**1. Introduction**

In order to ensure the security of the global food supply, a large amount of nitrogen fertilizer has been utilized in the past few decades. For example, the average nitrogen utilization has achieved the rate of 305 kg/hm2 in China [1]. Excessive bioavailable nitrogen such as nitrate nitrogen (NO− 3 ) and ammonia nitrogen (NH+ 4 ) in rivers will lead to water eutrophication and, consequently, damage the ecological balance of river systems. Particularly in coastal areas, nitrogen pollution in rivers is generally serious due to dense population and active industry and agriculture production. If it cannot be maintained under effective control, the excessive nitrogen will continue to enter the ocean and further affect the health of the coastal ecosystem and the sustainable utilization of marine resources.

Hyporheic zones of river ecosystem are water-saturated sediment (known as aquifer) below the riverbed and extending to the riparian areas on both sides. It is not only a key area for water exchange and solute migration between river and groundwater, but also an important place for microbial growth and metabolism. Mass exchange and energy transfer are frequent and biogeochemical reactions are complex within a hyporheic zone, which is of great significance to the structure, function, and health of river ecosystem [2–4]. Nitrogen

**Citation:** Cai, Y.; Xing, J.; Huang, R.; Ruan, X.; Zhou, N.; Yi, D. Occurrence Characteristics of Inorganic Nitrogen in Groundwater in Silty-Clay Riparian Hyporheic Zones under Tidal Action: A Case Study of the Jingzi River in Shanghai, China. *Appl. Sci.* **2022**, *12*, 7704. https://doi.org/ 10.3390/app12157704

Academic Editor: Bing Bai

Received: 20 May 2022 Accepted: 28 July 2022 Published: 30 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is transported between rivers and their riparian zones through hyporheic exchange and transformed between different chemical forms (e.g., NH+ 4 , NO− 3 , nitrite nitrogen (NO− 2 ), and N2) through biogeochemical reactions [5]. Therefore, to realize the effective control of nitrogen pollution in rivers, it is necessary to fully comprehend nitrogen migration and transformation in groundwater in hyporheic zones.

Nitrogen in groundwater in a hyporheic zone varies geographically, and its form changes temporally, which forms a complex dynamic cycle [6–9]. Some hydrological events (e.g., rainstorm, flooding, reservoir discharge, and tidal action) can change the recharge relationship between river water and groundwater, consequently influencing nitrogen migration in hyporheic zones [10–13]. Shibata et al. carried out onsite monitoring to study the water exchange and the change in nitrogen concentration in the hyporheic zone during the rainstorm. It was found that the increase in nitrogen concentration in the hyporheic zone was mainly caused by the rainwater infiltration through the overlying unsaturated zone rather than the lateral seepage from the river [14]. The research conducted by Singh et al. indicated that the flood process extended the mixing range of surface water and groundwater and had a significant impact on the mass cycle in the riverbed hyporheic zone [15]. Sawyer et al. studied the impact of the reservoir operation on the lateral hyporheic exchange, and the results showed that the impact range could extend to 1–5 m away from the shore [16]. Unlike the hydrological events mentioned above, tides exhibit periodicity, and the tidal period is usually about 12 or 24 h. This makes the hyporheic exchange more frequent and the biogeochemical process in the hyporheic zone more complex [17]. In recent years, there has been growing concern over the hyporheic zones of tidal rivers. Musial et al. quantified the tide-driven hyporheic exchange fluxes across the bank and the bed and deduced that the tidal bank storage in the sandy hyporheic zone might remove nutrients from rivers [18]. The geochemical measurements and numerical simulation performed by Barnes et al. showed that it was possible that the hyporheic zone with permeable sediment and low organic matter content could be a source of nitrate to the tidal river [19].

Hydrogeological conditions and ecological factors could affect the nitrogen cycle in hyporheic zones [20–22]. McGarr et al. investigated the hyporheic exchange process in a sand–gravel mixed riverbed and pointed it out that the pore water flowed preferentially through the sediment of high-permeability, which affected nitrogen transport pathways [23]. The sediment heterogeneity could result in the existence of tiny anoxic blocks within the aerobic zone near the shore, thus affecting nitrogen processing [24]. The retention time of river water is usually extended in the low-permeability riverbed sediment, which is beneficial to the full reaction between nitrogen and dissolved oxygen (DO) [25]. In addition, the migration ability of NH+ 4 in the hyporheic zone may be limited to a certain extent due to the electrostatic attraction by soil particles [26]. Eco-environmental factors mainly include the reactant concentration, redox potential (Eh), temperature (T), and pH [27–29]. The nitrogen-related biogeochemical reactions such as nitrification and denitrification are greatly governed by DO concentration and Eh in hyporheic zones [30]. Dissolved organic carbon (DOC) is another key factor controlling the nitrogen cycle, and increasing DOC concentration could promote denitrification [31]. Zarnetske et al. found that DOC and DO could be preferentially consumed in the near-shore hyporheic zone [32]. Additionally, temperature could affect the microbial activity, reaction rate, and DO concentration, consequently influencing the process of nitrogen cycle in the hyporheic zone [33,34]. Some studies demonstrated that pH showed a positive correlation with NO− 3 concentration and a negative one with NH+ 4 concentration [35].

Although the nitrogen cycle in hyporheic zones has been extensively studied, there is little work aiming at the case of low-permeability riparian hyporheic zones of tidal rivers. At present, the mechanism underlying how tides drive nitrogen migration and transformation in riparian hyporheic zones with low permeability sediment is not very clear. The main objective of the study was to characterize the spatiotemporal variations of various inorganic nitrogen concentrations in groundwater in poorly permeable hyporheic zones of tidal rivers and discuss the potential reason why such variations existed. A representative transect was chosen to carry out the drilling, monitoring, and sampling. By means of onsite monitoring and lab analysis, the response of the riparian groundwater level to the tide-driven fluctuation of the river stage was studied, the spatiotemporal variations of inorganic nitrogen concentrations in the groundwater in the riparian hyporheic zone were analyzed, and the potential influencing factors were discussed. The results are expected to enrich the theory of river hyporheic zones and provide a scientific basis for nitrogen pollution treatment of tidal rivers.


*Article* **Impact of DST (Daylight Saving Time) on Major Trauma: A European Cohort Study**

**André Nohl 1,2,3,4,\*, Christine Seelmann 5, Robert Roenick 6, Tobias Ohmann 5, Rolf Lefering 7, Bastian Brune 3,8, Veronika Weichert 4,9, Marcel Dudda 3,4,8,9 and The TraumaRegister DGU 10,†**

**Abstract:** (1) Background: Approximately 73 countries worldwide implemented a daylight saving time (DST) policy: setting their clocks forward in spring and back in fall. The main purpose of this practice is to save electricity. The aim of the present study was to find out how DST affects the incidence and impact of seriously injured patients. (2) Methods: In a retrospective, multi-center study, we used the data recorded in the TraumaRegister DGU® (TR-DGU) between 2003 and 2017 from Germany, Switzerland, and Austria. We compared the included cases 1 week before and after DST. (3) Results: After DST from standard time to summertime, we found an increased incidence of accidents of motorcyclists up to 51.58%. The result is consistent with other studies. (4) Conclusion: However, our results should be interpreted as a tendency. Other influencing factors, such as time of day and weather conditions, were not considered.

