Human-Specific Neuropeptide S Receptor Variants Regulate Fear Extinction in the Basal Amygdala of Male and Female Mice Depending on Threat Salience.

BACKGROUND: A nonsynonymous single nucleotide polymorphism in the neuropeptide S receptor 1 (NPSR1) gene (rs324981) results in isoleucine-to-asparagine substitution at amino acid 107. In humans, the ancestral variant (NPSR1 I107) is associated with increased anxiety sensitivity and risk of panic disorder, while the human-specific variant (NPSR1 N107) is considered protective against excessive anxiety. In rodents, neurobiological constituents of the NPS system have been analyzed in detail and their anxiolytic-like effects have been endorsed. However, their implication for anxiety and related disorders in humans remains unclear, as rodents carry only the ancestral NPSR1 I107 variant. METHODS: We hypothesized that phenotypic correlates of NPSR1 variants manifest in fear-related circuits in the amygdala. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-mediated gene editing to generate a "humanized" mouse strain, in which individuals express either NPSR1 I107 or NPSR1 N107. RESULTS: Stimulation of NPSR1 evoked excitatory responses in principal neurons of the anterior basal amygdala with significant differences in magnitude between genotypes, resulting in synaptic disinhibition of putative extinction neurons in the posterior basal amygdala in mice expressing the human-specific hypofunctional N107 but not the ancestral I107 variant. N107 mice displayed improved extinction of conditioned fear, which was phenocopied after pharmacological antagonism of NPSR1 in the anterior basal amygdala of I107 mice. Differences in fear extinction between male and female mice were related to an interaction of Npsr1 genotype and salience of fear training. CONCLUSIONS: The NPS system regulates extinction circuits in the amygdala depending on the Npsr1 genotype, contributing to sex-specific differences in fear extinction and high anxiety sensitivity of individuals bearing the ancestral NPSR1 I107 variant.

Impairment of frequency-specific responses associated with altered electrical activity patterns in auditory thalamus following focal and general demyelination.

Multiple sclerosis is characterized by intermingled episodes of de- and remyelination and the occurrence of white- and grey-matter damage. To mimic the randomly distributed pathophysiological brain lesions observed in MS, we assessed the impact of focal white and grey matter demyelination on thalamic function by directing targeted lysolecithin-induced lesions to the capsula interna (CI), the auditory cortex (A1), or the ventral medial geniculate nucleus (vMGN) in mice. Pathophysiological consequences were compared with those of cuprizone treatment at different stages of demyelination and remyelination. Combining single unit recordings and auditory stimulation in freely behaving mice revealed changes in auditory response profile and electrical activity pattern in the thalamus, depending on the region of the initial insult and the state of remyelination. Cuprizone-induced general demyelination significantly diminished vMGN neuronal activity and frequency-specific responses. Targeted lysolecithin-induced lesions directed either to A1 or to vMGN revealed a permanent impairment of frequency-specific responses, an increase in latency of auditory responses and a reduction in occurrence of burst firing in vMGN neurons. These findings indicate that demyelination of grey matter areas in the thalamocortical system permanently affects vMGN frequency specificity and the prevalence of bursting in the auditory thalamus.

Directional spread of activity in synaptic networks of the human lateral amygdala.

Spontaneous epileptiform activity has previously been observed in lateral amygdala (LA) slices derived from patients with intractable-temporal lobe epilepsy. The present study aimed to characterize intranuclear LA synaptic connectivity and to test the hypothesis that differences in the spread of flow of neuronal activity may relate to spontaneous epileptiform activity occurrence. Electrical activity was evoked through electrical microstimulation in acute human brain slices containing the LA, signals were recorded as local field potentials combined with fast optical imaging of voltage-sensitive dye fluorescence. Sites of stimulation and recording were systematically varied. Following recordings, slices were anatomically reconstructed using two-dimensional unitary slices as a reference for coronal and parasagittal planes. Local spatial patterns and spread of activity were assessed by incorporating the coordinates of electrical and optical recording sites into the respective unitary slice. A preferential directional spread of evoked electrical signals was observed from ventral to dorsal, rostral to caudal and medial to lateral regions in the LA. No differences in spread of evoked activity were observed between spontaneously and non-spontaneously active LA slices, i.e. basic properties of evoked synaptic responses were similar in the two functional types of LA slices, including input-output relationship, and paired-pulse depression. These results indicate a directed propagation of synaptic signals within the human LA in spontaneously active epileptic slices. We suggest that the lack of differences in local and in systemic information processing has to be found in confined epileptiform circuits within the amygdala likely involving well-known "epileptic neurons".

