Wayne B. Rowe

Hypothalamic-pituitary-adrenal activity in aged, cognitively impaired and cognitively unimpaired rats

There is a tendency for increased hypothalamic-pituitary-adrenal (HPA) activity with age in the rat, and the resulting elevations in circulating glucocorticoid levels have been implicated in the occurrence of hippocampal pathology and memory deficits. In the experiments reported here, we examined whether HPA dysfunction is selectively associated with cognitive impairments in a population of aged rats. Fifty-eight 23-27-month-old male Long-Evans rats were screened for spatial memory impairments using the Morris swim maze, and 2 groups of aged animals were selected; aged, cognitively impaired (AI) animals whose performance was significantly different (greater than 2 SD) from that of 6-month-old controls and aged, cognitively unimpaired (AU) animals whose performance was comparable to that of the young controls (a difference of less than 0.5 SD). Twenty-eight percent of the animals tested were designated as AI and 20% as AU. Histological analysis of a subset of these animals showed that, while both AU and AI animals showed neuron loss in the pyramidal cell fields of the hippocampus, the loss was significantly greater in the AI animals. The AI animals showed clear evidence of increased HPA activity. Thus, basal ACTH and corticosterone levels were significantly higher in the AI animals compared with both AU animals and young controls, especially during the dark phase of the cycle. The AI, AU, and young animals exhibited comparable corticosterone levels during a 20-min immobilization stress; however, following the termination of the stressor, corticosterone levels in AI animals were significantly elevated compared with both AU animals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Hippocampal Expression Analyses Reveal Selective Association of Immediate-Early, Neuroenergetic, and Myelinogenic Pathways with Cognitive Impairment in Aged Rats

Although expression of some genes is known to change during neuronal activity or plasticity, the overall relationship of gene expression changes to memory or memory disorders is not well understood. Here, we combined extensive statistical microarray analyses with behavioral testing to comprehensively identify genes and pathways associated with aging and cognitive dysfunction. Aged rats were separated into cognitively unimpaired (AU) or impaired (AI) groups based on their Morris water maze performance relative to young-adult (Y) animals. Hippocampal gene expression was assessed in Y, AU, and AI on the fifth (last) day of maze training (5T) or 21 d posttraining (21PT) and in nontrained animals (eight groups total, one array per animal; n = 78 arrays). ANOVA and linear contrasts identified genes that differed from Y generally with aging (differed in both AU and AI) or selectively, with cognitive status (differed only in AI or AU). Altered pathways/processes were identified by overrepresentation analyses of changed genes. With general aging, there was downregulation of axonal growth, cytoskeletal assembly/transport, signaling, and lipogenic/uptake pathways, concomitant with upregulation in immune/inflammatory, lysosomal, lipid/protein degradation, cholesterol transport, transforming growth factor, and cAMP signaling pathways, primarily independent of training condition. Selectively, in AI, there was downregulation at 5T of immediate-early gene, Wnt (wingless integration site), insulin, and G-protein signaling, lipogenesis, and glucose utilization pathways, whereas Notch2 (oligodendrocyte development) and myelination pathways were upregulated, particularly at 21PT. In AU, receptor/signal transduction genes were upregulated, perhaps as compensatory responses. Immunohistochemistry confirmed and extended selected microarray results. Together, the findings suggest a new model, in which deficient neuroenergetics leads to downregulated neuronal signaling and increased glial activation, resulting in aging-related cognitive dysfunction.

Neonatal handling alters serotonin (5-HT) turnover and 5-HT2 receptor binding in selected brain regions: relationship to the handling effect on glucocorticoid receptor expression.

Neonatal handling permanently alters hypothalamic-pituitary-adrenal responses to stress. This effect is, in part, mediated by a handling-induced increase in forebrain glucocorticoid receptor gene expression. The effect of postnatal handling on glucocorticoid receptor expression appears to be mediated by an increase in serotonin (5-HT) activity, acting via a 5-HT2 receptor with a high affinity for 5-HT (i.e. the 5-HT2H receptor). In the present study we examined the nature of the effects of handling on the relevant 5-HT systems. We found that: (1) handling increases 5-HT turnover in regions of the neonatal rat brain where glucocorticoid receptor expression is altered (i.e. the hippocampus and frontal cortex), but not in regions where glucocorticoid receptor expression in unaffected (e.g. hypothalamus and amygdala); (2) handling has no long-term effects on hippocampal or frontal cortex 5-HT turnover, and is actually associated with a decrease in 5-HT concentrations; and (3) handling does not alter 5-HT2 receptor density in the hippocampus or frontal cortex in neonates (although there are surprising effects on 5-HT2 receptor density in the frontal cortex of adult animals). Taken together these data provide further evidence for the importance of 5-HT in mediating the effects of handling on the development of glucocorticoid receptor expression, but suggest that the role of 5-HT is unique to early development; differences in glucocorticoid receptor expression in adult handled and non-handled animals are not associated with long-term differences in either 5-HT levels or 5-HT2 receptors.

