Lawrence R. Williams

Hypophagia is induced by intracerebroventricular administration of nerve growth factor

In previous experiments examining the stimulatory effect of intracerebroventricular (icv) NGF treatment on basal forebrain choline acetyltransferase (ChAT) activity, many of the rats treated with the maximally effective dose of NGF appeared gaunt compared to the vehicle-treated control animals. The present experiments determined that icv infusion of NGF at a dose of 1.2 micrograms/day causes a significant reduction in food consumption during the entire period of treatment compared to untreated and vehicle-treated animals. Male rats infused with NGF lost an average of about 30 g of body wt during the first week after the start of infusion and did not gain appreciable weight during the second week of NGF treatment. The hypophagic effect of NGF is dose-dependent, centrally mediated, and reversible. There is no correlation between the stimulatory effect of NGF on basal forebrain ChAT and the inhibitory effect of NGF on weight gain. A therapeutic dose of NGF for Alzheimers patients comparable to the rat dose for maximally stimulating central cholinergic neurons would approach 1 mg/day. If such therapy is applied, the potential exits for induction of hypophagia as a side effect of the NGF administration.

Exogenous Nerve Growth Factor Increases the Activity of High-Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats

Abstract: The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium‐sensitive high‐affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats, in 24‐month‐old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4‐month‐old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4‐month‐old animals, NGF treatment at 1.2 μg/ day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle‐treated 4‐month‐old animals; vehicle treatment had no effect on the activity of either marker. In 24‐month‐old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24‐month‐old control animals. Synaptosomal HACU in 24‐month‐old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values. Treatment of 24‐month‐old rats with NGF for 4 weeks produced further increases in ChAT activity and HACU in striatum and frontal cortex; in hippocampus, the 4‐week treatment did increase HACU, but ChAT activity was not further increased over that produced by 2 weeks of NGF administration. These data indicate an age‐related differential regulation of ChAT activity and HACU between specific brain areas. In addition, NGF stimulated phenotypic expression of these cholinergic markers in both young adult and aged rat brain; there might be an age‐associated differential sensitivity of particular brain regions to exogenous NGF.

Use of a Biophysical-Kinetic Model to Understand the Roles of Protein Binding and Membrane Partitioning on Passive Diffusion of Highly Lipophilic Molecules Across Cellular Barriers

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

Axotomy-dependent stimulation of choline acetyltransferase activity by exogenous mouse nerve growth factor in adult rat basal forebrain.

Transection of the adult rat dorsal fornix and fimbria (F-F) induced a sensitivity of the cholinergic neurons in the medial septum and diagonal band (MS/DB) to exogenous mouse nerve growth factor (mNGF). Continuous infusion of mNGF for two weeks after complete unilateral F-F aspiration resulted in a stimulation of choline acetyltransferase (ChAT)-specific activity in precise micro-dissections of the MS/DB ipsilateral to the transection to a level that was 200% higher than that measured in normal adult animals. This supranormal stimulation of ChAT activity reached plateau levels after 10 days of NGF infusion and was dose-dependent with an E.D.50 equal to 120 ng/day. Administration of mNGF had no effect on the ChAT activity in the MS/DB of normal animals or animals with a unilateral transection of only the supracallosal dorsal septo-hippocampal pathway. Partial transection experiments indicated that a predominent pathway for cholinergic neurons potentially sensitive to exogenous mNGF runs in the paramedian F-F. Administration of mNGF also induced a stimulation of ChAT activity in dissections of the caudate-putamen both ipsi- and contralateral to the infusion cannula. This indicates that unlike the cholinergic projection neurons of the MS/DB, adult cholinergic striatal interneurons are sensitive to exogenous NGF without prior axotomy.

