Transplantation of neural progenitor cells in chronic spinal cord injury.

Previous studies demonstrated that neural progenitor cells (NPCs) transplanted into a subacute contusion injury improve motor, sensory, and bladder function. In this study we tested whether transplanted NPCs can also improve functional recovery after chronic spinal cord injury (SCI) alone or in combination with the reduction of glial scar and neurotrophic support. Adult rats received a T10 moderate contusion. Thirteen weeks after the injury they were divided into four groups and received either: 1. Medium (control), 2. NPC transplants, 3. NPC+lentivirus vector expressing chondroitinase, or 4. NPC+lentivirus vectors expressing chondroitinase and neurotrophic factors. During the 8 weeks post-transplantation the animals were tested for functional recovery and eventually analyzed by anatomical and immunohistochemical assays. The behavioral tests for motor and sensory function were performed before and after injury, and weekly after transplantation, with some animals also tested for bladder function at the end of the experiment. Transplant survival in the chronic injury model was variable and showed NPCs at the injury site in 60% of the animals in all transplantation groups. The NPC transplants comprised less than 40% of the injury site, without significant anatomical or histological differences among the groups. All groups also showed similar patterns of functional deficits and recovery in the 12 weeks after injury and in the 8 weeks after transplantation using the Basso, Beattie, and Bresnahan rating score, the grid test, and the Von Frey test for mechanical allodynia. A notable exception was group 4 (NPC together with chondroitinase and neurotrophins), which showed a significant improvement in bladder function. This study underscores the therapeutic challenges facing transplantation strategies in a chronic SCI in which even the inclusion of treatments designed to reduce scarring and increase neurotrophic support produce only modest functional improvements. Further studies will have to identify the combination of acute and chronic interventions that will augment the survival and efficacy of neural cell transplants.

Functional role of exercise-induced cortical organization of sensorimotor cortex after spinal transection.

Spinal cord transection silences neuronal activity in the deafferented cortex to cutaneous stimulation of the body and untreated animals show no improvement in functional outcome (weight-supported stepping) with time after lesion. However, adult rats spinalized since neonates that receive exercise therapy exhibit greater functional recovery and exhibit more cortical reorganization. This suggests that the change in the somatotopic organization of the cortex may be functionally relevant. To address this issue, we chronically implanted arrays of microwire electrodes into the infragranular layers of the hindlimb somatosensory cortex of adult rats neonatally transected at T8/T9 that received exercise training (spinalized rats) and of normal adult rats. Multiple, single neuron activity was recorded during passive sensory stimulation, when the animals were anesthetized, and during active sensorimotor stimulation during treadmill-induced locomotion when the animal was awake and free to move. Our results demonstrate that cortical neurons recorded from the spinalized rats that received exercise 1) had higher spontaneous firing rates, 2) were more likely to respond to both sensory and sensorimotor stimulations of the forelimbs, and also 3) responded with more spikes per stimulus than those recorded from normal rats, suggesting expansion of the forelimb map into the hindlimb map. During treadmill locomotion the activity of neurons recorded from neonatally spinalized rats was greater during weight-supported steps on the treadmill compared with the neuronal activity during nonweight supported steps. We hypothesize that this increased activity is related to the ability of the animal to take weight supported steps and that, therefore, these changes in cortical organization after spinal cord injury are relevant for functional recovery.

5-HT precursor loading, but not 5-HT receptor agonists, increases motor function after spinal cord contusion in adult rats.

Serotonergic (5-HT) receptors are upregulated following spinal cord transection. Stimulation by administration of serotonergic receptor agonists has been successful in improving hindlimb function. We tested whether this strategy would be successful in incomplete injury models (moderate or severe thoracic contusion) where descending projections are partially spared which should produce less denervation-induced receptor upregulation. Adult rats received midthoracic moderate (MOD: 25 mm drop) or severe (SEV: 50 mm drop) contusion injuries. Distribution of 5-HT and its transporter and expression of 5-HT(2C) receptors were evaluated in lumbar spinal cord and motor response to 5-HT receptor activation was assessed using open field locomotion (BBB) score, percent weight supported treadmill stepping (%WS) and evaluation of hindlimb muscle activation (tremor and serotonin syndrome). 5-HT immunostaining 3 months post-contusion revealed few 5-HT fibers caudal to the severe contusion, and more spared caudal to the moderate contusion. The distribution of 5-HT transporter paralleled 5-HT staining, but was more greatly reduced. Thus serotonin reuptake may be less efficient in the injured spinal cord. Immunostaining for the 5-HT(2C) receptor in the dorsal and ventral horns at L5 showed significant upregulation in SEV, compared to sham or MOD rats. Neither 5-HT(2C) nor 5-HT(1A) receptor agonists, alone or in combination, nor the serotonin transporter inhibitor d-fenfluramine modified BBB scores or %WS in either group. Despite the increased sensitivity of post-synaptic targets, agonist treatment did not improve function in SEV rats. We conclude that selective 5-HT(2C) or 5-HT(1A) receptor activation was not effective in improving hindlimb function after incomplete lesions. In contrast, the 5-HT precursor 5-hydroxytryptophan (L-5-HTP), which leads to activation of all classes of 5-HT receptors, increased both %WS and hindlimb activity in the MOD group. While no side effects were observed in normal or MOD rats, SEV rats displayed hindlimb tremors and 33% mortality, indicating hypersensitivity to the precursor.

Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat.

OBJECTIVE: The myelin protein Nogo inhibits axon regeneration by binding to its receptor (NgR) on axons. Intrathecal delivery of an NgR antagonist (NEP1-40) promotes growth of injured corticospinal axons and recovery of motor function following a dorsal hemisection. The authors used a similar design to examine recovery and repair after a lesion that interrupts the rubrospinal tract (RST). METHODS: Rats received a lateral funiculotomy at C4 and NEP1-40 or vehicle was delivered to the cervical spinal cord for 4 weeks. Outcome measures included motor and sensory tests and immunohistochemistry. RESULTS: Gait analysis showed recovery in the NEP1-40-treated group compared to operated controls, and a test of forelimb usage also showed a beneficial effect. The density of labeled RST axons increased ipsilaterally in the NEP1-40 group in the lateral funiculus rostral to the lesion and contralaterally in both gray and white matter. Thus, rubrospinal axons exhibited diminished dieback and/or growth up to the lesion site. This was accompanied by greater density of 5HT and calcitonin gene-related peptide axons adjacent to and into the lesion/matrix site in the NEP1-40 group. CONCLUSIONS: NgR blockade after RST injury is associated with axonal growth and/or diminished dieback of severed RST axons up to but not into or beyond the lesion/matrix site, and growth of serotonergic and dorsal root axons adjacent to and into the lesion/matrix site. NgR blockade also supported partial recovery of function. The authors' results indicate that severed rubrospinal axons respond to NEP1-40 treatment but less robustly than corticospinal, raphe-spinal, or dorsal root axons.

Combined effects of neurotrophin secreting transplants, exercise, and serotonergic drug challenge improve function in spinal rats.

OBJECTIVES: To determine the effects of neurotrophin-secreting transplants combined with exercise and serotonergic drug challenges on recovery of hindlimb function in rats with midthoracic spinal cord transection injuries. METHODS: Spinalized animals received transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor and neurotrophin-3 and daily cycling exercise. Hindlimb movement in an open-field test (BBB) was scored weekly. Serotonin agonists were used monthly to further stimulate motor function. Axonal growth was quantified in the transplant and at L5 using immunocytochemical markers. Weights of hindlimb muscles were used to assess muscle atrophy. RESULTS: Neurotrophin-secreting transplants stimulated axonal growth, and cycling prevented muscle atrophy, but individual treatments did not improve motor scores. Combined treatments resulted in improvements in motor function. Serotonergic agonists further improved function in all groups, and transplant groups with exercise achieved weight-supporting levels following drug treatment. CONCLUSION: Combined treatments, but not individual treatments, improved hindlimb function.

Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery of function.

Delivery of neurotrophic factors in acute models of spinal cord injury in adult rats can rescue axotomized neurons, promote axonal growth, and partially restore function. The extent to which repair and recovery of function can be achieved after chronic injury has received less attention. In the companion paper we show that transplanting fibroblasts genetically modified to produce neurotrophic factors into chronic (6-week) hemisection injuries results in sprouting, partial neuroprotection, but only limited regeneration. Here we describe functional consequences of this treatment using a series of behavioral tests. Adult rats received a complete unilateral C3/C4 hemisection and recovery from the injury was assessed over 5 weeks. At 6 weeks postoperative, the experimental group received grafts of a combination of fibroblasts modified to secrete BDNF or NT-3. The operated control groups received grafts of either gelfoam or gelfoam with fibroblasts expressing GFP into the lesion site. Behavioral recovery in the three groups was assessed over the next 10 weeks. Severe deficits with no recovery in any of the groups were observed in several tests (BBB, limb preference, narrow beam, horizontal rope test) that measure primarily motor function. Recovery was observed in the grid test, a measure of sensorimotor function, and the von Frey test, a measure of response to mechanical stimulation, but there were no differences between the operated control or experimental groups. Both groups also showed recovery from heat-induced hyperalgesia, with the experimental group exhibiting greater recovery than the operated control groups. In this test, delivery of neurotrophic factors from transplanted fibroblasts does not worsen responses to nociceptive stimuli and in fact appears to reduce hypersensitivity. Our data also demonstrate that additional damage to the spinal cord upon placement of a graft further compromises behavioral recovery for locomotor and postural function. Additional therapeutic interventions will be necessary to provide greater levels of recovery after chronic injuries.

Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration.

Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.

Prenatal cocaine exposure produces consistent developmental alterations in dopamine-rich regions of the cerebral cortex.

Administration of cocaine to pregnant rabbits produces robust and long-lasting anatomical alterations in the dopamine-rich anterior cingulate cortex of offspring. These effects include increased length and decreased bundling of layer III and V pyramidal neuron dendrites, increases in parvalbumin expression in the dendrites of interneurons, and increases in detectable GABAergic neurons. We have now examined multiple cortical regions with varying degrees of catecholaminergic innervation to investigate regional variations in the ability of prenatal cocaine exposure to elicit these permanent changes. All regions containing a high density of tyrosine hydroxylase-immunoreactive fibers, indicative of prominent dopaminergic input, exhibited alterations in GABA and parvalbumin expression by interneurons and microtubule-associated protein-2 labeling of apical dendrites of pyramidal neurons. These regions included the medial prefrontal, entorhinal, and piriform cortices. In contrast, primary somatosensory, auditory and motor cortices exhibited little tyrosine hydroxylase staining and no measurable cocaine-induced changes in cortical structure. From these data we suggest that the presence of dopaminergic afferents contributes to the marked specificity of the altered development of excitatory pyramidal neurons and inhibitory interneurons induced by low dose i.v. administration of cocaine in utero.

Chlordiazepoxide-induced spatial learning deficits: dose-dependent differences following prenatal malnutrition.

