MORPHOLOGICAL DETERMINANTS OF CELL-TO-CELL VARIATIONS IN ACTION POTENTIAL DYNAMICS IN SUBSTANTIA NIGRA DOPAMINERGIC NEURONS

ABSTRACTAction potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. AP shape is also used to distinguish neuronal populations, as it greatly varies between neuronal types. For instance, AP duration ranges from hundreds of microseconds in cerebellar granule cells to 2-3 milliseconds in substantia nigra pars compacta (SNc) dopaminergic (DA) neurons. While most of this variation seems to arise from differences in the subtypes of voltage- and calcium-gated ion channels expressed, a few studies suggested that dendritic morphology may also affect AP shape. However, AP duration also displays significant variability in a same neuronal type, while 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 SNc DA neurons. 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 complexities of the ABD and the non-ABDs. Dendrotomy simulation and experiments 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 somato-dendritic compartment. In addition, dendritic morphology also modulated AP back-propagation efficiency in response to barrages of EPSCs in the ABD. In line 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 STATEMENTAction 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, due to the presence of sodium channels in the somato-dendritic 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.

Transcriptional profiling of sequentially generated septal neuron fates

The septum is a ventral forebrain structure known to regulate innate behaviors. During embryonic development, septal neurons are produced in multiple proliferative areas from neural progenitors following transcriptional programs that are still largely unknown. Here, we use a combination of single cell RNA sequencing, histology and genetic models to address how septal neuron diversity is established during neurogenesis. We find that the transcriptional profiles of septal progenitors change along neurogenesis, coinciding with the generation of distinct neuron types. We characterize the septal eminence, an anatomically distinct and transient proliferative zone composed of progenitors with distinctive molecular profiles, proliferative capacity and fate potential compared to the rostral septal progenitor zone. We show that Nkx2.1-expressing septal eminence progenitors give rise to neurons belonging to at least three morphological classes, born in temporal cohorts that are distributed across different septal nuclei in a sequential fountain-like pattern. Our study provides insight into the molecular programs that control the sequential production of different neuronal types in the septum, a structure with important roles in regulating mood and motivation.

Kv3 channels contribute to the excitability of sub-populations of spinal cord neurons in lamina VII

Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Cre-Dependent Anterograde Transsynaptic Labeling and Functional Imaging in Zebrafish Using VSV With Reduced Cytotoxicity

The small size and translucency of larval zebrafish (Danio rerio) have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory (vglut2a positive) and inhibitory (gad1b positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5–8 days post fertilization (2–5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.

Resurgent Sodium Current in Neurons of the Cerebral Cortex

In the late ’90, Dr. Indira Raman, at the time a postdoctoral fellow with Dr. Bruce Bean, at Harvard University, identified a new type of sodium current, flowing through the channels that reopens when the membrane is repolarized. This current, called “resurgent Sodium current,” was originally identified in cerebellar Purkinje neurons and has now been confirmed in around 20 different neuronal types. Since moving to Northwestern University in 1999 to establish her own research group, Dr. Raman has dedicated great efforts in identifying the mechanisms supporting the resurgent Sodium current and how its biophysical properties shape the firing of the different cell types. Her work has impacted greatly the field of cellular neurophysiology, from basic research to translation neuroscience. In fact, alterations in the resurgent sodium currents have been observed in several neuropathologies, from Huntington’s disease to epilepsy. In this Perspective we will focus on the current knowledge on the expression and function of the resurgent Sodium current in neurons of the cerebral cortex and hippocampus. We will also briefly highlight the role of Dr. Raman’s as teacher and mentor, not only for her pupils, but for the whole scientific community.

