Naples High-excitability Rats Research Articles

Striatal dynamics as determinants of reduced gambling vulnerability in the NHE rat model of ADHD

The Naples High-Excitability (NHE) is a validated rat strain to model for a mesocortical variant of Attention Deficit Hyperactivity Disorder (ADHD). NHE rats' brains have a tuned-down cortical and a potentiated limbic loop (Zoratto et al., 2017). ADHD and comorbid pathological gambling (PG) involve similar deficits of prefrontal-striatal dialogue. This work aimed to understand if NHE rats (compared to normal random-bred rats, NRB) can be a useful model for gambling vulnerability in ADHD. Experiment 1 evaluated gambling proneness in NHE rats, namely attraction/avoidance in nose-poking for a “Large & Luck-Linked” (LLL) reward (versus a “Small & Sure” one, SS), when the probability of LLL delivery was progressively reduced. Experiment 2 assessed (by phMRI) differential responsivity of ventral (vStr) versus dorsal (dStr) striatum following a methylphenidate (MPH, 4 mg/kg I.P.) challenge. In NHE rats, reduced attraction by secondary cues (associated with uncertain, rarefying LLL delivery) comes along with little or no activation of dStr and enhanced activation of vStr by MPH. Together, such evidences from NHE rats indicate distinctive roles of ventral (enhanced value given to actual primary reward) and dorsal (lower encoding of repeated stimulus-reward associations into a habit) striatum. In conclusion, the dynamics of reward systems could link an attention deficit with a decreased vulnerability to pathological gambling.

Enhanced limbic/impaired cortical-loop connection onto the hippocampus of NHE rats: Application of resting-state functional connectivity in a preclinical ADHD model

Due to a hyperfunctioning mesocorticolimbic system, Naples-High-Excitability (NHE) rats have been proposed to model for the meso-cortical variant of attention deficit/hyperactivity disorder (ADHD). Compared to Naples Random-Bred (NRB) controls, NHE rats show hyperactivity, impaired non-selective attention (Aspide et al., 1998), and impaired selective spatial attention (Ruocco et al., 2009a, 2014). Alteration in limbic functions has been proposed; however, resulting unbalance among forebrain areas has not been assessed yet. By resting-state functional Magnetic-Resonance Imaging (fMRI) in vivo, we investigated the connectivity of neuronal networks belonging to limbic vs. cortical loops in NHE and NRB rats (n=10 each). Notably, resting-state fMRI was applied using a multi-slice sagittal, gradient-echo sequence. Voxel-wise connectivity maps at rest, based on temporal correlation among fMRI time-series, were computed by seeding the hippocampus (Hip), nucleus accumbens (NAcc), dorsal striatum (dStr), amygdala (Amy) and dorsal/medial prefrontal cortex (PFC), both hemispheres. To summarize patterns of altered connection, clearly directional connectivity was evident within the cortical loop: bilaterally and specularly, from orbital and dorsal PFCs through dStr and hence towards Hip. Such network communication was reduced in NHE rats (also, with less mesencephalic/pontine innervation). Conversely, enhanced network activity emerged within the limbic loop of NHE rats: from left PFC, both through the NAcc and directly, to the Hip (all of which received greater ventral tegmental innervation, likely dopamine). Together with tuned-down cortical loop, this potentiated limbic loop may serve a major role in controlling ADHD-like behavioral symptoms in NHE rats.

Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals.

Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE)line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30mg/kg) or vehicle for 15days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal DA by indicating an enhancement of selective attention and working memory in a deficit model.

Prepuberal Stimulation of 5-HT7-R by LP-211 in a Rat Model of Hyper-Activity and Attention-Deficit: Permanent Effects on Attention, Brain Amino Acids and Synaptic Markers in the Fronto-Striatal Interface

The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates.

