Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey

PROJECTIONS OF CHOLINERGIC AND NON-CHOLINERGIC NEURONS OF THE BRAINSTEM CORE TO RELAY AND ASSOCIATIONAL THALAMIC NUCLEI IN THE CAT AND MACAQUE MONKEY

Several regulatory in the control

systems are thought to bc inwlvcd of fcx-cbr:un neuronal

lirs~ one was implicated cortwal lie\cci

xztication

. an effect that uas originally

and to hc mediated modulatory

the txilnstem l’orelxxin ;ICCCSS to

the

~ortlcall\-prcljccting dclincatcd

by thalamic

01 hc-

formation

nuclei.‘“’ Later

nuclei

aggregates.

ccrchral

on.

the basal

While

arise

in

nuclei. the brainsrem reticular

the rather

latlc~ wll-

Ihrmation

i\

ab 2 diffuse

Lrntil

brainstcni

reticular

quite

and

recently

neurons

appcarcd

!Xl3\_

1‘rwm the upper

hrachial

;Lrca] 10 the lateral

and latei-al

posterior

niidbrain

inlcctions

rat.”

Other

clear

gl-oup~.

\entrobasal (VA

VL)

hrain\reni

(LP)‘”

major

with

Ihalaniic

sensory

(VH)

and \\cre

ircticular

and motor

In Ihc

~h:~larn~c

gcnlculatc

belicvcd

alti‘rcnts.

aI‘tcr \lniil;ll

aniinoawi~

\entroantcrioI~ also

pet-I(I.(i)”

n~iclc~ 01‘c;il. the

unlabcleti

trilialcd

around

tcrmcd

pcn~cul~~~c

such ax the medial

nuclei

region

[henxl‘tcr

nuclei rclnained

aLlto1-0 ealing

In

brainstcni

conjunctiwm

COIC‘IO

Although

SLUWdCd

the brachium

sanlc thalamic

rct~cular

groups.”

tracing

(PB)

(11‘

lo hc rc-

and some mcd~al ndc~

from the brainstcm nuclear

radiographic projections

nct~d. target\

Lhcrc arc no ohv~c>ub aii;ll-

that

omical pathway\

ill-detincci

the rhalam~c wcrc lhought

?tricted to the intralaminar

iii ajo r

both having LI direct

cortex.

systems

and

regarded

Moreover.

since iI

systems hake been rccognired:

monoamincrgic

cholinergic

reticular

still

The

in the classical concept

10 arise in the brainstcm

two other

activities.

III]-

(\lCi).

\cnlrolatcral IO hc dc\ old

Slncc

the

01’

curt-cnl

knowlcdgc lacks cvidcncc 01‘ thcsc rc~iculothniamic projections. the facilitatory effects of brainstcni ruticLI~W stimulation upon synaptic transmission through sensory and motor thalamic relay rlucici’hii2i’ rcmaincd something of a mystery and awaited the ~larj~~ati(~n of the underlying pathways as well as thctr chemical signature. Nevcrthelcss, there was indirect evidence suggesting that at least some relay and associational thalamic nuclei rcccive cholincrgic affercnts from the upper brainstem core. For example, the spontaneous release of a~etyl~holin~ (ACh) was mcasurcd in the VA-X, VB and mediodorsal (MD) nucici of- cat” and monkey” and was found to increase after brainstem reticular stimulation.” In addition. the acetylcholinesterase (AChE)-containing fiber systems originating in the brainstem were found to extend to intralamjnar but also to MC. Lci and parts of the lateral and ventral thalamic nuclear group~.~‘.~” Marc importantly. the AChE staining in thcsc thalamic nuclei disappeared after lesion of the dorsal part ot the midbrain reticular formation or nucleus cuneiformis,“” an arca that is now known to partially overlap with the cholinergic PB area of the pedunculopontine nucleus.” x6 Furth~rrn~)r~, lesions of the latcrodorsal @mental (LDT) choiincrgic nucleus at the midbrain-pontine junction produced significant decreases in AChE and choline acctyltransferase (ChAT) activities in some thalamic nuclei. particularly the antcroventral (AV) and lateral part of the MD nuclei.” Despite some specificity problems related to AChE as a marker of cholinoccptivity, the degree of AChE staining of thalamic nuclei fits remarkably well with the distribution of ChAT-immunoreactive fibers in various thalamic territories. Figure 1 illustrates the distribution of AChE activity in the thaiami of the cat and the rhesus monkey. This map is similar to that

AC AD-AM- AV BC BP CeM CL CM DR F FTC HL IC ICC LC LDT LG LP MD MG MTB OP

anterior commissure anterodorsal-anteromedial

anteroventrai

thalamic nuclei brachium conjunctivum hrachium pontis central medial thalamic nucleus central lateral thalamic nucleus centrum medianum thalamic nucleus dorsal raphe nucleus fornix central tegmental field lateral habenula inferior colliculus inferior colliculus commissure locus coeruleus iaterodorsdl tegmental nucleus dorsal part of the lateral geniculate thalamic nucleus lateral posterior thalamic nucleus mediodorsal thalamic nucleus medial geniculate thalamic nucleus mammillothalamic bundle

dcscribcd 111 previous studies of \;OXXY, ~h;t~:rmri nuclei in r;tt.“,l’ cat” and nronkc>.” it csscntiall~ shows heavy staining in the rcticulsr (RE). ultralaminar centrum medianum- parafascicular r(‘M Pt.) complex and central lateral--paracentral~ccntrni mcdial (CL PC-CeM) wing. and some midline nuclei. In addition, tnoderate to heavy 4Chfl staining appears in associational and relay nuclei. such ;I> the anterodorsal--anteromedial---anter~~ventr~lt (AD AM ~AV). MD, VA. LP, lateral part 01’ pulvinar (PUL) and LC nuclei. With the exception of the AD nucleus, all thatamic nuclei that stain for AChE wcrc also found to be innervated by ChAT-immunoreactive tibers in a recent study in rat.“’ Both FIN. I and the localization of ChAT-immunoreactivc fibers in the rat”’ indicate that the VI.,, VI3 and MG nuclei are very lightly stained (as compared to sotnc 01 adjacent nuclei) or seemingly devoid of ~hoiin~r~i~ afferents. A series of questions arose from the data mcntioned above. The first relates to the origin of the cholinergic innervation of the thalamus for which two possible sources should be considered: the choline& nuclei at the midbrain-pontine junction and the basal forebrain nuclei. While cholinergic neurons in the substantia innominata and diagonal band nuclei project to RE and MD nuclei,7J other thalamic nuclei receive very few, if any, projections from the basal the origin of cholinergic forebrain. Therefore. &erents to most thalamic nuclei should bc starched within the upper brdinstem core. Since 1983 three studies, all conducted in rat, have combined retrograde tracing and AChE histochemistrylh or ChAT to reveal brainstem choliimmunohistochemistryti.” nergic projections to the thalamus. As also mentioned elsewhere,“” the targc injections of tracer in two of thcsc studies precluded the precise d~lin~atiol~ ot various thalamic targets of brainstem cholinergic

