Temporal Embryonic Origin Critically Determines Cellular Physiology in the Dentate Gyrus.

The dentate gyrus, the entry gate to the hippocampus, comprises 3 types of glutamatergic cells, the granule, the mossy and the semilunar granule cells. Whereas accumulating evidence indicates that specification of subclasses of neocortical neurons starts at the time of their final mitotic divisions, when cellular diversity is specified in the Dentate Gyrus remains largely unknown. Here we show that semilunar cells, like mossy cells, originate from the earliest stages of developmental neurogenesis and that early born neurons form age-matched circuits with each other. Besides morphology, adult semilunar cells display characteristic electrophysiological features that differ from most neurons but are shared among early born granule cells. Therefore, an early birthdate specifies adult granule cell physiology and connectivity whereas additional factors may combine to produce morphological identity.

Spatial Embryonic Origin Delineates GABAergic Hub Neurons Driving Network Dynamics in the Developing Entorhinal Cortex.

Coordinated neuronal activity is essential for the development of cortical circuits. GABAergic hub neurons that function in orchestrating early neuronal activity through a widespread net of postsynaptic partners are therefore critical players in the establishment of functional networks. Evidence for hub neurons was previously found in the hippocampus, but their presence in other cortical regions remains unknown. We examined this issue in the entorhinal cortex, an initiation site for coordinated activity in the neocortex and for the activity-dependent maturation of the entire entorhinal-hippocampal network. Using an unbiased approach that identifies "driver hub neurons" displaying a high number of functional links in living slices, we show that while almost half of the GABAergic cells single-handedly influence network dynamics, only a subpopulation of cells born in the MGE and composed of somatostatin-expressing neurons located in infragranular layers, spontaneously operate as "driver" hubs. This indicates that despite differences in the origin of interneuron diversity, the hippocampus and entorhinal cortex share similar developmental mechanisms for the establishment of functional circuits.

An Optogenetic Approach for Investigation of Excitatory and Inhibitory Network GABA Actions in Mice Expressing Channelrhodopsin-2 in GABAergic Neurons.

UNLABELLED: To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo SIGNIFICANCE STATEMENT: We report a novel optogenetic approach for investigating excitatory and inhibitory GABA actions in mice with conditional expression of channelrhodopsin-2 in GABAergic interneurons. This approach shows a developmental excitatory-to-inhibitory switch in the actions of GABA on glutamatergic neurons in neocortical and hippocampal slices from neonatal mouse pups in vitro, but also reveals inhibitory GABA actions in the neonatal mouse neocortex and hippocampus in vivo.

Extrinsic control of the early postnatal CA1 hippocampal circuits.

The adult CA1 region of the hippocampus produces coordinated neuronal dynamics with minimal reliance on its extrinsic inputs. By contrast, neonatal CA1 is tightly linked to externally generated sensorimotor activity, but the circuit mechanisms underlying early synchronous activity in CA1 remain unclear. Here, using a combination of in vivo and ex vivo circuit mapping, calcium imaging, and electrophysiological recordings in mouse pups, we show that early dynamics in the ventro-intermediate CA1 are under the mixed influence of entorhinal (EC) and thalamic (VMT) inputs. Both VMT and EC can drive internally generated synchronous events ex vivo. However, movement-related population bursts detected in vivo are exclusively driven by the EC. These differential effects on synchrony reflect the different intrahippocampal targets of these inputs. Hence, cortical and subcortical pathways act differently on the neonatal CA1, implying distinct contributions to the development of the hippocampal microcircuit and related cognitive maps.

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

The rapid developmental rise of somatic inhibition disengages hippocampal dynamics from self-motion.

