1Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194; 2Torii Nutrient-stasis Project, Exploratory Research for Advanced Technology, Research Development Corporation of Japan, Technowave 100, Kanagawa-ku, Yokohama 221-0031; and 3Basic Research Laboratories, Central Research Laboratories, Ajinomoto Company, Kawasaki-ku, Kawasaki 210-8681, Japan
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ABSTRACT |
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Tamura, Ryoi,
Takashi Kondoh,
Taketoshi Ono,
Hisao Nishijo, and
Kunio Torii.
Effects of Repeated Cold Stress on Activity of Hypothalamic
Neurons in Rats During Performance of Operant Licking Task.
J. Neurophysiol. 84: 2844-2858, 2000.
The present
study investigated the effects of repeated cold stress on single neuron
activity in the lateral hypothalamic area (LHA) and medial hypothalamic
area (MHA) of behaving rats. The rats were trained to lick a protruding
spout in response to one of several cue-tone stimuli (CTSs) to ingest
water, or amino acid, NaCl or glucose solution. Following this
training, the rats were raised under either stressed (repeated
temperature changes between 3 and 24°C) or control (24°C)
condition for 2 mo. During this period, neuronal activity was recorded
in the LHA and MHA. For rats raised under the stressed condition, mean
spontaneous firing rate of LHA neurons was significantly greater than
for rats under the control condition. More LHA neurons in the stressed
rats responded, with an accompanying decrease in activity (inhibitory
response), to CTSs than in the control rats. During extinction
learning, some LHA neurons enhanced or reversed the responses to CTSs
in the stressed rats, whereas no LHA neurons showed such response changes in the control rats. In contrast to the effects of the stressed
condition on LHA neuron activity, mean spontaneous firing rate of MHA
neurons in the stressed rats was significantly smaller than in the
control rats. Fewer MHA neurons in the stressed rats responded to CTSs
and/or ingestion of sapid solutions. The preceding results suggested
that repeated cold stress produces a specific pattern of changes in
spontaneous activity and responses to sensory stimuli in LHA and MHA
neurons; this could underlie the behavioral changes induced by repeated
cold stress such as hyperphagia and hyper-reactivity to sensory stimuli.
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INTRODUCTION |
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Repeated cold stress is a type
of chronic cold stress in which environmental temperature changes
rapidly and frequently several times within a day (Hata et al.
1984a). Animals exposed to chronic cold stress display various
abnormalities including hyperphagia, decrease in weight gain,
hyperalgesia, low blood pressure, decreased acetylcholine (ACh)
response in the isolated duodenum, suppression of antibody formation,
hypersensitivity in the immediate immunoreaction, and alteration in
hypothalamo-pituitary-adrenal and sympathoadrenal systems (Akana
and Dallman 1997
; Bing et al. 1998
;
Fukuhara et al. 1996a
,b
; Hanson et al.
1996
; Hata et al. 1984a
,b
, 1988a
;
Kawanishi et al. 1997
; Kondoh et al.
1996
; Kvetnansky et al. 1995
; Leung and
Horwitz 1976
; Morrison 1981
; Namimatsu et
al. 1992
; Okano et al. 1993
; Snyder and
Stricker 1985
).
The hypothalamus, which is a center of motivational behaviors as well
as autonomic, hormonal, and immune responses, is considered to be one
of the most important brain areas in producing various symptoms under
stressed conditions. In repeated cold stress, monoamine and ACh levels
in the hypothalamus change more rapidly than in other brain areas
(Hata et al. 1987a); interleukin-1
(IL-1
) gene expression is enhanced in the medial hypothalamic area (MHA) and
suppressed in the lateral hypothalamic area (LHA) during repeated cold
stress (Tagoh et al. 1995
). Stress-induced changes in
cytokine and neurotransmitter levels in the hypothalamus likely produce changes in neuronal activity in the hypothalamus. However, the effects
of repeated cold stress on neuronal activity in the hypothalamus have
not yet been examined in vivo.
When investigating the neuronal mechanism involved in
motivational behaviors, we believe it is important to examine changes in neuronal activity recorded in awake, behavior performing animals. Therefore we have developed an experimental system for awake rats and
several behavioral tasks including a cue-tone discrimination task
(Nakamura and Ono 1986; Nishijo and Norgren 1990
,
1991
; Ono et al. 1985
, 1986
). The cue-tone
discrimination task requires the rat to discriminate different cue-tone
stimuli (CTSs) that indicate different sapid solutions and to lick a
protruding spout. This method (the experimental system and task) has
allowed us to link the activity of neurons in the hypothalamus and
other brain regions to ongoing consummatory behaviors and to stimuli that predict the availability of gustatory rewards (Muramoto et al. 1993
; Nakamura and Ono 1986
; Nishijo
et al. 1998
; Ono et al. 1986
; Oyoshi et
al. 1996
; Takenouchi et al. 1999
; Uwano
et al. 1995
; Yonemori et al. 2000
). By testing
responses to a variety of different taste solutions, it is possible to
determine whether neuronal responses to licking (and/or associated cue
tones) are generalized across different taste stimuli, i.e.,
nondifferential responses to all the test stimuli, or are linked to a
particular class of tastant, i.e., differential (or specific) responses
to one or some of the stimuli. Indeed, using this method, we have found
both nondifferential neurons and differential neurons in the
hypothalamic areas involved in the control of food intake (Tabuchi et al. 1991
). Furthermore the method can also
be used to identify effects of a particular pathological condition on neuronal activity (Tabuchi et al. 1991
). A pathological
condition could affect food intake in a generalized way such as
hyperphagia or anorexia, in a specific way such as allotriophagy or
selective taste aversion, or in a way that mixes the two. Such an
effect of pathological condition on food intake may be reflected as
changes in population of hypothalamic neurons. For example, deficiency of a requisite amino acid (lysine), leading to a specific preference for this amino acid (Torii 1987
), increases the number
of LHA neurons that respond specifically to lysine solution and/or its associated CTS in rats (Tabuchi et al. 1991
).
