In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.

Distinct patterns of Fos immunoreactivity in striatum and hippocampus induced by different kinds of novelty in mice.

In this study the immediate-early gene Fos was used to investigate the response to different novel stimuli in a wide array of brain regions including the hippocampus, the rhinal cortex, the frontal cortex and different components of the striatal complex. Independent groups of CD-1 mice were exposed to three different novelty conditions: (1) novel environment (empty open field); (2) complex novel environment (i.e. open field containing objects); and (3) identity-based detection of novel objects. We observed that a complex novel environment and a knowledge-based novelty modulated Fos levels in both the dorsal and the ventral components of the striatum, while Fos immunoreactivity in the medial temporal lobe was only increased after exposure to novel environments, regardless of their complexity. Finally, we observed a strong increase of Fos levels in the prefrontal cortex in all the three novel conditions examined, indicating a major involvement of this structure in novelty assessment. Overall the present study demonstrates that distinct brain regions are recruited in different kinds of novelty and emphasizes the role of the striatal complex in processing complex novel information.

microRNAs Modulate Spatial Memory in the Hippocampus and in the Ventral Striatum in a Region-Specific Manner.

MicroRNAs are endogenous, noncoding RNAs crucial for the post-transcriptional regulation of gene expression. Their role in spatial memory formation, however, is poorly explored. In this study, we analyzed learning-induced microRNA expression in the hippocampus and in the ventral striatum. Among miRNAs specifically downregulated by spatial training, we focused on the hippocampus-specific miR-324-5p and the ventral striatum-specific miR-24. In vivo overexpression of the two miRNAs demonstrated that miR-324-5p is able to impair memory if administered in the hippocampus but not in the ventral striatum, while the opposite is true for miR-24. Overall, these findings demonstrate a causal relationship between miRNA expression changes and spatial memory formation. Furthermore, they provide support for a regional dissociation in the post-transcriptional processes underlying spatial memory in the two brain structures analyzed.

NMDA and AMPA antagonist infusions into the ventral striatum impair different steps of spatial information processing in a nonassociative task in mice.

Most of the research on ventral striatal functions has been focused on their role in modulating reward and motivation. More recently, a possible role of this structure in cognitive functions has been suggested. However, very little information is available on the involvement of the nucleus accumbens in the different stages of the consolidation process. In this study, the effect of focal injections of AP-5 and DNQX, competitive antagonists at the NMDA and AMPA receptors, respectively, was examined in a nonassociative task designed to estimate the ability of mice to react to spatial changes. The task consists of placing the animals in an open field containing five objects; after three sessions of habituation, their reactivity to object displacement was examined 24 hr later. AP-5 injections administered after training impaired the ability of mice to detect the spatial novelty but did not affect response when injected 120 min after training or before testing. On the contrary, DNQX did not affect response when administered immediately or 120 min after training but did impair spatial discrimination when administered before training or testing. These data demonstrate a double dissociation between glutamate receptor subtypes, such that accumbens NMDA receptors are important for consolidation and not ongoing discrimination of spatial information, whereas AMPA receptors have an opposite role in these processes.

Studies on a new series of THA analogues: effects of the aromatic residues that line the gorge of AChE.

A series of N-monoalkylsubstituted 1,2,3,4-tetrahydro-9-aminoacridines have been prepared after modelling simulation of the AChE-inhibitor complex. Molecular modelling has predicted a number of hydrophobic residues to be involved in the catalytic mechanism of this interaction between the binding sites of AChE and this series of aminoacridines. In these compounds the acridine moiety becomes sandwiched between the rings of PHE330 and TRP84. In particular, the alkyl chain shows the important role of aromatic groups as binding sites. Their in vitro inhibitory properties (enzyme from Electrophorus electricus) confirm the aromatic groups as a general and significant characteristic of the mechanism of AChE inhibition.

Effects of lesions to the glutamatergic afferents to the nucleus accumbens in the modulation of reactivity to spatial and non-spatial novelty in mice.

