Cy oa chi ec u e and Myeloa chi ec u e o he
sheep (O is a ies) audi o y co ex
Camille Plucho 1, Mélody Mo isse1, Ma yse Meu isse1, Jean-Ma ie G aïc2, Elodie
Chaillou1, Sco A. Lo e1
1INRAE, CNRS, Uni e si é de Tou s, PRC, 37380, Nouzilly, F ance
2Depa men o Compa a i e Biomedicine and Food Science, Uni e si y o
Pado a, Legna o
E-mail add esses, elephone numbe , ORCID, add ess o co esponding au ho s
[email p o ec ed] ; 02.47.42.78.53; ORCID: h ps://o cid.o g/0009-0005-7532-6624
[email p o ec ed] ; 02.47.42.75.00; ORCID: h ps://o cid.o g/0000-0001-7416-9210
INRAE Cen e Val de Loi e
37380 Nouzilly
F ance
E-mail add esses and ORCID o co-au ho s
[email p o ec ed] ; ORCID: h ps://o cid.o g/0000-0002-5577-8982
[email p o ec ed] ; ORCID: h ps://o cid.o g/0000-0002-1974-8356
melody[email p o ec ed]
ma y[email p o ec ed]
Sho unning i le: Sheep audi o y co ex neu oana omy
1
Acknowledgmen s
We hank F édé ic Le y o help ul discussions in designing his esea ch, Ma ie-Line
Ca eau o echnical suppo , and Louise and Mau ine Guillo eau, s uden s om Uni e si y
Bachelo o Technology o hei pa icipa ion in he s udy. This wo k bene i ed om he
equipmen and expe ise o he Imaging acili y "Pla eau d’Image ie Cellulai e" (PIC) o
UMR-PRC (h p://doi.o g/10.17180/a ap-gj59). We acknowledge he inancial suppo o he
Agence Na ionale de la Reche che (SheepVoice MRI: ANR-20-CE20-0001-01) and he Ins i u
Na ional de Reche che pou l’Ag icul u e, l’Alimen a ion e l’En i onnemen Dépa emen
Physiologie Animale e Sys èmes d’Ele age.
Da a Sha ing and Da a A ailabili y
The da a gene a ed du ing he cu en s udy a e a ailable in he associa ed Zenodo
eposi o y: 10.5281/zenodo.14824250. The eposi o y includes comp essed (quali y 70%)
e sions o he scanned sec ions wi h hei anno a ions (.czi ile o ma ) and sp eadshee s
con aining laye hickness and cell diame e measu emen s. Comp essed iles a e being made
a ailable, a he han uncomp essed, o comply wi h he size limi o a Zenodo eposi o y. The
quali y o he comp essed images is mo e han su icien o eplica e he cu en esul s;
howe e , uncomp essed e sions can be ob ained om he au ho s.
E hics app o al s a emen
This esea ch was conduc ed in compliance wi h F ench and Eu opean guidelines o he
housing and ca e o animals used o scien i ic pu poses (Eu opean Union Di ec i e
2010/63/EU). In acco dance wi h he 3R p inciples o animal esea ch, we chose o acqui e he
necessa y biological issue om animals ha we e ea ed and eu hanized, independen ly o he
p esen s udy; no expe imen al p ocedu es we e conduc ed on any li e animals.
2
Con lic o in e es disclosu e
The au ho s ha e no ele an inancial o non- inancial in e es s o disclose.
3
Abs ac
The audi o y co ex is cen al o audi o y pe cep ion, bu i s de ailed s uc u al and unc ional
o ganiza ion in sheep (O is a ies) has no been ho oughly in es iga ed. In his s udy, we sough
o add ess his gap by p o iding an in-dep h o e iew o he cy oa chi ec u e and
myeloa chi ec u e o he sheep audi o y co ex, using c esyl iole s aining and he
neu ochemical ma ke s myelin basic p o ein and pa albumin. Tissue samples om ou sheep
we e used o cha ac e ize co ical laye s, cellula composi ion, and myelina ion pa e ns,
e ealing a six-laye ed o ganiza ion wi h a ia ions in cell densi y and dis ibu ion. Myelin basic
p o ein s aining highligh ed myelina ed egions, p o iding insigh s in o he myeloa chi ec u e,
while pa albumin s aining iden i ied he dis ibu ion o GABAe gic in e neu ons, p o iding
indica ions o he po en ial loca ion o he p ima y audi o y co ex. These indings deepen ou
knowledge o he audi o y co ex in sheep, a key model o in es iga ing senso y p ocessing in
la ge mammals. The s uc u al and unc ional o ganiza ion o he o ine audi o y co ex aligns
wi h indings in o he mammals, sugges ing ha bo h a e conse ed ac oss species and
suppo ing he idea o e olu iona y conse a ion in audi o y p ocessing mechanisms. Howe e ,
u u e unc ional s udies, using audi o y s imula ion pa adigms, a e necessa y o ully
unde s and he unc ional o ganiza ion o his impo an senso y egion.
