The role of nitric oxide in the object recognition memory

Behavioural Brain Research 285 (2015) 200–207

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The role of nitric oxide in the object recognition memory Nikolaos Pitsikas ∗ Department of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Panepistimiou 3 (Biopolis), 415000 Larissa, Greece

h i g h l i g h t s • Nitric oxide (NO) is an intra- and inter-cellular messenger. • Experimental evidence suggests its involvement in recognition memory. • The object recognition task is sensitive to NO.

a r t i c l e

i n f o

Article history: Received 2 May 2014 Received in revised form 2 June 2014 Accepted 5 June 2014 Available online 13 June 2014 Keywords: Nitric oxide Recognition memory Object recognition Object location

a b s t r a c t The novel object recognition task (NORT) assesses recognition memory in animals. It is a non-rewarded paradigm that it is based on spontaneous exploratory behavior in rodents. This procedure is widely used for testing the effects of compounds on recognition memory. Recognition memory is a type of memory severely compromised in schizophrenic and Alzheimer’s disease patients. Nitric oxide (NO) is sought to be an intra- and inter-cellular messenger in the central nervous system and its implication in learning and memory is well documented. Here I intended to critically review the role of NO-related compounds on different aspects of recognition memory. Current analysis shows that both NO donors and NO synthase (NOS) inhibitors are involved in object recognition memory and suggests that NO might be a promising target for cognition impairments. However, the potential neurotoxicity of NO would add a note of caution in this context. © 2014 Elsevier B.V. All rights reserved.

1. Introduction 1.1. Recognition memory Recognition memory stems from a series of neural processes by which a subject becomes aware that a stimulus has been previously experienced, with recognition as the behavioral outcome of these processes. This type of memory requires that the perceived characteristics of the events are discriminated, identified, and compared with the memory of the characteristics of previously experienced events [73]. Importantly, recognition memory is a type of memory that is impaired in schizophrenia [15,22] and Alzheimer’s disease patients [72]. Twenty-six years ago Ennaceur and Delacour [24] introduced a new memory paradigm the novel object recognition task (NORT). NORT is a non-spatial recognition memory task, does not involve at all the learning of a rule since it is based on the spontaneous predisposition of rodents to explore novel objects. In this test, thus, the

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ability of rodents to recognize a set of novel stimuli in an otherwise familiar environment is considered as a measure of its recognition memory [24]. The standard form of this test involves exposing a rodent to two identical copies of an object (sample trial) for 2–10 min. After a certain delay (intertrial interval), the rodent is then exposed to a novel object and an identical copy of the familiar object (choice trial). Objects can be made of different material (glass, plastic, metal) can have different shape (f.i., cubes, pyramids, cylinders) should have a comparable size and could not be displaced by the rodents. Efforts should be made to equate the pairings of objects in order to avoid any unintentionally induced preference or bias. Attention should be paid to the object odors. Thus, the objects should carefully cleaned before being used for another animal [26]. Exploration of objects was defined as the followings: directing the nose toward the object at a distance of 2 cm or less and/or touching the object with the nose. Turning around or sitting on the object was not considered exploratory behavior. Successful recognition is displayed by the rodent spending a greater amount of time exploring the novel object during the choice trial [24]. Animal’s behavior is directly measured by an observer in the testing room or video-recorded and subsequently analyzed.

N. Pitsikas / Behavioural Brain Research 285 (2015) 200–207

Ennaceur and colleagues developed a novel version of this procedure, named the object location task (OLT), aiming to evaluate spatial recognition memory in rodents [25]. Spatial memory is the ability of an organism to acquire a cognitive representation of location in space and the ability to effectively navigate the environment [3]. This task assesses the ability of rodents to discriminate the novelty of the object locations but not the objects itself because the behavioral testing arena is already familiar to the animals [19,25]. During the sample trial of this new paradigm, similarly to NORT, rodents are exposed to two identical objects. After a certain delay, animals are re-exposed to the same two objects, one of which has been displaced to a new location within the apparatus. The definition of exploration is provided above in the context of describing the NORT protocol. Successful recognition is displayed by the animal spending a greater amount of time exploring the object in the new location during the choice trial. Reportedly, in this context, the duration of the retention interval is of high importance for both the above described recognition memory tasks. The performance of the animals deteriorates as the delay between the sample and the choice trial increases [4]. Moreover, one major challenge in memory research is the question whether a “deficit” is due to an “unspecific” effect on sensory, motor, and/or motivational systems, or actually reflects an effect on the neurobiological substrate of the memory system under question. “Unspecific” effects of the experimental manipulations, such as the application of drugs, brain lesions, genetic manipulations, etc. on these recognition memory paradigms can be potentially detected by a detailed analysis of rodent’s behavior in terms of the frequency of contacts with the objects, the time spent in exploring the objects, the distance travelled, the number of rearings, abnormal posture, defecation, etc. [18]. In addition, different studies indicate that the prefrontal cortex, the hippocampus, the parahippocampal regions of the temporal lobe (namely the perirhinal, entorhinal and postrhinal cortices) are brain structures implicated in recognition memory [26,40,79]. Both of these recognition memory tasks do not involve explicit reward or punishment but rely on the natural curiosity of rodents and preference for novelty [69], which do not appear to be influenced by reinforcement/response contingencies [18]. These paradigms are quite similar to procedures used in humans and should have a significant level of construct and predictive validity and probably reflect episodic memory [26]. Moreover, with suitable manipulations, these recognition memory paradigms can evaluate different stages of memory formation such as encoding, storage and retrieval of information. Therefore, either the NORT or the OLT are used for testing putative memory enhancing compounds. 1.2. Nitric oxide (NO) Nitric oxide (NO), a small, short-lived, and highly diffusible gas, is an important intra- and inter-cellular messenger in the brain [31]. NO originally was identified as endothelium-derived relaxing factor (EDRF) mediating relaxation of blood vessels [28]. NO plays essential roles in the regulation of a wide range of physiological processes, including cellular immunity [34], vascular tone [51], and neurotransmission [30]. 1.2.1. Synthesis of NO NO is originated by the conversion of l-arginine to l-citrulline, with the release of NO. The enzymatic oxidation of l-arginine to l-citrulline occurs in the presence of oxygen (O2 ) and nicotinamide adenine dinucleotide phosphate (NADPH) with flavin adenine dinucleotide (FAD) flavin mononucleotide (FMN), henme, thiol and tetrahydrobiopterin as cofactors [38,52]. The enzyme responsible for the generation of NO is NO synthase (NOS). Three NOS isoforms encoded on different distinct genes have

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been described: neuronal NOS (nNOS, NOS type I) being the isoform found in neuronal tissues, inducible NOS (iNOS, NOS type II) being the isoform which can be synthesized following induction by proinflammatory cytokines or endotoxin and endothelial (eNOS, NOS type III) being the isoform expressed in endothelial cells [12]. nNOS and eNOS are constitutively expressed and dependent on the presence of calcium (Ca2+ ) ions and calmodulin to function, whereas the activity of iNOS is Ca2+ independent [for review, see [13]]. NO is formed following activation of glutamate receptors, mainly the N-methyl-d-aspartate (NMDA) subtype. After this activation, Ca2+ is transiently increased in the cytosol and forms a complex with calmodulin that binds to and activates nNOS [38]. Glial cells (astrocytes and microglia) synthesize NO after the transcriptional expression of a Ca2+ independent iNOS isoform [48].

