Emilie A. Marcus

The Role of GSK3β in Regulating Neuronal Differentiation inXenopus laevis

The serine threonine protein kinase encoded by the shaggy locus has been implicated in neurogenesis in Drosophila. In vertebrates, the shaggy homolog, GSK3beta, is involved in early pattern formation, specifically in setting up the dorsal ventral axis. In the present study we have cloned the Xenopus homolog of the shaggy kinase and show (1) that GSK3beta is expressed in the right time and place to play a role in primary neurogenesis in Xenopus; (2) that overexpression of wild-type GSK3beta leads to a decrease in the number of primary neurons; (3) that inhibition of endogenous GSK3beta activity with overexpression of a dominant negative GSK3beta construct leads to an increase in the number of primary neurons; and (4) that GSK3beta inhibits the ability of neurogenin and NeuroD to produce ectopic tubulin expression, but does not inhibit the ability of neurogenin to produce ectopic NeuroD. On the basis of these data we propose that GSK3beta inhibits the function of NeuroD and therefore prevents neuronal differentiation at a relatively late stage in the developmental pathway.

A Cellular Analysis of Inhibition in the Siphon Withdrawal Reflex of Aplysia

Recent behavioral experiments examining the siphon withdrawal reflex of Aplysia have revealed inhibitory effects of strong tail shock, a stimulus commonly used as an unconditioned stimulus in studies of associative and nonassociative learning in Aplysia. We utilized a reduced preparation to perform a cellular analysis of tail shock- induced inhibition in the siphon withdrawal reflex. First, we carried out behavioral studies that showed that the reduced preparation exhibits a siphon withdrawal reflex to water jet stimuli, and that tail shock produces inhibitory behavioral effects comparable to those in the intact animal: (1) strong shock produces transient inhibition of nonhabituated responses, and (2) a habituated response is facilitated by weak shock, but not by strong shock, suggesting that increasing tail shock intensity recruits the inhibitory process that competes with facilitation of habituated reflexes. Next, we carried out cellular studies that showed that the amplitude of the complex EPSP in siphon motor neurons elicited by water jet stimuli to the siphon also exhibits the inhibitory patterns produced by tail shock: (1) the nondecremented complex EPSP (a neural correlate of a nonhabituated siphon withdrawal reflex) is significantly inhibited 90 sec after strong tail shock and recovers to preshock levels 10 min later, and (2) the decremented complex EPSP (a neural correlate of a habituated reflex) is significantly facilitated by weak shock, but is not facilitated by strong shock. In addition to the complex EPSP, we simultaneously examined the monosynaptic connection between siphon sensory neurons and siphon motor neurons. The monosynaptic EPSP does not show the pattern of inhibitory modulation by tail shock exhibited by the siphon withdrawal reflex and the complex EPSP: (1) the nondecremented monosynaptic EPSP is not inhibited 90 sec after strong shock, but tends to be above preshock levels; and (2) the decremented monosynaptic EPSP is facilitated by weak as well as strong tail shock. Our results suggest that an important component of the inhibitory process triggered by strong tail shock is mediated by neural elements presynaptic to the siphon motor neurons. Because modulation of the monosynaptic connection between identified siphon sensory and siphon motor neurons does not parallel the tail shock-induced inhibitory patterns observed in the siphon withdrawal reflex and in the complex EPSP, other synaptic connections are likely to play an important role in mediating tail shock-induced inhibition in the siphon withdrawal reflex.

Properties of Ectopic Neurons Induced byXenopusNeurogenin1 Misexpression

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3beta, which inhibits NeuroD function in vivo, blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.

A comparison of the mechanistic relationships between development and learning in Aplysia.

Publisher Summary This chapter discusses the cellular and molecular mechanisms of learning in Aplysia and uses it as the basis for comparison with the three principle stages of neuronal development: differentiation, neurite outgrowth, and synapse formation. The comparison of development and learning in Aplysia reveals a striking number of mechanistic similarities between these two processes. These observations lend substantial support to the hypothesis that growth mechanisms involved in the development of the nervous system persist into the adult where they subserve learning and memory. The adaptive properties of the adult nervous system represent a combination of two classes of mechanisms, one that includes retained developmental processes and the other that appears to be specifically related to adult plasticity; the distinguishing feature between the two classes is the dependence on the cell growth.