Keywo ds:
audi o y co ex - o ine - pa albumin - myelin basic p o ein - in e neu ons - pos e io ec osyl ian
gy us
4
In oduc ion
In mammals, he audi o y co ex is gene ally loca ed bila e ally in he pa ie o- empo al
egions o he b ain. I is a hie a chically o ganized s uc u e, consis ing o se e al dis inc
subdi isions, each specialized in di e en aspec s o audi o y pe cep ion (Kaas, 2011). Cen al
o hese subdi isions, he p ima y audi o y co ex is esponsible o he ini ial co ical p ocessing
o audi o y in o ma ion.
His ological echniques, such as immunohis ochemis y and neu al acing, ha e p o ided
aluable insigh s in o he cy oa chi ec u e and myeloa chi ec u e o he audi o y co ex ac oss
a ious mammalian species (human: Hacke e al., 2001; non-human p ima e: Hacke e al.,
2001; mouse: Ande son e al., 2009; ca : Wine & P ie o, 2001; abbi : De Venecia e al., 1998;
e e : Bajo e al., 2007; gleaning ba : Ma in del Campo e al., 2014; A ican wild dog:
Chenge anai e al., 2020). These s udies consis en ly show ha he audi o y co ex is o ganized
in o six dis inc co ical laye s, wi h a ia ions in hickness, cellula composi ion and dis ibu ion
(i.e., py amidal neu ons, in e neu ons, and glial cells, depending on he co ical laye s).
Due o hei social na u e, sheep (O is a ies) use senso y modali ies such as hea ing and
ision o communica ion and en i onmen al awa eness, making hem an in e es ing model o
s udying how he ana omy o mammalian b ains is se up o p ocess such senso y in o ma ion.
The sheep mo o and soma osenso y co ices ha e been ex ensi ely s udied (Bagley, 1922;
Dinopoulos e al., 1985; Gie hmuehlen e al., 2014; Johnson e al., 1974; King, 1911; Pe u o e
al., 2019; Simpson & King, 1911; Woolsey & Fai man, 1946). The p ima y isual co ex o sheep
has also ecei ed some a en ion (Cla ke e al., 1976; Cla ke & Whi e idge, 1976; Ebinge ,
1975; Ka amanlidis e al., 1979) and consis s o a six-laye ed o ganiza ion (G aïc e al., 2022),
simila o ha obse ed in o he mammals (ho ses, monkeys, bo lenose dolphins: G aïc e al.,
2022). Howe e , he cy oa chi ec u e o he sheep audi o y co ex emains la gely unexplo ed.
5
To da e, only wo s udies ha e in es iga ed he loca ion o he sheep audi o y co ex. In he
i s s udy, Michaloudi and colleagues (1986) injec ed a ho se adish pe oxidase e og ade
ace in o se e al a eas o he pos e io ec osyl ian gy us, in he pa ie al a ea o Rose (1942).
When he ace was injec ed in o he an e io pa o he pos e io ec osyl ian gy us, labeled
cells we e ound exclusi ely in he en al di ision o he medial genicula e nucleus (MGN ), he
inal subco ical elay o audi o y in o ma ion be o e he audi o y co ex. Based on his speci ic
connec ion, hey concluded ha his injec ion si e co esponded o he sheep audi o y co ex.
Decades la e , Sahas abuddhe and colleagues (2021) con i med he audi o y unc ion o his
egion by eco ding co ical su ace local ield po en ials in esponse o audi o y s imuli.
Toge he , hese indings sugges ha he sheep audi o y co ex is loca ed in b ain a eas
homologous o hose o o he mammals (Kaas, 2011). In e es ingly, he sheep has been used as
a model o hea ing loss using audi o y b ains em esponses in he e al and pe ina al phase
(Cook e al., 1987; G i i hs e al., 1994; Pie son e al., 1994, 1995), o middle ea his ology
(Robe o e al., 1989), bu no inqui y has been made u he up he unc ional chain.
