Morphological Determinants of Cell-to-Cell Variations in Action Potential Dynamics in Substantia Nigra Dopaminergic Neurons.

Action potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. As it greatly varies between neuronal types, AP shape is also used to distinguish neuronal populations. For instance, AP duration ranges from hundreds of microseconds in cerebellar granule cells to 2-3 ms in SNc dopaminergic (DA) neurons. While most of this variation across cell types seems to arise from differences in the voltage- and calcium-gated ion channels expressed, a few studies suggested that dendritic morphology also affects AP shape. AP duration also displays significant variability in a same neuronal type, although the determinants of these variations are poorly known. Using electrophysiological recordings, morphological reconstructions, and realistic Hodgkin-Huxley modeling, we investigated the relationships between dendritic morphology and AP shape in rat SNc DA neurons from both sexes. In this neuronal type where the axon arises from an axon-bearing dendrite (ABD), the duration of the somatic AP could be predicted from a linear combination of the ABD and non-ABDs' complexities. Dendrotomy experiments and simulation showed that these correlations arise from the causal influence of dendritic topology on AP duration, due in particular to a high density of sodium channels in the somatodendritic compartment. Surprisingly, computational modeling suggested that this effect arises from the influence of sodium currents on the decaying phase of the AP. Consistent with previous findings, these results demonstrate that dendritic morphology plays a major role in defining the electrophysiological properties of SNc DA neurons and their cell-to-cell variations.SIGNIFICANCE STATEMENT Action potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. AP shape (e.g., duration) greatly varies between neuronal types but also within a same neuronal type. While differences in ion channel expression seem to explain most of AP shape variation across cell types, the determinants of cell-to-cell variations in a same neuronal type are mostly unknown. We used electrophysiological recordings, neuronal reconstruction, and modeling to show that, because of the presence of sodium channels in the somatodendritic compartment, a large part of cell-to-cell variations in somatic AP duration in substantia nigra pars compacta dopaminergic neurons is explained by variations in dendritic topology.

Robustness to Axon Initial Segment Variation Is Explained by Somatodendritic Excitability in Rat Substantia Nigra Dopaminergic Neurons.

In many neuronal types, axon initial segment (AIS) geometry critically influences neuronal excitability. Interestingly, the axon of rat SNc dopaminergic (DA) neurons displays a highly variable location and most often arises from an axon-bearing dendrite (ABD). We combined current-clamp somatic and dendritic recordings, outside-out recordings of dendritic sodium and potassium currents, morphological reconstructions and multicompartment modeling on male and female rat SNc DA neurons to determine cell-to-cell variations in AIS and ABD geometry, and their influence on neuronal output (spontaneous pacemaking frequency, action potential [AP] shape). Both AIS and ABD geometries were found to be highly variable from neuron to neuron. Surprisingly, we found that AP shape and pacemaking frequency were independent of AIS geometry. Modeling realistic morphological and biophysical variations helped us clarify this result: in SNc DA neurons, the complexity of the ABD combined with its excitability predominantly define pacemaking frequency and AP shape, such that large variations in AIS geometry negligibly affect neuronal output and are tolerated.SIGNIFICANCE STATEMENT In many neuronal types, axon initial segment (AIS) geometry critically influences neuronal excitability. In the current study, we describe large cell-to-cell variations in AIS length or distance from the soma in rat substantia nigra pars compacta dopaminergic neurons. Using neuronal reconstruction and electrophysiological recordings, we show that this morphological variability does not seem to affect their electrophysiological output, as neither action potential properties nor pacemaking frequency is correlated with AIS morphology. Realistic multicompartment modeling suggests that this robustness to AIS variation is mainly explained by the complexity and excitability of the somatodendritic compartment.

Nutritional status-dependent endocannabinoid signalling regulates the integration of rat visceral information.

