Bénédicte Amilhon

Impairment of SLC17A8 Encoding Vesicular Glutamate Transporter-3, VGLUT3, Underlies Nonsyndromic Deafness DFNA25 and Inner Hair Cell Dysfunction in Null Mice

Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3−/− mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H+-dependent carriers (named VGLUT1–3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT1A-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

Developmentally regulated expression of VGLUT3 during early post-natal life.

Three subtypes of vesicular glutamate transporters, named VGLUT1-3, accumulate glutamate into synaptic vesicles. In this study, the post-natal expression of VGLUT3 was determined with specific probes and antiserums in the rat brain and compared with that of VGLUT1 and VGLUT2. The expression of VGLUT1 and VGLUT2 increases linearly during post-natal development. In contrast, VGLUT3 developmental pattern appears to have a more or less biphasic profile. A first peak of expression is centered around post-natal day 10 (P10) while the second one is reached in the adult brain. Between P1 and P15, VGLUT3 is observed in the frontal brain (striatum, accumbens, and hippocampus) and in the caudal brain (colliculi, pons and cerebellum). During a second phase extending from P15 to adulthood, the labeling of the caudal brain fades away. The adult pattern is reached at P21. We further analyzed the transient expression of VGLUT3 in the cerebellum and found it to correspond to a temporary expression in Purkinje cells. At P10 VGLUT3 immunoreactivity was present both in the soma and terminals of Purkinje cells (PC), where it colocalized with the vesicular inhibitory amino acid transporter (VIAAT). In agreement with data from the literature [Gillespie, D.C., Kim, G., Kandler, K., 2005. Inhibitory synapses in the developing auditory system are glutamatergic. Nat. Neurosci. 8, 332-338], our results suggest that during the first 2 weeks of post-natal life PC may have the potential to transiently release simultaneously GABA and glutamate.

Parvalbumin Interneurons of Hippocampus Tune Population Activity at Theta Frequency.

Hippocampal theta rhythm arises from a combination of recently described intrinsic theta oscillators and inputs from multiple brain areas. Interneurons expressing the markers parvalbumin (PV) and somatostatin (SOM) are leading candidates to participate in intrinsic rhythm generation and principal cell (PC) coordination in distal CA1 and subiculum. We tested their involvement by optogenetically activating and silencing PV or SOM interneurons in an intact hippocampus preparation that preserves intrinsic connections and oscillates spontaneously at theta frequencies. Despite evidence suggesting that SOM interneurons are crucial for theta, optogenetic manipulation of these interneurons modestly influenced theta rhythm. However, SOM interneurons were able to strongly modulate temporoammonic inputs. In contrast, activation of PV interneurons powerfully controlled PC network and rhythm generation optimally at 8 Hz, while continuously silencing them disrupted theta. Our results thus demonstrate a pivotal role of PV but not SOM interneurons for PC synchronization and the emergence of intrinsic hippocampal theta.

Optogenetic Activation of Septal Glutamatergic Neurons Drive Hippocampal Theta Rhythms

The medial septum and diagonal band of Broca (MS-DBB) has an essential role for theta rhythm generation in the hippocampus and is critical for learning and memory. The MS-DBB contains cholinergic, GABAergic, and recently described glutamatergic neurons, but their specific contribution to theta generation is poorly understood. Here, we examined the role of MS-DBB glutamatergic neurons in theta rhythm using optogenetic activation and electrophysiological recordings performed in in vitro preparations and in freely behaving mice. The experiments in slices suggest that MS-DBB glutamatergic neurons provide prominent excitatory inputs to a majority of local GABAergic and a minority of septal cholinergic neurons. In contrast, activation of MS-DBB glutamatergic fiber terminals in hippocampal slices elicited weak postsynaptic responses in hippocampal neurons. In the in vitro septo-hippocampal preparation, activation of MS-DBB glutamatergic neurons did increase the rhythmicity of hippocampal theta oscillations, whereas stimulation of septo-hippocampal glutamatergic fibers in the fornix did not have an effect. In freely behaving mice, activation of these neurons in the MS-DBB strongly synchronized hippocampal theta rhythms over a wide range of frequencies, whereas activation of their projections to the hippocampus through fornix stimulations had no effect on theta rhythms, suggesting that MS-DBB glutamatergic neurons played a role in theta generation through local modulation of septal neurons. Together, these results provide the first evidence that MS-DBB glutamatergic neurons modulate local septal circuits, which in turn contribute to theta rhythms in the hippocampus. SIGNIFICANCE STATEMENT The current paper explores the functional role of septal glutamatergic neurons, a recently identified population, in theta rhythm generation in the hippocampus. Using optogenetics and electrophysiology, we have explored the functional connectivity of these neurons in vitro, as well as their influence on theta rhythms both in vitro and in vivo, and show that this neuronal population can powerfully drive theta rhythms through intraseptal connections. These results strongly suggest a role of glutamatergic neurons in theta rhythm generation and may therefore be important for learning and memory.

Resilience to chronic stress is mediated by noradrenergic regulation of dopamine neurons.

