The effects of drug detection training on behavioral reactivity and blood neurotransmitter levels in drug detection dogs: A preliminary study

Journal of Veterinary Behavior (2012) 7, 11-20

RESEARCH

The effects of drug detection training on behavioral reactivity and blood neurotransmitter levels in drug detection dogs: A preliminary study Jacopo Rivaa, Stefano P. Marellia, Veronica Redaellia, Gianpietro P. Bondiolottib, Elisabetta Sforzinic, Michele Matteo Santorod, Corrado Carenzia, Marina Vergaa, Fabio Luzia a

Department of Animal Science, University of Milan, Milan, Italy; Department of Pharmacology, Chemotherapy and Toxicology, University of Milan, Milan, Italy; c Med Vet, Libero professionista, Broni (PV), Italy; and d Guardia di Finanza, Servizio Cinofili, Castiglione del Lago (PG), Italy. b

KEYWORDS: working dog; stress; behavioral reactivity; blood neurotransmitters

Abstract The aim of the present study was to analyze the effects of drug detection training on behavior and blood neurotransmitter levels in drug detection dogs so as to investigate some variables influencing dog reactivity and responsiveness to training. In all, 20 dogs were sampled out of the Guardia di Finanza canine population. All the subjects were born, reared, housed, and trained in the same facility and followed the same training sessions. Dogs’ behavioral reactivity was scored according to a standardized working dogs test to evaluate natural dog attitudes. Plasma samples were analyzed by the high-performance liquid chromatography method to evaluate adrenaline, noradrenaline, L-3,4-dihydroxyphenylalanine, homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol acid (MHPG), 5-hydroxyindole acetic acid (5-HIAA), and 5-hydroxytryptamine (5-HT) levels. 5-HT and 5-HIAA were also analyzed from platelets. The analysis was carried out considering training, breed, and sex as independent variables. From a behavioral point of view, significant differences were recorded before and after training in ‘‘sociability,’’ ‘‘playfulness,’’ ‘‘predatory instinct,’’ and ‘‘aggressiveness’’ scores. Lower levels of platelet 5-HT and 5-HIAA were found after training. Plasma L-3,4-dihydroxyphenylalanine levels differed between sexes, with males showing higher concentrations. These results underline the importance of complete and objective evaluations protocols of the dogs before, during, and after drugs search training to determine effective and successful selection strategies and training procedures. Ó 2012 Elsevier Inc. All rights reserved.

Introduction

Address for reprint requests and correspondence: Stefano P. Marelli, PhD, Department of Animal Science, University of Milan, via Celoria, Medicine 10, 20133 Milan, Italy; Tel: 139-02-503-18028; Fax: 139-02-503-18030. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2011.04.002

The social importance of drug detection dogs is recognized worldwide. Because of this, the effect of training on dogs’ welfare and behavior needs to be understood to optimize the effectiveness of training methods and to improve the quality of working dogs (Rooney et al., 2007,

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2009; Gaines et al., 2008; Haverbaeke et al., 2008a; Leighton, 2009). Early selection of dogs with a particular aptitude to become a working dog has attracted international attention from both scientists and operational personnel (Taylor and Mills, 2006; Vanderloo, 2009). Knowledge of specific reactivity characteristics related to learning and stress may supply tools for tailoring training programs while also subjectively enhancing attitudes of those who work with the dogs (Diederich and Giffroy, 2009). Such changes will reduce the costs of the training period while increasing the quality of the final product: a well trained, easy to handle dog. Standardized methods to characterize working dog behavior have been studied and applied by different authors. A strong scientific approach to the development, conduction, and evaluation of behavioral tests as a potential factor influencing dog welfare has been described by Taylor and Mills (2006). Svartberg and colleagues have applied behavioral tests to investigate personality traits in domestic dogs and the consistency of these traits, including the effects of artificial selection on breed-typical behavior in show and working dogs (Svartberg and Forkman, 2002; Svartberg et al., 2005; Svartberg, 2005, 2006). Clear phenotyping plays a key role in every selection plan, as described by Overall et al. (2009) who investigated the link between dog behavior and breed/genotype. Arvelius et al. (2009) characterized the behavior of 2,700 Border Collies using a defined scale (Herding Behavior Characterization) to investigate the heritability of herding behavior traits. The requirements of canine behavioral tests include standardization and validation of results by always considering the ‘‘field situation’’ (Diederich et al., 2009). The link between personality traits and performance in military working dogs has been studied using temperament measurements to predict performance in explosive detection dogs (Gosling and Hilliard, 2009). Haverbeke et al. (2008b) studied the training methods of military dog handlers and their results emphasize the importance of handler–dog interaction and training procedures. The importance of the behavior test in police dogs was shown by Erikson (2009) who focused on 3 critical points: dog suitability for a tryout period, training methods, and behaviors needing deeper investigation. These methods provide the basis on which to accurately evaluate the ontogeny of a particular behavioral trait, allowing for genetic investigation of the trait of interest (Ruefenacht et al., 2002; Gosling and Hilliard, 2009; Wilsson, 2009). The organism interaction with its environment is regulated by neurochemical interactions, which include 5-hydroxytryptamine (5-HT) and catecholamines which regulate several behavioral systems, and roles of the dopaminergic and serotoninergic systems in the neurotransmission of the stress response (Puglisi-Allegra and Cabib, 1990; Le Moal and Simon, 1991).

