The Japanese Journal of Psychiatry
`and Neurology, Vol. 48, No.
`l,
`l994
`
`Intracranial Self-Stimulation and Locomotor Traces as Indicators
`
`for Evaluating and Developing Antipsychotic Drugs
`
`Morikuni Takigawa, M.D., Hiroshi Fukuzako, M.D.,‘ Kenichi Ueyarna, M.D."
`
`and Hidefumi Tominaga, M.D.'
`
`Health Service Center, Kagoshima University, Kagoshima
`‘Department ofNe1tropsychiotry, Faculty of Medicine,
`Kagoshima University, Kagoshima
`
`Abstract: When chlorpromazine (CPZ) and lithium chloride (LiCl) are compared, the former
`suppresses both rat's intracranial self-stimulation (ICSS) and methamphetamine (MAP)-
`induced hyperactivity. On the other hand, the latter suppresses only MAP-induced abnormal
`hyperactivity but hardly suppresses a purpose-oriented ICSS associated with the reward system.
`Therefore, LiCl inhibits abnormal hyperactivity induced by MAP. but it does not suppress
`physiological motivation. Using the two types of antipsychotic drugs, the authors propose a
`method of combining the ICSS and locomotor activity together with its traces. These proposals
`are useful indicators for evaluating and developing the new antipsychotic drugs which are used
`clinically for psychotic patients and for understanding the drug-induced akinesia and anhedonia.
`
`Key Words:
`
`selfistimulction. locomotor truce. indicator, anhedonia. cnt1',ru'ychot:'c drug
`
`Jpn J Psychiatry Neurol 48: 127-132, 1994
`
`INTRODUCTION
`
`When electrodes are implanted in the rat
`brain reward system around the median fore-
`brain bundle, which passes through the lateral
`hypothalamus” *5, the rat tends to push the
`lever repeatedly causing continuous electrical
`stimulation of its brain reward system. This
`phenomenon was discovered in 1954 by Olds
`and Miner” and is called “ICSS” or “brain-
`stimulation reward.” Neuroleptics, which
`affect human emotion, are known to affect
`ICSS." Fluorescent
`histological
`studies
`
`Received for publication on Oct. 5, 1993.
`Mailing address: Morikuni Takigawa, M.D.,
`Department of Health Service Center, Kagoshima
`University, 1-21-24 Koorimoto, Kagoshima 890,
`Japan.
`
`suggested a relationship between the sites of
`ICSS and the catecholamine-associated nerv-
`
`ous projection.1° 13 Following these findings,
`ICSS has gained importance from a neuro-
`pharmacological viewpoint.
`The etfectiveness of lithium salt
`
`in the
`
`treatment of depression was first reported in
`1949 by Cade-.5 Later, Schou et at.“ re-
`evaluated the antidepressant action of lithium
`salt. At present, this drug is indispensable in
`the treatment of manic-depressive psychosis.
`When compared to conventional neurolep-
`tics of the phenothiazine,
`lithium can be
`characterized by a more natural sedating
`action and a slower manifestation of thera-
`
`peutic effect. However, there are many open
`questions regarding the etfect of this drug.
`As part of a series of behavioral pharmaco-
`
`1of6
`
`Alkermes, Ex. 1038
`
`1 of 6
`
`Alkermes, Ex. 1038
`
`

`
`128
`
`M. Takigawa er al.
`
`logical studies of the action mechanism of
`lithium salt, the authors recently studied the
`effect of lithium chloride (LiCl) on ICSS, and
`compared it with the phenothiazine used
`clinically for endogenous psychosis.
`
`SUBJECTS AND METHODS
`
`Male Wistar rats, with a mean weight of
`300 g, were used with 20 rats for ICSS assess-
`ment and 20 rats for behavioral observation.
`
`Under anesthesia with intraperitoneal so-
`dium pentobarbital
`(50-60 mg/kg; Nem-
`butal), bipolar electrodes for stimulation of
`ICSS were implanted into the lateral hypo-
`thalamus (LH), with reference to the brain
`atlas of Ktienig and K.lippel.9 One week later,
`each rat was trained for ICSS for 5 days using
`a Skinner box (28><16><3l cm). Biphasic
`rectangular waves, with a pulse width of 0.2
`msec and of a frequency of 80 Hz, were
`applied to the LH.” The stimulus constant
`current was monitored on an oscilloscope
`(Cs-1562, Trio). The effect of continuous
`LiCl and/or phenothiazine treatment on
`ICSS and MAP-induced hyperactivity was
`examined as follows.