Emotional and risk seeking behavior after prepuberal subchronic or adult acute stimulation of 5-HT7-Rs in naples high excitability rats

We report here the results of studies aimed to investigate the involvement of serotonin receptor 7 subtype (5-HT7-R) in the modulation of emotional response in Naples High-Excitability (NHE) rat, a validated model for hyperactivity and impaired attention. A range of dosages (0.0, 0.125, 0.250, or 0.500 mg/kg) of LP-211, a selective agonist of 5-HT7-Rs, has been evaluated in animals at different age (adolescence and adulthood). Male NHE and random bred (NRB) control rats were tested in an Elevated Zero-Maze (EZM) after LP-211 treatment in two different regimens: at the issue of adolescent, subchronic exposure (14 intraperitoneal [i.p.] injections, once/day, pnd 31-44, tested on pnd 45--Exp. 1) or as adult, acute effect (15 min after i.p. injection--Exp. 2). Adolescent, subchronic LP-211 at 0.500 mg/kg dosage increased the frequency of head-dips only in NHE rats. Drug effect on time spent and entries in open EZM quadrants were revealed with adult, acute administration of 0.125 mg/kg LP-211 (both strains), indicating a tendency toward anxiolytic effects. In conclusion, data demonstrate that subchronic stimulation of 5-HT7-Rs during prepuberal period increases novelty-seeking/risk-taking propensity in NHE adults. These sequels are revealing increased disinhibition and/or motivation to explore in the NHE rats, which are characterized by a hyperactive dopaminergic system. These data may open new perspectives in studying mechanism of risk-seeking behavior.

Prepuberal subchronic methylphenidate and atomoxetine induce different long-term effects on adult behaviour and forebrain dopamine, norepinephrine and serotonin in Naples High-Excitability rats

The psychostimulant methylphenidate and the non-stimulant atomoxetine are two approved drugs for attention-deficit hyperactivity disorder (ADHD) therapy. The aim of this study was to investigate the long-term effects of prepuberal subchronic methylphenidate and atomoxetine on adult behaviour and the forebrain neurotransmitter and metabolite content of Naples High-Excitability (NHE) rats, a genetic model for the mesocortical variant of ADHD. Male NHE rats were given a daily intraperitoneal injection (1.0mg/kg) of methylphenidate, atomoxetine or vehicle from postnatal day 29 to 42. At postnatal day 70–75, rats were exposed to spatial novelty in the Làt and radial (Olton) mazes. Behavioural analysis for indices of horizontal, vertical, non-selective (NSA) and selective spatial attention (SSA) indicated that only methylphenidate significantly reduced horizontal activity to a different extent, i.e., 39 and 16% respectively. Moreover methylphenidate increased NSA as assessed by higher leaning duration. The high-performance liquid chromatography (HPLC) tissue content assessment of dopamine, norepinephrine, serotonin and relative metabolites in the prefrontal cortex (PFC), cortical motor area (MC), dorsal striatum (DS), ventral striatum (VS), hippocampus and mesencephalon indicated that methylphenidate decreased (i) dopamine, DOPAC, norepinephrine, MHPG, 5-HT and 5-HIAA in the PFC, (ii) dopamine, DOPAC, HVA, serotonin, 5-HIAA in the DS, (iii) dopamine, DOPAC, HVA and MHPG (but increased norepinephrine) in the VS and (iv) norepinephrine, MHPG, serotonin and 5-HIAA in the hippocampus. Atomoxetine increased dopamine and decreased MHPG in the PFC. Like methylphenidate, atomoxetine decreased dopamine, DOPAC, HVA, serotonin and 5-HIAA in the DS, but decreased MHPG in the VS. These results suggest that methylphenidate determined long-term effects on behavioural and neurochemical parameters, whereas atomoxetine affected only the latter.

