- Email: [email protected]
Non-synaptic dendritic spines in neocortex
Neuroscience 145 (2007) 464 – 469
NON-SYNAPTIC DENDRITIC SPINES IN NEOCORTEX J. I. ARELLANO,a,b A. ESPINOSA,c A. FAIRÉN,c R. YUSTEd* AND J. DEFELIPEa*
still valid, and this question has unfortunately not been directly addressed. Early reports on cortical spines were based on single section ultrastructural analysis, a method that does not allow the identification of non-synaptic spines (Gray, 1959a,b; Colonnier, 1968; Jones and Powell, 1969; Peters and Kaiserman-Abramof, 1969). Serial section reconstructions have provided contradictory results: while some studies reported that all spines established synaptic contacts (Miller and Peters, 1981; White and Hersch, 1982; Hersch and White, 1982; Mates and Lund, 1983), other studies have reported occasional non-synaptic spines in neocortex (Trachtenberg et al., 2002; White and Rock, 1980; Hersch and White, 1981; Benshalom and White, 1986; Spacek, 1982; Knott et al., 2006) and hippocampus (Chicurel and Harris, 1992; Trommald and Hulleberg, 1997; Sorra and Harris, 1998). However, these non-synaptic spines have not been documented. Moreover, in their pioneering study, White and Rock (1980) admitted that they might establish synapses but that these were not detected “due to an unfavourable plane of section.” Thus, it is possible that all spines were indeed synaptic. Finally, a key criterion to identify non-synaptic spines relies on the absence of postsynaptic density (PSD). Unfortunately, most techniques used to label neurons produce intense, homogeneous intracellular labeling that masks PSDs (such as the deposit produced by the chromogen 3,3= diaminobenzidine tetrahydrochloride), particularly when membranes are sectioned tangentially. This makes the identification of axospinous synapses difficult and the detection of non-synaptic spines impractical. This, together with the intrinsic difficulty of obtaining complete serial ultrathin sections to achieve full reconstructions of spines, could explain why the question of whether some spines lack a synaptic contact is still outstanding. We aimed to resolve this question through the ultrastructural analysis of a large number of completely reconstructed neocortical spines, using gold-toned Golgi staining, a method that enables the visualization of postsynaptic densities.
a
Departamento de Neuroanatomia y Biologia Celular, Instituto Cajal (CSIC), Ave. Dr. Arce, 37, 28002 Madrid, Spain
b
Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA
c
Instituto de Neurociencias de Alicante, (CISC and Universidad Miguel Hernández), 03550 San Juan de Alicante, Spain
d
Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Abstract—A long-held assumption states that each dendritic spine in the cerebral cortex forms a synapse, although this issue has not been systematically investigated. We performed complete ultrastructural reconstructions of a large (nⴝ144) population of identified spines in adult mouse neocortex finding that only 3.6% of the spines clearly lacked synapses. Nonsynaptic spines were small and had no clear head, resembling dendritic filopodia, and could represent a source of new synaptic connections in the adult cerebral cortex. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: pyramidal neurons, excitatory synapses, serial section reconstructions.
Dendritic spines are the major targets of excitatory connections in the cerebral cortex and they also compartmentalize calcium and could implement local learning rules (Shepherd, 1996). Spines are motile (Matus, 2000) and can emerge in response to the synaptic stimulation, so they could mediate structural plasticity in the nervous system (Bonhoeffer and Yuste, 2002). Moreover, in adult neocortex in vivo, spines have been reported to be plastic (Trachtenberg et al., 2002), although other reports indicate that they are stable (Grutzendler et al., 2002). Nevertheless, it is still unclear what the relationship is between the formation or motility of spines with their establishment or not of synaptic junctions. Crucial to these questions is the basic issue of whether or not all spines have a synapse or whether non-synaptic spines exist. Since Gray (1959a) first demonstrated using electron microscopy that dendritic spines established synaptic contacts, numerous ultrastructural studies have described the synaptic features of dendritic spines. Nevertheless, as Gray (1959a) wrote: “At present it cannot be stated that all spines are sites of synaptic contact.” Almost 50 years later, this statement is
EXPERIMENTAL PROCEDURES All experiments were performed in accordance with the guidelines established by the European Union regarding the use and care of laboratory animals. Every effort was made to minimize the number of animals used and their suffering. Two ICR male mice (2–3 months old) were anesthetized with ketamine–xylazine, and perfused with 4% paraformaldehyde and 1% glutaraldehyde in 0.12 M PB. Animals were left overnight at 4 °C and their brains were removed the next morning and washed in several changes of 0.12 M PB. The crania were placed in a Kopf stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) and the brains were repositioned. Blocks between 2 and 3 mm thick were trimmed, these containing the primary visual cortex according to
*Corresponding author. Tel: ⫹34-91-585-4735; fax: ⫹34-91-5854754 (J. DeFelipe), Tel: ⫹1-212-854-2354; fax: ⫹1-212-865-8246 (R. Yuste). E-mail address: [email protected] (J. DeFelipe), rmy5@columbia. edu (R. Yuste). Abbreviation: PSD, postsynaptic density.
