**2.2 Clinical manifestations**

Diabetes can damage different parts of the peripheral nervous system with distal symmetric polyneuropathy (DSP) being the most common presentation. The symptoms are symmetric and with predominant sensory symptoms over motor involvement. Sensory symptoms such as numbness, tingling, and pain are common in DSP patients. These characteristics begin in the feet and spread proximally in a length-dependent pattern known as stocking-and-glove distribution [24]. Other patterns of injury include small-fiber predominant neuropathy, radiculo-plexopathy and autonomic neuropathy, among others.

DSP has an effect on the physical and emotional well-being of patients. Sensory loss caused by DSP often causes trouble walking, which can lead to falls. DSP is one of major risk factors for falls in diabetic patients along with retinopathy and vestibular dysfunction. Diabetic DSP patients are 2–3 times more likely than diabetics without neuropathy to fall. Diabetes is the leading cause of lower extremity amputations, with a 15-fold increase in the likelihood of this life-changing complication. Moreover, 80,000 lower extremity amputations are performed each year in patients with diabetes [25–27].

Neurogenic pain, numbness, a lack of control of voluntary movements, and a susceptibility to foot ulceration that contributes to infections and toe or foot amputations are all signs of diabetic neuropathy. Diabetic patients have a 15-fold higher risk of toe or foot amputations than nondiabetic patients [28].

According to Toronto Consensus Panel on Diabetic Neuropathy, DPN is defined as a symmetrical, length-dependent sensorimotor polyneuropathy that develops in the background of long-standing hyperglycemia, associated de-arrangements, and cardiovascular risk factors [29]. Different studies reported that some patients with prediabetes develop neuropathic complications, whereas others reported little evidence of neuropathy even after long-standing diabetes. This observation confirms the involvement of genetic etiological factors associated with the development of DPN [30].

Neuropathic pain is one of the major disabling symptoms of patients with DSP. It is estimated that diabetic neuropathic pain (DNP) develops in 10–20% of the diabetic population overall and can be found in 40–60% with documented neuropathy. Like other types of neuropathic pain, DNP is characterized by burning, electric, and stabbing sensations with or without numbness [31–33]. It is characteristically more severe at night often resulting in sleep disturbance. Together with painful symptoms during the day, this often leads to a reduction in quality of life. In one study, the burden of painful DPN was reported to be significant, resulting in persistent discomfort following polypharmacy and high resource usage, as well as limitations in everyday activities and dissatisfaction with treatments that were frequently deemed ineffective. DPN that is chronic, persistently painful, and highly distressing is linked to severe depression, anxiety, and sleep loss [34, 35]. Other types of diabetic neuropathies includes small-fiber polyneuropathy, mononeuropathy, mononeuropathy multiplex, radiculopathy, plexopathy (diabetic amyotrophy), autonomic neuropathy, and treatment-induced neuropathy.


2. Pharmacokinetics of catechins

Minor constituents include caffeine, theobromine, and theophylline. Among green tea catechins, EGCG is the most abundant and biologically active based on animal and human studies [1, 2].

Health benefits are evident for green tea catechins including anticancer [4] and antimicrobial activities [5]. Cancer prevention has been found in the colon, duodenum, esophagus, stomach, large intestine, liver, lung, mammary glands, and skin [6, 7]. There were in vitro anticancer effects on adult T-cell leukemia (ATL) caused by a latent infection of human retrovirus HTLV-1 [8]. Drinking green tea could reduce HTLV-1 provirus load in asymptomatic HTLV-1 carriers [9]. Although the underlying mechanisms for cancer-prevention is not well understood, their antioxidation, free radical scavenger activities [10], and NF-κB inhibitory effect [11] have been

Although biological and intervention studies have indicated various beneficial effects, results of clinical studies are not conclusive [12, 13]. Inconsistent findings may be attributed to variations of methodologies and study conditions such as differences in infusion techniques, consumption behavior, production methods, compositions of green tea constituents, and absorption profiles, among different study cohorts. Pharmacokinetics of green tea catechins have been studied using defined green tea catechin extract (GTE), Polyphenon E, and EGCG with variable doses of administration [14, 15]. Oral bioavailability of tea catechins in human plasma was found having 5 to 50 times lower than the level needed to exert biological activities in in vitro systems [16, 17]. Other studies showed very high free EGCG peak plasma concentrations, 300, 1970, and 2020 ng/mL, after ingestion of 3, 5, or 7 capsules of Sunphenon DCF-1 (containing 225, 375, and 525 mg EGCG), which is a green tea extract obtained by a defined protocol [18, 19]. Dosage affects the bioavailability of catechins. Many studies showed large

The biological activity is attributed to its structure moiety and hydroxyl groups [3].

18 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

attributed as the major factors.

Figure 1. Structures of catechins.

It is essential to know the effective concentrations and forms of catechins present in plasma and tissues after ingestion. Pharmacokinetics is a study of the absorption, distribution, metabolism, elimination, and bioavailability of catechins following administration. In brief, after oral administration of green tea or extract, catechins are absorbed from the small intestine and remaining excess catechins pass to the colon. In the small intestine, catechins are conjugated with glucuronic acid, sulfate or by O-methylation. Some catechins with secreted bile in an enterohepatic recirculation process pass into the colon and are degraded into different flavonoid rings by resident microorganisms. The catabolized phenolic acids can be reabsorbed into the circulation and excreted into urine. Catechins, their conjugated metabolites, and a large amount of catabolized small phenolic acids can be distributed to various organs and tissues, absorbed by tissue cells, further metabolized, and perform various biological actions (Figure 2).

Figure 2. Schematic diagram showing the absorption, distribution, metabolism, elimination of catechins in the body. Thick and fill arrows indicate the flow of large amount of colon metabolites. Thin and hollow arrows depict low amount of present catechins flowing in the body.


