**3. Long-Term Potentiation (LTP) and neurogenesis**

It is well established that new neurons are produced continuously in the SVZ and the subgra‐ nular zone (SGZ) of the dentate gyrus (DG) during adulthood [11-13]. Given this fact, many researchers have focused their attention on discovering how to stimulate these resident precursors to enhance neurogenesis. Environmental enrichment and voluntary running [14] are now widely accepted ways to enhance neurogenesis.

Long-term potentiation (LTP) is induced by brief high-frequency tetanization of excitatory afferents and is observed as a long-lasting enhancement in the efficacy of synaptic transmission [15]. LTP is considered to be a cellular model of learning [16] and memory [17]. Moreover, LTP is speculated to be one possible mechanism of enhanced motor function recovery resulting from electrical stimulation to the cortex after stroke [18]. Based on the accumulated evidence of hippocampal neurogenesis, the relationship among hippocampus-related function, LTP, and neurogenesis has been examined [19-22]. For example, environmental enrichment and running enhanced hippocampus-related memory function as well as LTP. More importantly, electrophysiological reports have demonstrated that recently created neurons contribute to synaptic plasticity [23] and that LTP correlates with neural plasticity, compensating for ischemia-induced damage [24]. These findings support the hypothesis that enhancing neurogenesis may be useful in the restoration and repair of a damaged brain and emphasizes the importance of finding a more effective way to stimulate neurogenesis. As mentioned above, a number of reports are available that describe the relationship between neurogenesis and LTP but these mainly focus on neurogenesis-dependent changes in LTP [19, 21, 22]. Only a few reports have shown that LTP per se can enhance neurogenesis [25-27].

Neural activity was recently demonstrated to stimulate a large number of hippocampal latent precursors, including a self-renewing stem cell population that only becomes activated following depolarization [28]. Given a number of recent reports linking LTP and hippocampal neurogenesis, we examined whether LTP is able to enhance proliferation of the neural precursors and neurogenesis in the adult mouse dentate gyrus. In this study, we stimulated the perforant pathway unilaterally with several stimulation protocols and analyzed hippo‐ campal precursor numbers using both the neurosphere assay and BrdU immunostaining. In the neuropshere assay, we found that induction of late-LTP, but not early-LTP, was required to activate latent precursors in the mouse dentate gyrus and that neurosphere number was positively correlated with LTP magnitude (Fig.5 & Fig.6). In the BrdU immnostaining, proliferation and differentiation in the dentate gyrus was significantly enhanced in the only mice in which LTP was successfully induced. In contrast, protocols that failed to induce LTP, induced only early LTP; used low-frequency stimulation (LFS) or included a pharmacological blocker of LTP (CPP: (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid), all failed to activate neurogenesis (Fig.7). These findings demonstrate that LTP can activate latent neural precursor cells in the adult mouse dentate gyrus, leading to increased neurogenesis [29].

Regenerative Medicine for Neurological Diseases with the Use of Electrical Stimulation http://dx.doi.org/10.5772/55612 751

penumbra, enhanced neuronal differentiation, and suppressed microglial activation in the

It is well established that new neurons are produced continuously in the SVZ and the subgra‐ nular zone (SGZ) of the dentate gyrus (DG) during adulthood [11-13]. Given this fact, many researchers have focused their attention on discovering how to stimulate these resident precursors to enhance neurogenesis. Environmental enrichment and voluntary running [14]

Long-term potentiation (LTP) is induced by brief high-frequency tetanization of excitatory afferents and is observed as a long-lasting enhancement in the efficacy of synaptic transmission [15]. LTP is considered to be a cellular model of learning [16] and memory [17]. Moreover, LTP is speculated to be one possible mechanism of enhanced motor function recovery resulting from electrical stimulation to the cortex after stroke [18]. Based on the accumulated evidence of hippocampal neurogenesis, the relationship among hippocampus-related function, LTP, and neurogenesis has been examined [19-22]. For example, environmental enrichment and running enhanced hippocampus-related memory function as well as LTP. More importantly, electrophysiological reports have demonstrated that recently created neurons contribute to synaptic plasticity [23] and that LTP correlates with neural plasticity, compensating for ischemia-induced damage [24]. These findings support the hypothesis that enhancing neurogenesis may be useful in the restoration and repair of a damaged brain and emphasizes the importance of finding a more effective way to stimulate neurogenesis. As mentioned above, a number of reports are available that describe the relationship between neurogenesis and LTP but these mainly focus on neurogenesis-dependent changes in LTP [19, 21, 22]. Only a few

Neural activity was recently demonstrated to stimulate a large number of hippocampal latent precursors, including a self-renewing stem cell population that only becomes activated following depolarization [28]. Given a number of recent reports linking LTP and hippocampal neurogenesis, we examined whether LTP is able to enhance proliferation of the neural precursors and neurogenesis in the adult mouse dentate gyrus. In this study, we stimulated the perforant pathway unilaterally with several stimulation protocols and analyzed hippo‐ campal precursor numbers using both the neurosphere assay and BrdU immunostaining. In the neuropshere assay, we found that induction of late-LTP, but not early-LTP, was required to activate latent precursors in the mouse dentate gyrus and that neurosphere number was positively correlated with LTP magnitude (Fig.5 & Fig.6). In the BrdU immnostaining, proliferation and differentiation in the dentate gyrus was significantly enhanced in the only mice in which LTP was successfully induced. In contrast, protocols that failed to induce LTP, induced only early LTP; used low-frequency stimulation (LFS) or included a pharmacological blocker of LTP (CPP: (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid), all failed to activate neurogenesis (Fig.7). These findings demonstrate that LTP can activate latent neural precursor cells in the adult mouse dentate gyrus, leading to increased neurogenesis [29].

