Melanie A. Woodin

Coincident Pre- and Postsynaptic Activity Modifies GABAergic Synapses by Postsynaptic Changes in Cl− Transporter Activity

Coincident pre- and postsynaptic activation is known to induce long-term modification of glutamatergic synapses. We report here that, in both hippocampal cultures and acute hippocampal slices, repetitive postsynaptic spiking within 20 ms before and after the activation of GABAergic synapses also led to a persistent change in synaptic strength. This synaptic modification required Ca2+ influx through postsynaptic L-type Ca2+ channels and was due to a local decrease in K+-Cl- cotransport activity, effectively reducing the strength of inhibition. Thus, GABAergic synapses can detect and be modified by coincident pre- and postsynaptic spiking, allowing the level of inhibition to be modulated in accordance to the temporal pattern of postsynaptic excitation.

Neurons Are Recruited to a Memory Trace Based on Relative Neuronal Excitability Immediately before Training

Memories are thought to be sparsely encoded in neuronal networks, but little is known about why a given neuron is recruited or allocated to a particular memory trace. Previous research shows that in the lateral amygdala (LA), neurons with increased CREB are selectively recruited to a fear memory trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability to bias neuronal recruitment, although this has not been shown experimentally. Here we use several methods to increase neuronal excitability and show this both biases recruitment into the memory trace and enhances memory formation. Moreover, artificial activation of these neurons alone is a sufficient retrieval cue for fear memory expression, showing that these neurons are critical components of the memory trace. These results indicate that neuronal memory allocation is based on relative neuronal excitability immediately before training.

Role of activity-dependent regulation of neuronal chloride homeostasis in development.

The polarity of neurotransmission mediated by the gamma-amino butyric acid (GABA) type A receptor depends crucially on intracellular chloride concentration, which is largely determined by the expression and function of cation/chloride co-transporters. Recent evidence shows how both activity and neurotrophic factors can affect GABAergic transmission in the mammalian central nervous system through their effects on the neuron-specific chloride-extruding transporter KCC2. In particular, GABAergic neurotransmission early in development, sustained neuronal activity in mature networks and brain-derived neurotrophic factor each modulate the expression or function of KCC2. The resulting changes in intracellular chloride concentration alter the nature or strength of fast GABAergic neurotransmission, profoundly affecting the development and function of neuronal networks.

Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent “Hypothesis and Theory Article,” the excitatory action of GABA in early brain development is suggested to be “an experimental artefact” (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

Significance Glioblastoma is an incurable brain tumor. It is characterized by intratumoral phenotypic and genetic heterogeneity, but the functional significance of this heterogeneity is unclear. We devised an integrated functional and genomic strategy to obtain single cell-derived tumor clones directly from patient tumors to identify mechanisms of aggressive clone behavior and drug resistance. Genomic analysis of single clones identified genes associated with clonal phenotypes. We predict that integration of functional and genomic analysis at a clonal level will be essential for understanding evolution and therapeutic resistance of human cancer, and will lead to the discovery of novel driver mechanisms and clone-specific cancer treatment. Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

Inhibitory synaptic plasticity: spike timing-dependence and putative network function

While the plasticity of excitatory synaptic connections in the brain has been widely studied, the plasticity of inhibitory connections is much less understood. Here, we present recent experimental and theoretical findings concerning the rules of spike timing-dependent inhibitory plasticity and their putative network function. This is a summary of a workshop at the COSYNE conference 2012.

Spike-timing dependent plasticity in inhibitory circuits

Inhibitory circuits in the brain rely on GABA-releasing interneurons. For long, inhibitory circuits were considered weakly plastic in the face of patterns of neuronal activity that trigger long-term changes in the synapses between excitatory principal cells. Recent studies however have shown that GABAergic circuits undergo various forms of long-term plasticity. For the purpose of this review, we identify three major long-term plasticity expression sites. The first locus is the glutamatergic synapses that excite GABAergic inhibitory cells and drive their activity. Such synapses, on many but not all inhibitory interneurons, exhibit long-term potentiation (LTP) and depression (LTD). Second, GABAergic synapses themselves can undergo changes in GABA release probability or postsynaptic GABA receptors. The third site of plasticity is in the postsynaptic anion gradient of GABAergic synapses; coincident firing of GABAergic axons and postsynaptic neurons can cause a long-lasting change in the reversal potential of GABAA receptors mediating fast inhibitory postsynaptic potentials. We review the recent literature on these forms of plasticity by asking how they may be triggered by specific patterns of pre- and postsynaptic action potentials, although very few studies have directly examined spike-timing dependent plasticity (STDP) protocols in inhibitory circuits. Plasticity of interneuron recruitment and of GABAergic signaling provides for a rich flexibility in inhibition that may be central to many aspects of brain function. We do not consider plasticity at glutamatergic synapses on Purkinje cells and other GABAergic principal cells.

Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize ECl during development

In the mature CNS, coincident pre‐ and postsynaptic activity decreases the strength of γ‐aminobutyric acid (GABA)A‐mediated inhibition through a Ca2+‐dependent decrease in the activity of the neuron‐specific K+‐Cl– cotransporter KCC2. In the present study we examined whether coincident pre‐ and postsynaptic activity can also modulate immature GABAergic synapses, where the Na+‐K+‐2Cl– (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl–]i). Dual perforated patch‐clamp recordings were made from cultured hippocampal neurons prepared from embryonic Sprague–Dawley rats. These recordings were used to identify GABAergic synapses where the reversal potential for Cl– (ECl) was hyperpolarized with respect to the action potential threshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within ± 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of ECl by 10.03 ± 1.64 mV, increasing the strength of synaptic inhibition. Blocking the inward transport of Cl– by NKCC1 with bumetanide (10 µm) hyperpolarized ECl by 16.14 ± 4.8 mV, and prevented this coincident activity‐induced shift of ECl. The bumetanide‐induced hyperpolarization of ECl occluded furosemide, a K+‐Cl– cotransporter antagonist, from producing further shifts in ECl. Together, this indicates that brief coincident pre‐ and postsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as a mechanism underlying inhibitory synaptic plasticity.

Patterns across multiple memories are identified over time

Memories are not static but continue to be processed after encoding. This is thought to allow the integration of related episodes via the identification of patterns. Although this idea lies at the heart of contemporary theories of systems consolidation, it has yet to be demonstrated experimentally. Using a modified water-maze paradigm in which platforms are drawn stochastically from a spatial distribution, we found that mice were better at matching platform distributions 30 d compared to 1 d after training. Post-training time-dependent improvements in pattern matching were associated with increased sensitivity to new platforms that conflicted with the pattern. Increased sensitivity to pattern conflict was reduced by pharmacogenetic inhibition of the medial prefrontal cortex (mPFC). These results indicate that pattern identification occurs over time, which can lead to conflicts between new information and existing knowledge that must be resolved, in part, by computations carried out in the mPFC.

Neto2 is a KCC2 interacting protein required for neuronal Cl− regulation in hippocampal neurons

KCC2 is a neuron-specific K+–Cl− cotransporter that is essential for Cl− homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl− extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.