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Complex patterns of [125I]ω-conotoxin GVIA binding site expression during postnatal rat brain development
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 78 (1994) 131-136
Short Communication
Complex patterns of [125I]o-conotoxin GVIA binding site expression during postnatal rat brain development F. Filloux *, Aaron Schapper, Scott R. Naisbitt, Baldomero M. Olivera, J. Michael Mclntosh Departments Of Neurolo,w, Pediatrics, Psychiato' and Biology, UnicersiO' o[ Utah, Salt Lake City, UT 84132, USA (Accepted 23 November 19931
Abstract
In the mature CNS, N-type calcium channels regulate neurotransmitter release. The role of these channels in developing brain is less clear. Study of [~25I]w-conotoxin GVIA binding sites in developing rat brain using autoradiography reveals that putative N-type channels appear and disappear in complex temporal-spatial profiles including: (1) gradual increase to adult levels (cerebral cortex); (2) substructure differentiation (cerebellum); (3) transient expression (pons); and, (4) selective depletion (medulla). Transient expression of N-type calcium channels may influence specific neurodevelopmental processes.
Key words: [125I]w-Conotoxin GVIA; N-type calcium channel; Autoradiograpby, Rat brain development
Nervous system development includes 'progressive' processes (e.g. neurulation, induction, cell proliferation, migration, synaptogenesis and myelination) as well as 'regressive' phenomena whereby early redundancies in CNS connectivity are eliminated [7,26]. For example, neuronal death (apoptosis) and synapse elimination are crucial to the formation of a normally functioning nervous system [7,18,26]. Defects in these latter processes have been proposed as contributing to various pathological conditions, as diverse as schizophrenia [5] and Sudden Infant Death Syndrome [4]. In view of the central importance of intracellular calcium ion concentration ([Ca2+]i) to the control of nervous system function in general, cell constituents which modulate [Ca2+]i may be involved in these 'regressive" phenomena [18]. Receptor-operated Ca 2+ channels, particularly N-methyl-D-aspartate (NMDA) type glutamate-activated Ca 2+ channels, have the potential to influence nervous system plasticity [6]. However, other molecular constituents also participate in regulating [Ca2+]~ including voltage-gated Ca 2+ channels (VGCCs), of which several subtypes (L, N, T and P) have been described [16,21,22,30]. In particular,
* Corresponding author. Research Laboratory, Western Institute of Neuropsychiatry, 501 Chipeta Way, Salt Lake City, U T 84108. USA. Fax: ( 1) (801) 582-8471. Elsevier Science B.V.
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roles for N-type Ca 2+ channels in synaptogenesis [32] and neuronal migration [15] have recently been proposed. The peptide w-conotoxin GVIA (w-CgTx) from the marine snail Conus geographus is known to block presynaptic Ca 2+ channels [8,10,29], and thus radiolabeled [8,9,14,20,31] and fluorescent [15] derivatives of the peptide are potential markers for synapses bearing such presynaptic ion channels. Within the mammalian nervous system this ligand is believed to selectively bind the N-type subset of VGCCs [29]. We have used ¢o-CgTx to investigate the postnatal ontogeny of N-type Ca 2+ channels in rat brain. The results indicate that, in contrast to L-type channels which seem to increase steadily to adult levels throughout the brain in a spatially uniform pattern [9,12,23], the appearance of wCgTx-labeled channels is more complex both in time and space. In particular, transiently expressed ~oCgTx-sensitive channels undergo a striking disappearance from most regions of the hindbrain. Sprague-Dawley rats of varying postnatal ages (1 day, 3 days, and 1, 2, 3 and 6 weeks and adult) were utilized independent of sex. Animals were deeply anesthetized with pentobarbital (60 m g / k g ip) and perfused transcardially with phosphate-buffered saline containing 5% dextrose (4°C, pH 7.4). Brains were promptly removed from the skull after decapitation and immediately frozen by immersion into isopentane (-65°C).
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qg. 1. [125I]~o-CgTx G V I A binding to sections of rat brain of varying ages. Slide-mounted transverse or sagittal tissue sections of rat brain of varying postnatal ages were labeled with t25I]w-CgTx (see text for details). These plates represent photomicrographs of original autoradiograms taken under brightfield illumination using a Wild Photomacroscope (Wild Heerbrugg. ;witzerland). Areas appearing darker indicate greater density ot [ I25I]w-CgTx binding. Autoradiograms are from 1-day-old (a,b,c) 2-week-otd (d~e,f), and 6-week-old (g.h.i) animals. (The pattern ~t ~ weeks of age is indistinguishable from that in mature rat brain.) Non-specific binding ~not shown) was very low at all development ages and represented less than ll)~ of total binding in all egions exhibiting significant densities of binding sites. Note the overall caudal to rostral redistribution of o)-CgTx binding sites with dense hindbrai~l hut minimal cerebral c~rtex binding at one lay of age tc). and the reverse (dense cortical binding, but light brainstem binding) al six weeks of age (i). Higher power images at the level of the medulla (a,d,g) and pons (P) and cerebellum Ce) (b.e,h) are also shown. At 6 weeks of age, binding sites within the hindbrain have virtually disappeared (plates g,h) with the exeepti~m of restricted regions such as the spinal tract of the Vth rama nerve (Sp5, plate g), the nucleus of the solitary tract (Sot. plate g), and the substantia nigra reticulata ISNr. plate i). In contrast, differentiation of the ccrebellar cortex i~ evident earlic~ e.g.. see frame e~, Ac. nucleus accumbens: cc, corpus callosum- CPu, caudate-putamen: egr, gr and tool" external granular layer, granule cell la~er and molecula~ laye~ of ce~'ebellum. espectively; Ctx. cerebral cortex: Hi, hippocampus; Io. inferior olivary complex; OT, olfactory tubercle: SC. superior colliculus. Bars = 2 mm.
