Early cellular and synaptic changes in dopaminoceptive forebrain regions of juvenile mice following gestational exposure to valproate.

Gestational exposure of mice to valproic acid (VPA) is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). In the present study we hypothesized that the reduction of dopaminergic source neurons of the VTA, followed by perturbed growth of the mesotelencephalic dopamine pathway (MT), should also modify pattern formation in the dopaminoceptive target regions (particularly its mesoaccumbens/mesolimbic portion). Here, we investigated VPA-evoked cellular morphological (apoptosis-frequency detected by Caspase-3, abundance of Ca-binding proteins, CaBP), as well as synaptic proteomic (western blotting) changes, in selected dopaminoceptive subpallial, as compared to pallial, regions of mice, born to mothers treated with 500 mg/kg VPA on day 13.5 of pregnancy. We observed a surge of apoptosis on VPA treatment in nearly all investigated subpallial and pallial regions; with a non-significant trend of similar increase the nucleus accumbens (NAc) at P7, the age at which the MT pathway reduction has been reported (also supplemented by current findings). Of the CaBPs, calretinin (CR) expression was decreased in pallial regions, most prominently in retrosplenial cortex, but not in the subpallium of P7 mice. Calbindin-D 28K (CB) was selectively reduced in the caudate-putamen (CPu) of VPA exposed animals at P7 but no longer at P60, pointing to a potency of repairment. The VPA-associated overall increase in apoptosis at P7 did not correlate with the abundance and distribution of CaBPs, except in CPu, in which the marked drop of CB was negatively correlated with increased apoptosis. Abundance of parvalbumin (PV) at P60 showed no significant response to VPA treatment in any of the observed regions we did not find colocalization of apoptotic (Casp3+) cells with CaBP-immunoreactive neurons. The proteomic findings suggest reduction of tyrosine hydroxylase in the crude synaptosome fraction of NAc, but not in the CPu, without simultaneous decrease of the synaptic protein, synaptophysin, indicating selective impairment of dopaminergic synapses. The morpho-functional changes found in forebrain regions of VPA-exposed mice may signify dendritic and synaptic reorganization in dopaminergic target regions, with potential translational value to similar impairments in the pathogenesis of human ASD.

Avian models for brain mechanisms underlying altered social behavior in autism.

The current review is an update on experimental approaches in which birds serve as model species for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). The discussion is focused on deficiencies of social behavior, from social interactions of domestic chicks, based on visual and auditory cues, to vocal communication in songbirds. Two groups of pathogenetic/risk factors are discussed: 1) non-genetic (environmental/epigenetic) factors, exemplified by embryonic exposure to valproic acid (VPA), and 2) genetic factors, represented by a list of candidate genes and signaling pathways of diagnostic or predictive value in ASD patients. Given the similarities of birds as experimental models to humans (visual orientation, vocal learning, social cohesions), avian models usefully contribute toward the elucidation of the neural systems and developmental factors underlying ASD, improving the applicability of preclinical results obtained on laboratory rodents. Furthermore, they may predict potential susceptibility factors worthy of investigation (both by animal studies and by monitoring human babies at risk), with potential therapeutic consequence.

D-Aspartate consumption selectively promotes intermediate-term spatial memory and the expression of hippocampal NMDA receptor subunits.

D-Aspartate (D-Asp) and D-serine (D-Ser) have been proposed to promote early-phase LTP in vitro and to enhance spatial memory in vivo. Here, we investigated the behavioural effects of chronic consumption of D-Asp and D-Ser on spatial learning of mice together with the expression of NMDA receptors. We also studied the alterations of neurogenesis by morphometric analysis of bromo-deoxyuridine incorporating and doublecortin expressing cells in the hippocampus. Our results specify a time period (3-4 h post-training), within which the animals exposed to D-Asp (but not D-Ser) show a more stable memory during retrieval. The cognitive improvement is due to elimination of transient bouts of destabilization and reconsolidation of memory, rather than to enhanced acquisition. D-Asp also protracted reversal learning probably due to reduced plasticity. Expression of GluN1 and GluN2A subunits was elevated in the hippocampus of D-Asp (but not D-Ser) treated mice. D-Asp or D-Ser did not alter the proliferation of neuronal progenitor cells in the hippocampus. The observed learning-related changes evoked by D-Asp are unlikely to be due to enhanced proliferation and recruitment of new neurones. Rather, they are likely associated with an upregulation of NMDA receptors, as well as a reorganization of receptor subunit assemblies in existing hippocampal/dentate neurons.

