Amyloid precursor protein knockout mice show age-dependent deficits in passive avoidance learning.

Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD), but its role in cognition has been relatively little studied. APP knockout (KO) animals have been described previously and show deficits in grip strength, reduced locomotor activity and impaired learning and memory in a conditioned avoidance test and the Morris water-maze. In order to further investigate the in vivo function of APP and its proteolytic derivatives, we tested APP KO mice and age-matched wild type controls at two different ages, 3 and 8 months, in a range of behavioural tests measuring neuromuscular, locomotor and cognitive functions. These tests included the acquisition of a passive avoidance response as a measure of long-term memory of an aversive experience, and spontaneous alternation in a Y-maze, regarded as a measure of spatial short-term memory. The absence of APP expression in APP KO mice was confirmed at the protein level using hippocampal tissue in Western blotting. APP KO mice displayed deficits in forelimb grip strength and locomotor activity in agreement with previous studies. In the Y-maze test used for spontaneous alternation behaviour, APP KO animals did not exhibit reduced alternation rates. On the other hand, in the passive avoidance test, APP KO mice showed an age-related deficit in retention of memory for an aversive experience. The results suggest that APP and/or its proteolytic derivatives may play a role in long-term memory in adult brain and/or may be required during the development and maintenance of neuronal networks involved in this type of memory.

GABA(B(1)) receptor isoforms differentially mediate the acquisition and extinction of aversive taste memories.

Conditioned taste aversion (CTA) is a form of aversive memory in which an association is made between a consumed substance and a subsequent malaise. CTA is a critical mechanism for the successful survival, and hence evolution, of most animal species. The role of excitatory neurotransmitters in the neurochemical mechanisms of CTA is well recognized; however, less is known about the involvement of inhibitory receptor systems. In particular, the potential functions of metabotropic GABA(B) receptors in CTA have not yet been fully explored. GABA(B) receptors are metabotropic GABA receptors that are comprised of two subunits, GABA(B(1)) and GABA(B(2)), which form heterodimers. The Gabbr1 gene is transcribed into two predominant isoforms, GABA(B(1a)) and GABA(B(1b)), which differ in sequence primarily by the inclusion of a pair of sushi domains (also known as short consensus repeats) in the GABA(B(1a)) N terminus. The behavioral function of mammalian GABA(B(1)) receptor isoforms is currently unknown. Here, using a point mutation strategy in mice, we demonstrate that these two GABA(B(1)) receptor isoforms are differentially involved in critical components of CTA. In contrast to GABA(B(1b))-/- and wild-type mice, GABA(B(1a))-/- mice failed to acquire CTA. In contrast, GABA(B(1b))-/- mice robustly acquired CTA but failed to show any extinction of this aversion. The data demonstrate that GABA(B) receptors are involved in both the acquisition and extinction of CTA; however, receptors containing the GABA(B(1a)) or the GABA(B(1b)) isoform differentially contribute to the mechanisms used to learn and remember the salience of aversive stimuli.

A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition.

Influences of the habenular complex on electrophysiological and neurochemical aspects of brain functioning are well known. However, its role in cognition has been sparsely investigated until recently. The habenular complex, composed of medial and lateral subdivisions, is a node linking the forebrain with midbrain and hindbrain structures. The lateral habenula is the principal actor in this direct dialogue, while the medial habenula mostly conveys information to the interpeduncular nucleus before this modulates further regions. Here we describe neuroanatomical and physiological aspects of the habenular complex, and its role in cognitive processes, including new behavioral, electrophysiological and imaging findings. Habenular complex lesions result in deficits in learning, memory and attention, some of which decline during repeated testing, while others become worse, consistent with multiple roles in cognition. The habenular complex is particularly responsive to feedback about errors. Electrophysiological studies indicate a role in metaplasticity, the modulation of neuroplasticity. These studies thus reveal important roles of the habenular complex in learning, memory and attention.

Specific roles of GABA(B(1)) receptor isoforms in cognition.