Although some behavioral studies have reported that repeated cold
stress produces abnormalities in food intake such as hyperphagia and a
specific preference for histidine solution (Hata et al. 1988; Kita et al. 1979
; Kondoh et al.
1996
), little is known, at neuronal level, as to how repeated
cold stress affects the CNS that is involved in the control of food
intake (such as the LHA, MHA, and paraventricular nucleus). In the
present study, therefore we addressed this issue as the principal
purpose, using the method described in the preceding text. As an
additional purpose, we also investigated the effect of repeated cold
stress on plastic changes of hypothalamic neurons accompanied with
learning process because repeated cold stress is known to produce
impairment in learning (Yago et al. 1992
; Yoneda
et al. 1992
). For these goals, rats were raised under a control
or stress condition for 2 mo. Single neuronal activity was recorded
from the LHA and MHA in these rats during the performance of the
cue-tone discrimination task, and changes in neuronal activity were
compared between these two conditions.
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METHODS |
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Subjects
Thirty male albino Wistar rats (SLC, Hamamatsu, Japan), weighing
200-220 g at the beginning of the experiments, were used. The rats
were divided into two groups: control (n = 15) and
stressed (n = 15). Each rat was housed individually in
a wire-meshed cage with free access to powdered food. The food, in
which the main constituents were starch and wheat gluten, was made
isonitrogenous and isocaloric to 20% purified whole-egg protein by
adding a mixture of essential L-amino acids (Tabuchi et al.
1991; Torii et al. 1987
). Tap water was also
accessible ad libitum in the home cage except during the training and
recording sessions. The rats were housed in a room where temperature
(24 ± 1°C), relative humidity (60 ± 10%), and
light (09:00-21:00) were automatically controlled except during the
period of the stress exposure.
Surgery
Twenty minutes after an injection of atropine (0.1 mg/kg ip),
each rat was anesthetized with pentobarbital sodium (50 mg/kg ip) and
mounted in a stereotaxic apparatus with its skull level between the
bregma and lambda suture points. The cranium was exposed and five
stainless steel screws (2-mm diam) were threaded into holes in the
skull to serve as anchors for cranioplastic acrylic. Stainless steel
wires were soldered onto two screws to serve as a ground. Two
concentric bipolar electrodes (20-30 k at 1,000 Hz) for
intracranial self-stimulation (ICSS) reward were implanted in the
lateral hypothalamic medial forebrain bundle (A,
4.5 from bregma; L,
±1.4; V, 8.6) according to the atlas of Paxinos and Watson
(1986)
. The concentric bipolar electrode consisted of an outer
stainless steel tube (0.3-mm diam) and an inner wire (0.1-mm diam,
enamel coated). The outer tube was insulated by polyurethane except for
the tip area (about 0.1 mm long); the enamel insulation of the inner
wire was removed at the tip (about 0. 3 mm long). The cranioplastic
acrylic was built up on the skull and molded around the conical ends of
two sets of double stainless steel rods (fake ear bars) that had a
single steel bar on one end and two bars on the other end. Once the
cement had hardened, these bars were removed, leaving a negative
impression of the double end on each side of the acrylic block. During
subsequent surgery, training sessions and recording sessions, the
double end of these fake ear bars was pressed into the indentations in
the acrylic block while the single end was inserted into the normal ear
bar slot in the stereotaxic apparatus and rigidly attached to it. Hence
these artificial ear bars served the same purpose as regular ear bars
but could be used in the unanesthetized animals because they did not
involve painful insertion into the ear canal. A short length of
27-gauge stainless steel tubing was embedded in the cranioplastic
acrylic near bregma to serve as a reference coordinate pin during
chronic recording. After surgery, an antibiotic (gentamicine sulfate,
Gentacin Injection, Schering-Plough, Osaka, Japan) was administered
topically and systematically (8 mU im).
After recovery (7-10 days) from the cranioplastic surgery mentioned in
the preceding text and the following task training (2 wk; see next
section), the rats were reanesthetized (pentobarbital sodium, 40 mg/kg
ip) and mounted in the stereotaxic device using the fake ear bars. A
hole (2-3 mm diam) for chronic recording was drilled through the
cranioplastic cap and the underlying skull over the intended recording
site (A, 1.5 to
3.0 from bregma; L, 1.5 to 2.5; V, 7.5 to 9.5 for
the LHA, and A,
2.3 to
3.3 from bregma; L, 0.3 to 1.0; V, 8.0 to
10.0 for the MHA). The exposed dura matter was covered with
hydrocortisone ointment (Rinderon-VG ointment, Shionogi, Osaka, Japan).
The hole was covered with sterile cotton and sealed with epoxy glue.
The preceding surgical procedure was performed under aseptic conditions. All rats were treated in strict compliance with the "Guiding Principles for Research Involving Animals and Human Beings" recommended by The American Physiological Society, and with the "Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences" recommended by The Physiological Society of Japan.
Training and task paradigm
Prior to the cranioplastic surgery, the rats were acclimated by
handling and accustomed to being placed into a small restraining cage
that was constructed with stainless steel rods. Following recovery from
the cranioplastic surgery, the rats were placed on a 20- to 22-h
water-deprivation regimen. During task training, the rat was placed in
the restraining cage with the head fixed rigidly and painlessly in the
stereotaxic devise by the fake ear bars. While restrained, the rat had
access to a spout from which the rat learned to take fluids. The rat
was also trained to lick the spout to get ICSS rewards. Licking was
signaled by a photoelectric sensor triggered when the tongue crossed an
infrared beam. The parameters for ICSS (0.5-s train of 100 Hz, 0.3-ms
capacitor-coupled negative square wave pulses, 50-150 µA) were
similar to those in our previous reports (Nakamura and Ono
1986; Nishijo et al. 1998
; Oyoshi et al.
