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Postnatal development of the noradrenergic system in the dorsal lateral geniculate nucleus of the rat
Developmental Brain Research 149 (2004) 79 – 83 www.elsevier.com/locate/devbrainres
Short communication
Postnatal development of the noradrenergic system in the dorsal lateral geniculate nucleus of the rat Maria Latsari, John Antonopoulos *, Ioanna Dori, Maria Chiotelli, Athanasios Dinopoulos Department of Anatomy, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Accepted 19 December 2003
Abstract The noradrenergic innervation of the developing dorsal lateral geniculate nucleus of the rat was examined with light and electron microscopic immunocytochemistry. At birth, few, relatively thick, noradrenergic fibers innervated the nucleus. Their density was steadily increased and they became thinner, tortuous, and varicose with the progression of age. Only a minority (11 – 15%) of labeled varicosities made synaptic contacts. Most of these synapses were symmetrical and on dendritic shafts. The present findings demonstrate the establishment of the anatomical relationships between noradrenergic afferents and neurons of the dorsal lateral geniculate nucleus during development and may help to understand the role of noradrenaline in the processing of visual information. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Noradrenaline; Monoaminergic system; Development; Dorsal lateral geniculate nucleus; Synapse
The dorsal division of the lateral geniculate nucleus (dLGN) relays information from the retina to the visual cortex, whereas the ventral division (vLGN) conveys information to subcortical targets. Relay neurons in the dLGN that project to the visual cortex use glutamate, whilst interneurons use GABA [5,15]. The dLGN also receives cortical and subcortical afferents [21]. Prominent among the subcortical afferents are the noradrenergic fibers [17] arising from cells in the locus coeruleus [8]. Most of these unmyelinated fibers ascend in the dorsal tegmental bundle through the mesencephalon and the zona incerta into the medial forebrain bundle. Noradrenergic branches from this bundle give rise to highly branched networks in many thalamic and metathalamic areas including the ventrobasal complex, the medial geniculate nucleus and the LGN [11,14]. In the dLGN, the noradrenergic fibers establish mainly symmetrical synapses with dendritic shafts and spines, outside the synaptic glomeruli [17]. In this nucleus, noradrenaline (NA) excites the relay neurons and inhibits
* Corresponding author. Tel.: +30-31-999874; fax: +30-31-999842. E-mail address: [email protected] (J. Antonopoulos). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.12.005
interneurons [7], modifying the responsiveness of these target neurons to convergent inputs carried by other afferent fibers [19]. In the present study, an antiserum against dopamine-hhydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to NA, was used to provide a detailed description of the noradrenergic innervation of the developing dLGN with the light and the electron microscope. We also examined whether noradrenergic synapses change with age and whether they participate in the formation of the synaptic glomeruli during development. A total of 23 Wistar albino rats of the following ages were used: postnatal day 0 (P0; day of birth; n=3), P7 (n=5), P14 (n=5), P21 (n=7) and P90 (adult; n=3). Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) for the care and use of laboratory animals. All efforts were made to minimize animal suffering and reduce the number of animals used. The rats were perfused, under deep ether anesthesia, through the heart, first with a small amount of saline and then with a fixative solution containing 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were subsequently
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removed from the skulls, postfixed for 2 –24 h and processed accordingly for light or electron microscopic immunocytochemistry. A commercially available polyclonal antibody, produced in rabbits, was purchased from Incstar USA and used at a dilution 1:2000 (for details see Ref. [9]). Noradrenergic synapses formed by labeled varicosities were identified by the presence of pre- and postsynaptic membrane specializations, a visible synaptic cleft, and the accumulation of synaptic vesicles in the presynaptic profile. These synapses were classified as symmetrical or asymmetrical and the postsynaptic neuronal elements were identified. The aim of this analysis was to ascertain whether there were changes in the nature of the noradrenergic synapses during development of the dLGN. Detailed quantitative analysis was carried out with the electron microscope. Specifically, counts of the number of synapses per 200 noradrenergic varicosities were made in single ultrathin sections of the dLGN, in each of three animals, at P7, P14 and P21. In the newborn, quantitative analysis was not performed due to the scarcity of immunoreactive varicosities. Our aim was to determine the percentages of labeled varicosities involved in synaptic contacts in single ultrathin sections at different stages of development. In order to ensure that any fluctuations in the proportion of synapses during development were not due to changes in the size of noradrenergic varicosities or of their synaptic appositions, we measured the lengths of a number of randomly selected synaptic junctions from those used in the synaptic counts and also the diameters (long axes) of the noradrenergic varicosities in the same samples. Furthermore, the relative synaptic frequency (%) observed in single sections (m) was converted to relative synaptic frequency (%) for whole varicosities (e) by means of a stereological formula, as it has been previously described [9].
