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Title: Mechanism of Morphine Addiction by Inhibiting the soluble Guanylate Cyclase-Nitric Oxide (sGC-
NO) Pathway

Article Type: Regular Article

Keywords: Opioids, LTPGABA, Memory, Ventral Tegmental Area.

Corresponding Author: Mr. Karan Bhatt, M. Tech

Corresponding Author's Institution: KSKV Kachchh University

First Author: Karan Bhatt, M. Tech

Order of Authors: Karan Bhatt, M. Tech; Amitesh Kumar, M. Tech, Ph. D

Abstract: Ample evidence has shown that morphine influences learning and memory and thereby
causing addiction. Various studies have shown that it decreases the inhibitory GABAergic synaptic
transmission (LTPGABA) via the soluble guanylate
cyclase (sGC) and nitric oxide (NO) pathway. But still it is unclear on how does morphine inhibit the
sGCNO pathway. In this study, we show the mechanism of LTPGABA inhibition by morphine with the
help of a mathematical model. A two step model of sGC activation is used, where morphine inhibits NO
during the first step and consequently blocks sGC activation. Here, morphine binding on opioid
receptors blocks the binding of retrogradely travelling NO to sGC and hence its activation. As a result,
LTPGABA is not produced which increases the chances of addiction
manifold. Alongwith the mechanism, the dependence of morphine inhibition on major parameters such
as morphine dissociation, morphine concentration, NO removal & rate of inhibition and its effect on
addiction is also shown.
Cover Letter

To
The Editor
Mathemati al Bios ien es
The mole ular me hanism whi h results in morphine addi tion has not been
de iphered till now. Various studies are arried out with a view to nd this par-
ti ular me hanism. As stated by Kauer and Marletta (2007), it is possible that
the mole ular me hanisms behind various types of drug addi tion may share
similar pathway; thus identifying even one of the pathway may pave way for the
treatment of addi tion resulting due to variety of drugs of abuse. In this parti -
ular study, a novel me hanism of morphine addi tion is proposed with the help
of mathemati al modelling. The me hanism is validated by the experimental
results obtained by Nugent et al. (2007) and Niehaus et al. (2009). Compu-
tational approa h based upon Fortran on Linux platform is used to solve the
mathemati al model. This paper will help resear hers understand the mole ular
me hanism behind morphine addi tion whi h might have impli ations in drug
abuse treatment.
Yours Faithfully,
Amitesh Kumar
Karan Bhatt

1
*Manuscript

1
2
3
4 Me hanism of Morphine Addi tion by Inhibiting the soluble
5
6 Guanylate Cy lase-Nitri Oxide (sGC-NO) Pathway
7
8
9 a,c b
Karan Bhatt , Amitesh Kumar
10 a
11 National Institute of Te hnology, Rourkela, India

12 b
National Institute of Te hnology, Rourkela, India
13
c
14 KSKV Ka h hh University, Bhuj-Ka h hh, India
15
16 Mar h 6, 2015
17
18
19 1
20
21
22
23 Abstra t

24
Ample eviden e has shown that morphine inuen es learning and memory and thereby ausing ad-
25
26 di tion. Various studies have shown that it de reases the inhibitory GABAergi synapti transmission
27 (LT PGABA ) via the soluble guanylate y lase (sGC ) and nitri oxide (N O) pathway. But still it is un-
28 lear on how does morphine inhibit the sGC N O pathway. In this study, we show the me hanism of
29 LT PGABA inhibition by morphine with the help of a mathemati al model. A two step model of sGC a ti-
30
31
vation is used, where morphine inhibits N O during the rst step and onsequently blo ks sGC a tivation.
32 Here, morphine binding on opioid re eptors blo ks the binding of retrogradely travelling N O to sGC
33 and hen e its a tivation. As a result, LT PGABA is not produ ed whi h in reases the han es of addi tion
34 manifold. Alongwith the me hanism, the dependen e of morphine inhibition on major parameters su h
35 as morphine disso iation, morphine on entration, N O removal & rate of inhibition and its ee t on
36
37 addi tion is also shown.
38 Keywords: Opioids, LT PGABA , Memory, Ventral Tegmental Area.

