**4. Spine plasticity**

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

**3. Signaling events during spine formation and maintenance**

Motility of dendritic spines is regulated by the dynamic balance between G-actin and F-actin [39]. F-actin consists of two pools, a large dynamic pool in the tip of the spine head and a small stable pool in the base of spine [6] [40]. With LTP induction, the stable F-actin is severed into short segments and reorganized to expand the spine [41]. Thus, the dynamics of actin cytoskeleton controls dendritic spine morphological remodeling and plenty of signaling mol-

Spine morphology is regulated by actin binding and cytoskeleton proteins. Drebrin was the first identified to modify dendritic spines since overexpression of drebrin in cultured neurons increased the length of spines [45]. Spines in the drebrin knockout mice exhibited normal morphology but altered plasticity [46]. Takahashi et al. reported that drebrin entered filopodia and formed an actin filament cluster to recruit postsynaptic components (including scaffolding protein PSD95), and this process enables the transition from filopodia to mature spines. Based on this observation, filopodia are classified into two types, an immature diffuse-type and a mature cluster-type. A filopodium with a drebrin cluster, whose maximum intensity was higher than twice the average intensity of the filopodium, was classified as a cluster-type filopodium. Otherwise, it was classified as a diffuse-type filopodium. The clustertype filopodia were likely to be converted to mature spines [47]. In addition, overexpression of drebrin in neurons caused F-actin to accumulate in the growth cone, whereas knockdown of drebrin reduced F-actin level [48]. Drebrin binds to F-actin to generate thick bundles of F-actin [49], and drebrin also competes with other actin binding proteins such as ADF/cofilin

Besides drebrin, other actin-binding proteins including myosin II, Abi-1 and spinophilin regulate actin polymerization in the dendritic spines. Myosin II belongs to the family of molecular motors which is highly expressed in dendritic spines, and regulates dendritic spine morphology and

cortex [17].

ecules participate in this process [42–44].

150 Neuroplasticity - Insights of Neural Reorganization

which depolymerizes F-actin [50, 51].

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 morphology/dimension, especially the size of spine head, have been widely reported, and are believed to be associated with changes in the strength of synapses that reside on these altered spines. In this chapter, we will focus on alterations of spine dimension.

#### **4.1. Changes in spine morphology**

Due to the heterogeneity of spine size/morphology, the most convincing way to demonstrate that spine morphology is altered is to compare the same set of spines before and after a manipulation, such as synaptic plasticity-inducing stimuli in brain slices or learning *in vivo*. By using time-lapse two-photon imaging on the same set of spines, Yang et al. found new spines were formed after *in vivo* experience in the form of sensory or motor, and a fraction of these newly appeared spines persisted for months after the experience. More importantly, of the appearance of these new spines is specifically related to the *in vivo* experience or training [66]. In addition, Hayashi-Takagi et al. showed that motor learning on rotarod led to an enhanced *Arc* signaling, together with an expansion of a subset of spines in the motor cortex. By expressing a photoactivatable GTPase Rac1 in spines, they further showed that prolonged photo-activation of Rac1 resulted in reversal of spine expansion and loss of motor memory. This is a striking demonstration that potentiated synapses and enlarged spines are likely the underlying biological substrates of stored memory and formed memory can be erased by reversing these changes [67].

Many studies have examined spine modifications with the induction of long-term potentiation (LTP) and long-term depression (LTD), since this procedure allows the examination of the same set spines associated with fast, large and long-lasting changes in synaptic strength. These two forms of synaptic plasticity refer to the increase and decrease in synaptic strength respectively, and are generally regarded as the cellular basis of synaptic modifications underlying developmental remodeling of neuronal connections, and learning and memory function in the adult brain [68]. When studied in brain slices (acute or organotypic culture), LTP or LTD is induced by stimulation of the presynaptic inputs with distinct patterns. In some studies, changes in the synaptic strength were also monitored, and thus changes in synaptic function and spine morphology can be related to each other in the same set of spines/synapse, or even a single synapse/spine [34, 69–71]. In general, spines exhibit the capacity of bi-directional changes in that spine enlargement is observed with LTP while spine shrinkage with LTD [72–74] (**Figure 2**). Either uncaging of caged glutamate onto a single spine [75] or electrical stimulation of a population of synapses [70, 71] had confirmed the above observations. Uncaging of glutamate directly enhances postsynaptic AMPAR function, and since it bypasses presynaptic release, and thus has provided the unambiguous evidence that postsynaptic changes can underlie the expression of LTP [69, 75]. These observations further indicate that morphological and functional changes are likely driven by the same stimuli or process (see below).

reported that the number of AMPAR in spines is of a large range. In addition, mushroom spines are enriched with AMPARs, compared to the low distribution of them in the thin spines and filopodia. These observations support a strong relationship between number of AMPAR and volume of spines [77]. In addition, it provides direct evidence that mushroom spines are func-

**Figure 2.** Bi-directional modification of spine size by synaptic plasticity. (A) Recording, synaptic stimulation and fluorescence imaging of the same set of synapses and spines in acute hippocampal slices. (B) Spine enlargement associated with LTP induction by theta burst stimulation, this enlargement is persistent and long-lasting. In addition, enlargement was restricted to spines close (upper) but not far away from the stimulation site (lower). (C) Change in spine volume before and after LTP induction and its requirement of activation of NMDARs since it was prevented by bath application of NMDAR antagonists APV and MK-801. (D) Increase in synaptic strength (EPSPs) as a result of LTP induction. LTP occurred in two phases, an initial rapid increase (indicated by 2 on the plot) and a slower gradual increase (indicated by 3). LTP was also sensitive to NMDAR blockade. (E) Spine shrinkage associated with LTD, it is persistent and long-lasting. (F) Shrinkage of spines occurred to those spines close to, but not to those far away from the stimulating electrode, or those had not received any stimulation. Compared to the almost instantaneous enlargement of spine heads after LTP, spine shrinkage develops slowly and takes much longer to reach a plateau. (G) Low frequency stimulation led

Dendritic Spine Modifications in Brain Physiology http://dx.doi.org/10.5772/intechopen.73326 153

Synapses can change their strength by the activity patterns that they receive, and this modification allows synaptic strength to be adjusted to better suit the need for adaptation. Originally put forwarded by Donald Hebb and later adopted as "fire together, wire together" model, the current model of synaptic modification states that neurons sharing spike activity have increased connections between these two partners [78]. After the discovery of LTP in 1973, this activity-driven increase in synaptic strength has been extensively studied, both in acute brain slices and *in vivo* [68]; and has been used widely as a tool to induce synaptic modification in order to study the associated processes. Various molecules have been identified to be required for the induction and expression of LTP [68]. In general, Ca2+ entry or elevated intracellular Ca2+ concentration is required to convert electrical activity into intracellular signaling that determines the direction of synaptic changes. Usually a large but transient elevation in

tionally mature while thin spines and filopodia are not.

**4.2. Synaptic plasticity**

to depression of EPSPs. Taken from [34, 71].

In general, there is a good correlation between the strength of a given synapse (measured by electrophysiological responses) and the size of spine. Electrophysiological responses are further determined by the number/density of AMPA receptors at a given synapse. Takumi et al. found a linear relationship between AMPAR density and the diameter of PSD [76]. Matsuzaki et al.