Regarding 5-HT2C receptor, Vimala et al. (2014) highlight that epilepsy affects negatively cognitive function, producing depression, anxiety, etc. Mentioning among other issues that agomelatine is a novel antidepressant acting as melatonin MT1 and MT2 receptor agonist and 5-HT2C receptor antagonist; producing reduction in the depolarization-evoked release of glutamate, strong neuroprotective action and possible antioxidant effects (Vimala et al., 2014); producing hippocampal neuronal cell survival and neurogenesis, neuroprotective effect in hippocampus and frontal cortex and the antioxidant potential may contribute to the protective action of agomelatine against epilepsy induced memory decline (Vimala et al., 2014). In addition, Walker and Foley (2010) report that administration of the 5-HT2C inverse agonist mianserin impaired autoshaped operant response on day 2 than any other agent tested. In addition, decreasing the length of the acquisition session to 1-h augmented the difficulty of the autoshaping task further modulating the consolidation effects produced by the 5-HT2C ligands (Walker and Foley, 2010). Moreover, Li et al. (2015b) report that repeat exposition to 2.856 GHz microwaves (averaging 5–30 mW/cm2 ) affects spatial learning and

#### TABLE 5 | 5HT3 receptor antagonist, neuroprotection.

AD, Alzheimer's disease; MCI, middle cognitive impairment; MWM, Morris water maze.

memory function, morphology structure of the hippocampus, electroencephalogram (EEG) and neurotransmitter content (amino acid and monoamine); including expression of 5- HT1A 2A, and 2C receptors. Li et al. (2015b) demonstrated that chronic exposure to microwave could induce dose-dependent deficit of spatial learning and memory and inhibition of brain electrical activity, the degeneration of hippocampus neurons, and the disturbance of neurotransmitters; including hippocampal and cortical expression of 5-HT1A and 5-HT2C receptors.

Importantly, 5-HT2A/2B/2C receptors are useful detecting learning and memory changes and drug effects. Aloyo et al. (2009) remind us of inverse agonism at 5-HT2A and 5-HT2C receptors.

#### 5-HT3 Receptor

5-HT3 receptor antagonists (e.g., tropisetron, ondansetron) have a long dated antiamnesic effects, including attenuation of ageassociated memory impairment (e.g., Costall and Naylor, 1992; see also Shimizu et al., 2013). Recent evidence, from preclinical studies suggests that the interaction between amyloid-β peptides (Aβ) and the α7 nicotinic acetylcholine receptor (α7 nAChR) (Hashimoto, 2015) (**Table 5**). And tropisetron is also a α7 nAChR agonist and 5-HT3 receptor antagonist; binding to amyloid precursor protein and enhancing memory in AD patients (**Table 5**). Importantly, 5-HT3 receptor antagonists have been useful in treatments such as chemotherapy-induced emesis to neuroprotection (Fakhfouri et al., 2014; Hashimoto, 2015). Certainly, subtypes of 5-HT3 receptor exist (Thompson, 2013); and their mechanisms are complex. For instance, Kozuska et al. (2014) deal with the multiple salt bridges in the intracellular domain of the 5HT3A receptor and these interactions increase the overall rigidity of the receptor, stabilize its low conducting state and affect the ligand cooperativity; suggesting that the allosteric effects of these regions on the receptor may be involved in a possible "reverse" allosteric modulation of 5HT3 receptor. In addition, it should be noted that agonist- and antagonistinduced up-regulation of surface 5-HT3A receptor (Morton et al., 2015).

Moreover, Kondo et al. (2014) studied 5-HT3A receptor subunit-deficient (htr3a-/-) mice revealing loss of exerciseinduced hippocampal neurogenesis and antidepressant effects, but not of learning enhancement (**Table 5**). Kondo et al. (2014) conclude that the 5-HT3 receptor is the critical target of 5-HT action in the brain following exercise, being indispensable for hippocampal neurogenesis and antidepressant effects induced by exercise.

#### 5-HT4 receptor

It should be noted that earlier evidence indicated that 5-HT4 receptor decreased in AD (see Eglen et al., 1995). Activation of 5-HT4 receptor has pro-cognitive effects on memory tasks (e.g., Bockaert et al., 2011; Peñas-Cazorla and Vilaró, 2014; Ramirez et al., 2014; Claeysen et al., 2015). Notably, Madsen et al. (2011) observe cerebral 5-HT4 receptor up-regulation starts at a preclinical stage of dementia and it continues while dementia is still at a mild stage and these authors speculate that this upregulation may be a compensatory effect of decreased levels of interstitial 5-HT, increase acetylcholine release or to counteract Aβ accumulation and improved cognitive function. Hippocampal 5-HT4 receptor expression correlates inversely with human memory (Haarh et al., 2013; **Table 6**). Also, old rats have decreased 5-HT4 receptor expression and poor memory relative to adult (**Table 6**).

In addition, evidence suggests that serotonergic activity, via 5-HT4 receptors in hippocampal, striatum, and cortical areas, mediates memory function and provides further evidence for a complex and regionally specific regulation over 5-HT receptor expression during memory formation (Manuel-Apolinar et al., 2005).

Segu et al. (2010) report adaptive changes in cholinergic systems, which may circumvent the absence of 5-HT4 receptor to maintain long-term memory under baseline conditions. In contrast, despite of adaptive mechanisms, the absence of 5-HT4 receptor aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HT4 receptor mediates a tonic influence on ChAT activity and muscarinic receptors remain to be determined (Segu et al., 2010). Restivo et al. (2008)

#### TABLE 6 | 5HT4 receptor.

AD, Alzheimer's disease.

highlight that pharmacological modulation of synaptic efficacy is a prominent target in the identification of promnesic compounds and that pre-training administration of the 5-HT4 receptor partial agonist SL65.0155 enhances olfactory discrimination and potentiates learning-induced dendritic spine growth in the mouse hippocampus; without affecting spine density in the pseudo-trained mice and, by itself, it does not promote spine growth. Likewise, the 5-HT4 receptor antagonist RS39604 prior to SL65.0155 prevents both improved memory and additional formation of spines; thus confirming the 5-HT4 receptor specificity of the observed effects (Restivo et al., 2008); and these authors conclude that 5-HT4 receptor stimulation selectively increases experience-dependent structural plasticity in learningactivated hippocampal circuits.

