Oliver Stork

Postnatal development of a GABA deficit and disturbance of neural functions in mice lacking GAD65.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is believed to play an essential role for GABA synthesis in the central nervous system. Using mice with targeted disruption of the GAD65 gene (GAD65(-/-) mice) we investigated the contribution of GAD65 to GABA synthesis in different brain areas during postnatal development and in adulthood. In the amygdala, hypothalamus and parietal cortex of GAD65(+/+) mice an increase of GABA levels was observed during postnatal development, most prominently between the first and second month after birth. This increase appeared to be dependent on GAD65, as it was delayed by 2 months in GAD65(+/-) mice and was not observed in GAD65(-/-) mice. Likely as a consequence of their GABA deficit, adult GAD65(-/-) mice showed a largely abnormal neural activity with frequent paroxysmal discharges and spontaneous seizures. They furthermore displayed increased anxiety-like behaviour in a light/dark avoidance test and reduced intermale aggression, as well as a reduced forced-swimming-induced immobility indicative of an antidepressant-like behavioural change. Adult GAD65(+/-) mice did not show behavioural disturbances except for a reduced aggressive behaviour that was comparable to that in GAD65(-/-) mice. We conclude that GAD65-mediated GABA synthesis may be crucially involved in control of emotional behaviour and indispensable for a tonic inhibition that prevents the development of hyperexcitability in the maturating central nervous system. Aggressive, and possibly other social behaviour may be especially prone to regulation through GAD65-mediated GABA synthesis.

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.

Recovery of emotional behaviour in neural cell adhesion molecule (NCAM) null mutant mice through transgenic expression of NCAM180

In the present study we further investigate functions of the neural cell adhesion molecule (NCAM) in the mature central nervous system and its implications for animal behaviour. To this end we generated transgenic mice expressing the major NCAM isoform with the largest cytoplasmic domain, NCAM180, under control of a promoter for the small form neurofilament gene. Transgenic mice were also bred with mice deficient in endogenous NCAM (Ncam–/– mice) so that effects of NCAM180 could be analysed in the presence and absence of endogenous NCAM. While overexpression of transgenic NCAM180 was without apparent behavioural or morphological effect, its expression in Ncam–/– mice counteracted NCAM ablation‐induced aggressive, anxiety‐like and antidepressant‐like behaviour. It furthermore prevented a hypersensitivity of Ncam–/– mice to the anxiolytic serotonin1A (5‐HT1A) receptor agonist buspirone. Such recovery of emotional behaviour and behavioural 5‐HT1A response occurred in spite of misdevelopment of the olfactory bulb and hippocampus that is characteristic of Ncam–/– mice, and without an apparent change in the expression of 5‐HT1A binding sites in the brain. Hippocampus‐ and amygdala‐dependent learning, though disturbed in Ncam–/– mice, remained unaffected by the transgenic NCAM180. We suggest an involvement of NCAM180‐mediated cell recognition processes in the serotonergic modulation of emotional behaviour in adult mice.

Increased Intermale Aggression and Neuroendocrine Response in Mice Deficient for the Neural Cell Adhesion Molecule (NCAM)

Mice deficient for the neural cell adhesion molecule (NCAM) show morphological and behavioural abnormalities in the adult form, including a reduced size of the olfactory bulb, reduced exploratory behaviour, and deficits in spatial learning. Here we report increased aggressive behaviour of both homozygous (NCAM ‐/–) and heterozygous (NCAM +/–) male mutant mice towards an unfamiliar male intruding into their home cage. While plasma testosterone concentrations did not differ between genotypes before or after behavioural testing, corticosterone levels were higher in mutant residents than in wild‐type (NCAM +/+) residents 30 min after encountering the intruder. Levels of c‐fos mRNA, analysed to monitor neuronal activation, were similar in primary output structures of the olfactory bulb in NCAM‐deficient and NCAM +/+ mice, but were increased in brain areas of the limbic system in both NCAM –/– and NCAM +/– mutant mice after the behavioural test. These results indicate that abnormalities in social behaviour correlate with enhanced neuronal activity in limbic brain areas and result in increased social stress in NCAM‐deficient mice.

Identification of a Neuropeptide S Responsive Circuitry Shaping Amygdala Activity via the Endopiriform Nucleus

Neuropeptide S (NPS) and its receptor are thought to define a set of specific brain circuits involved in fear and anxiety. Here we provide evidence for a novel, NPS-responsive circuit that shapes neural activity in the mouse basolateral amygdala (BLA) via the endopiriform nucleus (EPN). Using slice preparations, we demonstrate that NPS directly activates an inward current in 20% of EPN neurons and evokes an increase of glutamatergic excitation in this nucleus. Excitation of the EPN is responsible for a modulation of BLA activity through NPS, characterized by a general increase of GABAergic inhibition and enhancement of spike activity in a subset of BLA projection neurons. Finally, local injection of NPS to the EPN interferes with the expression of contextual, but not auditory cued fear memory. Together, these data suggest the existence of a specific NPS-responsive circuitry between EPN and BLA, likely involved in contextual aspects of fear memory.

Generalisation of conditioned fear and its behavioural expression in mice.

Mice are favourite subjects in molecular and genetic memory research and frequently studied with classical fear conditioning paradigms that use an auditory cue (conditioned stimulus, CS(+)) to predict an aversive, unconditioned stimulus (US). Yet the conditions that control fear memory specificity and generalisation and their behavioural expression in such conditioned mice have not been analysed systematically. In the current study we addressed these issues in the most widely used mouse strain of behavioural genetics, C57Bl/6. In keeping with findings in other species we demonstrate the dependence of fear memory generalisation on training intensity (i.e. both US intensity and the number of CS(+) and US applied) after both excitatory (explicitly paired presentation of CS(+) and US) and inhibitory training (explicitly unpaired presentation of CS(+) and US). Furthermore, inhibitory overtraining was associated with changes of uncued anxiety-like behaviour in a light/dark exploration test, indicative of an emotional sensitisation reaction as consequence of a lack of US predictability. Together our results describe the qualitatively and quantitatively different increases of defensive behaviour in response to conditioned stimuli of different salience and identify training conditions that lead to fear memory generalisation and emotional sensitisation in C57Bl/6 inbred mice.

Critical role of the 65-kDa isoform of glutamic acid decarboxylase in consolidation and generalization of Pavlovian fear memory

Evidence suggests that plasticity of the amygdalar and hippocampal GABAergic system is critical for fear memory formation. In this study we investigated in wild-type and genetically manipulated mice the role of the activity-dependent 65-kDa isozyme of glutamic acid decarboxylase (GAD65) in the consolidation and generalization of conditioned fear. First, we demonstrate a transient reduction of GAD65 gene expression in the dorsal hippocampus (6 h post training) and in the basolateral complex of the amygdala (24 h post training) during distinct phases of fear memory consolidation. Second, we show that targeted ablation of the GAD65 gene in Gad65(-/-) mice results in a pronounced context-independent, intramodal generalization of auditory fear memory during long-term (24 h or 14 d) but not short-term (30 min) memory retrieval. The temporal specificity of both gene regulation and memory deficits in Gad65 mutant mice suggests that GAD65-mediated GABA synthesis is critical for the consolidation of stimulus-specific fear memory. This function appears to involve a modulation of neural activity patterns in the amygdalo-hippocampal pathway as indicated by a reduction in theta frequency synchronization between the amygdala and hippocampus of Gad65(-/-) mice during the expression of generalized fear memory.

Reduction of extracellular GABA in the mouse amygdala during and following confrontation with a conditioned fear stimulus.

In this study we examined conditioned fear-induced changes of extracellular gamma-aminobutyric acid (GABA) levels in the mouse amygdala with the in vivo microdialysis technique. Confrontation of freely behaving mice with a previously conditioned fear stimulus was accompanied by a pronounced reduction of extracellular GABA levels, indicative of a reduced GABA release and/or increased GABA uptake from the extracellular space. Reduced GABA levels were still observed in the amygdala several hours after the presentation of the conditioned stimulus. Moreover, stimulus-specific and generalized aspects of this GABA response could be distinguished according to their magnitude and time course in different behavioural groups. Our observations suggest that changes of GABAergic transmission may be involved in retrieval and expression of conditioned fear and the modulation of emotional state through the amygdala.

Deficiency of the 65 kDa Isoform of Glutamic Acid Decarboxylase Impairs Extinction of Cued But Not Contextual Fear Memory

Extinction procedures are clinically relevant for reducing pathological fear, and the mechanisms of fear regulation are a subject of intense research. The amygdala, hippocampus, and prefrontal cortex (PFC) have all been suggested to be key brain areas in extinction of conditioned fear. GABA has particularly been implicated in extinction learning, and the 65 kDa isoform of glutamic acid decarboxylase (GAD65) may be important in elevating GABA levels in response to environmental signals. Extinction of conditioned fear was examined in Gad65−/− mice while recording local field potentials from the amygdala, hippocampus, and PFC simultaneously while monitoring behavior. Gad65−/− mice showed generalization of cued fear, as reported previously, and impaired extinction of cued fear, such that fear remained high across extinction training. This endurance in cued fear was associated with theta frequency synchronization between the amygdala and hippocampus. Extinction of contextual fear, however, was unaltered in Gad65−/− mice when compared with wild-type littermates. The data imply that GAD65 plays a critical role in regulating cued fear responses during extinction learning and that, during this process, GABAergic signaling is involved in modulating synchronized activity between the amygdala and hippocampus. In view of the more pronounced effect on cued versus contextual fear extinction, these influences may rely more on GABAergic mechanisms in the amygdala.

Dissociated theta phase synchronization in amygdalo- hippocampal circuits during various stages of fear memory

The amygdala and the hippocampus are critically involved in the formation and retention of fear memories. However, their precise contribution to, and their interplay during, fear memory formation are not fully understood. In the present study we investigated network activities in the amygdalo‐hippocampal system of freely behaving mice at different stages of fear memory consolidation and retention. Our data show enhanced theta phase synchronization in this pathway during the retrieval of fear memory at long‐term (24 h post‐training), but not short‐term (2 min, 30 min and 2 h post‐training) stages, following both contextual and auditory cued conditioning. However, retrieval of remotely conditioned fear (30 days post‐training) failed to induce an increase in synchronization despite there still being memory retention. Thus, our data indicate that the amygdalo‐hippocampal interaction reflects a dynamic interaction of ensemble activities related to various stages of fear memory consolidation and/or retention, and support the notion that recent and remote memories are organized through different network principles.