Investigation of ethanol-induced impairment of spatial memory in γ2 heterozygous knockout mice

GABA_A receptors, the major inhibitory receptors in the mammalian central nervous system, are affected by a number of drug compounds, including ethanol. The pharmacological effects of certain drugs have been shown to be dependent upon specific GABA_A receptor subunits. Because benzodiazepines and ethanol have similar effect signatures, it has been hypothesized that these drugs share the γ2-containing GABA_A receptors as a mechanism of action. To probe the involvement of the γ2 subunit in ethanol's actions, spatial memory for the Morris water maze task was tested in γ2 heterozygous knockout mice and wild type littermate controls following ethanol administration at the following doses: 0.0, 1.25, 1.75, and 2.25 g/kg. While baseline learning and memory were unaffected by reduction of γ2 containing GABA_A receptors, ethanol dose-dependently impaired spatial memory equally in γ2 heterozygous knockouts and wild type littermate controls.     

Neuroprotective effect of alpha-asarone on spatial memory and nitric oxide levels in rats injected with amyloid-β(25–35)

The chemical α-asarone is an important active substance of the Acori graminei rhizome (AGR). It has pharmacological effects that include antihyperlipidemic, antiinflammatory, and antioxidant activity. Our aim was to study the effects α-asarone on nitric oxide (NO) levels in the hippocampus and temporal cortex of the rat after injection of the fraction 25–35 from amyloid-β (Aβ_(25–35)). In addition we examined the working spatial memory in an eight-arm radial maze. Our results showed a significant increase of nitrites in the hippocampus and temporal cortex of Aβ_(25–35)-treated rats. Other evidence of neuronal damage was the expression of a glial-fibrillar-acid protein and a silver staining. There were impairments in the spatial memory evaluated in the eight-arm radial maze. We wanted to determine whether α-asarone improves the memory correlated with NO overproduction and neuronal damage caused by the injection of Aβ_(25–35) into rats. Then animals received a 16-day treatment of α-asarone before the Aβ_(25–35) injection. Our results show a significant decrease of nitrite levels in the hippocampus and temporal cortex, without astrocytosis and silver-staining cells, which correlates with memory improvement in the α-asarone-treated group. Our results suggest that α-asarone may protect neurons against Aβ_(25–35)–caused neurotoxicity by inhibiting the effects of NO overproduction in the hippocampus and temporal cortex.     

Activity-dependent scaling of GABAergic excitation by dynamic Cl- changes in Cajal-Retzius cells

To unravel the functional implications of activity-dependent Cl^? changes during early stages of neuronal development, we determined which changes in the GABA reversal potential (E _GABA) and GABAergic rheobase shifts were induced by episodes of GABA_A receptor activation using gramicidin-perforated patch-clamp recordings from Cajal?CRetzius cells in tangential cortical slices of newborn mice. Under this condition, focal application of the GABA_A agonist muscimol (10???M) depolarized the membrane by 15?±?0.8?mV (n?=?35). Such subthreshold GABAergic depolarizations considerably reduced the rheobase, corresponding to an excitatory action. After repetitive focal muscimol applications (50 pulses at 0.5?Hz) a significant reduction of E _GABA and an attenuation of the excitatory GABAergic rheobase shift were observed, while the GABAergic membrane conductance and the absolute value of the rheobase were unaltered after the muscimol pulses. Bath application of 100???M carbachol induced bursts of spontaneous GABAergic postsynaptic potentials. Both, E _GABA and the excitatory GABAergic rheobase shift was significantly reduced after such barrage of carbachol-induced GABAergic postsynaptic potentials, while neither the GABAergic membrane conductance nor the absolute value of the rheobase was affected under this condition. Both results indicate that GABAergic activity itself can limit the excitatory effects of GABA_A receptor activation, which supports the hypothesis that the low capacity of the Cl^? homeostasis in immature neurons could be a substrate for synaptic scaling and homeostatic plasticity.     

