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Both schizophrenia (SZ) and substance abuse (SA) exhibit significant heritability. Moreover, N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathophysiology of both SZ and SA. We hypothesize that the high prevalence of comorbid SA in SZ is due to dysfunction of NMDARs caused by shared risk genes. We used transgenic mice with a null mutation of the gene encoding serine racemase (SR), the enzyme that synthesizes the NMDAR co-agonist d-serine and an established risk gene for SZ, to recreate the pathology of SZ. We determined the effect of NMDAR hypofunction resulting from the absence of d-serine on motivated behavior by using intracranial self-stimulation and neurotransmitter release in the nucleus accumbens by using in vivo microdialysis. Compared with wild-type mice, SR-/- mice exhibited similar baseline intracranial self-stimulation thresholds but were less sensitive to the threshold-lowering (rewarding) and the performance-elevating (stimulant) effects of cocaine. While basal dopamine (DA) and glutamate release were elevated in the nucleus accumbens of SR-/- mice, cocaine-induced increases in DA and glutamate release were blunted. γ-Amino-butyric acid efflux was unaffected in the SR-/- mice. Together, these findings suggest that the impaired NMDAR function and a consequent decrease in sensitivity to cocaine effects on behavior are mediated by blunted DA and glutamate responses normally triggered by the drug. Projected to humans, NMDAR hypofunction due to mutations in SR or other genes impacting glutamatergic function in SZ may render abused substances less potent and effective, thus requiring higher doses to achieve a hedonic response, resulting in elevated drug exposure and increased dependence/addiction.

Glycine and/or D-serine are obligatory coagonists of the N-methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Muller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

There is substantial evidence implicating N-methyl-d-aspartate receptors (NMDARs) in memory and cognition. It has also been suggested that NMDAR hypofunction might underlie the cognitive deficits observed in schizophrenia since morphological changes, including alterations in the dendritic architecture of pyramidal neurons in the prefrontal cortex (PFC), have been reported in the schizophrenic brain post mortem. Here, we used a genetic model of NMDAR hypofunction, a serine racemase knockout (SR−/−) mouse in which the first coding exon of the mouse serine racemase gene has been deleted, to explore the role of d-serine in regulating cognitive functions as well as dendritic architecture. SR −/− mice exhibited a significantly disrupted representation of the order of events in distinct experiences as revealed by object recognition and odor sequence tests; however, SR −/− animals were unimpaired in the detection of novel objects and in spatial displacement, and showed intact relational memory in a test of transitive inference. In addition, SR −/− mice exhibited normal sociability and preference for social novelty. Neurons in the medial PFC of SR−/− mice displayed reductions in the complexity, total length, and spine density of apical dendrites. These findings demonstrate that d-serine is important for specific aspects of cognition, as well as in regulating dendritic morphology of pyramidal neurons in the mPFC. Moreover, they suggest that NMDAR hypofunction might, in part, be responsible for the cognitive deficits and synaptic changes associated with schizophrenia, and highlight this signaling pathway as a potential target for therapeutic intervention.

We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1(-/+)) and compared those results with wild-type (WT) littermate controls (GlyT1(+/+)). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N-[3-([1,1-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1(-/+) retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole-cell recordings from ganglion cells while bath applying D-serine or D-serine + NMDA. The interpretation of these studies was simplified by blocking post-synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D-serine in the GlyT1(-/+) mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1(-/+) animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D-serine as the endogenous coagonist.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in high concentrations in the brain. Using whole-cell recordings of CA1 pyramidal neurons in acute hippocampal slices, we found that either (i) the application of exogenous NAAG or (ii) an increase of endogenous extracellular NAAG, caused by the inhibition of its catabolic enzyme glutamate carboxypeptidase II (GCP II), resulted in a significant reduction in the amplitude of the isolated NMDA receptor (NMDAR) component of the evoked excitatory postsynaptic current (EPSC). Conversely, reduction of endogenous extracellular NAAG caused by either (i) perfusion with a soluble form of pure human GCP II or (ii) affinity purified antibodies against NAAG, enhanced the amplitude of the isolated NMDAR current. Bath application of GCP II inhibitor induced a progressive loss of spontaneous NMDAR miniatures. Furthermore, NAAG blocked the induction of long-term potentiation at Schaffer collateral axons-CA1 pyramidal neuron synapses. All together, these results suggest that NAAG acts as an endogenous modulator of NMDARs in the CA1 area of the hippocampus.

