Andreas Draguhn

Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro

Coherent oscillations, in which ensembles of neurons fire in a repeated and synchronous manner, are thought to be important in higher brain functions. In the hippocampus, these discharges are categorized according to their frequency as theta (4–10 Hz), gamma (20–80 Hz) and high-frequency (∼200 Hz) discharges, and they occur in relation to different behavioural states. The synaptic bases of theta and gamma rhythms have been extensively studied, but the cellular bases for high-frequency oscillations are not understood. Here we report that high-frequency network oscillations are present in rat brain slices in vitro, occurring as a brief series of repetitive population spikes at 150–200 Hz in all hippocampal principal cell layers. Moreover, this synchronous activity is not mediated through the more commonly studied modes of chemical synaptic transmission, but is in fact a result of direct electrotonic coupling of neurons, most likely through gap-junctional connections. Thus high-frequency oscillations synchronize the activity of electrically coupled subsets of principal neurons within the well-documented synaptic network of the hippocampus.

Functional properties of recombinant rat GABAA receptors depend upon subunit composition.

GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor (GABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. Large whole-cell currents were observed in cells expressing the alpha 1 beta 2, alpha 1 gamma 2, and alpha 1 beta 2 gamma 2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of beta 2 and gamma 3 subunits. GABA-activated currents in cells expressing GABARs with the beta 2 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of alpha 1 gamma 2 subunits. When the gamma 2 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alpha 1 beta 2 gamma 2 subunits compared with GABARs consisting of alpha 1 beta 2 or alpha 1 gamma 2 subunit combinations.

Disruption of ClC-3, a Chloride Channel Expressed on Synaptic Vesicles, Leads to a Loss of the Hippocampus

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co‐expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations.

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the α13 subunits of G proteins (Gα13). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Gα13 both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Gα13 might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.

Axo-Axonal Coupling: A Novel Mechanism for Ultrafast Neuronal Communication

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2

gamma-Aminobutyric acid receptor (GABAAR) channels in different neurons display heterogeneous functional properties. Molecular cloning revealed a large number of GABAAR subunits that assemble into GABAAR subtypes with different functional properties, suggesting that the subunit combination determines the functional properties of the receptor. In this study, the subunit composition of GABAARs is related to a functional distinction between Zn2(+)-sensitive and Zn2(+)-insensitive receptor subtypes. GABAARs reconstituted in transiently transfected fibroblasts from combinations of cDNAs encoding alpha and beta subunits are potently blocked by Zn2+. The presence of a gamma subunit in any combination with the other subunits leads to the formation of GABAARs that are almost insensitive to Zn2+. These data provide a structural correlate to the functional heterogeneity of the action of Zn2+ on GABAARs in native membranes and show that Zn2+ insensitivity of GABA-activated currents indicates the presence of a gamma-subunit in the assembled GABAAR channel.

High-frequency population oscillations are predicted to occur in hippocampal pyramidal neuronal networks interconnected by axoaxonal gap junctions.

In hippocampal slices, high-frequency (125-333 Hz) synchronized oscillations have been shown to occur amongst populations of pyramidal neurons, in a manner that is independent of chemical synaptic transmission, but which is dependent upon gap junctions. At the intracellular level, high-frequency oscillations are associated with full-sized action potentials and with fast prepotentials. Using simulations of two pyramidal neurons, we previously argued that the submillisecond synchrony, and the rapid time-course of fast prepotentials, could be explained, in principle, if the requisite gap junctions were located between pyramidal cell axons. Here, we use network simulations (3072 pyramidal cells) to explore further the hypothesis that gap junctions occur between axons and could explain high-frequency oscillations. We show that, in randomly connected networks with an average of two gap junctions per cell, or less, synchronized network bursts can arise without chemical synapses, with frequencies in the experimentally observed range (spectral peaks 125-182 Hz). These bursts are associated with fast prepotentials (or partial spikes and spikelets) as observed in physiological recordings. The critical assumptions we must make for the oscillations to occur are: (i) there is a background of ectopic axonal spikes, which can occur at low frequency (one event per 25 s per axon); (ii) the gap junction resistance is small enough that a spike in one axon can induce a spike in the coupled axon at short latency (in the model, a resistance of 273 M omega works, with an associated latency of 0.25 ms). We predict that axoaxonal gap junctions, in combination with recurrent excitatory synapses, can induce the occurrence of high-frequency population spikes superimposed on epileptiform field potentials.

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co‐expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high‐affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.

Amyloid β Oligomers (Aβ1–42 Globulomer) Suppress Spontaneous Synaptic Activity by Inhibition of P/Q-Type Calcium Currents

Abnormal accumulation of soluble oligomers of amyloid β (Aβ) is believed to cause malfunctioning of neurons in Alzheimers disease. It has been shown that Aβ oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of Aβ oligomer-induced synaptic modifications by using a recently described stable oligomeric Aβ preparation called “Aβ1–42 globulomer.” Synthetically prepared Aβ1–42 globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that Aβ1–42 globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that Aβ1–42 globulomer, at concentrations as low as 8 nm, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of Aβ1–42 globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by Aβ1–42 globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimers disease patients. The inhibitory effect of Aβ1–42 globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimers disease.