The p esen s udy aims o p o ide a de ailed desc ip ion o he cy oa chi ec u e and
myeloa chi ec u e o he sheep audi o y co ex, using he localiza ion es ablished by he wo
a o emen ioned s udies (Michaloudi e al., 1986; Sahas abuddhe e al., 2021). Fo his pu pose,
we employed c esyl iole (‘Nissl’) s aining o de ine he numbe and hickness o co ical laye s,
as well as he composi ion and dis ibu ion o cells wi hin each laye . Addi ionally, we assessed
he dis ibu ion o Myelin Basic P o ein (MBP), a ma ke o myelina ed ibe s, o in es iga e he
co ical myeloa chi ec u e (Je ey e al., 1990). We also in es iga ed he neu ochemical
p ope ies o he sheep audi o y co ex by quan i ying he dis ibu ion o Pa albumin (PV), a
ma ke o GABAe gic in e neu ons, which has been used o delinea e he p ima y audi o y
co ex in a ious species (mice: C uikshank e al., 2001; abbi s: McMullen e al., 1994; ge bils:
Budinge e al., 2000; ca s: Wallace e al., 1991; and monkeys: Jones e al., 1995, Kaas &
6
Hacke , 2000, Kosaki e al., 1997). Toge he , hese app oaches p o ide aluable insigh s in o
he s uc u al and unc ional o ganiza ion o he sheep audi o y co ex.
Ma e ials and me hods
Tissue sampling
This esea ch was conduc ed in compliance wi h F ench and Eu opean guidelines o he
housing and ca e o animals used o scien i ic pu poses (Eu opean Union Di ec i e
2010/63/EU). In acco dance wi h he 3R p inciples o animal esea ch, we chose o acqui e he
necessa y biological issue om animals ha we e ea ed and eu hanized, independen ly o he
p esen s udy; no expe imen al p ocedu es we e conduc ed on any li e animals. The b ains o
ou Ile-de-F ance sheep (3 ewes: 13507, 13403, 13155; and 1 am: 03133), be ween wo and
h ee yea s o age, we e collec ed a a local slaugh e house (UEPAO,
h ps://doi.o g/10.15454/1.5573896321728955E12; ag eemen numbe G37–175–2). The sheep
we e adminis e ed an in a enous injec ion o ke amine (5 mL). A e con i ming he loss o
consciousness (i.e., absence o pupilla y and palpeb al e lexes), hey we e slaugh e ed by a
licensed bu che . Then, he heads we e immedia ely pe used in he ca o id a e ies wi h sodium
ni i e a 37°C (2L pe sheep, 1% in sodium chlo u e solu ion 0,9%), ollowed by 4%
pa a o maldehyde a 4°C (4L pe sheep, in phospha e bu e ed saline (PBS), 0.1M, pH 7.4). The
b ain was ex ac ed om he skull and pos - ixed wi h 4% pa a o maldehyde o 24h o 48h. I
was washed in se e al PBS ba hs o emo e excess pa a o maldehyde, and imme sed in a
20% suc ose c yop o ec an solu ion in PBS o p e en he o ma ion o ice c ys als du ing
subsequen eezing. Each b ain was cu in hal sagi ally, h ough he in e hemisphe ic issu e.
Then, each hemisphe e was sec ioned in o a co onal o ien a ion block o app oxima ely wo
cen ime e s co e ing he audi o y co ex a ea (i.e., pos e io ec osyl ian gy us, Michaloudi e al.,
1986). The bounda ies o he audi o y co ex we e de ined as ollows: an e io ly by he syl ian
7
issu e and he an e io ec osyl ian gy us; pos e io ly by he sup asyl ian sulcus and he
an e io pa o he pos e io syl ian gy us; en ally by he syl ian issu e; and do sally by he
sup asyl ian sulcus (Figu e 1). The blocks we e ozen wi h d y ice (- 70°C) on he mic o ome
s age, hen cu in o 40 µm hick co onal sec ions using a ozen mic o ome (The mo Fishe
Scien i ic, Sliding Mic o ome Mic om HM 430, Ge many). The co onal sec ions we e s o ed a 4
deg ees in PBS con aining 0.1% sodium azide, un il he espec i e s aining p o ocols we e
pe o med.
Figu e 1. Iden i ica ion o he audi o y co ex (dashed ed ci cle) wi hin he block delimi ed by he
wo e ical ed lines. La e al iew o he le hemisphe e o a sheep b ain. Th ee sec ions o he
audi o y co ex (An e io (A); Medial (M) ; Pos e io (P)) pe hemisphe e, a compa able le els om one
animal o ano he , we e chosen o he analysis o each animal. Syl ian Fissu e (SF), an e io Ec osyl ian
8
Gy us (aEG), pos e io Ec osyl ian Gy us (pEG), Sup asyl ian Sulcus (SSS) and pos e io Syl ian Gy us
(pSG).
