Amy B. MacDermott

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

Neurite outgrowth is a fundamental step in establishing proper neuronal connections in the developing central nervous system. Dynamic control of outgrowth has been attributed to changes in growth cone Ca²⁺levels in response to extracellular cues. Here we have investigated a possible role for Ca²⁺permeable kainate (KA) receptors in regulating neurite outgrowth of nociceptive‐like dorsal root ganglion (DRG) neurons. To identify KA receptor subunits likely to be involved, we used quantitative RT‐PCR on acutely dissociated DRG and dorsal horn neurons. DRG neurons expressed more GluK1, particularly the GluK1b spice variant, than dorsal horn neurons. Conversely, dorsal horn neurons expressed more GluK2, particularly GluK2a, than DRG neurons. Further, an RNA editing assay indicated that the majority of GluK1 and GluK2 mRNA transcripts in DRG were unedited. Imaging Ca²⁺transients following application of a KA receptor agonist to DRG and dorsal horn co‐cultures revealed increases in intracellular Ca²⁺in the growth cones of DRG neurons. In the majority of cases, this increase in Ca²⁺was partly or completely blocked by Joro spider toxin (JSTX), an antagonist for Ca²⁺‐permeable AMPA and KA receptors. Treatment of DRG/dorsal horn co‐cultures with KA for 18 hours suppressed neurite outgrowth while application of the rapidly desensitizing KA receptor agonist SYM 2081, the competitive AMPA/KA receptor antagonist, CNQX, and JSTX or philanthotoxin enhanced neurite outgrowth and prevented KA effects on neurite outgrowth. Thus, Ca²⁺entry through KA receptors at the growth cone of DRG neurons may be an important regulator of neurite outgrowth. © 2011 Wiley Periodicals, Inc. Develop Neurobiol 71: 818–835, 2011

Disinhibition in the dorsal horn accompanies peripheral nerve injury and causes the development of hypersensitivity to mild stimuli. This demonstrates the critical importance of inhibition in the dorsal horn for maintaining normal sensory signaling. Here we show that disinhibition induces a novel polysynaptic low‐threshold input onto lamina I output neurons, suggesting that inhibition normally suppresses a preexisting pathway that probably contributes to abnormal pain sensations such as allodynia. In addition, we show that a significant proportion of superficial dorsal horn inhibitory neurons are activated by low‐threshold input. These neurons are well situated to contribute to suppressing low‐threshold activation of pain output neurons in lamina I. We further discuss several aspects of inhibition in the dorsal horn that might contribute to suppressing pathological signaling.

Background: NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg²⁺sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter.Results: Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg²⁺sensitive receptors and the NR2C/D containing NMDA receptors with less Mg²⁺sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations.Conclusions: Stronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.

INTRODUCTION: Dorsal root ganglion (DRG) neurons grow readily alone or in coculture with embryonic dorsal horn (DH) neurons and even appear to enhance survival of the DH cells. Furthermore, DRG neurons grow long neurites in the presence of nerve growth factor, forming many glutamatergic synapses onto DH neurons, if present. Otherwise (i.e., in monocultures), no synapses can be detected electrophysiologically. When preparing cocultures, use DRGs already removed from the same embryos used to prepare DH neurons, but after dissecting the dorsal horns; the DRGs are less sensitive to delay in dissection and dissociation, as long as they are left in culture medium. Microisland and mass cultures require different proportions of DRG and dorsal horn neurons. Synaptic interactions in the microisland culture of DRG and DH neurons are more intense than mass culture because the growing, branching DRG axons are confined to a limited area, and thus make many synapses onto each target DH neuron.

