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Brain Research 883 (2000) 125–130
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Short communication
Depletion of brain norepinephrine does not reduce spontaneous
ambulatory activity of rats in the home cage
James W. Murrough, Katherine A. Boss-Williams, Milburn S. Emery, Robert W. Bonsall,
Jay M. Weiss*
Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Emory West Campus, 1256 Briarcliff Road, NE Atlanta,
GA 30322, USA
Accepted 15 August 2000
Abstract
6-Hydroxydopamine (6-OHDA) lesions of brain noradrenergic neurons and terminals were made in rats to assess the importance of
forebrain norepinephrine (NE) for mediating circadian patterns of spontaneous ambulatory activity that rats show in the home cage.
6-OHDA was injected intracranially into the fibers of the ascending noradrenergic dorsal and ventral bundle pathways or infused into the
lateral ventricle or both. Rats living in a 12 / 12 h light / dark cycle exhibit a marked increase in ambulatory activity during the dark period
in comparison to the light period and a ‘W-shaped’ pattern of activity during the 12 h of the dark phase. Results showed that near-total
depletion of brain NE did not impair the capacity to generate normal patterns of spontaneous ambulatory activity that occur in the home
cage. In the animals that sustained the most complete NE lesions, the amounts of activity generated at times of peak activity were
exaggerated in comparison to the control animals, which is consistent with the possibility that NE in the brain exerts a moderating
influence on behavior. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Monoamines and behavior
Keywords: Motor activity; Norepinephrine; 6-hydroxydopamine; Ambulation; Circadian rhythm
Altered activity of noradrenergic neurons in the brain
has long been thought to be critically involved in the
pathophysiology of depression. The ‘catecholamine hypothesis of depression,’ first put forth over 30 years ago,
proposed that depression arose from a deficiency of
norepinephrine (NE) in the brain [4,22,23]. Although
shortcomings of this formulation became evident soon
after it was proposed, evidence nevertheless has continued
to accumulate indicating that brain NE is important in
depression (reviewed in Grant and Weiss [12]). The
present study examined the effect of reducing brain NE on
spontaneous motor activity of rats. Because altered motor
activity is one of the most common changes seen in
depression, the relationship between brain NE and motor
*Corresponding author. Tel.: 11-404-712-9771; fax: 11-404-7129755.
E-mail address: [email protected] (J.M. Weiss).
activity has been of considerable interest since the catecholamine hypothesis of depression was proposed.
Despite the close connection between brain NE and
motor activity suggested by the catecholamine hypothesis
of depression, basic research has by no means provided
unambiguous evidence showing that alteration of brain NE
will affect motor activity. Experimental manipulation of
brain NE, most commonly by making lesions of brain
noradrenergic neurons, often has failed to produce a
change in motor activity of animals [7,19,27]. A recent
study using a genetic manipulation to investigate the role
of NE (i.e., elimination of the gene for dopamine betahydroxylase) also reported no significant effect on spontaneous motor activity of mice unable to synthesize NE [28].
On the other hand, other studies have reported that NE
depletion leads to a decrease in motor activity in a novel
environment, suggesting a role for brain NE with respect to
motor activity under these conditions [3,16,21]. In addi-
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02850-X
126
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
tion, early studies found that catecholamine-depleting
drugs could reduce behavioral responses that are highly
dependent on motor activity in rats (e.g., [17,18]). And,
finally, a strong correlation between regional brain NE
depletion and decreased motor activity has been shown in
the context of a widely-studied animal model of depression
[24,25,29,30].
In the present study, we investigated the influence of
brain NE on diurnal changes in spontaneous ambulatory
motor activity of rats. Freely-behaving rats, living in a 12 h
on / 12 h off light / dark cycle, exhibit a pattern of spontaneous motor activity such that the animals’ ambulatory
activity is markedly elevated during the dark period of the
day in contrast to the light period when animals are much
less active. Moreover, animals show a characteristic pattern of activity during the ‘active’ dark phase. This pattern
shows an upward spike in ambulatory activity at the
beginning of the dark phase, after which activity then
declines, only to increase somewhat around the middle of
the dark period, after which it declines again, followed by
marked increase leading up to the end of the dark period
(i.e., a ‘W-shaped’ pattern). In this study, we examined
whether destruction of NE neurons and terminals in the
brain would affect these patterns of spontaneous ambulatory activity.
To alter brain NE, lesions of the ascending noradrenergic pathways — the dorsal noradrenergic bundle (DNB)
and ventral noradrenergic bundle (VNB) — were made
using the neurotoxin 6-hydroxydopamine (6-OHDA). Subjects were male Sprague–Dawley rats weighing approximately 400 g at time of the study. Sixteen animals were
assigned to one of three lesion groups (i.e., the axonal,
ventricular, or compound lesion group) or the control
group (n54 per group). Animals were anesthetized with
1–3% halothane mixed with oxygen and placed into a
stereotaxic instrument where surgery was performed. The
‘axonal’ lesion group received bilateral microinjections of
6-OHDA hydrochloride (Sigma) aimed at fibers of the
DNB and VNB just anterior to the locus coeruleus (LC).
Axons arising from NE-containing cells of the LC comprise the DNB, which provides over 70% of the NE in the
rat brain including all the NE in the neocortex and
hippocampus [9,13,15]. The VNB arises from noradrenergic neurons caudal to the LC and terminates in various
regions of the diencephelon, providing particularly rich
innervation to the hypothalamus [5,11]. After an incision
was made in the skin covering the skull and the skin
retracted, two small holes were drilled into the skull to
introduce a 26-gauge cannula bilaterally into the brain.
