Katarina E. Leão

OLM interneurons differentially modulate CA3 and entorhinal inputs to hippocampal CA1 neurons

The vast diversity of GABAergic interneurons is believed to endow hippocampal microcircuits with the required flexibility for memory encoding and retrieval. However, dissection of the functional roles of defined interneuron types has been hampered by the lack of cell-specific tools. We identified a precise molecular marker for a population of hippocampal GABAergic interneurons known as oriens lacunosum-moleculare (OLM) cells. By combining transgenic mice and optogenetic tools, we found that OLM cells are important for gating the information flow in CA1, facilitating the transmission of intrahippocampal information (from CA3) while reducing the influence of extrahippocampal inputs (from the entorhinal cortex). Furthermore, we found that OLM cells were interconnected by gap junctions, received direct cholinergic inputs from subcortical afferents and accounted for the effect of nicotine on synaptic plasticity of the Schaffer collateral pathway. Our results suggest that acetylcholine acting through OLM cells can control the mnemonic processes executed by the hippocampus.

Altered sodium currents in auditory neurons of congenitally deaf mice

Sodium currents are essential for action potential generation and propagation in most excitable cells. Appropriate tuning of these currents can be modulated both developmentally and in response to activity. Here we use a mouse model of congenital deafness (dn/dn– asymptomatic deafness associated with hair cell degeneration) to investigate the effect of lack of activity in the expression of Na+ currents in neurons from the medial nucleus of the trapezoid body (MNTB). Patch‐clamp recordings show that at postnatal day (P) 14, both normal and deaf mice display a significant amount of persistent and resurgent Na+ currents. However, the persistent current is greater in deaf mice than in normal mice, and resurgent current kinetics are slower in deaf mice. At P7, resurgent currents are not present in either group. MNTB immunohistochemistry demonstrates that Nav1.1 subunits are expressed postsynaptically in both P14 normal and deaf mice, while postsynaptic Nav1.6 staining was only observed in deaf mice. Labelling of Nav1.6 subunits in different age groups revealed that at younger ages (P7), both normal and deaf mice express this protein. Nav1.6 staining was not observed in MNTB neurons of P28 normal mice, whereas it is maintained in deaf mice cells until much later (P28). At P7, none of the groups displayed resurgent currents (despite the detection of Nav1.6 subunits at this age group); this suggests that factors other than alpha subunits are important for modulating these currents in MNTB cells. Our results emphasize the importance of activity during development in regulating Na+ channels.

Hyperpolarization-activated currents are differentially expressed in mice brainstem auditory nuclei

The hyperpolarization‐activated cation current (Ih) may influence precise auditory processing by modulating resting membrane potential and cell excitability. We used electrophysiology and immunohistochemistry to investigate the properties of Ih in three auditory brainstem nuclei in mice: the anteroventral cochlear nucleus (AVCN), the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO). Ih amplitude varied considerably between these cell types, with the order of magnitude LSO > AVCN > MNTB. Kinetically, Ih is faster in LSO neurons, and more active at rest, compared with AVCN and MNTB cells. The half‐activation voltage is −10 mV more hyperpolarized for AVCN and MNTB cells compared with LSO neurons. HCN1 immunoreactivity strongly labelled AVCN and LSO neurons, while HCN2 staining was more diffuse in all nuclei. The HCN4 subunit displayed robust membrane staining in AVCN and MNTB cells but weak labelling of the LSO. We used a dynamic clamp, after blocking Ih, to reinsert Ih to the different cell types. Our results indicate that the native Ih for each cell type influences the resting membrane potential and can delay the generation of action potentials in response to injected current. Native Ih increases rebound depolarizations following hyperpolarizations in all cell types, and increases the likelihood of rebound action potentials (particularly in multiple‐firing LSO neurons). This systematic comparison shows that Ih characteristics vary considerably between different brainstem nuclei, and that these differences significantly affect the response properties of cells within these nuclei.