Marchetti et al. (2011) have also highlighted that in developing rats as well as in rats ranging from 3 to 9 months of age, significant modifications of 5-HT4 receptor expression have been observed (for references see Marchetti et al., 2011). These same authors propone that the poor memory formation observed in aged rats (Marchetti et al., 2011). And corresponding decreases in 5-HT4 receptor expression in brain areas (e.g., hippocampus, amygdala, etc.) involved in memory formation, could explain improved memory, dendritic spines (Restivo et al., 2008), neuronal excitability and release of the neurotransmitter acetylcholine (Ach) (see Segu et al., 2010; Marchetti et al., 2011; Peñas-Cazorla and Vilaró, 2014). Clearly, 5-HT4 receptor is useful neural marker of dysfunctional and memory formation as well as therapeutic target. Moreover, studying 5-HT expression during memory formation is giving new fresh insights (e.g., Haahr et al., 2013). Importantly, Haahr et al. (2013) report that hippocampal 5-HT4 receptor expression correlates inversely with human memory performance.

#### 5-HT5

As mentioned above, Cavallaro (2008) reported that passive avoidance memory involves expression of several 5-HT receptors, including 5-HT5A. 5-HT5 receptor occurs in brain areas implicated in learning and memory. Post-training administration of the 5-HT5A receptor antagonist SB-6995516 decreased CR during short-term (STM; 1.5-h; at 0.1 mg/kg) and long-term memory (LTM; 24-h; at 3.0 mg/kg). Moreover, considering that there are no selective 5-HT5A receptor agonists, next, diverse doses of the serotonin precursor l-tryptophan were studied during STM and LTM, showing that l-tryptophan (5–100 mg/kg) facilitated performance, particularly at 50 mg/kg. In interactions experiments, l-tryptophan (50 mg/kg) attenuated the impairment effect induced by SB-699551 (either 0.3 or 3.0 mg/kg) (Gonzalez et al., 2013). All together this evidence suggests that the blockade of 5-HT5A receptor appear to be able to impair STM and LTM (24 h) in autoshaping task, while its stimulation might facilitate it. Of course further investigation is necessary, meanly with selective 5-HT5A compounds (Gonzalez et al., 2013). Interestingly, Yamazaki et al. (2014, 2015) reported that a 5-HT5A receptor antagonist ameliorates positive symptoms and cognitive impairment in animal models of schizophrenia and in aged rats and induced-amnesia. An analogous case is observed regarding 5-HT1A partial agonists (see above).

Returning to 5-HT5 receptor, Karimi et al. (2013) report that it has long been known that hippocampal spatial memory and the ability to navigate through space are sexually dimorphic traits among mammals, and numerous studies have shown that these traits can be altered by means of sex hormone manipulation. Male and female rat pups were injected with estradiol and testosterone respectively, at early stage of their lives to examine the effect of sex hormone manipulation on mRNA expression of Slc9a4, Nr3c2, Htr5b, and Mas1; among other results, these authors report that expressions of these genes are strongly influenced by sex hormones in both the frontal cortex and hippocampus, especially in male hippocampus, in which expression of all genes were up-regulated. Htr5b was the gene that was affected only in the males (Karimi et al., 2013). Hence, considering the pharmacological evidence mentioned above, probably learning and memory might be affected in these animals.

### 5-HT6 receptor

Diverse 5-HT6 receptor antagonists produce promnesic and/or antiamnesic effects in conditions, such as memory formation, age-related cognitive impairments; memory deficits in models of diseases such as schizophrenia, PD and AD (e.g., King et al., 2008; Claeysen et al., 2015). However, not all papers report promnesic and/antiamnesic effects of 5-HT6 receptor antagonists (e.g., Thur et al., 2014) (**Table 7**); probably related to timing, drug and memory task used. Memory, aging, and AD modify 5-HT6 receptors and signaling cascades; and 5-HT6 drugs modulate memory, which is accompanied with neural changes. Indeed, in an elegant work Eskenazi et al. (2015) manipulated selectively overexpression of 5-HT6 receptor in either direct or indirect pathway striatal mediumspiny neurons (dMSN and iMSN, respectively), revealing that increased 5-HT6 receptor expression in iMSNs delays instrumental learning and in DLS facilitates behavioral flexibility after habitual responding. It should be noted that 5-HT6 receptor expression decreases during memory (e.g., Huerta-Rivas et al., 2010; Ramirez et al., 2014). In addition, de Bruin and Kruse (2015) suggest that cognition could be improved by 5-HT6 receptor antagonists, by increasing the number of NCAM PSA-immunoreactive neurons in the dendate gyrus, inhibit mTOR and Fyn-tyrosine kinase and interact with DARPP-32.

Notably, 5-HT6 receptor antagonists are, among, serotonergic therapies for cognitive symptoms in AD (e.g., Ramirez et al., 2014). Indeed, Wilkinson et al. (2014) report safety and efficacy of idalopirdine, a 5-HT6 receptor antagonist, in patients with moderate AD. In addition, 5-HT6 receptor is providing new insights about plasticity (Dayer et al., 2015). For example, at early stages of neuronal development, expression of 5-HT6 receptor constitutively regulates the activity of the cyclin-dependent kinase (Cdk) 5 and, through this mechanism, controls cellular processes involved in circuit formation (e.g., neuronal migration, neurite outgrowth). In addition, 5-HT6 receptor modulates developmental targets, including Fyn, Jab1, and mammalian target of rapamycin (mTOR). In therapeutic terms such as blockade of pathological over-activation of the mTOR pathway induced by early life insults in rodents and normalizes the associated social and episodic memory deficits. It should be noted that 5-HT6 receptor and Cdk5; and the latter mediates neuronal differentiation (e.g., hippocampus, striatum) in an agonist-independent manner (Seo and Tsai, 2014). In addition, Ha et al. (2015) report that 5-HT6 receptor directly interacts with SNX14 (protein-coupled receptors/regulators of G protein signaling), which regulates internalization; degradation of 5-HT6 receptor and cAMP production. This finding might be related to the evidence that 5-HT6 receptor agonists and antagonists modulate cAMP production and improve memory formation (e.g., Meneses et al., 2011c). We do not know yet why 5- HT6 receptor agonists and antagonists (e.g., Woods et al., 2012) may facilitate memory or may reverse amnesia in some memory tasks. However, 5-HT6 receptor inverse agonist might be useful (e.g., Hostetler et al., 2014; but see also Benhamú et al., 2014).

AD, Alzheimer's disease; Cdk5, cyclin-dependent kinase; mTOR, mammalian target of rapamycin; dMSN, direct or indirect, iMSM pathway medium-spiny neurons.

#### 5-HT7 Receptor

Recently Nikiforuk (2015) is providing perspectives of 5-HT7 receptor in the search fortreatments for CNS disorders: including normal and dysfunctional serotonin-induced phase shifting of the circadian rhythm control of memory as well as locomotor and exploratory activity, anxiety, depression; and Guseva et al. (2014) about molecular mechanisms responsible for the 5-HT7 receptormediated signaling. Gasbarri and Pompili (2014) noted that 5- HT7 receptor antagonism might have antiamnesic effects (see also Horisawa et al., 2013). Gasbarri et al. (2008) suggested that 5- HT7 receptor blockade had procognitive effect, when the learning task implicated a high degree of difficulty. Others report that 5- HT7 receptor agonists facilitate memory and have antiamnesic effects (**Table 8**); remaining clarifying why of the paradoxical effects.

Notably, Saroja et al. (2014), highlight that although evidence about monoamine receptor (MAR) biochemistry and pharmacology in aging exists, work on MAR complexes rather than subunits is limited; in consequence, MAR complexes in hippocampi of three different age groups (3–12 and 18 months) in mice and to link MAR changes to spatial memory retrieval in the water maze were determine (**Table 8**). MAR complexes were separated in order to show the pattern of dopamine and 5-HT1A and 5-HT7 receptors and colocalizations (Saroja et al., 2014). For instance, D1-D2 and 5-HT7 receptors containing receptor complex levels decreased with age while 5-HT1A receptorcontaining complex was increasing. D1, 5-HT7, and 5-HT1A receptor complex correlated with good retrieval memory in the water maze; hence, individual monoamine receptors are linked to spatial memory and are modulated by age. However, Beaudet et al. (2015) mention that changes in the level of transcription of the 5-HT7 receptor mRNA did not account for the age-related difference observed at the protein level, at least in hippocampal CA3 region; besides, 5-HT7 receptor might also be putatively subjected, across aging, to modifications in their affinity or to changes in their coupling to G-proteins or other signaling

MWM, Morris Water Maze; MAR, monoamine receptor complex (i.e., D1, D2, and 5-HT7 containing receptors).

pathways. Notably, Beaudet et al. (2015) suggest that **a** decreased expression of 5-HT7 receptor in CA3 hippocampal could account for impairments of the shift between spatial strategies across aging (**Table 8**).

Moreover, when the time-course (0–120 h) of autoshaped CR is progressive; then mRNA 5-HT1A or 5-HT7 receptors expression is monotonically augmented or decreased in prefrontal cortex, hippocampus and raphe nuclei, respectively (Perez-Garcia and Meneses, 2009). Hence, 5-HT1A and 5-HT7 receptors expression might be regulated by the level of memory formation and to be brain areas dependent. Moreover, the cyclic adenosine monophosphate (cAMP) is a second messenger and a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory (e.g., Kandel, 2001). And progressive time-course of memory formation in an autoshaping learning task (Pérez-García and Meneses, 2008); shows that ex-vivo cAMP production from trained and over-trained groups compared to untrained ones, the former group had the highest levels of cAMP and the latter rats showed increased production but less relative to trained rats. Importantly these changes varied according with normal memory or amnesia and brain areas; hence cAMP production is important in the signaling case in mammalian memory formation (Pérez-García and Meneses, 2008).

The above findings should be considered in the context that apparently 5-HT7 receptor agonists and antagonist (e.g., Nikiforuk, 2015) might facilitate memory formation and/or have anti-amnesic effects. Other interesting recent finding is that according with Rojas et al. (2014) serotonin regulates neurite outgrowth through 5-HT1A and 5-HT7 receptors in cultured hippocampal neurons. Certainly, De Filippis et al. (2015) highlight that promnesic effects of the 5-HT7 receptor agonist LP-211 treatment strongly depend on the basal level of performance. Notably, Ruocco et al. (2014) report that 5-HT7 receptor stimulation improves selective spatial attention and produces permanent changes in several neural markers, including expression of glutamatergic receptors and dopamine transporter (DAT).

Very importantly, 5-HT7 receptor can form heterodimers with 5-HT1A receptors both in-vitro and in-vivo (see Guseva et al., 2014) and according with these authors, from the functional point of view, heterodimerization decreases Giprotein coupling of 5-HT1A receptor and attenuates receptormediate deactivation of G-protein-gated potassium (GIRK) channels, without substantial changes in the coupling of 5- HT7 receptor to the Gs-protein. Moreover, heterodimerization significantly facilitated internalization of 5-HT1A receptor, while internalization kinetics of 5-HT7 receptor was decelerated upon heterodimerization (see Guseva et al., 2014).

