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Morphological development of dendritic spines on rat cerebellar Purkinje cells
Int. J. Devl Neuroscience 29 (2011) 515–520
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Morphological development of dendritic spines on rat cerebellar Purkinje cells D.A. Velázquez-Zamora a,b , M. Martínez-Degollado a,b , I. González-Burgos a,b,∗ a b
Laboratorio de Psicobiología, División de Neurociencias, CIBO, IMSS, Guadalajara, Jal., Mexico Depto. de Biol. Cel. y Mol., CUCBA, Universidad de Guadalajara, Guadalajara, Jal., Mexico
a r t i c l e
i n f o
Article history: Received 13 December 2010 Received in revised form 30 March 2011 Accepted 15 April 2011 Keywords: Cerebellum Purkinje neurons Dendritic spines Filopodia Plasticity
a b s t r a c t The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions. © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Several genetic and epigenetic cerebellar abnormalities can affect motor development (see Schmahmann, 2004, for review). Under normal conditions, the vermal and hemispheric posterior cerebellar regions are implicated in several aspects of motor coordination including timing, velocity, force of movement (Horne and Butler, 1995), muscle tone (Andre et al., 2005), locomotion (Apps and Lidierth, 1989), and the control and correction of movement (Barlow, 2002). Indeed, both the posterior cerebellar vermis and cerebellar hemispheres are functionally activated during guided exercise training (Holschneider et al., 2007) suggesting that adaptive modifications in synaptic transmission may involve plastic changes in dendritic spines (González-Burgos et al., 2011). Development of the cerebellar cortex continues in postnatal animals, in parallel with motor development (Swinny et al., 2005). In the rat, 25–80% of granule cells migrate from the outer to the inner granular layer in the second and third postnatal weeks (Altman,
∗ Corresponding author at: Laboratorio de Psicobiología, División de Neurociencias, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada # 800, Col. Independencia 44340, Guadalajara, Jal., Mexico. Tel.: +52 33 36683000x31950; fax: +52 33 36181756. E-mail address: [email protected] (I. González-Burgos). 0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2011.04.005
1969). Their axons, from the parallel fibers, form stable synapses with the dendritic spines of Purkinje cells in the outer molecular layer from the second post-natal week until the end of the migratory process (Altman, 1972a,b,c). In fact, even after migration has terminated, both granule cell maturation (Altman, 1972c) and synaptogenesis between parallel fibers and Purkinje cells can continue up to post-natal day 30 (Altman, 1972b), though parallel fibers continue to increase in length until day 90 (Lauder, 1979). Spinogenesis involves the active participation of filopodial structures (Papa et al., 1995). Such filopodia can either constitute spine precursors (Dailey and Smith, 1996; Portera-Cailliau et al., 2003) or serve as axonal guides, mediating the formation of nonfunctional synaptic contacts between axon fibers and the dendritic shaft (Zhang and Benson, 2000) as the filopodia retract (Fiala et al., 1998; Harris, 1999). These premature synapses induce the formation of spines, whose maturation and morphology (Lippman and Dunaevsky, 2005; Tada and Sheng, 2006) are dependent on the nature of the synaptic stimulation (Zhang and Benson, 2000). In one-week old rats, 50% of the spiny structures correspond to filopodia, which disappear as the number of spines increases (Papa et al., 1995). Dendritic spines are postsynaptic sites for excitatory inputs (Gray, 1959). The mature dendritic spines on adult cerebellar Purkinje cells can be classified as thin, stubby, mushroom, branched or double spines, depending on their shape (González-Burgos, 2009; Harris et al., 1989). Other unclassified spines (Lee et al., 2005)
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Fig. 1. Schematic composition showing the cytoarchitectonic criteria for spine counting. Thin (t), mushroom (m), stubby (s), wide (w), branched (b), double (d), and thorn-like (th) spines, as well as filopodia (f) (arrows), were counted in 50 m from 3 to 4 dendritic branchlets (arrows) of cerebellar Purkinje cells (P) of rats. Scale bar: Spines: 5 m., P: 50 m.
may correspond to “thorns” (Sotelo and Dusart, 2009). As there are several critical periods in Purkinje cell synaptogenesis and presynaptic activity defines dendritic spine shape (see González-Burgos, 2009 for review), the geometry of Purkinje cell spines may vary during development. Hence, using the Golgi technique, we have characterized and quantified the changes in dendritic spine morphology at critical stages of Purkinje cell development.