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F. Fillowc et al. / Developmental Brain Research 78 (19941 131-136
They were stored at - 7 0 ° C in an airtight container until use. Ten /,m-thick sagittal or transverse/coronal sections encompassing brainstem through forebrain were cut at - 17°C with a cryostat microtome and were thaw-mounted on chrome-alum-coated microscope slides. These tissue sections were stored at -70°C. Five brains were sampled per developmental time point. Labeling with [t25 l]to-CgTx (2196 C i / m m o l ) was performed as previously described [14,20]. Specifically, slide-mounted tissue sections were warmed to room temperature and incubated for 30 min with 100-300 >1 (depending on size of tissue section) of 5 mM H E P E S / T r i s buffer (25°C, pH 7.4) containing 120 mM sucrose, 100 mM NaC1, 0.2 m g / m l lysozyme and 200 pM [tzSI]w-CgTx which was synthesized and iodinated as previously described [8,20]. Adjacent tissue sections were incubated similarly, but with the addition of 3 mM neomycin [11] to determine non-specific binding. Slides were then washed (3 × 10 min each) in fresh H E P E S / T r i s buffer (4°C) containing 160 mM NaCI, 1.5 mM CaC1 z, 1 m g / m l BSA, and 0.05% polyoxyethylene sorbitan monolaurate. Slides were dipped in distilled H 2 0 (4°C) for 1 s, dried promptly with a stream of cool, dry air, and stored in the presence of desiccant at 4°C overnight. Dried slides were then apposed to Hyperfilm /3-Max (Amersham, Arlington Hts., ILl, and film was developed after a 24-h exposure. Resultant autoradiograms were digitized and density of [t2sI]~o-CgTx binding quantitated with BRAIN software [19] (Drexel University, Philadelphia, PAl, by comparing grey levels of the tissue-generated autoradiograms to those produced by 125I plastic polymer standards (Amersham, Arlington Heights, ILl. Autoradiograms were photographed (Fig. 1) using a Wild Photomacroscope (Wild-Heerbrugg, Switzerland). The kinetics and pharmacological specificity of [tzSI]w-CgTx binding to neonatal tissue were examined in additional transverse, slide-mounted sections of 1day-old brainstem (medulla/pons) animals. Tissue sections were labeled as described above with 50 pM [IzsI]to-CgTx, while competition with varying concentrations of unlabeled w-CgTx (5 pM to 5 nM) was performed. Slides were rinsed as specified, tissue was wiped from the slides using filter discs (Whatman), and retained radioactivity measured with a gamma counter (Packard Multi-Prias 1, Downers Grove, Ill. Scatchard transformations of the data were accomplished using Inplot (GraphPad Software, San Diego, CA). Alternatively, competition with Ca z+ channel ligands exhibiting differing VGCC selectivity was performed (Table 1): namely, with 250 nM MVIIA (an N-channel-specific agent [34]), 200 nM agatoxin-IVA (a P-channel antagonist [22]) and 5 ~ M Nifedidine (an L-channel blocker). Quantitation was again performed using gamma counting.
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Table 1 Competition of calcium channel antagonists with specific [~251]~oCgTx binding to neonatal brainstem Competing agent (concentration)
Average cpms
c4 Displacement
None w-MVIIA (250 nM) w-lVA (200 nM) Nifedipine (5 # M )
2412 153 2616 2868
94 I) I)
Serial slide-mounted transverse sections of l-day-old rat brainstem (medulla/pons) were labeled with 157 pM [l>l]w-CgTx with or without the additional presence of other Ca e + channel-active agents. The concentration of each competing agent was chosen in order to achieve nearly 1(11)c2~ occupancy of the corresponding Ca e ' channel binding sites. Non-specific binding was defined in the presence of excess (250 nM) unlabeled eo-CgTx. Each data point represents the average CPMS (total binding-ram-specific) of six determinations: the experiment was repeated once with the same results. ~o-MVIIA, ~o-conotoxin MVIIA: to-IVA, w-agaloxinqVA.
Autoradiograms developed from sections of rat brain from different developmental stages are shown in Fig. 1. In the neonatal brain (Fig. la-c), the most densely labeled area is the hindbrain, with the cerebral cortex exhibiting very light labeling. However, later in development, the cerebral cortex has clearly become much more heavily labeled (Fig. if, i). The overall progression from caudal to rostral and from 'inside to outside' within cerebral cortex (as depicted in Fig. 1) is roughly parallel to the anatomical progression of other aspects of brain development [3,13] such as neuronal migration and synaptogenesis. However, closer inspection of Fig. 1 demonstrates that in addition to the general increase in labeling of more rostral regions of brain, there are selected areas where the intensity of labeling decreases with age, suggesting that there may be transient expression of some ~o-CgTx binding sites within certain locations. In particular, this seems to be true of the brainstem from medulla to mesencephalon. For example, plates lb, e and h depict sections at the level of the pons and cerebellum. It is clear that at postnatal day 1 (Fig. lb), the pons is more intensely labeled than is the primordial cerebellum, which shows somewhat lower, generalized labeling. No distinct regional heterogeneity of labeling within either of these structures is evident at this time. However, as development progresses, the cerebellum differentiates into a molecular layer which becomes more intensely labeled and a granular layer which has a relatively lower density of binding sites (Fig. le,h). There is also progressive expansion of the molecular layer (compare 2 week [Fig. lc] vs. 6 week [Fig. lh] sections), which coincides with the elaboration of Purkinje cell dendritic arborizations and with the progressive accumulation of layered parallel fibers within this structure [2]. In contrast to the cerebellum,
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F. Filloux et aL / Det,elopmental Brain Research 78 (1994) 131~130
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Postnatal age (days) Fig. 2. Quantitative comparison of [125I]o)-CgTx binding within the cerebral cortex, cerebellum (molecular layer) and pons. Autoradiograms of sagittal, coronal and transverse sections were quantified using video-based image-analysis (see text). While w-CgTx binding sites increase rapidly during development in the molecular layer of the cerebellum (©) and cerebral cortex ( zx ), there is a steady decline in binding sites within the ports (e). Scatchard analyses (not shown) confirm that these changes are in part attributable to changes in co-CgTx receptor number. Each data point represents the average of 3-5 animals per time point. Values at 42 days postnatal age are the same as those of mature adult rat brain. Bars indicate the S.E.M
labeling of the ports gradually decreases with development (Fig. lb,e,h). The intensity of labeling of the cortex, the molecular layer of the cerebellum and the ports are compared in Fig. 2. Over the time period examined, an increase in the intensity of labeling of both the cerebral cortex and of the molecular layer of the cerebellum is evident, with an accompanying decrease in the intensity of labeling of the pons. Clearly the pons, which had the highest density of sites at postnatal day 1, has become significantly depleted of binding sites by 6 weeks. A similar pattern of w-conotoxin site elimination is seen in the medulla (Fig. la,d,g); however, reduction in binding sites appears to be selective, with some regions retaining a high labeling density. Although w-CgTx binding sites are lost throughout the medullary reticular formation, loci such as the spinal tract of the trigeminal nucleus, the nucleus solitarius and to a lesser extent the inferior olive remain relatively intensely labeled (Fig. lg). These results demonstrate that the changes in density of w-conotoxin binding sites as a function of development are complex and highly region-specific. Competition studies with ligands selective for different VGCC subtypes indicate that [~25I]~o-CgTx binding to neonatal brainstem sites appears to be directed toward N-type Ca 2+ channels as defined pharmacologically (Table 1). Specifically, no displacement of specific []2SI]o)-CgTx by saturating concentrations of the P- or L-channel ligands w-agatoxin-IVA [22] and nifedipine is evident, whereas the N-channel ligand w-conotoxin MVIIA [34] competes effectively with binding to these brainstem sites. On the other hand,
