Biology subjects

Wierenga, C. J.

Publications and source records attributed to Wierenga, C. J..

10 recordsLinked to original sources

bioRxiv2024

Distinct modulation of Ih by synaptic potentiation in excitatory and inhibitory neurons

Selective modifications in the expression or function of dendritic ion channels regulate the propagation of synaptic inputs and determine the intrinsic excitability of a neuron. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open upon membrane hyperpolarization and conduct a depolarizing inward current (Ih). HCN channels are enriched in the dendrites of hippocampal pyramidal neurons where they regulate the integration of synaptic inputs. Synaptic plasticity can bidirectionally modify dendritic HCN channels in excitatory neurons depending on the strength of synaptic potentiation. In inhibitory neurons, however, the dendritic expression and modulation of HCN channels is largely unknown. In this study, we systematically compared the modulation of Ih by synaptic potentiation in hippocampal CA1 pyramidal neurons and stratum Radiatum (sRad) interneurons. Ih properties were similar in inhibitory and excitatory neurons and contributed to resting membrane potential and action potential firing. We found that in sRad interneurons, HCN channels were downregulated after synaptic plasticity, irrespective of the strength of synaptic potentiation. This suggest differential regulation of Ih in excitatory and inhibitory neurons, possibly signifying their distinct role in network activity. Significance statementLearning reflects a change in the way information is processed in neuronal circuits. This occurs via changes in synaptic connections and via alterations of intrinsic excitability of neurons. Here we examined how synaptic changes affect properties of HCN channels, which are important ion channels for intrinsic excitability. We found that strong synaptic potentiation leads to opposite changes in HCN channels in CA1 pyramidal neurons and sRad interneurons. We speculate that this reflects their differential role in the CA1 network. An upregulation of HCN channels in pyramidal neurons results in a decrease in their excitability, which limits overall network excitation. In contrast, sRad interneurons show downregulation of Ih, and therefore an increased excitability after strong synaptic activation, which will strengthen feedforward inhibition and sharpen activity patterns.

neuroscience↗

bioRxiv2023

Opportunities for risk-taking during play alters cognitive performance and prefrontal inhibitory signalling in rats of both sexes

Social play behaviour is a rewarding activity that can entail risks, thus allowing young individuals to test the limits of their capacities and to train their cognitive and emotional adaptability to challenges. Here, we tested in rats how opportunities for risk-taking during play affect the development of cognitive and emotional capacities and medial prefrontal cortex (mPFC) function, a brain structure important for risk-based decision-making. Male and female rats were housed socially or social play-deprived (SPD) between postnatal day (P)21 and P42. During this period, half of both groups were daily exposed to a high-risk play environment. Around P85, all rats were tested for cognitive performance and emotional behaviour after which inhibitory currents were recorded in layer 5 pyramidal neurons in mPFC slices. We show that playing in a high-risk environment altered cognitive flexibility in both sexes, and improved behavioural inhibition in males. High-risk play altered anxiety-like behaviour in the elevated plus maze in males and in the open field in females, respectively. SPD affected cognitive flexibility in both sexes and decreased anxiety-like behaviour in the elevated plus maze in females. We found that synaptic inhibitory currents in the mPFC were increased in male, but not female, rats after high-risk play, while SPD lowered PFC synaptic inhibition in both sexes. Together, our data show that exposure to risks during play affects the development of cognition, emotional behaviour and inhibition in the mPFC. Furthermore, our study suggests that the opportunity to take risks during play cannot substitute for social play behaviour.

neuroscience↗

bioRxiv2023

Treatment with furosemide indirectly increases inhibitory transmission in the developing hippocampus

During the first two postnatal weeks intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing {gamma}-aminobutyric acid (GABA) responses. GABAergic depolarization in the immature brain is crucial for the formation and maturation of excitatory synapses, but when GABAergic signaling becomes inhibitory it no longer promotes synapse formation. Here we examined the role of chloride transporters in developing postnatal hippocampal neurons using furosemide, an inhibitor of the chloride importer NKCC1 and chloride exporter KCC2 with reported anticonvulsant effects. We treated organotypic hippocampal cultures made from 6 to 7-day old mice with 200 M furosemide from DIV1 to DIV8. Using perforated patch clamp recordings we observed that the GABA reversal potential was depolarized after acute furosemide application, but after a week of furosemide treatment the GABA reversal potential but was more hyperpolarized compared to control. Expression levels of the chloride cotransporters were unaffected after one week furosemide treatment. This suggests that furosemide inhibited KCC2 acutely, while prolonged treatment resulted in (additional) inhibition of NKCC1, but we cannot exclude changes in HCO3-. We assessed the effects of accelerating the GABA shift by furosemide treatment on inhibitory synapses onto CA1 pyramidal cells. Directly after cessation of furosemide treatment at DIV9, inhibitory synapses were not affected. However at DIV21, two weeks after ending the treatment, we found that the frequency of inhibitory currents was increased, and VGAT puncta density in stratum Radiatum was increased. In addition, cell capacitance of CA1 pyramidal neurons was reduced in furosemide-treated slices at DIV21 in an activity-dependent manner. Our results suggest that furosemide treatment indirectly promoted inhibitory transmission, possibly by increasing activity-independent GABA release.

neuroscience↗

bioRxiv2023

Delaying the GABA shift indirectly affects membrane properties in the developing hippocampus

During the first two postnatal weeks intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing {gamma}-aminobutyric acid (GABA) responses. The postnatal GABA shift is delayed in rodent models for neurodevelopmental disorders and in human patients, but the impact of a delayed GABA shift on the developing brain remain obscure. Here we examine the direct and indirect consequences of a delayed postnatal GABA shift on network development in organotypic hippocampal cultures made from 6 to 7-day old mice by treating the cultures for one week with VU0463271, a specific inhibitor of the chloride exporter KCC2. We verified that VU treatment delayed the GABA shift and kept GABA signaling depolarizing until day in vitro (DIV) 9. We found that the structural and functional development of excitatory and inhibitory synapses at DIV9 was not affected after VU treatment. In line with previous studies, we observed that GABA signaling was already inhibitory in control and VU-treated postnatal slices. Surprisingly, fourteen days after the VU treatment had ended (DIV21), we observed an increased frequency of spontaneous inhibitory post-synaptic currents in CA1 pyramidal cells, while excitatory currents were not changed. Synapse numbers and release probability were unaffected. We found that dendrite-targeting interneurons in the stratum Radiatum had an elevated resting membrane potential, while pyramidal cells were less excitable compared to control slices. Our results show that depolarizing GABA signaling does not promote synapse formation after P7, and suggest that postnatal intracellular chloride levels indirectly affect membrane properties in a cell-specific manner. Significance StatementDuring brain development the action of neurotransmitter GABA shifts from depolarizing to hyperpolarizing. This shift is a thought to play a critical role in synapse formation. A delayed shift is common in rodent models for neurodevelopmental disorders and in human patients, but its consequences for synaptic development remain obscure. Here, we delayed the GABA shift by one week in organotypic hippocampal cultures and carefully examined the consequences for circuit development. We find that delaying the shift has no direct effects on synaptic development, but instead leads to indirect, cell type-specific changes in membrane properties. Our data call for careful assessment of alterations in cellular excitability in neurodevelopmental disorders.

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bioRxiv2023

Social play behavior shapes the development of prefrontal inhibition in a region-specific manner

Experience-dependent organization of neuronal connectivity is critical for brain development. We recently demonstrated the importance of social play behavior for the developmental fine-tuning of inhibitory synapses in the medial prefrontal cortex (mPFC) in rats. When these effects of play experience exactly occur and if this happens uniformly throughout the prefrontal cortex is currently unclear. Here we report important temporal and regional heterogeneity in the impact of social play on the development of excitatory and inhibitory neurotransmission in the mPFC and the orbitofrontal cortex (OFC). We recorded in layer 5 pyramidal neurons from juvenile (postnatal day (P)21), adolescent (P42) and adult (P85) rats after social play deprivation (SPD; between P21-P42). The development of these PFC subregions followed different trajectories. On P21, inhibitory and excitatory synaptic input was multiple times higher in the OFC than in the mPFC. SPD did not affect excitatory currents, but reduced inhibitory transmission in both mPFC and OFC. Intriguingly, the reduction occurred in the mPFC during SPD, while the reduction in the OFC only became manifested after SPD. These data reveal a complex interaction between social play experience and the specific developmental trajectories of prefrontal subregions.

