Involvement of nitric oxide in learning & memory processes

Abstract

Nitric oxide (NO), synthesized from the amino acid, L-arginine by nitric oxide synthase (NOS) has received attention as a neurotransmitter in the brain. NO has been found to induce cognitive behaviour in experimental animals. In order to show evidence for the involvement of NO in learning and memory processes, the reports indicating the effects of its precursor, donors, and inhibitors of its synthesis in mammals, birds, fishes and invertebrates have been reviewed. Further, learning and memory impairment occurring in man and animals due to defective NO activity in the brain due to pathological conditions such as epilepsy, stress, diabetes and side effects of therapeutic agents and reversal of this condition by L-arginine and NO donors have been included. In addition, the reports that indicate ageing-induced impairment of cognition that is known to occur in Alzheimer's disease due to deposition of the toxic protein, beta amyloid and the effect of L-arginine and NO donors in preventing dementia in these patients have been reviewed.

Full text

Introduction
It has been known for many years that NO which
is a gas made up of two most common gases in the
atmosphere, occurs in the biological system. NO
crosses cell membranes freely and plays a role as a
neurotransmitter in the brain. The function of NO
in the hypothalamus has largely been implicated in
learning process and in memory formation1. In order
to establish evidence for the involvement of NO in
learning and memory processes, the experimental
ndings that demonstrated synthesis of NO and the
neuronal action of NO at the time when experimental
animals were trained to learn and then to remember a
specic task, were reviewed in this article. In addition,
Involvement of nitric oxide in learning & memory processes
Vanaja Paul & Perumal Ekambaram
Department of Pharmacology & Environmental Toxicology, Dr A.L.M. Postgraduate Institute of
Basic Medical Sciences, University of Madras, Chennai, India
Received April 8, 2009
Nitric oxide (NO), synthesized from the amino acid, L-arginine by nitric oxide synthase (NOS) has
received attention as a neurotransmitter in the brain. NO has been found to induce cognitive behaviour
in experimental animals. In order to show evidence for the involvement of NO in learning and memory
processes, the reports indicating the effects of its precursor, donors, and inhibitors of its synthesis in
mammals, birds, shes and invertebrates have been reviewed. Further, learning and memory impairment
occurring in man and animals due to defective NO activity in the brain due to pathological conditions
such as epilepsy, stress, diabetes and side effects of therapeutic agents and reversal of this condition by
L-arginine and NO donors have been included. In addition, the reports that indicate ageing-induced
impairment of cognition that is known to occur in Alzheimer’s disease due to deposition of the toxic
protein, beta amyloid and the effect of L-arginine and NO donors in preventing dementia in these
patients have been reviewed.
Key words L-arginine - learning and memory processes - nitric oxide (NO) - NOS
the cognitive effects of agents that increase or decrease
NO concentration in the hypo-thalamus were also
included. Further, the reports indicating the benecial
effects of NO elevating agents in alleviating cognitive
disorder caused by pathological conditions, by the
deposition of endogenous substance like beta amyloid
in the brain of patients with Alzheimer’s disease (AD)
and the toxicities of therapeutic agents were also
included.
NO as a neurotransmitter and its involvement in
learning and memory processes
Studies in experimental animals have well
documented the synthesis of NO in the brain, and its
Review Article
Indian J Med Res 133, May 2011, pp 471-478
471