Acute and repeated exposures to stressful stimuli have been shown to increase release and the turnover of 5-HT in specific regions of the brain involved in behavioral and physiological responses to stressors. It has been hypothesized that physiological resistance to repeated stress is associated with an increase of 5-HT release in the hippocampus and that dysregulation of 5-HT release may contribute to the pathogenesis of depression (Robertson et al., 2005; Jiansong et al., 2008; Hedlund, 2009) and anxiety-related behavioral disorders in dogs (Reisner et al., 1996; King et al., 2000; Overall et al., 2001; Seksel and Lindeman, 2001; Overall and Dunham, 2002; Riva et al., 2008). The hippocampus has been proposed to mediate stress adaptation through dense serotoninergic innervations from the dorsal raphe, a pathway that is linked to mood and cognitive function. Stress adaptation allows animals to become tolerant to chronic aversive stimuli (Herman and Cullinan et al., 1997). It is possible, but unconfirmed, that plasma levels of the evaluated neurotransmitters may reflect what is happening at the brain level. Some authors describe a statistically significant correlation between some assessment of serotoninergic level, including that of its metabolite 5-hydroxyindole acetic acid (5-HIAA) in the cerebrospinal fluid (CSF), and blood levels in subhuman primates. This finding suggests that measurements in blood may be used as a peripheral indicator of central 5-HT (Stahl et al., 1982; Da Prada et al., 1988; Yan et al. 1993; Coccaro et al., 1997). Our research focused on analyzing the effects of drug detection training on behavior and blood neurotransmitter levels so as to investigate some variables influencing dog reactivity and responsiveness to training (Riva et al., 2007; Marelli et al., 2008)

Materials and methods Subjects In all, 20 dogs were selected as representative samples of the Guardia di Finanza canine population. These consisted of 18 German shepherd (GS) dogs and 2 Labrador retrievers (LR). There were 11 males and 9 females, and all dogs were intact. The age of the subjects ranged between 12 and 24 months. All of them were born, reared, housed, and trained in the same facility. Dogs were individually housed in an indoor–outdoor kennel. Diet and feeding regimen were the same for all the subjects (standard commercial dry adult dog diet, Crude Protein: 23.5%; metabolizable energy: 13.8 Mj/kg; twice a day). All the dogs followed the same training sessions. All of them were declared to be in good health after nonremarkable physical and laboratory examinations. This study was conducted over a period of 6 months. The dogs were tested twice: before and after drug detection training.

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Drug detection training: effect on behavioral reactivity and neurotransmitters levels

Measures Behavioral evaluation The behavioral reactivity of the subjects was scored (Table 1) according to a standardized test by Svartberg (2005, modified) that was specifically designed for working dogs to evaluate natural dog aptitudes. Dogs were rated for 7 variables analyzed across 8 situations: sociability (eagerness, cooperation, reaction to physical contact), playfulness (eagerness, cooperation, reaction to physical contact), activity level (passive situation, activity level), chase (chasing, grabbing), aggressiveness (interest toward stranger, aggressive behavior), metallic noise (startle reaction, exploratory behavior, avoidance behavior, approach behavior), and gunshot (avoidance reaction). The dog’s reaction to each test was scored on a scale ranging from 1 (fearful/over-reactive) to 5 (confident/playful) according to Svartberg’s protocol (2005) by considering the intensity of dogs’ behavioral reaction. The reactions of all the subjects were scored by a behavioral specialist (same person, tested for reliability).