**Keywords:** DST; daylight saving time; major trauma


**The Extracellular Matrix in the Nervous System: The Good and the Bad Aspects**

Elena Vecino and Jessica C. F. Kwok

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62527

#### **Abstract**

The study of extracellular matrix (ECM) in the nervous system has longed been focused on the molecules promoting growth and migration. This is well supported by the work in the developing nervous system. However, the discovery of Nogo and chondroitin sulphate proteoglycans (CSPGs) in the injured nervous system in late 1980s has shifted some of the focus to inhibitory molecules. One of the biggest hurdles in neural regeneration is the formation of glial scar and the highly up-regulated inhibitory molecules present in the area. Apart from Nogo and CSPGs, other myelin-associated inhibitors, tenascins and semaphorins have been found associated with neuronal inhibition. Together with the identification of their receptors, we now have a better understanding on the mechanism of how these molecules control and limit regenera‐ tion in the central nervous system (CNS). Recent focus has been put on designing strategies in neutralizing these inhibitions for promoting regeneration after injury, and some are showing promising results. Moreover, latest studies also show that rehabili‐ tation in injured animal models demonstrated drastic remodeling of ECM favoring regeneration. This review shall discuss all these different aspects and the importance of matrix remodeling in the CNS and the implication of ECM in some retinal pathologies.

**Keywords:** retina, regeneration, perineuronal nets, chondroitin sulfate, integrins


**1. Introduction**

In the last decade, we observed a massive upsurge of studies in the field of extracellular vesicles (EVs) [1]. As it is known now, EVs can be loaded with different therapeutic molecules and transport them to recipient cells with little interrogation by the immune system. This property of EVs prompts new possibilities for treatment in various clinical settings [2–4]. In this chapter, we review the biology of EVs as a universal cellular component from a broader perspective, and afterward provide an updated view on red blood cell extracellular vesicles (RBCEVs), their merits and potential applications in therapeutics [5].

### **2. Overview of extracellular vesicles**

#### **2.1 History of extracellular vesicles**

Wolf was the first to discover small procoagulant structures derived from activated platelets in human blood and named them "platelet dust" in 1967. He separated the small structures by ultracentrifugation and further characterized them using an

electron microscopy [6]. In 1987, Johnstone further studied the formation of such vesicles in the duration of sheep reticulocytes maturation *in vitro*. He was able to identify more activities and characteristics of the vesicles. However, he did not name the small vesicles or discover how they were generated in detail [7]. Both of these findings were important milestones in the field, which allowed for further studies on the function of these small vesicles. Today, we call these small vesicles as EVs. Valadi and colleagues were the first who discovered the natural delivery of microRNAs and mRNAs in EVs in mast cells. Later on, nucleic acid transport via EVs was also observed in many other cell types as an essential manner of intercellular communication [8–10]. We now have a much more profound understanding in the field of EVs due to the continuous efforts of various scientists throughout many decades.

#### **2.2 Biogenesis and compositions of extracellular vesicles**

EVs are a heterogeneous class of cell-derived structures with a lipid bilayer membrane, which comprise exosomes, microvesicles, and apoptotic bodies. They are either of the endosomal origins or are shed from the plasma membrane under physiological and pathological conditions. Additionally, they are present in almost all biological fluids, such as blood, urine, breast milk, cerebrospinal fluid, saliva, semen, etc. [11–17]. Further characterizations are based on the different sizes and biogenesis of EVs. Exosomes generally range from 50 to 150 nm in diameter and are secreted from endosomal multivesicular bodies, whereas microvesicles are larger vesicles ranging from 100 to 500 nm in diameter and are formed through a budding or exocytosis process of the plasma membrane [11, 18–23]. Apoptotic bodies are much larger, ranging from 800 to 5000 nm in diameter, and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Hence, apoptotic bodies represent the fragments of dying cells and differ from exosomes and microvesicles in property (**Figure 1**) [17–22]. In this chapter, we will collectively term both exosomes and microvesicles as EVs with apoptotic bodies excluded.

The components of EVs are mainly proteins, lipids, and nucleic acids. However, due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary slightly [11, 24–26]. Proteins that are associated with endocytic pathways can be usually found in EVs, such as flotillin and annexin. Some of the biogenesis-associated proteins, such as Tsg101 and Alix, and common tetraspanins, such as CD9 and CD81, are commonly used as EVs markers with CD63 which is mostly regarded as a marker of exosomes. However, currently, there lack well-defined protein markers to distinguish exosomes and microvesicles [11, 24–26]. Lipid components of EVs include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, and so on, which can be found in plasma membrane as well. As microvesicles are formed by budding from plasma membrame, the lipid composition of microvesicles resembles that of plasma membrane of the cells more while exosomes are of higher levels in sphingomyelin, cholesterol, and phosphatidylserine [27–29]. It is noteworthy that many nucleic acid species are highly enriched in EVs. The lipid bilayer structure of EVs acts as a natural shelter against degrading nucleases in the extracellular environment and protects the nucleic acid cargo under adverse conditions such as long-term storage and multiple freeze-thaw cycles. In the recent decade, reports have it that many mRNAs, microRNAs, and other non-coding RNAs are discovered in EVs (**Figure 1**) [30–32].

#### **2.3 Intercellular communication mediated by extracellular vesicles**

As EVs are abundant and widely distributed in biological fluids and carry bioactive cargo, they influence various biological processes of the donor and recipient

**123**

**Figure 1.**

*The Biology and Therapeutic Applications of Red Blood Cell Extracellular Vesicles*

cells [33]. The intercellular communication can occur between cells by transferring EVs that act as an exchange mediator of proteins, lipids, and RNAs. Thus, EVs have a fundamental role to play in important biological processes such as the exchange of surface membrane and horizontal RNA transport between neighboring and remote cells [18]. This aspect is being extensively investigated in cancers [34], neurodegenerative diseases [35], autoimmune disorders [36], aging [37], and so on. The bioactive cargo encapsulated by EVs contain valuable information from the source

*Biogenesis and composition of extracellular vesicles. Extracellular vesicles (EVs) are composed of exosomes, microvesicles, and apoptotic bodies. Exosomes are typically of endosomal origins and are the smallest among them with 50 to 150 nm in diameter. Microvesicles are larger in size from 100 to 500 nm in diameter and are generated through an outward budding or exocytosis of the plasma membrane. Apoptotic bodies are usually the largest ranging from 800 to 5,000 nm in diameter and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Major components of EVs are lipids, proteins, and nucleic acids. Due to different*

