Stuart C. Mangel

Dendritic compartmentalization of chloride cotransporters underlies directional responses of starburst amacrine cells in retina

The mechanisms in the retina that generate light responses selective for the direction of image motion remain unresolved. Recent evidence indicates that directionally selective light responses occur first in the retina in the dendrites of an interneuron, i.e., the starburst amacrine cell, and that these responses are highly sensitive to the activity of Na-K-2Cl (NKCC) and K-Cl (KCC), two types of chloride cotransporter that determine whether the neurotransmitter GABA depolarizes or hyperpolarizes neurons, respectively. We show here that selective blockade of the NKCC2 and KCC2 cotransporters located on starburst dendrites consistently hyperpolarized and depolarized the starburst cells, respectively, and greatly reduced or eliminated their directionally selective light responses. By mapping NKCC2 and KCC2 antibody staining on these dendrites, we further show that NKCC2 and KCC2 are preferentially located in the proximal and distal dendritic compartments, respectively. Finally, measurements of the GABA reversal potential in different starburst dendritic compartments indicate that the GABA reversal potential at the distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. These results thus demonstrate that the differential distribution of NKCC2 on the proximal dendrites and KCC2 on the distal dendrites of starburst cells results in a GABA-evoked depolarization and hyperpolarization at the NKCC2 and KCC2 compartments, respectively, and underlies the directionally selective light responses of the dendrites. The functional compartmentalization of interneuron dendrites may be an important means by which the nervous system encodes complex information at the subcellular level.

A circadian clock in the fish retina regulates dopamine release via activation of melatonin receptors

Although many biochemical, morphological and physiological processes in the vertebrate retina are controlled by a circadian (24 h) clock, the location of the clock and how the clock alters retinal function are unclear. For instance, several observations have suggested that dopamine, a retinal neuromodulator, may play an important role in retinal rhythmicity but the link between dopamine and a clock located within or outside the retina remains to be established. We found that endogenous dopamine release from isolated goldfish retinae cultured in continuous darkness for 56 h clearly exhibited a circadian rhythm with high values during the subjective day. The continuous presence of melatonin (1 nm) in the culture medium abolished the circadian rhythm of dopamine release and kept values constantly low and equal to the night‐time values. The selective melatonin antagonist luzindole (1  μm) also abolished the dopamine rhythm but the values were high and equal to the daytime values. Melatonin application during the late subjective day introduced rod input and reduced cone input to fish cone horizontal cells, a state usually observed during the subjective night. In contrast, luzindole application during the subjective night decreased rod input and increased cone input. Prior application of dopamine or spiperone, a selective dopamine D2‐like antagonist, blocked the above effects of melatonin and luzindole, respectively. These findings indicate that a circadian clock in the vertebrate retina regulates dopamine release by the activation of melatonin receptors and that endogenous melatonin modulates rod and cone pathways through dopamine‐mediated D2‐like receptor activation.

Dopamine mediates circadian clock regulation of rod and cone input to fish retinal horizontal cells

A circadian (24‐hour) clock regulates the light responses of fish cone horizontal cells, second order neurones in the retina that receive synaptic contact from cones and not from rods. Due to the action of the clock, cone horizontal cells are driven by cones in the day, but primarily driven by rods at night. We show here that dopamine, a retinal neurotransmitter, acts as a clock signal for the day by increasing cone input and decreasing rod input to cone horizontal cells. The amount of endogenous dopamine released from in vitro retinae was greater during the subjective day than the subjective night. Application of dopamine or quinpirole, a dopamine D2‐like agonist, during the subjective night increased cone input and eliminated rod input to the cells, a state usually observed during the subjective day. In contrast, application of spiperone, a D2‐like antagonist, or forskolin, an activator of adenylyl cyclase, during the subjective day reduced cone input and increased rod input. SCH23390, a D1 antagonist, had no effect. Application of Rp‐cAMPS, an inhibitor of cAMP‐dependent protein kinase, or octanol, an alcohol that uncouples gap junctions, during the night increased cone input and decreased rod input. Because D2‐like receptors are on photoreceptor cells, but not horizontal cells, the results suggest that the clock‐induced increase in dopamine release during the day activates D2‐like receptors on photoreceptor cells. The resultant decrease in intracellular cyclic AMP and protein kinase A activation then mediates the increase in cone input and decrease in rod input.