included several females, all males from each litter were preserved. Then, male rats were randomly selected from each of the seven litters to form the three experimental groups of different ages: 21-day (21; n = 7), 30-day (30; n = 7), and 90-day (90; n = 7). The pain and discomfort of the animals were minimized in all experimental procedures, which were performed in accordance with the NIH guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996 revision) and that were approved by the Research Ethics Committee of the Instituto Mexicano del Seguro Social, México. 2.2. Golgi study
2. Materials and methods 2.1. Animals Twenty one male Sprague-Dawley rats born to 7 dams were used in the present study. Subjects were housed under standard conditions on regular 12-h light–dark cycles (07:00–19:00 h), with 45–50% environmental humidity, a temperature of 22 ± 2 ◦ C, and free access to food and water. The dams were housed individually and after birth, litters were restricted to 8 pups until weaning (day 21). Since some litters
Upon reaching the age, six animals per group were anaesthetized with 30 mg/kg of ketamine intramuscular and 50 mg/kg of sodium pentobarbital i.p. Then, they were perfused with 200 ml of a washing phosphate-buffered solution (PB, 0.01 M, pH 7.4) containing 1000 IU/L of the anticoagulant sodium heparin and 1 g/L of the vasodilator procaine hydrochloride (Feria-Velasco and Karnovsky, 1970). This was followed by perfusion with 200 ml of 4% formaldehyde solution in PB. Both solutions were perfused at a rate of 11.5 ml/min. The rat’s brain was then removed and placed in 100 ml of fresh fixative solution for 48 h. Tissue corresponding to the posterior
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Fig. 2. Photomicrographs of the vermal region of the posterior cerebellar cortex of rats at day 21 (A), 30 (B) and 90 (C). Note the clearly differentiated cytoarchitecture of the molecular (m), Purkinje cell (P) and granule (g) layer. Scale bar: 100 m. cerebellar vermis (Voogd, 2004) was dissected out and impregnated according to a modified version of the Golgi technique (González-Burgos et al., 1992). Sagittal slices (75 m thick) were mounted on one slide per animal and eight well-impregnated, clearly visible Purkinje cells were selected from each rat for study. To assess the reliability of the counting of dendritic protrusions, a “blind” study was initially performed and an index of reliability was calculated: number of agreements − number of disagreements/number of agreements. Once a minimum of 0.95 in reliability was reached, the quantification of dendritic filopodia and spines was performed again using a “blind” procedure. Dendritic protrusions were quantified in terms of the density of filopodial structures (González-Burgos, 2009; Portera-Cailliau and Yuste, 2001), and the density of thin, stubby, mushroom, wide, branched, double (González-Burgos, 2009; Harris et al., 1989) and unclassified (Lee et al., 2005) spines or “thorns” (Sotelo and Dusart, 2009). These were counted along 50 m from terminal dendritic branchlets distal to the soma (Fig. 1); since distal dendritic branchlets measure frequently less than 50 m in length, 3–4 dendritic segments per cell were selected and, in addition, randomly. Counting was performed by direct observation at 2000× using a magnification changer coupled to a light microscope. The corresponding values from six cells per animal were averaged, and the average of the six animals per group was used for statistical comparisons. 2.3. Histology One rat per group of age was used to monitor the maturation of the cerebellar cortex. These animals were anaesthetized, perfused and post-fixed as described above. The cerebellar vermis was dissected out, processed histologically and 10-m thick sagittal slices were stained with cresyl violet.
No significant difference was observed between days 21st and 30th (Fig. 3). 3.3. Dendritic spines 3.3.1. Spine density Dendritic spine density differed significantly between the three groups studied (F = 11.963, p < 0.003) (Fig. 4). Spines were more abundant at day 90 than at day 21st (p < 0.006) or 30th (p < 0.008). No significant differences were observed between days 21st and 30th (Fig. 5). 3.3.2. Spine types In Purkinje cells, significant differences in the relative abundance of thorns (F = 32.586, p < 0.0001) and of thin (F = 69.064, p < 0.0001), mushroom (F = 17.255, p < 0.0001), stubby (F = 6.771, p < 0.008) and wide (F = 3.593, p < 0.05) spines were detected. While no differences were observed between rats aged days 21 and 30, at day 90 both thin and mushroom spines were more abundant than at day 21 (p < 0.0001, and p < 0.0001, respectively) or 30 (p < 0.0001, and p < 0.0001, respectively). By contrast, thorns were less abun-
2.4. Statistics Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey post hoc tests, and Pearson’s correlation coefficient. Significance was established at p < 0.05.
