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 2 days 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, 0.30 mg/kg) or vehicle for 15 days 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 (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/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.

Behavioral alterations and changes in Ca/calmodulin kinase II levels in the striatum of connexin36 deficient mice.

Gap junctions (GJ) are intercellular channels which directly connect the cytoplasm of adjacent cells. GJ allow direct cell-to-cell communication via the diffusion of ions, metabolites and second messengers such as IP(3). The connexin36 (Cx36) protein has been detected in GJ between interneurons of the hippocampus, cerebral cortex, striatum, amygdala, the inferior olive, cerebellum and other brain structures, such as the olfactory bulb. Cx36 knockout (Cx36 KO) mice display changes in synchronous network oscillations in the hippocampus, neocortex and inferior olive and exhibit impaired spatial alternation and one-trial object recognition in a Y-maze. Here, we further characterized the behavioral changes induced by Cx36 deficiency in the mouse by using different behavioral measures and experimental procedures. Additionally, we examined the effects of Cx36 deficiency on acetylcholine esterase (AChE) activity and calcium calmodulin kinase II alpha (CaMKII) protein levels in the striatum. The homozygous Cx36 KO mice displayed increased locomotion and running speed in the open-field, reduced object exploration and impaired one-trial object-place recognition. Furthermore, they exhibited more anxiety-like behavior as compared to the heterozygous controls in the light-dark box. Homozygous Cx36 KO mice exhibited reduced CaMKII levels in the striatum as compared to the heterozygous mice. AChE activity in the striatum was not significantly different between groups. The present results suggest that Cx36 deficiency in the mouse leads to reduced CaMKII levels in the striatum and behavioral changes in open-field activity, anxiety-related behavior in the light-dark box and one-trial object-place recognition.

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.

Neuronal histamine and the interplay of memory, reinforcement and emotions.

The biogenic amine histamine is an important neurotransmitter-neuromodulator in the central nervous system that has been implicated in a variety of biological functions including thermo- and immunoregulation, food intake, seizures, arousal, anxiety, reward and memory. The review of the pertinent literature indicates that the majority of findings are compatible with the appraisal that the inhibition of histaminergic neurotransmission impairs learning and memory formation, decreases cortical activation and arousal, has a suppressive effect on behavioral measures of fear and anxiety, exponentiates the rewarding effects of drugs of abuse and intracranial brain stimulation. In contrast, the stimulation of histaminergic neurotransmission can ameliorate learning and memory impairments that are associated with various experimental deficit models and pathological conditions. Clinical investigations with patients suffering from neurodegenerative diseases such as Alzheimer's and Parkinson's disease demonstrate pathological alterations in the brain's histaminergic system, which, in some cases are correlated with the severity of cognitive deficits. The role of the brain's histamine system in episodic memory formation and the potential of histamine-related drugs to ameliorate cognitive deficits in early stages of neurodegenerative diseases are discussed.

Histamine H1 receptor knockout mice exhibit impaired spatial memory in the eight-arm radial maze.

BACKGROUND AND PURPOSE: In the mammalian brain, histaminergic neurotransmission is mediated by the postsynaptic histamine H1 and H2 receptors and the presynaptic H3 autoreceptor, which also acts as a heteroreceptor. The H1 receptor has been implicated in spatial learning and memory formation. However, pharmacological and lesion studies have revealed conflicting results. To examine the involvement of histamine H1 receptor in spatial reference and working memory formation, H1 receptor knockout mice (KO) were tested in the eight-arm radial maze. Previously, we found that the H1 receptor-KO mice showed reduced emotionality when confronted with spatial novelty. As it is known that emotions can have an impact on spatial learning and memory performance, we also evaluated H1 receptor-KO mice in terms of emotional behaviour in the light-dark box. EXPERIMENTAL APPROACH: Mice lacking the H1 receptor and wild-type mice (WT) were tested for spatial reference and working memory in an eight-arm radial maze with three arms baited and one trial per day. Emotional behaviour was measured using the light-dark test. KEY RESULTS: The H1 receptor-KO mice showed impaired spatial reference and working memory in the radial maze task. No significant differences between H1 receptor-KO and WT mice were observed in the light-dark test. CONCLUSIONS AND IMPLICATIONS: The spatial memory deficits of the H1 receptor-KO mice might be due to the reported changes in cholinergic neurochemical parameters in the frontal cortex and the CA1 subregion of the hippocampus, to impaired synaptic plasticity in the hippocampus, and/or to a dysfunctional brain reward/reinforcement system.

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.

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.

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.

Episodic-like and procedural memory impairments in histamine H1 Receptor knockout mice coincide with changes in acetylcholine esterase activity in the hippocampus and dopamine turnover in the cerebellum.

We investigated episodic-like (ELM) and procedural memory (PM) in histamine H1 receptor knockout (H1R-KO) mice. In order to relate possible behavioral deficits to neurobiological changes, we examined H1R-KO and wild-type (WT) mice in terms of acetylcholine esterase (AChE) activity in subregions of the hippocampus and AChE and tyrosine hydroxylase (TH) expression in the striatum. Furthermore, we analyzed acetylcholine (ACh), 5-HT and dopamine (DA) levels, including metabolites, in the cerebellum of H1R-KO and WT mice. The homozygous H1R-KO mice showed impaired ELM as compared with the heterozygous H1R-KO and WT mice. The performance of homozygous H1R-KO mice in the ELM task was primarily driven by familiarity-based memory processes. While the homozygous H1R-KO mice performed similar to the heterozygous H1R-KO and WT mice during the acquisition of a PM, as measured with an accelerating rotarod, after a retention interval of 7 days their performance was impaired relative to the heterozygous H1R-KO and WT mice. These findings suggest that, both, ELM and long-term PM are impaired in the homozygous H1R-KO mice. Neurochemical assays revealed that the H1R-KO mice had significantly lower levels of AChE activity in the dentate gyrus (DG) and CA1 subregions of the hippocampus as compared with the WT mice. The homozygous H1R-KO mice also displayed significantly reduced dihydroxyphenylacetic acid (DOPAC) levels and a reduced DOPAC/DA ratio in the cerebellum, suggesting that the DA turnover in the cerebellum is decelerated in homozygous H1R-KO mice. In conclusion, homozygous H1R-KO mice display severe long-term memory deficits in, both, ELM and PM, which coincide with changes in AChE activity in the hippocampus as well as DA turnover in the cerebellum. The importance of these findings for Alzheimer's (AD) and Parkinson's disease (PD) is discussed.