A circadian clock regulates the pH of the fish retina

Although it is generally accepted that the acid/base ratio of tissue, as represented by the pH, is strictly regulated to maintain normal function, recent studies in the nervous system have shown that neuronal activity can result in significant shifts in pH. In the vertebrate retina, many cellular phenomena, including neuronal activity, are regulated by a circadian clock. We thus investigated whether a circadian clock regulates the pH of the retina. pH‐sensitive microelectrodes were used to measure the extracellular pH of the in vitro goldfish retina superfused with a bicarbonate‐based Ringer solution in the subjective day and night; that is, under conditions of constant darkness. These measurements demonstrated that a circadian clock regulates the pH of the vertebrate retina so that the pH is lower at night compared to the day. This day‐night difference in retinal pH was observed at two different values of Ringer solution pH, indicating that the circadian phenomenon is independent of the superfusion conditions. The circadian‐induced shift in pH was several times greater than light‐induced pH changes and large enough to influence synaptic transmission between retinal neurons. These findings indicate that a circadian clock regulates the pH of the vertebrate retina. Thus, an intrinsic oscillator in neural tissue may modulate metabolic activity and pH as part of normal daily function.

Absence of circadian clock regulation of horizontal cell gap junctional coupling reveals two dopamine systems in the goldfish retina

In fish and other vertebrate retinas, although dopamine release is regulated by both light and an endogenous circadian (24‐hour) clock, light increases dopamine release to a greater extent than the clock. The clock increases dopamine release during the subjective day so that D2‐like receptors are activated. It is not known, however, whether the retinal clock also activates D1 receptors, which display a much lower sensitivity to dopamine in intact tissue. Because activation of the D1 receptors on fish cone horizontal (H1) cells uncouples the gap junctions between the cells, we studied whether the clock regulates the extent of biocytin tracer coupling in the goldfish retina. Tracer coupling between H1 cells was extensive under dark‐adapted conditions (low scotopic range) and similar in the subjective day, subjective night, day, and night. An average of approximately 180 cells were coupled in each dark‐adapted condition. However, bright light stimulation or application of the D1 agonist SKF38393 (10 μM) dramatically reduced H1 cell coupling. The D2 agonist quinpirole (1 μM) or application of the D1 antagonist SCH23390 (10 μM) and/or the D2 antagonist spiperone (10 μM) had no effect on H1 cell coupling in dark‐adapted retinas. These observations demonstrate that H1 cell gap junctional coupling and thus D1 receptor activity are not affected by endogenous dopamine under dark‐adapted conditions. The results suggest that two different dopamine systems are present in the goldfish retina. One system is controlled by an endogenous clock that activates low threshold D2‐like receptors in the day, whereas the second system is controlled by light and involves activation of higher threshold D1 receptors. J. Comp. Neurol. 467:243–253, 2003.

Retinal pH Reflects Retinal Energy Metabolism in the Day and Night

The extracellular pH of living tissue in the retina and elsewhere in the brain is lower than the pH of the surrounding milieu. We have shown that the pH gradient between the in vitro retina and the superfusion solution is regulated by a circadian (24-h) clock so that it is smaller in the subjective day than in the subjective night. We show here that the circadian changes in retinal pH result from a clock-mediated change in the generation of H+ that accompanies energy production. To demonstrate this, we suppressed energy metabolism and recorded the resultant reduction in the pH difference between the retina and superfusate. The magnitude of the reduction in the pH gradient correlated with the extent of energy metabolism suppression. We also examined whether the circadian-induced increase in acid production during the subjective night results from an increase in energy metabolism or from the selective activation of glycolysis compared with oxidative phosphorylation. We found that the selective suppression of either oxidative phosphorylation or glycolysis had almost identical effects on the dynamics and extent of H+ production during the subjective day and night. Thus the proportion of glycolysis and oxidative phosphorylation is maintained the same regardless of circadian time, and the pH difference between the tissue and superfusion solution can therefore be used to evaluate total energy production. We conclude that circadian clock regulation of retinal pH reflects circadian regulation of retinal energy metabolism.

The generation of directionally selective responses in the retina.