`
`injected
`LiCl, dissolved in saline, was
`intraperitoneally into rats at a dose of 25
`or 50 mg/kg. Treatment with LiCl or
`phenothiazine was done daily (at about 1:00
`p.m.)
`for 8-consecutive days. Neuroleptics
`
`have been usually effective clinically several
`days after treatment. On the 9th day of the
`drug treatment, we examined the frequency
`of ICSS following 15 minutes after adminis-
`tration of sham saline treatment for every 30-
`minute period and these frequencies of ICSS
`were compared with those of 9-consecutive
`saline treatment as shown in Table I. And
`
`then using a similar time schedule as above,
`the spontaneous activity of the rats during a
`30-minute period was examined by using an
`activity monitor (Automex, Columbus In-
`strument) and was also compared with the
`saline activity. The statistical significance of
`inter-group differences in the ICSS frequency
`and the activity was tested by Tukey Test.
`After the last LiCl administration, blood
`samples were taken from the rats for determi-
`nation of the serum lithium levels by atomic
`absorption spectrometry.
`In a group of
`animals in the ICSS study,
`1 mA of direct
`current was applied to the tip of the elec-
`trodes for about 30 seconds at the end of the
`
`experiment. Then, using thionine staining, the
`tip of the electrodes within the LH was con-
`firmed.
`
`RESULTS
`
`Eject of CPZ and LECI on ICSS
`
`The rats which showed the stable bar—push-
`
`Table 1 :
`
`30min
`
`60min
`
`846.?.'2‘l4].2
`
`ICSS (MiSD, N=20) of the Rats Treated with CPZ and LiCl, Respectively
`and q(5,95) Values (Given in Parentheses), Compared with ICSS Counts
`for Saline Using the Tukey Multiple Comparison Test
`Saline
`CPZ
`CPZ
`LiCl
`LiCl
`1.0 ml/kg
`0.5 mg/kg
`1.0 mg/kg
`25.0 mg/kg
`50.0 mg/kg
`828.9114-2.3
`20l.9i34.6"'
`l3.0:“4-.9"
`959.2i22l.6'
`736.2‘: 103.?
`(223105)
`(284526)
`(4.5719)
`(3.2526)
`185.T+3S.8“‘
`9.8i3.l"'
`968.8ilSl.l"
`';"a"9_3i 86.1
`(29.0677)
`(368029)
`(53693)
`(23639)
`6S.lLl4-.8"
`T.0:‘_‘2.5"'
`986.6i2l0.5“
`804.34: 85.3
`(304608)
`(317070)
`(11650)
`(1.332?)
`63.3:l3.3"
`6.61‘2.l"
`945.lTi‘l'.r'T.l'
`740.21 84.0
`(322343)
`(3411066)
`(4-.6610)
`(19205)
`ICSS: lnlracraniai self-stimulation, CPZ: Chlorpromazine. LiCl: Lithium chloride.
`": p<0.Dl. ‘: p-(0.05.
`
`90min
`
`853.0: 122.5
`
`120 min
`
`833.Til36.D
`
`2of6
`
`Alkermes, Ex. 1038
`
`2 of 6
`
`Alkermes, Ex. 1038
`
`

`
`Self-Stimulation and Locomotor Traces as Indicators
`
`129
`
`ing behavior were regarded as ICSS positive.
`The mean stimulus current in 20 ICSS posi-
`tive rats were 352 ,ttA (200—4-50 ,uA).
`Table 1 shows the means (M) and standard
`deviation (SD) of
`ICSS counts per 30
`minutes. They were counted every 30 minutes
`from 30 to 120 minutes after treatment by
`CPZ with doses of 0.5 and 1.0 mg/kg, and by
`LiCl with doses of 25.0 and 50.0 mg/kg. The
`mean counts of CPZ and LiCl groups across
`the 20 rats were compared with saline using
`the Tukey multiple comparison test between
`saline and CPZ, or saline and LiCl groups.
`The values in the parentheses of Table 1 are
`the q (5,95)9 values of the Tukey test. The
`notation q (5,95) indicates that the number
`of degrees of free-dom=5 and number of
`samples=95.
`(Levels of significance of the
`Tukey test: P< 0.01 and P < 0.05). In order to
`examine the effect of different doses,
`the
`Tukey test was also performed on the groups
`of different CPZ and LiCl. CPZ showed an
`
`Saline (1.0 mlfkg)
`
`200
`
`290
`
`E 200
`8
`
`J5 +
`
`30
`
`80
`
`min
`
`l2l]
`
`CPZ {1.0 mgfkg)
`
`15+
`
`3{}
`
`5|]
`
`min
`
`l2[]
`
`LiCl (50.0 mgfltg)
`
`30
`15 *
`treatment
`
`50
`
`min
`
`120
`
`1: The example of cumulative fre-
`Fig.