Stereotaxic coordinates were used for placement of the
cannulae. For axonal lesions, the coordinates for cannula
location were as follows: with nose piece 210.08; anterior /
posterior (AP) 22.6 mm from lambda; medial / lateral
(ML)61.2 mm from midline; dorsal / ventral (DV) 27.0
mm and 28.0 mm from dura matter. 6-OHDA was freshly
dissolved in artificial cerebrospinal fluid (aCSF) and
delivered through the cannula into each side of the brain. A
cannula was first lowered into one side of the brain to
28.0 mm, targeting axons of the VNB and 1 ml of
6-OHDA (4 mg / ml) was injected over a period of half a
minute. After a wait of 1 min, the cannula was raised to
27.0 mm, targeting axons of the DNB and a second 1 ml
injection of 6-OHDA was performed, followed by a wait
of 1 min and then the cannula was removed. Thirty
minutes after completion of the injection on one side of the
brain, the procedure was repeated on the other side of the
brain (the 30-min delay between injections was imposed to
promote animal survival). The ‘ventricular’ lesion group
received a microinfusion of 6-OHDA into the lateral
ventricle. For this group, the surgical procedure was the
same as described above except that a single hole was
drilled in the skull for insertion of a single 26-gauge
cannula. Stereotaxic coordinates used for this condition
were as follows: with nose piece level with skull (23.38),
AP 21.0 mm from bregma; ML61.5 mm; DV 23.5 mm
from skull (variable). After the cannula was lowered at this
location, placement in the ventricle was confirmed by the
observation of cerebrospinal fluid rising in a small length
of silastic tubing attached to the cannula. 6-OHDA introduced into the ventricle normally would be taken up into
dopamine (DA)-containing cells and terminals as well as
NE-containing neurons and would also destroy DA cells
and terminals. To prevent lesioning of DA terminals and
cells, the DA uptake blocker GBR 12909 (Sigma) was
infused through the ventricular cannula just prior to
infusion of the 6-OHDA. GBR 12909 was dissolved in
warm (378C) aCSF (1 mg / ml) and 6 ul was infused at a
rate of 2 ml / min. Following a wait of 15 min to allow time
for the drug to bind to DA transporters, 4 ml of 6-OHDA
(40 mg / ml) was then infused at a rate of 2 ml / min. to
complete the ventricular infusion procedure. The ‘compound’ lesion group underwent both the axonal lesion
procedure and the ventricular lesion procedure. For this
group, first the axonal lesion was carried out and then,
after a recovery period of 2 weeks, the ventricular lesion
procedure was conducted. All doses and surgical procedures used for the compound lesion group were the same
as described above for the axonal and ventricular lesion
groups. For the control group, these animals were anesthetized with halothane for the amount of time that surgery
normally required but no surgery was performed (control
animals were subjected to minimal manipulation so that
their subsequent ambulatory behavior would not be affected by any experimental procedure and thus would constitute normal activity for a rat).
Four weeks after surgery was initiated, animals were
placed individually into standard polycarbonate laboratory
cages (45325320 cm). Each of these cages was mounted
within a photocell assembly (ANA 1219, Riverpoint
Electronics) that permitted the continuous monitoring of
the activity of the animal in the cage. The photocell
assemblies were connected to a computer which recorded
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
and tabulated the number of interruptions of the photocells
by each animal. Animals were housed directly on bedding
and received ad libitum food and water. Five days was
allowed for acclimation to individual housing, followed by
7 days of monitoring the animals’ nocturnal spontaneous
motor activity. On each day, the recording apparatus was
activated 2 h before onset of the dark phase and recording
ended 5 h after lights on. Ambulatory motor activity was
expressed as ambulation counts per hour, an ambulation
count being defined as interruption of a photocell beam
that had not been broken in the previous four beam breaks
and thus represented horizontal locomotion by the animal.
At the conclusion of the study, the animals were
sacrificed and brains removed. The following brain regions
were dissected and retained for analysis: LC region of
brain stem (LC), hypothalamus (HYP), hippocampus
(HIPP), prefrontal cortex (PFC) and striatum / nucleus
accumbens (STR). Brain samples were frozen at 2858C
for analysis of catecholamine content using high pressure
liquid chromatography (HPLC). Tissue samples were
analyzed for NE content to assess the extent of the lesion;
striatal tissue was principally analyzed for DA to assess
whether DA-containing terminals had been protected from
destruction by infusion of GBR 12909 prior to icv infusion
of 6-OHDA. Tissues were homogenized in 0.1 M perchloric acid containing an internal standard. Catecholamines were separated using reverse-phase, ion–pair
HPLC and were quantified by an electrochemical detector
(ESA Coulochem II) attached to a computerized data
acquisition system (Perkin Elmer Turbochrom 4). Protein
content of each sample was measured by the Lowry
technique so that catecholamine content could be expressed as pg NE (or DA) per mg tissue protein.
As expected, all lesion groups (axonal, ventricular,
compound) showed marked depletion of NE in brain
regions known to be major targets of the ascending
noradrenergic pathways, i.e., cortex and hippocampus for
the DNB and hypothalamus for the VNB. The results are
shown in Table 1. 6-OHDA administered icv was found to
be somewhat more effective for depleting NE in the
terminal regions of the DNB than was 6-OHDA aimed at
127
the axon bundles, although a difference between these
techniques was not observed in the hypothalamus. Hypothalamic NE proved somewhat more resistant to neurotoxic
depletion than did forebrain NE, consistent with previous
reports [20]. The compound lesion group showed the
largest reduction of NE, indicating that the ventricular and
axonal lesion techniques had cumulative effects. In all
animals of the compound lesion group, NE was undetectable in the hippocampus and cortical NE was almost
equally reduced. The compound lesion technique also was
able to deplete hypothalamic NE to an average of 10% of
control, with two animals of the group found to have less
than 10% of the control level of NE remaining. DA levels
were not reduced by either the axonal or ventricular lesion
techniques as estimated by DA content in striatum / nucleus
accumbens, while the compound lesion technique produced a small reduction in DA (i.e., 30%) that is not
considered to be functionally effective [6,14].
