**2. Development of dendritic spines during brain development**

The relative sequence of synapse and spine genesis during brain development is still in debate. Some evidences suggest that spine genesis lags behind synapse genesis. Fiala et al. showed that axonal fibers made synaptic contacts with long filopodia, which subsequently were transformed into mature spines [25]. Durand et al. reported during the first postnatal week in rats, synapses on the excitatory neurons are functional and plastic in the absence of dendritic spines [26]. Hence synaptic function and plasticity can take place without spines. After induction of long-term enhancement of synaptic connections in area CA1, new spines appeared on the postsynaptic dendrite [27]. Maletic-Savatic found that with axonal inputs activation, these small filopodia-like protrusions enlarged and became dendritic spines [28]. For those filopodia that do not connect with axonal inputs, they did not turn into mature spines and were absorbed back into dendrites [12]. Interestingly, increase of spine synapse might inhibit the mobility of nearby filopodia on the same dendrite and diminish the formation of synapses [29]. These results indicate that synapse formation or strengthening promotes the formation or maturation of spines, and lend support for the notion that synapse genesis occurs prior to spine genesis.

are highly enriched in spine heads, while microtubules are found mostly in the dendritic shafts [4, 5]. Actin filaments inside spines are of two pools, G-actin and F-actin. G-actin is a monomer form of actin and F-actin is the polymer form, while they are found in the tip and base of spines, respectively [6]. Actin filaments bind to the scaffold proteins such as PSD-95 and Shanks, which anchor receptors and signaling molecules at the synapses [7]. Actin filaments are dynamic since actin monomer dissociates from the pointed end while new actin monomers are recruited into

Spines are dynamic features in that they are in a constant motion (termed morphing), and their sizes fluctuate around a mean value [9, 10]. Dendritic filopodia are highly motile and flexible, and their lifetime is on the order of minutes to hours [11, 12]. This high motility may allow filopodia to explore the space around them in search for potential presynaptic inputs to form connections [13]. To understand this dynamic nature and to monitor these changes accurately, two-photon imaging has become an invaluable tool. With two-photon imaging, spine morphology and dynamics can be studied in much more details using time-lapse and repetitive imaging. This approach has revealed spine modifications under physiological or pathological conditions or events [14–16] and has greatly advanced our understanding of spine function and allowed in-depth study on the underlying structure–function relationship. During brain development dendritic spines are dynamic in their genesis and elimination, while in adolescence spines show much higher elimination than formation which results in a net spine loss or pruning. In contrast, the rate of spine genesis and elimination in adult is much lower and about equal, and this balance maintains the stability of spine density [17, 18]. Spine is considered as a unique calcium compartment, because the transfer of electrical charge is limited by the spine neck. The length of spine neck controls the degree of interaction between spines and their parent dendrite. In general, short spines and parent dendrites show similar responses to glutamate, while long spines exhibit faster and larger responses [19]. Spine plasticity is evidenced by their rapid (on the order of seconds) and persistent (for months to years) changes in response to physiological or pathological stimuli. Large spines has been suggested to be the site of stable long-term memory storage [1] while filopodia are considered by most to be an immature form of spine. Filopodia may transform into mature spines or are eliminated [20]. Hence, we define spine plasticity in two forms: alterations in their morphology/size and alterations in their density. We note that both forms of plasticity reflect modification of synaptic connections. In neurodegenerative and psychiatric diseases, spine density and spine morphology are altered, and changes in spine density and morphology may at least partially account for altered brain functions in these diseases [1, 21–24]. Therefore, better understand-

the barbed end. Certain actin-binding proteins regulate this dynamic process [8].

148 Neuroplasticity - Insights of Neural Reorganization

ing of spine pathology may provide better therapeutic intervention.

**2. Development of dendritic spines during brain development**

In this chapter, we will discuss signaling mechanisms underlying the formation and maintenance of spines, plasticity of spine morphology and its relationship to modification of synaptic strength.

The relative sequence of synapse and spine genesis during brain development is still in debate. Some evidences suggest that spine genesis lags behind synapse genesis. Fiala et al. Do spines form from filopodia, or from existing synapses on the dendritic shafts? In mature cultures, some stable spines could emerge without going through the dynamic filopodia stage [12]. The series sample analysis in young hippocampal area CA1 also revealed that most of synapses are on dendritic shafts, with rare synapses on stubby and mushroom spines [3]. Despite all this, the transformation from dendritic shaft synapses to spines has not been supported by direct observations [30]. On the other hand, several *in vitro* studies revealed that during the initial 1–2 weeks in culture, the long and headless filopodia bear no synaptic contacts associated with the presynaptic axons. Over the subsequent 4 weeks, these dynamic filopodia turned into stable, mushroom-like spines [29, 31, 32]. Fiala et al. also found that in the hippocampal CA1, synapses were present on both filopodia and dendrites. From PN1 to PN12, the number of shaft synapses and filopodia synapses was decreased, while the number of stubby and spine synapses was significantly increased [25]. Thus, it is likely that during early development, shaft synapses are the dominant form of synaptic contact. With development, and likely the need for increasing contact area, spine synapse replace shaft synapses to become the major form of synapses, at least in the adult cortex. Recently, shaft synapses are shown to define the locations where dendritic spines are formed [30] (**Figure 1**), providing more evidence for the transition from shaft synapse to spine synapse as a major process in synapse formation and maturation.

The initial surge of spine genesis leads the generation of more spines than what eventually is retained in the adult brain, and pruning of excessive spines after spine genesis allows a better adaptation to the environment [33]. This pruning process could be evoked by lowfrequency glutamatergic stimulation and requires activation of NMDA receptors [34–36].

**Figure 1.** Dendritic spines are derived from filopodia with the assistance of shaft synapses. (1) presynaptic axon forms synapses with dendritic shaft. (2) a dendritic protrusion occurs adjacent to the dendritic shaft synapse. (3) the dendritic protrusion contacts with the presynaptic axon and eventually a mushroom dendritic spine is generated. Modified from [30].

In young adolescent mice (1-month old), within a 2 week period of time, 13–20% of total spines were eliminated with 5–8% formed in the barrel, motor and frontal cortices, and this imbalance led to a significant spine loss in many brain regions. However, in the adult mice (4–6 months old), 3–5% of spines were eliminated and formed in 2 weeks [18]. Most evidences support that dendritic spines are stable in the adulthood [37, 38]. Grutzendler et al. reported that spines in the primary visual cortex of young adolescence have a turnover rate of 27% per month but this rate dropped to only 4% in the adult [15]. In contrast, Trachtenberg et al. found that adult spines are highly dynamic with about 20% turnover per day in the mouse barrel cortex [16]. This discrepancy is likely due to differences in the methodology in that the use of cranial window in the latter study triggered inflammatory responses in the brain which resulted in elevated turnover rates. It should be pointed out that spine turnover rate differs in various brain regions. For example, Holtmaat et al. reported that spines turn over more slowly (both generation and elimination) in the visual cortex than in the somatosensory cortex, with the fraction of transient spines (lifetime ≤ 4 days) also lower in visual cortex [17].

synaptic plasticity [52]. Blockade of myosin II with shRNA suppressed the formation of mushroom-like spines and increased the presence of filopodia [53]. Abi-1 is a member of the c-Abl tyrosine interactor (Abi) protein family, which interacts with scaffolding proteins and F-actin in the spines [54]. Knocking down of Abi-1 by RNAi shifted spines to an immature form [55]. Spinophilin has an actin-binding domain at its N terminus and can bundle F-actin filaments [56].

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Actin polymerization is regulated by actin binding proteins, whose active and inactive states are regulated by small GTPases. Of the Rho family of small GTPases, three are most actively involved in spine morphogenesis, RhoA, Rac1 and Cdc42. These three GTPases are distinguished by two opposite activities: RhoA inhibits whereas Rac1 and Cdc42 promote spine growth. Tashiro et al. reported that in hippocampal neurons, Rac1 increased spine density but reduced spine length, while RhoA decreased both spine density and spine length [58]. Interestingly, RhoA and Cdc42 play opposite roles in stress fiber formation by controlling the phosphorylation of myosin light chain. RhoA inhibits myosin phosphatase via the Rho kinase while Rac1 and Cdc42 activate it via the serine/threonine kinase PAK [59, 60]. Thus, Rac1 and RhoA might have opposite effects on the same target proteins and hence opposite effects in regulating spine density. Similarly, Nakayama et al. found that Rac1 is essential for the maintenance of dendritic spines while

Receptor tyrosine kinases also regulate spine morphology. Among them, the erythropoietinproducing hepatocellular carcinoma (Eph) receptors have unique activity on synapse. They consist of type A and type B receptor subclasses based on their binding capability to Ephrin A and Ephrin B ligands. Moeller et al. reported that activation of EphB2 in the cultured hippocampal neurons led to shortening of filopodia [62]. Furthermore, activation of EphB likely phosphorylates guanine exchange factors (GEFs) such as kalirin7, which further stimulates Rho family GTPases Rac1 and Cdc42 [63]. Opposite to EphB2, activation of EphA4 by its ligand, ephrin-A3, located in the perisynaptic processes of astrocytes, decreased spine length and density. Loss of EphA4 led to spine elongation and disorganization [64]. Similarly, in the hippocampus of ephrin-A3-null mice, EphA4 phosphorylation was decreased and abnormal spine elongation was observed [65]. Thus, either loss of EphA4 or ephrin-A3 induces identical dendritic spine deficits. In summary, we have reviewed three types of important and representative signaling molecules in spine function. The first signaling pathway is mediated by actin binding proteins, the second is the family of small GTPases (including Rac1, RhoA and Cdc42, which determine the activity states of actin binding proteins), and the third is receptor tyrosine kinases. The absence or malfunctioning of the above three signaling pathways leads to altered spine mor-

Spine plasticity may be exhibited in two forms—changes in spine density and spine dimension. Change in spine density reflects modification of connection density between the presynaptic and postsynaptic neurons, which happens most commonly during brain development (increase, decrease/pruning) and aging/degeneration (decrease). Changes in spine

Knockout of spinophilin in mice increased the presence of filopodia [57].

enhanced RhoA activity led to significant simplification of dendrites [61].

phogenesis and function.

**4. Spine plasticity**