PAG PB PBG PC PP PUL RE RFB RN SC SUB VA VB VL VM VPL VPM V3 V4 WM 21 5M

periaqueductal gray peribrachial area parabigeminal nucleus par¢ral thalamic nucleus pes pedunculi pulvinar thalamic nucleus reticular thalamic nucleus retroflex bundle red nucleus superior collicuius subthalamic nucleus ventroanterror thafamic nucieus ventrobasal thalamic nucleus ventrolateral thalamic nucleus ventromedial thalamic nucleus ventral posterolateral thalamic nucleus ventrat posteromedial thalamic nucleus third ventricle fourth ventricle white matter zona incerta mesencephalic nucleus of the 5th nerve

CAT

MONKEY

Fig. I. Distribution of acctylcholinesterase activity in thalamic nuclei in cat and macaque monkey. Staming accordmg to Gomori’s technique. Frontal sections. Four levels rostra1 to caudal (.4 D) In cat. and two levels (A and B) in monkey. In this and following figurec. horirontal bars indicate mm.

cells. The more recent study”’ in4vcd locali& infusions of fluorescent tracers into AD-AV. RE, MD and LG. The common conclusion of all these recent investigations is that the cholinergic inncrvation of the rat thalamus arises in the pedunculopontine and LDT nuclei, also termed groups Ch5 and Ch6 in the nomenclature used by Mesulam and his colleagues? It should be emphasized that the great majority of physiological studies aimed at investigating the brainstem control of behavioral state and the ACh influences on thalamic neurons have employed the cat’h.h’.” (set also Discussion). Thus, a major goal of the present study was to reveal the brainstem--thalamic cholinergic projections in cat. Another question pertains to those major relay sensory and motor thalamic nuclei (MC, VB, VA-VL, VM) which were not analysed in previous studies dealing with brainstem cholinergic projections and are not known to receive afferents from the brainstem reticular formation. These and other specific thalamic nuclei are the first relays where synaptic transmission is significantly enhanced by brainstem reticular stimulation or during electroencephalogram (EEG)-desynchronized behavioral stateshS.” In VA-VL and VM nuclei, the brainstem control seems to be exerted through chohnergic mechanisms since electrophoretically applied ACh alters the cells’ burst responses into single-spikes?.” mimicking the change from EEG-synchronized to EEG-desynchronized epochs. “‘J Finally, because of the great development of associational thalamic nuclei in primates, we also investigated the brainstem cholinergic projections to the MD and PUL -LP nuclei of the macaque monkey.

the htgh paraformaldehydc conccntratton 01 tiu\ Itu,rt~\e is not opttmal for WGA HRP htstochcnnstry. pi1<)1 experiments have shown that tixatices with suc!r hrglt panformaldehyde and low glutaraldehyde concentratmn pr<, mote ChAT immunoreactivity w/ith the monocirmal an~rbody (Boehringer, Mannheim, F.R.G.) used in the present study without significant decrease in WGA HRP activity. Following perfusion, the brains were removed and placed in phosphate buffer contaming 10% sucrose and IO”,~, paraformaldehyde for 4 h at 4 C, and then transfcrrcd tn a phosphate buffer with 10% sucrose for at least I7 h at 4 C’. The brains wcrc then sectioned on a freezing microtome at 40 pm in the frontal plane. Three series of rections were serially collected (one section out of four) and kept in phosphate buffer (0.1 M. pH 7.4) at 4 C while awaiting further processing. (a) The first series of sections was processed accordmg to the tetramethylbenzidine (TMB) procedure”’ to reveal HRP and counterstained by means of Neutral Red. (b) The second series was exclusively collected from the level of the injection site and, for comparison purposes, processed according to the 3.3’.diaminobenzidme~ tetrahydrochloride (DAB) method’H for WGA HRP histochemistrv. IC) The third series was used for the concurrent visualization of the retrogradely transported WGA HRP and C’hAT immunohistochemistry. Those sections were first processed according to the TMB method and stabilization procedurc.“.i” Thereafter. brainstem sections were incubated with rat monoclonal antibody against ChAT and processed according to the avidin biotinperoxidase complex (ABC) technique.” The localization of different types of neurons (HRPpositive, ChAT-positive, and double-labeled for both HRP and ChAT) was performed by means of a camera lucida OI a computer-assisted microscope. The microscope stage was coupled to an X Y plotter through a microcomputer permitting storage of cell coordinates and plotting of their locations according to different magnification factors. To calculate percentages of HRP-labeled neurons at various levels of the upper brainstem reticular formation, we counted all HRP-positive elements on 27 sections of the rostra1 brainstem core, 3 sections for each of the 9 mm from the stereotaxic planes anterior 4 to posterior 4. The counted HRP-positive neurons were located in the territory of the rostra1 reticular formation (that is the central tegmental EXPERIMENTAL PROCEDURES field” and its dorsal extensions, namely the deep layer of the including the The experimental material consisted of 20 adult cats of supertor colliculus and nucleus cuneiformis). both sexes (2.5-3.5 kg) and two adult Macacu .v$r~ancr PB area and the LDT nucleus. We did not include in the female monkeys (6.5 and 7 kg). In both spectes, injections of calculations labeled neurons in structures that arc outside the classical reticular territories, such as the superficial and horseradish peroxidase conjugated with wheat germ agglutiintermediate layers of the superior colliculus. substantia nin (WGA-HRP; Sigma; 10% in phosphate buffer 0.01 M, nigra, parabigeminal nucleus, inferior collilculus. dorsal and pH 7.4) were performed under anesthesia with ketamine ventral nuclei of the lateral lemniscus. dorsal and lateral hydrochloride (40 mg/kg, i.m.) using a 0.5-p] Hamilton parts of the periaqueductal gray (PAG) (we counted, howsyringe. ever, the LDT nucleus which is embedded in the ventral part Cat experiments of the PAG), raphe nuclei and locus coeruleus. The detailed Unilateral 0.005-~1 injections of WGA-HRP were diresults of WGA HRP injections in visual (LG and LP) rected in a dorsoventral or oblique direction into the MC thalamic nuclei will be reported elsewhere. We use hem (n = I), LG (n = 2), VB (n = 2), LP (n = 3), VA--VL those results to compare the HRP labeling of brainstem (n = 3), VM (n = 2) MD (n = 2) and AV-AM (n = 2) reticular neurons after LG and LP injections to that occurthalamic nuclei by using coordinates from a stereotaxic atlas ring after injections in other thalamic nuclei. of cat brains Three control injections (each of 0.01 ~1) were Monkey r.ywiment.s made into the pericruciate cortex, the suprasylvian cortex, and the posteroventral hippocampus. For thalamic injecAs no stereotaxic atlas exists for Macucu sylz~~nu. coorditions we allowed a period of IS-25 min between the end of nates were extrapolated from an atlas of the rhesus monkey an injection and the removal of the needle from the brain brain.h’ In the first monkey, several 0.005-~1 injecttons of in order to minimize WGA-HRP diffusion along needle WGA HRP were directed to cover the whole extent of the trajectories. After a 48-h survival time, the cats were anesPUL LP nuclear complex. In the second monkey. \cvcral thetized with pentobarbital, artificially venttlated, and per0.005-111 injections of WGA HRP were made into the MD fused with 500 ml of a 0.9% saline solution followed by 3 1 nucleus. All technical procedures were similar to those of freshly prepared fixative (2% paraformaldehyde~a. 1“%I described for cat experiments. However, since we observed glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) and 2 1 of in a pilot experiment that the Boehringer monoclonal phosphate buffer containing 10% sucrose at 4 C. Although antibody was less effective than in experiments on cats. wc

used thr li~~mut~onucle~r rat ~~~oi~oclo~~~l antibody (diluted hetwcen I : 100 and I :NO) in monkey experiments. The locahjraticnl of dilrercnt types of labeled neurcms was made with a computer-assisted microscope for the PUL LP injcc-

irr7d noti -chtrlit7c~r,qii~ twurot1.s irr tlw iat

tion and with camera

two

lucida for the MD

injectlon.