Mean (6standard error) hourly ambulatory activity of
the different groups during the 19 h of the day when
activity was recorded is shown in Fig. 1, with the activity
of each lesion group plotted against the control group.
These data were analyzed by analysis of variance (twoway [group3h] with repeated measures across the hour
factor) which was carried out for each of the three panels
shown in the Fig. 1. No analysis generated an effect of
group that approached significance, thereby indicating that
depletion of brain NE by these lesion techniques did not
cause a change in spontaneous motor activity in comparison to control animals. The total nocturnal ambulatory
counts for each of the lesion groups, which was compared
to the control group by t-test, showed no difference that
approached significance. Moreover, inspection of Fig. 1
shows that even the ‘W-shaped’ pattern of dark phase
activity that occurs in normal rats was intact in the
lesioned animals. The only potential departure from this
was seen in the ventricular lesion condition, where a
somewhat reduced elevation in the first hour of activity
after onset of the dark period was seen. However, the lack
of an overall effect of group in the analysis of variance that
compared this group with the control condition, which was
Table 1
Effects of three different 6-OHDA lesion techniques on brain catecholamine levels a
Groups
Norepinephrine
Dopamine
HIPP
Control
Ventricular
Axonal
Compound
a
PFC
HYP
LC
STR
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
14836117
15615
4286157
0
1
29
0
19166118
203689
4326177
1267
11
23
1
66276498
22306220
18086442
6926165
34
27
10
11 7766458
334061099
20196630
9836251
28
17
8
41 87764864
39 44362165
52 67963394
29 27063806
94
125
70
The data are given as group means6S.E.M. The control group was non-lesioned. The ventricular group received 6-OHDA injected into the lateral
ventricle; the axonal group received 6-OHDA injected into the fibers of the dorsal and ventral bundle ascending noradrenergic pathways; the compound
group received both treatments. Sensitivity of the HPLC electrochemical detection was 2.0–3.0 pg / mg protein for hippocampal and cortical
norepinephrine.
128
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
Fig. 1. Mean (6S.E.M.) hourly ambulatory activity of animals with
lesions of the noradrenergic neurons in the brain in comparison to
non-lesioned animals. Activity data was collected throughout a 12 / 12 h
light / dark cycle over a period of 7 days; monitoring began 2 h prior to
onset of the dark phase and ended 5 h after onset of the light phase. Each
panel compares the non-lesioned animals (control group) to one of three
lesion groups (n54 / group). The top panel shows animals who received
microinfusion of 6-OHDA into the lateral ventricle (ventricular lesion
group); the middle panel shows animals who received microinjections of
6-OHDA into the fibers of ascending noradrenergic projections (axonal
lesion group); the bottom panel shows animals who were subjected to
both lesion procedures (compound lesion group). No statistically significant differences were found. The horizontal black bar designates duration
of the dark phase.
noted above, means that the difference at this single time
point is statistically attributable to chance; also, the failure
to see a similar change in any other lesioned group
indicates that this was in all likelihood a chance occurrence. In summary, not only did lesioned rats fail to show
any overall difference in ambulatory activity from control
rats but they also showed an unchanged pattern of nocturnal activity; i.e., like the control (normal) rats, lesioned rats
showed a marked increase in activity at the onset of the
dark period, followed by a decline and then another
smaller peak in activity toward the middle of the dark
period, followed finally by another large increase in
activity leading up to onset of the light period. Finally,
ambulatory activity during hours of light also was similarly
low in all groups.
What is indicated by these data is that forebrain NE is
not necessary for the rat to generate a normal pattern of
ambulatory activity in a home cage situation during light
and dark phases of the day. Lesions of NE neurons and
terminals that virtually eliminated NE from the forebrain
neither reduced total spontaneous motor activity nor
impaired ability to generate increases in nocturnal ambulatory activity that characterizes normal rat behavior. But
insofar as the technique used for affecting NE in the
present study was to make a lesion that would reduce brain
NE, an important issue to consider is whether compensatory increases in the function of noradrenergic systems in
the brain (such as upregulation of postsynaptic adrenergic
receptors) might have overcome effects of decreased
transmission of brain NE. Thus it might be argued that,
despite reduced NE levels in the brain, noradrenergic
neurotransmission in the lesioned animals nevertheless was
functioning well enough to mask motor deficits that will
result from the depletion of NE. With regard to this issue,
studies that have assessed compensation after experimentally-produced reduction of catecholamine content in brain
agree that compensation indeed occurs but also indicate
that compensation sufficient to return functional responses
to normal generally requires 10% or more of the normal
amount of NE or DA to be present [6,8,10,14]. In regard to
the present experiment, the study of Curet and Montigny
[8] is particularly relevant. These investigators examined
electrophysiological responses of hippocampal neurons
following electrical stimulation of the locus coeruleus
(which will cause NE to be released in the hippocampus).