The GABA(B) receptor is a heterodimer of GABA(B(1)) and GABA(B(2)) subunits. There are two isoforms of the GABA(B(1)) subunit: GABA(B(1a)) and GABA(B(1b)). Recent studies with mutant mice suggest a differential role for the two GABA(B(1)) isoforms in behavioural processes. As pharmacological and genetic studies have implicated GABA(B) receptors in cognition we investigated the behaviour of GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice in different types of cognitive paradigms. GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice were both impaired relative to wildtype controls in a continuous spontaneous alternation behaviour test of working spatial memory. In contrast to the reported phenotype of GABA(B(1))(-/-) mice, however, neither GABA(B(1a))(-/-) nor GABA(B(1b))(-/-) mice were deficient in a passive avoidance task. On the other hand, GABA(B(1a))(-/-) mice were impaired in familiar and novel object recognition. We conclude that GABA(B(1)) isoforms contribute differentially to GABA(B) receptor-mediated cognitive processes.

Cholinergic changes in the APP23 transgenic mouse model of cerebral amyloidosis.

Alzheimer's Disease (AD) is a neurodegenerative disorder that is characterized by extracellular deposits of amyloid-beta peptide (Abeta) and a severe depletion of the cholinergic system, although the relationship between these two events is poorly understood. In the neocortex, there is a loss of cholinergic fibers and receptors and a decrease of both choline acetyltransferase (ChAT) and acetylcholinesterase enzyme activities. The nucleus basalis of Meynert (NBM), which provides the major cholinergic input to the neocortex, undergoes profound neuron loss in AD. In the present study, we have examined the cholinergic alterations in amyloid precursor protein transgenic mice (APP23), a mouse model of cerebral beta-amyloidosis. In aged APP23 mice, our results reveal modest decreases in cortical cholinergic enzyme activity compared with age-matched wild-type mice. Total cholinergic fiber length was more severely affected, with 29 and 35% decreases in the neocortex of aged APP23 mice compared with age-matched wild-type mice and young transgenic mice, respectively. However, there was no loss of cholinergic basal forebrain neurons in these aged APP23 mice, suggesting that the cortical cholinergic deficit in APP23 mice is locally induced by the deposition of amyloid and is not caused by a loss of cholinergic basal forebrain neurons. To study the impact of cholinergic basal forebrain degeneration on cortical amyloid deposition, we performed unilateral NBM lesions in adult APP23 mice. Three to 8 months after lesioning, a 38% reduction in ChAT activity and significant cholinergic fiber loss were observed in the ipsilateral frontal cortex. There was a 19% decrease in Abeta levels of the ipsilateral compared with contralateral frontal cortex with no change in the ratio of Abeta40 to Abeta42. We conclude that the severe cholinergic deficit in AD is caused by both the loss of cholinergic basal forebrain neurons and locally by cerebral amyloidosis in the neocortex. Moreover, our results suggest that disruption of the basal cholinergic forebrain system does not promote cerebral amyloidosis in APP23 transgenic mice.

Redistribution of GABAB(1) protein and atypical GABAB responses in GABAB(2)-deficient mice.

GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.

Selective neuron loss in the paraventricular nucleus of hypothalamus in patients suffering from major depression and bipolar disorder.

The structural basis for depressive disorders remains unknown. Studies using neuroimaging and postmortem brain tissue indicate that anatomic substrates may contribute to major depression disorder (MDD) and bipolar disorder (BD). The present study used design-based stereology to assess neuron loss in 2 well-defined hypothalamic structures, the paraventricular nucleus (PVN) and supraoptic nucleus (SON), in clinically well-studied cases with severe depression. The left or right diencephalon was blocked from 26 brains removed at autopsy from age-matched controls (5 male/3 female) and patients with MDD (6 male/5 female) and BD (5 male/2 female). Serial sections were cut at an instrument setting of 60 microm through the entire PVN and SON from left hypothalamus and 8 to 10 sections per brain were systematic-random sampled and stained with cresyl violet. A trained operator blind to clinical diagnosis used computerized stereology to estimate total neuron numbers in PVN and SON. The results revealed a selective, robust reduction of approximately 50% in total neuron number in the PVN for major depression and bipolar cases compared with age-matched controls, with no differences in neuron numbers in the SON. This selective neuronal loss in the PVN appears to identify an important neurobiologic substrate for the behavioral manifestations of depression.

Bilateral lesions of the habenula induce attentional disturbances in rats.