0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.12.015
464
J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469 the coordinates of Paxinos and Franklin (2001: between ⫺0.38 and 4.21 mm to Bregma, and 2 and 3 mm to the midline). The tissue contained in the blocks, but outside of these limits, was not considered for analysis. Trimmed blocks were embedded in 4% agar. Rapid Golgi staining was performed by immersion of the blocks in 2.4% potassium dichromate and 0.2% osmium tetroxide in darkness at 18 °C, for 4 days, followed by a 0.75% silver nitrate immersion for 1 day, also in darkness. Brains were dehydrated in an ascending glycerol series into anhydrous glycerol, cut into 120 m thick sections, and gold toned (Fairén et al., 1977). Sections were dehydrated in increasing series of ethanol followed by absolute acetone and embedded in araldite resin on siliconcoated slides. Araldite embedded sections were cured at 60 °C for 48 h. Three gold-toned pyramidal cells from layer 2/3 of visual cortex were chosen for the present study. Selection was based on the quality of the Golgi impregnation, preservation of the dendritic arbor and isolation from other impregnated neurons. Selected pyramidal cells were digitalized with Neurolucida (MBF Biosciences, Williston, VT, USA) software to obtain a three-dimensional model of the dendritic arbor. Small pieces of the section
465
containing the neuron of interest were removed from the slide with the help of a razor blade, and glued to an araldite block using cyanoacrylate glue under a dissecting microscope. After trimming the block, ultrathin serial sections (50 –70 nm thick) were cut in a Reichert ultramicrotome with a diamond knife. Twenty series (19 –34 ultrathin sections each) of dendritic segments were obtained. Between consecutive series, photomicrographs were obtained to ascertain those parts of the impregnated neuron that were to be considered in the ultrathin sections. Using a modified microtome chuck (Fairén et al., 1977), the block could be repositioned exactly in the microtome, in such a way that no significant loss of material occurred between the series. The section were collected on formvar-coated nickel slot grids, stained for 1 h with 1% uranyl acetate in bidistilled water, and for 30 min with lead citrate. Digital pictures of selected segments from basal and apical dendrites located at 12.5–127 m from the soma were captured at 30,000⫻ and 60,000⫻ in a JEOL 150 transmission electron microscope, equipped with a SIS Megaview III CCD digital camera. Ten series were analyzed, ranging from 21 to 52 sections. Serial
Fig. 1. Examples of spines forming synapses (A–F). The gold-particles allow clear distinguishing of the PSDs (red arrows) when present. Notice the small size of the PSD (60 nm) in the head of a small spine in B (asterisk). E and F are consecutive sections of a spine head to illustrate a PSD cut tangentially. Scale bar⫽560 nm in A; 350 nm in B and C, 300 nm in D; 280 nm in E and F.
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J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469
analysis of dendritic spines was achieved with the help of Reconstruct software (Fiala, 2005).
RESULTS Adult mice were perfused and their brains were impregnated using the rapid Golgi method before carrying out gold toning (Fairén et al., 1977). This procedure was followed to perform a detailed correlative light and electron microscopy analysis
of a selected sample of dendritic spines from layers II–III pyramidal cells. In this analysis distinct characteristics of the spines were defined, such as the distance to the soma or the dendritic order of the parent dendrite. The advantage of performing gold-toning of Golgi-stained material is that a very fine deposit of opaque particles is scattered on the impregnated and labeled neurons. Thus, labeling does not obscure the PSD enabling the synaptic contacts of the dendritic
Fig. 2. Non-synaptic spine. (A–H) Electron micrographs showing serial sections (70 nm thick) through a gold-toned Golgi-impregnated spine (arrow) lacking a PSD arising from a dendrite (ds). An axon terminal (asterisk) can be observed adjacent to the labeled spine head. ax1 And ax2, are unlabeled and Golgi-impregnated axon terminals, respectively, forming asymmetrical synapses with unlabeled dendritic spines. Note the absence of a PSD in the labeled spine, observed just after it first appears and before it disappears (arrows in B and G, respectively). Scale bar⫽350 nm.
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467
Fig. 3. Reconstructions of electron micrographs from serial sections of dendritic segments to illustrate the distribution of some non-synaptic spines (blue) indicated by arrows. The remaining spines establish synaptic contacts (red, PSD). (A, B) Basal dendrites; (C) apical dendrite. Scale bar⫽2000 nm.
spines to be clearly identified (Fig. 1), even when PSDs are cut tangentially (Fig. 1E, F). Hence, all morphological types of spines that are labeled with the Golgi method can be systematically examined at both the light and electron microscope level. Because of this, this method allows reconstruction of spines from a selected population of neurons and thus enables a more focused dataset than studies of randomly chosen spines from unstained samples.
Three pyramidal cells from layers II–III of the visual cortex were chosen based mainly on the quality of the gold-toning of the Golgi-impregnated neurons, the preservation of their dendritic arbor and their isolation from other impregnated neurons. Strings of serial sections were obtained and digitally photographed in the electron microscope (Fig. 2), and these were then reconstructed in three dimensions to obtain morphological data of dendritic spines (Figs. 3, 4).
Fig. 4. Morphology of the reconstructed non-synaptic spines (A) and some examples of reconstructed synaptic spines (B). Note the absence of a clear head in the non-synaptic spines. Red, PSD. Scale bar⫽250 nm.