In part of the study, responses were assessed in rats whose
NE had been depleted by intraventricular infusion of 6OHDA; electrophysiological measurement was made 14–
21 days after the NE lesioning procedure had been carried
out. In comparison to naturally-occurring physiological
stimuli that activate LC neurons, electrical stimulation of
LC is a very strong stimulus for NE release, so that this
technique is likely to ‘test the limits’ by recruiting whatever NE is still available for release. In animals that had
been infused with 6-OHDA, the normal influence of LC
stimulation on hippocampal neurons was not diminished
until NE depletion in the hippocampus was found to reach
90%, but with depletions above this level, the influence of
stimulation began to decline and no influence at all of LC
stimulation was seen (i.e., no evidence of any compensation being present) when NE depletion was measured as
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
being total (i.e., 100%). Thus, Curet and Montigny reported that substantial depletions of NE (at least 90%)
were needed to alter the influence of NE on hippocampal
cells that was produced by electrically stimulating the LC,
but even a strong stimulus for release such as this had no
effect at all when NE depletion was judged to be complete.
In the present study, NE lesions were quite extensive, with
the compound lesion technique producing near-total depletion of NE in the forebrain. In this regard, Fig. 2 shows the
individual activity data of the two animals in the compound lesion group that were found to have the most
complete lesions in the study. For these two subjects
(animals [7 and [9), no NE at all was detected in
prefrontal cortex and hippocampus (assay sensitivity for
these regions was less than 3.0 pg / ml protein, which was
less than 0.2% of control levels) and the reduction in
hypothalamic NE was 95% and 92% respectively. Examination of Fig. 2 reveals that these animals showed undiminished elevation of ambulatory activity in response to
the onset of darkness and also showed a similar ‘W’
pattern of ambulatory activity during the dark period as
was shown by control animals. These results in animals
whose NE depletions were so large as to preclude the
possibility of any significant compensation indicate, together with the other findings shown in Fig. 1, that
forebrain NE is not necessary for the ambulatory behavior
assessed in this study.
The results shown in Fig. 2 also may shed some light on
the function of forebrain NE. Rather than showing a failure
to respond, the two lesioned subjects whose data are shown
in Fig. 2 can be seen to manifest exaggerated ambulatory
responses at times when nocturnal ambulatory activity
normally increases. In evaluating these results, it should be
Fig. 2. Hourly ambulatory activity of two individual rats with virtually
complete ablation of central NE compared with the mean (6S.E.M.)
activity of the control group (n54). In the case of the individual animals,
error bars represent variation in hourly activity recorded on 7 days; the
small magnitude of error bars indicates that the pattern of activity shown
was stable from one day to the next. The horizontal black bar designates
duration of the dark phase within a 12 / 12 h light / dark cycle.
129
considered that the data shown for the two lesioned
animals in Fig. 2 are means (6SEs) calculated from
recordings made over 7 days, so that elevated activity at a
particular hour was not an isolated occurrence on a single
day but, instead, represents a consistent daily pattern.
Therefore, animal [7 consistently showed (a) an exaggerated response to the onset of darkness, as well as (b) a
large increase in activity near the end of the dark period
which also occurred slightly earlier in time than did the
usual pre-light increase in activity shown by normal
animals. Animal [9 also showed a consistent exaggeration
of ambulatory activity peaks in producing the ‘W-shaped’
pattern that characterizes dark-phase activity. Thus,
eliminating forebrain NE in these animals seems to have
removed a moderating influence on ambulatory activity; as
a result, the animals responded in an exaggerated manner
to the excitatory stimuli, both internal and external, that
give rise to ambulatory activity at the time points where
the exaggerated responses were seen. In a comprehensive
review of basic research relating to the function of dorsal
bundle noradrenergic pathway that originates in the LC,
Robbins, Everitt and colleagues [19] concluded that the
functioning of this system allows an animal to respond in a
discriminating way to the environment (pg 146). While the
theoretical formulation of Robbins and Everitt emphasizes
how NE influences attention and stimulus processing, their
formulation seems consistent with the possibility that
forebrain NE serves to moderate behavioral responses in
many situations.
When evaluating the results described here, these findings should not be interpreted as demonstrating that action
of NE in the brain does not affect motor activity. First, the
present study assessed spontaneous ambulatory activity in
the home cage and did not examine motor responses that
occur within brief periods of time and / or in reaction to
challenging or novel events. Second and perhaps most
important, the study described here utilized lesion techniques to assess the influence of forebrain NE on behavior.
While this methodology has a long history with respect to
the question of how NE is related to motor activity, it has
distinct drawbacks. Long-term reduction or removal of
brain NE, which is what the technique brings about, does
not reproduce how a change in NE normally occurs to
exert its influence; in physiological function, transient
increases or decreases in synaptic NE concentration are
what occur to affect function. As a consequence, such
reduction or removal of a substance that acts acutely,
particularly one that appears to exert a modulatory influence, ultimately may not prove to be a good method for
assessing its effects. This may explain why techniques that
acutely perturb NE neurotransmission (e.g., [1,2,26,31])
have observed effects on motor behavior with more
regularity than studies in which brain NE has been
removed by lesion techniques. Such observations argue
strongly against overgeneralization of the present findings
to conclude that brain NE does not affect motor activity.
130
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
But regardless of the possibility that making acute perturbations of NE action in the brain may reveal appropriate
physiological functions of brain NE that are not seen when
NE is chronically removed from the brain, the present
results do indicate that the presence of NE in the forebrain
is not required for the rat to generate normal diurnal
patterns of ambulatory activity.
[14]
[15]
[16]
Acknowledgements
The authors gratefully acknowledge the assistance of
Sandra Parks in preparation of this manuscript. This
research was supported in part by grant MH56602 from the
National Institute of Mental Health.