Impact of Life History on Fear Memory and Extinction.

Behavioral profiles are strongly shaped by an individual's whole life experience. The accumulation of negative experiences over lifetime is thought to promote anxiety-like behavior in adulthood ("allostatic load hypothesis"). In contrast, the "mismatch hypothesis" of psychiatric disease suggests that high levels of anxiety-like behavior are the result of a discrepancy between early and late environment. The aim of the present study was to investigate how different life histories shape the expression of anxiety-like behavior and modulate fear memory. In addition, we aimed to clarify which of the two hypotheses can better explain the modulation of anxiety and fear. For this purpose, male mice grew up under either adverse or beneficial conditions during early phase of life. In adulthood they were further subdivided in groups that either matched or mismatched the condition experienced before, resulting in four different life histories. The main results were: (i) Early life benefit followed by late life adversity caused decreased levels of anxiety-like behavior. (ii) Accumulation of adversity throughout life history led to impaired fear extinction learning. Late life adversity as compared to late life benefit mainly affected extinction training, while early life adversity as compared to early life benefit interfered with extinction recall. Concerning anxiety-like behavior, the results do neither support the allostatic load nor the mismatch hypothesis, but rather indicate an anxiolytic effect of a mismatched early beneficial and later adverse life history. In contrast, fear memory was strongly affected by the accumulation of adverse experiences over the lifetime, therefore supporting allostatic load hypothesis. In summary, this study highlights that anxiety-like behavior and fear memory are differently affected by specific combinations of adverse or beneficial events experienced throughout life.

Neuronal correlates of sustained fear in the anterolateral part of the bed nucleus of stria terminalis.

As part of the extended amygdala network, the bed nucleus of the stria terminalis (BNST) was shown to be critically involved in processing sustained fear responses to diffuse and unpredictable threats. However, neuronal activity patterns in relation to sustained components of the fear response remain elusive, so far. We used a fear training paradigm with unpredictable pairing of conditioned and unconditioned stimuli allowing distinction between phasic and sustained components of conditioned fear, and recorded single units in the anterolateral part of the BNST (BNSTal) in freely behaving mice. An objective, non-biased cluster-analysis was performed for each identified single unit on specific waveform-, activity-, stimulus-dependent and LFP-related parameters. The analysis revealed three distinct neuronal subpopulations of biphasic-, sustained fear on- and fear off-neurons. Results show that activities of biphasic- and sustained fear on-neurons temporally coincide with the shift from phasic to sustained components of the fear response. Presentation of non-conditioned auditory stimuli resulted in a variety of neuronal responses in BNSTal with no indication of biphasic response profiles. It is suggested that fear conditioning sharpens neuronal response profiles in BNSTal with biphasic-cells signaling phasic and sustained fear. These results confirm the pivotal role of BNST in processing sustained fear on the neuronal level, thereby complementing pharmacological experimental animal and human imaging data.

Distinct state anxiety after predictable and unpredictable fear training in mice.

Sustained fear paradigms in rodents have been developed to monitor states of anxious apprehension and to model situations in patients suffering from long-lasting anxiety disorders. A recent report describes a fear conditioning paradigm, allowing distinction between phasic and sustained states of conditioned fear in non-restrained mice. However, so far no prospective studies have yet been conducted to elucidate whether induction of phasic or sustained fear can affect states of anxiety. Here, we used CS (conditioned stimulus) and US (unconditioned stimulus) pairing with predictable and unpredictable timing to induce phasic and sustained fear in mice. State anxiety during various fear response components was assessed using the elevated plus-maze test. Training with unpredictable CS-US timing resulted in CS-evoked sustained components of fear (freezing), while predictable CS-US timing resulted in rapid decline. Data suggested the influence of training procedure on state anxiety which is dependent on progression of conditioned fear during fear memory retrieval. Animals trained with unpredictable CS-US timing showed an unchanged high anxiety state throughout behavioral observation. In contrast, mice trained with predictable CS-US timing showed anxiolytic-like behavior 3 min after CS onset, which was accompanied by a fast decline of the fear conditioned response (freezing). Further systematic studies are needed to validate the phasic/sustained fear model in rodents as translational model for anxiety disorders in humans.