*biogenesis mechanisms, the compositions of exosomes and microvesicles do vary.*

*DOI: http://dx.doi.org/10.5772/intechopen.81758*

*The Biology and Therapeutic Applications of Red Blood Cell Extracellular Vesicles DOI: http://dx.doi.org/10.5772/intechopen.81758*

#### **Figure 1.**

*Erythrocyte*

electron microscopy [6]. In 1987, Johnstone further studied the formation of such vesicles in the duration of sheep reticulocytes maturation *in vitro*. He was able to identify more activities and characteristics of the vesicles. However, he did not name the small vesicles or discover how they were generated in detail [7]. Both of these findings were important milestones in the field, which allowed for further studies on the function of these small vesicles. Today, we call these small vesicles as EVs. Valadi and colleagues were the first who discovered the natural delivery of microRNAs and mRNAs in EVs in mast cells. Later on, nucleic acid transport via EVs was also observed in many other cell types as an essential manner of intercellular communication [8–10]. We now have a much more profound understanding in the field of EVs

due to the continuous efforts of various scientists throughout many decades.

EVs are a heterogeneous class of cell-derived structures with a lipid bilayer membrane, which comprise exosomes, microvesicles, and apoptotic bodies. They are either of the endosomal origins or are shed from the plasma membrane under physiological and pathological conditions. Additionally, they are present in almost all biological fluids, such as blood, urine, breast milk, cerebrospinal fluid, saliva, semen, etc. [11–17]. Further characterizations are based on the different sizes and biogenesis of EVs. Exosomes generally range from 50 to 150 nm in diameter and are secreted from endosomal multivesicular bodies, whereas microvesicles are larger vesicles ranging from 100 to 500 nm in diameter and are formed through a budding or exocytosis process of the plasma membrane [11, 18–23]. Apoptotic bodies are much larger, ranging from 800 to 5000 nm in diameter, and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Hence, apoptotic bodies represent the fragments of dying cells and differ from exosomes and microvesicles in property (**Figure 1**) [17–22]. In this chapter, we will collectively term both

**2.2 Biogenesis and compositions of extracellular vesicles**

exosomes and microvesicles as EVs with apoptotic bodies excluded.

**2.3 Intercellular communication mediated by extracellular vesicles**

As EVs are abundant and widely distributed in biological fluids and carry bioactive cargo, they influence various biological processes of the donor and recipient

The components of EVs are mainly proteins, lipids, and nucleic acids. However, due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary slightly [11, 24–26]. Proteins that are associated with endocytic pathways can be usually found in EVs, such as flotillin and annexin. Some of the biogenesis-associated proteins, such as Tsg101 and Alix, and common tetraspanins, such as CD9 and CD81, are commonly used as EVs markers with CD63 which is mostly regarded as a marker of exosomes. However, currently, there lack well-defined protein markers to distinguish exosomes and microvesicles [11, 24–26]. Lipid components of EVs include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, and so on, which can be found in plasma membrane as well. As microvesicles are formed by budding from plasma membrame, the lipid composition of microvesicles resembles that of plasma membrane of the cells more while exosomes are of higher levels in sphingomyelin, cholesterol, and phosphatidylserine [27–29]. It is noteworthy that many nucleic acid species are highly enriched in EVs. The lipid bilayer structure of EVs acts as a natural shelter against degrading nucleases in the extracellular environment and protects the nucleic acid cargo under adverse conditions such as long-term storage and multiple freeze-thaw cycles. In the recent decade, reports have it that many mRNAs, microRNAs, and other non-coding RNAs are discovered in EVs (**Figure 1**) [30–32].

**122**

*Biogenesis and composition of extracellular vesicles. Extracellular vesicles (EVs) are composed of exosomes, microvesicles, and apoptotic bodies. Exosomes are typically of endosomal origins and are the smallest among them with 50 to 150 nm in diameter. Microvesicles are larger in size from 100 to 500 nm in diameter and are generated through an outward budding or exocytosis of the plasma membrane. Apoptotic bodies are usually the largest ranging from 800 to 5,000 nm in diameter and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Major components of EVs are lipids, proteins, and nucleic acids. Due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary.*

cells [33]. The intercellular communication can occur between cells by transferring EVs that act as an exchange mediator of proteins, lipids, and RNAs. Thus, EVs have a fundamental role to play in important biological processes such as the exchange of surface membrane and horizontal RNA transport between neighboring and remote cells [18]. This aspect is being extensively investigated in cancers [34], neurodegenerative diseases [35], autoimmune disorders [36], aging [37], and so on. The bioactive cargo encapsulated by EVs contain valuable information from the source

of diseases, which can serve as robust biomarkers in diagnostics and status snapshots in treatment monitoring [38, 39]. The endogenous property of transporting molecules by EVs inspires researchers to utilize them as a superb delivery platform of therapeutic agents as well [40].


*2.2. Signal Transduction of Na,K-ATPase by CTS*

It is noteworthy that low-dose ouabain treatment activates the specific signaling without affecting the intracellular ion concentrations [21]. Mounting evidence has shown that Na,K-ATPase is engaged in the cardiac glycoside-mediated signaling cascades [22]. In the context of the signal-transducing functions of Na,K-ATPase, this pump is capable of communicating with signaling molecules by ligand-induced conformational changes within the signalosome [10]. Ouabain binding to the Na,K-ATPase in caveolae activates not only inositol triphosphate receptor (IP3R)-mediated Ca2+ release from S/ER but also Src kinase-mediated signal cascades [8]. CTS-induced conformational changes occurring within the caveolar sodium pump α1 subunit, which is a negative regulator of Src, allow the activation of Src [9].

Currently, Na,K-ATPase complex with Src is regarded as a unique receptor for CTS [9]. Activated Src triggers the execution of downstream signaling cascades, such as epidermal growth factor receptor (EGFR) transactivation-mediated Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) 1/2 pathways [23], inducing gene activation [10]. Src is known to be a key player in the Na,K-ATPase-mediated inter-receptor crosstalk with EGFR [24]. Activated as well are phosphoinositide 3-kinase (PI3K) [25] and EGFR-transactivated generation of reactive oxygen species (ROS), and the latter explains the clinical role of Na,K-ATPase as an oxidant amplifier contributing to the progression of cardiac fibrosis [9]. In addition, Src activates the phospholipase C (PLC) pathway [26] that also contributes to the IP3-induced Ca2+ release [10]. The signaling pathways of the sodium transporter are assumed to be implicated in hypertension, renal disease, diabetes, metabolic disease, and cardiovascular diseases. When there are alterations in the signaling pathways, signaling receptor function would change, causing hypertension, cardiac hypertrophy, tissue fibrosis, or cancer [9].

