i0. S. Holiingworth, M. W. Marshall, and E. Robson, "Ionic currents and charge movements in organ-cultured rat skeletal muscle," J. Physiol., 357, 369-386 (1984).

ii. J. R. Kal!o and R. A. Steinhardt, "The regulation of extrajunctional acetylcholine re- ceptors in the denervated rat diaphragm muscle in culture," J. Physiol., 344, 433-452 (1983).

12. R. P. Kernan, "Chloride-dependent sodium influx into rat skeletal muscle fibers measured with ion-selective microelectrodes," J. Physiol., 371, 146 (1986).

13. P. T. Paiade and R. L. Barchi, "Characteristics of the chloride conductance in muscle fibers of the rat diaphragm," J. Gen. Physiol., 69, No. 3, 325-342 (1977).

14. J. M. Russell, "Cation coupled chloride influx in squid axon: role of potassium and stoichiometry of the transport process," J. Gen. Physiol., 81, No. 6, 909-925 (1983).

DISTRIBUTION OF RETROGRADELY PRIMULENE-LABELED DOPAMINE-

CONTAINING NEURONS FORMING SPINAL PROJECTIONS

N. Z. Doroshenko and V. A. Maiskii UDC 612.826.4:611.814

The distribution of primuline-labeled dopamine-containing neurons in the rat forebrain was investigated by combining catecholamine fluorescence and retro- grade neuronal labeling techniques following injection of fluorochrome into the upper thoracic spinal cord. It was found that only diencephalic neurons of catecholamine-containing group All, located in the dorsal hypothalamus and the caudal thalamus send out direct projections to the spinal cord. Cells of nonidentified transmitter category were also primuline-labeled in this area. An average of 173 ± 4 catecholamine-containing neurons were revealed in group All, of which 86 were retrogradely labeled with primuline. Numbers of labeled dopamine-containing neurons increased, traveling in a rostro-caudal direction. The functional role which may be played by the dopaminergic diencephalo-spinal neuronal system described is discussed.

INTRODUCTION

Catecholaminergic neurons are found in the brain of different animal species; the loca- tion of these has been well documented in the brain structures of rats. The same neurons have been classed into groups AI-AI6 in this animal species [6, 8]° Catecholamine-contain- ing neurons project to nearly all sections of the central nervous system, including the spi- nal cord. Catecholaminergic nerve endings of three types: noradrenaline, adrenaline, and dopamine-containing, have been discovered over the entire extent of the spinal cord.

Analysis of the quantitative distribution of catecholamine levels in the gray matter of the rat thoracic spinal cord using isotopicaliy-labeled catecholamine metabolic enzymes revealed that the highest concentration seen in this area is reached by noradrenaline, at 754 ± 56 ng/100 mg protein; dopamine and adrenalin concentrations measured 76 ± 1 ng/100 mg and under 17 ng/100 mg protein, respectively [9]. Initial findings on the distribution of cells found to be sources of catecholaminergic innervation of the spinal cord have been ob- tained using up-to-date neuroanatomical research techniques for tracing connections between chemically identifiable neurons [i, 5, 21]. It was shown [7, 12] that neurons belonging to dopaminergic groups All and AI3 located in the forebrain project to the spinal cord. In the gray matter of the spinal cord, the densest network of dopaminergic terminals was discovered in the intermediolateral nucleus and areas adjoining the central canal, as well as in the superficial layers of th dorsal horn [17]. This distribution pattern of dopaminergic termi- nals in the spinal cord taken together with findings from physiological experiments would

S. V. Kurashov Medical institute, Ministry of Pubiic Health of the RSFSR, Kazan' .... Trans- lated from Neirofiziologiya, Vol. 19, No. 6, pp. 771-779, November-December, 1987. Original article submitted October 30, 1986.