The purpose of this study was to compare the effects of selective lesions of the three main sources of limbic afferents to the nucleus accumbens-fornix, prelimbic cortex and amygdala-with those induced by N-methyl-D-aspartate receptor blockage in this structure, in a non-associative task designed to estimate the ability of rodents to encode spatial and non-spatial relationships between discrete stimuli. The task consists of placing mice in an open field containing five objects and, after three sessions of habituation, examining their reactivity to object displacement (spatial novelty) and object substitution (object novelty). Focal administrations of the competitive N-methyl-D-aspartate antagonist DL-2-amino-5-phosphonopentanoic acid (0.1 microg/side) induced a selective impairment in the ability of mice to react to the spatial change. Lesions to the different structures affect the response of mice to spatial and non-spatial novelty in different ways. In particular, while fornix lesions induced a decrease in re-exploration of the displaced objects, prelimbic cortex lesions enhanced the exploration of both displaced and non-displaced objects. Finally, the basolateral amygdala lesions did not induce any impairment in the detection of the displaced objects but decreased the latencies to approach novel objects. It is concluded that N-methyl-D-aspartate receptor blockage in the nucleus accumbens subsumes the effects of the three lesions. Some hypotheses on the role of glutamatergic transmission in the accumbens on information processing are briefly discussed.

Locomotor activity induced by the non-competitive N-methyl-D-aspartate antagonist, MK-801: role of nucleus accumbens efferent pathways.

We have recently shown that focal administration of dizocilpine hydrogen maleate (MK-801, a non-competitive N-methyl-D-aspartate antagonist) within the nucleus accumbens increases locomotor activity in a dopamine-independent manner. The purpose of this study was to investigate the neural network underlying locomotor stimulation induced by N-methyl-D-aspartate receptor blockade in the accumbens. In the first experiment, we examined the effect of different doses (1, 5 and 25 nmol) of the active and inactive enantiomers of the N-methyl-D-aspartate antagonist, (+)- and (-)-MK-801, respectively, focally administered in the nucleus accumbens. Only the active enantiomer induced a significant increase in locomotor activity; furthermore, the effect induced by the two highest doses of (+)-MK-801 was significantly different from that induced by (-)-MK-801. In the second part of the study, we performed ibotenic acid lesions to the major output nuclei of the accumbens, the ventral pallidum, mediodorsal thalamus, ventrolateral/ventromedial thalamus and pedunculopontine tegmental nucleus, to observe their effect on locomotor activity induced by focal (+)-MK-801 (25 nmol) administration into the accumbens. None of the lesions had any effect on spontaneous locomotor activity. Hyperactivity induced by accumbens MK-801 administrations was unaffected by ibotenic acid lesions of the pedunculopontine tegmental nucleus, while lesions of the mediodorsal thalamus induced only a partial inhibition. In contrast, ibotenic acid lesions of the ventral pallidum and ventrolateral/ventromedial thalamus completely blocked the motor response induced by accumbens MK-801. These data indicate that the intact mediodorsal thalamus, which has been proposed as a part of the loop that relays accumbens information to the prefrontal cortex, does not seem to be a structure of primary importance in MK-801 locomotor activity. On the contrary, the motor nuclei of the thalamus appear to play a more relevant role, suggesting that different neural substrates may mediate dopamine and glutamate functional output from the nucleus accumbens.

Distinct pattern of c-fos mRNA expression after systemic and intra-accumbens amphetamine and MK-801.

Pharmacological manipulation of both dopamine and glutamate systems affects motor responses in laboratory animals. The two systems, however, seem to act in opposite ways, since direct or indirect activation of dopamine receptors induces similar stimulatory effects to those seen following blockade of N-methyl-D-aspartate receptors. In the present study we compared the pattern of c-fos activation induced by systemic and intra-accumbens administration of the non-competitive N-methyl-D-aspartate antagonist MK-801 and the indirect dopamine agonist amphetamine. Systemic MK-801 induced c-fos mRNA expression in the motor cortex and preferentially in the motor thalamus, i.e. ventrolateral nucleus. Systemic amphetamine, on the other hand, enhanced c-fos mRNA expression in the shell of the accumbens and in limbic thalamic nuclei such as the anteroventral and anterodorsal nuclei. The main effect observed after intra-accumbens administrations of either drug was enhanced c-fos expression in the thalamus, somewhat similar to what seen following systemic administration. In fact also in this case there was a preferential activation of the limbic thalamus by amphetamine and the motor thalamus by MK-801. The present results confirm that different neural substrates underlie behavioral effects induced by systemic administrations of N-methyl-D-aspartate receptor antagonists and dopamine agonists. Further they suggest that intra-accumbens manipulation of the two neural systems could affect different efferent pathways from this structure activating different thalamic targets.

Some central effects in mice of compounds related to nicotine.

1. Some hydroxy-, amino-, and methoxy- phenylalkyltrimethylammonium compounds, beta-pyridylmethyl- dimethylamine and pyrrolidine, and beta-pyridylethyltrimethylammonium, were tested on avoidance learning in mice and their effects were compared with those of (-)-nicotine.2. The o- and m- hydroxybenzyl-, o-hydroxyphenethyl- and m-hydroxyphenylpropyl- trimethylammonium compounds improved performance; (-)-nicotine, in one-quarter of the dose, had similar effects. The m- and p-hydroxyphenethyl-, o-hydroxyphenylpropyl- and o- and p- aminobenzyl, and o-, m-, and p- aminophenethyl-trimethylammonium compounds impaired performance.3. (-)-Nicotine and m-hydroxyphenylpropyltrimethylammonium appeared also to enhance memory consolidating processes.4. The central actions of some of the compounds suggest that the possibility that they can penetrate into the central nervous system should not be ruled out even though they are quaternary salts.5. No correlation was found between the effects of the compounds on avoidance learning and on the frog rectus muscle. Though the differences may be due to differences in access to the central nervous system, it is also possible that the receptors associated with learning processes are different from those in the frog rectus and possibly more specialized.

Effects of the NMDA-antagonist, MK-801, on stress-induced alterations of dopamine dependent behavior.

The effects of pretreatment with the non-competitive NMDA antagonist (+)MK-801 on the behavioral alterations induced by repeated restraint stress were investigated. Repeatedly stressed (restraint stress 2 h a day x 10 days) mice showed enhanced sensitivity to the inhibitory effects of a low dose of direct dopamine agonist, apomorphine (0.25 mg/kg), on climbing behavior. On the other hand, no changes were observed for the stimulatory effect of the high dose of apomorphine (3 mg/kg) on this behavioral response. Mice pretreated with MK-801 (0.15 mg/kg) before the stressful experience did not show altered response to the low dose of apomorphine (0.25 mg/kg). Finally, ten daily injections with 0.15 mg/kg MK-801 did not affect the behavioral response to the low dose of apomorphine, but enhanced the stimulatory effect of the high dose of the dopaminergic agonist on climbing behavior. Therefore, it is possible that the protective action of MK-801 against stress-induced behavioral alteration is due to changes in sensitivity of postsynaptic receptors.

Chronic treatment with MK-801 affects the behavioral response to both D1 and D2 dopamine agonist in the one-trial inhibitory avoidance.

Post-training administration of the N-methyl-D-aspartate (NMDA) antagonists CPP (0.5 and 1.0 mg/kg) and MK-801 (0.25 and 0.5 mg/kg) impaired, in a dose dependent fashion, the one-trial inhibitory avoidance response in NMRI mice. The D1 dopamine (DA) agonist SKF 38393 (10 and 20 mg/kg) and the D2 agonist quinpirole (0.5 and 1.0 mg/kg) instead facilitate the response in the same behavioral paradigm. Sub-chronic blockade of NMDA receptors with MK-801 (0.25 mg/kg once a day for 14 days) did not change the response to both competitive (CPP) and non-competitive (MK-801) NMDA antagonists. The same chronic treatment with MK-801 induced an increased response to both SKF 38393 and quinpirole. These data suggest that repeated administration of MK-801 induce an upregulation of both D1 and D2 DA receptors without affecting NMDA receptors.

Effects of heroin, alone or in combination with other drugs, on the locomotor activity in two inbred strains of mice.

The locomotor activity of C57Bl/6J and DBA/2J mice was studied, under the influences of heroin, amphetamine, strychnine, or ethanol, and of combinations of the opiate with each one of the other drugs. Heroin treatment was followed by the typical "running fit" in the C57 mice, while the DBA strain was unaffected. Amphetamine enhanced the activity in the C57 strain only. The combination of heroin with amphetamine or ethanol increased the locomotor activity only in the DBA strain, while heroin + strychnine exerted a clear stimulating effect on the activity of the C57 mice. The strychnine + heroin mixture was more toxic than heroin alone when the lethal doses (LD50) were determined in the 2 strains.

Differential effects of opiate agonists-antagonists on morphine-induced hyperexcitability and analgesia in mice.

The effects of two opiate agonists-antagonists, butorphanol (4.0 and 8.0 mg/kg) and buprenorphine (0.1 and 1.0 mg/kg), were assessed on locomotor activity and analgesia in DBA/2 and C57BL/6 mice. Different behavioral effects were evident in these strains, which might be characterized by different reactions to the effects of opiates and by differences in endorphin distributions and opiate receptor populations. In particular, buprenorphine acted as an agonist-antagonist to morphine in both strains while a dissociation of butorphanol effects was evident, depending on the strain considered. The clinical implications of these findings are discussed.

Effect of intra-accumbens dopamine receptor agents on reactivity to spatial and non-spatial changes in mice.

RATIONALE: Some evidence suggests an involvement of nucleus accumbens in spatial learning. However, it is controversial whether the mesoaccumbens dopaminergic pathways play a specific role in the acquisition of spatial information. OBJECTIVE: The goal of these experiments was to investigate the effect of dopaminergic manipulations in the nucleus accumbens on a non-associative task designed to estimate the ability to encode/transmit spatial and non-spatial information. METHODS: The effects of focal administrations of the D1 and D2 dopamine receptor antagonists, SCH 23390 (6.25, 12.5, 50 ng/side) and sulpiride (12.5, 50, 100 ng/side), respectively, and dopamine (DA; 1.25 and 2.5 microg/side) into the nucleus accumbens were studied on reactivity to spatial and non-spatial changes in an open field with objects. RESULTS: Both SCH 23390 and sulpiride impaired reactivity to spatial change. However, several differences were found in the effects induced by the two DA antagonists. SCH 23390 did not affect locomotor activity and only slightly impaired exploration of the novel object. On the contrary, the D2 antagonist, induced a general, dose-dependent, impairment on all variables measured. Local administration of DA increased locomotor activity, but did not affect reactivity to spatial and non-spatial changes. CONCLUSIONS: These results demonstrate a facilitatory role of mesoaccumbens dopamine in the acquisition of spatial information. Moreover, they suggest that nucleus accumbens D1 DA receptors, play a more selective role in the modulation of spatial learning than accumbens D2 DA receptors.

Role of dopaminergic system in reactivity to spatial and non-spatial changes in mice.

RATIONALE: While experimental evidence shows that dopamine (DA) systems have an important role in locomotor function and in motivation, their role in the reactivity to spatial and non-spatial novelty is less well established. OBJECTIVE: In this study, we investigated the effects of dopaminergic pharmacological manipulation on the capability of CD1 mice to encode spatial and non-spatial information in an open field with objects. METHODS: The effects of systemic administration of: (1) selective D1 and D2 antagonists (SCH23390, 0.015 mg/kg or 0.020 mg/kg; sulpiride, 10 mg/kg or 20 mg/kg); (2) direct and indirect DA agonists (apomorphine, 1 mg/kg or 2 mg/kg; amphetamine, 1 mg/kg or 2 mg/kg) were studied. RESULTS: On the one hand, systemic administration of either D1 or D2 antagonists induced a selective impairment in the detection of spatial change but did not affect reaction to non-spatial novelty. On the other hand, amphetamine induced a selective decrease in exploratory activity in the first three sessions. This decrease did not affect the ability of mice to react to spatial change, but a dose-dependent decrease was observed in reactivity to non-spatial novelty. Such an effect does not seem to be due to amphetamine-induced hyperactivity or to non-DA mechanisms, since apomorphine induced a similar neophobic profile, without affecting locomotion. CONCLUSIONS: Taken together, these results demonstrate that manipulations of DA transmission affect reactivity to spatial and non-spatial novelty. In particular, we suggest that these two behavioral responses are modulated in opposite ways by the DA system.

Brain dopamine receptor plasticity: testing a diathesis-stress hypothesis in an animal model.

A wealth of clinical data supports a major role of genetic liability as well as of altered brain dopamine (DA) functioning in different types of behavioural disturbances. Genetic influence on behaviour involves multiple genes, rather than one or two major genes, as well as non-genetic sources of variance. Thus, in recent years, increasing attention has been devoted to the involvement of stressful experiences (life events) in the development and expression of psychopathology. Moreover, a diathesis-stress hypothesis has been proposed, which suggests that the environmental factors (stress) are not specific for a given pathology, whereas genetic factors (diathesis) are. Results obtained in an animal model offer support to this hypothesis. Indeed, mice of the C57BL/6 and DBA/2 inbred strains are equally susceptible to stress but develop different behavioural disturbances related to different alterations of brain DA receptors. Moreover, quantitative trait loci (QTL) associations in the C57 (B) x DBA (D) recombinant inbred (RI) strains indicate a number of provisional QTLs influencing the behavioural effect of stress. Finally, the results of this analysis suggest the involvement of regulatory factors related to stress response and neural or synaptic plasticity in the control of brain DA receptor plasticity.

N-methyl-D-aspartate receptors in the nucleus accumbens are involved in detection of spatial novelty in mice.

The aim of this study was to investigate the role played by intra-accumbens N-methyl-D-aspartate (NMDA) receptors in spatial information encoding. For this purpose, the effect of local administration of both competitive (AP-5) and non-competitive (MK-801) NMDA antagonists was assessed in a task designed to estimate the ability of rodents to encode spatial relationships between discrete stimuli. The task consists of placing mice in an open field containing five objects and, after three sessions of habituation, examining their reactivity to object displacement (spatial novelty) and object substitution (object novelty). The results show that both doses of MK-801 (0.15 and 0.3 microg/side) induced a selective impairment in the capability of mice to detect spatial novelty. A similar effect was obtained by injecting the low dose of the competitive antagonist AP-5 (0.1 microg/side), whereas the high dose (0.15 microg/side) abolished detection of both spatial and object novelty. Taken together, these results show that intra-accumbens injections of low doses of competitive and non-competitive NMDA antagonists can produce selective deficits in processing spatial information resembling those observed after hippocampal damage. Moreover, the fact that pharmacological treatments spare memory processes involved in habituation suggests that NMDA antagonists may interfere with the formation of spatial representations rather than producing memory deficits per se.

Effects of corticotropin releasing factor and sauvagine on social behavior of isolated mice.

Corticotropin releasing factor (CRF) and sauvagine (SVG) when injected ICV both reduced aggressive behavior and sociability while increasing defensive behavior in isolated DBA/2 mice interacting with a group-housed intruder. SVG was more effective than CRF in producing such behavioral effects. These results add further evidence to the similarity between CRF and SVG, and are discussed in terms of the involvement of these peptides in emotional reactivity in the laboratory mouse.

Effects of sauvagine on behavioural arousal of mice.

The effects of natural and synthetic sauvagine on locomotor activity and ECS-induced seizures were studied in DBA/2 mice. A dose-dependent activity depression was evident following the administration of both compounds. Moreover they exerted a protective effect against ECS-induced seizures. This effect was naloxone-reversible, suggesting the involvement of endogenous opioids. In both series of experiments natural sauvagine was more effective than the synthetic compound.

Nonhuman behavioral models in the genetics of disturbed behavior.

The development of the association method in which genetic markers match quantitative traits had led to quantitative trait loci (QTL) interval mapping. The association method has been extensively used in animal behavior genetics. Animal research allows more suitable linkage studies and detailed assessment of cellular and subcellular components of the central nervous system that may play a crucial role in the development susceptibility to behavioral disorders. Moreover, experimental designs in the laboratory setting allow genotype x environment interactions to be controlled, thus possibly providing more information on the role of nongenetic factors in gene expression. Experimental results are discussed which indicate that animal studies will provide a sort of test for hypotheses arising in clinical settings, allowing gene-product and product-behavior pathways to be examined at molecular levels when the gene accounts for a very small amount of genetic variance. In such a perspective, new molecular biology approaches and behavior genetics in nonhuman species could provide useful tools in the assessment of the genetic as well as nongenetic factors that lead to psychopathology.