Faculty Bibliography

We examined developmental changes in dopaminergic (DA) neurons of rat pups between postnatal (P) days 3 and 21. DA cell bodies and dendrites grew progressively between P3-15. Voltage-sensitive sodium channels were present in axons at P11, but the ring-like DA axon terminals appeared only during the third postnatal week. The density of ring terminals increased markedly between P15 and P21. The vesicular monoamine transporter (VMAT2) was absent before P13 and became concentrated in DA ring terminals after P17. A steady increase in VMAT2-containing rings around AII amacrine cells occurred during the third postnatal week. The presynaptic membrane protein SNAP-25 colocalized with DA terminals, but several other presynaptic proteins tested, including synaptotagmin I, synapsin, bassoon, syntaxin, and synaptogyrin, appeared not to be associated with DA neurons. Our study shows that the somatodendritic compartment of DA neurons matures before the DA axon terminals do. Maturation of DA axons during the third postnatal week corresponds to the period of onset of visual function

We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABA(A) and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves

We investigated parvalbumin immunoreactivity (PA-IR) in the retinas of rats maintained on a 12:12 h light:dark cycle, or after being placed in constant darkness for 24-72 h. Retinas were harvested at zeitgeber and circadian times 02:00, 06:00, 10:00, 14:00, 18:00 and 22:00 h. PA-IR was found primarily in retinal amacrine cells of the AII subtype. In a light/dark cycle, PA-IR showed a clear rhythm, with a low near zeitgeber time (ZT) 10:00 h and a peak near ZT 18:00 h. The ratio of immunofluorescence intensities at these timepoints was >15-fold. When animals were kept in complete darkness for 1-3 days, the rhythm of PA-IR was still preserved, but was progressively reduced in amplitude. The rhythm of PA-IR inferred from immunohistochemical data was confirmed by Western blots. We conclude that PA-IR in the rat retina shows an underlying circadian rhythm that is enhanced by cyclic light. The regulation may involve translocation of the protein between cell compartments and/or new protein synthesis

This review summarizes the experimental evidence in support of dopamine's role as a chemical messenger for light adaptation. Dopamine is released by a unique set of amacrine cells and activates D1 and D2 dopamine receptors distributed throughout the retina. Multiple dopamine-dependent physiological mechanisms result in an increased signal flow through cone circuits and a diminution of signal flow through rod circuits. Dopamine also has multiple trophic roles in retinal function related to circadian rhythmicity, cell survival and eye growth. In a reciprocal way, the health of the dopaminergic neurons depends on their receiving light-driven synaptic inputs. Dopamine neurons appear early in development, become functional in advance of the animal's onset of vision and begin to die in aging animals. Some diseases affecting photoreceptor function also diminish day/night differences in dopamine release and turnover. A reduction in retinal dopamine, as occurs in Parkinsonian patients, results in reduced visual contrast sensitivity

We obtained intracellular recordings from transient, On-Off amacrine and ganglion cells of the turtle retina. We tested the ability of neurotransmitter agonists and antagonists to modify the responses to light stimuli. The metabotropic glutamate agonist, 2-amino-phosphonobutyric acid (APB), selectively blocked On responses, whereas the amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor antagonist, GYKI, blocked both On and Off responses. Although GYKI appeared to block excitation completely, suggesting an absence of N-methyl-d-aspartate (NMDA)-mediated responses, it was found that in the presence of ionotropic gamma-aminobutyric acid (GABA) blockers, the excitatory postsynaptic potential (EPSP) was prolonged. The late component of the EPSP was blocked by the NMDA antagonist, D-2-amino-5-phosphopentanoic acid (D-AP5). Picrotoxin (PTX) and bicuculline (BCC) induced a mean hyperpolarization of -6.4 mV, suggesting a direct effect of GABA on transient amacrine and ganglion cells, since antagonism of a GABA-mediated inhibition of release of glutamate by bipolars would depolarize third-order neurons. The acetylcholine agonist, carbachol, or the nicotinic agonist, epibatidine, depolarized all On-Off neurons. This action was blocked by d-tubocurarine. Cholinergic inputs to On-Off neurons increase their excitability without altering the pattern of light responsiveness

