Jay R. Simon

Determination of the Equilibrium Dissociation Constants and Number of Glycine Binding Sites in Several Areas of the Rat Central Nervous System, Using a Sodium‐Independent System

Abstract Parameters affecting the binding of [3H]glycine to membrane fractions isolated from the cerebral cortex, midbrain, cerebellum, medulla oblongata, and spinal cord of the rat were investigated in a Na+‐free medium. A [3H]glycine binding assay was established in which the binding was specific, saturable, pH‐sensitive, and reversible. Conditions were chosen in an effort to minimize binding to glycine uptake sites. From data on specific [3H]glycine binding, Scatchard plots were prepared and the KD and Bmax values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (KD: 44, 211 nM; Bmax: 361, 1076 fmol/mg protein) and spinal cord (KD: 19, 104nM;Bmax: 105, 486 fmol/mg protein). The ranges of the KD and Bmax values for the other three areas studied were 59 to 144 nM and 882 to 3401 fmol/mg protein, respectively. When the glycine content of each area, expressed as fmol/neuron, was plotted against the respective K0 (high affinity), a negative correlation was found (r=−0.90; p < 0.05). A similar negative correlation was found between the glycine content and Bmax (r=−0.88; p <0.05). Hill plots indicated a slope of essentially 1.0 for all areas. GABA, taurine, strychnine, diazepam, bicuculline, and imipramine had little or no effect on [3H]glycine binding.

Dorsomedial hypothalamic GABA dysfunction produces physiological arousal following sodium lactate infusions

Since impairing gamma-aminobutyric acidA (GABAA) receptor-mediated inhibition in the dorsomedial hypothalamus (DMH) of rats elicits a panic-like response, experiments were conducted to test if rats with GABA dysfunction in the DMH would be vulnerable to precipitation of a panic-like response after intravenous sodium lactate infusions. Rats were implanted with unilateral infusion cannula into the DMH which were connected with Alzet minipumps that chronically infused (3.5 nmol/microliter /h) either a-CSF (vehicle), dl-(racemic), l-(active) or d-(inactive) isomers of allylglycine (AG), an inhibitor of GABA synthesis. Another group of rats had l-allylglycine pumps implanted in the paraventricular nucleus of the hypothalamus (PVN) as anatomical controls. Animals were tested in the social interaction (SI) test and given sodium lactate infusions (10 ml/kg/15 min) before Alzet pump implantations and on days 4, 7, and 14 after pump placement. Rats were also tested in the elevated plus-maze on treatment day 4. Chronic impairment of GABA function in the DMH and not PVN resulted in rats being more anxious in the SI test on treatment days 4, 7, and 14 and in the elevated plus-maze on day 4 compared to a-CSF and d-AG infusions. Further, rats with GABA dysfunction in the DMH, and not PVN, exhibited significant increases in heart rate and blood pressure following IV sodium lactate infusions. There were significant decreases in DMH glutamic acid decarboxylase activity and GABA content in rats receiving 7 days of dl-AG or l-AG infusions. These results indicate that chronic reduction of GABA function in the DMH leads to the development of panic-like disorder in this animal model.

Intrahippocampal transplants of septal cholinergic neurons : high-affinity choline uptake and spatial memory function

Recent studies have demonstrated that intrahippocampal cholinergic septal grafts can ameliorate deficits in spatial memory function and hippocampal cholinergic neurochemical activity in animals with disruptions of the septohippocampal system. However, no study has determined if the restoration of spatial memory function is correlated to the restoration of cholinergic activity, as measured by high-affinity choline uptake (HACU). The present study was designed to determine if such a correlation between behavioral and neurochemical restoration exists. Male Sprague-Dawley rats received either sham lesions (SHAM), bilateral lesions of the septohippocampal pathway (LES), or bilateral lesions along with intrahippocampal septal grafts (SG). After 8 months, rats were tested for their ability to perform spatial reference, spatial navigation and working memory tasks. Upon completion of the behavioral testing, neurochemical activity of the hippocampus was measured by HACU. The results indicate that animals in the SG group had significantly higher behavioral scores and hippocampal HACU rates than animals in the LES group. Regression analysis indicates that a significant correlation exists between performance on each behavioral task and HACU rates. These results demonstrate that hippocampal cholinergic activity, as measured by HACU, correlates significantly with performance on tests of spatial memory function.

Restoration of High Affinity Choline Uptake in the Hippocampal Formation Following Septal Cell Suspension Transplants in Rats with Fimbria-Fornix Lesions

Abstract: High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria‐fornix lesions. Nine‐14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria‐fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus. HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and pic‐rotoxin. These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria‐fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system. The activation of HACU by picrotoxin, a γ‐aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus. Lesions of the fimbria‐fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus. This increase in binding was not significantly ameliorated by intrahippo‐campal grafts of cholinergic neurons.

Intrahippocampal transplants of septal cholinergic neurons: choline acetyltransferase activity, muscarinic receptor binding, and spatial memory function.

Recent studies have demonstrated that intrahippocampal cholinergic septal grafts can ameliorate deficits in spatial memory function and hippocampal cholinergic neurochemical activity in animals with disruptions of the septohippocampal pathway. Further studies have revealed that hippocampal cholinergic activity, as measured by high affinity choline uptake, correlates significantly with performance on tests of spatial memory function. The present study was designed to examine the effect of cholinergic septal grafts on reversing deficits in hippocampal choline acetyltransferase activity and on normalizing muscarinic receptor binding in animals with lesions of the septohippocampal system, and to examine the correlations between these cholinergic parameters and performance of spatial memory tasks. The results of this study indicated that in animals with lesions plus septal grafts, hippocampal ChAT activity was restored significantly and muscarinic receptor binding was normalized to a level not different from the control animals. Regression analyses indicated that ChAT activity was significantly correlated with performance on spatial reference memory, spatial navigation and spatial working memory, while muscarinic receptor binding correlated significantly with spatial reference memory performance.