1.2.2. Main physiological targets of NO NO has been described as an unconventional neurotransmitter because it is not stored in synaptic vesicles and not released upon membrane depolarization but released immediately after its synthesis. NO does not mediates its action by binding to membrane associated receptors but diffuses to adjacent neurons and acts directly to intracellular components [31].The most prominent natural target of NO is soluble guanylyl cyclase (sGC). NO acts as a messenger, activating sGC [2] and participating in the transduction signaling pathway involving cyclic guanosine monophosphate (cGMP). cGMP, in turn, activates cGMP-dependent protein kinase (PKG), which may affect additional second messenger systems. cGMP can also directly activate other protein kinases, such as the cyclic adenosine monophosphate (cAMP)-dependent kinase PKA [49]. Metabolism of cGMP by phosphodiesterase (PDE) suppresses or terminates NO/sCG signaling [37]. Thus, NO is similar to conventional transmitters that act via second messengers to activate protein kinases which may in turn affect transcription factors and protein synthesis. In this context, current literature indicates that the cGMP should probably no longer be considered the only target of the action of NO. Alternative cGMP-independent mechanisms have recently been proposed. One reaction which is gaining prominence is the S-nitrosylation of various proteins such as the NMDA receptor; the caspases 1–4 and 6–8; the cyclic nucleotide-gated (CNG) channels; the large conductance Ca2+ -activated potassium (BKCa ) channels and the ryanodine receptor Ca2+ release (RyR) channels. Depending upon the protein species, S-nitrosylation can either inhibit or up-regulate their activity. The aforementioned opened by S-nitrosylation channels and the enzyme mono(ADP-ribosyl) transferase are amongst the cGMP-independent mechanisms by which NO may exerts its action [23]. NO is involved in synaptic activity, neural plasticity and memory functions. It promotes also survival and differentiation of neurons and exerts long-lasting effects through regulation of transcription factors and modulation of gene expression. NO potentially acts among the above described mechanisms, depending on the concentration, with low concentrations being neuroprotective and mediate physiological signaling (e.g., neurotransmission or vasodilatation), whereas higher concentrations mediate immune/inflammatory actions and are neurotoxic [13,14]. Because of its mobility, unconstrained by cell membranes, NO can act across a broad volume and its actions are limited by inactivation (e.g., scavenging or degradation). It has long been postulated that NO can also could act as a retrograde messenger at the synapse, mediating transmission from target neurons back onto the synapse and regulating synaptic plasticity, but the same properties also enable NO to signal to any local compartment and to cells that lack synaptic activity or NOS expression [74].

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1.2.3. NO and cognition Long-term potentiation (LTP) and long-term depression (LTD) are activity-dependent forms of modified transmission efficacy at synapses [6]. This type of neuronal plasticity, first shown in the hippocampus, meanwhile has been found to occur in various brain regions thus being widespread phenomenon in the central nervous system (CNS). In addition, learning and memory occur as a result of changes in the efficacy of synaptic transmission. Soon after the discovery of NO synthesis in brain tissue, it has been suggested that NO acts as a retrograde messenger which influences synaptic transmission in the presynaptic cell and promotes synaptic plasticity [41,71]. NO’s implication in cognition is well documented. Behavioral investigations have demonstrated that blockade of NOS, the key synthesizing enzyme, by different NOS inhibitors inhibit learning and these learning deficits could be counteracted by diverse NO donors [for review see [65]].

2. NO and recognition memory 2.1. NO donors and object recognition An overview of the literature regarding the effects of NO donors on object recognition memory is provided in Table 1. NO donors are synthetic chemical reagents that release NO continuously over a period of time, under physiological conditions. NO donors are in use in clinical management of cardiovascular diseases. Among NO donors, molsidomine has a high bioavailability, a long-lasting duration of action [8], likely crosses the blood brain barrier (BBB) [43,68] and increases its permeability [47]. Intraperitoneal (i.p.) administration, either before or just after the sample trial, or before the choice trial, of molsidomine (at 4 but not at 2 mg/kg) counteracted delay-dependent deficits in the NORT in control rats [55]. In line with these findings, the novel NO donor (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 5(nitrooxy)butyl ester) NCX-2057 (10 mg/kg, i.p.) attenuated delaydependent deficits in the same recognition memory paradigm in rats [11]. These results suggest that both molsidomine and NCX2057 are able to affect distinct aspects of recognition memory such as acquisition, storage and retrieval of information. Pre- or post-training administration of molsidomine (4 but not 2 mg/kg, i.p.) antagonized performance deficits in the NORT induced by the non-selective NOS inhibitor N␻ -nitro-larginine-methylester (l-NAME) (30 mg/kg, i.p.) in rats [56,57]. Further, intrahippocampal coinfusion of the NO donor S-nitrosoN-acetylpenicillamine (SNAP) (5 ␮g) and the pKG activator 8-Br-cGMP (75 ␮g) were found to attenuate performance deficits induced by infusion of the NOS inhibitor N␻ -nitroarginine-2,4-ldiaminobutyric amide di(trifluoroacetate) l-NN (1 ␮g) in the NORT [29]. These findings clearly indicate the involvement of NO in recognition memory. Systemic administration of molsidomine (2 and 4 m/kg, i.p.) and NCX-2057 (3 and 10 mg/kg, i.p.) reversed the cholinergic muscarinic receptor antagonist scopolamine (0.2 mg/kg, s.c.)-induced performance deficits in the NORT bespeaking the involvement of the nitrergic system in the cholinergic modulation of memory [11,54]. Further, when this NO donor was applied at the dose of 4 mg/kg, either before or just after the sample phase of the object recognition procedure, it reversed recognition memory deficits produced by the GABAB receptor agonist baclofen (4 mg/kg, i.p.). These results support a functional interaction between the NO-ergic and the GABA-ergic system [58]. Moreover, post-training administration of molsidomine (2–4 mg/kg, i.p.) attenuated performance deficits induced by the 5-HT1A receptor agonist R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) (0.3 mg/kg, i.p.) in the NORT. These findings

