453
Forlano et al. 2014 ). In addition to projecting to the saccule, the OE is reciprocally
connected to the vocal prepacemaker nucleus (VPP) of the vocal pattern generator
(VPG) and receives a corollary discharge when the VPG is activated (Chagnaud and
Bass 2013 ).
3.2.4 Saccule
Because TH-ir fi bers could be seen within the efferent bundle (EB) that includes
the lateral OE axons which connect to nerve VIII (Figs. 3a, b and 5d–f ), we investigated TH-ir within the saccule. Using double-label immunofl uorescence for TH
and otoferlin, labeled by the hair cell specifi c antibody (HCS-1), we were able to
characterize TH-ir distribution within sections of the saccular epithelium (Forlano
et al. 2014 ). Several large caliber TH-ir fi bers are seen within the saccular branch
of VIII proximal to the sensory macula. These fi bers course past ganglion cells,
bifurcate heavily, and mostly appear to form terminals and punctate-like structures
proximal to the base of hair cells and within the layer that includes support cell
nuclei (Figs. 6b, c and 7b ).
3.3 Origin of CA Fiber Innervation
The origin of CA efferents to the saccule was revealed by identifying a small number
of large, pear-shaped neurons in the diencephalon that were TH-ir and also consistently labeled with neurobiotin after saccular backfi lls (Fig. 6d–f ) (Forlano et al.
2014 ). These TH-ir neurons reside in the periventricular posterior tuberculum (TPp)
and are known to be dopaminergic in teleosts and homologous to DA A11 neurons
in other vertebrates (Filippi et al. 2010 ; Kastenhuber et al. 2010 ; Ryu et al. 2006 ;
Tay et al. 2011 ). All neurobiotin-fi lled TPp cells were TH-ir and backfi lled cells
were found on either side of the third ventricle regardless of which saccule was
labeled, implying both ipsi-and contralateral projection patterns. We estimate that
about only 5–10 % of ~300 TH-ir TPp neurons project to the saccule in midshipman. As mentioned above, TH-ir fi bers of the same caliber seen in the saccular
branch of VIII are also seen in LL nerves and it is highly likely that same TPp neurons innervate lateral line and other inner ear end organs (Forlano et al. 2014 ).
Indeed, a study in larval zebrafi sh showed that these DA diencephalic neurons project to lateral line (Bricaud et al. 2001 ), and tract tracing studies in goldfi sh identifi ed
neurons in a similar position project to lateral line nerves (Puzdrowski 1989 ; Zottoli
and Van Horne 1983 ). Prominent descending TH-ir tracts originating from TPp are
obvious from sagittal and horizontal sections through the midshipman brain, and
these fi bers appeared to target the rostral and caudal OE in single-labeled tissue
(Figs. 4d and 5 ). Whether the same DA neurons in TPp project to both the saccule
and OE somata (and dendrites) is unknown at this time, but it is likely that much of
the CA innervation of the auditory hindbrain originates from TPp. These projections form the bulk of the prominent medial longitudinal CA tract (mlct) and this
tract courses through the SOv and some fi bers from this tract appear to branch off
Neuroanatomical Evidence for Catecholamines as Modulators of Audition…
Forlano et al. 2014 ). In addition to projecting to the saccule, the OE is reciprocally
connected to the vocal prepacemaker nucleus (VPP) of the vocal pattern generator
(VPG) and receives a corollary discharge when the VPG is activated (Chagnaud and
Bass 2013 ).
3.2.4 Saccule
Because TH-ir fi bers could be seen within the efferent bundle (EB) that includes
the lateral OE axons which connect to nerve VIII (Figs. 3a, b and 5d–f ), we investigated TH-ir within the saccule. Using double-label immunofl uorescence for TH
and otoferlin, labeled by the hair cell specifi c antibody (HCS-1), we were able to
characterize TH-ir distribution within sections of the saccular epithelium (Forlano
et al. 2014 ). Several large caliber TH-ir fi bers are seen within the saccular branch
of VIII proximal to the sensory macula. These fi bers course past ganglion cells,
bifurcate heavily, and mostly appear to form terminals and punctate-like structures
proximal to the base of hair cells and within the layer that includes support cell
nuclei (Figs. 6b, c and 7b ).
3.3 Origin of CA Fiber Innervation
The origin of CA efferents to the saccule was revealed by identifying a small number
of large, pear-shaped neurons in the diencephalon that were TH-ir and also consistently labeled with neurobiotin after saccular backfi lls (Fig. 6d–f ) (Forlano et al.
2014 ). These TH-ir neurons reside in the periventricular posterior tuberculum (TPp)
and are known to be dopaminergic in teleosts and homologous to DA A11 neurons
in other vertebrates (Filippi et al. 2010 ; Kastenhuber et al. 2010 ; Ryu et al. 2006 ;
Tay et al. 2011 ). All neurobiotin-fi lled TPp cells were TH-ir and backfi lled cells
were found on either side of the third ventricle regardless of which saccule was
labeled, implying both ipsi-and contralateral projection patterns. We estimate that
about only 5–10 % of ~300 TH-ir TPp neurons project to the saccule in midshipman. As mentioned above, TH-ir fi bers of the same caliber seen in the saccular
branch of VIII are also seen in LL nerves and it is highly likely that same TPp neurons innervate lateral line and other inner ear end organs (Forlano et al. 2014 ).
Indeed, a study in larval zebrafi sh showed that these DA diencephalic neurons project to lateral line (Bricaud et al. 2001 ), and tract tracing studies in goldfi sh identifi ed
neurons in a similar position project to lateral line nerves (Puzdrowski 1989 ; Zottoli
and Van Horne 1983 ). Prominent descending TH-ir tracts originating from TPp are
obvious from sagittal and horizontal sections through the midshipman brain, and
these fi bers appeared to target the rostral and caudal OE in single-labeled tissue
(Figs. 4d and 5 ). Whether the same DA neurons in TPp project to both the saccule
and OE somata (and dendrites) is unknown at this time, but it is likely that much of
the CA innervation of the auditory hindbrain originates from TPp. These projections form the bulk of the prominent medial longitudinal CA tract (mlct) and this
tract courses through the SOv and some fi bers from this tract appear to branch off
Neuroanatomical Evidence for Catecholamines as Modulators of Audition…