C esyl iole s aining
Fo each block, one ou o wen y sec ions we e moun ed on gela in glass slides and s ained
wi h c esyl iole (Nissl p o ocol) o isualize he co ical ana omy along he an e io -pos e io
axis o he sheep audi o y co ex. Fi s , sec ions we e imme sed in a 0.5% c esyl iole solu ion
o 15 o 20 minu es and hen insed wice wi h wa e o emo e excess dye. The sec ions we e
hen dehyd a ed in h ee successi e ba hs o 95°C alcohol o wo o se en minu es and h ee
successi e ba hs a 100°C alcohol o wo o se en minu es. Finally, hey we e imme sed in
h ee successi e ba hs o oluene o a leas i e minu es and co e slipped wi h Depex and
scanned 48 hou s la e .
Immunohis ochemis y
Double labeling wi h pa albumin (PV) and Myelin Basic P o ein (MBP) was pe o med on
he sec ions adjacen o hose used o c esyl iole s aining. Sec ions we e pe meabilized in
PBS-T i on-Azide-BSA 1% (PBSTA-BSA) o one hou a oom empe a u e. Then, hey we e
incuba ed in he p ima y an ibodies solu ion (monoclonal mouse an i-PV and monoclonal a
an i-MBP) o 48 hou s a 37°C (Table 1). Then, sec ions we e insed in ou successi e ba hs o
PBS o en minu es. They we e incuba ed o h ee hou s a 4°C wi h he seconda y an ibodies
solu ion, con aining donkey an i-mouse-CY3 and donkey an i- a -488 (Table 1). Then, sec ions
we e insed in ou successi e ba hs o PBS o en minu es. The sec ions we e moun ed on
slides and o en-d ied a 37°C o a leas one day. They we e co e slipped unde
Fluo omoun -G® (Sou he n Bio echnology, Bi mingham, AL), p o ec ed om ligh and scanned
48 hou s la e .
15
Figu e 4. Nissl-s ained sec ion o he sheep audi o y co ex. A- Iden i ica ion o six co ical laye s (I,
II, III, IV, V, VI) and he whi e ma e (WM); B- Glial cells (pink a owhead) and Cajal-Re zius cells (b own
a owhead) o laye I; Sphe oid g anula cells (g een a owhead) and py amidal cells (black a owhead) o
laye s II (C), III (D), IV (E), V (F) and VI (G); La ge py amidal cells o laye V (un illed black a owhead);
G’- Spindle-shaped g anula cells o laye VI (un illed g een a owhead). No e ha glial cells we e p esen
in all six laye s (pink a owheads). Da a shown is om he ex e nal ROI, o he an e io sec ion, o he le
hemisphe e, o animal 03133. Scale ba s: 200 µm (A); 20 µm (B-G’).
Based on his cellula o ganiza ion, he hickness o each o he six co ical laye s ac oss
sec ions, o each ROI and hemisphe e o he ou animals was measu ed (Figu e 5). Laye s II
and IV we e he hinnes (131 µm ± 30; 181 µm ± 45, espec i ely) and exhibi ed he lowes
a iabili y. These wo laye s ep esen ed 7.9 and 10.9 % o he o al hickness, espec i ely.
Laye I was hicke (282 µm ± 71) and showed sligh ly mo e a iabili y han laye s II and IV. The
mean hicknesses o laye s III and V we e simila (342 µm ± 79; 341 µm ± 88, espec i ely)
while laye VI was he hickes (376 µm ± 136) o all laye s. The a iabili y obse ed in he
16
hicknesses o laye s I, III, V, and VI did no appea o be linked o any speci ic c i e ion, such as
ROIs, sec ions, hemisphe es, o animals. O e all, he mean pe cen age o o al co ical
hickness o laye s I, III, V and VI was simila , anging om 17.1 o 22.8 % (Table 2).
Figu e 5. Thickness (µm) o he six co ical laye s in he sheep audi o y co ex. Thickness o each
co ical laye was measu ed o he an e io , medial and pos e io sec ions wi hin he h ee ROIs (IT - IM -
ET, indica ed by di e en shapes). Measu emen s we e pe o med o bo h hemisphe es (indica ed by
poin size), in ou animals (dis inguished by colo ). No clea , consis en ends in laye hickness we e
obse ed ac oss ei he animal, hemisphe e, sec ion o ROI.
