**3.4 The object recognition test**

In laboratory, cognitive tasks have shown a good reliability in many experimental models of human neurodegenerative diseases. Specifically, a lot of laboratory studies have shown that the object recognition task in rodents is highly sensitive to psychoactive drug. For example, this is the case of drugs such as acetylcholinesterase inhibitors (AChEIs) which can improve object memory performance in rats [Prickaerts et al., 2002; Hornick et al., 2008; Goh et al., 2009]. In fact, in rats these ACh enhancers can reverse drug-induced memory impairments [Bejar et al., 1999; van der Staay and Bouger, 2005; Yamada et al., 2005]. This has encouraged researchers that such drugs may also be useful in treating memory impairments in patients with dementia. On the other hand, to date, clear evidence for a reliable memory enhancing effect of these drugs in humans is lacking and controversial [Snyder et al., 2005; Wezenberg et al., 2005]; that might probably be related to the discrepancy between the large numbers of animal studies and only a limited number of human studies showing memory enhancing effects of these drugs.

Object discrimination requires the integrity of cortical cholinergic system; in rodents the cortex-hippocampus circuitry consents to distinguish individual objects such as different shapes [Hauser et al., 2009].

The novel object test or object recognition test (ORT) was first described by Ennaceur and Delacour (1988). Rats or mice are exposed first to two identical objects and then one of the objects is replaced by a new object. The time spent exploring each of the objects is measured. The test has become popular for assessing the effects of amnesic drugs in rodents in general and, after that, to test new compounds enhancing attention and memory [Bartolini et al., 1996]. The test is based on spontaneous behavior with no reinforcement such as food or shock. Non-amnesic animals will spend more time exploring the novel object than the familiar one. An absence of any difference in exploration time can be interpreted as a memory defect or, in case an amnesic drug is tested, a non-effective drug.

Although the novel object recognition task has shown high sensibility and it can be a simple approach to test new potential antidementia drugs, researchers need a stronger experimental tools to test *in vivo* pharmacological activity before clinical trials. From our point of view, an electrophysiological approach together with novel object recognition task can probably be useful.

## **3.5 An experimental model of AD**

Discovering the cause of Alzheimer's disease should imply the ultimate hope of developing safe and effective pharmacological treatments [Francis et al., 1999].

Most researches on working memory, carried out in experimental models of AD, have been modelled on those conducted in physiological studies of monkeys.

Neural Basis of Object Recognition 11

Scopolamine impairs object recognition and increases theta frequency in the EEG. In this experimental model it is suggested that scopolamine likely caused a decrement in arousal. However, the effects of scopolamine on mnemonic paradigms can be characterized as disrupting acquisition and encoding information rather than retrieval processes. Most experiments used a relatively low dose of scopolamine (ranging 0.1 to 0.2 mg/kg). In fact, it must be noted that high doses of the muscarinic antagonist may not only have an effect on the muscarinic receptors, but also on the nicotinic receptors [Schmeller et al., 1994,1995]. Methyl–scopolamine, which only differs from scopolamine in that it does not cross the

Therefore, to target this aim an *in vivo* study, using our model of AD, was designed to test pharmacological properties of new compounds. With this purpose, a set of experiments was planned to evaluate them on cortex- and hippocampus-dependent memory. Attention, learning and working memory, with respect to cortical and hippocampal EEG theta rhythm, recorded during novel object recognition task in animals with lesion,of the nucleus basalis of Meynert, were studied. In NBM-lesioned animals, compared with control, an increased theta power in the cortex and a reduced theta rhythm oscillation in the hippocampus was found. These EEG changes were correlated with a worse performance in learning and memory tasks. In rats with damaged NBM, novel compounds were able to restore EEG architecture, producing cortical desynchronization and reduction in theta power [Rispoli et al., 2004a, 2006, 2008], while in the hippocampus the drugs increased theta oscillation and reduced the impairment in attention/working memory in the behavioural tasks [Rispoli et

Here, we report data supporting this experimental model of AD in testing nwe compounds

Fig. 3. Stereotaxic lesion of the Nucleus Basalis of Meynert.

blood brain barrier, is generally used as a control.

al., 2011, data in progress].

as putative antidementia drugs.