KESI’LTS

Til~ll[ii?ti~

~~f’b~~~itl.~~~~}?l t.cii<~dtrr ~.lf~liii~~~~~l(’

p~i~j~J(,li(~~l,~

Retrogradely regions

labeled

by the presence or absence of cholincrRostrally.

small

after i,?jections

to distinct relay and associational

of the injections

loci

part of the VB complex did not

the

adjacent

encroach

upon

eroventral

wing of the RE nucleus (Fig. 2A). The MG

LG.

LP

and

injection was located within the posterovcntral of this nucleus (Fig. invade

the VB,

2B). The VM

MD

injections

As shown

p;iper.lc the injection procedure with

aspect did not con-

PC nucleus (Fig. ZC). All three 2 are illustrated

in Fig.

procedure.

injection

post-

or even the irnmcdiately

tiguous intralaminar

in Fig.

with

non-cholincr@c

than after DAB

I of the companion

the TMB

cstimatcs the actual territory

procedure

over-

where the uptake of the

(around

the

were local-

HRP

For

example.

there

was

ir?jections in various after

injections

massive

mammillary

into

retrograde

cell

following

thalamic

nuclei.

AV-AM

nuclei.

labeling

in

the

nuclei and in layer VI of many fields in

the cingulate gyrus. but the precruciatc

by retrograde

HRP

to

pcdunculopontinc tcrmcd

.gyrus was not

culopontinc

sylvlan areas 5 and Sa after LP injections

I of the coronal

in areas 3 and iqjection

(Fig.

tion. As dcpictcd

accompanied

neurons in layer VI was

by antcrograde

all th:ilamic

injections

neurons wcrc rctrogradcly of the RE nuclear complex. RE

nucleus.

other that

intcrnuclcar

solving

the RE

nuclei.

in

We

(PB)

nucleus. In

cat.

therefore

preferred

arca which

comprises

of the cell g-oup

Experimental

Procedures) HRP

LP) thalamic

dis-

the

term

the rostra1 the

brainstem

rcttc-

on 27 sections (see much higher

in associative (MD

and

nuclei than after injections into sensory VI.) rela! nuclei. The

nucleus is a special case since, in addltlon

cortical

function.

it projects

arcas (see Discussion)

ticipates

in

gcncralized

o\cr

and

to it\

~\~lcsprcatf

thuc also par-

thaiamocnrtical

proccs\ca.

The actual numbers of HRP-positive

brainstem

ular neurons arc 7090 (for the MD

injections).

(VM).

X20 (LP).

(1-G).

168 (MC;)

include

ih

pedu~

that surrounti~

was pcnerally

injections

LG. VB) :md motor (VA

VM

part

the

conjunctivum

after WGA

620 (AV and

AM). 17

3”

(VB).

the ma_jor ipsilateral

tralateral

(VA

1343

VI.).

These

and

rctic170

numhct-x

the minor

con-

reticul~~tli~~l~~rnicprojection.

The striking

difference

between the rctrogradc

ccl1

With the cxccption of the

that after injections

in relay nuclei. results from two

labeled

supports other

neurons

in

the idea-‘J,“i

than

those In-

are the exccption’i MD

rathet injcc-

labcling in choll-

larger A4

nuclei”

and

and in the

posterior

hypo-

tcgmentum:

number

tcgmcntal

of

field

area and

LDT

part

and

labeled

of

in lnidbr~lin

A.?).

(P3

hand

anterior

injections.

as compared

to

factors: a higher pr~~p~~rtit~llof labeled neurons in the contralateral

innominata thalamus.”

ap-

retrogradcl>

the caudal In

The total number of HRP-posltivc

rostra1

the

nucleus.

ular neurons which was counted

nergic and n~~~l-ch~~lincr~ic neurons of the substantin of

of

MD

projections.

and diagonal

majority;

i~~belin~ seen after

lions. WC found massive rctrogradc

part

C‘hAT

sectors

than the rule in the th~~l~~~?lus. hollowing

dorsal

uith neuron5

nucleus is not a cytoarchitccturally

peribrachial

(except AV -AM”” -I).

labeled in different

which

nucleus.

lahcling

part of layer III.

we did not detect

thalamic

injcc-

in Fig. 3A and B. the retrograde

layer IV ;md the supervening After

.?A). a VB

.gyrus after a VA- VI.

of corticothalamlc

typically

(Fig.

3B). and at the limit between areas 6

end 4 in the prccruciate labeling

supra-

gyrus after

tho

nucleus while

territory.‘”

role in motor

in anterior

combined

cholinergic

~~r~br~ichi~i

f-nirly

occurred

transport

represent

(MG.

labeling

gray, later-alI> IO

nucleus (Fig. 4). A\ indlc;ttcd

immunohistochemistq.

labeled. thus indicating that the adjacent VM and CL nuclei were not involved in the AV~-AM injection. localized

to the Icvcl

nucleus) that is located withln

the dorsal raphe (DR)

brachium

WGA

and cxtcnd\

rostrally

the periaqucductal-pcriventriclilar

differential

labcling

conjunctivum)

dccussation

cell group (the LDT

three-quarters

of retrograde

) neuronb ap-

of the locus coeruleus (LO and subcoerulcu~ C;~IIdally. where it merges dorsomedially into (h) ;I ~cond

iled within distinct nuclear limits was provided by the pattern

51/c

(plane P4). two agprc-

(400~ hOO ,0 m’

brachium

from the brachium

tinct

evidence that the injections

(soma

between plant .41 and

labeled cells in the PB area and the LDT

enzvme occurs.” ,&ditional

plana

some studies. the rostra1 perib~~chi~~l zrea is tcrmcd

staining. This is in keeping

that

neurons

junction

gates of medium-sired

peared

the TMB

sites were larger with the TMB

the indication

stcreotaxic

pear: (a) ZI lirst cell group is located in the PB arca

thalamic

that the core of the deposit in the ventral (VPL)

at frontal

burn’). More caudally.

the midbrain-pontinc

posterolateral

in cot-c

A4 and A3. the central tegmental field consists mainI>

We report here the results obtained nuclei. The close examination

reticular

gic neurons.

X0-400

Indicates

conccntrntod

brainstem

charactcrircd

ol

confined

cells were

of the upper

the

rostra]

reticular

nucleus (A2 of

the

cspcciall!

neurons

in

labeling

midhr:lin

PZ).

pontinc

observed

nucleus. anti the negligible

much (plane\

fields outhide the PB ;14 ucll

reticular

P4). This profile stands in contrast

sclectite

2

the central

in

PB

:I\

111 the

li~rniatlon

with the quite

;trcrt and

lahclin,c at plant\

I.I)T

A3

G

.

Fig. 3. Anterogradc and rotqrade labeling in anterior suprasyivim gqrus (A! and coronal prus (B) t>l HRP injecttons nn LP and VB nucia. rcspecti\el~. TMB procedure. Descriptitln I,, Icsl.

cat after WGA

and P4. after injections in sensory (MC, and motor

(VA--VL)

E‘ig. 111depicting that

nuclei (compare

the labeling

in Pig. X depicting

iniection).

With

injections

led

mldhraln

pontinc

uniform

dislrib~ition

thus lacking

after MD

injection

to relay massive:

junction

nuclei.

only

labeling

at

(planes P3-P4)

planes Al

at plane Al

for I.<;.

PI

\,ation

the

and to a

in

nuclei.

for

VH

and

MD.

from the caudal

The

(c) The

microphotographs

small

hluc

f1RP

In

granules

proximal

dendrites.

playing

a

f:~g. h depict

throughout

in

both

light-brown

the cell body and its proccsxes. and finall> cells containing

can be drawn by examining

of iahelcd neurons in the brainstem

tion of these three types of cells in Figs 7

(3)

The

Fig. 5. The sourcc values that wcrc used for these

(HRP

cclmputcr-generated

HRP-pohitivr:

tindIng\:

(a) With

graphs

the exception

that rccclvcs around from A4

indicate

of the MD

16 “6 of its brainstem

A3 Icccls. other thalamic

than

C’?;, (MC;.

VM)

irwn

associational

VH,

three

II)

rostra1

and motor

VA

(LG.

VL

total

bctwecn A9 and PL! uerc ahn\c

Vi_,

Sensory.

;I rcglgm conccntratcd

(Al

3 mm

tht

from

PI).

from The

plane 0 ~‘OI

(78%) after

numhct

cells counted in the brainstcm

in sensory nuch.

aflercnts

around

neurons

1I.

~i~~~~bi~-i~~h~l~~i

formation

nuclei receive he-

atfercnts arc at frontal

ot

the distrlbu-

injection5

twecn 52 and 75 %, of their hrainstcm peaks i>I‘brainstcm

per~~nt~g~~

The I’olln~ing

nucleus

VA

levels. (b)

+ ChAT)

and C’hAT.

affcrcnts

nuclei rtccivc ICSS

or S 10%

these most

sahcnt

HRP

hoth retrogradcl~

conclusions

in

n~xlct

XZ”/o after nucleus.

as well as

illjections

Thcsc

neurons indicate labeled

in motor

high

70”11 aticr

that I\ VH (77”~)). MC; VA

in AV

into

VL. ~h3”~,) and

the assc)ciati\e

perccntagcs found

(,O”i, VM

ARI nuclei (61”o): ami

within

\.i
01‘ double-labclod

that the o\crwhclmlng

I~~LII-OI~Sarc

ot

rctlculat

and LG (X7”;,) nuclei: they Nc’rc around Injections

(58%)

cik

i~nrn~~n(~st~~~~~i~ig

nine Ic~cls hctwccn A4 and P4 from the total number depicted

and

~CLIWI~

WGA

diagrams

typical

porikarqa

transported

reticuiar core arc

inner-

neurons con(;lining

the ChAT-positive

diffuse

IIIICICLI~

VM

P3- P4 lc\tl~;.

examples of WGA-HRP-labeled

the double-labeled

The perccntagcs or labeled neurons at each of the

lildi~~lt~d in the siinlni~lri~ing

and ;II

)“‘% of its brain~tcm receives ~~ppr(~xilli~tcty 4_,

VM

PI seen after injections

VL.

to

the peaked lo~~~li~~tion in PB and LDT

nuclei xound

LP and VA

plane

of labeling between A4 and Pf.

all sensory and in VA-VL

MC.

in

the results of the VA-VL

regard to

LG and VW) left column

malorit)

the choh&pic

01‘ PR

54

0.1 Fig. 4. Features of retrograde cell labeling in peribrachial (PB) and laterodorsal (LDT) nuclear grculps at the midbmin-pontine junction after an MD injection in the cat. Arrow in (A) points to the same bl ood vessel as indicated in the enlarged photograph in (8). (C) is an adjacent more posterior section

k4

13 *2 *i 0 Rostrocaudal

P,

P2 F-3 P4 level

15 10

5 0

A4 A.3 12 Ai 0 Rostrocaudal

and LOT much

brainstem

Iwer

into the MD

ccl1 groups (see Figs 7-9).

value (7X%)

fhund

nucleus ic explained

ol’ rctrogradely

labeled

neurons

after

Pl

The

the injection

by a high amount within

PZ

P3

w

level

tcrritortcs

without

cholincr-gic

neurons.

fields outside the limits ofthc Fig.

in br;~instcm rcticulat PH and LDT

nwlcl

(WC

IO).

(b) The abow

values rcprcscnt the pcrccntagc~ 01

Fig. 6. Microphotographs showing examples of the three cell types analysed in the cat material processed according to TMB procedure combined with ChAT immunohistochemistry: HRP-positive neurons (black arrows): ChAT-positive neurons (open arrows); and double-labeled elements (triangles). Al and A2 arc low-power photographs depicting retrogradely labeled cells in LDT nucleus after an MD injection in the cat: the area marked by rectangle in (A21 is enlarged in (A3). (Bl) and (B2) depict do~ibie-ladled and simple cholinergic cells in the PB area after a VM injection.

double-ladled neurons from the total number of HRP-positive cells found between planes A2 and P2. After most injections, labeled neurons in the PB area were much more numerous than in the LDT nucleus.

partly because of the greater extent of the Former compared to the latter. The ratios between the numbers of double-labeled neurons in PB area and those in LDT nucleus varied as a function of the

WC;.4

HRP

injections

in ditti-rent

thalamic

nuclei.

15: VH: X) than in motor (VM: 4: VA VL: 2) nuclei. The That

ratio:, \+crc higher

ratios

hetwccn

injccticw

111sensory (LG:

PH- and

in MD.

LDT-lahclcd

LP and AV

40: MG:

ncuronx

AM

nuclei

al’tct

wrc

5. 2

;~nd t . rcspccti~et~.

(i) As expected. the att‘crcnt pro_jcctions were found to ;iriw

predominanll~

rcticuI,ii.

nuClC1.

prolcctionh

in the ipsilalcral the

Howcc\er.

\+CI-C \urprlsingly

high.

hrainw3~~ contralatcral

The

number

01‘

dloublc-labclcct cells in contralateral PH arw I.DT nuckus amounts to 37”~) (AV -AM), 35”,,

(1-I’).

-li”u (MC;). 35”~ (L(i).