Gestational Exposure to Sodium Valproate Disrupts Fasciculation of the Mesotelencephalic Dopaminergic Tract, With a Selective Reduction of Dopaminergic Output From the Ventral Tegmental Area.

Gestational exposure to valproic acid (VPA) is known to cause behavioral deficits of sociability, matching similar alterations in human autism spectrum disorder (ASD). Available data are scarce on the neuromorphological changes in VPA-exposed animals. Here, we focused on alterations of the dopaminergic system, which is implicated in motivation and reward, with relevance to social cohesion. Whole brains from 7-day-old mice born to mothers given a single injection of VPA (400 mg/kg b.wt.) on E13.5 were immunostained against tyrosine hydroxylase (TH). They were scanned using the iDISCO method with a laser light-sheet microscope, and the reconstructed images were analyzed in 3D for quantitative morphometry. A marked reduction of mesotelencephalic (MT) axonal fascicles together with a widening of the MT tract were observed in VPA treated mice, while other major brain tracts appeared anatomically intact. We also found a reduction in the abundance of dopaminergic ventral tegmental (VTA) neurons, accompanied by diminished tissue level of DA in ventrobasal telencephalic regions (including the nucleus accumbens (NAc), olfactory tubercle, BST, substantia innominata). Such a reduction of DA was not observed in the non-limbic caudate-putamen. Conversely, the abundance of TH+ cells in the substantia nigra (SN) was increased, presumably due to a compensatory mechanism or to an altered distribution of TH+ neurons occupying the SN and the VTA. The findings suggest that defasciculation of the MT tract and neuronal loss in VTA, followed by diminished dopaminergic input to the ventrobasal telencephalon at a critical time point of embryonic development (E13-E14) may hinder the patterning of certain brain centers underlying decision making and sociability.

Valproate Exposure in ovo Attenuates the Acquisition of Social Preferences of Young Post-hatch Domestic Chicks.

Embryonic exposure to valproic acid (VPA) is known to produce sociability deficits, resembling human autistic phenotypes, in several vertebrate species. Animals living in groups prefer the proximity of peers and have the ability to perceive and to respond to social signals for modifying behavior. Chicks of Galliform birds, known to display early preference behaviors, have been used extensively for adaptive learning studies. Young precocial birds seem to be useful models also for studying the effect of embryonic VPA treatment. Here, domestic chicken eggs were injected with sodium valproate (200 µl of 35 µmol/L solution) or with vehicle (distilled water) on the 14th day of incubation. After hatching, the chicks were tested for one-trial passive avoidance learning at day 1, vocalization due to isolation as a measure of stress level (day 2), approach preference to large versus small groups of age-matched conspecifics (day 5), and to those with normal versus blurred head features (day 7). In addition, we tested the preference of birds to conspecifics reared in group versus those reared in isolation (day 9), as well as the preference of chicks to familiar versus non-familiar conspecifics (day 21). Our findings confirm previous reports concerning an adverse effect of VPA on embryonic development, including a tendency for aborted or delayed hatching and, occasionally, for locomotor disorders in a small percentage of birds (eliminated from later studies). Otherwise, VPA treatment did not impair motor activity or distress level. Memory formation for the aversive stimulus and discrimination of colors were not impaired by VPA treatment either. Innate social predispositions manifested in approach preferences for the larger target group or for the birds with natural facial features remained unaffected by VPA exposure. The most prominent finding was attenuation of social exploration in VPA-exposed birds (expressed as the frequency of positional switches between two stimulus chicks after the first choice), followed by a deficit in the recognition of familiar conspecifics, unfolding at the end of the third week. Social exploration and recognition of familiar individuals are the key elements impaired at this stage. The results underline the importance of early social exploration in ASD.

Selective neuronal death following exposure to methylenedioxypyrovalerone is accompanied by an inhibition of NMDA receptor NR2B subunit expression.