#### *2.3. Ouabain-Induced Hypertension and Related Diseases*

Ouabain, a well-known positive ionotropic agent in the heart, is also an endogenous adrenal cortical hormone because it can be synthesized in the adrenal glands [27]. It is known that the infusion of ouabain for a prolonged period in rodents [4–6], elevation of endogenous ouabain [28], and ouabain-like factors [29] in the circulation are associated with the development of hypertension. The elevation of endogenous ouabain also facilitates the proliferation of myocardial and smooth muscle cells [27]. Circulating endogenous ouabain inhibits the activity of the ouabain-sensitive Na,K-ATPase α2 subunit [4] and inhibition of the sodium pump by cardiac glycosides, such as ouabain, is associated with the pathogenesis of hypertension [17], cataract [18], and diabetic diseases [16], through the elevation of the intracellular Ca2+ concentrations.

#### **3. TCTP, as an Intracellular Na,K-ATPase Suppressor**

TCTP, alternatively referred to as fortilin, p23, and histamine-releasing factor (HRF), is a highly conserved multifunctional 172-amino-acid protein that is under a high degree of regulation both at transcriptional and translational levels [30]. TCTP plays fundamental roles in the regulation of cell cycle progression, apoptosis, autophagy, survival, stress responses, growth and development, and cytokine-like activities, among others [30]. Given the multiplicity of roles it plays, dysregulation of TCTP can lead to cardiovascular and metabolic diseases including systemic and pulmonary arterial hypertension [31], atherosclerosis, diabetes, carcinogenesis, and allergic and inflammatory disorders [32].

Our research group has identified TCTP as a cytoplasmic repressor for Na,K-ATPase [33]. In contrast to ouabain that binds to the outer part of the sodium pump, TCTP interacts with the cytosolic domain of Na,K-ATPase [33]. Searching for the cytoplasmic molecules that are potentially associated with the Na,K-ATPase α subunit, we screened a rat skeletal muscle DNA library using a yeast two-hybrid system. We found that the third large cytoplasmic domain (CD3) of Na,K-ATPase α1 and α2 isoforms interacts with the rat TCTP [33].

While studying the interaction of TCTP with the sodium pump in HeLa cells, we discovered the inhibitory effect of TCTP on Na,K-ATPase activity. Interaction of rat TCTP with the sodium pump does not affect the mRNA and protein levels of the Na,K-ATPase α subunit, indicating a relatively short-term regulation by TCTP [33]. TCTP inhibited the sodium pump activity in a dose-dependent manner [33]. In addition, the C-terminal region (102–172 residues) of rat TCTP was the moiety involved in the inhibition of pump activity of Na,K-ATPase [33]. These observations suggest that TCTP is possibly playing a promotive role in hypertension and raise the question of whether TCTP elevates cytosolic Ca2+ in vivo [33]. Interaction of the sodium pump with TCTP is now generally accepted to have important roles not only in the biological functions and signaling but also in the pathophysiology of human diseases [34–36].

It has been shown that TCTP plays either protective or causative roles in certain diseases in some conditions, but it is clear that the dysregulation of TCTP contributes to the development of various pathophysiological processes [37], as shown in several genetically altered mouse models (Table 1). Conventional or tissue/cell-specific TCTP-overexpressing transgenic mice (TCTP-TG) were used to study the gain-of-function of TCTP. Conversely, heterozygous TCTP-deficient mice (TCTP+/−), in which TCTP gene is deficient causing a loss of function, were studied because of embryonic lethality of homozygous TCTP knockout mice (TCTP−/−) [38]. This review focuses on the roles of TCTP in hypertension and related diseases, including atherosclerosis, cataracts, and other metabolic disorders that have been studied in genetically engineered animal models.

**Table 1.** Genetically modified mouse models that exhibit elevated or reduced expressions of TCTP and its phenotypes and pathophysiological significances in vivo.

↑ indicates increase; ↓ indicates decrease.

#### **4. Pathophysiology of Hypertension in TCTP-TG**

Animal models have been used for conducting etiological studies of genetic hypertension. These include spontaneous hypertensive rats (SHRs), Dahl salt-sensitive rats, and transgenic models generated by overexpression of particular genes [53]. To address the questions raised regarding the pathophysiological consequences of TCTP-mediated Na,K-ATPase inhibition in vivo, we constructed transgenic mice overexpressing TCTP (TCTP-TG). It was established in either C57BL/6×CBA hybrid or C57BL/6N inbred backgrounds using pCAGGS-TCTP cDNA construct consisting of cytomegalovirus enhancer (CMV-IE) and chicken β-actin promotor (Figure 1A).

**Figure 1.** Generation of TCTP-TG and TCTP-TG/ApoE KO mice. (**A**) Schematic representation of the gene construct used for TCTP transgenic overexpression in mice. The TCTP transgene of pCAGGS-TCTP cDNA containing CMV-IE enhancer and chicken β-actin promoter constructed in the transgenic expression vector pCAGGS was used to generate the transgenic mice. (**B**) Strategy of crossbreeding for generation of nTG/ApoE KO and TCTP-TG/ApoE KO mice exhibiting identical genetic backgrounds.

Their phenotypic characteristics are partly affected by the genetic background of TCTP-TG because, for example, cataractogenesis was profound in C57BL/6N background mice [41] while both types of transgenic mice show hypertensive properties regardless of their genetic backgrounds [39,41,44]. Because of the decreased fertility of inbred offspring, TCTP-TG was generated in C57BL/6×CBA hybrid strains in the majority of our investigations. For the study regarding atherogenesis, TCTP-TG was subjected to crossbreeding to generate apolipoprotein (ApoE)-deficient TCTP-TG (ApoE-KO/TCTP-TG) with a hybrid background (Figure 1B).

#### *4.1. Systemic Arterial Hypertension by Na,K-ATPase Inhibition in TCTP-TG*

Both C57BL/6×CBA and C57BL/6N mice overexpressing TCTP showed phenotypes of hypertension and hybrid mice were used in the study. The gender-related dimorphism regarding BP was observed, also reported in heterozygous female TGR(mRen2)27 transgenic rats [54], possibly due to the differences in hormonal changes or vascular responsiveness between female and male TCTP-TG mice occurring with ageing [39]. At approximately 6 weeks after birth, systemic arterial hypertension developed in both male and female TCTP-TG but after 19–20 weeks female TCTP-TG did not exhibit a significant difference in BP compared with non-transgenic (nTG) mice [39]. Therefore, heterozygous male TCTP-TG mice were studied for further investigations.