The habenular nuclear complex is a major influence on brainstem cell groups that influence attention, but its role in attentional performance has not previously been explored. The present study investigated how habenula lesions affect attentional function as assessed by the 5-choice serial reaction time task (5-CSRTT) in male Lister-Hooded rats. Rats were pretrained in the 5-CSRTT before receiving discrete bilateral lesions of the habenula or a sham procedure. In test sessions immediately following recovery from surgery, lesioned rats showed a marked increase in premature responding. Over the course of testing this increase of premature responding declined in magnitude. In contrast, choice accuracy showed no impairment during the earliest postsurgery test sessions but progressively deteriorated over the course of testing. These opposite time courses strongly imply that different mechanisms mediate these two effects of the habenula lesion. Differential effects of drug treatment on these effects further supported this view. Thus, D-amphetamine (0.2 mg/kg s.c.) increased premature responding without affecting choice accuracy. On the other hand, haloperidol (0.01-0.03 mg/kg i.p.) decreased premature responding without significantly affecting choice accuracy. The results are consistent with the view that elevated premature responding in habenula-lesioned animals is mediated by increased dopaminergic activity, whereas impaired choice accuracy is not. Implications of these findings for the hypothesis that habenula dysfunction is involved in cognitive symptoms of schizophrenia are discussed.

Impaired cognitive performance in rats after complete epithalamus lesions, but not after pinealectomy alone.

In the midbrain, the epithalamus comprises the habenular nuclei and the pineal gland. Based on evidence including imaging studies in schizophrenia patients, several investigators have postulated that dysfunction of this structure is causally involved in symptoms of schizophrenia. Recently, we showed that bilateral habenula lesions in the rat induced some schizophrenia-like behavioural changes, namely memory and attention impairments, but unaltered social interaction in a brief encounter and prepulse inhibition (PPI) of the startle reflex. Here, the possible involvement of the pineal gland in the same behaviours was assessed, by examining them in two series of experiments. In the first, these behaviours were examined in pinealectomized rats compared to sham-operated controls. In the second, they were examined in rats with combined lesion of habenula plus pinealectomy compared to sham-operated controls, to examine whether pinealectomy induced further deficits when combined with habenula damage. Lesions of habenula were confirmed histologically and neurochemically by reduction of choline acetyltransferase in the interpeduncular nucleus. Pinealectomy was confirmed post mortem by careful visual inspection. Pinealectomy induced no deficits in any test, while combined lesions led to the same pattern of deficits as previously observed after habenula lesion, i.e. marked memory impairment in the Morris water maze without affecting the amount of social interaction or PPI of the startle reflex. Thus, loss of pineal function causes no deficits in these behaviours and does not alter the qualitative pattern of deficits resulting from habenula damage.

Gastrin-releasing peptide signaling plays a limited and subtle role in amygdala physiology and aversive memory.

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe(6), Leu-NHEt(13), des-Met(14))-Bombesin (6-14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders.

Selective effects of benzodiazepines on the acquisition of conditioned taste aversion compared to attenuation of neophobia in C57BL/6 mice.

INTRODUCTION: The effects of pre-conditioning administration of anxiolytic benzodiazepines on the acquisition of a conditioned taste aversion (CTA) and on the acquisition of attenuation of neophobia (AN) were investigated in C57BL/6 mice. MATERIALS AND METHODS: A CTA was induced by injecting lithium chloride (LiCl; 6 mEq x kg(-1)) 30 min after the animal had imbibed a novel 0.5% saccharin solution. In other animals, neophobia was attenuated by a single access to the novel 0.5% saccharin solution, followed only by injection of saline. RESULTS AND DISCUSSION: Pre-conditioning administration of chlordiazepoxide (CDZ; 6-24 mg x kg(-1), i.p.) and alprazolam (0.3-1 mg x kg(-1), p.o.) resulted in a CTA that did not differ initially from that observed in vehicle-treated controls, but which showed faster extinction. The acquisition of AN was impaired only after the higher doses of CDZ (12-24 mg x kg(-1), i.p.) or alprazolam (1 mg x kg(-1), i.p.). The results show that in this test, altered acquisition of an aversive CTA memory by anxiolytic benzodiazepines is reflected in more rapid extinction. Moreover, at low doses, these drugs showed selectivity for weakening CTA learning compared to AN learning. Evidence is discussed that selective weakening of aversive memory formation is a clinically relevant effect of anxiolytic benzodiazepines.

Increased exploratory activity of APP23 mice in a novel environment is reversed by siRNA.