**Figure 2.** Bi-directional modification of spine size by synaptic plasticity. (A) Recording, synaptic stimulation and fluorescence imaging of the same set of synapses and spines in acute hippocampal slices. (B) Spine enlargement associated with LTP induction by theta burst stimulation, this enlargement is persistent and long-lasting. In addition, enlargement was restricted to spines close (upper) but not far away from the stimulation site (lower). (C) Change in spine volume before and after LTP induction and its requirement of activation of NMDARs since it was prevented by bath application of NMDAR antagonists APV and MK-801. (D) Increase in synaptic strength (EPSPs) as a result of LTP induction. LTP occurred in two phases, an initial rapid increase (indicated by 2 on the plot) and a slower gradual increase (indicated by 3). LTP was also sensitive to NMDAR blockade. (E) Spine shrinkage associated with LTD, it is persistent and long-lasting. (F) Shrinkage of spines occurred to those spines close to, but not to those far away from the stimulating electrode, or those had not received any stimulation. Compared to the almost instantaneous enlargement of spine heads after LTP, spine shrinkage develops slowly and takes much longer to reach a plateau. (G) Low frequency stimulation led to depression of EPSPs. Taken from [34, 71].

reported that the number of AMPAR in spines is of a large range. In addition, mushroom spines are enriched with AMPARs, compared to the low distribution of them in the thin spines and filopodia. These observations support a strong relationship between number of AMPAR and volume of spines [77]. In addition, it provides direct evidence that mushroom spines are functionally mature while thin spines and filopodia are not.

#### **4.2. Synaptic plasticity**

morphology/dimension, especially the size of spine head, have been widely reported, and are believed to be associated with changes in the strength of synapses that reside on these altered

Due to the heterogeneity of spine size/morphology, the most convincing way to demonstrate that spine morphology is altered is to compare the same set of spines before and after a manipulation, such as synaptic plasticity-inducing stimuli in brain slices or learning *in vivo*. By using time-lapse two-photon imaging on the same set of spines, Yang et al. found new spines were formed after *in vivo* experience in the form of sensory or motor, and a fraction of these newly appeared spines persisted for months after the experience. More importantly, of the appearance of these new spines is specifically related to the *in vivo* experience or training [66]. In addition, Hayashi-Takagi et al. showed that motor learning on rotarod led to an enhanced *Arc* signaling, together with an expansion of a subset of spines in the motor cortex. By expressing a photoactivatable GTPase Rac1 in spines, they further showed that prolonged photo-activation of Rac1 resulted in reversal of spine expansion and loss of motor memory. This is a striking demonstration that potentiated synapses and enlarged spines are likely the underlying biological substrates of stored memory and formed memory can be erased by

Many studies have examined spine modifications with the induction of long-term potentiation (LTP) and long-term depression (LTD), since this procedure allows the examination of the same set spines associated with fast, large and long-lasting changes in synaptic strength. These two forms of synaptic plasticity refer to the increase and decrease in synaptic strength respectively, and are generally regarded as the cellular basis of synaptic modifications underlying developmental remodeling of neuronal connections, and learning and memory function in the adult brain [68]. When studied in brain slices (acute or organotypic culture), LTP or LTD is induced by stimulation of the presynaptic inputs with distinct patterns. In some studies, changes in the synaptic strength were also monitored, and thus changes in synaptic function and spine morphology can be related to each other in the same set of spines/synapse, or even a single synapse/spine [34, 69–71]. In general, spines exhibit the capacity of bi-directional changes in that spine enlargement is observed with LTP while spine shrinkage with LTD [72–74] (**Figure 2**). Either uncaging of caged glutamate onto a single spine [75] or electrical stimulation of a population of synapses [70, 71] had confirmed the above observations. Uncaging of glutamate directly enhances postsynaptic AMPAR function, and since it bypasses presynaptic release, and thus has provided the unambiguous evidence that postsynaptic changes can underlie the expression of LTP [69, 75]. These observations further indicate that morphological and functional changes are likely driven

In general, there is a good correlation between the strength of a given synapse (measured by electrophysiological responses) and the size of spine. Electrophysiological responses are further determined by the number/density of AMPA receptors at a given synapse. Takumi et al. found a linear relationship between AMPAR density and the diameter of PSD [76]. Matsuzaki et al.

spines. In this chapter, we will focus on alterations of spine dimension.

**4.1. Changes in spine morphology**

152 Neuroplasticity - Insights of Neural Reorganization

reversing these changes [67].

by the same stimuli or process (see below).

Synapses can change their strength by the activity patterns that they receive, and this modification allows synaptic strength to be adjusted to better suit the need for adaptation. Originally put forwarded by Donald Hebb and later adopted as "fire together, wire together" model, the current model of synaptic modification states that neurons sharing spike activity have increased connections between these two partners [78]. After the discovery of LTP in 1973, this activity-driven increase in synaptic strength has been extensively studied, both in acute brain slices and *in vivo* [68]; and has been used widely as a tool to induce synaptic modification in order to study the associated processes. Various molecules have been identified to be required for the induction and expression of LTP [68]. In general, Ca2+ entry or elevated intracellular Ca2+ concentration is required to convert electrical activity into intracellular signaling that determines the direction of synaptic changes. Usually a large but transient elevation in Ca2+ concentration induces LTP while a small but much longer elevation in Ca2+ concentration results in LTD [79]. This Ca2+ entry can be through opening NMDA receptors, voltagegated Ca2+ channels or metabotropic glutamate receptors [68]. Increase in postsynaptic kinase activity is usually required for LTP while phosphatase activity required for LTD. After a long debate, it is now generally agreed that synaptic modifications are expressed in the postsynaptic neurons, except in a few specific cases (such as mossy fiber LTP in the hippocampal CA3 region) [80, 81]. Postsynaptic changes involve the translocation of AMPARs and their phosphorylation state, in that LTP is associated with translocation of AMPARs to synapse and/ or increased phosphorylation of AMPARs while LTD with endocytosis of AMPARs and/or dephosphorylation of AMPARs [82–85].

#### **4.3. Relationship between spine and synapse plasticity**

Since both increase in the synaptic strength and enlargement of dendritic spine occur with LTP, an obvious question is whether changes in synaptic physiology/function are casually related to changes in spine morphology/structure. More specifically, are these two processes driven by the same initial process? Does the occurrence or persistence of one process require the occurrence/presence of the other? It is now well established that influx of Ca2+ through synaptic NMDARs during LTP induction drives AMPAR phosphorylation and/or insertion [68], and polymerization of action filaments inside spines which drives enlargement of spine heads [74] (**Figure 3**). Thus, the initial changes in function and structure are driven by the same signaling process. This initial increase in synaptic response and spine volume occurs very rapidly (less than 1 min) [71].

