Voltage sensitive calcium channels mark a critical period in mouse neurodevelopment

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ht. J. Devl Neuroscience,Vol. 11, No. 1, pp. 17-24, 1993

07365748193.%.00+0.00 Pergamon Press Ltd @ 1993ISDN

Printedin Great Britain.



of Pediatrics and Physiology, University of Utah, Salt Lake City, UT 84142, U.S.A.

(Received 3 August 1992; in revised form 16 November 1992; accepted 17 November 1992)

Abstract-Voltage sensitive calcium channel (VSCC) probes ‘251-w-GVIA Conotoxin (o-GVIA), ( +)-[5-methyl-3H]-PNZOO-l10 (3H-PNZOO), and 3H-Nimodipine were bound to developing Swiss Webster mouse whole brain from postnatal days 3 to 24. ‘*‘I-o-GVIA binding, thought to be presynaptic, showed a 50% increase between days 11 and 14. 3H-dihydropyridine binding, thought to be postsynaptic, showed spike patterns when measured developmentally. 3H-PN200 binding showed a > 150% increase between days -11 and 15. 3H-Nimodipine binding showed a > 100% increase between days 11 and 14. Deuolarization-induced *‘Ca fluxes also increased between davs 8 and 16 bv >500%. The dramatic increases indicated by these binding data correspond to a critical period described by Himwich (M. Rev. Newobiol. 4, 117, 1962) between postnatal days 11 and 14 in Swiss Webster mice; during this critical period, dendrites exhibit rapid outgrowth, sensory modalities come on line, EEG patternsmature, and the cortex reaches adult proportions. We conclude from these data that the increase in VSCC activity parallels a critical period in the development of the central nervous system in Swiss Webster mice. Key words: w-Conotoxin, dihydropyridine,

calcium, ion channel

~imwich described a critical period in Swiss Webster mouse neurodevelopment.15 As the immature mouse opens its eyes between postnatal days 11 and 14, the cortical width and weight reaches adult proportions; brain waves mature to almost adult patterns, cochlear microphonics and evoked potentials are initiated; and dendritic proliferation is set in full motion.“’ It is as if the electrical system has been switched ‘on line’ and is now ready to receive sensory input. Hafemann and Unsworth showed that binding with Tetrodotoxin increases during a similar developmental period, between postnatal day 8 and 14.” Wasterlain et al. also showed that in rats postnatal days 9-18, synaptic connections were most vulnerable to seizure induced reduction.42 All of these studies suggest that in the development of rodent brain, the critical period is most likely linked to synapse formation. The development of neuronal calcium channels is essential to the onset of whole brain electrical activity in the central nervous system.” VSCC have been associated with neurotransmitter release,8,40 action potential propagation,z7 long term potentiation,13 synaptic plasticity,24 and genetic regulation of neurons in the central nervous system.29,31 o-GVIA, shown to produce a persistent block of VSCC in both central and peripheral cultured mouse neurons,” is a 27 amino acid neuropeptide toxin isolated from the venom of the Conus geogruphus marine snai1.33Azimi and Litzinger showed a developmentally specific pattern of binding around chick hatching with the VSCC probe 12SI-~-GVIA.1 This ‘~5I-w-GVIA binding differed from the binding during development with a second VSCC probe, the 1,4 dihydropy~dines.” This study examines the VSCC in developing Swiss Webster mouse whole brain by comparing binding throughout the development of three VSCC probes: ‘251-w-GVIA, 3H-Nimodipine, and 3H-PN200 which is the 1,4 dihydropyridine with the greatest affinity for neurons.30*41 Potassium depolarized 45Ca uptake in synaptosomes was added as a physiological correlate to the ‘%o-GVIA binding. This work has been presented in part at the Child Neurology Societylo and the Society for Neuroscience.‘* EXPERIMENTAL


Toxin preparation and radioactive labels Synaptic w-GVIA was prepared as previously reported by Rivier et al.” and was iodinated as described by Cruz and Olivera.’ ( +)-[5-methyl-3H]-PN2~-l10 was purchased from NEN *Author to whom correspondence should be addressed. Abbreviutionx BCA, bicinchoninic acid; SHT, sucrose, HEPES-Tris buffer; VSCC, voltage sensitive calcium channel; u-GVIA, w-GVIA Conotoxin; ‘H-PN200, (+)-[5-methyl-3H]-PN200-l10.