Intranasal application of dopamine reduces activity and improves attention in Naples High Excitability rats that feature the mesocortical variant of ADHD

Based on findings of a profound action of intranasally applied dopamine (DA) on dopamine release in the striatum, we examined the possibility that intranasal application of DA would influence indices of attention and activity in juvenile male rats of the Naples High Excitability line. This rat model features the main aspects of Attention Deficit/Hyperactivity Disorder (ADHD). Juvenile NHE rats received an intranasal application of either DA (0.075 mg/kg, 0.15 mg/kg and 0.3 mg/kg) or vehicle into both nostrils daily for 15 days. On day 14, 1 h after treatment, they were tested in the Làt maze, and one day later, in the eight arm radial maze. Activity in the Làt maze: The highest dose of DA (0.3 mg/kg) decreased horizontal (HA) and vertical (VA) activity during the first 10 min of the test. No effect was found for rearing duration (RD), which indexes non-selective attention (NSA). Activity in the radial maze: No treatment effects were found for HA and VA components, and for RD. Attention indices: The intermediate dose of DA (0.15 mg/kg) significantly improved the number of arms visited before the first repetitive arm entry in the radial maze, an index of selective spatial attention (SSA). In conclusion, intranasal application of DA reduced hyperactivity at the highest dose used, whereas the intermediate dose improved attention in an animal model of ADHD. These results suggest the potential of employing intranasal DA for therapeutic purposes.

Non-selective and selective spatial attention are differentially affected by acute and subchronic methylphenidate in the Naples high-excitability rats

Non-selective and selective spatial attention are differentially affected by acute and subchronic methylphenidate in the Naples high-excitability rats

Elevated forebrain excitatory l-glutamate, l-aspartate and d-aspartate in the Naples high-excitability rats

The Naples high-excitability (NHE) rats are thought to model the mesocortical variant of attention-deficit hyperactivity disorder (ADHD). The aim of this study was to investigate forebrain level of l-glutamate, l-aspartate and d-aspartate, in NHE vs. Naples random bred (NRB) control rats. Thus, prepuberal NHE and NRB rats were daily handled in the 5th and 6th week of postnatal life. Then rats were exposed to two spatial novelties i.e. a Làt and a Olton maze for 10 min. Amino acids were detected by HPLC in the prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that all amino acids were higher in NHE than in NRB rats. This, in turn, may explain the behavioural hyperactivity and attention deficit of this animal model of ADHD.

Excitatory amino acids in the forebrain of the Naples high-excitability rats: neurochemical and behavioural effects of subchronic d-aspartate and its diethyl ester prodrug

The excitatory amino acids (EAA) l-glutamate ( l-Glu), l-aspartate ( l-Asp) and d-aspartate ( d-Asp) are thought to play a neurotransmitter/neuromodulator role in neuronal communications. Recently, a high level of EAA l-Glu, d- and l-Asp isomers has been found in the forebrain of Naples high-excitability (NHE) rat line that models the mesocortical variant of Attention-Deficit Hyperactivity Disorder (ADHD). The aim of this study was to assess the functions of d-Asp using two forms, i.e. free d-Asp or d-Asp diethyl ester (DEE) as prodrug, on brain and behaviour. Thus, prepuberal rats were given, for two weeks daily, an i.p. injection of d-Asp or DEE or vehicle. Then rats were exposed to two spatial novelties i.e. Làt and radial Olton maze. Behaviour was monitored for indices of activity, non-selective attention (NSA), selective spatial attention (SSA) and emotional reactivity. l-Glu and d- and l-Asp were detected by HPLC in cognitive and non-cognitive brain areas such as prefrontal cortex, striatum, hippocampus and hypothalamus. Results indicate that subchronic d-Asp or DEE (i) reduced EAA levels in the NHE and increased it in the random-bred controls (NRB) rats, (ii) in the Làt-maze d-Asp increased horizontal activity in NHE but DEE decreased it in NRB rats, (iii) in the Olton maze d-Asp and DEE decreased vertical activity in NHE and NRB rats respectively, (iv) d-Asp impaired attention only in NRB decreasing number of arms visited before first repetition. Therefore, data demonstrate differential effects of prepuberal subchronic d-Asp and DEE that may be related to different basal EAA levels in NHE and NRB rats.