indicate that a NO component modulates the effects of the serotonergic 5-HT1A receptor on recognition memory [59]. As we have already mentioned that recognition memory is impaired in schizophrenic patients [15,22]. In this context, cognitive deficits produced by dysfunctions of the glutamatergic and the dopaminergic system are typical of schizophrenic patients [35]. Post-training administration of molsidomine (4 mg/kg, i.p.) counteracted performance deficits induced by the NMDA receptor antagonist MK-801 (0.1 mg/kg, i.p.) in the NORT in rats [61]. Similarly, post-training injection of different NO donors such as molsidomine (2–4 mg/kg, i.p.) and sodium nitroprusside (SNP) (0.3 and 1 mg/kg, i.p.) reversed recognition memory deficits produced by the mixed D1/D2 dopaminergic agonist apomorphine (1 mg/kg, i.p.) in rats in the same recognition memory procedure [33]. As a whole, these findings indicate that NO donors attenuated recognition memory impairments in different animal models of cognitive impairments associated with schizophrenia and propose a functional interaction between the NO and the glutamatergic and dopaminergic system, respectively. Post-training co-administration of sub-threshold doses of molsidomine (1 mg/kg, i.p.) and the non-competitive NMDA receptor antagonist memantine (3 mg/kg, i.p.), a drug approved for the treatment of severe Alzheimer Disease [67,70], counteracted extinction of recognition memory in the rats [63]. The present findings indicate that a NO component modulates the effects of memantine on recognition memory. In addition, molsidomine (4 mg/kg, i.p.) was found efficacious to reverse age-related impairments in the NORT in the 24 month-old rat suggesting that the integrity of the NO-ergic system may be important in brain aging processes [60]. Finally in the spatial version of the object recognition task, namely the OLT, pre-training administration of molsidomine (2–4 mg/kg, i.p.) attenuated spatial recognition memory deficits produced either by l-NAME (30 mg/kg, i.p.) or scopolamine (0.2 mg/kg, s.c.). These results demonstrated that this NO donor affects the encoding of spatial information, that NO is involved in spatial recognition memory and that an NO component modulates the effects of the cholinergic system on spatial memory [64]. The mechanism underlying effects of NO donors on learning and memory is still matter of investigation. At the moment there is no information that would support the hypothesis that NO donors can enhance cognition by improving cerebral blood flow [53]. Several lines of evidence indicate that NO donors are capable to modulate the release of different neurotransmitters, which play an important role in cognition, such as glutamate, acetylcholine, serotonin and GABA [65]. Importantly, NO donors were found to promote LTP which is considered the electrophysiological correlate of memory [1]. 2.2. NOS inhibitors and object recognition An overview of the literature regarding the effects of NOS inhibitors on object recognition memory is provided in Table 2. NOS inhibitors are compounds which can decrease NO concentrations in the brain. Peripheral post-training acute administration of the selective nNOS inhibitor 7-nitroindazole (7-NI) (10–30 mg/kg, i.p.) and the non-selective NOS inhibitor l-NAME (30 mg/kg, i.p.) injected either pre-or post-training) impaired rats’ performance in the NORT [56,57,66]. Similarly, pre-training acute administration of l-NAME (30 mg/kg, i.p.) elicited spatial recognition memory deficits evidenced in the OLT [62,64]. A single injection of the NOS inhibitors 7-NI (15, 30 and 45 mg/kg, i.p.) and 1-(2-trifluoromethylphenyl) imidazole (TRIM) (10, 30 and 50 mg/kg, i.p.) in mice produced opposite results. TRIM did not modify rodents’ recognition memory abilities, at any dose tested, whereas 7-NI (30 mg/kg) disrupted animals performance in the NORT [50]. Interestingly, 7-NI, at the highest dose (45 mg/kg)

