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Responses in the avian midbrain, thalamus and forebrain evoked by click stimuli
EXPERIMENTAL
iVEL?ROLOGY
18,
276-286
Responses in the Forebrain AMY Department
of
L.
Biology,
(1967)
Avian Midbrain, Evoked by Click
HARMAN Virginia
AND
Polytechnic
Received
E.
RICHARD
March
Institute, 15,
Thalamus Stimuli
and
PHILLIP+ Blacksburg,
Virginia
24061
1967
Electrophysiological studies of the brain potentials of urethane-anesthetized chickens revealed regions in midbrain, ‘tween brain and forebrain that responded to clicks. Response latencies ranged from 2-6 msec in the lateral lemniscus and the region of nucleus isthmi, pars principalis, to 2-10 msec in nucleus mesencephalicus lateralis, pars dorsalis. In the thalamus shortest latencies were found in the vicinity of tractus nucleus ovoidalis (4-12 msec), those in nucleus ovoidalis (lo-12 msec) were intermediate, and those in the dorsal nuclear complex (lo-16 msec) exhibited longest delays. Nucleus rotundus was not activated by clicks. In the paleostriatum latencies were 9-10 msec, in caudal neostriatum 11-32 msec, and 5.5 msec or more in the frontal neostriatum.
Introduction The conspicuous vocalizations of birds have often drawn the attention of poet and biologist alike. Although the hearing abilities of birds seem to be similar to those of mammals, little is known about the anatomy and physiology of avian auditory systems. The cochlea of birds contrasts with that of mammals in being only about a tenth as long and in lacking the pillars and arches of the mammalian organ of Corti (8). Auditory fibers of the eighth cranial nerve terminate in nucleus angularis and nucleus magnocellularis (3)) and evoked potential recording has revealed very short-latency responsesin nucleus laminaris as well (4, 9). Boord and Rasmussen(3) however, were unable to find degeneration in this last nucleus after auditory nerve section. More centrally, Ariens-Kappers, Huber and Crosby (2) described projections from the cochlear nuclei to nucleus mesencephalicus lateralis, pars dorsalis (MLD), giving fibers to the nucleus of the lateral 1 This research was submitted in partial fulfillment for the degree of Master of Science in Bio!opy at Virginia Polytechnic Institute, Blacksburg, and supported in part by NSF research grant (GB 768) to Phillips. We gratefully acknowledge the facilities and funds contributed to the study by the Department of Biology and by the Virginia Agricultural Experiment Station, and we thank Mrs. Peggy Winesett for her technical assistance. The authors’ addresses are: Department of Biology, Frostburg State College, Frostburg, Maryland 21532 and Department of Poultry Science, St. Paul, Minnesota 5.5101, respectively. 276
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lemniscus, nucleus semilunaris and nuclei isthmi principalis (pars magnoand parvocellularis) on the way. They considered the MLD homologous to the inferior colliculus of mammals on these grounds, but Erulkar (4) doubted this homology because he was unable to record evoked activity regularly from MLD. Karten (personal communication), however, used the Nauta stain for degenerating axons and found that the major projection from the cochlear nuclei was to MLD with no major input to the isthmi complex. Papez (7) proposed an auditory path from the midbrain to nucleus ovoidalis of the thalamus, but again, Erulkar (4) was unable to record evoked responses to clicks in this or any other thalamic nucleus, although he did find responses in the caudal neostriatum. The most conclusive evidence on this point was furnished by Karten (6) who traced degeneration from MLD lesions to both ipsi- and contralateral ovoid nuclei by way of tractus nucleus ovoidalis and the dorsal supraoptic commissure. Adamo and King ( 1) also reported click-evoked responses from the forebrain, but from the surface of the “Wulst” rather than from caudal neostriatum. The present investigation provides information on latencies, amplitudes and localization of click evoked responses in various midbrain, diencephalic and forebrain auditory areas. It is hoped that this will serve as a step toward better understanding of auditory function in the behavior of the class Aves. Methods Experiments were performed on fifty-eight adult female white leghorn chickens anesthetized with urethane (3.0-3.5 g; iv). The head of the animal was immobilized in a Baltimore stereotaxic instrument with hollow ear bars, and was leveled as described by van Tienhoven and Juhasz ( 10) ; their atlas of the chicken brain was used to determine the locations of structures to be explored, and their nomenclature is followed throughout. The coordinates to be used during the experiment were marked on the skull. This portion of the skull was removed with a small trephine (3.4 mm in diameter) and the dura mater cut just enough to allow the electrodes to enter the brain. Cotton soaked with a warm saline solution was placed over the exposed areas of the brain to prevent drying. Cloaca1 temperature remained at 41.5 C from beginning to end of even the longest experiments. More than 3 000 sites along 180 tracks were explored. Bipolar concentric electrodes made of 26-gauge stainless steel hypodermic tubes, each with a central, enamel-insulated Nilstain wire projecting 1 mm beyond the end of the tube were used. The outer electrode was insulated with Insl-X except the terminal 0.5 mm; and 0.5 mm of the core electrode was also scraped bare. The outside diameter of the electrode was approximately 400 u. Clicks were produced by 0.05msec rectangular pulses from a Grass S-4
278
HARMAN
AND
PHILLIPS
stimulator to a crystal earphone. Except when the effects of increased intensity on the response were being observed, constant voltage was used. Rubber tubing was connected between the earphones and the hollow ear bars to prevent vibration artifacts and to restrict the stimulus to one ear as much as possible. Evoked potentials were displayed on a Tektronix 565 oscilloscope driven by a 2A61 plug-in preamplifier. The recordings were so arranged that positive potentials were up. Intervals between stimuli were 1 set or longer except when the object was to examine the effect of stimulus frequency on response. The usual procedure was to record in 0.5mm steps along each electrode track. Permanent records were made with a Tektronix C-12 oscilloscope camera. The location of each electrode track was verified histologically. The brains were perfused through the carotid arteries with saline followed by 10% buffered formalin solution containing 1 $X potassium ferrocyanide. Frozen sections 50 p thick were cut, and only those sections needed to determine the position of the exploring electrode track were retained. Half of these sections were stained with cresyl violet and the other half were mounted in glycerol. Glycerol mounting caused a minimum of distortion, making measurements more accurate. The exact location of the electrode tip was determined by passing a direct current at 10 v for 3 set through the electrode at the end of the track, and at another known site, usually 5 mm up from the bottom of the track, to give a Prussian blue reaction with the potassium ferrocyanide. Results
iMidbrain. Figure 1 shows the midbrain sites responding to clicks. In the region of nucleus semilunaris contralateral to the stimulated ear, the responses had latencies of 2 to 4 msec with amplitudes of 20 to 40 pv. Ipsilateral clicks evoked potentials with 5 to 6-msec latent periods and 80- to 350~pv amplitudes. Just dorsomedial to the area (atlas coordinates: A 1.5-1.75; L 3.5-4.0; V 253.0) latencies ranged from 4 to 6 msec with an average amplitude of 160 pv. Small responses of inconsistent latency and duration were found at the atlas coordinates of A 3.5-4.0, L 1.5-2.5, and V 1.5-2.5. This is the general area of tractus bulbothalamicus. Nucleus isthmi pars principalis parvocellularis was entered nine times and was readily activated by clicks delivered to either ear. The lateral part of the nucleus responded to ipsilateral clicks with latencies of 6 to 9 msec and amplitudes ranging from 25 to 40 pv. Earlier responses at 3 and 4 msec were sometimes seen. In the medial part, evoked potentials were much more complicated. The most stable part of the response was a SO- to 400-uv wave at 10 msec. When the magnocellular part of the nucleus responded,
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FIG. I. Diagrammatic representation of auditory areas with examples of clickCalibration units are 50 KY and 10 msec. evoked potentials from various structures. A, archistriatum; Dm, dorsomedial nucleus of thalamus; H, hypecstriatum; LFB, lateral forebrain bundle; MLD, nucleus mesencephalicus latetalis, pars dorsalis; N, neostriatum; 0, nucleus ovoidalis; OC, optic chiasma; Pa, paleostriatum augmentatum; Pp, pa.leoS, septal area, Vertical shading roughly outstciatum primitivum; R, nucleus rotundus; lines regions where responses were recorded.
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latencies were 5 to 6 msec, but activity was not regularly evoked in it by clicks. The torus semicircuiaris was entered twenty-three times and was usually responsive to auditory stimuli but several electrodes failed to discloseevoked activity in it. All fourteen of those that encountered responsesthere penetrated the large-celled MLD. Ipsilateral clicks evoked potentials in this nucleus at 4- to 6-mseclatency with amplitudes ranging from 10 to 100 pv. A slower wave that followed at 6 to 10 msec had an amplitude between 50 and 250 pv. In somecasesa still slower wave was seenwith a much longer duration than the earlier ones. This lo- to 25l.rv wave came 10 to 16 msec after the stimulus. Contralateral clicks elicited lo- to 2.5yv waves peaking at 6 msec.Clicks presented to both ears at the same time evoked potentials consisting of a 40-pv wave at 3 msec, a lO+v wave at 5 msec, and a 9O+v wave peaking at 8 msec. The shorter latency in responseto bilateral rather than to unilateral clicks to either ear suggestsspatial summation of crossed and uncrossedfibers. The ability of midbrain auditory pathways to conduct repetitive impulses was tested by using paired clicks. Figure 2 shows the time required for recovery of the amplitude of the secondof a pair of evoked responsesin the mesencephalon:when the interval between clicks was 1.6 msecthere was no responseto the second click, but when the interval was 3.6 msec, response to the secondclick was again maximal. At an interval of 2.6 msec the shape of the first stimulus responsewas affected although a second spike could not be seen. The time required for recovery of amplitude was similar in all areas of the mesencephalon. The effects of repetitive stimulation at different frequencies and those of increased loudnesswere investigated. Amplitudes of responsesgenerally decreased with increased stimulus frequency. The large evoked potentials in MLD and in nucleus semilunaris were still seeneven at stimulus frequencies above lOO/sec. Stimulus frequencies below lO/sec resulted in waxing and waning of responseamplitudes. Louder clicks gave larger responsesup to a point. Increasing the loudnessgreatly, then reducing it to a much lower level sometimesproduced a responsewhich was not otherwise observed. Thalamus. In the thalamus, potentials evoked by either contralateral or ipsilateral stimulation usually were simple, monophasicresponses.When other components were observed, they were very inconsistent and faded rapidly with repetitive stimulation. The amplitudes of these slower componentswere usually not as great as those of the main part of the response. Initial waves in nuclei dorsolateralis and dorsomedialis had both the highest amplitudes and the longest latencies found in the thalamus. Clicks presented to the contralateral ear produced a SO- 120~pv wave in the
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AUDITORY
281
RESPONSES
lateral part of the area with latencies ranging from 10 to 16 msec (most frequently 12 to 14 msec), very similar to the 12- to 20-msec latencies and 20- to 170+4v amplitudes of responses to ipsilateral clicks. In medial parts, responses were more complex than those in lateral areas. Initial responses were 50 to 100 pv at 14 to 16 msec. The other components had smaller amplitude (10 to 60 uv) at latencies of 20 to 36 msec with durations of 14 msec or longer. Responses to ipsilateral stimuli tended to have slightly
30.0
III
A fi
IV
A
mr.3.
B
L
50m.r.
FIG. 2. Differences in responses of the mesencephalon and caudal neostriatum when tested for absolute refractory period. I and II are negative potentials taken from the nucleus mesencephalicus lateralis pars dorsalis. III, IV, and V are potentials from the caudal neostriatum. Vertical lines indicate the stimulus artifacts. A: response to a single click. B: responses to paired clicks.
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longer latencies and lower amplitudes than those to contralateral clicks, but the ranges overlapped broadly. The responsesrecorded in the nucleus ovoidalis to contralateral stimulation had shorter latencies and were less complex than those in the dorsal nuclei; latencies ranged from 10 to 12 msec, amplitudes from 50 to 80 yv (Fig. 1) . On the lateral edge of the nucleus ovoidalis ipsilateral clicks produced waves with 9- to 12-mseclatencies and IO- to lOO+v amplitudes. In the region 1.5 to 2.0 mm ventral to the nucleus ovoidalis (in or very near tractus nucleusovoidalis) latencies were from 6 to 11 msecwhen evoked by either ipsilateral or contralateral stimuli. Amplitudes ranged from 30 to 150 nv. Just ventral to this (A 5.0-5.5; V 2-3; L I.53.0), evoked potentials had short latencies (4.5 to 7.0 msec), and relatively high amplitudes (6.5 to 175 nv). They faded rapidly at stimulation frequencies from 10 to 20/set, and no responsecould be seen over 20/set frequencies. The region from which these responseswere evoked would appear to be that of connections between mesencephalonand supraoptic commissure.These tracts are afferent to nucleus ovoidalis as indicated by the latencies and by their degeneration after mesencephaliclesions (6). Similar responseswere observed medially and ventrally to the nucleus rotundus, which itself did not respond to clicks. These short-latency responseswere not over 40 to 50 lrv and most of them were smaller. Other responseswith much longer latencies were observed in this area although they were not often seen. Their amplitudes were low (10 to 30 uv) with latencies of 14 to 17 msec. These responses probably were of the fibers from mesencephalonto ovoidalis. In the lateral forebrain bundle, at atlas coordinates of A 7.5-9.0, V 5.5-6, and L 2-3, responseshad latent periods of 8 to 12 msec and amplitudes of 30 to 100 uv, but the responseswere not sharp. These responseswere much more consistent to contralateral than to ipsilateral stimulation. Repetitive clicks up to 8/set elicited responses.Above 8 click/set, no responsescould be seen. The second of a pair of clicks elicited no responseat intervals less than 10 msec. Forebrain. Responseswere evoked more frequently from the paleostriatum primitivum than from the paleostriatum augumentatum. The responsesin the paleostriatum primitivum to either ipsilateral or contralateral stimuli were waves of II- to 17-mseclatency (mostly 12 to 13 msec) with 35 to 1lo-nv amplitudes (Fig. 1). In the caudal neostriatum stimulation of the opposite ear evoked potentials consisting of an initial wave at 13- to 17-mseclatency (most frequently 14 to 16 msec) having a 25 to 250~nv amplitude. Slower waves were recorded with latencies of 18 to 40 msecand in most casessmall early potentials were recorded at 8 to 13 msec. When both ears were stimulated simultaneously, potentials had latencies of 13 to 15 msec, amplitudes of 65 to 110 pv, and
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AUDITORY
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lasted 20 to 30 msec. Duration was longer than when either ear was stimulated alone. Potentials evoked in the caudal neostriatum faded more rapidly with increased frequency than did those in the mesencephalon and diencephalon. Figure 3 shows the effect of increasing stimulus intensity and frequency on the response amplitude. Neither ipsilateral nor contralateral clicks evoked
80 60 40 20 0 1
3
5
7
10
CLICKS PER SECOND FIG. 3. Graph showing the relationship of stimulus intensity and frequency on the response amplitude. Dotted line: 50-v stimulus intensity. Solid line: 15-v intensity. Abscissa: click frequency per second. Ordinate: amplitude of response in microvolts. Data from the neostriatum.
a second response when the interval between the clicks was less than 7 msec (Fig. 2) compared to 3.6 msec for full restoration of response in midbrain. Increased intensity seemed either to inhibit or to obscure some of the components of the response. In the frontal neostriatum (Fig. 1)) some responses to contralateral stimulation were negative waves with 8- to ll-msec latency and lo- to 250yv amplitudes. Others were positive waves with latencies at 8 to 12 msec and lOO- to IlO-pv amplitudes. These waves had slower components at 14- to 25msec latency with amplitudes of 40 to 110 pv. Sometimes shorter
284
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latency (5.5 to 8 msec, 50 to 70 pv) negative potentials preceded these positive waves. Silent regions of the forebrain are shown in Fig. 1. Discussion
The auditory pathway in birds appears to run from the cochlear nuclei primarily to nucleus mesencephalicus lateralis pars dorsalis with possibly some fibers to the isthmi complex. We regularly recorded evoked responses to clicks in the isthmic region of chickens as did Erulkar (4) in pigeons. Karten, however, found no important projection to this area in his degeneration studies of pigeon brain (personal communication). A possible explanation of this apparent difference is suggested by the large size of the electrodes used in both studies compared to the small nuclei, and the fact that the lateral lemniscus fibers course right above the nuclei, especially on their medial sides. Very likely any such electrode recording from the isthmi complex would also be at least partially in the lemniscal fibers and so record activity there. Unit records from microelectrodes would be necessary to prove auditory activation of the isthmic nuclei in the face of the negative anatomical evidence. From the midbrain nuclei, auditory information is transmitted to nucleus ovoidalis (lo- to 12-msec latency from click) by way of tractus nucleus ovoidalis and the dorsal thalamic nuclei, and from these thalamic relays on to paleostriatum and neostriatum (12-16 msec) by way of the lateral forebrain bundle. Some responses were also recorded in the supraoptic commissure: these were probably from the fibers described by Karten (6) crossing from MLD to contralateral nucleus ovoidalis by way of tractus nucleus ovoidalis or possibly from those described by Huber and Crosby (5) from nucleus isthmi. Our demonstration of click-evoked responses in nucleus ovoidalis combined with Karten’s (6) demonstration of the path from MLD to ovoidalis confirms the suggestion by Papez ( 7) that the ovoidalis (“spiriformis” of Papez) is a thalamic relay for acoustic impulses in birds. Erulkar’s (4) failure to evoke responses in the thalamus of pigeons is puzzling but may have been a result of his technique of aspirating the hemispheres for recording from lower structures. We left the brain intact except for the electrode insertion itself. Species differences between pigeons and chickens seem unlikely to be responsible for the presence of thalamic responses in our birds and their absence in Erulkar’s. Similarity between chickens and pigeons is indicated by the comparable response latencies in both species and by the fact that Karten’s anatomical studies in pigeons show auditory connections where we recorded them in chickens. Numerous instances where responses of 100 pv or more abruptly appeared or disappeared with electrode movements of 0.5 mm or less furnished evidence for limited
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pickup by our electrodes. The sharply restricted areas from which these potentials could be detected supports the interpretation that potentials originated close to the points where we recorded them. Presence of potentials in the dorsal thalamic nuclei suggests that more than one path may project to the forebrain, a suggestion made more probable by the widely varying latencies of the responses that we recorded in various parts of the striatum. Karten’s (6) failure to find degeneration in the dorsal thalamic nuclei after MLD lesions supports the probability that more synapses (based on longer latencies) occur in the auditory input to these nuclei than to ovoidalis. Figure 4 diagrams possible pathways consistent with our data and with known anatomy. Presence of large potentials in
/
Tr. N. ovoidalis
N. ovoidalis
FIG. 4. diagrammed,
Diagrammatic although
summary at all levels
of main features of auditory path. Only one side is both sides of the brain are activated by either ear.
frontal neostriatum and hyperstriatum with latencies of only 5 to 11 msec seemsto require that an appreciable number of acoustic fibers reach there without synapse in the thalamus where latencies were already lo-17 msec. Tractus striotegmentalis (2) runs approximately through the active points, but no auditory connections have been described for it, and our data do not permit us to assignthe responsesto it. Experimental degeneration studies will be needed to clarify the details. Confirmation that conduction is toward the striatum in the paths between midbrain nuclei and neostriatum was provided by a few experiments in which two pairs of electrodes were lowered into the brain, one in the midbrain and one in caudomedial neostriatum, until each encountered clickevoked responses.Then each pair was used in turn for stimulating and for recording. In all casespotentials could be evoked in the neostriatum by midbrain stimulation but not the reverse, indicating both that normal conduc-
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tion is afferent to neostriatum from these midbrain points and that at least one synapse is interposed in this path. Comparison of the neostriate latencies (14 to 17 msec) for click-evoked responses with those evoked by midbrain stimulation (9 msec) plus the latency for click activation of MLD (6 to 7 msec) shows them to be similar, supporting the contention that we were indeed testing a normal auditory pathway. The shorter latencies and greater ability of midbrain and thalamic than of forebrain sites to follow repetitive stimuli is further evidence that these former areas were activated by sensory impulses not by reflex discharges from the striatum. Adamo and King (1) reported click-evoked responses from the surface of the “Wulst” in chickens under chloralose. This is paralleled by our records from the rostra1 pole of the forebrain (Fig. 1). We did not record for the first 3 or 4 mm on most tracks so our failure to find surface activity may not reflect an actual lack of responses, or their records may reflect distant. pickup of deeper activity. References 1. 2.
3. 4.
5.
6. 7. 8.
9. 10.
ADAMO, N. J., and R. L. KING. 1965. Electrical responses to auditory stimulation in the chicken telencephalon. Anat. Record 151: 317. ARI~?NS-KAPPERS, C. V., G. C. HUBER, and E. C. CROSBY. 1936. “The Comparative Anatomy of the Nervous System of Vertebrates Including Man.” Macmillan, New York. BOORD, R. L., and G. L. RASMUSSEN. 1963. Projection of the cochlear and lagenar nerves on the cochlear nuclei of the pigeon. J. Co@. Neural. 120: 463-475. ERULKAR, S. D. 1955. Tactile and auditory areas in the brain of the pigeon. An experimental study by means of evoked potentials. J. Camp. Neural. 103: 421457. HUBER, G. C., and E. C. CROSBY. 1929. The nuclei and fiber paths of the avian diencephalon, with considerations of telencephalic centers and connections. .7. Camp. Neural. 43: l-25.5. KARTEN, H. 1966. Efferent projections of the nucleus mesencephalicus lateralis, pars dorsalis (MLD) in the pigeon (Columba Zivia). Anat. Rec. 154: 365. PAPU, J. W. 1929. “Comparative Neurology.” Crowell, New York. (Reprinted by Hafner, New York.) PUMPHREY, R. J. 1961. Sensory mechanisms: Hearing, pp. 69-86. In “Biology and Comparative Physiology of Birds.” A. J. Marshall Led.]. Academic Press, New York. STOPP, P. E., and I. C. WHITFIELD. 1961. Unit responses from brain-stem nuclei in the pigeon. J. Physiol. London 158: 165-177. VAN TIENHOVEN, A., and L. P. JUHASZ. 1962. The chicken telencephalon, diencephalon, and mesencephalon in stereotaxic coordinates. 1. Camp. Neural. 118: 185-198.
iVEL?ROLOGY
18,
276-286
Responses in the Forebrain AMY Department
of
L.
