Sharmila Venugopal

Ca2+ signaling in astrocytes from Ip3r2-/- mice in brain slices and during startle responses in vivo

Intracellular Ca2+ signaling is considered to be important for multiple astrocyte functions in neural circuits. However, mice devoid of inositol triphosphate type 2 receptors (IP3R2) reportedly lack all astrocyte Ca2+ signaling, but display no neuronal or neurovascular deficits, implying that astrocyte Ca2+ fluctuations are not involved in these functions. An assumption has been that the loss of somatic Ca2+ fluctuations also reflects a similar loss in astrocyte processes. We tested this assumption and found diverse types of Ca2+ fluctuations in astrocytes, with most occurring in processes rather than in somata. These fluctuations were preserved in Ip3r2−/− (also known as Itpr2−/−) mice in brain slices and in vivo, occurred in end feet, and were increased by G protein–coupled receptor activation and by startle-induced neuromodulatory responses. Our data reveal previously unknown Ca2+ fluctuations in astrocytes and highlight limitations of studies that used Ip3r2−/− mice to evaluate astrocyte contributions to neural circuit function and mouse behavior.

Local circuit input to the medullary reticular formation from the rostral nucleus of the solitary tract

The intermediate reticular formation (IRt) subjacent to the rostral (gustatory) nucleus of the solitary tract (rNST) receives projections from the rNST and appears essential to the expression of taste-elicited ingestion and rejection responses. We used whole cell patch-clamp recording and calcium imaging to characterize responses from an identified population of prehypoglossal neurons in the IRt to electrical stimulation of the rNST in a neonatal rat pup slice preparation. The calcium imaging studies indicated that IRt neurons could be activated by rNST stimulation and that many neurons were under tonic inhibition. Whole cell patch-clamp recording revealed mono- and polysynaptic projections from the rNST to identified prehypoglossal neurons. The projection was primarily excitatory and glutamatergic; however, there were some inhibitory GABAergic projections, and many neurons received excitatory and inhibitory inputs. There was also evidence of disinhibition. Overall, bath application of GABA(A) antagonists increased the amplitude of excitatory currents, and, in several neurons, stimulation of the rNST systematically decreased inhibitory currents. We have hypothesized that the transition from licks to gapes by natural stimuli, such as quinine monohydrochloride, could occur via such disinhibition. We present an updated dynamic model that summarizes the complex synaptic interface between the rNST and the IRt and demonstrates how inhibition could contribute to the transition from ingestion to rejection.

Modulation of inhibitory strength and kinetics facilitates regulation of persistent inward currents and motoneuron excitability following spinal cord injury

Spasticity is commonly observed after chronic spinal cord injury (SCI) and many other central nervous system disorders (e.g., multiple sclerosis, stroke). SCI-induced spasticity has been associated with motoneuron hyperexcitability partly due to enhanced activation of intrinsic persistent inward currents (PICs). Disrupted spinal inhibitory mechanisms also have been implicated. Altered inhibition can result from complex changes in the strength, kinetics, and reversal potential (E(Cl(-))) of γ-aminobutyric acid A (GABA(A)) and glycine receptor currents. Development of optimal therapeutic strategies requires an understanding of the impact of these interacting factors on motoneuron excitability. We employed computational methods to study the effects of conductance, kinetics, and E(Cl(-)) of a dendritic inhibition on PIC activation and motoneuron discharge. A two-compartment motoneuron with enhanced PICs characteristic of SCI and receiving recurrent inhibition from Renshaw cells was utilized in these simulations. This dendritic inhibition regulated PIC onset and offset and exerted its strongest effects at motoneuron recruitment and in the secondary range of the current-frequency relationship during PIC activation. Increasing inhibitory conductance compensated for moderate depolarizing shifts in E(Cl(-)) by limiting PIC activation and self-sustained firing. Furthermore, GABA(A) currents exerted greater control on PIC activation than glycinergic currents, an effect attributable to their slower kinetics. These results suggest that modulation of the strength and kinetics of GABA(A) currents could provide treatment strategies for uncontrollable spasms.