Increased GABAergic Efficacy of Central Amygdala Projections to Neuropeptide S Neurons in the Brainstem During Fear Memory Retrieval.

The canonical view on the central amygdala has evolved from a simple output station towards a highly organized microcircuitry, in which types of GABAergic neurons in centrolateral (CeL) and centromedial (CeM) subnuclei regulate fear expression and generalization. How these specific neuronal populations are connected to extra-amygdaloid target regions remains largely unknown. Here we show in mice that a subpopulation of GABAergic CeL and CeM neurons projects monosynaptically to brainstem neurons expressing neuropeptide S (NPS). The CeL neurons are PKCδ-negative and are activated during conditioned fear. During fear memory retrieval, the efficacy of this GABAergic influence on NPS neurons is enhanced. Moreover, a large proportion of these neurons (~50%) contain prodynorphin and somatostatin, two neuropeptides inhibiting NPS neurons. We conclude that CeL and CeM neurons inhibit NPS neurons in the brainstem by GABA release and that efficacy of this connection is strengthened upon fear memory retrieval. Thereby, this pathway provides a possible feedback mechanism between amygdala and brainstem routes involved in fear and stress coping.

Directional theta coherence in prefrontal cortical to amygdalo-hippocampal pathways signals fear extinction.

Theta oscillations are considered crucial mechanisms in neuronal communication across brain areas, required for consolidation and retrieval of fear memories. One form of inhibitory learning allowing adaptive control of fear memory is extinction, a deficit of which leads to maladaptive fear expression potentially leading to anxiety disorders. Behavioral responses after extinction training are thought to reflect a balance of recall from extinction memory and initial fear memory traces. Therefore, we hypothesized that the initial fear memory circuits impact behavioral fear after extinction, and more specifically, that the dynamics of theta synchrony in these pathways signal the individual fear response. Simultaneous multi-channel local field and unit recordings were obtained from the infralimbic prefrontal cortex, the hippocampal CA1 and the lateral amygdala in mice. Data revealed that the pattern of theta coherence and directionality within and across regions correlated with individual behavioral responses. Upon conditioned freezing, units were phase-locked to synchronized theta oscillations in these pathways, characterizing states of fear memory retrieval. When the conditioned stimulus evoked no fear during extinction recall, theta interactions were directional with prefrontal cortical spike firing leading hippocampal and amygdalar theta oscillations. These results indicate that the directional dynamics of theta-entrained activity across these areas guide changes in appraisal of threatening stimuli during fear memory and extinction retrieval. Given that exposure therapy involves procedures and pathways similar to those during extinction of conditioned fear, one therapeutical extension might be useful that imposes artificial theta activity to prefrontal cortical-amygdalo-hippocampal pathways that mimics the directionality signaling successful extinction recall.

Activation of neuropeptide S-expressing neurons in the locus coeruleus by corticotropin-releasing factor.

A recently discovered neurotransmitter system, consisting of neuropeptide S (NPS), NPS receptor, and NPS-expressing neurons in the brain stem, has received considerable interest due to its modulating influence on arousal, anxiety and stress responsiveness. Comparatively little is known about the properties of NPS-expressing neurons. Therefore in the present study, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP) in NPS neurons was used to characterize the cellular and functional properties of NPS-expressing neurons located close to the locus coeruleus. Particular emphasis was on the influence of corticotropin-releasing factor (CRF), given previous evidence of stress-related activation of the NPS system. Upon acute immobilization stress, an increase in c-fos expression was detected immunocytochemically in brain stem NPS-EGFP neurons that also expressed the CRF receptor 1 (CRF1). NPS-EGFP neurons were readily identified in acute slice preparations and responded to CRF application with a membrane depolarization capable of triggering action potentials. CRF-induced responses displayed pharmacological properties indicative of CRF1 that were mediated by both a reduction in membrane potassium conductance and an increase in a non-specific cation conductance different from the hyperpolarization-activated cation conductance Ih, and involved protein kinase A signalling. In conclusion, stress exposure results in activation of brain stem NPS-expressing neurons, involving a CRF1-mediated membrane depolarization via at least two ionic mechanisms. These data provide evidence for a direct interaction between the CRF and the NPS system and thereby extend previous observations of NPS-modulated stress responsiveness towards a mechanistic level.