Table 1 Effects of NO donors on recognition memory. Species and strain

Drug

Action

Dose range

Route

Infusion stage

ITI

Task

Effect

Reference

CD-COBS rat

Molsidomine Scopolamine Molsidomine l-NAME Molsidomine

NO donor Ach antagonist NO donor NOS inhibitor NO donor

2, 4 mg/kg 0.2 mg/kg 2, 4 mg/kg 30 mg/kg 2, 4 mg/kg

i.p. Acute s.c. Acute i.p. Acute i.p. Acute i.p. Acute

Pre-sample

1h

NORT

Reversed scopolamine-induced-deficits

[54]

Post-sample

1h

NORT

[57]

24 h

NORT

Molsidomine l-NAME Molsidomine Baclofen Molsidomine 8-OH-DPAT Molsidomine Molsidomine MK-801 Molsidomine Memantine Molsidomine Scopolamine l-NAME SNAP 8-Br-cGMP l-NN NCX-2057 Scopolamine NCX-2057 Molsidomine SNP

NO donor NOS inhibitor NO donor GABAB agonist NO donor 5-HT1A agonist NO donor NO donor NMDA blocker NO donor NMDA blocker NO donor Ach antagonist NOS inhibitor NO donor pKG activator NOS inhibitor NO donor Ach antagonist NO donor NO donor NO donor

i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute i.p. Acute s.c. Acute i.p. Acute Intrahipp. Intrahipp. Intrahipp. i.p. Acute s.c. Acute i.p. Acute i.p. Acute i.p. Acute

1h

NORT

[56]

Pre-sample, post-sample Post-sample

1h

NORT

3h

NORT

Reversed (4 mg/kg) l-NAME-induced deficits Reversed (4 mg/kg) baclofen-induced deficits Reversed 8-OH-DPAT-induced-deficits

Post-sample Post-sample

1h 3h

NORT NORT

[60] [61]

Post-sample

24 h

NORT

Pre-sample

20 min

OLT

Reversed (4 mg/kg) age-related deficits Reversed (4 mg/kg) MK-801-induced deficits Coadministration of mosidomine and memantine reversed delay-dependent deficits Reversed scopolamine and l-NAME-induced deficits

Pre-sample

1h

NORT

Coinfusion of SNAP and 8-Br-cGMP reversed l-NN deficits

[29]

Pre-sample

1h

NORT

Reversed scopolamine-induced-deficits

[11]

Post-sample Post-sample

24 h 3h

NORT NORT

Reversed (10 mg/kg)-delay-dependent deficits Molsidomine and SNP reversed apomorphine-induced deficits

[11] [33]

Apomorphine

D1/D2 DA agonist

2, 4 mg/kg 30 mg/kg 2, 4 mg/kg 4 mg/kg 2, 4 mg/kg 0.3mg/kg 2, 4 mg/kg 2, 4 mg/kg 0.1 mg/kg 1 mg/kg 3 mg/kg 2, 4 mg/kg 0.2 mg/kg 30 mg/kg 5 ␮g 75 ␮g 1 ␮g 3, 10 mg/kg 0.2 mg/kg 1–30 mg/kg 2, 4 mg/kg 0.3, 1 mg/kg 1 mg/kg