The cellular location and rhythmic expression of Period 1 (Per1) circadian clock gene were examined in the retina of a Per1::GFP transgenic mouse. Mouse Per1 (mPer1) RNA was localized to inner nuclear and ganglion cell layers but was absent in the outer nuclear (photoreceptor) layer. Green fluorescent protein (GFP), which was shown to colocalize with PER1 protein, was found in a few subtypes of amacrine neuron, including those containing tyrosine hydroxylase, calbindin, and calretinin, but not in cholinergic amacrine cells. A small subset of ganglion cells also contained GFP immunoreactivity (GFP-IR), but the melanopsin-containing subtype, which projects to the suprachiasmatic nuclei (SCN), lacked GFP-IR. Although the intensity of GFP-IR varied among the populations of amacrine cells at each time point that was examined, both diurnal and circadian rhythms were found for the fraction of neurons showing strong GFP-IR, with peak expression between Zeitgeber/circadian (ZT/CT) times 10 and 14. In SCNs that were examined in the same mice used for the retinal measures, the peak in GFP-IR also occurred at approximately ZT/CT 10. Our results are the first to demonstrate a circadian rhythm of a biological clock component in identified neurons of a mammalian retina

We examined synaptic transmission between rods or cones and horizontal cells, using perforated patch recording techniques in salamander retinal slices. Experimental conditions were established under which horizontal cells received nearly pure rod or pure cone input. The response-intensity relation for both photoreceptors and horizontal cells was described by a Michaelis-Menten function with an exponent close to 1. A dynamic model was developed for the transduction from photoreceptor voltage to postsynaptic current. The basic model assumes that: (i) photoreceptor light-evoked voltage controls Ca2+ entry according to a Boltzmann relation; (ii) the rate of glutamate release depends linearly on the voltage-gated Ca2+ current (ICa) in the synaptic terminal; (iii) glutamate concentration in the synaptic cleft reflects the balance of release and reuptake in which reuptake obeys first order kinetics; (iv) the binding of glutamate to its receptor and channel gating are fast compared with glutamate kinetics in the synaptic cleft. The good fit to the model confirms that these are the key features of synaptic transmission from rods and cones. The model accommodated changes in kinetics induced by the glutamate uptake blocker, dihydrokainate. The match between model and response was not improved by including an estimate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor desensitization or by making glutamate uptake voltage dependent.

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain

Activation of D2-like dopamine receptors in rods with quinpirole stimulates L-type calcium currents (ICa). This result appears inconsistent with studies showing that D2-like dopamine receptor activation diminishes rod signals in second-order retinal neurons. Since small reductions in [Cl-] can inhibit photoreceptor ICa, we tested the hypothesis that enhancement of ICa with the D2/D4 receptor agonist, quinpirole, increases calcium-activated chloride currents (ICl(Ca)) causing an efflux of Cl- from rods that would provide a negative feedback inhibition of ICa. In agreement with studies from Xenopus, quinpirole reduced rod input to second-order neurons of tiger salamander retina without significantly altering rod voltage responses. Quinpirole also diminished the amplitude of depolarization-evoked increases in [Ca2+]i measured with Fura-2 in rods, a finding consistent with inhibition of synaptic transmission from rods. Electrophysiological and Cl(-)-imaging experiments indicated ECl in rods is approximately -20 mV. Quinpirole enhanced ICl(Ca) and elicited an efflux of Cl- at the resting potential. A similar Cl- efflux was produced by extracellular replacement of 24 mM Cl- with CH3SO4- and this low Cl- solution inhibited Ca2+ responses to a similar degree as quinpirole did. When ICl(Ca) was inhibited with niflumic acid, quinpirole enhanced both ICa and depolarization-evoked increases in [Ca2+]i. Furthermore, with niflumic acid, quinpirole no longer inhibited rod inputs into horizontal and bipolar cells. These results suggest an initial enhancement of ICa by quinpirole is followed by a stimulation of Cl- currents, including ICl(Ca). The net result is a Cl- efflux that inhibits depolarization-evoked increases in [Ca2+]i and synaptic transmission from rods