Cohort profile: the MUNICH Preterm and Term Clinical study (MUNICH-PreTCl), a neonatal birth cohort with focus on prenatal and postnatal determinants of infant and childhood morbidity.

PURPOSE: The MUNICH Preterm and Term Clinical (MUNICH-PreTCl) birth cohort was established to uncover pathological processes contributing to infant/childhood morbidity and mortality. We collected comprehensive medical information of healthy and sick newborns and their families, together with infant blood samples for proteomic analysis. MUNICH-PreTCl aims to identify mechanism-based biomarkers in infant health and disease to deliver more precise diagnostic and predictive information for disease prevention. We particularly focused on risk factors for pregnancy complications, family history of genetically influenced health conditions such as diabetes and paediatric long-term health-all to be further monitored and correlated with proteomics data in the future. PARTICIPANTS: Newborns and their parents were recruited from the Perinatal Center at the LMU University Hospital, Munich, between February 2017 and June 2019. Infants without congenital anomalies, delivered at 23-41 weeks of gestation, were eligible. FINDINGS: Findings to date concern the clinical data and extensive personal patient information. A total of 662 infants were recruited, 44% were female (36% in preterm, 46% in term). 90% of approached families agreed to participate. Neonates were grouped according to gestational age: extremely preterm (<28 weeks, N=28), very preterm (28 to <32 weeks, N=36), late preterm (32 to <37 weeks, N=97) and term infants (>37+0 weeks, N=501). We collected over 450 data points per child-parent set, (family history, demographics, pregnancy, birth and daily follow-ups throughout hospitalisation) and 841 blood samples longitudinally. The completion rates for medical examinations and blood samples were 100% and 95% for the questionnaire. FUTURE PLANS: The correlation of large clinical datasets with proteomic phenotypes, together with the use of medical registries, will enable future investigations aiming to decipher mechanisms of disorders in a systems biology perspective. TRIAL REGISTRATION NUMBER: DRKS (00024189); Pre-results.

Early postnatal switch in GABAA receptor α-subunits in the reticular thalamic nucleus.

The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.

Selectivity of direct and network-mediated stimulation of the retinal ganglion cells with epi-, sub- and intraretinal electrodes.

OBJECTIVE: Intra-retinal placement of stimulating electrodes can provide close and stable proximity to target neurons. We assessed improvement in stimulation thresholds and selectivity of the direct and network-mediated retinal stimulation with intraretinal electrodes, compared to epiretinal and subretinal placements. APPROACH: Stimulation thresholds of the retinal ganglion cells (RGCs) in wild-type rat retina were measured using the patch-clamp technique. Direct and network-mediated responses were discriminated using various synaptic blockers. MAIN RESULTS: Three types of RGC responses were identified: short latency (SL, τ < 5 ms) originating in RGCs, medium latency (ML, 3 < τ < 70 ms) originating in the inner nuclear layer and long latency (LL, τ > 40 ms) originating in photoreceptors. Cathodic epiretinal stimulation exhibited the lowest threshold for direct RGC response and the highest direct selectivity (network/direct thresholds ratio), exceeding a factor of 3 with pulse durations below 0.5 ms. For network-mediated stimulation, the lowest threshold was obtained with anodic pulses in OPL position, and its network selectivity (direct/network thresholds ratio) increased with pulse duration, exceeding a factor of 4 at 10 ms. Latency of all three types of responses decreased with increasing strength of the stimulus. SIGNIFICANCE: These results define the optimal range of pulse durations, pulse polarities and electrode placement for the retinal prostheses aiming at direct or network-mediated stimulation of RGCs.

Endogenous positive allosteric modulation of GABA(A) receptors by diazepam binding inhibitor.

Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABAARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABAARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together, these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking ("endozepine") roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a therapy for epilepsy and other neurological disorders.

Upper threshold of extracellular neural stimulation.

It is well known that spiking neurons can produce action potentials in response to extracellular stimulation above certain threshold. It is widely assumed that there is no upper limit to somatic stimulation, except for cellular or electrode damage. Here we demonstrate that there is an upper stimulation threshold, above which no action potential can be elicited, and it is below the threshold of cellular damage. Existence of this upper stimulation threshold was confirmed in retinal ganglion cells (RGCs) at pulse durations ranging from 5 to 500 µs. The ratio of the upper to lower stimulation thresholds varied typically from 1.7 to 7.6, depending on pulse duration. Computational modeling of extracellular RGC stimulation explained the upper limit by sodium current reversal on the depolarized side of the cell membrane. This was further confirmed by experiments in the medium with a low concentration of sodium. The limited width of the stimulation window may have important implications in design of the electro-neural interfaces, including neural prosthetics.

Synaptogenesis of electrical and GABAergic synapses of fast-spiking inhibitory neurons in the neocortex.

Parvalbumin-expressing fast-spiking (FS) cells are interconnected via GABAergic and electrical synapses and represent a major class of inhibitory interneurons in the neocortex. Synaptic connections among FS cells are critical for regulating network oscillations in the mature neocortex. However, it is unclear whether synaptic connections among FS interneurons also play a central role in the generation of patterned neuronal activity in the immature brain, which is thought to underlie the formation of neocortical circuits. Here, we investigated the developmental time course of synaptogenesis of FS cell in mouse visual cortex. In layer 5/6 (L5/6), we recorded from two or three FS and/or pyramidal (PYR) neurons to study the development of electrical and chemical synaptic interactions from postnatal day 3 (P3) to P18. We detected no evidence for functional connectivity for FS-FS or FS-PYR pairs at P3-P4. However, by P5-P6, we found that 20% of FS pairs were electrically coupled, and 24% of pairs were connected via GABAergic synapses; by P15-P18, 42% of FS pairs had established functional electrical synapses, and 47% of FS pairs were connected via GABAergic synapses. FS cell GABAergic inhibition of pyramidal cells showed a similar developmental time line, but no electrical coupling was detected for FS-PYR pairs. We found that synaptogenesis of electrical and GABAergic connections of FS cells takes place in the same period. Together, our results suggest that chemical and electrical connections among FS cells can contribute to patterned neocortical activity only by the end of the first postnatal week.

Giant spontaneous depolarizing potentials in the developing thalamic reticular nucleus.

The thalamic reticular nucleus (nRt) provides a major source of inhibition in the thalamo-cortical circuit and is critically involved in the generation of spindle oscillations. Here we describe the properties of thalamic giant depolarizing potentials (tGDPs) that were observed in nRt during early development. tGDPs persisted in presence of ionotropic glutamate antagonists but were completely abolished by GABA(A)R antagonist SR 35591. tGDPs occurred primarily between p3 and p8 (in 30-50% of cells) and occasionally up until p15. tGDPs lasted 0.4-3 s with peak conductances of 2-13 nS and occurred at frequencies between 0.02 and 0.06 Hz. We used mice with a benzodiazepine-insensitive alpha3 subunit [alpha3(H126R)] to probe for the identity of the GABA receptors responsible for tGDP generation. Benzodiazepine enhancement of tGDP amplitude and duration persisted in nRt neurons in alpha3(H126R) mice, indicating that the GABA(A)Rs containing alpha3 are not critical for tGDP generation and suggesting that tGDPs are mediated by GABA(A)Rs containing the alpha5 subunit, which is transiently expressed in nRt neurons in early postnatal development. Furthermore we found that exogenous GABA application depolarized nRt neurons younger than p8, indicating elevated [Cl(-)](i) at this developmental stage. Taken together, these data suggest that in immature nRt, long-lasting depolarizing responses mediated by GABA receptors could trigger Ca(2+) entry and play a role in functional development of the spindle-generating circuitry.

Intrinsic and synaptic dynamics interact to generate emergent patterns of rhythmic bursting in thalamocortical neurons.

Rhythmic inhibition entrains the firing of excitatory neurons during oscillations throughout the brain. Previous work has suggested that the strength and duration of inhibitory input determines the synchrony and period, respectively, of these oscillations. In particular, sleep spindles result from a cycle of events including rhythmic inhibition and rebound bursts in thalamocortical (TC) neurons, and slowing and strengthening this inhibitory input may transform spindles into spike-wave discharges characteristic of absence epilepsy. Here, we used dynamic clamp to inject TC neurons with spindle-like trains of IPSCs and studied how modest changes in the amplitude and/or duration of these IPSCs affected the responses of the TC neurons. Contrary to our expectations, we found that prolonging IPSCs accelerates postinhibitory rebound (PIR) in TC neurons, and that increasing either the amplitude or duration of IPSCs desynchronizes PIR activity in a population of TC cells. Tonic injection of hyperpolarizing or depolarizing current dramatically alters the timing and synchrony of PIR. These results demonstrate that rhythmic PIR activity is an emergent property of interactions between intrinsic and synaptic currents, not just a passive reflection of incoming synaptic inhibition.

Mice deficient in microtubule-associated protein MAP1B show a distinct behavioral phenotype and altered retina function.

We investigated mice deficient for the microtubule-associated protein MAP1B, a cytoskeletal element highly expressed in the developing nervous system, for altered performance in behavior, learning, and memory. Using the multiple T-maze, the open field and the Morris water maze we found that mice homozygous for a deletion of the MAP1B gene demonstrate impaired locomotor activity most likely correlated to a lack of physical endurance in general. In contrast, there were no significant differences in cognitive function and memory retention. In addition, we performed electroretinography and observed a reduction of the a-wave amplitude in response to single flash, white light stimulation. Taken together, these data provide further evidence for an important role of MAP1B in synaptic neurotransmission.