22”,,

(MI>)

and

prc>lcction

IX”,)

3i”o

(VA

VL)

(VB). of

2X”,<) (VM). the

and

ipsilateral

Fig. 8. Cholinergic and non-cholinergic brainstem neurons projecting to the right VAT-VL thalamic complex of cat (A-C), three levels. At each level, left column depicts the total number of retrogradely labeled neurons as found on five sections after TMB procedure counterstained with Neutral Red. Right column depicts the same levels. with three cell types (symbols in AZ), as found on two sections after TMB procedure combined with ChAT immunohistochemist~. Camera lucida focaiizat~on.

campus fed to retrograde labeling in the PB area, LDT nucleus or in the more rostra1 non-cholinergic central tegmental field (see Discussion).

above description may imply that some of the HRPpositive and double (HRP + ChAT)-labeled neurons seen in the medial part of the PB nucleus in Fig. 12 actually belong to the LDT nucleus. Brainstem reticulur praje~t~~~nsto ~u~t~inur-iutera~ The MD injection (Fig. I3A) also led to intense posterior and mediodorsal thalamic nuclei in the man labeling of various brainstem structures. Several kq differences could be revealed by comparing the results In addition to the expected retrograde labeling in following the injections in the two explored thalamic the intcrmcdiate and deep layers of the superior nuclei of monkey, the PUL-LP and the MD. Firstly, colliculus,2.48 the injection into PUL-LP nuclei led to we found intense anterograde labeling in the massive cell labeling in the caudal part of the midsuperficial layer, and light anterograde labeling in the brain reticular formation, particularly in the medial intermediate part, of the ipsilateral superior colliculus and ventral aspects of the PB area (Fig. 12). HRPafter the MD injection (Fig. 13B). This was not seen positive neurons were disclosed further ventroin the case of the PUL-LP injection. Concerning the medially to the brachium within the dorsal part of the MD-tectal projection revealed here, we are just puzmidbrain-pontine tegmentum. Surprisingly, the numzled and cannot link it to any known circuitry and ber of labeled neurons found contralaterally after the function. Secondly, numerous neurons in the dorsal PUL-LP injection was as large as that found ipsiand lateral parts of the periaqueductal gray were laterally. Around 45% of all HRP-positive elements retrogradely labeled after the MD injection. Such a were also ChAT-positive (Fig. 12). The medial part prominent thalamic projection of the periaqueductal of the PB territory merges with the other (LDT) gray was not found after the PUL-LP injection. Both cholinergic nucleus that is embedded in the perithe anterograde labeling in the superior colliculus and aqueductal gray. Since we could not clearly disthe retrograde labeling in the periaqueductal gray tinguish the limits between PB and LDT nuclei, the after the injection into the right MD nucleus were

Bramstem

cholmergic

projections

to thalamx

rela)

nuclei

I ‘ig. 0 C‘holinergic and non-chollnergic brainstem neurons projecting to the right VM nucleus of cat (we injection site’ in Fig. 3C). Same type of graph as m the preceding figure. Here. each drawing 111the left or right column represents one section. Localization by means of a computer-aswted microscope. The areas dchm~tcd bq black rectangles III (82) and ((‘2) arc vhoun at a higher magniticallon

strictly

ipsilateral

labeling lateral

(Fig.

13B). Thirdly.

in rho cholinergic part

the retrograde

of its dorsal

and ventral

neurons project to Lirtually

every sensory and motor

in the

thalamic

relay nucleus in the cat as well as to asso-

aspects (Fig.

ciational

nuclei in both cat and macaque monkey.

PB area prevailed

13C EI. Few, labeled cells wet-c found in the medial

shall now discuss the evidence

part of the PH. adjacent

target of ascending

mu

iIll

PUI.

which

wus

1 P injection.

to the periaqueductal intensely

More

than

labeled 50%

gray,

after

of all

the

HRP-

and particularly

I b) this projection

positive neurons in the PB nucleus were also ChAT-

towards

posltivc

widespread

axons from

the cholinergic

The

present study provides the first detailed

ysis shtlwing that hralnstem

cholincrgic

anal-

PB and LDT

cholincrgic

brainstcm

the monoamine-containing aggregates.

Finally.

and is more and

projections

relay nuclei; (c) the thalamic LDT

the brainstem

complexes

neocortical

WC

(a) the major core.

ones. is the thalamus:

is bilateral

associational

that:

massive

nuclei

than

projections

with

to specific of PB and

neurons exceed those of (DR

abnd Lc‘) neuronal

w e elahorntc

about

the

rote

Fig. IO. Cholinergic and non-cholinergic brainstem neurons projecting to the right MD nucleus of cat. Same type of graph as in Fig. 8 (each drawing represents labeled cells as found in five sections in the left column, and in two sections in the right column). Camera lucida localization.

played by the brainstem-thalamic cholinergic and non-choiiner~c projections in tonic and phasic events of behavioral states of vigilance.

Ascending bra~nstem ch~~~~er~icaxons m~~du~atecortical excitability through a thalamic relay At variance with serotonergic (DR) and catecholaminergic (LC) nuclei which give rise to widespread projections to neo- and allocortical areas,j9.“’ the axons arising in cholinergic PB and LDT nuclei at the midbrain-pontine junction and in the more rostra1 central tegmental mesencephalic field are overwhelmingly relayed in the thalamus. This point should be emphasized because, with the exception of some reports about cortical projections of brainstem cholinergic neurons,78 most experiments in both cats and rats have determined that direct cortical projections from brainstem core areas other than monoaminergic nuclei are extremely sparse. Indeed, after large injections of retrograde tracers in the occipital cortex of cat, no labeled neurons were found in the rostra1 reticular formation.7h Corroboratively, serotonin and tyrosine hydroxylase immunohisto-

chemistry combined with the retrograde transport of tracers injected in the cat’s visual cortex showed double-labeled cells in DR and LC monoaminergic nuclei, but no non-monoaminergic cell was retrogradely labeled in the midbrain and pontine reticular fields.57 No evidence of a cortical projection was obtained in autoradiographic studies in cat following injections of tritiated amino acids in midbrain and pontine reticular fo~at~on9.‘* or in PB and surrounding areas.‘* The control injections we made in the neocortex and the hippocampus of cat produced no labeling in the chohnergic PB and LDT nuclei as well as the more rostra1 reticular core. Similarly, a WGA-HRP injection into the dorsal neocortex of rat did not result in retrogradely labeled cells in the PB3’ (see Fig. 4L in that paper). Furthermore, an autoradiographic study in rat mentioned that midbrain reticular projections to the cerebral cortex are sparse and difficult to visualize.” In contrast to the dense antcrograde labeling in the dorsal thalamus after WGA-HRP injections in the rat LDT nucleus, only “isolated” labeled fibers were found in the cortex where they were confined to the medial (infraiimbic) area.”

# 8% - PUL-LP r ChAT

HRP

l.

Double

: i

-..

/

_

Fig. 13. Brainstem tegmental projections to right MD nucleus in the monkey. (A) Injection site. (B) Retrograde labeling in dorsal and lateral parts of PAG and antcrogradc labeling in stratum superficiaie of the SC, ipsilateral to the injection site. (C) The BC and dorsal part of the PB area (PBd) indicates the same area as depicted at higher magnification in (D). (D) Dorsal and lateral are indicated by arrowheads (D and L). {E) The rectangle is enlarged to ahow in more detail the retrogradely labeled neurons in the ventromedial part of the PB area TMB procedure. 62

by this

brainstem

iological

remain

show

dau

and

amus

all major

(VA-VL) substrate

of synaptic

associational

(MD

nuclei

tories

(VM

more

transmission LP)

project

over

cell labeling specific

are congruent

with

;I most

tibcrs

that

the

highest

cozltic\

arc

found

in

sired

that

lcszcr

RE

and

rcccivc band

a cholincrgic nuclei

and

nuclei

In f’acl. the more hrainstcm

core

counted

Ihr

gic PB

after

reticular

f’ormation.