`quentry of ICSS after treatment with CPZ and
`LiCl. (CPZ: Chlorpromazine, LiCl: Litl1iun1
`chloride.)
`
`obvious inhibition effect on ICSS. Compared
`to the saline, the ICSS of CPZ treated groups
`(0.5 and 1.0 mg/kg) decreased significantly
`with the passage of time. On the other hand,
`the ICSS of LiCl treated groups presented
`dilferent results for different doses. ICSS in-
`
`creased slightly for the dose of 25.0 mg/kg
`whereas it showed an insignificant variation
`for 50.0 mg/kg dose.
`Fig.
`1 shows the example of cumulative
`frequency of ICSS. When compared to the
`frequency recorded for saline, the ICSS fre-
`quency decreased markedly after treatment
`with CPZ (1.0 mg/kg). On the other hand, in
`the rats treated with LiCl (50.0 mg/kg), the
`depressive effect on ICSS was not observed.
`
`Effect of CPZ and LECI
`on Rat's Hyperactivity Induced by MAP
`
`When the rats were placed in an open field,
`they began to move slowly along the edge of
`the square space and occasionally stood up.
`The
`authors
`treated MAP to get
`the
`hyperactivity of the rats which remained sta-
`tionary in this experiment. Table 2 shows a
`locomotor count of behavior for each drug
`during a 30-minute period. In the rats treated
`with 1.0 mg/kg MAP, the locomotor count
`began to remain stationary after treatment.
`As shown in Table 2, MAP induced-
`hyperactivity of the rat was inhibited by pre-
`treatment with CPZ and LiCl
`in a dose-
`
`dependent manner. The degree of suppression
`was much stronger for CPZ than LiCl. The
`mean counts of the CPZ and LiCl groups
`across the 20 rats were compared with MAP
`using the Tukey multiple comparison test be-
`tween the MAP and CPZ or the MAP and
`
`LiCl groups. The values in the parentheses of
`Table 2 are the q(5,95) values of the Tukey
`test.
`
`determined
`serum lithium level,
`The
`around one hour after the last LiCl admin-
`
`istration, was 0.558 mEq/liter for the 25.0
`mg/lcg group and 1.29 rnE¢1/liter for the 50.0
`mg/kg group, respectively.
`Fig. 2 shows the locomotor traces of rat,
`after treatment with saline, MAP, CPZ and
`
`3of6
`
`Alkermes, Ex. 1038
`
`3 of 6
`
`Alkermes, Ex. 1038
`
`

`
`130
`
`M. Takigawa et at.
`
`Table 2: The Locomotor Counts (ML SD, N=20) of the Rats Pretreated with CPZ
`and LiCl, Respectively, and q(5.95) Values (Given in Parentheses), Compared
`with MAP-Induced Counts Using the Tukey Multiple Comparison Test
`
`
`MAP
`1.0 mg/kg
`
`30 min
`
`58':-'.3i'l'l.4
`
`60min
`
`564.1’: 73.2
`
`90min
`
`53:.6t5s.4
`
`120 min
`
`S0l'.'l':59.9
`
`CPZ
`0.5 mg/kg
`
`96.2+lfi.3"
`(404031)
`s7.2-- 9.6"‘
`(484279)
`54.0: 6.0“
`(559250)
`36.0i 3.9"
`(553314)
`
`CPZ
`1.0 mg/kg
`
`36.1L5.4'"
`[45.34'.t6)
`24.1t3.7“"
`(526234)
`12312.2"
`(60.80'?9)
`11.6 " 2.1"
`(5s.s772)
`
`LiCl
`25.0 mg/kg
`
`576.3+48.l
`(09050)
`293.6+61.1"‘
`(263896)
`235.1:58.1“
`(343139)
`l28.9:S6.l"‘
`(449560)
`
`LiCl
`50.0 mg/kg
`
`4l6.8i34.l"‘
`(143271)
`147.1:36.9“
`(406502)
`92.3 1.21.6“
`($1.331?)
`'?0.9i 18.4"
`(513395)
`
`MAP: Methamphetamine. ' ' : p<0.01_
`
`In the rat pre-
`type.
`increased peripheral
`treated with 1.0 mg/kg CPZ, however,
`the
`increased peripheral type of MAP was dis-
`turbed due to CPZ action and changed into a
`trace showing an irregular type. In the rat 30
`minutes pretreated by LiCl (50 mg/kg),
`it
`was accompanied by a marked decrease in the
`MAP-induced hyperactivity but showed a rel-
`atively regular peripheral
`type similar
`to
`saline.