Intrinsic Membrane Properties of Pre-oromotor Neurons in the Intermediate Zone of the Medullary Reticular Formation

Neurons in the lower brainstem that control consummatory behavior are widely distributed in the reticular formation (RF) of the pons and medulla. The intrinsic membrane properties of neurons within this distributed system shape complex excitatory and inhibitory inputs from both orosensory and central structures implicated in homeostatic control to produce coordinated oromotor patterns. The current study explored the intrinsic membrane properties of neurons in the intermediate subdivision of the medullary reticular formation (IRt). Neurons in the IRt receive input from the overlying (gustatory) nucleus of the solitary tract and project to the oromotor nuclei. Recent behavioral pharmacology studies as well as computational modeling suggest that inhibition in the IRt plays an important role in the transition from a taste-initiated oromotor pattern of ingestion to one of rejection. The present study explored the impact of hyperpolarization on membrane properties. In response to depolarization, neurons responded with either a tonic discharge, an irregular/burst pattern or were spike-adaptive. A hyperpolarizing pre-pulse modulated the excitability of most (82%) IRt neurons to subsequent depolarization. Instances of both increased (30%) and decreased (52%) excitability were observed. Currents induced by the hyperpolarization included an outward 4-aminopyridine (4-AP) sensitive K+ current that suppressed excitability and an inward cation current that increased excitability. These currents are also present in other subpopulations of RF neurons that influence the oromotor nuclei and we discuss how these currents could alter firing characteristics to impact pattern generation.

Homeostatic Dysregulation in Membrane Properties of Masticatory Motoneurons Compared with Oculomotor Neurons in a Mouse Model for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motoneuron disease with presently no cure. Motoneuron (MN) hyperexcitability is commonly observed in ALS and is suggested to be a precursor for excitotoxic cell death. However, it is unknown whether hyperexcitability also occurs in MNs that are resistant to degeneration. Second, it is unclear whether all the MNs within homogeneous motor pools would present similar susceptibility to excitability changes since high-threshold MNs innervating fast fatigable muscle fibers selectively degenerate compared with low-threshold MNs innervating fatigue resistant slow muscle fibers. Therefore, we concurrently examined the excitability of ALS-vulnerable trigeminal motoneurons (TMNs) controlling jaw musculature and ALS-resistant oculomotor neurons (OMNs) controlling eye musculature in a well studied SOD1G93A ALS mouse model using in vitro patch-clamp electrophysiology at presymptomatic ages P8–P12. Our results show that hyperexcitability is not a global change among all the MNs, although mutant SOD1 is ubiquitously expressed. Instead, complex changes occur in ALS-vulnerable TMNs based on motor unit type and discharge characteristics. Firing threshold decreases among high-threshold TMNs and increases in a subpopulation of low-threshold TMNs. The latter group was identified based on their linear frequency–current responses to triangular ramp current injections. Such complex changes in MN recruitment were absent in ALS-resistant OMNs. We simulated the observed complex changes in TMN excitability using a computer-based jaw closer motor pool model. Model results suggest that hypoexcitability may indeed represent emerging disease symptomology that causes resistance in muscle force initiation. Identifying the cellular and molecular properties of these hypoexcitable cells may guide effective therapeutic strategies in ALS.

Differential contributions of somatic and dendritic calcium-dependent potassium currents to the control of motoneuron excitability following spinal cord injury

The hyperexcitability of alpha-motoneurons and accompanying spasticity following spinal cord injury (SCI) have been attributed to enhanced persistent inward currents (PICs), including L-type calcium and persistent sodium currents. Factors controlling PICs may offer new therapies for managing spasticity. Such factors include calcium-activated potassium (KCa) currents, comprising in motoneurons an after-hyperpolarization-producing current (IKCaN) activated by N/P-type calcium currents, and a second current (IKCaL) activated by L-type calcium currents (Li and Bennett in J neurophysiol 97:767–783, 2007). We hypothesize that these two currents offer differential control of PICs and motoneuron excitability based on their probable somatic and dendritic locations, respectively. We reproduced SCI-induced PIC enhancement in a two-compartment motoneuron model that resulted in persistent dendritic plateau potentials. Removing dendritic IKCaL eliminated primary frequency range discharge and produced an abrupt transition into tertiary range firing without significant changes in the overall frequency gain. However, IKCaN removal mainly increased the gain. Steady-state analyses of dendritic membrane potential showed that IKCaL limits plateau potential magnitude and strongly modulates the somatic injected current thresholds for plateau onset and offset. In contrast, IKCaN had no effect on the plateau magnitude and thresholds. These results suggest that impaired function of IKCaL may be an important intrinsic mechanism underlying PIC-induced motoneuron hyperexcitability following SCI.