1996
; Uwano et al. 1995
).
In the initial training (1-2 h daily for 3-5 days), the rat was allowed to freely lick a spout placed close to the mouth. Later the spout was retracted, and the rat was trained to lick at the spout when it was extended to within close range of the mouth for 2.0 s. The positive reinforcement was a drop of glucose solution or an ICSS reward to facilitate the licking behavior.
In the following training (2-4 h daily for 9-11 days), a 2.0-s cue
tone preceded extension of the spout. The cue tone (about 80 dB), with
a different frequency corresponding to each solution, was delivered by
a mid range speaker 1 m above the rats; subsequently, the
associated solution flowed from the tip of the spout for 2.0 s
during spout extension if the rat licked the spout. The cue tones and
their associated solutions were: 1,000 Hz, 0.2 M L-lysine HCl; 2,350 Hz, 0.15 M monosodium L-glutamate (MSG); 3,500 Hz, 0.05 M
L-arginine; 5,400 Hz, 0.5 M glycine; 8,750 Hz, 0.15 M NaCl (saline); and 1,600 Hz, distilled water. The concentrations of the
sapid solutions were determined according to our previous study in
awake rats (Tabuchi et al. 1991). Training with each solution was carried out separately in one block of 10 trials. We did
not use ICSS as rewards in the training period of this cue-tone
association learning to minimize the interaction of ICSS with learning
process as well as to avoid any interaction of ICSS with repeated cold
stress. The rats were trained for progressively longer periods. After
an adaptation period of several days, most rats accepted the restraint
for up to 4 h per day without struggling. Finally, the total
number of trials per day reached 400-500 in 4 h. Throughout the
training and recording periods, the rat was permitted to ingest 30 ml
of fluid per session in the restraining cage. If a rat failed to take a
total volume of 30-ml fluid in the training, tap water was given when
the rat was returned to its home cage so that the final volume of total
intake was 30 ml.
Stress exposure
After the surgery and task training, the rats in the stressed
group were housed individually for 2 mo in a repeated cold-stress apparatus (modified M-9000 incubator made by Advantec Toyo, Tokyo) with
a built-in heater and cooler that could be controlled by an adjustable
self-timer except when neuronal activity was recorded. The
environmental temperature in this apparatus alternated four times
between 24 and 3°C at 1 cycle/2 h from 10:00 to 18:00; it was kept
at
3°C from 18:00 until 10:00 the following morning. It took about
30 min to change the temperature from
3 to 24°C. The time constant
(the time needed to go two-thirds of the remaining distance to the end
point) was 7.1 min. It took about 1 h to change the temperature
from 24 to
3°C. The time constant was 23.3 min. Recording sessions
(for detail, see Electrophysiological recording) in the
stressed group began 7-8 days after the onset of stress exposure; at
this time point, several abnormalities reportedly reach a steady state
(Hata et al. 1984a
, 1986
, 1987a
; Hori et al.
1993
). Electrophysiological recording was performed in each rat
for 2-4 h every other day, and after the recording, the rat was
returned to its cage in the repeated cold-tress apparatus.
Electrophysiological recording
After being placed in the restraining cage, the head was fixed rigidly and painlessly by the fake ear bars. The hydrocortisone ointment in the skull hole was removed, and the rat's dura mater was incised with a fine needle for electrode insertion under local anesthesia (a drop of 2% lidocaine jelly).
Extracellular single neuronal activity was recorded from the LHA and
MHA through glass microelectrodes (1-2 µm diam at tip) filled with 4 M NaCl (2-4 M at 1,000 Hz). The recording electrode was
stereotaxically inserted stepwise with a pulse motor-driven manipulator
(SM-21, Narishige, Tokyo) into various parts of the LHA and MHA. During
recording, one of the two ICSS electrodes served as an indifferent
electrode. Since the rat usually stayed quietly in the restraining cage
while receiving available rewards, movement artifacts were negligible.
Furthermore the differential recording effectively eliminated, if any,
the movement artifacts produced during licking; this allowed us to
record neuronal activity stably for enough time to analyze data.
Extracellular neuronal activity was passed through a high-input
impedance preamplifier made of a dual-channel field-effect transistor
(2SK18A, Toshiba, Japan), passed through a main amplifier (MEG-6100,
Nihon Kohden, Japan), monitored on an oscilloscope, and recorded on a
magnetic tape by a data recorder (XR-9000, TEAC, Japan). Lick signals
and computer-generated synchronizing trigger signals that represented the onset of the trials were also stored on the same magnetic tape for
later off-line analysis. Neuronal activity was processed through a
window discriminator. The analog signal and discriminator output were
monitored continuously on the oscilloscope during the analysis.
The cue tones and their associated solutions were the same as those used in the training. Some neurons were additionally tested with a 330-Hz tone associated with 0.05-M L-histidine and a 440-Hz tone associated with 0.3-M glucose. Inter-trial intervals were 25-50 s, and 5 to 10 successive trials were performed for each solution. To avoid interference of the preceding different kind of solution, data in initial two trials were discarded when solution was changed. If a neuron responded to CTSs, extinction test (the CTS was presented but the rewarding solution was not given to the rat) and re-association test (the CTS was again coupled with the rewarding solution) were carried out in additional 20-30 and 10-15 trials, respectively.