As with arterial BP elevation, an increase in left ventricular systolic pressure (LVSP) was found in TCTP-TG mice [39]. The thickening of the left ventricular posterior walls was observed in 15- to 16-month-old male TG mice, possibly caused by the sustained elevation of BP, but TCTP-TG did not exhibit the significant cardiac dysfunction [39]. TCTP-TG showed the characteristics of hypertension: first, the alteration of vascular functions that exhibit enhanced contractility of aortic rings devoid of endothelial layer, and secondly, augmented sensitivity to vasoconstrictor stimuli such as norepinephrine (NE) and serotonin (5-HT). Moreover, vascular relaxation reactivity following sodium nitroprusside (SNP) treatment after NE-induced contraction was decreased, indicating the reduced sensitivity to vasodilators [39]. Impaired vasodilatory responses accompanied by the hypercontractile profile in the vasculature isolated from TCTP-TG mice aberrantly elevated the BP [39].

As expected, vascular smooth muscle cells (VSMCs) isolated from TCTP-TG showed suppressed activity of Na,K-ATPase α1 and α2 isoforms that are the predominant Na,K-ATPase in mouse aorta, confirming the TCTP-induced sodium pump inhibition in VSMCs [39]. The expression of α1 in VSMCs from TCTP-TG (TG-VSMCs) was elevated, whereas that of α2 did not show any significant changes [39]. Because transient TCTP overexpression in HeLa cells did not affect the Na,K-ATPase expression, as observed in our earlier studies [33], elevated α1 expression in TCTP-TG mice suggests a potential compensatory mechanism due to the prolonged pump inhibition in vivo [39].

Then, we confirmed that inhibition of Na,K-ATPase activity in TG-VSMCs induces augmented intracellular Ca2+ mobilization, possibly through the reverse mode of NCX. Upon 5-HT stimuli, TG-VSMCs exhibited elevated Ca2+ transients that were mobilized from the ER [39]. Of interest, ouabain treatment at a concentration that blocks almost all the activity of Na,K-ATPase did not further enhance the 5-HT-evoked Ca2+ transients in TG-VSMCs whereas that of non-transgenic mice was elevated by ouabain, compared with untreated cells [39]. This phenomenon can be explained by the fact that ouabain may have negligible impact on TCTP-TG whose homeostasis has already been established by TCTP-induced Na,K-ATPase inhibition [39]. Likewise, VSMCs of TCTP-TG showed increased sensitivity to vasoconstrictor, elevated levels of Ca2+ in the resting state, and increased capacity for intracellular Ca2+ mobilization, and ultimately enhanced contraction by stimuli, accompanied by reduction in vasorelaxant-induced relaxation [39].

Taking these observations together, we delineated the pathophysiology of TCTPinduced hypertension using TCTP-TG, as depicted in Figure 2. Repression of Na,K-ATPase by TCTP overexpression results in the rise of cytosolic Na+ level, and subsequent elevation of cytosolic Ca2+ mobilization and Ca2+ storage, possibly through the reversion action of NCX, ultimately inducing the hyper-contractility of VSMC in TCTP-TG. Clearly, based on the fact that high blood pressure implies the dysfunctions in cardiac and/or arterial parameters [55], non-significant cardiac malfunctions indicate that TCTP-TG-induced hypertension is mediated by vascular malfunctions, as shown by elevated contractility and reduced relaxation in VSMC from TCTP-TG mice. This phenomenon is similar to that of ouabain treatment-induced hypertension exhibiting augmented vascular reactivity to vasopressors in renal artery of rats [56], confirming that TCTP can be viewed as an endogenous ouabain-like entity. Based on the observation that TCTP+/− and wild-type mice did not show a significant difference in systolic BP [45], overexpression of TCTP might be essential in inducing arterial systemic hypertension in vivo.

**Figure 2.** TCTP overexpression-induced consequences in transgenic TCTP-overexpressing (TCTP-TG) mice. Transgenic overexpression of TCTP led to the Na,K-ATPase inhibition, which results in the development of hypertension through elevated cytosolic Ca2+ levels and vascular contractility and tone. Such an increase in Ca2+ can also occur in the lens epithelial cells of TCTP-TG, at this time, inducing the activation of proteolytic enzyme and lens swelling, all of which affect the cataractogenic processes. RhoA/Rho kinase activation and resultant MYPT-1 phosphorylation in TCTP-TG appears to partly contribute to the increased sensitivity of myofibrils upon constrictor stimuli, thereby increasing the contractility of VSMC in TCTP-TG (↑ indicates increase).

#### *4.2. Upregulation of RhoA/Rho Kinase Signaling in TCTP-TG*

The contractility of smooth muscle cells is largely determined by the phosphorylation status of the regulatory myosin light chain (MLC) whose activity is under regulation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) [57]. Vasoconstrictors like Ang II binding to G-protein coupled receptors (GPCR) activates Gq/11, which in turn activates phospholipase C (PLC)-β and elevates Ca2+ concentration, thereby stimulating MLCK activity and induces G12/13 to activate RhoA/Rho kinase and, in turn, inhibits MLCP. A myosin binding subunit, the myosin phosphatase target unit (MYPT-1) of MLCP, is one of the most important substrates of Rho kinase [57]. Both pathways together converge into the MLC phosphorylation and enhance the contraction of VSMCs and myofibrils [57]. As a downstream mediator of Ang II signaling, a small GTP-binding protein, RhoA, which maintains MLC phosphorylation in VSMCs, regulates Ca2+ sensitivity of the myofilaments through MLCP inhibition [58,59].

It is generally accepted that Ca2+ sensitivity of myofilaments is under regulation by RhoA/Rho kinase-induced MLCP inhibition [58,59] and that dysregulation of RhoA/Rho kinase signaling results in the hypercontractile properties of VSMC, thereby contributing to the pathogenesis of hypertension [57]. Upregulation of RhoA/Rho kinase signaling pathway has been described in cardiovascular diseases, such as systemic and pulmonary hypertension, atherosclerosis, heart failure, and stroke [57,60] and it is deeply involved in cardiovascular and renal pathophysiology [61]. Consistently, dysregulation of RhoA/Rho kinase pathway has been reported in several hypertensive rat models, including SHRs, renal hypertensive rats, and deoxycorticosterone acetate (DOCA) salt-induced hypertensive rats [58].

We found that TCTP-induced RhoA/Rho kinase upregulation is involved in the hypertensive phenotype of TCTP-TG [40]. Increased contraction in the aorta from TCTP-TG under lower concentrations of K+ [39] implies the potential dysregulation of pathways regulating Ca2+ sensitization, such as RhoA/Rho kinase pathway. When we examined these alterations in aortas isolated from TCTP-TG mice, elevated RhoA expression and phosphorylated MLC (p-MLC) were confirmed, whereas aortas obtained from heterozygous mice containing a deleted allele of TCTP (TCTP+/−) showed a decrease in RhoA expression and p-MLC in their aortas [40].