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J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469
Table 1. Morphological parameters of synaptic and non-synaptic dendritic spines that were completely reconstructed (n⫽144) Spine type
Spine length (m)
Spine surface (m2)
Spine volume (m3)
Head volume (m3)
Neck volume (m3)
Synaptic spines Non-synaptic spines Difference (%)
1.28⫾0.04 0.89⫾0.09 30.3
1.26⫾0.05 0.62⫾0.09 50.6
0.09⫾0.01 0.03⫾0.01 67.0
0.07⫾0.01 —
0.03⫾0.002 —
Of a total of 365 spines tracked in the serial sections, 144 spines were completely reconstructed. It was essential for the purpose of our study that the spines be completely reconstructed (i.e. where every single section that revealed the spine was recovered and photographed), thus, 221 not complete spines were ruled out of our analysis. Of the 144 completely reconstructed spines, 103 were located on basal dendrites, 31 were on apical dendrites and 10 were on dendrites of undetermined origin. Six of the 144 spines were ramified while 138 were simple. All the ramified spines presented two branches and each branch (with or without a clear head) displayed a PSD. Of the simple spines, 133 established a single synapse that could be classified as asymmetrical in most cases. Five spines of the simple spines clearly lacked a PSD as well as other typical characteristics of synaptic junctions, they were classified as non-synaptic spines (Fig. 2). These non-synaptic spines displayed in all cases axonal boutons directly apposed to the spine membrane, although these boutons did not show electron-dense presynaptic densities. Also, electron-dense synaptic cleft material could not be distinguished either. Thus, these five spines lacked PSDs, presynaptic densities and cleft material, and they were thus identified as non-synaptic. Non-synaptic spines were significantly smaller than synaptic spines (Table 1). Specifically, non-synaptic spines were ⬃30% shorter in total spine length and their volume was approximately one third of the volume of synaptic spines. This difference in volume was mainly due to differences in the head region, which was poorly differentiated in nonsynaptic spines (Fig. 4). Regarding the distribution of the spines, one of these non-synaptic spines belonged to an unidentified impregnated dendrite, whereas the remaining four were located in the basal (n⫽2) and apical pyramidal dendrites (n⫽2), at a distance of 36, 47, 112 and 118 m from the soma, respectively. Finally, glial processes were directly apposed to these spines, but in all cases they covered only a small portion of the total surface of the spines.
DISCUSSION The study of dendritic spines is currently an exciting area of research in neurosciences. Not only are spines the major targets of excitatory connections in the cerebral cortex, but they are also motile and highly plastic structures that appear to be key elements in learning and memory. Dendritic spines were first discovered by Cajal (1888, 1890) and he suggested that they were points of contact with axon terminals. Thus, it is somewhat surprising that despite the many studies performed since then, certain
fundamental questions relating to dendritic spines remain unresolved, such as whether all spines establish a synaptic contact or not. The results presented here essentially confirm the long-held assumption that one spine⫽one synapse, since the vast majority of spines establish synaptic contacts. However, we unambiguously demonstrate that a small number of non-synaptic spines exist in the neocortex (3.6%), in agreement with earlier studies on the existence of non-synaptic spines in various regions of the cerebral cortex (White and Rock, 1980; Hersch and White, 1981, 1982; Spacek, 1982; Chicurel and Harris, 1992; Sorra and Harris, 1998). For example, using a similar approach, Hersch and White (1981) reported non-synaptic spines (2.4%) on pyramidal cells from layers V and VI of the mouse somatosensory cortex. Furthermore, in our study, nonsynaptic spines were thin and lacked clear heads, somewhat resembling the filopodia found at earlier developmental stages (Portera-Cailliau et al., 2003). Indeed, our results would imply that filopodia, which lack clear heads, also lack synaptic contacts. At the same time, although all spines in our sample with prominent heads were indeed synaptic, we did occasional encounter synaptic spines that lack clearly differentiated heads (Fig. 4). Different hypotheses can be formulated about the possible function of non-synaptic spines. These spines could represent “fossil” spines that failed to establish a synaptic contact during development and that then remained static in their dendritic location. This possibility seems to be unlikely, since a number of studies have shown that dendritic protrusions (filopodia and spines) are highly dynamic during development (Portera-Cailliau et al., 2003). In addition, recent studies using in vivo imaging of dendritic spines have shown that spine formation and elimination are maintained in adult mice and that the population of spines that are particularly plastic is one that resembles filopodia (Trachtenberg et al., 2002). Thus, it seems more likely that non-synaptic spines correspond to transient structures that are emerging or disappearing, as occurs throughout the adult life of the animal. They could arise via two different mechanisms. First, it is possible these spines had established a synaptic contact that later disappeared because the presynaptic axon terminal either degenerated or its contact with the spine disappeared. The resulting axon terminal could form a synapse with another element or it could fail to re-establish a synapse. Alternatively, these non-synaptic spines may represent newly formed spines capable of establishing new synaptic contacts. Finally, while the percentage of non-synaptic spines is relatively small, there are in fact many such spines given
J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469
that each pyramidal neuron has thousands of spines (see Konur et al., 2003 and Ballesteros-Yáñez et al., 2006 for spine counts in mouse neocortical cells). Indeed, nonsynaptic spines could be important functionally since their effect could accumulate over time. If non-synaptic spines have a short lifespan and eventually establish synaptic contacts, a small percentage of non-synaptic spines at any given time could give rise to a much larger percentage of synaptic contacts. Thus, they might represent an important source of new synaptic connections, contributing to the plasticity of adult cortical circuits. Acknowledgments—J.A., J.D., A.E. and A.F. are supported by Spanish Ministry of Education and Science (grants BFU200613395 and BFU2004-04660, respectively). J.A. and R.Y. were supported by the NEI (EY11787) and the John Merck Fund. R.Y. thanks J.D. for hosting him.
