Markus Fendt

Rare pathogenic mutations in the human BSN gene, which encodes the presynaptic cytomatrix protein Bassoon – a key organizer of the neurotransmitter release apparatus – have been linked to neuropsychiatric conditions including schizophrenia. Here we began to dissect the potential contribution of Bsn deficiency in distinct populations of mouse (fore)brain neurons on behavioral and anatomical alterations resembling schizophrenia-like phenotypes. To this end, conditional knockout mice lacking Bassoon in subsets of inhibitory synapses ( Bsn ^Dlx5/6 cKO), in forebrain excitatory synapses ( Bsn ^Emx1 cKO), or at dopaminergic release sites ( Bsn ^DAT cKO) were generated. The mice were assessed using prepulse inhibition, a paradigm that models sensorimotor gating, which is often impaired in schizophrenia, and a fear/safety learning paradigm. Further, anxiety-related behaviors were evaluated and magnetic resonance imaging (MRI) was performed to assess anatomical brain alterations. Our data reveal synapse-specific effects of the Bassoon deficiency. Sensory gating deficits were observed in both Bsn ^Dlx5/6 cKO and Bsn ^Emx1 cKO mice. Bsn ^Dlx5/6 cKO mice further demonstrated impaired discrimination between conditioned fear and safety – a condition frequently observed in individuals with schizophrenia. By contrast, Bsn ^DAT cKO mice displayed enhanced discrimination between conditioned fear and safety. All mouse strains showed elevated anxiety-like behavior in the light/dark box. Structural MRI revealed increased brain volumes in Bsn ^Dlx5/6 cKO and Bsn ^Emx1 cKO mice, with the most pronounced changes in the hippocampus and cortex. Previous studies have shown that Bsn ^Dlx5/6 cKO mice display autistic-like behaviors, altered hippocampal network activity and epilepsy. Our findings support the notion that deficits in Bsn -mediated functions, particularly in interneurons, may contribute to the etiology of neurodevelopmental and neuropsychiatric conditions. Highlights: – Loss of Bassoon in inhibitory or excitatory synapses induces macrocephaly and prepulse inhibition deficits. – Selective ablation of Bsn in inhibitory or dopaminergic neurons exerts opposite effects on fear/safety learning. – All three conditional Bsn mutants – lacking Bassoon in inhibitory, excitatory or dopaminergic neurons – exhibit an anxiety-like phenotype. Graphical

Fear conditioning provides a basis for understanding how animals perceive threats. Unlike delay conditioning, trace fear conditioning requires associating events across a temporal gap and depends on the dorsal hippocampus. However, the role of the dorsal hippocampal dopamine D2 receptors in trace fear learning is relatively unexplored. In this study, we first recorded the expression patterns of fear across multiple trials in delay and trace fear conditioning (5 s and 10 s trace interval) using the fear-potentiated startle paradigm. We found that the 5 s trace group showed a generalized fear response, whereas the 10 s trace group showed a temporally specific response peaking near the expected time of the footshock, which was used for subsequent experiments. Our exploratory analysis suggested that the contextual fear was higher in trace than in delay conditioning. We then mapped the distribution of dopaminergic fibres and D2 receptors in the hippocampus using immunohistochemistry with specific antibodies against tyrosine hydroxylase and D2 receptors. This was followed by the intracranial administration of the D2 receptor antagonist raclopride into the dorsal hippocampus to assess its effect on trace fear learning. Raclopride disrupted the timed trace response that was otherwise present in the saline treated group, while leaving general startle intact. These findings indicate that dorsal hippocampal D2 receptors contribute to trace fear conditioning.

