Altered brain arginine metabolism in schizophrenia.

Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease.

Association of aberrant neural synchrony and altered GAD67 expression following exposure to maternal immune activation, a risk factor for schizophrenia.

A failure of integrative processes within the brain, mediated via altered GABAergic inhibition, may underlie several features of schizophrenia. The present study examined, therefore, whether maternal immune activation (MIA), a risk factor for schizophrenia, altered inhibitory markers in the hippocampus and medial prefrontal cortex (mPFC), while also altering electroencephalogram (EEG) coherence between these regions. Pregnant rats were treated with saline or polyinosinic:polycytidylic acid mid-gestation. EEG depth recordings were made from the dorsal and ventral hippocampus and mPFC of male adult offspring. Glutamic decarboxylase (GAD67) levels were separately assayed in these regions using western blot. GAD67 expression was also assessed within parvalbumin-positive cells in the dorsal and ventral hippocampus using immunofluorescence alongside stereological analysis of parvalbumin-positive cell numbers. EEG coherence was reduced between the dorsal hippocampus and mPFC, but not the ventral hippocampus and mPFC, in MIA animals. Western blot and immunofluorescence analyses revealed that GAD67 expression within parvalbumin-positive cells was also reduced in the dorsal hippocampus relative to ventral hippocampus in MIA animals when compared with controls. This reduction was observed in the absence of parvalbumin-positive neuronal loss. Overall, MIA produced a selective reduction in EEG coherence between the dorsal hippocampus and mPFC that was paralleled by a similarly specific reduction in GAD67 within parvalbumin-positive cells of the dorsal hippocampus. These results suggest a link between altered inhibitory mechanisms and synchrony and, therefore point to potential mechanisms via which a disruption in neurodevelopmental processes might lead to pathophysiology associated with schizophrenia.

Potential involvement of NOS and arginase in age-related behavioural impairments.

The present study investigated age-related changes in nitric oxide synthase (NOS) and arginase, which shares a substrate with NOS, in the hippocampus and parahippocampal region and the relationship between NOS/arginase and age-associated behavioural impairments. Aged rats (24 months old) displayed reduced exploratory activity, enhanced anxiety, poorer spatial learning and memory, and impaired object recognition memory relative to the young adults (4 months old). There were significant increases in total NOS activity in the aged hippocampus and perirhinal, postrhinal and temporal cortices and a dramatic decrease in endothelial NOS expression in the aged postrhinal cortex. Activity and protein expression of inducible NOS were not detected in any region from either group and a significant increase in total arginase activity was found in the aged perirhinal cortex. Multiple regression analysis revealed significant correlations between NOS/arginase and behavioural measures in both groups. The present findings provide further support for a contribution of nitric oxide to the normal aging process and suggest a potential involvement of arginase in aging and learning and memory.

Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex.

Increasing evidence suggests that nitric oxide (NO), generated by nitric oxide synthase (NOS) from l-arginine, plays an important role in the ageing process. The present study, for the first time, investigates age-related changes in NOS and arginase, an enzyme that shares a common substrate with NOS, in the prefrontal cortex of rats assessed with and without prior behavioural testing. A significant increase in total NOS activity was found in the prefrontal cortex in aged (24-month-old) as compared with young (4-month-old) rats. Western blotting revealed that there were no significant differences between young and aged rats in neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression. Inducible isoform of NOS (iNOS), in terms of activity and protein expression, was not detected in either group. Total arginase activity and arginase I and II protein expression did not differ between the young and aged groups. The present findings support the contribution of NOS/NO to ageing but question the importance of iNOS in the normal ageing process.

Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus.

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.

Prefrontal cortex lesions augment the location-related firing properties of area TE/perirhinal cortex neurons in a working memory task.

It has previously been proposed that prefrontal cortex may have some role in keeping temporal cortex-based representations "on-line" during a working memory task. To test this hypothesis, the effects of electrolytic prefrontal cortex lesions on the firing of area TE and perirhinal cortex (PRC) neurons were examined while rats performed a delayed non-match to position task in the T-maze. The behavioural performance of control (n = 4) and lesioned (n = 4) animals were similar during this task, and many neurons displayed a statistically significant location-related variation in firing rate during the sample (44/56 neurons) and test (39/56 neurons) phases. Units from prefrontal-lesioned animals (82%) were more likely to display a significant variation in firing across the maze compared to controls (50%; P < 0.01), and to have more discrete location-related properties (50% of neurons) compared to the control (5%) group (P < 0.0005). This finding suggests that prefrontal cortex normally modulates the transmission and/or processing of spatial information in area TE/PRC during a working memory task. Modulation could be mediated through direct connections between the structures or via prefrontal control of subcortical structures. This finding has implications for our understanding of prefrontal-temporal involvement in memory and cognitive disorders.

