Brain Research Bulktin, Vol. 18, 191-203. Copyright 0 Pergamon Journals Ltd., 1987. F’rinted in the U.S.A. 0361-9230/W$3.

0361-9230/W$3.00 + .OO

Phaseolus vulgaris Leuco-Agglutinin

Tracing of Intrahypothalamic
Connections of the Lateral, Ventromedial,
Dorsomedial and Paraventricular
Hypothalamic Nuclei in the Rat
G. J. TER HORST AND P. G. M. LUITEN

Department of Animal Physiology, State University of Groningen

P.O. Box 14. 9750 AA Haren, The Netherlands

Received 16 May 1986

TER HORST, G. J. AND P. G. M. LUITEN. Phaseolus vulgaris leuco-ugglutinin tracing of intrahypothalamic connec-
tions of the lateral, venrromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat. BRAIN RES BULL
18(2) 191-203, 1987.-Intrahypothalamic connections of the lateral (LHA), ventromedial (VMH), dorsomedial (DMH) and
paraventricular (PVN) hypothalamic nuclei were studied with anterograde transport of iontophoreticalIy injected
Phaseolus vulgaris leuco-agglutinin and the immunocytochemical detection of labeled structures. The LHA was found to
give rise to a minor projection in the VMH, whereas the VMH in reverse maintains few connections with the ventromedial
part of the tuberal LHA. Tracer deposits in both the LHA and VMH resulted in anterograde terminal labeling in the DMH.
The DMH, in turn, donates a small number of projections to the LHA and VMH. The main projection of the DMH is aimed
at the parvocellular paraventricular nucleus. Direct outflow pathways from the VMH to the PVN were not found, but lectin
injections in the LHA on the other hand gave rise to terminal labeling in both the parvocellular and magnocellular divisions
of the PVN. The PVN in turn was found to give only minor reciprocal projections to the LHA, DMH and VMH. These
findings indicate that the main stream of connections in the hypothalamus runs from the LHA and VMH to the DMH, and
from the DMH to the PVN. The identified circuitry patterns were discussed with respect to the role of the hypothalamus in
the control of homeostasis and metabolic regulation, and more specifically in relation to the modulation of the hormone
release from the pancreas and adrenal glands.

Lateral hypothalamic area Ventromedial hypothalamic nucleus Dorsomedial hypothalamic nucleus

Paraventricular nucleus PHA-L tracing Intrahypothalamic connections Pancreas hormone release
Homeostatic control

ONE of the major functions ascribed to the hypothalamus in at all and VMH lesioned rats overeat and become obese
the mammalian brain is to regulate the homeostatic proc- [28,31].
esses of the internal environment. These homeostatic control One of the major mechanisms by which the hypothalamus
functions are achieved by a variety of autonomic, endocrine exerts its influence on feeding and body weight appears to be
and behavioral responses in which the hypothalamus is con- the modulation of hormone release from the pancreas. Pre-
sidered to play a key role. ceding anatomical investigations already have revealed de-
With respect to the control of ingestive behavior and scending connections of the hypothalamus to the pre-
metabolic regulation it was shown that experimental manipu- ganglionic cell groups of the lower brainstem and spinal cord
lations of the lateral hypothalamic area (LHA) and the ven- that innervate the pancreas [24, 31, 41, 44, 451.
tromedial hypothalamic nucleus (VMH) have opposite be- Intrahypothalamic connections related to the regulation of
havioral effects. These observations resulted in the intro- feeding and metabolic homeostasis, however, are still far
duction of the “dual-center” theory for feeding control (for from clear and remain a matter of discussion. A few
review see [28]). Electrical stimulation of the lateral hypotha- anatomical studies, employing the retrograde transport of
lamic area induces eating, whereas the ongoing meal is ter- horseradish peroxidase (HRP) report direct connections be-
minated by stimulation of the ventromedial nucleus. In le- tween the LHA and VMH [I, 17, 181, but Luiten and Room
sion studies these antagonistic effects are even more dra- [23] were unable to reveal this anatomical relationship. The
matic. Animals with bilateral damage to the LHA do not eat latter authors, on the other hand, indicated reciprocal con-