Pre-sample; post-sample, pre-retrieval Pre-sample

Reversed (4 mg/kg) l-NAME-induced-deficits Reversed (4 mg/kg)-delay-dependent deficits

CD-COBS rat CD-COBS rat

CD-COBS rat Wistar rat CD-COBS aged rat Wistar rat Wistar rat Wistar rat

Wistar rat

Wistar rat Wistar rat Wistar rat

[58] [59]

[63] [64]

N. Pitsikas / Behavioural Brain Research 285 (2015) 200–207

CD-COBS rat

[55]

i.p. Acute

Abbreviations: Ach, acetylcholine; DA, dopamine; GABA, ␥-aminobutyric acid; 5-HT, serotonin; ITI, intertrial interval; intrahipp., intrahippocampal; i.p, intraperitoneally; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NORT, novel object recognition task; NOS, nitric oxide synthase; OLT, object location task; pKG, protein kinase cGMP-dependent; s.c, subcutaneously.

203

204

Table 2 Effects of NOS inhibitors and antagonists on recognition memory. Drug

Action

Dose range

Route

Infusion stage

ITI

Task

Effect

Reference

Tyron-maze bright rat Wistar rat

7-NI l-NA

NOS inhibitor NOS inhibitor

10, 30 mg/kg 3, 10, 30 ␮g/kg

Post-sample Post-sample

1h 1h

NORT NORT

Impairment Impairment (10 and 30 ␮g/kg)

[66] [7]

Wistar rat

l-NA

NOS inhibitor

10, 30 ␮g/kg

Pre-sample

1h

NORT

No effect

[7]

CD-COBS rat CD-COBS rat Wistar rat Wistar rat

l-NAME l-NAME l-NAME l-NN

NOS inhibitor NOS inhibitor NOS inhibitor NOS inhibitor

10, 30 mg/kg 10, 30 mg/kg 10, 30 mg/kg 0.01, 0.1, 1 ␮g

i.p. Acute Intrahipp. acute Intrahipp. acute i.p. Acute i.p. Acute i.p. Acute Intrahipp. acute

1h 1h 20 min 1h

NORT NORT OLT NORT

Impairment (30 mg/kg) Impairment (30 mg/kg) Impairment (30 mg/kg) Impairment (1 ␮g)

[57] [56] [62] [29]

Wistar rat

l-NAME

NOS inhibitor

1, 3, 10 mg/kg

i.p. Acute

24 h

NORT

Reversed (1–3 mg/kg) delay-dependent deficits

[9]

Wistar rat

l-NAME MK-801

1, 3, 10 mg/kg 0.1 mg/kg

i.p. Acute i.p. Acute

Post-sample Pre-sample Pre-sample Pre-sample Pre-retrieval Pre-sample Post-sample Pre-retrieval Post-sample

1, 3 h

NORT

Reversed MK-801 and ketamine-induced deficits

[10]

3 mg/kg

i.p. Acute

25 mg/kg 25 mg/kg

i.p. Acute i.p. Acute

Pre-sample

5 min

NORT

Impaired D. alfa-treated mice’ performance

[36]

i.p. Acute

Pre-retrieval

1h

NORT

7-NI (30 and 45 mg/kg) but not TRIM impaired mice’ performance

[50]

i.p. Chronic i.p. Chronic

Pre-sample Pre-sample

1h 24 h

NORT NORT

[32]

i.p. Acute i.p. Acute i.p. Acute Intraperihn

Post-sample

3h

NORT

Impairment (3 mg/kg) Reversed (1 and 10 mg/kg) delay-dependent deficits l-NAME and 7-NI reversed apomorphine-induced deficits

Pre-sample Pre-sample

20 min 24 h

NORT

No effect Impairment

[76]

STOP null mouse

l-NAME D. alfa

Swiss mouse

TRIM

NOS inhibitor NMDA blocker NMDA blocker NOS inhibitor Erythropoietin analog NOS inhibitor

