induced an increase of latency to the first episode of the different sleep stages (intermediate stage and slow wave sleep). NT 1-8 (30 and 90 micrograms in awake rats, 10 and 90 micrograms for sleep-wakefulness cycles) was inactive in all these experiments. Thus, it seems that all these effects can be linked to neurotensin receptors; indeed only fragments which recognize receptors possess an EEG activity.

Effect of gamma-butyrolactone on the retrograde axonal transport of neurotensin in dopaminergic neurones.

gamma-Butyrolactone is a drug known to inhibit the flow of electrical impulses in dopaminergic neurones and to prevent dopamine release in the striatum. In this study, we investigated the effects of this compound on the retrograde transport of neurotensin in the nigrostriatal dopaminergic pathway. The amount of radioactivity measured in the substantia nigra after injection of iodinated neurotensin into the striatum and the time course of its accumulation were not found to be modified in gamma-butyrolactone-treated rats. We therefore conclude that the retrograde axonal transport of neurotensin in the nigrostriatal pathway is not affected by reduced dopaminergic firing or by inhibition of the striatal release of dopamine.

Differential dopaminergic regulation of the neurotensin striatonigral and striatopallidal pathways in the rat.

Recently the existence of a neurotensin striatonigral pathway strongly up-regulated by methamphetamine has been demonstrated. The aim of the present study was to investigate, using immunohistochemistry and radioimmunoassay, the modulation of this pathway by dopamine antagonists. Rats were injected either with methamphetamine alone or together with the dopamine D1 receptor antagonist, SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzapine hydrochloride), or with the dopamine D2 receptor antagonist, sulpiride. Both techniques showed that this neurotensin striatonigral pathway is regulated by dopamine D1 receptors, since SCH 23390 totally prevented the methamphetamine-induced increase in neurotensin-like immunoreactivity, both in the striatum and in the substantia nigra pars reticulata. Conversely, sulpiride was unable to counteract the effect of methamphetamine in these two areas, suggesting that dopamine D2 receptors are not involved in the regulation of this neurotensin pathway. On the other hand, neurotensin-like immunoreactivity was markedly increased in striatal cell bodies and in the globus pallidus after treatment with sulpiride, indicating that this pathway is mainly regulated by dopamine D2 receptors.

Reduction of the amount of neurotensin retrogradely transported in dopaminergic neurons of senescent rats.

It is now clearly established that fast anterograde axonal transport can be altered during ageing, both in the central and the peripheral nervous systems, but no information is yet available concerning the modifications of fast retrograde axonal transport during senescence. In the present paper, we report the changes occurring in the retrograde axonal transport of neurotensin in dopaminergic neurons of old rats. When iodinated neurotensin was injected into the striatum, a diminution of approximately 50% in the amount of the labelling measured in the ipsilateral substantia nigra was observed in senescent rats by comparison with young adult rats. Nevertheless, the rate of neurotensin transport was not modified. Our results clearly indicate that less neurotensin is transported from the nerve terminals towards the cell bodies in senescent rats which may have possible consequences for dopaminergic neurons.

Evidence for upregulation of galanin synthesis in rat glial cells in vivo after colchicine treatment.

The localization of galanin (GAL) and GAL mRNA was studied in rat brain after colchicine or vinblastine treatment using in situ hybridization and immunohistochemistry. GAL-like immunoreactivity was found in glial cells, presumably activated microglia, in the cortex, caudate nucleus and septum, mainly on the injection side. GAL mRNA expression was found in small cells in the same areas with an overlapping distribution, including the adjacent white matter. The results suggest that the glial cells initiate synthesis of the peptide GAL in response to intraventricular injection of high doses of the 2 mitosis inhibitors.