472 INDIAN J MED RES, MAY 2011
role in a variety of neuronal functions including learning
and memory processes, cortical arousal, nociception,
food intake, penile erection, yawning, blood vessel
dilatation and immune response1. NO is synthesized
in the brain upon demand as in cognitive condition
for which NO activity is required. Neurons synthesize
NO as a response to the activation of N-methyl-D-
aspartate (NMDA) receptors by the excitatory amino
acid glutamate. NO is generated in the neuronal
cells as a co-product of the conversion of the semi-
essential amino acid L-arginine to L-citrulline by the
enzyme nitric oxide synthase (NOS) with calcium and
calmodulin as cofactors. Three distinct NOS have been
identied in the hippocampus, cortex, cerebellum,
corpus striatum and medulla of rat brain. NOS from
endothelial cells (eNOS) and neurons (nNOS) are
constitutively expressed and the action of these enzymes
are stimulated by an increase in intracellular calcium.
NO produced by these enzymes act as a neuronal
messenger. NO synthesized by calcium-independent
induction NOS (iNOS) mediates immune function1.
Although it plays an important role in cell
signaling in the brain, NO has been described as an
unconventional neurotransmitter, because it is not
stored in synaptic vesicles and not released upon
membrane depolarization but released as soon it is
synthesized. NO does not mediate its action by binding
to membrane associated receptors but diffuses from
one neuron to another and acts directly on intracellular
components. NO function as a neurotransmitter by
stimulating soluble guanylyl cyclase to form the second
messenger molecule, cyclic guanosine monophosphate
(cGMP) in the target cells1. Studies on various forms
of synaptic plasticity in the brain have provided insight
into the cellular and molecular mechanisms for learning
and memory processes. Long-term potentiation (LTP),
a homosynaptic plasticity2 and long-term depression
(LTD), a heterosynaptic plasticity3 are two major forms
of activity dependent synaptic plasticity in the brain.
NO-cGMP pathway has been implicated in the induction
of hippocampal LTP and LTD which are known to be
the predominant mechanisms of learning and memory
processes. LTP in the hippocampus is the primary
experimental model for investigating the synaptic basis
of learning and memory in vertebrates2. Expression of
LTD-like synaptic plasticity in the hippocampus has
been suggested to underlie certain forms of motor
learning and visual recognition memory4. NO acts as
a retrograde messenger for the induction of LTP and
LTD in the hippocampus3.
NO formed in the hippocampus has been
suggested to have a role in learning and memory
processes because the activity of NMDA receptor
which is an initiator of the reaction that produces NO
from L-arginine, has been activated in this region of
the brain at the time of consolidation of the acquired
avoidance task in chicks5 and in rats6. For further study,
the rates of synthesis of NO and induction of LTP and
LTD have been determined in the hippocampus of
experimental animals that have been trained to learn
and then to consolidate the acquired maze traversing,
exploring, avoidance and object recognizing tasks.
Since neuronal NO has a very short half-life (5 sec), the
level of its metabolite, nitrite or the activity of NOS has
been measured in the hippocampus of animals during
learning process. Learning of spatial task by rats was
found to be accompanied by an elevation of nitrite
in the hippocampus6. The activity of NOS was found
to be increased by 45 per cent in the hippocampus
immediately after acquisition of an avoidance task in
rats7. Further, spatial memory was accompanied by an
increase in the activity of NOS in the hippocampus of
rats8. These ndings and an increased expression of
nNOS in the hippocampus during learning of odour in
sheep9 and in mice10 provide evidence for a correlation
between learning process and an activation of nNOS in
the hippocampus. In addition, an increased formation
of NO at the time when rats were learning foot-shock
avoidance task was accompanied by an induction of
LTP and LTD in the hippocampus7.
Alteration in learning and memory processes by
agents that increase/decrease NO concentration in
the brain
In order to demonstrate adequate evidence for the
involvement of NO in learning and memory processes,
the effects of the agents that are known to increase/
decrease NO concentration in the brain regions have
been tested in experimental animals. L-arginine, the
precursor of NO11 , and the donors of NO such as sodium
nitro-prusside (SNP)12, S-nitroso-N-acetylpenicillamine
(SNAP)13 and molsidomine14 are known to increase the
concentration of NO in the brain regions of rats. The
antagonists of NMDA receptors, dizocipline (MK-801)
and AP5 have been found to inhibit NO synthesis in
the brain15. The synthetic analogues of L-arginine that
are known to decrease NO concentration by inhibiting
nNOS and eNOS are N-nitro-L-arginine methyl
ester (L-NAME) and N-nitro-L-arginine (L-NA),
N-monomethyl-L-arginine (L-NMMA)1, and the
nitro-indazole compound, 7-nitroindazole (7-NI)16 are