3. Results 3.1. Maturation of the cerebellar cortex The molecular, Purkinje cell and granular layers were clearly distinguishable at each of the three time points studied. Notably, the outer granular layer was absent in each group (Fig. 2). 3.2. Filopodia The density of filopodia differed significantly between the three age groups studied (F = 8.932, p < 0.003). Filopodia were less abundant at day 90th than both day 21st (p < 0.006) and 30th (p < 0.006).
Fig. 3. Comparison of filopodial structures counted in the Purkinje cells of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: day 90 vs. 21; b: day 90 vs. 30.
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Fig. 4. Photomicrographs of representative Purkinje cell apical dendritic branchlets of rats at days 21 (A), 30 (B) and 90 (C). Note the greater spine density in C compared with A and B. Scale bar: 10 m.
dant at day 90 than at 21 (p < 0.0001) or 30 (p < 0.0001). Stubby and wide spines were less abundant at day 90 than at day 30 (p < 0.006 and p < 0.04, respectively), whereas the number of branched and double spines remained unchanged at all three ages studied (Fig. 6). 3.4. Filopodia-dendritic spine development Variations in filopodia density through development correlated negatively with the corresponding values of dendritic spine density (r = −0.501, p < 0.03) (Fig. 7). 4. Discussion The posterior cerebellar cortex is strongly implicated in several aspects of movement coordination. The cerebellar cortex receives extracerebellar inputs from mossy fibers and climbing fibers from the spinal cord and inferior olivary complex, respectively. Mossy fibers carry sensory and cortical information to the cerebellar cortex via synapses with the dendrites of granule cells and axonal terminals of Golgi cells forming the cerebellar glomeruli. Granule cell axons or parallel fibers then establish glutamatergic excitatory synaptic contacts with the dendritic spines of Purkinje cells. Both exteroceptive, and in particular propioceptive sensory information from the spinal cord, is received by the cerebellar cortex via clim-
Fig. 5. Graph comparing the spine density counted in the Purkinje neurons’ dendritic branchlets of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: day 90 vs. 21; b: day 90 vs. 30.
bing fibers (Ghez and Thach, 2000; Voogd, 2004). These extend to the Purkinje cell dendritic arbor where they establish excitatory glutamatergic synaptic contacts with thorns on the shafts of the primary and secondary dendrites of Purkinje neurons (Sotelo and Dusart, 2009). The cerebellum matures postnatally and in the rat, granule cells from the outer granular layer migrate downward from the bottom and up towards the developing inner granular layer during the second-to-third post-natal week, leading to the formation of the molecular layer (Altman, 1969). During this migratory process, granule cells “slip down” from their own axon that bifurcates into two orthogonal ramifications: the parallel fibers. These fibers extend transversely (Rakic, 1971), forming excitatory synaptic connections with the dendritic spines of Purkinje cells, and from days 21 to 30 until approximately post-natal day 90 (Lauder, 1979), they make connections that mature in the upper molecular layer (Altman, 1972b; Sotelo and Dusart, 2009). By contrast, excitatory synaptic contacts in the lower dendritic arbor of Purkinje neurons form between dendritic thorns and climbing fibers (Sotelo and Dusart, 2009). Thus, we performed a cytoarchitectonic study of the dendritic spines on the apical arbor of Purkinje cells, at different critical periods during Purkinje cell synaptogenesis. Dendritic spines were denser at day 90 than at days 21 and 30, whereas the converse occurred with filopodial structures. This is in agreement with previous studies that describe the direct participation of filopodia in dendritic spine development, with filopodia retracting as spines form (Fiala et al., 1998; Harris, 1999; Papa et al., 1995). These findings are also supported by the continued growth of parallel fibers reported until approximately post-natal day 90 (Lauder, 1979), suggestive of active synaptogenesis between parallel fibers and the dendritic spines of Purkinje cells. Though not significantly, dendritic spine density increased at day 30 when compared to day 21, in line with previous reports of increased synapse formation of between parallel fibers and Purkinje cell dendritic spines between the days 21 and 30 of post-natal development (Altman, 1972b). Both the association and specificity of synaptic information are favoured by spine proximity (Harris and Kater, 1994). Thus, the increase in spine density at day 90 may be related to an increased capacity to integrate synaptic impulses to Purkinje neurons, enabling the more effective motor patterns required for highly coordinated movements and motor adjustments to be developed. There were more thin and mushroom spines at day 90, which have been strongly linked to learning and memory, respectively, in studies of cognitive processes (Bourne and Harris, 2007; Kasai et al., 2003; Matsuzaki, 2007). However, the cerebellar region studied in this work, the posterior vermis, is associated with motor coordination rather than cognition, suggesting different roles for these types of spines in Purkinje cell development in this region, as suggested recently (González-Burgos et al., sent). Spine neck width has long been associated with the efficiency of synaptic transmission: the narrower the spine neck, the stronger the synapse (Koch and Zador, 1993; Koch et al., 1992). As both thin and mushroom spines have narrow necks, their increase at 90 days strongly suggests a close relationship between synaptic efficiency and cerebellar maturation. Together with the development of inhibitory contacts on Purkinje cells mediated by stellate, basket and Golgi cells, the increased presence of such spines would be indicative of a system capable of highly efficient transmission of excitatory impulses to spines, as well as inhibitory modulation of these same stimuli. Such a system would provide the cerebellum with the level of control of movement coordination required to mediate the highly efficient motor responses characteristic of this structure when mature. The number of stubby and wide spines increased slightly between days 21 and 30, but had decreased significantly by day 90. With their thicker necks, these spines would not restrict the ion-
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Fig. 6. Comparative graphs showing the density of thin (t), mushroom (m), stubby (s), wide (w), branched (b), double (d) and thorn-like (th) spines counted in the Purkinje neurons’ dendritic branchlets of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: 90th vs. 21st day; b: 90th vs. 30th day.