17
Table 2. Co ical o ganiza ion o he sheep audi o y co ex
C esyl iole
Myelin Basic P o ein
Pa albumin
Laye
Mean
hickness
Cell
Mean cell diame e
(µm)
Fibe
Cell
Mean cell diame e
(µm)
Fibe
µm
%
Type
Densi y
Min
Max
Densi y
O ien a ion
ela i e o he
co ical su ace
Densi y
Type
Min
Max
Densi y
O ien a ion ela i e
o he co ical
su ace
I
282
±71
17.1
±4.3
Glial
Mode a e
6.2±1.4
11±2.5
Low
Pa allel
Ø
Ø
Ø
Ø
Low
Pe pendicula
Cajal
Low
12.1±2.2
18.8±3
Pa allel
II
131
±30
7.9
±1.8
G anula
High
13.6±2.6
20.4±3.5
Low
Pe pendicula
Low
Sphe oïd
15.4±3
19.2±3.7
Mode a e
Pe pendicula
Polygonal
18±5.6
22±4.5
Py amidal
Low
13.7±2.6
21.3±3.3
Pa allel
Spindle-shaped
22.3±2
22.8±2.4
III
342
±9
20.7
±4.8
G anula
Mode a e
14.9±2.2
21.5±3.1
Mode a e
/ High
Pe pendicula
High
Sphe oïd
14.8±1.8
18.9±2
High
Pe pendicula
Polygonal
18±2.2
24.5±3.6
Py amidal
Mode a e
14.6±2.2
21.8±3
Pa allel
Spindle-shaped
21.9±3.3
24±2.8
IV
181
±45
10.9
±2.7
G anula
Mode a e
14.4±2.2
19.7±2.5
Mode a e
/ High
Pe pendicula
Mode a e
Sphe oïd
14.5±2.5
17.4±2.6
High
Pe pendicula
Polygonal
17.8±2.7
25±4.1
Py amidal
Mode a e
13.3±2
19.6±3
Pa allel
Spindle-shaped
20.4±2.9
23.1±4.1
V
341
±88
20.6
±5.3
G anula
Mode a e
14.5±2
20.8±2.9
High
Pe pendicula
High
Sphe oïd
15±1.9
19.2±3
High
Pe pendicula
Py amidal
Mode a e
14.1±2.2
19.6±3
Polygonal
20.2±3.1
27.7±3.8
Pa allel
La ge
Py amidal
Low
23.2±3.8
33.4±5.2
Spindle-shaped
20.5±3.2
24.1±3.7
VI
376
±136
22.8
±8.2
G anula
Mode a e
14.5±2.3
19.8±2.7
Mode a e
Pe pendicula
Mode a e
Sphe oïd
16.6±3.5
20.4±3
Mode a e
Pe pendicula
Polygonal
21.1±3.8
26.1±4.5
Py amidal
Mode a e
13.9±2.2
20.7±3.3
Pa allel
Spindle-shaped
23.1±3.2
25.2±3.3
18
MBP immunos aining
In he sheep audi o y co ex, MBP-i myelin was obse ed o all animals, hemisphe es, sec ions
and ROIs. MBP-i myelin was p esen in all six co ical laye s (Figu e 6). MBP immunos aining
e ealed ibe s o ien ed pa allel o he co ical su ace in he uppe pa o laye I (Figu e 6B’), whe eas
ibe s o ien ed pe pendicula o he co ical su ace we e obse ed a e sing laye s II o VI (Figu e
6B).
Figu e 6. O ien a ion o ibe s and cells in he sheep audi o y co ex. A- C esyl iole s aining e ealed cells
aligned pe pendicula o he co ical su ace; B- MBP s aining highligh ed ibe s wi h a simila pe pendicula
o ien a ion (black a owheads) and B’ pa allel ibe s in he uppe pa o laye I (un illed whi e a owhead); C- PV
s aining e ealed ewe pe pendicula ibe s and cell bodies, and C’- ibe s pa allel o he co ical su ace (un illed
whi e a owhead) in he uppe pa o laye I. Da a shown is om he ex e nal ROI, o he pos e io sec ion, o he
le hemisphe e, o animal 13507. Black a owheads in A-, B- & C- indica e equi alen loca ions and we e
de ined based on he ibe s in B-. Scale ba s: 200 µm (A, B & C); 50 µm (B’ & C’).