Dendritic spine heads accumulate F-actin during the rapid expansion phase of synaptic modification. Potentiation of single synapse/spine with uncaging of glutamate led to a significant expansion of the spine head and a shortening and widening of the spine neck [86, 87]. Spine expansion takes place rapidly after LTP, as fast as it can be measured (~ 20 sec after LTP induction) [71]. F-actin concentration inside the spine head rises, together with the entry of actinsevering, actin-depolymerizing/−polymerizing, actin-capping proteins, while actin-stabilizing proteins leave the spines [6, 39, 86, 88–90]. Actin-depolymerizing agent, cofilin, is highly elevated in spines during this initial process [89]. Interestingly, unlike the expression of LTP, this initial spine expansion did not require postsynaptic exocytosis or PKA signaling [71], suggesting the involvement of different signaling pathways in spine enlargement than that supports LTP. After this initial rapid expansion, the next phase of events lasts up to 1 h, with spine head volume decreased from the initial increase, but still larger than the pre-LTP baseline. In addition, the total actin concentration in the spine may drop to the baseline level [89].

and Linden demonstrated that LTD expression and spine changes in cerebellar Purkinje cells could be induced independently of each other, and induction of one did not affect the other [92]. Wang et al. reported that trafficking of AMPARs to and away from PSDs was activityindependent and not associated with alterations in spine size. The significance of this finding

**Figure 3.** Time-dependent reversal of synaptic and spine modification. (A) Spine shrinkage induced by low frequency stimulation (LFS) can be readily reversed by subsequent high frequency stimulation (HFS). (B) Spine enlargement induced by HFS is also readily reversed by subsequent LFS. (C) Spine enlargement induced by TBP can be reversed by LFS only if LFS is given within a time window of about 15 min after TBP. (D) this critical reversal window also holds for reversing TBP-induced LTP. (E) LFS given outside this reversal window does not affect spine size. (F) LFS given outside

Dendritic Spine Modifications in Brain Physiology http://dx.doi.org/10.5772/intechopen.73326 155

One interesting and important feature of synaptic modification is its reversibility. This reversibility is defined by reversal of synaptic modification after its induction [34, 93]. There are a few aspects to this reversal: (1) reversal applies to both LTP and LTD, and spine enlargement and spine shrinkage [34, 71] (**Figure 4**). More specifically, low frequency stimulation reverses LTP and spine enlargement, while high frequency stimulation reverses LTD

requires further investigation [70].

the reversal window does not reverse LTP either. Taken from [34, 71].

LTD is associated with the shrinkage of spines and removal of synaptic AMPARs via internalization [34, 36, 39]. During the induction of LTD with low frequency synaptic stimulation, Ca2+ influx through the activated NMDARs is required for both LTD and spine shrinkage [34, 70]. Ca2+ entry through synaptic NMDARs leads to the activation of calcineurin which is also required for both LTD and spine shrinkage, while activation of protein phosphatase 2A is required for LTD expression but not spine shrinkage, while elevated cofilin activity is required for spine shrinkage but not LTD [34, 39, 70, 91]. Consistent with the above conclusion, Sdrulla

Ca2+ concentration induces LTP while a small but much longer elevation in Ca2+ concentration results in LTD [79]. This Ca2+ entry can be through opening NMDA receptors, voltagegated Ca2+ channels or metabotropic glutamate receptors [68]. Increase in postsynaptic kinase activity is usually required for LTP while phosphatase activity required for LTD. After a long debate, it is now generally agreed that synaptic modifications are expressed in the postsynaptic neurons, except in a few specific cases (such as mossy fiber LTP in the hippocampal CA3 region) [80, 81]. Postsynaptic changes involve the translocation of AMPARs and their phosphorylation state, in that LTP is associated with translocation of AMPARs to synapse and/ or increased phosphorylation of AMPARs while LTD with endocytosis of AMPARs and/or de-

Since both increase in the synaptic strength and enlargement of dendritic spine occur with LTP, an obvious question is whether changes in synaptic physiology/function are casually related to changes in spine morphology/structure. More specifically, are these two processes driven by the same initial process? Does the occurrence or persistence of one process require the occurrence/presence of the other? It is now well established that influx of Ca2+ through synaptic NMDARs during LTP induction drives AMPAR phosphorylation and/or insertion [68], and polymerization of action filaments inside spines which drives enlargement of spine heads [74] (**Figure 3**). Thus, the initial changes in function and structure are driven by the same signaling process. This initial increase in synaptic response and spine volume occurs

Dendritic spine heads accumulate F-actin during the rapid expansion phase of synaptic modification. Potentiation of single synapse/spine with uncaging of glutamate led to a significant expansion of the spine head and a shortening and widening of the spine neck [86, 87]. Spine expansion takes place rapidly after LTP, as fast as it can be measured (~ 20 sec after LTP induction) [71]. F-actin concentration inside the spine head rises, together with the entry of actinsevering, actin-depolymerizing/−polymerizing, actin-capping proteins, while actin-stabilizing proteins leave the spines [6, 39, 86, 88–90]. Actin-depolymerizing agent, cofilin, is highly elevated in spines during this initial process [89]. Interestingly, unlike the expression of LTP, this initial spine expansion did not require postsynaptic exocytosis or PKA signaling [71], suggesting the involvement of different signaling pathways in spine enlargement than that supports LTP. After this initial rapid expansion, the next phase of events lasts up to 1 h, with spine head volume decreased from the initial increase, but still larger than the pre-LTP baseline. In addi-

tion, the total actin concentration in the spine may drop to the baseline level [89].

LTD is associated with the shrinkage of spines and removal of synaptic AMPARs via internalization [34, 36, 39]. During the induction of LTD with low frequency synaptic stimulation, Ca2+ influx through the activated NMDARs is required for both LTD and spine shrinkage [34, 70]. Ca2+ entry through synaptic NMDARs leads to the activation of calcineurin which is also required for both LTD and spine shrinkage, while activation of protein phosphatase 2A is required for LTD expression but not spine shrinkage, while elevated cofilin activity is required for spine shrinkage but not LTD [34, 39, 70, 91]. Consistent with the above conclusion, Sdrulla

phosphorylation of AMPARs [82–85].

154 Neuroplasticity - Insights of Neural Reorganization

very rapidly (less than 1 min) [71].

**4.3. Relationship between spine and synapse plasticity**

**Figure 3.** Time-dependent reversal of synaptic and spine modification. (A) Spine shrinkage induced by low frequency stimulation (LFS) can be readily reversed by subsequent high frequency stimulation (HFS). (B) Spine enlargement induced by HFS is also readily reversed by subsequent LFS. (C) Spine enlargement induced by TBP can be reversed by LFS only if LFS is given within a time window of about 15 min after TBP. (D) this critical reversal window also holds for reversing TBP-induced LTP. (E) LFS given outside this reversal window does not affect spine size. (F) LFS given outside the reversal window does not reverse LTP either. Taken from [34, 71].

and Linden demonstrated that LTD expression and spine changes in cerebellar Purkinje cells could be induced independently of each other, and induction of one did not affect the other [92]. Wang et al. reported that trafficking of AMPARs to and away from PSDs was activityindependent and not associated with alterations in spine size. The significance of this finding requires further investigation [70].