39
40
41
42
1 Introdu tion
43 Morphine is one of the most powerful analgesi and painrelieving agent. Unfortunately its nonmedi al
44
45 use and its abuse is in reasing day by day [1, thereby adding to the global drug problem. Addi tion is
46 aused by powerful and long lasting memories of drug experien e. As shown by Joy e et al. [2 opiate
47 toleran e and dependen e requires protein synthesis. This happens be ause of an essential role of synapti
48
plasti ity in triggering long-term adaptations in regions innervated by dopamine (DA) neurons of the Ventral
49
50 Tegmental Area (VTA) [3. Opioids su h as morphine are hypothesized to indu e addi tion by taking part
51 in the synapti plasti ity of the reward learning pro ess at the mesolimbi dopamine system [38. The
52
1
53
Corresponding Author: Karan Bhatt; Tel: +91-9408133293, email: karan.bhatthotmail. o.in
54
55
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58 1
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 net ee t on the synapti plasti ity of the DA system depends on the ex itatory and inhibitory inputs.
1 Several studies have shown that there is an in rease in the synapti transmission of ex itatory inputs on DA
2 neurons following drug exposure [915. In addition, the drugs are also found to depress inhibitory synapti
3
4 plasti ity on the DA neurons in the VTA [1618. Thus, these drugs promote ex itatory transmission or blo k
5 inhibitory transmission or perform both the a tions simultaneously and therefore inuen e long-term storage
6 of reward-related memories in the VTA that may lead to addi tion [8, 19. These hanges in the synapti
7
8 plasti ity are seen due to hanges in the LTP and LTD of both the ex itatory as well as inhibitory neurons.
9 By releasing GABA from the presynapti neurons onto GABAA re eptors present at the postsynapti DA
10 neurons, inhibitory inter neurons oppose postsynapti ex itation and limit the spread of neural a tivity by
11
12 generating LT PGABA [20, 21. This long-lasting potentiation of GABAergi synapses onto DA neurons in
13 the VTA (LT PGABA ) is blo ked by in vivo administration of morphine [18, 22 whi h seems to be the major
14 ause for addi tion.
15
16 Administration of in vivo morphine ompletely blo ked the ability of the inhibitory synapses to undergo
17 LT PGABA within 2 hours and 24 hours prior to brain sli e preparation, but not after 5 days [18, 22. A single
18 administration of morphine, therefore, potentiates ex itatory synapti transmission [10 while, at the same
19
20 time, it prevents a omplementary in rease in inhibitory transmission that normally ould have ounterbal-
21 an ed the in reased ex itation. Thus, blo kade of LT PGABA by morphine indu es long lasting ex itability of
22 DA neurons whi h ontributes to the reinfor ing ee ts of opioids and development of addi tion. LT PGABA
23
is heterosynapti ; it is initiated by glutamate release onto the N-Methyl-D-aspartate (NMDA) re eptors on
24
25 the postsynapti DA neuron. NMDA re eptor indu es in reased uptake of Ca2+ whi h a tivates nitri oxide
26 (N O) synthase leading to the produ tion of intra ellular N O, whi h then travels retrogradely to presynapti
27
GABAergi neurons and a tivates soluble guanylate y lase (sGC ). Further downstream pro essing leads to
28
29 in reased levels of y li Guanosine Monophosphate (cGM P ) and Protein Kinase G (P KG), responsible for
30 promoting long-lasting potentiation of GABA release at these synapses [18. Morphine-indu ed blo kade of
31
LT PGABA spe i ally ae ts the sGC cGM P P KG pathway, presumably at the level of sGC [18. Inter-
32
33 estingly, a tivation of sGC with a sGC a tivator in sli es from morphine-treated rats is also able to indu e
34 LT PGABA, providing indire t eviden e for the presen e of adequate levels of sGC in morphine-treated sli es
35 to produ e enough cGM P and thus mimi LT PGABA [22. Whether morphine dire tly or indire tly intera ts
36
37 with sGC to disrupt LT PGABA is still not known. In this study, we show the me hanism of LT PGABA
38 inhibition in the presen e of morphine whi h in turn is responsible for addi tion. The present study also
39 emphasizes on the roles of various parameters su h as morphine on entration, rate of inhibition, rate of
40
41 morphine removal and rate of N O removal from the ell on addi tion.
42
43
44
45
2 Methods
46
47
2.1 The model
48 sGC is a heterodimeri hemoprotein omposed of and subunits and the heme moeity is the N O binding
49
50 site [23. sGC is a tivated by as mu h as 300-fold when N O binds to the heme ofa tor. The a tivation
51 of sGC by N O is ompli ated [24. The rea tion between sGC and N O is shown by a two step model as
52 shown in Ballou et al. [25. The binding of N O is very fast, yielding initially a 6- oordinate ferrousnitrosyl
53
54 (6C sGC N O) spe ies that would then de ay to the nal 5- oordinate omplex (5C sGC N O) via
55 one of the two pro esses; the rst one N O-dependent and the se ond one N O-independent. The model uses
56 N O as a atalyst in the se ond step su h that the rate depends on the N O on entration, but N O is not
57
58 2
59
60
61
62
63
64
65
 K
M