KEY POINTS: Vagal sensory inputs transmit information from the viscera to brainstem neurones located in the nucleus tractus solitarii to set physiological parameters. These excitatory synapses exhibit a CB1 endocannabinoid-induced long-term depression (LTD) triggered by vagal fibre stimulation. We investigated the impact of nutritional status on long-term changes in this long-term synaptic plasticity. Food deprivation prevents LTD induction by disrupting CB1 receptor signalling. Short-term refeeding restores the capacity of vagal synapses to express LTD. Ghrelin and cholecystokinin, respectively released during fasting and refeeding, play a key role in the control of LTD via the activation of energy sensing pathways such as AMPK and the mTOR and ERK pathways. ABSTRACT: Communication form the viscera to the brain is essential to set physiological homoeostatic parameters but also to drive more complex behaviours such as mood, memory and emotional states. Here we investigated the impact of the nutritional status on long-term changes in excitatory synaptic transmission in the nucleus tractus solitarii, a neural hub integrating visceral signals. These excitatory synapses exhibit a CB1 endocannabinoid (eCB)-induced long-term depression (LTD) triggered by vagal fibre stimulation. Since eCB signalling is known to be an important component of homoeostatic regulation of the body and is regulated during various stressful conditions, we tested the hypothesis that food deprivation alters eCB signalling in central visceral afferent fibres. Food deprivation prevents eCB-LTD induction due to the absence of eCB signalling. This loss was reversed by blockade of ghrelin receptors. Activation of the cellular fuel sensor AMP-activated protein kinase or inhibition of the mechanistic target of rapamycin pathway abolished eCB-LTD in free-fed rats. Signals associated with energy surfeit, such as short-term refeeding, restore eCB-LTD induction, which in turn requires activation of cholecystokinin receptors and the extracellular signal-regulated kinase pathway. These data suggest a tight link between eCB-LTD in the NTS and nutritional status and shed light on the key role of eCB in the integration of visceral information.

Kv4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii.

In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

Anandamide, cannabinoid type 1 receptor, and NMDA receptor activation mediate non-Hebbian presynaptically expressed long-term depression at the first central synapse for visceral afferent fibers.

Presynaptic long-term depression (LTD) of synapse efficacy generally requires coordinated activity between presynaptic and postsynaptic neurons and a retrograde signal synthesized by the postsynaptic cell in an activity-dependent manner. In this study, we examined LTD in the rat nucleus tractus solitarii (NTS), a brainstem nucleus that relays homeostatic information from the internal body to the brain. We found that coactivation of N-methyl-D-aspartate receptors (NMDARs) and type 1 cannabinoid receptors (CB1Rs) induces LTD at the first central excitatory synapse between visceral fibers and NTS neurons. This LTD is presynaptically expressed. However, neither postsynaptic activation of NMDARs nor postsynaptic calcium influx are required for its induction. Direct activation of NMDARs triggers cannabinoid-dependent LTD. In addition, LTD is unaffected by blocking 2-arachidonyl-glycerol synthesis, but its induction threshold is lowered by preventing fatty acid degradation. Altogether, our data suggest that LTD in NTS neurons may be entirely expressed at the presynaptic level by local anandamide synthesis.

Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons.

Substantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized in vitro by a spontaneous tonic regular activity (pacemaking activity), a broad action potential (AP) and a biphasic postinhibitory response. The transient A-type current (IA) is known to play a crucial role in this electrical phenotype, and so far, this current was considered to be carried exclusively by Kv4.3 potassium channels. Using Kv4.3-/- transgenic mice, we demonstrate that the constitutive loss of this channel is associated with increased exploratory behavior and impaired motor learning at the behavioral level. Consistently, it is also associated with a lack of compensatory changes in other ion currents at the cellular level. Using antigen retrieval (AR) immunohistochemistry, we then demonstrate that Kv4.2 potassium channels are also expressed in SNc DA neurons, although their contribution to IA appears significant only in a minority of neurons (∼5-10%). Using correlative analysis on recorded electrophysiological parameters and multicompartment modeling, we then demonstrate that, rather than its conductance level, IA gating kinetics (inactivation time constant) appear as the main biophysical property defining postinhibitory rebound delay and pacemaking frequency. Moreover, we show that the hyperpolarization-activated current (IH) has an opposing and complementary influence on the same firing features.

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.

Silent synapses in developing rat nucleus tractus solitarii have AMPA receptors.