To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral–CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/−) as compared to wild type (WT) littermates using whole-cell patch-clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc-induced antagonism in N-methyl-d-aspartate receptor (NMDAR) currents of GlyT1+/− mice. Moreover, the ratio α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission.

Numerous clinical studies demonstrate that subanesthetic doses of dissociative anesthetics, which are noncompetitive antagonists at the NMDA receptor, replicate in normal subjects the cognitive impairments, negative symptoms, and brain functional abnormalities of schizophrenia. Postmortem and genetic studies have identified several abnormalities associated with schizophrenia that would interfere with the activation of the glycine modulatory site on the NMDA receptor. Placebo-controlled clinical trials with agents that directly or indirectly activate the glycine modulatory site consistently reduce negative symptoms and frequently improve cognition in patients with chronic schizophrenia who are receiving concurrent typical antipsychotics. Thus, there is convincing evidence that hypofunction of a subset of NMDA receptors may contribute to the symptomatic features of schizophrenia.

Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two- to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over-expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

Cerebellar granule cells (CGC) deprived of serum or trophic factors develop sensitivity to kainate neurotoxicity that is mediated by the α-amino-3-hydroxy-5-methyl-isoxazole proprionic acid (AMPA) subtypes of glutamate receptors (GluR). The L-type voltage-gated calcium channel (L-type VGCC) blocker nifedipine increases the potency of kainate 50-fold. Thus, one goal of this laboratory is to determine the underlying protective mechanism triggered by calcium influx through this channel. The cell-permeable heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine effected complete protection against kainate treatment in the presence of nifedipine, as did the iron chelator deferoxamine. The chelatable heavy metal pool decreased ∼70% immediately following treatment with kainate, but did not change following kainate/nifedipine treatment. Tetramethylrhhodamine ethyl ester (TMRE) fluorescence, an indicator of mitochondrial membrane potential, decreased ∼70% following kainate treatment but displayed a more modest decrease (∼15%) when CGC were treated with kainate/nifedipine. Reactive oxygen species (ROS) formation decreased in CGC immediately following kainate treatment but was slightly elevated following kainate/nifedipine treatment. Electron microscopic examinations of the CGC indicated severe swelling and distortion of mitochondria immediately following kainate/nifedipine treatment and the appearance of mitochondrial herniations, whorls, and bridges 2 h later, features that were rarely observed following kainate treatment. These results support the hypothesis that calcium entry through L-type VGCCs protects CGC during kainate treatment by lowering the chelatable heavy metal pool and the mitochondrial membrane potential, thereby mitigating the formation of ROS. Synapse 43:30–41, 2002. © 2001 Wiley-Liss, Inc.

Receptor supersensitivity is an important concept for understanding neurotransmitter and receptor dynamics. Traditionally, detection of receptor supersensitivity has been performed using autoradiography or positron emission tomography (PET). We show that use of magnetic resonance imaging (MRI) not only enables one to detect dopaminergic supersensitivity, but that the hemodynamic time course reflective of this fact is different in different brain regions. In rats unilaterally lesioned with intranigral 6-hydroxydopamine, apomorphine injections lead to a large increase in hemodynamic response (cerebral blood volume, CBV) in the striato-thalamo-cortico circuit on the lesioned side but had little effect on the intact side. Amphetamine injections lead to increases in hemodynamic responses on the intact side and little on the lesioned side in the same animals. The time course for the increase in CBV after either amphetamine or apomorphine administration was longer in striatum and thalamus than in frontal cortex. 11C-PET studies of ligands which bind to the dopamine transporter (2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 1,5-naphthalnendisulfonate, WIN 35, 428 or CFT) and D2 receptors (raclopride) confirm that there is a loss of presynaptic dopamine terminals as well as upregulation of D2 receptors in striatum in these same animals. Pharmacologic MRI should become a sensitive tool to measure functional supersensitivity in humans, providing a complementary picture to that generated using PET studies of direct receptor binding. Synapse 36:57–65, 2000. © 2000 Wiley-Liss, Inc.