Role of low and high-voltage activated Ca2+-dependent K+ currents in the control of alpha-motoneuron discharge and its implication in hyperreflexia

Specificity of calcium-activated potassium (K+) currents to different sources of calcium has been noted in many neurons (e.g.1). Recently, in spinal alpha-motoneurons (α-MN), it was shown that the low-voltage activated L-type calcium currents (also known as persistent calcium currents) activate an exclusive subset of small conductance K+ currents (SKL)2. The SKL currents were distinct from the medium after-hyperpolarization (mAHP) producing N/P-type calcium activated K+ currents (SKAHP currents). The same study further suggested that an enhancement of persistent calcium current often observed after chronic spinalization can in part be due to reduced availability of the SKL channels albeit mAHP remained unchanged. While mAHP has been suggested to be integral in controlling motoneuron firing frequencies and grading L-Ca activation, the role of SKL currents in motoneuron discharge is unknown. The goal of this study is to characterize the influence of SKAHP and SKL currents on motoneuron firing frequencies. Here we test the hypothesis that SKAHP and SKL currents play differential roles in the control of persistent inward currents that are key determinants of motoneuron excitability.

Role of inhibition in the suppression of α-motoneuron hyper-excitability following chronic spinal cord injury

Introduction The monosynaptic spinal stretch reflex consists of glutamatergic Ia muscle spindle afferents synapsing on αmotoneuron (α-MN) dendrites. Also, renshaw cells (RC) mediate a direct recurrent inhibition of α-MNs potentially via GABAA and glycinergic receptors. The RC synapses are confined to α-MN dendrites. Several studies have implicated a GABAB receptor mediated pre-synaptic inhibition of the Ia terminal during reflex generation. Supra-spinal inputs further modulate the efficacy of the synaptic inputs to the α-MN, e.g. brainstem nuclei exert a tonic monoaminergic inhibition on RCs. Following spinal cord injury (SCI), hyper-reflexia and motor spasticity occur with concomitant α-MN hyper-excitability. The hyper-excitability has largely been attributed to an enhancement of dendritic persistent inward currents (PICs), while inhibitory pathways may also play a role. However, the effect of a combination of PIC enhancement and changes in inhibitory inputs on α-MN excitability is yet unclear [1]. In this study, we use a network model for the monosynaptic stretch reflex with RC-type recurrent inhibition of the αMN to test the hypothesis that GABAergic inputs are essential for suppressing α-MN hyper-excitability after chronic SCI.

Early dysregulation of trigeminal motor pool excitability in a mouse model for neurodegenerative motoneuron disease

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative motoneuron (MN) where in fast fatigable motor units (MUs) of vulnerable motor pools preferentially degenerate followed by fast fatigue resistant and slow MUs . Excitability is a key endogenous mechanism of MN neuroprotection [1] and therefore we hypothesize that pre-symptomatic excitability indicates impending disease development. Using a transgenic mouse model for ALS, we performed in vitro patchclamp electrophysiology in ALS-vulnerable trigeminal motoneurons (TMNs) retrogradely labeled from jaw closer muscles at P8-12. We proposed a novel k-means clustering approach to classify TMNs into putative fast fatigable (PFF), fast fatigue resistant (PFR) and slow (PS) MUs based on rheobase and input resistance. Interestingly, hyper-excitability was noted in PFF and PFR

A self-organizing neural network for neuromuscular control

Adaptive technology holds great promise for sensorimotor rehabilitation in people suffering from spinal cord injury, neuromuscular disease and stroke. With a long-term goal of developing adaptive technology for diagnosis and rehabilitation of neuromuscular dysfunction, we begin the development of a self-organizing neural network (SNN) that compensates for reduced neural drive. We suggest that the self-organizing architecture that adds or deletes nodes online to generate suitable compensatory muscle excitation (Figure ​(Figure1A)1A) is an apt mechanism to emulate the motor pool behavior of recruitment and de-recruitment of motor units during muscle force generation. Using a virtual muscle [1] resembling the human biceps brachii, we demonstrate the augmentation of neural excitation by the SNN to compensate for abnormal muscle force due to change in the number of motor units. Figure 1 A. Schematic showing the virtual muscle-SNN system; Φ1, Φ2, .. Φn are radial basis functions and w1, w2, ..wn are weights for summation. B. Simulation of normal (Slow-Fast motor unit ratio - 2:4), abnormal (Slow-Fast motor unit ...