Subtype- and Layer-Specific Developmental Gradients of Postnatal GABAergic Neurons in the Rodent Barrel Cortex

Developmental Mechanisms Underlying the Generation of Cortical Interneuron Diversity

1. The functional properties of low-voltage-activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole-cell patch-clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12). 2. It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I-II) and deep (V-VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III-IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 microM) and were insensitive to Ni2+ (25 microM). 3. The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high-voltage-activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F- applied through the patch pipette were observed. 4. The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin-Huxley equation with an inactivation time constant of 24 +/- 6 ms. The steady-state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half-inactivation, -86.9 mV; steepness coefficient,3.4 mV. 5. It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another ('slow') type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cells is different.

The pore-forming alpha subunits of voltage-gated potassium channels in neurons and other excitable cells are expressed in association with accessory beta subunits. These subunits both promote insertion of channel complexes into surface membranes and influence their electrophysiological properties. As part of an effort to understand the regulation of voltage-gated potassium channels during development, we cloned the mouse homolog of the rat Kvbeta1 potassium channel subunit. Kvbeta1 subunits are known to associate preferentially with Shaker (Kv1)-related alpha subunits. We then used a digoxigenin-tagged cRNA probe and in situ hybridization techniques to visualize the appearance of Kvbeta1 mRNA transcripts during late embryonic and early neonatal development of the mouse brain. We detected Kvbeta1-specific labeling of cells in hippocampus, cerebral cortex, caudate putamen, colliculus, and cerebellum. In hippocampus, we observed Kvbeta1 mRNA in CA3 pyramidal neurons at the earliest time examined, embryonic day 16 (E16). Between E16 and postnatal day 7 (P7), cell labeling increased uniformly across the pyramidal neurons of Ammon's horn (CA1, CA2, and CA3). Subsequently, between P7 and P22, regional differences characteristic of mature hippocampus appeared-intense labeling of neurons in CA3 and CA1, and less in CA2. In cortex, labeling of cells in the subplate and cortical plate layers was observed at E16. During development, the intensity of this labeling increased, and labeled cells persisted into the adult stage in the deep cortical layer (VIb) formed from subplate neurons. Additional labeling of scattered solitary cells in cortical layers II-VIa emerged between P3 and P7 and was prominent in mature cortex. In caudate putamen, Kvbeta1-labeled cells were observed at P1 and were restricted to the lateral and rostral half of the caudate. During development, labeling expanded caudally and medially and eventually filled the mature caudate putamen. In colliculus, a small population of inferior colliculus cells showed labeling at P7, and additional labeling of scattered cells appeared during development. In superior colliculus, labeling was observed only in the adult deep gray layer. In cerebellum, intense labeling was observed in Purkinje cells at all stages between P1 and adult. Labeling was also seen in granule neurons in the external granule layer at early postnatal stages and in the inner granule layer beginning at P7.

It has been proposed that assembly of the final NMDA receptor complex may be modified by prenatal ethanol exposure, resulting in long-term alterations of NMDA receptor pharmacology. We investigated the effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus. Female Sprague-Dawley rats were chronically intoxicated for 4 weeks with a 10% (v/v) ethanol solution administered throughout pregnancy and lactation. Hippocampus and cerebellum were isolated from pups (postnatal days 1-28) of the ethanol-exposed and ad libitum groups. Our results, using a semiquantitative RT-PCR technique, showed a selective effect of ethanol exposure on the various NMDA receptor subunits. Ethanol exposure significantly increased the levels of NR1(1XX), NR1(X11) and NR2(D) mRNAs on postnatal days 7 and 14 and decreased the level of NR2(C) on postnatal day 1. Immunoblot analyses demonstrated that NR2(D) protein levels were increased on postnatal day 7 after ethanol exposure. However, the developmental profile of mRNAs encoding for NR2(A-B), NR3(L/S), GBP and Gly/TCP-BP subunits were not affected. Moreover, no significant effects of ethanol exposure were observed on the developmental transition from expression of NR1(0XX) to NR(1XX) splice variants occurring in the cerebellum on postnatal day 19. Unexpectedly, [(3) H]MK-801 binding experiments showed that ethanol exposure increased the B (max) values of high-affinity sites on postnatal days 14 and 28, with no change of K (d) values. These findings indicate that prenatal and/or postnatal ethanol exposure alters the hippocampal levels of mRNAs encoding for certain subunits and the density of high-affinity [(3) H]MK-801 binding sites. As these subunits have been shown to modulate the functional properties of NMDA receptors, these results suggest that this altered expression could be involved in the neurodevelopmental disorders associated with fetal ethanol exposure.

GABAergic Circuit Development and Its Implication for CNS Disorders

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Prenatal exposure to ethanol causes a delay in the developmental expression of neurofilament epitopes in cerebellum

Decision letter: Interacting rhythms enhance sensitivity of target detection in a fronto-parietal computational model of visual attention

Editor's evaluation: Interacting rhythms enhance sensitivity of target detection in a fronto-parietal computational model of visual attention

Subspecies of protein kinase C in the rat spinal cord

Adenosine A2A receptor modulates microglia-mediated synaptic pruning of the retinogeniculate pathway during postnatal development

Optogenetic and pharmacogenetic techniques have been effective to analyze the role of neuronal activity in controlling oligodendroglia lineage cells in behaving juvenile and adult mice. This kind of studies is also of high interest during early postnatal (PN) development since important changes in oligodendroglia dynamics occur during the first two PN weeks. Yet, neuronal manipulation is difficult to implement at an early age because high-level, specific protein expression is less reliable in neonatal mice. Here, we describe a protocol allowing for an optogenetic stimulation of neurons in awake mouse pups with the purpose of investigating the effect of neuronal activity on oligodendroglia dynamics during early PN stages. Since GABAergic interneurons contact oligodendrocyte precursor cells (OPCs) through bona fide synapses and maintain a close relationship with these progenitors during cortical development, we used this relevant example of neuron-oligodendroglia interaction to implement a proof-of-principle optogenetic approach. First, we tested Nkx2.1-Cre and Parvalbumin (PV)-Cre lines to drive the expression of the photosensitive ion channel channelrhodopsin-2 (ChR2) in subpopulations of interneurons at different developmental stages. By using patch-clamp recordings and photostimulation of ChR2-positive interneurons in acute somatosensory cortical slices, we analyzed the level of functional expression of ChR2 in these neurons. We found that ChR2 expression was insufficient in PV-Cre mouse at PN day 10 (PN10) and that this channel needs to be expressed from embryonic stages (as in the Nkx2.1-Cre line) to allow for a reliable photoactivation in mouse pups. Then, we implemented a stereotaxic surgery to place a mini-optic fiber at the cortical surface in order to photostimulate ChR2-positive interneurons at PN10. In vivo field potentials were recorded in Layer V to verify that photostimulation reaches deep cortical layers. Finally, we analyzed the effect of the photostimulation on the layer V oligodendroglia population by conventional immunostainings. Neither the total density nor a proliferative fraction of OPCs were affected by increasing interneuron activity in vivo, complementing previous findings showing the lack of effect of GABAergic synaptic activity on OPC proliferation. The methodology described here should provide a framework for future investigation of the role of early cellular interactions during PN brain maturation.

Ca 2+-permeable AMPA receptors mediate induction of test pulse depression of naive synapses in rat visual cortical slices at early postnatal stage

Experimental data on developmental elimination of retrosplenial cortex GABAergic interneurons in a mouse model of ethanol exposure during the last trimester of human pregnancy

Kv4.3, an A-type K+ channel, is the only channel molecule showing anterior-posterior (A-P) compartmentalization in the granular layer of mammalian cerebellum known so far. Kv4.3 mRNA has been detected from the posterior but not anterior granular layer in adult rat cerebellum. To characterize this A-P compartmentalization further, we examined Kv4.3 protein expression in rat cerebellum by immunohistochemistry at the embryonic, early postnatal and adult stages. Specificity of the Kv4.3 antibody was confirmed by both Western blot and immunoprecipitation analysis. In adulthood, Kv4.3 was detected from the somatodendritic domain of posterior granule cells, with a restriction boundary in the vermal lobule VI extending laterally to the hemispheric crus 1 ansiform lobules. At the early postnatal stage, this A-P pattern first appeared on postnatal day 8, when significant numbers of granule cells had migrated into the posterior granular layer and started to express Kv4.3. Similar Kv4.3 expression in the somatodendritic domain of post-migratory neurons in the cerebellum was also observed in basket cells, stellate cells, a subset of GABAergic deep neurons, Lugaro cells and, probably, deep Lugaro cells. However, none of them showed A-P compartmentalization. Strikingly, we found Kv4.3 in several clusters of migrating Purkinje cells with mediolateral compartmentalization. These Purkinje cells no longer expressed Kv4.3 after completing the migration. By contrasting the expression in migrating and post-migratory neurons, our results suggest that Kv4.3 may play an important role in the development of cerebellum, as well as in the mature cerebellum.