NMDA-only synapses, called silent synapses, are thought to be the initial step in synapse formation in several systems. However, the underlying mechanism and the role in circuit construction are still a matter of dispute. Using combined morphological and electrophysiological approaches, we searched for silent synapses at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibers. Silent synapses were detected at birth by using electrophysiological recordings and minimal stimulation protocols. However, anatomical experiments indicated that nearly all, if not all, NTS synapses had AMPA receptors. Based on EPSC fluctuation measurements and differential blockade by low-affinity competitive and noncompetitive glutamate antagonists, we then demonstrated that NTS silent synapses were better explained by glutamate spillover from neighboring fibers and/or slow dynamic of fusion pore opening. Glutamate spillover at immature NTS synapses may favor crosstalk between active synapses during development when glutamate transporters are weakly expressed and contribute to synaptic processing as well as autonomic circuit formation.

Diversity of Axonal and Dendritic Contributions to Neuronal Output.

Our general understanding of neuronal function is that dendrites receive information that is transmitted to the axon, where action potentials (APs) are initiated and propagated to eventually trigger neurotransmitter release at synaptic terminals. Even though this canonical division of labor is true for a number of neuronal types in the mammalian brain (including neocortical and hippocampal pyramidal neurons or cerebellar Purkinje neurons), many neuronal types do not comply with this classical polarity scheme. In fact, dendrites can be the site of AP initiation and propagation, and even neurotransmitter release. In several interneuron types, all functions are carried out by dendrites as these neurons are devoid of a canonical axon. In this article, we present a few examples of "misbehaving" neurons (with a non-canonical polarity scheme) to highlight the diversity of solutions that are used by mammalian neurons to transmit information. Moreover, we discuss how the contribution of dendrites and axons to neuronal excitability may impose constraints on the morphology of these compartments in specific functional contexts.

The excitatory amino acid carrier 1 (EAAC1) in the rat nucleus of the solitary tract: subcellular localization suggests no major role in glutamate clearance.

The excitatory amino acid carrier 1 (EAAC1) is a sodium-dependent glutamate transporter widely found in the mammalian brain and mainly localized in the somatodendritic compartment of neurons. The present study was performed to determine whether EAAC1 is present in the rat nucleus of the solitary tract (NST, a sensory brainstem nucleus involved in visceroception) and to document its subcellular localization. Using fluorescent immunolabeling, peroxidase immunostaining and quantitative immunogold labeling, we showed that both intracellular and plasma membrane-associated pools of EAAC1 transporters existed in dendrites of NST neurons. Although plasma membrane-associated transporters were more concentrated in the vicinity of synapses, no labeling was found at the axon-dendrite interface, suggesting that EAAC1 was not (or barely) expressed in this portion of dendritic membrane. Using computer simulation, we next showed that the ability of EAAC1 to efficiently take up synaptically released glutamate was very low outside the axon-dendrite interface. These data suggest that EAAC1 transporters present on NST dendrites may play a minor role if any in glutamate clearance.

A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem.

Cerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the 'transient receptor potential (TRP)' superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors. Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.

Activation of N-methyl-d-aspartate Receptors Induces Endogenous Rhythmic Bursting Activities in Nucleus Tractus Solitarii Neurons: An Intracellular Study on Adult Rat Brainstem Slices.

A brainstem slice preparation and intracellular recording techniques were used to examine the effects of N-methyl-d-aspartate (NMDA) application on neurons within the swallowing area of the nucleus tractus solitarii (NTS). According to their cellular properties, NTS neurons were classified into type I and type II neurons. The most striking difference was the occurrence of delayed excitation in type I but not in type II neurons, when they were depolarized from membrane potentials more negative than -60 mV. Bath application of NMDA (30 - 60 microM) elicited depolarization and triggered stable repetitive firing in all the NTS neurons but one. During the NMDA-induced depolarization, hyperpolarization below -60 mV elicited, in some type I neurons, a rhythmic bursting pattern. The duration of the bursts (300 - 1000 ms) and their frequency (0.5 - 2 Hz) depended on the membrane potential. With hyperpolarizations below -75 mV, rhythmic bursting was converted into rhythmic single discharges, a pattern elicited directly in the other type I neurons. In all cases, rhythmic patterns were superimposed on cyclic depolarizations of the membrane potential characterized by an initial ramp-shaped phase. In type II neurons, rhythmic bursting discharges, superimposed on rhythmic oscillations of the membrane potential, were also obtained upon hyperpolarization during the NMDA-induced depolarization. In all type I neurons tested, NMDA-induced cyclic ramp-shaped depolarizations continued after addition of tetrodotoxin to the medium. Rhythmic bursting was not elicited by bath application of kainate (10 - 20 microM). Application of d-2-amino-5-phosphonovalerate (50 microM) blocked NMDA-induced depolarizations without modifying those elicited by kainate, which were selectively depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM). Moreover, removal of Mg2+ from the medium suppressed NMDA-induced cyclic depolarizations. Results demonstrate that both NMDA and non-NMDA receptors are present in NTS neurons and that selective activation of NMDA receptors induced rhythmic bursting and/or rhythmic single discharges. Rhythmic patterns were not driven by synaptic mechanisms but originated from endogenous properties of NTS neurons activated by NMDA. Thus, NTS neurons can be considered as conditional pacemakers. According to the location of the neurons, the conditional properties shown in these in vitro experiments might be involved in vivo in the generation of rhythmic motor activities set up at the NTS level, such as swallowing.

Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics.

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.

Glutamatergic synapses in the rat nucleus tractus solitarii develop by direct insertion of calcium-impermeable AMPA receptors and without activation of NMDA receptors.

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current-voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR2 subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR2 subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.

Early expression of AMPA receptors and lack of NMDA receptors in developing rat climbing fibre synapses.

Whether nascent glutamatergic synapses acquire their AMPA receptors constitutively or via a regulated pathway triggered by pre-existing NMDA receptor activation is still an open issue. Here, we provide evidence that some glutamatergic synapses develop without expressing NMDA receptors. Using immunocytochemistry, we showed that synapses between developing rat climbing fibres and Purkinje cells expressed GluR2-containing AMPA receptors as soon as they were formed (i.e. on embryonic day 19) but never carried detectable NMDA receptors. This was confirmed by electrophysiological recordings. Excitatory synaptic currents were recorded in Purkinje cells as early as P0. However, no NMDA receptor-mediated component was found in either spontaneous or evoked synaptic responses. In addition, we ruled out a possible role of extrasynaptic NMDA receptors by showing that AMPA receptor clustering at nascent climbing fibre synapses was not modified by chronic in utero NMDA receptor blockade.

Successive episodes of synapses production in the developing rat nucleus tractus solitarii.

In the rat nucleus tractus solitarii (NTS), synaptogenesis is thought to occur both pre- and postnatally. The present study was performed to precisely define the timetable of synapse formation in the NTS after birth. Changes in synapse morphology and densities were analyzed between postnatal day 3 (P3) and P28 using electron microscopy and ethanol phosphotungstic acid (E-PTA) staining. The proportion of morphologically immature synapses was high at P3 (38%) and P14 (30%) and low (8-14%) at the other ages investigated (P7, P21, and P28). Synaptic density significantly increased between P7 and P14 (60%) and between P21 and P28 (54%), but did not significantly change between P3 and P7 and between P14 and P21. Mean synaptic diameter also increased over the first postnatal month. Significant increases in synaptic size occurred between P3 and P7 (28%) and between P14 and P21 (15%). The present data indicate that, in the NTS, synaptogenesis occurs over a protracted period of time and involves distinct successive episodes of synapse production.

Synaptic localization of the glutamate receptor subunit GluR2 in the rat nucleus tractus solitarii.

The GluR2 subunit controls several key features of the alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor including calcium permeability, rectification and gating. In the present study, electrophysiological recordings and immunocytochemistry were used to document the synaptic localization of GluR2 in the rat nucleus tractus solitarii (NTS). Synaptic responses recorded in NTS neurons exhibited linear current-voltage relationships suggestive of GluR2-containing AMPA receptor responses. Furthermore, after antigen retrieval GluR2 immunolabelling in the NTS mainly consisted of small puncta. Double-labelling experiments showed that these GluR2 puncta were apposed to glutamatergic synaptic terminals identified by type II vesicular glutamate transporter immunoreactivity. These results indicate that NTS glutamatergic synapses are endowed with AMPA receptors which contain the GluR2 subunit and are therefore likely to be both calcium-impermeable and slowly desensitizing.