Early electrophysiological brain oscillations recorded in preterm babies and newborn rodents are initially mostly driven by bottom-up sensorimotor activity and only later can detach from external inputs. This is a hallmark of most developing brain areas, including the hippocampus, which, in the adult brain, functions in integrating external inputs onto internal dynamics. Such developmental disengagement from external inputs is likely a fundamental step for the proper development of cognitive internal models. Despite its importance, the developmental timeline and circuit basis for this disengagement remain unknown. To address this issue, we have investigated the daily evolution of CA1 dynamics and underlying circuits during the first two postnatal weeks of mouse development using two-photon calcium imaging in non-anesthetized pups. We show that the first postnatal week ends with an abrupt shift in the representation of self-motion in CA1. Indeed, most CA1 pyramidal cells switch from activated to inhibited by self-generated movements at the end of the first postnatal week, whereas the majority of GABAergic neurons remain positively modulated throughout this period. This rapid switch occurs within 2 days and follows the rapid anatomical and functional surge of local somatic GABAergic innervation. The observed change in dynamics is consistent with a two-population model undergoing a strengthening of inhibition. We propose that this abrupt developmental transition inaugurates the emergence of internal hippocampal dynamics.

Ozone inhalation activates stress-responsive regions of the CNS.

Ozone (O(3)), a major component of air pollution, has considerable impact on public health. Besides the well-described respiratory tract inflammation and dysfunctions, there is accumulating evidence indicating that O(3) exposure affects brain functions. However, the mechanisms through which O(3) exerts toxic effects on the brain remain poorly understood. This work aimed at precisely characterizing CNS neuronal activation after O(3) inhalation using Fos staining in adult rat. We showed that, together with lung inflammation, O(3) exposure caused a sustained time- and dose-dependent neuronal activation in the dorsolateral regions of the nucleus tractus solitarius overlapping terminal fields of lung afferents running in vagus nerves. Furthermore, we highlighted neuronal activation in interconnected central structures such as the caudal ventrolateral medulla, the parabrachial nucleus, the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular hypothalamic nucleus. In contrast, we did not detect any neuronal activation in the thoracic spinal cord where lung afferents running in spinal nerves terminate. Overall, our results demonstrate that O(3) challenge evokes a lung inflammation that induces the activation of nucleus tractus solitarius neurons through the vagus nerves and promotes neuronal activation in stress-responsive regions of the CNS.

CA1 pyramidal cell diversity is rooted in the time of neurogenesis.

Cellular diversity supports the computational capacity and flexibility of cortical circuits. Accordingly, principal neurons at the CA1 output node of the murine hippocampus are increasingly recognized as a heterogeneous population. Their genes, molecular content, intrinsic morpho-physiology, connectivity, and function seem to segregate along the main anatomical axes of the hippocampus. Since these axes reflect the temporal order of principal cell neurogenesis, we directly examined the relationship between birthdate and CA1 pyramidal neuron diversity, focusing on the ventral hippocampus. We used a genetic fate-mapping approach that allowed tagging three groups of age-matched principal neurons: pioneer, early-, and late-born. Using a combination of neuroanatomy, slice physiology, connectivity tracing, and cFos staining in mice, we show that birthdate is a strong predictor of CA1 principal cell diversity. We unravel a subpopulation of pioneer neurons recruited in familiar environments with remarkable positioning, morpho-physiological features, and connectivity. Therefore, despite the expected plasticity of hippocampal circuits, given their role in learning and memory, the diversity of their main components is also partly determined at the earliest steps of development.

Mixed GABA-glycine synapses delineate a specific topography in the nucleus tractus solitarii of adult rat.

Using combined morphological and electrophysiological approaches, we have determined the composition of inhibitory synapses of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. Immunohistochemical experiments demonstrate that, in adult rat, GABA axon terminals are present throughout the NTS while mixed GABA-glycine axon terminals are strictly located to the lateral part of the NTS within subnuclei surrounding the tractus solitarius. Purely glycine axon terminals are rare in the lateral part of the NTS and hardly detected in its medial part. Electrophysiological experiments confirm the predominance of GABA inhibition throughout the NTS and demonstrate the existence of a dual inhibition involving the co-release of GABA and glycine restricted to the lateral part of NTS. Since GABA(A) and glycine receptors are co-expressed postsynaptically in virtually all the inhibitory axon terminals throughout the NTS, it suggests that the inhibition phenotype relies on the characteristics of the axon terminals. Our results also demonstrate that glycine is mostly associated with GABA within axon terminals and raise the possibility of a dynamic regulation of GABA/glycine release at the presynaptic level. Our data provide new information for understanding the mechanisms involved in the processing of visceral information by the central nervous system in adult animals.

Perinatal development of inhibitory synapses in the nucleus tractus solitarii of the rat.

The nucleus tractus solitarii (NTS) plays a key role in the central control of the autonomic nervous system. In adult rats, both GABA and glycine are used as inhibitory neurotransmitter in the NTS. Using a quantitative morphological approach, we have investigated the perinatal development of inhibitory synapses in the NTS. The density of both inhibitory axon terminals and synapses increased from embryonic day 20 until the end of the second postnatal week (postnatal day 14). Before birth, only GABAergic axon terminals developed and their number increased during the first postnatal week. Mixed GABA/glycine axon terminals appeared at birth and their number increased during the first postnatal week. This suggests the development of a mixed GABA/glycine inhibition in parallel to pure GABA inhibition. However, whereas GABAergic axon terminals were distributed throughout the NTS, mixed GABA/glycine axon terminals were strictly located in the lateral part of the NTS. Established at birth, this specific topography remained in the adult rat. From birth, GABA(A) receptors, glycine receptors and gephyrin were clustered in inhibitory synapses throughout the NTS, revealing a neurotransmitter-receptor mismatch within the medial part of the NTS. Together these results suggest that NTS inhibitory networks develop and mature until postnatal day 14. Developmental changes in NTS synaptic inhibition may play an important role in shaping neural network activity during a time of maturation of autonomic functions. The first two postnatal weeks could represent a critical period where the impact of the environment influences the physiological phenotypes of adult rats.

Hippocampal hub neurons maintain distinct connectivity throughout their lifetime.

The temporal embryonic origins of cortical GABA neurons are critical for their specialization. In the neonatal hippocampus, GABA cells born the earliest (ebGABAs) operate as 'hubs' by orchestrating population synchrony. However, their adult fate remains largely unknown. To fill this gap, we have examined CA1 ebGABAs using a combination of electrophysiology, neurochemical analysis, optogenetic connectivity mapping as well as ex vivo and in vivo calcium imaging. We show that CA1 ebGABAs not only operate as hubs during development, but also maintain distinct morpho-physiological and connectivity profiles, including a bias for long-range targets and local excitatory inputs. In vivo, ebGABAs are activated during locomotion, correlate with CA1 cell assemblies and display high functional connectivity. Hence, ebGABAs are specified from birth to ensure unique functions throughout their lifetime. In the adult brain, this may take the form of a long-range hub role through the coordination of cell assemblies across distant regions.

Assemblies of Perisomatic GABAergic Neurons in the Developing Barrel Cortex.

The developmental journey of cortical interneurons encounters several activity-dependent milestones. During the early postnatal period in developing mice, GABAergic neurons are transient preferential recipients of thalamic inputs and undergo activity-dependent migration arrest, wiring, and programmed cell-death. Despite their importance for the emergence of sensory experience and the role of activity in their integration into cortical networks, the collective dynamics of GABAergic neurons during that neonatal period remain unknown. Here, we study coordinated activity in GABAergic cells of the mouse barrel cortex using in vivo calcium imaging. We uncover a transient structure in GABAergic population dynamics that disappears in a sensory-dependent process. Its building blocks are anatomically clustered GABAergic assemblies mostly composed by prospective parvalbumin-expressing cells. These progressively widen their territories until forming a uniform perisomatic GABAergic network. Such transient patterning of GABAergic activity is a functional scaffold that links the cortex to the external world prior to active exploration. VIDEO ABSTRACT.

Distribution of zinedin in the rat brain.

