Willem Hendrik Gispen

Cognition and synaptic plasticity in diabetes mellitus

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits are paralleled by neurophysiological and structural changes in the brain. In animal models of diabetes, impairments of spatial learning occur in association with distinct changes in hippocampal synaptic plasticity. At the molecular level these impairments might involve changes in glutamate-receptor subtypes, in second-messenger systems and in protein kinases. The multifactorial pathogenesis of diabetic encephalopathy is not yet completely understood, but clearly shares features with brain ageing and the pathogenesis of diabetic neuropathy. It involves both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. Treatment with insulin might therefore not only correct hyperglycaemia, but could also directly affect the brain.

The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structure-activity studies.

Abstract Intraventricular administration of synthetic ACTH-like peptides in the rat induces excessive grooming, stretching and yawning. The present study demonstrates that induction of excessive grooming is dose-dependent and independent of the endocrine system. Structure-activity studies show that ACTH1–24, ACTH1–16-NH2, ACTH1–16, α-MSH and βp-MSH are equipotent. Although the presence of the sequence ACTH5–10 in the peptides studies seems of importance in the induction of excessive grooming, it appeared that C-terminal elongation is necessary for the expression of the activity. Administration of [D-Phe7] ACTH4–10 and [D-Phe7] ACTH1–10 results in appreciable grooming activity of the rat. However, substitution of a D-arginine at the 8 position did not alter the activity of ACTH4–10. The structure-activity relationship of these peptides on grooming activity of the rat is compared to that known for retardation of avoidance extinction. Although some similarities exist, it is concluded that the expression of excessive grooming and retardation of avoidance activity is mediated through different mechanisms.

Automated Quantitative Gait Analysis During Overground Locomotion in the Rat: Its Application to Spinal Cord Contusion and Transection Injuries

Analysis of locomotion is an important tool in the study of peripheral and central nervous system damage. Most locomotor scoring systems in rodents are based either upon open field locomotion assessment, for example, the BBB score or upon foot print analysis. The former yields a semiquantitative description of locomotion as a whole, whereas the latter generates quantitative data on several selected gait parameters. In this paper, we describe the use of a newly developed gait analysis method that allows easy quantitation of a large number of locomotion parameters during walkway crossing. We were able to extract data on interlimb coordination, swing duration, paw print areas (total over stance, and at 20-msec time resolution), stride length, and base of support: Similar data can not be gathered by any single previously described method. We compare changes in gait parameters induced by two different models of spinal cord injury in rats, transection of the dorsal half of the spinal cord and spinal cord contusion injury induced by the NYU or MASCIS device. Although we applied this method to rats with spinal cord injury, the usefulness of this method is not limited to rats or to the investigation of spinal cord injuries alone.

Water maze learning and hippocampal synaptic plasticity in streptozotocin diabetic rats: effects of insulin treatment

Streptozotocin-diabetic rats express deficits in water maze learning and hippocampal synaptic plasticity. The present study examined whether these deficits could be prevented and/or reversed with insulin treatment. In addition, the water maze learning deficit in diabetic rats was further characterized. Insulin treatment was commenced at the onset of diabetes in a prevention experiment, and 10 weeks after diabetes induction in a reversal experiment. After 10 weeks of treatment, insulin-treated diabetic rats, untreated diabetic rats and non-diabetic controls were tested in a spatial version of the Morris water maze. Next, hippocampal long-term potentiation (LTP) was measured in vitro. To further characterize the effects of diabetes on water maze learning, a separate group of rats was pre-trained in a non-spatial version of the maze, prior to exposure to the spatial version. Both water maze learning and hippocampal LTP were impaired in diabetic rats. Insulin treatment commenced at the onset of diabetes prevented these impairments. In the reversal experiment, insulin treatment failed to reverse established deficits in maze learning and restored LTP only partially. Non-spatial pre-training abolished the performance deficit of diabetic rats in the spatial version of the maze. It is concluded that insulin treatment may prevent but not reverse deficits in water maze learning and LTP in streptozotocin-diabetic rats. The pre-training experiment suggests that the performance deficit of diabetic rats in the spatial version of the water maze is related to difficulties in learning the procedures of the maze rather than to impairments of spatial learning.