Additionally, overexpression of TCTP increased RhoA expression and activated its downstream signaling, including MYPT-1 and MLC phosphorylation in primary cultured VSMC. In contrast, knockdown of TCTP expression in VSMCs showed suppression of RhoA expression as well as Rho kinase signaling pathways [40]. Inhibition of Rho kinase signaling can be an alternative approach in the modulation of TCTP-induced hypertension. It is evident that dysregulation of RhoA-related Ca2+ sensitization mechanism partly underlies the hypertensive phenotypes of TCTP-TG (Figure 2).

## *4.3. Elevation of the Expression of Peroxiredoxin 3 (Prx3) and Heat Shock Protein 25 (Hsp25) in TCTP-TG*

A proteomic analysis to delineate the potential pathophysiological changes in TCTP-TG mice of C57BL/6×CBA strain showed differentially expressed proteins involving reactive oxygen species (ROS) metabolism, fatty acids/amino acids metabolism, energy metabolism, and cytoskeletal organization in heart tissue of TCTP-TG compared with those of control mice [62]. Of note, the expression of a mitochondrial antioxidant enzyme, peroxiredoxin 3 (Prx3) showed decrease in 9-week-old TCTP-TG mice, whereas cytosolic Prx2 expression was elevated in 19-week-old TCTP-TG [62]. Because mitochondrial oxidative stress takes part in the pathogenesis of hypertension [63–65], altered expression of these enzymes implies the potential relation of ROS in TCTP-induced hypertension [62]. The protective and anti-inflammatory roles of Prx3 in various cell types were suggested [66] and Prx3 overexpression protects the heart from the ventricular remodeling and heart failure following myocardial infarction by attenuating mitochondrial oxidative stress [67]. Additionally, the higher Hsp25 expression in 9-week-old TCTP-TG mice, also indicates the possible role of ROS in TCTP-induced hypertension. Stress-inducible Hsp25 is upregulated in the kidney isolated from the rats with prolonged Ang II administration, and its expression was reported to be involved in the regulation of BP [68]. Studies on the potential roles of Prx3 and Hsp25 in the heart of TCTP-TG mice are an area of interest for the further studies.

#### **5. Pathophysiology of Cataractogenesis in TCTP-TG**

There is a 2.13 times greater risk of having a cataract extraction in hypertensive patients, especially in the 60–69 year age group, compared with non-hypertensives individuals [69]. A meta-analysis also indicates that hypertension enhances the risk of cataracts [70]. In salt-sensitive hypertensive Dahl rats, the incidence of cataracts was 30–35% and it can be predicted by the suppression of Na,K-ATPase, suggesting a defect in this pump in hypertensive rats [71–73].

Cataractogenesis, an impaired lens transparency, results from the dysregulation of ionic and fluid homeostasis [41], which is tightly regulated by Na,K-ATPase in the lens cells [20,74]. Elevated sodium and calcium concentrations or digitalis-like compounds have been found in human cataractous lenses [75]. Sodium pump inhibitors like ouabain induce the elevation of the sodium level with a concomitant decrease in the potassium level in the lens, and such osmotic imbalance leads to the accumulation of fluids in the lens, and then cells swell and degrade, resulting in the formation of fluid droplets, the cause to opacify [76–78]. In addition to fluid-induced cell death, elevated sodium levels due to the Na,K-ATPase repression induce the rise in cytosolic level and transient of Ca2+, which activates Ca2+-dependent proteolytic enzymes, cellular integrity impairment and apoptosis, eventually resulting in the loss of lens transparency [76–78].

Of interest, the elevation of BP is not necessarily the only effect of TCTP-induced Na,K-ATPase inhibition in the TCTP-TG because an elevated incidence rate of cataracts was found in TCTP-TG mice of C57BL/6N inbred strain, compared to that of non-TG (7.38% vs. 1.47%) at 10 weeks after birth [41]. Similar to the majority types of human cataracts, TCTP-TG showed mixed types of nuclear, cortical, and anterior subcapsular opacification [41]. For still unknown reasons, this phenotype was not prominent in C57BL6×CBA hybrid TCTP-TG although both strains of TCTP-TG elicited hypertension as a pathophysiological consequence of Na,K-ATPase inhibition [41]. This mode of cataractogenesis operates in human lens epithelial cells because TCTP overexpression induced an increase in cytosolic Ca2+ mobilization upon histamine stimuli via sodium pump inhibition, which might induce the proteolytic activity of enzymes (Figure 2). The transient overexpression of TCTP in lens epithelial cells did not affect the expression of the Na,K-ATPase α-subunit [41].

Interestingly, TCTP appears to affect the normal eye development because some TCTP-TG mice showed abnormal eye development, including unusual sizing of the eyes, and even absence of the eye [41]. This intriguing observation emphasizes the need to identify the fundamental roles of TCTP in the developmental processes of the eyes.


**2.3 Synthesis of [(***n***-C4H9)4N]4[α-PW11{Al(OH2)}O39]**

Cs7[γ-PW10O36]19H2O (2.00 g; 0.538 mmol) was dissolved in water (600 mL) at 40 °C, and solid Al(NO3)39H2O (0.250 g, 0.666 mmol) was added to the colorless clear solution. After stirring for 1 h at 40 ºC, a solid [(*n*-C4H9)4N]4Br (12.14 g; 37.7 mmol) was added to the solution, followed by stirring at 25 °C for 3 days. The white precipitate was collected on a glass frit (G4) and washed with water (ca. 1 L). At this stage, a crude product was obtained in a 1.662 g yield. The crude product (1.662 g) was dissolved in acetonitrile (10 mL), followed by filtering through a folded filter paper (Whatman #5). After the product was left standing for a week at 25 °C, colorless platelet crystals were formed. The obtained crystals weighted 0.752 g (the yield calculated considering that [mol of [(*n*-C4H9)4N]4[α-PW11{Al(OH2)}O39]/[mol of Cs7[γ-PW10O36]19H2O] 100 was 36.9%). The elemental analysis results were as follows: C, 20.73; H, 4.00; N, 1.58; P, 0.84; Al, 0.77; W, 54.6; Cs, <0.1%. The calculated values for [(*n*-C4H9)4N]4[α-PW11{Al(OH2)}O39] = C64H146AlN4O40PW11: C, 20.82; H, 3.99; N, 1.52; P, 0.84; Al, 0.73; W, 54.77; Cs, 0%. A weight loss of 2.16% was observed in the product during overnight drying at room temperature under 10-3–10-4 Torr before the analysis, thereby suggesting the presence of two weakly solvated or adsorbed acetonitrile molecules (2.18%). TG/DTA under atmospheric conditions showed a weight loss of 31.0% with an exothermic peak at 337 °C was observed in the temperature range from 25 to 500 °C; our calculations indicated the presence of four [(C4H9)4N]+ ions, two acetonitrile molecules, and a water molecule (calcd. 28.4%). The results were as follows: IR soectroscopy results (KBr disk): 1078s, 964s, 887s, 818s, 749m, 702w, 518w cm–1; 31P NMR (25C, acetonitrile-*d*3): δ -12.5; 27Al NMR (25 C, acetonitrile-*d*3): δ 16.1; 183W NMR (25 C, acetonitrile-*d*3): -56.2 (2W), -93.1 (2W), -108.6 (2W), -115.8 (2W), -118.5 (1W), -153.9 (2W).