(see Fig.

caque monkey.

numerous

was prc\iously The ular AV

Inrgc cells

AM

after

widcsprcad tion

projections

nucleus

is known memory.

of the frontal cinyulate

and

ciational

PUL-LP

cxtrastriatc role

III

complex

relaying

exerts The limblc ‘The

”

cffccts

of

which

were

brainstcm

found core.“’

and

inncrvatc

visual

of cat.“’ (c)

to

be directly

The

brainstem

the

cortex”

cleus.

LP

excited reticular

zone.

also

thalamic brainstem

in

with to

tmns

and

nuclei

of

nu-

t! rosinc

l>DT

75

(V.4

VL)

85”~~~01‘ their

and PI

of’ cortical the

ditfcrcnccs

thalamic

cortical nuclei

labeling

is not due to spurious

an injection

in

5) contrast7 arcas

injected hctwccn

lahcling support the

two

involvcmcnt

into

the Phi\

aficl- injcctlon\

(see I;lg.

to

whcrc

f11llh dc\clop.

labcling

nuclei

linked

cell

Al

nuclei

labeling

thcsc

15 that the

stud!

and motor

fI_om an arca conccntratcd

and retrograde

aficr

to

of the

the parahrachial

in this

hrainstcm

in \;irious

f’cv,

part

num her

planes

thalamic

brainbtcm

the posterior

VB)

alrcrents

Again.

pcri-

as compared

rcccivc about

bc reciprocally

regrade

of’ this

at-c relativcl)

nuclei

the differcntlal

nuclei.

and

t-at”

neurons.”

monotonous

so diverse

cat.” part

LG.

PB

hcrc

cholincrgic

area in the present

rostral

findings

the stereotaxic

cholinergic

of

of PB

(MC;.

core

between

VM (d)

rcla!

rcprotcd

of’ thcsc

termed

rc-

+ ChAT)-lahclcd

atlases

great

One of the major

quite

(HRP

Only

:I

scnsorl

documcntcd

pcrlkarya.

elements.

the

cot-c ncurony

ccl1 group5

the

to hc in much

to monoamincl-tic

of cat. wjhcrc thcrc

contains

groups

I.”

known

thalamic the

antc-

af’tcl- indccthe fact that cholincrgic of adjacent

one nucJc:it- goup.

entire

neurons in

to

ventral douhlc-

much hcavict

brainstem

designation

/one

cholinergic

its and

the

in

and

nucleus,”

were also

catccholaminc-containing

the

“.‘-

layer

ahnost

reticular

investigated

of

Besides

influxes,

to

asso-

associational

on cortical

project

brainstem

clcctl-ophysiologicallly

the MD

to a cluster

anterior

the motor

actions

nuclei

cortex.“’

(a) In addi-

(b) The

fastigiocortical

dcpolari/ing AV-AM

and thcil

origin

projects

gyrus

beyond

and

more complex

in associative

descrihcd

01‘

rctropradc

was reported

the boundaries

corresponds

brachial

latcrul

compared

of double

hydroxylasc-positive

retic-

VM

chohnergic

with The

the

7 -I I in the cat. The

projections,

pcribrachial

lobe as well as the orbital.

areas and to more

projects

LP.

their

of MD

cortIccs.“”

;lrc;lb in the suprasylvlan nucleus

MD.

the cortex.

;lxons

insular

paper

brainstem

to be involved

large arcas

ac-

fcuturc

aspects of ollaction.“’

I” The

not

This

information

upon

as

monkcy.ih

major

with

sensory

to i! s role in sonic

spatial

into

in well

in

of the DR

in the PB and LDT

nuclei.

labeling

Figs

to

that

idcntilicatlon with

neurons

the

investigation

injected

part

in

afl’crcnts

rat. the only conslstcnt

distribution

the

in cat.”

injections

LC

in

cat and ma-

injection.

tits

an

I)R thalamtc

bc seen

HRP

frotn

and The

can

of’thc

rostra1

neurons

and contralatcral

of’ HRP-labeled

than relaying

DR

cells in the periaqucductal

nuclei is in keeping

function\

is

In

to the cat 1-C; nucleus. ccntly.

and LDT

afiel-

of the brainstem

elements

of brainstem

projections

also

fibcrs

diffcrencc

study

complex.”

same as in our

PB

LC

the immunohistochemical

the tnost

to :I

and pointine

in both

the MD

described

number

and.

of the cholincr-

ipsi-

Moreover,

gray wcrc labclcd aficr

and

Thcrclbrc.

midbrain

on both

IO).

LP

labeling

the diagonal

in_jcction

ol‘thc

PUL

after

be empha-

but by the massive

parts

similar HRP

nuclei

\a+

ccl1 labeling

MD

the

A

of’an

transport

in the brainstcm.

contribution

nuclcl.

in non-cholincrgic sides

the

by a grater

and LDT

stained

from

important

figures

thillamic

which

with cholincrgic

does not caclusively originate

injections.

of HRP-labclcd

scrotonin-containing

and in the noradrcnergic

core

of’ ChAT-

innominata.

innervation

nucleus

number

of‘cholincrgic

in presumably

data

preparation)

Inncr\ation

substantia

the rich thalamic

These

nuclciYJ

(in

po-

than

larger

the limits

combined

intralaminar

thalamic

AM

nuclei

to be investigated.

a much

within

scrotonin-containing

nuclei. “’ It should

MD

AV

found

tcrri-

of

AV.

We

involved

remain

times

thalamus

densities

effects

pro.iecting

cortical

study

rat

in these

and diffusely

as well as the transmitter(s)

to eight

nuclei.

recent the

associational

nuclei

arc

nuclei and

in the brainstem

MD.

thalamic

cxtcnt,

wide

relay

in

showed

(RE)

thalamic

the other

neurons

into

relay nuclc~

thalamic

rc-

the thal-

injections

led to three

into

reticular

through

WGA-HRP

injections

immunorcactivc

projections

to thalamic

upon

LG.

nuclei

of the reticular-induced

and AV-AM)

rctrogradc

than

relay

These

and PUL

which

phys-

(MC.

sensory

thalamic

(see Introduction).

those

and their

core afTeren&

the structural tcntiation

pathway

projections

to be elucidated.

that

motor

ceivc bruinstcm

cholineryc

-for&rain

actions

Our

VB)

Bralnstem

wet-e

neurons from

the

actions

The

transition

chronization

to

from cithcr

(EEG-desynchronized REM)

sleep is associated

quiet

sleep

wakefulncs~ or with

with or

EEG

syn-

paradoxical

rapid-cyc-movctiicnt. enhanced

cxcltahihtk

of‘ thalamocorticaf and corticofugal neurons. as tested by using antidromic or monosynaptic volleys.“” While spinal reflexes are inhibited during paradoxical sleep.“’ forebrain neurons arc more ready to respond to either external stimuli (as in waking) or internal drives (as in REM sleep) whether or not an overt motor response is generated. This is the meaning of brain activation in both waking and REM sleep.“’ Both chofinergic PB and LDT groups and noncholinergic neurons in other &Ids of the upper brainstem reticular formation have been found to project towards relay and associational thafamic nuclei. The increased responsiveness of thaiamocorticaf neurons during waking and REM sleep is paralleled by equally increased firing rates and tonic discharges, in both these behavioral states, of neurons recorded from PB and LDT nuclei” as well as from more rostra1 sites of the midbrain reticular formation.~~ Moreover, stimulations applied to the chofinergic PB area at the midbrain-pontine junction and more anteriorly to the non-cholincrgic central tegmentaf field, both induce direct tonic depolarization of thalamocorticaf neurons, blockade of their long-lasting and cyclic inhibitory periods. and enhanced probability of responses to incoming volleys.*.‘” 5’~h’,hfi.hX The transmitter actions involved in such operations are more complex than those of ACh alone, which was previously assumed to account entirely for these activating effects. Indeed, previous failure to antagonize completely the effects of midbrain reticular stimulation with various cholinergic blockers may be ascribed to co-localization of peptides in brainstem cholinergic LDT neurons7x.7y and to yet unknown transmitters of the rostra] midbrain reticular neurons. The disclosure of the transmitters used by neurons located in the very large territory of the central tegmental field (at planes A4-A3) where numerous retrogradeiy labeled neurons were found especially after injections in VM and MD nuclei is an important task in future investigations. The virtually ubiquitous thafamic projections of cholinergic PB and LDT neurons in the cdt raise questions that should be further elucidated. In this and the following” study we found that the percentages of double-labeled (ChAT + HRP) neurons from the total number of ChAT-positive cells in PB