The sensitivity of prenatally protein-malnourished rats to the amnestic properties of the benzodiazepine (BZ) receptor agonist, chlordiazepoxide (CDP), was studied in the male offspring of rats provided with a protein-deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy. Rats were tested during acquisition of the submerged platform version of the Morris water maze task using three systemic doses of CDP (3.2, 5.6, and 7.5 mg/kg i.p.) at two ages (day 30 and day 90). At 30 days, prenatally malnourished rats showed less sensitivity to the amnestic effect of the 5.6-mg/kg dose when compared with well-nourished controls by displaying shorter swim paths during acquisition and a more selective search of the target quadrant upon removal of the platform (probe trial). At 90 days, prenatally malnourished rats again showed less sensitivity to CDP at a dose of 5.6 mg/kg, but more sensitivity to the 3.2-mg/kg dose (indicated on the probe trial). No obvious relationship was identified between the nutritional group differences in behavioral sensitivity to CDP at 90 days and their BZ receptor density in the hippocampus or medial septum. It can be concluded that prenatal malnutrition alters the amnestic response to CDP in a dose-dependent and developmentally specific manner, thus providing further support for functional changes within the GABAergic system subsequent to malnutrition.

Differential Effects of Prenatal Protein Malnutrition and Prenatal Cocaine on Radial Arm Maze Performance in Adult Male Rats.

The effects of prenatal cocaine and protein malnutrition were examined on acquisition of the radial arm maze in adult male Sprague-Dawley rats whose mothers were provided with a 6% casein, a 25% casein or a standard chow diet and cocaine (30mg/kg) or saline injections beginning 5 weeks prior to mating and continuing to parturition. Rats were tested using an 8-arm radial maze with 4 baited arms and were required to collect all 4 food pellets within 5 min to complete a trial. Subjects were tested for 1 trial/day until they met criterion for successful acquisition of the task. Criterion was attained when the rat collected 3 out of the 4 food pellets within their first 4 arm entries within a trial (while still completing the trial) with this level of performance being maintained for 3 consecutive trials. The results showed dissociation between the effects of prenatal protein malnutrition and prenatal cocaine. Prenatally protein malnourished adult male rats required a greater number of trials to criterion, made more reference memory (but not working memory) errors, and required a longer time to complete each trial when compared with control males. However, rats with prenatal cocaine exposure showed no significant impairments in the radial arm maze. These results contrast with our previous findings using the Morris maze task in which adult male subjects exhibited impaired acquisition following prenatal cocaine while there were no effects following prenatal protein malnutrition. Thus, the radial arm maze and water maze procedures appear to engage different processes that are differentially sensitive to the prenatal insults.

Effects of diet on sensitization to cocaine-induced stereotypy in female rats.

The progressive increase in cocaine-induced stereotyped behavior that accompanies repeated cocaine injections (sensitization) was examined in rats consuming different diets. Adult female Sprague-Dawley rats were fed one of three diets: low protein (6% casein), adequate protein (25% casein), or a standard chow diet. Following 1 week of adaptation to the diets, the rats were injected every 3-4 days with either cocaine (30 mg/kg, IP) or saline, and the total amount of stereotypy was measured over a 90-min interval following each of four injections. Cocaine-induced stereotypy peaked at 40-50 min following each injection, after which it declined for all diet groups. With repeated injections, the total amount of stereotypy increased in all diet groups. By the fourth injection, the low protein diet group (6% casein) exhibited a slower onset and a possibly prolonged duration of cocaine-induced stereotypy when compared with the two adequate protein diet groups (25% casein and chow). Interestingly, the rats in the two purified diet groups (6% casein and 25% casein) exhibited significantly more stereotypy across injections than those in the chow diet group. Weight differences did not explain the differences in stereotypy present among the diet groups. This study concludes that diet significantly alters the pattern of cocaine-induced stereotypy in female rats, especially after repeated exposure.

Development of spatial navigation following prenatal cocaine and malnutrition in rats: lack of additive effects.

The effects of prenatal cocaine exposure and protein malnutrition on the development of spatial navigation were assessed in rats. Sprague-Dawley dams were fed a low-protein (6% casein), adequate protein (25% casein), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg i.p. two times per week prior to mating and then 30 mg/kg s.c. daily from day 3 to 18 of pregnancy) or saline injections. All litters were fostered on the day of birth to saline-injected mothers fed either the 25% casein diet or the chow diet. Gestation length was decreased by prenatal cocaine exposure whereas litter size was reduced by prenatal malnutrition. On postnatal days 21, 25, 30, or 70, rats were tested for their ability to locate a submerged platform in a Morris water maze. In well-nourished rats, prenatal cocaine increased the mean distance swum during acquisition over days 21-30, a difference that was abolished in rats with prenatal malnutrition. In the absence of drug exposure (saline groups), prenatal malnutrition was itself associated with longer swim paths. Neither prenatal insult affected the accuracy of the spatial navigation at these ages, as determined by their search pattern when the platform was removed. On postnatal day 25, rats raised on the chow diet exhibited superior performance to that of rats raised on the 25% casein diet, but by day 30 these two well-nourished groups were comparable. At day 70, prenatal cocaine impaired spatial performance on the first session, in well-nourished rats only. Thus, these results provide no support for the hypothesis that prenatal cocaine and protein malnutrition combine to produce a greater effect on behavioral development than either insult alone.

Effects of cocaine-induced seizures during pregnancy in the rabbit.

The effects of chronic administration of cocaine to pregnant rabbits on maternal seizures and on pregnancy outcome were studied. Cocaine (2, 3 or 4 mg/kg/injection) or saline was administered, I.V., twice daily, from gestation Day 8 (G8) to G29. There were no significant differences in maternal weight gain or pregnancy outcome between saline control animals and animals given a cocaine dose of 2, 3 or 4 mg/kg/injection. Generalized tonic-clonic seizures (GTCSs) were occasionally elicited by the highest dose (4 mg/kg). There were significant individual differences in vulnerability to cocaine-elicited GTCSs in animals given 4 mg/kg/injection. Of this group, 18% were classified as having high vulnerability to seizures, and they experienced a range from 3 to 27 GTCSs. Postnatal mortality of their offspring was significantly increased. The incidence and temporal patterns of GTCSs elicited by chronic, I.V. cocaine in rabbits, at the doses used, are similar to those reported in human cocaine use. These GTCSs may involve different mechanisms from seizures elicited in other animal studies, in which high doses of cocaine are administered I.P. or S.C. Nevertheless, in our animal model, the GTCSs elicited by prenatal cocaine exposure had no detectable effects on pregnancy outcome (except in the highly vulnerable subgroup).

Differential tolerance to the effects of chlordiazepoxide on unpunished and punished operant responding following chronic treatment.

Differential development of tolerance to the effects of benzodiazepines (BZs) is a common clinical phenomenon. To examine whether the development of tolerance to the response-suppressant and antipunishment effects of BZs were differentially regulated in animals, male Sprague-Dawley rats were treated chronically with either the BZ receptor agonist chlordiazepoxide (CDP, 25 mg/kg, IP, b.i.d.) or saline for 15 weeks and examined under a multiple schedule of operant behavior. Chronic administration of CDP produced tolerance to its suppressive effects on unpunished responding (RI 80 s) but no tolerance to its enhancing effects on punished responding. This conclusion is supported by three observations. First, repeated priming with CDP produced tolerance to its response-suppressive effects in the RI 80-s schedule and revealed increases in punished responding. Second, baseline levels for punished responding remained elevated over the 15-week treatment period. Third, tolerance developed to the response-suppressant effects of CDP under the RI 80-s schedule, as indicated by a sixfold shift to the right in the dose-response curves for rats treated chronically with CDP when compared to saline-treated controls. However, tolerance did not develop to the antipunishment effects of CDP, as indicated by no differences in the dose-response curves for punished responding. Discontinuation of chronic treatment disrupted unpunished responding only on the first day, and reversed the increase in punished responding. Taken together, these results indicate that differential regulation occurs for the development of tolerance to the response-suppressant and antipunishment effects of BZs.