Physiological parameters Blood samples for assessment of neurotransmitter levels were collected before and after each behavioral test, with a 2-hour rest time between the initial blood collection and the subsequent behavior test. Samples were collected from the cephalic vein by venipuncture, 5 mL of blood collected in the morning and immediately mixed with 10 ml (vol/vol) of 1% disodium ethylenediaminetetraacetate. The blood was centrifuged at 120 ! g

Table 1

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(15 minutes at 20 C); later the plasma was centrifuged at 2,700 ! g (15 minutes at 4 C) (Da Prada and Picotti, 1979; Bondiolotti et al., 1987). The platelet pellet was washed with saline and analyzed with the highperformance liquid chromatography (HPLC) method. Plasma samples were analyzed by HPLC to evaluate adrenaline, noradrenaline, L-3,4-dihydroxyphenylalanine (L-DOPA), homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol acid (MHPG), 5-HIAA, and 5-HT levels. Platelets were analyzed for levels of 5-HT and 5-HIAA.

HPLC analyses Monoamines and their derivatives were measured using liquid chromatography with electrochemical detection. For the analysis of the catecholamines, platelet pellets were homogenized with 0.3N perchloric acid and plasma deproteinized (1/1 v/v) with 0.6N perchloric acid containing dihydroxybenzylamine as the internal standard. Catecholamines were extracted onto alumina before the injection. For the analysis of 5-HT and 5-HIAA, 100 mL of plasma was deproteinized with an equal volume of 0.6N perchloric acid containing 3-metoxytyramine as an internal standard, 50 mL of supernatant was then injected in the chromatograph. Evaluation of chromatographed products was by coulometric detection. HPLC separation was performed on a reverse-phase analytical column, as described elsewhere (Lollis et al., 1979; Alleva et al., 1998). The protein content of platelets was assayed using a microassay with bovine serum albumin as a standard (Lowry et al., 1951).

Traits and subtests for behavior reactivity scoring

Subtest

Trait

a 5 reaction to greeting b 5 cooperation c 5 reaction to physical contact d 5 interest e 5 cooperation f 5 reaction to physical contact g 5 activity level h 5 chase proneness i 5 grabbing proneness j 5 chase proneness k 5 grabbing proneness l 5 interest in the stranger m 5 aggressive behavior n 5 startle reaction o 5 exploratory behavior p 5 avoidance behavior q 5 approach behavior r 5 avoidance behavior

Sociability (friendly stranger)

Playfulness

Activity level (passive situation) Predatory instinct (chase)

aggressiveness (threatening stranger) curiosity (metallic noise)

Reaction to unexpected occurrence (gunshot)

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Calculation and statistical analysis Statistical analysis was carried out using Kruskal–Wallis nonparametric analysis of variance and the general linear model (Type III sums of squares). Student t test was applied to the calculations of the least square means difference, and the level of significance was set at 3 different levels: P % 0.05, P % 0.01, and P % 0.001. Training, breed, and sex were considered as independent variables (SAS Institute, 2008).

Results Behavioral reactivity The effect of training on behavior reactivity scores is reported in Table 2. Significant differences occur before and after training in scores for ‘‘sociability,’’ ‘‘playfulness,’’ ‘‘predatory instinct,’’ and ‘‘aggressiveness.’’ Lower scores after training were found for ‘‘reaction to stranger friendly contact’’ (P % 0.05) and ‘‘cooperation’’ (P % 0.01), both subtests of the assessment of ‘‘sociability.’’ ‘‘Cooperation’’ (P % 0.05) and ‘‘reaction to physical contact’’ (P % 0.01) scores were negatively influenced by drug search training, and reflected lower ‘‘playfulness’’ scores after training. The studied population showed lower scores after training both for ‘‘grabbing proneness’’ (2 repetitions) and ‘‘chase proneness’’ (second repetition) (P % 0.05), predatory instinct according to Svartberg (2005) was evaluated with 2 repetitions for both subtests (Table 1).