ACTH-induced excessive grooming in the rat

Excessive grooming is a remarkable behavioral response for several reasons. Probably the most important is that it represents an almost unique behavioral assay for the injection of the longer ACTH fragments. Fractions of a microgram of ACTH1–24 or ACTH1–16 produce extensions of grooming bout duration to an extent that the animal does little else during the entire observation period. The specificity of the response is remarkable, as well. Few other neuropeptides induce excessive grooming, and those that do (e.g. [7-d-phe]ACTH4–10) are only of limited effectiveness. Also of special interest is the limited number of brain regions in which lesions influence the response. Only two such regions have been found: the substantia nigra and the hippocampus. A link to naturally occurring events is now provided by the observations which indicate that certain forms of environmental manipulations also lead to excessive grooming. These are conditions of mild stress, the most completely studied being that of ‘novelty’. While providing a connection to real world events, the study of stress-induced excessive grooming has also led to certain difficult questions. In the study of stress conditions that produce excessive grooming, only certain ones have been found to be effective. These include handling, noise, transport, novelty, the anticipation of footshock and water immersion. Of these, water immersion has the most pronounced effects. From our pharmacologic studies, water immersion produces a type of grooming that is distinctly different from either that produced by novelty or other mild stress conditions and also different from that produced by central ACTH administration. For example, the excessive grooming induced by water immersion is not reduced by either substantia nigra or hippocampal lesions, neither is it reduced by the systematic administration of clonidine. On the other hand, these treatments and procedures reduce both novelty and ACTH-induced excessive grooming. The stress procedures which do not induce excessive grooming also bear consideration. They include the exposure to ether fumes, bodily restraint, and electrical footshock. In fact, the latter reduces excessive grooming. Putting aside, for a moment, the effects of water immersion, stressors seem to fall into two classes: those that produce excessive grooming after their termination and those that do not. It seems to us that this could come about in two ways: (1) the stressors that do elicit grooming may produce a different type of distribution of ACTH release in the brain than those that do not; or (2) all stressors may produce ACTH release in a similar fashion, but those that fail to elicit grooming may also elicit antagonistic activity, possibly involving the brain monoamine systems. While there is little evidence to support a differential pattern of release as a consequence of different stress conditions, the possibility cannot be ruled out. The second possibility, the activation of incompatible neural systems by certain stressors, seems more likely. If this is the case, then it could be proposed that the amount of excessive grooming displayed by an animal is positively related to the amount of ACTH affecting certain brain regions involved with the production of excessive grooming, and negatively related to the activity in opposing systems. ACTH could reach the target areas of the central nervous system from the anterior pituitary (de Kloet and de Wied, 1980; Mezey, et al., 1978) or from central ACTH networks. Beagley (1976) felt that this electrical stimulation at higher intensities may have activated an excitatory system for grooming that did not achieve behavioral expression because of the simultaneous induction of an opposing, inhibitory process. He proposed that the grooming system, however, had a longer afterdischarge than the opposing processes. Thus, when inhibition or the competing response tendencies declined in activity, grooming would be observed. Bolles (1960) came to a similar conclusion. He had found that the induction of motivation for food or water by deprivation decreased grooming as did the induction of fear. He concluded that these ‘motivational’ procedures did not induce grooming directly but that grooming-occurred after the conclusion of goal-directed acts or highly motivated episodes, presumably when competing neural mechanisms declined in activity. The relatively high levels of excessive grooming seen after water immersion seem to be quite different in some aspects from either novelty- or ACTH-induced grooming. Their resistance to lesions effects and pharmacological intervention suggest a different component or mechanism has been involved. It may be that this is related to increased ‘maintenance’ grooming (an attempt to remove water from the fur), but if this were the case, it would be necessary to account for the fact that from 1 to 3 min of water immersion is necessary to induce the effect. Exposure to water for less than one min fails to elicit the effect. It is also difficult to explain why the soiling and disturbances of the fur in the restraint procedures fails to elicit a similar amount of grooming on the basis of a maintenance hypothesis. The involvement of opiate-sensitive systems in the grooming response is based on the ubiquitous blocking effect of naloxone on all excessive grooming, including that induced by water immersion. It is certainly not a response common to all of the known endogenous opiates since the enkephalins and some endorphin fragments are ineffective in producing excessive grooming. In fact, the excessive grooming induced by β-endorphin is qualitiatively different from that produced by ACTH. β-endorphin seems to activate the animals into more behavioral activities including grooming, while ACTH acts to extend the duration of grooming episodes. The neurochemical mechanism by which ACTH induces the grooming behavior is not understood. However, the recent isolation of an ACTH-sensitive protein kinase from rat brain synaptosomal plasma membranes suggests that ACTH may modulate trans-synaptic neuronal activities by altering the degree of phosphorylation of certain synaptic proteins. The insensitivity of this protein kinase to endorphins and the unique ACTH structure-activity correlation between the induction of excessive grooming and inhibition of protein phosphorylation support this notion. Since excessive grooming is found after the termination of several stressors, it is possible that it is related to restorative neural and chemical processes. ACTH could be thought of and acting as an initiating factor in the mobilization of the organism to meet the emergency situation while at the same time initiating processes aimed at re-establishing a stable, internal equilibrium. The grooming response, itself, has been considered to play a deactivating role in animal behavior (for example see Delius, 1970; Delius, et al., 1976; Jolles et al., 1979a). It is also possible to cast the behavioral observations into a related but different context which removes some of the objections that can be made to the view that grooming should only occur before relatively inactive states. This would be that excessive grooming is a correlate of a central state in which behavioral activation may be present but without concomittant demands for fight or flight. This central state may be correlated with a number of classes of responses and the ones selected for exhibition would depend on environmental opportunities, the animals past history and genetic predispositions. Grooming may occur after extremely arousing conditions. This suggests that these acts may be correlated with pleasurable experiences. The slow body stretching and paw extensions found in cats by MacLean (1957) would be one example as would the signs of sexual excitation observed. Bertolini, et al. (1975) report similar signs of sexual arousal after injection of ACTH1–24 into the lateral ventricle of the rabbit. However, the presumed sexual arousal reflects an activation of the mechanisms related to sexual behavior without an enhanced motivation to copulate. During the peptide-induced stimulation the animals do not seek copulation. For example, the injection of the peptide into the ventricles of male rats does not increase the percentage of males copulating with females. It is as if the animal is ‘tuned in’ to its own pleasurable bodily reactions that may include those of a sexual nature. Bertolini et al. also provided evidence that the sexual activation in male rats or rabbits instituted by ACTH1–24 depends on the integrity of the testes. However, the excessive grooming observed in rats after the intraventricular injection of ACTH1–24 or β-endorphin, in the amounts used in our laboratories, as well as the grooming induced by novelty does not concentrate on the genital area. Few signs of sexual arousal can be observed. However, it is possible that a ‘pleasurable’, positive affective state is induced by these treatments and that at greater magnitudes the pleasurable state could facilitate signs of sexual arousal. This would suggest that in the rat the central state induced by i.c.v. administration of ACTH1–24 is closely limited to the grooming response but is may be tied to other, many more varied responses in other species.