1
2 Morphine X (Inhibitor)
3
K
4 NO
K
I
5 NO
NO NO
6
7 K
1
K
3
8 sGC 6CsGCNO 5CsGCNO
9 K (Transient) K (Active)
2 4
10
11
12 K
5
13 K
6
14
15
16 Figure 1: Rea tion s heme of the model showing sGC -N O a tivation and morphine inhibition. The a tivation
17 me hanism of sGC -N O omplex is two step as shown in Ballou et al. [25. Morphine is assumed to a tivate
18 a set of inhibitory mole ules X , whi h inhibit the formation of 6C sGC N O omplex and thereby the
19 a tivation of 5C sGC N O omplex. In the se ond step of a tivation, N O is used as a atalyst su h
20 that the rate of a tivation depends on it but it is not onsumed in the step. It is assumed that morphine
21 disso iates from the opioid re eptors at zeroth order and N O is also removed from the ell at zeroth
22 order at their respe tive rate onstants.
23
24
25 onsumed in this step. It is assumed that morphine a tivates a set of mole ules X inside the ell by binding
26 to opioid re eptor on the neuronal ell membrane of the presynapti GABAergi neurons. The mole ule
27
28 X ae ts the sGC -N O pathway by inhibiting the formation of 6C sGC N O omplex as shown in Fig.
29 1.
30 The mode of addi tion is as follows: The neuronal transmission of the postsynapti dopamine neurons in
31
32 the VTA is ontrolled by two opposite transmissions whi h leads to the regulation of reward related memo-
33 ries. One is ex itatory transmission from the presynapti glutamatergi neurons and other is the inhibitory
34 transmission by presynapti GABAergi neurons. LT PGABA produ ed by the inhibitory GABAergi neu-
35
36 rons ounterbalan es the ex itatory neuronal transmission of glutamatergi neurons on the postsynapti DA
37 neurons and thus regulates its saturation. LT PGABA is produ ed in the presynapti GABAergi neurons
38 by the sGC cGM P P KG pathway whi h is a tivated by the retrogradely travelling N O from the post-
39
synapti DA neurons. N O binds to the ina tive sGC and forms an a tive omplex required for a tivating
40
41 P KG whi h results in the release of GABA mole ules from the presynapti neuron. This mole ules bind to
42 GABAA re eptors on the postsynapti neuron and de rease the postsynapti neuronal ex itability of the DA
43
neurons for a long time whi h results in inhibition of reward related memory formation. In the presen e of
44
45 morphine, a tive sGC N O omplex formation is inhibited whi h stops further downstream pro essing and
46 hen e LT PGABA. Thus LT PGABA is not produ ed whi h might have ontributed in preventing the ex itatory
47 synapti plasti ity indu ed by morphine and may lead to reward related memory resulting in addi tion.
48
49
50 2.2 Mathemati al Formulation
51
52 In this model, mass a tion kineti s is used to solve the bio hemi al equations. sGC is a tivated when N O
53 binds to it and forms 5C sGC N O omplex by a two step method. Assuming that sGC a tivation follows
54
55 mass a tion kineti s, the rate equations are expressed by Eq. 1, Eq. 2and Eq. 3:
56
57
58 3
59
60
61
62
63
64
65
 1
dS
2 = K1 S N + K2 St + K6 Sa (1)
3 dt
4
5
6
dSt
7 = K 1 S N K 2 St K 3 St N + K 4 Sa N K 5 St (2)
8 dt
9
10
11 dSa
12 = K 3 St N + K 5 St K 4 Sa N K 6 Sa (3)
dt
13
14 where S , N , St and Sa are the on entrations of sGC , N O, 6C sGC N O and 5C sGC N O
15
16 omplex. K1 and K2 are the rate onstants of 6C sGC N O formation and degradation respe tively,
17 K3 and K4 are the rate onstants of 5C sGC N O formation and degradation respe tively by enzymati
18 atalysis while K5 is the rate onstant of natural 5C sGC N O formation and K6 is the rate onstant for
19
20 5C sGC N O disso iation into free sGC and N O.
21 The inhibition of 5C sGC N O omplex formation by morphine via mole ule X is des ribed by hill's
22 kineti s as shown in Eq. 4:
23
24
25 1
26 F = [I] 2
(4)
27 1 + (K i
)
28
29 where F is the fra tion of sGC inhibited by inhibitor, I is the on entration of inhibitor (X) and Ki
30 is the logarithmi on entration of inhibitor at whi h the rate of 6C sGC N O formation be omes half.
31
32 Following morphine inhibition, the hange in the on entration of sGC and 6C sGC N O is shifted as
33 des ribed by Eq. 5 and Eq. 6:
34
35
36 dS
37 = K1 S N F + K2 St + K6 Sa (5)
dt
38
39
40 dSt
41 = K 1 S N F K 2 St K 3 St N + K 4 Sa N K 5 St (6)
dt
42
43 The hill's oe ient for the inhibition is taken as 2. The rate of disso iation of morphine from opioid
44
re eptors is of enzymati nature as shown in Eq. 7. The rate of disso iation of N O from the ell is given
45
46 by zeroth order rea tion in Kharitonov et al.[26, but as NO an never be negative we also use enzymati
47 disso iation given in Eq. 8 and Eq. 9:
48
49
50 KM M O
51 DM = (7)
M O + KE
52
53 and
54
55
56 RN O = KN O (8)
57
58 4
59
60
61
62
63
64
65
 1
KN O N O
2 RN O = (9)
3 N O + KN E
4
5 where DM is the rate of disso iation of morphine from opioid re eptors and RN O is the rate of removal
6 of N O from the ell while KM and KN O are the rate onstants of their disso iation and removal respe tively.
7 The numeri al simulations were arried out by impli it method. Parameters for the standard system are
8
9 given in Table 1. In the following gures where one parameter varies, all others are xed a ording to the
10 table, unless otherwise stated. For those parameters in the table without experimental referen es, we hose
11 values that were in a reasonable range as provided in the literature for similar hemi al rea tions.
12
13
14
15 3 Results & Dis ussion
16
17 3.1 Morphine blo ks the produ tion of LT PGABA
18
19 Previous nding suggests that 24 hours after in vivo exposure to morphine, LT PGABA is ompletely blo ked
20 [18, but the me hanism of morphine inhibition is still unknown. A more detailed understanding of the pro ess
21
22 by whi h morphine alters LT PGABA ould provide valuable information about the ee ts of morphine on
23 the VTA and the brain reward ir uit. We, therefore, propose a me hanism of morphine inhibition whi h is
24 able to blo k LT PGABA. The simulations are arried out in the presen e of morphine. In the model, sGC
25
and N O bind together and form an a tive omplex whi h plays a major role in generating LT PGABA. It
26
27 is observed that in the presen e of morphine, sGC is unable to bind with N O and, hen e, it does not form
28 an intermediate 6C sGC N O omplex. Hen e, the formation of a tive 5C sGC N O omplex is
29
also inhibited. As shown in g. 2, a lag phase is observed before sGC starts binding with N O to form an
30
31 intermediate 6C sGC N O omplex. Due to this, a delay in the formation of 5C sGC N O omplex
32 is observed. As a result, further downstream, a tivation of cGM P and P KG is also paused, and hen e the
33
generation of LT PGABA is blo ked in the presen e of morphine [22, 27. Thus, administration of morphine
34
35 ompletely blo ks the ability of the GABAergi synapses to undergo LT PGABA . A single administration of
36 morphine is, therefore, able to potentiate ex itatory synapti transmission [10, while at the same time it
37 prevents a omplementary in rease in inhibitory transmission that normally would have ounterbalan ed the
38
39 in reased ex itation. Thus, blo kade of LT PGABA by morphine prevents DA neuron inhibition that might
40 be able to reverse or prevent synapti plasti ity at ex itatory terminals indu ed by drugs of abuse, and hen e
41 ontribute to the development of addi tion.
42
43
44 3.2 Sustenan e of LT PGABA independent of NO
45
46 Sustained a tivity of protein kinases su h as protein kinase C (P KC ) and Ca2+ / almodulin kinase type II
47 (CaM KII ) have been proposed to be involved in maintenan e and expression of LTP [28, 29. We also tested,
48
49 here, whether onstitutive release of N O is ne essary to sustain LT PGABA or, instead, a brief exposure is
50 su ient to persistently enhan e GABA release? From g. 3a and g. 3b, it is observed that sGC remains
51 a tive for a long period of time even after N O has depleted from the presynapti GABAergi neuron. As
52
53 a result, it is able to a tivate further downstream mole ules, i.e. cGM P and P KG whi h are ne essary
54 for generating LT PGABA. Thus, even a brief exposure of N O in the presynapti GABAegi neurons is able
55 to indu e long lasting LT PGABA whi h ounterbalan es the ex itatory synapti potentiation indu ed by
56
57
58 5
59
60
61
62
63
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 1
2
3
4
5
6
7 Table 1: Parameters used in the model:
8 Parameter Symbol Value Unit Referen e
9 Total on entration of S 0.5 M [25
10
sGC
11
Initial on entration of N 0.75 M [25
12
NO
13
14 Initial on entration of MO 0.1 1.0 M This paper
15 Morphine
16 Rate onstant for K1 1.55 108 M 1 s1 [25
17 6C sGC N O
18 formation
19 Rate onstant for K2 1.0 102 s1 [25
20 6C sGC N O
21 degradation
22 Rate onstant for K3 3.3 105 M 1 s1 [25
23 enzymati
24 6C sGC N O turnover
25 Rate onstant for K4 1.0 103 s1 [25
26 5C sGC N O
27 dea tivation
28 Rate onstant for natural K5 5.0 104 s1 [25
29
6C sGC N O turnover
30
Rate onstant for K6 1.0 103 s1 [25
31
32 5C sGC N O
33 disso iation
34 Rate onstant for KM 3.0 1010 1.0 109 s1 This paper
35 morphine disso iation
36 Rate onstant for N O KN O 7.5 1011 2.5 1010 s1 This paper
37 removal
38 Logarithmi Ki 1.0 1010 5.0 1010 M This paper
39 on entration of inhibitor
40 when the velo ity of
41 6C sGC N O
42 formation be omes half
43 Equivalent onstant for KE 1.0 107 M This paper
44 the enzymati
45 disso iation of morphine
46 Equivalent onstant for KN E 1.0 107 M This paper
47 the enzymati
48
disso iation of NO
49
* Spe i values are given in gure legends.
50
51
52
53
54
55
56
57
58 6
59
60
61
62
63
64
65
 5E-07
1
2 Morphine
Free sGC
3 4E-07
4
5