The Slow Afterhyperpolarization in Hippocampal CA1 Neurons Covaries with Spatial Learning Ability in Aged Fisher 344 Rats

Rodents commonly exhibit age-related impairments in spatial learning tasks, deficits widely thought to reflect cellular or synaptic dysfunction in the hippocampus. Using whole-cell recordings, we examined the afterhyperpolarization (AHP) in CA1 pyramidal cells in hippocampal slices from young (4-6 months of age) and aged (24-26 months of age) Fisher 344 male rats that had been behaviorally characterized in the Morris water maze. The slow AHP (sAHP) recorded from learning-impaired aged rats (AI) was significantly larger than that seen in either age-matched unimpaired rats or young controls. Among aged rats, sAHP amplitude was inversely correlated with both acquisition and probe performance in the water maze. Action potential parameters among the three groups were similar, except for spike accommodation, which was more pronounced in the AI group. Intracellular application of the cAMP analog 8-CPT-cAMP suppressed the sAHP but failed to reveal any age- or performance-related differences in the medium AHP. 8-CPT-cAMP abolished the age-related difference in spike accommodation, whereas instantaneous firing frequency was unchanged. Calcium spikes were of similar amplitude in all three groups but were broader and had significantly larger tails in aged rats; these age-related changes could be mimicked in young neurons after exposure to BayK8644. The calcium spike among aged rats correlated with task acquisition in the maze but, unlike the sAHP, failed to correlate with probe performance. This is the first demonstration that sAHP amplitude covaries with spatial learning ability in aged rats, implying that CA1 excitability strongly influences certain aspects of cognitive function. Our findings also indicate that multiple processes, in addition to elevated calcium influx, conspire to induce cognitive decline during aging.

Dopamine D1, receptor ligands modulate cognitive performance and hippocampal acetylcholine release in memory-impaired aged rats

The possible modulation by D1 drugs of learning abilities of a population of aged memory-impaired animals was investigated in the present study. The level of D1/[3H]SCH 23390 receptors was first examined by quantitative autoradiography to ascertain if cognitive deficits seen in these animals could be related to alterations in the levels of these receptors. No significant differences in [3H]SCH 23390 binding were observed in any of the brain areas examined between young, and aged memory-unimpaired and aged memory-impaired animals. However, the cognitive deficits of the aged-impaired rats were modulated by D1 drugs. The D1 agonists SKF 38393 and SKF 81297 (3.0 mg/kg, i.p.) significantly reduced the latency period to find a hidden platform in the Morris Water Maze, reflecting improved cognitive functions, while the D1 antagonist SCH 23390 (0.05 mg/kg, i.p.) had no overall significant effect. Moreover, the D1 agonist SKF 38393 increased, whereas the antagonist inhibited, in vivo hippocampal acetylcholine release. Taken together, these results suggest that functional hippocampal acetylcholine-dopamine interactions exist in aged memory-impaired rats. More importantly, the cognitive deficits seen in the aged-impaired rats can be attenuated by stimulations of D1 receptors, hence suggesting an alternative approach to alleviate the cognitive deficits seen in the aged brain.

Distribution and levels of [125I]IGF-I, [125I]IGF-II and [125I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats

The insulin-like growth factors (IGF-I and IGF-II) and insulin are localized within distinct brain regions and their respective functions are mediated by specific membrane receptors. High densities of binding sites for these growth factors are discretely and differentially distributed throughout the brain, with prominent levels localized to the hippocampal formation. IGFs and insulin, in addition to their growth promoting actions, are considered to play important roles in the development and maintenance of normal cell functions throughout life. We compared the anatomical distribution and levels of IGF and insulin receptors in young (five month) and aged (25 month) memory-impaired and memory-unimpaired male Long Evans rats as determined in the Morris water maze task in order to determine if alterations in IGF and insulin activity may be related to the emergence of cognitive deficits in the aged memory-impaired rat. In the hippocampus, [125I]IGF-I receptors are concentrated primarily in the dentate gyrus (DG) and the CA3 sub-field while high amounts of [125I]IGF-II binding sites are localized to the pyramidal cell layer, and the granular cell layer of the DG. [125I]insulin binding sites are mostly found in the molecular layer of the DG and the CA1 sub-field. No significant differences were found in [125I]IGF-I. [125I]IGF-II or [125I]insulin binding levels in any regions or laminae of the hippocampus of young vs aged rats. and deficits in cognitive performance did not relate to altered levels of these receptors in aged memory-impaired vs aged memory-unimpaired rats. Other regions. including various cortical areas, were also examined and failed to reveal any significant differences between the three groups studied. It thus appears that IGF-I, IGF-II and insulin receptor sites are not markedly altered during the normal ageing process in the Long Evans rat, in spite of significant learning deficits in a sub-group (memory-impaired) of aged animals. Hence. recently reported changes in IGF-I receptor messenger RNA levels in aged memory-impaired rats are apparently not reflected at the level of the translated protein.

RG3487, a Novel Nicotinic α7 Receptor Partial Agonist, Improves Cognition and Sensorimotor Gating in Rodents

Neuronal nicotinic α7 acetylcholine receptors (α7nAChRs) are expressed primarily in the brain and are implicated in modulating many cognitive functions (e.g., attention, working and episodic memory). Not surprisingly, much effort has been committed to the development of molecules acting at α7nAChRs as potential therapies for a variety of central nervous system diseases (e.g., Alzheimers). N-[(3S)-1-azabicyclo[2.2.2]oct-3-yl]-1H-indazole-3-carboxamide hydrochloride (RG3487) binds potently to the human α7nAChR (Ki = 6 nM), in which it acts as a partial agonist (63–69% of acetylcholine) as assessed by whole-cell patch-clamp recordings in both oocytes and QM7 cell lines. RG3487 activates human α7nAChRs with an EC50 of 0.8 μM (oocytes) and 7.7 μM (QM7 cells). RG3487 also exhibits antagonist properties at the serotonin 3 receptor [IC50 = 2.8 nM (oocytes), 32.7 nM (N1E-115 cells)]. In vivo, RG3487 improved object recognition memory in rats after acute [minimally effective dose (MED) 1.0 mg/kg p.o.] or repeated (10 day) administration at brain and plasma concentrations in the low-nanomolar range. Spatial learning deficits in age-impaired rats were reversed after RG3487 administration (MED: 0.03 mg/kg i.p.) as evaluated in the Morris water maze task. In the prepulse inhibition (PPI) of startle model of sensorimotor gating, RG3487 improved apomorphine-induced deficits in PPI performance (MED: 0.03 mg/kg i.p.) and reversed phencyclidine-induced impairments in an attentional set-shifting model of executive function (MED: ≤0.03 mg/kg i.p.). Cumulative evidence from these studies indicates RG3487 is a novel and potent α7nAChR partial agonist that improves cognitive performance and sensorimotor gating.

Reactivity to novelty in cognitively-impaired and cognitively-unimpaired aged rats and young rats

Two distinct populations of aged, Long-Evans rats can be identified on the basis of performance in the Morris water maze task. Aged (24 month) unimpaired rats perform similarly to young (six month) animals. Aged, impaired rats display latencies to find the submerged platform greater than two standard deviations from the mean of the young animals. A hallmark of efficient cognitive processing is the ability to cope with environmental change. Consequently, the present studies were conducted to assess if aged, impaired animals display differential reactivity to repeated exposure to novel stimuli. Reactivity was assessed by examining the degree of (i) consumption of a novel gustatory/olfactory stimulus (sweetened milk), (ii) pain inhibition induced by exposure to a novel hot-plate (48.5 degrees C) apparatus and (iii) exploratory behaviour in an elevated plus maze and a novel open field. Aged, impaired rats exhibited lower milk consumption on day one and protracted reactivity (lower consumption over days two to eight) in comparison to aged, unimpaired and young animals. Aged, impaired rats were more reactive to novelty on the hot plate test (as indicated by longer paw lick latencies); this novelty-induced pain inhibition did not habituate in aged, impaired rats following repeated plate exposures. The degree of exploratory behaviour in both the plus maze and the open field was reduced in aged, impaired rats. This effect was not entirely a consequence of deficient affective mechanisms, as measures of anxiety (e.g., time in open arms, time in inner squares) were not different among aged impaired, aged unimpaired and young animals. These results are the first to demonstrate that behavioural deficits observed in aged, impaired animals extend beyond the impairments observed in the water maze. This behavioural profile is attributed, in part, to heightened anxiety. In addition, the impairments observed in aged, impaired animals may also reflect a reduced sensitivity to the positive incentive properties of novel stimuli.