Table 2 Effect of training on behavior reactivity scores; (means 6 SD), N 5 20 Test (Table 1)

Before training

a b c d e f g h i j k l m n o p q r

4.25 4.50 4.00 3.65 3.55 3.95 2.40 4.00 4.20 4.55 4.65 4.10 1.30 1.70 2.10 3.5 2.00 2.00

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.02 0.83 1.34 1.23 1.57 1.28 1.27 1.38 1.40 0.83 0.75 0.91 0.57 0.80 1.29 1.54 1.34 0.86

After training 3.37 3.37 3.53 2.89 2.42 2.53 2.53 3.05 2.89 3.32 3.37 2.47 1.37 1.84 1.95 3.79 1.63 1.53

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.16 1.30 1.31 1.63 1.64 1.71 1.26 1.65 1.73 1.80 1.77 1.31 0.83 1.07 1.18 1.40 1.12 0.77

P ,0.05 ,0.01 NS NS ,0.05 ,0.01 NS NS ,0.05 ,0.05 ,0.05 ,0.001 NS NS NS NS NS NS

The ‘‘aggressiveness’’ trait was influenced by training only with respect to the ‘‘interest in the stranger’’ subtest, and a lower score was recorded after training (P % 0.01). Table 3 reports the results of statistical analysis using sex as the independent variable. Only the ‘‘startle reaction’’ to the metallic noise revealed significantly different scores between males and females, with males displaying more ‘‘shy/reactive behavior’’ than females (P % 0.05). The significant effect of breed on the subtest is reported in Table 4. Lower scores for GS than for LR were found for ‘‘cooperation’’ and ‘‘reaction to physical contact’’ in the sociability evaluation with a friendly stranger. The ‘‘playfulness’’ scores were also higher in LR, as were those for ‘‘interest,’’ ‘‘cooperation,’’ and ‘‘reaction to physical contact’’ subtests (P % 0.05).

Blood neurotransmitters levels Lower levels of platelet 5-HT and 5-HIAA were found after training (5-HT: 548.69 6 103.39 vs. 127.07 6 103.80 ng/mg protein; P % 0.01 [Figure 1]; 5-HIAA: 3.72 6 1.10 vs. 0.46 6 1.10 ng/mg protein; P % 0.05 [Figure 2]). Plasma L-DOPA levels differed between sexes (1.75 6 0.16 vs. 1.24 6 0.17 ng/mg protein; P % 0.05), with males having higher concentrations (Figure 3).

Discussion Behavioral reactivity analysis revealed significant differences for various tests. Training results in lower scores for components of ‘‘sociability’’ (‘‘reaction to greeting’’ and ‘‘cooperation’’), ‘‘predatory instinct’’ (‘‘grabbing proneness’’ and ‘‘chase proneness’’), and ‘‘aggressiveness’’ (‘‘interest in the stranger’’). Detection-trained dogs in this study seem to show a less interactive and a more reactive behavior. These results reinforce those reported by Marshall-Pescini et al. (2009) who evaluated dogs using problem solving and sociocognitive tasks. In their study, dogs with a high training level were less dependent on their handlers for problem solutions and more proactive in problem solving situations, emphasizing the effect of training, regardless of type, in behavior performance differentiation. Training seems to influence behavioral characteristics considered to be important in working dog attitude (Svartberg, 2005), but training sessions can also be stressful for a dog (Beerda et al., 1997). Female dogs in our study scored high for the startle reaction subtest, and they were also more curious than males. Behavioral reactivity differences between males and females have been described by different authors (Bradshaw et al., 1996; Bradshaw and Goodwin, 1999; Takeuchi and Mori, 2006; Notari and Goodwin, 2007). These studies all suggest that females score higher than males for ability to be trained for obedience purposes, level of affection demand, and ease of

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Drug detection training: effect on behavioral reactivity and neurotransmitters levels

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Table 3 Effect of sex on behavior reactivity scores: only tests with significant differences have been reported (means 6 SD), N 5 20 Test