0090-2977/87/1906-0559512.50 © 1988 Plenum Publishing Corporation 559

indicate that the descending dopaminergic neuronal system may exert an influence on the spinal sympathetic reflex and conduction of impulses produced by nociceptive stimuli [14, 16]. A detailed picture of how the descending dopaminergic neuronal system is organized must be formed in order to understand the central mechanisms underlying supraspinal control over sympathetic and sensory functions of the organism. For these reasons the study in hand set out to investigate the distribution of catecholamine-containing neurons in the rat midbrain and diencephalon sending out projections to the upper thoracic segments of the spinal cord on the one hand and make a quantitative analysis of the sources of dopaminergic supraspinal projections on the other, Techniques of primuline retrograde axonal transport and creating highly specific catecholamine fluorescence were combined, utilizing formaldehyde-catechol- amine complex formation in the liquid phase [2, i0].

METHODS

Experiments were conducted on 20 white rats of both sexes weighing 150-250 g. The ani- mals were injected with 0.5-4 ~i 10% primuline solution (Primuline O, Reichert, Austria, prepared in a 2% aqueous dimethylsulfoxide solution) into one half of the spinal cord at thoracic levels Th1-Th 3 under nembutal-induced anesthesia (35 mg/kg). In 5 days the animals were perfused transcardially with appropriate solutions for producing good fixation of the brain at the same time as forming stable fluorescent, catecholamine-formaldehyde complexes in the liquid phase [2]. Substances potentiating formation and accumulation of catechol- amines in neurons were administered prior to perfusion so as to intensify fluorescence of dopamine-containing structures: nailamide (i00 mg/kg), a monoamine oxidase inhibitor, with- in 5 h and L-DOPA (i00 mg/kg) within 1 h. Serial brain slices 30 ~m thick were prepared in a freezing microtome from embedded midbrain and hypothalamic tissue; after drying on slides and clearing in toluol, they were enclosed in a nonfluorescent medium (Epon 812). Pieces of embedded brain tissue were impregnated with soft petroleum jelly in a vacuum at a temper- ature of 55°C in some experiments and 5- to 10-~m thick slices were then prepared from the paraffin wax blocks, so that the neurons could be made more fluorescent and stand out better against background fluorescence. Sections were examined using a fluorescence microscope at an activating wavelength of 360-400 ran. Retrogradely-transported primuline accumulated in the cells in the form of granules glowing with gold fluorescence. Catecholamine-containing neurons could be identified by the bright green fluorescence of the cytoplasm. Primuline-labeled dopamine- containing neurons could easily be distinguished on the brain slices by the granules glowing gold against the background of green diffuse fluorescence of the cytoplasm.

Labeled and unlabeled dopamine-containing neurons belonging to group All were spotted on all brain slices but neuronal fragments measuring less than i0 ~m were not taken into ac- count. Abercrombie's estimation was used to rectify the error of counting the same cell twice over on two separate adjoining brain slices [3].

The following abbreviations appear on the sketches contained in the diagrams: AHA, anterior hypothalamic area; Ar, arcuate nucleus; CA, anterior commissure; CI, internal cap- sule; CO, optic chiasma; CP, caudate-putamen; DMN, dorsomedial hypothalamic nucleus; Fx, fornix tract; GP, globus pallidus; LHA, lateral hypothalamic area; 124, medial iemniscus; ME, median eminence; MFB, medial forebrain bundle; ML, lateral mamiliary nucleus; MM, mamillary body; MP, posterior mamillary body; MT, mamillo-thalamic tract; NR, red nucleus; Ped, cereb- ral peduncle; PMV, ventral premamillary nucleus; PVH, paraventricular thalamic nucleus; RCA, retrochiasmatic area; SNC, compact section of the substantia nigra; SNR, reticular part of substantia nigra; SO, supraoptic nucleus; SLY, supramamillary nucleus; TO, optic tract; ~H, ventromediai hypothalamic nucleus; VT, ventral thalamus; VTA, ventral tegmental area; ZI, zona incerta; III, third ventricle.

RESULTS

Microspectrofluorometric analysis of the brain slices revealed green fluorescent neurons in all known midbrain dopaminergic groups. Retrogradely primuline-labeled dopamine-contain- ing neurons were only observed in the diencephalon, but not in the midbrain (substantia nigra and ventral tegmentum).