Immunoreactive fibers were observed in nuclei of the thalamus including the ventrobasal complex, the ventrolateral nucleus, the lateral posterior nucleus and the laterodorsal nucleus as well as in the metathalamus. Here our description will be restricted to the LGN. At P0, few short, almost unbranched, and relatively thick immunoreactive fibers were found to innervate the dLGN. Sparse labeled fibers of the same morphology were also found in the vLGN. At the end of the first postnatal week, a substantial increase in the number of immunoreactive fibers was observed in the dLGN, whereas the number of labeled fibers did not change substantially in the vLGN. This difference in the density of noradrenergic innervation between the dorsal and the ventral division of the LGN was observed throughout development. At the end of the second postnatal week, the number of the noradrenergic fibers was further increased and their distribution pattern was similar to the pattern observed in the adult. Overall, at this developmental stage, immunoreactive fibers were finer, more varicose and more tortuous compared with those at birth. At the end of the third postnatal week, the density and the morphology of noradrenergic fibers were similar to those observed in the adult (Figs. 1 and 2a – c). In the adult, the morphology and the density of the noradrenergic fibers matched closely to those described by Papadopoulos and Parnavelas [17]; immunoreactive fibers were thin, tortuous, highly branched and varicose and formed a dense plexus throughout the dLGN (Fig. 2d). Electron microscopic examination showed that the immunoreactive product was localised in axonal varicosities or intervaricose segments. Noradrenergic varicosities were identified as round or elongated dilations, containing numerous synaptic vesicles and usually one or two mitochondria. Intervaricose segments were thin, unmyelinated axonal
Fig. 1. Darkfield photomicrographs showing the density and distribution pattern of the noradrenergic innervation of the dorsal lateral geniculate nucleus of the rat at various postnatal ages (a, P0; b, P7; c, P14; d, P21). dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet. Scale bar = 100 Am in d (applies to a – d).
M. Latsari et al. / Developmental Brain Research 149 (2004) 79–83
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Fig. 2. Darkfield photomicrographs showing the density and distribution pattern of the noradrenergic innervation of the dorsal lateral geniculate nucleus of the rat at various postnatal ages (a, P0; b, P7; c, P21; d, adult). Scale bar = 60 Am in d (applies to a – d).
profiles occasionally containing mitochondria and axially oriented microtubules. At birth, very few noradrenergic varicosities were found in the dLGN. They were closely apposed to dendritic shafts or somata without any glial processes intervening between them. At times they formed symmetrical synapses with small or medium dendritic shafts. At the end of the first postnatal week, although noradrenergic varicosities were more numerous than at P0, few of them were engaged in synaptic contacts (Fig. 3a). At the end of the second postnatal week, labeled synapses were predominantly axodendritic of the symmetrical variety (Fig. 3b), however a few asymmetrical axospinous synapses were also encountered. At the end of the third postnatal week the
density of the noradrenergic varicosities was further increased, but the synaptic contacts formed by these varicosities remained few in number. Elements postsynaptic to noradrenergic varicosities were often found to receive mainly asymmetrical synapses from a number of unlabeled axons. Labeled synapses were never found to participate in the formation of the synaptic glomeruli of the dLGN. At this age and in the adult, noradrenergic varicosities were involved in synaptic relationships similar to those observed at P14. The results of the quantitative analysis of the synaptic frequency in the developing dLGN are summarized in Table 1. As shown in this table, the proportion of labeled vari-
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Fig. 3. Electron photomicrographs illustrating noradrenergic varicosities in the dorsal lateral geniculate nucleus of the rat at P7 (a) and P14 (b). Arrows point to an asymmetrical synapse (a) or a symmetrical synapse (b) formed with unlabeled dendrites. Scale bar = 0.3 Am in b (applies to a,b).