19
No clea di e ences in MBP immunos aining ac oss hemisphe es, sec ions, and ROIs we e
obse ed o laye s I, II, V, and VI. Laye s I and II consis en ly con ained a low densi y o MBP-i
myelin, while laye VI exhibi ed a mode a e densi y, and laye V a high densi y. In con as , laye s III
and IV showed g ea e a iabili y in hei MBP-i densi y. Laye III con ained ei he a mode a e o high
densi y o MBP-i bu wi h a no able p e alence o high densi y (Figu e 7A). Laye IV con ained
mode a e o high MBP-i myelin densi y, wi h mode a e densi y mainly in ex e nal ROIs (Figu e 7B),
high densi y p edominan ly in in e nal ROIs, while in e media e ROIs had an equal dis ibu ion o
mode a e o high MBP-i myelin densi y.
Figu e 7. MBP-i myelin dis ibu ion in he in e nal (A) and ex e nal (B) ROIs o he an e io sec ion o he
sheep audi o y co ex. A- In e nal ROI: low densi y o MBP-i myelin in laye s I and II, mode a e densi y in laye
VI, and high densi y in laye s III-V; B- Ex e nal ROI: low densi y o MBP-i myelin in laye s I - II, mode a e densi y
in laye s IV and VI, and high densi y in laye s III and V. Co ical laye s, de ined using c esyl iole -s ained
sec ions, we e p ecisely ep oduced on o he immunos ained sec ions. Da a shown is om he le hemisphe e o
animal 13507. Scale ba s: 200 µm (A & B).
20
PV immunos aining
PV-i immunos aining e ealed ibe s and cell bodies dis ibu ed h oughou he sheep audi o y
co ex. In gene al, a do so en al g adien o s aining was obse ed, wi h he highes densi y being
obse ed in he do sal pa o he pos e io ec osyl ian gy us be ween he in e nal and in e media e
ROIs (Figu e 8, Supplemen a y Figu e 1). The do so en al g adien dec eased ab up ly in o he
sup asyl ian sulcus, wi h a nea ly comple e absence o cell body s aining a e he in e nal ROI. On
he ou e co ical su ace o he pos e io ec osyl ian gy us, he g adien dec eased mo e g adually,
wi h he ex e nal ROI exhibi ing an almos comple e absence o cell body s aining (Figu e 8D).
Howe e , sho PV-i ibe s we e s ill obse ed in his ROI despi e he lack o s ained cell bodies.
In he majo i y o he in e nal and in e media e ROIs, medium o long ibe s we e obse ed in
laye s I and II, whe eas laye s III, IV, V and VI con ained p edominan ly long ibe s (Figu e 8E). The
highes densi y o PV-i ibe s was obse ed in laye s III, IV and V, ollowed by laye s II and VI, and he
lowes in laye I. Ac oss all six co ical laye s, ibe s o ien ed bo h pe pendicula and pa allel o he
co ical su ace we e obse ed, wi h mo e pe pendicula (Figu e 8E). Fibe s wi h di e se o ien a ions
we e also obse ed in he uppe pa o laye I (Figu e 6C’). In addi ion, la ge baske -like s uc u es
we e obse ed a ound uns ained cell bodies in he uppe pa o laye V, wi h a mean diame e anging
om 34.8 µm ± 8.7 o 42.9 µm ± 8.3 (Figu e 8H).
PV-i cell bodies we e obse ed in laye s II, III, IV, V, and VI, bu no in laye I (Figu e 8B-C). They
we e GABAe gic in e neu on cell bodies, including sphe oid, polygonal, and spindle-shaped (Table 2,
Figu e 8F-I). The densi y o PV-i cell bodies a ied ac oss sec ions and ROIs. The highes median
densi y was ound in he in e media e ROI (65.9 cells/mm²), ollowed by he in e nal ROI (50.2
cells/mm²), and he lowes densi y in he ex e nal ROI (17.2 cells/mm², Figu e 8B-D).