7-NI

NOS inhibitor

Wistar rat Wistar rat

l-NAME l-NAME

NOS inhibitor NOS inhibitor

10, 30, 50 mg/kg 15, 30, 45 mg/kg 1, 3, 10 mg/kg 1, 3, 10 mg/kg

Wistar rat

l-NAME 7-NI Apomorphine NPA

NOS inhibitor NOS inhibitor DA agonist NOS antagonist

1, 3 mg/kg 1, 3 mg/kg 1 mg/kg 2 ␮M

Ketamine

Dark Agouti rat

i.p. Acute

[33]

Abbreviations: DA, dopamine; intrahipp., intrahippocampal; intraperhin, intraperirhinal; i.p, intraperitoneally; ITI, intertrial interval; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NORT, novel object recognition task; NOS, nitric oxide synthase; OLT, object location task; s.c, subcutaneously.

N. Pitsikas / Behavioural Brain Research 285 (2015) 200–207

Species and strain

N. Pitsikas / Behavioural Brain Research 285 (2015) 200–207

tested impaired animals’ performance in the NORT but also induced hypomotility in the mice [50]. Lastly, l-NAME (25 mg/kg, i.p.) abolished the beneficial effect produced by administration of the erythropoietin analog darbepoetin alpha (25 mg/kg, i.p.) in a mouse model of schizophrenia in the NORT [36]. Intrahippocampal infusion of the non-selective NOS inhibitor N␻ -nitro-l-arginine (l-NA) (10–30 ␮g) just after but not before the sample phase impaired rats’ performance in the NORT. Since treatment with 30 ␮g l-NA has been found to inhibit hippocampal NOS almost completely and lasts longer than 2 h, it was concluded that hippocampal NOS inhibition might induced a state-dependent performance deficit [7]. Intrahippocampal application of the selective nNOS inhibitor l-NN (1 ␮g) disrupted rats’ performance in the NORT [29]. Reportedly, infusion of the NOS antagonist NG -propyl-larginine (NPA) (2 mM) in the perirhinal cortex impaired encoding of long-term but not of short-term visual recognition memory in the rat [76]. Importantly, peripheral acute administration of NOS inhibitors, at a lower dose range than that described above, led to opposite findings. Specifically, l-NAME (1, 3 and 10 mg/kg, i.p.) did not affect animals’ performance in the NORT. In addition, this NOS inhibitor (1 and 3 but not 10 mg/kg, i.p.), injected either before or just after the sample trial or before the choice trial, counteracted delay-dependent impairments in the NORT in the normal rat. These findings indicate that l-NAME modulated different aspects of memory such as encoding, storage and retrieval [9]. In line with the above results, l-NAME (1–3 mg/kg, i.p.) reversed recognition memory deficits produced by the NMDA receptor antagonists MK-801 (0.1 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.) in the NORT in rats [10]. Further, either l-NAME (1–3 mg/kg, i.p.) or 7-NI (1–3 mg/kg, i.p.) reversed performance deficits induced by the DA agonist apomorphine (1 mg/kg, i.p.) in the NORT in rats [33]. Collectively, these results indicate the beneficial action of acute systemic administration of different NOS inhibitors on recognition memory deficits produced by dysfunction of the glutamatergic and dopaminergic system. Interestingly, repeated systemic administration of l-NAME (1, 3, 10 mg/kg, once a day, for 5 consecutive days) differentially affected animals’ recognition memory abilities. Specifically, this NOS inhibitor, at the dose of 3 mg/kg, disrupted rats’ performance in the NORT, while when it was injected at 1 and 10 mg/kg, counteracted delay-dependent deficits in the control rat in the same recognition memory paradigm. These results suggest a complex involvement of l-NAME in recognition memory [32]. The mechanism(s) of action underlying the effects of high doses of NOS inhibitor on memory is still a matter of investigation. Current literature, reportedly, refers to conflicting data concerning the role of NOS inhibitors on cognition and on LTP. NOS inhibitors were found to inhibit or partially block hippocampal LTP. In contrast, other studies did not support this proposition [65]. In addition, evidence of the reduction produced by NOS inhibitors on hippocampal NO concentration has been provided in several studies [65]. The most reliable observed effect of NOS inhibitors on memory has been impairment of acquisition of a new behavior rather than disruption of previously learned behavior [78]. It has been suggested, however, that in some circumstances, it is difficult to dissociate learning impairment from unspecific factors (f.i., hypomotilty and increase of blood pressure) which might have altered animals’ performance treated with high doses of NOS inhibitors in different memory paradigms [21]. The involvement of non-specific factors in the amnestic action of high doses of NOS inhibitors on recognition memory seems however, to be unlikely. Specifically, it has been demonstrated that the effects produced by high doses of these compounds either on non-spatial and spatial recognition memory were unrelated to their effects on motility or on blood pressure [50,56,57,62,64,66].