cosities forming synapses increased from 10.88% at P7 to 14.86% at P21. However, the statistical analysis and evaluation of the synaptic frequencies in single sections has not revealed any significant difference between ages examined (Chi square tests: v2=1.688, df=2, level of significance a=5%). The mean length of noradrenergic synaptic junctions, estimated by measuring the lengths of their active zones, was found to be 0.23F0.01 Am at P7, 0.23F0.03 Am at P14 and 0.24F0.02 Am at P21. The mean diameter of noradrenergic varicosities, estimated by measuring their long axis, was found to be 0.68F0.07 Am at P7, 0.69F0.1 Am at P14 and 0.68F0.04 Am at P21. These results indicate that the proportion of labeled varicosities forming synapses remained low during development and in the adult and that the mean length of the synaptic junctions and the mean diameter of varicosities were not changed substantially in all ages examined. The present light microscopic analysis shows that the ingrowth and the arborisation of noradrenergic axons to the dLGN are correlated with its development showing gradual increase in density. The differences in the density of innervation and the distribution pattern of the noradrenergic fibers described by Papadopoulos and Parnavelas [17] in the adult between the dLGN and the vLGN were apparent as early as at birth and remained the same throughout postnatal development. Indeed, the density of noradrenergic fibers was considerably higher in the dLGN, whereas in the vLGN only sparse fibers were observed in all ages examined. The steady increase in the density of the noradrenergic fibers in the dLGN during the first weeks of life was not followed by a similar increase in synaptic contacts. The quantitative analysis showed that the synaptic incidence was increased with age, but with a considerably lower rate. The first appearance of synaptic glomeruli at P4 [1] or eye opening did not affect the synaptic incidence and the pattern of the noradrenergic innervation. The low proportion of synaptic contacts formed by noradrenergic varicosities is a constant finding in many areas of the developing and mature brain including cortical areas (Ref. [9] and references therein),
thalamic nuclei [14], nuclei of the basal forebrain, as well as the lateral and the medial septum [our unpublished data]. The present and other ultrastructural findings show that the noradrenergic afferents form mainly symmetrical axodendritic synapses (see Refs. [9,17], our unpublished data for the septal area). Therefore, NA acts on neurons of the LGN at least in part through conventional synapses; however, as shown here the majority of the noradrenergic varicosities do not form synapses. This finding leads to the assumption that NA may act through transmission by diffusion [2]. The hypothesis of a non-synaptic modulation of neuronal activity by NA has been also suggested by Nothias et al. [14] for other nuclei of the rat thalamus. Electrical stimulation of the locus coeruleus and iontophoretic application of NA have been shown to produce a slow membrane depolarization of neurons in the dLGN [7,19] and reduce excitability of interneurons [6,7]. Since the majority of noradrenergic synapses are of the symmetrical variety, these findings further support the view that NA may act mainly on distal targets and not only on synaptic sites formed by noradrenergic terminals. The excitatory effect is mediated by a1-adrenergic receptors [6], which are present in large numbers in the dLGN [4,16]. The lack of noradrenergic varicosities in the synap-
Table 1 Synaptic frequency of NA varicosities in the dLGN1 Age
NA varicosities Synapses
Non-synapses
P7 P14 P21
22 25 31
578 575 569
m (%)
e (%)
3.70a 4.00a 5.20a
10.88 11.76 14.86
m (%), relative synaptic frequency in single ultrathin sections; e (%), relative synaptic frequency extrapolated from single sections to whole varicosities; a, relative frequencies in the same column with a superscript in common do not differ significantly (Pz0.05). NA, noradrenergic; dLGN, dorsal lateral geniculate nucleus; P, postnatal day. 1 Three animals per age were used and 200 NA varicosities in each animal were examined for synaptic relationships.
