Pierre Meyrand

NKCC1 cotransporter inactivation underlies embryonic development of chloride-mediated inhibition in mouse spinal motoneuron

Early in development, GABA and glycine exert excitatory action that turns to inhibition due to modification of the chloride equilibrium potential (ECl) controlled by the KCC2 and NKCC1 transporters. This switch is thought to be due to a late expression of KCC2 associated with a NKCC1 down‐regulation. Here, we show in mouse embryonic spinal cord that both KCC2 and NKCC1 are expressed and functional early in development (E11.5–E13.5) when GABAA receptor activation induces strong excitatory action. After E15.5, a switch occurs rendering GABA unable to provide excitation. At these subsequent stages, NKCC1 becomes both inactive and less abundant in motoneurons while KCC2 remains functional and hyperpolarizes ECl. In conclusion, in contrast to other systems, the cotransporters are concomitantly expressed early in the development of the mouse spinal cord. Moreover, whereas NKCC1 follows a classical functional extinction, KCC2 is highly expressed throughout both early and late embryonic life.

Descending 5-Hydroxytryptamine Raphe Inputs Repress the Expression of Serotonergic Neurons and Slow the Maturation of Inhibitory Systems in Mouse Embryonic Spinal Cord

Spontaneous synchronous rhythmic activities are a common feature of immature neuronal networks. Although the mechanisms underlying such activities have been studied extensively, whether they might be controlled by modulatory information remains questionable. Here, we investigated the role of descending serotonergic (5-HT) inputs from the medulla to the spinal cord in the maturation of rhythmic activity. We found that in spinal cords maintained, as a whole, in organotypic culture without the medulla, the maturation of spontaneous activity is similar to that found in spinal cords developed in utero. Interestingly, in organotypic cultures without the medulla (i.e., devoid of descending inputs), numerous intraspinal neurons expressed 5-HT, unlike in spinal cords cultivated in the presence of the medulla or matured in utero. We demonstrated that this 5-HT expression was specifically dependent on the absence of 5-HT fibers and was repressed by 5-HT itself via activation of 5-HT1A receptors. Finally, to verify whether the expression of 5-HT intraspinal neurons could compensate for the lack of descending 5-HT fibers and play a role in the development of spontaneous activity, we blocked the 5-HT synthesis usingp-chlorophenylalanine methyl ester in cultures devoid of the medulla. Surprisingly, we found that this pharmacological treatment did not prevent the development of spontaneous activity but accelerated the maturation of intraspinal inhibition at the studied stages. Together, our data indicate that descending 5-HT raphe inputs (1) repress the expression of spinal serotonergic neurons and (2) slow the maturation of inhibitory systems in mouse spinal cord.

Sequential developmental acquisition of cotransmitters in identified sensory neurons of the stomatogastric nervous system of the lobsters, Homarus americanus and Homarus gammarus.

We studied the developmental acquisition of three of the cotransmitters found in the gastropyloric receptor (GPR) neurons of the stomatogastric nervous systems of the lobsters Homarus americanus and Homarus gammarus. By using wholemount immunocytochemistry and confocal microscopy, we examined the distribution of serotonin‐like, allatostatin‐like, and FLRFNH2‐like immunoreactivities within the stomatogastric nervous system of embryonic, larval, juvenile, and adult animals. The GPR neurons are peripheral sensory neurons that send proprioceptive information to the stomatogastric and commissural ganglia. In H. americanus, GPR neurons of the adult contain serotonin‐like, allatostatin‐like, and Phe‐Leu‐Arg‐Phe‐amide (FLRFNH2)‐like immunoreactivities. In the stomatogastric ganglion (STG) of the adult H. americanus and H. gammarus, all of the serotonin‐like and allatostatin‐like immunoreactivity colocalizes in neuropil processes that are derived exclusively from ramifications of the GPR neurons. In both species, FLRFNH2‐like immunoreactivity was detected in the STG neuropil by 50% of embryonic development (E50). Allatostatin‐like immunoreactivity was visible first in the STG at approximately E70–E80. In contrast, serotonin staining was not clearly visible until larval stage I (LI) in H. gammarus and until LII or LIII in H. americanus. These data indicate that there is a sequential acquisition of the cotransmitters of the GPR neurons. J. Comp. Neurol. 408:318–334, 1999.

SEQUENTIAL DEVELOPMENTAL ACQUISITION OF NEUROMODULATORY INPUTS TO A CENTRAL PATTERN-GENERATING NETWORK

The activity of the adult stomatogastric ganglion (STG) depends on a large number of aminergic and peptidergic modulatory inputs. Our aim is to understand the role of these modulatory inputs in the development of the central pattern‐generating networks of the STG. Therefore, we analyze the developmental and adult expressions of three neuropeptides in the stomatogastric nervous system of the lobsters Homarus americanus and Homarus gammarus by using wholemount immunocytochemistry and confocal microscopy. In adults, red pigment‐concentrating hormone (RPCH)‐like, proctolin‐like, and a tachykinin‐like immunoreactivity are present in axonal projections to the STG. At 50% of embryonic development (E50), all three peptides stain the commissural ganglia and brain, but only RPCH‐ and proctolin‐like immunoreactivities stain axonal arbors in the STG. Tachykinin‐like immunoreactivity is not apparent in the STG until larval stage II (LII). The RPCH‐immunoreactive projection to the STG consists of two pairs of fibers. One pair stains for RPCH immunoreactivity at E50; the second RPCH‐immunoreactive pair does not stain until about LII. One pair of the RPCH fibers double labels for tachykinin‐like immunoreactivity. The adult complement of neuromodulatory inputs is not fully expressed until close to the developmental time at which major changes in the STG motor patterns occur, suggesting that neuromodulators play a role in the tuning of the central pattern generators during development. J. Comp. Neurol. 408:335–351, 1999.