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Figu e 8. Dis ibu ion o PV-i cell bodies and PV-i myelin in he sheep audi o y co ex. A- The
do so en al g adien o PV s aining in he an e io sec ion o he igh hemisphe e o animal 13507, wi h he
h ee ROIs (IT: in e nal, IM: in e media e, ET: ex e nal) indica ed in blue. The highes densi y o PV s aining was
obse ed in he do sal pa o he pos e io ec osyl ian gy us (whi e a owhead). The g adien dec eased
ab up ly in o he sup asyl ian sulcus (SSS) and mo e g adually along he ou e co ical su ace (pEG). B-D-
Dis ibu ion o PV s aining in he h ee ROIs. E- PV-i ibe o ien a ion and leng h, e ealing long ( illed g ey
a owhead) and medium (un illed g ey a owhead) pe pendicula ibe s, medium ( illed pu ple a owhead) and
sho (un illed pu ple a owhead) pa allel ibe s. F-I: GABAe gic PV-i cell bodies; F- polygonal in e neu on wi h
axonal p ojec ions (pink a owhead); G- spindle-shaped in e neu on (g een a owhead); H) baske -like s uc u e
(blue a owhead) and I- sphe oid in e neu on (o ange a owhead). NOTE: The highes densi y o PV was
obse ed in he IM ROI, ollowed by he IT ROI, and no PV-i cell-bodies in he ET ROI. Scale ba s: 2000 µm (A);
200 µm (B-D); 50 µm (E); 10 µm (F-I).
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The densi y o PV-i cell bodies a ied by laye , wi h he highes densi y in laye s III and V, ollowed
by a mode a e densi y in laye s IV and VI, a low densi y in laye II, and no in e neu ons in laye I
(Figu e 8C). In de ail, laye II consis ed o a spa se dis ibu ion o sphe oid in e neu ons (mean
diame e : 15.4 µm ± 3 - 19.2 µm ± 3.7), along wi h a e polygonal and spindle-shaped in e neu ons
(mean diame e : 18 µm ± 5.6 - 22 µm ± 4.5; 22.3 µm ± 2 - 22.8 µm ± 2.4, espec i ely). In laye s III
and IV, polygonal (mean diame e : 18 µm ± 2.2 - 24.5 µm ± 3.6; 17.8 µm ± 2.7 - 25 µm ± 4.1,
espec i ely), wi h axonal p ojec ions, and sphe oid in e neu ons (mean diame e : 14.8 µm ± 1.8 - 18.9
µm ± 2; 14.5 µm ± 2.5 - 17.4 µm ± 2.6, espec i ely) we e p esen . Spindle-shaped in e neu ons
(mean diame e : 21.9 µm ± 3.3 - 24 µm ± 2.8; 20.4 µm ± 2.9 - 23.1 µm ± 4.1, espec i ely) we e also
occasionally obse ed in hese laye s. In laye s V and VI, polygonal in e neu ons (mean diame e :
20.2 µm ± 3.1 - 27.7 µm ± 3.8; 21.1 µm ± 3.8 - 26.1 µm ± 4.5, espec i ely) we e p esen , along wi h a
smalle numbe o sphe oid in e neu ons (mean diame e : 15 µm ± 1.9 - 19.2 µm ± 3; 16.6 µm ± 3.5 -
20.4 µm ± 3, espec i ely) and a e spindle-shaped in e neu ons (mean diame e : 20.5 µm ± 3.2 - 24.1
µm ± 3.7; 23.1 µm ± 3.2 - 25.2 µm ± 3.3, espec i ely).
Discussion
In his s udy, we p o ided a de ailed desc ip ion o he cy oa chi ec u e and myeloa chi ec u e o
he sheep audi o y co ex, shedding ligh on i s s uc u al o ganiza ion. PV s aining u he con ibu ed
o unde s anding i s unc ional p ope ies by highligh ing key GABAe gic in e neu ons. These indings
deepen ou knowledge o he audi o y co ex in sheep, an impo an model o in es iga ing senso y
p ocessing in la ge mammals.
The mean co ical hickness o he sheep audi o y co ex (1649 µm, min: 1280 µm; max: 2116 µm)
was consis en wi h he co ical hickness o o he egions o he sheep b ain epo ed in p e ious
s udies. Among hese egions, he p ima y isual co ex has been epo ed o be he hinnes (1488
µm; G aïc e al., 2022), ollowed by he audi o y co ex and he o bi o on al co ex (1697 µm; Ge ussi
e al., 2022). In con as , he mo o co ex exhibi s he g ea es hickness (1838 µm; Pe u o e al.,
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2019). Mo eo e , he six-laye lamina o ganiza ion obse ed in he sheep audi o y co ex is consis en
wi h indings in he audi o y co ices o o he mammals ( e iewed in, Kaas, 2011). This sugges s a
somewha conse ed s uc u al o ganiza ion, and po en ially common audi o y p ocessing
mechanisms, ac oss mammalian species.
