Tracing the auditory pathways to electrophysiologically characterized neurons with HRP and Fos double-labeling technique
Ying Qian, Min Wu t, Philip H.-S. Jen *
Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA
Accepted 11 June 1996
Abstract
By combining HRP histochemistry with Fos immunocytochemistry, we demonstrate in this study that electrophysiologically characterized auditory neurons can be double-labeled with HRP and Fos after iontophoretic injection of HRP into the recording site. Neurons which projected fibers to the recording site were labeled with HRP and were Fos-like immunoreactive. This double-labeling technique in combination with electrophysiological recording offers the possibility to determine the fiber projections between sound- activated neurons which are identified either electrophysiologically and/or immunocytochemically.
Since the introduction of horseradish peroxide (HRP) as a neuroanatomical tracer in the 1970s, HRP histochemistry has been a powerful method for tracing connections of neurons in the central nervous system [5,8]. Similarly, Fos immunocytochemistry has been used as a rapid and sensi- tive marking technique for identification of stimulus- activated neurons in the central nervous system since its inception a decade ago [9-11,14]. By combining HRP histochemistry with Fos immunocytochemistry and using bats as a model mammalian auditory system, we demon- strate here that sound-activated pontine neurons, which were characterized electrophysiologically, contained HRP reaction product and showed Fos-like immunoreactivity after iontophoretic injection of HRP into the recording site. Colocalization of HRP reaction product and Fos-like im- munoreactivity was also observed in neurons which pro- jected fibers to the recording site. Thus this double-label- ing technique in combination with electrophysiological recording offers the possibility to determine the fiber projections between sound-activated neurons which are identified either electrophysiologically and/or immunocy- tochemically. This novel technique can be a very powerful tool to trace neural circuits between stimulus-activated
1 Present address: Department of Surgery/Otolaryngology, Yale Uni- versity, New Haven, CT 06510, USA.
neurons not only in the auditory system but also in other sensory systems.
The auditory projections to electrophysiologically char- acterized pontine neurons from 4 big brown bats, Eptesi-
cus fuscus were traced by using this double-labeling tech- nique. The procedures for preparing each bat for recording sound-activated responses from pontine neurons were the same as in previous reports [2,15]. A glass microelectrode (impedance: 2 - 4 Mg2) filled with a 10% HRP-WGA in Tris buffer (pH 7.4)-KC1 (0.5 M) solution was used to record pontine neurons which responded to acoustic stimu- lus. A representative pontine neuron discharged tonically to a 4 ms tone burst with a shortest latency of 7.5 ms (Fig. 1A,N). The number of impulses increased monotonically with stimulus intensity (Fig. 1B) and the V-shaped fre- quency threshold curve showed a best frequency of 69 kHz and a minimum threshold of 66 dB SPL with a Q10 value of 3.4 (Fig. 1C). After determining these auditory response properties, 10% HRP-WGA in Tris buffer (pH 7.4)-KC1 (0.5 M) solution was iontophoretically ejected into the recording site (pulsed positive direct current, 0.07 Hz, 500-700 namp, 40-60 min). At 48 h survival time, the bat was placed inside a 10 × 10 × 10 cm 3 cage made of aluminum mesh in a sound proof room and kept in total darkness for 2 h to adapt to the new environment. The bat was then stimulated with 50 kHz, 79 dB SPL sounds (4 ms, 0.5 ms rise-decay times delivered at 2 pulses/s) for 45 min. At the end of sound stimulation, the bat was deeply
242 Y. Qian et al./Brain Research 731 (1996) 241-245
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Fig. 1. The response pattern shown in post-stimulus-time histograms (A, X), intensity-rate function (B) and frequency threshold curve (C) of a pontine neuron determined with sound stimulation (4 ms duration, 0.5 ms rise-decay times). The stimulus intensity (dB SPL) used and number (No) of impulses per 16 stimuli obtained in A and P/ are shown within each panel. Note that the number of impulses of this pontine neuron monotonically increased with sound intensity (B). The neuron had a V-shaped frequency threshold curve. The upper solid curve in C is the frequency characteristics curve of the loudspeaker.
anaesthetized with Nembutal and perfused transcardially with phosphate buffered saline (PBS) (0.1 M PBS, pH 7.4)
fol lowed by 2% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4). The brain was then removed and sect ioned
for H R P - W G A histochemistry [8,19]. The HRP react ion
product was stabil ized with 0.05% DAB in 0.02% CoC12 before be ing further processed with Fos immunocy tochem- istry [13]. Briefly, all bra in sections were first incubated in
1.5% normal goat serum in PBS for 1 h before be ing reacted with the pr imary ant ibody, a polyclonal rabbit IgG
to the Fos protein (c-fos 4: cat # S C - 5 2 , Santa Cruz Biotechnology) at a di lut ion of 1:100 in PBS overnight.
These sections were then processed immunocy tochemi -
cally with the avidin-biot in-peroxidase method (Vector
A B C kit cat # PK-4001) at the room temperature. Three control exper iments were performed: (1) the brain sections
of a bat which was injected with H R P - W G A in the brain
but without sound s t imulat ion were processed for Fos immunoreact iv i ty ; (2) the brain sections of a bat without sound s t imulat ion were processed for Fos immunoreac t iv-
ity; (3) the brain sections of another bat with sound stimulation were immunocytochemically processed with goat serum rather than the primary antibody for Fos. All brain sections were examined under the light microscope.
Fos-like immunoreactivity were not found in the brain sections of the control bats. Fig. 2 shows HRP and/or Fos-like immunoreactive positive neurons in the injection site and five other brain centers in the experimental bats. Neurons within the central core of the injection site (Fig. 2B dotted circle) and some neurons in the surrounding halo (e.g. Fig. 2B1, C1) were both labeled with HRP and
showed Fos-l ike immunoreact iv i ty . These neurons con-
tained black granules against b rownish purple background. The appearance of these double- labeled neurons ranged
from a light granulari ty of the soma to a dense staining of
the entire neuron (i.e. neurons wi thin the central core of
the inject ion site, Fig. 2B dotted circles). However, other neurons in the surrounding halo only showed Fos-l ike immunoreac t iv i ty (Fig. 2B2, B3; C2, C3). These single- labeled neurons showed lightly brownish purple color. In
Fig. 2. Photomicrographs showing HRP and/or Fos-like immunoreactive positive neurons in the injection site of a recorded pontine neuron and five brain centers of the big brown bat, Eptesicus fuscus. After studying the auditory response properties of the recorded pontine neuron, WGA-HRP was iontophoretically ejected into the recording site. At 48 h survival time, the bat was stimulated with 50 kHz, 79 dB SPL sound under free field conditions before being sacrificed for HRP histochemical and Fos immunocytochemical processing. A, a coronal section of the brain showing the HRP injection site in the pontine nuclei (rectangle). B, photomicrographs showing the central core of the injection site (dotted circle) and three neurons in the surrounding halo. All neurons in the central core and some neurons in the surrounding core of the injection site were either both HRP and Fos-like immunoreactive positive (arrow, B1) or Fos-like immunoreactive positive only (arrow heads, B2 and B3). They are shown in high magnification in C1, C2 and C3. D-H show representative double-labeled neurons (HRP and Fos-like immunoreactive, arrow) and single-labeled (Fos-like immunoreactive only, arrow head) neurons in the inferior colliculus (D1, D2 vs. D3), the auditory cortex (El vs. E2), the contralateral pontine nucleus (F1 vs. F2), the fastigial nucleus (G1 vs. G2) and the dentate nucleus (H1 vs. H2, H3). While all these neurons show sound-activated Fos-like immunoreactivity, only the double-labeled neurons project fibers to the recording site of the pontine neuron. All these single-labeled or double-labeled neurons can be easily distinguished from those neurons which contained neither HRP product nor Fos immunoreactivity (e.g. E3, open arrow). All neurons in C-H should refer to the scale bar in H. CG, central gray; IC, inferior colliculus; PN, pontine nuclei.
Y. Qian et al./Brain Research 731 (1996) 241-245 243
contrast, neurons which contained neither HRP product nor HRP and Fos-like immunoreactive positive neurons or Fos-like immunoreactivity are extremely pale and they can Fos-like immuuoreactive positive only neurons were also be barely discerned under the light microscope (Fig. 2E3, observed and easily distinguished from each other in the open arrow), ventrolateral portion of the ipsilateral inferior colliculus
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244 Y. Qian et al . / Brain Research 731 (1996) 241-245
Fig. 3. Photomicrographs of neurons shown in Fig. 2 were scanned with a Macintosh computer (8100/80AV) with Adobe Photoshop 3.0 to increase the contrast between the HRP product and Fos-like immunoreactivity. Note that the increased contrast between the HRP (dark) and Fos-like immunoreactivity (gray) in doubleqabeled neurons is so great that they can be easily distinguished from those single-labeled (Fos-like immunoreactive only) neurons (see Fig. 2 for legends).
Y. Qian et al. / Brain Research 731 (1996) 241-245 245
(Fig. 2D1, D2 vs. D3), layers I I I - V of the ipsilateral auditory cortex (Fig. 2El vs. E2), the ventrolateral portion of the caudal contralateral pontine nucleus (Fig. 2F1 vs. F2), the contralateral fastigial nucleus (Fig. 2G1 vs. G2) and contralateral dentate nucleus (Fig. 2H1 vs. H2, H3). To increase the contrast between the HRP product (dark) and Fos-l ike immunoreactivity (gray), photomicrographs of neurons shown in Fig. 2 were scanned with a Macintosh computer ( 8100 /80AV) with Adobe Photoshop 3.0. The increased contrast between the HRP product and the Fos- like immunoreactivity contained within the double-labeled neurons was so sharp that they can be easily distinguished from other single-labeled (Fos-l ike immunoreactive only) neurons (Fig. 3B1 vs. B2, B3; CI vs. C2, C3; D1, D2 vs. D3; E1 vs. E2; F1 vs. F2; G1 vs. G2; H1 vs. H2, H3).
Our study showed that the greatest number of double- labeled neurons were found in the ipsilateral inferior col- liculus and the contralateral pontine nucleus. Within each brain center, there were more single-labeled neurons (Fos- l ike immunoreactive positive only) than double-labeled neurons (both HRP and Fos-l ike immunoreactive positive). Although all Fos-l ike immunoreactive positive neurons in these five brain centers were activated by sound stimula- tion, only the double-labeled neurons projected fibers to the recording site of the electrophysiologically character- ized pontine neuron.
Previous double-labeling techniques were either com- bining tracers study with transmitter identification [ 16-18], uses of autoradiographic and peroxidase markers for detec- tion of neuronal pathways [12], immunocytochemical la- beling for ultrastructural studies of synaptic interaction and identification of neurons containing transmitters and neu- ropeptides [1,3,4,6] or combining tracers study with Fos immunocytochemistry to examine fiber projections of stimulus-activated neurons to target cells [7]. The present double-labeling technique with HRP and Fos offers the possibil i ty to determine the fiber projections between sound-activated neurons in which target neurons can be characterized electrophysiologically. This double-labeling technique can be a powerful tool to trace neural pathways between stimulus-activated neurons in all sensory systems.
Acknowledgements
This work is supported by the National Institute on Deaf and Other Communicat ion Disorders, National Insti- tute of Health (DC 247). We thank Drs. G. Summers and A. McClel lan for reading the early version of this manuscript.
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