Differential prepuberal handling modifies behaviour and excitatory amino acids in the forebrain of the Naples High-Excitability rats

Naples High-Excitability (NHE) rats model the mesocortical variant of Attention-Deficit Hyperactivity Disorder (ADHD). Recently, a high level of excitatory amino acids (EAA) has been found in the forebrain of NHE rats. The aim of this study was to verify the effect of postnatal stimulation in prepuberal rats on forebrain EAA. Thus, prepuberal NHE and Naples Random Bred (NRB) control rats were daily handled (PS) or they were left undisturbed throughout (NO-PS). One hour after the last stimulation, PS and NO-PS rats were exposed to a spatial novelty in a Làt-maze and one day later to a non-reinforced Olton maze. In both tests the horizontal (HA) and vertical (frequency – VA and duration of rearing – RD) components of behaviour indexed activity and non-selective attention (NSA). Moreover, in the Olton maze the position of the number of arms visited before first repetition (FE) and to criterion (NVTC), indexed selective spatial attention (SSA). Amino acids were detected by HPLC in prefrontal cortex (PFC), striatum (STR), hippocampus (HPC) and hypothalamus (HYP). Results indicate that (i) in the Làt-maze, only for HA, NO-PS NHE rats were more active than PS, (ii) in the Olton maze NO-PS rats of both lines showed shorter rearing durations than PS, (iii) EAA level was higher in NHE than in NRB rats and (iv) NO-PS vs. PS treatment increased level of EAA across the forebrain in both rat lines. In contrast in NHE NO-PS rats l-glutamate ( l-Glu) decreased in HYP and l-aspartate ( l-Asp) decreased in HPC. In conclusion, postnatal stimulation in prepuberal rats significantly affects forebrain excitatory amino acids and behaviour in NHE line. Thus EAA are modulated by genetic determinants and environmental (epigenetic) factors.

Galactosylated dopamine enters into the brain, blocks the mesocorticolimbic system and modulates activity and scanning time in Naples high excitability rats

Pathological conditions, such as Parkinson’s disease and attention deficit hyperactivity disorder, have been linked to alterations of specific dopamine (DA) pathways. However, since exogenous DA does not cross the blood–brain barrier, DA levels can be modulated e.g. by DA precursors or DA reuptake blockers. Hereby histochemical, analytical and behavioral evidence shows that a galactosylated form of DA (GAL-DA) carries DA into the brain, thus modulating activity and nonselective attention in rats. To this aim adult male rats of the Naples high-excitability (NHE) and random bred controls (NRB) lines were given a single i.p. injection of GAL-DA (10 or 100 mg/kg). Three hours later the behavior was videotaped and analyzed for horizontal activity, orienting frequency and scanning duration. The dose of 100 mg/kg of GAL-DA reduced by 25% the horizontal activity in NHE rats, mainly in the first part of the testing period. No effect was observed on orienting frequency or on scanning duration. However, GAL-DA 100 mg/kg was associated with longer rearing episodes in the second part of the testing period in NHE rats. In parallel experiments histochemistry with a galactose-specific lectin showed 10% increase in galactose residues into the striatum between 0.5 and 3.0 h. To quantify the level of GAL-DA, its metabolite DA-succinate and DA in the prefrontal cortex, neostriatum, and cerebellum, rats were killed 2.0 h after the injection of prodrug. Mass high performance liquid chromatography (HPLC) was used for analysis of GAL-DA and DA succinate whereas electrochemical HPLC for DA. Both HPLC techniques demonstrate that GAL-DA carries and releases DA into the brain. Specifically 100 mg/kg of GAL-DA increased DA level in the striatum in the NHE rats only. Moreover, DA in the mesencephalon (MES) was correlated positively with striatal and prefrontal cortex DA in NHE rats. In contrast DA in the MES was negatively correlated with striatal DA in NRB. GAL-DA disrupted these correlations in both rat lines. Thus, this new DA prodrug may modify DA neurotransmission and might have a potential clinical application.