Transparency in authors’ contributions and responsibilities to promote integrity in scientific publication

In keeping with the growing movement in scientific publishing toward transparency in data and methods, we propose changes to journal authorship policies and procedures to provide insight into which author is responsible for which contributions, better assurance that the list is complete, and clearly articulated standards to justify earning authorship credit. To accomplish these goals, we recommend that journals adopt common and transparent standards for authorship, outline responsibilities for corresponding authors, adopt the Contributor Roles Taxonomy (CRediT) (docs.casrai.org/CRediT) methodology for attributing contributions, include this information in article metadata, and require authors to use the ORCID persistent digital identifier (https://orcid.org). Additionally, we recommend that universities and research institutions articulate expectations about author roles and responsibilities to provide a point of common understanding for discussion of authorship across research teams. Furthermore, we propose that funding agencies adopt the ORCID identifier and accept the CRediT taxonomy. We encourage scientific societies to further authorship transparency by signing on to these recommendations and promoting them through their meetings and publications programs.

Development of behavior and learning inAplysia

A set of fundamental issues in neuroethology concerns the neural mechanisms underlying behavior and behavioral plasticity. We have recently analyzed these issues by combining a simple systems approach in the marine molluscAplysia with a developmental analysis aimed at examining the emergence and maturation of different forms of behavior and learning. We have focussed on two kinds of questions: 1) How are specific neural circuits developmentally assembled to mediate different types of behaviors? and 2) how is plasticity integrated with these circuits to give rise to different forms of learning? From our analysis of the development of learning and memory inAplysia, several themes have emerged: 1) Different forms of learning emerge according to different developmental timetables. 2) Cellular analogs of learning have the same developmental timetables as their respective forms of behavioral learing. 3) An analysis of non-decremented responses prior to the emergence of sensitization reveals a novel inhibitory process on both behavioral and cellular levels. 4) Sensitization emerges simultaneously in diverse response systems, suggesting an underlying general process. 5) A widespread proliferation of central neurons occurs in the same developmental stage as the emergence of sensitization, raising the possibility that some aspect of the trigger for neuronal proliferation may also contribute to the expression of sensitization.

Ontogenetic analysis of learning in a simple system.

Over the past two decades significant progress has been made in our understanding of the neural mechanisms contributing to several forms of learning through the use of simple invertebrate model systems (Byrne, 1987; Carew & Sahley, 1986; Hawkins, Clark & Kandel, 1987). The power afforded by a simple systems approach arises mainly from the fact that the neurons in many invertebrate systems are large (in some cases 100-1000 times larger than vertebrate neurons) and therefore are readily accessible for biophysical and biochemical analyses. Moreover, neural circuits in invertebrates are often considerably simpler than those of vertebrates, sometimes consisting of as few as 20-30 cells (Selverston & Moulins, 1987). With several orders of magnitude fewer neurons, the central nervous system (CNS) of many invertebrates can be at least partially mapped as a series of identifiable cells with unique positions, sizes, transmitter phenotypes, and biophysical properties (Kandel, 1976; Kuffler, Nicholls & Martin, 1984). One result of this reduction in complexity is that in many cases, a single neuron can contribute significantly to a given behavior. Thus, it is often possible to assign specific cells unique roles in the generation of a particular behavioral response. In this chapter, we will discuss recent experiments in which we have combined an invertebrate simple system strategy with a developmental approach in order to examine at a mechanistic level how different forms of learning emerge and are assembled through ontogeny. The overall strategy of this work has been to characterize behaviorally when different forms of learning are first expressed in development and then to try to elucidate the synaptic, biophysical, and biochemical events that are added to the neural circuit underlying the behavior to give rise to the expression of the learning. The defensive gill and siphon withdrawal reflex of the marine mollusc Aplys~a offers several advantages for a developmental analysis of learning and its underlying cellular and biochemical mechanisms. It is known from work in adult animals that this reflex can exhibit a variety of forms of learning, ranging from simple nonassociative habituation, dishabituation, and sensitization to more complex higher order Pavlovian conditioning and operant conditioning (Carew, 1987). Many of these different forms of learning exist in both a short-term form (lasting minutes to hours) and a long-term form (lasting days to weeks). Moreover, the reflex is known to be