carrying current flow from glutamate receptors located in the postsynaptic density to the parental dendrite, thus diminishing the strength of the synaptic signal (Koch and Zador, 1993; Koch et al., 1992). Thus, stubby and wide spines would transmit synaptic impulses less effectively than narrow necked spines, such as thin or mushroom spines. A role for stubby and wide spines in the regulation of cell excitability has been proposed (González-Burgos, 2009; Feria-Velasco et al., 2002; Pérez-Vega et al., 2000) which, in addition with the maturation of inhibitory contacts, provides
the cerebellum a highly efficient control of movement coordination. The Purkinje cell dendritic arbor reaches its maximum length by post-natal day 30 (Berry and Bradley, 1976). From post-natal day 19, the thorn structures of the lower dendritic arbor synapse with climbing fibers (Morara et al., 2001), whereas parallel fibers form synapses with spines located in the apical dendritic branchlets up until post-natal day 30 (Sotelo and Dusart, 2009). Thus, synaptogenesis at spines occurs later than at thorn structures. This concords
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Fig. 7. Graph showing the negative correlation (r = −0.501, p < 0.03) between filopodia and dendritic spine density, through development.
with the significant decrease in distal thorn structures observed at day 90 when compared to days 21 and 30, and the overall increase in dendritic spines observed with maturation. In summary, in the animals studied here spine density was seen to increase with age. Specifically, this involved an increase in thin and mushroom spines, while the stubby and wide types diminished during the same period. These findings suggest a greater efficiency and specificity in processing excitatory inputs in adulthood, as well as a finer regulation of these stimuli. This is in line with the postnatal increase in the control of motor performance seen with age, and supports the proposed link between motor development and cerebellar cortex maturation (Swinny et al., 2005). To our knowledge, this is the first study to quantify the postnatal development of Purkinje cell spines. The findings have important implications for future studies on the morphophysiology of Purkinje cell dendritic spines during development, and for studies of cerebellar dysfunction. References Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136, 269–293. Altman, J., 1972a. Postnatal development of cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145, 353–397. Altman, J., 1972b. Postnatal development of cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Comp. Neurol. 145, 399–463. Altman, J., 1972c. Postnatal development of cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145, 465–513. Andre, P., Pompeiano, O., Manzoni, D., 2005. Adaptive modification of the cats vestibulospinal reflex during sustained and combined roll tilt of the whole animal and forepaw rotation: cerebellar mechanisms. Neuroscience 132, 811–822. Apps, R., Lidierth, M., 1989. Simple spike discharge patterns of Purkinje cells in the paramedical lobule of the cerebellum during locomotion in the awake cat. Neurosci. Lett. 102, 205–210. Barlow, J.S., 2002. The Cerebellum and Adaptive Control. Cambridge University Press, Cambridge. Berry, M., Bradley, P., 1976. The growth of the dendritic trees of Purkinje cells in the cerebellum of the rat. Brain Res. 112, 1–35. Bourne, J., Harris, K.M., 2007. Do thin spines learn to be mushroom spines that remember? Curr. Opin. Neurobiol. 17, 1–6. Dailey, M.E., Smith, S.J., 1996. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994. Feria-Velasco, A., Del Angel-Meza, A.R., González-Burgos, I., 2002. Modification of dendritic development. In: Azmitia, E.C., De Felipe, J., Jones, E.G., Rakic, P., Ribak, C.E. (Eds.), Changing Views of Cajal’s Neuron. Progress in Brain Research Series, vol. 136. Elsevier, USA, pp. 135–143.