REFERENCES Ballesteros-Yáñez I, Benavides-Piccione R, Elston GN, Yuste R, DeFelipe J (2006) Density and morphology of dendritic spines in mouse neocortex. Neuroscience 138:403– 409. Benshalom GJ, White EL (1986) Quantification of thalamocortical synapses with spiny stellate neurons in layer IV of mouse somatosensory cortex. J Comp Neurol 253:303–314. Bonhoeffer T, Yuste R (2002) Spine motility. Phenomenology, mechanisms, and function. Neuron 35:1019 –1027. Cajal SR (1888) Estructura de los centros nerviosos de las aves. Rev Trim Histol Norm Patol 1:1–10. Cajal SR (1890) Textura de las circunvoluciones cerebrales de los mamíferos inferiores. Nota preventiva. Gac Méd Catalana 1: 22–31. Colonnier M (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res 9:268 –287. Chicurel ME, Harris KM (1992) Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J Comp Neurol 325:169 –182. Fairén A, Peters A, Saldanha J (1977) A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J Neurocytol 6:311–337. Fiala JC (2005) Reconstruct: a free editor for serial section microscopy. J Microsc 218:52– 61. Gray EG (1959a) Axo-somatic and axo-dendritic synapses of the cerebral cortex: An electron microscopic study. J Anat 83: 420 – 433. Gray EG (1959b) Electron microscopy of synaptic contacts on dendritic spines of the cerebral cortex. Nature 183:1592–1594. Grutzendler J, Kasthuri N, Gan WB (2002) Long-term dendritic spine stability in the adult cortex. Nature 420:812– 816.
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Hersch SM, White EL (1981) Quantification of synapses formed with apical dendrites of Golgi-impregnated pyramidal cells: variability in thalamocortical inputs, but consistency in the ratios of asymmetrical to symmetrical synapses. Neuroscience 6:1043–1051. Hersch SM, White EL (1982) A quantitative study of the thalamocortical and other synapses in layer IV of pyramidal cells projecting from mouse SmI cortex to the caudate putamen nucleus. J Comp Neurol 211:217–225. Jones EG, Powell TPS (1969) Morphological variation in the dendritic spines of the neocortex. J Cell Sci 5:509 –529. Knott GW, Holtmaat A, Wilbrecht L, Welker E, Svoboda K (2006) Spine growth precedes synapse formation in the adult neocortex in vivo. Nat Neurosci 9:1117–1124. Konur S, Rabinowitz D, Fenstermaker V, Yuste R (2003) Systematic regulation of spine head diameters and densities in pyramidal neurons from juvenile mice. J Neurobiol 56:95–112. Mates S, Lund J (1983) Spine formation and maturation of type 1 synapses on spiny stellate neurons in primate visual cortex. J Comp Neurol 221:91–97. Matus A (2000) Actin-based plasticity in dendritic spines. Science 290:754 –758. Miller M, Peters A (1981) Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons. J Comp Neurol 203:555–573. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates, 2nd edition. San Diego: Academic Press. Peters A, Kaiserman-Abramof I (1969) The small pyramidal neuron of the rat cerebral cortex. The synapses upon dendritic spines. Z Zellforsch Mikrosk Anat 100:487–506. Portera-Cailliau C, Pan DT, Yuste R (2003) Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia. J Neurosci 23:7129 –7142. Shepherd G (1996) The dendritic spine: a multifunctional integrative unit. J Neurophysiol 75:2197–2210. Sorra K, Harris K (1998) Stability in synapse number and size at 2 hr after long-term potentiation in hippocampal area CA1. J Neurosci 18:658 – 671. Spacek J (1982) “Free” postsynaptic-like densities in normal adult brain: their occurrence, distribution, structure and association with subsurface cisterns. J Neurocytol 11:693–706. Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda K (2002) Long-term in vivo imaging of experiencedependent synaptic plasticity in adult cortex. Nature 420: 788 –794. Trommald M, Hulleberg G (1997) Dimensions and density of dendritic spines from rat dentate granule cells based on reconstructions from serial electron micrographs. J Comp Neurol 377:15–28. White EL, Rock MP (1980) Three-dimensional aspects and synaptic relationships of a Golgi-impregnated spiny stellate cell reconstructed from serial thin sections. J Neurocytol 9:615– 636. White EL, Hersch SM (1982) A quantitative study of thalamocortical and other synapses involving the apical dendrites of corticothalamic projection cells in mouse SmI cortex. J Neurocytol 11: 137–157.