Chrna2-Martinotti Cells Synchronize Layer 5 Type A Pyramidal Cells via Rebound Excitation

Martinotti cells are the most prominent distal dendrite–targeting interneurons in the cortex, but their role in controlling pyramidal cell (PC) activity is largely unknown. Here, we show that the nicotinic acetylcholine receptor α2 subunit (Chrna2) specifically marks layer 5 (L5) Martinotti cells projecting to layer 1. Furthermore, we confirm that Chrna2-expressing Martinotti cells selectively target L5 thick-tufted type A PCs but not thin-tufted type B PCs. Using optogenetic activation and inhibition, we demonstrate how Chrna2-Martinotti cells robustly reset and synchronize type A PCs via slow rhythmic burst activity and rebound excitation. Moreover, using optical feedback inhibition, in which PC spikes controlled the firing of surrounding Chrna2-Martinotti cells, we found that neighboring PC spike trains became synchronized by Martinotti cell inhibition. Together, our results show that L5 Martinotti cells participate in defined cortical circuits and can synchronize PCs in a frequency-dependent manner. These findings suggest that Martinotti cells are pivotal for coordinated PC activity, which is involved in cortical information processing and cognitive control.

Sensorimotor function is modulated by the serotonin receptor 1d, a novel marker for gamma motor neurons.

Gamma motor neurons (MNs), the efferent component of the fusimotor system, regulate muscle spindle sensitivity. Muscle spindle sensory feedback is required for proprioception that includes sensing the relative position of neighboring body parts and appropriately adjust the employed strength in a movement. The lack of a single and specific genetic marker has long hampered functional and developmental studies of gamma MNs. Here we show that the serotonin receptor 1d (5-ht1d) is specifically expressed by gamma MNs and proprioceptive sensory neurons. Using mice expressing GFP driven by the 5-ht1d promotor, we performed whole-cell patch-clamp recordings of 5-ht1d::GFP⁺ and 5-ht1d::GFP⁻ motor neurons from young mice. Hierarchal clustering analysis revealed that gamma MNs have distinct electrophysiological properties intermediate to fast-like and slow-like alpha MNs. Moreover, mice lacking 5-ht1d displayed lower monosynaptic reflex amplitudes suggesting a reduced response to sensory stimulation in motor neurons. Interestingly, adult 5-ht1d knockout mice also displayed improved coordination skills on a beam-walking task, implying that reduced activation of MNs by Ia afferents during provoked movement tasks could reduce undesired exaggerated muscle output. In summary, we show that 5-ht1d is a novel marker for gamma MNs and that the 5-ht1d receptor is important for the ability of proprioceptive circuits to receive and relay accurate sensory information in developing and mature spinal cord motor circuits.

Sound stimulation modulates high-threshold K+ currents in mouse auditory brainstem neurons

The auditory system provides a valuable experimental model to investigate the role of sensory activity in regulating neuronal membrane properties. In this study, we have investigated the role of activity directly by measuring changes in medial nucleus of the trapezoid body (MNTB) neurons in normal hearing mice subjected to 1‐h sound stimulation. Broadband (4–12 kHz) chirps were used to activate MNTB neurons tonotopically restricted to the lateral MNTB, as confirmed by c‐Fos‐immunoreactivity. Following 1‐h sound stimulation a substantial increase in Kv3.1b‐immunoreactivity was measured in the lateral region of the MNTB, which lasted for 2 h before returning to control levels. Electrophysiological patch‐clamp recordings in brainstem slices revealed an increase in high‐threshold potassium currents in the lateral MNTB of sound‐stimulated mice. Current‐clamp and dynamic‐clamp experiments showed that MNTB cells from the sound‐stimulated mice were able to maintain briefer action potentials during high‐frequency firing than cells from control mice. These results provide evidence that acoustically driven auditory activity can selectively regulate high‐threshold potassium currents in the MNTB of normal hearing mice, likely due to an increased membrane expression of Kv3.1b channels.

Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCsα2) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCsα2 properties confirmed that neurons genetically marked by the Chrna2‐Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole‐cell patch‐clamp recordings revealed that RCsα2 constitute an electrophysiologically stereotyped population with a resting membrane potential of −50.5 ± 0.4 mV and an input resistance of 233.1 ± 11 MΩ. We identified a ZD7288‐sensitive hyperpolarization‐activated cation current (Ih) in all RCsα2, contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCsα2 to express small calcium‐activated potassium currents (ISK) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCsα2 can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.

Synchronization through nonreciprocal connections in a hybrid hippocampus microcircuit.