Synaptic dysfunction and resulting imbalance of excitatory vs. inhibitory transmission are fundamental aspects of autism spectrum disorders (ASD). Here we addressed the role of inhibitory synapse disturbances in ASD employing mice with a conditional ablation in GABAergic forebrain interneurons of the presynaptic active zone scaffolding protein Bassoon (Bsn), a key factor in synaptic development, activity and maintenance. The conditional Bsn gene knockout resulted in a reduction of synaptic vesicles and reduced synaptic efficacy of affected inhibitory synapses as well as diminished GABAergic inhibition of hippocampal pyramidal neurons. Bsn mutants further displayed a reconfiguration of the hippocampal synaptic network in vivo, as seen in subfield-specific changes of inhibitory synaptic markers and reduced parvalbumin interneuron labeling, culminating in disturbance of hippocampal network activity patterns and profound mitochondrial and metabolic dysregulation. Importantly, the mutant mice developed behavioral abnormalities reminiscent of ASD including widespread social behavioral deficits and novelty-induced hyperarousal with altered motor behavior, increased anxiety and epileptiform activity. Bsn conditional knockout mice thus provide strong evidence for a causal involvement of GABAergic synapses in the emergence of ASD-related behavioral and physiological phenotypes.

Cognitive deficits are a major contributor to disability in numerous neuropsychiatric and neurodegenerative disorders, yet effective pharmacological treatments remain limited. Ferulic acid eicosyl ester (FAE-20), a natural constituent of the plant Rhodiola rosea , has previously been identified as an enhancer of simple forms of Pavlovian conditioning in flies, bees, and mice. Here, we investigated whether FAE-20 has further potential to enhance cognitive flexibility, working memory, or spatial learning in mice, and explored potential neurobiological mechanisms underlying such enhancement. Cognitive flexibility was assessed using the attentional set-shifting task (ASST). Subchronic FAE-20 treatment significantly improved ASST performance in both male and female young adult mice, indicating enhanced cognitive flexibility. In contrast, no effects were observed on spatial working memory, assessed by spontaneous alternations in the Y-maze, or on spatial learning in the Barnes maze in either young or aged mice. Notably, FAE-20 enabled spatial learning in the Barnes maze in a subgroup of aged mice that failed to learn the task under vehicle treatment. Histological analyses using c-Fos immunohistochemistry as a marker of neural activity and doublecortin expression and spine density as markers of hippocampal plasticity revealed sex-specific effects on components of the ascending arousal system. FAE-20 increased the activation of orexinergic neurons in the lateral hypothalamus of male mice, whereas it reduced the activity of cholinergic neurons in the laterodorsal tegmental nucleus of females. No effects were detected on hippocampal neurogenesis or dendritic spine density. These findings suggest that the cognitive effects of FAE-20 are selective, depending on the cognitive demands of the task and the baseline cognitive abilities of the animals, and may be mediated, at least in part, by modulation of arousal-related neural circuits. Highlights: FAE-20 enhanced cognitive flexibility in young adult mice Effects of FAE-20 were strongest in demanding cognitive tasks Aged poor learners benefited from FAE-20 treatment FAE-20 activated hypothalamic orexin neurons in male mice FAE-20 modulated ascending arousal systems in a sex-specific manner

Cognitive deficits in executive functions such as working memory and cognitive flexibility are central to many brain disorders, yet effective treatments remain limited. Dysfunction of GluN2A-containing NMDA receptors (NMDAR) contributes to these impairments, and positive allosteric modulators (PAM) that selectively enhance GluN2A function offer a promising strategy to rescue cognitive deficits while preserving physiological neurotransmission. However, it remains unclear whether PAM-driven GluN2A potentiation can rescue mechanistically distinct, pharmacologically induced impairments and whether the efficacy of GluN2A-PAMs differs across executive domains and sexes. Here, we assessed the effects of GNE-5729, a brain-penetrant GluN2A-selective PAM, on executive functions in mice. GNE-5729 showed no overall effect on working memory performance in the Y-maze, but improved performance in mice with low baseline spontaneous alternations, indicating a baseline-dependent effect. It also rescued working memory deficits induced by dl-amphetamine and scopolamine, which disrupt neuromodulatory regulation while leaving NMDAR channels functionally accessible. In contrast, GNE-5729 failed to reverse MK-801-induced impairments, consistent with MK-801’s non-competitive pore blockade of NMDAR. Notably, GNE‑5729 effects showed sex‑dependent patterns, with female mice displaying more pronounced effects. GNE-5729 had no effects on cognitive flexibility in the attentional set-shifting task (ASST) and did not alter prefrontal GluN2A expression, though it disrupted the association between endogenous GluN2A levels and ASST performance observed in controls. Together, these findings indicate that GNE-5729 exerts effects under conditions of reduced baseline performance or disrupted neuromodulatory signaling and demonstrates differential effects across executive domains, supporting a state-dependent therapeutic profile of GluN2A-targeted modulation.

