Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation.
ABSTRACT Glutamatergic excitatory neurotransmission is dependent on glutamate release from presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Here, we show that VGLUT2 (Slc17a6) is required for life ex utero. Vglut2 null mutant mice die immediately after birth because of the absence of respiratory behavior. Investigations at embryonic stages revealed that neural circuits in the location of the pre-Bötzinger (PBC) inspiratory rhythm generator failed to become active. However, neurons with bursting pacemaker properties and anatomical integrity of the PBC area were preserved. Vesicles at asymmetric synapses were fewer and malformed in the Vglut2 null mutant hindbrain, probably causing the complete disruption of AMPA/kainate receptor-mediated synaptic activity in mutant PBC cells. The functional deficit results from an inability of PBC neurons to achieve synchronous activation. In contrast to respiratory rhythm generation, the locomotor central pattern generator of Vglut2 null mutant mice displayed normal rhythmic and coordinated activity, suggesting differences in their operating principles. Hence, the present study identifies VGLUT2-mediated signaling as an obligatory component of the developing respiratory rhythm generator.
- SourceAvailable from: Julien Bouvier[Show abstract] [Hide abstract]
ABSTRACT: La respiration est une activité rythmique motrice bilatéralement synchronisée présente chez le fœtus et vitale à la naissance. Au cours du développement, des interneurones dans le tronc cérébral s'organisent en réseaux qui génèrent et modulent la commande rythmique respiratoire. Ils y forment deux oscillateurs, actifs chez l'embryon et vitaux à la naissance : l'oscillateur parafacial et le complexe preBötzinger. Les mécanismes qui gouvernent l'assemblage et contraignent la fonction de ces réseaux sont étudiés dans le laboratoire avec des outils génétiques, des techniques histologiques, électrophysiologiques et d'imagerie chez la souris. Etudiant le développement du complexe preBötzinger, nous montrons que des progéniteurs neuraux exprimant le gène à homéobox Dbx1 donnent naissance aux interneurones qui constituent le cœur de l'oscillateur. Ces interneurones (i) sont glutamatergiques et nécessaires à la genèse du rythme, (ii) expriment le gène Robo3 requis pour que leurs axones croisent la ligne médiane et conditionnent la synchronisation de l'activité. Cette étude illustre et affine les liens existant entre les schémas de régionalisation du tube neural et l'émergence de modules fonctionnels en permettant de proposer une signature transcriptionnelle des neurones rythmogènes principaux de la respiration.Mémoire de Thèse. 06/2013;
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ABSTRACT: Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.Neuron 03/2011; 69(6):1147-59. · 15.77 Impact Factor
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ABSTRACT: The medial prefrontal cortex (mPFC) and the dorsal hippocampus (DHC) play significant roles in stimulant-induced neurobehavioral effects. Methamphetamine (MAP)-induced hyperactivity has been reported to be involved in the regulation of the glutamatergic system. The present study examined whether the glutamatergic and GABAergic systems in the mPFC and DHC were involved in MAP-induced hyperactivity in mice. A combined kainic acid (KA) or N-methyl-d-aspartate (NMDA) lesion and microdialysis technique targeting both the mPFC and DHC were used. The results showed that both KA- and NMDA-induced lesions of the mPFC facilitated MAP-induced hyperactivity, while neither KA- nor NMDA-induced lesions of the DHC had a similar effect. MAP increased the extracellular glutamate (Glu) levels in the mPFC and reduced Glu levels in the DHC. GABA levels in both of these regions were reduced. A KA or NMDA lesion of the mPFC inhibited the Glu reduction in the DHC, and the same lesion of the DHC inhibited the Glu increase in the mPFC induced by MAP. A NMDA lesion of the mPFC blocked GABA reduction in the DHC, but a lesion of DHC enhanced the GABA decrease in the mPFC induced by MAP. Furthermore, a NMDA lesion of DHC increased the vesicular glutamate transporter-2 (VGLUT2) expression in the mPFC following MAP-administration. These findings indicate that glutamatergic as well as GABAergic systems in these two regions are involved in MAP-induced hyperactivity. Moreover, there may be an inhibitory role in these two regions, especially mediated by NMDA receptors, in MAP-induced abnormal behavior and neurotransmission responses.Behavioural brain research 06/2012; 232(1):44-52. · 3.22 Impact Factor