On basis of these data, in the last decades many attempts have been done to alter central cholinergic neurotransmission. The two major approaches contemplate substances pharmacologically altering central cholinergic neurotransmission or toxins, directly injected in brain and disrupting cholinergic system. Commonly, the aim is to produce highly selective lesions of cholinergic neurons with none or marginal effects on other neurons [Torres et al., 1994; Perry et al., 2001].

Our group is involved in preclinical research investigating new therapeutical approaches to AD (Fig. 2). To this purpose, in the last decade, we developed an experimental model of Alzheimer's disease to investigate the pharmacological effects of drugs with putative antidementia activity. Original compounds, likely thought to enhance central cholinergic activity, were designed, synthesized and firstly studied in our molecular modeling laboratory; after that, their pharmacological properties on both EEG brain activity and novelty object recognition were tested; finally, the relation between the EEG architecture and performance measures was studied too.

Fig. 2. Laboratory for Preclinical Researches in Neuropharmacology and Neurodegenerative Diseases at the Department of Pharmacological Sciences, University *Magna Græcia* of Catanzaro. Surgery room (left) and Behavioural Lab (right) with Noldus Ethovision® XT 8.0 apparatus for novel object recognition are here depicted.

In this AD model, we selectively damaged portion of NBM which targets the frontal cortex, producing in rat a significant deficit in attention and working memory (Fig. 3), [Rispoli et al., 2004a,b, 2006, 2008]. Further, in this experimental model, attention, learning and working memory can be evaluated monitoring cortico-hippocampal qEEG activity during object recognition task [Rispoli et al., 2011, data in progress].

The brain lesion produces a significant reduction of cholinergic neuronal population in the NBM (45%; *p*<0.01 vs control; Fig. 3, panel B). Immunohistochemistry was performed to quantify the neuronal loss in the NBM by ChAT immunoreactive neurons. Quantitative analysis of ChAT-positive neurons in NBM was carried out using a computerized image analysis system (Axiophot Zeiss microscope equipped with a Vidas Kontron system). Notably no spontaneous recovering of ChAT immunoreactive neurons has been found by us, not even after several weeks post NBM lesion.

To validate our AD model, we compared it with other well validated experimental models producing dysfunction in cognitive processes: the scopolamine-induced amnesia, a classical pharmacological model of amnesia, and that in which cholinergic neurons in the basal forebrain are subjected to immunolesion by IgG-saporin.

10 Advances in Object Recognition Systems

On basis of these data, in the last decades many attempts have been done to alter central cholinergic neurotransmission. The two major approaches contemplate substances pharmacologically altering central cholinergic neurotransmission or toxins, directly injected in brain and disrupting cholinergic system. Commonly, the aim is to produce highly selective lesions of cholinergic neurons with none or marginal effects on other neurons

Our group is involved in preclinical research investigating new therapeutical approaches to AD (Fig. 2). To this purpose, in the last decade, we developed an experimental model of Alzheimer's disease to investigate the pharmacological effects of drugs with putative antidementia activity. Original compounds, likely thought to enhance central cholinergic activity, were designed, synthesized and firstly studied in our molecular modeling laboratory; after that, their pharmacological properties on both EEG brain activity and novelty object recognition were tested; finally, the relation between the EEG architecture

Fig. 2. Laboratory for Preclinical Researches in Neuropharmacology and Neurodegenerative

In this AD model, we selectively damaged portion of NBM which targets the frontal cortex, producing in rat a significant deficit in attention and working memory (Fig. 3), [Rispoli et al., 2004a,b, 2006, 2008]. Further, in this experimental model, attention, learning and working memory can be evaluated monitoring cortico-hippocampal qEEG activity during