In a previous study, methylenedioxypyrovalerone (MDPV), a designer drug of the cathinone family, caused selective enhancement of Caspase3 immunoreactive (Casp3+) apoptotic cells in the nucleus accumbens (NAc) of 7­day­old mice. To further elaborate on the mechanism underlying MDPV­elicited apoptosis, here, we investigated the appearance of Casp3+ cells in developing neural tube explants of E12.5 mice, following MDPV treatment in vitro. Apoptotic cells appeared in large number in the pallium as radial progenitor cells and multipolar neurons, and in the subpallium including the future NAc, both in control and MDPV treated specimens. MDPV did not cause gross morphological changes in the neural tube or in the abundance of Casp3+ cells, based on a visual impression, though quantification was not attempted. We also studied the changes in NMDA receptor (NMDAR) protein subunits NR1 and NR2B in the NAc of 7­day­old MDPV treated and control mice, using western blotting of tissue obtained by selective dissection. In MDPV treated animals, expression of NR2B was lower than in the control animals, whereas expression of NR1 did not differ significantly from controls. The findings indicate that, during early postembryonic development, downregulation of the NR2B receptor subunit (at this time predominant in the NMDAR) is accompanied by a decreased viability of neurons. Decreased viability is expressed, in this case, as enhanced susceptibility to stimulation by MDPV - essentially a robust dopaminergic agent, potently affecting the neurons of the NAc. The findings are likely relevant to dopaminergic/NMDAR interactions and a potential pro­survival role of the NR2B subunit in critical phases of neural development.

Brain Distribution and Sexually Dimorphic Expression of Amylin in Different Reproductive Stages of the Zebra Finch (Taeniopygia guttata) Suggest Roles of the Neuropeptide in Song Learning and Social Behaviour.

The expression of the recently identified neuropeptide, amylin, is restricted in rodents to the postpartum preoptic area and may play a role in the control of parental behaviours and food intake. These processes are substantially different between bird and rodent parents as birds do not lactate but often show biparental care of the offspring. To establish the presence and role of amylin in the bird brain, in the present study, we investigated the distribution of amylin in brains of adult male and female zebra finches in three different reproductive stages (i.e. paired without young, incubating eggs or provisioning nestlings) and in unpaired control birds living in same sex flocks. Amylin mRNA was identified in the hypothalamus of zebra finch by RT-PCR, which was also used to produce probes for in situ hybridisation. Subsequently, in situ hybridisation histochemistry was performed in brain sections, and the labelling signal was quantified and compared between the groups. Amylin showed a much wider brain distribution than that of rodents. A strong and, in some regions, sexually dimorphic label was found in the striatum and several brain regions of the social behavioural network in both males and females. Many regions responsible for the learning of birdsong also contained amylin-positive neurons, and some regions showed sex differences reflecting the fact that vocalisation is sexually dimorphic in the zebra finch: only males sing. Area X (Ar.X), a striatal song centre present only in males, was labelled in paired but not unpaired male. Ar.X, another song centre, the lateral part of the magnocellular nucleus of the anterior nidopallium (lMAN) also contained amylin and had higher amylin label in paired, as opposed to unpaired birds. The wider distribution of amylin in birds as compared to rodents suggests a more general role of amylin in social or other behaviours in avian species than in mammals. Alternatively, parental care in birds may be a more complex behavioural trait involving a wider set of brain regions. The sex differences in song centres, and the changes with reproductive status suggest a participation of amylin in social behaviours and related changes in the singing of males.

Age-related and function-dependent regional alterations of free L- and D-aspartate in postembryonic chick brain.

D-aspartate (D-Asp) modulates adult neural plasticity and embryonic brain development by promoting cell proliferation, survival and differentiation. Here, developmental changes of the excitatory amino acids (EAAs) L-Glu, L-Asp and D-Asp were determined during the first postembryonic days, a time window for early learning, in selected brain regions of domestic chickens after chiral separation and capillary electrophoresis. Extracellular concentration (ECC) of EAAs was measured in microdialysis samples from freely moving chicks. ECC of D-Asp (but not L-EAAs) decreased during the first week of age, with no considerable regional or learning-related variation. ECC of L-Asp and L-Glu (but not of D-Asp) were elevated in the mSt/Ac in response to a rewarding stimulus, suggesting importance of Asp-Glu co-release in synaptic plasticity of basal ganglia. Potassium-evoked release of D-Asp, with a protracted transient, was also demonstrated. D-Asp constitutes greater percentage of total aspartate in the extracellular space than in whole tissue extracts, thus the bulk of D-Asp detected in tissue appears in the extracellular space. Conversely, only a fraction of tissue L-EAAs can be detected in extracellular space. The lack of changes in tissue D-Asp following avoidance learning indicates a tonic, rather than phasic, mechanism in the neuromodulatory action of this amino acid.