Differential regulation of glutamic acid decarboxylase gene expression after extinction of a recent memory vs. intermediate memory.

Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65 isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD). However, a detailed analysis of changes in gene expression of GAD in the subregions comprising the extinction network has not been undertaken. Here, we report changes in gene expression of the GAD65 and GAD67 isoforms of GAD, as measured by relative quantitative real-time RT-PCR, in subregions of the amygdala, hippocampus, and prefrontal cortex 24-26 h after extinction of a recent (1-d) or intermediate (14-d) fear memory. Our results show that extinction of a recent memory induces a down-regulation of Gad65 gene expression in the hippocampus (CA1, dentate gyrus) and an up-regulation of Gad67 gene expression in the infralimbic cortex. Extinguishing an intermediate memory increased Gad65 gene expression in the central amygdala. These results indicate a differential regulation of Gad gene expression after extinction of a recent memory vs. intermediate memory.

Prevention of stress-impaired fear extinction through neuropeptide s action in the lateral amygdala.

Stressful and traumatic events can create aversive memories, which are a predisposing factor for anxiety disorders. The amygdala is critical for transforming such stressful events into anxiety, and the recently discovered neuropeptide S transmitter system represents a promising candidate apt to control these interactions. Here we test the hypothesis that neuropeptide S can regulate stress-induced hyperexcitability in the amygdala, and thereby can interact with stress-induced alterations of fear memory. Mice underwent acute immobilization stress (IS), and neuropeptide S and a receptor antagonist were locally injected into the lateral amygdala (LA) during stress exposure. Ten days later, anxiety-like behavior, fear acquisition, fear memory retrieval, and extinction were tested. Furthermore, patch-clamp recordings were performed in amygdala slices prepared ex vivo to identify synaptic substrates of stress-induced alterations in fear responsiveness. (1) IS increased anxiety-like behavior, and enhanced conditioned fear responses during extinction 10 days after stress, (2) neuropeptide S in the amygdala prevented, while an antagonist aggravated, these stress-induced changes of aversive behaviors, (3) excitatory synaptic activity in LA projection neurons was increased on fear conditioning and returned to pre-conditioning values on fear extinction, and (4) stress resulted in sustained high levels of excitatory synaptic activity during fear extinction, whereas neuropeptide S supported the return of synaptic activity during fear extinction to levels typical of non-stressed animals. Together these results suggest that the neuropeptide S system is capable of interfering with mechanisms in the amygdala that transform stressful events into anxiety and impaired fear extinction.

Social defeat: impact on fear extinction and amygdala-prefrontal cortical theta synchrony in 5-HTT deficient mice.

Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA) and infralimbic region of the medial prefrontal cortex (mPFC) in male 5-HTT wild-type (+/+), homo- (-/-) and heterozygous (+/-) mice. Naive males and experienced losers (generated in a resident-intruder paradigm) of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated by both the 5-HTT gene activity and social experiences in adulthood, accompanied by corresponding alterations of the theta activity in the amygdala-prefrontal cortex network.

Patterns of coupled theta activity in amygdala-hippocampal-prefrontal cortical circuits during fear extinction.

Signals related to fear memory and extinction are processed within brain pathways involving the lateral amygdala (LA) for formation of aversive stimulus associations, the CA1 area of the hippocampus for context-dependent modulation of these associations, and the infralimbic region of the medial prefrontal cortex (mPFC) for extinction processes. While many studies have addressed the contribution of each of these modules individually, little is known about their interactions and how they function as an integrated system. Here we show, by combining multiple site local field potential (LFP) and unit recordings in freely behaving mice in a fear conditioning paradigm, that theta oscillations may provide a means for temporally and functionally connecting these modules. Theta oscillations occurred with high specificity in the CA1-LA-mPFC network. Theta coupling increased between all areas during retrieval of conditioned fear, and declined during extinction learning. During extinction recall, theta coupling partly rebounded in LA-mPFC and CA1-mPFC, and remained at a low level in CA1-LA. Interfering with theta coupling through local electrical microstimulation in CA1-LA affected conditioned fear and extinction recall depending on theta phase. These results support the hypothesis that theta coupling provides a means for inter-areal coordination in conditioned behavioral responsiveness. More specifically, theta oscillations seem to contribute to a population code indicating conditioned stimuli during recall of fear memory before and after extinction.

Impaired extinction of fear and maintained amygdala-hippocampal theta synchrony in a mouse model of temporal lobe epilepsy.