Although the phenomenon of directional selectivity has been of great interest to sensory physiologists for more than three decades, the cellular mechanisms that underlie it are still unknown. Directionally selective (DS) neurons, which have been described in the retina, in various cortical areas and in the peripheral nervous system, respond well to stimulus motion in one (preferred) direction, but respond little or not at all to motion in the opposite (null) direction. In the vertebrate retina, the neurotransmitter γ-aminobutyric acid (GABA) mediates null-direction inhibition of On-Off DS ganglion cells (Caldwell et al. 1978). These DS ganglion cells also receive substantial synaptic contact from acetylcholine (ACh)-containing amacrine cells, called ‘starburst’ amacrine cells due to their regularly spaced, evenly radiating dendrites. Although it has been proposed that the excitatory transmitter ACh mediates preferred-direction facilitation (Vaney, 1990; Borg-Graham & Grzywacz, 1991), clear evidence that this is so has been lacking. For example, nicotinic ACh receptor blockade with curare reduces preferred-direction responses without eliminating directional selectivity (Ariel & Daw, 1982). Furthermore, targeted laser ablation of starburst amacrine cells does not reveal any asymmetric contribution to the DS ganglion cell light response (He & Masland, 1997). The study of Grzywacz et al. (1998) in this issue of The Journal of Physiology provides the first clear evidence that nicotinic ACh input mediates the DS light responses of rabbit On-Off DS ganglion cells. The authors show that the contribution of ACh to DS responses depends on the stimulus configuration. Nicotinic blockade eliminates directional selectivity to drifting sine- and square-wave gratings, but as reported previously (Ariel & Daw, 1982), does not eliminate directional selectivity to isolated, moving bar stimuli. Thus, an asymmetric nicotinic input to DS ganglion cells extends the range of stimuli that can elicit directional responses to include moving textures, that is to those stimuli with multiple peaks in their spatial luminance profile. In fact, the directional responses of simple cells in cat striate cortex also depend on whether bar or grating stimuli are used (Casanova et al. 1992). Directionally selective responses in the rabbit retina are thus constructed piecemeal from an asymmetric nicotinic input that provides preferred-direction facilitation and from an asymmetric GABA input that provides null-direction inhibition (see Fig. 1). The ACh input to DS ganglion cells also depends on a separate GABA input from an unidentified amacrine cell (Massey et al. 1997). The two asymmetric inputs appear to act in parallel, providing On-Off DS ganglion cells with the ability to respond directionally to both low and high spatiotemporal frequency stimuli via GABA and ACh inputs, respectively. Although the actual functional role of On-Off DS retinal ganglion cells is at present unclear, the broad stimulus range over which these cells respond and the fact that their axons synapse onto cells in the lateral geniculate nucleus and superior colliculus suggest that the computation of directionality to many aspects of the visual scene is important for visual processing and behaviour. Figure 1 A simplified model of the generation of directional responses by rabbit On-Off directionally selective (DS) ganglion cells Grzywacz et al. (1998) were able to show that a nicotinic ACh input mediates directional selectivity presumably because GABA-mediated null-direction inhibition exhibits a slow decay time and is spatially wide (Amthor & Grzywacz, 1993). As a result, null-direction inhibition is ineffective at higher spatial and temporal frequencies and the blockade of nicotinic ACh receptors when grating stimuli were used revealed the presence of the asymmetric ACh input. The findings of Grzywacz et al. (1998) thus also suggest that the GABA input that regulates the asymmetric ACh input to DS cells (Massey et al. 1997) is separate from and operates on a faster time scale than GABA-mediated null-direction inhibition. Previous studies (Ariel & Daw, 1982; He & Masland, 1997) may not have found evidence of an asymmetric ACh input to DS cells simply because only bar stimuli were used. Consequently, these studies may have been examining primarily the slower/spatially wide GABA-mediated null-direction inhibition. The findings of Grzywacz et al. (1998) are thus a reminder that, in spite of over three decades of experimentation, the light response repertoire of DS ganglion cells had been insufficiently characterized. Several questions still remain unanswered. First, the exact nature of the ACh- and GABA-mediated asymmetries is not understood at the subcellular/anatomical level. Second, the roles of NMDA receptors (Tjepkes & Amthor, 1998) and calcium channels (Jensen, 1995) in the generation of directional responses also remain unclear. Finally, directional responses can be induced from non-directional ganglion cells in the amphibian retina by GABAB receptor activation (Pan & Slaughter, 1991). This finding raises the possibility that directionality may be a relatively common attribute of ganglion cells, but one that is usually non-functional. The generation of DS responses in the vertebrate retina may thus be more complicated than previously appreciated.