`
`DISCUSSION
`
`This paper compared the effect of CPZ and
`LiCl, using both ICSS and locomotor counts
`and its traces as an indicator of evaluating the
`effects of these drugs.
`The results show that CPZ significantly
`inhibit not only the MAP-induced hyper-
`activity but also ICSS which is associated
`with the reward system. It is known that the
`neuroleptics such as CPZ is effective for psy-
`chomotor excitation, hallucination and delu-
`sion. But those drugs have averse elfects and
`suppress both the moderate consciousness
`and normal motivation of the patients, so that
`the patients look blank, or sleepy and seem to
`be atfected by the drug-induced akinesia and
`anhedonia.3 These suggest that neuroleptics
`tend to inhibit even the normal physiological
`vivid emotion.
`
`Saline (1.0 ml,-’kg)
`
`MAP (1-0 mgfkgl
`_ _ .
`__,
`
`
`
`LiCl (50.0 mgfkg)
`
`{increased peripheral type)
`
`
`
`(peripheral type)
`
`CPZ (1.0 mglkg)
`
`
`
`{irregular type)
`
`(P¢'«1'iPh5Ta1 W139}
`
`Fig. 2: The increased traces of periphral
`type affected by MAP are suppressed after 30,
`min pretreatment of CPZ and LiCI.
`(MAP:
`Methamphetamine.)
`
`respectively. In the 1.0 ml/kg saline
`LiCl,
`treatment group, the rats moved slowly and
`the trace of behavior in this group of rats
`showed a peripheral type.
`In the 1.0 mg/kg MAP treatment group,
`the movement was increased and the rats
`
`frequently moved along the edge of the open
`field with hyperactivity showing a trace of an
`
`is
`it
`When we develop the neuroleptics,
`necessary to consider not only the sedation
`
`4of6
`
`Alkermes, Ex. 1038
`
`4 of 6
`
`Alkermes, Ex. 1038
`
`

`
`Self-Stimulation and Locomotor Traces as Indicators
`
`131
`
`effects for psychomotor excitation, hallucina-
`tion and/or delusion, but also the effect for
`the
`physiological motivation which
`is
`associated with the reward System.” '5
`Wake amines are known to exert acute
`
`CNS-stimulating effects for excitation, eleva-
`tion of the threshold for antihypnotic action,
`and reduction in fatigue.‘ Therefore, we
`assessed the effect of 8-day lithium salt treat-
`ment on the elevation of spontaneous activity
`induced by 1.0 mg/kg MAP treatment in rats.
`Berggren et
`(11.2 and Engel and Berggrenfi
`reported that
`intraperitoneal
`treatment of
`mice with 300 mg/kg or
`less of LiCl
`suppressed the hyperactivity induced by 3.0
`mg/kg or 7.8 mg/kg d-amphetamine, al-
`though they did not study the eifect of contin-
`uous lithium treatment. Furukawa et af.B
`
`compared the effect of intraperitoneal treat-
`ment and one- to three-week treatment with
`
`of LiCl in mice. In their study, hyperactivity
`induced by 2.0 or 5.0 mg/kg of d-am-
`phetamine was suppressed by the repeated
`LiCl treatment in a dose-dependent manner.
`Borison et at.‘ reported that an 8-day treat-
`ment of mice with 45.0 mg/kg of LiCl
`suppressed hyperactivity induced by 5 mg/kg
`of d-amphetamine.
`In the present study,
`hyperactivity induced by 1.0 mg/kg MAP
`was suppressed to some degree by an 8-day
`LiCl treatment in a dose-dependent manner.
`This finding is consistent with the reports of
`Furukawa et al.3 and Borison et all Lithium
`
`is known to suppress amphetamine-
`salt
`induced CNS stimulation in humans .1 7
`
`Ramsey et of.” reported that in rats, an 8-day
`treatment with 2 mEq/kg Li3CO3 (i.p.) did
`not significantly alter the frequency of ICSS
`when compared to rats treated with saline.
`Until now, not much research has been
`carried out at the same time comparing the
`effect of lithium salt on both ICSS and MAP-
`
`induced hyperactivity. In the present study,
`the frequency of ICSS was not significantly
`altered by the 8-day intraperitoneal treatment
`with LiCl (1.29 mEq/liter). This finding is
`consistent with the result of Ramsey er a1.”In
`the present study even when the repeated
`
`suppressed MAP-
`treatment
`lithium salt
`induced abnormal hyperactivity to some
`degree, the operant self-stimulation due to the
`brain stimulation reward system was not
`suppressed. This suggests that LiCl will not
`suppress normal motivation although it will
`sedate abnormal excitation. This is the reason
`
`why LiCl, when clinically used, will not
`induce the patient to drug-induced akinesia
`and anhedoniaa, which is an adverse side
`effect of neuroleptics such as chlorpromazine,
`etc.