Injury-induced class 3 semaphorin expression in the rat spinal cord

In this study we evaluate the expression of all members of the class 3 semaphorins and their receptor components following complete transection and contusion lesions of the adult rat spinal cord. Following both types of lesions the expression of all class 3 semaphorins is induced in fibroblast in the neural scar. The distribution of semaphorin-positive fibroblasts differs markedly in scars formed after transection or contusion lesion. In contusion lesions semaphorin expression is restricted to fibroblasts of the meningeal sheet surrounding the lesion, while after transection semaphorin-positive fibroblast penetrate deep into the center of the lesion. Two major descending spinal cord motor pathways, the cortico- and rubrospinal tract, continue to express receptor components for class 3 semaphorins following injury, rendering them potentially sensitive to scar-derived semaphorins. In line with this we observed that most descending spinal cord fibers were not able to penetrate the semaphorin positive portion of the neural scar formed at the lesion site. These results suggest that the full range of secreted semaphorins contributes to the inhibitory nature of the neural scar and thereby may inhibit successful regeneration in the injured spinal cord. Future studies will focus on the neutralization of class 3 semaphorins, in order to reveal whether this creates a more permissive environment for regeneration of injured spinal cord axons.

Phosphorylation of B 50 protein by calcium activated, phospholipid dependent protein kinase and B 50 protein kinase

Abstract: B‐50 is a brain‐specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B‐50 protein. Only calcium‐activated, phospholipid‐dependent protein kinase (kinase C) and B‐50 protein kinase were able to use B‐50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B‐50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B‐50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B‐50 protein kinase is a calcium‐dependent, phospholipidstimulated protein kinase of the same type as kinase C.

Long-lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment.

Behavioural experience (e.g. chronic stress, environmental enrichment) can have long‐lasting effects on cognitive functions. Because activity‐dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as hippocampus, we tested whether behaviour has also long‐lasting effects on synaptic plasticity by examining the induction of long‐term potentiation (LTP) and long‐term depression (LTD) in slices of hippocampal CA1 obtained from rats either 7–9 months after social defeat (behavioural stress) or 3–5 weeks after 5‐week exposure to environmental enrichment. Compared with age‐matched controls, defeated rats showed markedly reduced LTP. LTP was even completely impaired but LTD was enhanced in defeated and, subsequently, individually housed (during the 7–9‐month period after defeat) rats. However, increasing stimulus intensity during 100‐Hz stimulation resulted in significant LTP. This suggests that the threshold for LTP induction is still raised and that for LTD lowered several months after a short stressful experience. Both LTD and LTP were enhanced in environmentally enriched rats, 3–5 weeks after enrichment, as compared with age‐matched controls. Because enrichment reduced paired‐pulse facilitation, an increase in presynaptic release, facilitating both LTD and LTP induction, might contribute to enhanced synaptic changes. Consistently, enrichment reduced the number of 100‐Hz stimuli required for inducing LTP. But enrichment may also actually enhance the range of synaptic modification. Repeated LTP and LTD induction produced larger synaptic changes in enriched than in control rats. These data reveal that exposure to very different behavioural experiences can produce long‐lasting effects on the susceptibility to synaptic plasticity, involving pre‐ and postsynaptic processes.

ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated32P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10−10 to 10−4 M. In a number of protein bands, a biphasic effect of ACTH1–24 was observed: in concentrations of 10−4–10−5 M, a reduced amount of32P was found; in concentrations of 10−6–10−7 M, hardly any effect could be detected, whereas consistently at concentrations around 10−8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.

Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function: Beneficial effects of melanocortins

A procedure for placing a crush lesion in the sciatic and tibial nerve of the rat based on anatomical landmarks is described. These crush lesions are used to study the process of regeneration of peripheral nervous tissue and the beneficial effects of melanocortins on speed and quality of nerve regeneration. A new precise and rapid method for testing the return of sensory function by a locally applied electric stimulus is discussed.