Download Properties of ventromedial hypothalamic neurons with axons

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BrainResearch
Exp Brain Res (1982) 46:292-300
9 Springer-Verlag 1982
Properties of Ventromedial Hypothalamic Neurons
with Axons to Midbrain Central Gray*
Y. Sakuma ~ and D.W. Pfaff
The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
Summary. In female rats anesthetized with urethane,
151 neurons in and around the ventromedial nucleus
of the hypothalamus were identified by antidromic
activation as having axonal projection to the
mesencephalic central gray at the midcollicular level.
Identified neurons were most numerous in the rostral
part and at the borders of the nucleus.
Antidromic spike latencies, constant for a given
cell to stimulation with fixed intensity at a low
repetition rate, had a wide range across cells
(1.4-41.5 ms). In 37 cells, gradual increases in
stimulus intensity allowed sudden discrete latency
decreases as large as 9.8 ms. These may reflect
activation of separate axonal branches of terminal
arborizations.
Eleven among 43 tested cells were antidromically
driven from the dorsal longitudinal fasciculus (DLF)
at the diencephalic-mesencephalic junction as well as
from the central gray. Latencies of DLF responses
were always shorter than those from central gray.
From this and collision experiments between central
gray-evoked and DLF-evoked antidromic spikes, it
was concluded that at least one quarter of mesencephalic projections from the ventromedial nucleus
descend through DLF. The mean conduction velocity
of these axons was 0.8 m/s, indicating that they
belong to thin unmyelinated C-group fibers.
Thirty percent of the cell population studied
received excitatory input from the cortical or medial
nucleus of the amygdala. Four cells were identified as
having projections both to the central gray and t h e
amygdala.
* Supported by NIH grant HD-05751 and by institutional grant
from the Rockefeller Foundation for the study of reproductive
biology
1 Present address: Dept. of Physiology, Niigata University School
of Medicine, Niigata 951, Japan
Offprint requests to: D.W. Pfaff (address see above)
Estrogen treatment of ovariectomized female rats
caused no major changes in antidromic latency,
absolute refractory period or resting activity of these
identified hypothalamic neurons. However, the
stimulation threshold for antidromic activation was
significantly lower in the estrogen-treated animals.
Axons to the central gray from ventromedial
hypothalamic neurons provide for hypothalamic bias
on brain stem reflex paths, for reproductive and
other behaviors.
Key words: Ventromedial nucleus - Hypothalamus Antidromic activation - Central gray - Midbrain Amygdala
Introduction
The ventromedial nucleus of the hypothalamus
(VMN) is important for the control of several
autonomic and behavioral activities (Clemente and
Chase 1973; Shimazu and Ogasawara 1975). Among
these, the lordosis reflex in estrogen-primed female
rodents depends on VMN (Pfaff and Sakuma 1979a,
b), an effect mediated by projections descending to
the dorsal midbrain including the central gray (CG).
Deficits in lordosis follow knife cuts in the trajectories of VMN-CG projections (Manogue 1979).
VMN output apparently biases activity in reflex paths
for lordosis completed at or below the mesencephaIon (Sakuma and Pfaff 1979a, b). Extracellular
recording in female rats revealed that electrical
stimulation of VMN can enhance electrical excitability in CG neurons (Sakuma and Pfaff 1980a, b),
probably via heavy, direct descending projections
(Krieger et al, 1979). Thus, in the present experiments we aimed to characterize VMN neurons with
axons projecting directly to the CG.
O014-4819/82/0046/0292/$ 1.80
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
Methods
Surgical Preparation
Experiments were carried out on 26 adult ovariectomized SpragueDawley female rats. The animals weighed between 275 and 410 g.
Thirteen of 26 rats were used without replacement therapy for the
ovariectomy; the other 13 were implanted with an estrogen pellet
(Progynon, Schering), subcutaneously 10-14 days before recording. Before they were anesthetized for surgical procedures, all of
the estrogen-treated rats showed a strong lordosis reflex to
pressure on rump-tail base-perineum skin, while none of the
untreated animals did.