Distribution of hypothalamic dopamine-containing neurons projecting (and those not pro- jecting) to the spinal cord is illustrated in Figs. 1 and 2. It will be seen from examining frontal planes of the brain slices that only neurons of the most caudal of diencephalic dop- aminergic groups (All) proved to be sources of spinal projections; no primuline-labeled neur- ons were to be seen in rostral groups AI2-AI4. Large numbers of cells of uncertain transmit- ter category were retrogradely labeled in the hypothalamus, apart from dopamine-containing

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Fig. i. Distribution of dopamine-containing and primuline (Pr)- labeled neurons in the rat hypothalamus after injecting fluoro- chrome into the upper thoracic spinal cord. Sketches 1-7: frontal planes of brain slices following in a rostro-caudal di- rection; AI2-AI4: dopaminergic neuronal groups. Circles denote dopamine-containing neurons; dots: Pr-labeled cells of known transmitter category; asterisks: Pr-labeled dopamine-containing neurons. Framed area (sketches 4 and 7) appear on photomicro- graphs (Figs. 3b and 3a, respectively). See Methods section for abbreviations.

cells. These were located in the paraventricular nucleus, lateral hypothalamus, and the ret- rochiasmatic area, as well as the zona incerta.

Dopamine-containing neurons constituting group All were observed extensively in the dor- sal and posterior hypothalamus and in the periventricular gray matter of the caudal thalamus. On frontal brain slices, neurons belonging to this'group could be distinguished from dop- amine-containing neurons of other diencephalic groups by their shape and dimensions (Fig. 3). These cells measured up to 20-30 ~m in diameter, were triangular or multipolar in shape, and often their fluorescent neurites could be traced over considerable distances from the neuronal soma. In a rostral direction, neurons of this group without clearly defined bounda- ries were replaced by dopamine-containing group AI3 cells. The latter form a compact group located along the mamillothalamic tract in the medial section of the zona incerta. Group AI3 cells were usually oval-shaped but occasionally spherical with neurites tending in a mediolateral direction which could be traced for short distances from the cell body. At 15 pm, measurements of these neurons were smaller than those of group All cells but exceeded group AI2 and AI4 neurons (located in the arcuate and periventricular hypothalamic nuclei) in size.

Numbers of group All neurons were counted in four animals. Taking the correlation fac- tor [3] into account, the overall number of group All cells in one half-brain equalled 173 ± 4. Most neurons of this group accumulated fluorochrome following injection of 4 ul primuline into the upper cervical segments of the spinal cord. The percentage of labeled group All

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dopamine-containing cells found in these animals equalled 64-86%° No differences were seen between the quantities of labeled dopamine-containing neurons distributed in the two halves of the brain, since the diffusion zone of fluorochrome had apread to the opposite half.

Non-dopamine-containing as well as dopamine-containing neurons were labeled in brain structures where group All cells were located. A typical sample distribution of labeled cells in the paraventricular area is shown in Fig. 2. Photomicrographs of the neurons sketched on the section shown in Fig. 2 (frame 4) are given in Figs. 3d and 4a. A certain regular pattern was noted in the distribution of labeled and unlabeled dopamine-containing neurons. Unlabeled neurons predominated in the rostral section of the brain, while numbers of labeled cells rose traveling in a rostro-caudal direction. Almost all dopamine-containing neurons accumulated retrograde marker in the caudal thalamus; numbers of labeled cells of unidenti- fied transmitter category also increased in this area. Labeled dopamine-containing neurons hardly differed in shape and size from those of dopamine-containing cells which had not ac- cumulated fluorochrome.

The catecholamine fluorescence technique used also enabled us to detect catecholaminer- gic fibers and varicosities on such fibers on brain slices. A sample of fluorescent eate- cholaminergic fibers in the median eminence is shown on photomicrograph 3b of the hypotha- lamic site corresponding to the brain site framed on the frontal plane brain slice (Fig. i, sketch 4).