`
`This research has shown that both the ICSS
`
`and locomotor traces are very useful indica-
`tors for the development of the new anti-
`psychotic drugs and evaluation of its effects.
`
`REFERENCES
`
`l. Angrist, B. and Gershon, S.: Variable attenua-
`tion of amphetamine effects by lithium. Am J
`Psychiatry 136: 806-810, 1979.
`2. Berggren, U., Tallstedt, L., Ahlenius, S. and
`Engel,
`I .: The effect of lithium on am-
`phetamine-indueed iocomotor
`stimulation.
`Psychopharmacology 59: 41-45, 1978.
`3. Bermanzohn, P.C. and Siris, S.G.: Akinesia:
`A syndrome
`common
`to parkinsonism,
`retarded depression, and negative symptoms
`of schizophrenia. Compr Psychiatry 33: 221-
`232, 1992.
`4. Borison, R. and Diamond, B.l.: Lithium pre-
`vention of amphetamine-induced “manic” ex-
`citement and of reserpine-induced “depres-
`sion” in mice: Possible role of 2-pheny-
`lethylamine. Psychopharmacology 59: 259-
`262. 1978.
`5. Cade, I.F.J.: Lithium salt in the treatment of
`psychotic excitement. Med I Australia 36:
`349-352, 1949.
`6. Engel, I. and Berggem, U.: Effects of lithium
`on behavior and central monoamines. Acta
`Psychiat Scand Suppl 61: 133-143, 1980.
`7. Flemenbaum, A.: Does lithium block the
`effects of amphetamine? A report of three
`cases. Am J Psychiatry 131: 820-321, 1974.
`8. Furukawa, T., Ushizima,
`I. and Ono, N.:
`Modifications by lithium of behavioral
`re-
`sponses
`to methamphetamine
`and tetra-
`benazine. Psychopharmacologia (Berl.) 42:
`
`5of6
`
`Alkermes, Ex. 1038
`
`5 of 6
`
`Alkermes, Ex. 1038
`
`

`
`M. Takigawa er al.
`
`132
`
`10.
`
`11.
`
`12.
`
`243-243, 1975.
`Kiienig, I.F.R. and Klippel, R.A.: The Rat
`Brain: A Stereotaxjc Atlas of the Forebrain
`and Lower Parts of the Brain Stem. Williams
`and Wilkins, Baltimore, 1963.
`Lindvall, O. and Bjiirklund, A.: The organiza-
`tion of the ascending catecholaine neuron sys-
`tems in the rat brain. Acta Physiol Scand
`Suppl-112: 1-48, 1974.
`Olds, J.: Differentiation of reward systems in
`the brain by self—stimulation technics.
`In:
`Electrical Studies on the Unanesthetized
`
`(Ed.), 0‘Doherty DS,
`Brain. Ramey E.R.
`Hoeber, New York. pp 17-51, 1960.
`Olds, J. and Milner, P.: Positive reinforce-
`ment produced by electrical stimulation of
`septal area and other regions of rat brain. J
`Comp Physiol Psychol 47: 419-427, 1954.
`Ramsey, T.A., Mendels, J., Hamilton, C. and
`
`14.
`
`15.
`
`16.
`
`Frazer, A.: The efi'ect of lithium carbonate on
`self-stimulating behavior in the rat. Life sci-
`ences 11: 1'73-TF9, 1912.
`Rolls, E.T.: The brain and Reward. Pergamon
`Press, Oxford, 1975.
`Routtenberg, A.: The reward system of the
`brain. Sci Am 239: 154-164, 1978.
`Schou, M., Juel-Nielsen, N., Strotngren, E.
`and Voldby, H.: The treatment of manic
`psychoses by the administration of lithium
`salt. J Neurol Neurosurg Psychiat 17: 250-
`260, 1954.
`. Takigawa, M., Robertson, A. and Mogenson,
`G.J.: Brain stimulation reward associated
`with stimulation of the supracallosal bundle.
`Experimental Neurology 58: 81-94, 1978.
`Ungerstedt, U.: Stereotaxic mapping of the
`monoamine pathways in the rat brain. Acta
`Physiol Scand Suppl 367: 1-48, 1971.
`
`6of6
`
`Alkermes, Ex. 1038
`
`6 of 6
`
`Alkermes, Ex. 1038