M. .I. Litzinger

ct ICI

Research Products. Nitrendipine was a gift from Miles Laboratories. Amersham, and ‘H-Nimodipine from NEN.

‘“Ca was purchased from

Membrane preparation

Membranes were prepared from Swiss Webster mice asphyxiated in a closed container with dry ice. The mice were immediately decapitated and their brains were placed in sucrose, HEPES-Tris (SHT) buffer pH 7.4, and kept at 0°C. The tissue was homogenized with a Teflon glass homogenizer (Wheaton) on a table top drill press at 3000 rpm (nine strokes) and centrifuged at 3600 rpm for 10 min in a Sorvall SS-34 rotor to remove non-central nervous system debris, e.g. red blood cells. The supernatant was centrifuged again at 12,500 rpm for 20 min in the same rotor, in order to collect neuronal material in a pellet. The pellet was then re-suspended in the same buffer (SHT pH 7.4). Preparations that were not immediately bound were frozen for later assay.’ o-GVZA binding

o-GVIA binding was carried out at saturation both with (non-specific binding) and without (total binding) 5 ~.LMunlabelled w-GVIA (approximately 100 times the concentration necessary to displace 50% of ligand binding)‘,’ for 30 min on ice. Incubation was followed by a room temperature 30 min incubation of all samples with 1251-o-GVIA.5 The specific activity of “‘1-w-GVIA ranged from 0.152 to 0.157 mCi/nmol. All ‘251-o-GVIA samples were counted on a Brinkman gamma counter with an efficiency of 80%. Displacement binding was carried out utilizing the same procedures as listed above. ‘251-w-GIVA was displaced by increasing amounts of unlabeled o-GVIA in mice postnatal days 7, 42 and 90. Dihydropyridine


PN200 and Nimodipine binding were performed in dim light at 0°C. Tissue was incubated for 30 min with (non-specific binding) and without (total binding) Nitrendipine (10 ~.LM)or Nimodipine (20 p.M) respectively in standard Nitrendipine buffer (0.54 M KCl, 0.15 M CaC&-2H20, 100 mM HEPES-Tris, pH 7.4). This was followed by a 90 min incubation of all samples with 3H-PN200 (specific activity between 74.1 and 73.8 Ci/mmol) or 3H-Nimodipine (specific activity between 152 and 149 Ci/mmol). Binding was done with 10 nM tritiated dihydropyridines. There is still a question within the literature of an unsaturable low affinity site for the 1,4 dihydropyridines.22 All 3H samples were counted on a Beckman liquid scintillation counter with an efficiency of 40%. Displacement binding was carried out according to the same procedures listed above. Binding occurred. 3H-PN200 was displaced by increasing amounts of unlabeled Nimodipine in mice postnatal days 7 and 42. Filtration

Filtration for all ligands was carried out as per Cruz et al. 4 in all assays. Glass fiber filters (Watman GF/C) were used on a Millipore apparatus. Synaptosomes

Experiments have shown that purification of crude membranes by sucrose gradients does not increase 45Ca uptake per milligram of protein.i4 Therefore, all 45Ca experiments were performed using brain tissue/synaptosomes as prepared above, except that the final pellet was re-suspended in S-saline (S-saline: 145 mM NaCl, 5 mM KCl, 1.4 mM MgLc2, 1.2 mM NaH2P04, 10 mM glucose and 20 mM HEPES-Tris, pH 7.4). Synaptosomal experiments were performed in a Twenty microliters of brain tissue preparation was added to each reaction standard method. 1~14,34 tube. Eighty microliters of S-saline was added to bring the volume to 100 ~1. One hundred microliters of either ‘low K+’ (20 pCi/ml 45Ca++, 45 mM NaCl, 5 mM NaCl, 5 mM KCl, 1.4 mM MgC12, 1.2 mM NaH2P04 10 mM glucose, 1.0 mM CaCl,, and 20 mM HEPES-Tris, pH 7.4) or ‘high K+’ (20 &i/ml 45Cal++, 137 mM KCl, 1.4 mM MgC12, 1.2 mM NaH2P04, 100 mM glucose, 1.0 mM CaCl*, and 20 mM HEPES-Tris, pH 7.4) was added to each sample. Uptake was terminated after approximately l-2 set by the addition of 30 mM EGTA in 120 mM NaCl, 5 mM KC1 at pH 7.6 (adjusted with Tris base). All samples were immediately filtered with Watman