DISCUSSION

All (or nearly all) catecholamine-containing neurons constituting diencephalic group All are dopamine-containing cells [18, 19]. The number of group All neurons we counted (173 ± 4 cells) was comparable with like findings (130 cells) of other workers [18] using the ALPHA technique of catecholamine-containing structure imaging, which would imply that the technique we employed of inducing catechoiamine fluorescence possessed a high degree of sensitivity. It was found during our experiments that of all the dopamine-containing cells in the brain, only group All diencephalic neurons project to the spinal cord. Other workers have also come to the same conclusion [7, 12, 18]. Bessingand Chalmers did discover, however, that group AI3 neurons also project to the cervical segments of the spinal cord [4]. We our- selves observed single dopamine-containing neurons retrogradely labeled by introducing prim- uline into the spinal cord; these were located close to the brain region where group AI3 cells are

562

Fig. 3. Photomicrographs of dopaminergic neuronal groups (a-d) in the rat diencephalon, a) Primuline (Pr)-labeled neurons of unidentified transmitter type marked by normal arrows and dop- amine-containing Pr-labeled cells by double-headed arrows. Framed area in (d) illustrated in photomicrograph (a), Fig. 4. Remaining notations as for Fig. i.

concentrated. They differed from the spherical and smaller group AI3 cells by their tri- angular shape and larger size. In common with other writers [7], we classed these neurons as group All cells and assume that group AI3 neurons do not put out spinal projections.

We detected single cells with green fluorescence only in the paraventricular nucleus. Similar results were also obtained by other workers using histochemical fluorescence tech- niques. Swanson et al., on the other hand, detected over 500 tyrosine hydroxylase-labeled (presumably dopamine-containing) cells in this nucleus on one side [20]. According to their findings, some of these cells project to the spinal cord. These quantitative differences have not yet been satisfactorily explained [18].

Up to 86~ of catecholamine-containing group All neurons were labeled following admin- istration of large doses of primuline to the upper thoracic spinal cord during our experi- ments. Other writers give lower levels of 20-40~ only in their published work [18]. The quantity of fluorochrome administered, a 4-~i i0~ solution, was one order of magnitude greater in our research, however. Fluorochrome uptake at the terminals is known to be depen- dent on concentration and overall amounts of the agent present in the tissue° The primuline injection site extended over two segments of the spinal cord, and fluorochrome diffusion was observed in the gray matter to either side. This made it possible to label neurons forming direct and crossed projections; the latter could make up i0~ of the total [18]o Findings exist [18] showing that dopamine-containing group All neurons have long axons which send out fine collaterals to many segments of the spinal cord. When primuline is injected into two segments of the spinal cord, fluorochrome spreads to the greater terminal area and hence ac- cumulates in fairly large quantities in the neuronal somata.

Group Allneurons, aswell as spinal cells, are known[7] to form projections to structures of the hypothalamus itself. Findings indicating that a proportion of neurons belonging to this group have T-shaped branching axons forming ascending and descending arbors would indi-

563

Fig. 4. Photomicrographs of Primuline (Pr) retrogradely-labeled and unlabeled catecholamine (Ca)-containing neurons of diencephal- ic group AI (a-d). a) Pr-labeled neurons of unidentified trans- mitter type marked by normal arrows, dopamine-containing Pr-label- ed neurons by double-headed and (simply) dopamine-containing neur- ons by triple-headed arrows; b-d) dopamine-containing neurons (Ca) and Pr-labeled dopamine-containing neurons (Ca + Pr).

cate the existence of dopamine-containing neurons forming both intra- and extrahypothalamic projections.

The functional role of the diencephalo-spinal dopaminergic neuronal system has not yet been conclusively established. It may be postulated that this neuronal system contributes to maintaining regulation of sympathetic tonus and conduction of impulses produced by noci- ceptive stimuli as well as helping to activate the spinal generators of rhythmic motion on the basis of separate data from physiological experiments and the topographical distribution of dopaminergic terminals in the spinal cord gray matter. The dopaminergie supraspinal neuro- nal system apparently performs a modulatory role at spinal level. Dopamine has been shown to raise background activity level in the sympathetic renal nerve and evoked activity in white connective arborizations [15, 16]. Electrical stimulation of dorsal and posterior hypotha- lamic sites containing locations of dopamine-containing neurons projecting to the spinal cord likewise produces a rise in sympathetic tonus [ii]. It has also been established [14] that the diencephalo-spinal dopaminergic neuronal system exerts inhibitory effects on conduction of signals produced by nociceptive stimuli.

LITERATURE CITED

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