Concentration (M)
6
7 3E-07
8
9
10
11 2E-07
12
13
14
15 1E-07
16
17
18
19 0
0 500 1000 1500 2000 2500
20
Time (s)
21
22
23 Figure 2: Inhibition of sGC N O omplex formation by morphine. When morphine is present then there
24 is inhibition of the sGC N O omplex formation and as soon as morphine is ompletely disso iated from
25 opioid re eptor, sGC be omes ompletely saturated with N O to form sGC N O omplex whi h results in
26 sGC a tivation and hen e LT PGABA generation. Initial morphine on entration, M O = 5.0 107M, initial
27 sGC on entration, S = 5.0 107 M and Rate onstant for morphine disso iation, KM = 5.0 1010 s1 .
28
29
30 presynapti glutamatergi neurons on the postsynapti DA neurons. Therefore, when morphine inhibits N O
31 to bind with sGC to form an a tive omplex and blo ks LT PGABA, the ex itatory potentiation is not reversed
32 and so the behavioural responses towards the drugs of abuse are modied in the mesolimbi dopamine system
33
34 whi h ould lead to the development of addi tion. These results are similar to the one obtained by Nugent
35 et al. [27, where an N O s avenger, PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, 300 M )
36 was added after the N O donor eli ited synapti potentiation, but the potentiation did not de ay ba k to
37
38 ontrol values. Although there is no overall ee t of the rate of disso iation of N O on the nal a tivity of
39 sGC as it remains a tive for a long time after N O ompletely disso iates, it is important to learn the exa t
40 me hanism by various empiri al methods be ause some of the earlier resear h by Kharitonov et al. [26 shows
41
42 that disso iation of N O is zeroth order whi h seems improbable as N O an never be negative inside the ell.
43
44
45
3.3 KM and MO ae t the initiation of 6C sGC NO omplex formation
46 It is found that in vivo morphine administration entirely blo ked LT PGABA [18. GABAA synapses in VTA
47
48 sli es from rats that had re eived morphine 24 hours earlier did not exhibit LT P , but after 5 days the
49 ee t of morphine was nonexistent. So, we examined whether the hanges in morphine on entration ae ts
50 the inhibition of LT PGABA. The morphine on entration at any given time depends on KM , the rate of
51
52 disso iation of morphine from the opioid re eptor and M O, the initial on entration of morphine bound
53 to the opioid re eptors present on the ell membrane of presynapti GABAergi ells. As shown in g.
54 4a, it is observed that when the value of KM de reases then the rate of morphine disso iation also de reases
55
whi h results in a longer lag phase for initiating 6C sGC N O omplex formation. As a result, a delay is
56
57
58 7
59
60
61
62
63
64
65
 5E-07
1
2 NO
3 Active sGC
4 4E-07
5
6