The brain lesion produces a significant reduction of cholinergic neuronal population in the NBM (45%; *p*<0.01 vs control; Fig. 3, panel B). Immunohistochemistry was performed to quantify the neuronal loss in the NBM by ChAT immunoreactive neurons. Quantitative analysis of ChAT-positive neurons in NBM was carried out using a computerized image analysis system (Axiophot Zeiss microscope equipped with a Vidas Kontron system). Notably no spontaneous recovering of ChAT immunoreactive neurons has been found by

To validate our AD model, we compared it with other well validated experimental models producing dysfunction in cognitive processes: the scopolamine-induced amnesia, a classical pharmacological model of amnesia, and that in which cholinergic neurons in the basal

Diseases at the Department of Pharmacological Sciences, University *Magna Græcia* of Catanzaro. Surgery room (left) and Behavioural Lab (right) with Noldus Ethovision® XT 8.0

[Torres et al., 1994; Perry et al., 2001].

and performance measures was studied too.

apparatus for novel object recognition are here depicted.

object recognition task [Rispoli et al., 2011, data in progress].

us, not even after several weeks post NBM lesion.

forebrain are subjected to immunolesion by IgG-saporin.

Fig. 3. Stereotaxic lesion of the Nucleus Basalis of Meynert.

Scopolamine impairs object recognition and increases theta frequency in the EEG. In this experimental model it is suggested that scopolamine likely caused a decrement in arousal. However, the effects of scopolamine on mnemonic paradigms can be characterized as disrupting acquisition and encoding information rather than retrieval processes. Most experiments used a relatively low dose of scopolamine (ranging 0.1 to 0.2 mg/kg). In fact, it must be noted that high doses of the muscarinic antagonist may not only have an effect on the muscarinic receptors, but also on the nicotinic receptors [Schmeller et al., 1994,1995]. Methyl–scopolamine, which only differs from scopolamine in that it does not cross the blood brain barrier, is generally used as a control.

Therefore, to target this aim an *in vivo* study, using our model of AD, was designed to test pharmacological properties of new compounds. With this purpose, a set of experiments was planned to evaluate them on cortex- and hippocampus-dependent memory. Attention, learning and working memory, with respect to cortical and hippocampal EEG theta rhythm, recorded during novel object recognition task in animals with lesion,of the nucleus basalis of Meynert, were studied. In NBM-lesioned animals, compared with control, an increased theta power in the cortex and a reduced theta rhythm oscillation in the hippocampus was found. These EEG changes were correlated with a worse performance in learning and memory tasks. In rats with damaged NBM, novel compounds were able to restore EEG architecture, producing cortical desynchronization and reduction in theta power [Rispoli et al., 2004a, 2006, 2008], while in the hippocampus the drugs increased theta oscillation and reduced the impairment in attention/working memory in the behavioural tasks [Rispoli et al., 2011, data in progress].

Here, we report data supporting this experimental model of AD in testing nwe compounds as putative antidementia drugs.

Neural Basis of Object Recognition 13

attraction to objects in NBM-lesioned animals. These differences were not due to decreased exploration, motivation, or locomotion, but they likely were due to decresed cholinergic

Fig. 5. Typical example of video tracking showing performance of rat with NBM lesion in

Performance in **A.** control animal (intact and sham-operated); **B.** NBM lesioned rat and **C**. NBM lesioned animal treated with AC1. Note the increased traces in T2 around the novel

Rats were equipped with neocortical electrodes to record EEG from cerebral cortex while an other electrode was implanted into the dorsal hippocampus to register hippocampal theta activity, since previous work has shown the last brain area to be involved in object

In intact as well as in NBM-lesioned rats, EEG activity, derived from neocortex and hippocampus, was continuously monitored and recorded when animals were exposed to

novel object recognition (Noldus Ethovision® XT 8.0).

recognition [Prickaerts et al., 2002; Broadbent et al., 2004].

**3.7 EEG recording** 

familiar and novel environments.

object in control (A) and NBM lesioned and AC1 treated animal (C).

transmission arising from the NBM.