Cholinergic markers in aged cognitively impaired long-evans rats

Aged Long-Evans rats (24-25 months old) were classified into cognitively impaired or unimpaired subgroups based on their performances in the Morris Swim Maze task compared to young controls. Using quantitative in vitro receptor autoradiography, we investigated the status of various cholinergic markers in these two groups and in young adults (six months) animals. The apparent density of [3H]pirenzepine (muscarinic M1) sites was similar in the three groups of rats in various cortical areas, subfields of the hippocampus, medial septum and striatum. Similarly, choline acetyltransferase activity and the density of [3H]hemicholinium-3 (high-affinity choline uptake) and [3H]cytisine (nicotinic) binding sites were also unchanged in the brain regions studied between the aged cognitively impaired, unimpaired and young adult rats. In contrast, significant increases in [3H]AF-DX 384 (muscarinic M2) binding density were observed in various cortical areas and in the molecular layer of the dentate gyrus of aged cognitively impaired versus unimpaired rats and in few cortical regions of old as compared to young animals. Therefore, a selective alteration in the regulation of putative M2 receptor sites is apparent, particularly in the aged cognitively impaired rats. Increases in M2 binding sites could lead to a decrease in the capacity to release acetylcholine, as some of the M2 receptors are believed to act as negative autoreceptors. This could influence cognitive functions as selective M2 blockers have recently been reported to facilitate spatial memory in aged impaired rats [Doods et al. (1993) Life Sci. 52, 497-503: Quirion et al. (1995) J. Neurosci. 15, 1455-1462.

Serotonergic regulation of type II corticosteroid receptor binding in hippocampal cell cultures: evidence for the importance of serotonin-induced changes in cAMP levels.

The development of the hypothalamic-pituitary-adrenal response to stress is profoundly altered by environmental events. One target for environmental regulation within the hypothalamic-pituitary-adrenal axis is the hippocampal type II corticosteroid (or glucocorticoid) receptor system, which mediates the negative-feedback effects of glucocorticoids on hypothalamic-pituitary-adrenal activity. Thus, adult rats handled early in life show increased hippocampal type II corticosteroid receptor density and increased sensitivity to the inhibitory effects of circulating glucocorticoids on post-stress hypothalamic-pituitary-adrenal activity. Both effects persist throughout life. The effects of handling on type II corticosteroid receptor development are, at least in part, mediated by changes in hippocampal 5-hydroxytryptamine turnover. Moreover, 5-hydroxytryptamine can regulate type II corticosteroid receptor density in cultured hippocampal cells, providing a paradigm for examining the neurochemical mechanisms by which environmental stimuli might regulate neural differentiation. In the present studies, we examined the intracellular mechanisms underlying the effects of 5-hydroxytryptamine on type II corticosteroid receptors ([3H]RU 28362 binding) in hippocampal cell cultures. cAMP, but not cGMP, levels in cultured hippocampal cells were significantly increased by the addition of 5-hydroxytryptamine to the medium. The cAMP response to 5-hydroxytryptamine was biphasic: an initial increase in cAMP levels occurred in response to nanomolar 5-hydroxytryptamine concentrations (EC50 = 7.2 nM), while a second increase was apparent at low micromolar concentrations. 5-Hydroxytryptamine also increased [3H]RU 28362 binding (EC50 = 4.5 nM) with a maximal effect at a concentration of 10 nM. There was no further increase in [3H]RU 28362 binding even with higher, micromolar concentrations of 5-hydroxytryptamine.(ABSTRACT TRUNCATED AT 250 WORDS)