Surgery was performed under light urethane anesthesia (1.2 g/
kg body weight, given intraperitoneally in 0.5 g/ml solution). The
animal was tracheotomized and put in a stereotaxic frame in a
prone position, with the incisor bar put 5 mm above the center of
the ear bars. Craniotomy was made in the parietal area and the
dura was removed. The exposed cerebral cortex was covered with
warm agar solution in saline (4%). Rectal temperature was
maintained between 35 and 38~ C. Electroencephalogram (EEG)
was recorded from the frontal cortex through a pair of stainless
steel screw electrodes on the dura, and the electrocardiogram was
monitored throughout the recording session.
Coaxial bipolar electrodes were constructed from 30 gauge
hypodermic tubing and stainless steel wire 120 am in diameter.
Except at the tip, electrodes were insulated with Epoxilite 6001
and had a DC resistance of approximately 75 KfL These electrodes
were used in the CG to stimulate axons from VMN cells
antidromically. In 19 animals an additional electrode was placed in
the cortical or medial nucleus of the amygdala (AMY); 11 animals
had another electrode in the dorsal longitudinal fasciculus at the
diencephalic-mesencephalic junction (DLF). Stereotaxic coordinates were: 5.8 mm posterior to the bregma (P), 0.7 mm lateral to
the midline (L), and 4.5 mm below the surface of the dura (D) for
the CG; for the AMY, P 1.0 mm, L 3.5 mm, D 8.5 mm, and for the
DLF, P 3.0 ram, L 0.2 mm, D 6.3 mm. These electrodes were
inserted into the brain through holes in the skull and dura and
fixed to the skull with dental cement.
Stimulation and Recording Procedures
The VMN was systematically explored while biphasic pulses to the
CG were applied at 0.5 Hz. For the reduction of stimulus artifact,
each negative rectangular pulse (0.2 ms duration) was followed by
a positive pulse of 0.1 ms duration. By adjusting the intensity of
the second pulse, artifact caused by polarization of the electrode
tip could be minimized. Current intensity of the pulse was
monitored on an oscilloscope with differential inputs as a voltage
across a calibrated 500 ~ resistor in the stimulation circuit. Intensity
of the current is referred to in terms of the leading, negative pulse
of each biphasic pulse-pair, and was in a range of 750-1,000 tta.
Although the positive pulse compensation current practically does
not change the efficacy of the leading, stimulating pulse (Asanuma
et al. 1976), negative monophasic pulses were delivered when
determining the threshold of each cell for antidromic activation.
Electrical stimulation with above parameters did not desynchronize the EEG nor did it produce body movement.
Recordings of extracellular potentials were by glass
micropipettes constructed from Pyrex tubes of 2 mm outer
diameter and filled with 0.5 M sodium acetate solution. Pontamine
Sky Blue 6BX was added to make a 2% solution of the dye, to
allow marking of recording sites by iontophoretic ejection. The
DC resistance of the pipette was 10-30 MQ, 20 MQ being an optimal
value. Potentials were recorded between the electrode and a
293
chlorided silver plate under the skin in the temporal area.
Extracellular potentials were amplified and displayed with conventional methods, with a bandpass of 300-10,000 Hz. Microelectrode
tracks were made in a series in the transverse planes approximately
0.2 mm posterior to the bregma. Five or six electrode penetrations, with a separation of 100 or 200 ~m, were usually made in
each preparation.
Histological Procedures
Following completion of each penetration which had identified
cells, cathodal (electrode negative) current of 3-5 ~xa was passed
via the micropipette at two points in each track, at the deepest and
the shallowest points with antidromic potentials. The dye deposit
thus produced was about 100 ~tm in diameter and could be readily
recognized during histological processing. At the termination of
each experiment, the animal was given an overdose of Nembutal
and perfused through the heart with 10% formalin. The brain was
removed and fixed in formalin, and frozen serial sections (100 ~tm)
were made in the frontal plane. The sections which contained dye
spots were stained with cresyl violet, and midbrain sections with
lesions caused by penetration of stimulation electrodes were
stained with luxol fast blue and cresyl violet. Recording sites were
determined in reference to the dye spots, and stimulation sites
were identified from their electrode tracks.