83:2468-2470, 2000. ;
Jean-René Cazalets, Marie Gardette and Gérard Hilaire
in the MAOA-Deficient Mouse
Locomotor Network Maturation Is Transiently Delayed
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Locomotor Network Maturation Is Transiently Delayed in the
JEAN-RENE´ CAZALETS, MARIE GARDETTE, AND GE´RARD HILAIRE
Centre National de la Recherche Scientifique, Laboratoire de Neurobiologie et Mouvements, 13402 Marseille Cedex 9,
Cazalets, Jean-Rene ´, Marie Gardette, and Ge ´rard Hilaire. Loco-
motor network maturation is transiently delayed in the MAOA-defi-
cient mouse. J. Neurophysiol. 83: 2468–2470, 2000. In vivo and in
vitro experiments were performed in control (C3H) and monoamine
oxidase A (MAOA)-deficient (Tg8) neonatal mice to determine
whether MAOA deficiency affected spinal locomotor network matu-
ration. Comparing the swimming behaviors at birth in C3H mice with
those in Tg8 mice revealed a delayed role for the hindlimbs in Tg8
swimming, even though adult swimming behavior was acquired at
postnatal day 14 (P14) in both strains. Analyzing the locomotor
network activity in vitro showed that serotonin (5-HT) induced and
modulated locomotor-like discharges in hindlimb ventral roots of
C3H but not Tg8 neonates. The Tg8 network began, however, to be
affected by 5-HT at P11. Thus both in vivo and in vitro results argue
for a transient delay of locomotor network maturation in the Tg8
I N T R O D U C T I O N
Serotonin (5-HT) is a widely distributed neuromodulator
that has a role in numerous functions in adulthood (Vogt 1982)
and that modulates rhythmic activities of the locomotor (Caza-
lets et al. 1992; Sqalli-Houssaini et al. 1993) and the respira-
tory (Hilaire and Duron 1999) networks at birth. Moreover, as
suggested by its early expression and its role in the control of
cell division, differentiation, growth, and synaptogenesis
(Lauder 1993; Lauder and Krebs 1978; Levitt et al. 1997;
Whitaker-Azmitia 1991), 5-HT may affect CNS maturation
(Cases et al. 1996; Chubakov et al. 1986; Mooney et al. 1998;
Yan et al. 1997a,b). Therefore, besides its role in modulating
the locomotor network, 5-HT might also play a role in its
maturation, as suggested by the transient difficulties in swim-
ming abilities observed at birth in rats prenatally treated with
para-chlorophenylalanine (Nakajima et al. 1998).
In the MAOA-deficient (Tg8) neonatal mouse, which was
created from the C3H/HeJ (C3H) strain by disrupting the gene
encoding monoamine oxidase A (MAOA), the enzyme that
degrades 5-HT, the lack of MAOA activity results in 5-HT
levels 10 times larger than those in C3H neonates (Cases et al.
1995; Lajard et al. 1999). To determine whether MAOA defi-
ciency affects locomotor network maturation, we used in vivo
and in vitro approaches to compare the locomotor activity
produced by Tg8 neonates with that produced by C3H neo-
nates. Both results suggest a transient delay in the maturation
of the Tg8 locomotor network, which provides increasing
evidence for a possible role of 5-HT in mammalian CNS
M E T H O D S
In the in vivo experiments, the swimming behavior of 35 C3H and
24 Tg8 pups was observed daily from postnatal day 0 to postnatal day
7 (P0–P7). Each pup was gently immersed in a warmth-regulated
water bath (37 ? 1°C) for a 30 s trial during which its motor activity
was observed and classified in one of three swimming patterns, H0,
H1, or H2, depending on whether it used 0, 1, or 2 hindlimbs for
swimming (see RESULTS). The observer did not know which strain was
being observed. In addition, seven Tg8 and six C3H mice from two
different litters of each strain were again tested from P13 to P15.
Results were analyzed with statistical software SYSTAT in multidi-
mensional contingency tables as the occurrence of the H0, H1, and H2
swimming patterns versus the strains and the classes of age. The
multidimensional contingency tables were treated as log-linear mod-
els (Fienberg 1980).
The in vitro experiments were performed as reported previously for
newborn rats (Cazalets et al. 1992; Sqalli-Houssaini et al. 1993).
