M.-N. Castel

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.

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.

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.

Axonal transport of receptors. A major criterion for presynaptic localization.

Interneuronal communication through chemical transmission involves the releasc of transmitters from nerve terminals and their action on postsynaptic receptors. Owing to the distance of nerve terminals from the neuronal cell body, traffic systems or transport mechanisms are required in order to allow neuronal constituents or informative molecules to reach all the intracellular compartments, including nerve terminals. It is interesting to note that the sequence of intracellular events (FIGURE 1) that had been firmly established for numerous neurotransmitters, and on which the concept of neurotransmission is founded, also seems to be valid for presynaptic receptors. The five different stages, or dynamic events, in neurotransmission are very similar for both transmitters and receptors:

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.

Axonal Transport of Dopamine-containing Vesicles Labelled In Vivo with [3H] Reserpine

Axonal transport of the vesicular monoamine transporter was assayed in the rat brain by in vivo binding of the specific ligand [3H] reserpine. Because of the marked localization of reserpine binding sites in dopaminergic cell bodies and nerve terminals, the dopaminergic nigrostriatal pathway was chosen for the study of the axonal transport of the monoamine carrier present in the membrane of synaptic vesicles. When labelled reserpine was injected into the substantia nigra, a delayed accumulation of radioactivity in the ipsilateral striatum was observed ∼4 h after the injection. Similarly, injection into the right striatum was followed by a substantial accumulation of radioactivity in the ipsilateral substantia nigra, which was prevented by peripheral injection of unlabelled reserpine or tetrabenazine. This process was rapid and dependent on microtubules. In senescent rats, the amount of retrogradely transported [3H] reserpine was significantly reduced. These results demonstrate that labelled reserpine may be used to monitor in vivo fast axonal transport in central neurons.

Striatal Injection of Neurotensin Increases Tyrosine Hydroxylase mRNA in Substantia Nigra

It has been demonstrated that labeled neurotensin (NT) injected into the rat striatum is retrogradely transported in dopaminergic neurons to the ipsilateral substantia nigra (SN) through a NT receptor-dependent mechanism. I However, the physiological role for this transported material in the cell body remains to be elucidated. The fact that NT remains intact in the cell body for at least two hours after its transport* and since electron microscopic studies) indicated that 17% of the silver grains detected in the SN pars compacta (SNC) were associated with neuronal nuclei suggest that NT might be involved in the regulation of gene expression in dopaminergic neurons. To test this hypothesis, tyrosine hydroxylase messenger RNA (TH mRNA) expression was evaluated by in situ hybridization histochemistry in the SNC after the injection of NT into the striatum. Adult male Sprague-Dawley rats were injected with 16 nmol of NT, its receptoractive fragment (NT8-13) or its receptor-inactive fragment (NT1-8) into the striatum. The opposite side, serving as control, was injected with saline. In situ hybridization experiments were performed with a 3%-labeled cDNA probe for TH mRNA on brain sections that were dipped in NTB2 Kodak emulsion for two weeks. Nonspecific cDNA probe pBR322 was used for the control. The quantification of TH mRNA was performed with a computer-based image analysis system (HISTO 200) from Biocom (France) in a 0.25 mm2 area positioned at the same level on the right and the left SNC on the same slice. Labeled cells detected on the right and left sides were compared using pooled data obtained from different determinations and subjected to Students paired t-test. Four hours after the intrastriatal injection of NT, a highly significant increase was observed in the number of cells expressing a detectable level of TH mRNA in the SNC of the NT injected side comparing to the control side (+ 39%, p < 0.001, TABLE 1). A similar effect was observed with NT8-13, the receptor-active fragment of NT (+ 39%, p < 0.05, TABLE 1). However, when animals were injected with the

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.

Effect of γ-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.