The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Muller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells.

The cytoplasmic copper-zinc superoxide dismutase (Cu, Zn SOD; SOD-1) is an abundant and well-conserved intracellular antioxidant enzyme which has been implicated in a number of oxidative stress mediated phenomena, especially Down Syndrome, in which SOD-1 activity is increased due to triplication of chromosome 21 containing the gene and, in hereditary amyotrophic lateral sclerosis, in which the gene is mutated. Overexpression of SOD-1 could theoretically, therefore, lead to increased vulnerability to oxidative stress in two distinct manners: increasing steady-state hydrogen peroxide levels or increasing toxic side reactions. We used two mouse neuronal culture systems--one in which the murine chromosome containing SOD-1 is triplicated and one in which human SOD-1 is a transgene--to test the effect of overexpression of this enzyme on antioxidant status in general and specifically on glutamate mediated oxidative stress. We found that SOD-1 overexpression increases antioxidant status at the same time it decreases vulnerability to glutamate.

Using choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry, we investigated regional and laminar differences in cholinergic innervation in the cerebral cortex, hippocampus, amygdala, and thalamus of mice. In mice, unlike rats, the patterns of ChAT-immunostained and AChE-positive fibers are virtually identical in the cortex and are organized in a trilaminar pattern with cholinergic processes prominent in layers I and IV and within the lower portion of layer V and upper segment of layer VI. ChAT-immunoreactive cells were not seen in cortex. In the amygdala, the basolateral nucleus showed the highest density of cholinergic processes. In the hippocampus, a thin, dense band of ChAT-labeled processes was present in the inner segment of the molecular layer of the dentate gyrus and within the stratum oriens of CA1-3, adjacent to the basal aspect of pyramidal cells. Within the thalamus, anteroventral, mediodorsal (lateral portion), intralaminar, and reticular nuclei showed high densities of cholinergic processes. The results of this study provide the basis for examining the effects of transgenes and age on forebrain cholinergic systems.

N-acetylated alpha-linked acidic dipeptidase (NAALADase) is a membrane bound enzyme that cleaves glutamate from the endogenous neuropeptide N-acetyl-aspartyl-glutamate (NAAG). We report the immunocytochemical localization of NAALADase in rat brain and kidney by using specific anti-NAALADase antiserum. NAALADase-immunoreactivity (NAALADase-IR) was widely distributed, abundant in neuropil, absent from neuronal cytoplasm, and displayed regional heterogeneity. Staining was selectively enriched in several structures previously reported to contain NAAG-immunoreactivity (NAAG-IR) including the amygdala, caudate-putamen, central gray, dorsal raphe, globus pallidus, hippocampus, hypothalamus, locus coerulus, medial and lateral geniculate, olfactory bulb, periaqueductal gray, solitary nucleus, spinal trigeminal nucleus, substantia nigra, superior colliculus, and thalamus. Staining within these structures was enriched in neuropil; no intracellular staining was detected, even after colchicine treatment. In addition, NAALADase-IR was observed in some NAAG-containing fiber tracts including the corpus callosum, fornix, habenular commissure, solitary tract, stria medularis, and stria terminalis. The co-localization of NAALADase-IR and NAAG-IR support the hypothesis that NAALADase is responsible for the catabolism of NAAG in vivo. NAALADase-IR was also detected in brain regions that, to date, have not revealed NAAG-IR, including the bed nucleus of the stria terminalis and the median eminence. In addition, NAALADase-IR was detected in the rat kidney cortex, specifically in the brush border of the proximal convoluted tubules. The observation that NAALADase-IR was more widespread than NAAG-IR suggests that NAALADase may also be involved in the catabolism of other structurally related neural and renal peptides.