(Accepted 1 December 2006) (Available online 16 December 2006)
NON-SYNAPTIC DENDRITIC SPINES IN NEOCORTEX J. I. ARELLANO,a,b A. ESPINOSA,c A. FAIRÉN,c R. YUSTEd* AND J. DEFELIPEa*
still valid, and this question has unfortunately not been directly addressed. Early reports on cortical spines were based on single section ultrastructural analysis, a method that does not allow the identification of non-synaptic spines (Gray, 1959a,b; Colonnier, 1968; Jones and Powell, 1969; Peters and Kaiserman-Abramof, 1969). Serial section reconstructions have provided contradictory results: while some studies reported that all spines established synaptic contacts (Miller and Peters, 1981; White and Hersch, 1982; Hersch and White, 1982; Mates and Lund, 1983), other studies have reported occasional non-synaptic spines in neocortex (Trachtenberg et al., 2002; White and Rock, 1980; Hersch and White, 1981; Benshalom and White, 1986; Spacek, 1982; Knott et al., 2006) and hippocampus (Chicurel and Harris, 1992; Trommald and Hulleberg, 1997; Sorra and Harris, 1998). However, these non-synaptic spines have not been documented. Moreover, in their pioneering study, White and Rock (1980) admitted that they might establish synapses but that these were not detected “due to an unfavourable plane of section.” Thus, it is possible that all spines were indeed synaptic. Finally, a key criterion to identify non-synaptic spines relies on the absence of postsynaptic density (PSD). Unfortunately, most techniques used to label neurons produce intense, homogeneous intracellular labeling that masks PSDs (such as the deposit produced by the chromogen 3,3= diaminobenzidine tetrahydrochloride), particularly when membranes are sectioned tangentially. This makes the identification of axospinous synapses difficult and the detection of non-synaptic spines impractical. This, together with the intrinsic difficulty of obtaining complete serial ultrathin sections to achieve full reconstructions of spines, could explain why the question of whether some spines lack a synaptic contact is still outstanding. We aimed to resolve this question through the ultrastructural analysis of a large number of completely reconstructed neocortical spines, using gold-toned Golgi staining, a method that enables the visualization of postsynaptic densities.
a
Departamento de Neuroanatomia y Biologia Celular, Instituto Cajal (CSIC), Ave. Dr. Arce, 37, 28002 Madrid, Spain
b
Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA
c
Instituto de Neurociencias de Alicante, (CISC and Universidad Miguel Hernández), 03550 San Juan de Alicante, Spain
d
Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Abstract—A long-held assumption states that each dendritic spine in the cerebral cortex forms a synapse, although this issue has not been systematically investigated. We performed complete ultrastructural reconstructions of a large (nⴝ144) population of identified spines in adult mouse neocortex finding that only 3.6% of the spines clearly lacked synapses. Nonsynaptic spines were small and had no clear head, resembling dendritic filopodia, and could represent a source of new synaptic connections in the adult cerebral cortex. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: pyramidal neurons, excitatory synapses, serial section reconstructions.
Dendritic spines are the major targets of excitatory connections in the cerebral cortex and they also compartmentalize calcium and could implement local learning rules (Shepherd, 1996). Spines are motile (Matus, 2000) and can emerge in response to the synaptic stimulation, so they could mediate structural plasticity in the nervous system (Bonhoeffer and Yuste, 2002). Moreover, in adult neocortex in vivo, spines have been reported to be plastic (Trachtenberg et al., 2002), although other reports indicate that they are stable (Grutzendler et al., 2002). Nevertheless, it is still unclear what the relationship is between the formation or motility of spines with their establishment or not of synaptic junctions. Crucial to these questions is the basic issue of whether or not all spines have a synapse or whether non-synaptic spines exist. Since Gray (1959a) first demonstrated using electron microscopy that dendritic spines established synaptic contacts, numerous ultrastructural studies have described the synaptic features of dendritic spines. Nevertheless, as Gray (1959a) wrote: “At present it cannot be stated that all spines are sites of synaptic contact.” Almost 50 years later, this statement is
EXPERIMENTAL PROCEDURES All experiments were performed in accordance with the guidelines established by the European Union regarding the use and care of laboratory animals. Every effort was made to minimize the number of animals used and their suffering. Two ICR male mice (2–3 months old) were anesthetized with ketamine–xylazine, and perfused with 4% paraformaldehyde and 1% glutaraldehyde in 0.12 M PB. Animals were left overnight at 4 °C and their brains were removed the next morning and washed in several changes of 0.12 M PB. The crania were placed in a Kopf stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) and the brains were repositioned. Blocks between 2 and 3 mm thick were trimmed, these containing the primary visual cortex according to
*Corresponding author. Tel: ⫹34-91-585-4735; fax: ⫹34-91-5854754 (J. DeFelipe), Tel: ⫹1-212-854-2354; fax: ⫹1-212-865-8246 (R. Yuste). E-mail address: [email protected] (J. DeFelipe), rmy5@columbia. edu (R. Yuste). Abbreviation: PSD, postsynaptic density.