### Factors Responsible for Inconsistencies among Laboratories

BOX 1 | Factors responsible for inconsistencies among laboratories. Certainly, a number of factors might be produce similar results or be responsible for some inconsistencies among laboratories studying memory; which are

complex and multi focal; which should provide an analytic framework offering key clues. Indeed, analysis of memory should include behavioral tasks, type of memory, the dynamic hierarchy of neural markers and brain areas involved in memory formation (e.g., Euston et al., 2012; Eskenazi et al., 2015) vs. no training, amnesia, anti-amnesic effects or forgetting (e.g., see below). Likewise, the species and the nature of behavioral task (e.g., appetitively or aversively motivated), curves of behavioral acquisition (i.e., multi-trial or two trials task) or patterns of behavioral responses (progressive vs. all or none response), cognitive demand (easy or difficult task), timing of drug administration (pre-training, post-training or pretest) and kind of drug (e.g., agonist or antagonist), protocols of training and testing together with neurobiological markers (e.g., Duewer et al., 1995; Patton, 1995) accompanying mnemonic processes deserve attention. Among the behavioral memory tasks available (e.g., Peele and Vincent, 1989; Myhrer, 2003; Lynch, 2004); importantly, the implementation of new instruments for measuring memory in behavioral tasks assists in gaining deeper insight into learning and memory processes (e.g., Cook et al., 2004; Walker et al., 2011; Markou et al., 2013; Leger et al., 2014; Wolf et al., 2014).

#### Neural Transporters, Memory, Forgetting and Drugs

Notwithstanding neurotransmission systems are related to memory formation, amnesia and/or therapeutic targets for memory alterations, the role of transporters γ-aminobutyric acid (GABA, GAT1), glutamate (neuronal glutamate transporter excitatory amino acid carrier; EACC1), dopamine (DAT) and serotonin (SERT) is poorly understood. Emerging evidence indicates that memory formation (short- and long-term memory; STM and LTM, respectively) in a Pavlovian/instrumental autoshaping (see **Box 1**) is associated to up-regulation of prefrontal cortex GAT1 and EAAC1, striatal SERT, DAT and EACC1; while, hippocampal EACC1, GAT1, and SERT are downregulated (Tellez et al., 2012a,b; **Table 9**; **Figure 1**). Moreover, pharmacological analysis shows that methamphetamine (METH)- induced amnesia down-regulated SERT, DAT, EACC1, and GAT1 in hippocampus and the GAT1 in striatum; no-changes are observed in prefrontal cortex. Fluoxetine (antidepressant, 5-HT uptake inhibitor) improved memory consolidation (particularly LTM), which is associated to DAT, GAT1 (prefrontal cortex) up-regulation, but GAT1 (striatum) and SERT (hippocampus) down-regulation. Fluoxetine plus METH prevented amnesia, which was associated to DAT, EACC1 and GAT1 (prefrontal cortex), SERT and DAT (hippocampus) and EACC1 or DAT (striatal) up-regulation.

#### Memory Formation/Forgetting and SERT Expression

Forgetting in Pavlovian/instrumental autoshaping is associated to up-regulation of GAT1 (PFC and HIP) and DAT (PFC) while SERT (HIP) is down-regulated; no-changes are observed in striatum (**Table 9**). Methamphetamine alone not affected forgetting but up-regulates hippocampal DAT and EACC, prefrontal cortex DAT and striatal GAT1 or EACC1. Fluoxetine alone prevents forgetting, which is associated to striatal GAT1 and hippocampal DAT up-regulation, but prefrontal cortex GAT1 down-regulation. Fluoxetine plus METH prevent forgetting, which is associated to hippocampal DAT, prefrontal cortex SERT and striatal GAT1, DAT, or SERT up-regulation,

#### BOX 2 | Autoshaping tasks.

 According with Holland et al. (2014), "autoshaping" or "sign-tracking" phenomenon has recently attracted considerable attention as a platform for studying individual differences in impulsivity, drug sensitization, and other traits associated with vulnerability to drug addiction. Autoshaping has been also used for detecting effects induced by memory, amnesia, drugs, genetic variations, aging and neural markers (e.g., Tomie et al., 2003, 2012; Vanover et al., 2004; Rodriguez et al., 2008; Fitzpatrick et al., 2013; Markou et al., 2013; Talpos et al., 2014). Notably, autoshaping is an associative automatized learning task (see below), and during memory consolidation of Pavlovian/instrumental autoshaping learning task, dentate gyrus, hippocampal CA1, basolateral amygdaloid nucleus and prefrontal cortex are require (see below). It should be noted that an important innovation, and growingly popular method of assessing cognitive functions is the automated touchscreen platform (e.g., Abela et al., 2013; Talpos et al., 2014; Delotterie et al., 2015), used for diverse cognitive tasks, comparable those in employ in human subjects (Horner et al., 2013), including autoshaping (e.g., Gallistel et al., 2014; Talpos et al., 2014; Silverman et al., 2015).

Autoshaping learning tasks involve classical and instrumental conditioning (i.e., stimulus-stimulus and stimulus-responding conditioning). It should be mentioned that long-lasting memories are most efficiently formed by multiple training sessions separated by appropriately timed intervals. Autoshaping meets this criterion and it allows modeling of behavioral situations requiring integration of information obtained from sign- and goal-tracking settings; representing memory of self-taught settings (Meneses, 2013, 2014). Certainly, autoshaping tasks (Pavlovian or instrumental; and Pavlovian/instrumental may produce initial modest and/or variable levels of conditioned responses (CR). Importantly, memory formation, amnesia and forgetting in Pavlovian/instrumental paradigms are accompanied by changes in neural markers, including 5-HT, glutamate, dopamine, and GABA transporters expression levels (Tomie et al., 2003; Tellez et al., 2012a,b), 5-HT receptor expression and cAMP production (Meneses, 2013). Certainly, forgetting as therapeutic targets for dysfunctional memory it has been little explored. As above mentioned, similar results, including pharmacological and neurobiological changes to those reported in autoshaping have been described in other memory behavioral tasks (for review see King et al., 2008; Marcos et al., 2008; Da Silva Costa-Aze et al., 2012; Reichel et al., 2012; Woods et al., 2012; Haahr et al., 2013; Freret et al., 2014; Nasehi et al., 2014b; Seyedabadi et al., 2014; Subramaniyan et al., 2014; Wilkinson et al., 2014; Delotterie et al., 2015; Sase et al., 2015; Westrich et al., 2015).

