Activity-dependent phosphorylation of tyrosine hydroxylase in dopaminergic neurons of the rat retina

Abstract

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.

Figure 1.

Influence of light and dark on TH-P. Vertical pairs (a/d, b/e, etc.) show identical fields immunostained with panTH or one of three phosphospecific TH antibodies, as labeled. The top two rows are from a retina removed from a fully dark-adapted rat, and the bottom two rows are from a retina taken from a rat exposed to room light for 20 min. Dark or light does not affect panTH immunostaining, whereas light greatly increases phosphospecific TH (THP19, THP31, and THP40) immunoreactivity, particularly in the DA plexus of fine processes and rings. The dim background staining in e and f are blood vessels. Scale bar in e applies to all panels.

Figure 2.

Extent of phosphospecific TH-IR in the DA plexus. a, Double immunostaining with anti-THP19 (red) and anti-panTH (green). Colocalization is represented by yellow. The DA plexus consists of fine processes containing multiple varicosities. A portion of a DA dendrite is indicated by an arrow. Note that not all elements of the plexus are immunoreactive for the THP19 antibody. b, Colocalization of anti-THP19 (red) and anti-THP40 (green) in the DA plexus, using the Zenon technique (see Materials and Methods). Almost all of the varicosities (seen here as small, variously shaped profiles) colocalize the two phosphospecific antibodies. A rare exception is illustrated by the arrow. ^* indicates a portion of a DA dendrite. Scale bar applies to both panels.

Figure 3.

Colocalization of TH and panNa-IR in DA neurons. a, Vertical section of retina immunostained with an anti-panNa antibody. Note panNa-IR in DA cell body (asterisks) and rings (arrows) in outer portion of the IPL and ganglion cell axons (ax). b, The identical field immunostained for TH-IR. ^* indicates DA perikaryon. The DA plexus of rings also is visible. c, Merged image showing colocalization of TH- and panNa-IR in DA cell body (^*) and DA plexus. Arrows indicate colocalization in the corresponding three axon terminal rings of a.

Figure 4.

Voltage-clamp profiles of rat retinal spiking neurons. Whole-cell patch-clamp records were obtained from neurons in the ganglion cell layer in rat retinal slices. Neurons were held at -70 mV and then stepped in 20 mV depolarizing increments to +70 mV. Left, In slices from control retina (n = 8), the voltage-clamp profile consists primarily of a rapid inward current and a slower, sustained outward current. Right, In retinal slices from a TTX-treated eye (n = 6), the fast inward current is selectively blocked

Figure 5.

Effect of TTX on TH-P immunoreactivity. Left-right pairs (a/b, etc.) show identical fields immunostained with anti-panTH (left member of the pair) and one of three anti-phosphoTH antibodies, as labeled. a/b, e/f, i/j, Left eye injected in darkness with bicuculline (100 μm) and strychnine (20 μm). c/d, g/h, k/l, Right eye of same rat injected in darkness with bicuculline, strychnine, and TTX (15 μm). Bicuculline plus strychnine alone induced THP in DA cell bodies, dendrites, and DA plexus (b, f, j). In the presence of TTX, bicuculline, plus strychnine, THP is weak or absent in the DA plexus (d, h, l) but is present in DA cell bodies and dendrites. TTX did not affect anti-panTH-IR (compare a,e,i with c,g,k). Scale bar in i applies to all panels. gly, Glycine.

Figure 6.

VMAT2 immunoreactivity in rat retinal DA neurons. Anti-VMAT2-IR alone (a) and combined anti-VMAT2 and anti-panTH-IR (b). Note the patch of anti-VMAT2-IR in cell body (oval in a) but otherwise mainly in rings. Two representative corresponding rings in a and b are indicated by arrows. c, Colocalization of anti-VMAT2- and anti-panTH-IR in the DA plexus. Top arrow indicates colocalization in an axonal varicosity, and bottom arrow indicates colocalization in a ring. Note that not all varicosities show anti-VMAT2-IR. d, Arrows indicate colocalization of anti-VMAT2-IR and anti-panTH-IR in varicosities within the INL. Circles surround two varicosities that lack anti-VMAT2-IR. e, Similar colocalization of anti-VMAT2-IR and anti-panTH-IR in varicosities found in the middle of the IPL (arrows). The contrast of this photo was increased to make the small varicosities more visible, resulting in an overexposure of the DA cell body (asterisks). Scale bars (in d): a, b, 10 μm; c-e, 5 μm.

Figure 7.

Effects of veratridine on TH-P immunoreactivity. Horizontal pairs (a/b, c/d) illustrate identical fields immunostained for panTH (left) or anti-serine 19 phosphotyrosine hydroxylase (right). a/b, Eye received sham injection in darkness. Note absence of THP19 in DA plexus (b). c/d, Left eye injected with veratridine (100 μm) in darkness. Veratridine induces strong THP19 in the DA plexus (d). Scale bar in d is for all panels.