Debra E. Bramblett

The Transcription Factor Bhlhb4 Is Required for Rod Bipolar Cell Maturation

Retinal bipolar cells are essential to the transmission of light information. Although bipolar cell dysfunction can result in blindness, little is known about the factors required for bipolar cell development and functional maturation. The basic helix-loop-helix (bHLH) transcription factor Bhlhb4 was found to be expressed in rod bipolar cells (RB). Electroretinograms (ERGs) in the adult Bhlhb4 knockout (Bhlhb4(-/-)) showed that the loss of Bhlhb4 resulted in disrupted rod signaling and profound retinal dysfunction resembling human congenital stationary night blindness (CSNB), characterized by the loss of the scotopic ERG b-wave. A depletion of inner nuclear layer (INL) cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development. Other retinal cell populations including photoreceptors were unaltered. The timing of RB cell depletion in the Bhlhb4(-/-) mouse suggests that Bhlhb4 is essential for RB cell maturation.

Genetic Dissection of Rod and Cone Pathways in the Dark-Adapted Mouse Retina

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.

Direct rod input to cone BCs and direct cone input to rod BCs challenge the traditional view of mammalian BC circuitry

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by early anatomical studies is that bipolar cells in mammalian retinas receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Trα−/−], connexin36 knockout [Cx36−/−], and transcription factor beta4 knockout [Bhlhb4−/−]), we show that a subpopulation of rod DBCs (DBCR2s) receives substantial input directly from cones and a subpopulation of cone DBCs (DBCC1s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-DBCC and cone-DBCR synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma.

Relative contributions of rod and cone bipolar cell inputs to AII amacrine cell light responses in the mouse retina

AII amacrine cells (AIIACs) are crucial relay stations for rod‐mediated signals in the mammalian retina and they receive synaptic inputs from depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) as well as from other amacrine cells. Using whole‐cell voltage‐clamp technique in conjunction with pharmacological tools, we found that the light‐evoked current response of AIIACs in the mouse retina is almost completely mediated by two DBC synaptic inputs: a 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX)‐resistant component mediated by cone DBCs (DBCCs) through an electrical synapse, and a DNQX‐sensitive component mediated by rod DBCs (DBCRs). This scheme is supported by AIIAC current responses recorded from two knockout mice. The dynamic range of the AIIAC light response in the Bhlhb4−/− mouse (which lacks DBCRs) resembles that of the DNQX‐resistant component, and that of the connexin36 (Cx36)−/− mouse resembles the DNQX‐sensitive component. By comparing the light responses of the DBCCs with the DNQX‐resistant AIIAC component, and light responses of the DBCRs with the DNQX‐sensitive AIIAC component, we obtained the input–output relations of the DBCC→AIIAC electrical synapse and the DBCR→AIIAC chemical synapse. Similar to other glutamatergic chemical synapses in the retina, the DBCR→AIIAC synapse is non‐linear. Its highest voltage gain (approximately 5) is found near the dark membrane potential, and it saturates for presynaptic signals larger than 5.5 mV. The DBCC→AIIAC electrical synapse is approximately linear (voltage gain of 0.92), consistent with the linear junctional conductance found in retinal electrical synapses. Moreover, relative DBCR and DBCC contributions to the AIIAC response at various light intensity levels are determined.

Pancreatic Islet Development

Publisher Summary Appropriately, research with emphasis on the endocrine pancreas has grown; in fact, a virtual explosion of discoveries, regarding pancreas development, has occurred over the past few years. The goal in this field is to identify the molecular events that govern pancreas functionality and the genetic components that delineate these events. Ultimately, these findings will lead to the better treatment and prevention of pancreatic disease. This chapter discusses many of the recent findings related to the molecular and developmental biology of the pancreas. The emphasis of the most recent studies, and consequently this survey is the specification and differentiation of pancreatic endocrine cells during ontogeny. In terms of gene regulation, much is now known about how pancreatic hormone genes are regulated. Many of the promoter elements that dictate islet-cell-specific gene expression have been characterized and many of the protein factors that mediate these activation and repression events have been identified. The chapter discusses a wide range of topics, ranging from pancreatic gene regulation to pancreatic islet development. Factors determined to influence islet-cell differentiation and the signaling factors that may specify the pancreatic cell fate have been discussed in the chapter. Pancreatic disorders can and do arise from the defects at all levels of pancreas development, involving inappropriate transcriptional regulation, cellular differentiation, and extracellular and intracellular signaling, as well as enzymatic dysfunction and organogenesis.

Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes

Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα(-/-) mice, but indistinguishable from controls in Cx36(-/-) and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα(-/-) mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36(-/-) mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night visions ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways.

A role for bHLH transcription factors in retinal degeneration and dysfunction.

The basic helix loop helix (bHLH) transcription factors collectively mediate cellular differentiation in almost every type of tissue including the retina (Murre et al. 1989; Jan and Jan 1993; Cepko 1999). Class A factors are ubiquitously expressed throughout mammalian tissue, while the expression of class B factors are cell type specific. These factors have both a DNA binding domain and helix loop helix domain (HLH) protein dimerization domain. Class B factors usually heterodimerize with the ubiquitously expressed, bHLH factors, such as E12/E47. Because of their importance during photoreceptor development, bHLH factors are candidate genes for photoreceptor degeneration. We have examined the roles of two bHLH factors, both which are expressed during retinal development, but also share the property of continued expression in the adult retina.

A Laboratory for Education in Molecular Medicine: a Dedicated Resource for Medical Student Research

The Paul L. Foster School of Medicine in El Paso, Texas seated its inaugural class in 2009 and introduced a highly-integrated pre-clinical curriculum that provides our students with a solid introduction to the scientific principles of medicine, medical skills, early clinical experiences, ethics and professionalism. To further enhance their undergraduate training, all students additionally complete a scholarly concentration requirement called the Scholarly Activity and Research Program (SARP). Students can choose a wide variety of topics for this faculty-mentored activity; however, about two-thirds of the students choose projects relating to basic, clinical or translational research. To broaden the on-campus opportunities for students in these areas we have developed a research laboratory, called the Laboratory for Education in Molecular Medicine (LEMM), that is fully-dedicated for mentored SARP projects. This ‘community’ laboratory is housed in the Department of Medical Education and represents a unique model for the establishment and development of viable research projects. We discuss the evolution of the LEMM, its current organization and the challenges and opportunities in maintaining and growing this valuable resource.