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Morphological development of dendritic spines on rat cerebellar Purkinje cells D.A. Velázquez-Zamora a,b , M. Martínez-Degollado a,b , I. González-Burgos a,b,∗ a b
Laboratorio de Psicobiología, División de Neurociencias, CIBO, IMSS, Guadalajara, Jal., Mexico Depto. de Biol. Cel. y Mol., CUCBA, Universidad de Guadalajara, Guadalajara, Jal., Mexico
a r t i c l e
i n f o
Article history: Received 13 December 2010 Received in revised form 30 March 2011 Accepted 15 April 2011 Keywords: Cerebellum Purkinje neurons Dendritic spines Filopodia Plasticity
a b s t r a c t The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions. © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Several genetic and epigenetic cerebellar abnormalities can affect motor development (see Schmahmann, 2004, for review). Under normal conditions, the vermal and hemispheric posterior cerebellar regions are implicated in several aspects of motor coordination including timing, velocity, force of movement (Horne and Butler, 1995), muscle tone (Andre et al., 2005), locomotion (Apps and Lidierth, 1989), and the control and correction of movement (Barlow, 2002). Indeed, both the posterior cerebellar vermis and cerebellar hemispheres are functionally activated during guided exercise training (Holschneider et al., 2007) suggesting that adaptive modifications in synaptic transmission may involve plastic changes in dendritic spines (González-Burgos et al., 2011). Development of the cerebellar cortex continues in postnatal animals, in parallel with motor development (Swinny et al., 2005). In the rat, 25–80% of granule cells migrate from the outer to the inner granular layer in the second and third postnatal weeks (Altman,
∗ Corresponding author at: Laboratorio de Psicobiología, División de Neurociencias, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada # 800, Col. Independencia 44340, Guadalajara, Jal., Mexico. Tel.: +52 33 36683000x31950; fax: +52 33 36181756. E-mail address: [email protected] (I. González-Burgos). 0736-5748/$36.00 © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2011.04.005
1969). Their axons, from the parallel fibers, form stable synapses with the dendritic spines of Purkinje cells in the outer molecular layer from the second post-natal week until the end of the migratory process (Altman, 1972a,b,c). In fact, even after migration has terminated, both granule cell maturation (Altman, 1972c) and synaptogenesis between parallel fibers and Purkinje cells can continue up to post-natal day 30 (Altman, 1972b), though parallel fibers continue to increase in length until day 90 (Lauder, 1979). Spinogenesis involves the active participation of filopodial structures (Papa et al., 1995). Such filopodia can either constitute spine precursors (Dailey and Smith, 1996; Portera-Cailliau et al., 2003) or serve as axonal guides, mediating the formation of nonfunctional synaptic contacts between axon fibers and the dendritic shaft (Zhang and Benson, 2000) as the filopodia retract (Fiala et al., 1998; Harris, 1999). These premature synapses induce the formation of spines, whose maturation and morphology (Lippman and Dunaevsky, 2005; Tada and Sheng, 2006) are dependent on the nature of the synaptic stimulation (Zhang and Benson, 2000). In one-week old rats, 50% of the spiny structures correspond to filopodia, which disappear as the number of spines increases (Papa et al., 1995). Dendritic spines are postsynaptic sites for excitatory inputs (Gray, 1959). The mature dendritic spines on adult cerebellar Purkinje cells can be classified as thin, stubby, mushroom, branched or double spines, depending on their shape (González-Burgos, 2009; Harris et al., 1989). Other unclassified spines (Lee et al., 2005)
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Fig. 1. Schematic composition showing the cytoarchitectonic criteria for spine counting. Thin (t), mushroom (m), stubby (s), wide (w), branched (b), double (d), and thorn-like (th) spines, as well as filopodia (f) (arrows), were counted in 50 m from 3 to 4 dendritic branchlets (arrows) of cerebellar Purkinje cells (P) of rats. Scale bar: Spines: 5 m., P: 50 m.
may correspond to “thorns” (Sotelo and Dusart, 2009). As there are several critical periods in Purkinje cell synaptogenesis and presynaptic activity defines dendritic spine shape (see González-Burgos, 2009 for review), the geometry of Purkinje cell spines may vary during development. Hence, using the Golgi technique, we have characterized and quantified the changes in dendritic spine morphology at critical stages of Purkinje cell development.