Results
Identification of Cells
A t o t a l o f 151 n e u r o n s w i t h m e s e n c e p h a l i c p r o j e c t i o n
w e r e i d e n t i f i e d in o r a d j a c e n t to t h e v e n t r o m e d i a l
n u c l e u s o f t h e h y p o t h a l a m u s ( V M N ) in 26 rats.
Identification was made by antidromic activation
f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n in t h e m e s e n c e p h a l i c
c e n t r a l g r a y at m i d c o l l i c u l a r l e v e l s ( C G ) . F e a t u r e s o f
a n t i d r o m i c p o t e n t i a l s a r e i l l u s t r a t e d in Fig. 1. T h e
responses had discrete, all-or-none thresholds. The
antidromic spike latency was constant when the
s t i m u l a t i o n was g i v e n w i t h f i x e d s u p r a t h r e s h o l d
i n t e n s i t y at a l o w r e p e t i t i o n r a t e o f 0:5 H z . O f
151 cells, 58 ( 3 8 . 4 % ) h a d a n a n t i d r o m i c s t i m u l u s
t h r e s h o l d o f less t h a n 200 ~a, w h i l e 77 ( 5 1 . 0 % ) h a d
t h r e s h o l d s b e t w e e n 200 a n d 750 ~a.
Antidromic Action Potentials
The waveform of the antidromic action potentials
w a s b i p h a s i c , w i t h t h e initial d e f l e c t i o n p o s i t i v e w i t h
r e s p e c t to a n i m a l g r o u n d . P e a k - t o - p e a k a m p l i t u d e o f
t h e a n t i d r o m i c a c t i o n p o t e n t i a l s w a s n o less t h a n
0.6 m V , a n d m o s t w e r e in t h e r a n g e o f 2 - 5 i n V .
T h e l a r g e s t a m p l i t u d e o b s e r v e d in this s t u d y w a s
14.0 m V . A n i n f l e c t i o n w a s s e e n in t h e rising p h a s e
o f 38 a n t i d r o m i c r e s p o n s e s . U n l i k e t h o s e r e c o r d e d
f r o m C G cells in r e s p o n s e to m e d u l l a r y s t i m u l a t i o n
( S a k u m a a n d P f a f f 1980b, c), t h e p r o p a g a t i o n o f
294
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
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Fig. 1A, B. Antidromic action potentials evoked by central gray
stimulation. A Cell 293-8. C ) response to suprathreshold stimuli at
a repetition rate of 0.5 Hz; (~) response to paired pulse stimuli
(arrows) at an interval of 2.0 ms; (~) blockage of antidromic
response by spontaneously generated spike (arrow head, T).
Antidromic response to the second pulse ~ f t e r an interval of 5.0
ms) showed that the neuron was not lost; 4~)response to triggered
stimuli with a delay larger than the critical value was not blocked
by the spontaneous action potential. Repetition rates of antidromic stimuli are indicated in trace A ( ~ ) - ( ~ ) . Note the
fluctuation of latency at or above repetition rate of 10 Hz. An
arrow head in (~) indicates the response at the onset of the
repetitive stimulation. One-to-one relation between the stimulus
pulse and response was not preserved when the repetition rate
exceeded 50 Hz. B Antidromic responses from two cells were
recorded simultaneously, indicating high density of cells in the
ventromedial hypothalamus with central gray projection. The
neuron with the larger potential had spontaneous activity, and
blockage by collision of the orthodromic (V) with the antidromic
response is shown (B_(~)). All calibrations are 2 ms and 0.5 mV.
Calibrations in A ~ are common for the rest of traces in A.
Except for A C ) - ~ ) , all traces are superimposed five times
antidromic action potential into the somatodendritic
complex was rarely blocked in these VMN cells.
Antidromically-evoked potentials were cancelled
by collision with spontaneously or orthodromically
generated spikes. When these spikes were used to
trigger an antidromic stimulus, the critical delay at
which the triggered stimulus was effective approximated the value of the antidromic latency plus 0.5
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500
1,000
Stimulus intensity (pa)
Fig. 2. A An antidromic response that showed discrete shifts in
latency according to stimulus intensity. Numbers accompanying
each trace denote stimulus intensity in ~a. B Discrete nature of
changes in antidromic spike latency as a function of stimulus
intensity is illustrated for six cells
ms. With shorter delay, antidromic responses were
always absent. The extra 0.5 ms is presumed to be the
refractory period of the axon at the site of stimulation. Absence of spontaneous or orthodromic activity
in 106 (70.2%) of these cells precluded the ability to
observe cancellation of the antidromic action potential. In such cells, the presence of constant latency
spikes in response to suprathreshold stimuli, and the
ability for these neurons to follow paired stimuli at
short intervals (2-3 ms) were considered to indicate
their antidromic nature.
Variations in Antidromic Spike Latencies
Across cells, latencies for antidromic responses
ranged from 1.4 to 41.5 ms (mean 13.3 ms + 6.8
S.D., Standard Deviation) at threshold. For a given
cell, alterations in stimulus intensity, repetition rate
in stimuli trains or interstimulus interval of paired
stimuli could cause two types of latency variation. In
37 of 151 antidromic responses, gradual increases in
the antidromic stimulus intensity induced two to four
discrete shortenings, "latency jumps", in the antidromic spike latencies (Fig. 2). Inversely, gradual
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
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Fig. 3. A Antidromic response in a neuron with an absolute
refractory period of 1.3 ms. Interspike intervals remained constant
at 2.4 ms, despite reductions in the interstimulus intervals down to
1.3 ms. Arrow heads: stimulus artifacts. B Relations between
interstimulus intervals and antidromic interspike intervals for
seven cells
decrease caused discrete prolongations in the latencies. However, at any constant suprathreshold intensity, the latency was constant to stimuli delivered at
0.5 Hz. Discrete reductions in antidromic spike
latency were between 0.1 and 9.8 ms (mean 1.9 ms),
and could be induced by changes in the antidromic
stimulus as small as 10 ~a.
In another type of latency variation (Fig. 3), if
stimulus pairs were presented every 2 s, across
interstimulus intervals of 5-20 ms some V M N cells
responded to the second stimulus with reduced
latencies. As the interstimulus interval was gradually
shortened into the absolute refractory period, an
increase in the antidromic spike latency to the second
shock resulted. Fluctuations in antidromic spike
latency thus induced ranged f r o m 0.2 to 2 ms.
Histological Localization
Locations of 151 antidromically activated cell bodies
are shown in Fig. 4 in drawings of frontal sections
through the medial basal hypothalamus. Stimulation
sites in the C G are also given. In addition to the
V M N , some cells are located in the retrochiasmatic
and periventricular areas of the hypothalamus,
anterior hypothalamus, paraventricular nucleus, and
dorsomedial nucleus. The largest population (n = 76)
was found within the boundary of the VMN.
Fig. 4. A Locations of 151 cells in and around the ventromedial
nucleus of the hypothalamus (VMN) that were antidromically
activated from the central gray of the mesencephalon (CG).
B Stimulation sites are indicated. Numbers accompanying
sketches of frontal sections of the hypothalamus and brainstem
denote distances from the bregma in mm. Abbreviations. AHA,
anterior hypothalamus; ARC, arcuate nucleus; DMN, dorsomedial nucleus; FX, fornix; IC, inferior colliculus; MLF, medial
longitudinal fasciculus; OT, optic tract; PVA, periventricular area;
PVN, paraventricular nucleus; RCA, retrochiasmatic area; SC,
superior colliculus; SCP, superior cerebellar peduncle; V3, third
ventricle; III, oculomotor nucleus; Vm, mesencephalic trigeminal
nucleus
Within the V M N nuclear boundary, a cluster of
antidromically-driven cells is located at the rostral tip
of VMN. Several cells were noted at the shell of the
nucleus.