Concentration (M)
7
8 3E-07
9
10
11
12 2E-07
13
14
15
16 1E-07
17
18
19
20 0
0 2000 4000 6000 8000 10000
21 Time (s)
22 (a)
23
24
25
26 NO
27 Active sGC
28 6E-07
29
30
Concentration (M)

31
32
33 4E-07
34
35
36
37
38 2E-07
39
40
41
42
43
0
44 0 2000 4000 6000 8000 10000
45 Time (s)
46 (b)
47
48 Figure 3: (a) sGC remains a tivated even after N O is depleted from the ell. Although N O is ompletely
49 removed from the ell, it is observed that a tivated sGC i.e. 5C sGC N O takes a long time to ome
50 ba k to its ina tive form i.e. sGC. It assists in a tivating further downstream mole ules su h as cGM P and
51 P KG whi h results in LT PGABA . Thus it is shown that N O is not required to sustain LT PGABA. Initial
52 N O on entration, N = 5.0 107 M and Rate onstant for N O removal, KN O = 1.0 1010 s1 . (b) Even
53 though the rate of disso iation be omes enzymati , the overall result is the same. It is observed here that sGC
54 remains a tivated for a long time after N O ompletely disso iates whi h results in sustenan e of LT PGABA.
55 Initial N O on entration, N = 7.5 107 M and Rate onstant for N O removal, KN O = 1.0 1010 s1 .
56
57
58 8
59
60
61
62
63
64
65
 5E-07
1 Lower rate
2 Normal rate
3 Higher rate
4
4E-07
5
6
7
8 3E-07

sGC (M)
9
10
11
12 2E-07
13
14
15
16 1E-07
17
18
19
20
21 0 500 1000 1500 2000
Time (s)
22
(a)
23
24
5E-07
25
26
27
28
4E-07
29
30
31
32 3E-07
sGCa (M)

33
34
35
36 2E-07
37
38
39
40 1E-07
41
42 Lower rate
43 Normal rate
Higher rate
44
45 0 500 1000 1500 2000
Time (s)
46
47 (b)