saturation analyses (graphs not shown) suggest that neonatal w-CgTx-sensitive sites may be somewhat distinct in that they exhibit a higher affinity for [~2~l]~oCgTx under the conditions employed than do adult forebrain binding sites ( K ~ = 2 3 0 pM for neonatal brainstem sites vs. 570 pM for adult forebrain sites). The above findings describe complex developmental changes in rat brain [~25I]o)-CgTx binding sites. At least four different developmental patterns are seen: (A) some regions (e.g., the cerebral cortex) show a continuous increase in labeling intensity until mature levels are achieved; (B) other regions, such as the pons, exhibit transient expression, with virtually all binding sites disappearing by adulthood; (C) the cerebellum begins with a relatively uniform distribution of sites, but with progressive elaboration of cerebellar structures the molecular layer increases in labeling intensity relative to the granule cell layer; and (D) finally, in regions such as the medulla, there may be selective [t25I]w-CgTx GVIA binding site depletion. Sites present within the medullary reticular formation during the neonatal period are lost while intense labeling of regions such as the spinal tract of the trigeminal nerve remains. A similar pattern occurs in rostral mesencephalon at the level of the substantia nigra reticulata (SNr) where generalized binding at 1 day of age (Fig. lc) is succeeded by loss of binding sites throughout most of the midbrain with the exception of the SNr and the superficial layer of the superior colliculus where intense co-CgTx binding persists into adulthood (Fig. If, i). It is likely, but remains to be proven, that [~251]coCgTx binding sites in neonatal brain represent N-type Ca 2+ channels. Autoradiographic studies in mature rat brain suggest that w-CgTx GVIA binding sites exhibit a distribution distinct from that of brain L-channels [14,20,31]. Neonatal brainstem [125I]w-CgTx binding sites demonstrate resistance to L- and P-type [22] channel blockers and sensitivity to the N-channel agent, ~o-conotoxin MVI1A [34] (see Table 1). On the other hand, neonatal w-CgTx binding sites exhibit a greater affinity for [~2Sl]~o-CgTx than do adult forebrain binding sites ( K D = 230 pM and 570 pM, respectively). Although this modest difference in affinity may be due to non-specific factors such as variation in tissue constituents at distinct developmental ages, it is also conceivable that these affinity differences may reflect unique structural properties of the neonatally expressed N-channels. More specific molecular characterization of neonatal calcium channels will be required to resolve such possibilities. The major role for N-channels within the mature central nervous system appears to be that of regulating Ca2+-dependent release of neurotransmitters [10,29], and thus, [~2Sl]w-CgTx binding should be concentrated in synaptic areas [14,31]. In developing brain, a 'per-
F. Filloux et al. / Developmental Brain Research 78 (1994) 131-136
missive' role for N-channels in neuronal migration has been recently proposed by Komuro and Rakic [15]. These investigators have demonstrated that N-channel (but not L- or T-channel) blockade appears to markedly slow the rate of granule cell migration in the cerebellar slice [15]. In addition, rhodamine-conjugated w-CgTx binding demonstrated the temporal appearance of Nchannels just prior to onset of neuronal migration [15]. The accumulation of cerebral cortical oJ-CgTx binding sites as shown herein (i.e., between postnatal days 1 and 14) is somewhat delayed relative to neuronal migration within the cortex which, in the rat, takes place largely between gestational day 14 and the first few days of extrauterine life (although migrations to the outer cortical layers continue to a lesser degree until postnatal day 10) [3,13]. Thus, the temporal pattern of the appearance of w-CgTx binding sites may in fact better correspond to the earliest phases of rapid cortical synaptogenesis [1,13]. Likewise, the appearance of dense w-CgTx binding within the molecular layer of the cerebellum identified in this study coincides with the ramification of bipolar cell processes (incipient parallel fibers) within the molecular layer along with the exuberant growth of Purkinje cell dendritic arborizations [2]. Failure of granule cell migration and parallel fiber elaboration as occurs in the w e a v e r mutant mouse is accompanied by minimal levels of w-conotoxin binding within this cerebellar layer [17]. These observations are consistent with the observed temporal correspondence between CNS synaptogenesis and increases in numbers of N-type Ca 2+ channels [32]. The transient expression of putative N-type Ca 2~channels observed in discrete regions, especially within the brainstem, is somewhat unexpected but of great interest. It is in marked contrast to the ontogeny of L-type VGCCs. During development L-type Ca channels (as measured by dihydropyridine binding) appear to gradually increase to adult levels throughout the brain regardless of neuroanatomical location [9,12,23]; transient brainstem expression of L-channels has not been described. On the other hand, the loss of w-CgTx binding sites within the hindbrain appears to be accompanied in some cases by parallel changes in neurotransmitter receptor systems [25,28,33]. Thus, muscarinic acetylcholine [28] and G A B A A [33] receptors have higher densities in the brainstem during early development; brainstem nicotine binding sites which are present in early development almost completely disappear during adulthood [25]. The coincidence between loss of binding sites for the presynaptic marker, w-CgTx on the one hand, and of neurotransmitter receptors on the other may be due to selective elimination of specific types of synapses in the hindbrain during postnatal ontogeny. Depletion of oJ-CgTx-sensitive sites could conceivably even play a causative role in postsynaptic receptor elimination, since removal of
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presynaptic input is known to cause more rapid turnover of postsynaptic receptors (as in the case of nicotinic acetylcholine receptors at the neuromuscular junction; see ref. 27). Therefore, reduction in synaptic transmitter release engendered by loss of N-type Ca 2 + channels at nerve terminals could provide one mechanism triggering the loss of postsynaptic receptors in selected hindbrain synapses. N-type Ca 2+ channel depletion in the hindbrain may have important functional correlates. It has been previously reported that intracerebral injection of wCgTx produces respiratory arrest in neonatal animals, but not in mature animals [24]. These results are consistent with disappearance of w-CgTx binding sites in the respiratory control regions within the reticular formation of the hindbrain. Aberrant development of brainstem Ca 2+ channels could thus produce a disturbance of respiratory control, a possibility potentially relevant to the understanding of Sudden Infant Death Syndrome (SIDS) [4]. Although ion channel pruning may coincide with synapse elimination in some cases, it may not necessarily do so in all instances. The requirement for N-type Ca 2+ channels in regulation of neuronal migration [15] could also lead to their transient expression. Once cell migration is completed, the N-type Ca -,+ channels which participate in the process may become obsolete, and therefore turn over. Thus, using methods capable of greater resolution, it will be of interest to determine which of the N-type Ca 2+ channel depletion events are correlated with synaptic elimination or neuronal cell death, and which represent ion channel turnover for other purposes such as neuronal cell migration. The striking complexity of N-channel ontogeny relative to that of L-channels warrants further investigation, particularly to clarify potential roles of N-type Ca 2+ channels in diverse neurodevelopmental processes. Kevin Ryujin and Michael Totzke provided technical assistance and Nancy Kurtzeborn expert preparation of the manuscript. Thanks is also due to D. Yoshikami, W. Gray and M. Jacobson lk)r critical review. This work was supported by NIH Grants HD 00912 (F.F.), PO1 GM48677 (B.M.O.) and K20 MH0(1929 (J.M.M.). [1] Aghajanian, G.K. and Bloom, F.E., The fl~rmation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Res., 6 (1967) 716-727. [2] Altman, J., Postnatal development of the cerebellar cortex in the rat. 1I. Phases in the maturation of Purkinje cells of the molecular layer, J. Comp. Neurol., 145 (1972) 399-464. [3] Angevine, J.B. and Sidman, R.L., Autoradiographic sludy of cell migration during histogenesis of cerebral cortex in the mouse, Nature, 192 (1961) 766-768. [4] Becker, L.E., Neural maturational delay as a link in the chain of events leading to S1DS, Can. J. Neurol. Sci., 17 (1990) 361-371. [5] Benes, F.M. and Bird, E.D., An analysis of the arrangement of neurones in the cingulate cortex of schizophrenic patients, Are. Gen. Psychiat., 44 (1987) 608-616.