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Conversely, NOS inhibitors applied acutely, at a low dose range, displayed a beneficial action on recognition memory. The mechanism(s) through which low doses of NOS inhibitors exert their facilitatory action on learning and memory is not yet clarified. The facilitation of learning produced by l-NAME may have been mediated through its action on glutamatergic transmission at the NMDA receptor. In particularly, NO is an intra- and inter-cellular messenger and by a negative feedback mechanism modulates the NMDA receptor activity. NO can inhibit NMDA receptors located either on NOS neurons or on nearby neurons while procedures that decrease NO potentiate the excitatory effects of NMDA [44,45]. It has been suggested that the excitations of the NMDA receptors, with the consequent mobilization of intracellular Ca2+ promotes the formation of NO through activation of NOS. The NO so formed can then diffuse to reversibly nitrosylate NMDA receptors and thus decrease excitation [39,42]. Therefore, blocking NO production would have removed this feedback effect and thus maintained the excitation of the NMDA receptor during testing [20]. It still under investigation the mechanism(s) by which NOS inhibitors attenuated recognition memory deficits produced by dysfunction of the glutamatergic and the DAergic systems. In this context, it has been suggested that l-NAME abolished psychotomimetic effects of phencyclidine (PCP), including cognitive deficits, by reducing phencyclidine PCP-induced increase in cGMP production in the medial prefrontal cortex of the mouse brain [27]. Since also apomorphine increases cGMP content in the brain [5] and in analogy to what was observed with PCP [27] it could be imagined that inhibiting NOS activity may decrease cGMP and abolish memory deficit due to dopaminergic dysfunction. Sub-chronic peripheral administration of low doses l-NAME however, produced discrepant results and suggesting therefore, a complex involvement of this NOS inhibitor in cognition [32]. There is no clear explanation for this finding. This impairing effect of repeated application of 3 mg/kg l-NAME on recognition memory was not seen at the lowest (1 mg/kg) and at the highest (10 mg/kg) dose, revealing thus, a U-shaped dose–effect curve. Ushaped curves are a consistent feature of administration of drugs affecting numerous neurotransmitters and hormonal systems [46]. At present, the biological bases of U dose–response relationship are unknown, although receptor fatigue or tachyphylaxis [17] has been suggested as one possible mechanism to account for the failure of higher doses to produce an asymptotic behavioural response [46]. In this context, we cannot entirely rule out issues related to the pharmacokinetic profile of l-NAME. l-NAME is de-esterified to its active metabolite l-NA within 4 h [77] which, in turn, displays a long half-life (20 h) [75]. Because the current studies employed a multiple exposure to l-NAME, treatment somehow might contribute to the observed deficit in memory performance. Further, studies are needed however, to elucidate this issue. In contrast, sub-chronic application of l-NAME (1 and 10 mg/kg) counteracted delay-dependent recognition memory deficits. One possible hypothesis to explain this result is that l-NAME acts through different mechanisms operating at different doses. For instance, we cannot exclude that l-NAME, at this low dose range, might interfere with the cGMP system rather than with NOS activity.

3. Conclusions The object recognition task is a sensitive procedure to detect alteration in animal behavior. This recognition memory paradigm is able to assess distinct memory components such as encoding, storage and retrieval and is widely used to evaluate the influence of different compounds on recognition memory.

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