#### Behavioral parameters during STM and LTM

In addition to measuring CR in autoshaping, head-pokes (HP) during each training/testing session and head-pokes during CS (HP-CS) have recorded. These parameters provide information about exploration activity (HP) and food- intake motivation (Tellez et al., 2012a). For instance, as CR becomes progressive, HP-CS provides information on the association of CS-US and CR-US.

#### Maximum level of CR

It should be noted that as animals present different levels of CR, these values are normalized and the maximal CR level attained for each rat at 48 h is considered as 100% of performance. This value is then used to calculate the proportion or percentage of CR observed at 1.5, 24, and 216 h and the data of 1.5 h and 24 h are used as illustration; and multiple comparisons, including memory, forgetting, time vs. treatments for all behavioral parameters (Meneses and Tellez, 2015).

#### Memory, amnesia and forgetting and neural transporters analysis

As already mentioned autoshaping procedures produce variable levels of CR and a number of laboratories have been using autoshaping. It should be noted that, reproducibility among studies is important and expected that to vary (e.g., Marcus, 2014).

but prefrontal cortex GAT1 down-regulation. Together these results show that forgetting provokes primarily hippocampal and prefrontal cortex transporters changes; it represents a cognitive process hardly modifiable and its prevention could causes different transporters expression patterns. Notably, together the results suggest that: (1) memory formation, amnesia and antiamnesic effects are associated to specific patterns of transporters expression; (2) STM and LTM, forgetting and anti-forgetting effects are associated to specific patters of transporters expression and brain areas; (3) amnesia and forgetting affect different brain areas and produce differential patters of transporter expression. Hence, in pharmacological and neuroanatomical terms, amnesia and forgetting differ.

#### Neural Transporters and Brain Functions and Dysfunctions

It should be noted that neural transporters regulate intrasynaptic levels of neurotransmitter, which allows a global picture of synapses. Moreover, diverse evidence indicates that memory formation, forgetting, amnesia, and/or anti-amnesic effects can also be modulated by changes in the expression of neurotransmitter transporters (e.g., Schmitt and Hiemke, 2002; Chen et al., 2011; Reichel et al., 2012; Yang et al., 2013). Hence, a brief overview of evidence involving GAT1, EAAT1, SERT, and DAT as well other neurobiological markers regarding memory and other cerebral functions is include.

#### GAT 1

Attention deficit/hyperactivity disorder (ADHD) is featured by hyperactivity, impaired sustained attention, impulsivity, and usually varying degrees of dysfunctional learning and memory (see also Meneses et al., 2011b) and motor incoordination (Yang et al., 2013). Importantly, Yang et al. (2013) report that GAT1 gene knockout (KO) mouse (GAT1−/−) is hyperactive and exhibit impaired memory performance (Morris water maze). KO GAT1 mice have low levels of attentional focusing and increased impulsivity; the hyperactivity in these KO mice is reduced by both methylphenidate and amphetamine; Yang et al. (2013) suggest that GAT1 KO mouse is a new animal model for ADHD studying and GAT1 may be a new target to treat ADHD. Schmitt and Hiemke (2002) note that GABA is cleaved from the synaptic cleft by uptake (see Hu and Quick, 2008), via specific transporters and inhibition of such transporters increases the effectiveness of physiologically released GABA. Increased GABAergic neurotransmission has an impact on learning and memory. Indeed, tiagabine, a GABA-transporter inhibitor, impaired learning (Morris water-maze) and retrieval (only at the probe trial; Schmitt and Hiemke, 2002). But,

TABLE 9 | Neural transporters during STM and LTM, amnesia (methamphetamine), forgetting, (fluoxetine) improved LTM, (fluoxetine) anti-forgetting effects and anti-amnesic (fluoxetine plus methamphetamine) effects.

STM, short-term memory; LTM, long-term memory; GAT1, GABA transporter 1; DAT, dopamine transporter; SERT, serotonin transporter; EACC1, glutamate transporter 1; PFC, prefrontal cortex; HIP, hippocampus.

Sałat et al. (2015) note that tiagabine slightly decreased memory but did not augment that induced by scopolamine. According with Shi et al. (2012), homozygous GAT1(−/−) mice exhibit impaired hippocampus-dependent learning and memory; and they evaluated the impact of endogenous reduced GABA reuptake on cognitive behaviors. Learning and memory of heterozygous GAT1(+/−) mice was determined in passive avoidance and Morris water maze; showing that GAT1(+/−) mice displayed increased learning and memory, decreased anxiety-like behaviors, and highest synaptic plasticity relative to wild-type and homozygous GAT1(−/−) mice; and authors conclude that a moderate reduction in GAT1 activity is associated to learning and memory facilitation (Shi et al., 2012); which is consistent, in part, with GAT1 reduced and increased expression in autoshaping amnesia, forgetting and improved memory as well as anti-amnesic and anti-forgetting effects (see **Table 10**). In addition, Pang et al. (2011) testing the GABAergic immunotoxin; GAT1-saporin (GAT1-SAP), report no alterations in spatial reference memory. But GAT1-SAP impaired the platform location in a delayed match to position test (changing daily the platform location). In the active avoidance task, intraseptal GAT1-SAP impaired extinction but not acquisition (Pang et al., 2011). In contrast, GAT1-Saporin into the medial septum/vertical limb of the diagonal band (MS/VDB) spared mnemonic function and use of environmental cues; however, self-movement cue processing was compromised (Köppen et al., 2013).

#### EAAT1

According with Chen et al. (2011), an imbalance of neurotransmitters (e.g., glutamate, acetylcholine, dopamine, and serotonin) has been proposed as the neurobiological basis of behavioral symptoms of AD, hence they are hypothesizing that altered reuptake of neurotransmitters by vesicular glutamate transporters (VGLUTs), excitatory amino acid transporters (EAATs), the vesicular acetylcholine transporter (VAChT), SERT or DAT. Examining protein and mRNA levels of these transporters in post-mortem prefrontal cortex from patients and matched non-AD controls, Chen et al. (2011)

#### TABLE 10 | GABA transporter GAT1.

ADHD, attention deficit and hyperactivity disorder; KO, knock out; MWM, Morris water maze; PA, passive avoidance.

#### TABLE 11 | Glutamate transporter 1 and markers.

AD, Alzheimer's disease; VGLUTs, Vesicular glutamate transporters; MDMA, methylenedioxymethamphetamine.

found that protein and mRNA levels of VGLUTs, EAAT1-3, VAChT, and SERT are reduced in AD, without changing DAT (**Table 11**). Chen et al. (2011) conclude that the reduced VAChT expression could contribute to cholinergic deficit in AD and altered neurotransmitter transporters could contribute to the pathophysiology of AD; which are potential targets for therapy (Chen et al., 2011).

Likewise, Kindlundh-Högberg et al. (2010) investigated the effect of intermittent 3,4-methylenedioxy-metamphetamine (MDMA; ecstasy) administration upon gene-transcript expression of the glutamate transporters (EAAT1, EAAT2- 1, EAAT2-2), glutamate receptor subunits of AMPA (GluR1, GluR2, GluR3), glutamate receptor subunits of NMDA (NR1, NR2A, and NR2B), and metabotropic glutamate receptors (mGluR1, mGluR2, mGluR3, mGluR5); showing increased cortical expression of GluR2, mGluR1, mGluR5, NR1, NR2A, NR2B, EAAT1, and EAAT2-2 (Kindlundh-Högberg et al., 2010). In the caudate putamen, mRNA levels of GluR3, NR2A, and NR2B receptor subunits are increased; in contrast, GluR1 is reduced in the hippocampus but in the hypothalamus GluR1, GluR3, mGluR1, and mGluR3 expression is increased (Kindlundh-Högberg et al., 2010; see also Carmona et al., 2009); concluding that repeated MDMA administration is associated with changes in gene-transcript expressions of glutamatergic NMDA and AMPA receptor subunits, metabotropic receptors and transporters in brain areas mediating learning and memory (Kindlundh-Högberg et al., 2010). In addition, decreased expression of vesicular glutamate transporter 1 (VGLUT1+/−) respect to wild-type (WT) mice occur with chronic mild stress (CMS)-induced, affecting several functions and impairing recognition memory. In addition, Heo et al. (2012) detect hippocampal glutamate transporter 1 (GLT-1) complex expression during training and memory in the Multiple T-maze.

#### SERT

Reichel et al. (2012) report that control rats spent more time interacting with the objects in the changed locations. In contrast, contingent or non-contingent methamphetamine (meth) disrupted object-in-place (OIP) task performance as seen by similar amounts of time spent with all objects, regardless of location. While only acute meth binge produced signs of neurotoxicity, both meth regimens decreased SERT in the perirhinal cortex and hippocampus. Only meth self-administration resulted in a selective decrease in NET. Meth-induced changes in SERT function in the OIP circuitry may underlie memory deficits independently of overt neurotoxic effects (Reichel et al., 2012). It should be noted that SERT is reduced in AD (Chen et al., 2011; Claeysen et al., 2015).

Parrott (2013) highlights that decreased SERT (hippocampus, parietal cortex, and prefrontal cortex expression) in abstinent Ecstasy/MDMA users is associated to dysfunctional declarative and prospective memory. Even the children of mothers who take Ecstasy/MDMA during pregnancy have psychomotor impairments (Parrott, 2013). In addition, Thomasius et al. (2006) report reduced SERT expression, which might be a transient effect of heavy ecstasy use, since it partially recovered as the users reduced their MDMA use; though this parameter may not necessarily be a valid indicator of the number or integrity of serotonergic neurons. Importantly, ex-ecstasy users' verbal memory show no sign of improvement even after over 2.5 years of abstinence and thus may represent persistent functional consequences of MDMA neurotoxicity; alternative causes such as pre-existing group differences cannot be excluded (Thomasius et al., 2006). In addition, AD and drugs of abuse like dmethamphetamine (METH) or MDMA have been associated to decrements in the SERT expression and memory deficits; thus supporting the notion that the SERT plays a key role in both normal and pathological states (e.g., Line et al., 2014). Particularly, the s allele of the polymorphic regulatory region of the SERT or 5-HTT gene promoter is associated with reduced 5-HTT expression and vulnerability to psychiatric disorders, including anxiety and depression. Moreover, the l allele increases 5-HTT expression and is generally considered protective (Line et al., 2014). However, Line et al. (2014) suggest that 5-HTT over-expression results in a reduced sensitivity to both positive and negative reinforcers, and produces some maladaptive effects, supporting recent suggestions that l allele homozygosity may

AD, Alzheimer's disease; MDMA, methylenedioxymethamphetamine; CER, conditioned emotional response.

#### TABLE 13 | Dopamine transporter DAT.

be a potential risk factor for disabling psychiatric traits (Line et al., 2014). In contrast, increased 5-HTT expression reduces negative cognitive bias for stimuli with uncertain outcomes (McHugh et al., 2015). And Brigman et al. (2010) report that fluoxetine-treated C57BL/6J mice made fewer errors than controls during the early phase of learning reversal when perseverative behavior is relatively high and 5-HTT null mice made fewer errors than controls in completing the reversal task (**Table 12**). And these authors suggest that inactivating 5-HTT improves reversal learning, which is relevant for the pathophysiology and treatment of neuropsychiatric disorders characterized by executive dysfunction (Brigman et al., 2010) and possibly post-traumatic stress disorder.