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[6] Collingridge, G.L. and Singer, W., Excitatory amino acid receptors and synaptic plasticity. In D. Lodge and G. Collingridge (Eds.), The Pharmacology of Excitatory Amino Acids. A TiPS Special Report, Elsevier, Amsterdam, 1990, pp. 42-48. [7] Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfied, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265. [8] Cruz, L.J. and Olivera, B.M., Calcium channel antagonists: w-conotoxin defines a new high affinity site, J. Biol. Chem., 23 (1986) 6230-6233. [9] Dooley, D.J., Lickert, M., Lupp, A. and Osswald, H., Distribution of [z25I]to-conotoxin GVIA and [3H]isradipine binding sites in the central nervous system of rats of different ages, Neurosci. Lett., 93 (1988) 318-323. [10] Dooley, D.J., Lupp, A. and Hertting, G., Inhibition of central neurotransmitter release by to-conotoxin GVIA, a peptide modulator of the N-type voltage-sensitive calcium channel, NaunynSchmiedeberg's Arch. Pharmacol., 336 (1987) 467-470. [11] Filloux, F., Olivera, B.M. and McIntosh, J.M. Neomycin displacement of [lzsI]w-conotoxin GVIA binding is not uniform across neuroanatomical regions: evidence from autoradiographic studies, Soc. Neurosci. Abstr., 18 (1992) 970. [12] Galewski, S., Skangiel-Kramska, J., Pomorski, P. and Kossut, M., Voltage-dependent L-type calcium channels in the development and plasticity of mouse barrel cortex, Dev. Brain Res., 67 (1992) 293-300. [13] Jacobson, M., Developmental Neurobiology, 3rd edn., Plenum, New York, 1991, pp. 250-278, 311-345. [14] Kerr, L.M., Filloux, F., Olivera, B.M., Jackson, H. and Wamsley, J.K., Autoradiographic localization of calcium channels with [125I]w-conotoxin in rat brain, Eur. J. Pharmacol., 146 (1988) 181-183. [15] Komuro, H. and Rakic, P., Selective rote of N-type calcium channels in neuronal migration, Science, 257 (1992) 806-809. [16] Llinas, R., Sugimori, A., Hillman, D.E. and Cherksey, B., Distribution and functional significance of the P-type voltage dependent Ca 2÷ channels in the mammalian central nervous system, Trends Neurosci., 15 (1992) 351-355. [17] Maeda, N., Wada, K., Yuzaki, M. and Mikoshiba, K., Autoradiographic visualization of a calcium channel antagonist, [1251]~0-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver), Brain Res., 489 (1989) 21-30. [18] Mattson, M.P., Cellular signaling mechanisms common to the development and degeneration of neuroarchitecture. A review, Mech. Aging Det:., 50 (1989) 103-157. [19] McEachron, D.L., Gallistel, C.C., Tretiak, O. and Eilbert, J.L., The analytic and functional accuracy of a videodensitometry system, Z Neurosci. Methods, 25 (1988) 63-74.
[20] Mclntosh, J.M., Adams, M.E., Olivera, B.M. and Filloux, F., Autoradiographic localization of the binding of calcium channel antagonist, [~zsI]to-agatoxin IliA, in rat brain. Brain Res., 594 (1992) 109-114. [21] Miller, R.J., Multiple calcium channels and neuronal function, Science, 235 (1987) 46-52. [22] Mintz, I.M., Venema, V.J., Swiderek, K., Lee, T., Bean, B.P. and Adams, M.E., P-type calcium channels blocked by the spider toxin (w-Aga-IVA), Nature, 355 (1992) 827-829. [23] Mourre, C., Cerbera, P. and Lazdunski, M., Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na + channels, Ca2+-dependent K + channels and slow Ca 2 + channels identified as receptors for tetrodotoxin, apamine and (-)-desmethoxy-verapamil, Brain Res., 417 (1987) 21-32. [24] Myers, R.A., Mclntosh, J.M., Imperial, J., Williams, R.W., Oas, T., Haack, J.A., Hernandez, J.-F., Rivier, J., Cruz, L.J. and Olivera B.M., Peptides from Conus venoms which affect Ca 2+ entry into neurons, J. Toxicol. Toxin Rer., 9 (1990) 179-202. [25] Naeff, B., Schlumpf, M. and Lichtensteiger, W., Pre- and postnatal development of high-affinity [3H]nicotine binding sites in rat brain regions: an autoradiographic study, Dev. Brain Res., 68 (1992) 163-174. [26] Purves, D. and Lichtman, J.W., Elimination of synapses in the developing nervous system, Science, 210 (1980) 153-157. [27] Salpeter, M., Cooper, D.L. and Levitt-Gilmour, T., Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction, J. Cell Biol., 103 (1986) 1399-1403. [28] Schlumpf, M., Palacios, JM., Cortes, R. and Lichtensteiger, W., Regional development of muscarinic cholinergic binding sites in the prenatal rat brain, Neuroscience, 45 (199t) 347-357. [29] Sher, F. and Clementi, F., ~o-Conotoxin-sensitive voltage-operated calcium channels in vertebrate cells, Neuroscience 42 (1991) 301-307. [30] Snutch, T.P. and Reiner, P.B., Ca 2+ channels: diversity of form and function, Current Opin. Neurobiol., 2 (1992) 247-253. [31] Takemura, M., Kiyama, H., Fukui, H., Tokyama, M. and Wada, H., Distribution of the o~-conotoxin receptor in rat brain. An autoradiographic mapping, Neuroscience, 32 (1989) 405-416. [32] Vigers, A.J. and Pfenninger, K.H., N-type and L-type calcium channels are present in nerve growth cones. Numbers increase on synaptogenesis, Dec. Brain Res., 60 (1991) 197-203. [33] Xia, Y. and Haddad, G.G., Ontogeny and distribution of GABA A receptors in rat brainstem and rostral brain regions, Neuroscience, 49 (1992) 973-989. [34] Yoshikami, D., Bagabaldo, Z. and Olivera, B.M., The inhibitory effects of omega-conotoxins on Ca channels and synapses, Ann. N.Y. Acad. Sci., 560 (1989) 230-248.