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tic glomeruli of the dLGN shows that the visual processing might be affected by extraglomerular noradrenergic synapses on relay neurons or interneurons, or by the non-synaptic release of NA. The pattern of distribution of the noradrenergic fibers in the LGN suggests that NA may play a major role in the processing of visual information to the cortex. On the other hand, serotonergic and dopaminergic fibers are preferentially distributed in the vLGN and in the intergeniculate leaflet [3,18] where they form asymmetrical synapses with dendritic shafts. Serotonin and dopamine are probably involved in functions associated with the vLGN and the intergeniculate leaflet (e.g. brightness, black/white discrimination [10], entrainment of circadian rhythms [20]). Finally, NA has been implicated in the generation of rapid-eye-movement sleep in rats [12], but its depletion does not alter the sleepstate-related patterns of activity in neurons of the dLGN [13].
Acknowledgements The authors thank Dr. Ch. Batzios for help with the statistical analysis and T. Zlatis for technical assistance. The Hellenic Ministry of Development and the Hellenic State Fellowship Foundation supported this work. Grant number: PENED 1995.
References [1] N. Aggelopoulos, J.G. Parnavelas, S. Edmunds, Synaptogenesis in the dorsal lateral geniculate nucleus of the rat, Anat. Embryol. 180 (1989) 243 – 257. [2] L. Descarries, P. Se´gue´la, C. Watkins, Nonjunctional relationships of monoamine axon terminals in the cerebral cortex of adult rat, in: K. Fuxe, L.F. Agnati (Eds.), Volume Transmission in the Brain, Raven Press, New York, 1991, pp. 53 – 62. [3] A. Dinopoulos, I. Dori, J.G. Parnavelas, Serotonergic innervation of the lateral geniculate nucleus of the rat during postnatal development: a light and electron microscopic immunocytochemical analysis, J. Comp. Neurol. 363 (1995) 532 – 544. [4] L.S. Jones, L.L. Gauger, J.N. Davis, Anatomy of brain alpha1-adrenergic receptors: in vitro autoradiography with [125I]-heat, J. Comp. Neurol. 231 (1985) 190 – 208. [5] T. Kaneko, N. Mizuno, Immunohistochemical study glutaminase-containing neurons in the cerebral cortex and thalamus of the rat, J. Comp. Neurol. 267 (1998) 590 – 602.