known to inhibit NO synthesis in the brain. The results
of the studies carried out with these compounds on the
cognitive behaviour of different species of animals
have been shown in the Table.
These ndings provide strong support to the
concept that NO plays a vital role in both learning
process and memory of the learnt task.
Since NO is known to relax blood vessels and to
increase blood supply to the brain1, this action of NO
can also be assessed to have a role in inducing neuronal
activity. In this context, a decrease in NO synthesis
following an inhibition of NOS activity is likely to
result in vasoconstriction and a decrease in perfusion
into the brain. This effect of NOS inhibitors can be
proposed for an impairment of learning and memory
processes in animals treated with these compounds.
Interestingly, 7-NI which failed to affect cerebral blood
ow because of its inhibitory action on neuronal specic
NOS16, impaired learning and memory processes in
rats28, chicks31 and in shes9. These ndings clearly
show that not an inhibition of cerebral blood ow but
an impairment of neuronal action of NO is responsible
for the cognitive decits produced by the inhibitors of
NO synthesis.
The physiological effects of NO predominate
when NO is produced in sufcient concentration.
Interestingly, a several fold increase by L-arginine
of NO concentration in the brain has resulted in an
impairment of retention of acquired task in rats32.
Larger doses of SNP7 and molsidomine14 also impaired
avoidance and maze learning tasks, respectively in rats.
Excess formation of NO has been suggested for these
effects of NO donors, because an increased synthesis
of NO has been found to produce neurotoxicity due to
accumulation of its toxic metabolite, peroxynitrite1. In
support of this nding, the memory impairing action of
higher doses of L-arginine was prevented by L-NAME
and 7-NI32.
Table. Effect of agents that increase/decrease NO concentration in the brain
Drug Animal Experiment Result Reference
1. Precursor of NO
L-arginine Rats Learning and memory of avoidance task Facilitated 11, 12
2. NO donors
SNP Rats Learning and memory of avoidance task Facilitated 12
Chicks Learning and memory of avoidance task Facilitated 5
Rats Ageing-induced memory impairment Prevented 12
SNAP Rats Memory of avoidance task Facilitated 7
Molsidomine Rats Learning of maze performance and object recognition Facilitated 14
3. Inhibitors of NO synthesis
MK-801, AP5 Gold sh Learning of avoidance task Impaired 15
MK-801 Mice Learning of avoidance task Impaired 17
L-NAME Rats Learning and memory of maze performance Impaired 18, 19
Rabbit Eye-blink conditioning Impaired 18
Chicks Learning of avoidance task Impaired 20
Rats Learning and memory of avoidance task Impaired 21
Rats Learning of avoidance task Impaired 34
Octopus vulgaris Learning of avoidance task Impaired 22
Aplysia Learning of avoidance task Impaired 23
Honey bee Learning of olfactory discrimination Impaired 24
L-NA Rats Learning and memory of avoidance task Impaired 25
Rats Learning and memory of maze performance Impaired 26
Rats Learning and memory of maze performance Impaired 29
L-NMMA Rats Memory of avoidance task Impaired 16, 27
7-NI Rats Learning and memory of avoidance task Impaired 28
Rats Learning and memory of maze performance Impaired 29
Chicks Learning of avoidance task Impaired 31
4. Polychlorinated
biphenyls
Rats Memory of avoidance task Impaired 30
PAUL & EKAMBARAM: NITRIC OXIDE & COGNITION 473