Among he six co ical laye s, laye II was he hinnes , which is consis en wi h o he egions o he
sheep b ain (G aïc e al., 2022; Rose, 1942) and in he co ex o o he ce a iodac yls (G aïc e al.,
2024; Ho e al., 1999). In addi ion, his laye exhibi ed he highes cell densi y, consis en wi h indings
in mammals (Wine , 1985). Laye IV was also ela i ely hin bu i s bounda ies we e mo e challenging
o delinea e om adjacen laye s. These obse a ions mi o indings in o he p ima y senso y a eas,
such as he p ima y isual co ex o sheep (G aïc e al., 2022; Rose, 1942), whe e laye IV is p esen
bu di icul o iden i y.
Immunos aining o MBP and PV e ealed he highes ibe densi y in laye s III o V, which is
consis en wi h s udies in o he mammals, whe e hese laye s a e known o ha bo dense p ojec ions
(Ho e al., 1999; Jones e al., 1995; Kaas, 2011; Wallace e al., 1991). The dense MBP-i myelin and
PV-i ibe s sugges a de eloped ne wo k o connec ions media ed by in e neu ons, acili a ing bo h
local p ocessing, co ico-co ical, and halamo-co ical communica ion wi hin he sheep audi o y co ex.
In e es ingly, we iden i ied baske -like s uc u es exclusi ely in he uppe pa o laye V using PV
immunos aining. These s uc u es, based on hei dis inc i e mo phology and size, likely su ound
la ge py amidal cells. Such la ge py amidal cells we e obse ed in he uppe pa o laye V wi h c esyl
iole s aining. This a angemen sugges s ha he baske -like s uc u es may modula e he ac i i y o
py amidal cells h ough GABAe gic synapsing, po en ially playing a pi o al ole in egula ing exci a o y
ou pu om laye V, which was p e iously epo ed in o he mammals (Ho e al., 1999). Addi ionally,
long axonal p ojec ions we e obse ed ex ending in o laye II, which may co espond o he p ojec ions
o la ge py amidal neu ons. This ana omical con igu a ion sugges s a complex in e play be ween
inhibi o y and exci a o y elemen s, wi h baske -like s uc u es po en ially con ibu ing o local inhibi o y
egula ion o exci a o y py amidal cells, c ucial o audi o y in o ma ion p ocessing.
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PV s aining has al eady been used as an indica o o he loca ion o he p ima y audi o y co ex
(Kaas & Hacke , 2000). In ou s udy, high densi y o PV-i cell bodies was obse ed in he do sal pa
o he pos e io ec osyl ian gy us, wi h s aining dec easing en ally, bo h in o he sup asyl ian sulcus
and along he ou e co ical su ace. These obse a ions sugges ha he sheep p ima y audi o y
co ex is loca ed in he do sal pos e io ec osyl ian gy us, maybe be ween he in e nal and
in e media e ROIs o he an e io and medial sec ions, which b oadly co esponds o acing esul s
(Michaloudi e al., 1986). Howe e , u he unc ional s udies a e needed o con i m he p ecise
loca ion o he p ima y audi o y co ex in sheep.
Conclusion
In his s udy, we delinea ed six co ical laye s wi hin he sheep audi o y co ex in he an e io pa
o he pos e io ec osyl ian gy us, wi h dis inc cell ypes, dis ibu ion, and ibe o ganiza ion.
Addi ionally, we obse ed inc eased pa albumin s aining in he do sal pa o his egion, which may
co espond o he p ima y audi o y co ex. These indings a e consis en wi h exis ing li e a u e on he
audi o y co ex in sheep and o he mammalian species. Toge he , hese esul s sugges ha bo h he
s uc u al and unc ional o ganiza ion o he audi o y co ex a e conse ed ac oss species, suppo ing
he idea o an e olu iona y conse a ion o audi o y p ocessing mechanisms in mammals.
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Supplemen a y ma e ial
Supplemen a y Figu e 1. Do so en al g adien o PV s aining in he sheep audi o y
co ex. The h ee sec ions (an e io , medial, pos e io ) a e ep esen ed o bo h hemisphe es o
animals 03133 and 13507. The h ee ROIs (IT, IM, ET) a e indica ed in blue o each sec ion. A
do so en al g adien in PV s aining densi y is obse ed, wi h he highes densi y in he do sal
pa o he pos e io ec osyl ian gy us (whi e a owhead). The g adien dec eases ab up ly in o
he sup asyl ian sulcus (SSS) and mo e g adually along he ou e co ical su ace (pEG). Scale
ba a bo om igh applied o all sec ions: 2000 µm.