Newborn mice (5 C3H and 5 Tg8 pups at P0–P5 and 3 Tg8 pups at
P11) were decapitated under ether anesthesia and their spinal cords
were dissected and cut at the level of T8. The motor output from the
lumbar ventral roots was recorded, amplified, and stored. Up to three
spinal cords were pooled in the same experimental chamber so that
simultaneous recordings could be performed. The spinal cords were
continuously superfused with oxygenated saline into which drugs
could be added (Sigma Chemicals). The mean period (? SE) of
ventral root rhythmic activity was estimated from 60 cycles, and
differences between the means were taken as significant at P ? 0.05
R E S U L T S
Rodents are immature right after birth and exhibit a very
poor motor repertoire. Locomotor activity can be observed,
however, when postural constraints are removed [Cazalets et
al. 1990 (swimming experiments); McEwen et al. 1997 (air-
stepping experiments)]. Behavioral data were collected in vivo
using mouse swimming activity as a criterion of locomotor
network maturation. Figure 1 shows that swimming pattern
development is different in C3H and Tg8 strains. At birth
(P0–P1), most of the Tg8 pups presented the H0 pattern (no
rhythmic hindlimb movements); they either floated motion-
lessly or with smooth uncoordinated leg movements, or they
presented alternated movements of the forelimbs while their
hindlimbs were extended backward. In the same age class, 23%
of the C3H pups already presented the H1 pattern; they showed
alternated rhythmic forelimb movements while one hindlimb
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(and only one) moved in synchrony with the contralateral
forelimb. The second hindlimb was maintained extended back-
ward. In the P2–P3 age class, 75% of the Tg8 pups still
presented the H0 pattern whereas 46% of the C3H pups were
displaying the H1 pattern and 24% already showed the H2
pattern, wherein they swam with all four legs and presented
rhythmic and alternated movements of their forelimbs and
hindlimbs. In the P4–P5 age class, 21% of the Tg8 neonates
still exhibited the H0 pattern, 36% acquired the H1 pattern, and
43% acquired the H2 pattern. Most of the C3H pups (85%),
however, already displayed the H2 pattern. By P6–P7, almost
all the pups of the two strains showed the H2 pattern. Finally,
the adult swimming pattern, wherein alternated rhythmic hind-
limb movements are evident while immobile forelimbs are
extended forward, was present by P14 in both strains. To swim,
rats and mice first use only their forelimbs, then both forelimbs
and hindlimbs, and finally only hindlimbs. In C3H and Tg8
pups, statistical treatment revealed that acquisition of the
swimming pattern was age- and strain-dependent (P ? 0.001);
Tg8 pups were less able to use their hindlimbs for swimming
than C3H pups up to P6–P7.
In vitro experiments were carried out on spinal cord prepa-
rations similar to the one used to analyze locomotor-like ac-
tivity in neonatal rats (Cazalets et al. 1992; Sqalli-Houssaini et
al. 1993). As in rats, the mouse isolated locomotor network
was quiescent when the spinal cord was superfused with nor-
mal saline (no activity in ventral roots), but could be activated
by adding drugs to the saline, which led to rhythmic motor
command of the hindlimbs (Fig. 2). In C3H preparations (n ?
5), 5-HT (5 ? 10?5M) induced alternating rhythmic bursts of
action potentials in the left and right ventral roots (Fig. 2A1;
mean burst period ? SE, 7.1 ? 0.5 s). In contrast, 5-HT (from
10?6to 10?3M) never induced rhythmic or tonic activity in
Tg8 preparations from P1 to P7 (n ? 5) (Fig. 2B1). To show
that the lack of activity in the Tg8 spinal cord during 5-HT bath
application was not caused by a general disruption of all
neuronal activity, we tested the action of other transmitters.
N-methyl-D, L-aspartate (NMA, 2 ? 10?5M), an N-methyl-D-
aspartate (NMDA) receptor agonist known to induce locomo-
tor-like activity in the newborn rat (Cazalets et al. 1992), also
induced rhythmic bursting in all the tested C3H preparations
(Fig. 2A2; n ? 5) and in three-fifths of the Tg8 preparations
(Fig. 2B2). In the two remaining Tg8 preparations, NMA only
induced tonic discharges in ventral roots. The burst period of
the NMA-induced activity, although irregular, was signifi-
cantly shorter in C3H (1.6 ? 0.6 s) than in Tg8 preparations
(2.6 ? 0.1 s). In C3H mouse (Fig. 2A3), as in rat preparations
(Sqalli-Houssaini et al. 1993), adding 5-HT (5 ? 10?5M) to
the NMA-containing saline modified and improved the motor
rhythm. The burst period of the NMA-induced activity was
significantly lengthened (2.5 ? 0.1 s, n ? 5, Fig. 2A3). In
contrast, in three Tg8 preparations, adding 5-HT had no effect
over NMA alone (Fig. 2B3, 2.4 ? 0.1 s). Because MAOA
swimming patterns in control (C3H) and mono-
amine oxidase A (MAOA)-deficient (Tg8) pups.