PURPOSE: The relationship between epilepsy and fear has received much attention. However, seizure-modulated fear and physiologic or structural correlates have not been examined systematically, and the underlying basics of network levels remain unclear to date. Therefore, this project was set up to characterize the neurophysiologic basis of seizure-related fear and the contribution of the amygdala-hippocampus system. METHODS: The experimental strategy was composed of the following steps: (1) use of the mouse pilocarpine model of temporal lobe epilepsy (TLE); (2) behavioral analyses of anxiety states in the elevated plus maze test, light-dark avoidance test, and Pavlovian fear conditioning; and (3) probing neurophysiologic activity patterns in amygdala-hippocampal circuits in freely behaving mice. RESULTS: Our results displayed no significant differences in basic anxiety levels comparing mice that developed spontaneous recurrent seizures (SRS) and controls. Furthermore, conditioned fear memory retrieval was not influenced in SRS mice. However, during fear memory extinction, SRS mice showed an extended freezing behavior and a maintained amygdala-hippocampal theta frequency synchronization compared to controls. DISCUSSION: These results indicate specific alterations in conditioned fear behavior and related neurophysiologic activities in the amygdala-hippocampal network contributing to impaired fear memory extinction in mice with TLE. Clinically, the nonextinguished fear memories may well contribute to the experience of fear in patients with TLE.

Alteration of NMDA receptor-mediated synaptic interactions in the lateral amygdala associated with seizure activity in a mouse model of chronic temporal lobe epilepsy.

PURPOSE: Because results from both animal models and human temporal lobe epilepsy (TLE) have pointed to synaptic network alterations in the amygdala, we have tested the hypothesis that glutamatergic transmission in the lateral amygdala (LA) is critically involved. METHODS: Using the pilocarpine mouse model, LA slices were prepared ex vivo in the recurrent phase of TLE (Pilo group), and LA projection neurons (PNs) were recorded using patch-clamp techniques. Intrinsic and synaptic properties of LA PNs were analyzed and compared with those in age-matched saline-injected controls. RESULTS: Only mild changes were observed in intrinsic properties of LA PNs, whereas both spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs) were significantly increased in Pilo as compared to saline controls. This difference was sensitive to AP5, but persisted during action of NBQX, indicating mediation by N-methyl-d-aspartate (NMDA) receptors. Moreover, these changes were associated with an increase in frequency but not amplitude of mEPSCs, indicative of a contribution of presynaptic mechanisms. DISCUSSION: In conclusion, dynamic changes seem to occur in glutamatergic transmission within the amygdala during TLE, to which a functional upregulation of presynaptic NMDA receptors in LA PNs makes a significant contribution.

Neuropeptide S: a transmitter system in the brain regulating fear and anxiety.

The recently discovered Neuropeptide S (NPS) and its cognate receptor represent a highly interesting system of neuromodulation with unique physiological effects. On one hand, NPS increases wakefulness and arousal. On the other, NPS produces anxiolytic-like effects by acutely reducing fear responses as well as modulating long-term aspects of fear memory, such as attenuation of contextual fear or enhancement of fear extinction. The main sources of NPS in the brain are a few clusters of NPS-producing neurons in the brainstem. NPS binds to a G-protein-coupled receptor that is highly conserved among vertebrates and stimulates mobilization of intracellular Ca(2+) as well as activation of protein kinases. In synaptic circuits within the amygdala, which are important for processing of acute fear as well as formation and expression of fear memories, NPS causes increased release of the excitatory transmitter glutamate, especially in synaptic contacts to a subset of GABAergic interneurons. Polymorphisms in the human NPS receptor gene have been associated with altered sleep behavior and panic disorder. In conclusion, the NPS system displays a unique physiological profile with respect to the specificity and time course of its actions. These functions could provide interesting opportunities for both basic research and clinical applications.

Neuropeptide S-mediated control of fear expression and extinction: role of intercalated GABAergic neurons in the amygdala.

