Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memorydependent phosphorylation event on calcium-calmodulin-dependent kinase II ␣ (CaMKII␣) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKII␣ activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior.

Repetitive synaptic stimulation overcomes the ability of astrocytic processes to clear glutamate from the extracellular space, allowing some dendritic segments to become submerged in a pool of glutamate, for a brief period of time. This dynamic arrangement activates extrasynaptic NMDA receptors located on dendritic shafts. We used voltage-sensitive and calcium-sensitive dyes to probe dendritic function in this glutamate-rich location. An excess of glutamate in the extrasynaptic space was achieved either by repetitive synaptic stimulation or by glutamate iontophoresis onto the dendrites of pyramidal neurons. Two successive activations of synaptic inputs produced a typical NMDA spike, whereas five successive synaptic inputs produced characteristic plateau potentials, reminiscent of cortical UP states. While NMDA spikes were coupled with brief calcium transients highly restricted to the glutamate input site, the dendritic plateau potentials were accompanied by calcium influx along the entire dendritic branch. Once initiated, the glutamate-mediated dendritic plateau potentials could not be interrupted by negative voltage pulses. Activation of extrasynaptic NMDA receptors in cellular compartments void of spines is sufficient to initiate and support plateau potentials. The only requirement for sustained depolarizing events is a surplus of free glutamate near a group of extrasynaptic receptors. Highly non-linear dendritic spikes (plateau potentials) are summed in a highly sublinear fashion at the soma, revealing the cellular bases of signal compression in cortical circuits. Extrasynaptic NMDA receptors provide pyramidal neurons with a function analogous to a dynamic range compression in audio engineering. They limit or reduce the volume of “loud sounds” (i.e., strong glutamatergic inputs) and amplify “quiet sounds” (i.e., glutamatergic inputs that barely cross the dendritic threshold for local spike initiation). Our data also explain why consecutive cortical UP states have uniform amplitudes in a given neuron.

Neurons derived from human embryonic stem cells hold promise for the therapy of neurological diseases. Quality inspection of human embryonic stem cell-derived neurons has often been based on immunolabeling for neuronal markers. Here we put emphasis on their physiological properties. Electrophysiological measurements were carried out systematically at different stages of neuronal in vitro development, including the very early stage, neuroepithelial rosettes. Developing human neurons are able to generate action potentials (APs) as early as 10 days after the start of differentiation. Tyrosine hydroxylase (TH)-positive (putative dopaminergic, DA) neurons tend to aggregate into clumps, and their overall yield per coverslip is relatively low (8.3%) because of areas void of DA neurons. On the same in vitro day, neighboring neurons can be in very different stages of differentiation, including repetitive AP firing, single full-size AP, and abortive AP. Similarly, the basic electrophysiological parameters (resting membrane potential, input resistance, peak sodium, and peak potassium currents) are scattered in a wide range. Visual appearance of differentiating neurons, and number of primary and secondary dendrites cannot be used to predict the peak sodium current or AP firing properties of cultured neurons. Approximately 13% of neurons showed evidence of hyperpolarization-induced current (I(h)), a characteristic of DA neurons; however, no neurons with repetitive APs showed I(h). The electrophysiological measurements thus indicate that a standard DA differentiation (dibutyryl cyclic AMP-based) protocol, applied for 2-5 weeks, produces a heterogeneous ensemble of mostly immature neurons. The overall quality of human neurons under present conditions (survival factors were not used) begins to deteriorate after 12 days of differentiation.

Molecular genetic studies are typically performed on homogenized biological samples, resulting in contamination from non-neuronal cells. To improve expression profiling of neurons we combined patch recordings with single-cell PCR. Two iPSC lines (healthy subject and 22q11.2 deletion), were differentiated into neurons. Patch electrode recordings were performed on 229 human cells from Day-13 to Day-88, followed by capture and single-cell PCR for 13 genes: ACTB, HPRT, vGLUT1, βTUBIII, COMT, DISC1, GAD1, PAX6, DTNBP1, ERBB4, FOXP1, FOXP2, and GIRK2. Neurons derived from both iPSC lines expressed βTUBIII, fired action potentials, and experienced spontaneous depolarizations (UP states) ~2 weeks before vGLUT1, GAD1 and GIRK2 appeared. Multisite calcium imaging revealed that these UP states were not synchronized among hESC-H9-derived neurons. The expression of FOXP1, FOXP2 and vGLUT1 was lost after 50 days in culture, in contrast to other continuously expressed genes. When gene expression was combined with electrophysiology, two subsets of genes were apparent; those irrelevant to spontaneous depolarizations (including vGLUT1, GIRK2, FOXP2 and DISC1) and those associated with spontaneous depolarizations (GAD1 and ERBB4). The results demonstrate that in the earliest stages of neuron development, it is useful to combine genetic analysis with physiological characterizations, on a cell-to-cell basis.