Genetic abnormalities in amyloid precursor protein (APP) are associated with Down's syndrome and familial Alzheimer's disease where hallmark plaques contain A beta peptides derived from APP. Both APP and its derivatives are implicated in neurodegenerative processes and may play important physiological and pathophysiological roles in synaptic function. Here, we show that young APP23 transgenic mice overexpressing human APP with the Swedish double mutation display altered novelty seeking behavior before the age of plaque onset. Using short interfering RNA (siRNA) targeted against APP, we investigate the direct contribution of APP and its derivatives to this behavioral deficit. After validating siRNAs targeting human APP in vitro, siRNAs were infused directly into the brain of APP23 mice for 2 weeks. Behavioral analysis shows that infusion of siRNA targeted against APP completely reverses increased exploratory activity in APP23 mice. Collectively, these data suggest that excessive APP and/or its derivatives, causes a hyperactive phenotype in APP23 mice when placed in a novel environment, which is fully reversible and not linked to plaque deposits.

Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice.

The cleavage-product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer's disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down-regulated mouse APP protein levels (APP-siRNA) were identified using an APP plasmid-siRNA co-transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP-siRNAs into the ventricular system for 2 weeks also down-regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2-CA3 regions of the hippocampus. Mice treated with the most active APP-siRNA showed a significant reduction in spontaneous alternation rate in the Y-maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus-dependent spatial working memory.

Habenula lesions cause impaired cognitive performance in rats: implications for schizophrenia.

Cognitive impairment is a prominent feature of schizophrenia. Currently there is no well-accepted explanation of the aetiology of this disorder, but recent evidence indicates that dysfunction of the habenula may be involved. We therefore examined whether habenula lesions in Sprague-Dawley rats cause behavioural changes resembling those of schizophrenia. Rats received either habenula lesions, a sham operation or a small lesion of the overlying dorsal hippocampus as a check that effects observed were not due to incidental damage to this structure. As there are alterations of social behaviour, sensorimotor gating and cognition in schizophrenia, we examined comparable behaviours. Social interaction time was measured during a 5-min encounter with a novel juvenile conspecific. Prepulse inhibition of an acoustic startle response, as an index of sensorimotor gating, was measured with prepulses of various amplitudes, and spatial cognitive performance was assessed in the Morris water maze task. Histological analysis showed that habenula lesions substantially damaged both medial and lateral habenula bilaterally while largely sparing neighbouring structures. Assay of choline acetyltransferase (ChAT) in the interpeduncular nucleus terminal region of the habenulo-interpeduncular tract, showed marked reduction (by 80%) in habenula-lesioned animals. Habenula-lesioned rats, but not the control group with small dorsal hippocampus lesions, showed marked impairment of Morris maze performance compared to the sham-operated control group. Social interaction time and prepulse inhibition were not significantly altered in either lesion group. The results are consistent with a role of the habenula in cognition, and with the view that pathology of the habenula may contribute to the cognitive impairments of schizophrenia.

Antidepressant and anxiolytic-like effects in mice lacking the group III metabotropic glutamate receptor mGluR7.

Glutamatergic neurotransmission has been strongly implicated in the pathophysiology of affective disorders, such as major depression and anxiety. Of all glutamate receptors, the role of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8) in such disorders is the least investigated because of the lack of specific pharmacological tools. To this end, we examined the behavioural profiles of mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) in animal models of depression and anxiety. mGluR7-/- mice were compared with wild-type (mGluR7+/+) littermates and showed substantially less behavioural immobility in both the forced swim test and the tail suspension test. Both behavioural paradigms are widely used to predict antidepressant-like activity. Further, mGluR7-/- mice displayed anxiolytic activity in four different behavioural tests, i.e. the light-dark box, the elevated plus maze, the staircase test, and the stress-induced hyperthermia test, while their cognitive performance was normal in the passive avoidance paradigm. Analysis of locomotor activity in a novel environment demonstrated that mGluR7-/- mice were slightly more active in the initial minutes following placement in the chamber only. Together, these data suggest that mGluR7 may play a pivotal role in mechanisms that regulate behavioural responses to aversive states. Therefore, drugs acting at mGluR7 may provide novel treatments for psychiatric disorders such as depression and anxiety.

Behavioral characterization of the novel GABAB receptor-positive modulator GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): anxiolytic-like activity without side effects associated with baclofen or benzodiazepines.

The role of GABAB receptors in various behavioral processes has been largely defined using the prototypical GABAB receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABAB receptors in these disorders is largely unclear. We recently identified GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABAB receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital- and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABAB receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines.