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Functional laminar and columnar organization of the auditory centers in echolocating Japanese greater horseshoe bats
\'eutos~wmeletter~ 88(I988) 17 20 Elsevier Sclentlh¢ Publishers Ireland Ltd
17
NSL 0S262
Functional laminar and columnar organization of the auditory centers in echolocating Japanese greater horseshoe bats Ikuo Tamguchl l, Oklo Aral 2 and N o z o m u S m t o 2 'D~patlm~tt o/ Imlllorl Dt~order~ Medual Re~eardl In~ltlule Toledo Medt~alandD, ntal ~ m ~ ~l~ l o a l o rJapan ~ and'Department o/ Pln ~tolo~ Dol~h~o ['nn'et~lt~ S~hool o/ l~I~dt¢ltu Fo~hn,,t fJapan ~ (Received 14 December 1987 Accepted 7 Januar'v 1988} k~ ~ i, otd~
Audltor~, center Echolocation Bat
During echolocation bats coordinate b emplo) the xocahzahon system to emit orientation sounds and the audltor,¢ s~stem to hear returning echoes The brain m these ammals should be lunctxonall', organized to process such btologlcalb ~lgmheant sounds Here we utilized the autoradlographlc 2-deox>-[~aC]glu~o~ I DG) method It) ~Jsuahze functional organlzdtlon of the auditory centers o1 echolocating Japanese greater horseshoe bats Rhmolophu~/trrumequmum nippon Autoradlographlc color images made lrom lht_ brains ol echolocating bats resealed a functional laminar structure m the inferior ¢olhculus Furthermore a lun¢honal columnar organl7allon appeared in the audltor'v cortex during echolocation These laminar and eolunmar organlTatlon were not observed in the brain ol resting bats
Echolocation sounds and hearmg of the Japanese greater horseshoe bats have been recently studmd [10] This species emits an ortentauon sound (pulse) from the nostril which consist of a long constant frequency (CF) component followed by a short downward frequency-modulated (FM) component The duration of a pulse is about 30 ms and the CF o f a restmg pulse is about 65 5 kHz Their hearmg is most sensmve up to around 66 kHz, shghtly higher than the CF When the bats are smnulated by a frequency near the optimal frequency as a mimetic echo, they vocahze frequently orientation sounds Four bats were examined during echolocation, and two bats were examined ~ hen they' were restmg 2-Deoxy-t)-[L4C]glucose (DG, Ne~ England Nuclear, spec act 5~ mCi/mmol) was ex aporated, and reconstituted tn sterile sahne DG was administered mtrapentoneally as a pulse with 2 pCl m 0 2 ml normal sahne to the alert and unanesthettzed ammal [7] After rejection, each antmal was hung from a toe-hold m a small btrd-~.age m a sound-proof room wtth or without sound sttmulatlon Eighty mm alter the DG rejection, the antmal was ktlled by mtraperttoneal admlnlstratmn ol a (orle~pondem~ I Tamguchl Department of Audnory Disorders Medical Research Institute Tokyo Medical and Dental Umverslt', 1-5-45 "Yushlma Bunkyo-ku Tokyo 113 Japan 0:;04-~,940 88 $ IB ~0 @ 1988 Else'~ler Scmntfllc Publishers Ireland Ltd
18 large dose of sodium pentobarbltal The brain was quickly removed and placed m embedding medium (Lab-Tek Products, Tlssue-Tek II) in a small metal contalncJ and frozen in acetone cooled with dry ice The frozen brain was mounted with embedding medium on a cryobtat (Nakagav~a N A 300) maintained at - 15 C, and frozen sections 30/tm thick were cut Sections were picked up on glass shdes and dried at room temperature The sections were then exposed to single emulsmn X-ray film (Sakura M A R G . ~H-type) m an X-ray him cassette for 4 days, and the film was developed The optacal density of the autoradlographs was measured by a den~atometer (Joel JCR-80) with an aparture of 0 2 mm, which was controlled by a computer (Digital Equipment Corporatlom PDP 11-40) The reading pitch of light transmittance was 0 1 mm The dagatal data of optical density were transformed to 9 dafl'erent colors Then the color image was displayed on the face of a color monitor to be photographed To stimulate the bats to vocahzatlon, a tone burst of 66 kHz was generated with a function generator (NFB, FG-163) through an electronic swatch Rise-fall time, duration and interval of the tone bursts were I, 30 and 60 ms, respectively The electronic switch was triggered manually to present the stimulus until the bats began to vocahze The tone bursts were amphfied by a power amplifier (Technics, SU-V8) and dehvered to the ammals through a tweeter (Techmchs, 10H 1000, 3 dB from 4 to 100 kHz) which was placed 1 m in front of the animal The sound pressure level was fixed at 60 dB SPL whach was calibrated with a heterodyne analyzer (Bruel and Kjaer) and a one-eight-inch condenser macrophone (Bruel and Kjaer, 4183) posmoned close to the animal's head Fig 1A shows the autoradmgraphac color images made at the level of the inferior colhculus A laminar structure m the central nucleus of the inferior colhculus was revealed in echolocating bats~ but such a structure was not readdy detected m resting bats The laminar structure represents a tonotoplcal organization whach has been shown in the inferior colhculus of other mammals [1,6] The lowest and active lamina may consast of a cluster of neurons that respond to the optimal frequency around 66 kHz A less acnve lamina was also observed between the active ones The neurons in the less active lamina may optimally respond to a frequency slightly lower than 66 kHz, but their actavlty Is suppressed during echolocation Fig 1B shows the autoradmgraph~c color amages made from a coronal sectmn at the level of the auditory cortex D G uptake Into the auditory cortex was increased by echolocation Furthermore, functional columns approximately vertical to the cortical surface appeared during echolocation They are arranged alternately active and less active and are on the average about 0 3 mm apart Such a structure IS very similar to the ocular dominance columns found m the visual cortex of other mammals [2, 3] An electrophyslologlcal study on the auditory cortex of a European subspecies, Rhmolophuv [errumequmumferrumequmum has suggested that the CF and FM components in pulse and echo are processed separately in two subdivisions In the auditory area [5] More subdivisions in the auditory cortex have been found in the mus-
19
A
1 O0
75::,
!
25
[lg 1 A color images of D G autoradiographs of coronal sections taken at the level of the inferior colhculus A laminar structure Is clear m the central nucleus of the inferior colhculus of the echolocating bat (right) as indicated b~ the arrow, but not m the resting bat (left) B color ,mages of DG autoradlographs o[ coronal sectmns taken at the level of the auditory cortex A columnar organization m the auditor,; cortex appears m the echolocating bat (left) as indicated by the arrov, q-he columns in the right auditor,, ar~.a are obscure due to small tissue fold,, No columnar orgamzatmn appears in the resting bat (lelt) I hr. relall'~e optical den',ltms are shown b~ the vertical columns in color on the right side
tached
bat whose
ortentatton
sounds
our study, the bats were stimulated and
returning
echo
from
unchanged
other m regard to the CF component
consist of the CF and FM
components
by a tone burst of 66 kHz, an ortentatmn surroundmgs
These
If there are separate
sttmuh
[8]
In
sound
are close to each
subdtvxslons for process-
20
mg the specific parameters revolved in the snmuh, active regmns should appear discretely Several cortmal columns shown tn this study may correspond to actwe regmns m such subdivisions The suppressed regmn lateral to the actwe regmn ~s observed m both inferior colhculus and auditory cortex during echolocatmn The electrophyslologmal evidence for inhibition durmg vocallzatmn has been reported m the auditory system o f bats [9] and other mammals [4] Our results may suggest that vocalization suppresses the au&tory centers spacmlly m a lammar or columnar manner Inputs to such functional structures may be sent from the sensory feedback via the auditory pathways or from the vocahzatton centers We thank