The high density of the cells with C G projection
in the rostral part of V M N is exemplified by an
observation that, in one preparation, all ten cells
encountered during 438 pm advancement of the
electrode responded antidromically to the C G stimulation, with distances between cells of 21-99 btm.
Descending Projection Through
Dorsal Longitudinal Fasciculus
Forty-three neurons with C G projections were tested
by electrical stimulation of the dorsal longitudinal
fasciculus (DLF) at the diencephalic-mesencephalic
junction (Fig. 5). Eleven (25.6%) of them were
antidromically activated from the D L F with a latency
of 1.4-8.0 ms, as well as from the CG.
Three types of observations indicated that D L F
stimulation activated axons of V M N cells passing
through the D L F on their way to the CG. First,
antidromic spike tatencies from the D L F were always
shorter than those from the C G (mean difference 4.6
ms). Second, in no cells did changes in the intensity
of a D L F stimulus cause latency jumps (an indication
of terminal arborizations, see Discussion). Third,
296
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
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antidromic response latencies from these two sites.
This was seen in all of the eight cells tested, showing
that the action potential evoked by DLF stimulation
collided with the CG-evoked potential (Fig. 5).
When the DLF stimulation followed the CG shocks
with delays approximating the value of the latency
difference, DLF responses were blocked and only the
CG stimulation yielded an antidromic response. In
this time sequence, the CG-evoked potential passed
down the DLF site just prior to the DLF stimulation,
and the refractory period following the CG-evoked
excitation caused failure of the DLF response.
In eight cells which were antidromically activated
from both the CG and DLF, the conduction time
between these two points (for a distance of 3.3 ram)
ranged from 1.4 to 9.5 ms. Thus, the conduction
velocity in the axons of these cells did not exceed
2.4 m/s, indicating that these VMN axons are thin
unmyelinated C-group fibers.
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Transsynaptic Inputs from Amygdala
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Fig. 5. A Recordings from a cell which showed antidromic
responses to central gray (C or CG) and dorsal longitudinal
fasciculus (D or DLF) stimulation. Numbers accompanying each
trace denote interstimulus intervals in ms. When the interval was
shorter than the absolute value of the difference in antidromic
latencies from the two sites, one of the two responses was blocked.
B Relation between the interstimulus interval and the blockage of
antidromic responses (indicated by half-open circles) is shown for
eight cells. Broken lines demarcate the area in which the interstimulus intervals are shorter than the differences in the antidromic spike latencies. C Nine stimulation sites in the DLF, from
which 11 hypothalamic cells with CG projections were antidromically activated. Number in each panel denotes distance from the
bregma in mm. Abbreviations. FR, fasciculus retroflexus; H,
habenula; IP, interpeduncular nucleus; LM, medial lemniscus;
MTT, mammillothalamic tract; PC, posterior commissure; SM,
stria medullaris; SN, substantia nigra; V, ventricle
and most decisive, was the blockage of CG responses
when the interstimulus intervals between the CG and
DLF shocks were shorter than the difference in
Forty-seven cells with CG projections were tested
with electrical stimulation in the medial or cortical
nucleus of the amygdala (AMY) at 0.5 Hz (Fig. 6).
Fourteen (29.8%) responded to AMY stimulation
with orthodromic excitation. Current intensity of
effective stimulation was between 160 and 400 va.
Stimulation with these parameters did not induce
EEG seizure in the frontal cortex. The mean latency
of the initial onset of excitatory responses was 19.0 +_
9.7 (S.D.) ms, with a range of 8.3-34.4 ms. The
latency in each facilitation fluctuated over a range of
1.5-7.8 ms, when the stimulation was repeated at 0.5
Hz for 15 s. A sustained increase in discharge rate
could be induced in this group of cells, when AMY
stimulation was given at 10 Hz. Analysis of AMY
effects on VMN in the cat by means of evoked
potentials suggested a role for interneurons at the
lateral edge of VMN (Murphy and Renaud 1969).