191
 I’).! ‘I‘ER HORST AND ILUITHN

TABLE 1 MF.‘I‘HOI~

ABBREVIATlONS
Iontophoretic injections of PHA-L were made in the var--
A. AHA anterior hypothalamic area ious hypothalamic nuclei of 128 male Wistar rats (300 g). The
AR arcuate nucleus experimental animals were anesthetized with an intraperito-
Cl internal capsule neal injection of sodium pentobarhital (30 mg/kg) and ;I
DM, DMH dorsomedial hypothalamic nucleus simultaneous intramuscular injection of Hypnorm (Duphar)
F fomix (0.4 ml/kg), and placed in a Kopf stereotaxic apparatus ad-
LH lateral hypothalamic nucleus justed to the coordinate system of Paxinos and Watson [30].
MA medial amygdaloid nucleus
Bevelled glass micropipettes with tip diameters of IO to 20
mt mamillothalamic tract
PF perifornical area +rn were filled with a solution containing 2.5% PHA-L (Vec-
PH posterior hypothalamic area tor Labs.) in tris-buffered saline (TBS, pH-7.4) and posi-
PMD dorsal premamillary nucleus tioned in the hypothalamus stereotactically. A Midgard CS-3
PMV ventral premamillary nucleus constant current source supplied a pulsed positive DC-
PV paraventricular hypothalamic nucleus current of 4-7 PA to the pipette for 30 minutes. Following
PVM magnocellular paraventricular nucleus iontophoresis the pipette was left in situ for IO minutes to
PVP parvocellular paraventricular nucleus avoid loss of tracer in the pipette track.
RE reuniens nucleus After a 7 days post-operative survival time the animals
TO optic tract
were reanesthetized with an overdose of sodium pentobarbi-
VM, VMH ventromedial hypothalamic nucleus
v 111 third cerebral ventricle tal (100 mg/kg), and fixed by transcardial perfusion with a
Zl zona incerta solution containing 0.5% paraformaldehyde, 2.5% glutardal-
dehyde and 4%’sucrose in 0.05 M phosphate buffer (pH=-7.4).
After storage overnight in 3@Z sucrose in 0.05 M phosphate
buffer (pH=7.4) at 4”C, the brains were cut in 40 pm sections
on a cryostat-microtome. The free floating sections were
nections of the LHA and VMH with the dorsomedial hypo- thoroughly rinsed in tris-buffered saline (TBS: pH==7.4) and
thalamic nucleus (DMH), which is a nucleus also known to incubated for 48 hours at room temperature with goat-anti-
participate in control mechanisms of food intake [3-61. PHA-L (1:2000) (Vector Labs.) dissolved in tris buffer to
Moreover, various investigators have shown that the which 0.5% Triton X-100 and 0.5 M NaCl was added (TBS-
paraventricular nucleus (PVN) is the most effective site T). Subsequently the sections were rinsed thoroughly in
within the hypothalamus for adrenergic stimulation of inges- TBS-T again and incubated with rabbit-anti-goat IgG (1:200)
tive behavior [ll, 21, 221. From autoradiographic and HRP (Sigma) for I2 to 24 hours. Following a next rinse in TBS-T
tracing studies it appears that the paraventricular nucleus the sections were transferred for 4 hours to a solution con-
receives efferents from the ventromedial [19, 20, 32, 35, 421, taining goat peroxidase-anti-peroxidase complex (1:400)
dorsomedial [35,42] and lateral hypothalamic areas [33, 35, (Dako), soaked in tris-HCI solution (pH=7.6) and reacted for
421. The use of retrograde transport of HRP and fluorescent I hour in a tris-buffered solution containing 40 mg di-
markers has revealed the outgoing connections of the PVN aminobenzidine (DAB) and 0.8 ml 1.5% H,O, per 100 ml.
towards the preganglionic cell groups of the lower brainstem Finally, the sections were mounted. counterstained with cre-
and spinal cord which innervate the pancreas [41,45]. ayl violet and covet-slipped. The labeling was examined by
The intrahypothalamic organization of the connectivity using both light- and dark-field microscopy and results were
between the above mentioned nuclei still remains to be drawn in representative sections of the atlas of Paxinos and
elucidated [31], partly because of the technical limitations of Watson [30].
the neuroanatomical tracing techniques employed so far. In- The PHA-L immunocytochemical tracing procedure de-
jection size, non-specific background artifacts, tracer diffu- scribed here is a modified version of the method originally
sion and the different staining procedures that are used for described by Gerfen and Sawchenko [IO].
HRP and other retrograde transport methods all contribute
to often contradictory results. Anterograde tracing methods
such as transport of tritiated amino acids proved to be un-
RESULTS
suitable for investigating short-distance connections in a
complex structure like the mammalian hypothalamus. The LHA, VMH, DMH and PVN are not homogeneous
In this study the intrahypothalamic connections of LHA, nuclear structures but consist of several subdivisions each
VMH, DMH and PVN are re-investigated with the with a particular cell density and cell diameter. For the hypo-
anterograde Phaseofus vulgaris leuco-agglutinin (PHA-L) thalamus in general we have used the topographic arrange-
immunocytochemical tracing technique. The PHA-L method ment as indicated by Paxinos and Watson [30]. With regard
proved to be very effective for the identification of short to the parcellation of the paraventricular complex the no-
distance projections, as is described in preceding papers [ 10, menclature of Swanson and Sawchenko [42] is referred to.
43, 44, 461. The immunocytochemical PHA-L procedure is The following divisions are used throughout this paper: the
not hampered by haphazard spread of injected tracer or as- parvocellular (PVP) and magnocellular (PVM) paraventricu-
pecific debris and precipitates. It is also possible to make lar nucleus, the anterior (aDMH) and posterior (pDMH) dor-
small iontophoretic tracer injections that are restricted to a somedial hypothalamic nucleus, and the anterior (aVMH),
nuclear area under study. This anterograde tracing technique medial (mVMH) and posterior (pVMH) ventromedial hypo-
yields completely filled axons that can be traced from labeled thalamic nucleus. Moreover, the posterior cell groups of the
cell body to the target area where the characteristic var- DMH and VMH are composed of a dorsal, a compact or
icosities and terminal boutons appear indicating the site of central, and a ventral subdivision. The lateral hypothalamic
presynaptic terminations [lo]. area (LHA) is more easily defined by reference to the struc-
INTRAHYF’OTHALAMIC CONNECTlONS