Exogenous nerve growth factor stimulates choline acetyltransferase activity in aging Fischer 344 male rats

The effect of age and exogenous nerve growth factor (NGF) infusion on choline acetyltransferase (ChAT) specific activity is examined in microdissections of cerebral and hippocampal cortices, and the cholinergic nuclei of the medial septum and diagonal band of Broca (MS/DB), the nucleus basalis magnocellularis (NBM), and striatum of Fischer 344 male rats. Significant, 20% losses in ChAT activity are found in the MS/DB and striatum of 24-month-old rats (n = 21) compared to 4-month-old animals, but there is no apparent loss of enzyme activity in the NBM. Loss of ChAT activity in the MS/DB is only observed in animals older than 19 months of age, while a striatal deficit is found in animals older than 7 months. Treatment for 2 weeks with NGF at 1.2 micrograms/day results in significant 70% increases of ChAT activity in the MS/DB and striatum of 24-month-old rats compared to untreated and vehicle-treated 4-month-old rats, but does not stimulate activity in the NBM. Sensitivity of ChAT activity in the MS/DB and striatum to exogenous NGF increases with age. These experiments indicate that in the MS/DB, NBM, and striatum of Fischer 344 male rat there is an age-associated, differential regulation of ChAT enzyme activity and sensitivity to exogenous NGF.

Induction of cobalt accumulation by excitatory amino acids within neurons of the hippocampal slice

Computer-assisted image analysis was used to establish the dose response of excitatory amino acid (EAA) analogs on the induction of cobalt accumulation within pyramidal and granule cell neurons in 400 microns slices of gerbil hippocampus. Slices were incubated 20 min at 22 degrees C in a solution containing 5 mM CoCl2 and 0-1,000 microM EAA analog. The cobalt was visualized by development in (NH4)2S, and the slices were digitized for quantitative densitometry. Kainic acid (KA) had the largest effect and induced cobalt accumulation in the dentate gyrus and CA1, 180% and 150% above control, respectively, with an ED50 = 30 microM. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) induced accumulations of cobalt in CA1 and hilar neurons 130% above control with an ED50 = 30 microM, but had little effect on dentate granule cells. The accumulations induced by KA and AMPA were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovaleric acid (AP5) or voltage-dependent calcium channel blockers. N-Methyl-D-aspartate (NMDA) induced accumulation in the dentate and CA1 150% above control in a pattern similar to KA, but with an ED50 of 100 microM. The accumulation was blocked by both AP5 and CNQX. These data indicate that cobalt-permeable, receptor-activated divalent cation channels are differentially distributed within the gerbil hippocampus, and have differential sensitivities to non-NMDA agonists. The localization of KA-activated, cobalt-permeable channels appears to be coincident with the flop form of the AMPA-selective calcium-permeable glutamate receptor-activated channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat aorta isografts possess nerve regeneration-promoting properties in silicone Y chambers

Recent investigations using synthetic entubation materials have demonstrated that nerve regeneration across a gap requires peripheral nervous tissue as a distal component within the chamber. Regeneration fails if the distal end of the regeneration chamber remains open to the environment, or is filled with tendon or skin. These results are contrary to those of Weiss and Taylor (1944) who used homologous aorta as a chamber material and found that regeneration was successful across a gap regardless of the distal tissue, even in the branches of the aorta that were closed or open. In the present report, silicone Y chambers were implanted with the distal branch remaining open, or filled with pieces of nerve, tendon, or isologous aorta. A homogenous fibrin matrix formed in all the chamber branches by 1 week postimplantation. At 4 weeks, regeneration across a 10-mm gap failed in the chamber branches that were open or filled with tendon. However, regeneration was successful in those branches containing aorta or nerve. These experiments strongly support the hypothesis that aorta tissue, like peripheral nervous tissue, is a source of regeneration-promoting materials. The results provide a probable explanation for and a reconciliation of the discrepancies between the earlier observations of Weiss and Taylor and those of more recent investigators.

Chapter 30: Nerve growth factor affects the cholinergic neurochemistry and behavior of aged rats

Publisher Summary The major central cholinergic systems include (1) the projection neurons of the medial septum and diagonal band of Broca (MS/DB) to the hippocampus; (2) the projection neurons of the nucleus basalis magnocellularis (NBM) to the amygdala and cerebral cortex; (3) the inter-neurons of the striatum. Although the basal forebrain projection neurons have been implicated in learning and memory, and the striatal inter-neurons in motor behaviors, it still is not clear how and to what extent the central cholinergic neurons are involved in specific behaviors. Similarly, relatively little is known about the cellular and molecular mechanisms that regulate animal behaviors mediated by the central cholinergic systems. Although acetylcholine (ACh) was the first neurotransmitter discovered in the central nervous system, it remains unclear how the synthesis, storage, and release of ACh are regulated, or how ACh neurochemistry might be altered by environmental stimuli or synaptic experience, both of which would impact on cholinergically mediated behaviors. The central cholinergic systems are particularly dysfunctional in Alzheimers disease. Thus, our animal research on the regulation of central cholinergic transmission and behavior has focused on the deficits in the systems associated with senescence and the potential amelioration of age-related deficits by the administration of the neurotrophic protein, nerve growth factor (NGF). This chapter describes the current understanding of the regional regulation of ACh synthesis and release in young and old rats, and illustrates several correlations between age-related and NGF-induced alterations in cholinergic neurochemistry, electrophysiology, morphology, and behavior.

Prediction of cerebral ischemia by ophthalmoscopy after carotid occlusion in gerbils.

Background and Purpose: The Mongolian gerbil provides a unique model of unilateral focal cerebral ischemia because of the lack of posterior communicating arteries in all gerbils as well as an absence of an anterior communicating artery in approximately 20% of the gerbil population. It is unclear how to identify unequivocably the subpopulation of animals that would suffer a severe focal cerebral ischemia after unilateral carotid occlusion. Methods: Ninety-three male gerbils were exposed to unilateral occlusion of the right common carotid artery. The severity of neuronal loss was evaluated histologically in gerbils selected as having significant focal ischemia based on either behavioral criteria (i.e., the demonstration of stereotypical behavior within 1 hour after occlusion) or ophthalmoscopic criteria (i.e., interruption of the retinal arterial perfusion within 10 minutes of carotid ligation as assessed with an ophthalmoscope). After 3 hours of unilateral carotid occlusion, cerebral blood flow was reinstated for 24 hours before fixation for histological analysis. The viability of the CA1 region of the hippocampus, lateral cortex, and medial cortex was scored on a scale of 0–4 based on the percentage of apparent neuronal loss (e.g., 0, no damage; 4, >75% damage (the Viability Index). Results: Twenty-eight percent of the gerbils met the behavioral selection criteria, and 17% met the ophthalmoscopic criteria. In the specimens selected by behavioral criteria (n=7), 30% demonstrated no evidence of postischemic neuronal loss; the mean±SEM Viability Index scores for CA1, lateral cortex, and medial cortex were 1.6±0.6, 1.0±0.3, and 0.3±0.2, respectively. Of the animals selected by ophthalmoscopic criteria (n = 12), 100% had severe ischemic tissue damage to the ipsilateral hemisphere; the Viability Index scores for CA1, lateral cortex, and medial cortex were 3.5±0.1, 3.1±0.2, and 1.2±0.2, respectively; all scores were significantly larger than those observed in the behaviorally selected group. Conclusions: Selection of animals by ophthalmoscopic criteria provides a reliable, consistent method to predict animals with severe focal cerebral ischemia.

Sensitivity of Fischer 344 × Brown Norway hybrid rats to exogenous NGF: Weight loss correlates with stimulation of striatal choline acetyltransferase

Nerve growth factor (NGF) infusion into normal Fischer 344 x Brown Norway (F344/BN) hybrid male rats for 2 weeks resulted in a dose-dependent stimulation of choline acetyltransferase (ChAT) enzyme activity to 70% above control values in both the basal forebrain and striatum, and a statistically significant 10% loss in animal weight. There was a significant correlation between weight gain and stimulation of striatal ChAT activity, but not with stimulation of basal forebrain ChAT. Thus, unlike some other rat strains, the normal F344/BN rat is sensitive to exogenous NGF, and can be used to study the efficacy of NGF on normal central cholinergic neurons. The NGF effect on weight gain may be mediated by cholinergic stimulation of the nucleus accumbens.