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[6] Y. Kayama, Ascending, descending and local control of neuronal activity in the rat lateral geniculate nucleus, Vis. Res. 25 (1985) 339 – 347. [7] Y. Kayama, T. Negi, M. Sugitani, K. Iwama, Effects of locus coeruleus stimulation on neuronal activities of dorsal lateral geniculate nucleus and perigeniculate reticular nucleus of the rat, Neuroscience 7 (1982) 655 – 666. [8] L.F. Kromer, R.Y. Moore, A study of the organization of the locus coeruleus projections to the lateral geniculate nuclei in the albino rat, Neuroscience 5 (1980) 255 – 271. [9] M. Latsari, I. Dori, J. Antonopoulos, M. Chiotelli, A. Dinopoulos, Noradrenergic innervation of the developing and mature visual and motor cortex of the rat brain: a light and electron microscopic immunocytochemical analysis, J. Comp. Neurol. 445 (2002) 145 – 158. [10] C.R. Legg, A. Cowey, The role of the ventral lateral geniculate nucleus and posterior thalamus in intensity discrimination in rats, Brain Res. 123 (1977) 261 – 273. [11] O. Lindvall, A. Bjo¨rklund, A. Nobin, U. Stenevi, The adrenergic innervation of the rat thalamus as revealed by glyoxylic acid fluorescence method, J. Comp. Neurol. 154 (1974) 317 – 348. [12] G.A. Marks, J. Farber, M. Rubinstein, H.P. Roffwarg, Demonstration of ponto-geniculo-occipital waves in the albino rat, Exp. Neurol. 69 (1980) 648 – 677. [13] G.A. Marks, S.G. Speciale, H.P. Roffwarg, Sleep state-specific neuronal activity in rat dorsal lateral geniculate nucleus is not altered by local serotonin and norepinephrine depletion, Exp. Brain Res. 74 (1989) 402 – 410. [14] F. Nothias, B. Onteniente, F. Roudier, M. Peschanksi, Immunocytochemical study of serotoninergic and noradrenergic innervation of the ventrobasal complex of the rat thalamus, Neurosci. Lett. 95 (1988) 59 – 63. [15] P.T. Ohara, A.R. Lieberman, S.P. Hunt, J.-Y. Wu, Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat: immunohistochemical studies by light and electron microscopy, Neuroscience 8 (1983) 189 – 212. [16] J.M. Palacios, D. Hoyer, R. Cortes, a1 adrenoreceptors, Brain Res. 419 (1987) 65 – 75. [17] G.C. Papadopoulos, J.G. Parnavelas, Distribution and synaptic organization of serotoninergic and noradrenergic axons in the lateral geniculate nucleus of the rat, J. Comp. Neurol. 294 (1990) 345 – 355. [18] G.C. Papadopoulos, J.G. Parnavelas, Distribution and synaptic organization of dopaminergic axons in the lateral geniculate nucleus of the rat, J. Comp. Neurol. 294 (1990) 356 – 361. [19] M.A. Rogawski, G.K. Aghajanian, Modulation of lateral geniculate neurone excitability by noradrenaline microiontophoresis or locus coeruleus stimulation, Nature (Lond.) 287 (1980) 731 – 734. [20] B. Rusak, J.H. Meijer, M.E. Harrington, Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract, Brain Res. 493 (1989) 283 – 291. [21] A.J. Sefton, B. Dreher, Visual system. in: G. Paxinos (Ed.), The Rat Nervous System, 2nd ed., Academic Press, San Diego, 1995, pp. 833 – 898.
Short communication
Postnatal development of the noradrenergic system in the dorsal lateral geniculate nucleus of the rat Maria Latsari, John Antonopoulos *, Ioanna Dori, Maria Chiotelli, Athanasios Dinopoulos Department of Anatomy, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Accepted 19 December 2003
Abstract The noradrenergic innervation of the developing dorsal lateral geniculate nucleus of the rat was examined with light and electron microscopic immunocytochemistry. At birth, few, relatively thick, noradrenergic fibers innervated the nucleus. Their density was steadily increased and they became thinner, tortuous, and varicose with the progression of age. Only a minority (11 – 15%) of labeled varicosities made synaptic contacts. Most of these synapses were symmetrical and on dendritic shafts. The present findings demonstrate the establishment of the anatomical relationships between noradrenergic afferents and neurons of the dorsal lateral geniculate nucleus during development and may help to understand the role of noradrenaline in the processing of visual information. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Noradrenaline; Monoaminergic system; Development; Dorsal lateral geniculate nucleus; Synapse
