Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons.

Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.

Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion.

BACKGROUND: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K+ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA). OBJECTIVE: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion. DESIGN AND PATIENTS: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out. RESULTS: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected. CONCLUSIONS: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

Human endogenous retrovirus-K contributes to motor neuron disease.

The role of human endogenous retroviruses (HERVs) in disease pathogenesis is unclear. We show that HERV-K is activated in a subpopulation of patients with sporadic amyotrophic lateral sclerosis (ALS) and that its envelope (env) protein may contribute to neurodegeneration. The virus was expressed in cortical and spinal neurons of ALS patients, but not in neurons from control healthy individuals. Expression of HERV-K or its env protein in human neurons caused retraction and beading of neurites. Transgenic animals expressing the env gene developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex, decreased synaptic activity in pyramidal neurons, dendritic spine abnormalities, nucleolar dysfunction, and DNA damage. Injury to anterior horn cells in the spinal cord was manifested by muscle atrophy and pathological changes consistent with nerve fiber denervation and reinnervation. Expression of HERV-K was regulated by TAR (trans-activation responsive) DNA binding protein 43, which binds to the long terminal repeat region of the virus. Thus, HERV-K expression within neurons of patients with ALS may contribute to neurodegeneration and disease pathogenesis.

Derivation of neural stem cells from human adult peripheral CD34+ cells for an autologous model of neuroinflammation.

Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.

Synaptic plasticity by antidromic firing during hippocampal network oscillations.

Learning and other cognitive tasks require integrating new experiences into context. In contrast to sensory-evoked synaptic plasticity, comparatively little is known of how synaptic plasticity may be regulated by intrinsic activity in the brain, much of which can involve nonclassical modes of neuronal firing and integration. Coherent high-frequency oscillations of electrical activity in CA1 hippocampal neurons [sharp-wave ripple complexes (SPW-Rs)] functionally couple neurons into transient ensembles. These oscillations occur during slow-wave sleep or at rest. Neurons that participate in SPW-Rs are distinguished from adjacent nonparticipating neurons by firing action potentials that are initiated ectopically in the distal region of axons and propagate antidromically to the cell body. This activity is facilitated by GABA(A)-mediated depolarization of axons and electrotonic coupling. The possible effects of antidromic firing on synaptic strength are unknown. We find that facilitation of spontaneous SPW-Rs in hippocampal slices by increasing gap-junction coupling or by GABA(A)-mediated axon depolarization resulted in a reduction of synaptic strength, and electrical stimulation of axons evoked a widespread, long-lasting synaptic depression. Unlike other forms of synaptic plasticity, this synaptic depression is not dependent upon synaptic input or glutamate receptor activation, but rather requires L-type calcium channel activation and functional gap junctions. Synaptic stimulation delivered after antidromic firing, which was otherwise too weak to induce synaptic potentiation, triggered a long-lasting increase in synaptic strength. Rescaling synaptic weights in subsets of neurons firing antidromically during SPW-Rs might contribute to memory consolidation by sharpening specificity of subsequent synaptic input and promoting incorporation of novel information.

Repeated cocaine exposure increases fast-spiking interneuron excitability in the rat medial prefrontal cortex.

The medial prefrontal cortex plays a key role in cocaine addiction. However, how chronic cocaine exposure affects cortical networks remains unclear. Most studies have focused on layer 5 pyramidal neurons (the circuit output), while the response of local GABAergic interneurons to cocaine remains poorly understood. Here, we recorded from fast-spiking interneurons (FS-IN) after repeated cocaine exposure and found altered membrane excitability. After cocaine withdrawal, FS-IN showed an increase in the number of spikes evoked by positive current injection, increased input resistance, and decreased hyperpolarization-activated current. We also observed a reduction in miniature excitatory postsynaptic currents, whereas miniature inhibitory postsynaptic current activity was unaffected. We show that, in animals with cocaine history, dopamine receptor D(2) activation is less effective in increasing FS-IN intrinsic excitability. Interestingly, these alterations are only observed 1 wk or more after the last cocaine exposure. This suggests that the dampening of D(2)-receptor-mediated response may be a compensatory mechanism to rein down the excitability of FS-IN.

Enhanced intrinsic excitability in basket cells maintains excitatory-inhibitory balance in hippocampal circuits.

The dynamics of inhibitory circuits in the cortex is thought to rely mainly on synaptic modifications. We challenge this view by showing that hippocampal parvalbumin-positive basket cells (PV-BCs) of the CA1 region express long-term (>30 min) potentiation of intrinsic neuronal excitability (LTP-IE(PV-BC)) upon brief repetitive stimulation of the Schaffer collaterals. LTP-IE(PV-BC) is induced by synaptic activation of metabotropic glutamate receptor subtype 5 (mGluR5) and mediated by the downregulation of Kv1 channel activity. LTP-IE(PV-BC) promotes spiking activity at the gamma frequency (∼35 Hz) and facilitates recruitment of PV-BCs to balance synaptic and intrinsic excitation in pyramidal neurons. In conclusion, activity-dependent modulation of intrinsic neuronal excitability in PV-BCs maintains excitatory-inhibitory balance and thus plays a major role in the dynamics of hippocampal circuits.

The role of hyperpolarization-activated cationic current in spike-time precision and intrinsic resonance in cortical neurons in vitro.

Hyperpolarization-activated cyclic nucleotide modulated current (I(h)) sets resonance frequency within the θ-range (5­12 Hz) in pyramidal neurons. However, its precise contribution to the temporal fidelity of spike generation in response to stimulation of excitatory or inhibitory synapses remains unclear. In conditions where pharmacological blockade of I(h) does not affect synaptic transmission, we show that postsynaptic h-channels improve spike time precision in CA1 pyramidal neurons through two main mechanisms. I(h) enhances precision of excitatory postsynaptic potential (EPSP)--spike coupling because I(h) reduces peak EPSP duration. I(h) improves the precision of rebound spiking following inhibitory postsynaptic potentials (IPSPs) in CA1 pyramidal neurons and sets pacemaker activity in stratum oriens interneurons because I(h) accelerates the decay of both IPSPs and after-hyperpolarizing potentials (AHPs). The contribution of h-channels to intrinsic resonance and EPSP waveform was comparatively much smaller in CA3 pyramidal neurons. Our results indicate that the elementary mechanisms by which postsynaptic h-channels control fidelity of spike timing at the scale of individual neurons may account for the decreased theta-activity observed in hippocampal and neocortical networks when h-channel activity is pharmacologically reduced.

Axon physiology.

Axons are generally considered as reliable transmission cables in which stable propagation occurs once an action potential is generated. Axon dysfunction occupies a central position in many inherited and acquired neurological disorders that affect both peripheral and central neurons. Recent findings suggest that the functional and computational repertoire of the axon is much richer than traditionally thought. Beyond classical axonal propagation, intrinsic voltage-gated ionic currents together with the geometrical properties of the axon determine several complex operations that not only control signal processing in brain circuits but also neuronal timing and synaptic efficacy. Recent evidence for the implication of these forms of axonal computation in the short-term dynamics of neuronal communication is discussed. Finally, we review how neuronal activity regulates both axon morphology and axonal function on a long-term time scale during development and adulthood.

Presynaptic GABAA receptors enhance transmission and LTP induction at hippocampal mossy fiber synapses.

Presynaptic GABA(A) receptors (GABA(A)Rs) occur at hippocampal mossy fiber synapses. Whether and how they modulate orthodromic signaling to postsynaptic targets is poorly understood. We found that an endogenous neurosteroid that is selective for high-affinity delta subunit-containing GABA(A)Rs depolarized rat mossy fiber boutons, enhanced action potential-dependent Ca(2+) transients and facilitated glutamatergic transmission to pyramidal neurons. Conversely, blocking GABA(A)Rs hyperpolarized mossy fiber boutons, increased their input resistance, decreased spike width and attenuated action potential-dependent presynaptic Ca(2+) transients, indicating that a subset of presynaptic GABA receptors are tonically active. Blocking GABA(A)Rs also interfered with the induction of long-term potentiation at mossy fiber-CA3 synapses. Presynaptic GABA(A)Rs therefore facilitate information flow to the hippocampus both directly and by enhancing LTP.

Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices.

Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.

Downregulation of dendritic I(h) in CA1 pyramidal neurons after LTP.

Hyperpolarization-activated (h)-channels occupy a central position in dendritic function. Although it has been demonstrated that these channels are upregulated after large depolarizations to reduce dendritic excitation, it is not clear whether they also support other forms of long-term plasticity. We show here that nearly maximal long-term potentiation (LTP) induced by theta-burst pairing produced upregulation in h-channel activity in CA1 pyramidal neurons. In contrast, moderate LTP induced by spike-timing-dependent plasticity or high-frequency stimulation (HFS) downregulated the h-current (I(h)) in the dendrites. After HFS-induced LTP, the h-conductance (G(h)) was reduced without changing its activation. Pharmacological blockade of I(h) had no effect on LTP induction, but occluded EPSP-to-spike potentiation, an input-specific facilitation of dendritic integration. Dynamic-clamp reduction of G(h) locally in the dendrite mimicked the effects of HFS and enhanced synaptic integration in an input-selective way. We conclude that dendritic I(h) is locally downregulated after induction of nonmaximal LTP, thus facilitating integration of the potentiated input.

Spike timing-dependent plasticity: a learning rule for dendritic integration in rat CA1 pyramidal neurons.

Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation (LTP) or depression (LTD) based on presynaptic activity patterns. It is, however, not clear whether synaptic plasticity induced by temporal pairing of pre- and postsynaptic activity is also associated with synergistic modification in dendritic integration. We show here that the spike timing-dependent plasticity (STDP) rule accounts for long-term changes in dendritic integration in CA1 pyramidal neurons in vitro. Positively correlated pre- and postsynaptic activity (delay: +5/+50 ms) induced LTP and facilitated dendritic integration. Negatively correlated activity (delay: -5/-50 ms) induced LTD and depressed dendritic integration. These changes were not observed following positive or negative pairing with long delays (> +/-50 ms) or when NMDA receptors were blocked. The amplitude-slope relation of the EPSP was facilitated after LTP and depressed after LTD. These effects could be mimicked by voltage-gated channel blockers, suggesting that the induced changes in EPSP waveform involve the regulation of voltage-gated channel activity. Importantly, amplitude-slope changes induced by STDP were found to be input specific, indicating that the underlying changes in excitability are restricted to a limited portion of the dendrites. We conclude that STDP is a common learning rule for long-term plasticity of both synaptic transmission and dendritic integration, thus constituting a form of functional redundancy that insures significant changes in the neuronal output when synaptic plasticity is induced.