0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.12.015
464
J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469 the coordinates of Paxinos and Franklin (2001: between ⫺0.38 and 4.21 mm to Bregma, and 2 and 3 mm to the midline). The tissue contained in the blocks, but outside of these limits, was not considered for analysis. Trimmed blocks were embedded in 4% agar. Rapid Golgi staining was performed by immersion of the blocks in 2.4% potassium dichromate and 0.2% osmium tetroxide in darkness at 18 °C, for 4 days, followed by a 0.75% silver nitrate immersion for 1 day, also in darkness. Brains were dehydrated in an ascending glycerol series into anhydrous glycerol, cut into 120 m thick sections, and gold toned (Fairén et al., 1977). Sections were dehydrated in increasing series of ethanol followed by absolute acetone and embedded in araldite resin on siliconcoated slides. Araldite embedded sections were cured at 60 °C for 48 h. Three gold-toned pyramidal cells from layer 2/3 of visual cortex were chosen for the present study. Selection was based on the quality of the Golgi impregnation, preservation of the dendritic arbor and isolation from other impregnated neurons. Selected pyramidal cells were digitalized with Neurolucida (MBF Biosciences, Williston, VT, USA) software to obtain a three-dimensional model of the dendritic arbor. Small pieces of the section
465
containing the neuron of interest were removed from the slide with the help of a razor blade, and glued to an araldite block using cyanoacrylate glue under a dissecting microscope. After trimming the block, ultrathin serial sections (50 –70 nm thick) were cut in a Reichert ultramicrotome with a diamond knife. Twenty series (19 –34 ultrathin sections each) of dendritic segments were obtained. Between consecutive series, photomicrographs were obtained to ascertain those parts of the impregnated neuron that were to be considered in the ultrathin sections. Using a modified microtome chuck (Fairén et al., 1977), the block could be repositioned exactly in the microtome, in such a way that no significant loss of material occurred between the series. The section were collected on formvar-coated nickel slot grids, stained for 1 h with 1% uranyl acetate in bidistilled water, and for 30 min with lead citrate. Digital pictures of selected segments from basal and apical dendrites located at 12.5–127 m from the soma were captured at 30,000⫻ and 60,000⫻ in a JEOL 150 transmission electron microscope, equipped with a SIS Megaview III CCD digital camera. Ten series were analyzed, ranging from 21 to 52 sections. Serial
Fig. 1. Examples of spines forming synapses (A–F). The gold-particles allow clear distinguishing of the PSDs (red arrows) when present. Notice the small size of the PSD (60 nm) in the head of a small spine in B (asterisk). E and F are consecutive sections of a spine head to illustrate a PSD cut tangentially. Scale bar⫽560 nm in A; 350 nm in B and C, 300 nm in D; 280 nm in E and F.
466
J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469
analysis of dendritic spines was achieved with the help of Reconstruct software (Fiala, 2005).
RESULTS Adult mice were perfused and their brains were impregnated using the rapid Golgi method before carrying out gold toning (Fairén et al., 1977). This procedure was followed to perform a detailed correlative light and electron microscopy analysis
of a selected sample of dendritic spines from layers II–III pyramidal cells. In this analysis distinct characteristics of the spines were defined, such as the distance to the soma or the dendritic order of the parent dendrite. The advantage of performing gold-toning of Golgi-stained material is that a very fine deposit of opaque particles is scattered on the impregnated and labeled neurons. Thus, labeling does not obscure the PSD enabling the synaptic contacts of the dendritic
Fig. 2. Non-synaptic spine. (A–H) Electron micrographs showing serial sections (70 nm thick) through a gold-toned Golgi-impregnated spine (arrow) lacking a PSD arising from a dendrite (ds). An axon terminal (asterisk) can be observed adjacent to the labeled spine head. ax1 And ax2, are unlabeled and Golgi-impregnated axon terminals, respectively, forming asymmetrical synapses with unlabeled dendritic spines. Note the absence of a PSD in the labeled spine, observed just after it first appears and before it disappears (arrows in B and G, respectively). Scale bar⫽350 nm.
J. I. Arellano et al. / Neuroscience 145 (2007) 464 – 469
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Fig. 3. Reconstructions of electron micrographs from serial sections of dendritic segments to illustrate the distribution of some non-synaptic spines (blue) indicated by arrows. The remaining spines establish synaptic contacts (red, PSD). (A, B) Basal dendrites; (C) apical dendrite. Scale bar⫽2000 nm.
spines to be clearly identified (Fig. 1), even when PSDs are cut tangentially (Fig. 1E, F). Hence, all morphological types of spines that are labeled with the Golgi method can be systematically examined at both the light and electron microscope level. Because of this, this method allows reconstruction of spines from a selected population of neurons and thus enables a more focused dataset than studies of randomly chosen spines from unstained samples.
Three pyramidal cells from layers II–III of the visual cortex were chosen based mainly on the quality of the gold-toning of the Golgi-impregnated neurons, the preservation of their dendritic arbor and their isolation from other impregnated neurons. Strings of serial sections were obtained and digitally photographed in the electron microscope (Fig. 2), and these were then reconstructed in three dimensions to obtain morphological data of dendritic spines (Figs. 3, 4).
Fig. 4. Morphology of the reconstructed non-synaptic spines (A) and some examples of reconstructed synaptic spines (B). Note the absence of a clear head in the non-synaptic spines. Red, PSD. Scale bar⫽250 nm.