The dorsal division of the lateral geniculate nucleus (dLGN) relays information from the retina to the visual cortex, whereas the ventral division (vLGN) conveys information to subcortical targets. Relay neurons in the dLGN that project to the visual cortex use glutamate, whilst interneurons use GABA [5,15]. The dLGN also receives cortical and subcortical afferents [21]. Prominent among the subcortical afferents are the noradrenergic fibers [17] arising from cells in the locus coeruleus [8]. Most of these unmyelinated fibers ascend in the dorsal tegmental bundle through the mesencephalon and the zona incerta into the medial forebrain bundle. Noradrenergic branches from this bundle give rise to highly branched networks in many thalamic and metathalamic areas including the ventrobasal complex, the medial geniculate nucleus and the LGN [11,14]. In the dLGN, the noradrenergic fibers establish mainly symmetrical synapses with dendritic shafts and spines, outside the synaptic glomeruli [17]. In this nucleus, noradrenaline (NA) excites the relay neurons and inhibits
* Corresponding author. Tel.: +30-31-999874; fax: +30-31-999842. E-mail address: [email protected] (J. Antonopoulos). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.12.005
interneurons [7], modifying the responsiveness of these target neurons to convergent inputs carried by other afferent fibers [19]. In the present study, an antiserum against dopamine-hhydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to NA, was used to provide a detailed description of the noradrenergic innervation of the developing dLGN with the light and the electron microscope. We also examined whether noradrenergic synapses change with age and whether they participate in the formation of the synaptic glomeruli during development. A total of 23 Wistar albino rats of the following ages were used: postnatal day 0 (P0; day of birth; n=3), P7 (n=5), P14 (n=5), P21 (n=7) and P90 (adult; n=3). Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) for the care and use of laboratory animals. All efforts were made to minimize animal suffering and reduce the number of animals used. The rats were perfused, under deep ether anesthesia, through the heart, first with a small amount of saline and then with a fixative solution containing 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were subsequently
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removed from the skulls, postfixed for 2 –24 h and processed accordingly for light or electron microscopic immunocytochemistry. A commercially available polyclonal antibody, produced in rabbits, was purchased from Incstar USA and used at a dilution 1:2000 (for details see Ref. [9]). Noradrenergic synapses formed by labeled varicosities were identified by the presence of pre- and postsynaptic membrane specializations, a visible synaptic cleft, and the accumulation of synaptic vesicles in the presynaptic profile. These synapses were classified as symmetrical or asymmetrical and the postsynaptic neuronal elements were identified. The aim of this analysis was to ascertain whether there were changes in the nature of the noradrenergic synapses during development of the dLGN. Detailed quantitative analysis was carried out with the electron microscope. Specifically, counts of the number of synapses per 200 noradrenergic varicosities were made in single ultrathin sections of the dLGN, in each of three animals, at P7, P14 and P21. In the newborn, quantitative analysis was not performed due to the scarcity of immunoreactive varicosities. Our aim was to determine the percentages of labeled varicosities involved in synaptic contacts in single ultrathin sections at different stages of development. In order to ensure that any fluctuations in the proportion of synapses during development were not due to changes in the size of noradrenergic varicosities or of their synaptic appositions, we measured the lengths of a number of randomly selected synaptic junctions from those used in the synaptic counts and also the diameters (long axes) of the noradrenergic varicosities in the same samples. Furthermore, the relative synaptic frequency (%) observed in single sections (m) was converted to relative synaptic frequency (%) for whole varicosities (e) by means of a stereological formula, as it has been previously described [9].