The gene encoding growth-associated protein 43 (Gap43), a neuronal phosphoprotein associated with axonal outgrowth and synaptic plasticity, is located on mouse chromosome 16 (MMU16). We examined the developmental expression of Gap43 in normal, trisomy 16 (Ts16), and trisomy 19 (Ts19) mouse brain using northern blot analysis and in situ hybridization as a first step toward understanding the neurobiologic consequences of increased gene dosage on brain development. Gap43 expression was detected by in situ hybridization throughout the mesencephalon, rhombencephalon, spinal cord, and first branchial arch in whole embryos as early as day 10 of gestation (E10). By E15, Gap43 expression was localized to cells in the retina, the olfactory bulbs, and anterior olfactory structures, the cortical plate, the basal telencephalon, diencephalon, midbrain, hindbrain, and spinal cord. Northern blot analysis detected a three-fold increase in Gap43 mRNA levels in the brains of normal mice between E12-E18. At E15, Gap43 mRNA levels were increased 35-40% in Ts16 mouse brain and decreased 10% in Ts19 mouse brain, relative to euploid littermate controls. Using in situ hybridization we found that overexpression of Gap43 occurred in the diencephalon, medial and lateral basal telencephalon, and cortical plate region in Ts16 mice relative to littermate controls. Thus, the degree of overexpression of Gap43 mRNA in Ts16 mice is consistent with that expected from gene dosage effects.

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.

Assays for synaptosomal high-affinity uptake activity (glutamate, gamma-aminobutyric acid, norepinephrine), neurotransmitter synthesizing enzymes (choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase), and endogenous neurotransmitters were performed in cats with advanced inherited GM1 gangliosidosis. A significant reduction in uptake activity, ranging from 24 to 77% of control, was demonstrated in motor, occipital, and cerebellar brain regions. This reduction was unassociated with comparable alterations in neurotransmitter levels or synthesizing enzyme activity. We hypothesize that the defect of neurotransmitter inactivation is part of an overall abnormality of synaptic membrane function that could contribute to the neurological symptoms seen in the hereditary gangliosidoses.

Intrastriatal injection of either kainic acid (0.35 micrograms) or ibotenic acid (7.0 micrograms) in the mouse causes a profound and selective degeneration of striatal neurons accompanied by a secondary astrocytic response. The kainate injection (0.35 micrograms) resulted in significant decrements in the striatal levels of phosphocreatine and ATP by 30 min. a progressive reduction in adenosine phosphates between 30 min and 48 h, and a decrease in energy charge; whereas lactate levels increased by 44% at 2 h, glucose levels fell by 56%. Two hours after intrastriatal injection of ibotenic acid (7.0 micrograms) similar alternations in striatal high-energy phosphates and glucose disposition were found. Prior decortication protected against the neurotoxic effects of kainate in the mouse striatum and prevented the alterations in high-energy phosphates at 2 h although lactate levels increased by 212%. These findings in vivo are consistent with the hypothesis that the neurotoxic effects of acidic excitatory amino acids involve a profound activation of energy consumption by affected neurons.

— Dopamine-β-hydroxylase (DBH) was purified from rat adrenal medulla by a series of steps including sedimentation of membranes, extraction with n-butanol, ammonium sulfate fractionation, gel chromatography and ion-exchange chromatography. Disk gel electrophoresis revealed two protein bands, both of which were active. Antiserum was prepared against homogeneously purified bovine adrenal and rat adrenal DBH: Ouchterlony immunodiffusion, enzyme neutralization and complement fixation tests demonstrated that the respective homologous antisera were monospecific and of high titer. Antiserum to bovine DBH was only 2- to 3-fold more potent than pre-immune serum in inhibition of rat DBH activity. Complement fixation tests demonstrate that antiserum to bovine DBH has a 25,000-fold lower immunoreactivity with rat DBH than with bovine DBH.