One interesting and important feature of synaptic modification is its reversibility. This reversibility is defined by reversal of synaptic modification after its induction [34, 93]. There are a few aspects to this reversal: (1) reversal applies to both LTP and LTD, and spine enlargement and spine shrinkage [34, 71] (**Figure 4**). More specifically, low frequency stimulation reverses LTP and spine enlargement, while high frequency stimulation reverses LTD and spine shrinkage. (2) There is a critical time window only during which reversal can occur [34, 71, 93, 94] (**Figure 4**). In hippocampal slices, the window for LTP reversal is about 15–30 min [71]. (3) The typical stimuli that can induce reversal are not capable altering basal synaptic strength or spine dimension [71, 93, 95, 96].

then move laterally into spines/PSDs. Thus, with LTP induction, two rapid processes (within 30 sec) occurs independent of each other: spine expansion which requires actin polymerization and activation of NMDARs but not postsynaptic PKA activation, and delivery of AMPARs to the perisynaptic regions which requires activation of NMDARs and postsynaptic PKA signaling but not actin polymerization. The next 15 min or so (reversal time window) determines whether LTP and spine expansion can be stabilized into a long-term change. During this period, translocation of the newly delivered perisynaptic AMPARs stabilized spine expansion, while removal of these receptors led to collapse of enlarged spines [71]. On the other hand, reversal of spine enlargement also removed these perisynaptic AMPARs. Hence, there is a mutual interaction between perisynaptic AMPARs and spine enlargement in that the presence of one is required to sustain the other (**Figure 3**). Yang et al. found that postsynaptic PKC activity is required for the translocation of perisynaptic AMPARs to synapse, and in the absence of PKC activity, these AMPARs remain perisynaptic. Importantly, as long as the perisynaptic AMPARs are present, both LTP and spine expansion exist in a labile state in that they can either be reverted to the baseline state (no plasticity), or they can enter a stabilized state of persistent increase in synaptic strength and spine size (persistent plasticity). Low frequency synaptic stimulation given within a 15 min "grace period" post-LTP induction reversed spine expansion and removed AMPARs from the perisynaptic regions and hence blocked the conversion of short-term plasticity to a long-

Dendritic Spine Modifications in Brain Physiology http://dx.doi.org/10.5772/intechopen.73326 157

**Figure 5.** Two-step model for coordinated expression of synaptic potentiation and spine enlargement. TBP triggers two initial processes, spine enlargement and perisynaptic delivery of AMPARs. These two processes occur simultaneously and mostly independent of each other. In the absence of any disturbance, perisynaptic AMPARs translocate into synapse, which stabilizes both AMPARs and spine enlargement, and this leads to persistent potentiation of synaptic responses and spine enlargement. On the other hand, when low frequency synaptic stimulation (LFS) is given within this "grace period" when both processes are in a liable state, it readily removes perisynaptic AMPARs. As a result, synaptic potentiation is aborted and spine enlargement collapses, and no LTP and spine enlargement. This model shows the interaction between functional and structural aspects of synapse modification is critical to the persistency and long-

term one (**Figure 5**) [71, 82].

lasting occurrence of synaptic modification.

Although it is generally believed that expression of LTP requires the addition of synaptic AMPARs, some evidences suggest that these newly added AMPARs are not delivered directly into the PSDs inside spines, but rather they are either delivered to regions outside synapses (i.e., the perisynaptic regions; [82] or onto dendritic shaft [83]. These AMPARs

**Figure 4.** Actin polymerization is required for spine expansion and maintenance of perisynaptic AMPA receptors. (A) Actin depolymerizing agent Latrunculin A (LatA) added before and during TBP abolished spine expansion. (B) Bath perfusion of LatA before and during TBP impaired enhancement of synaptic response (EPSPs). (C) Bath perfusion of LatA 2 min after TBP did not disrupt the delivery of AMPARs to perisynaptic site, as revealed by the increased in responses to application of glutamate transporter blocker TBOA (open symbols). (D) Bath perfusion of LatA 2 min after TBP did not affect spine enlargement, indicating that persistent spine enlargement does not require persistent actin polymerization. (E) Bath perfusion of LatA 2 min after TBP prevented the occurrence of gradual increase in EPSP enhancement which requires the synaptic addition of new AMPARs. (F) Addition of LatA 2 min after TBP removed the newly delivered perisynaptic AMPARs as revealed by the absence of increase in response to TBOA. Taken from [83].

then move laterally into spines/PSDs. Thus, with LTP induction, two rapid processes (within 30 sec) occurs independent of each other: spine expansion which requires actin polymerization and activation of NMDARs but not postsynaptic PKA activation, and delivery of AMPARs to the perisynaptic regions which requires activation of NMDARs and postsynaptic PKA signaling but not actin polymerization. The next 15 min or so (reversal time window) determines whether LTP and spine expansion can be stabilized into a long-term change. During this period, translocation of the newly delivered perisynaptic AMPARs stabilized spine expansion, while removal of these receptors led to collapse of enlarged spines [71]. On the other hand, reversal of spine enlargement also removed these perisynaptic AMPARs. Hence, there is a mutual interaction between perisynaptic AMPARs and spine enlargement in that the presence of one is required to sustain the other (**Figure 3**). Yang et al. found that postsynaptic PKC activity is required for the translocation of perisynaptic AMPARs to synapse, and in the absence of PKC activity, these AMPARs remain perisynaptic. Importantly, as long as the perisynaptic AMPARs are present, both LTP and spine expansion exist in a labile state in that they can either be reverted to the baseline state (no plasticity), or they can enter a stabilized state of persistent increase in synaptic strength and spine size (persistent plasticity). Low frequency synaptic stimulation given within a 15 min "grace period" post-LTP induction reversed spine expansion and removed AMPARs from the perisynaptic regions and hence blocked the conversion of short-term plasticity to a longterm one (**Figure 5**) [71, 82].

and spine shrinkage. (2) There is a critical time window only during which reversal can occur [34, 71, 93, 94] (**Figure 4**). In hippocampal slices, the window for LTP reversal is about 15–30 min [71]. (3) The typical stimuli that can induce reversal are not capable altering basal

Although it is generally believed that expression of LTP requires the addition of synaptic AMPARs, some evidences suggest that these newly added AMPARs are not delivered directly into the PSDs inside spines, but rather they are either delivered to regions outside synapses (i.e., the perisynaptic regions; [82] or onto dendritic shaft [83]. These AMPARs

**Figure 4.** Actin polymerization is required for spine expansion and maintenance of perisynaptic AMPA receptors. (A) Actin depolymerizing agent Latrunculin A (LatA) added before and during TBP abolished spine expansion. (B) Bath perfusion of LatA before and during TBP impaired enhancement of synaptic response (EPSPs). (C) Bath perfusion of LatA 2 min after TBP did not disrupt the delivery of AMPARs to perisynaptic site, as revealed by the increased in responses to application of glutamate transporter blocker TBOA (open symbols). (D) Bath perfusion of LatA 2 min after TBP did not affect spine enlargement, indicating that persistent spine enlargement does not require persistent actin polymerization. (E) Bath perfusion of LatA 2 min after TBP prevented the occurrence of gradual increase in EPSP enhancement which requires the synaptic addition of new AMPARs. (F) Addition of LatA 2 min after TBP removed the newly delivered perisynaptic AMPARs as revealed by the absence of increase in response to TBOA. Taken from [83].

synaptic strength or spine dimension [71, 93, 95, 96].

156 Neuroplasticity - Insights of Neural Reorganization

**Figure 5.** Two-step model for coordinated expression of synaptic potentiation and spine enlargement. TBP triggers two initial processes, spine enlargement and perisynaptic delivery of AMPARs. These two processes occur simultaneously and mostly independent of each other. In the absence of any disturbance, perisynaptic AMPARs translocate into synapse, which stabilizes both AMPARs and spine enlargement, and this leads to persistent potentiation of synaptic responses and spine enlargement. On the other hand, when low frequency synaptic stimulation (LFS) is given within this "grace period" when both processes are in a liable state, it readily removes perisynaptic AMPARs. As a result, synaptic potentiation is aborted and spine enlargement collapses, and no LTP and spine enlargement. This model shows the interaction between functional and structural aspects of synapse modification is critical to the persistency and longlasting occurrence of synaptic modification.