Identified tuberoinfundibular neurons also have been
proven to respond to amygdala stimulation (Renaud
1976a).
Simultaneous Projections to CG and A M Y
Antidromic responses to AMY stimulation were
obtained in 4 among 47 cells with CG projection. In
three cells, antidromic spike latencies from the CG
were longer than those from the AMY (mean difference 8.3 ms), and in one cell the latency of the AMY
response was 11.7 ms longer than that from the CG.
The antidromic action potentials evoked in these
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
297
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Fig. 6. A Ranges of latency variations in the transsynaptic responses of ten cells to amygdalar (AMY) stimulation are indicated by open
bars. Except for three cells in the anterior hypothalamus (AHA) or in periventricular hypothalamus (PVA), all were located in the
ventromedial nucleus. B An example of transsynaptic response to AMY stimulation. C Antidromic response to the central gray stimulation
(1) and its blockade (2) by an orthodromically-evoked spike (arrow head 9 in 2, 3) for the cell shown in B. D AMY stimulation at 10 Hz
(solid bars, with stimulus intensity indicated) induced sustained discharges in the same cell when delivered at 200 ~a for 25 s. The activation
lasted for 60 s after the end of stimulation
cells collided when A M Y stimulation followed or
preceded C G stimulation within a range shorter than
the sum of antidromic spike latencies from the two
sites in each cell. As the intervals between the C G
and A M Y stimulations were shortened from this
value, either of two responses was blocked. This was
seen when the absolute value of interstimulus intervals was less than the summed values of the latencies,
regardless of the order of the stimuli (minus sign at
the ordinate in Fig. 7 indicates that A M Y stimuli
precede to C G stimuli). We expected the neuronal
soma to be located at some distance from the point
where the main axon bifurcates into the two branches
directed to the C G and AMY. This hypothesis was
tested by applying the following equation to evaluate
the conduction time (t) between the neuronal soma
and the branching point of the parent axon (Shinoda
et al. 1977; Ferreyra Moyano and Mo!ina 1980): t =
(Lc + LA - dmax + R)/2; where Lc and L A represent
antidromic spike latencies from the C G and AMY,
r e s p e c t i v e l y ; dmax, the maximal interstimulus interval
for cancellation of the response with the shorter
latency; R, the refractory period of the axon at the
stimulation site on the shorter branch of the two
(presumed to be 0.5 ms). The calculated values of t
are between 1.9 and 2.9 ms.
Somatosensory Stimulation
During recording from all cells, somatosensory stimuli were tried, to look for unit responses, including
cutaneous stimuli which would trigger lordosis
behavior in awake females. VMN cells without
resting discharge were not activated by somatosensory input. Ceils with spontaneous activity did not
show prompt or specific responses to lordosis-relevant stimuli, even when they responded well to trains
of AMY stimuli.
Estrogenic Effects
The 151 antidromic responses were made up of 77
responses from ovariectomized estrogen-treated ani-
298
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Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
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Fig. 7. A Responses of a cell which was activated antidromically
from the amygdala (A) as well as from the central gray (C).