K Ktmura and K Mlyamoto at D o k k y o Umverslty School o f Medicine for allowmg us to use the facthttes to make the autoradmgraphs and to process the color images 1 Clopton, B M and Wlnheld J A Tonotoplt- organization in the inferior colhculus ol the. rat Brain Res 56 (197~) 355 358 2 Hubel D H and Wmsel T N , Laminar and columnar distribution of genlculo-cortmal hbers m the macaque monkey, J Comp Neurol 146(1972)421 450 3 Kenned), C Des Roslers, M H Sakurada O , Shlnohara, M , Relvlch M , Jehle, J W and Sokoloff L, Metabolic mapping of the primary visual system of the monkey b) means of the autoradlographlt. [14C]deox)glucose technique, Proc Natl Acad Scl U S A , 73 (1976) 42304234 4 Multer-Preuss, P and Ploog D , Inhlbmon ot auditory cortical neurons during phonatlon Brain Res 215(1981)61 76 5 Oswald, J , Tonotop~cal orgamzatlon and pure tone response character~stms ot single umts m the auditory cortex of the greater horsesho~ bat J Comp Physlol, 155 (1984) 821 834 6 Rose, J E Greenwood D D , Goldberg, J M and Hind, J E , Some discharge characteristics ot single neurons m the inferior colhculus ol the cat 1 Tonotopmal orgamzatmn, relanon oI spike-counts to tone intensity, and firing patterns of single elements, J Neurophysml 26 (1963) 29,g 320 7 Sokoloff, L Rewlch M , Kennedy C , Des Roslers M H , Patlak, C S, Pettlgrew, K D , Sakurada O and Shmohara, M , The p4C]deoxyglucose method for the measurement of local cerebral glucose utfllzatmn theory, procedure, and normal values m the conscmus and anesthetized albino rat, J Neurochem, 28 (1977) 897 916 8 Suga, N Specmhzatmn of the auditory system for receptmn and processing of spemes-speofic sounds F~d Proc ~;7(1978) 2:~42 2~54 9 ~,uga N and Shlmozawa T Site ol neural attenuation ot responses to ~elt-vocallTed ~ounds m ~.dmio ~.atmgbat Science 183 (1974) 1211-1213 10 TamguLhl, I Echolocation sounds and hearing of the greater Japanese horseshoe bat (Rhtnolophus tetrum¢qumummppon) J Comp Phvsml 156(1985) 185 188
17
NSL 0S262
Functional laminar and columnar organization of the auditory centers in echolocating Japanese greater horseshoe bats Ikuo Tamguchl l, Oklo Aral 2 and N o z o m u S m t o 2 'D~patlm~tt o/ Imlllorl Dt~order~ Medual Re~eardl In~ltlule Toledo Medt~alandD, ntal ~ m ~ ~l~ l o a l o rJapan ~ and'Department o/ Pln ~tolo~ Dol~h~o ['nn'et~lt~ S~hool o/ l~I~dt¢ltu Fo~hn,,t fJapan ~ (Received 14 December 1987 Accepted 7 Januar'v 1988} k~ ~ i, otd~
Audltor~, center Echolocation Bat
During echolocation bats coordinate b emplo) the xocahzahon system to emit orientation sounds and the audltor,¢ s~stem to hear returning echoes The brain m these ammals should be lunctxonall', organized to process such btologlcalb ~lgmheant sounds Here we utilized the autoradlographlc 2-deox>-[~aC]glu~o~ I DG) method It) ~Jsuahze functional organlzdtlon of the auditory centers o1 echolocating Japanese greater horseshoe bats Rhmolophu~/trrumequmum nippon Autoradlographlc color images made lrom lht_ brains ol echolocating bats resealed a functional laminar structure m the inferior ¢olhculus Furthermore a lun¢honal columnar organl7allon appeared in the audltor'v cortex during echolocation These laminar and eolunmar organlTatlon were not observed in the brain ol resting bats
Echolocation sounds and hearmg of the Japanese greater horseshoe bats have been recently studmd [10] This species emits an ortentauon sound (pulse) from the nostril which consist of a long constant frequency (CF) component followed by a short downward frequency-modulated (FM) component The duration of a pulse is about 30 ms and the CF o f a restmg pulse is about 65 5 kHz Their hearmg is most sensmve up to around 66 kHz, shghtly higher than the CF When the bats are smnulated by a frequency near the optimal frequency as a mimetic echo, they vocahze frequently orientation sounds Four bats were examined during echolocation, and two bats were examined ~ hen they' were restmg 2-Deoxy-t)-[L4C]glucose (DG, Ne~ England Nuclear, spec act 5~ mCi/mmol) was ex aporated, and reconstituted tn sterile sahne DG was administered mtrapentoneally as a pulse with 2 pCl m 0 2 ml normal sahne to the alert and unanesthettzed ammal [7] After rejection, each antmal was hung from a toe-hold m a small btrd-~.age m a sound-proof room wtth or without sound sttmulatlon Eighty mm alter the DG rejection, the antmal was ktlled by mtraperttoneal admlnlstratmn ol a (orle~pondem~ I Tamguchl Department of Audnory Disorders Medical Research Institute Tokyo Medical and Dental Umverslt', 1-5-45 "Yushlma Bunkyo-ku Tokyo 113 Japan 0:;04-~,940 88 $ IB ~0 @ 1988 Else'~ler Scmntfllc Publishers Ireland Ltd
18 large dose of sodium pentobarbltal The brain was quickly removed and placed m embedding medium (Lab-Tek Products, Tlssue-Tek II) in a small metal contalncJ and frozen in acetone cooled with dry ice The frozen brain was mounted with embedding medium on a cryobtat (Nakagav~a N A 300) maintained at - 15 C, and frozen sections 30/tm thick were cut Sections were picked up on glass shdes and dried at room temperature The sections were then exposed to single emulsmn X-ray film (Sakura M A R G . ~H-type) m an X-ray him cassette for 4 days, and the film was developed The optacal density of the autoradlographs was measured by a den~atometer (Joel JCR-80) with an aparture of 0 2 mm, which was controlled by a computer (Digital Equipment Corporatlom PDP 11-40) The reading pitch of light transmittance was 0 1 mm The dagatal data of optical density were transformed to 9 dafl'erent colors Then the color image was displayed on the face of a color monitor to be photographed To stimulate the bats to vocahzatlon, a tone burst of 66 kHz was generated with a function generator (NFB, FG-163) through an electronic swatch Rise-fall time, duration and interval of the tone bursts were I, 30 and 60 ms, respectively The electronic switch was triggered manually to present the stimulus until the bats began to vocahze The tone bursts were amphfied by a power amplifier (Technics, SU-V8) and dehvered to the ammals through a tweeter (Techmchs, 10H 1000, 3 dB from 4 to 100 kHz) which was placed 1 m in front of the animal The sound pressure level was fixed at 60 dB SPL whach was calibrated with a heterodyne analyzer (Bruel and Kjaer) and a one-eight-inch condenser macrophone (Bruel and Kjaer, 4183) posmoned close to the animal's head Fig 1A shows the autoradmgraphac color images made at the level of the inferior colhculus A laminar structure m the central nucleus of the inferior colhculus was revealed in echolocating bats~ but such a structure was not readdy detected m resting bats The laminar structure represents a tonotoplcal organization whach has been shown in the inferior colhculus of other mammals [1,6] The lowest and active lamina may consast of a cluster of neurons that respond to the optimal frequency around 66 kHz A less acnve lamina was also observed between the active ones The neurons in the less active lamina may optimally respond to a frequency slightly lower than 66 kHz, but their actavlty Is suppressed during echolocation Fig 1B shows the autoradmgraph~c color amages made from a coronal sectmn at the level of the auditory cortex D G uptake Into the auditory cortex was increased by echolocation Furthermore, functional columns approximately vertical to the cortical surface appeared during echolocation They are arranged alternately active and less active and are on the average about 0 3 mm apart Such a structure IS very similar to the ocular dominance columns found m the visual cortex of other mammals [2, 3] An electrophyslologlcal study on the auditory cortex of a European subspecies, Rhmolophuv [errumequmumferrumequmum has suggested that the CF and FM components in pulse and echo are processed separately in two subdivisions In the auditory area [5] More subdivisions in the auditory cortex have been found in the mus-
19
A
1 O0
75::,
!