Instability in the place field location of hippocampal place cells after lesions centered on the perirhinal cortex.

The perirhinal cortex appears to play a key role in memory, and the neighboring hippocampus is critically involved in spatial processing. The possibility exists, therefore, that perirhinal-hippocampal interactions are important for spatial memory processes. The purpose of the present study was to investigate the contribution of the perirhinal cortex to the location-specific firing ("place field") of hippocampal complex-spike ("place") cells. The firing characteristics of dorsal CA1 place cells were examined in rats with bilateral ibotenic acid lesions centered on the perirhinal cortex (n = 4) or control surgeries (n = 5) as they foraged in a rectangular environment. The activity of individual place cells was also monitored after a delay period of either 2 min, or 1 or 24 hr, during which time the animal was removed from the environment. Although the perirhinal cortex lesion did not affect the place field size or place cell firing characteristics during a recording session, it was determined that the location of the place field shifted position across the delay period in 36% (10 of 28) of the cells recorded from lesioned animals. In contrast, none of the place cells (0 of 29) recorded from control animals were unstable by this measure. These data indicate that although the initial formation of place fields in the hippocampus is not dependent on perirhinal cortex, the maintenance of this stability over time is disrupted by perirhinal lesions. This instability may represent an erroneous "re-mapping" of the environment and suggests a role for the perirhinal cortex in spatial memory processing.

Excitotoxic lesions of the pre- and parasubiculum disrupt object recognition and spatial memory processes.

Rats with bilateral ibotenic acid lesions centered on the pre- and parasubiculum and control rats were tested in a series of spatial memory and object recognition memory tasks. Lesioned rats were severely impaired relative to controls in both the reference and working memory versions of the water maze task and displayed a delay-dependent deficit in a delayed nonmatch to place procedure conducted in the T-maze. Lesioned rats also displayed reduced exploration in a novel environment, and performance was altered in an object recognition procedure as compared with the control group. These findings indicate that the pre- and parasubiculum plays an important role in the processing of both object recognition and spatial memory.

The effect of excitotoxic lesions centered on the hippocampus or perirhinal cortex in object recognition and spatial memory tasks.

Rats with bilateral ibotenic acid lesions centered on the hippocampus (HPC) or perirhinal cortex (PRC) and sham-operated controls were tested in a series of object recognition and spatial memory tasks. Both HPC and PRC rats displayed reduced habituation in a novel environment and were impaired in an object-location task. HPC rats were severely impaired in both the reference and working-memory versions of the water maze and radial arm maze tasks. In contrast, although PRC rats displayed mild deficits in the reference memory version of the water maze and radial arm maze tasks, they were markedly impaired in the working-memory version of both the tasks. These findings demonstrate that under certain conditions both the HPC and PRC play a role in the processing of spatial memory. Further investigation of these conditions will provide important new insights into the role of these structures in memory processes.

A low cost, high precision subminiature microdrive for extracellular unit recording in behaving animals.

A new design for an inexpensive and reliable subminiature microdrive for unit recording in the freely moving animal is presented. The 'Scribe' microdrive is (a) of a small size and low weight, (b) allows for precise advancement of the electrodes, (c) permits stable unit recordings over time, (d) is simple to install, and (e) is economical to construct. These advantages are a result of its simple, single screw-based drive system and the ready availability of component parts. The Scribe microdrive is a small diameter device suitable for multi-site, multi-electrode applications.

Intrinsic theta-frequency membrane potential oscillations in layer III/V perirhinal cortex neurons of the rat.

The firing of a proportion of neurons in the in vivo perirhinal cortex, a brain region involved in object recognition memory, has recently been shown to be synchronized with hippocampal theta activity. The purpose of the present study was to determine whether neurons located in perirhinal cortex have intrinsic properties that might encourage their participation in theta activity. To these ends, current clamp recordings were made from 98 neurons located in layer III/V of the in vitro rat perirhinal cortex. The intrinsic properties of these neurons were investigated, and a subset of 61 neurons were tested for the presence of membrane potential oscillations at threshold levels of depolarization. Thirty-nine percent of these neurons displayed a theta-frequency membrane potential oscillation (MPO; mean frequency = 8.6 Hz). When depolarized past spike threshold, these neurons tended to fire in clusters, with a within-cluster interspike interval close to the peak to peak interval of the MPOs. Neurons that did not generate MPOs generated nonaccomodating action potential trains with a frequency that spanned the theta range. Biocytin staining indicated that MPOs could be generated in cells with both pyramidal and nonpyramidal morphology. These findings demonstrate that a large proportion of perirhinal neurons exhibit intrinsic properties that could assist in the entrainment and synchronization of theta-frequency oscillations. These properties may enhance the communication of information between the perirhinal cortex, entorhinal cortex, and hippocampus.

Immediate early gene transcription and synaptic modulation.

Long-term changes in gene expression appear to be critical to the formation of memory, but little is known about its stimulus- transcription coupling. Numerous studies in the last decade, by focusing on unraveling this signal transduction pathway, have investigated the potential role of the immediate-early genes in this process. The krox family of immediate-early gene proteins are of particular interest because they may be involved in stabilizing the synaptic modifications that underlie hippocampal long-term potentiation (LTP). A potential upstream mediator of krox induction is cyclic AMP-responsive element binding protein (CREB), a posttranslationally activated transcription factor that has been implicated in numerous memory paradigms. In this study we investigated whether the activation of CREB by phosphorylation may have a role in the development of rat perforant- path-stimulated LTP and associated dentate granule cell krox-24 mRNA expression. Contrary to what was expected, we failed to show any difference in the levels of phosphorylated CREB after LTP or following endogenous synaptic facilitation stimulated by novelty. Using these same model systems we also investigated the protein levels of brain- derived neurotrophic factor (BDNF), another immediate-early gene that is induced following a durable form of LTP. However, BDNF protein was not induced within the hippocampus after LTP and was transiently decreased following novel environmental stimulation.

The effect of excitotoxic lesions centered on the perirhinal cortex in two versions of the radial arm maze task.

Rats with bilateral ibotenic acid lesions centered on the perirhinal cortex and sham-operated controls were tested in 2 versions of a spatially guided radial arm maze task. Lesioned rats made significantly more errors and required more sessions to reach criterion relative to controls in the standard radial maze task. When they were tested in a delayed nonmatch to sample version of this task, lesioned rats made more errors during the predelay phase and at both the 30-s and 10-min delays of the postdelay phase. These findings provide further support for the hypothesis that the role of the perirhinal cortex in object recognition memory may include reference to some spatial aspect of the environment.

Synchronous modulation of perirhinal cortex neuronal activity during cholinergically mediated (type II) hippocampal theta.

The perirhinal cortex (PRC) plays a major role in memory processes. This role may be influenced by activity in the adjacent entorhinal cortex (EC) and hippocampus (HPC), particularly during the processing of spatial information. In the current experiment we sought to determine whether the cholinergically mediated (type II) theta rhythm, which is a prominent electrophysiological feature of both HPC and EC activity, influenced neuronal firing in the PRC of urethane-anesthetized rats. When the spontaneous firing activity of single units recorded in PRC was related to theta recorded from the hippocampal fissure, it was determined that the firing of 50/163 (31%) PRC neurons exhibited a statistically significant phase relationship (mean phase angle = 188 degrees) to HPC theta. Thirty-three (66%) of these neurons tended to fire near the trough, and 17 near the peak, of this activity. These data indicate that a high proportion of PRC neurons participate in hippocampal-entorhinal theta activity. This activity may support information transmission and storage within and between these structures.

Excitotoxic lesions centered on perirhinal cortex produce delay-dependent deficits in a test of spatial memory.

Rats with bilateral electrolytic or ibotenic acid lesions that were centred in perirhinal cortex displayed a significant delay-dependent deficit on a delayed nonmatch to position task in the T maze. Although the removal of prominent extramaze visual cues did not affect the performance of these rats, rotating the maze between the sample and test phases did, indicating that rats were using a spatial strategy. Interestingly, a further group of rats with hippocampal and perirhinal damage displayed deficits that may reflect a dysfunction in the use of inertial cues. These results suggest that both electrolytic and excitotoxic lesions of perirhinal cortex produce spatial memory impairments but that these impairments are qualitatively different than those exhibited following hippocampal damage.

Lesions of perirhinal cortex produce spatial memory deficits in the radial maze.

Rats with bilateral electrolytic lesions of perirhinal cortex (PRC) or sham control (SHAM) lesions were tested in spatial reference and working memory tasks in the radial arm maze. In experiment 1, one arm of the maze was baited and always located in a fixed position relative to the extra-maze environment. PRC lesioned animals made a significantly greater number of errors than did SHAM animals during initial training in this reference memory task and exhibited a delay-dependent impairment on trial 5 in a series when a delay period of 5, 60, 120, or 240 s was inserted between trials 4 and 5. In experiment 2, when a second group of the animals was tested on the standard radial arm maze working memory task, the performance of the PRC group was markedly impaired relative to controls. These data demonstrate that electrolytic PRC lesions result in a deficit in both spatial reference and spatial working memory tasks. These effects are interpreted as being consistent with the idea that PRC plays an important role in episodic memory processes. These processes may include the storage of information, which is required for the performance of spatial tasks.

Perirhinal cortex contributions to performance in the Morris water maze.

Rats with bilateral, electrolytic lesions of perirhinal cortex (PRC), lateral entorhinal cortex (LEC), or combined lesions (PRLE) were impaired relative to controls (sham) during initial acquisition in the Morris water maze, although all groups were eventually able to learn to locate the platform. A further deficit in the performance of PRC and PRLE, but not LEC groups, was evident, however, when a probe trial was conducted 2 min (but not 24 hours) after training. When the performance of sham- and PRC-lesioned rats was tested with variable memory delays inserted between training and probe trials, lesioned rats displayed an increase in the rate of forgetting for information made available during the training trial. This finding suggests that the PRC normally stores information regarding the cue-platform relationship and transfers this information to related structures during water maze performance.

Parallel involvement of perirhinal and lateral entorhinal cortex in the polysynaptic activation of hippocampus by olfactory inputs.

It has previously been shown that olfactory input to the hippocampus (HPC) is mediated polysynaptically via the lateral entorhinal cortex (LEC), the site of origin of the lateral perforant pathway (LPP). Because previous anatomical studies have shown that olfactory projections also terminate in perirhinal cortex and that this latter region projects directly to the hippocampus, we investigated the role of perirhinal cortex (PRC) in the mediation of the olfactory-hippocampal potential in the rat. Single-pulse stimulation of the lateral olfactory tract (LOT) resulted in a long onset latency (12-20 ms) evoked response in the dentate gyrus of the ipsilateral hippocampal formation. LOT-HPC potentials were rapidly and completely abolished following the microinfusion of procaine into the LPP, suggesting that they are ultimately mediated via this pathway. In support of this finding, current source density analysis indicated that the LOT-HPC response was generated by a current sink at the outer molecular layer of both dorsal and ventral blades of the dentate gurus. Electrolytic and ibotenic acid lesions of PRC produced a significant decrease in the amplitude of LOT-HPC potentials when testing was conducted 4-7 days postlesion. Lesions of LEC produced similar effects and combined lesions of LEC and PRC resulted in an almost complete eradication of the potential, suggesting that parallel entorhinal-hippocampal and perirhinal-hippocampal pathways are involved. These data suggest, therefore, that a portion of the olfactory input to the hippocampus is mediated via polysynaptic connections routed through perirhinal cortex. Because recent research has suggested that PRC plays an important role within the temporal lobe memory system, this connectivity may be important for olfactory memory processes.

Current source density analysis of the potential evoked in hippocampus by perirhinal cortex stimulation.

Previous anatomical research has demonstrated that the perirhinal cortex (PRC) projects to the dorsal hippocampal CA1 field. We have recently presented data (Liu and Bilkey, Hippocampus 1996; 6:125-135) which suggests that this pathway courses via the lateral perforant path (LPP). In the present study, laminar profiles of the average evoked potentials and current source density (CSD) analysis were used to study the input from the perirhinal cortex to the dorsal hippocampus in the urethane-anaesthetized rat. Stimulation of the lateral perforant path activated a current sink in the stratum lacunosum-moleculare of CA1 and the outer molecular layer of the dentate gyrus with an onset latency of 3.5 ms. Stimulation of the perirhinal cortex produced a very similar sink-source pattern with an onset latency of 4.0 ms. Higher-intensity stimulation of lateral entorhinal cortex also produced a similar pattern with an onset latency of 4.5 ms. Electrolytic lesions of PRC conducted 4-5 days prior to testing resulted in a major decrease (58%) in the amplitude of the LPP-elicited potentials and a corresponding reduction across the whole source-sink pattern. A similar result was observed following ibotenic acid lesions of PRC. In contrast, similar-sized electrolytic lesions of lateral entorhinal cortex produced a much smaller (16%) decrease in potential amplitude and little change in the source-sink pattern. These data provide further support for the hypothesis that perirhinal cortex projects to both the dentate gyrus and CA1 regions of the hippocampus via the lateral perforant path.