New Developments in Behavioral Pharmacology

*Jonathan Cueto-Escobedo, Fabio García-García, Caio Maximino and Juan Francisco Rodríguez-Landa*

### **Abstract**

Behavioral pharmacology research has been a cornerstone in the understanding of the processes that underlie the behavior of living organisms as well as the biological basis of the behavioral, emotional, and cognitive disorders that affect humans. The findings in this area have helped to explore the potential therapeutic effects of several substances for the treatment of the mentioned disorders. The present chapter brings an extremely brief introduction to this vast area. First, we try to put in context behavioral pharmacology and its relevance and then show some brief examples of how this discipline has developed over the years. Second, we review the concept of a "research model" in preclinical behavioral pharmacology, given the importance of animal models and tests in this area, followed by a brief review of the recent advances using zebra fish as a valuable tool of research. Third, more specific examples are aborded, such as the findings on sleep disorders and those related to sexual hormones and menopause.

**Keywords:** behavioral pharmacology, psychopharmacology, psychoactive drugs, behavioral models

### **1. Introduction**

Every time academics talk about the evolution of human societies and the advance of humanity, language is always mentioned, followed by different pieces of technology that allowed us to change the world. Few times, medicine is mentioned, and within the same area of knowledge, pharmacology is even more frequently omitted. But without the development of pharmacology as a science founded in systematic research, the capacities of medical sciences and therapeutics would be very limited. Knowledge in pharmacology allows us to understand that there exist chemical substances with very specific structures and properties which, in controlled doses, can interact with the normal physiology of our organism in order to produce effects that improve our health, known as therapeutic effects; but if the doses are insufficient or excessive, the effects will be useless or harmful (toxic), respectively [1]. These substances responsible for the actions of medicines are named as active compounds.

Most of the active compounds used in medicine were consumed together with the organism which contained them, most frequently plants. As chemistry advanced, scientists succeed in isolating these compounds and described their chemical structure. In consequence, laboratories started to synthesize these

substances and others with a similar structure that should be tested in research laboratories before using them to treat diseases in humans [2].

Nowadays, pharmacological research has grown beyond treatments for infectious agents, covering diseases related to the alteration of the normal functioning of the central nervous system (CNS). There are medications to treat disorders such as depression, anxiety, chronic pain, attention deficit and hyperactivity disorder, epilepsy, and Parkinson's disease, and new drugs are desperately sought to stop Alzheimer's disease. On the other hand, one of the most important current health problems is related to the addictive behaviors triggered by the consumption of certain substances and the side effects of these addictions: respiratory and cardiovascular diseases in the case of tobacco, metabolic diseases in the case of alcoholism and addictive consumption of refined sugars, infectious diseases in the case of injected drugs, and many others that are not mentioned here. Without losing sight of the fact that addiction is itself a disease of the nervous system with devastating effects *per se* on the patient's quality of life. In several countries, prescription of different therapeutic agents acting on the CNS to treat psychiatric disorders, such as antidepressants, antipsychotics, and stimulants, has increased [3, 4] as in the case of methylphenidate and amphetamines in different countries such as United States [5] and the Netherlands [6]. The same way, antidepressant users have increased markedly around the world in countries such as Norway, Sweden, and Denmark [7], among others. Additionally, the use of different substances of abuse such as tobacco [8] and marijuana has increased in the population [9]. Also, the development of new technologies and products has a significant impact on mental health as the discovery of Internet addiction [10] and the addictive consumption of refining sugar [11, 12], which impacts on the behavior of subjects. All these make important the continuous development of behavioral pharmacology in order to cope with the challenges in mental health.

#### **2. Development of behavioral pharmacology**

Behavioral pharmacology, also known as psychopharmacology, has developed as an interdisciplinary science that comprises fields such as neuroethology, neurochemistry, pharmacology and neuropharmacology, psychophysiology, neurophysiology, experimental analysis of behavior, and several other fields related to neurosciences [13]. Behavioral pharmacology is founded on systematic research with precise methods for assessing and interpreting the effects of chemical, hormones, and drugs on the behavior in humans and experimental animals in order to establish its potential as therapeutic agents or pharmacologic tools to explore how the brain functions and the underlying neurobiological mechanism of cognition, emotions, and behavior. Behavioral pharmacology must thus be an integral component of many neuroscience research programs [14].

In this sense, the development of behavioral pharmacology comprises the development of areas as pharmacology and psychology, experimental analysis of behavior, and recently neuroscience. For a historical review, see [14–16]. However, research in behavioral pharmacology can be summarized in: (1) the development of procedures to screen pharmacological agents for potential clinical effectiveness. (2) Perfecting behavioral techniques to explore the mechanisms of action of behaviorally active drugs and using these chemicals and drugs as tools for the analysis of complex behaviors (i.e., when drugs reinforce behavior and when drugs serve as discriminative stimuli) [16] (see **Table 1**). Therefore, drugs are not only a subject of study, because of its behavioral effects but are also a piece of technology that helps to elucidate how behaviors are controlled by living organisms.

**9**

**3. Measuring behavior**

**Table 1.**

Behavior is a biological property of organisms, which remarks on the significance of the study of drug-behavior interactions [15]. Maybe, a great example of the impact of behavior beyond psychology is the research by ethologists K. Lorenz, N. Tinbergen, and K. von Frisch, which focused on the analysis of behavior in several species including fish, insects, and birds, and the importance of which made them worthy of the Nobel price of medicine in 1973 "*for their discoveries concerning*

**Year Description Reference**

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

1936 Selye H. described the impact of several types of adverse stimuli on animal health, in the form of a syndrome characterized by three phases: alarm, adaptation, and exhaustion, which can lead to death if stimuli are maintained. This syndrome was later named as the stress response which has been intensively studied and strongly associated with the impairment of brain function in

animals or the development of mental disorders in humans

1972 The first study to administrate Delta-9-tetrahydrocannabinol in humans to test

1977 The forced swim test is proposed as a behavioral tool to explore the effects of

1986 Elevated plus maze is developed as a tool to measure anxiety-like behaviors of the rat and test substances with potential anxiolytic effects

1988 Modafinil was prescribed for the first time for the treatment of narcolepsy and

ovariectomized rats (OVX). These rats were more sensitive to stress, which was associated with a low concentration of steroid hormones. This effect was