Synaptic dysfunction and resulting imbalance of excitatory vs. inhibitory transmission are fundamental aspects of autism spectrum disorders (ASD). Here we addressed the role of inhibitory synapse disturbances in ASD employing mice with a conditional ablation in inhibitory forebrain interneurons of the presynaptic active zone scaffolding protein Bassoon (Bsn), a key factor in synaptic development, activity and maintenance. The conditional Bsn gene knock out resulted in a reduction of synaptic vesicles and reduced synaptic efficacy of affected inhibitory synapses as well as diminished GABAergic inhibition in hippocampal pyramidal neurons. Bsn mutants further displayed a reconfiguration of the hippocampal synaptic network in vivo , as seen in subfield-specific changes of inhibitory synaptic markers and reduced numbers of parvalbumin interneurons, culminating in disturbance of hippocampal network activity patterns and profound mitochondrial and metabolic dysregulation. Importantly, the mutant mice developed behavioral abnormalities reminiscent of ASD including widespread social behavioral deficits and novelty-induced hyperarousal with altered motor behavior, increased anxiety and epileptiform activity. Bsn conditional knock out mice thus provide strong evidence for a causal involvement of GABAergic synapses in the emergence of ASD-related behavioral and physiological phenotypes.

Cognitive flexibility and working memory are important executive functions mediated by the prefrontal cortex and can be impaired by circadian rhythm disturbances such as chronic jet lag (CJL) or shift work. In the present study, we used mice to investigate whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such impairment, and (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Mice were exposed to either standard light-dark conditions or simulated CJL consisting of series of advance time shifts. Experiment (1) investigated the effects of a mild CJL protocol on cognitive flexibility using the attentional set shifting task. Experiment (2) used a stronger CJL protocol and examined CJL effects on the orexin system utilizing c-Fos and orexin immunohistochemistry. Experiment (3) tested whether nasal orexin application can rescue CJL-induced deficits in cognitive flexibility and working memory, the latter by measuring spontaneous alternation in the Y-maze. The present data show that CJL (1) impairs cognitive flexibility and (2) reduces the activity of orexin neurons in the lateral hypothalamus. (3) Nasal administration of orexin A rescued CJL-induced deficits in working memory and cognitive flexibility. These findings suggest that executive function impairments by circadian rhythm disturbances such as CJL are caused by dysregulation of orexinergic input to the prefrontal cortex. Compensation of decreased orexinergic input by nasal administration of orexin A could be a potential therapy for CJL- or shift work-induced human deficits in executive functions.

Cognitive flexibility and working memory are important executive functions mediated by the prefrontal cortex and can be impaired by circadian rhythm disturbances such as chronic jet lag (CJL) or shift work. In the present study, we used mice to investigate whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such impairment, and (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Mice were exposed to either standard light-dark conditions or simulated CJL consisting of series of advance time shifts. Experiment (1) investigated the effects of a mild CJL protocol on cognitive flexibility using the attentional set shifting task. Experiment (2) used a stronger CJL protocol and examined CJL effects on the orexin system utilizing c-Fos and orexin immunohistochemistry. Experiment (3) tested whether nasal orexin application can rescue CJL-induced deficits in cognitive flexibility and working memory, the latter by measuring spontaneous alternation in the Y-maze. The present data show that CJL (1) impairs cognitive flexibility and (2) reduces activity of orexin neurons in the lateral hypothalamus. (3) Nasal administration of orexin A rescued CJL-induced deficits in working memory and cognitive flexibility. These findings suggest that executive functions impairments by circadian rhythm disturbances such as CJL are caused by dysregulation of orexinergic input to the prefrontal cortex. Compensation of decreased orexinergic input by nasal administration of orexin A could be a potential therapy for CJL- or shift work-induced human deficits in executive functions.