Neuronal activity was monitored by a workstation (Masscomp 6300, Concurrent Nippon, Tokyo) on-line during recording. Peristimulus events (prestimulus, 3 s; poststimulus, 12 s) were stored to display rasters on each trial and accumulated to display peristimulus histograms by the workstation. Recording was performed from two rats a day around 18:00-24:00 (2-4 h/rat).
Data analysis
Both neuronal and licking data in each trial of the task were counted for three phases: a pretrial control phase (2 s), a CTS phase (2 s), and a licking phase (2 s). One-way ANOVA test was performed for discharge rates of these three phases to test main effect of phase. Excitatory or inhibitory neuronal responses were determined by a post hoc pairwise comparison (Tukey test) between the discharge rate in the pretrial control phase and that in the CTS or licking phase.
Neurons that responded during CTS or licking were labeled as cue-tone- or licking-related, respectively. Neurons that did not show a change in activity compared with the pretrial control phase were labeled as nonresponsive. Neurons that showed significantly different responses to different CTSs and/or to the licking of different solutions were labeled as differential. Neurons that showed similar responses to different CTSs and/or during licking of different solutions were labeled as nondifferential. Responsive (nondifferential and differential) neurons were further subclassified as neurons that responded to CTS only (CTS type), licking only (licking type), or both CTS and licking (CTS and licking type). Percentages of categorized neurons were compared by Fisher's exact probability test. Mean spontaneous firing rates were compared by Student's t test.
All results are expressed as the means ± SE. Significance level employed for all tests was P < 0.05.
Histology
After the last recording session, each rat was reanesthetized
with pentobarbital sodium (50 mg/kg ip). Several iron deposits were
made stereotaxically around the recorded sites in the brain by passing
a positive current (20 µA, 30 s) through a stainless steel
electrode (0.2-mm diam, polyurethane insulated except for 0.1 mm at the
tip). Rats were then given a further overdose of anesthetic and
perfused transcardially with heparinized 0.9% saline followed by l0%
buffered formalin containing 2% potassium ferrocyanide. The brain was
removed, and cut into 50-µm frontal sections with a freezing
microtome. Sections were stained with cresyl violet. All marking sites
were verified microscopically. The location of each recording site was
then calculated from the stereotaxic coordinates of the recording
electrode and the coordinates of the marking electrodes, and plotted on
the atlas of Paxinos and Watson (1986).
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RESULTS |
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Mortality, food intake, and body weight change during repeated cold stress
All the rats in the stressed group (n = 15) survived
during the exposure to repeated cold stress. During the stress, food intake increased from the first day of stress exposure and reached a
maximum on the second day. The food intake of the stressed group was
about 50% higher than that of the control group throughout the rest of
the stress period. In contrast to food intake, body weight decreased on
the first day of stress exposure: It was about 200% of the change in
the control group. However, it increased on the second day and was
increased throughout the rest of the stress period although the rate of
body weight gain in the stressed group was smaller than that in the
control group. More specifically, the reduced body weight gain
gradually recovered during the first week of the stress exposure and
reached a steady state: during this steady state, the body weight gain
in the stressed rats was one-third to half of that in the control rats.
LHA neurons
ANALYZED NEURONS. The activity of 421 neurons was recorded in the rat LHA (215 in the control rats; 206 in the stressed rats). Each neuron was tested with six standard liquids (distilled water and lysine, MSG, arginine, glycine, and NaCl solutions) and the associated CTSs. Eight neurons from the control rats and 55 neurons from the stressed rats were further tested with histidine solution and its associated CTS. Twenty-four neurons from the control rats and 132 neurons from the stressed rats were also tested with glucose solution and its associated CTS. As no neurons responded preferentially to histidine or glucose solution in the present study, all the neurons sampled were classified based on the responses to the six standard liquids and the associated CTSs. Table 1 summarizes responsiveness of the 421 LHA neurons.
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RESPONSES OF NONDIFFERENTIAL NEURONS DURING CTS AND LICKING PHASES. Of 215 neurons recorded in the control rats and 206 neurons in the stressed rats, 117 (54.4%) and 139 (67.5%), respectively, were classified as nondifferential neurons. The percentage of nondifferential neurons in the stressed rats was significantly larger than that in the control rats (Fisher's exact probability test, P < 0.01) (Table 1). As described in METHODS, these nondifferential neurons were further divided into three subclasses (CTS, licking, and CTS and licking types). The percentage of CTS type was significantly larger in the stressed rats (18.0%, 37/206) than in the control rats (10.2%, 22/215; P < 0.05). The percentage of licking type was significantly smaller in the stressed rats (7.3%, 15/206) than in the control rats (13.0%, 28/215; P < 0.05). The percentage of CTS and licking type was significantly larger in the stressed rats (42.2%, 87/206) than in the control rats (31.2%, 67/215; P < 0.05).
Of the CTS-and-licking-type nondifferential neurons, 63 in the control rats and 75 in the stressed rats changed activity in the same response direction (excitatory or inhibitory) during the CTS and licking phases. The remaining 4 neurons in the control rats and 12 in the stressed rats changed activity in opposite response directions during these two phases. Figure 1 shows two examples of CTS-and-licking-type nondifferential neurons (recorded in the stressed rats) displaying activity changes in opposite response directions. Activity of the neuron shown in Fig. 1A decreased during the CTS phase and increased during the licking phase in a nondifferential manner [ANOVA, F(5, 39) = 2.05, P = 0.09, and F(5, 39) = 1.46, P = 0.22, respectively]. Activity of this neuron also decreased after the cessation of licking behavior. In contrast, activity of the neuron shown in Fig. 1B increased during the CTS phase and decreased during the licking phase in a nondifferential manner [F(4, 21) = 0.90, P = 0.48, and F(4, 21) = 2.42, P = 0.08, respectively]. The percentage of CTS-and-licking-type nondifferential neurons with activity changes in opposite response directions was significantly larger in the stressed rats (5.8%, 12/206) than in the control rats (1.9%, 4/215; P < 0.05).