Certainly, SERT is providing useful information as neural marker and therapeutic target. For instance, Wallace et al. (2014) report that vortioxetine, a novel, multimodal-acting antidepressant, is a 5-HT3, 5-HT7, and 5-HT1D receptor antagonist, a 5-HT1B receptor partial agonist, a 5-HT1A receptor agonist, and inhibits the 5-HT transporter. This drug changes the expression of multiple genes involved in neuronal plasticity by antidepressant treatment, which is associated with improved cognitive function and a reduction in depression-like behavior in middle-aged mice (Li et al., 2015c).

Hence, the SERT expression seems to be a reliable neural marker related to memory mechanisms, its alterations and potential treatment (Meneses, 2013). Resulting crucial determining the pharmacological, neural and molecular mechanisms associated to these changes and therapeutic targets. For instance, Sheline et al. (2014a) report that serotonin signaling suppresses generation of amyloid-β (Aβ) in-vitro and in animal models of AD and healthy individuals. In fact, in an aged transgenic AD mouse model the antidepressant citalopram (5-HT uptake inhibitor) in dose-dependent manner decreased Aβ in cerebrospinal fluid, suggesting AD prevention trials (Sheline et al., 2014a,b).

#### DAT

According with Mereu et al. (2013), modafinil (MOD) and its R-enantiomer (R-MOD) are used for narcolepsy and sleep disorders; and also employed, off-label used as cognitive

#### References

enhancers in individuals with mental disorders, including substance abusers that demonstrate impaired cognitive function. Their mechanisms of action include inhibition of dopamine (DA) reuptake via the DAT in diverse brain areas (Mereu et al., 2013; **Table 13**). Importantly, memantine (MEM), a dual antagonist of NMDA and alpha7 receptors, is neuroprotector against MDMA in rats, and it also prevents MDMA effect on SERT functionality and METH effect on DAT (Escubedo et al., 2009). Moreover, Söderqvist et al. (2012) have noted that dopamine plays an important role not only in dysfunctional working memory (WM) but also for improving it, including variation in DAT1, improving WM and fluid intelligence in preschool-age children following cognitive training; concluding with the role of dopamine in determining cognitive plasticity (Söderqvist et al., 2012). Ruocco et al. (2014) report that 5-HT7 receptor stimulation (low doses) was associated to among other findings reduced horizontal activity and (at higher dose) increased selective spatial attention, the DAT levels were decreased (low dose), and modulated expression of NMDA receptors.

It should be noted that, before the perspective of the absence of effective treatments for dysfunctional memory and regardless the mechanisms; environmental interventions and exercise (physical and cognitive) seem offer feasible approaches (e.g., Mora, 2013; Mo et al., 2015).

#### Conclusions

Of course if the above findings are replicated over time, across countries and in different experimental settings, they might provide insights about serotonin and other neurotransmission systems presenting convergent changes in diverse neural markers and signaling; thus, allowing the study of different brain functions and dysfunctions, including memory. Hence, diverse approaches might support the translatability of using neural markers and cerebral functions and dysfunctions (e.g., memory formation, AD, MCI). Likewise, hypothesis and theories (e.g., Borroto-Escuela et al., 2015) might provide appropriate limits and perspectives of the diversity of evidence. Certainly, at least, 5-HT1A, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors as well as SERT seem to be useful as neural markers and therapeutic targets.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Meneses. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The Movable Heritage of Drăghia

*Patricia Săsăran, Ancuța Țenter, Bety Burghele, Marin Cotețiu and Loreley-Diana Jianu*

#### **Abstract**

The movable heritage of the Drăghia village, Maramureș County, is still around today thanks to a handful of people who diligently tended to the aged objects forsaken long ago. The old wooden church, built in 1706, is today a museum in which some of the age-old ritual objects, as well as other type of objects, are conserved. The decorations of the church, an important aspect over time, include various objects which today can be considered movable heritage. In this regard, the icons or paintings were highlighted by decorating them with decorative towels, woven by the village's women. Other cloths, rugs and mats were also used for "dressing" the church. We will open the list of movable heritage objects with the key to the church, and tell its over 300-years old story. The icons, religious books, royal doors, banners, thurible, the two choir pews, the church bells, and the priest robes will complete our study. On the other hand, you can find an old shirt, a towel, a bench or some other object kept out of respect for their ancestors in almost every house in the village. The Radu families have impressive collections, having establishes a so-called personal museum.

**Keywords:** movable heritage, church, religious books, key, icons, table cloth

#### **1. Introduction**

Patrimonial artifacts of Drăghia Village, Maramureș county, exist today only through the benevolence of some people that are aware of their value and managed to take care of their family's heritage until today, despite the modernity taking over [1].

The village's church, built in 1706, has long developed into a museum, where one can have a glimpse of various objects used in old Christian rituals. Embellishment of churches was an important aspect in the old days. The icons and the paintings were framed and exposed with special handwoven textiles made by the women of the village. In the same time, carpets and other types of local made textiles were used for covering the floors or furniture in the church. Beside the church, almost in every house one can still find an old traditional shirt (ie), a hemp towel, a bench or several other objects kept out of respect for their ancestors' tradition. Most impressive are the collections coming from Radu family and Blenche family, that managed to create a small private museum.

The key, the icons, the old religious books, the altar doors, the church' flags, the censer, the two lecterns, the bells or the priest clothing will be part of our study.

#### **2. The key**

The first object in the series of patrimonial objects presented herein is the key of the wooden churches' front door. We'll take a moment to tell the 300 years key story.

The key from the old church is a specific one for the area. It is a long iron key, with a fixed part of 39 cm and a mobile part of 10 cm, acting upon a wooden closing system. An encrusted 19 cm wooden beam is set in motion by the key, therefore blocking the door. A mechanism that works flawless for more than 300 years (**Figure 1**).

#### **Figure 1.** *The key.*