INTRODUCTION: Primary cultures of embryonic dorsal horn (DH) neurons can be prepared using mass cultures, microisland cultures, or macroisland cultures. In all cases, long-term survival of neurons is enhanced by the presence of astrocytes. After the feeder layer of astrocytes has become nearly or fully confluent, it is ready to receive freshly dissociated neurons. Embryonic dorsal root ganglion (DRG) neurons can also be added to the cultures if DRG/DH neuron cocultures are desired. These cultures are suitable for electrophysiological, molecular, and immunocytochemical studies. DH neurons, including both excitatory and inhibitory neurons, form synapses with themselves (autapses) and with each other. DRG neurons form synapses onto DH neurons. Finding synaptically connected pairs of neurons is most readily achieved when the cells are grown on microislands. This protocol describes the dissociation, plating, and maintenance of DH neuron cultures using a feeder layer of astrocytes.

INTRODUCTION: Survival of central nervous system neurons in culture is usually greatly improved if the neurons are plated on top of a layer of confluent astrocytes. Under these conditions, neurons attach more firmly and develop a more readily identifiable bidimensional neuritic tree than in culture conditions where primary cells are plated on top of collagen, laminin, or other cell-free substrates. Although dissociated cells from any central nervous system region will include astrocytes and other glia, these generally do not support the survival of the primary neurons as well as preplated astrocyte feeder layers. For example, plating dorsal root ganglion (DRG)/dorsal horn (DH) cocultures directly onto collagen or laminin results in a very low number of attached DH neurons, and those have stunted neurites. Furthermore, although a higher number of DRG neurons do attach without astrocytes, this attachment is weaker, as demonstrated by the tendency of those neurons (or at least their neurites) to blow off the coverslip when a local superfusion system is directed at them. Neonatal rat cortex provides abundant astrocytes that can be cultured readily. The protocol presented here selects for type I astrocytes that grow in a monolayer with contact inhibition.

INTRODUCTION: This protocol describes methods for preparing glass coverslips to use with three different types of dissociated neuronal cultures: mass culture, microisland culture, and macroisland culture. In mass culture, the entire glass coverslip is precoated with poly-D-lysine and laminin and then plated with astrocytes followed by dissociated neurons. In this system, the plated neurons tend to form an extensive and widespread network of synapses. However, depending on the origin of the neurons, proximity might not be a good predictor of the presence of synapses between any two cells. Microisland culture aims to increase the probability of identifying neurons synaptically connected to each other; its basic principle is to restrict spatially the choice of connections. This is done by plating neurons on astrocyte-seeded collagen dots on a background of a nonadhesive agarose substrate. Synaptic interactions between dorsal horn (DH) neurons or between dorsal-root ganglion (DRG) and DH neurons on the microislands are reliably found using this system, whereas they are much harder to find in mass culture. The macroisland approach is similar, but generates larger islands. It was developed specifically to improve neuronal survival in DRG/DH cocultures while still providing a high probability of connection between DRG and DH neurons. For DH-DH synapses however, microislands are still the best system.

NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers typically composed of two NR1 and two of four NR2 subunits: NR2A-2D. Mice lacking specific NR2 subunits show deficits in pain transmission yet subunit location in the spinal cord remains unclear. We have combined electrophysiological and pharmacological approaches to investigate the composition of functional NMDA receptors expressed by lamina I, substance P receptor-expressing (NK1R+) neurons, as well as NK1R- neurons. Under low Mg²⁺ conditions (100 μM), the conductance of NMDA receptors at −90 mV (g(−90 mV)) with NR2A or NR2B subunits (NR2A/B) is low compared to conductance measured at the membrane potential where the inward current is maximal or maximal inward current (MIC) (ratio of ~0.07 calculated from Kuner and Schoepfer, 1996). For NR2C or NR2D subunits (NR2C/D), the ratio is higher (ratio ~0.4). NK1R+ and NK1R- neurons express NMDA receptors that give ratios ~0.28 and 0.16, respectively, suggesting both types of subunits are present in both populations of neurons, with NK1R+ neurons expressing a higher percentage of NR2C/D type NMDA receptors. This was confirmed using EAB318, an NR2A/B preferring antagonist, and UBP141, a mildly selective NR2C/D antagonist to increase and decrease the g(−90 mV)/g(MIC) ratios in both subpopulations of neurons.