GABAC receptors are functionally expressed in the intermediate zone and regulate radial migration in the embryonic mouse neocortex

Radial neuronal migration in the cerebral cortex depends on trophic factors and the activation of different voltage- and ligand-gated channels. To examine the functional role of GABA_C receptors in radial migration we analyzed the effects of specific GABA_A and GABA_C receptor antagonists on the migration of BrdU-labeled neurons in vitro using organotypic neocortical slice cultures. These experiments revealed that the GABA_A specific inhibitor bicuculline methiodide facilitated neuronal migration, while the GABA_C specific inhibitor (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic-acid (TPMPA) impeded migration. Co-application of TPMPA and bicuculline methiodide or the unspecific ionotropic GABA receptor antagonist picrotoxin both impeded migration, suggesting that the GABA_C receptor mediated effects dominate. Addition of the specific GABA_C receptor agonist cis-4-aminocrotonic acid (CACA) also hampered migration, indicating that a physiological GABAergic stimulation is required for appropriate function. RT-PCR experiments using specific probes for GABA_C receptor mRNA and Western blot assays using an antibody directed against rho subunits revealed the expression of GABA_C receptor mRNA and translated GABA_C receptor protein in the immature cortex. Microfluorimetric Ca²⁺ imaging in neurons of identified cortical layers using Calcium Green revealed the functional expression of GABA_A and GABA_C receptors in the intermediate zone, while only GABA_A receptor mediated responses were observed in the upper cortical plate. In summary, these results demonstrate that activation of GABA_C receptors is a prerequisite for accurate migration and that GABA_C receptors are functionally expressed in the intermediate zone.     

Fos expression in the rostral thalamic nuclei and associated cortical regions in response to different spatial memory tests

Using the quantification of the Fos protein as an indicator of neuronal activation, we studied the involvement of the rostral thalamic nuclei and associated structures in different spatial memory tasks in two experiments. In both experiments, tasks were matched for sensorimotor factors but differed in their spatial and mnemonic demands. In Experiment 1, matched groups of rats either ran in a standard eight-arm radial maze or ran up and down just one arm of the maze while the number of runs and rewards were matched across pairs of rats. In Experiment 2, both groups were trained on the eight-arm radial maze but in different rooms. On the test day, one group was moved so that both groups now performed the same radial-maze task in the same room but for one group the extramaze cues were novel. There were significant increases in Fos in all three of the anterior thalamic nuclei (anterodorsal, anteroventral and anteromedial) as well as the adjacent nucleus reuniens and rostral reticular thalamic nucleus, in both the eight-arm versus one-arm condition (Experiment 1) and the novel room versus familiar room condition (Experiment 2). There were no significant differences in the mediodorsal thalamic nucleus in either experiment. The more spatially demanding task in each experiment also resulted in increased Fos expression in the subicular complex (postsubiculum, presubiculum and parasubiculum), as well as in the prelimbic cortex. Performing the standard radial-arm maze task also produced significant Fos increases in both rostral and caudal levels of the retrosplenial cortex when compared to rats running up and down a single arm in the same maze (Experiment 1); performing the task in a novel room did not, however, result in any further Fos increases in this region (Experiment 2). The specificity of the changes in levels of Fos was shown by a lack of any consistent difference in levels in six control sites.The present results reveal a group of anatomically related structures that work together in the intact rat brain during tasks that tax allocentric spatial working memory.     

Cholinergic mediation of spatial memory in the preweanling rat: application of the radial arm maze paradigm

These experiments assessed spatial memory capabilities in the developing rat by the radial arm maze paradigm. Subjects were trained and tested in the maze beginning at 16 days until they were 25 days of age. Results showed that animals that received training performed significantly better than those naive to the task, and better than chance, which suggests an early capacity for this type of learning. The second experiment investigated the neural mechanism that underlies spatial memory at this age as measured by the radial arm maze. In order to distinguish between the working and the reference memory components of the task, a modification of the basic radial arm maze paradigm was used. Subjects trained from 16 days received drug injections of saline, methylscopolamine, scopolamine, or arecoline prior to testing at age 25 days. Results indicated that central cholinergic antagonism severely impairs working memory while sparing reference memory. This finding is consistent with the existing literature that suggests a role for acetylcholine in adult learning and memory, specifically in working memory. Most important, these experiments document that (a) the radial arm maze paradigm can be used effectively for the developmental study of learning and memory in the rat and (b) cholinergic system(s) mediate working memory at an early age.     

Working memory impairment and reduced hippocampal and prefrontal cortex c-Fos expression in a rat model of cirrhosis

Hepatic encephalopathy (HE) is a frequent neurological complication observed in patients with liver malfunction. Previous studies have shown memory impairment in these patients. In order to investigate brain substrates of spatial working memory impairment in chronic HE, neuronal expression of c-Fos protein was studied in an experimental model of cirrhosis. Control and cirrhotic rats were trained on a spatial working memory task in the Morris water maze (MWM). Differences between groups were found in the working memory task. Cirrhotic rats were unable to locate the platform in the retention trial. Neuronal activation, measured by c-Fos protein, was compared between groups. No differences were found in c-Fos expression of control and cirrhotic rats that were not tested in the MWM. Working memory task produced increase in c-Fos positive cells in dorsal hippocampus, CA1 and CA3, and prefrontal cortex in control group compared to thioacetamide group or naïve, which only swam in the maze during a similar time. These findings suggest that cirrhotic rats show spatial working memory impairment that could be linked to dysfunction in neuronal activity in prefrontal cortex and hippocampus.     

The Effects of the Lurcher Mutation on Object Localization, T-Maze Discrimination, and Radial Arm Maze Tasks

The Lurcher mutation is characterized by degeneration of the cerebellar cortex and cerebellar ataxia. The mutants were compared to littermate controls of the same background strain in three spatial tasks: 1) left-right discrimination in a water-filled T-maze, a reference memory task requiring a win-stay strategy; 2) the radial arm maze, a working memory task requiring a win-shift strategy; 3) object localization, a reference memory task requiring the use of cognitive mapping. Lurcher mutants were impaired in the object localization and radial arm maze tasks but not in the left-right discrimination task. These results indicate that trial-independent tasks and reference memory tasks using cognitive mapping may be particularly vulnerable to cerebellar degeneration.     

Hippocampal function required for nonspatial working memory

Summary The hippocampus has been implicated as a brain structure necessary for normal memory functions and normal spatial behavior. Both these explanations are consistent with the results of previous experiments demonstrating an impairment of choice accuracy following fimbria-fornix lesions on an elevated radial arm maze because the test procedure required working memory and permitted cognitive mapping. In the present experiment, rats were tested in an enclosed radial arm maze in which each arm had a distinctive set of discriminative stimuli and the test procedure required working memory but prevented cognitive mapping. Rats were tested preoperatively, given fimbria-fornix lesions, and then tested postoperatively. The rats with lesions performed at chance levels and showed no evidence of recovery of function. These results are consistent with the hypothesis that the poor choice accuracy in the elevated radial arm maze task arose because of the working memory requirements of that task, rather than its cognitive mapping characteristics, and support interpretations of hippocampal function that emphasize memory processing.The research described here was supported in part by a Research Grant MH 24213 from the National Institute of Mental Health and by a Biomedical Research Support Grant from the Johns Hopkins University     

Basal and hypercapnia-altered cerebrovascular perfusion predict mild cognitive impairment in aging rodents

With increasing age, a subset of otherwise healthy individuals undergoes impairments in learning and memory that have been termed mild cognitive impairment (MCI). The enhanced neuronal activity associated with learning and memory requires increased cerebral blood flow (CBF) to specific brain regions. However, the interactions between cerebral blood flow and MCI remain unclear. In this study, we address whether baseline or hypercapnia-induced (increased blood CO₂ levels) changes in CBF are modified with age, and whether these measures are predictive of cognitive status in rodents. Adult and aged rats were evaluated using a hippocampally-dependent task in a water maze. Aged rats were classified as memory-impaired or memory-intact based on performance comparisons with adult rats. Cerebral blood flow was assessed using flow-alternating inversion recovery (FAIR) magnetic resonance imaging (MRI), before and after breathing 10% CO₂. The transition period between CO₂ concentrations was examined with blood oxygen level dependent (BOLD) MRI. Separation of aged animals into memory-intact and impaired categories revealed increased basal perfusion in the dorsal hippocampus of memory-impaired versus memory-intact aged animals. Linear regression revealed that higher hippocampal perfusion was correlated with impaired memory in aged animals, and a logistic regression indicated that hippocampal perfusion predicted spatial memory ability. Several brain regions of aged rats demonstrated an attenuation of the perfusion increase normally observed in adult rats under hypercapnia. Memory-impaired animals were the primary contributor to this effect, as their perfusion response to hypercapnia was significantly reduced compared to adult animals. Aged, memory-intact animals were not significantly different from adults. BOLD MRI demonstrated a reduced response in aged animals to hypercapnia, with impaired animals being the primary contributor to the effect. A logistic regression model based on basal and hypercapnia perfusion correctly predicted cognitive status in 83.3% of animals tested. Our results indicate that age-related changes in vascular reactivity and perfusion are important contributing factors in memory impairment.     

Memory performance and scopolamine: Hypoactivity of the thalamus revealed by cytochrome oxidase histochemistry

Spatial memory learning is related to the functioning of a neuronal circuit composed of cortical, hippocampal and diencephalic brain regions. The Morris water maze (MWM) is frequently used to assess spatial memory in rats. In this study, the neuronal functional activity of some brain limbic system regions after a memory task in adult male Wistar rats injected with scopolamine (1.0 mg/kg, i.p.) was assessed using cytochrome oxidase (COx) histochemistry. The rats were trained following a working memory schedule in the MWM. A trained group injected with saline and an untreated control group were examined to compare changes in COx activity in the dorsal hippocampus, anterior thalamus, mammillary nuclei, prefrontal cortex and ventral tegmental area. The scopolamine-treated group showed an impairment of spatial learning. Also, a decrease in COx activity was found in this group as compared to the saline group in the anteroventral and anteromedial thalamic nuclei. Overall, these findings suggest that memory deficits induced by scopolamine may be due to impairment of the cholinergic function in the anterior thalamic nuclei.     

Effects of scopolamine on radial maze performance in rats

The behaviour of male Wistar rats given repeated daily injections of scopolamine (0.5 mg/kg) over a 14 day acquisition period was compared with that of controls treated with isotonic saline on an 8-arm radial maze. Following this were 2 test days when the group learning the task under scopolamine received saline and vice-versa. Scopolamine significantly impaired acquisition of the radial maze task, but animals rapidly improved when tested without scopolamine. Animals trained on the task without scopolamine were significantly impaired when treated with it. Those animals using non-spatial strategies were relatively resistant to the effects of scopolamine. The results are discussed in terms of the similarity of effect of hippocampal lesions and anticholinergic drug treatment, and in relation to recent theories concerning the role of the hippocampus in spatial memory.     

Lesions of specific and nonspecific thalamic nuclei affect prefrontal cortex-dependent aspects of spatial working memory

Three studies compared lesions of specific mediodorsal (MD) and nonspecific midline/intralaminar (M/IL) and ventromedial (VM) thalamic nuclei placed to spare the anterior nuclei. Lesions of MD, M/IL, or VM impaired delayed matching trained with retractable levers, a measure of spatial memory affected by prefrontal cortical lesions. The effects of the MD lesion increased at longer retention intervals and thus appeared delay dependent. The effects of M/IL and VM lesions were delay independent. Even when combined, these lesions had no effect on varying choice radial maze delayed nonmatching, a task sensitive to hippocampal or anterior thalamic (but not prefrontal) lesions. These results demonstrate effects of MD, M/IL, and VM lesions distinct from the contributions of hippocampus or anterior thalamus to spatial memory.     

Correlation between cognitive deficits and Abeta deposits in transgenic APP+PS1 mice

Doubly transgenic mAPP+mPS1 mice (15-16 months) had impaired cognitive function in a spatial learning and memory task that combined features of a water maze and a radial arm maze. Nontransgenic mice learned a new platform location each day during 4 consecutive acquisition trials, and exhibited memory for this location in a retention trial administered 30 min later. In contrast, transgenic mice were, on average, unable to improve their performance in finding the hidden platform over trials. The cognitive performance of individual mice within the transgenic group were inversely related to the amount of Abeta deposited in the frontal cortex and hippocampus. These findings imply that mAPP+mPS1 transgenic mice develop deficits in cognitive ability as Abeta deposits increase. These data argue that radial arm water maze testing of doubly transgenic mice may be a useful behavioral endpoint in evaluating the functional consequences of potential AD therapies, especially those designed to reduce Abeta load.     

Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion

Hippocampal NMDA receptors (NMDARs) and NMDAR-dependent synaptic plasticity are widely considered crucial substrates of long-term spatial memory, although their precise role remains uncertain. Here we show that Grin1(ΔDGCA1) mice, lacking GluN1 and hence NMDARs in all dentate gyrus and dorsal CA1 principal cells, acquired the spatial reference memory water maze task as well as controls, despite impairments on the spatial reference memory radial maze task. When we ran a spatial discrimination water maze task using two visually identical beacons, Grin1(ΔDGCA1) mice were impaired at using spatial information to inhibit selecting the decoy beacon, despite knowing the platform's actual spatial location. This failure could suffice to impair radial maze performance despite spatial memory itself being normal. Thus, these hippocampal NMDARs are not essential for encoding or storing long-term, associative spatial memories. Instead, we demonstrate an important function of the hippocampus in using spatial knowledge to select between alternative responses that arise from competing or overlapping memories.     

Delay-dependent modulation of memory retrieval by infusion of a dopamine D1 agonist into the rat medial prefrontal cortex

Dopamine (DA) in the medial prefrontal cortex (PFC) can modulate the short-term retention of information and other executive functions. The present study examined whether administration of a DA D1 agonist into the PFC could have differential effects on memory retrieval in circumstances in which memory was either excellent or poor. Separate groups of rats were trained on a delayed version of the radial maze task. On the test day, the delay between the phases was either 30 min or 12 hr. Infusions of the D1 receptor agonist SKF 81297 (0.05, 0.10, or 0.20 microg/0.5 microl) into the PFC before the test phase improved memory retrieval after a 12-hr delay but disrupted performance after a 30-min delay. These data suggest that D1 receptor activity can exert differential effects over PFC function, depending on the strength of the memory trace. When memory is decremented by an extended delay, activation of PFC DA D1 receptors by an agonist can improve cognitive function.     

Perirhinal cortex and anterior thalamic lesions: comparative effects on learning and memory

Three learning and memory tasks were used to compare the effects of neurotoxic anterior thalamic nuclei (ATN) and perirhinal cortex (PRC) lesions in rats. Rats with ATN lesions showed impaired spatial memory in a 12-arm radial maze, whereas rats with PRC lesions showed intact spatial memory, despite the use of minimal pretraining and extensive within-session delays (to 40 rain). PRC, but not ATN, lesions produced impairments on a configural learning task using complex visual-tactile cues in the radial maze. Neither ATN nor PRC lesions consistently affected spontaneous object recognition across extended sample-test delays (to 40 min). These findings confirm the differential involvement of the ATN and PRC in learning and memory.     

Pre- and postsynaptic localization of GABAB receptors in the basal ganglia in monkeys

GABAergic neurotransmission involves ionotropic GABA_A and metabotropic GABA_B receptor subtypes. Although fast inhibitory transmission through GABA_A receptors activation is commonly found in the basal ganglia, the functions as well as the cellular and subcellular localization of GABA_B receptors are still poorly known. Polyclonal antibodies that specifically recognize the GABA_BR1 receptor subunit were produced and used for immunocytochemical localization of these receptors at the light and electron microscope levels in the monkey basal ganglia. Western blot analysis of monkey brain homogenates revealed that these antibodies reacted specifically with two native proteins corresponding to the size of the two splice variants GABA_BR1a and GABA_BR1b. Preadsorption of the purified antiserum with synthetic peptides demonstrated that these antibodies recognize specifically GABA_BR1 receptors with no cross-reactivity with GABA_BR2 receptors. Overall, the distribution of GABA_BR1 immunoreactivity throughout the monkey brain correlates with previous GABA_B ligand binding studies and in situ hybridization data as well as with recent immunocytochemical studies in rodents. GABA_BR1-immunoreactive cell bodies were found in all basal ganglia nuclei but the intensity of immunostaining varied among neuronal populations in each nucleus. In the striatum, interneurons were more strongly stained than medium-sized projection neurons while in the substantia nigra, dopaminergic neurons of the pars compacta were much more intensely labeled than GABAergic neurons of the pars reticulata. In the subthalamic nucleus, clear immunonegative neuronal perikarya were intermingled with numerous GABA_BR1-immunoreactive cells. Moderate GABA_BR1 immunoreactivity was observed in neuronal perikarya and dendritic processes throughout the external and internal pallidal segments. At the electron microscope level, GABA_BR1 immunoreactivity was commonly found in neuronal cell bodies and dendrites in every basal ganglia nuclei. Many dendritic spines also displayed GABA_BR1 immunoreactivity in the striatum. In addition to strong postsynaptic labeling, GABA_BR1-immunoreactive preterminal axonal segments and axon terminals were frequently encountered throughout the basal ganglia components. The majority of labeled terminals displayed the ultrastructural features of glutamatergic boutons and formed asymmetric synapses. In the striatum, GABA_BR1-containing boutons resembled terminals of cortical origin, while in the globus pallidus and substantia nigra, subthalamic-like terminals were labeled.Overall, these findings demonstrate that GABA_B receptors are widely distributed and located to subserve both pre- and postsynaptic roles in controlling synaptic transmission in the primate basal ganglia.     

Altered GABAA receptor subunit expression and pharmacology in human Angelman syndrome cortex

The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the β₃-α₅-γ₃ GABA_A receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABA_A receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of β₃/β₂ and α₅/α₁ subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage-clamp analysis of GABA_A receptor currents was then performed. Studies of GABA_A receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the α₁-selective benzodiazepine-site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABA_A receptor affinity and modulation by neurosteroids were unchanged. This shift in GABA_A receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder.