Circuits and Synapses: Hypothesis, Observation, Controversy and Serendipity – An Opinion Piece

More than a century of dedicated research has resulted in what we now know, and what we think we know, about synapses and neural circuits. This piece asks to what extent some of the major advances – both theoretical and practical – have resulted from carefully considered theory, or experimental design: endeavors that aim to address a question, or to refute an existing hypothesis. It also, however, addresses the important part that serendipity and chance have played. There are cases where hypothesis driven research has resulted in important progress. There are also examples where a hypothesis, a model, or even an experimental approach – particularly one that seems to provide welcome simplification – has become so popular that it becomes dogma and stifles advance in other directions. The nervous system rejoices in complexity, which should neither be ignored, nor run from. The emergence of testable “rules” that can simplify our understanding of neuronal circuits has required the collection of large amounts of data that were difficult to obtain. And although those collecting these data have been criticized for not advancing hypotheses while they were “collecting butterflies,” the beauty of the butterflies always enticed us toward further exploration

Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts

Characterization of neurons in the CA2 subfield of the adult rat hippocampus.

The hippocampal cornu ammonis 2 (CA2) region is unique in being the only CA region receiving inputs from the hypothalamic supramammillary nucleus, of importance in modulating hippocampal theta rhythm, and is seizure resistant in temporal lobe epilepsy. CA2 has, however, been little studied, possibly because of its small size and difficulty encountered in defining its borders. To investigate the properties of CA2 interneurons, intracellular recordings with biocytin filling were made in adult hippocampal slices. Two types of basket cells were identified. A minority resembled those in CA1, with fast spiking behavior, vertically oriented dendrites, and axons confined to the region of origin. In contrast, the majority of parvalbumin-immunopositive CA2 basket and bistratified cells had long, horizontally oriented, sparsely spiny dendrites extending into all CA subfields in stratum oriens, adapting firing patterns and a pronounced "sag" in voltage responses to hyperpolarizing current, indicative of I(h). Broad CA2 basket cells innervated all three CA subfields and could thus provide CA1 and CA2 with feedforward and CA3 with feedback inhibition. In contrast, CA2 bistratified cell axons displayed striking subfield preference, innervating stratum oriens and stratum radiatum of CA2 and CA1 but stopping abruptly at the CA2/CA3 border, implying feedforward inhibition of CA2 and CA1. These unique features suggest that CA2 is more than a transitional region between CA1 and CA3. The pronounced slow sag current of many CA2 interneurons may contribute to coordination of pyramidal cell firing during theta, whereas the fast spiking behavior of a smaller population of interneurons supports more localized gamma

Educational needs of epileptologists regarding psychiatric comorbidities of the epilepsies: a descriptive quantitative survey.

Psychiatric disorders are relatively frequent comorbidities in epilepsy and they have an impact on morbidity, mortality, and quality of life. This is a report from the Task Force on Education of the ILAE Commission on Neuropsychiatry based on a survey about educational needs of epileptologists regarding management of the psychiatric comorbidities of epilepsy. The Task Force designed a quantitative questionnaire to survey the self-perceived confidence of child and adult epileptologists and psychiatrists in managing major psychiatric comorbidities of epilepsy to identify: (1) critical areas of improvement from a list of skills that are usually considered necessary for effective management of these conditions, and (2) the preferred educational format for improving these skills. A total of 211 respondents from 36 different countries participated in the survey. Confidence and usefulness scores suggest that responders would most value education and training in the management of specific clinical scenarios. Child neurologists identified major Axis I disorders, such as mood and anxiety disorders, while adult neurologists identified attention deficit hyperactivity disorder, intellectual disabilities, and autistic spectrum disorder as key areas. Both adult and child neurologists identified screening skills as the priority. Psychiatrists mainly valued specific training in the management of psychiatric complications of epilepsy surgery or psychiatric adverse events of antiepileptic drugs. Sessions during congresses and face-to-face meetings represent the preferred educational format, while e-learning modules and review papers were chosen by a minority of respondents. Results of this survey identify key areas for improvement in managing the psychiatric comorbidities of epilepsy and suggest specific strategies to develop better training for clinicians involved in epilepsy care

Complete genome sequences of two Citrobacter rodentium bacteriophages, CR8 and CR44b

© 2014 Toribio et al. The complete genomes of two virulent phages infecting Citrobacter rodentium are reported here for the first time. Both bacteriophages were isolated from local sewage treatment plant effluents. Genome analyses revealed a close relationship between both phages and allowed their classification as members of the Autographivirinae subfamily in the T7-like genus

Pharmacokinetics of high-dose oral thiamine hydrochloride in healthy subjects

Background: High dose oral thiamine may have a role in treating diabetes, heart failure, and hypermetabolic states. The purpose of this study was to determine the pharmacokinetic profile of oral thiamine hydrochloride at 100 mg, 500 mg and 1500 mg doses in healthy subjects. Methods: This was a randomized, double-blind, single-dose, 4-way crossover study. Pharmacokinetic measures were calculated. Results: The $AUC_{0-10 hr}$ and $C_{max}$ values increased nonlinearly between 100 mg and 1500 mg. The slope of the $AUC_{0-10 hr}$ vs dose, as well as the $C_{max}$ vs dose, plots are steepest at the lowest thiamine doses. Conclusion: Our study demonstrates that high blood levels of thiamine can be achieved rapidly with oral thiamine hydrochloride. Thiamine is absorbed by both an active and nonsaturable passive process

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses.

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABAA receptors (GABAA Rs) themselves - the essential functional postsynaptic components of GABAergic synapses - can be sufficient to initiate formation of synaptic contacts, a novel co-culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2-GABAA Rs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse-like contacts in these co-cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle-luminal domain-specific antibody; and they supported spontaneous and action potential-driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N-methyl-d-aspartate receptor-expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin-2 was co-expressed with GABAA Rs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABAA Rs can promote the adhesion of inhibitory axons and the development of functional synapses

Target and temporal pattern selection at neocortical synapses.

We attempt to summarize the properties of cortical synaptic connections and the precision with which they select their targets in the context of information processing in cortical circuits. High-frequency presynaptic bursts result in rapidly depressing responses at most inputs onto spiny cells and onto some interneurons. These 'phasic' connections detect novelty and changes in the firing rate, but report frequency of maintained activity poorly. By contrast, facilitating inputs to interneurons that target dendrites produce little or no response at low frequencies, but a facilitating-augmenting response to maintained firing. The neurons activated, the cells they in turn target and the properties of those synapses determine which parts of the circuit are recruited and in what temporal pattern. Inhibitory interneurons provide both temporal and spatial tuning. The 'forward' flow from layer-4 excitatory neurons to layer 3 and from 3 to 5 activates predominantly pyramids. 'Back' projections, from 3 to 4 and 5 to 3, do not activate excitatory cells, but target interneurons. Despite, therefore, an increasing complexity in the information integrated as it is processed through these layers, there is little 'contamination' by 'back' projections. That layer 6 acts both as a primary input layer feeding excitation 'forward' to excitatory cells in other layers and as a higher-order layer with more integrated response properties feeding inhibition to layer 4 is discussed

Distribution of interneurons in the CA2 region of the rat hippocampus.

The CA2 region of the mammalian hippocampus is a unique region with its own distinctive properties, inputs and pathologies. Disruption of inhibitory circuits in this region appears to be linked with the pathology of specific psychiatric disorders, promoting interest in its local circuitry, its role in hippocampal function and its dysfunction in disease. In previous studies, CA2 interneurons, including a novel subclass of CA2 dendrite-preferring interneurons that has not been identified in other CA regions, have been shown to display physiological, synaptic and morphological properties unique to this sub-field and may therefore play a crucial role in the hippocampal circuitry. The distributions of immuno-labeled interneurons in dorsal CA2 were studied and compared with those of interneurons in CA1 and CA3. Like those in CA1 and CA3, the somata of CA2 parvalbumin-immunoperoxidase-labeled interneurons were located primarily in Stratum Pyramidale (SP) and Stratum Oriens (SO), with very few cells in Stratum Radiatum (SR) and none in Stratum Lacunosum Moleculare (SLM). There was, however, a greater proportion of GAD-positive cells were immunopositive for PV in SP in CA2 than in CA1 or CA3. CA2 SP also contained a larger density of somatostatin-, calbindin-, and VIP-immunopositive somata than CA1 and/or CA3. Like those in CA1 and CA3, CCK-immunopositive somata in CA2 were mostly located in SR. Reelin- and NPY- immunolabeled cell bodies were located in all layers of the three CA regions. However, a higher density of Reelin-positive somata was found in SP and SR of CA2 than in CA1 or CA3