Numbers accompanying each trace (superimposed five times)
denote intervals between the two stimuli in ms. When the
interstimulus interval was shorter than the sum of the values of
antidromic spike latencies from two sites, one of the two responses
was blocked. B Relation between the interstimulus interval and
the blockage of antidromic responses (indicated by half-open
circles) is shown for four cells. Broken lines demarcate the area in
which the interstimulus intervals are shorter than the sum of
antidromic spike latencies from the two sites. C Fifteen stimulation
sites in the cortical (CO) and medial (M) nuclei of the amygdala
(AMY), from which four antidromic responses were obtained, in
addition to orthodromic responses (shown in Fig. 6), by
hypothalamic ceUs with central gray projections. Number in each
panel denotes distance from bregma in mm. Abbreviations. B,
basal complex; CE, central nucleus; CL, daustrum; LV, lateral
ventricle; OT, optic tract
mals and 74 responses from ovariectomized untreated control preparations. No differences were found
between these two types of preparations in mean
antidromic spike latencies. Comparisons between the
two types of preparations were also made o n
thresholds for antidromic activation, absolute refractory period, and spontaneous activity (Table 1). A
statistically significant difference between the two
The present electrophysiological study demonstrates
that electrical stimulation of the CG elicits antidromic responses in VMN neurons. Antidromic
responses were recorded as well from neurons in the
retrochiasmatic and anterior hypothalamic areas,
dorsomedial and paraventricular nuclei of the
hypothalamus. This is evidence that neurons in these
structures give rise to direct descending projections
to the stimulated sites in the CG. The stimulation
points correspond to the course of descending axons
indicated by morphological studies utilizing tritiated
amino acid autoradiography (Krieger et al. 1979).
Injection of horseradish peroxidase in the CG produced retrogradely labelled cells in the VMN and
other hypothalamic nuclei, showing that axons of
these cells terminate in the CG (Morrell et al. 1978).
Antidromic spike latencies were constant when
the CG stimulation was given at a fixed, suprathreshold intensity, at a low repetition rate of 0.5 Hz.
Under certain circumstances, such as varying intensity or high repetition rate of the antidromic stimuli,
latencies varied (Mayer 1979; MacLeod and Mayer
1980). The "latency jump" (Mayer 1979), a discrete
shift in antidromic spike latencies in response to
gradual changes in the stimulus intensity, was seen in
about 25% of antidromic responses in the present
study. Such latency jumps have been observed in the
spinothalamic tract cells (Price et al. 1978), medial
preoptic cells with CG projection (MacLeod and
Mayer 1980), and CG cells with medullary projection
(Sakuma and Pfaff 1980c). These observations are
interpreted to suggest either that the axon pursues a
tortuous course through the stimulated site (Cross et
al. 1975) or that there are terminal arborizations of
axons or axon collaterals with different thresholds for
activation due to their membrane characteristics or
their distance from the stimulation site (Barker et
al. 1971; Renaud 1976b; Sakuma and Pfaff 1980c).
Anatomical descriptions confirm that mesencephalic
projections from the hypothalamus do indeed form
terminals in the CG (Conrad and Pfaff 1976a, b;
Krieger et al. 1979). Thus the latency jumps in the
antidromic responses from the VMN may be additional evidence that the axons of these neurons
terminate in the CG. It is noteworthy that none of
the neurons identified antidromically in the paraventricular nucleus showed a latency jump. Anatomical
studies have traced descending axons of this nucleus
through the CG to the spinal cord (Hancock 1976;
Hosoya 1980).
Y. Sakuma and D.W. Pfaff: HypothalamicProjections to Central Gray
299
Table 1. Comparisonsbetween single unit recordings from control ovariectomized(OVX) and estrogen-treated (OVX + E) rats
OVX
OVX+ E
Antidromic
latency, ms
Threshold*,
~ta
Refractory
periodc, ms
No. of active
cells
Mean discharge
rated, Hz
13.3 +7.1 a
(74)b
13.3 + 6.6
(77)
489 + 311
(63)
264 + 167
(67)
2.1 + 1.1
(22)
1.7 + 0.8
(32)
23
(74)
22
(77)
1.2 + 3.2
(23)
1.7 + 4.0
(22)
standard deviations; b number of observations; c absolute refractoryperiod; d mean dischargerate of cells with spontaneous discharges
* P < 0.001, one way analysisof variance
The reduction in the latency of response to the
second pulse of a pair was noted when interpulse
intervals were between 10 and 200 ms (Bliss and
Rosenberg 1974; Gardner-Medwin 1972). This reduction is caused by supernormal conduction in
axons, which has been associated with activation of a
metabolic pump (Bliss and Rosenberg 1974), or
changes in the extracellular environment (GardnerMedwin 1972). A causal relation between the supernormality and a dense package of thin unmyelinated
axons which may be activated synchronously by
electrical stimulation, is suggested for cerebellar
parallel fibers (Gardner-Medwin 1972). In contrast
to the supernormal conduction, a prolonged latency
of response was observed to the second of a pair of
pulses at intervals close to the refractory period (less
than 3 ms in the present experiments). Similar
observations were reported on antidromic responses
of hypothalamic cells to median eminence stimulation (Renaud 1976b).
Pathways taken by projections from VMN to CG
include the periventricular and the supraoptic commissure systems (Krieger et al. 1979). The present
observation that eleven among 43 cells were antidromically invaded following stimulation of both the
CG and the D L F suggests that in at least a quarter of
VMN cells with CG projection, axons descend in the
DLF, the caudal continuation of the periventricular
system. When D L F stimuli followed the CG shocks
with delays shorter than the difference between the
antidromic spike latencies from the two sites,
responses to the CG stimulation were always
blocked. This indicated that both stimuli activated
the parent axon of the VMN neuron, which
descended in the D L F and terminated in the CG.
A strong facilitatory transsynaptic input from the
AMY was demonstrated in about 30% of the neurons
which were antidromically invaded from the CG. The
variation in latency of each transsynaptic response
was as small as 1.5 ms, implying that the responses
are mono- or oligosynaptic. It can be concluded that
there exists a neural circuit which carries descending
impulses from the A M Y to the CG via the VMN. It
is of interest that estradiol-concentrating cells are
found in all of these three structures (Pfaff and
Keiner 1973).
We have shown that electrical stimulation of the
VMN had facilitatory effects on the lordosis reflex of
the female rat, an essential element in female
copulatory behavior (Pfaff and Sakuma 1979a). Subsequent demonstration of an immediate, large facilitation of the lordosis reflex from the CG (Sakuma
and Pfaff 1979a) prompted us to speculate that the
VMN may exert an estrogen-dependent tonic bias on
the reflex circuitry for lordosis in the CG (Sakuma
and Pfaff 1979b). The VMN neurons identified in the
present study could include the neural substrate for
the VMN-induced behavioral activation. Certain preoptic and hypothalamic perykarya contain luteinizing
hormone-releasing hormone (LH-RH) (Barry 1979;
Silverman et al. 1979), and dense networks of LHR H immunoreactive fibers were traced caudally to
the CG (Lipositz and Srt~il6 1980). Facilitation of the
lordosis reflex by infusion of L H - R H into the CG
(Sakuma and Pfaff 1980d) raises the idea that preoptic or hypothalamic axons release L H - R H in the CG
when excited, and are related to the VMN-induced
facilitation of lordosis. Estrogen supplement to the
ovariectomized female rats is known to increase
hypothalamic content of L H - R H (Araki et al. 1975).
The resting discharge rates of these antidromically-identified cells were not influenced by estrogen.
There are estrogen-concentrating cells in VMN
(although not so many in the anterior subdivision) of
the rat (Pfaff and Keiner 1973) and other species
(Morrell et al. 1975; Rees et al. 1980). Also, recording from cells not antidromically identified, Bueno
and Pfaff (1976) found increases in single unit activity
due to estrogen, analogous to Dyer's diestrus-proestrus comparison (1973). Either estrogen-concentrating and estrogen-sensitive cells were too small a
fraction of our antidromically identified neuron sample for us to show an effect on mean discharge rate,
or the Bueno and Pfaff results were due to cells not
projecting to CG. If the latter, we must consider the
hypothesis that estrogen influences transmitted from
hypothalamus to midbrain do not depend entirely on
estrogen-altered electrical activity but may also rely
300
Y. Sakuma and D.W. Pfaff: Hypothalamic Projections to Central Gray
on estrogen-altered biosynthesis and transport ot
modulatory substances such as LH-RH and other
peptides.
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Received June 24, 1981