48
49 Figure 4: (a) Initiation of sGC omplex formation depending on the value of KM . Depending on the value of
50 KM , a lag phase is observed before the sGC N O omplex formation is initiated. At lower values of KM , a
51 longer lag phase is observed and as the value of KM in reases, the lag phase de reases and free sGC is qui kly
52 saturated into its a tivated state. (b) Dependen e of sGC a tivation on the rate of morphine disso iation.
53 With in rease in the value of KM , morphine disso iates qui kly and hen e the inhibition by morphine redu es
54 whi h results in redu ed lag phase. Thus sGC is qui kly onverted to its a tive form by forming omplex
55 with N O. Lower rate= 3.0 1010 , normal rate= 5.0 1010 and higher rate= 1.0 109 .
56
57
58 9
59
60
61
62
63
64
65
 observed in the a tivation of 5C sGC N O omplex whi h a ounts for the redu ed rate of cGM P and
1 P KG a tivation. So, a prolonged inhibition of LT PGABA is observed due to whi h there is a de rease in
2 inhibitory transmission to the postsynapti DA neurons, whi h in turn in reases the han es of addi tion.
3
4 But when the value of KM is in reased, morphine disso iates from the opioid re eptors at a faster rate and
5 hen e the inhibition on sGC also vanishes rapidly. The initiation of 6C sGC N O omplex formation is
6 started qui kly whi h results in early formation of 5C sGC N O omplex and thereby de reased inhibition
7
8 on LT PGABA. Therefore, the inhibitory transmission generated from the presynapti GABAergi ells ould
9 ountera t the ex itatory transmission on the postsynapti DA neurons and redu e the han es of addi tion.
10 As 5C sGC N O is the a tive form of enzyme, sGC whi h is used for subsequent downstream pathways
11
12 [23, we have also studied the ee t of KM on the rate of formation of 5C sGC N O omplex. From g.
13 4b, it is observed that with de rease in the value of KM , morphine binds to the opioid re eptors for a
14 longer duration of time whi h results in in reased inhibition of sGC . So, the initiation of sGC a tivation
15
16 takes more time and a longer lag phase is observed. As a result, cGM P and P KG a tivation slows down and
17 LT PGABA is inhibited for a prolonged duration. This results in in reased probability of addi tion be ause of
18 the absen e of inhibitory synapti transmission whi h ould ountera t the ex itatory synapti transmission.
19
20 Another interesting observation made from g. 4b is that with de rease in the value of KM , the maximum
21 amount of 5C sGC N O omplex that an be a tivated also de reases. This happens be ause as the value
22 of KM de reases, morphine remains bound to the opioid re eptors for a long time. As a result, sGC
23
is unable to bind N O for a prolonged time. So, the maximum on entration of 5C sGC N O de reases
24
25 with a de rease in KM . This shows that along with the duration of inhibition of sGC, KM also inuen es
26 the maximum amount of 5C sGC N O omplex that an be formed. Thus, it an be inferred that KM is
27
able to ontrol the duration and amount of LT PGABA inhibition.
28
29 A re ent study by Kitumura et al. [30 revealed that there is a marked hange in behavioural sensitization
30 at the mesolimbi dopamine system between the investigator-administered drug (passive administration) and
31
self-administered drug (a tive administration). These hanges o ur due to time ourse over whi h drugs
32
33 are administered and their amount of administration [19. Therefore, we studied here the ee t of dierent
34 on entrations of initial morphine bound to the opioid re eptors i.e. M O on the inhibition of LT PGABA
35 be ause the amount of drug also plays a very important role in development of addi tive behaviour. It is
36
37 observed from g. 5a that the ee t of M O is almost similar to that of KM . When the morphine on entration
38 in reases, omplete withdrawal of morphine from the opioid re eptors takes more time. So, morphine
39 is able to inhibit the binding of sGC with N O for a longer duration and a longer lag phase for initiating
40
41 6C sGC N O omplex formation is observed. As a result, the a tive omplex of sGC , i.e. 5C sGC N O,
42 is generated slowly; due to whi h the a tivation of cGM P and P KG also slows down. This implies that there
43 is an in reased inhibition of LT PGABA whi h an play an important role in developing addi tion. As shown in
44
45 g. 5b, it is observed that with the in rease in morphine on entration, there is an in rease in 5C sGC N O
46 inhibition by morphine and so the amount of time taken for sGC to get a tivated also in reases. As a result,
47 further downstream pathway for LT PGABA generation is blo ked for a prolonged duration. So, LT PGABA is
48
49 also inhibited for a long duration of time with an in rease in morphine on entration whi h usually results
50 in in reased han es of addi tion.
51
52
53
54
55
56
57
58 10
59
60
61
62
63
64
65
 5E-07
1
2
3
4 4E-07
5
6
7
8 3E-07

sGC (M)
9
10
11
12 2E-07
13
14
15
16 1E-07
Lower concentration
17 Normal concentration
18 Higher concentration
19
20 0 500 1000 1500 2000
21 Time (s)
22 (a)
23
24 5E-07
25
26 Lower concentration
27 Normal concentration
28 4E-07 Higher concentration
29
30
31
32 3E-07
sGCa (M)