Biology,
(1967)
Avian Midbrain, Evoked by Click
HARMAN Virginia
AND
Polytechnic
Received
E.
RICHARD
March
Institute, 15,
Thalamus Stimuli
and
PHILLIP+ Blacksburg,
Virginia
24061
1967
Electrophysiological studies of the brain potentials of urethane-anesthetized chickens revealed regions in midbrain, ‘tween brain and forebrain that responded to clicks. Response latencies ranged from 2-6 msec in the lateral lemniscus and the region of nucleus isthmi, pars principalis, to 2-10 msec in nucleus mesencephalicus lateralis, pars dorsalis. In the thalamus shortest latencies were found in the vicinity of tractus nucleus ovoidalis (4-12 msec), those in nucleus ovoidalis (lo-12 msec) were intermediate, and those in the dorsal nuclear complex (lo-16 msec) exhibited longest delays. Nucleus rotundus was not activated by clicks. In the paleostriatum latencies were 9-10 msec, in caudal neostriatum 11-32 msec, and 5.5 msec or more in the frontal neostriatum.
Introduction The conspicuous vocalizations of birds have often drawn the attention of poet and biologist alike. Although the hearing abilities of birds seem to be similar to those of mammals, little is known about the anatomy and physiology of avian auditory systems. The cochlea of birds contrasts with that of mammals in being only about a tenth as long and in lacking the pillars and arches of the mammalian organ of Corti (8). Auditory fibers of the eighth cranial nerve terminate in nucleus angularis and nucleus magnocellularis (3)) and evoked potential recording has revealed very short-latency responsesin nucleus laminaris as well (4, 9). Boord and Rasmussen(3) however, were unable to find degeneration in this last nucleus after auditory nerve section. More centrally, Ariens-Kappers, Huber and Crosby (2) described projections from the cochlear nuclei to nucleus mesencephalicus lateralis, pars dorsalis (MLD), giving fibers to the nucleus of the lateral 1 This research was submitted in partial fulfillment for the degree of Master of Science in Bio!opy at Virginia Polytechnic Institute, Blacksburg, and supported in part by NSF research grant (GB 768) to Phillips. We gratefully acknowledge the facilities and funds contributed to the study by the Department of Biology and by the Virginia Agricultural Experiment Station, and we thank Mrs. Peggy Winesett for her technical assistance. The authors’ addresses are: Department of Biology, Frostburg State College, Frostburg, Maryland 21532 and Department of Poultry Science, St. Paul, Minnesota 5.5101, respectively. 276
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lemniscus, nucleus semilunaris and nuclei isthmi principalis (pars magnoand parvocellularis) on the way. They considered the MLD homologous to the inferior colliculus of mammals on these grounds, but Erulkar (4) doubted this homology because he was unable to record evoked activity regularly from MLD. Karten (personal communication), however, used the Nauta stain for degenerating axons and found that the major projection from the cochlear nuclei was to MLD with no major input to the isthmi complex. Papez (7) proposed an auditory path from the midbrain to nucleus ovoidalis of the thalamus, but again, Erulkar (4) was unable to record evoked responses to clicks in this or any other thalamic nucleus, although he did find responses in the caudal neostriatum. The most conclusive evidence on this point was furnished by Karten (6) who traced degeneration from MLD lesions to both ipsi- and contralateral ovoid nuclei by way of tractus nucleus ovoidalis and the dorsal supraoptic commissure. Adamo and King ( 1) also reported click-evoked responses from the forebrain, but from the surface of the “Wulst” rather than from caudal neostriatum. The present investigation provides information on latencies, amplitudes and localization of click evoked responses in various midbrain, diencephalic and forebrain auditory areas. It is hoped that this will serve as a step toward better understanding of auditory function in the behavior of the class Aves. Methods Experiments were performed on fifty-eight adult female white leghorn chickens anesthetized with urethane (3.0-3.5 g; iv). The head of the animal was immobilized in a Baltimore stereotaxic instrument with hollow ear bars, and was leveled as described by van Tienhoven and Juhasz ( 10) ; their atlas of the chicken brain was used to determine the locations of structures to be explored, and their nomenclature is followed throughout. The coordinates to be used during the experiment were marked on the skull. This portion of the skull was removed with a small trephine (3.4 mm in diameter) and the dura mater cut just enough to allow the electrodes to enter the brain. Cotton soaked with a warm saline solution was placed over the exposed areas of the brain to prevent drying. Cloaca1 temperature remained at 41.5 C from beginning to end of even the longest experiments. More than 3 000 sites along 180 tracks were explored. Bipolar concentric electrodes made of 26-gauge stainless steel hypodermic tubes, each with a central, enamel-insulated Nilstain wire projecting 1 mm beyond the end of the tube were used. The outer electrode was insulated with Insl-X except the terminal 0.5 mm; and 0.5 mm of the core electrode was also scraped bare. The outside diameter of the electrode was approximately 400 u. Clicks were produced by 0.05msec rectangular pulses from a Grass S-4
278
HARMAN
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PHILLIPS
stimulator to a crystal earphone. Except when the effects of increased intensity on the response were being observed, constant voltage was used. Rubber tubing was connected between the earphones and the hollow ear bars to prevent vibration artifacts and to restrict the stimulus to one ear as much as possible. Evoked potentials were displayed on a Tektronix 565 oscilloscope driven by a 2A61 plug-in preamplifier. The recordings were so arranged that positive potentials were up. Intervals between stimuli were 1 set or longer except when the object was to examine the effect of stimulus frequency on response. The usual procedure was to record in 0.5mm steps along each electrode track. Permanent records were made with a Tektronix C-12 oscilloscope camera. The location of each electrode track was verified histologically. The brains were perfused through the carotid arteries with saline followed by 10% buffered formalin solution containing 1 $X potassium ferrocyanide. Frozen sections 50 p thick were cut, and only those sections needed to determine the position of the exploring electrode track were retained. Half of these sections were stained with cresyl violet and the other half were mounted in glycerol. Glycerol mounting caused a minimum of distortion, making measurements more accurate. The exact location of the electrode tip was determined by passing a direct current at 10 v for 3 set through the electrode at the end of the track, and at another known site, usually 5 mm up from the bottom of the track, to give a Prussian blue reaction with the potassium ferrocyanide. Results