**3. Long-Term Potentiation (LTP) and neurogenesis**

are now widely accepted ways to enhance neurogenesis.

reports have shown that LTP per se can enhance neurogenesis [25-27].

ischemic penumbra [10].

750 Regenerative Medicine and Tissue Engineering

**Figure 5.** LTP enhances proliferation of neural precursors. A neurosphere formation assay was performed 2 days after stimulation. Under normal K+ conditions, only the LTP(+) group revealed a significant increase in neurosphere number from the stimulated hemisphere relative to the control hemisphere. Furthermore, a significantly greater number of neurospheres was generated from the stimulated hippocampi of the LTP(+) group compared with the simulated hip‐ pocampi of the CPP group and LTP(-) group. When CPP was administered to block the NMDA receptor, neurosphere formation was significantly reduced compared with the LTP(+) group. low: low K+, high: high K+. \*\**p* < 0.01, \*\*\**p* < 0.001. This figure was modified and/or reproduced from the original article [29].

**Figure 6.** Late-LTP but not early-LTP can activate hippocampal precursors in vitro. (a) At the end of recording, the magnitude of potentiation in the late-LTP group was significantly greater than that in the early-LTP group. (b) Follow‐ ing the induction of late-LTP, a significant increase in the number of neurospheres was observed in the low K+ condi‐ tion compared with the early-LTP group. (c) The magnitude of LTP, calculated 60 min after HFS, showed a significant positive correlation (r = 0.879, n = 15, *p* < 0.0001, Pearson's correlation) with the number of neurospheres grown in vitro, after hippocampi were dissected 2 days after HFS. The arrow indicates HFS, error bars indicate SEM, low: low K+, high: high K+. \*\**p* < 0.01, \*\*\**p* < 0.001. Abbreviations: fEPSP, field extracellular postsynaptic potential; HFS, high-fre‐ quency stimulation. This figure was modified and/or reproduced from the original article [29].

Regenerative Medicine for Neurological Diseases with the Use of Electrical Stimulation http://dx.doi.org/10.5772/55612 753

**Figure 7.** Proliferation and differentiation were significantly enhanced in the LTP(+)-stimulated hippocampus. (a) The number of BrdU-positive cells per length of the dentate gyrus (mm) was significantly enhanced in stimulated hippo‐ campi of the LTP(+) group compared to the unstimulated hemisphere. Furthermore, there was a greater number of BrdU-positive cells following LTP induction compared with the CPP group and LFS group. (b) Following LTP induction, the number of BrdU/DCX double-positive cells per perimeter length of the dentate gyrus (mm) was significantly en‐ hanced in the stimulated hippocampus compared with the control hippocampus. BrdU/DCX-double labeling also re‐ vealed a significantly greater number of neurons in the stimulated hippocampi of the LTP(+) group compared with the stimulated hippocampi of the CPP and the LFS groups. (c) Representative maximum intensity projection image of BrdU (red) and DCX (green) immunostaining reconstructed from 40 optical sections of the LTP-stimulated hippocampus of the LTP(+) group. Inset shows a higher magnification. Scale bar: 50 mm in main image, 20 mm for inset. \**p* < 0.05, \*\**p* < 0.01. Abbreviations: C, control side; DCX, doublecortin; S, stimulated side. This figure was modified and/or repro‐ duced from the original article [29].

In the future, these findings can be clinically applied to individuals suffering from depression, Alzheimer's disease, and age-related cognitive decline explained by neuronal loss. In fact, electrical convulsive seizure is clinically performed for severe depression and this therapy showed increase of the proliferation and neuronal differnetiation of hippocampal precursors in a depression model of animals [30-32]. For Alzheimer's disease, a previous report showed that transcranial direct current stimulation enhanced aspects of memory performance in both healthy controls and individuals with Alzheimer's disease [33]. However, an operation for

**Figure 6.** Late-LTP but not early-LTP can activate hippocampal precursors in vitro. (a) At the end of recording, the magnitude of potentiation in the late-LTP group was significantly greater than that in the early-LTP group. (b) Follow‐ ing the induction of late-LTP, a significant increase in the number of neurospheres was observed in the low K+ condi‐ tion compared with the early-LTP group. (c) The magnitude of LTP, calculated 60 min after HFS, showed a significant positive correlation (r = 0.879, n = 15, *p* < 0.0001, Pearson's correlation) with the number of neurospheres grown in vitro, after hippocampi were dissected 2 days after HFS. The arrow indicates HFS, error bars indicate SEM, low: low K+, high: high K+. \*\**p* < 0.01, \*\*\**p* < 0.001. Abbreviations: fEPSP, field extracellular postsynaptic potential; HFS, high-fre‐

quency stimulation. This figure was modified and/or reproduced from the original article [29].

752 Regenerative Medicine and Tissue Engineering

electrode insertion is needed, and this is invasive for patients. To reduce invasiveness, transcranial magnetic stimulation (TMS), which is currently clinically applied to patients with severe depression, would solve this problem. For age-related cognitive decline, TMS is currently the main therapeutic option; however, more evidence needs to be collected. Contin‐ uous learning and exercise is the best method to overcome age-related cognitive decline.

In the future, we need to clarify the mechanism of electrical stimulation and neurogenesis in more detail. In the electrical stimulation conducted for cerebral infarction, we found that the optimal stimulation parameter was low frequency; however, in this LTP experiment, neuro‐ genesis was enhanced by HFS that can induce LTP. Disease model (cerebral infarction) or healthy model is a crucial difference between these two experiments. This difference in animal condition might be one possible explanation for this discrepancy in optimal stimulation parameters. As written above, we found the therapeutic potentials of electrical stimulation; however, further studies and ethical consensus are required for its application in a clinical setting because electrical stimulation has the potential to modulate the human mind and cognition.