33
34
35
36 2E-07
37
38
39
40 1E-07
41
42
43
44 0 500 1000 1500 2000
45 Time (s)
46 (b)
47
48 Figure 5: (a) Dependen e of initial morphine on entration on the initiation of sGC N O omplex formation.
49 As the initial on entration of morphine is de reased, the initiation of omplex formation started rapidly
50 be ause of de reased inhibition. Thus, a smaller lag phase is observed whi h in reases with the in rease
51 in initial morphine on entration. (b) The a tivation of sGC is dependent on the initial on entration
52 of morphine. When the initial on entration of morphine is in reased, the inhibition persists for a longer
53 duration and therefore sGC be omes a tive after some time. Thus, a longer lag phase is observed in ase of
54 sGC a tivation whi h de reases with the de rease in initial morphine on entration. Lower on entration=
55 1.0 107 , normal on entration= 5.0 107 and higher on entration= 1.0 106 .
56
57
58 11
59
60
61
62
63
64
65
 3.4 Regaining initial sGC on entration, after 5C sGC NO disso iation, de-
1
2
pends on KN O
3 Addi tion is not triggered instantaneously upon a ute exposure to drugs of abuse. It involves omplex
4
neural adaptations that develop with repeated drug exposure at dierent time intervals ranging from hours
5
6 to days to months. Resear h work to date suggests that during exposure to opiates, LT PGABA is blo ked
7 by the opiate via inhibition of sGC to bind with retrogradely travelling N O and form an a tive omplex
8
[18. To indu e addi tion by repeated drug exposure, sGC has to be present in free form in the presynapti
9
10 GABAergi neurons. Here, we have studied the ee t of KN O on the model be ause it ae ts the time taken
11 for free sGC to regain its initial on entration after it has formed an a tive omplex, i.e. 5C sGC N O.
12
Depending on the rate of N O removal from the ell, the omplex between sGC and N O is broken down
13
14 and free sGC regains its initial on entration in the neuron. This free sGC , again, does not form omplex
15 be ause of nonavailability of free N O in the GABAergi neuron. From g. 6a, it is observed that when the
16 value of KN O de reases then sGC remains in its a tive form for a long period of time. During this time, if
17
18 morphine is re-exposed then it is not able to inhibit LT PGABA generation be ause it only blo ks free sGC
19 and N O omplex formation; but here sGC is not available in free form. Therefore, with redu tion in the
20 value of KN O , the primary LT PGABA generated ounterbalan es the ex itatory synapti transmission of the
21
22 presynapti glutamatergi neurons developed be ause of drug exposure and hen e redu es the han es of
23 addi tion. So, it an be on luded that as the value of KN O de reases, it takes a long time for free sGC
24 to shoot up to its initial value and hen e upon re-exposure to morphine, sGC is not available in free form
25
26 to get inhibited again. Thus, morphine is not able to blo k LT PGABA again and this redu es the han es
27 of addi tion. An interesting observation is made from g. 6b, whi h shows that with in rease in the value
28 of KN O , the maximum amount of sGC found in a tive form de reases. As the value of KN O in reases, the
29
30 rate of N O removal also in reases and the on entration of a tive sGC omplex de reases from the ell at
31 a faster rate. This happens be ause, with in rease in the rate of removal of N O, i.e. KN O , the rate of
32 formation of a tive sGC de reases as free N O is not present in the ell to bind with free sGC. Therefore,
33
sGC remains in its ina tive form; as a result, the maximum amount of sGC found in a tive form at any
34
35 given time in the neuron de reases. At the same time, this ina tive form of sGC is ready to be inhibited
36 after morphine re-exposure whi h in turn results in LT PGABA blo kade. This blo kade serves to enhan e the
37
han es of addi tion in the mesolimbi orti al system. But when the value of KN O de reases, sGC remains
38
39 in its a tive form for a prolonged time, be ause N O takes longer time to deplete from the ell. Therefore,
40 upon re-exposure of morphine, there is unavailability of free sGC in the neuron whi h results in nonblo kade
41
of LT PGABA and eventually de reases the han es of addi tion. It is also observed that the amount of sGC
42
43 that is onverted to a tive form also depends on the value of KN O . After a ertain in rease in its value, the
44 maximum amount of sGC , that has been onverted into a tive form, de reases be ause the rate of removal of
45 N O ex eeds the rate of formation of 5C sGC N O and, before the disso iated N O from 5C sGC N O
46
47 is again able to form omplex with free sGC , it is removed from the ell and hen e sGC is not onverted
48 ba k to its a tive form.
49
50
51 3.5 Ki hanges the nature of morphine inhibition
52
53 Although morphine inhibits sGC to bind with retrogradely travelling N O and form an a tive omplex but
54 the type of inhibition, i.e. omplete inhibition or partial inhibition, is dependent on the rate of inhibition. In
55 the ase of omplete inhibition, sGC will not form omplex with N O, even when morphine is present in very
56
57
58 12
59
60
61
62
63
64
65
 5E-07
1
2
3
4 4E-07
5
6
7
8 3E-07

sGC (M)
9
10
11
12 2E-07
13
14
15
16 1E-07
Lower rate
17 Normal rate
18 Higher rate
19
20 0 2000 4000 6000 8000 10000
21 Time (s)
22 (a)
23
24 5E-07
25
26 Lower rate
27 Normal rate
28 4E-07 Higher rate

29
30
31
32 3E-07
sGCa (M)