The above results are consist with a model (**Figure 5**) in which (1) the expression of functional plasticity (LTP) and structural plasticity (spine enlargement) are initially two independent processes only share the activation of NMDARs; (2) these two processes then enter an interactive state that the continuous presence of one is required for the persistence of the other; (3) the above state is liable in that interference (such as low frequency stimulation) can revert both changes back to the baseline; (4) once both processes are stabilized, synapse modification has entered a state resistant to reversal. We like to note that during the "grace period" coordinated changes in synaptic plasticity (function and structure) are cross-checked to ensure that they do occur together, and in the situation only one such process occurs (perhaps can be viewed as a mistake), the other process will be aborted albeit in process. This double-proof mechanism is essential to ensure that only appropriate changes are allowed to be sustained, and may thus be especially important in face of the highly dynamic nature of synaptic modifications, such as those occurring during early neural development [94].

and structure. Extensive progress has been made on our understanding of the structure and function of spine which vastly has advanced our understanding of neuronal and synaptic communication and plasticity. In addition, changes in spine density and dimension may serve as a marker of pathological processes and hence have potential therapeutic/diagnostic

Dendritic Spine Modifications in Brain Physiology http://dx.doi.org/10.5772/intechopen.73326 159

This work was supported by grants from Peking University Shenzhen Graduate School and grants from Shenzhen government (ZDSYS201504301539161 and KQTD2015032709315529).

School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate

[1] Tackenberg C, Ghori A, Brandt R. Thin, stubby or mushroom: Spine pathology in

[2] Peters A, Kaiserman-Abramof IR. The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines. The American Journal of Anatomy. 1970;**127**

[3] Harris KM, Jensen FE, Tsao B. Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: Implications for the maturation of synaptic physiology and long-term potentiation. The Journal of

[4] Cohen RS, Chung SK, Pfaff DW. Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe. Cellular and Molecular

Alzheimer's disease. Current Alzheimer Research. 2009;**6**(3):261-268

values.

**Acknowledgements**

**Competing interests**

**Author details**

Jun Ju and Qiang Zhou\*

School, Shenzhen, China

(4):321-355

Neuroscience. 1992;**12**(7):2685-2705

Neurobiology. 1985;**5**(3):271-284

**References**

The authors declare that they have no competing interests.

\*Address all correspondence to<EMAIL_ADDRESS>
Sustained reduction in synaptic strength may eventually lead to the loss of synaptic connections, and this loss is manifested as a reduction in spine density. Spine loss appears to be a protracted process and hence it is difficult to study. Even if monitoring changes in the same set of synapses/spines, it is usually more difficult to exclude the possibility that the reduced synaptic function and spine number is caused by deterioration of the health of the preparation, or by some other unknown or uncontrolled processes that occur randomly during the long period (>hours) between LTD induction and spine loss. Nonetheless, a few studies have examined this process. By using organotypical slices and monitoring both presynaptic boutons and spines, Becker et al. showed that LTD induction increased the turnover rate of presynaptic boutons and resulted in decreased synaptic contacts between the pre- and post-synaptic sites. Although presynaptic boutons and postsynaptic spines disappear at much greater rate after LTD, there is no particular pattern to follow, since disappearance of either presynaptic boutons or spines could occur prior to the other [97]. Therefore, the above observations suggest that the mismatch between presynaptic and postsynaptic sites is more likely a key factor in the elimination of synapse, while the exact sequence might not play much role.


**Author details**

**Table 5**. From the discussion in **Table 5**, we can notice that F-N enhances the

*Thermal characteristics of PVOH and SBR-based composites reinforced with F-N layers of* Opuntia

30–120 Evaporation of residual moisture

*Invasive Species - Introduction Pathways, Economic Impact, and Possible Management Options*

120–380 Degradation of hemicelluloses in lignocellulosic fibers and the elimination

380–520 Decomposition of chain segments of PVOH molecules and lignocellulosic

30–300 Evaporation of absorbed moisture and residual water in SBR latex 300–570 Volatilization of SBR (styrene derivative) and lignocellulosic fibers

of hydroxide groups from PVOH in the form of water molecules

compounds (cellulose and lignin)

(cellulose and lignin)

but also as a solution to alleviating the uncertainty of the petroleum supply.

*Opuntia* (Cactaceae) is an alternative and sustainable plant for fiber production for semiarid and arid regions. It is in large quantities and considered as a useful source in most countries without forests. Indeed, natural fibers derived from Tunisian *Opuntia* (Cactaceae) plants prove their greatest potentials for use in paper manufacturing by applying two pulping processes which affects the pulp properties and paper characteristics and composite applications (as reinforcement in thermoplastic polymers) because of their excellent characteristics such as low density, high specific stiffness, good mechanical properties, biodegradability, eco-friendliness, and good thermal resistance. *Opuntia* fibers can be a good reinforcement candidate for high-performance biodegradable polymer composites. This study has paved the way of proposing this xerophyte genus as a suitable new resource of non-woody fibers at different fields and as an environmentally friendly alternative to green

The authors would like to express their deep gratitude to Mohamed Naceur Belgacem, Professor and Director of the Grenoble INP-Pagora and Agefpi, for his

The biodegradability potential (BP) (soil-burial test) of the matrix and produced composites were obtained by the mass retention technique, following the procedure outlined in literature [60, 61], and the results are given in the previous work [57]. The evolution of BP vs. time for the different materials after soil burial decreases gradually and tends to 93 and 86.6%, respectively, for PVOH/F-N and SBR/F-N [57]. These values are higher than those reported for PVOH/palm kernel shell powder bio-composites (20%) [62] and PVOH/corn starch films (40%) [63]. It should be mentioned that cellulosic fibers from *Opuntia* (Cactaceae) plant, PVOH, and SBR are biodegradable in nature, in which they may serve as a source of energy and carbon for specific microorganisms [64, 65]. From this study, eco-friendly *Opuntia*-derived fiber-reinforced polymer (thermoplastic) composites would be the materials for near future not only as a solution to the growing environmental threat

thermal properties of the used thermoplastic polymers.

*(Cactaceae) trunk obtained from TGA measurements.*

**Composites T (°C) Discussion**

**4. Conclusions**

PVOH/F-N [57]

SBR/F-N [57]

**Table 5.**

product.

**152**

**Acknowledgements**

Faten Mannai1 , Ramzi Khiari4,5,6 and Younes Moussaoui2,3\*

1 Material Environment and Energy Laboratory (UR14ES26), Faculty of Sciences of Gafsa, University of Gafsa, Tunisia

2 Organic Chemistry Laboratory (LR17ES08), Faculty of Sciences of Sfax, University of Sfax, Tunisia

3 Faculty of Sciences of Gafsa, University of Gafsa, Tunisia

4 Faculty of Sciences, UR13 ES 63, Research Unity of Applied Chemistry and Environment, University of Monastir, Monastir, Tunisia

5 Department of Textile, Higher Institute of Technological Studies of Ksar Hellal, Tunisia

6 University of Grenoble Alpes CNRS, Grenoble, France

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Flood Risk Prediction and Management

**3**

**Chapter 1**

**Abstract**

**1. Introduction**

Management

*Guangwei Huang*

New Frontiers in Flood Risk

understanding of, and new approaches to, flood risk management.

and (3) exposure assessment. Mathematically, it can be expressed:

or community such as water supply system.