and LDT groups arc around 230”/0 when all m~ccuxi thalamic nuclei are considered. This ra~scs the ~OSSIbility that individual PB and LDT chofincrgic cclfs have axons that branch to different thalamic nuclei. The presumed extensive coffaterafizat~on of brainstem reticular neurons (which challenges our previous view, based on antidromic invasion, that few midbrain reticular neurons have ascending bifurcating axon9) can now be tested by using ChAT immunohistochemistry in retrograde transport experiments with multiple fluorescent tracers injected in various thalamic nuclei. The widespread thafamic projections of PB and LDT neurons are clearly involved in the distribution of ponto-genicufo-occipital waves, the major phasic component of REM sleep and the probable physiological correlate of oneiric behavior. PB and LDT neurons with physiologicaffy identified projections to LG. PUL or intralaminar CL nucleus discharge spike bursts reliably preceding the ponto-geniculo-occipital waves.“’ It is known that. while the original term indicated their presence in the visual pathway, pontogeniculo-occipital waves largely transcend this scnsory system and are disseminated in many thalamic nuclei as well as in cortical areas outside the visual cortex.“‘.” Ponto-geniculo-occipital waves can be triggered by stimulating the upper brainstem core when the cat is in REM sleep or during the transition to REM sleep, thus suggesting a selective gate opening during this behavioral state.’ The same brainstem reticular area is involved in active eye movements related to alerting reactions in waking. Thus. in addition to the tonic activation processes in thalamocortical neurons, as reflected in the enduring BEG desynchronization and the associated cellular events of the relay mode, brainstem reticular neurons generate phasic ev$nts that underly orienting reactions to stimuli from the outside world during the alert state and they project the internally generated activity to the forebrain during REM sleep. At,k-nct,~(e~~~mrnts---This work was supported by grants from the Medical Research Council of Canada (MT--3689 and MT-5781) to M.S. and A.P. D. Pare and Y. Smith arc graduate students in our laboratories. We thank C. Harvey. A. Madariaga. D. Drofet and L. Bertrand for their assistance.

REFERENCES 1. Aggleton J. P. and Mishkin M. (1983) Memory impairments following restricted medial thalamic lesions in monkeys. Expi Brain Res. 52, 199-209. 2. Benevento L. A. and Faffon J. H. (1975) The ascending projections of the superior cofliculus in the rhesus monkey (Macaca mulatra). J. cony. Neurol. 160, 339.-361. 3. Bizzi E. and Brooks D. C. (1963) Functional connections between pontine reticular formation and lateral geniculate nucleus during deep sleep. 2rch.s’ ital. Bio/. 101, 66W580. 4. Berman A. L. (1968) The Bruin Szem of the Cur, p. 175. The University of Wisconsin Press. Madison. 5. Berman A. L. and Jones E. G. (1982) ?he T~~i~~~~~asd Busul Tele~ce~h~lo~ qf rhe Car. p. 164. The University of

Wisconsin Press, Madison. 6. Consofazione A.. Priestfey J. V. and Cueflo A. C. (1984) Scrotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique. Brain Res. 322,233-243. 7. De Lima A. D. and Singer W. (1987) The brainstem projection to the lateral geniculate nucleus in the cat: identification of cholinergic and monoaminergic elements. J. camp. Neural. 259, 92- 12I.

Bralnstem

cholinergic

projections

to thalamic

X. DeschPnes M.. Hu B. and Stcriadc M. (19X6) The effect of mcsencephalic

relay nuclei

reticular

hi

stimulation

dorsal lateral geniculate nucleus. Sock. Nwrwci. Ahstr. 12, X54. 9. Edwards S. B. and DeOlmos J. S. (1976) Autoradiographic studies of the projections

on relay neurons of the

of the midbrain

reticular

formatron: ascending projections of nucleus cuneiformis. J. c’omp. Nwrol. 165, 417 43 I. IO. t-ilion M.. Lamarre Y. and Cordeau J. P. (1971) Neuronal discharges of the ventrolatcral nucleus of the thalamus during sleep and wakefulness. II. Evoked activity. E.xp/ Bruin RCA. 12, 499 508. I I. Glenn L. L., Hada J.. Roy J. P., Deschines M. and Sterlade M. (19X2) Antrrogradc tracer and ticld potential analy\~r of the neocortical layer I proJection from nucleus vcntralis medialis of the thalamus in cat. ~Vwrawwrr~ o 7. I X6 I I X77 II-. Grayblel A. M. (1977) Direct and indirect preoculomotor pathways of the hralnstem: an autoradlogr;lphli dud! 01 the pontine reticular formation in the cat. J. camp. Newt/. 175, 37 7X. I?. Grayhel A. M. and Bcrson D. M. (1980) Histochemical identification and aft‘ercnt connections of ch~~line acetyltransferase and tyrosine hydronylasc Immunohistochemistry .I, c’~,n?p. kcwv/. 261. I5 31. 23. Jona B. E. and Yang T. Z ( 1985) The elrcrcnt prqectlcms from the reticular formation and the locu\ cocrulcu\ \~udlcd h) anlcrogradc and retrograde axonal transport in tht, rat. J c,,nr/~ Vc,urc,/ 242, 56 97 24. Jones E.
3X 30 40 31 37