25
[lg 1 A color images of D G autoradiographs of coronal sections taken at the level of the inferior colhculus A laminar structure Is clear m the central nucleus of the inferior colhculus of the echolocating bat (right) as indicated b~ the arrow, but not m the resting bat (left) B color ,mages of DG autoradlographs o[ coronal sectmns taken at the level of the auditory cortex A columnar organization m the auditor,; cortex appears m the echolocating bat (left) as indicated by the arrov, q-he columns in the right auditor,, ar~.a are obscure due to small tissue fold,, No columnar orgamzatmn appears in the resting bat (lelt) I hr. relall'~e optical den',ltms are shown b~ the vertical columns in color on the right side
tached
bat whose
ortentatton
sounds
our study, the bats were stimulated and
returning
echo
from
unchanged
other m regard to the CF component
consist of the CF and FM
components
by a tone burst of 66 kHz, an ortentatmn surroundmgs
These
If there are separate
sttmuh
[8]
In
sound
are close to each
subdtvxslons for process-
20
mg the specific parameters revolved in the snmuh, active regmns should appear discretely Several cortmal columns shown tn this study may correspond to actwe regmns m such subdivisions The suppressed regmn lateral to the actwe regmn ~s observed m both inferior colhculus and auditory cortex during echolocatmn The electrophyslologmal evidence for inhibition durmg vocallzatmn has been reported m the auditory system o f bats [9] and other mammals [4] Our results may suggest that vocalization suppresses the au&tory centers spacmlly m a lammar or columnar manner Inputs to such functional structures may be sent from the sensory feedback via the auditory pathways or from the vocahzatton centers We thank K Ktmura and K Mlyamoto at D o k k y o Umverslty School o f Medicine for allowmg us to use the facthttes to make the autoradmgraphs and to process the color images 1 Clopton, B M and Wlnheld J A Tonotoplt- organization in the inferior colhculus ol the. rat Brain Res 56 (197~) 355 358 2 Hubel D H and Wmsel T N , Laminar and columnar distribution of genlculo-cortmal hbers m the macaque monkey, J Comp Neurol 146(1972)421 450 3 Kenned), C Des Roslers, M H Sakurada O , Shlnohara, M , Relvlch M , Jehle, J W and Sokoloff L, Metabolic mapping of the primary visual system of the monkey b) means of the autoradlographlt. [14C]deox)glucose technique, Proc Natl Acad Scl U S A , 73 (1976) 42304234 4 Multer-Preuss, P and Ploog D , Inhlbmon ot auditory cortical neurons during phonatlon Brain Res 215(1981)61 76 5 Oswald, J , Tonotop~cal orgamzatlon and pure tone response character~stms ot single umts m the auditory cortex of the greater horsesho~ bat J Comp Physlol, 155 (1984) 821 834 6 Rose, J E Greenwood D D , Goldberg, J M and Hind, J E , Some discharge characteristics ot single neurons m the inferior colhculus ol the cat 1 Tonotopmal orgamzatmn, relanon oI spike-counts to tone intensity, and firing patterns of single elements, J Neurophysml 26 (1963) 29,g 320 7 Sokoloff, L Rewlch M , Kennedy C , Des Roslers M H , Patlak, C S, Pettlgrew, K D , Sakurada O and Shmohara, M , The p4C]deoxyglucose method for the measurement of local cerebral glucose utfllzatmn theory, procedure, and normal values m the conscmus and anesthetized albino rat, J Neurochem, 28 (1977) 897 916 8 Suga, N Specmhzatmn of the auditory system for receptmn and processing of spemes-speofic sounds F~d Proc ~;7(1978) 2:~42 2~54 9 ~,uga N and Shlmozawa T Site ol neural attenuation ot responses to ~elt-vocallTed ~ounds m ~.dmio ~.atmgbat Science 183 (1974) 1211-1213 10 TamguLhl, I Echolocation sounds and hearing of the greater Japanese horseshoe bat (Rhtnolophus tetrum¢qumummppon) J Comp Phvsml 156(1985) 185 188