Male

Female

P

n

1.55 6 0.80

2.06 6 1.03

,0.05

housetraining. Rooney and Bradshaw (2004) found that males and females across different breeds differed primarily with respect to aggression: male dogs were more aggressive to other dogs. No differences were reported for search dog ability. Although the sample size of the 2 breeds in our study was unbalanced, we could still observe that the 2 LR were more sociable and playful than the GS. The link between breed and behavior reactivity has been investigated by many authors (Bradshaw et al., 1996; Bradshaw and Goodwin, 1999; Overall and Dunham, 2002; Ruefenacht et al., 2002; Svarberg, 2005, 2006; Takeuchi and Mori, 2006; Duffy et al., 2008; Overall et al., 2009). Behavioral predictability of breeds is considered a power tool in training optimization and costs reduction in working dogs trained for assistance and military purpose. Our results are in accordance with those reported for behavioral profiles of purebred dogs in Japan, the United States of America, and the United Kingdom (Takeuchi and Mori; 2006). LR are usually highly ranked with respect to playfulness level, and they are most frequently ranked in favorable trait clusters. Neurotransmitter concentration and receptor sensitivity determine the contribution of serotoninergic function to

Table 4 Effect of breed on behavior reactivity scores (means 6 SD); N 5 20 Test

German shepherd

a b c d e f g h i j k l m n o p q r

3.71 3.83 3.63 3.11 2.80 3.06 2.51 3.40 3.46 3.83 3.91 3.17 1.26 1.74 1.94 3.66 1.77 1.71

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

1.18 1.22 1.33 1.45 1.66 1.63 1.27 1.59 1.72 1.54 1.52 1.36 0.61 0.89 1.19 1.43 1.17 0.83

Labrador retriever 4.75 5.00 5.00 4.75 4.75 5.00 2.00 4.74 4.50 5.00 5.00 4.50 2.00 2.00 2.75 3.50 2.25 2.25

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.50 0.00 0.00 0.50 0.50 0.00 1.15 0.50 1.00 0.00 0.00 1.00 1.15 1.41 1.50 1.91 1.89 0.96

P NS ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 NS NS NS NS NS NS NS NS NS NS NS NS

Figure 1 Platelets 5-HT variables (6SD), N 5 20, before (1) and after (2) training: 548.69 6 103.39 ng/mg protein versus 127.07 6 103.80 ng/mg protein (P % 0.01). For a colored version of this figure, the reader is referred to the Web version of this article.

central fatigue. Changes in neurotransmitter concentrations are the focus of much work on the effects of acute bouts of exercise on serotoninergic function. Amounts of 5-HT have been reported to increase during resting (Brown et al., 1979) and decrease after exercise (Acworth et al., 1986; Hoffmann et al., 1994), with increased turnover of brain 5-HT found in endurance-trained rats (Dey et al., 1992). Other research has focused on understanding the relationship between cognitive and physical functions, such as motor exercise, and neurobiochemical variables. Anxiety, depression, learning, and memory are some of the physiological functions where serotonin (5-HT), a biogenic amine, is involved (Ogren, 1985; Buhot et al., 2000). Reports on effects of exercise training on receptors sensitivity are rare. The few available studies have shown that training sessions induced a decrease in receptor sensitivity in animals (Dey, 1994; Seguin et al., 1998) and human beings (Jakeman et al., 1994; Broocks et al., 1999). An adaptive downregulation or desensitization of central serotonin receptors has been hypothesized to be the result of a repeated increase in 5-HT concentration in the brain associated with exercise training, according to the description by Shankaran et al. (2007) who describe the adaptation downregulation as the process during which

Figure 2 Platelets 5-HIAA variables (6SD), N 5 20, before (1) and after (2) training: 3.72 6 1.10 ng/mg protein versus 0.46 6 1.10 ng/mg protein (P % 0.05). For a colored version of this figure, the reader is referred to the Web version of this article.

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Journal of Veterinary Behavior, Vol 7, No 1, January/February 2012 L-DOPA PLASMA