Dysfunctions in dopamine systems and ADHD: evidence from animals and modeling.

Animal models are useful for characterizing neural substrates of neuropsychiatric disorders. Several models have been proposed for the study of Attention Deficit Hyperactivity Disorder (ADHD). The models can be divided into various groups: (i) genetically derived hyperactivity/ inattention, (ii) animal models showing symptoms after pharmacological intervention, and (iii) those based on spontaneous variations in a random population. Spontaneously hypertensive (SHR) and Naples High Excitability (NHE) rats show behavioral traits featuring the main aspects of ADHD in humans but show different changes in dopamine (DA) systems. In fact, the enzyme tyrosine hydroxylase is hyperexpressed in NHE rats and hypoexpressed in SHR. The DA transporter is hyperexpressed in both lines, although in the SHR, DAT activity is low (reduced DA uptake). The DA levels in the striatum and prefrontal cortex are increased in the juvenile SHR, but are decreased in handled young and non-handled older animals. The mRNA of the D1 DA receptor is upregulated in the prefrontal cortex of SHR and downregulated in NHE. The D2 DA receptors are likely to be hypofunctioning in SHR, although the experimental evidence is not univocal, whereas their mRNA is hyperexpressed in NHE. Thus, in SHR both the mesocortical and mesolimbic DA pathways appear to be involved, whereas in NHE only the mesocortical system. To understand the effects of methylphenidate, the elective ADHD drug treatment in humans, in a dysfunctioning DA system, we realized a simple mathematical model of DA regulation based on experimental data from electrophysiological, cyclic voltammetry, and microdialysis studies. This model allows the estimation of a higher firing frequency of DA neurons in SHR rats and suggests that methylphenidate increases attentive processes by regulating the firing rate of DA neurons.

Involvement of norepinephrine in the control of activity and attentive processes in animal models of attention deficit hyperactivity disorder.

Functional and morphological studies in children affected by Attention Deficit Hyperactivity Disorder (ADHD) suggest a prefrontal cortex (PFc) dysfunction. This cortical region is regulated by subcortical systems including noradrenergic (NEergic), dopaminergic (DAergic), cholinergic, serotonergic, and histaminergic pathways. A wealth of data in humans and in animal models demonstrates altered dopamine (DA) regulation. Drugs that modulate norepinephrine (NE) transmission are also effective in ADHD patients, thus leading to the hypothesis of a NEergic disorder. This review covers the regulation of PFc functions by NE and the interaction between the NE and DA systems, as suggested by pharmacological, electrophysiological, morphological, and gene knock out (KO) studies. A negative feedback between NE and DA neurons emerges from KO studies because KO mice showing increased (NE transporter (NET) KO) or decreased (DBH and VMAT2 KO) NE levels are respectively associated with lower and higher DA levels. Locomotor activity can be generally predicted by the DA level, whereas sensitivity to amphetamines is by NE/DA balance. Some animal models of ADHD, such as spontaneously hypertensive rats (SHR), show alterations in the PFc and in the DA system. Evidence about a correlation between the NE system and hyper-locomotion activity in such animals has not yet been clarified. Therefore, this review also includes recent evidence on the behavioral effects of two NET blockers, reboxetine and atomoxetine, in two animal models of ADHD: SHR and Naples High Excitability rats. As these drugs modulate the DA level in the PFc, certain effects are likely to be due to a rebalanced DA system. We discuss the significance of the results for theories of ADHD and make suggestions for future experimentation.