included several females, all males from each litter were preserved. Then, male rats were randomly selected from each of the seven litters to form the three experimental groups of different ages: 21-day (21; n = 7), 30-day (30; n = 7), and 90-day (90; n = 7). The pain and discomfort of the animals were minimized in all experimental procedures, which were performed in accordance with the NIH guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996 revision) and that were approved by the Research Ethics Committee of the Instituto Mexicano del Seguro Social, México. 2.2. Golgi study
2. Materials and methods 2.1. Animals Twenty one male Sprague-Dawley rats born to 7 dams were used in the present study. Subjects were housed under standard conditions on regular 12-h light–dark cycles (07:00–19:00 h), with 45–50% environmental humidity, a temperature of 22 ± 2 ◦ C, and free access to food and water. The dams were housed individually and after birth, litters were restricted to 8 pups until weaning (day 21). Since some litters
Upon reaching the age, six animals per group were anaesthetized with 30 mg/kg of ketamine intramuscular and 50 mg/kg of sodium pentobarbital i.p. Then, they were perfused with 200 ml of a washing phosphate-buffered solution (PB, 0.01 M, pH 7.4) containing 1000 IU/L of the anticoagulant sodium heparin and 1 g/L of the vasodilator procaine hydrochloride (Feria-Velasco and Karnovsky, 1970). This was followed by perfusion with 200 ml of 4% formaldehyde solution in PB. Both solutions were perfused at a rate of 11.5 ml/min. The rat’s brain was then removed and placed in 100 ml of fresh fixative solution for 48 h. Tissue corresponding to the posterior
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Fig. 2. Photomicrographs of the vermal region of the posterior cerebellar cortex of rats at day 21 (A), 30 (B) and 90 (C). Note the clearly differentiated cytoarchitecture of the molecular (m), Purkinje cell (P) and granule (g) layer. Scale bar: 100 m. cerebellar vermis (Voogd, 2004) was dissected out and impregnated according to a modified version of the Golgi technique (González-Burgos et al., 1992). Sagittal slices (75 m thick) were mounted on one slide per animal and eight well-impregnated, clearly visible Purkinje cells were selected from each rat for study. To assess the reliability of the counting of dendritic protrusions, a “blind” study was initially performed and an index of reliability was calculated: number of agreements − number of disagreements/number of agreements. Once a minimum of 0.95 in reliability was reached, the quantification of dendritic filopodia and spines was performed again using a “blind” procedure. Dendritic protrusions were quantified in terms of the density of filopodial structures (González-Burgos, 2009; Portera-Cailliau and Yuste, 2001), and the density of thin, stubby, mushroom, wide, branched, double (González-Burgos, 2009; Harris et al., 1989) and unclassified (Lee et al., 2005) spines or “thorns” (Sotelo and Dusart, 2009). These were counted along 50 m from terminal dendritic branchlets distal to the soma (Fig. 1); since distal dendritic branchlets measure frequently less than 50 m in length, 3–4 dendritic segments per cell were selected and, in addition, randomly. Counting was performed by direct observation at 2000× using a magnification changer coupled to a light microscope. The corresponding values from six cells per animal were averaged, and the average of the six animals per group was used for statistical comparisons. 2.3. Histology One rat per group of age was used to monitor the maturation of the cerebellar cortex. These animals were anaesthetized, perfused and post-fixed as described above. The cerebellar vermis was dissected out, processed histologically and 10-m thick sagittal slices were stained with cresyl violet.
No significant difference was observed between days 21st and 30th (Fig. 3). 3.3. Dendritic spines 3.3.1. Spine density Dendritic spine density differed significantly between the three groups studied (F = 11.963, p < 0.003) (Fig. 4). Spines were more abundant at day 90 than at day 21st (p < 0.006) or 30th (p < 0.008). No significant differences were observed between days 21st and 30th (Fig. 5). 3.3.2. Spine types In Purkinje cells, significant differences in the relative abundance of thorns (F = 32.586, p < 0.0001) and of thin (F = 69.064, p < 0.0001), mushroom (F = 17.255, p < 0.0001), stubby (F = 6.771, p < 0.008) and wide (F = 3.593, p < 0.05) spines were detected. While no differences were observed between rats aged days 21 and 30, at day 90 both thin and mushroom spines were more abundant than at day 21 (p < 0.0001, and p < 0.0001, respectively) or 30 (p < 0.0001, and p < 0.0001, respectively). By contrast, thorns were less abun-
2.4. Statistics Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey post hoc tests, and Pearson’s correlation coefficient. Significance was established at p < 0.05.
3. Results 3.1. Maturation of the cerebellar cortex The molecular, Purkinje cell and granular layers were clearly distinguishable at each of the three time points studied. Notably, the outer granular layer was absent in each group (Fig. 2). 3.2. Filopodia The density of filopodia differed significantly between the three age groups studied (F = 8.932, p < 0.003). Filopodia were less abundant at day 90th than both day 21st (p < 0.006) and 30th (p < 0.006).