33
34
35
36 2E-07
37
38
39
40 1E-07
41
42
43
44 0 2000 4000 6000 8000 10000
45 Time (s)
46 (b)
47
48 Figure 6: (a) The rate of free sGC restoraton to its initial on entration depends on the rate of N O removal.
49 With a de rease in the value of KN O , N O remains in the ell for a longer amount of time and sGC is able to
50 bind with it and form omplex. Therefore, sGC is not available in free form and it rea hes its initial value
51 after a prolonged time. (b) Formation of a tive sGC i.e. 5C sGC N O depends on the rate of N O removal
52 from the ell. With a in rease in the value of KN O , the period of time for whi h sGC remains in a tive form
53
de reases be ause the disso iated N O is qui kly removed from the ell before it ould again omplex with
54
free sGC to be ome a tivated; and after a riti al in rease in KN O , the maximum amount of sGC found in
55
56 a tive form also de reases. Lower rate= 7.5 1011, normal rate= 1.0 1010 and higher rate= 2.5 1010.
57
58 13
59
60
61
62
63
64
65
 small amount. While in the ase of partial inhibition, sGC will partially bind with N O to form an a tive
1 omplex but at redu ed on entrations. Thus, during omplete inhibition, even though initial morphine
2 on entration bound to the opioid re eptors is low, it will lead to in reased inhibition; on the other hand,
3
4 during partial inhibition, even in the presen e of ex ess morphine, sGC would initiate binding with N O. We
5 studied here the ee t of the rate of inhibition on the nature of morphine inhibition. This rate of inhibition
6 (KI ) is inversely proportional to the logarithmi on entration of inhibitor when the rate of 6C sGC N O
7
8 formation is halved (Ki ). It is observed from g. 7a that when the value of Ki de reases then the rate of
9 inhibition in reases and the rea tion between sGC and N O is not initiated for a long duration of time due to
10 whi h downstream mole ules su h as cGM P and P KG also take a long time to get a tivated. So, LT PGABA
11
12 is generated after a long gap. Thus, ex itatory synapti transmission from the presynapti glutamatergi
13 neurons is not ounterbalan ed whi h ould result in in reased han es of addi tion. But when the value
14 of Ki in reases then the rea tion between sGC and N O is initiated qui kly and so LT PGABA is qui kly
15
16 produ ed whi h is able to nullify the ee t of ex itatory transmission on the postsynapti DA neurons. As
17 a result, the han es of addi tion are redu ed. It is interesting to note that unlike KM , Ki does not hange
18 the time taken for the rea tion to rea h omplete saturation as seen in g. 7a, whi h signies that either
19
20 Ki or KI only ae ts the amount of time sGC would be inhibited to rea t with N O and form an a tive
21 omplex but not the rate at whi h they both will rea h saturation. As shown in g. 7b, as the value of
22 Ki in reases, sGC be omes a tive rapidly be ause KI de reases and the rate of inhibition de reases whi h
23
results in rapid a tivation of cGM P and P KG. Therefore, LT PGABA is generated at the earliest whi h
24
25 de reases the han es of addi tion. It is also observed that there is no hange in the time taken for a tive
26 sGC to rea h its maximum value. This happens be ause Ki only ae ts the initial time when sGC starts
27
forming the omplex with N O and get onverted into a tive sGC i.e. 5C sGC N O.
28
29
30
31 4 Con lusion
32
33 In this study, a novel me hanism of morphine inhibition on LT PGABA is proposed. It is observed that sGC
34 is inhibited to form omplex with N O in the presen e of morphine whi h ultimately results in LT PGABA
35
36 inhibition. Here, it is shown that morphine is able to a tivate an intra ellular mole ule X whi h binds to the
37 N O binding site on sGC and thus prevents sGC N O omplex formation. This inhibition is shown to be
38 dependent on the initial morphine on entration (M O), disso iation rate of morphine from the opioid
39
40 re eptors (KMO ), rate of N O removal from the ell (KN O ) and the rate of morphine inhibition (KI ). KM
41 and M O regulate the amount of time sGC is inhibited by X and unable to bind to N O and form an a tive
42 omplex, thereby a tivating cGM P and P KG whi h indu es LT PGABA. KN O ontrols the rate at whi h
43
free sGC rea hes its initial on entration. Depending on the rate of N O removal, N O is available for the
44
45 disso iated sGC to form an a tive omplex again and produ e LT PGABA . Ki is only responsible for the
46 nature of morphine inhibition. It only regulates the amount of time sGC remains in its free form at the start
47
but does not play any role in the time taken for 5C sGC N O to rea h saturation. Although an inhibition
48
49 me hanism of morphine is proposed, we remain un lear about the mole ular pro esses taking pla e behind
50 this me hanism. Advan es in this dire tion would enhan e our understanding of the interplay of mole ular
51
and network properties in determining the basi me hanism behind morphine addi tion.
52
53
54
55
56
57
58 14
59
60
61
62
63
64
65
 5E-07
1
2
3
4E-07
4
5
6
7 3E-07

sGC (M)
8
9
10
11 2E-07
12
13
14 Lower rate
Normal rate
15 1E-07 Higher rate
16
17
18
19
20 0 200 400 600 800 1000
Time (s)
21
22 (a)

23
24 5E-07
25
26 Lower rate
27 Normal rate
4E-07 Higher rate
28
29
30
31
3E-07
sGCa (M)

32
33
34
35 2E-07
36
37
38
39 1E-07
40
41
42
43
44 0 200 400 600 800 1000
45 Time (s)
46 (b)

47
48 Figure 7: (a) Dependen e of the rate of morphine inhibition on the sGC N O omplex formation. Ki
49 is inversely proportional to the rate of inhibition and as its value in reases, the rate of morphine inhibition
50 de reases and sGC qui kly initiates a tive omplex formation with N O. It is observed that Ki does not ae t
51 the time taken for sGC to rea h saturation be ause for the same on entration of morphine and its rate of
52 disso iation, Ki only ae ts the type of inhibition whether sGC will qui kly initiate omplex formation with
53 N O or will remain in free form for a longer duration. (b) Dependen e on the type of sGC a tivation on the
54 rate of morphine inhibition. As Ki is inversely proportional to the rate of morphine inhibition, when its value
55 in reases the rate of sGC a tivation also in reases. It only ae ts the type of a tivation and not the time in
56 whi h 5C sGC N O would rea h saturation be ause of the same amount of morphine and its disso iation
57 rate. Lower rate= 1.0 1010 , normal rate= 2.5 1015 10
and higher rate= 5.0 1010 .
58
59
60
61
62
63
64
65
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62
63
64
65