Glycine Release from Radial Cells Modulates the Spontaneous Activity and Its Propagation during Early Spinal Cord Development

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.

Central inputs mask multiple adult neural networks within a single embryonic network

It is usually assumed that, after construction of basic network architecture in embryos, immature networks undergo progressive maturation to acquire their adult properties. We examine this assumption in the context of the lobster stomatogastric nervous system. In the lobster, the neuronal population that will form this system is at first orgnanized into a single embryonic network that generates a single rhythmic pattern. The system then splits into different functional adult networks controlled by central descending systems; these adult networks produce multiple motor programmes, distinctively different from the single output of the embryonic network. We show here that the single embryonic network can produce multiple adult-like programmes. This occurs after the embryonic network is silenced by removal of central inputs, then pharmacologically stimulated to restore rhythmicity. Furthermore, restoration of the flow of descending information reversed the adult-like pattern to an embryonic pattern. This indicates that the embryonic network possesses the ability to express adult-like network characteristics, but descending information prevents it from doing so. Functional adult networks may therefore not necessarily be derived from progressive ontogenetic changes in networks themselves, but may result from maturation of descending systems that unmask pre-existing adult networks in an embryonic system.

Ontogenetic alteration in peptidergic expression within a stable neuronal population in lobster stomatogastric nervous system

In the adult lobster, Homarus gammarus, the stomatogastric ganglion (STG) contains two well‐defined motor pattern generating networks that receive numerous modulatory peptidergic inputs from anterior ganglia. We are studying the appearance of extrinsic peptidergic inputs to these networks during ontogenesis. Neuron counts indicate that as early as 20% of development (E20) the STG neuronal population is quantitatively established. By using immunocytochemical detection of 5‐bromo‐2′‐deoxyuridine incorporation, we found no immunopositive cells in the STG by E70. We concluded that the STG neuronal population remains quantitatively stable from mid‐embryonic life until adulthood.

BioMEA™: A versatile high-density 3D microelectrode array system using integrated electronics

Microelectrode arrays (MEAs) offer a powerful tool to both record activity and deliver electrical microstimulations to neural networks either in vitro or in vivo. Microelectronics microfabrication technologies now allow building high-density MEAs containing several hundreds of microelectrodes. However, dense arrays of 3D micro-needle electrodes, providing closer contact with the neural tissue than planar electrodes, are not achievable using conventional isotropic etching processes. Moreover, increasing the number of electrodes using conventional electronics is difficult to achieve into compact devices addressing all channels independently for simultaneous recording and stimulation. Here, we present a full modular and versatile 256-channel MEA system based on integrated electronics. First, transparent high-density arrays of 3D-shaped microelectrodes were realized by deep reactive ion etching techniques of a silicon substrate reported on glass. This approach allowed achieving high electrode aspect ratios, and different shapes of tip electrodes. Next, we developed a dedicated analog 64-channel Application Specific Integrated Circuit (ASIC) including one amplification stage and one current generator per channel, and analog output multiplexing. A full modular system, called BIOMEA, has been designed, allowing connecting different types of MEAs (64, 128, or 256 electrodes) to different numbers of ASICs for simultaneous recording and/or stimulation on all channels. Finally, this system has been validated experimentally by recording and electrically eliciting low-amplitude spontaneous rhythmic activity (both LFPs and spikes) in the developing mouse CNS. The availability of high-density MEA systems with integrated electronics will offer new possibilities for both in vitro and in vivo studies of large neural networks.

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.

Myogenic oscillatory activity in the pyloric rhythmic motor system of Crustacea

Summary1.The dorsal dilator muscle of the pylorus of the shrimpPalaemon (Fig. 1) is innervated, as in lobsters, by two electrically coupled excitatory motorneurons, the pyloric dilator (PD) neurons located in the stomatogastric ganglion (Fig. 2). The PD motorneurons are conditional oscillators and, when bursting, they rhythmically drive the pyloric dilator muscle (Fig. 3).2.When isolated the pyloric dilator muscle can undergo spontaneous rhythmic contractions with associated electrical membrane events. This spontaneous rhythmic activity is not of neural origin since: (1) it cannot be correlated with any injury discharge in the cut motor nerve; (2) it remains unaffected by bath application of tetrodotoxin to suppress neuronal spiking (Fig. 4).3.Electrical stimulation of the motor nerve or membrane depolarization with injected current shows that an otherwise quiescent pyloric dilator muscle can express either a non-oscillatory state (Fig. 5A, C, E) or an oscillatory state (Fig. 5 B, D, F). It is always possible to switch from the non-oscillatory. state to the oscillatory state by bath application of dopamine (Fig. 6); it is concluded that the muscle is a conditional oscillator.4.In its oscillatory state the pyloric dilator muscle displays properties characteristic of endogenous oscillators. These include: (1) phasic response to tonic stimulation; (2) voltage-dependence of the cycle frequency of the rhythmic activity (Fig. 7); (3) ability of cycling to be reset by a brief stimulus (Fig. 8); (4) ability to be entrained by repetitive stimuli (Fig. 9).5.The pyloric dilator neuromuscular system ofPalaemon thus appears to consist of a conditional motorneuronal oscillator (PD) and a conditional muscle oscillator; the second can be entrained by the first (Fig. 10). The functional implications of such a neuromuscular control system are tested (Fig. 11) and discussed.