VSCC in mouse neurodevelopment

GF/C filters and washed with 3 x 200 ~1 ‘Na+ wash’ (145 mM NaCl, 5 mM KCl, 1.4 mM MgC12, 1 .O mM CaCl, and 20 mM HEPES-Tris, pH 7.4). Filters were dried and placed overnight in 4 ml of Beckman Ready Safe scintillation fluid. Samples were then counted for 1 min each in a Beckman scintillation counter with an efficiency of 85%. Data analysis

Specific binding of each ligand was calculated by subtracting non-specific from total binding. Both conditions, non-specific and total binding, were run in the same assay to assure accurate comparison. Data were averaged and the means plotted plus or minus the standard deviation (error bars). Two sample t-tests were applied to test significant differences in the data through development in the binding of o-GVIA and synaptosome calcium fluxes. Analysis of variance (ANOVA) was used to test if the increase on days 14-16 is significant in the dihydropyridine binding. Regression analysis was performed on each different age in the displacement curves. All statistical analyses were carried out on Dee Station using the statistical package SAS. Protein determination

Protein concentrations (BCA) method.38

of tissue preparations

were calculated using Smith’s bicinchoninic acid

RESULTS Previous studies of brain maturation and sodium channel development have shown a critical period of development in Swiss Webster mice’2*‘5 and other rodents.‘7,42 In order to determine how VSCC development behaves during this critical period, binding of non-competitive3’*@’ o-GVIA and dihydropyridines was carried out. Ca++ uptake assays were added as physiological correlates for the presynaptic VSCC binding experiments. o-GVZA



Binding with ‘251-w-GVIA was carried out to show the development of N-type VSCC in the mammalian brain. Although some authors have reported the N-type VSCC as being both pre- and postsynaptic,18 recently many data have shown that it exists predominantly on the presynaptic terminal of the neuronal axon.3*26,37There was a dramatic, >50% increase in binding sites between postnatal days 11 and 14 (Fig. 1). Values for the binding of ‘251-w-GVIA from postnatal


oo+ . 5





Postnatal Days Fig. 1. The binding of ‘%GVIA to brain preparation of the developing mouse. Each data point is the average plus and minus the standard deviation of six-eight reactions from four assays. Tissue from two to three mice was combined for each assay. Each reaction was carried out at saturation. Total binding for each data point was determined by incubating crude brain preparation with iZSI-GVIA. Non-specific binding was determined concomitantly by pre-incubating the brain preparation with unlabeled GVIA before the addition of ‘251-GVIA.


M. J. Litzinger et ul.

0.8 / 0.6t






/ -7










Log of unlabeled w-GVIA [M] Fig. 2. The percent binding of ‘25f-GVIA after pre-incubation with increasing concentrations of unlabeled GVIA. Brain tissue preparations from mice ages 7, 42 and 90 days were assayed. Each determination is the percent of specific binding after pre-incubation with unlabeled toxin. Each data point is the average of three-four reactions from two assays. Tissue from two to three mice was combined for each assay. Total binding was determined by incubating crude brain preparation with a saturating amount of ‘2”f-o-GVIA. Non-specific binding was determined in parallel by pre-incubating the preparation with unlabeled toxin before the addition of the radiolabeled toxin.

days 3 to 11 have a mean of 0.2243 pmol/mg. Values for the binding of ‘251-w-GVIA from postnatal days 14 to 23 have a mean of 0.3509 pmollmg. The increase from the values on or before day 11 to the values on day 14 and after is significant (P 100% increase followed by a rapid decrease on day 11. Displacement binding with the 1,4 dihydropyridine, Nimodipine was carried out at ages 10 and 42 days (Fig. 4). Based on a Multivariate Test (P~O.1929), no significant differences between the young and adult Swiss Webster mice were found. This finding is in agreement with Kazazoglou’s study in rodents where Kd’s for the binding of dihydropyridines remained constant throughout development of neuronal tissue. 26This indicates that the affinity of the 3H-Nimodipine binding site does not change through development. This displacement data suggests that the increases in binding seen through development reflect a change in binding site number and potentially