Immunoreactive fibers were observed in nuclei of the thalamus including the ventrobasal complex, the ventrolateral nucleus, the lateral posterior nucleus and the laterodorsal nucleus as well as in the metathalamus. Here our description will be restricted to the LGN. At P0, few short, almost unbranched, and relatively thick immunoreactive fibers were found to innervate the dLGN. Sparse labeled fibers of the same morphology were also found in the vLGN. At the end of the first postnatal week, a substantial increase in the number of immunoreactive fibers was observed in the dLGN, whereas the number of labeled fibers did not change substantially in the vLGN. This difference in the density of noradrenergic innervation between the dorsal and the ventral division of the LGN was observed throughout development. At the end of the second postnatal week, the number of the noradrenergic fibers was further increased and their distribution pattern was similar to the pattern observed in the adult. Overall, at this developmental stage, immunoreactive fibers were finer, more varicose and more tortuous compared with those at birth. At the end of the third postnatal week, the density and the morphology of noradrenergic fibers were similar to those observed in the adult (Figs. 1 and 2a – c). In the adult, the morphology and the density of the noradrenergic fibers matched closely to those described by Papadopoulos and Parnavelas [17]; immunoreactive fibers were thin, tortuous, highly branched and varicose and formed a dense plexus throughout the dLGN (Fig. 2d). Electron microscopic examination showed that the immunoreactive product was localised in axonal varicosities or intervaricose segments. Noradrenergic varicosities were identified as round or elongated dilations, containing numerous synaptic vesicles and usually one or two mitochondria. Intervaricose segments were thin, unmyelinated axonal
Fig. 1. Darkfield photomicrographs showing the density and distribution pattern of the noradrenergic innervation of the dorsal lateral geniculate nucleus of the rat at various postnatal ages (a, P0; b, P7; c, P14; d, P21). dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; IGL, intergeniculate leaflet. Scale bar = 100 Am in d (applies to a – d).
M. Latsari et al. / Developmental Brain Research 149 (2004) 79–83
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Fig. 2. Darkfield photomicrographs showing the density and distribution pattern of the noradrenergic innervation of the dorsal lateral geniculate nucleus of the rat at various postnatal ages (a, P0; b, P7; c, P21; d, adult). Scale bar = 60 Am in d (applies to a – d).
profiles occasionally containing mitochondria and axially oriented microtubules. At birth, very few noradrenergic varicosities were found in the dLGN. They were closely apposed to dendritic shafts or somata without any glial processes intervening between them. At times they formed symmetrical synapses with small or medium dendritic shafts. At the end of the first postnatal week, although noradrenergic varicosities were more numerous than at P0, few of them were engaged in synaptic contacts (Fig. 3a). At the end of the second postnatal week, labeled synapses were predominantly axodendritic of the symmetrical variety (Fig. 3b), however a few asymmetrical axospinous synapses were also encountered. At the end of the third postnatal week the
density of the noradrenergic varicosities was further increased, but the synaptic contacts formed by these varicosities remained few in number. Elements postsynaptic to noradrenergic varicosities were often found to receive mainly asymmetrical synapses from a number of unlabeled axons. Labeled synapses were never found to participate in the formation of the synaptic glomeruli of the dLGN. At this age and in the adult, noradrenergic varicosities were involved in synaptic relationships similar to those observed at P14. The results of the quantitative analysis of the synaptic frequency in the developing dLGN are summarized in Table 1. As shown in this table, the proportion of labeled vari-
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Fig. 3. Electron photomicrographs illustrating noradrenergic varicosities in the dorsal lateral geniculate nucleus of the rat at P7 (a) and P14 (b). Arrows point to an asymmetrical synapse (a) or a symmetrical synapse (b) formed with unlabeled dendrites. Scale bar = 0.3 Am in b (applies to a,b).