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RESPONSES OF DIFFERENTIAL NEURONS DURING CTS AND LICKING PHASES. Eighteen (8.4%, 18/215) neurons in the control rats and 11 (5.3%, 11/206) in the stressed rats responded differentially during one or a few CTSs and/or licking of one or a few sapid solutions (differential neurons; Table 1). These differential neurons were further classified into three subcategories (CTS, licking, and CTS and licking types) as were the nondifferential neurons. The proportion of differential neurons did not differ significantly between the two groups.
Figure 2 shows two examples of differential neurons with responses to MSG in the control (A) and stressed (B) rats. The neuron shown in Fig. 2A was a CTS and licking type. It showed increased activity during the CTS associated with MSG solution and licking of MSG solution (Fig. 2Aa; Tukey test after ANOVA, Ps < 0.01) but did not respond during any phase of lysine or water trials (Fig. 2A, c and e). Neuronal activation during licking of MSG solution was also observed when CTS was absent, as shown in Fig. 2Ab (P < 0.01). This suggests that the response during licking was not simply due to a prolonged response or afterdischarge to CTS but was rather due to the licking of the MSG solution itself. Although MSG is a sodium salt, the neuronal activity was suppressed both during the CTS associated with saline and licking of saline (Fig. 2Ad; Ps < 0.01). This suggests that the neuronal activation by ingestion of MSG solution was not due to the presence of sodium but rather to glutamate or the complex taste of sodium plus glutamate. The neuron in Fig. 2B recorded from a stressed rat was the licking type. It showed increased activity during the ingestion of MSG solution (Fig. 2Ba; P < 0.01) but not during the ingestion of other sapid solutions (Fig. 2B, b-f).
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CUE-TONE-RELATED AND LICKING-RELATED NEURONS. Ninety-nine neurons (46.0%, 99/215) in the control rats and 133 neurons (64.6%, 133/206) in the stressed rats responded to one or more CTSs (cue-tone-related neurons; Table 1). The percentage of the cue-tone-related neurons was significantly larger in the stressed rats than in the control rats (P < 0.01). Of these cue-tone-related neurons, 50 (23.3%, 50/215) in the control rats and 53 (25.7%, 53/206) in the stressed rats showed an increase in activity during CTSs, whereas the remaining 49 (22.8%, 49/215) and 80 (38.8%, 80/206), respectively, showed a decrease in activity during the CTS phase. The percentage of the cue-tone-related neurons with decreased activity was significantly larger in the stressed rats than in the control rats (P < 0.05). In contrast, the percentage of the cue-tone-related neurons with increased activity did not differ statistically between the two groups. In the control rats, the percentage of the cue-tone-related neurons with increased activity (50.5%, 50/99) was similar to that of the neurons with decreased activity (49.5%, 49/99). In the stressed rats, the percentage of the cue-tone-related neurons with decreased activity (60.2%, 80/133) was significantly larger than that of the neurons with increased activity (39.9%, 53/133; P < 0.05). One hundred and eleven neurons (51.6%, 111/215) in the control rats and 113 neurons (54.9%, 113/206) in the stressed rats responded during licking of one or more sapid solutions (licking-related neurons). The percentage of the licking-related neurons in the stressed rats was similar to that in the control rats. Of the licking-related neurons, 53 (24.7%, 53/215) in the control rats and 51 (24.8%, 51/206) in the stressed rats showed increased activity during licking, whereas the remaining 58 (27.0%, 58/215) in the control rats and 62 (30.1%, 62/206) in the stressed rats showed decreased activity during licking. In the control rats, the percentage of the licking-related neurons with increased activity (47.7%, 53/111) was similar to that of the neurons with decreased activity (52.3%, 58/111). In the stressed rats, the percentage of the licking-related neurons with increased activity (45.1%, 51/113) was also similar to that of the neurons with decreased activity (54.9%, 62/113). Thus the results indicated that the increase in the number of responsive neurons in the stressed rats was due to a selective increase in the number of neurons that responded to CTSs with decreased activity.
To analyze the effect of stress stage on neuronal activity, we compared the relative number of cue-tone-related neurons and licking-related neurons during earlier recording sessions (days 8-29 in the stress exposure) with these values during later sessions (days 30-60). The percentage of cue-tone-related neurons recorded in the stressed rats during the earlier sessions (62.6%, 67/107) was similar to that during the later sessions (66.7%, 66/99). The percentage of licking-related neurons during the earlier sessions (49.5%, 53/107) did not differ significantly from that during the later sessions (60.6%, 60/99; P > 0.05).SPONTANEOUS FIRING RATE. Spontaneous firing rates of LHA neurons in the two groups are shown as histograms in Fig. 3. The spontaneous firing rates ranged from 0.7 to 62.9 spikes/s (n = 215) in the control rats and from 2.8 to 53.3 spikes/s (n = 206) in the stressed rats. In the stressed rats, distribution of spontaneous firing rates was clearly shifted to the right relative to the control rats. The peak of the histogram was 4-6 spikes/s in the control rats, and 10-12 spikes/s in the stressed rats. The mean spontaneous firing rate in the stressed rats (17.0 ± 0.71) was 34% higher than that in the control rats (12.6 ± 0.66; Student's t test, P < 0.01). In the stressed rats, the mean spontaneous firing rate in the later recording sessions (15.9 ± 1.02, n = 99) did not differ significantly from that in the earlier sessions (17.9 ± 1.02, n = 107).