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids

Abstract— Crude synaptosomal (P2) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X‐irradiation treatment and from the cerebella of control animals. In the P2 fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2‐fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3‐fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P2 fraction isolated from the X‐irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X‐irradiation treatment. The uptake of 1.0 μm‐l‐[3H]glutamate and l‐[3H]aspartate was reduced approx 20% by X‐irradiation treatment, whereas the uptake of 1.0 μm‐[3H]GABA and [3H]taurine was unchanged. A more detailed kinetic analysis of l‐[3H]glutamate uptake revealed there was a 20% decrease in the Vmax value with X‐irradiation treatment and no change in the apparent Km value.

Regional distribution of cholinergic parameters within the rat striatum

The topographical distribution of choline acetyltransferase, muscarinic receptor binding and high affinity choline uptake was studied in 21 separate areas of the rat striatum. The areas of the nucleus chosen represented dorsolateral, dorsomedial, ventrolateral and ventromedial regions along the rostrocaudal aspect of the striatum, such that a three-dimensional distribution of the cholinergic parameters could be obtained. Within any given rostrocaudal section, no significant dorsoventral differences were noted for any of the cholinergic parameters. On the other hand, marked differences were found in a comparison of the medial and lateral striatum. Choline acetyltransferase, muscarinic receptor binding and high affinity choline uptake were more concentrated in the lateral striatum than the medial striatum, and the magnitude of this medio-lateral disparity increased from rostral to caudal regions of the nucleus. The lateral striatum exhibited no significant rostrocaudal variations in the cholinergic parameters; however, the medial portion of the striatum did exhibit differences along its rostrocaudal extent, with the rostral-most sections being enriched relative to the more caudal sections. These results suggest that the cholinergic system in the striatum is heterogeneously distributed within this nucleus, with the lateral portion possessing a greater cholinergic innervation than the medial portion. They further suggest that future neurochemical studies of cholinergic alterations in the striatum should include a consideration of the possibility of regional effects within the nucleus rather than treating the striatum as a homogeneous tissue.

Neurochemical studies of the nucleus of the solitary tract, dorsal motor nucleus of the vagus and the hypoglossal nucleus in rat: Topographical distribution of glutamate uptake, GABA uptake and glutamic acid decarboxylase activity

The topographical distribution of glutamate uptake, GABA uptake, and GAD activity was studied in caudal, intermediate and rostral areas of the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMN) and the of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMN) and the hypoglossal nucleus (n.XII). Within the NTS and n.XII, all three neurochemical parameters exhibited increasing activity from caudal to rostral regions. The distribution pattern for glutamate uptake within the DMN was qualitatively similar to the other nuclei studied, whereas GABA uptake and GAD activity were found to be homogeneously distributed within the DMN. The NTS also exhibited a medial-lateral heterogeneity for glutamate and GABA uptake, with the medial aspect of this nucleus containing significantly higher uptake than the lateral aspect.

A possible role for tyrosine kinases in the regulation of the neuronal dopamine transporter in mouse striatum

The present investigation was undertaken to test the hypothesis that a reduction in the activity of protein tyrosine kinases would result in an alteration in dopamine transport. Genistein, a broad-spectrum inhibitor of protein tyrosine kinases, inhibited dopamine uptake into mouse striatal homogenates with an IC50 of 18 microM. The inhibition by genistein was rapid, reversible and somewhat selective, in that genistein did not inhibit the uptake of choline or GABA under similar conditions. Kinetic analyses indicated that genistein was a non-competitive inhibitor. Another protein tyrosine kinase inhibitor, tyrphostin 23, also inhibited transport but was significantly less potent than genistein. Tyrphostin 25 and lavendustin A were without major effect on dopamine uptake. In addition, the inactive structural analog of genistein, genistein, had no significant effect on dopamine uptake. The inhibition of dopamine transport by 50 microM genistein was accompanied by a reduction in the level of a 110-kDa band of tyrosine phosphoprotein. It is suggested that protein tyrosine kinases play a role in the cascade of events which ultimately lead to regulation of neuronal dopamine transport.

Distribution of choline acetyltransferase, acetylcholinesterase, muscarinic receptor binding, and choline uptake in discrete areas of the rat medulla oblongata

Quantitative measurements were made of choline acetyltransferase (CAT) activity, acetylcholinesterase (AChE) acitivity and cholinergic muscarinic receptor binding ([3H]QNB) in eight areas of a cross-section of the rat medulla oblongata. A fourth cholinergic parameter, high-affinity choline uptake, was measured in three groups of these areas. CAT, AChE and [3H]QNB binding were found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus; the lowest value was in the area which contains the inferior olive and the corticospinal tract. The distribution of AChE and CAT acitivities varied approximately 7- to 10-fold among the eight regions examined, whereas that of the muscarinic receptor varied only about 4-fold. The Na+-dependent high-affinity choline uptake varied approximately 20-fold from the region with the lowest activity (inferior olivary nucleus and corticospinal tract) to that with the highest activity (tissue areas containing the dorsal motor nucleus, hypoglossal nucleus, nucleus of the solitary tract and nucleus cuneatus). The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied.