Immunocytochemical evidence for direct connections between neurotensin-containing axons and dopaminergic neurons in the rat ventral midbrain tegmentum

Brain Research, 479 (1989)402-406 Elsevier

402 BRE 23351

Immunocytochemicai evidence for direct connections between neurotensin-containing axons and dopaminergic neurons in the rat ventral midbrain tegmentum John Woulfe and Alain Beaudet Neuroanatomy Laboratory, MontrealNeurological Institute, Montreal, Que. (Canada) (Accepted 18 October 1988)

Key words: Neurotensin;Dopamine; Tyrosinehydroxylase;Substantia nigra; Ventral tegmental area; Immunocytochemistry; Diaminobenzidine; Benzidine dihydrochloride

A light- and electron-microscopicdouble antigen localizationtechnique was employedto study cellular relationshipsbetween neurotensin (NT)-containingaxons and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. Direct c o n tacts were observedbetween NT-immunoreactiveaxon terminalsand the dendrites and perikarya of neurons containingthe dopamine biosyntheticenzyme, tyrosinehydroxylase(TH). NT-positiveterminals were also found to con~,actunlabeled dendrites and neuronal somata. These results reveal an ultrastructur~isubstrate through which endogenous NT might influence the activityof dopaminergic neurons in the ventral midbraintegmentum Neurotensin (NT), a tridecapeptide originally isolated and characterized from bovine hypothalamus by Carraway and Leeman 3, exhibits a widespread and heterogeneous distn'bution throughout the mammarian CNS 4'10'11'16'24'27'35where it is believed to play a transmitter role ts,26,36. Relatively high concentrations of endogenous NT have been detected by radioimmunoassay within the substantia nigra (SN) and the ventral tegmental area (VTA) of the midbrain 7, ~3.22,24. Within these two regions, NT has been localized immunohistochemically to both neuronal perikarya and axon terminals t4a6. NT-containing perikarya, a proportion of which also contain dopamine ~ L , ~ , are primarily concentrated in the VTA. NT axon terminals form a dense plexus throughout the ventral tegmentum, surrounding A9 and A10 DA neurons in the SN, pars compacta (SNC) and VTA, respectivelyTM. The morphological proximity of NT-containing axon terminals to DA perikarya and dendrites ~4 has suggested the existence of functional interactions between NT and DA in the SNC and VTA. Several lines of evidence support this hypothesis. NT, ap-

plied microiontophoretically in vivo or peffused onto tissue slices in vitro, elicits a marked and selective activation of DA neurons in the SNC and adjacent VTA i'll'J2. Bilateral intra-VTA injections of NT stimulate both DA release and the appearance of DA metabolites in the nucleus accumbens, a major site of •termination of VTA-efferent axons 17,t8,19. Similarly, intranigral microinjection of NT evokes an increase in the release of DA and the concentration of DA metabolites in the striatum, a major projection site of nigral DA efferents 29. The demonstration of specific, high-affinity NT binding sites on DA neurons in the SNC and VTA 2,3°'a4 suggests that some of these effects may be mediated through a direct action by NT on DA neurons. It is still unknown, however, whether NT-containing axon terminals establish synaptie connections with DA neurons in the SNC and VTA. In order to address this question and to further elucidate the ultrastructural substrate underlying NT-DA interactions in the rat ventral mesencephalic tegmenturn, we have employed a sequential double antigen localization technique 25 as an approach to examining cellular relationships between NT- and DA-contain-

Correspondence: J. Woulfe, NeuroanatomyLaboratory, Montreal Neurological Institute, McGill University,380i UniversityStreet, Montreal, Que., Canada, H3A 2B4. 0006-8993/89i$03.50 © 1989Elsevier Science PublishersB.V. (BiomedicalDivision)

403 ing elements in the SNC and VTA. Adult male Sprague-Dawley rats, 200.-225 g b. wt., were perfused transaorticaUy with 600 ml of 4% paraformaldehyde, 0.08% glutaraldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.2-7.4). The brain was removed from the skull and block~ of the midbrain were placed in the same fixative for 2 h at 25 °C. Coronal sections through the midbrain were cut at 30~tm on a Vibratome (Lancer) and incubated in 0.1 M Tris-buffered saline (TBS), pH 7.6, containing 1% sodium borohydride. Following a 30 rain preincubation i.a normal goat ~erum (3% in TBS), sections were incubated in an antiserum to NT (Immunonuclear; diluted 1:6000) for 48 h at 4 °C. They were then inc~:bated in a goat anti-rabbit immunoglobulin (ICN; 1:50) for 40 rain at 25 °C, followed by a rabbit PAP complex (Dako; 1:50) for 40 min at 25 °C. The bound peroxidase was visualized by reaction in a solution of 0.05% diaminobenzidine (DAB; Sigma) and 0.01% H202 in 0.1 M Tris-HCI buffer. The sections were then rinsed thoroughly in TBS and incubated sequentially in a mouse monoclonal antiserum to tyrosine hydroxylase (TH; Boehringer-Mannheim; clone 2/40/15; 40 ~g/ml) overnight at 4 °C, a goat anti-mouse immunoglobulin (1:20) for 30 min at 25 °C, and a mouse monoclonal PAP complex (ICN; clone P6/38; 1:500) for 30 min at 25 °C. Following several rinses in 0.01 M phosphate buffer,

pH 6.6, the sections were incubated in 0.01 M phosphate buffer containing 0.01% benzidine dihydrochloride (BDHC; Sigma), 0.025% sodium nitroferricyanide (Sigma) and 0.005% H202 for 15 min. After several thorough washes in 0.01 M phosphate buffer, sections were postfixed for I h in 1% osmium tetroxide, dehydrated in graded ethanols, embedded in Epon between plastic coverslips and examined under the light microscope. Regions of interest were selected for EM processing and were trimmed and blocked accordingly. Ul~rathin sections were collected on copper gr~ds, counterstained with uranyl acetate and lead citrate, and examined with a JEOL JEM-100CX I! electron microscope. On light microscopic examination, TH-immunoreactive (BDHC-positive) neurons were detected throughout the rostrocaudal extent of the ventral tegment~m in a pattern of distribution reminiscent of that reported previously s. Specifically, BDHC-positive perikarya were detected within the retrorubra! nucleus of Swanson 33 (group A8 of Dahlstrom and Fuxe s, the SNC (group A9), and the VTA (including its paranigral and parabrachial subdivisions as well as its associated nuclei, the interfascicular and linear raphe; group A10). Because the DA biosynthetic enzyme TH has been extensively acknowledged as a reliable indicator of the dopaminergic identity of neurons in the VTA and SNC, it is reasonable to assume

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Fig. 1. Two ~XFF-immunoreactive terminals ( ~ and 3"2) directly abut a ~-I-imm~noreacti~e dendrite (Den). In this plane of section, neither apposition exhibits identifiable junctional specializations. Bar = 1.0/~m. Fig. 2. A TH-immunoreactive perikaryon, identified by the presence of large BDHC crystals (arrows) is contacted by a NT-immunoreactive (DAB-positive) axon terminal. The terminal is filled with small, clear synaptic vesicles and forms an atypical symmetric synapse characterized by the presence of multiple, small, presynaptic DAB deposits (arrowheads). Bar = 1.0/~m.

404

that the BDHC-labelled elements identified in the present study were indeed DA-containing. Intermingled with these TH-immunoreactive elements, NT-immunoreactive (DAB-positive) terminals formed a dense plexus extending from the pars lateralis of the SN (SNL) laterally, to the V T A medially. Both the SNL and SNC exhibited relatively dense NT terminal immunoreactivity whereas the pars reticulata was essentially devoid of NT-containing processes. Within the VTA, NT-immunoreactive terminals were densely distributed across the paranigral and parabrachial subdivisions as well as within the interfascicular and linear raphe nuclei. Within both the VTA and SNC, NT-immunoreacttve terminal boutons were identified in close proximity to THpositive perikarya and dendrites. These results are largely consistent with those of earlier immunohistochemical studies 14'~6. However, in contrast to these studies in which colch~c'ne pretreatment was employed, no NT-containing perikarya were detected in the ventral midbrain tegmentum of the non-colchicinized animals used in the present study. On electron microscopic examination of ultrathin sections through the SNC and VT A, NT immunoreactivity (DAB labeling) was identified in axon terminals and, occasionally, axonal segments. The NT immtmoreactive terminals ranged from 0.6 to 2.0 # m in diameter and were predominantly round, flattened, or ellipsoidal in shape (Figs. 1-4). They contained densely packed, round, agranular vesicles with a few interspersed dense-core vesicles. Many of them were identified in direct apposition to BDHC-labeled (TH-immunoreactive) dendrites (Fi~s. 1 and 3) and, less frequently, BDHC-positive somata (Fig. 2). Single TH-immunoreactive dendrites were occasionally contacted by multiple NT-immunoreactive terminals (Fig. 1 and 3). In such cases, the labeled terminals were usually interspersed among numerous unlabeled ones. In some instances however, two apposed, DAB-labeled terminals were identified in contact with a single dendrite (Fig. 3). Only a small proportion of contacts involving NT-labeied terminals exhibited conventional synaptic specializations in the plane of section sampled. These were usually of the asymmetric variety with clear pre- and postsynaptic membrane specializations (Fig. 3). Frequently however, a discrete atypical symmetric differentiation was detected at the site of contact (Fig.

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Four NT-immunoreactive terminals and several unlabeled terminals surround a BDHC-labeled (arrows; TH-immunoreactive) nigral dendrite (Den). In this plane of section, one of the labeled terminals (T~) forms a synaptic junction whereas the other 3 lack any observable membrane specialization. Note that terminals T~ and "i"2 are directly adjacent to one another whereas T2, T3, and 1"4 are separated by unlabeled terminals. Bar = 0.5pro.

Fig. 3.

Fig. 4. One unlabeled and two NT-immunoreactive terminals (T l and Tz) contact an unlabeled dendrite (Den). in this plane of section, the unlabeled terminal is seen to establish an asymmetric synapse (arrow) whereas the other two exhibit no clearcut junctional specialization. Note the presence of a thin, presynaptic DAB deposit (arrowhead) in one of the labeled terminals (T2). Bar = 0 . 5 / ~ m . 2). This specialization was characterized by the presence of a thin density along the internal face of the 'presynaptic' junctional surface. The density was sometimes continuous but often interrupted, giving the appearance of multiple presynaptic DAB deposits (Fig, 2). In addition to direct contacts between NT-immunoreactive axon terminals and TH-immunoreactive

405 perikarya and dendrites~ many appositions were identified between NT-positive varicosities and unlabeled elements. A smal~ proportion of these exhibited well-delineated asymmetric junctional complexes, but the majority showed either no synaptic density or an atypical symmetric specialization identical to that described in reference to terminals apposed to TH-immunoreactive elements (Fig. 4). The present results reveal that a proportion of NTcontaining varicosities identified in the SNC and the VTA is in direct apposition to TH-immunopositive perikarya and dendrites. Some of these established conventional synaptic connections with the adjacent dopaminergic elements. Others appear to exhibited either no, or only atypical, junctional specializations in the plane of section examined. Whether the latter also establish conventional synapses in other crosssectional planes could not be ascertained in the present analysis performed on random thin sections. However, in view of the low incidence of synapses identified in planes of section in which the two elements were seen in contact (which should represent the most favorable planes of sampling), it would appear unlikely that they do. Functionally, this issue appears rather academic since NT receptors have recently been shown by electron microscopic radioautography to be more or less uniformly distributed over the surface of nigral perikarya and dendrites, rather than being confined to synaptic specializations 2. Whether NT released from terminals showing highly differentiated junctions acts on more restricted postsynaptic targets and/or plays a different role than that released at less differentiated contact zones remains to be elucidated. The direct inne~vation of DA neurons by NT-containing axons de,~nonstrated in the present study provides a morphological substrate for functional interactions between endogenous NT and DA in the midbrain tegmentum. These may include, in particular~ the locomotor and thermoregulatory effects that have been reported after bilateral microinjection of NT into the VTA 17'18and the locomotor effects associated with intranigral injection of NT 29. The identifi1 Andrade, R. and Aghajanian, G.K., Neurotensin selectively activates dopaminergic neurons of the substantia nigra, Soc. Neurosci. Abstr., 7 (1981)573. 2 Beaudet, A., Leonard, K., Vial, M., Moyse, E., Kitabgi, P., Vincent, J.P. and Rost6ne, W., Electron microscopic

cation of synaptic contacts between NT-immunoreactive axons and immunonegative perikarya and dendrites suggests that NT may also be in a position to influence the activity of non-DA neurons within the SN and VTA. The extent to which NT terminals contact these non-DA targets remains difficult to assess however, since many TH-immunoreactive dendrites may not have exhibited BDHC crystals in the plane of section in which they were sampled (see Fig. 2). Worthy of note is the fact that many, if not all, DAB-positive terminals detected in the present study may contain the peptide neuromedin-N (NN) in addition to, or even in lieu of, NT. NT and NN share a common genetic and molecular precursor 9~ but is has been suggested that their synthesis may be differentially regulated in the CNS 5. Since the antiNT antibody employed in the present study primarily recognizes epitopes comprising the carboxy-terminal sequence of the NT molecule n which shares a 4amino acid sequence homology with NN 28, it is reasonable to suspect that it may cross-react to some degree with the latter. Several lines of evidence support the contention that NN, like NT, influences the activity of mesotelencephalic DA neurons. In fact, following intra-VTA administration, NN has been reported to be more potent than NT at increasing spontaneous motor activity and, like NT, to elevate DA metabolism in the nucleus accumbens 2°. Furthermore, it has been demonstrated that NN competes with NT for the occul:a~cy of presumptive NT receptors and that, like NT, it stimulates cGMP formation and inositol phosphate hydrolysis6'12'21. Therefore, in the light of the present results, it is tempting to postulate that these two commonly-derived neuropeptides both act directly on ventral tegmental DA neurons, perhaps each through one of the different types of junctional specializations observed between these two categories of chemically distinct elements. The technical assistance of Kathleen Leonard, Charles Hedge and Colin Holmes is gratefully acknowledged. Parkinson Foundation of Canada. localization of neurotensin receptor in the substantia nigra of the rat, Soc. Neurosci. Abstr., 13 (1987)563. 3 Carraway, R. and Leeman, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem., 248 (1973)6854-6861.

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