The basal forebrain-cortex connections of the rat were topographically mapped by retrograde tracer methods; and their contribution to the cholinergic innervation of the cortex was assessed by excitotoxin lesions placed in the rostral and caudal aspects of the complex. Discrete injections of tracer into frontal cortex labeled the prominent multipolar acetylcholinesterase (AchE)-positive cells of the ventromedial globus pallidus. Injections of tracer into the parietal cortex labelled cells in the ventral globus pallidus, the underlying substantia innominata, and the lateral hypothalamus. Separate injections of Fast Blue and Nuclear Yellow in the frontal and in the parietal cortex resulted in double-labeled cells in the ventral globus pallidus, which indicates that at least some of these cells may possess collateralizing axons. The cingulate cortex is innervated predominantly by neurons in the nucleus of the horizontal limb of the diagonal band. The occipital cortex was also shown to receive a projection primarily from the nucleus of the horizontal limb of the diagonal band. The hippocampal formation is innervated primarily by cells located in the vertical limb of the diagonal band and in the medial septum. Consistent with the results of the retrograde tracing studies, excitotoxin lesions affecting the diagonal band and medial septum decreased choline acetyltransferase (CAT) activity up to 40% on the occipital cortex and by 64% in the hippocampus, but did not affect CAT activity in the rostral neocortex. In contrast, ibotenate lesions of the caudal ventral globus pallidus and substantia innominata caused decreases in CAT activity in the frontal cortex of up to 65% without affecting enzyme activity in the hippocampal formation. The results of the present study provide details on the topographic organization of the cortical projections originating in the basal forebrain complex and indicate that these neurons are the predominant source of cortical cholinergic innervation.

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.

The distribution of presynaptic markers for cholinergic and GABAergic neurons in the various layers of the lateral neocortex was determined with a technique in which sections of increasing depth horizontal to the pial surface were obtained on a frozen-stage microtome and analyzed for the markers. The specific activities of choline acetyltransferase and acetylcholinesterase had an uneven distribution in the cortex with lowest levels of activity associated with lamina II and III, whereas glutamate decarboxylase activity was highest in lamina II, III, and IV. Fetally-induced hypoplasia of the cortex resulted in an enrichment in both choline acetyltransferase and acetylcholinesterase activity in all layers with no significant change in the activity of glutamate decarboxylase. Lesion of the nucleus basalis-cortical cholinergic pathway resulted in marked reductions in the activities of acetylcholinesterase and choline acetyltransferase in all cortical layers and an elimination of the uneven distribution of these markers within the cortex. These results provide further evidence that acetylcholinesterase activity is specifically associated with cortical cholinergic afferents and support the existence of cortical intrinsic cholinergic neurons.

A prospective clinical and biochemical study on the effects of treatment with haloperidol has been performed in seven patients with Tourette syndrome. Pretreatment cerebrospinal fluid levels of homovanillic acid (CSF HVA) were significantly reduce in all patients, whereas 5-hydroxyindoleacetic acid was reduced in only two. With haloperidol treatment, symptoms decreased in all cases (21 to 88%) and clinical improvement was associated with an increased level of CSF HVA, often returning to the normal range. Optimal therapeutic response was found with serum levels of haloperidol between 1 and 4 ng/ml; however, disturbing side effects also occurred within this range. These results support the hypothesis that Tourette syndrome may result from a supersensitivity of dopaminergic receptors.

There is convincing evidence that acidic amino acids, in particular L-glutamate, or substances containing them serve as the major excitatory neurotransmitters in the brain. At least three distinct receptors mediate the excitatory effects of this class of neurotransmitters. Pharmacological studies with agonists and antagonists of these receptors suggest that they may mediate the neurodegenerative consequences of Huntington's disease, status epilepticus, and hypoxemia, and that glutamate receptor antagonists have clinical potential as anticonvulsants, analgesics, and neuroprotective agents.