Prenatal cocaine but not prenatal malnutrition affects foster mother-pup interactions in rats.

The separate and combined effects of prenatal cocaine exposure and malnutrition on mother-pup interactions in rats were assessed daily from postnatal day 2 to day 21. Sprague-Dawley dams were fed a diet of low protein content (6% casein), an isocaloric diet of adequate protein content (25% casein, control), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg IP two times per week prior to mating and then 30 mg/kg SC daily from days 3 to 18 of pregnancy) or saline injections. Litters were fostered on the day of birth to control mothers (i.e., nondrug-exposed dams fed the control or chow diet). Foster mothers fed the 25% casein diet showed increased contact with cocaine-exposed pups compared with nondrug-exposed pups in the second postnatal week but lower levels as the pups approached weaning. Passive nursing was increased in dams caring for prenatally malnourished, cocaine-exposed pups compared with those caring for similar pups with no drug exposure. Chow-fed mothers did not differ in their behavior towards pups with or without prenatal cocaine treatment. Prenatal cocaine and malnutrition independently compromised birth weight and various reflexive milestones but the attainment of physical milestones was affected only by prenatal cocaine. There were no additive effects of the two prenatal insults on any measure of mother-pup interaction or pup development.

The effects of cocaine exposure prior to and during pregnancy in rats fed low or adequate protein diets.

Sprague-Dawley rats were fed a diet of low protein content (6% casein), an isocaloric diet of adequate protein content (25% casein), or a laboratory chow diet for 5 weeks prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine (30 mg/kg IP two times per week prior to mating and 30 mg/kg or 40 mg/kg SC daily from days 3 to 18 of pregnancy) or saline injections. Cocaine produced a greater reduction in food intake during pregnancy in the malnourished group compared with the other two diet groups. The effect of cocaine on food intake was minimal in chow-fed rats. Weight gain in pregnancy was reduced by cocaine in a dose-dependent manner, and by malnutrition. Both prenatal cocaine and malnutrition impaired skeletal maturation of the pups, but there was no additive effect of the two insults on this measure. Litter size was significantly reduced by the 40 mg/kg, but not by the 30 mg/kg dose of cocaine across all diet groups. Consequently, the 40 mg/kg dose of cocaine proved to be fetotoxic in this model. Birth weight was significantly reduced by prenatal malnutrition but not by prenatal cocaine. Gestation length was unaffected by either insult. Hence, the ability to detect a diet x drug interaction was dependent upon the variable being measured.

Differential regulation of the behavioral effects of chlordiazepoxide.

Chronic administration of the benzodiazepine (BZ) receptor agonist chlordiazepoxide (CDP) produced tolerance to its motor-impairing effects but little or no tolerance to its hypothermic effects or to its amnesic effects in the radial arm maze. Male Sprague-Dawley rats were pretreated for 14 days with CDP (25 mg/kg, b.i.d., IP) or saline, and chronic treatment was maintained throughout the experiments. Tolerance was evaluated by constructing dose-response curves to CDP following chronic administration of either CDP or saline. Tolerance developed to only certain behavioral effects of CDP. Tolerance developed to the motor-impairing effects of CDP as assessed in three different procedures: rotarod, spontaneous locomotor activity, and acquisition of the step-through inhibitory avoidance response. In contrast, tolerance did not develop to the hypothermic effects of CDP. Tolerance to the amnesic effects of CDP was contingent upon the behavioral procedure. For example, tolerance developed to reductions of retention latency in the step-through inhibitory avoidance response, but not to impairment of the acquisition of radial arm maze performance. These results are consistent with the effects of chronic BZ administration in humans and demonstrate a parallel regulation of drug effects, potentially mediated by regional differences in BZ receptor subtype regulation or composition.

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L.

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [(3)H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K(0.5) = 2.8 x 10(-7) molar. The K(0.5) for NPA under these conditions is 2 x 10(-8) molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [(3)H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [(3)H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.