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Table 1. Morphological parameters of synaptic and non-synaptic dendritic spines that were completely reconstructed (n⫽144) Spine type
Spine length (m)
Spine surface (m2)
Spine volume (m3)
Head volume (m3)
Neck volume (m3)
Synaptic spines Non-synaptic spines Difference (%)
1.28⫾0.04 0.89⫾0.09 30.3
1.26⫾0.05 0.62⫾0.09 50.6
0.09⫾0.01 0.03⫾0.01 67.0
0.07⫾0.01 —
0.03⫾0.002 —
Of a total of 365 spines tracked in the serial sections, 144 spines were completely reconstructed. It was essential for the purpose of our study that the spines be completely reconstructed (i.e. where every single section that revealed the spine was recovered and photographed), thus, 221 not complete spines were ruled out of our analysis. Of the 144 completely reconstructed spines, 103 were located on basal dendrites, 31 were on apical dendrites and 10 were on dendrites of undetermined origin. Six of the 144 spines were ramified while 138 were simple. All the ramified spines presented two branches and each branch (with or without a clear head) displayed a PSD. Of the simple spines, 133 established a single synapse that could be classified as asymmetrical in most cases. Five spines of the simple spines clearly lacked a PSD as well as other typical characteristics of synaptic junctions, they were classified as non-synaptic spines (Fig. 2). These non-synaptic spines displayed in all cases axonal boutons directly apposed to the spine membrane, although these boutons did not show electron-dense presynaptic densities. Also, electron-dense synaptic cleft material could not be distinguished either. Thus, these five spines lacked PSDs, presynaptic densities and cleft material, and they were thus identified as non-synaptic. Non-synaptic spines were significantly smaller than synaptic spines (Table 1). Specifically, non-synaptic spines were ⬃30% shorter in total spine length and their volume was approximately one third of the volume of synaptic spines. This difference in volume was mainly due to differences in the head region, which was poorly differentiated in nonsynaptic spines (Fig. 4). Regarding the distribution of the spines, one of these non-synaptic spines belonged to an unidentified impregnated dendrite, whereas the remaining four were located in the basal (n⫽2) and apical pyramidal dendrites (n⫽2), at a distance of 36, 47, 112 and 118 m from the soma, respectively. Finally, glial processes were directly apposed to these spines, but in all cases they covered only a small portion of the total surface of the spines.
DISCUSSION The study of dendritic spines is currently an exciting area of research in neurosciences. Not only are spines the major targets of excitatory connections in the cerebral cortex, but they are also motile and highly plastic structures that appear to be key elements in learning and memory. Dendritic spines were first discovered by Cajal (1888, 1890) and he suggested that they were points of contact with axon terminals. Thus, it is somewhat surprising that despite the many studies performed since then, certain
fundamental questions relating to dendritic spines remain unresolved, such as whether all spines establish a synaptic contact or not. The results presented here essentially confirm the long-held assumption that one spine⫽one synapse, since the vast majority of spines establish synaptic contacts. However, we unambiguously demonstrate that a small number of non-synaptic spines exist in the neocortex (3.6%), in agreement with earlier studies on the existence of non-synaptic spines in various regions of the cerebral cortex (White and Rock, 1980; Hersch and White, 1981, 1982; Spacek, 1982; Chicurel and Harris, 1992; Sorra and Harris, 1998). For example, using a similar approach, Hersch and White (1981) reported non-synaptic spines (2.4%) on pyramidal cells from layers V and VI of the mouse somatosensory cortex. Furthermore, in our study, nonsynaptic spines were thin and lacked clear heads, somewhat resembling the filopodia found at earlier developmental stages (Portera-Cailliau et al., 2003). Indeed, our results would imply that filopodia, which lack clear heads, also lack synaptic contacts. At the same time, although all spines in our sample with prominent heads were indeed synaptic, we did occasional encounter synaptic spines that lack clearly differentiated heads (Fig. 4). Different hypotheses can be formulated about the possible function of non-synaptic spines. These spines could represent “fossil” spines that failed to establish a synaptic contact during development and that then remained static in their dendritic location. This possibility seems to be unlikely, since a number of studies have shown that dendritic protrusions (filopodia and spines) are highly dynamic during development (Portera-Cailliau et al., 2003). In addition, recent studies using in vivo imaging of dendritic spines have shown that spine formation and elimination are maintained in adult mice and that the population of spines that are particularly plastic is one that resembles filopodia (Trachtenberg et al., 2002). Thus, it seems more likely that non-synaptic spines correspond to transient structures that are emerging or disappearing, as occurs throughout the adult life of the animal. They could arise via two different mechanisms. First, it is possible these spines had established a synaptic contact that later disappeared because the presynaptic axon terminal either degenerated or its contact with the spine disappeared. The resulting axon terminal could form a synapse with another element or it could fail to re-establish a synapse. Alternatively, these non-synaptic spines may represent newly formed spines capable of establishing new synaptic contacts. Finally, while the percentage of non-synaptic spines is relatively small, there are in fact many such spines given
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that each pyramidal neuron has thousands of spines (see Konur et al., 2003 and Ballesteros-Yáñez et al., 2006 for spine counts in mouse neocortical cells). Indeed, nonsynaptic spines could be important functionally since their effect could accumulate over time. If non-synaptic spines have a short lifespan and eventually establish synaptic contacts, a small percentage of non-synaptic spines at any given time could give rise to a much larger percentage of synaptic contacts. Thus, they might represent an important source of new synaptic connections, contributing to the plasticity of adult cortical circuits. Acknowledgments—J.A., J.D., A.E. and A.F. are supported by Spanish Ministry of Education and Science (grants BFU200613395 and BFU2004-04660, respectively). J.A. and R.Y. were supported by the NEI (EY11787) and the John Merck Fund. R.Y. thanks J.D. for hosting him.