**Keywords:** vulnerability, framework, flooding time, flood duration, Ec0-DRR

Flood disaster management includes flood risk assessment, risk mitigation, preparedness, and emergency response and rehabilitation efforts. It can also be classified into before, during, and after event activities. A flood risk assessment is an assessment of the risk of flooding from all flooding mechanisms and consists of three components: (1) hazard identification, (2) vulnerability analysis,

Risk = Hazard × Vulnerability × Exposure (1)

According to UN-ISDR [1], hazard can be defined as a dangerous phenomenon, substance, human activity, or condition that may cause loss of life, injury or other health impacts, property damage, loss of livelihoods and services, social and economic disruption, or environmental damage. It can be quantified by a probability of occurrence within a specified period of time and within a given area and given intensity. The term exposure is used to indicate elements subject to potential damage due to a hazard. Elements here may be referred to population, houses, facilities, or physical and life infrastructure essential to the functioning of a society

There are many aspects of vulnerability, related to physical, social, economic, and environmental conditions (see, for example, Birkmann [2]). Therefore, vulnerability can be defined in a number of different ways from as simple a notion as the degree of damage to an object exposed to a given hazard, to a more sophisticated one such as the characteristics and circumstances of a community, system, or asset

Flood risk management has been studied extensively and intensively in many academic fields from civil engineering, sociology, economics, culture, and even psychology. However, the fact that flooding accounts for a greater number of damaging events than any other type of natural events worldwide on an yearly scale proves that our understanding of flooding is still insufficient, flawed, and fragmented. This chapter intends to shed new light on a number of issues that deserve more comprehensive study in order to advance flood risk management. As a result, a new two-layer framework of vulnerability is proposed, which can lead to a better


**2.1.6 Cryoprotection**

Liquid medium (MS) supplemented with 0.2, 0.4 M, 0.07 M, 0.14 M, sucrose, 2.0 M, 0.64 M, 1.28 M, 3.23 M glycerol, 2.42 M ethylene glycol, 0.017 M raffinose, 0.64 M, 1.28 M and 1.92 M DMSO, in varied combinations, plant vitrification solution II (PVS2), (Sakai *et al*., 1990) and half strength PVS2 were used to cryoprotect nodal cutting explants for 5, to 40 minutes. Explants used were obtained from shoots grown on 0.1 M mannitol for 2-3 weeks. Excised explants were cultured on 0.3 M mannitol for 72 hours, and cryoprotected in cryovials, using 1 ml cryoprotectant solution (the 1 ml cryoprotectant was decanted and replace with 0.5 ml during cryoprotection). To enhance explant cryoprotection, dehydration over activated silica gel for 60 minutes either before or after cryoprotection was also investigated. Following cryoprotection, the cryoprotectant solution was decanted and explants were washed three times with rehydration solution (described above). However, explants subjected to cooling, (LN or freezing to -70°C) were immediately on retrieval, rewarmed in water bath at 40oC for two - three minutes. Following rewarming, tissues were then allowed to stay for 30 min in the rehydration solution before blotting dry, and cultured on growth medium. Incubation was in continuous dark conditions till signs of growth and development were observed. Developing cultures were transferred to 16 h photoperiod.


*2.7. Color Response and the Leaching Rate under Different pH Buffers of Films*

The film samples were immersed in plastic Petri dishes containing 15 mL of buffer solutions (2–10). During the different time intervals, the exudation rate was determined by calculating the concentration of the anthocyanin leaching solution, and the color response was captured using a camera at the beginning time.


2.3.2. Joint Position Sense Test

The SYSTEM 3 PRO dynamometer (Biodex Medical Systems, Shirley, NY, USA) was used to conduct a joint position sense test measuring active and passive repositioning. The dynamometer exhibited medium-to-high reliability in an assessment of joint kinesthesia ability (r = 0.6–0.8) [20]. The participants laid in a supine position while blindfolded, and the involved ankle was placed on the ankle inversion-eversion footplate of the dynamometer with a plantar flexion of 15◦. Three reference angles were established for ankle inversion, neutral ankle position, and ankle eversion (15◦, 0◦, and 10◦, respectively), and the participants were asked to actively and passively reproduce the angle. The absolute value of the joint angle error represents the actual difference between the reference angle and the matching angle [21].

In active repositioning, the involved ankle was first placed in a neutral position and then moved to the inversion or eversion reference angle for 10 s. The participants were asked to actively reproduce the angle three times; the corresponding angles produced by the participants were then recorded. In passive repositioning, the involved ankle was also first placed in a neutral position and then moved to the inversion or eversion reference angle for 10 s. The involved ankle was then passively inverted and everted through a full range of motion by using 5◦/s of angular velocity and stopped at the original reference angle by using a hand-held switch. Three passive repositioning trials were completed to reproduce the reference angle, and the corresponding angles were recorded. The average absolute values of the joint angle errors were subsequently analyzed.

#### 2.3.3. Isokinetic Strength Test

The SYSTEM 3 PRO dynamometer was also used to conduct the isokinetic strength test. The dynamometer exhibited high reliability (ICC coefficients = 0.87–0.96) and was effective in measuring the isokinetic strength of ankle joints [22]. Ankle invertor and evertor muscle strength was measured in terms of concentric contraction (CON) and eccentric contraction (ECC) at velocities of 30◦/s and 120◦/s, respectively [23]. Prior to testing, the participants warmed up for 10 min using general range-of-motion exercises for ankle joints. The participants sat in a chair with the backrest at a seatback tilt of 70◦, and their trunks and pelvises were fixed with straps. The involved leg was fixed with a strap, and the involved foot was secured to ankle attachments with two straps. The tested ankle was positioned with 20◦ of plantar flexion, and the rotational axis of the dynamometer was leveled at the subtalar joint. Three repetitions were performed at velocities of 30◦/s and 120◦/s with 1-min rest intervals between each repetition [24]. The CON and ECC of the ankle inversion and eversion were calculated as peak torque normalized according to body weight. The respective ratios of inversion and eversion for ECC or CON at 30◦/s and 120◦/s were also calculated.

### *2.4. Statistical Analysis*

Statistical analysis was performed using SPSS version 25.0 (SPSS, Chicago, IL, USA). The Shapiro–Wilk test was used to verify the normality of the data to ensure the normal distribution of all assessed variables (*p* > 0.05). Descriptive statistics were used, and all data are presented as the mean ± standard deviation. An analysis of variance (ANOVA) and chi-squared test were used for continuous and categorical variables, respectively, to compare the differences within groups. The results of the SEBT, joint position sense test, and isokinetic strength test were analyzed using a two-way repeated-measures ANOVA (three groups × two times) followed by a Bonferroni post hoc test. Effect size (d) was classified according to the scale of Cohen [25] into very small (<0.2), small (0.2–0.5), medium (0.5–0.8), and large (>0.8), and the effects of Groups A and B were determined to respectively compare Group C. Multivariable linear regression analysis was used to evaluate the association of main outcome measurements between the two training groups and the non-training group. Multivariable modelling was performed with R2 and β coefficients and specified as

the change values of assessment variables for the prediction. The α level for all statistical analyses was set at 0.05.