Single-nucleus transcriptomic atlas of spinal cord neuron in human

Despite the recognized importance of spinal cord in sensory processing, motor behaviors and/or neural diseases, the underlying neuronal clusters remain elusive. Recently, several studies attempted to define the neuronal types and functional heterogeneity in spinal cord using single cell and/or single-nucleus RNA-sequencing in varied animal models. However, the molecular evidence of neuronal heterogeneity in human spinal cord has not been established yet. Here we sought to classify spinal cord neurons from human donors by high-throughput single-nucleus RNA-sequencing. The functional heterogeneity of identified cell types and signaling pathways that connecting neuronal subtypes were explored. Moreover, we also compared human results with previous single-cell transcriptomic profiles of mouse spinal cord. As a result, we generated the first comprehensive atlas of human spinal cord neurons and defined 18 neuronal clusters. In addition to identification of the new and functionally-distinct neuronal subtypes, our results also provide novel marker genes for previously known neuronal types. The comparation with mouse transcriptomic profiles revealed an overall similarity in the cellular composition of spinal cord between the two species. In summary, these results illustrate the complexity and diversity of neuronal types in human spinal cord and will provide an important resource for future researches to explore the molecular mechanism underlying several spinal cord physiology and diseases.

Time in Neurogenesis: Conservation of the Developmental Formation of the Cerebellar Circuitry

The cerebellum is a conserved structure of vertebrate brains that develops at the most anterior region of the alar rhombencephalon. All vertebrates display a cerebellum, making it one of the most highly conserved structures of the brain. Although it greatly varies at the morphological level, several lines of research point to strong conservation of its internal neural circuitry. To test the conservation of the cerebellar circuit, we compared the developmental history of the neurons comprising this circuit in three amniote species: mouse, chick, and gecko. We specifically researched the developmental time of generation of the main neuronal types of the cerebellar cortex. This developmental trajectory is known for the mammalian cell types but barely understood for sauropsid species. We show that the neurogenesis of the GABAergic lineage proceeds following the same chronological sequence in the three species compared: Purkinje cells are the first ones generated in the cerebellar cortex, followed by Golgi interneurons of the granule cell layer, and lately by the interneurons of the molecular layer. In the cerebellar glutamatergic lineage, we observed the same conservation of neurogenesis throughout amniotes, and the same vastly prolonged neurogenesis of granule cells, extending much further than for any other brain region. Together these data show that the cerebellar circuitry develops following a tightly conserved chronological sequence of neurogenesis, which is responsible for the preservation of the cerebellum and its function. Our data reinforce the developmental perspective of homology, whereby similarities in neurons and circuits are likely due to similarities in developmental sequence.

Angiotensin II Inhibits the A-type K+ Current of Hypothalamic Paraventricular Nucleus Neurons in Rats with Heart Failure: Role of MAPK-ERK1/2 Signaling

ABSTRACTANGII-mediated sympathohumoral activation constitutes a key pathophysiological mechanism in heart failure (HF). While the hypothalamic paraventricular nucleus (PVN) is recognized as a major site mediating ANGII effects in HF, the precise mechanisms by which ANGII influences sympathohumoral outflow from the PVN remain unknown. ANGII activates the ubiquitous intracellular MAPK signaling cascades and recent studies revealed a key role for ERK1/2 MAPK signaling in ANGII-mediated sympathoexcitation in HF rats. Importantly, ERK1/2 was reported to inhibit the transient outward potassium current (IA) in hippocampal neurons. Given that IA is a critical determinant of the PVN neuronal excitability, and that downregulation of IA in the brain has been reported in cardiovascular disease states, including HF, we investigated here whether ANGII modulates IA in PVN neurons via the MAPK-ERK pathway, and, whether these effects are altered in HF rats. Patch-clamp recordings from identified magnocellular neurosecretory (MNNs) and presympathetic (PS) PVN neurons revealed that ANGII inhibited IA in both PVN neuronal types, both in sham and HF rats. Importantly, ANGII effects were blocked by inhibiting MAPK-ERK signaling as well as by inhibiting EGFR, a gateway to MAPK-ERK signaling. While no differences in basal IA magnitude were found between sham and HF rats under normal conditions, MAPK-ERK blockade resulted in significantly larger IA in both PVN neuronal types in HF rats. Taken together, our studies show that ANGII-induced ERK1/2 activity inhibits IA and increases the excitability of presympathetic and neuroendocrine PVN neurons, contributing to the neurohumoral overactivity than promotes progression of the HF syndrome.GRAPHICAL ABSTRACT