REFERENCES Ballesteros-Yáñez I, Benavides-Piccione R, Elston GN, Yuste R, DeFelipe J (2006) Density and morphology of dendritic spines in mouse neocortex. Neuroscience 138:403– 409. Benshalom GJ, White EL (1986) Quantification of thalamocortical synapses with spiny stellate neurons in layer IV of mouse somatosensory cortex. J Comp Neurol 253:303–314. Bonhoeffer T, Yuste R (2002) Spine motility. Phenomenology, mechanisms, and function. Neuron 35:1019 –1027. Cajal SR (1888) Estructura de los centros nerviosos de las aves. Rev Trim Histol Norm Patol 1:1–10. Cajal SR (1890) Textura de las circunvoluciones cerebrales de los mamíferos inferiores. Nota preventiva. Gac Méd Catalana 1: 22–31. Colonnier M (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res 9:268 –287. Chicurel ME, Harris KM (1992) Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J Comp Neurol 325:169 –182. Fairén A, Peters A, Saldanha J (1977) A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J Neurocytol 6:311–337. Fiala JC (2005) Reconstruct: a free editor for serial section microscopy. J Microsc 218:52– 61. Gray EG (1959a) Axo-somatic and axo-dendritic synapses of the cerebral cortex: An electron microscopic study. J Anat 83: 420 – 433. Gray EG (1959b) Electron microscopy of synaptic contacts on dendritic spines of the cerebral cortex. Nature 183:1592–1594. Grutzendler J, Kasthuri N, Gan WB (2002) Long-term dendritic spine stability in the adult cortex. Nature 420:812– 816.
469
Hersch SM, White EL (1981) Quantification of synapses formed with apical dendrites of Golgi-impregnated pyramidal cells: variability in thalamocortical inputs, but consistency in the ratios of asymmetrical to symmetrical synapses. Neuroscience 6:1043–1051. Hersch SM, White EL (1982) A quantitative study of the thalamocortical and other synapses in layer IV of pyramidal cells projecting from mouse SmI cortex to the caudate putamen nucleus. J Comp Neurol 211:217–225. Jones EG, Powell TPS (1969) Morphological variation in the dendritic spines of the neocortex. J Cell Sci 5:509 –529. Knott GW, Holtmaat A, Wilbrecht L, Welker E, Svoboda K (2006) Spine growth precedes synapse formation in the adult neocortex in vivo. Nat Neurosci 9:1117–1124. Konur S, Rabinowitz D, Fenstermaker V, Yuste R (2003) Systematic regulation of spine head diameters and densities in pyramidal neurons from juvenile mice. J Neurobiol 56:95–112. Mates S, Lund J (1983) Spine formation and maturation of type 1 synapses on spiny stellate neurons in primate visual cortex. J Comp Neurol 221:91–97. Matus A (2000) Actin-based plasticity in dendritic spines. Science 290:754 –758. Miller M, Peters A (1981) Maturation of rat visual cortex. II. A combined Golgi-electron microscope study of pyramidal neurons. J Comp Neurol 203:555–573. Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates, 2nd edition. San Diego: Academic Press. Peters A, Kaiserman-Abramof I (1969) The small pyramidal neuron of the rat cerebral cortex. The synapses upon dendritic spines. Z Zellforsch Mikrosk Anat 100:487–506. Portera-Cailliau C, Pan DT, Yuste R (2003) Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia. J Neurosci 23:7129 –7142. Shepherd G (1996) The dendritic spine: a multifunctional integrative unit. J Neurophysiol 75:2197–2210. Sorra K, Harris K (1998) Stability in synapse number and size at 2 hr after long-term potentiation in hippocampal area CA1. J Neurosci 18:658 – 671. Spacek J (1982) “Free” postsynaptic-like densities in normal adult brain: their occurrence, distribution, structure and association with subsurface cisterns. J Neurocytol 11:693–706. Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda K (2002) Long-term in vivo imaging of experiencedependent synaptic plasticity in adult cortex. Nature 420: 788 –794. Trommald M, Hulleberg G (1997) Dimensions and density of dendritic spines from rat dentate granule cells based on reconstructions from serial electron micrographs. J Comp Neurol 377:15–28. White EL, Rock MP (1980) Three-dimensional aspects and synaptic relationships of a Golgi-impregnated spiny stellate cell reconstructed from serial thin sections. J Neurocytol 9:615– 636. White EL, Hersch SM (1982) A quantitative study of thalamocortical and other synapses involving the apical dendrites of corticothalamic projection cells in mouse SmI cortex. J Neurocytol 11: 137–157.
(Accepted 1 December 2006) (Available online 16 December 2006)