For C3H (A) and Tg8 (B) neonatal mice, the his-
tograms represent frequency of occurrence of H0,
H1, and H2 swimming patterns (depending on
whether pups swam with 0, 1, or 2 hindlimbs) vs.
postnatal day ages (white bars, P0–P1; light gray
bars, P2–P3; gray bars, P4–P5; black bars, P6–
P7). Frequency of occurrence of a given swim-
ming pattern at a given age is expressed as a
percentage of the total number of observations for
the given age class. Statistical comparison of A and
B values showed delayed ability of Tg8 mice to
swim with their hindlimbs.
Developmental changes of juvenile
in C3H isolated spinal cords, all the bath-applied transmitters [A2, 2 ? 10?5
M N-methyl-D, L-aspartate (NMA); A4, 10?5M noradrenaline (NA)], includ-
ing 5-HT (A1, 5 ? 10?5M), elicited characteristic rhythmic activities in
lumbar ventral roots that alternated between right and left sides. In addition,
5-HT (5 ? 10?5M) affected NMA-induced activity (A2–A3). B: in Tg8
isolated spinal cords, applications of 2 ? 10?5M NMA (B2) and 10?5M NA
(B4) elicited characteristic rhythmic activities in lumbar ventral roots, but
5-HT applications (5 ? 10?5M) did not induce rhythmic bursts (B1) and did
not affect the NMA-induced activity (B2–B3). The two spinal cords in A1 and
B1 were recorded simultaneously in the same bath. The recordings in B2–B4
were obtained from a single spinal cord different from B1. Horizontal scale
bar, 5 s.
Lack of serotonin (5-HT) responses in Tg8 isolated spinal cord. A:
2469MAOA DEFICIENCY AND LOCOMOTOR NETWORK MATURATION
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deficiency affects both noradrenaline and 5-HT levels, we
tested noradrenaline effects. In both Tg8 (n ? 5) and C3H (n ?
5) preparations, noradrenaline (10?5M) (Fig. 2, A4–B4) in-
duced slow rhythmic discharges in ventral roots. 5-HT (5 ?
10?5) was applied in three Tg8 preparations at P11 and in-
duced ventral root tonic discharges in each preparation. The
response was observed only once at the first trial, however, and
after washout with normal saline could not be elicited again.
This suggests that at least a partial recovery occurred with age
for the 5-HT response in Tg8. Because of the viability of the
preparation at this age, however, it could not be concluded if
recovery was complete or not.
D I S C U S S I O N
Both in vivo and in vitro results suggest that MAOA deficiency
transiently delays locomotor network maturation, although it can-
not be unambiguously established that the the same neuronal
changes underlie the perturbations observed in vivo and in vitro.
Tg8 neonates swim without their hindlimbs for a longer period
than C3H neonates, but both strains acquire the adult hindlimb
may partly explain the delayed onset of hindlimb use in Tg8
activity in C3H neonates, it had no effects in Tg8 neonates.
MAOA deficiency in Tg8 pups increases both noradrenaline and
5-HT levels by ?50% and 900%, respectively (Cases et al. 1995;
Lajard et al. 1999). It seems unlikely that this small noradrenaline
change could be responsible for the maturational differences we
observed, although this possibility cannot be totally rejected be-
cause noradrenaline effects persisted in Tg8 preparations. The
substantial 5-HT excess in Tg8 pups, which has already been
shown to be responsible for maturational differences in both the
thalamocortical projections (Cases et al. 1996) and the central
respiratory network (Bou et al. 1998a,b), is more likely to be
involved. This perinatal 5-HT excess may have induced alter-
ations in 1) the maturation of the locomotor network itself, 2) the
maturation of the descending central projections, and/or 3) the
expression of spinal 5-HT receptors. As of now, none of these
network development was already suggested (Nakajima et al.
1998) because 5-HT synthesis blockade by prenatal treatment
perturbs both 5-HT spinal innervation and swimming movements
in neonatal rats, with recovery observed by P20. It is noteworthy
that decrease (see Nakajima et al. 1998) and increase (present
study) in 5-HT levels both affect the maturation of the rodent
locomotor network, suggesting that normal 5-HT metabolism
during gestation is a requisite for normal maturation of mamma-
lian central networks. Future experiments will be performed to
determine if the delay in maturation can be relieved with 5-HT
antagonists administered during development.
The authors thank E. De Maeyer and I. Seif (Orsay, France) for a gift of C3H
and Tg8 mice. We gratefully acknowledge Professor Albert Berger (Seattle,
WA) for a valuable review of the manuscript.
Address for reprint requests: J.-R. Cazalets, CNRS, Laboratoire de Neuro-
biologie et Mouvements, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 9,
Received 21 September 1999; accepted in final form 20 December 1999.
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