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NEURONAL RESPONSES TO CTS IN EXTINCTION AND RE-ASSOCIATION TESTS. Of the 99 cue-tone-related neurons in the control rats and 133 in the stressed rats, 19 and 47, respectively, were further tested in the extinction and re-association trials. Of the 19 neurons recorded in the control rats, 9 (47%, 9/19) showed a decrease in response to CTS during the course of the extinction test, and recovered rapidly during the re-association test. The remaining 10 neurons (53%, 10/19) did not show a significant change in response to CTS in either the extinction or re-association test. Figure 4 shows representative plastic changes in neuronal and behavioral responses of the control rats during the pre-extinction, extinction, and re-association trials. In the pre-extinction trials (Fig. 4, Aa and Ba), the neuron showed decreased activity (inhibitory responses) both during the CTS associated with MSG solution and during ingestion of the MSG solution. In the early extinction trials (Fig. 4, Ab and Bb), the inhibitory responses during the CTS and licking phases abated. The licking behavior also gradually decreased in frequency until it disappeared on the second trial of the extinction test (trial 12). In the late extinction trials (Fig. 4, Ac and Bc), neuronal activity during the CTS and licking phases was similar to that during the pretrial control phase (no inhibitory responses). In the first trial of the re-association test (Fig. 4Ad, trial 31), the neuron did not show decreased activity during the CTS although the rat did lick the spout. However, from the 2nd to the 10th trials of the re-association test (trials 32-40), the neuron again showed decreased activity during the CTS as in the pre-extinction trials. The inhibitory neuronal response during the licking phase also recovered in the first trial of the re-association test, and this was observed throughout the remaining re-association trials. These changes in neuronal responses are shown in Fig. 4Ca quantitatively as line graphs. The magnitude of the inhibitory response during the CTS and licking phases gradually decreased over the course of the extinction test (trials 11-18) and reached a level close to the spontaneous firing rate (trials 19-30). In the re-association test, the inhibitory responses recovered rapidly to almost the same level as that in the pre-extinction trials. Mean response magnitudes during the CTS and licking phases were statistically compared between the four trial blocks (each block consisted of 10 trials, Fig. 4Cb): one pre-extinction block (block 1), two extinction blocks (blocks 2 and 3) and one re-association block (block 4). Mean response magnitude during the CTS phase was significantly smaller in block 3 than in block 1 (pre-extinction; Tukey test after 1-way ANOVA, P < 0.05). Mean response magnitude during the licking phase was significantly smaller in blocks 2 and 3 than in block 1 (Ps < 0.01). Mean response magnitudes during the CTS and licking phases in block 4 did not differ significantly from those in block 1 (Ps > 0.05).
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MHA neurons
ANALYZED NEURONS. The activity of 127 neurons was recorded in the MHA (67 in the control rats; 60 in the stressed rats). Each neuron was tested with the six standard liquids and the associated CTSs. Forty-two neurons from the control rats and 37 neurons from the stressed rats were further tested with histidine solution and its associated CTS. Fifty-eight neurons from the control rats and 43 neurons from the stressed rats were also tested with glucose solution and its associated CTS. In contrast to the LHA neurons mentioned in the preceding text, the percentage of MHA neurons that responded during the CTS and/or licking phase was relatively low. Of the 67 neurons recorded in the control rats and 60 recorded in the stressed rats, 9 (13.4%) and 3 (5.0%), respectively, were classified as nondifferential neurons. The remaining 58 (86.6%) and 57 (95.0%), respectively, were classified as nonresponsive neurons. No differential MHA neurons were recorded in the present study. Of the nine nondifferential neurons recorded in the control rats, one was CTS type, one was licking type, and the remaining seven were CTS and licking type. Of the three nondifferential neurons recorded in the stressed rats, one was licking type, and the other two were CTS and licking type.
SPONTANEOUS FIRING RATE. Spontaneous firing rates of MHA neurons in the two groups are shown as histograms in Fig. 7. Spontaneous firing rates ranged from 1.3 to 19.1 spikes/s (n = 67) in the control rats and from 1.1 to 8.9 spikes/s (n = 60) in the stressed rats. The mean spontaneous firing rate in the stressed rats (3.12 ± 0.22) was 36% lower than that in the control rats (4.87 ± 0.42; Student's t test, P < 0.01). Although the histogram peak appeared in the range of 2-4 spikes/s both in the control and stressed groups, the histogram distribution in the stressed group was shifted to the left relative to the control group.
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Recording sites
The recording sites for all the neurons sampled are shown in Fig. 8. Given that the total number of differential neurons was small, this type of neuron was included in the cue-tone-related and/or licking-related neurons. The recording sites in the stressed rats were almost identical to those in the control rats. The neurons that responded to CTSs and/or during licking with either increased or decreased activity were diffusely distributed in the LHA both in the control and stressed groups. In each atlas plane, the number of cue-tone-related LHA neurons showing inhibitory responses was larger in the stressed group than in the control group. The percentage of neurons showing inhibitory responses to CTS in the most anterior portion of the LHA (Fig. 8A, 7,200 µm) was significantly larger in the stressed rats (48.1%, 25/52) than in the control rats (13.2%, 7/53; Fisher's exact probability test, P < 0.01).
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The LHA neurons sampled in each group were divided into four populations according to the anterior-posterior (A-P) levels of their recording sites, and mean spontaneous firing rate was calculated for each population (Table 3). The mean spontaneous firing rates were higher in the stressed rats than in the control rats in all the A-P ranges except for the most anterior one (7,500-7,100 µm). The difference in the mean spontaneous firing rates between the control and stressed rats tended to be larger in the posterior ranges than in the anterior ranges.