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www.elsevier.com / locate / bres
Short communication
Depletion of brain norepinephrine does not reduce spontaneous
ambulatory activity of rats in the home cage
James W. Murrough, Katherine A. Boss-Williams, Milburn S. Emery, Robert W. Bonsall,
Jay M. Weiss*
Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Emory West Campus, 1256 Briarcliff Road, NE Atlanta,
GA 30322, USA
Accepted 15 August 2000
Abstract
6-Hydroxydopamine (6-OHDA) lesions of brain noradrenergic neurons and terminals were made in rats to assess the importance of
forebrain norepinephrine (NE) for mediating circadian patterns of spontaneous ambulatory activity that rats show in the home cage.
6-OHDA was injected intracranially into the fibers of the ascending noradrenergic dorsal and ventral bundle pathways or infused into the
lateral ventricle or both. Rats living in a 12 / 12 h light / dark cycle exhibit a marked increase in ambulatory activity during the dark period
in comparison to the light period and a ‘W-shaped’ pattern of activity during the 12 h of the dark phase. Results showed that near-total
depletion of brain NE did not impair the capacity to generate normal patterns of spontaneous ambulatory activity that occur in the home
cage. In the animals that sustained the most complete NE lesions, the amounts of activity generated at times of peak activity were
exaggerated in comparison to the control animals, which is consistent with the possibility that NE in the brain exerts a moderating
influence on behavior. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Monoamines and behavior
Keywords: Motor activity; Norepinephrine; 6-hydroxydopamine; Ambulation; Circadian rhythm
Altered activity of noradrenergic neurons in the brain
has long been thought to be critically involved in the
pathophysiology of depression. The ‘catecholamine hypothesis of depression,’ first put forth over 30 years ago,
proposed that depression arose from a deficiency of
norepinephrine (NE) in the brain [4,22,23]. Although
shortcomings of this formulation became evident soon
after it was proposed, evidence nevertheless has continued
to accumulate indicating that brain NE is important in
depression (reviewed in Grant and Weiss [12]). The
present study examined the effect of reducing brain NE on
spontaneous motor activity of rats. Because altered motor
activity is one of the most common changes seen in
depression, the relationship between brain NE and motor
*Corresponding author. Tel.: 11-404-712-9771; fax: 11-404-7129755.
E-mail address: [email protected] (J.M. Weiss).
activity has been of considerable interest since the catecholamine hypothesis of depression was proposed.
Despite the close connection between brain NE and
motor activity suggested by the catecholamine hypothesis
of depression, basic research has by no means provided
unambiguous evidence showing that alteration of brain NE
will affect motor activity. Experimental manipulation of
brain NE, most commonly by making lesions of brain
noradrenergic neurons, often has failed to produce a
change in motor activity of animals [7,19,27]. A recent
study using a genetic manipulation to investigate the role
of NE (i.e., elimination of the gene for dopamine betahydroxylase) also reported no significant effect on spontaneous motor activity of mice unable to synthesize NE [28].
On the other hand, other studies have reported that NE
depletion leads to a decrease in motor activity in a novel
environment, suggesting a role for brain NE with respect to
motor activity under these conditions [3,16,21]. In addi-
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02850-X
126
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
tion, early studies found that catecholamine-depleting
drugs could reduce behavioral responses that are highly
dependent on motor activity in rats (e.g., [17,18]). And,
finally, a strong correlation between regional brain NE
depletion and decreased motor activity has been shown in
the context of a widely-studied animal model of depression
[24,25,29,30].
In the present study, we investigated the influence of
brain NE on diurnal changes in spontaneous ambulatory
motor activity of rats. Freely-behaving rats, living in a 12 h
on / 12 h off light / dark cycle, exhibit a pattern of spontaneous motor activity such that the animals’ ambulatory
activity is markedly elevated during the dark period of the
day in contrast to the light period when animals are much
less active. Moreover, animals show a characteristic pattern of activity during the ‘active’ dark phase. This pattern
shows an upward spike in ambulatory activity at the
beginning of the dark phase, after which activity then
declines, only to increase somewhat around the middle of
the dark period, after which it declines again, followed by
marked increase leading up to the end of the dark period
(i.e., a ‘W-shaped’ pattern). In this study, we examined
whether destruction of NE neurons and terminals in the
brain would affect these patterns of spontaneous ambulatory activity.
To alter brain NE, lesions of the ascending noradrenergic pathways — the dorsal noradrenergic bundle (DNB)
and ventral noradrenergic bundle (VNB) — were made
using the neurotoxin 6-hydroxydopamine (6-OHDA). Subjects were male Sprague–Dawley rats weighing approximately 400 g at time of the study. Sixteen animals were
assigned to one of three lesion groups (i.e., the axonal,
ventricular, or compound lesion group) or the control
group (n54 per group). Animals were anesthetized with
1–3% halothane mixed with oxygen and placed into a
stereotaxic instrument where surgery was performed. The
‘axonal’ lesion group received bilateral microinjections of
6-OHDA hydrochloride (Sigma) aimed at fibers of the
DNB and VNB just anterior to the locus coeruleus (LC).
Axons arising from NE-containing cells of the LC comprise the DNB, which provides over 70% of the NE in the
rat brain including all the NE in the neocortex and
hippocampus [9,13,15]. The VNB arises from noradrenergic neurons caudal to the LC and terminates in various
regions of the diencephelon, providing particularly rich
innervation to the hypothalamus [5,11]. After an incision
was made in the skin covering the skull and the skin
retracted, two small holes were drilled into the skull to
introduce a 26-gauge cannula bilaterally into the brain.