12-week post-ovariectomy there is more anxiety-like behavior than a 3-week

condition that triggers despair behavior (immobility)

2005 This study explored the behavioral and neuronal response to stress in

sleep parameters, but some adverse effects were reported

experimental model of surgical post-menopause

2006 Anxiety-like behavior is dependent on the post-ovariectomy time frame. At

2016 The first systemic review and meta-analysis that discuss the effects of the orexin agonist Suvorexant for the treatment of insomnia. Suvorexant improved some

2019 In this study, it was identified that at 3-week post-ovariectomy appears anxietylike behavior, but from 6-week post-ovariectomy in addition to anxiety-like behavior, also increases depression-like behavior in rats, supporting an

the effects on sleep patterns is carried out. The results show a decrease in sleep onset latency. To date, there are controversial results about the positive effects

antidepressant drugs in rats and mice that are exposed to a stressful inescapable

The first step in all behavioral sciences has been to define what is behavior; it could seem an easy task, but historically many different definitions of behavior have been used by scientists over the time, and even the knowing of a unique definition is elusive and may be useless for every different area such as psychology, ethology, and experimental analysis of behavior, among others; for review see [26, 27]. As mentioned before, one of the directions of behavioral pharmacology was the development of procedures to screen the effects of pharmacological agents on specific behaviors under controlled environments. This approach allows scientists to work with operational definitions of specific behaviors, for example, exploration can be

*organization and elicitation of individual and social behaviour patterns*."

*New Developments in Behavioral Pharmacology DOI: http://dx.doi.org/10.5772/intechopen.93700*

the cannabis on sleep quality

idiopathic hypersomnia in patients

post-ovariectomy

*Emblematic research in behavioral pharmacology.*

prevented by restitution with 17-β estradiol

*New Developments in Behavioral Pharmacology DOI: http://dx.doi.org/10.5772/intechopen.93700*

*Behavioral Pharmacology - From Basic to Clinical Research*

challenges in mental health.

ence research programs [14].

**2. Development of behavioral pharmacology**

laboratories before using them to treat diseases in humans [2].

substances and others with a similar structure that should be tested in research

Nowadays, pharmacological research has grown beyond treatments for infectious agents, covering diseases related to the alteration of the normal functioning of the central nervous system (CNS). There are medications to treat disorders such as depression, anxiety, chronic pain, attention deficit and hyperactivity disorder, epilepsy, and Parkinson's disease, and new drugs are desperately sought to stop Alzheimer's disease. On the other hand, one of the most important current health problems is related to the addictive behaviors triggered by the consumption of certain substances and the side effects of these addictions: respiratory and cardiovascular diseases in the case of tobacco, metabolic diseases in the case of alcoholism and addictive consumption of refined sugars, infectious diseases in the case of injected drugs, and many others that are not mentioned here. Without losing sight of the fact that addiction is itself a disease of the nervous system with devastating effects *per se* on the patient's quality of life. In several countries, prescription of different therapeutic agents acting on the CNS to treat psychiatric disorders, such as antidepressants, antipsychotics, and stimulants, has increased [3, 4] as in the case of methylphenidate and amphetamines in different countries such as United States [5] and the Netherlands [6]. The same way, antidepressant users have increased markedly around the world in countries such as Norway, Sweden, and Denmark [7], among others. Additionally, the use of different substances of abuse such as tobacco [8] and marijuana has increased in the population [9]. Also, the development of new technologies and products has a significant impact on mental health as the discovery of Internet addiction [10] and the addictive consumption of refining sugar [11, 12], which impacts on the behavior of subjects. All these make important the continuous development of behavioral pharmacology in order to cope with the

Behavioral pharmacology, also known as psychopharmacology, has developed as an interdisciplinary science that comprises fields such as neuroethology, neurochemistry, pharmacology and neuropharmacology, psychophysiology, neurophysiology, experimental analysis of behavior, and several other fields related to neurosciences [13]. Behavioral pharmacology is founded on systematic research with precise methods for assessing and interpreting the effects of chemical, hormones, and drugs on the behavior in humans and experimental animals in order to establish its potential as therapeutic agents or pharmacologic tools to explore how the brain functions and the underlying neurobiological mechanism of cognition, emotions, and behavior. Behavioral pharmacology must thus be an integral component of many neurosci-

In this sense, the development of behavioral pharmacology comprises the development of areas as pharmacology and psychology, experimental analysis of behavior, and recently neuroscience. For a historical review, see [14–16]. However, research in behavioral pharmacology can be summarized in: (1) the development of procedures to screen pharmacological agents for potential clinical effectiveness. (2) Perfecting behavioral techniques to explore the mechanisms of action of behaviorally active drugs and using these chemicals and drugs as tools for the analysis of complex behaviors (i.e., when drugs reinforce behavior and when drugs serve as discriminative stimuli) [16] (see **Table 1**). Therefore, drugs are not only a subject of study, because of its behavioral effects but are also a piece of technology

that helps to elucidate how behaviors are controlled by living organisms.

**8**

#### **Table 1.**

*Emblematic research in behavioral pharmacology.*

## **3. Measuring behavior**

Behavior is a biological property of organisms, which remarks on the significance of the study of drug-behavior interactions [15]. Maybe, a great example of the impact of behavior beyond psychology is the research by ethologists K. Lorenz, N. Tinbergen, and K. von Frisch, which focused on the analysis of behavior in several species including fish, insects, and birds, and the importance of which made them worthy of the Nobel price of medicine in 1973 "*for their discoveries concerning organization and elicitation of individual and social behaviour patterns*."

The first step in all behavioral sciences has been to define what is behavior; it could seem an easy task, but historically many different definitions of behavior have been used by scientists over the time, and even the knowing of a unique definition is elusive and may be useless for every different area such as psychology, ethology, and experimental analysis of behavior, among others; for review see [26, 27]. As mentioned before, one of the directions of behavioral pharmacology was the development of procedures to screen the effects of pharmacological agents on specific behaviors under controlled environments. This approach allows scientists to work with operational definitions of specific behaviors, for example, exploration can be

*Behavioral Pharmacology - From Basic to Clinical Research*

measured by scoring ambulation, rearing or nose approaching to an object; sexual behavior can be measured by conditioned place preference, number of mounts, latency and number of ejaculations. All these behaviors are normally studied under controlled environments that are designed specifically to the required behavioral display and every feature of the environment; the experimental subjects or chemical agents with probed effects on humans have been studied in this environment with the purpose of establishing these manipulations as models of a specific behavior (see **Table 2**) as spatial learning and memory, or models of specific pathologies behaviorally expressed as is the case of anxiety [28], depression [29], obsessive compulsive disorder [30], Parkinson [31], epilepsy [32] or addictive behaviors [33], and sleep deprivation [34], among others.