ELSEVIER
Developmental Brain Research 78 (1994) 131-136
Short Communication
Complex patterns of [125I]o-conotoxin GVIA binding site expression during postnatal rat brain development F. Filloux *, Aaron Schapper, Scott R. Naisbitt, Baldomero M. Olivera, J. Michael Mclntosh Departments Of Neurolo,w, Pediatrics, Psychiato' and Biology, UnicersiO' o[ Utah, Salt Lake City, UT 84132, USA (Accepted 23 November 19931
Abstract
In the mature CNS, N-type calcium channels regulate neurotransmitter release. The role of these channels in developing brain is less clear. Study of [~25I]w-conotoxin GVIA binding sites in developing rat brain using autoradiography reveals that putative N-type channels appear and disappear in complex temporal-spatial profiles including: (1) gradual increase to adult levels (cerebral cortex); (2) substructure differentiation (cerebellum); (3) transient expression (pons); and, (4) selective depletion (medulla). Transient expression of N-type calcium channels may influence specific neurodevelopmental processes.
Key words: [125I]w-Conotoxin GVIA; N-type calcium channel; Autoradiograpby, Rat brain development
Nervous system development includes 'progressive' processes (e.g. neurulation, induction, cell proliferation, migration, synaptogenesis and myelination) as well as 'regressive' phenomena whereby early redundancies in CNS connectivity are eliminated [7,26]. For example, neuronal death (apoptosis) and synapse elimination are crucial to the formation of a normally functioning nervous system [7,18,26]. Defects in these latter processes have been proposed as contributing to various pathological conditions, as diverse as schizophrenia [5] and Sudden Infant Death Syndrome [4]. In view of the central importance of intracellular calcium ion concentration ([Ca2+]i) to the control of nervous system function in general, cell constituents which modulate [Ca2+]i may be involved in these 'regressive" phenomena [18]. Receptor-operated Ca 2+ channels, particularly N-methyl-D-aspartate (NMDA) type glutamate-activated Ca 2+ channels, have the potential to influence nervous system plasticity [6]. However, other molecular constituents also participate in regulating [Ca2+]~ including voltage-gated Ca 2+ channels (VGCCs), of which several subtypes (L, N, T and P) have been described [16,21,22,30]. In particular,
* Corresponding author. Research Laboratory, Western Institute of Neuropsychiatry, 501 Chipeta Way, Salt Lake City, U T 84108. USA. Fax: ( 1) (801) 582-8471. Elsevier Science B.V.
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roles for N-type Ca 2+ channels in synaptogenesis [32] and neuronal migration [15] have recently been proposed. The peptide w-conotoxin GVIA (w-CgTx) from the marine snail Conus geographus is known to block presynaptic Ca 2+ channels [8,10,29], and thus radiolabeled [8,9,14,20,31] and fluorescent [15] derivatives of the peptide are potential markers for synapses bearing such presynaptic ion channels. Within the mammalian nervous system this ligand is believed to selectively bind the N-type subset of VGCCs [29]. We have used ¢o-CgTx to investigate the postnatal ontogeny of N-type Ca 2+ channels in rat brain. The results indicate that, in contrast to L-type channels which seem to increase steadily to adult levels throughout the brain in a spatially uniform pattern [9,12,23], the appearance of wCgTx-labeled channels is more complex both in time and space. In particular, transiently expressed ~oCgTx-sensitive channels undergo a striking disappearance from most regions of the hindbrain. Sprague-Dawley rats of varying postnatal ages (1 day, 3 days, and 1, 2, 3 and 6 weeks and adult) were utilized independent of sex. Animals were deeply anesthetized with pentobarbital (60 m g / k g ip) and perfused transcardially with phosphate-buffered saline containing 5% dextrose (4°C, pH 7.4). Brains were promptly removed from the skull after decapitation and immediately frozen by immersion into isopentane (-65°C).
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qg. 1. [125I]~o-CgTx G V I A binding to sections of rat brain of varying ages. Slide-mounted transverse or sagittal tissue sections of rat brain of varying postnatal ages were labeled with t25I]w-CgTx (see text for details). These plates represent photomicrographs of original autoradiograms taken under brightfield illumination using a Wild Photomacroscope (Wild Heerbrugg. ;witzerland). Areas appearing darker indicate greater density ot [ I25I]w-CgTx binding. Autoradiograms are from 1-day-old (a,b,c) 2-week-otd (d~e,f), and 6-week-old (g.h.i) animals. (The pattern ~t ~ weeks of age is indistinguishable from that in mature rat brain.) Non-specific binding ~not shown) was very low at all development ages and represented less than ll)~ of total binding in all egions exhibiting significant densities of binding sites. Note the overall caudal to rostral redistribution of o)-CgTx binding sites with dense hindbrai~l hut minimal cerebral c~rtex binding at one lay of age tc). and the reverse (dense cortical binding, but light brainstem binding) al six weeks of age (i). Higher power images at the level of the medulla (a,d,g) and pons (P) and cerebellum Ce) (b.e,h) are also shown. At 6 weeks of age, binding sites within the hindbrain have virtually disappeared (plates g,h) with the exeepti~m of restricted regions such as the spinal tract of the Vth rama nerve (Sp5, plate g), the nucleus of the solitary tract (Sot. plate g), and the substantia nigra reticulata ISNr. plate i). In contrast, differentiation of the ccrebellar cortex i~ evident earlic~ e.g.. see frame e~, Ac. nucleus accumbens: cc, corpus callosum- CPu, caudate-putamen: egr, gr and tool" external granular layer, granule cell la~er and molecula~ laye~ of ce~'ebellum. espectively; Ctx. cerebral cortex: Hi, hippocampus; Io. inferior olivary complex; OT, olfactory tubercle: SC. superior colliculus. Bars = 2 mm.