Behavioural, pharmacological, morpho-functional molecular studies reveal a hyperfunctioning mesocortical dopamine system in an animal model of attention deficit and hyperactivity disorder

Clinical and experimental evidence suggest an involvement of dopamine systems, mainly the mesocorticolimbic one (MCL), in Attention-Deficit Hyperactivity Disorder (ADHD). However, it remains to be ascertained whether the systems are hyper- or hypo-functioning, for the implications of the functional state. Indeed, differential functional states of the MCL branches are suggested to be the neural substrate of different ADHD variants. This review covers published and unpublished data from the Naples-High Excitability (NHE) rat, an animal model of ADHD, featuring its main aspects, with no hypertension. Therefore, a multiple approach based on morphological studies of dopamine, norepinephrine, glutamate, acetylcholine and GABA systems, synaptic (Calcium/Calmodulin kinase II) and extrasynaptic (chondroitin sulphates) environments, and molecular biology and pharmacological studies on the dopamine system has been carried out. Morphological findings suggest dopamine neurons in the Ventral Tegmental Area (VTA) to be hypertrophic in NHE rats. The mesostriatal and mesolimbic dopamine branches appear to be normal in basal conditions. However, the striatal interface is probably defective following activation. Conversely, the prefrontal cortex, which represents the second main target of VTA dopamine neurons, has many alterations at the basal level. Therefore, the emerging picture is the association of a hyperinnervating and hyperfunctioning mesocortical branch of the dopamine system. Thus, the evidence gathered so far might improve our understanding of the neural substrates of neuropsychiatric disorders such as ADHD, schizophrenia and drug addiction.

Prenatal elevation of endocannabinois corrects the unbalance between dopamine systems and reduces activity in the Naples High Excitability rats

Several evidences suggest that endocannabinoids exert a neurotrophic effect on developing mesencephalic dopamine neurons. Since an altered mesocorticolimbic system seems to underlie hyperactivity and attention deficit in clinical and animal studies of attention deficit hyperactivity disorder (ADHD), prenatal elevation of anandamide has been induced in Naples high excitability (NHE) rats by inhibition of its reuptake. To this aim, pregnant NHE and random-bred females received a subcutaneous injection of AM-404 (1 mg/kg) or vehicle daily from E11 until E20. Young adult male offsprings were exposed to a spatial novelty (Làt-maze) for 30 min and the behavior was videotaped and analysed for indices of activity (travelled distance, rearing frequency) and attention (rearing duration). Moreover, morphological analysis of the brains was carried out that pertained to cytochrome oxydase as marker of metabolic activity and thyrosine hydroxylase as marker of the dopamine systems. The results indicate that prenatal AM-404 treatment significantly reduces activity by about 20% during the entire testing period and modifies the distribution of scanning times towards short duration episodes in the first part of the test only in NHE-treated rats. In addition, image analysis revealed a significant increase in relative optical density of TH+terminals in the dorsal striatum and substantia nigra of AM-404 treated NHE rats and minor changes in the dorsal cortex of AM-404 treated NRB rats. The data suggest a corrected unbalance between the two dopamine systems that apparently leads to reduced hyperactivity and modified scanning times in this animal model of ADHD. This, in turn, might open new strategies in the treatment of a subset of ADHD cases.

Impaired metabolic capacity in the perirhinal and posterior parietal cortex lead to dissociation between attentional, motivational and spatial components of exploration in the Naples High-Excitability rat