0.20 -

m PN200 l



in mouse neurodevelopment















Postnatal Days Fig. 3. The binding of 3H-PN200 and 3H-N~odipine to brain preparations of the developing mouse. Each data point is the average plus and minus the standard deviation of two-three reactionsfrom two assays. Tissue from two to three mice was combined for each assay. Each reaction was carried out at concentrations believed to saturate high aftinity binding sites. Total binding for each data point was determined by incubating crude brain preparation with 3H-PN200 or ‘H-Nimodipine. Non-specific binding was determined concomitantly by pre-incubating the brain preparation with unlabeled Nitrendipine or Nimodipine respectively before the addition of radiolabeled ligand.



-6 Log




of unlabeled Nimodipine [fvl]

Fig. 4. The percent binding of 3H-Nimodipine after pre-~cubation with increasing con~ntrations of unlabeled Nimodipine. Brain tissue preparations from mice ages 7 and 42 days were assayed. Each determination is the percent of specific binding after pre-incubation with unlabeled Nimodipine. Each data point is the average of two-three reactions from two assays. Tissue from two to three mice was combined for each assay. Total binding was determined by incubating crude brain preparation with an amount of 3H-Nimodipine thought to saturate high affinity sites. Non-specific binding was determined in parallel by pre-incubating the preparation with unlabeled Nimodipine before the addition of the %I-Nimodipine.

postsynaptic WCC number of structure. Low affinity binding sites with this probe have been reported by other authors. r6 Explanations range from the recemic nature of the ligand to low affinity states of the calcium channel. l6 Binding has been affected by depolarization, temperature and other kinetic variables.

Potassium depolarized synaptosomal uptake of “Ca was carried out on postnatal days 8 and 16 (Fig. 5), before and after the dramatic binding changes seen in Figs 1 and 3. Syuaptosome studies were done to verify that the physiological functions of the VSCC were consistent with the presynaptic binding data. Between days 8 and 16 there was a >500% increase in 45Ca uptake,


M. .I. Litzinger

et al.


Specific 45Ca” Flux


High K’



Low K+

Day 8

Day 16 Postnatal Day

Fig. 5. %a uptake into crude brain synaptosomes of the mouse. Each data point is the average plus and minus the standard deviation of six-ten reactions from two assays. Tissue from two to three mice was combined for each assay. The same crude brain preparations used in the binding studies were used for synaptosomal studies. Low K+ refers to “%a uptake in the present of 5 mM extracellular [K+] for 1 sec. High KC refers to “%a uptake in the presence of 70 mM extracellular [K+] for 1 set and defines the basal rate. Specific Ca flux is calculated by taking the difference of the flux at low K+ from the flux at high K+.

reflecting increases in binding sites around this period (Fig. 5). The difference in the 4’Ca uptake was significant (P 50% over levels recorded prior to day 11. The binding of postsynaptic VSCC probes, PN200 and Nimodipine, increased rapidly during the critical period, and then rapidly decreased. This decrease may correspond to the pruning of dendritic trees that occurs during this period. These probes, o-GVIA and the 1,Cdihydropyridines (PN200 and Nimodipine) appear to parallel pre- and postsynaptic patterns of development respectively in mouse brain. The pre- and postsynaptic calcium-related elements, such as neurotransmitter release and the regulation of intracellular calcium concentrations, may provide a basic link between environmental stimuli and neuronal organization and function.77’8719.31Might these channels have a functional role in synapse stabilization? This study is of interest in light of others’ recent findings concerning calcium’s role in the mechanisms regulating dendritic growth and synaptic stabilization.9~‘8~30~3z We have identified a window in the development of VSCC which may also be particularly critical for normal central nervous system maturation in the Swiss Webster mouse. In chick brain, data from o-Conotoxin’ and Tetrodotoxin” binding suggest that a critical period of dendritic proliferation and synapse formation occurs just prior to hatching. In mouse brain, the delayed timing of its maturation and the onset of sensory input could allow the environment to play a much greater role in the final stages of neurodevelopment, perhaps allowing for more plasticity, flexibility and evolutionary change. Acknowledgements-This work was supported by Research Grant HD 008867 from the National Institutes of Public Health and in part by the Miles Laboratory. Special thanks to the late Dr Dixon Woodbury for his support and suggestions.

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