period (Kurosawa et al., 1993; Meeusen et al., 1994; Wilson and Marsden, 1994). The benefits of exercise on brain health and function, particularly in aging human populations, have been described by many authors. Exercise participation has consistently emerged as a key indicator of improved cognitive function (Cotman and Berchtold, 2002; Kubesch et al., 2003; Meeusen et al., 2006). Furthermore, cardiovascular fitness improved by exercise is directly associated with cognition achievements in young adult men (Aberg et al., 2009). Yet, studies evaluating the effects of exercise on brain neurotransmitter levels are scarce in comparison with research about the peripheral adaptations to exercise. Because motor function, movement initiation, and control of locomotion are affected by brain neurotransmitter systems, it could be interesting to examine the effect of chronic exercise on neurotransmitters level fluctuation (Meeusen and De Meirleir, 1995; Meeusen et al., 2001, 2007). Different authors investigated the variations of levels of brain noradrenaline, serotonin (5-HT), and dopamine (DA) depending on exercise (Meeusen et al., 2001). This study reported that there is evidence in favor of changes in synthesis and metabolism of monoamines during exercise, even when great discrepancies in experimental protocols were found (Meeusen et al., 2001). Adaptation in central brain neurotransmitter function needs to be accurately studied, and such studies are difficult. Descriptions of an inhibitory role for the serotoninergic system and a facilitating role for the dopaminergic system in the central for the control of fatigue during prolonged exercise (Chaouloff, 1989) have been improved by recent studies involving pharmacological manipulation of brain serotoninergic activity in rats (Bailey et al., 1993) and human beings (Wilson and Maughan, 1992). Although the precise pathway of serotoninergic activity during exercise is unknown, studies on rats report that the synthesis and synaptic release of 5-HT is related to the availability of its precursor, tryptophan, in relation to other large neutral amino acids (Fernstrom and Wurtmann 1971; Bosch et al., 2007).

concentration ng/ml

2,5 2 1,5 1

1,75 1,24

0,5 0

1

2 samples

Figure 3 Plasma L-DOPA variables (6SD), N 5 20, before (1) and after (2) training: 1.75 6 0.16 ng/mg protein versus 1.24 6 0.17 ng/mg protein (P % 0.05). For a colored version of this figure, the reader is referred to the Web version of this article.

the activation of cell surface receptors causes the regulatory processes that restrict the duration of signaling. This decrease in receptor sensitivity is likely to offer protection from the fatiguing effects of an increase in 5-HT associated with acute bouts of exercise. Jakeman et al. (1994) describe a decrease in the neuroendocrine response to a 5-HT1a agonist in highly trained athletes as compared with sedentary controls, suggesting that a decrease in central serotonin receptor sensitivity was involved. The effects of acute and prolonged exercise on brain serotonin and 5-HIAA levels have been investigated by Chalouff et al. (1986, 1987) who found that a single running session did not change 5-HT level in the rats trained for 1 week, but that 5-HT was diminished in rats trained for 8 weeks, indicating a change in serotonin utilization. Increased and decreased levels and turnover of serotonin and 5-HIAA affected by acute and prolonged exercise studies have been described to be linked to the brain region of interest. Dey et al. (1992) described serotonin and 5-HIAA alterations following chronic exercise, which activated not only the synthesis but also the metabolism of serotonin in cerebral cortex. Such interactions are often reported. 5-HTand 5-HIAA levels in the limbic forebrain and brain stem decrease in the immediate postexercise

Table 5

Effect of training session on neurotransmitters concentration scores (means 6 SD); N 5 20

Neurotransmitters (ng/mg protein)

Before training

MHPG plasma HVA plasma 5-HIAA plasma 5-HT plasma NA plasma A plasma L-DOPA plasma 5-HIAA platelet 5-HT platelet

5.56 2.81 10.82 11.47 0.30 0.29 1.53 3.72 548.69

6 6 6 6 6 6 6 6 6

1.14 0.37 2.66 3.02 0.04 0.09 0.16 1.10 103.39

After training 5.76 2.47 8.85 7.93 0.26 0.27 1.46 0.46 127.07

6 6 6 6 6 6 6 6 6

1.14 0.37 2.67 3.03 0.04 0.09 0.17 1.10 103.80

P NS NS NS NS NS NS %0.05 %0.05 %0.01

MHPG, 3-methoxy-4-hydroxyphenylglycol acid; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindole acetic acid; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; A, adrenaline; L-DOPA, L-3,4-dihydroxyphenylalanine.

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Drug detection training: effect on behavioral reactivity and neurotransmitters levels

Exercise intensity, exercise duration, and training status of the individual influence the extent of the body’s response to chronic exercise (Mazzeo, 1991). To adjust to the disturbance in resting homeostasis induced by the exercise stimulus, several regulatory systems are called upon to return the body to a new level of homeostasis. Catecholamines and serotonin have powerful regulatory properties that exert control over critical physiological and metabolic functions: serotonin has been shown to modulate gastrointestinal motility, peripheral vascular tone, cerebral vascular tone, and platelet function and has been implicated in the pathophysiology of mood disorders, emesis, migraine, irritable bowel syndrome, and pulmonary and systemic hypertension (Mazzeo, 1991; Mohammad-Zadeh et al., 2008). Alterations in tissue neurotransmitters may be considered crude measures of activity that do not necessarily reflecting corresponding changes in synaptic release. Sarrias et al. (1990) found a relationship between the amount of 5-HT and related metabolites in human blood and CSF: the concentrations of indoleacetic acid in CSF and plasma were strongly correlated, suggesting that ‘‘in vivo’’ measures of 5-HT and related metabolites in plasma and platelets may be used as an index of serotoninergic function in affective disorders. Blood concentrations of catecholamines in many species, such as human beings, cats, mice and rabbits, are higher in the platelets than in plasma (Da Prada and Picotti, 1979; Picotti et al., 1984). The concern about the reliability of any index is because distribution of 5-HT and DA in platelets and plasma can be affected by numerous factors (Pletscher, 1968). In vitro findings on rat platelets show that 5-HT and DA compete for the same active transport mechanism/carrier, but 5-HT has a higher affinity than DA for this transport process (Gordon and Olverman, 1978). This competition is also likely to occur in vivo because it has been shown both in human beings and rabbits that concentrations measured in plasma were higher for 5-HT than for DA (Da Prada and Picotti, 1979). Moreover, neuronal and platelet proteins for 5-HT and DA have been shown to be encoded by the same genes (Ramamoorthy et al., 1993; Cook et al., 1994). These factors may explain why different concentrations of DA and 5-HT can be found for platelets and plasma. With respect to detection dogs, the fusion of behavioral and physiological observations allows a multifaceted analysis of the relationship between training sessions and the dogs’ behavioral and stress reactions. Different responses to stressors and their correlated behavioral characteristics characterize individual differences and subjective predispositions to cope in particular contexts (Batt et al., 2009). The plasma concentration values for noradrenaline, DA, and 5-HT and metabolites noted in this article are within the range noted by other researchers for dogs (Da Prada and Picotti, 1979; Roche et al., 2002), although few references exist for canine plasma levels of 5-HIAA, 3,4-dihydroxyphenylacetic acid (DOPAC), and L-DOPA (Riva et al., 2007, 2008).

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In this research, we found out a clear reduction (P % 0.05) in 5-HT and 5-HIAA platelet concentrations after training, plasma L-DOPA levels decreased significantly (P % 0.05) too (Table 5). Training session and ‘‘central fatigue’’ could be considered to be the main variables influencing concentrations of neurotransmitters: all the subjects lived in a standardized housing system, were fed a standard feed, and were handled according to standard procedures throughout the experimental trial. Chronic and long-lasting exercise could improve central serotoninergic activity and consequently the peripheral platelet activity causing a reduction in concentrations of platelet and plasma neurotransmitters. Serotonin release is considered to be influenced by the activity of other neurotransmitter systems (DA, gammaaminobutyric acid), as well as cerebral glucose availability (Berquet et al., 2002). Furthermore, the interaction between brain 5-HT and DA during prolonged exercise could play a regulative role in the onset of fatigue (Davis and Bailey,1997).

Conclusion Significant differences for some behavioral traits were noted in dogs trained for drug detection. Not all the applied subtest revealed significant effect of training on dogs reactivity, which may be because of the fact that drug search activity requires very specialized training protocols and dog selection is very strict; conversely, the test we used has been used for the evaluation of the dogs’ responses to environmental challenges and to control the situation in everyday life (Svartberg et al., 2005). Training increased the dogs’ ‘‘reactivity.’’ Female dogs experienced greater increases in ‘‘curiosity’’ with training than did male dogs. These results underline the importance of a complete and objective evaluation of the dogs using different traits to determine a selection strategy and effective training course for successful drug detection dogs. Based on decreases in blood platelets levels of 5-HT and 5-HIAA after prolonged exercise during drug search training, a relative increase in the release of serotonin associated with training may be hypothesized.

Acknowledgments The authors thank Colonnello Walter Di Mari of the ‘‘Guardia di Finanza’’ for their hospitality and their technical support during the trial.

References Aberg, M.A.I., Pedersen, N.L., Toren, K., Svartengren, M., Backstrand, B., Johnson, T., Cooper-Kuhn, C.M., Aberg, N.D., Nilsson, M., Kuhn, H.G., 2009. Cardiovascular fitness is associated with cognition in young adulthood. PNAS 106(49), 20906-20911.

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