This study aimed at investigating the neural substrates of spatial and non-spatial Behavioural components of exploration to novelty by a neurogenetic approach. Thus, functional imaging and Behavioural analysis were carried out in the Naples High-Excitability (NHE) rats, a model of hyperactivity and attention-deficit. Quantitative cytochrome oxidase (C.O.) histochemistry was used to measure the basal metabolic capacity of different forebrain structures. In parallel experiments, exploration in an 8-arm radial maze (Olton-maze) with extra-maze cues was used to measure attentional, motivational and spatial components of Behaviour after feeding rats’ ad-libitum or at a reduced diet. Functional imaging analysis: brains from naive rats were stained for quantitative C.O. histochemistry along with standards. NHE rats showed lower C.O. activity in perirhinal and posterior-parietal cortex (all layers) and cortical amygdala, and greater activity in entorhinal cortex (superficial layers). The outer granular cell layer of the dentate gyrus had greater activity in NHE. Behavioural analysis: at low and high motivational level, maze exploration was reinforced during shaping throughout and then only a single arm. The Behaviour was monitored by a CCD camera and videotaped. (i) There was no line difference in working memory during non reinforced maze exploration, independent of the motivational level; (ii) during shaping with all baited arms, there was no line difference in working memory, but NHE rats showed a very low or lower food consumption at low and high motivational level, respectively; (iii) rats showed a higher working memory in finding the single baited arm at high motivational level; (iv) NHE rats paid little attention towards reinforcement upon visiting the baited arm only at low motivational level. Thus, Behavioural and functional neuroimaging analysis suggests the neural substrates of spatial and non-spatial components of exploration to be underlined by different network operations in the neocortical and limbic cortices in the NHE rat lines. Therefore, they appear as an useful tool to the understanding of Attention-Deficit Hyperactivity Disorder (ADHD) in children. (Supported by Telethon–Italy grant E.513).

A morphometric evidence for a hyperfunctioning mesolimbic system in an animal model of ADHD

The hyperfunctioning dopamine hypothesis in the mesocorticolimbic (MCL) system has been addressed by a neurogenetic approach in model systems. Thus, a morphometric analysis was carried out on neurons of origin of Substantia Nigra (SN) and Ventral Tegmental Area (VTA) dopamine systems of the Naples High-Excitability (NHE), Low-Excitability (NLE) and control lines. Male adult rats were tested in a spatial novelty for indices of activity and non-selective attention. Mesencephalic coronal sections were processed for tyrosine hydroxylase (TH) immunohistochemistry and cytochromoxidase (C.O.) histochemistry. Image analysis in the rostro-caudal plane showed (i) a higher neuron size of TH+ elements in the VTA of NHE and NLE, across the entire structure in the NHE, and only in the middle portion in the NLE; (ii) a higher expression of TH in the neuropil of the VTA in NHE; (iii) a lower C.O. activity in both NLE and NHE; (iv) no differences in the SN. The larger neuron size in both NHE and NLE rats as compared with control rats, along with higher TH expression mainly in the NHE, in absence of any relevant alteration in the SN, reveals an unbalance between the two dopamine systems and a subsequent alteration in limbic (reward, motivation, sustained attention) functions. The decreased C.O. activity might be due to reduced feedback inhibition by striatal GABA neurons and interneurons leading to increased DA neuron firing. In conclusion, the increased behavioral activity and impaired attention observed in the NHE rats are associated to hyperfunctioning MCL system in this genetic model of Attention-Deficit Hyperactivity Disorder (ADHD).

Environmental factors during postnatal period modify activity and non-selective attention in the Naples High-Excitability rat

The involvement of epigenetic factors in the phenotypic expression of the neural systems underlying activity and attentive processes has been investigated in an animal model of hyperactivity and attention-deficit, the Naples High-Excitability rat (NHE). To this aim, male NHE pups have been reared in small (four) or normal litter size (nine) during the first 4 weeks of postnatal life. Both groups underwent a differential handling procedure occurring once, twice or four times a week. After weaning (28 days), rats were housed in groups of two and tested as young adults for activity and non-selective attention in a spatial novelty situation for three consecutive tests at 24-h intervals. The behaviour was videotaped and analysed off line for the frequency of corner crossings and rearings and duration of rearings. The results indicate that the increased maternal care and high fat diet induced by the small litter size produced long lasting effects on activity and duration of rearing episodes that indexes non-selective attention. These effects were complex as differential handling was beneficial only at low stimulation level. Thus, these findings suggest that epigenetic factors acting during critical periods of post-natal development may interact with genetic determinants that in turn influence the maturation of the neural systems controlling activity, orienting and scanning time.