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DISCUSSION |
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Effect of repeated cold stress on spontaneous firing rates of LHA and MHA neurons
In the present study, repeated cold stress increased the
spontaneous firing rates of LHA neurons and decreased those of MHA neurons. These changes can be interpreted to indicate that basal activity level of the LHA was increased and that of MHA was decreased under repeated cold stress. This interpretation is of interest when
considering the functional implication of these two brain areas. For
instance, hyperphagia is a unique characteristic of chronic cold stress
including repeated cold stress (Bing et al. 1998;
Hata et al. 1984a
,b
, 1988a
; Kondoh et al.
1996
; Leung and Horwitz 1976
; Morrison
1981
; Okano et al. 1993
; Snyder and
Stricker 1985
), which can be clearly distinguished from the
anorexia induced by severe acute stress conditions (Morley and
Levine 1982
; Shimizu et al. 1989
). Consistent
with the previous studies (Hata et al. 1984a
,b
, 1988a
;
Kondoh et al. 1996
; Okano et al. 1993
),
in the present study we also observed an increase in food intake in the rats exposed to repeated cold stress. Previous behavioral and neurophysiological studies have suggested that the LHA and ventromedial hypothalamic nucleus (VMH, a part of the MHA defined in the present study) control feeding behavior in opposing manners; the LHA is thought
to be a feeding center and the VMH together with the PVN are satiety
centers (Delgado and Anand 1953
; Leibowitz
1986
; Oomura et al. 1967
, 1969
; Shimizu
et al. 1987
; Smith 1956
; Winn et al. 1984
). Thus the present finding of increased spontaneous
activity in the LHA (feeding center) and decreased spontaneous activity in the VMH (satiety center) could be involved in the increase in food
intake observed in the stressed rats.
A variety of substances (nutrients and their metabolites,
neurotransmitters, hormones, etc.) are known to affect activity of LHA
and/or MHA neurons so as to control feeding behavior (for review, see
Leibowitz 1986; Oomura 1989
). Although it
is difficult to determine based on the present results what substances
are responsible for the activity changes in the hypothalamic neurons during repeated cold stress, several possibilities were suggested. When
rats are chronically exposed to a cold environment, the sympathoadrenal system and hypothalamo-pituitary-thyroid axis are activated to enable
adaptation to the cold environment (Fukuhara et al.
1996a
,b
), resulting in thermogenesis (energy expenditure) and
compensatory hyperphagia. Oomura et al. demonstrated the presence of
LHA neurons (glucose-sensitive neurons) that decrease activity and the
presence of VMH neurons (glucoreceptor neurons) that increase activity when glucose is directly applied to the membrane of these neurons (Oomura et al. 1969
, 1974
). Furthermore free fatty acids
stimulate glucose-sensitive neurons while suppressing glucoreceptor
neurons (Oomura et al. 1976
). Therefore a decrease in
blood glucose levels and an increase in blood free fatty acid levels
accompanied by increased energy expenditure during cold exposure could
increase the mean spontaneous firing rate of LHA neurons and decrease
that of MHA neurons. Bing et al. (1998)
reported that
chronic cold exposure decreases plasma leptin and insulin levels but
does not significantly change hypothalamic neuropeptide Y and plasma
cortisone levels. Leptin and insulin are transported from plasma to the hypothalamus through the blood-brain barrier (Banks et al.
1996
; King and Johnson 1985
; Pardridge
1986
; Schwartz et al. 1991
), and these peptides
in turn suppress food intake when centrally administered
(Campfield et al. 1995
; Woods et al.
1979
). In the LHA and MHA, many neurons have leptin receptors
(Elmquist et al. 1998
; Funahashi et al.
1999
; Hakansson et al. 1998
); LHA and
MHA neurons also have insulin receptors (Havrankova et al.
1979
; Unger et al. 1991
). These peptides inhibit
activity of LHA glucose-sensitive neurons and stimulate VMH
glucoreceptor (or glucose-responsive) neurons (Funahashi et al.
1999
; Oomura et al. 1976
). Therefore the
reported decrease in these peptides in plasma is consistent with the
changes in spontaneous activity of LHA and MHA neurons observed in the
present study. Furthermore we recently found that mRNA level of IL-1
decreased in the LHA while increasing in the MHA in mice exposed to
repeated cold stress (Tagoh et al. 1995
). IL-1
, which
has an inhibitory effect on food intake, suppresses the activity of
neurons in the LHA and VMH (Kuriyama et al. 1990
; Plata-Salamán et al. 1988
). Therefore the
site-specific change in IL-1
level (Tagoh et al.
1995
) is also consistent with the present finding of changes in
spontaneous activity of LHA and MHA neurons.
Absence of specific effect of repeated cold stress on responses of hypothalamic neurons to tastants
Pathological conditions may affect hypothalamic neuronal responses
in a tastant (or nutrient) specific manner. We have previously found
that deficiency of a requisite amino acid (lysine), leading to a
specific preference for this amino acid (Torii 1987),
increased the number of LHA neurons that responded specifically to
lysine solution and its associated CTS in rats (Tabuchi et al.
1991
). The levels of histamine and histamine turnover in the
hypothalamus change during cold stress (Taylor and Snyder
1971
). We recently found that repeated cold stress induces a
specific preference for histidine (Kondoh et al. 1996
).
Therefore we originally predicted that repeated cold stress would
change the proportion of differential neurons or increase the number of
neurons that specifically respond to histidine solution and its
associated CTS. We also thought that repeated cold stress might
preferentially affect responsiveness of hypothalamic neurons to glucose
solution and its associated CTS since cold exposure increases energy
expenditure and results in hyperphagia. However, the present results
were contrary to our prediction, at least for LHA neurons: the repeated
cold stress did not change the relative percentage of differential
neurons nor did it increase the number of LHA neurons that specifically responded to the glucose or histidine solution and its associated CTS.