iMidbrain. Figure 1 shows the midbrain sites responding to clicks. In the region of nucleus semilunaris contralateral to the stimulated ear, the responses had latencies of 2 to 4 msec with amplitudes of 20 to 40 pv. Ipsilateral clicks evoked potentials with 5 to 6-msec latent periods and 80- to 350~pv amplitudes. Just dorsomedial to the area (atlas coordinates: A 1.5-1.75; L 3.5-4.0; V 253.0) latencies ranged from 4 to 6 msec with an average amplitude of 160 pv. Small responses of inconsistent latency and duration were found at the atlas coordinates of A 3.5-4.0, L 1.5-2.5, and V 1.5-2.5. This is the general area of tractus bulbothalamicus. Nucleus isthmi pars principalis parvocellularis was entered nine times and was readily activated by clicks delivered to either ear. The lateral part of the nucleus responded to ipsilateral clicks with latencies of 6 to 9 msec and amplitudes ranging from 25 to 40 pv. Earlier responses at 3 and 4 msec were sometimes seen. In the medial part, evoked potentials were much more complicated. The most stable part of the response was a SO- to 400-uv wave at 10 msec. When the magnocellular part of the nucleus responded,
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FIG. I. Diagrammatic representation of auditory areas with examples of clickCalibration units are 50 KY and 10 msec. evoked potentials from various structures. A, archistriatum; Dm, dorsomedial nucleus of thalamus; H, hypecstriatum; LFB, lateral forebrain bundle; MLD, nucleus mesencephalicus latetalis, pars dorsalis; N, neostriatum; 0, nucleus ovoidalis; OC, optic chiasma; Pa, paleostriatum augmentatum; Pp, pa.leoS, septal area, Vertical shading roughly outstciatum primitivum; R, nucleus rotundus; lines regions where responses were recorded.
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latencies were 5 to 6 msec, but activity was not regularly evoked in it by clicks. The torus semicircuiaris was entered twenty-three times and was usually responsive to auditory stimuli but several electrodes failed to discloseevoked activity in it. All fourteen of those that encountered responsesthere penetrated the large-celled MLD. Ipsilateral clicks evoked potentials in this nucleus at 4- to 6-mseclatency with amplitudes ranging from 10 to 100 pv. A slower wave that followed at 6 to 10 msec had an amplitude between 50 and 250 pv. In somecasesa still slower wave was seenwith a much longer duration than the earlier ones. This lo- to 25l.rv wave came 10 to 16 msec after the stimulus. Contralateral clicks elicited lo- to 2.5yv waves peaking at 6 msec.Clicks presented to both ears at the same time evoked potentials consisting of a 40-pv wave at 3 msec, a lO+v wave at 5 msec, and a 9O+v wave peaking at 8 msec. The shorter latency in responseto bilateral rather than to unilateral clicks to either ear suggestsspatial summation of crossed and uncrossedfibers. The ability of midbrain auditory pathways to conduct repetitive impulses was tested by using paired clicks. Figure 2 shows the time required for recovery of the amplitude of the secondof a pair of evoked responsesin the mesencephalon:when the interval between clicks was 1.6 msecthere was no responseto the second click, but when the interval was 3.6 msec, response to the secondclick was again maximal. At an interval of 2.6 msec the shape of the first stimulus responsewas affected although a second spike could not be seen. The time required for recovery of amplitude was similar in all areas of the mesencephalon. The effects of repetitive stimulation at different frequencies and those of increased loudnesswere investigated. Amplitudes of responsesgenerally decreased with increased stimulus frequency. The large evoked potentials in MLD and in nucleus semilunaris were still seeneven at stimulus frequencies above lOO/sec. Stimulus frequencies below lO/sec resulted in waxing and waning of responseamplitudes. Louder clicks gave larger responsesup to a point. Increasing the loudnessgreatly, then reducing it to a much lower level sometimesproduced a responsewhich was not otherwise observed. Thalamus. In the thalamus, potentials evoked by either contralateral or ipsilateral stimulation usually were simple, monophasicresponses.When other components were observed, they were very inconsistent and faded rapidly with repetitive stimulation. The amplitudes of these slower componentswere usually not as great as those of the main part of the response. Initial waves in nuclei dorsolateralis and dorsomedialis had both the highest amplitudes and the longest latencies found in the thalamus. Clicks presented to the contralateral ear produced a SO- 120~pv wave in the
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lateral part of the area with latencies ranging from 10 to 16 msec (most frequently 12 to 14 msec), very similar to the 12- to 20-msec latencies and 20- to 170+4v amplitudes of responses to ipsilateral clicks. In medial parts, responses were more complex than those in lateral areas. Initial responses were 50 to 100 pv at 14 to 16 msec. The other components had smaller amplitude (10 to 60 uv) at latencies of 20 to 36 msec with durations of 14 msec or longer. Responses to ipsilateral stimuli tended to have slightly
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FIG. 2. Differences in responses of the mesencephalon and caudal neostriatum when tested for absolute refractory period. I and II are negative potentials taken from the nucleus mesencephalicus lateralis pars dorsalis. III, IV, and V are potentials from the caudal neostriatum. Vertical lines indicate the stimulus artifacts. A: response to a single click. B: responses to paired clicks.