Stereotaxic coordinates were used for placement of the
cannulae. For axonal lesions, the coordinates for cannula
location were as follows: with nose piece 210.08; anterior /
posterior (AP) 22.6 mm from lambda; medial / lateral
(ML)61.2 mm from midline; dorsal / ventral (DV) 27.0
mm and 28.0 mm from dura matter. 6-OHDA was freshly
dissolved in artificial cerebrospinal fluid (aCSF) and
delivered through the cannula into each side of the brain. A
cannula was first lowered into one side of the brain to
28.0 mm, targeting axons of the VNB and 1 ml of
6-OHDA (4 mg / ml) was injected over a period of half a
minute. After a wait of 1 min, the cannula was raised to
27.0 mm, targeting axons of the DNB and a second 1 ml
injection of 6-OHDA was performed, followed by a wait
of 1 min and then the cannula was removed. Thirty
minutes after completion of the injection on one side of the
brain, the procedure was repeated on the other side of the
brain (the 30-min delay between injections was imposed to
promote animal survival). The ‘ventricular’ lesion group
received a microinfusion of 6-OHDA into the lateral
ventricle. For this group, the surgical procedure was the
same as described above except that a single hole was
drilled in the skull for insertion of a single 26-gauge
cannula. Stereotaxic coordinates used for this condition
were as follows: with nose piece level with skull (23.38),
AP 21.0 mm from bregma; ML61.5 mm; DV 23.5 mm
from skull (variable). After the cannula was lowered at this
location, placement in the ventricle was confirmed by the
observation of cerebrospinal fluid rising in a small length
of silastic tubing attached to the cannula. 6-OHDA introduced into the ventricle normally would be taken up into
dopamine (DA)-containing cells and terminals as well as
NE-containing neurons and would also destroy DA cells
and terminals. To prevent lesioning of DA terminals and
cells, the DA uptake blocker GBR 12909 (Sigma) was
infused through the ventricular cannula just prior to
infusion of the 6-OHDA. GBR 12909 was dissolved in
warm (378C) aCSF (1 mg / ml) and 6 ul was infused at a
rate of 2 ml / min. Following a wait of 15 min to allow time
for the drug to bind to DA transporters, 4 ml of 6-OHDA
(40 mg / ml) was then infused at a rate of 2 ml / min. to
complete the ventricular infusion procedure. The ‘compound’ lesion group underwent both the axonal lesion
procedure and the ventricular lesion procedure. For this
group, first the axonal lesion was carried out and then,
after a recovery period of 2 weeks, the ventricular lesion
procedure was conducted. All doses and surgical procedures used for the compound lesion group were the same
as described above for the axonal and ventricular lesion
groups. For the control group, these animals were anesthetized with halothane for the amount of time that surgery
normally required but no surgery was performed (control
animals were subjected to minimal manipulation so that
their subsequent ambulatory behavior would not be affected by any experimental procedure and thus would constitute normal activity for a rat).
Four weeks after surgery was initiated, animals were
placed individually into standard polycarbonate laboratory
cages (45325320 cm). Each of these cages was mounted
within a photocell assembly (ANA 1219, Riverpoint
Electronics) that permitted the continuous monitoring of
the activity of the animal in the cage. The photocell
assemblies were connected to a computer which recorded
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
and tabulated the number of interruptions of the photocells
by each animal. Animals were housed directly on bedding
and received ad libitum food and water. Five days was
allowed for acclimation to individual housing, followed by
7 days of monitoring the animals’ nocturnal spontaneous
motor activity. On each day, the recording apparatus was
activated 2 h before onset of the dark phase and recording
ended 5 h after lights on. Ambulatory motor activity was
expressed as ambulation counts per hour, an ambulation
count being defined as interruption of a photocell beam
that had not been broken in the previous four beam breaks
and thus represented horizontal locomotion by the animal.
At the conclusion of the study, the animals were
sacrificed and brains removed. The following brain regions
were dissected and retained for analysis: LC region of
brain stem (LC), hypothalamus (HYP), hippocampus
(HIPP), prefrontal cortex (PFC) and striatum / nucleus
accumbens (STR). Brain samples were frozen at 2858C
for analysis of catecholamine content using high pressure
liquid chromatography (HPLC). Tissue samples were
analyzed for NE content to assess the extent of the lesion;
striatal tissue was principally analyzed for DA to assess
whether DA-containing terminals had been protected from
destruction by infusion of GBR 12909 prior to icv infusion
of 6-OHDA. Tissues were homogenized in 0.1 M perchloric acid containing an internal standard. Catecholamines were separated using reverse-phase, ion–pair
HPLC and were quantified by an electrochemical detector
(ESA Coulochem II) attached to a computerized data
acquisition system (Perkin Elmer Turbochrom 4). Protein
content of each sample was measured by the Lowry
technique so that catecholamine content could be expressed as pg NE (or DA) per mg tissue protein.
As expected, all lesion groups (axonal, ventricular,
compound) showed marked depletion of NE in brain
regions known to be major targets of the ascending
noradrenergic pathways, i.e., cortex and hippocampus for
the DNB and hypothalamus for the VNB. The results are
shown in Table 1. 6-OHDA administered icv was found to
be somewhat more effective for depleting NE in the
terminal regions of the DNB than was 6-OHDA aimed at
127
the axon bundles, although a difference between these
techniques was not observed in the hypothalamus. Hypothalamic NE proved somewhat more resistant to neurotoxic
depletion than did forebrain NE, consistent with previous
reports [20]. The compound lesion group showed the
largest reduction of NE, indicating that the ventricular and
axonal lesion techniques had cumulative effects. In all
animals of the compound lesion group, NE was undetectable in the hippocampus and cortical NE was almost
equally reduced. The compound lesion technique also was
able to deplete hypothalamic NE to an average of 10% of
control, with two animals of the group found to have less
than 10% of the control level of NE remaining. DA levels
were not reduced by either the axonal or ventricular lesion
techniques as estimated by DA content in striatum / nucleus
accumbens, while the compound lesion technique produced a small reduction in DA (i.e., 30%) that is not
considered to be functionally effective [6,14].
Mean (6standard error) hourly ambulatory activity of
the different groups during the 19 h of the day when
activity was recorded is shown in Fig. 1, with the activity
of each lesion group plotted against the control group.
These data were analyzed by analysis of variance (twoway [group3h] with repeated measures across the hour
factor) which was carried out for each of the three panels
shown in the Fig. 1. No analysis generated an effect of
group that approached significance, thereby indicating that
depletion of brain NE by these lesion techniques did not
cause a change in spontaneous motor activity in comparison to control animals. The total nocturnal ambulatory
counts for each of the lesion groups, which was compared
to the control group by t-test, showed no difference that
approached significance. Moreover, inspection of Fig. 1
shows that even the ‘W-shaped’ pattern of dark phase
activity that occurs in normal rats was intact in the
lesioned animals. The only potential departure from this
was seen in the ventricular lesion condition, where a
somewhat reduced elevation in the first hour of activity
after onset of the dark period was seen. However, the lack
of an overall effect of group in the analysis of variance that
compared this group with the control condition, which was
Table 1
Effects of three different 6-OHDA lesion techniques on brain catecholamine levels a
Groups
Norepinephrine
Dopamine
HIPP
Control
Ventricular
Axonal
Compound
a
PFC
HYP
LC
STR
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
pg/mg
protein
% of
control
14836117
15615
4286157
0
1
29
0
19166118
203689
4326177
1267
11
23
1
66276498
22306220
18086442
6926165
34
27
10
11 7766458
334061099
20196630
9836251
28
17
8
41 87764864
39 44362165
52 67963394
29 27063806
94
125
70
The data are given as group means6S.E.M. The control group was non-lesioned. The ventricular group received 6-OHDA injected into the lateral
ventricle; the axonal group received 6-OHDA injected into the fibers of the dorsal and ventral bundle ascending noradrenergic pathways; the compound
group received both treatments. Sensitivity of the HPLC electrochemical detection was 2.0–3.0 pg / mg protein for hippocampal and cortical
norepinephrine.
128
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
Fig. 1. Mean (6S.E.M.) hourly ambulatory activity of animals with
lesions of the noradrenergic neurons in the brain in comparison to
non-lesioned animals. Activity data was collected throughout a 12 / 12 h
light / dark cycle over a period of 7 days; monitoring began 2 h prior to
onset of the dark phase and ended 5 h after onset of the light phase. Each
panel compares the non-lesioned animals (control group) to one of three
lesion groups (n54 / group). The top panel shows animals who received
microinfusion of 6-OHDA into the lateral ventricle (ventricular lesion
group); the middle panel shows animals who received microinjections of
6-OHDA into the fibers of ascending noradrenergic projections (axonal
lesion group); the bottom panel shows animals who were subjected to
both lesion procedures (compound lesion group). No statistically significant differences were found. The horizontal black bar designates duration
of the dark phase.
noted above, means that the difference at this single time
point is statistically attributable to chance; also, the failure
to see a similar change in any other lesioned group
indicates that this was in all likelihood a chance occurrence. In summary, not only did lesioned rats fail to show
any overall difference in ambulatory activity from control
rats but they also showed an unchanged pattern of nocturnal activity; i.e., like the control (normal) rats, lesioned rats
showed a marked increase in activity at the onset of the
dark period, followed by a decline and then another
smaller peak in activity toward the middle of the dark
period, followed finally by another large increase in
activity leading up to onset of the light period. Finally,
ambulatory activity during hours of light also was similarly
low in all groups.
What is indicated by these data is that forebrain NE is
not necessary for the rat to generate a normal pattern of
ambulatory activity in a home cage situation during light
and dark phases of the day. Lesions of NE neurons and
terminals that virtually eliminated NE from the forebrain
neither reduced total spontaneous motor activity nor
impaired ability to generate increases in nocturnal ambulatory activity that characterizes normal rat behavior. But
insofar as the technique used for affecting NE in the
present study was to make a lesion that would reduce brain
NE, an important issue to consider is whether compensatory increases in the function of noradrenergic systems in
the brain (such as upregulation of postsynaptic adrenergic
receptors) might have overcome effects of decreased
transmission of brain NE. Thus it might be argued that,
despite reduced NE levels in the brain, noradrenergic
neurotransmission in the lesioned animals nevertheless was
functioning well enough to mask motor deficits that will
result from the depletion of NE. With regard to this issue,
studies that have assessed compensation after experimentally-produced reduction of catecholamine content in brain
agree that compensation indeed occurs but also indicate
that compensation sufficient to return functional responses
to normal generally requires 10% or more of the normal
amount of NE or DA to be present [6,8,10,14]. In regard to
the present experiment, the study of Curet and Montigny
[8] is particularly relevant. These investigators examined
electrophysiological responses of hippocampal neurons
following electrical stimulation of the locus coeruleus
(which will cause NE to be released in the hippocampus).
In part of the study, responses were assessed in rats whose
NE had been depleted by intraventricular infusion of 6OHDA; electrophysiological measurement was made 14–
21 days after the NE lesioning procedure had been carried
out. In comparison to naturally-occurring physiological
stimuli that activate LC neurons, electrical stimulation of
LC is a very strong stimulus for NE release, so that this
technique is likely to ‘test the limits’ by recruiting whatever NE is still available for release. In animals that had
been infused with 6-OHDA, the normal influence of LC
stimulation on hippocampal neurons was not diminished
until NE depletion in the hippocampus was found to reach
90%, but with depletions above this level, the influence of
stimulation began to decline and no influence at all of LC
stimulation was seen (i.e., no evidence of any compensation being present) when NE depletion was measured as
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
being total (i.e., 100%). Thus, Curet and Montigny reported that substantial depletions of NE (at least 90%)
were needed to alter the influence of NE on hippocampal
cells that was produced by electrically stimulating the LC,
but even a strong stimulus for release such as this had no
effect at all when NE depletion was judged to be complete.
In the present study, NE lesions were quite extensive, with
the compound lesion technique producing near-total depletion of NE in the forebrain. In this regard, Fig. 2 shows the
individual activity data of the two animals in the compound lesion group that were found to have the most
complete lesions in the study. For these two subjects
(animals [7 and [9), no NE at all was detected in
prefrontal cortex and hippocampus (assay sensitivity for
these regions was less than 3.0 pg / ml protein, which was
less than 0.2% of control levels) and the reduction in
hypothalamic NE was 95% and 92% respectively. Examination of Fig. 2 reveals that these animals showed undiminished elevation of ambulatory activity in response to
the onset of darkness and also showed a similar ‘W’
pattern of ambulatory activity during the dark period as
was shown by control animals. These results in animals
whose NE depletions were so large as to preclude the
possibility of any significant compensation indicate, together with the other findings shown in Fig. 1, that
forebrain NE is not necessary for the ambulatory behavior
assessed in this study.
The results shown in Fig. 2 also may shed some light on
the function of forebrain NE. Rather than showing a failure
to respond, the two lesioned subjects whose data are shown
in Fig. 2 can be seen to manifest exaggerated ambulatory
responses at times when nocturnal ambulatory activity
normally increases. In evaluating these results, it should be
Fig. 2. Hourly ambulatory activity of two individual rats with virtually
complete ablation of central NE compared with the mean (6S.E.M.)
activity of the control group (n54). In the case of the individual animals,
error bars represent variation in hourly activity recorded on 7 days; the
small magnitude of error bars indicates that the pattern of activity shown
was stable from one day to the next. The horizontal black bar designates
duration of the dark phase within a 12 / 12 h light / dark cycle.
129
considered that the data shown for the two lesioned
animals in Fig. 2 are means (6SEs) calculated from
recordings made over 7 days, so that elevated activity at a
particular hour was not an isolated occurrence on a single
day but, instead, represents a consistent daily pattern.
Therefore, animal [7 consistently showed (a) an exaggerated response to the onset of darkness, as well as (b) a
large increase in activity near the end of the dark period
which also occurred slightly earlier in time than did the
usual pre-light increase in activity shown by normal
animals. Animal [9 also showed a consistent exaggeration
of ambulatory activity peaks in producing the ‘W-shaped’
pattern that characterizes dark-phase activity. Thus,
eliminating forebrain NE in these animals seems to have
removed a moderating influence on ambulatory activity; as
a result, the animals responded in an exaggerated manner
to the excitatory stimuli, both internal and external, that
give rise to ambulatory activity at the time points where
the exaggerated responses were seen. In a comprehensive
review of basic research relating to the function of dorsal
bundle noradrenergic pathway that originates in the LC,
Robbins, Everitt and colleagues [19] concluded that the
functioning of this system allows an animal to respond in a
discriminating way to the environment (pg 146). While the
theoretical formulation of Robbins and Everitt emphasizes
how NE influences attention and stimulus processing, their
formulation seems consistent with the possibility that
forebrain NE serves to moderate behavioral responses in
many situations.
When evaluating the results described here, these findings should not be interpreted as demonstrating that action
of NE in the brain does not affect motor activity. First, the
present study assessed spontaneous ambulatory activity in
the home cage and did not examine motor responses that
occur within brief periods of time and / or in reaction to
challenging or novel events. Second and perhaps most
important, the study described here utilized lesion techniques to assess the influence of forebrain NE on behavior.
While this methodology has a long history with respect to
the question of how NE is related to motor activity, it has
distinct drawbacks. Long-term reduction or removal of
brain NE, which is what the technique brings about, does
not reproduce how a change in NE normally occurs to
exert its influence; in physiological function, transient
increases or decreases in synaptic NE concentration are
what occur to affect function. As a consequence, such
reduction or removal of a substance that acts acutely,
particularly one that appears to exert a modulatory influence, ultimately may not prove to be a good method for
assessing its effects. This may explain why techniques that
acutely perturb NE neurotransmission (e.g., [1,2,26,31])
have observed effects on motor behavior with more
regularity than studies in which brain NE has been
removed by lesion techniques. Such observations argue
strongly against overgeneralization of the present findings
to conclude that brain NE does not affect motor activity.
130
J.W. Murrough et al. / Brain Research 883 (2000) 125 – 130
But regardless of the possibility that making acute perturbations of NE action in the brain may reveal appropriate
physiological functions of brain NE that are not seen when
NE is chronically removed from the brain, the present
results do indicate that the presence of NE in the forebrain
is not required for the rat to generate normal diurnal
patterns of ambulatory activity.
[14]
[15]
[16]
Acknowledgements
The authors gratefully acknowledge the assistance of
Sandra Parks in preparation of this manuscript. This
research was supported in part by grant MH56602 from the
National Institute of Mental Health.
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