#### **3. Results**

Sixty-three female athletes (50 basketball players and 13 volleyball players) with dominant-leg CAI participated following the inclusion criteria in our study. The participants were randomly divided using a random number generator into Groups A, B, and C (all *n* = 21, Figure 1). After the study process, no participants dropped out, and no participants reported adverse reactions. All of the participants completed the study. The demographics are presented in Table 1, and no significant differences were observed within the three groups (all *p* > 0.05).

**Table 1.** Demographics of the participants.

Left, Lt; Right. Rt; Cumberland Ankle Instability Tool, CAIT.

The ANOVA outcomes are summarized in Table 2. In the results of multivariable linear regression (Table 3), the R2 values were 0.80 and 0.93 in Group A and B, respectively. Significant relationships between the change in values in active repositioning and 30◦/s of ECC ankle inversion were noted (*p* < 0.05). Results of the SEBT among the three groups are presented in Table 4. To calculate the composite score of SEBT, the main effects of group (F(2, 60) = 5.30, *p* = 0.03), time (F(2, 60) = 67.78, *p* = 0.001), and time × group (F(2, 60) = 17.84, *p* = 0.001) were observed. Post hoc tests indicated no significant differences in the SEBT within the groups before assessment in terms of composite score and individual directions (*p* > 0.05). Between Groups A and C, the anteromedial (*p* = 0.01, effect size: d = 1.25, 95% CI = 0.59–1.91), posterolateral (*p* = 0.03, effect size: d = 1.05, 95% CI = 0.41–1.70), and lateral (*p* = 0.03, effect size: d = 1.09, 95% CI = 0.44–1.74) directions in the SEBT were significantly different. However, no significant difference was observed in composite scores on the SEBT within the two groups (*p* > 0.05). Moreover, the results indicated that the SEBT composite score and individual directions were higher in Group B than in Group C (all *p* < 0.05). Within the two groups, a small effect size in composite score on the SEBT was observed (d = 2.34, 95% CI = 1.55–3.12), and very small to small effect sizes were observed for all individual directions (anterior, d = 1.70, 95% CI = 1.00–2.41; anterolateral, d = 1.53, 95% CI = 0.84–2.22; anteromedial, d = 1.88, 95% CI = 1.15–2.60; posteromedial, d = 1.21, 95% CI = 0.55–1.86; posterior, d = 1.74, 95% CI = 1.03–2.45; posterolateral, d = 2.13, 95% CI = 1.38–2.89; medial, d = 1.32, 95% CI = 0.65–1.99; and lateral, d = 2.18, 95% CI = 1.41–2.94).

**Table 2.** Outcome of ANOVA with the factors for the assessed variables.

COM: concentric contraction; ECC: eccentric contraction.

**Table 3.** The main outcome measurements in two training groups using linear regression analysis.

COM: concentric contraction; ECC: eccentric contraction.

**Table 4.** Results of star excursion balance test in three group.

\* *p* < 0.05, Group A vs. Group C, between-group using Bonferroni test; + *p* < 0.05, Group B vs. Group C, between-group using Bonferroni test.

The changes in active and passive repositioning data before and after assessment for the three groups are presented in Table 5. During active repositioning, for an ankle inversion of 15◦, the main effects of group (F(2, 60) = 3.35, *p* = 0.08), time (F(2, 60) = 12.37, *p* = 0.006), and time × group (F(2, 60) = 0.48, *p* = 0.63) were observed. For the neutral ankle position, the main effects of group (F(2, 60) = 6.59, *p* = 0.01), time (F(2, 60) = 12.26, *p* = 0.006), and time × group (F(2, 60) = 1.58, *p* = 0.25) were observed. For an ankle eversion of 10◦, the main effects of group (F(2, 60) = 0.51, *p* = 0.61), time (F(2, 60) = 8.27, *p* = 0.01), and time × group (F(2, 60) = 1.60, *p* = 0.25) were observed. During passive repositioning, for an ankle inversion of 15◦, neutral ankle position, and an ankle eversion of 10◦, no statistical significance was observed in the main effects of group, time, and time × group (*p* > 0.05).

**Table 5.** Results of joint position sense measurements in three group.

\* *p* < 0.05, Group A vs. Group C, between-group using Bonferroni test; + *p* < 0.05, Group B vs. Group C, between-group using Bonferroni test.

For active repositioning data, the within-group analysis of Group A revealed a significant decrease in neutral ankle position (*p* = 0.04) but no significant decreases for an ankle inversion of 15◦ (*p* = 0.16) and an ankle eversion of 10◦ (*p* = 0.13). In Group B, significant decreases for an ankle inversion of 15◦ (*p* = 0.01), neutral ankle position (*p* = 0.02), and an ankle eversion of 10◦ (*p* = 0.01) were observed. Post hoc tests indicated no significant differences within the three groups before assessment. Compared with Group C, Groups A and B exhibited significant decreases for an ankle inversion of 15◦, neutral ankle position, and an ankle eversion of 10◦ (*p* < 0.05). Very small to small effect sizes were observed for an ankle inversion of 15◦ (d = −0.97, 95% CI = −1.61 to −0.33), neutral ankle position (d = −2.18, 95% CI = −2.94 to −1.41), and an ankle eversion of 10◦ (d = −0.95, 95% CI = −1.59 to −0.31) after assessment between Groups A and C. Between Groups A and C, small to medium effect sizes were also observed for an ankle inversion of 15◦ (d = −0.90, 95% CI = −1.54 to −0.27), neutral ankle position (d = −2.20, 95% CI = −2.97 to −1.43), and an ankle eversion of 10◦ (d = −0.89, 95% CI = −1.52 to −0.25) after assessment.

The CON and ECC data before and after assessment for the three groups are presented in Table 6. For 30◦/s of CON ankle inversion, the main effects of group (F(2, 60) = 3.01, *p* = 0.10), time (F(2, 60) = 0.04, *p* = 0.82), and time × group (F(2, 60) = 4.49, *p* = 0.04) were observed. For 30◦/s of ECC ankle inversion, the main effects of group (F(2, 60) = 14.02, *p* = 0.002), time (F(2, 60) = 5.56, *p* = 0.04), and time × group (F(2, 60) = 0.71, *p* = 0.51) were observed. Post hoc tests indicated no significant differences within the three groups for 30◦/s of CON and ECC ankle inversion before assessment, but the number of ankle invertor muscle contractions in Group C was significantly lower than those in Groups A and B for 30◦/s of CON (*p* = 0.01 and *p* = 0.02, respectively) and 30◦/s of ECC (*p* = 0.01 and *p* = 0.001, respectively). Very small effect sizes were observed in 30◦/s of CON ankle inversion after assessment in Group A (d = 1.24, 95% CI = 0.58–1.90) and Group B (d = 1.13, 95% CI = 0.47–1.78) compared with Group C. Small effect sizes were also observed in 30◦/s of ECC ankle inversion after assessment in Group A (d = 1.15, 95% CI = 0.49–1.80) and Group B (d = 1.37, 95% CI = 0.69–2.04) compared with Group C. However, no significant main effects of group, time, and time × group (*p* > 0.05) were observed in 30◦/s of ECC and CON ankle eversion. In addition, for 120◦/s of CON and ECC ankle inversion or eversion, no statistical significance was observed in the main effects of group, time, and time × group (*p* > 0.05). Regarding the analysis of the ratios of inversion and eversion for ECC or CON at 30◦/s and 120◦/s, no isokinetic parameters were statistically significant after using two-way repeated-measures ANOVA (*p* > 0.05).