cosities forming synapses increased from 10.88% at P7 to 14.86% at P21. However, the statistical analysis and evaluation of the synaptic frequencies in single sections has not revealed any significant difference between ages examined (Chi square tests: v2=1.688, df=2, level of significance a=5%). The mean length of noradrenergic synaptic junctions, estimated by measuring the lengths of their active zones, was found to be 0.23F0.01 Am at P7, 0.23F0.03 Am at P14 and 0.24F0.02 Am at P21. The mean diameter of noradrenergic varicosities, estimated by measuring their long axis, was found to be 0.68F0.07 Am at P7, 0.69F0.1 Am at P14 and 0.68F0.04 Am at P21. These results indicate that the proportion of labeled varicosities forming synapses remained low during development and in the adult and that the mean length of the synaptic junctions and the mean diameter of varicosities were not changed substantially in all ages examined. The present light microscopic analysis shows that the ingrowth and the arborisation of noradrenergic axons to the dLGN are correlated with its development showing gradual increase in density. The differences in the density of innervation and the distribution pattern of the noradrenergic fibers described by Papadopoulos and Parnavelas [17] in the adult between the dLGN and the vLGN were apparent as early as at birth and remained the same throughout postnatal development. Indeed, the density of noradrenergic fibers was considerably higher in the dLGN, whereas in the vLGN only sparse fibers were observed in all ages examined. The steady increase in the density of the noradrenergic fibers in the dLGN during the first weeks of life was not followed by a similar increase in synaptic contacts. The quantitative analysis showed that the synaptic incidence was increased with age, but with a considerably lower rate. The first appearance of synaptic glomeruli at P4 [1] or eye opening did not affect the synaptic incidence and the pattern of the noradrenergic innervation. The low proportion of synaptic contacts formed by noradrenergic varicosities is a constant finding in many areas of the developing and mature brain including cortical areas (Ref. [9] and references therein),
thalamic nuclei [14], nuclei of the basal forebrain, as well as the lateral and the medial septum [our unpublished data]. The present and other ultrastructural findings show that the noradrenergic afferents form mainly symmetrical axodendritic synapses (see Refs. [9,17], our unpublished data for the septal area). Therefore, NA acts on neurons of the LGN at least in part through conventional synapses; however, as shown here the majority of the noradrenergic varicosities do not form synapses. This finding leads to the assumption that NA may act through transmission by diffusion [2]. The hypothesis of a non-synaptic modulation of neuronal activity by NA has been also suggested by Nothias et al. [14] for other nuclei of the rat thalamus. Electrical stimulation of the locus coeruleus and iontophoretic application of NA have been shown to produce a slow membrane depolarization of neurons in the dLGN [7,19] and reduce excitability of interneurons [6,7]. Since the majority of noradrenergic synapses are of the symmetrical variety, these findings further support the view that NA may act mainly on distal targets and not only on synaptic sites formed by noradrenergic terminals. The excitatory effect is mediated by a1-adrenergic receptors [6], which are present in large numbers in the dLGN [4,16]. The lack of noradrenergic varicosities in the synap-
Table 1 Synaptic frequency of NA varicosities in the dLGN1 Age
NA varicosities Synapses
Non-synapses
P7 P14 P21
22 25 31
578 575 569
m (%)
e (%)
3.70a 4.00a 5.20a
10.88 11.76 14.86
m (%), relative synaptic frequency in single ultrathin sections; e (%), relative synaptic frequency extrapolated from single sections to whole varicosities; a, relative frequencies in the same column with a superscript in common do not differ significantly (Pz0.05). NA, noradrenergic; dLGN, dorsal lateral geniculate nucleus; P, postnatal day. 1 Three animals per age were used and 200 NA varicosities in each animal were examined for synaptic relationships.
M. Latsari et al. / Developmental Brain Research 149 (2004) 79–83
tic glomeruli of the dLGN shows that the visual processing might be affected by extraglomerular noradrenergic synapses on relay neurons or interneurons, or by the non-synaptic release of NA. The pattern of distribution of the noradrenergic fibers in the LGN suggests that NA may play a major role in the processing of visual information to the cortex. On the other hand, serotonergic and dopaminergic fibers are preferentially distributed in the vLGN and in the intergeniculate leaflet [3,18] where they form asymmetrical synapses with dendritic shafts. Serotonin and dopamine are probably involved in functions associated with the vLGN and the intergeniculate leaflet (e.g. brightness, black/white discrimination [10], entrainment of circadian rhythms [20]). Finally, NA has been implicated in the generation of rapid-eye-movement sleep in rats [12], but its depletion does not alter the sleepstate-related patterns of activity in neurons of the dLGN [13].
Acknowledgements The authors thank Dr. Ch. Batzios for help with the statistical analysis and T. Zlatis for technical assistance. The Hellenic Ministry of Development and the Hellenic State Fellowship Foundation supported this work. Grant number: PENED 1995.
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