Figure 5.
Correlative electron microscopy with HRP-mCherry.
A
, A ribbon synapse in a 2 dpf wild-type larva is indistinguishable from those in older animals.
B
, The synapse in a 5 dpf wild-type larva exhibits the characteristic features of a ribbon synapse, including a presynaptic dense body or ribbon, a halo of tethered synaptic vesicles, and prominent presynaptic and postsynaptic densities.
C
, Expression of the HRP-mCherry protein in the neurolemma places the fluorescent mCherry component intracellularly and the HRP moiety extracellularly.
D
, A bright-field micrograph depicts an afferent terminal expressing HRP-mCherry within a neuromast. The densely labeled fiber, which is also depicted in
E
,
F
, and
H–M
, is visible through the plastic resin in which the specimen has been embedded.
E
, An optical section through a neuromast of a living Brn3c:gap43-GFP larva features hair cells expressing a membrane-localized form of GFP (green). An afferent fiber labeled with HRP-mCherry (red) innervates three of the hair cells. The region bracketed by arrowheads is examined in greater detail in
K
.
F
, In an optical section through the basal region of the same neuromast, arrowheads bracket a site that was later explored under the electron microscope (
L
,
M
).
G
, A transverse section of the PLL nerve in a wild-type 5 dpf larva demonstrates several afferent axons.
H
, In a transverse section through a PLL nerve, two afferent fibers that express HRP-mCherry (arrowheads) produce prominent electron density in the surrounding extracellular space. The weakly labeled fiber in the lower right did not innervate the neuromast depicted in
D–F
and
H–M
.
I
, A higher-magnification view of the labeled neuron at the top left of
H
illustrates a localized precipitate that does not damage nearby cells.
J
, An unlabeled afferent neuron lacking electron density synapses with a hair cell of the neuromast.
K
, A synaptic ribbon (arrowhead) in the region of membrane contact denoted by arrowheads in
E
verifies that the membrane contact observed by light microscopy represents an afferent synapse.
L
, This ribbon synapse occurs at the site of membrane apposition bracketed by arrowheads in
F
. In this instance, the neuron has become distorted and exhibits poor preservation of intracellular organelles.
M
, Viewed at higher magnification, the ribbon synapse in
L
illustrates the typical attributes of hair-cell afferent synapses. Scale bars:
A
,
B
,
I–K
,
M
, 100 nm;
D–F
, 5 μm;
G
,
H
,
L
, 500 nm.