FICI, I, G series of coronat bypoth~~i~ sections ~~~st~t~~~ tke

sourre of ~~t~~~tha~~ afferents to the post&or Vlvfff @ILI&~
are&. Areas in * b~~~~~~~~ wtricA contak (B) g.i% rise to a
hefty termirrat Meling, (0) produce a modePate terminal labeling,
and (-) maintain no direct connections to the VMH. The diagram
was constructed by plotting the injection sites of the 128 cases in a
stereotrrxic atlas of the hypothalamus. Every injection was, there-
fore, first classified in one of the foliowing three groups; deposits
giving rise to a dense, moderate or absence of t~~&~al lab&g in
the VMH. Hence, such a diagram gives t&e ~~~~~~~ foT the ap- a&%-& ~a~~A~~ tnberal ~~~~A~ and $L%llsterior Q&HA]. The
peat%nce of terminal tab&$ in the %%BHafter k?ctin deposits in the aLHA is cont~uons rostraIfy with the lateral preopticarea
~~~~~~a~arnus. and extends caudaify to the level of the rostra:al pole of the
ventrome?dM hypothalamic nucleus. The &HA is coextenw
sive with the ventromedial nucleus and the pLHA replaces
the tuberal division at the level of the mammiilary complex,
&aud&y the &HA merges with the ventrat &&mental area.
 ‘I‘ER HORST AND l,UlTEN

FIG. 3. Dark-field photomicrographs showing terminal labeling in the ventromedial hypothalamic nucleus after it
PHA-L injection in the anterior DMH (A) and in the AHA (B). Bar is 500 em.

to a nuclear area under study. This method is used because it maintain a small projection to the VMH. The lateral aspect
was found that the various subdivisions of LHA, VMH. of the aDMH innervates the ventral pVMH, whereas the
DMH, and PVN have different projection areas within the posterior DMH sends efferent fibers to the lateral and dor-
hypothalamus. In descriptions by means of efferent connec- somedial rim of the entire ventromedial nucleus. The projec-
tions of the various subdivisions of one nuclear area tions of the DMH to the nuclear core of the VMH, however.
intrahypothalamic relationships would not appear uniform, constitute only a minor part of the outflow pathways from
but in a variety representing the different functions of the the dorsomedial to the ventromedial nucleus. In Golgi prep-
individual nuclei. They can, however, more easily be shown arations it is shown that the dendritic trees of many cells
in the summary diagrams. Some more characteristic projec- within the VMH extend outside the nuclear core of this nu-
tion patterns in the hypothalamus are illustrated with indi- cleus. Such dendrites form a perinuclear fiber shell that is
vidual injections of PHA-L in the LHA, VMH, DMH, and characteristic for the VMH. In particular the injections of
PVN . PHA-L centered in the ventral part of the posterior DMH
lead to extensive projections to this perinuclear shell of the
ventromedial nucleus.
Intrcrhypothalamic Input to the Ventrornedial Hypothalamic,
The input from the parvocellular paraventricular nucleus
Nucleus (Fig. I) is found in the medial aspect of the VMH, at all longitudinal
In the VMH a few labeled terminals are found after injec- levels. The perinuclear fiber shell, on the other hand, is not
tions of PHA-L in the medial aLHA and in the central and innervated by the parvocellular PVN. The magnocellular
ventromedial tLHA. The medial aLHA and the central division of the PVN does not send efferent fibers to the
tLHA (Fig. 2) project to the dorsal aspect of the anterior and VMH.
medial VMH, whereas the ventromedial tLHA innervates The VMH receives additional inputs from the anterior
the ventral part of the posterior VMH. The main output hypothalamic area (AHA) and ventral premamillary nucleus
channel from the lateral hypothalamic area to the ventrome- (PMV). In terms of numbers of fibers, the largest input to the
dial nucleus is formed by the efferents from the ventromedial VMH originates in the AHA. Following tracer injections in
tLHA. The ventrolateral tLHA and the posterior LHA do the AHA the labeled varicose fibers and terminal boutons
not send efferents to the VMH. appear diffusely over the nuclear core, but very densely in
The medial aspect of the anterior and medial VMH is the perinuclear shell of the VMH (Fig. 3B). Iontophoretic
densely innervated by fibers from the medial aDMH (Fig. injections of PHA-L in the ventral premamillary nucleus give
3A). The remaining parts of the dorsomedial nucleus only rise to a circumscribed projection to the ventral aspect of the
INTRAHYPOTHALAMIC CONNECTIONS

/
‘.
OO”**
l . i_
c,/ ,
,‘;

((3.*
0.
. . . . ~~-
,.: O0 l ,’
.* O0 /I
o”_0
_--
-
__-
-
o.**=‘:
-
/ :(: :~
-_
o”
0
:.

i ‘, -,
--0
0.
_ - 0 0 .~ ..

i d _ ,d.”

FIG. 4. Sections of the hypothalamus showing the source of FIG. 5. Transverse sections of the rat hypothalamus illustrating the