Dopamine (DA) neurons in the ventral tegmental area (VTA) help mediate stress susceptibility and resilience. However, upstream mechanisms controlling these neurons remain unknown. Noradrenergic (NE) neurons in the locus coeruleus, implicated in the pathophysiology of depression, have direct connections within the VTA. Here we demonstrate that NE neurons regulate vulnerability to social defeat through inhibitory control of VTA DA neurons.

Reversal of theta rhythm flow through intact hippocampal circuits

Activity flow through the hippocampus is thought to arise exclusively from unidirectional excitatory synaptic signaling from CA3 to CA1 to the subiculum. Theta rhythms are important for hippocampal synchronization during episodic memory processing; thus, it is assumed that theta rhythms follow these excitatory feedforward circuits. To the contrary, we found that theta rhythms generated in the rat subiculum flowed backward to actively modulate spike timing and local network rhythms in CA1 and CA3. This reversed signaling involved GABAergic mechanisms. However, when hippocampal circuits were physically limited to a lamellar slab, CA3 outputs synchronized CA1 and the subiculum using excitatory mechanisms, as predicted by classic hippocampal models. Finally, analysis of in vivo recordings revealed that this reversed theta flow was most prominent during REM sleep. These data demonstrate that communication between CA3, CA1 and the subiculum is not exclusively unidirectional or excitatory and that reversed inhibitory theta signaling also contributes to intrahippocampal synchrony.

Experimentally constrained CA1 fast-firing parvalbumin-positive interneuron network models exhibit sharp transitions into coherent high frequency rhythms.

The coupling of high frequency oscillations (HFOs; >100 Hz) and theta oscillations (3–12 Hz) in the CA1 region of rats increases during REM sleep, indicating that it may play a role in memory processing. However, it is unclear whether the CA1 region itself is capable of providing major contributions to the generation of HFOs, or if they are strictly driven through input projections. Parvalbumin-positive (PV+) interneurons may play an essential role in these oscillations due to their extensive connections with neighboring pyramidal cells, and their characteristic fast-spiking. Thus, we created mathematical network models to investigate the conditions under which networks of CA1 fast-spiking PV+ interneurons are capable of producing high frequency population rhythms. We used whole-cell patch clamp recordings of fast-spiking, PV+ cells in the CA1 region of an intact hippocampal preparation in vitro to derive cellular properties, from which we constrained an Izhikevich-type model. Novel, biologically constrained network models were constructed with these individual cell models, and we investigated networks across a range of experimentally determined excitatory inputs and inhibitory synaptic strengths. For each network, we determined network frequency and coherence. Network simulations produce coherent firing at high frequencies (>90 Hz) for parameter ranges in which PV-PV inhibitory synaptic conductances are necessarily small and external excitatory inputs are relatively large. Interestingly, our networks produce sharp transitions between random and coherent firing, and this sharpness is lost when connectivity is increased beyond biological estimates. Our work suggests that CA1 networks may be designed with mechanisms for quickly gating in and out of high frequency coherent population rhythms, which may be essential in the generation of nested theta/high frequency rhythms.

Excitatory Inputs Determine Phase-Locking Strength and Spike-Timing of CA1 Stratum Oriens/Alveus Parvalbumin and Somatostatin Interneurons during Intrinsically Generated Hippocampal Theta Rhythm.

Theta oscillations are essential for learning and memory, and their generation requires GABAergic interneurons. To better understand how theta is generated, we explored how parvalbumin (PV) and somatostatin (SOM) interneurons in CA1 stratum oriens/alveus fire during hippocampal theta and investigated synaptic mechanisms underlying their behavior. Combining the use of transgenic mice to visually identify PV and SOM interneurons and the intact hippocampal preparation that can generate theta oscillations in vitro without cholinergic agonists, we performed simultaneous field and whole-cell recordings. We found that PV interneurons uniformly fire strongly phase-locked to theta, whereas SOM neurons are more heterogeneous with only a proportion of cells displaying tight phase-locking. Differences in phase-locking strength could be explained by disparity in excitatory inputs received; PV neurons received significantly larger EPSCs compared with SOM neurons, and the degree of phase-locking in SOM neurons was significantly correlated with the size of EPSCs. In contrast, IPSC amplitude did not differ between cell types. We determined that the local CA1 rhythm plays a more dominant role in driving CA1 interneuron firing than afferent inputs from the CA3. Last, we show that PV and strongly phase-locked SOM neurons fire near the peak of CA1 theta, under the tight control of excitatory inputs that arise at a specific phase of each theta cycle. These results reveal a fundamental mechanism of neuronal phase-locking and highlight an important role of excitation from the local network in governing firing behavior during rhythmic network states. SIGNIFICANCE STATEMENT Rhythmic activity in the theta range (3–12 Hz) is important for proper functioning of the hippocampus, a brain area essential for learning and memory. To understand how theta rhythm is generated, we investigated how two types of inhibitory neurons, those that express parvalbumin and somatostatin, fire action potentials during theta in an in vitro preparation of the mouse hippocampus. We found that the amount of excitatory input they receive from the local network determines how closely their spikes follow the network theta rhythm. Our findings reveal an important role of local excitatory input in driving inhibitory neuron firing during rhythmic states and may have implications for diseases, such as epilepsy and Alzheimers disease, which affect the hippocampus and related areas.