Members of the striatin family are scaffolding proteins involved in numerous signaling pathways principally in neurons. Zinedin is the only member of this protein family for which the brain distribution has not been determined so far. Here, we have validated a specific antibody against zinedin and used this tool to study the localization of zinedin at cellular and sub-cellular levels in the rat brain. Zinedin is primarily expressed in neurons of the hippocampus, cerebral cortex, olfactory bulb and caudate putamen nucleus. Like other members of the striatin family, zinedin displays a polarized distribution in the somato-dendritic compartment of neurons and is enriched in dendritic spines. The rostral expression of zinedin as well as its compartmented distribution in dendritic spines may have important implications not only for zinedin function but also in the physiology of dendritic spines of a particular subset of neurons.

Spike timing of dendrite-targeting bistratified cells during hippocampal network oscillations in vivo.

Behavior-contingent network oscillations bring about transient, functionally coherent neuronal assemblies in the cerebral cortex, including the hippocampus. Inhibitory input on and close to the soma is believed to phase intrinsic oscillations and output of pyramidal cells, but the function of GABA release to pyramidal cell dendrites remains unknown. We recorded the oscillation-locked spike timing of identified bistratified interneurons in rats. These cells mainly innervated small dendritic shafts of pyramidal cells co-aligned with the glutamatergic Schaffer collateral/commissural input. During theta oscillations, bistratified cells fired at a phase when, on average, pyramidal cell dendrites are most hyperpolarized. Interneurons targeting the perisomatic domain discharge at an earlier phase. During sharp wave-associated ripples, bistratified cells fired with high frequency and in-phase with basket cells, on average 1-2 ms after the discharges in pyramidal cell somata and dendrites. Our results indicate that bistratified cells rhythmically modulate glutamatergic input to the dendrites of pyramidal cells to actively promote the precise input/output transformation during network oscillations.

Mouse subthalamic nucleus neurons with local axon collaterals.

The neuronal population of the subthalamic nucleus (STN) has the ability to prolong incoming cortical excitation. This could result from intra-STN feedback excitation. The combination of inducible genetic fate mapping techniques with in vitro targeted patch-clamp recordings, allowed identifying a new type of STN neurons that possess a highly collateralized intrinsic axon. The time window of birth dates was found to be narrow (E10.5-E14.5) with very few STN neurons born at E10.5 or E14.5. The fate mapped E11.5-12.5 STN neuronal population included 20% of neurons with profuse axonal branching inside the nucleus and a dendritic arbor that differed from that of STN neurons without local axon collaterals. They had intrinsic electrophysiological properties and in particular, the ability to generate plateau potentials, similar to that of STN neurons without local axon collaterals and more generally to that of classically described STN neurons. This suggests that a subpopulation of STN neurons forms a local glutamatergic network, which together with plateau potentials, allow amplification of hyperdirect cortical inputs and synchronization of the STN neuronal population.

Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus.

Presynaptic metabotropic glutamate receptors (mGluRs) show a highly selective expression and subcellular location in nerve terminals modulating neurotransmitter release. We have demonstrated that alternatively spliced variants of mGluR8, mGluR8a and mGluR8b, have an overlapping distribution in the hippocampus, and besides perforant path terminals, they are expressed in the presynaptic active zone of boutons making synapses selectively with several types of GABAergic interneurons, primarily in the stratum oriens. Boutons labeled for mGluR8 formed either type I or type II synapses, and the latter were GABAergic. Some mGluR8-positive boutons also expressed mGluR7 or vasoactive intestinal polypeptide. Interneurons strongly immunopositive for the muscarinic M2 or the mGlu1 receptors were the primary targets of mGluR8-containing terminals in the stratum oriens, but only neurochemically distinct subsets were innervated by mGluR8-enriched terminals. The majority of M2-positive neurons were mGluR8 innervated, but a minority, which expresses somatostatin, was not. Rare neurons coexpressing calretinin and M2 were consistently targeted by mGluR8-positive boutons. In vivo recording and labeling of an mGluR8-decorated and strongly M2-positive interneuron revealed a trilaminar cell with complex spike bursts during theta oscillations and strong discharge during sharp wave/ripple events. The trilaminar cell had a large projection from the CA1 area to the subiculum and a preferential innervation of interneurons in the CA1 area in addition to pyramidal cell somata and dendrites. The postsynaptic interneuron type-specific expression of the high-efficacy presynaptic mGluR8 in both putative glutamatergic and in identified GABAergic terminals predicts a role in adjusting the activity of interneurons depending on the level of network activity.

Development of early-born γ-Aminobutyric acid hub neurons in mouse hippocampus from embryogenesis to adulthood.

Early-born γ-aminobutyric acid (GABA) neurons (EBGNs) are major components of the hippocampal circuit because at early postnatal stages they form a subpopulation of "hub cells" transiently supporting CA3 network synchronization (Picardo et al. [2011] Neuron 71:695-709). It is therefore essential to determine when these cells acquire the remarkable morphofunctional attributes supporting their network function and whether they develop into a specific subtype of interneuron into adulthood. Inducible genetic fate mapping conveniently allows for the labeling of EBGNs throughout their life. EBGNs were first analyzed during the perinatal week. We observed that EBGNs acquired mature characteristics at the time when the first synapse-driven synchronous activities appeared in the form of giant depolarizing potentials. The fate of EBGNs was next analyzed in the adult hippocampus by using anatomical characterization. Adult EBGNs included a significant proportion of cells projecting selectively to the septum; in turn, EBGNs were targeted by septal and entorhinal inputs. In addition, most EBGNs were strongly targeted by cholinergic and monoaminergic terminals, suggesting significant subcortical innervation. Finally, we found that some EBGNs located in the septum or the entorhinal cortex also displayed a long-range projection that we traced to the hippocampus. Therefore, this study shows that the maturation of the morphophysiological properties of EBGNs mirrors the evolution of early network dynamics, suggesting that both phenomena may be causally linked. We propose that a subpopulation of EBGNs forms into adulthood a scaffold of GABAergic projection neurons linking the hippocampus to distant structures. J. Comp. Neurol. 524:2440-2461, 2016. © 2016 Wiley Periodicals, Inc.

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.

Pioneer glutamatergic cells develop into a morpho-functionally distinct population in the juvenile CA3 hippocampus.

The developing CA3 hippocampus is comprised by highly connected hub neurons that are particularly effective in achieving network synchronization. Functional hub neurons were shown to be exclusively GABAergic, suggesting that the contribution of glutamatergic neurons to physiological synchronization processes at early postnatal stages is minimal. However, without fast GABAergic transmission, a different situation may prevail. In the adult CA3, blocking fast GABAergic transmission induces the generation of network bursts that can be triggered by the stimulation of single pyramidal neurons. Here we revisit the network function of CA3 glutamatergic neurons from a developmental viewpoint, without fast GABAergic transmission. We uncover a sub-population of early-generated glutamatergic neurons that impacts network dynamics when stimulated in the juvenile hippocampus. Additionally, this population displays characteristic morpho-physiological features in the juvenile and adult hippocampus. Therefore, the apparently homogeneous glutamatergic cell population likely displays a morpho-functional diversity rooted in temporal embryonic origins.

Pioneer GABA cells comprise a subpopulation of hub neurons in the developing hippocampus.

Connectivity in the developing hippocampus displays a functional organization particularly effective in supporting network synchronization, as it includes superconnected hub neurons. We have previously shown that hub network function is supported by a subpopulation of GABA neurons. However, it is unclear whether hub cells are only transiently present or later develop into distinctive subclasses of interneurons. These questions are difficult to assess given the heterogeneity of the GABA neurons and the poor early expression of markers. To circumvent this conundrum, we used "genetic fate mapping" that allows for the selective labeling of GABA neurons based on their place and time of origin. We show that early-generated GABA cells form a subpopulation of hub neurons, characterized by an exceptionally widespread axonal arborization and the ability to single-handedly impact network dynamics when stimulated. Pioneer hub neurons remain into adulthood, when they acquire the classical markers of long-range projecting GABA neurons.