The effects of L-arginine and NO donors have been
tested against learning and memory impairment caused
by the inhibitors of NO synthesis. In this study, the
memory impairing action of MK-801 was prevented
by both L-arginine and SNP in mice17. L-arginine
reverted learning and memory impairment produced by
L-NA22,25 and 7-NI28. Further, molsidomine prevented
L-NA from impairing acquisition and retention of
avoidance task in rats14. SNAP prevented L-NAME
from impairing long-term memory in aplysia23. The
effects of NOS inhibitors have also been tested on the
cognitive action of L-arginine. L-NAME prevented
L-arginine from facilitating learning and memory
processes of avoidance task in rats21. Moreover,
L-arginine-induced facilitation of spatial learning
was prevented by L-NAME33. All these experimental
ndings conrm that the mechanism mediated by NO
is involved in learning and memory processes.
Association between cholinergic neurons and NO in
learning and memory processes
NO mediated mechanisms have been assigned
a role in cortical perfusion and cognitive function.
Cholinergic transmission has also been associated
with cerebral blood ow and performance in learning
and memory tasks suggesting a link between
cholinergic and NO-mediated mechanisms34. In
view of this nding an interaction is likely to occur
between cholinergic and NO activities in learning
process and memory formation. Inhibition of NO
synthesis has been shown to cause a decrease in
acetylcholine (ACh) release and an impairment of
retention of conditioned response in rats35. Further,
antagonism of nicotinic receptors and NOS activity
has been found to impair formation of tactile
associative long-term memory in honey bees36.
Further, activation of muscarinic acetylcholine
receptors has been found to induce NO-dependent
LTP in rat medial prefrontal cortex37. The combined
action of the inhibitors of NO and Ach have also
been tested, and it was observed that the combined
action of L-NAME and scopolamine, the muscarinic
receptor antagonist resulted in an impairment of maze
learning in rats38. Further, NO donor molsidomine
has antagonized scopolamine and L-NAME-induced
memory impairment in rats39. These ndings clearly
indicate that cholinergic activity has an involvement
in cognitive effect of NO. However, NO formed as a
result of iNOS upregulation during hypoxia has been
found to interrupt memory process by inhibiting
acetylcholinergic activity40.
Involvement of other neurotransmitters in the
action of NO in learning and memory processes
The interaction of NO with dopamine, noradrenaline
(NA), gamma aminobutyric acid (GABA) and
5-hydroxytryptamine (5-HT) has also been investigated.
Dopamine and its agonist have enhanced cognitive
behaviour and have prevented L-NAME from impairing
learning and memory process in rats41. Alpha adrenergic
antagonist, phentolamine and 6-hydroxydopamine,
a depletory of NA, prevented SNP from promoting
memory formation in mice42. Blockade of GABA
activity has resulted in an impairment of olfactory
discrimination in honey bees24. Memory impairment
by L-NAME has been found to be accompanied by a
decrease in the conversion of 5-HT to its metabolite,
5-hydroxyindole acetic acid in the hippocampus of
rats43.
Endogenous substances and pathophysiological
factors that alter the action of NO on learning and
memory processes
The endogenously occurring analogues of
L-arginine such as methyl-L-arginine,dimethyl-L-
arginine and agmatine which are normally present in
the nervous tissue, have been found to inactivate both
nNOS and eNOS and to decrease NO production in
the brain1. The levels of these analogues are increased
in pathological conditions like chronic renal failure
and essential hypertension resulting in a decrease in
the production of NO and cGMP in the brain regions
of rats44. However, evidence for the role of these
endogenous substances on cognitive behaviour of
experimental animals is yet to be investigated.
Melanin concentrating hormone has been found
to increase NOS activity and the levels of NO and
cGMP in the hippocampus to prevent L-NAME from
producing amnesia in rats45. The neurosteroids, such as
pregnonolone and dehydrepiandrosterone have been
found to improve avoidance task and maze perfor-
mance and to inhibit MK-801 and ageing-induced
memory impairment in rats by increasing NO synthesis
in the brain46.
Diabetes has been found to decrease synthesis of
NO, induction of LTP and synaptic plasticity in the
hippocampus of rats47, suggesting that insulin deciency
and occurrence of blood sugar greater than normal
level can result in an inhibition of NO synthesis and
an impairment of cognitive behaviour. Administration
of insulin results in an induction of NO synthesis in
474 INDIAN J MED RES, MAY 2011