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bioRxiv2022

Using SuperClomeleon to measure changes in intracellular chloride during development and after early life stress

Intraneuronal chloride concentrations ([Cl-]i) decrease during development resulting in a shift from depolarizing to hyperpolarizing y-aminobutyric acid (GABA) responses via chloride-permeable GABAA receptors. This GABA shift plays a pivotal role in postnatal brain development, and can be strongly influenced by early life experience. Here, we assessed the applicability of the recently developed fluorescent SuperClomeleon (SClm) sensor to examine changes in [Cl-]i using two-photon microscopy in brain slices. We used SClm mice of both sexes to monitor the developmental decrease in neuronal chloride levels in organotypic hippocampal cultures. We could discern a clear reduction in [Cl-]i between DIV3 and DIV9 (equivalent to the second postnatal week in vivo) and a further decrease in some cells until DIV22. In addition, we assessed alterations in [Cl-]i in the medial prefrontal cortex (mPFC) of P9 male SClm mouse pups after early life stress (ELS). ELS was induced by limiting nesting material between P2 and P9. ELS induced a shift towards higher (i.e. immature) chloride levels in layer 2/3 cells in the mPFC. Although conversion from SClm fluorescence to absolute chloride concentrations proved difficult, our study underscores that the SClm sensor is a powerful tool to measure physiological changes in [Cl-]i in brain slices. Significance StatementThe reduction of intraneuronal chloride concentrations is crucial for brain development, as it ensures a shift from the initial excitatory action of the neurotransmitter GABA in immature neurons to the inhibitory GABA signaling in the adult brain. Despite the significance of chloride maturation, it has been difficult to study this phenomenon in experiments. Recent development of chloride sensors enable direct imaging of intracellular chloride signaling in neurons. Here we assessed the applicability of the SuperClomeleon chloride sensor to measure physiologically relevant changes in chloride levels using two-photon microscopy in cultured and acute brain slices. Although we also point out some limitations, we conclude that the SuperClomeleon sensor is a powerful tool to measure physiological changes in intracellular chloride.

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bioRxiv2021

Axonal CB1 receptors mediate inhibitory bouton formation via cAMP increase

Experience-dependent formation and removal of synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by formation of inhibitory synapses. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons, we found that CB1 receptor activation mediated formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs proteins using DREADDs. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate a novel role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.

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bioRxiv2020

An altered cognitive strategy associated with reduction of synaptic inhibition in the prefrontal cortex after social play deprivation in rats

Sensory driven activity during early life is critical for setting up the proper connectivity of the sensory cortices. Here we ask if social play behavior, a particular form of social interaction that is highly abundant during post-weaning development, is equally important for setting up connections in the developing prefrontal cortex (PFC). Young rats were deprived from social play with peers for 3 weeks during the period in life when social play behavior normally peaks (P21-42; SPD rats), followed by resocialization until adulthood. We recorded synaptic currents in L5 cells in slices from medial PFC of adult SPD and control rats and observed that inhibitory synaptic currents were reduced in SPD slices, while excitatory synaptic currents were unaffected. This was associated with a decrease in perisomatic inhibitory synapses from parvalbumin-positive GABAergic cells. In parallel experiments, adult SPD rats achieved more reversals in a probabilistic reversal learning task (PRL), which depends on the integrity of the PFC. They appeared to use a different cognitive strategy than controls. One hour of intense play during SPD did not prevent the decrease in inhibitory synaptic inputs and had only a limited effect on behavioral outcomes in the PRL. Our data demonstrate the importance of unrestricted social play for the development of inhibitory synapses in the PFC and cognitive skills in adulthood.

neuroscience↗

bioRxiv2020

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

Early neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

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bioRxiv2017

Molecular pathway underlying bouton stabilization by Semaphorin4D during inhibitory synapse formation

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high resolution two-photon microscopy in organotypic hippocampal slices to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilizing boutons rapidly recruited synaptic vesicles, which was followed by accumulation of postsynaptic gephyrin. Newly formed inhibitory synapses were complete and functional after 24 hours, as determined by electrophysiology and immunohistochemistry. We further showed that Sema4D signaling is regulated by network activity and can induce a local increase in bouton density, suggesting a possible role in circuit adaptation. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurred through rapid remodeling of actin, and this could be mimicked by the actin-depolymerizing drug Latrunculin B or by reducing ROCK activity. The intracellular signaling cascade required activation of the receptor tyrosine kinase MET, which is a well-known autism risk factor. Our immunohistochemistry data suggests that MET may be localized to presynaptic inhibitory axons. Together, our data yield important insights in the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for MET in inhibitory synapses.\n\nSignificance StatementGABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We make important steps in unraveling the molecular processes that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this process depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET in inhibitory synapses. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

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