In this study, we examined the neurochemical profiles of selected brain regions (cerebral hemispheres, diencephalon/brainstem) in fetal (day 14 to 18 gestation) trisomy 19 (Ts19) mice. The neurochemical characteristics we observed in Ts19 mice were quite different from those we observed previously in Ts16 mice. Choline acetyltransferase (ChAT) activity was reduced significantly in the cerebral hemispheres, but not in the brainstem/diencephalon, of the fetal Ts19 mouse brain, suggesting a selective vulnerability of telencephalic cholinergic neurons. Additionally, the activity of glutamic acid decarboxylase (GAD) was reduced significantly in both hemispheres and diencephalon/brainstem of late gestation Ts19 fetuses, suggesting a selective vulnerability of GABAergic neurons as well. While the levels of catecholaminergic and dopaminergic markers were reduced significantly at late gestational ages, the relative rate of turnover of dopamine (DA), measured by the ratio of DOPAC/DA, was elevated significantly in Ts19 mice. Neither reduction in the thickness of various cellular zones of the cerebral cortex nor reduced cell density of the cerebral cortex accounts for the alterations in neurochemical parameters observed in Ts19 mice. These results suggest that the effects of the triplication of specific genes on the respective chromosomes, rather than a generalized disruption of developmental homeostasis resulting from extra chromosomal material, may produce selective alterations in neurochemical and neuroanatomical markers observed in these two mouse trisomies.

An isocratic HPLC method to measure endogenous N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsilateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Lesions of the cerebral cortex alter striatal neuronal vulnerability to locally injected kainic acid. Whereas extensive lesions involving the frontal-parietal-occipital cortex are most effective, lesions limited to the frontal or to the dorsal-lateral parietal cortex offer partial protection. The extensive cortical lesions are associated with selective, marked reductions in the presynaptic markers for glutamatergic afferents in striatum. The protective effects of decortication appear between 6 and 24 hours after the lesion and are maintained up to 30 days after decortication. Whereas decortication results in only a transient reduction of specific receptor binding of [3H]kainic acid to striatal membranes, lesion of striatal intrinsic neurons with kainic acid causes a delayed but marked reduction in specific binding of the ligand. Coadministration of L-glutamic acid (1 mumole) with kainic acid (9 nmoles) partially restores the neurotoxic action of kainic acid in the decorticate striatum; GABA, alanine, and proline (1 mumole) are ineffective with regard to restoring kainate's toxicity for striatal GABAergic neurons. These results suggest that afferent input exerts a permissive effect on the neurotoxic action of kainic acid and that neurotoxicity may involve a cooperative interaction between kainic acid at specific receptors on vulnerable neurons and synaptically released endogenous neurotransmitters, in particular L-glutamic acid.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide localized to several putative glutamatergic neuronal systems, including the rodent optic pathway. To determine whether the peptide is released by depolarization, the superior colliculus of the rat was perfused with 2 microCi of [3H]NAAG, then with Krebs-bicarbonate buffer for 1 h, using a microdialysis system. Subsequently, 10-min fractions were collected and analyzed by HPLC for [3H]NAAG. Addition of 100 microM veratridine resulted in a several-fold increase in the evoked release of [3H]NAAG that was virtually abolished by coperfusion with Ca2+-free Krebs buffer containing 1 mM EGTA. When [3H]glutamate was used as the precursor, veratridine depolarization resulted in only an 80% increase in the release of [3H]NAAG. Prior enucleation of the right eye reduced the spontaneous release of [3H]NAAG by 50%, and the veratridine-evoked release by greater than 85%, from the left superior colliculus. These results suggest that NAAG is released upon depolarization and may serve as a neurotransmitter/neuromodulator in the optic tract.

The role that acetylcholine plays in processing sensory stimuli is beginning to be characterized; however, morphological correlates of cholinergic effects on activity patterns in sensory cortex are not available. To study this problem, unilateral neurotoxic lesions that depleted the necortex of acetylcholine were made in the basal forebrains of mice. The aim of these experiments was to study the effect of cholinergic depletion on stimulus-evoked activity in the barrel field of the mouse somatosensory cortex. One month following the lesion, 2-deoxyglucose (2DG) experiments were conducted on the lesioned and on normal mice while the animal received bilateral stimulation to the C3 whisker. The tissue was processed for acetylcholinesterase and cytochrome oxidase histochemistry and 2DG autoradiography. Evaluation of the column-like 2DG label evoked in the somatosensory cortex revealed that the activity on the lesioned side was significantly reduced in dimension and intensity from that in the normal hemisphere. On the normal side, the activated barrels averaged 641 microns in tangential width, were 76.5% above background in density, and extended from lamina I-V. On the lesioned side, the activated barrels were 485 microns in tangential width, 65.4% above background in density, and extended from lamina II-V. In other cortical regions, outside the stimulus-evoked barrel field, 2DG activity values were similar on the normal and lesioned side. Additionally, both the pattern and intensity of the cytochrome oxidase staining within the barrel field displayed no differences in either hemisphere. These studies suggest that acetylcholine plays a significant role in the processing of somatosensory information. Neurotoxic lesions that diminish cortical cholinergic innervation cause a reduction of stimulus-evoked activity levels, while underlying metabolic activity is either not affected or recovers over time.