Indeed no neurons displayed preferential responses to glucose or
histidine. Although to date we have no data to explain this dissociation between our original prediction and the present results, repeated cold stress may have affected the hypothalamus in a more "general" way than the lysine deficiency. Animals can use not only
carbohydrate but also fat and protein as energy sources, and thus LHA
neurons might not necessarily differentiate the gustatory stimuli
tested in the present study, while the specific deficiency of lysine
could affect the gustatory responses of LHA neurons in a more specific
way because of its specificity for nutrient deficiency. The water
deprivation regimen might also have abolished or masked specific
changes in neuronal activity related to hyperphagia since water
restriction reduces food intake; it could also affect any specific
preference for histidine, although we did not test this possibility in
the present study. Further studies are necessary to elucidate these points.
Effect of repeated cold stress on nondifferential neurons
In the present study, the relative percentage of nondifferential
LHA neurons was increased and that of nondifferential MHA neurons
decreased in stressed rats. Enhanced awareness of and responsiveness to
environmental stimuli by repeated cold stress may have played a role in
these changes. Rats exposed to repeated cold stress exhibit
hypersensitivity to external stimuli in galvanic skin response,
resting-arousal electrocorticograms with low-voltage fast waves, and
hyperactivity accompanied by increased defecation in an open-field test
(Hata et al. 1987b, 1988b
). Furthermore hyperalgesia is
a unique characteristic of repeated cold stress (Hata et al.
1988a
; Kawanishi et al. 1997
; Kita et al.
1979
) in contrast to the stress-induced analgesia caused by
acute stress (Bondnar et al. 1980
). These results
suggest that animals exposed to repeated cold stress are in a state of
hyperreactivity to external stimuli. Previous anatomical and
neurophysiological studies have suggested that the LHA is at the
rostral end of the ascending brain stem reticular formation
(Nieuwenhuys et al. 1982
), and that LHA neurons are
involved in information processing concerning not only internal but
also external environments to produce appropriate autonomic and
behavioral responses (Iwata et al. 1986
; Ono et al. 1981
). Furthermore electrical stimulation of VMH and DMH
induced pain inhibition (Rhodes and Liebeskind
1978
), while lesions of the VMH caused hyperalgesia
(Hata et al. 1988a
). The decreased nociceptive threshold
in repeated cold stress is increased by LHA lesions in rats
(Hata et al. 1988a
). The present findings and those of
previous studies suggest that altered activity of the hypothalamic
neurons in repeated cold stress is linked to hypersensitivity to
external sensory (auditory, nociceptive, etc.) stimuli.
Effect of repeated cold stress on changes in responsiveness of LHA neurons during extinction test
In the present study, some LHA neurons in the stressed rats showed
enhanced or reversed responses to CTSs in extinction trials. No such
response changes were ever observed in the control rats, a finding that
was consistent with the results of our previous studies on response
properties of LHA and VMH neurons (Nakamura and Ono
1986; Nishino et al. 1988
; Ono et al.
1986
, 1992
). In these studies, we regarded plastic changes in
neuronal responses to CTSs associated with rewarding or aversive
stimuli during the course of extinction and re-association tests as CTS
learning of positive and negative reinforcement.
Repeated cold stress is also known to produce impairment in passive
avoidance learning during consolidation phase in rats and mice
(Yago et al. 1992; Yoneda et al. 1992
). A
previous study from our laboratory demonstrated that dopamine (DA) is
involved in activity change of LHA neurons accompanied with CTS
learning of positive reinforcement, whereas ACh is involved in that of negative reinforcement (Ono et al. 1992
). Exposure to a
cold environment, including repeated cold stress condition
significantly affects the levels of hypothalamic DA and ACh
(Hata et al. 1987a
; Kita et al. 1986
;
Myers 1980
; Yoneda et al. 1992
).
Furthermore physostigmine, an acetylcholinesterase inhibitor, improves
impaired passive avoidance learning in rats exposed to repeated cold
stress (Yago et al. 1992
). The results of the present
study together with those of the previous studies suggest the
possibility that the stress-induced unusual (i.e., enhanced or
reversed) changes in LHA neuron responses are involved in the learning
deficits observed in the rat exposed to repeated cold stress. Repeated
cold stress, which produced changes in the levels of DA and ACh in the
hypothalamus, might have affected LHA neuron activity to abolish normal
plasticity in the formation and extinction of stimulus-reinforcement association.
In the present study, we did not observe any differences in the behavioral (licking) changes between the control and stressed groups during the course of the extinction and re-association tests in most of the recording sessions. This was likely due to the fact that the behavioral requirement in the extinction and re-association tests was very simple so that the rats, even under the stressed condition, readily learned how to behave in these tests; this would make it difficult to detect behavioral differences between the control and stressed rats during the course of these tests. It may be necessary to use different tasks with more complex learning requirements or naive rats to establish any relationship between learning-related changes in behavior and neuronal activity during repeated cold stress.
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ACKNOWLEDGMENTS |
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We thank Dr. M. J. Wayner (University of Texas) and Dr. M. Sugimori (New York University) for critical comments.
This work was supported in part by the Japanese Ministry of Education, Science, and Culture Grants-in-Aid for Scientific Research (08279105, 11308033, and 11680805).
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FOOTNOTES |
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Address for reprint requests: T. Ono, Dept. of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan (E-mail: onotake{at}ms.toyama-mpu.ac.jp).
Received 6 April 2000; accepted in final form 11 September 2000.
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REFERENCES |
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