Fig. 3. Comparison of filopodial structures counted in the Purkinje cells of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: day 90 vs. 21; b: day 90 vs. 30.
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Fig. 4. Photomicrographs of representative Purkinje cell apical dendritic branchlets of rats at days 21 (A), 30 (B) and 90 (C). Note the greater spine density in C compared with A and B. Scale bar: 10 m.
dant at day 90 than at 21 (p < 0.0001) or 30 (p < 0.0001). Stubby and wide spines were less abundant at day 90 than at day 30 (p < 0.006 and p < 0.04, respectively), whereas the number of branched and double spines remained unchanged at all three ages studied (Fig. 6). 3.4. Filopodia-dendritic spine development Variations in filopodia density through development correlated negatively with the corresponding values of dendritic spine density (r = −0.501, p < 0.03) (Fig. 7). 4. Discussion The posterior cerebellar cortex is strongly implicated in several aspects of movement coordination. The cerebellar cortex receives extracerebellar inputs from mossy fibers and climbing fibers from the spinal cord and inferior olivary complex, respectively. Mossy fibers carry sensory and cortical information to the cerebellar cortex via synapses with the dendrites of granule cells and axonal terminals of Golgi cells forming the cerebellar glomeruli. Granule cell axons or parallel fibers then establish glutamatergic excitatory synaptic contacts with the dendritic spines of Purkinje cells. Both exteroceptive, and in particular propioceptive sensory information from the spinal cord, is received by the cerebellar cortex via clim-
Fig. 5. Graph comparing the spine density counted in the Purkinje neurons’ dendritic branchlets of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: day 90 vs. 21; b: day 90 vs. 30.
bing fibers (Ghez and Thach, 2000; Voogd, 2004). These extend to the Purkinje cell dendritic arbor where they establish excitatory glutamatergic synaptic contacts with thorns on the shafts of the primary and secondary dendrites of Purkinje neurons (Sotelo and Dusart, 2009). The cerebellum matures postnatally and in the rat, granule cells from the outer granular layer migrate downward from the bottom and up towards the developing inner granular layer during the second-to-third post-natal week, leading to the formation of the molecular layer (Altman, 1969). During this migratory process, granule cells “slip down” from their own axon that bifurcates into two orthogonal ramifications: the parallel fibers. These fibers extend transversely (Rakic, 1971), forming excitatory synaptic connections with the dendritic spines of Purkinje cells, and from days 21 to 30 until approximately post-natal day 90 (Lauder, 1979), they make connections that mature in the upper molecular layer (Altman, 1972b; Sotelo and Dusart, 2009). By contrast, excitatory synaptic contacts in the lower dendritic arbor of Purkinje neurons form between dendritic thorns and climbing fibers (Sotelo and Dusart, 2009). Thus, we performed a cytoarchitectonic study of the dendritic spines on the apical arbor of Purkinje cells, at different critical periods during Purkinje cell synaptogenesis. Dendritic spines were denser at day 90 than at days 21 and 30, whereas the converse occurred with filopodial structures. This is in agreement with previous studies that describe the direct participation of filopodia in dendritic spine development, with filopodia retracting as spines form (Fiala et al., 1998; Harris, 1999; Papa et al., 1995). These findings are also supported by the continued growth of parallel fibers reported until approximately post-natal day 90 (Lauder, 1979), suggestive of active synaptogenesis between parallel fibers and the dendritic spines of Purkinje cells. Though not significantly, dendritic spine density increased at day 30 when compared to day 21, in line with previous reports of increased synapse formation of between parallel fibers and Purkinje cell dendritic spines between the days 21 and 30 of post-natal development (Altman, 1972b). Both the association and specificity of synaptic information are favoured by spine proximity (Harris and Kater, 1994). Thus, the increase in spine density at day 90 may be related to an increased capacity to integrate synaptic impulses to Purkinje neurons, enabling the more effective motor patterns required for highly coordinated movements and motor adjustments to be developed. There were more thin and mushroom spines at day 90, which have been strongly linked to learning and memory, respectively, in studies of cognitive processes (Bourne and Harris, 2007; Kasai et al., 2003; Matsuzaki, 2007). However, the cerebellar region studied in this work, the posterior vermis, is associated with motor coordination rather than cognition, suggesting different roles for these types of spines in Purkinje cell development in this region, as suggested recently (González-Burgos et al., sent). Spine neck width has long been associated with the efficiency of synaptic transmission: the narrower the spine neck, the stronger the synapse (Koch and Zador, 1993; Koch et al., 1992). As both thin and mushroom spines have narrow necks, their increase at 90 days strongly suggests a close relationship between synaptic efficiency and cerebellar maturation. Together with the development of inhibitory contacts on Purkinje cells mediated by stellate, basket and Golgi cells, the increased presence of such spines would be indicative of a system capable of highly efficient transmission of excitatory impulses to spines, as well as inhibitory modulation of these same stimuli. Such a system would provide the cerebellum with the level of control of movement coordination required to mediate the highly efficient motor responses characteristic of this structure when mature. The number of stubby and wide spines increased slightly between days 21 and 30, but had decreased significantly by day 90. With their thicker necks, these spines would not restrict the ion-
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Fig. 6. Comparative graphs showing the density of thin (t), mushroom (m), stubby (s), wide (w), branched (b), double (d) and thorn-like (th) spines counted in the Purkinje neurons’ dendritic branchlets of rats at days 21, 30 and 90. Mean ± SEM. p < 0.05. a: 90th vs. 21st day; b: 90th vs. 30th day.