The rates of accumulation of the catecholamine synthesizing and metabolizing enzymes proximal to a ligation on the sciatic nerve of the rat were studied. Dopamine-β hydroxylase (EC 1.14.2.1) and tyrosine hydroxylase (EC 1.14.3a) accumulated at a similar rapid rate, and catechol-O-methyl-transferase (EC 2.1.1.6), choline acetyltransferase (EC 2.3.1.6) and monoamine oxidase (EC 1.4.3.4) accumulated at the same slow rate, whereas DOPA decarboxylase (EC 4.1.1.26) accumulated at an intermediate rate. Based on clearance of the rapidly accumulating enzymes, absolute flow rates were estimated to be: 106-167 mm/24 h for tyrosine hydroxylase; 138-185 mm/24 h for dopamine-β-hydroxylase; and 36-86 mm/24 h for DOPA decarboxylase. In contrast, the mean rate of transport of the slowly accumulating enzymes (monomine oxidase, catechol-O-methyltransferase and choline acetyltransferase) was approximately 3 mm/24 h. Colchicine and vinblastine completely blocked the axonal transport of both the rapidly and slowly transported enzymes. Studies of the subcellular distribution of each enzyme failed to confirm the suggestion that particulate enzymes are transported rapidly and soluble enzymes slowly. Our results suggest that the transport and inactivation of dopamine-β-hydroxylase, DOPA decarboxylase, and tyrosine hydroxylase are under different controls than monoamine oxidase and catechol-O-methyltransferase.

Explants of substantia nigra and corpus striatum obtained from newborn rats were maintained in tissue culture for up to six days. Explants of substantia nigra exhibited a net increase in the ability to take up H3-dopamine, a process associated with the dopaminergic neurons; in contrast, the explants of corpus striatum showed a rapid loss in this ability to accumulate H3-dopamine. After three days in culture, the specific activity of tyrosine hydroxylase and monoamine oxidase had decreased 50% in explants of substantia nigra. A medium including fetal calf serum and chick embryo extracts was necessary for the increase in H3-dopamine uptake, and nerve growth factor had an inhibitory effect. Histofluorescent examination of nigral explants cultured for three days indicated morphologically normal dopaminergic neurons.

The uptake and storage of L-[3H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[3H]norepinephrine with a Km of 3 × 10 −7m was first demonstrable; the Km value did not vary during subsequent development. The Vmax. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l-[3H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.

The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [3 H-methyl]S-adenosylmethionine; and the [3H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.

Acetylcholinesterase (AChE) activity was measured in cerebrospinal fluid (CSF) samples from 36 individuals, including 12 persons with Alzheimer's disease, 12 normal controls, and 12 patients with other dementias. AChE activity also was measured in 47 normal subjects whose ages ranged from 20 to 84 to evaluate the effect of age on AChE activity. CSF from patients with senile dementia of the Alzheimer type showed significantly lower mean AChE activity than in age-matched controls and patients with other dementia syndromes. No correlation was found between duration of illness, age, or severity of illness (as measured by the Mini-Mental State Examination score) and CSF AChE activity in Alzheimer's disease. AChE increased significantly over the age range of 20 to 84. CSF AChE activity may prove to be a useful diagnostic test to confirm the clinical diagnosis of moderate to severe Alzheimer's disease.