the hippocampus and improvement of learning and
memory processes in rats48. In this study, L-NAME
prevented insulin-induced memory improvement
suggesting further that NO has a role in the cognitive
action of insulin.
The production of NO by nNOS was found to be
diminished in the hippocampus of stress-induced rats.
This was accompanied by a decit in learning and
memory processes in these animals49. Chronic brain
hypoperfusion decreased NOS activity, NO synthesis
and impaired memory formation in rats50. Hypoxia-
ischaemia was found to decrease the activity of nNOS in
the hippocampus and to delay acquisition of avoidance
task in rats51.
Memory impairment has been found to occur
in patients with epilepsy52. Epidemiological studies
have shown that several children with epilepsy have
learning difculties and memory impairment soon
after recovery from seizures53. Convulsion disorder
induced experimentally by picrotoxin (PCT) has
been found to impair the ability of rats to learn and
to remember shock-avoidance task28. Since, sustained
clonic convulsions are known to produce hypotension
and ischaemia resulting in neuronal death54, this
abnormality seems to be responsible for an impairment
of learning and memory processes in these animals.
However, there is evidence for a decreased synthesis
of NO in the hypothala-mus of rats that convulsed after
administration of PCT10. In this study, PCT-induced
convulsions was accompanied by memory impairment
and a decrease in the concentration of NO in the brain,
and NO increasing dose of L-arginine restored NO
concentration in the brain and reverted the memory
impairment in these animals.
Ageing on NO synthesis and learning and memory
processes
Comparative study carried out in young adult (3-4
month old) and aged (13-17 month old) rats have shown
that NO production is decreased with ageing because
serum L-arginine level and urinary excretion of nitrite
and nitrate have been found to be decreased (30-50%)
in aged rats in comparison to that of young rats55.
Behavioral study also showed that aged rats required
signicantly more trials than young ones to learn maze
task56. In this study, the function of glutamate-NO-
cGMP pathway was lesser in older animals than that was
measured in young animals suggesting that a decrease
in NO activity was responsible for impairment of
learning process in older animals. Further, the activity of
cGMP hydrolyzing enzyme, phosphodiesterase (PDE)
was found to be greater in the brain of aged (24 month
old)in comparison to that was measured in young (3
month old) rats57. This factor seems to be responsible
for a decreased activity of both cGMP and NOS and
a lesser synthesis of NO in the brain of aged rats.
Interestingly, sildenal, an inhibitor of PDE, has been
found to enhance memory for mation and to prevent
L-NAME from impairing foot-shock avoidance and
maze learning tasks in rats58. This nding has led these
investigators to suggest that sildenal may prevent
ageing-induced cognitive decline by modulating NO-
cGMP pathway.
AD is known to be associated with progressive
neuro-degeneration, resulting in disturbance of
learning, memory, thought, orientation, judgement and
eventually dementia. Neuronal damage that occurs in
AD has been found to result in an impairment of NO
synthesis and a decrease in NO containing neurons
in the hippocampus59. An inhibition of NO synthesis
has been found in this study, to impair vasodilatation
resulting in a decrease in blood ow and a reduction
in the availability of glucose and other nutrients which
are necessary for the continued neuronal activity
in the brain. Further, deposition of the neurotoxic
proteins such as microglia, apolipoprotein60, and beta
amyloid plaque61 in the brain tissue has been found
to be characteristic of AD. Although, accumulation
of microglia and apolipoprotein seems to have an
involvement in dementia associated with AD, these
agents are unlikely to impair cognition in these patients
by inhibiting the activity of NO in the brain60. On
the other hand, ageing-induced deposition of a toxic
protein beta amyloid, has been proposed to destroy
brain cells61, and to inhibit NO synthesis, and induction
of NMDA receptor-dependent LTP62 and to disrupt
synapses of the nerve cells which are responsible for
learning process and memory formation63. The protein
fragment, beta amyloid that occurs in the brain tissue of
individuals with AD tends to accumulate into clumps in
the brain61 and to impair LTP and synapses of the nerve
cells that are responsible for information processing
and memory formation by disrupting the activity of
NO in the brain64. Thus damage to LTP by beta amyloid
results in synaptic dysfunction, neuronal injury and
inhibition of long-term memory in these patients. Since
beta amyloid disrupts synaptic function, deposition of
amyloid plaques has been considered as a key player
in the development and progress of AD. In view of this
nding, agents that increase NO concentration in the
PAUL & EKAMBARAM: NITRIC OXIDE & COGNITION 475

brain are likely to prevent impairment of cognition in
AD patients. In support of this suggestion, L-arginine,
the precursor of NO59 and NO donors65 have been
found to produce therapeutic effects in patients with
age-related degenerative disease such as AD.
Involvement of NO in learning and memory
impairment caused by side effects of therapeutic
agents
The antiepileptic drugs such as phenobarbitone,
phenytoin and carbamazepine are known to produce
cognitive deterioration as a side effect66. The
anticonvulsant dose of phenobarbitone impaired
retention of acquired pole-climbing shock-avoidance
task in rats67. In this study, NO increasing dose of
L-arginine prevented the memory impairing action
of the anticonvulsant. This nding together with the
anticonvulsant effect of L-arginine and SNP against
PCT-induced convulsions in rats68 has led these
investigators to suggest that administration of L-arginine
or SNP along with phenobarbitone is likely to result in
a potent antiepileptic effect as well as prevention of
cognitive decit produced by both epilepsy and the
anticonvulsant.
Another therapeutic agent, morphine has been
found to impair learning and memory of avoidance
task in mice by decreasing NO synthesis in the brain69.
In this study, L-arginine prevented morphine from
producing cognitive decit.
Conclusion
The experimental ndings reviewed provide
sufcient evidence that NO activates the computational
ability of the brain. These ndings provide sufcient
support to the report that L-arginine, the precursor59
and donor65 of NO may play a prominent role in
the treatment of age-related degenerative disease
such as AD. L-arginine and NO donors may also be
effective in preventing cognitive disorder produced
by epilepsy, antiepileptic drugs, and diabetes. For
further investigation in the mechanism of action of
NO70, its interaction with enzymes, ion channels and
receptors may have to be investigated, to explore new
prospective on the mechanism of its cognitive action
in the brain.
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Reprint requests: Dr (Mrs) Vanaja Paul, F-1, Varalakshmi Castle, 3, Akbarabad II Street, Kodambakkam, Chennai 600 024, India
e-mail: vanaja.paul2008@gmail.com
478 INDIAN J MED RES, MAY 2011