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F. Fillowc et al. / Developmental Brain Research 78 (19941 131-136
They were stored at - 7 0 ° C in an airtight container until use. Ten /,m-thick sagittal or transverse/coronal sections encompassing brainstem through forebrain were cut at - 17°C with a cryostat microtome and were thaw-mounted on chrome-alum-coated microscope slides. These tissue sections were stored at -70°C. Five brains were sampled per developmental time point. Labeling with [t25 l]to-CgTx (2196 C i / m m o l ) was performed as previously described [14,20]. Specifically, slide-mounted tissue sections were warmed to room temperature and incubated for 30 min with 100-300 >1 (depending on size of tissue section) of 5 mM H E P E S / T r i s buffer (25°C, pH 7.4) containing 120 mM sucrose, 100 mM NaC1, 0.2 m g / m l lysozyme and 200 pM [tzSI]w-CgTx which was synthesized and iodinated as previously described [8,20]. Adjacent tissue sections were incubated similarly, but with the addition of 3 mM neomycin [11] to determine non-specific binding. Slides were then washed (3 × 10 min each) in fresh H E P E S / T r i s buffer (4°C) containing 160 mM NaCI, 1.5 mM CaC1 z, 1 m g / m l BSA, and 0.05% polyoxyethylene sorbitan monolaurate. Slides were dipped in distilled H 2 0 (4°C) for 1 s, dried promptly with a stream of cool, dry air, and stored in the presence of desiccant at 4°C overnight. Dried slides were then apposed to Hyperfilm /3-Max (Amersham, Arlington Hts., ILl, and film was developed after a 24-h exposure. Resultant autoradiograms were digitized and density of [t2sI]~o-CgTx binding quantitated with BRAIN software [19] (Drexel University, Philadelphia, PAl, by comparing grey levels of the tissue-generated autoradiograms to those produced by 125I plastic polymer standards (Amersham, Arlington Heights, ILl. Autoradiograms were photographed (Fig. 1) using a Wild Photomacroscope (Wild-Heerbrugg, Switzerland). The kinetics and pharmacological specificity of [tzSI]w-CgTx binding to neonatal tissue were examined in additional transverse, slide-mounted sections of 1day-old brainstem (medulla/pons) animals. Tissue sections were labeled as described above with 50 pM [IzsI]to-CgTx, while competition with varying concentrations of unlabeled w-CgTx (5 pM to 5 nM) was performed. Slides were rinsed as specified, tissue was wiped from the slides using filter discs (Whatman), and retained radioactivity measured with a gamma counter (Packard Multi-Prias 1, Downers Grove, Ill. Scatchard transformations of the data were accomplished using Inplot (GraphPad Software, San Diego, CA). Alternatively, competition with Ca z+ channel ligands exhibiting differing VGCC selectivity was performed (Table 1): namely, with 250 nM MVIIA (an N-channel-specific agent [34]), 200 nM agatoxin-IVA (a P-channel antagonist [22]) and 5 ~ M Nifedidine (an L-channel blocker). Quantitation was again performed using gamma counting.
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Table 1 Competition of calcium channel antagonists with specific [~251]~oCgTx binding to neonatal brainstem Competing agent (concentration)
Average cpms
c4 Displacement
None w-MVIIA (250 nM) w-lVA (200 nM) Nifedipine (5 # M )
2412 153 2616 2868
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Serial slide-mounted transverse sections of l-day-old rat brainstem (medulla/pons) were labeled with 157 pM [l>l]w-CgTx with or without the additional presence of other Ca e + channel-active agents. The concentration of each competing agent was chosen in order to achieve nearly 1(11)c2~ occupancy of the corresponding Ca e ' channel binding sites. Non-specific binding was defined in the presence of excess (250 nM) unlabeled eo-CgTx. Each data point represents the average CPMS (total binding-ram-specific) of six determinations: the experiment was repeated once with the same results. ~o-MVIIA, ~o-conotoxin MVIIA: to-IVA, w-agaloxinqVA.
Autoradiograms developed from sections of rat brain from different developmental stages are shown in Fig. 1. In the neonatal brain (Fig. la-c), the most densely labeled area is the hindbrain, with the cerebral cortex exhibiting very light labeling. However, later in development, the cerebral cortex has clearly become much more heavily labeled (Fig. if, i). The overall progression from caudal to rostral and from 'inside to outside' within cerebral cortex (as depicted in Fig. 1) is roughly parallel to the anatomical progression of other aspects of brain development [3,13] such as neuronal migration and synaptogenesis. However, closer inspection of Fig. 1 demonstrates that in addition to the general increase in labeling of more rostral regions of brain, there are selected areas where the intensity of labeling decreases with age, suggesting that there may be transient expression of some ~o-CgTx binding sites within certain locations. In particular, this seems to be true of the brainstem from medulla to mesencephalon. For example, plates lb, e and h depict sections at the level of the pons and cerebellum. It is clear that at postnatal day 1 (Fig. lb), the pons is more intensely labeled than is the primordial cerebellum, which shows somewhat lower, generalized labeling. No distinct regional heterogeneity of labeling within either of these structures is evident at this time. However, as development progresses, the cerebellum differentiates into a molecular layer which becomes more intensely labeled and a granular layer which has a relatively lower density of binding sites (Fig. le,h). There is also progressive expansion of the molecular layer (compare 2 week [Fig. lc] vs. 6 week [Fig. lh] sections), which coincides with the elaboration of Purkinje cell dendritic arborizations and with the progressive accumulation of layered parallel fibers within this structure [2]. In contrast to the cerebellum,
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F. Filloux et aL / Det,elopmental Brain Research 78 (1994) 131~130
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Postnatal age (days) Fig. 2. Quantitative comparison of [125I]o)-CgTx binding within the cerebral cortex, cerebellum (molecular layer) and pons. Autoradiograms of sagittal, coronal and transverse sections were quantified using video-based image-analysis (see text). While w-CgTx binding sites increase rapidly during development in the molecular layer of the cerebellum (©) and cerebral cortex ( zx ), there is a steady decline in binding sites within the ports (e). Scatchard analyses (not shown) confirm that these changes are in part attributable to changes in co-CgTx receptor number. Each data point represents the average of 3-5 animals per time point. Values at 42 days postnatal age are the same as those of mature adult rat brain. Bars indicate the S.E.M
labeling of the ports gradually decreases with development (Fig. lb,e,h). The intensity of labeling of the cortex, the molecular layer of the cerebellum and the ports are compared in Fig. 2. Over the time period examined, an increase in the intensity of labeling of both the cerebral cortex and of the molecular layer of the cerebellum is evident, with an accompanying decrease in the intensity of labeling of the pons. Clearly the pons, which had the highest density of sites at postnatal day 1, has become significantly depleted of binding sites by 6 weeks. A similar pattern of w-conotoxin site elimination is seen in the medulla (Fig. la,d,g); however, reduction in binding sites appears to be selective, with some regions retaining a high labeling density. Although w-CgTx binding sites are lost throughout the medullary reticular formation, loci such as the spinal tract of the trigeminal nucleus, the nucleus solitarius and to a lesser extent the inferior olive remain relatively intensely labeled (Fig. lg). These results demonstrate that the changes in density of w-conotoxin binding sites as a function of development are complex and highly region-specific. Competition studies with ligands selective for different VGCC subtypes indicate that [~25I]~o-CgTx binding to neonatal brainstem sites appears to be directed toward N-type Ca 2+ channels as defined pharmacologically (Table 1). Specifically, no displacement of specific []2SI]o)-CgTx by saturating concentrations of the P- or L-channel ligands w-agatoxin-IVA [22] and nifedipine is evident, whereas the N-channel ligand w-conotoxin MVIIA [34] competes effectively with binding to these brainstem sites. On the other hand,
saturation analyses (graphs not shown) suggest that neonatal w-CgTx-sensitive sites may be somewhat distinct in that they exhibit a higher affinity for [~2~l]~oCgTx under the conditions employed than do adult forebrain binding sites ( K ~ = 2 3 0 pM for neonatal brainstem sites vs. 570 pM for adult forebrain sites). The above findings describe complex developmental changes in rat brain [~25I]o)-CgTx binding sites. At least four different developmental patterns are seen: (A) some regions (e.g., the cerebral cortex) show a continuous increase in labeling intensity until mature levels are achieved; (B) other regions, such as the pons, exhibit transient expression, with virtually all binding sites disappearing by adulthood; (C) the cerebellum begins with a relatively uniform distribution of sites, but with progressive elaboration of cerebellar structures the molecular layer increases in labeling intensity relative to the granule cell layer; and (D) finally, in regions such as the medulla, there may be selective [t25I]w-CgTx GVIA binding site depletion. Sites present within the medullary reticular formation during the neonatal period are lost while intense labeling of regions such as the spinal tract of the trigeminal nerve remains. A similar pattern occurs in rostral mesencephalon at the level of the substantia nigra reticulata (SNr) where generalized binding at 1 day of age (Fig. lc) is succeeded by loss of binding sites throughout most of the midbrain with the exception of the SNr and the superficial layer of the superior colliculus where intense co-CgTx binding persists into adulthood (Fig. If, i). It is likely, but remains to be proven, that [~251]coCgTx binding sites in neonatal brain represent N-type Ca 2+ channels. Autoradiographic studies in mature rat brain suggest that w-CgTx GVIA binding sites exhibit a distribution distinct from that of brain L-channels [14,20,31]. Neonatal brainstem [125I]w-CgTx binding sites demonstrate resistance to L- and P-type [22] channel blockers and sensitivity to the N-channel agent, ~o-conotoxin MVI1A [34] (see Table 1). On the other hand, neonatal w-CgTx binding sites exhibit a greater affinity for [~2Sl]~o-CgTx than do adult forebrain binding sites ( K D = 230 pM and 570 pM, respectively). Although this modest difference in affinity may be due to non-specific factors such as variation in tissue constituents at distinct developmental ages, it is also conceivable that these affinity differences may reflect unique structural properties of the neonatally expressed N-channels. More specific molecular characterization of neonatal calcium channels will be required to resolve such possibilities. The major role for N-channels within the mature central nervous system appears to be that of regulating Ca2+-dependent release of neurotransmitters [10,29], and thus, [~2Sl]w-CgTx binding should be concentrated in synaptic areas [14,31]. In developing brain, a 'per-
F. Filloux et al. / Developmental Brain Research 78 (1994) 131-136
missive' role for N-channels in neuronal migration has been recently proposed by Komuro and Rakic [15]. These investigators have demonstrated that N-channel (but not L- or T-channel) blockade appears to markedly slow the rate of granule cell migration in the cerebellar slice [15]. In addition, rhodamine-conjugated w-CgTx binding demonstrated the temporal appearance of Nchannels just prior to onset of neuronal migration [15]. The accumulation of cerebral cortical oJ-CgTx binding sites as shown herein (i.e., between postnatal days 1 and 14) is somewhat delayed relative to neuronal migration within the cortex which, in the rat, takes place largely between gestational day 14 and the first few days of extrauterine life (although migrations to the outer cortical layers continue to a lesser degree until postnatal day 10) [3,13]. Thus, the temporal pattern of the appearance of w-CgTx binding sites may in fact better correspond to the earliest phases of rapid cortical synaptogenesis [1,13]. Likewise, the appearance of dense w-CgTx binding within the molecular layer of the cerebellum identified in this study coincides with the ramification of bipolar cell processes (incipient parallel fibers) within the molecular layer along with the exuberant growth of Purkinje cell dendritic arborizations [2]. Failure of granule cell migration and parallel fiber elaboration as occurs in the w e a v e r mutant mouse is accompanied by minimal levels of w-conotoxin binding within this cerebellar layer [17]. These observations are consistent with the observed temporal correspondence between CNS synaptogenesis and increases in numbers of N-type Ca 2+ channels [32]. The transient expression of putative N-type Ca 2~channels observed in discrete regions, especially within the brainstem, is somewhat unexpected but of great interest. It is in marked contrast to the ontogeny of L-type VGCCs. During development L-type Ca channels (as measured by dihydropyridine binding) appear to gradually increase to adult levels throughout the brain regardless of neuroanatomical location [9,12,23]; transient brainstem expression of L-channels has not been described. On the other hand, the loss of w-CgTx binding sites within the hindbrain appears to be accompanied in some cases by parallel changes in neurotransmitter receptor systems [25,28,33]. Thus, muscarinic acetylcholine [28] and G A B A A [33] receptors have higher densities in the brainstem during early development; brainstem nicotine binding sites which are present in early development almost completely disappear during adulthood [25]. The coincidence between loss of binding sites for the presynaptic marker, w-CgTx on the one hand, and of neurotransmitter receptors on the other may be due to selective elimination of specific types of synapses in the hindbrain during postnatal ontogeny. Depletion of oJ-CgTx-sensitive sites could conceivably even play a causative role in postsynaptic receptor elimination, since removal of
135
presynaptic input is known to cause more rapid turnover of postsynaptic receptors (as in the case of nicotinic acetylcholine receptors at the neuromuscular junction; see ref. 27). Therefore, reduction in synaptic transmitter release engendered by loss of N-type Ca 2 + channels at nerve terminals could provide one mechanism triggering the loss of postsynaptic receptors in selected hindbrain synapses. N-type Ca 2+ channel depletion in the hindbrain may have important functional correlates. It has been previously reported that intracerebral injection of wCgTx produces respiratory arrest in neonatal animals, but not in mature animals [24]. These results are consistent with disappearance of w-CgTx binding sites in the respiratory control regions within the reticular formation of the hindbrain. Aberrant development of brainstem Ca 2+ channels could thus produce a disturbance of respiratory control, a possibility potentially relevant to the understanding of Sudden Infant Death Syndrome (SIDS) [4]. Although ion channel pruning may coincide with synapse elimination in some cases, it may not necessarily do so in all instances. The requirement for N-type Ca 2+ channels in regulation of neuronal migration [15] could also lead to their transient expression. Once cell migration is completed, the N-type Ca -,+ channels which participate in the process may become obsolete, and therefore turn over. Thus, using methods capable of greater resolution, it will be of interest to determine which of the N-type Ca 2+ channel depletion events are correlated with synaptic elimination or neuronal cell death, and which represent ion channel turnover for other purposes such as neuronal cell migration. The striking complexity of N-channel ontogeny relative to that of L-channels warrants further investigation, particularly to clarify potential roles of N-type Ca 2+ channels in diverse neurodevelopmental processes. Kevin Ryujin and Michael Totzke provided technical assistance and Nancy Kurtzeborn expert preparation of the manuscript. Thanks is also due to D. Yoshikami, W. Gray and M. Jacobson lk)r critical review. This work was supported by NIH Grants HD 00912 (F.F.), PO1 GM48677 (B.M.O.) and K20 MH0(1929 (J.M.M.). [1] Aghajanian, G.K. and Bloom, F.E., The fl~rmation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Res., 6 (1967) 716-727. [2] Altman, J., Postnatal development of the cerebellar cortex in the rat. 1I. Phases in the maturation of Purkinje cells of the molecular layer, J. Comp. Neurol., 145 (1972) 399-464. [3] Angevine, J.B. and Sidman, R.L., Autoradiographic sludy of cell migration during histogenesis of cerebral cortex in the mouse, Nature, 192 (1961) 766-768. [4] Becker, L.E., Neural maturational delay as a link in the chain of events leading to S1DS, Can. J. Neurol. Sci., 17 (1990) 361-371. [5] Benes, F.M. and Bird, E.D., An analysis of the arrangement of neurones in the cingulate cortex of schizophrenic patients, Are. Gen. Psychiat., 44 (1987) 608-616.