intrahypothalamic input to the peripheral zone of the tLHA (shaded projection pattern of the medial VMH.
area). Hypothalamic areas containing (0) give rise to a heavy termi-
nal labeling, (0) produce a moderate terminal labeling, and (-)
maintain no direct connection to the LHA.

posterior VMH. Minor inputs from the PMV are found in the the pVMH was never shown to receive projections from the
perinuclear shell of the aVMH and mVMH. other parts of the ventromedial nucleus.
The ventromedial nucleus also maintains a large number
of intranuclear connections. This is evident from the appear-
Intruhypothulamic Input to the Lateral Hypothalmic
ante of labeled presynaptic endings and varicose “en
Area (Fig. 4)
passant” labeling within its nuclear boundaries after PHA-L
deposits in the VMH. With respect to the organization of the Injections of PHA-L in the various subdivisions of the
intranuclear connectivity within the VMH the ventral aspect ventromedial hypothalamic nucleus (Fig. 5), except for de-
of the posterior VMH has a special position. This division of posits in the dorsal aspect of the pVMH, all give rise to
 ‘TER HORST AND LU ITEN

FIG, 6. Transverse sections of the bypotha~~us showing the FIG. 7, Series of coronal hy~tha~amic sections which show the
labeled projections after a PI-IA-L injection in the medial part of the afferentation of the posterior DMH (shaded area). Hypothalamic
aDMH. areas containing (@) give rise to a heavy terminal labeling, (0)
produce a moderate terminal labeling, and (-) maintain no direct
connection to the DMH.

terminal Iabehng in the ventromediai part of the anterior and of terminaf labeling in the medial aLHA. The posterior part
tuberal LHA. Such projections appear lateral to the VMH in of the DMH, however, sends most of its efferents to the
the ventromedial aspect of the tLHA which, in part, also medial tLHA. Such projections to the tuberal LHA originate
contains dendritic trees of adjacent VMH neurons. for the greater part in the ventral section of the posterior
The medial part of the anterior LHA, and the mediai and DMH. The tuberal LHA division receives a small number of
ventral segments of the tuberal LHA are target areas for the efferents from the parvocellular paraventricdar nucleus.
dorsomedial h~th~~i~ neurons. Moreover, a topo- The Lateral hypothalamic area, like the VMH, atso main-
graphic arrangement is apparent for the DMH projections to tains extensive intranuclear connections. PHA-L injections
the lateral hypothalamic area. PHA-L injections in the into the anterior LHA often result in positively reacting ti-
anterior aspect of the DMH (Fig. 6) result in a dense pattern bers, carrying numerous labeled presynaptic boutons, in the
INTRAHYPOTHALAMIC CONNECTIONS

FIG. 8. Photomicrographs showing a small PHA-L injection in the posterior LHA (A: bar is 650 pm), terminal labeling in the PVP following a
tracer injection in the VMH (B: dark-field, bar is 250 km), projections in the PVN after PHA-L injection in the LHA (C: dark-field, bar is 200
pm), terminal labeling in the PMV following a tracer injection in the anterior DMH (D: dark-field, bar is 250 Fm), and a small size PHA-L
injection in the posterior VMH (E: bar is 500 Fm).
 IO8 1ER HORST AND LUIWN

medial peripheral zone of the tLHA and pLHA. Such intra- Apart from receiving input from other hypothalamic nu-
nuclear synaptic contacts are found in the central and lateral clei, the dorsomedial nucleus maintains intranuclear con-
aLHA, in the central tLHA and in the lateral pLHA follow- nections as well. Presynaptic terminal boutons of
ing tracer deposits in the inner core of the tuberal LHA. intranuclear origin appear extremely dense in the ventral
Lectin injections in the ventromedial part of the tuberal LHA pDMH. Moderate numbers of terminals occur in the aDMH
give rise to intranculear projections in the peripheral zone of and the dorsal pDMH. The compact part of the posterior
almost every anterior-posterior level of the lateral hypothal- DMH only contains a limited number of such intrinsic pro-
amus. Furthermore, intranuclear presynaptic endings are jections.
found in the medial parts of the aLHA and tLHA after injec- The dorsomedial hypothalamic nucleus receives inputs
tions in the posterior division of the LHA. from the ventral premamillary and anterior hypothalamic
The ventral premamillary and anterior hypothalamic nu- area, as well. Lectin injections in the PVM give rise to a
clei do not heavily contribute efferent connections to the minor projection in the aDMH and the medial pDMH.
lateral hypothalamic region. Fibers that originate from the Moreover, the PMV is found to be one of the few nuclei
PMV give rise to some input to the medial aLHA and tLHA. within the hypothalamus that specifically sends efferent fi-
Efferent fibers of the ventral premamillary nucleus, on their bers to the compact division of the posterior DMH. The
way to the amygdala complex, in addition produce a minor aDMH and ventral pDMH, in addition, are the recipients of a
number of terminal branches in the ventral aLHA and tLHA. moderate projection from the AHA.
The medial and ventral aLHA and tLHA receive a moderate In summary, the VMH is a major source of input to the
projection from the anterior hypothalamic area as well. dorsomedial nucleus. Moderate projections to the DMH, on
In conclusion, the VMH and the PVP are minor, and the the other hand, are provided by the lateral and paraventricu-
DMH only a moderate contributor of input to the LHA. lar hypothalamic nuclei.
Terminal labeling in the LHA appears for the major part in
the peripheral zone of this nucleus (Fig. 4). The remaining
central part only contains the PHA-L labeled fibers that are
traversing the LHA.
injections of PHA-L which are centered in the VMH (Fig.
8E) produce labeled projections in the areas that ventrally
and dorsally surround the paraventricular nucleus. In these
The injections of PHA-L in the anterior and medial VMH cases, labeled fibers are seen to traverse the parvocellular
and in the dorsal aspect of the posterior VMH gave rise to and magnocellular PVN that, however, do not carry terminal
extensive projections to the anterior dorsomedial hypotha- boutons (Fig. 8B). The aLHA and tLHA supply inputs to the
lamic nucleus. The pDMH is a target for neurons in the magnocellular division of the PVN. In particular the ventral
dorsomedial part of the mVMH and pVMH. The projections and ventromedial parts of the tLHA give rise to moderate
of the medial and posterior ventromedial nucleus to the projections to the PVM. The ventromedial tLHA donates
DMH. however, are not evenly distributed over the dorsal, efferents to the parvocellular part of the PVN as well (Fig.
compact and ventral divisions of the pDMH. After deposits 8C). The injections of PHA-L in the ventral part of the tLHA
of PHA-L in the dorsal mVMH and pVMH the major and in the pLHA result in a few labeled presynaptic endings
number of the presynaptic endings is found in the dorsal in the PVP.
aspect of the posterior DMH. The ventral sections of the The dorsomedial nucleus maintains a circumscribed and
pDMH. on the other hand, are heavily labeled following in- dense projection to the PVP. Such input to the PVP origi-
jections in the ventral part of the posterior VMH. Further- nates for a greater part in the ventral division of the posterior
more, it is evident that the compact division of the posterior DMH. The medial aspect of the magnocellular PVN, in ad-
DMH only receives a moderate input from the VMH. dition, is a target for efferents from the medial aDMH.
Labeled presynaptic endings occasionally appear in the Intranuclear connections by way of boutons en passant
compact area of pDMH. are also found within the paraventricular nucleus. Such in-
The greater part of the tracer deposits in the lateral hypo- trinsic projections occur in the parvocellular and magnocel-
thalamic area resulted in labeling of projections to the DMH. lular divisions of the PVN after tracer deposits in the PVP,
An elaborate terminal labeling in various divisions of the whereas they are identified only in the magnocellular part
dorsomedial nucleus follows on tracer deposits in the ven- following injections in the PVM. In comparison to the
tromedial tLHA. A circumscribed, but less abundant input intranuciear connections of the VMH, LHA and DMH.
to the aDMH and ventral pDMH originates from the remain- however, the terminal intranuclear labeling of the paraven-
ing segments of the lateral hypothalamus (aLHA, central tricular nucleus appears to be much less abundant.
tLHA and pLHA). Projections from the anterior LHA are The PVN also receives inputs from the ventral premamil-
found in the lateral part of the aDMH and ventral pDMH. lary nucleus and from the anterior hypothalamic area. Lectin
Terminal branches from the inner core of the tuberal LHA injections in these nuclei of the hypothalamus give rise to a
appear in the lateral part of the aDMH and ventral pDMH. widespread terminal labeling in the PVP.
The medial sections of the dorsomedial nucleus, on the other In survey, the parvocellular and magnocellular divisions
hand, are most heavily labeled after injections in the of the paraventricular nucleus receive inputs from the lat-
posterior LHA (Fig. 8A). The compact pDMH receives a eral, dorsomedial and ventral premamillary nuclei of the
small number of inputs only from the medial anterior and hypothalamus. The ventromedial hypothalamic nucleus,
tuberal lateral hypothalamic area. however. does not project to the PVN.
Efferent fibers from the parvocellular paraventrkular
nucleus terminate, for a part, in the lateral aspect of the
DISCUSSION
anterior DMH. A major projection from the PVP is found in
the ventral pDMH (Fig. 9). In the present investigation reciprocal connections were
INTRAHYPOTHALAMIC CONNECTIONS 199

FIG. 9. A series of transverse hypothalamic sections showing the FIG. 10. Consecutive transverse sections of the hypothalamus
labeled projections after a PHA-L injection in the parvocellular showing the inputs to the parvocellular paraventricular nucleus
paraventricular nucleus. (shaded area). Hypothalamic areas containing (0) give rise to a
heavy terminal labeling, (0) produce a moderate terminal labeling,
and (--_) maintained no direct connections to the PVP.

identified between the LHA and VMH. Furthermore, it was rat hypothalamus under study, except the ventral and lateral
shown that the main stream of connections in the hypothal- parts of the LHA, appear to project to the ventromedial
amus runs from the LHA and VMH to the DMH and from nucleus. The anterior DMH, ventromedial tLHA and PMV
the DMH to the parvocellular PVN (Fig. 11). In this section show fairly well developed outflow pathways to the VMH.
the data are compared with preceding anatomical investiga- Efferents from the dorsomedial nucleus and the parvocellu-
tions and discussed in relation to the role of the hypothala- lar PVN terminate in the dorsal aVMH and mVMH, whereas
mus in the control of homeostasis and metabolic regulation. the ventral pVMH receives projections from the ventrome-
dial tLHA and the PMV. Such ventral premamillary and
Anatomical Aspects paraventricular nucleus projections to the VMH already
Ventromedial hypothalamic nucleus. Most nuclei of the were described in retrograde transport studies [17,23].
 rER HORST AND 1,UITEN

present PHA-L experiments applied to the PMV revealed

the precise target areas for these PMV efferents in the ven-
tromedial part of the tuberal LHA.
[Link]~tlial hypothrrlrrtnic ~~r~c~lr~rs. The dorsomedial
nucleus receives projections from the ventromedial. lateral,
paravent~cular and ventral premamillary nuclei of the hypo-
thalamus. These afferent connections of the DMH were also
previously shown with the retrograde transport of horse-
radish peroxidase [ 17.231. In these latter investigations many
HRP labeled cells were found in the ventral premamillary
nucleus which is indicative of a well developed outflow
pathway from the PMV to the dorsomedial nucleus. Ion-
tophoretic injections of PHA-L in the PMV, however, only
caused tabeling of a minor number of presynaptic boutons in
the DMH. The contrast in results of the anatomical ap-
proaches may partly be explained by the diffusion of HRP
FLG. 11. Summary diagram of the intrahypothalamic connections from the DMH to the medial part of the perifomical region
identified in the present investigation. which is heavily innervated by the PMV.
The impressive projections from the DMH to the PVP
form the major output channel for the dorsomedial nucleus in
the hypothalamus and is one of the most conspicuous find-
In previous anterograde tracing studies it was shown that ings of the present study.
the dorsal and ventral divisions of the pVMH have different P~ir,,oc,c~ilt4lrrr
p(r1.(1).(,ttt1.;(.111(1t.
tu~clrus. The existence ot
efferent connections [19, 20, 321. In the present investigation projections from the LHA, DMH and PMV to the paraven-
not only additional unreported projections of the dorsal and tricular nucleus was previously reported by Sawchenko and
ventral pVMH were found, but also differences in the Swanson 1351who employed combined anterograde and ret-
intrahypoth~amic afferents to the dorsal and ventral VMH. rograde transport methods. Sawchenko and Swanson j3.51,
Efferent fibers from the LHA, except from the ventromedial however. also identified retrogradely labeled cells in the
tLHA, and Tom the anterior DMH terminate in the dorsal VMH after true blue injections in the PVP. which was sub-
segment of the pVMH. Fibers from the ventromedial tLHA stantiated in the same study by anterograde tracing with
and from the PMV, on the other hand, are aimed at the “H-Leu after injections in the VMH. Such a VMH-PVN con-
ventral division of the pVMH. Another difference in the nection. however. could not be identified in our PHA-1, trac-
connectivity of dorsal and ventral segments of the posterior ing material. A large contingent of labeled efferent fibers
VMH was found with respect to the int~nucle~ projections from the VMH could he observed traversing but not actually
of the ventromedial nucleus. The ventral aspect of the terminating within the paraventricular nucleus (Fig. 12).
posterior VMH never received any input from other regions Terminal branches from these fibers were identified in the
of the ventromedial nucleus in contrast to the dorsal section areas that ventrally and dorsally surround the paraventricu-
of the pVMH. lar nucleus. An explanation for the differences between
Lateral hypothalamic area. Evidence for a monosynaptic PHA-I. tracing. on the one hand, and ‘IH-Leu and true blue
connection between the ventromedial and lateral nuclei of tracing, on the other hand. may be found in the large size of
the hypoth~~us was first obtained with neurophysiologica~ the injections that were used in the auto~~djographic and
methods [28]. Anatomical studies, employing the retrograde fluorescent tracing experiments. Tritiated leucine injections
transport of HRP, did not supply unanimous information on of the ventromedial nucleus, ah reported by Sawchenko and
the interrelationship between VMH and LHA. In two inves- Swanson 1351. extended into the ventromedial part of the
tigations [1,18] some positively reacting cells in the lateral [Link] which is a region in the hypothalamus that project5 to
region of the VMH were identified after injections of HRP in the pamventricular nucleus. Identical problems occur with
the LHA. Luiten and Room (231, however, were unable to the true blue injections employed by these authors. Deposits
show such connections of the VMH with the same retro- of the fluorescent dyes gave rise to diffusion of the tracer
grade tracing technique. Conclusive evidence for a VMH into the target areas of the ventromedial nucleus that are
projection to the LHA has neither been obtained with au- situated immediately ventral and dorsal to the paraventricu-
toradiographic tracing of tritiated amino acids [7, 19, 20, 32, lar hypothalamic nucleu\.
341. These anterograde tract-tracing studies demonstrate the
existence of two pathways of the VMH to the LHA, one
coursing in a dorsolateral and the other in a lateral direction.
It remains unclear, however, whether these efferent fibers The aim of the present study was to gain further under-
carry labeled terminals in the lateral hypothalamic area. In standing on the intrahypothalamic circuitry involved in the
the present investigation some terminal presynaptic boutons maintenance of homeostatic processes of the internal en-
from the VMH were found in the ventromedial aspect of the vironment. In recent years evidence has accumulated which
tuberal LHA. The ventromedial tLHA also receives input shows the participation of the lateral [14. 28, 29, 39, 401,
from the dorsomedial hypothalamic nucleus, an efferent ventromedial [4, 7, 28, 29, 401, dorsomedial [3-5, 471 and
connection of the DMH that was previously demonstrated paraventricular hypothal~ic nuclei Ill, 21, 22,421 in these
with retrograde tracing methods 118,231. control mechanisms. Pa~icu~arly the lateral and ventrome-
Studies employing retrograde transport of HRP [ 1, 18,231 dial nuclei have received overwhelming attention because of
and degeneration methods [2] present evidence for efferent their role in the autonomic regulation of pancreas hormone
projections of the PMV to the lateral hypothalamic area. The secretion and feeding [6, 9, 14, 37, 38, 39, 471. The lateral
INTRAHYPOTHALAMIC CONNECTIONS 201

FIG. 12. Camera lucida drawing of PHA-L labeled fibers in the PVN after a lectin injection in the VMH. Insets show
the injection in the VMH and the position of the PVN in the hypothalamus.

hypothalamic area controls pancreas hormone release pre- splanchnic nerve, depending on the position of the lesion
dominantly via the parasympathetic autonomic division, within the lateral hypothalamic area. In anatomical studies
whereas the ventromedial nucleus acts via the sympathetic the efferent connections from the hypothalamus were
chain of the autonomic nervous system. In preceding papers demonstrated to medullary parasympathetic preganglionic
we have used this autonomic control of hormone release cell groups which innervate the pancreas [24, 25, 31,41,45],
from the pancreas as a model, in order to discuss the func- and to spinal preganglionic neurons which participate in the
tion of the descending pathways of the hypothalamus in sympathetic innervation of the pancreas [24, 25, 411 and ad-
homeostatic control mechanisms in general. Pancreas hor- renal glands [12]. The LHA and DMH maintain direct and
mone release, however, is not only regulated by autonomic indirect connections to the dorsal motor vagus (DMnX) and
mechanisms and blood-borne parameters, but is also influ- ambiguus (AMB) nuclei, whereas the VMH has only indirect
enced by the neuroendocrine pathway. In this paper, there- projections to these medullary parasympathetic cell groups.
fore, we will also pay some attention to the role of the iden- Direct projections to the DMnX and AMB also originate
tified hypothalamic connectivity in the release of adrenal from the parvocellular paraventricular nucleus. Moreover,
hormones that reportedly modulate pancreas hormone secre- the PVP sends efferent fibers to the sympathetic cell groups
tion (e.g., corticosterone and catecholamines) [8, 13, 15,261. of the thoracic spinal cord as well [24,41]. These anatomical
The participation of the autonomic nervous system in the data show that at least four different descending pathways
hypothalamic control of hormone release from the pancreas can relay information from the hypothalamus to the pre-
was shown by Yoshimatsu and co-workers [47]. These au- ganglionic parasympathetic and sympathetic cells for the in-
thors demonstrated changes in the electrical activity in the nervation of pancreas and adrenals. The PVP is the only
splanchnic and vagal nerve branches to the pancreas as a hypothalamic nucleus that maintains direct connections to
result of hypothalamic lesions. In summary, bilateral de- both the medullary and spinal autonomic centers.
struction of the VMH gave rise to increasing vagal and de- Our anatomical data show that the main stream of con-
creasing splanchnic nerve activity. Similar effects occurred nectivity in the hypothalamus runs from the LHA and VMH,
in DMH and PVN lesioned rats, but bilateral destruction of via the DMH, to the paraventricular nucleus (Fig. 11). Thus,
the LHA reduced the neuronal activity in the vagal nerve the efferents of the PVN to the preganglionic parasympathe-
and produced either increased or decreased responses in the tic and sympathetic cell groups form an additional indirect
 202 ‘TER HORST AND LUITFN

connection from the LHA and VMH to the autonomic cen- tar) [ 16.361, enable integration of autonomic and neuroen
ters in the medulla oblongata and spinal cord. docrine responses. lntegration of autonomic and neuroen-
The present study also revealed direct interconnections docrine output may be a necessary prerequisite for a fine
between the LHA and VMH and scarce projections from the tuning of hormone release from pancreas and adrenal glands,
DMH to the LHA and VMH. These anatomical data weaken and for adaptation to different arousal states of the organism.
a presumed role for the dorsomedial nucleus in maintaining a In conclusion, PHA-L, trdcing showed that the main
balance in the output of the ventromedial and lateral hypo- stream of connections in the hypothalamus runs from the
thalamic nuclei [4, 23. 291. It seems more likely that the LHA and VMH via the DMH to the PVN and that the LHA
DMH can modulate the output of the paraventricular nucleus and VMH maintain direct reciprocal connections to each
by the strong efferent connection to the PVN. This way, the other (Fig. 11). It was argued that these intrahypothalamic
DMH may contribute to the balance and the integration of connections are involved in the transfer of information from
autonomic and neuroendocrine responses in metabolic ho- the LHA and VMH to the autonomic centers of the lower
meostasis. Tracing of PVN efferents with double labeling of brainstem and spinal cord.
retrogradely transported fluorescent markers showed that
the PVP contains separate populations of cells for the pro-
jections to DMnX and spinal cord [41]. Such an intranuclear ACKNOWLEDGEMENTS
organization of the PVP suggests that a balance between
The authors wish to thank Dr. 9. Bohus for his useful comments
sympathetic and parasympathetic autonomic mechanisms al- during the preparation of the manuscript and Mrs. Joke Poelstra fat
ready can be coordinated within the paraventricular nucleus. typing this paper. This work was supported by a grant from the
Furthermore, the connections between magnocellular and Foundation for Medical Research FUNGO (grant number 13-46-36)
parvocellular PVN, which contains the releasing factors for which is subsidized by the Dutch Organization for the Advancement
pituitary hormones (for example corticotropin-releasing fac- of Pure Research (ZWO).

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