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex.

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

The histamine H1-receptor mediates the motivational effects of novelty.

Novelty-induced arousal has motivational effects and can reinforce behavior. The mechanisms by which novelty acts as a reinforcer are unknown. Novelty-induced arousal can be either rewarding or aversive dependent on its intensity and the preceding state of arousal. The brain's histamine system has been implicated in both arousal and reinforcement. Histamine and histamine-1-receptor (H1R) agonists induced arousal and wakefulness in humans and rodents, e.g. by stimulating cortical acetylcholine (ACh) release. The H1R has also been implicated in processes of brain reward via interactions with the nigrostriatal- and mesolimbic dopamine (DA) systems. We asked whether the motivational effects of novelty-induced arousal are compromised in H1R knockout (KO) mice. The H1R-KO mice failed to develop a conditioned place-preference induced by novel objects. Even though they still explore novel objects, their reinforcing value is diminished. Furthermore, they showed impaired novelty-induced alternation in the Y-maze. Rearing activity and emotional behavior in a novel environment was also altered in H1R-KO mice, whereas object-place recognition was unaffected. The H1R-KO mice had higher ACh concentrations in the frontal cortex and amygdala (AMY). In the latter, the H1R-KO mice had also increased levels of DA, but a lower dihydrophenylacetic acid/DA ratio. Furthermore, the H1R-KO mice had also increased tyrosine hydroxylase immunoreactivity in the basolateral anterior, basolateral ventral and cortical AMY nuclei. We conclude that the motivational effects of novelty are diminished in H1R-KO mice, possibly due to reduced novelty-induced arousal and/or a dysfunctional brain reward system.

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 mglkg 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.

Galactosilated dopamine increases attention without reducing activity in C57BL/6 mice.

Different strategies can be used to carry dopamine into the brain such as L-Dopa precursors or galactosilated form of DA (GAL-DA). The aim of this study was to investigate whether GAL-DA would reduce hyperactivity and increase non-selective attention (NSA) in a mouse model of attention deficit hyperactivity disorder (ADHD), as, i.e. C57BL/6 as did in NHE rats. Here we report that GAL-DA increases NSA in a spatial novelty in C57BL/6 mice. They received a single i.p. injection of GAL-DA (10 mg/kg or 100 mg/kg) or equimolar galactose vehicle. Another mouse strain the Swiss albino was introduced as inbred control group. Three hours after last injection mice were tested in a Làt-maze for 30-min. Behaviour was analyzed for horizontal (traveled distance) and vertical activity (orienting frequency and scanning durations) which shares cognitive and non-cognitive nature, respectively. Ten milligram per kilograms of GAL-DA, increases scanning duration in C57BL/6 mice. Thus a low dose of GAL-DA increases NSA without reducing hyperactivity in this mouse model of ADHD.

Transcription factor expression, RNA synthesis and NADPH-diaphorase across the rat brain and exposure to spatial novelty.

The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos, c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by (3)H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Làt-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6h after testing, and in the somatosensory cortex 24h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2h and disappeared 24h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

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.

Prenatal elevation of endocannabinoids 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.

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.

Selective inhibition of neuronal nitric oxide synthesis reduces hyperactivity and increases non-selective attention in the Naples High-Excitability rat.

The involvement of neuron-derived NO in the process of orienting and scanning times (non-selective attention: NSA) towards environmental stimuli has been investigated in the Naples High-Excitability rat (NHE), a putative animal model of Hyperactivity and Attention Deficit (ADHD). To this aim, orienting and scanning times have been monitored by the frequency and duration of rearing episodes, respectively. Adult male NHE rats were tested in a novelty situation (Làt-maze) for 30 min following single or repeated injections of the non competitive inhibitor 7-Nitroindazole (7-NINA) of the neuronal isoform of the enzyme nitric oxide synthase (n-NOS). In the acute experiments, rats received a single injection of 7-NINA (1 mg/kg) intraperitonealy in a saline vehicle (exp. 1, fast release) or subcutaneously in a lipid carrier, dimethyl sulfoxide (DMSO; exp. 2, slow release) or the vehicles alone as controls 30 min before testing. In the repeated injection experiments, rats received a subcutaneus injection of 1 mg/kg in DMSO or DMSO alone daily for 14 days, and tested 24 h after the last injection (exp. 3, slow release). The results showed a significant differential effect of the drug that was dependent on the release rate, i.p. saline-diluted 7-NINA increased the duration of individual rearing episodes whereas, both single and repeated subcutaneous DMSO-carried 7-NINA exerted an opposite effect. Thus, selective inhibition of n-NOS by an allosteric inhibitor that increases arginine availability without displacing the inhibitor from n-NOS, strengthens the hypothesized role of NO in NSA. These findings may shed light on the mechanism of action of drug treatment of and be useful in the treatment of ADHD in children.