Synchronization among neurons is thought to arise from the interplay between excitation and inhibition; however, the connectivity rules that contribute to synchronization are still unknown. We studied these issues in hippocampal CA1 microcircuits using paired patch clamp recordings and real time computing. By virtually connecting a model interneuron with two pyramidal cells (PCs), we were able to test the importance of connectivity in synchronizing pyramidal cell activity. Our results show that a circuit with a nonreciprocal connection between pyramidal cells and no feedback from PCs to the virtual interneuron produced the greatest level of synchronization and mutual information between PC spiking activity. Moreover, we investigated the role of intrinsic membrane properties contributing to synchronization where the application of a specific ion channel blocker, ZD7288 dramatically impaired PC synchronization. Additionally, background synaptic activity, in particular arising from NMDA receptors, has a large impact on the synchrony observed in the aforementioned circuit. Our results give new insights to the basic connection paradigms of microcircuits that lead to coordination and the formation of assemblies.

Modulation of dendritic synaptic processing in the lateral superior olive by hyperpolarization-activated currents

We have previously shown that mice lateral superior olive (LSO) neurons exhibit a large hyperpolarization‐activated current (Ih), and that hyperpolarization‐activated cyclic‐nucleotide‐gated type 1 channels are present in both the soma and dendrites of these cells. Here we show that the dendritic Ih in LSO neurons modulates the integration of multiple synaptic inputs. We tested the LSO neuron’s ability to integrate synaptic inputs by evoking excitatory post‐synaptic potentials (EPSPs) in conjunction with brief depolarizing current pulses (to simulate a second excitatory input) at different time delays. We compared LSO neurons with the native Ih present in both the soma and dendrites (control) with LSO neurons without Ih (blocked with ZD7288) and with LSO neurons with Ih only present peri‐somatically (ZD7288+ computer‐simulated Ih using a dynamic clamp). LSO neurons without Ih had a wider time window for firing in response to inputs with short time separations. Simulated somatic Ih (dynamic clamp) could not reverse this effect. Blocking Ih also increased the summation of EPSPs elicited at both proximal and distal dendritic regions, and dramatically altered the integration of EPSPs and inhibitory post‐synaptic potentials. The addition of simulated peri‐somatic Ih could not abolish a ZD7288‐induced increase of responsiveness to widely separated excitatory inputs. Using a compartmental LSO model, we show that dendritic Ih can reduce EPSP integration by locally decreasing the input resistance. Our results suggest a significant role for dendritic Ih in LSO neurons, where the activation/deactivation of Ih can alter the LSO response to synaptic inputs.

Developmental disruption of recurrent inhibitory feedback results in compensatory adaptation in the Renshaw cell-motor neuron circuit

When activating muscles, motor neurons in the spinal cord also activate Renshaw cells, which provide recurrent inhibitory feedback to the motor neurons. The tight coupling with motor neurons suggests that Renshaw cells have an integral role in movement, a role that is yet to be elucidated. Here we used the selective expression of the nicotinic cholinergic receptor α2 (Chrna2) in mice to genetically target the vesicular inhibitory amino acid transporter (VIAAT) in Renshaw cells. Loss of VIAAT from Chrna2Cre-expressing Renshaw cells did not impact any aspect of drug-induced fictive locomotion in the neonatal mouse or change gait, motor coordination, or grip strength in adult mice of both sexes. However, motor neurons from neonatal mice lacking VIAAT in Renshaw cells received spontaneous inhibitory synaptic input with a reduced frequency, showed lower input resistance, and had an increased number of proprioceptive glutamatergic and calbindin-labeled putative Renshaw cell synapses on their soma and proximal dendrites. Concomitantly, Renshaw cells developed with increased excitability and a normal number of cholinergic motor neuron synapses, indicating a compensatory mechanism within the recurrent inhibitory feedback circuit. Our data suggest an integral role for Renshaw cell signaling in shaping the excitability and synaptic input to motor neurons. SIGNIFICANCE STATEMENT We here provide a deeper understanding of spinal cord circuit formation and the repercussions for the possible role for Renshaw cells in speed and force control. Our results suggest that while Renshaw cells are not directly required as an integral part of the locomotor coordination machinery, the development of their electrophysiological character is dependent on vesicular inhibitory amino acid transporter-mediated signaling. Further, Renshaw cell signaling is closely associated with the molding of motor neuron character proposing the existence of a concerted maturation process, which seems to endow this particular spinal cord circuit with the plasticity to compensate for loss of the Renshaw cell in adult circuit function.