A deficient extinction of memory is particularly important in the regime of fear, where it limits the beneficial outcomes of treatments of anxiety disorders. Fear extinction is thought to involve inhibitory influences of the prefrontal cortex on the amygdala, although the detailed synaptic mechanisms remain unknown. Here, we report that neuropeptide S (NPS), a recently discovered transmitter of ascending brainstem neurons, evokes anxiolytic effects and facilitates extinction of conditioned fear responses when administered into the amygdala in mice. An NPS receptor antagonist exerts functionally opposing responses, indicating that endogenous NPS is involved in anxiety behavior and extinction. Cellularly, NPS increases glutamatergic transmission to intercalated GABAergic neurons in the amygdala via presynaptic NPS receptors on connected principal neurons. These results identify mechanisms of NPS in the brain, a key role of intercalated neurons in the amygdala for fear extinction, and a potential pharmacological avenue for treating anxiety disorders.

Ontogeny of the dopamine innervation in the nucleus accumbens of gerbils.

The postnatal maturation of immunohistochemically stained dopamine (DA) fibres was quantitatively examined in the core and shell subareas of the nucleus accumbens (NAC) of gerbils. Animals of different ages, ranging from juvenile [postnatal day (PD) 14, 30] to adolescent (PD70), adult (PD90, PD180, PD360) and ageing (PD540, PD720) were analysed. The timescale of the maturation of the accumbal DA innervation was regionally different, probably due to the different origin of DA fibres in the mesencephalon. Both the accumbal core, with DA afferents arising from the lateral ventral tegmental area (VTA) and the substantia nigra pars compacta, as well as the accumbal shell, with DA afferents arising from the medial VTA, show moderate DA fibre densities at PD14. The core displayed a significant decrease of the DA fibre density up to PD30 and a subsequent significant increase between PD70 and 90, whereas the shell solely showed an augmentation of the DA innervation between PD70 and 90. Our data suggest that the different maturation of the DA innervation in core and shell might reflect differences in the development of motor and limbic functions, mediated by the nigrostriate and the mesolimbic system, respectively.

Hemisphere-specific effects on serotonin but not dopamine innervation in the nucleus accumbens of gerbils caused by isolated rearing and a single early methamphetamine challenge.

The aim of this study was twofold: We examined whether serotonin (5-HT) and dopamine (DA) innervations of the nucleus accumbens are lateralised and whether the environment or the combination with an early pharmacological impact might interfere with the postnatal maturation of the monoaminergic innervation. Male gerbils were assigned to either enriched rearing (ER) or isolated rearing (IR). Animals from both rearing conditions additionally received a single dose of either methamphetamine [MA (50 mg/kg ip)] or saline on postnatal day 14. DA and 5-HT fibres of the adult animals (postnatal day 90-110) were immunocytochemically stained and fibre densities were quantified in nucleus accumbens core and shell of both the left and right hemisphere. Our data demonstrate that the DA and 5-HT innervation is not lateralised in saline-treated animals of both rearing conditions. IR increases the DA fibre density in both hemispheres of saline controls, whereas an additional MA treatment reverses this effect. In both ER and IR groups, MA provokes an excessive 5-HT fibre in growth of only the right hemisphere. The combination of IR with MA induces right-side asymmetries of the 5-HT fibre density in both the core and shell. From the data obtained, we conclude that the maturation of the monoaminergic innervation of the nucleus accumbens is vulnerable to postnatal stimuli. The subtle "innervation imbalance" observed in our studies is consistent with previously reported effects in other brain regions of this animal model and may be causative for behavioural disturbances.

Serotonin fibre densities in subcortical areas: differential effects of isolated rearing and methamphetamine.

Serotoninergic neurons interact with dopaminergic cells on all levels and are physiologically affected by both isolated rearing (IR) and a single early methamphetamine (MA) injection. We therefore checked for anatomical effects of both interventions by immunohistochemically staining serotonin fibres and assessing fibre densities in the caudate-putamen (CPu), nucleus accumbens (NAc) and amygdala of Mongolian gerbils. IR led to significantly increased 5-HT fibre densities in the dorsal part of the CPu and in the central and basolateral amygdala. No effects were seen in the ventral CPu, in the NAc and in the lateral amygdala. The early MA injection resulted in a denser 5-HT innervation in the dorsomedial and ventromedial CPu, in the NAc shell of animals reared in an enriched environment and in the NAc core of both rearing conditions, leaving the lateral CPu and the amygdala unaffected. Thus, the single pharmacological versus the environmental challenge exerts an almost complementary effect on the 5-HT innervation in different areas of the brain, which demonstrates that systemic interactions, e.g. with dopaminergic and glutamatergic afferents, must be taken into account when the seemingly uniform 5-HT projections are investigated.