carrying current flow from glutamate receptors located in the postsynaptic density to the parental dendrite, thus diminishing the strength of the synaptic signal (Koch and Zador, 1993; Koch et al., 1992). Thus, stubby and wide spines would transmit synaptic impulses less effectively than narrow necked spines, such as thin or mushroom spines. A role for stubby and wide spines in the regulation of cell excitability has been proposed (González-Burgos, 2009; Feria-Velasco et al., 2002; Pérez-Vega et al., 2000) which, in addition with the maturation of inhibitory contacts, provides
the cerebellum a highly efficient control of movement coordination. The Purkinje cell dendritic arbor reaches its maximum length by post-natal day 30 (Berry and Bradley, 1976). From post-natal day 19, the thorn structures of the lower dendritic arbor synapse with climbing fibers (Morara et al., 2001), whereas parallel fibers form synapses with spines located in the apical dendritic branchlets up until post-natal day 30 (Sotelo and Dusart, 2009). Thus, synaptogenesis at spines occurs later than at thorn structures. This concords
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Fig. 7. Graph showing the negative correlation (r = −0.501, p < 0.03) between filopodia and dendritic spine density, through development.
with the significant decrease in distal thorn structures observed at day 90 when compared to days 21 and 30, and the overall increase in dendritic spines observed with maturation. In summary, in the animals studied here spine density was seen to increase with age. Specifically, this involved an increase in thin and mushroom spines, while the stubby and wide types diminished during the same period. These findings suggest a greater efficiency and specificity in processing excitatory inputs in adulthood, as well as a finer regulation of these stimuli. This is in line with the postnatal increase in the control of motor performance seen with age, and supports the proposed link between motor development and cerebellar cortex maturation (Swinny et al., 2005). To our knowledge, this is the first study to quantify the postnatal development of Purkinje cell spines. The findings have important implications for future studies on the morphophysiology of Purkinje cell dendritic spines during development, and for studies of cerebellar dysfunction. References Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136, 269–293. Altman, J., 1972a. Postnatal development of cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145, 353–397. Altman, J., 1972b. Postnatal development of cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Comp. Neurol. 145, 399–463. Altman, J., 1972c. Postnatal development of cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145, 465–513. Andre, P., Pompeiano, O., Manzoni, D., 2005. Adaptive modification of the cats vestibulospinal reflex during sustained and combined roll tilt of the whole animal and forepaw rotation: cerebellar mechanisms. Neuroscience 132, 811–822. Apps, R., Lidierth, M., 1989. Simple spike discharge patterns of Purkinje cells in the paramedical lobule of the cerebellum during locomotion in the awake cat. Neurosci. Lett. 102, 205–210. Barlow, J.S., 2002. The Cerebellum and Adaptive Control. Cambridge University Press, Cambridge. Berry, M., Bradley, P., 1976. The growth of the dendritic trees of Purkinje cells in the cerebellum of the rat. Brain Res. 112, 1–35. Bourne, J., Harris, K.M., 2007. Do thin spines learn to be mushroom spines that remember? Curr. Opin. Neurobiol. 17, 1–6. Dailey, M.E., Smith, S.J., 1996. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994. Feria-Velasco, A., Del Angel-Meza, A.R., González-Burgos, I., 2002. Modification of dendritic development. In: Azmitia, E.C., De Felipe, J., Jones, E.G., Rakic, P., Ribak, C.E. (Eds.), Changing Views of Cajal’s Neuron. Progress in Brain Research Series, vol. 136. Elsevier, USA, pp. 135–143.
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