MoowEdle! S. and Grayhlel A. M. (19X3) The altrent and elfercnt c‘onncctlons of the fcllnc nucIcu\ tegrnsnt~ pcduiiculopontln~~\. par\ compacta. J. camp Lc~rrol. 217, 1X7 71 5 Morll\on J. Ci.. Gr/anna R.. Molliver M. E. and Covle J T. ( IY7X) The dlstrihution and orientation 01 nr>ratlrcncrq lihers in neocortex of the ral an Immunof~uorescencc~res~~n~e study. J. romp .Ymro/. 181. I7 30 M~~rw.71 <;. and Magoun II. W. (1949) Brain \tem rctlcular formation and act~\at~on of the I:E
43. Olivier A., Parent A. and Poirier L. J. (1970) ldenttfication of the thalamic nuclei on the basis of their cholme~~er;~\c content in the monkey. J. Anut. 106, 37 50. 44. Ottersen 0. P.. Fisher B. 0. and Storm-Mathisen J. (1983) Retrograde transport of I>-[?H] aspartate in thalamocortlc,~l neurones. Nc~urosci. Leti. 42, 19~ 24. 45. Par6 D., Smith Y., Parent A. and Steriade M. (1988) Projections of upper brainstem cholinergic and non-cholinerglc neurons of cat to intralaminar and reticular thalamic nuclei. Neuroscience 25, 69-86. 46. Park D., Steriade. M., Desch&nes M. and Oakson G. (1987) Physiological properties of anterior thalamic nuclei. a group devoid of inputs from reticular thalamic nucleus. J. NeurophJlsiol. 57, 1669%1685. 47. Parent A., Descarries L. and Beaudet A. (1981) Organization of ascending serotonin systems in the adult rat bram. A radiographic study after intraventricular administration of ‘H-5-hydroxytryptamine. Neuroscience 6, 115- 138. 48. Partlow G. D., Colonnier M. and Szabo J. (1977) Thalamic projections of the superior colliculus in the rhesus monkey, Maccrcu mulu~ta: a light and electron microscopic study. J. camp. Neural. 171, 285-318. 49. Phyllis J. W.. Tebecis A. K. and York D. H. (1968) Acetylcholine release from the feline thalamus. ./. Phurmuc 20, 477478. 50. Pompeiano 0. (1967) The neurophysiological mechanisms of postural and motor events during desynchronized sleep. Proc. Assoc. Res. New. Menr. Dis. 45, 351-423. 51. Purpura D. P. (1970) Operations and processes in thalamic and synaptically related neural subsystems. In The Neurosciences: Second Sru& Program (ed. Schmitt F. O.), pp. 458--470. Rockefeller Univ. Press. New York. 52. Robertson R. T. and Kaitz S. S. (1981) Thalamic connections with limbic cortex. I. Thalamocortical projections. J. camp. Neural. 195, 501-525. 53. Rodrigo-Angulo M. L. and Reinoso-Suarez F. (1982) Topographical organization of the brainstem affcrents to the lateral posterior-pulvinar thalamic complex in the cat. Neuroscience 7, 1495-1508. 54. Ropert N. and Steriade M. (1981) Input&output organization of the midbrain reticular core. J. Neurophysiol. 46, I7 3 1. 55. Rotter A. and Jacobowitz D. M. (1981) Neurochemical identification of cholinergic forebrain projection sites of the nucleus tegmentalis dorsalis lateralis. Buin Res. Bull. 6, 525-529. 56. Rye D. B., Saper C. B. and Wainer B. H. (1984) Stabilization of the tetramethylbenzidme (TMB) reaction product. J. Histochem. Cytochem. 32, 1145 1153. 57. Sakai K. (1985)Anatomical and physiological basis of paradoxical sleep. In Bruin Mechanisms of’Sleep (eds McGinty D. J.. Drucker-Colin R.. Morrison A. and Parmeggiani P. L.), pp. 11 I- 137. Raven, New York. 58. Satoh K. and Fibiger H. C. (1985) Distribution central choiinergic neurons in the baboon (Papio papro). II. A topographical atlas correlated with catecholamine neurons. J. romp. Neural. 236, 215--233. 59. Satoh K. and Fibiger H. C. (1986) Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections. J. romp. Neural. 253, 277-302. 60. Shute C. C. D. and Lewis P. R. (1967) The ascending cholinergic ascending reticular system: neocortical. olfactory and subcortical projections. Bruin 90, 497 520. 61. Simon R. P., Gershon M. D. and Brooks D. C. (1973) The role of the raphe nuclei in the regulation of ponto-geniculooccipital wave activity. Bruin Res. 58, 313-330. 62. Singer W. (1977) Control of thalamic transmission by corticofugal and ascending reticular pathways. Physiol. Rer. 57, 38&420. 63. Snider R. S. and Lee J. C. (1961) A Srereofaxic Atlas offhe Monkey Brain (Macaca Mulatta). University of Chicago Press, Chicago and London. 64. Sofroniew M. V., Priestley J. V. Consolazione A., Eckenstein F. and Cuello A. C. (1985) Cholinergic projections from the midbrain and pons to the thalamus in rat, identified by combined retrograde tracing and choline acetyltransferase imunohistochemistry. Bruin Res. 329, 213.-223. 65. Steriade M. (1970) Ascending control of thalamic and cortical responsiveness. Inl. Rea. Neurobiol. 12, 87 144. 66. Steriade M. (1984) The excitatory-inhibitory response sequence of thalamic and neocortical cells: state-related changes and regulatory systems. In Dynamic Aspects qf Neocorfical Function (eds Edelman G. M.. Gall W. E. and Cowan W. M.), pp. 107-157. Wiley, New York. 67. Steriade M., Apostol V. and Oakson G. (1971) Control of unitary activities in cerebellothalamic pathway during wakefulness and synchronized sleep. J. Neurophysiol. 34, 384413. 68. Steriade M. and Deschtnes M. (1984) The thalamus as a neuronal oscillator. Bruin Res. Rev. 8, l-63. 69. Steriade, M., Diallo A., Oakson G. and White-Guay B. (1977) Some synaptic inputs and ascending projections of lateralis posterior thalamic neurons. Bruin Res. 131, 39-53. 70. Steriade M. and Glenn L. L. (1982) The neocortical and caudate projections of intralaminar thalamic neurons and their synaptic excitation from the midbrain core. J. Neurophysiol. 48, 352-371. 71. Steriade M. and Hobson J. A. (1976) Neuronal activity during the sleep-waking cycle. Prog. Neurobiol. 6, 155-376. 72. Steriade M., Oakson G. and Ropert N. (1982) Firing rates and patterns of midbrain reticular neurons during steady and transitional states of the sleepwaking cycle. Expl Brain Res. 46, 37-51. 73. Steriade M., Parent A. and Hada J. (1984) Thalamic projections of nucleus reticularis thalami of cat: a study usmg retrograde transport of horseradish peroxidase and double fluorescent tracers. J. camp. Neural. 229, 531 547. 74. Steriade M., Parent A., Par6 D. and Smith Y. (1987) Cholinergic and non-cholinergic of cat basal forebram project to reticular and mediodorsal thalamic nuclei. Bruin Res. 408, 372-376. 75. Szerb J. C. (1967) Cortical acetylcholine release and electroencephalographic arousal. J. Physiol. 192, 329 ~345. 76. Tigges J. and Tigges M. (1985) Subcortical sources of direct projections to visual cortex. In Cerebra/ Caries (eds Peters A. and Jones E. G.), Vol. 3, pp. 351 378. Plenum, New York. 77. Velayos J. L. and Reinoso-Suarez F. (1982) Topographic organization of the brainstem afferents to the mediodorsal thalamic nucleus. J. camp. Neural. 206, 17 -27. 78. Vincent S. R., Satoh K.. Armstrong D. M. and Fibiger H. C. (1983) Substance P in the ascending cholinergic system. Nuture 306, 688.-69 I. 79. Vincent S. R., Satoh K., Armstrong D. M., Panula P., Vale W. and Fibiger H. C. (1986) Neuropeptides and NADPHdiaphorase activity in the ascending cholinergic reticular system of the rat. Neuroscience 17, 167-182. 80. Wilson P. M. (1985) A photographic perspective on the origin, form, course and relations of the acetylcholinesterasecontaining fibers of the dorsal tegmental pathway in the rat brain. Brain Res. Rev. 10, 85-l 18.

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