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longer latencies and lower amplitudes than those to contralateral clicks, but the ranges overlapped broadly. The responsesrecorded in the nucleus ovoidalis to contralateral stimulation had shorter latencies and were less complex than those in the dorsal nuclei; latencies ranged from 10 to 12 msec, amplitudes from 50 to 80 yv (Fig. 1) . On the lateral edge of the nucleus ovoidalis ipsilateral clicks produced waves with 9- to 12-mseclatencies and IO- to lOO+v amplitudes. In the region 1.5 to 2.0 mm ventral to the nucleus ovoidalis (in or very near tractus nucleusovoidalis) latencies were from 6 to 11 msecwhen evoked by either ipsilateral or contralateral stimuli. Amplitudes ranged from 30 to 150 nv. Just ventral to this (A 5.0-5.5; V 2-3; L I.53.0), evoked potentials had short latencies (4.5 to 7.0 msec), and relatively high amplitudes (6.5 to 175 nv). They faded rapidly at stimulation frequencies from 10 to 20/set, and no responsecould be seen over 20/set frequencies. The region from which these responseswere evoked would appear to be that of connections between mesencephalonand supraoptic commissure.These tracts are afferent to nucleus ovoidalis as indicated by the latencies and by their degeneration after mesencephaliclesions (6). Similar responseswere observed medially and ventrally to the nucleus rotundus, which itself did not respond to clicks. These short-latency responseswere not over 40 to 50 lrv and most of them were smaller. Other responseswith much longer latencies were observed in this area although they were not often seen. Their amplitudes were low (10 to 30 uv) with latencies of 14 to 17 msec. These responses probably were of the fibers from mesencephalonto ovoidalis. In the lateral forebrain bundle, at atlas coordinates of A 7.5-9.0, V 5.5-6, and L 2-3, responseshad latent periods of 8 to 12 msec and amplitudes of 30 to 100 uv, but the responseswere not sharp. These responseswere much more consistent to contralateral than to ipsilateral stimulation. Repetitive clicks up to 8/set elicited responses.Above 8 click/set, no responsescould be seen. The second of a pair of clicks elicited no responseat intervals less than 10 msec. Forebrain. Responseswere evoked more frequently from the paleostriatum primitivum than from the paleostriatum augumentatum. The responsesin the paleostriatum primitivum to either ipsilateral or contralateral stimuli were waves of II- to 17-mseclatency (mostly 12 to 13 msec) with 35 to 1lo-nv amplitudes (Fig. 1). In the caudal neostriatum stimulation of the opposite ear evoked potentials consisting of an initial wave at 13- to 17-mseclatency (most frequently 14 to 16 msec) having a 25 to 250~nv amplitude. Slower waves were recorded with latencies of 18 to 40 msecand in most casessmall early potentials were recorded at 8 to 13 msec. When both ears were stimulated simultaneously, potentials had latencies of 13 to 15 msec, amplitudes of 65 to 110 pv, and
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lasted 20 to 30 msec. Duration was longer than when either ear was stimulated alone. Potentials evoked in the caudal neostriatum faded more rapidly with increased frequency than did those in the mesencephalon and diencephalon. Figure 3 shows the effect of increasing stimulus intensity and frequency on the response amplitude. Neither ipsilateral nor contralateral clicks evoked
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CLICKS PER SECOND FIG. 3. Graph showing the relationship of stimulus intensity and frequency on the response amplitude. Dotted line: 50-v stimulus intensity. Solid line: 15-v intensity. Abscissa: click frequency per second. Ordinate: amplitude of response in microvolts. Data from the neostriatum.
