Faculty Bibliography

Evidence showing expression of endogenous opioids in the mammalian retina is sparse. In the present study we examined a transgenic mouse line expressing an obligate dimerized form of Discosoma red fluorescent protein (DsRed) under the control of the pro-opiomelanocortin promoter and distal upstream regulatory elements to assess whether pro-opiomelanocortin peptide (POMC), and its opioid cleavage product, beta-endorphin, are expressed in the mouse retina. Using double label immunohistochemistry we found that DsRed fluorescence was restricted to a subset of GAD-67-positive cholinergic amacrine cells of both orthotopic and displaced subtypes. About 50% of cholinergic amacrine cells colocalized DsRed and a large fraction of DsRed-expressing amacrine cells was positive for beta-endorphin immunostaining, whereas beta-endorphin-immunoreactive neurons were absent in retinas of POMC null mice. Our findings contribute to a growing body of evidence demonstrating that opioid peptides are an integral component of vertebrate retinas, including those of mammals.

We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters

Somatodendritic dopamine (DA) release in the substantia nigra pars compacta (SNc) shows a limited dependence on extracellular calcium concentration ([Ca(2+)](o)), suggesting the involvement of intracellular Ca(2+) stores. Here, using immunocytochemistry we demonstrate the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) that sequesters cytosolic Ca(2+) into the endoplasmic reticulum (ER), as well as inositol 1,4,5-triphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in DAergic neurons. Notably, RyRs were clustered at the plasma membrane, poised for activation by Ca(2+) entry. Using fast-scan cyclic voltammetry to monitor evoked extracellular DA concentration ([DA](o)) in midbrain slices, we found that SERCA inhibition by cyclopiazonic acid (CPA) decreased evoked [DA](o) in the SNc, indicating a functional role for ER Ca(2+) stores in somatodendritic DA release. Implicating IP(3)R-dependent stores, an IP(3)R antagonist, 2-APB, also decreased evoked [DA](o). Moreover, DHPG, an agonist of group I metabotropic glutamate receptors (mGluR1s, which couple to IP(3) production), increased somatodendritic DA release, whereas CPCCOEt, an mGluR1 antagonist, suppressed it. Release suppression by mGluR1 blockade was prevented by 2-APB or CPA, indicating facilitation of DA release by endogenous glutamate acting via mGluR1s and IP(3)R-gated Ca(2+) stores. Similarly, activation of RyRs by caffeine increased [Ca(2+)](i) and elevated evoked [DA](o). The increase in DA release was prevented by a RyR blocker, dantrolene, and by CPA. Importantly, the efficacy of dantrolene was enhanced in low [Ca(2+)](o), suggesting a mechanism for maintenance of somatodendritic DA release with limited Ca(2+) entry. Thus, both mGluR1-linked IP(3)R- and RyR-dependent ER Ca(2+) stores facilitate somatodendritic DA release in the SNc

Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA-IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA-IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium-dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage-dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA-IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA-IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function

The goal of the present study was to elucidate the role of DARPP-32 (dopamine- and cyclic adenosine 3'-5'-monophosphate-regulated phosphoprotein, 32 kDa) in retinal function. We examined mouse and rat retinas for the presence of DARPP-32 by immunocytochemistry. In both rodent retinas DARPP-32 immunoreactivity was localized to horizontal and AII amacrine neurons and to the Mueller glial cells, using immuno-double labelling. Additional unidentified neurons in the amacrine cell layer also showed DARPP-32 immunoreactivity. Using mice entrained to a 12-12 h light-dark cycle, we found that exposure to light presented during the dark phase significantly enhanced phosphorylation of DARPP-32 at threonine (Thr) 34 and phosphorylation of the ionotropic glutamate receptor subunit GluR1 at serine (Ser) 845, as measured by immunoblots. However, light also increased Ser 845-GluR1 phosphorylation in DARPP-32-knockout mice. When a dopamine D1 receptor antagonist was injected into the eye prior to light exposure, phosphorylation of both Thr 34-DARPP-32 and Ser 845-GluR1 was significantly reduced. These data indicate that DARPP-32 participates in dopamine-mediated modifications of retinal function. We also tested for a possible circadian rhythm of Thr 34- and Thr 75-DARPP-32 and Ser 845-GluR1 expression. No significant circadian rhythm of either DARPP-32 or GluR1 phosphorylation was found

Genetically modified mice lacking the beta2 laminin chain (beta2null), the gamma3 laminin chain (gamma3 null), or both beta2/gamma3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in gamma3 null mice. In beta2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Muller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Muller glial cell and/or the retinal ganglion cell

This essay looks at the historical significance of three APS classic papers that are freely available online: Naka K-I and Nye PW. Role of horizontal cells in organization of the catfish retinal receptive field. J. Neurophysiol 34:785-801, 1971. Marmarelis PZ and Naka K-I. Nonlinear analysis and synthesis of receptive-field responses in the catfish retina. II. One-input white-noise analysis. J. Neurophysiol 36: 619-633, 1973. Naka K-I, Marmarelis PZ, and Chan RY. Morphological and functional identifications of catfish retinal neurons. III. Functional identification. J. Neurophysiol 38: 92-131, 1975

We studied by immunocytochemistry and Western blots the identity and cellular distribution of voltage-gated calcium channels within dopaminergic neurons of the rat retina. The aim was to associate particular calcium channel subtypes with known activities of the neuron (e.g., transmitter release from axon terminals). Five voltage-gated calcium channels were identified: alpha1A, alpha1B, alpha1E, alpha1F, and alpha1H. All of these, except the alpha1B subtype, were found within dopaminergic perikarya. The alpha1B channels were concentrated at axon terminal rings, together with alpha1A calcium channels. In contrast, alpha1H calcium channels were most abundant in the dendrites, and alpha1F calcium channels were restricted to the perikaryon. The alpha1E calcium channel was present at such a low density that its cellular distribution beyond the perikaryon could not be determined. Our findings are consistent with the available pharmacological data indicating that alpha1A and alpha1B calcium channels control the major fraction of dopamine release in the rat retina

The molecular organization of ribbon synapses in photoreceptors and ON bipolar cells is reviewed in relation to the process of neurotransmitter release. The interactions between ribbon synapse-associated proteins, synaptic vesicle fusion machinery and the voltage-gated calcium channels that gate transmitter release at ribbon synapses are discussed in relation to the process of synaptic vesicle exocytosis. We describe structural and mechanistic specializations that permit the ON bipolar cell to release transmitter at a much higher rate than the photoreceptor does, under in vivo conditions. We also consider the modulation of exocytosis at photoreceptor synapses, with an emphasis on the regulation of calcium channels