Gestational Exposure to the Synthetic Cathinone Methylenedioxypyrovalerone Results in Reduced Maternal Care and Behavioral Alterations in Mouse Pups.

The member of synthetic cathinone family, methylenedioxypyrovalerone (MDPV), is a frequently used psychoactive drug of abuse. The objective of our study was to determine the effect of MDPV (administered from the 8th to the 14th day of gestation) on the behavior of neonatal and adolescent mice, as well as its effect on maternal care. We measured maternal care (pup retrieval test, nest building), locomotor activity (open field test), and motor coordination (grip strength test) of dams, whereas on pups we examined locomotor activity at postnatal day 7 and day 21 (open field test) and motor coordination on day 21 (grip strength test). On fresh-frozen brain samples of the dams we examined the expression of two important peptides implicated in the regulation of maternal behavior and lactation: tuberoinfundibular peptide 39 (TIP39) mRNA in the thalamic posterior intralaminar complex, and amylin mRNA in the medial preoptic nucleus. We detected decreased birth rate and survival of offspring, and reduced maternal care in the drug-treated animals, whereas there was no difference between the motility of treated and control mothers. Locomotor activity of the pups was increased in the MDPV treated group both at 7 and 21 days of age, while motor coordination was unaffected by MDPV treatment. TIP39 and amylin were detected in their typical location but failed to show a significant difference of expression between the drug-treated and control groups. The results suggest that chronic systemic administration of the cathinone agent MDPV to pregnant mice can reduce birth rate and maternal care, and it also enhances motility (without impairment of motor coordination) of the offspring.

Update on forebrain evolution: From neurogenesis to thermogenesis.

Comparative developmental studies provide growing understanding of vertebrate forebrain evolution. This short review directs the spotlight to some newly emerging aspects, including the evolutionary origin of the proliferative region known as the subventricular zone (SVZ) and of intermediate progenitor cells (IPCs) that populate the SVZ, neural circuits that originated within homologous regions across all amniotes, and the role of thermogenesis in the acquisition of an increased brain size. These data were presented at the 8th European Conference on Comparative Neurobiology.

Multiple amygdaloid divisions of arcopallium send convergent projections to the nucleus accumbens and neighboring subpallial amygdala regions in the domestic chicken: a selective pathway tracing and reconstruction study.

Retrograde tracing with choleratoxin B, injected into the nucleus accumbens (Ac) and bed nucleus of stria terminalis, lateral part (BSTL), yielded labeled perikarya in a ring-shaped area of arcopallium, including dorsal and hilar subdivisions, with a wedge-shaped node of dense accumulation in the amygdalopiriform area (APir). Also, the position of source neurons for this arcopallio-subpallial pathway was verified by anterograde tracing. Three subregions of arcopallium (amygdalopiriform, dorsal, hilar) were injected with dextran (10 kDa), and fibers and terminal fields were detected in Ac, BSTL and extended amygdala (EA). Most abundant projections to Ac arose from APir. The study enabled precise description of the main output fiber streams: the dorsal stream follows the dorsal border of arcopallium and, continuing in the ventral amygdalofugal tract, it traverses the EA and the BSTL before reaching the Ac. The ventral stream of fibers enters the EA along the ventral subpallial border and terminates in the basal nucleus and ventral pallidum. The course of the pathway was reconstructed in 3D. Retrogradely labeled arcopallial neurons were devoid of DARPP-32. DARPP-32 was present in the Ac but not the BSTL. No colocalization between the calcium binding proteins calbindin, parvalbumin and calretinin, and retrogradely labeled neurons was detected, despite a considerable territorial overlap. This finding further supports the excitatory nature of the arcopallial-accumbens pathway. Conjoint and convergent amygdalar input to EA, including BSTL, as well as to Ac subregions likely transmits fear and aggression related signals to both viscerolimbic (EA) and learned reward- and motivation-related (Ac) ventrobasal forebrain regions.

Effect of nucleus accumbens lesions on socially motivated behaviour of young domestic chicks.

Behaviour of young domestic chicks when isolated from conspecifics is influenced by two conflicting drives: fear of potential predator and craving for company. The nucleus accumbens (Ac) has been suggested to influence social behaviour, as well as motivation in goal-directed tasks. In this study, the Ac of 1-day-old domestic chicks was lesioned bilaterally, using radiofrequency method. Open field behaviour before and after presenting a silhouette of a bird of prey was recorded, followed by a behavioural test to measure group size preference and social motivation of chicks. Ac-lesioned individuals emitted more distress calls and ambulated more in the open field test, however, they reacted to the predatory stimulus very similarly to control chicks: their vocalization was reduced and the intergroup difference in motor activity also disappeared. There was no difference between the lesioned and control chicks in the latency to approach their conspecifics in the social motivation test, and both groups chose the larger flock (eight) of conspecifics over the smaller one (three). Concerning the role of Ac in social behaviour, a difference between lesioned and sham birds was evident here only in the absence of detectable stimulus (predator or conspecifics). These findings may reflect either decreased fear of exposure to predators or increased craving for conspecifics suggesting that the likely function of Ac is to modulate goal-driven, including socially driven, behaviours, especially when the direct stimulus representing the goal is absent. This is in harmony with the known promotion of impulsiveness by Ac lesions.

Distribution of vasotocin- and vasoactive intestinal peptide- like immunoreactivity in the brain of penduline tit

Penduline tits are songbirds, used as model animals in numerous studies of sexual conflict. Nevertheless, the distribution of neuropeptides in the brain of this avian species remains largely unknown. Here we present some of the first results on distribution of vasotocin (AVT) and vasoactive intestinal peptide (VIP) in the brain of this songbird species, using immunohistochemical mapping. The bulk of AVT-like cells are found in the hypothalamic supraoptic, paraventricular and suprachiasmatic nuclei, medial bed nucleus of the stria terminalis, and along the lateral forebrain bundle. Most AVT-like fibres course toward the median eminence, but also in the medial bed nucleus of the stria terminalis, preoptic area and lateral septum. Further terminal fields occur in the dorsal thalamus, ventral tegmental area and pretectal area. Most VIP-like cells are in the lateral septal organ and arcuate nucleus. VIP-like fibres are distributed extensively in the hypothalamus, preoptic area, lateral septum, nucleus of the diagonal band. They are also found in the medial bed nucleus of the stria terminalis, amygdaloid nucleus of taenia, robust nucleus of the arcopallium, caudo-ventral hyperpallium, nucleus accumbens and the brainstem. The results indicate a high degree of conservatism in both AVT and VIP neuropeptidergic systems across avian species, as well as the different vertebrate taxa. The anatomical distribution of AVT and VIP in the penduline tit supports an involvement of these peptides in reproductive behaviours and social decision making, widely studied in this wild bird species

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Neurotensin: revealing a novel neuromodulator circuit in the nucleus accumbens-parabrachial nucleus projection of the domestic chick.

Lower brainstem projections from nucleus accumbens (Ac) subregions to the parabrachial complex (PB), the nucleus of the solitary tract and the vagal motor nuclei have been described previously in the domestic chick by our group. Such projections, particulary those from the core and rostral pole regions of Ac have not been found in mammals or pigeons. Here we report on the presence of neurotensin (NT) in the neurons projecting from different Ac subnuclei, and also from the bed nucleus of stria terminalis, to the PB in the domestic chicken. The study is based upon correlated retrograde tracing (using Fast Blue) and NT immunohistochemistry, supplemented with regional charting and quantitative analysis of double-labeled neurons. The number of retrogradely labeled cells in Ac subdivisions reflects the size of FB tracer deposit, and the degree to which it extends to the medial PB. Of all Ac subregions, the core contained the largest amount of double-labeled cells. The findings demonstrate that the anatomical pathway through which the Ac can directly modulate taste-responsive neurons of the PB employs mainly neurotensin as a neuromodulator. The observed anatomical difference between mammals and birds is either a general taxonomic feature or it reflects feeding strategies specific for the domestic chick. The results are also relevant to a better understanding of the role of NT in food intake and reward-related behaviors in birds.

Distribution of vasotocin- and vasoactive intestinal peptide-like immunoreactivity in the brain of blue tit (Cyanistes coeruleus).

Blue tits (Cyanistes coeruleus) are songbirds, used as model animals in numerous studies covering a wide field of research. Nevertheless, the distribution of neuropeptides in the brain of this avian species remains largely unknown. Here we present some of the first results on distribution of Vasotocine (AVT) and Vasoactive intestinal peptide (VIP) in the brain of males and females of this songbird species, using immunohistochemistry mapping. The bulk of AVT-like cells are found in the hypothalamic supraoptic, paraventricular and suprachiasmatic nuclei, bed nucleus of the stria terminalis, and along the lateral forebrain bundle. Most AVT-like fibers course toward the median eminence, some reaching the arcopallium, and lateral septum. Further terminal fields occur in the dorsal thalamus, ventral tegmental area and pretectal area. Most VIP-like cells are in the lateral septal organ and arcuate nucleus. VIP-like fibers are distributed extensively in the hypothalamus, preoptic area, lateral septum, diagonal band of Broca. They are also found in the bed nucleus of the stria terminalis, amygdaloid nucleus of taenia, robust nucleus of the arcopallium, caudo-ventral hyperpallium, nucleus accumbens and the brainstem. Taken together, these results suggest that both AVT and VIP immunoreactive structures show similar distribution to other avian species, emphasizing evolutionary conservatism in the history of vertebrates. The current study may enable future investigation into the localization of AVT and VIP, in relation to behavioral and ecological traits in the brain of tit species.

Effect of synthetic cathinones: mephedrone, butylone and 3,4 methylene-dioxypyrovalerone (MDPV) on social separation induced distress vocalization, vigilance and postural control of young domestic chicks.

Designer drugs have become a distinct social problem and health hazard in Europe and US, and their abuse has increased dramatically in the last decade. Selective effects of these agents on animal behavioral parameters may help in better understanding of the potential risks of human drug abuse. In the present study, the effects of three different abusive agents of the cathinone family, mephedrone, butylone and 3,4 methylene-dioxypyrovalerone (MDPV) were tested in young domestic chicks, following administration of single intraperitoneal injections (10mg/bwt). Early maturing (precocial) birds are particularly suited for investigation of isolation stress-related behavioral response and stereotypic or targeted pecking. Both mephedrone and MDPV increased the frequency of distress calls of socially isolated birds as measured over a period of 10min. While this effect of mephedrone was only evident in the first half of observation period, an increase with MDPV was more lasting. Though increased non-distress vocalization, butylone failed to enhance distress calls probably due to a general adverse effect on muscle tone. Apart from its effect on distress vocalization, mephedrone did not alter the behavior of chicks. However, both butylone and MDPV showed prominent behavioral changes, which were examined in another set of long term experiments, over a period of 120min. Butylone caused hyperventilation and a robust impairment of postural control, whereas neither the wakeful activity level, nor the pecking frequency was significantly affected. Conversely, no hyperventilation or postural disorder was observed with MDPV, however, both waking state and pecking were significantly enhanced. The results may be relevant to potentially different and specific effects of cathinone drugs under stress-related conditions, as well as on other physiological and behavioral parameters, even in case of closely related compounds.

Apoptotic effects of the 'designer drug' methylenedioxypyrovalerone (MDPV) on the neonatal mouse brain.

The designer drug of cathinone family, methylenedioxypyrovalerone (MDPV), is a cheap and frequently used psychoactive drug of abuse. However, its mechanism of action, particularly its potential detrimental effect on the developing brain, is largely unknown, despite the fact that pregnant females may occur among the users. The objective of our study was to identify the brain areas sensitive for a possible apoptotic effect of the widely abused MDPV on the developing brain. To this end, we used a mouse model which can be compared with the human fetus of third trimester, considering the developmental stage of the brain. Litters of 7-day-old C57BL/6J mice were treated either with i.p. injection of 10mg/kg b.wt.of MDPV or vehicle (saline), and sacrificed after 24h. Similar dose of MDPV enhanced locomotor activity of pups. The brains were processed for anti-caspase 3 (Casp3) immunohistochemistry and the apoptotic cells were identified and counted. We found prominent increase in the number of apoptotic cells in the piriform cortex, retrosplenial area, hippocampus CA1 and nucleus accumbens, whereas the overall density of cells did not change significantly in these regions. The neurons of the nucleus accumbens appeared to be especially sensitive to MDPV: Casp3-immunoreactive cells marked out the core and shell regions of the accumbens. Highest percentage of apoptotic cells as compared to total cell density was also found in the nucleus accumbens. However, we did not observe the same effect on the brain of adult mice. Thus, MDPV did not seem to increase apoptosis in the mature nervous system. The results are in agreement with the assumption that cathinones (in particular MDPV) may adversely affect neural integrity in the developing CNS.