a second response when the interval between the clicks was less than 7 msec (Fig. 2) compared to 3.6 msec for full restoration of response in midbrain. Increased intensity seemed either to inhibit or to obscure some of the components of the response. In the frontal neostriatum (Fig. 1)) some responses to contralateral stimulation were negative waves with 8- to ll-msec latency and lo- to 250yv amplitudes. Others were positive waves with latencies at 8 to 12 msec and lOO- to IlO-pv amplitudes. These waves had slower components at 14- to 25msec latency with amplitudes of 40 to 110 pv. Sometimes shorter
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latency (5.5 to 8 msec, 50 to 70 pv) negative potentials preceded these positive waves. Silent regions of the forebrain are shown in Fig. 1. Discussion
The auditory pathway in birds appears to run from the cochlear nuclei primarily to nucleus mesencephalicus lateralis pars dorsalis with possibly some fibers to the isthmi complex. We regularly recorded evoked responses to clicks in the isthmic region of chickens as did Erulkar (4) in pigeons. Karten, however, found no important projection to this area in his degeneration studies of pigeon brain (personal communication). A possible explanation of this apparent difference is suggested by the large size of the electrodes used in both studies compared to the small nuclei, and the fact that the lateral lemniscus fibers course right above the nuclei, especially on their medial sides. Very likely any such electrode recording from the isthmi complex would also be at least partially in the lemniscal fibers and so record activity there. Unit records from microelectrodes would be necessary to prove auditory activation of the isthmic nuclei in the face of the negative anatomical evidence. From the midbrain nuclei, auditory information is transmitted to nucleus ovoidalis (lo- to 12-msec latency from click) by way of tractus nucleus ovoidalis and the dorsal thalamic nuclei, and from these thalamic relays on to paleostriatum and neostriatum (12-16 msec) by way of the lateral forebrain bundle. Some responses were also recorded in the supraoptic commissure: these were probably from the fibers described by Karten (6) crossing from MLD to contralateral nucleus ovoidalis by way of tractus nucleus ovoidalis or possibly from those described by Huber and Crosby (5) from nucleus isthmi. Our demonstration of click-evoked responses in nucleus ovoidalis combined with Karten’s (6) demonstration of the path from MLD to ovoidalis confirms the suggestion by Papez ( 7) that the ovoidalis (“spiriformis” of Papez) is a thalamic relay for acoustic impulses in birds. Erulkar’s (4) failure to evoke responses in the thalamus of pigeons is puzzling but may have been a result of his technique of aspirating the hemispheres for recording from lower structures. We left the brain intact except for the electrode insertion itself. Species differences between pigeons and chickens seem unlikely to be responsible for the presence of thalamic responses in our birds and their absence in Erulkar’s. Similarity between chickens and pigeons is indicated by the comparable response latencies in both species and by the fact that Karten’s anatomical studies in pigeons show auditory connections where we recorded them in chickens. Numerous instances where responses of 100 pv or more abruptly appeared or disappeared with electrode movements of 0.5 mm or less furnished evidence for limited
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pickup by our electrodes. The sharply restricted areas from which these potentials could be detected supports the interpretation that potentials originated close to the points where we recorded them. Presence of potentials in the dorsal thalamic nuclei suggests that more than one path may project to the forebrain, a suggestion made more probable by the widely varying latencies of the responses that we recorded in various parts of the striatum. Karten’s (6) failure to find degeneration in the dorsal thalamic nuclei after MLD lesions supports the probability that more synapses (based on longer latencies) occur in the auditory input to these nuclei than to ovoidalis. Figure 4 diagrams possible pathways consistent with our data and with known anatomy. Presence of large potentials in
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FIG. 4. diagrammed,
Diagrammatic although
summary at all levels
of main features of auditory path. Only one side is both sides of the brain are activated by either ear.
frontal neostriatum and hyperstriatum with latencies of only 5 to 11 msec seemsto require that an appreciable number of acoustic fibers reach there without synapse in the thalamus where latencies were already lo-17 msec. Tractus striotegmentalis (2) runs approximately through the active points, but no auditory connections have been described for it, and our data do not permit us to assignthe responsesto it. Experimental degeneration studies will be needed to clarify the details. Confirmation that conduction is toward the striatum in the paths between midbrain nuclei and neostriatum was provided by a few experiments in which two pairs of electrodes were lowered into the brain, one in the midbrain and one in caudomedial neostriatum, until each encountered clickevoked responses.Then each pair was used in turn for stimulating and for recording. In all casespotentials could be evoked in the neostriatum by midbrain stimulation but not the reverse, indicating both that normal conduc-
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tion is afferent to neostriatum from these midbrain points and that at least one synapse is interposed in this path. Comparison of the neostriate latencies (14 to 17 msec) for click-evoked responses with those evoked by midbrain stimulation (9 msec) plus the latency for click activation of MLD (6 to 7 msec) shows them to be similar, supporting the contention that we were indeed testing a normal auditory pathway. The shorter latencies and greater ability of midbrain and thalamic than of forebrain sites to follow repetitive stimuli is further evidence that these former areas were activated by sensory impulses not by reflex discharges from the striatum. Adamo and King (1) reported click-evoked responses from the surface of the “Wulst” in chickens under chloralose. This is paralleled by our records from the rostra1 pole of the forebrain (Fig. 1). We did not record for the first 3 or 4 mm on most tracks so our failure to find surface activity may not reflect an actual lack of responses, or their records may reflect distant. pickup of deeper activity. References 1. 2.
3. 4.
5.
6. 7. 8.
9. 10.
ADAMO, N. J., and R. L. KING. 1965. Electrical responses to auditory stimulation in the chicken telencephalon. Anat. Record 151: 317. ARI~?NS-KAPPERS, C. V., G. C. HUBER, and E. C. CROSBY. 1936. “The Comparative Anatomy of the Nervous System of Vertebrates Including Man.” Macmillan, New York. BOORD, R. L., and G. L. RASMUSSEN. 1963. Projection of the cochlear and lagenar nerves on the cochlear nuclei of the pigeon. J. Co@. Neural. 120: 463-475. ERULKAR, S. D. 1955. Tactile and auditory areas in the brain of the pigeon. An experimental study by means of evoked potentials. J. Camp. Neural. 103: 421457. HUBER, G. C., and E. C. CROSBY. 1929. The nuclei and fiber paths of the avian diencephalon, with considerations of telencephalic centers and connections. .7. Camp. Neural. 43: l-25.5. KARTEN, H. 1966. Efferent projections of the nucleus mesencephalicus lateralis, pars dorsalis (MLD) in the pigeon (Columba Zivia). Anat. Rec. 154: 365. PAPU, J. W. 1929. “Comparative Neurology.” Crowell, New York. (Reprinted by Hafner, New York.) PUMPHREY, R. J. 1961. Sensory mechanisms: Hearing, pp. 69-86. In “Biology and Comparative Physiology of Birds.” A. J. Marshall Led.]. Academic Press, New York. STOPP, P. E., and I. C. WHITFIELD. 1961. Unit responses from brain-stem nuclei in the pigeon. J. Physiol. London 158: 165-177. VAN TIENHOVEN, A., and L. P. JUHASZ. 1962. The chicken telencephalon, diencephalon, and mesencephalon in stereotaxic coordinates. 1. Camp. Neural. 118: 185-198.