**Table 6.** Results of isokinetic strength test in three groups.

\* *p* < 0.05, Group A vs. Group C, between-group using Bonferroni test; + *p* < 0.05, Group B vs. Group C, between-group using Bonferroni test; concentric contraction, CON; eccentric contraction, ECC.


**4. Discussion 4. Discussion**  Advanced economies experienced intense demographic changes leveraging sequential

Advanced economies experienced intense demographic changes leveraging sequential urbanization and suburbanization waves [71–75]. In Europe, multifaceted migration trends, new household structures and a particularly heterogeneous natural balance over space shaped regional population trends [76–79]. In these regards, understanding how demographic processes, metropolitan cycles and economic downturns interact shaping regional competitiveness and location attractiveness, may contribute to a refined understanding of local development mechanisms [80–85]. When comparing population dynamics over sequential waves of urban expansion and rural shrinkage [86–88], Greece is exemplificative of traditional Mediterranean societies with a polarized economy along urban–rural gradients that reflect territorial divides between coastal and inland districts. Despite a persistent spatial configuration at the country scale, such divides had reduced during the last 80 years, fortifying—especially between 1940 and 1980—the role of few urban nodes (Athens-Piraeus and Thessaloniki) inserted in broader continental (or global) networks. This process consolidated in the subsequent four decades (1980–2019) reflecting the West–East gradient in Greece and the importance of (low-density) coastal districts attracting new population and economic activities (Figure 8). urbanization and suburbanization waves [71–75]. In Europe, multifaceted migration trends, new household structures and a particularly heterogeneous natural balance over space shaped regional population trends [76–79]. In these regards, understanding how demographic processes, metropolitan cycles and economic downturns interact shaping regional competitiveness and location attractiveness, may contribute to a refined understanding of local development mechanisms [80–85]. When comparing population dynamics over sequential waves of urban expansion and rural shrinkage [86–88], Greece is exemplificative of traditional Mediterranean societies with a polarized economy along urban–rural gradients that reflect territorial divides between coastal and inland districts. Despite a persistent spatial configuration at the country scale, such divides had reduced during the last 80 years, fortifying—especially between 1940 and 1980—the role of few urban nodes (Athens-Piraeus and Thessaloniki) inserted in broader continental (or global) networks. This process consolidated in the subsequent four decades (1980–2019) reflecting the West–East gradient in Greece and the importance of (low-density) coastal districts attracting new population and economic activities (Figure 8).

**Figure 8.** Population growth rate (%) in Greek municipalities by time interval (**a**) 1940–1980; (**b**) 1981–2019).

A spatially heterogeneous population growth has been observed in Greece, being related with a comprehensive set of background predictors. Regional population growth in Greece has reflected mechanisms of local development leveraging spatial disparities (e.g., [12]). As a matter of fact, the empirical results of our study outlined a strong difference in population dynamics between urban and rural areas, especially from 1940 to 1980 [26]. Accessibility, as well as economic specialization and advanced productive functions were assumed to be significant factors shaping demographic dynamics in such decades, since they have alimented a compact and radio-centric settlement growth in metropolitan regions and rural-to-urban mobility determining rural shrinkage [89]. The following dynamics (1981–2019) were less associated with central cities, suggesting a less important role for agglomeration and scale factors. After a continuous increase of resident population, a less intense gap between urban and rural areas was observed since the early 1990s.

Population redistribution over wider regions was evident in Greece since the late 1990s, reflecting spatially heterogeneous socioeconomic transformations increasingly decoupled from traditional geographic gradients (e.g., urban–rural, coastal-inland). Since the 1990s, population increased in rural districts and low-density touristic coastal areas, especially large and

medium-sized islands, as well as lowland, internal districts devoted to intensive cropland. More intense urban-to-rural internal movements were specifically observed in the last decade as a result of progressive counter-urbanization and shrinkage of central cities associated with worse socioeconomic conditions in metropolitan regions, mainly in Athens and, partly, Thessaloniki [13,32,90]. However, the overall impact of these processes on the Greek urban hierarchy was rather modest [21]. In such a context, the possible role of unregistered or partially registered (i) emigration abroad and (ii) illegal international immigration should be evaluated further.

As a result of economic processes and mixed socio-demographic contexts, spatial heterogeneity in population growth rates was persistently observed in both urban and rural districts. However, this process was clearly asymmetric over time, being more intense in the second time interval (1980–2019) corresponding with late suburbanization, counter-urbanization and early re-urbanization and less intense in the first time interval (1940–1980) corresponding with urbanization and early suburbanization in Greece. Tapia et al. [91] investigated population trends in Spain at different time windows between 1860 and 1991 with the aim at testing if population at the beginning of each time window affects population growth during the same time window. According to the authors' results (p. 81) "while such a relationship between these two variables hardly existed during the second half of the nineteenth century, this link increased significantly between 1910 and 1970, although this trend was abruptly interrupted by the Civil War and the autarkic period that followed [92,93]. The intensity of this relationship decreased in the 1970s, a process that continued during the 1980s ( . . . ); agglomeration economies were stronger in medium-sized districts, especially from 1960 onwards".

In Greece, a slightly different spatial model was observed, based on the specific socioeconomic context and the distinctive development path. Population decline in urban, industrial and intensive agricultural districts was more intense than in rural districts experiencing long depopulation, suggesting that central locations display a lower demographic attractiveness than peripheral locations with dynamic economic sectors, e.g., tourism [94–96]. In these regards, a mix of factors that includes industrial decline, informal economies and dependence on external funds, especially in agriculture, determined population shrinkage in Southern Europe [97–102], suggesting a lower demographic resilience of the locations (mostly rural) experiencing this kind of development path.

A better knowledge of new demographic scenarios opens up a key reflection on the intimate mechanisms of long-term change in population redistribution over wider regions [7,103–106] and may delineate opportunities for regional development policy. In these regards, newly emergent issues such as resurging internal migration, declining immigration flows from abroad and re-approaching the lowest-low fertility levels, suggest a thorough rethinking of sustainable urbanization [107–111]. Planning strategies stimulating a polycentric expansion of human settlements should incorporate policy measures improving local development in regions exposed to demographic and economic stagnation [29,112,113]. A comparative, long-term analysis of population distribution over space proved to be a necessary tool identifying the emergent socioeconomic dynamics and delineating the most appropriate policies to face with [114,115]. A refined investigation of the negative impact of recession on local socioeconomic structures is therefore meaningful to shed light on future population dynamics in a post-crisis Mediterranean Europe.

Taken together, results of this study, at least for the first time interval (1940–1980), suggest how "being closely intertwined with the policy debate, the concept of agglomeration economies and its relation with spatial economic performance has maintained a central role" in regional science [116]. However, the empirical results for the most recent time horizon (1981–2019) reflect more complex demographic and settlement processes over space. According with Berliant and Wang [117], "while basic questions such as 'does urbanization cause growth or does growth cause urbanization?' or 'does supporting urbanization imply neglecting rural areas?' are still valid and need a more comprehensive research and immediate policy response, our study definitely contributes to a better understanding of the role of urbanization in population distribution and economic growth, informing policies that tackle the formidable challenges it poses".