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[6] Collingridge, G.L. and Singer, W., Excitatory amino acid receptors and synaptic plasticity. In D. Lodge and G. Collingridge (Eds.), The Pharmacology of Excitatory Amino Acids. A TiPS Special Report, Elsevier, Amsterdam, 1990, pp. 42-48. [7] Cowan, W.M., Fawcett, J.W., O'Leary, D.D.M. and Stanfied, B.B., Regressive events in neurogenesis, Science, 225 (1984) 1258-1265. [8] Cruz, L.J. and Olivera, B.M., Calcium channel antagonists: w-conotoxin defines a new high affinity site, J. Biol. Chem., 23 (1986) 6230-6233. [9] Dooley, D.J., Lickert, M., Lupp, A. and Osswald, H., Distribution of [z25I]to-conotoxin GVIA and [3H]isradipine binding sites in the central nervous system of rats of different ages, Neurosci. Lett., 93 (1988) 318-323. [10] Dooley, D.J., Lupp, A. and Hertting, G., Inhibition of central neurotransmitter release by to-conotoxin GVIA, a peptide modulator of the N-type voltage-sensitive calcium channel, NaunynSchmiedeberg's Arch. Pharmacol., 336 (1987) 467-470. [11] Filloux, F., Olivera, B.M. and McIntosh, J.M. Neomycin displacement of [lzsI]w-conotoxin GVIA binding is not uniform across neuroanatomical regions: evidence from autoradiographic studies, Soc. Neurosci. Abstr., 18 (1992) 970. [12] Galewski, S., Skangiel-Kramska, J., Pomorski, P. and Kossut, M., Voltage-dependent L-type calcium channels in the development and plasticity of mouse barrel cortex, Dev. Brain Res., 67 (1992) 293-300. [13] Jacobson, M., Developmental Neurobiology, 3rd edn., Plenum, New York, 1991, pp. 250-278, 311-345. [14] Kerr, L.M., Filloux, F., Olivera, B.M., Jackson, H. and Wamsley, J.K., Autoradiographic localization of calcium channels with [125I]w-conotoxin in rat brain, Eur. J. Pharmacol., 146 (1988) 181-183. [15] Komuro, H. and Rakic, P., Selective rote of N-type calcium channels in neuronal migration, Science, 257 (1992) 806-809. [16] Llinas, R., Sugimori, A., Hillman, D.E. and Cherksey, B., Distribution and functional significance of the P-type voltage dependent Ca 2÷ channels in the mammalian central nervous system, Trends Neurosci., 15 (1992) 351-355. [17] Maeda, N., Wada, K., Yuzaki, M. and Mikoshiba, K., Autoradiographic visualization of a calcium channel antagonist, [1251]~0-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver), Brain Res., 489 (1989) 21-30. [18] Mattson, M.P., Cellular signaling mechanisms common to the development and degeneration of neuroarchitecture. A review, Mech. Aging Det:., 50 (1989) 103-157. [19] McEachron, D.L., Gallistel, C.C., Tretiak, O. and Eilbert, J.L., The analytic and functional accuracy of a videodensitometry system, Z Neurosci. Methods, 25 (1988) 63-74.
[20] Mclntosh, J.M., Adams, M.E., Olivera, B.M. and Filloux, F., Autoradiographic localization of the binding of calcium channel antagonist, [~zsI]to-agatoxin IliA, in rat brain. Brain Res., 594 (1992) 109-114. [21] Miller, R.J., Multiple calcium channels and neuronal function, Science, 235 (1987) 46-52. [22] Mintz, I.M., Venema, V.J., Swiderek, K., Lee, T., Bean, B.P. and Adams, M.E., P-type calcium channels blocked by the spider toxin (w-Aga-IVA), Nature, 355 (1992) 827-829. [23] Mourre, C., Cerbera, P. and Lazdunski, M., Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na + channels, Ca2+-dependent K + channels and slow Ca 2 + channels identified as receptors for tetrodotoxin, apamine and (-)-desmethoxy-verapamil, Brain Res., 417 (1987) 21-32. [24] Myers, R.A., Mclntosh, J.M., Imperial, J., Williams, R.W., Oas, T., Haack, J.A., Hernandez, J.-F., Rivier, J., Cruz, L.J. and Olivera B.M., Peptides from Conus venoms which affect Ca 2+ entry into neurons, J. Toxicol. Toxin Rer., 9 (1990) 179-202. [25] Naeff, B., Schlumpf, M. and Lichtensteiger, W., Pre- and postnatal development of high-affinity [3H]nicotine binding sites in rat brain regions: an autoradiographic study, Dev. Brain Res., 68 (1992) 163-174. [26] Purves, D. and Lichtman, J.W., Elimination of synapses in the developing nervous system, Science, 210 (1980) 153-157. [27] Salpeter, M., Cooper, D.L. and Levitt-Gilmour, T., Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction, J. Cell Biol., 103 (1986) 1399-1403. [28] Schlumpf, M., Palacios, JM., Cortes, R. and Lichtensteiger, W., Regional development of muscarinic cholinergic binding sites in the prenatal rat brain, Neuroscience, 45 (199t) 347-357. [29] Sher, F. and Clementi, F., ~o-Conotoxin-sensitive voltage-operated calcium channels in vertebrate cells, Neuroscience 42 (1991) 301-307. [30] Snutch, T.P. and Reiner, P.B., Ca 2+ channels: diversity of form and function, Current Opin. Neurobiol., 2 (1992) 247-253. [31] Takemura, M., Kiyama, H., Fukui, H., Tokyama, M. and Wada, H., Distribution of the o~-conotoxin receptor in rat brain. An autoradiographic mapping, Neuroscience, 32 (1989) 405-416. [32] Vigers, A.J. and Pfenninger, K.H., N-type and L-type calcium channels are present in nerve growth cones. Numbers increase on synaptogenesis, Dec. Brain Res., 60 (1991) 197-203. [33] Xia, Y. and Haddad, G.G., Ontogeny and distribution of GABA A receptors in rat brainstem and rostral brain regions, Neuroscience, 49 (1992) 973-989. [34] Yoshikami, D., Bagabaldo, Z. and Olivera, B.M., The inhibitory effects of omega-conotoxins on Ca channels and synapses, Ann. N.Y. Acad. Sci., 560 (1989) 230-248.