William J. Shoemaker

Perinatal development of the endorphin- and enkephalin- containing systems in the rat brain

Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other.

Senescent changes in a neurobiological model system: Cerebellar Purkinje cell electrophysiology and correlative anatomy

A within-subjects design was used to assess age changes in cerebellar Purkinje neurons. Four groups of naive male Sprague-Dawley rats, aged 3, 10, 20, and 28 months. underwent single cell recording for electrophysiological assessment of Purkinje cell firing patterns, followed by perfusion for glyoxylic acid induced catecholamine fluorescence. Cerebellar sections were photographed first by fluorescence microscope for catecholamines, and 2-3 weeks later for quantification of lipofuscin autofluorescence. Finally, these same tissues were treated with cresyl violet and photographed a third time to permit quantitative estimates of age changes in the number of Nissl staining Purkinje neurons. Electrophysiological studies revealed significant effect of age on a number of Purkinje cell firing parameters: in particular, increasing numbers of aberrant, very slow-firing cells were encountered in older animals. These cells showed normal climbing fiber mediated burst activity, but spontaneous simple spike firing rates 3-5 times less than normal. Rats exhibiting the highest numbers of such abnormal cells also exhibited the poorest Nissl staining. Conversely, good Nissl staining of Purkinje neurons in an old rat was a reliable predictor of relatively normal Purkinje cell firing. Lipofuscin was found to accumulate measurably in Purkinje neurons by 20 months of age, and to increase significantly thereafter. Deposition of the substance occurred almost exclusively at the apical pole of the soma. Our data suggest, however, that accumulation of lipofuscin in Purkinje neurons, as well as its reported accumulation in the inferior olive, is not a primary cause of electrophysiological dysfunction. There was no apparent age change in glyoxylic acid induced catecholamine fluorescence nor, in separate pharmacological studies, could any senescent alteration in cerebellar catecholamine levels be found.

Catecholamines in mutant mouse cerebellum. Fluorescence microscopic and chemical studies.

Catecholamine-containing fibers have been examined in the cerebella of normal and hypocerebellar mutant mice using Falck-Hillarp and glyoxylic acid histofluorescence techniques. The amounts of norepinephrine and dopamine were also determined chemically in the same mutants. Green fluorescent fibers in cerebella of normal mice are similar in size and distribution to those described in the rat. Weanling and adult weaver, reeler and staggerer mice all manifest greatly increased specific catecholamine fluorescence per unit area in cerebellar cortex, but the patterns of fluorescent fibers are distinctive. In weaver, the fibers are of normal diameter, surround Purkinje cell bodies and appear to climb along major dendrites. In reeler, similar fibers form a plexus around cortical and non-cortical Purkinje cells; relatively normal fluorescent fiber patterns are present in well-organized cortical regions, while stouter disoriented fibers course through the shallow molecular layer in disorganized regions. Staggerer cerebellar cortex exhibits the greatest fluorescence with most terminals appearing as matted tangles adjacent cell bodies. Clearly defined fibers, however, can be distinguished in the molecular layer running perpendicular to the pia or for long distances in the coronal plane parallel to the pia. The major catecholamine determined chemically is norepinephrine. Reeler cerebella contain normal absolute levels and a doubled concentration of norepinephrine. In contrast, and despite the fluorescence findings, the total norepinephrine content of weaver and staggerer cerebella is significantly reduced and concentrations are not significantly different from normal.

Effects of chronic lithium treatment on dopamine receptors in the rat corpus striatum. II. No effect on denervation or neuroleptic-induced supersensitivity.

The influence of chronic dietary lithium administration was evaluated on dopamine receptor supersensitivity in the rat corpus striatum. Supersensitivity was induced with either unilateral destruction of dopamine-containing fibers in the nigrostriatal pathway or with 3 weeks of treatment with haloperidol (HAL). Both treatments elevated [3H]spiroperidol binding sites, but in neither case was this increase in ligand binding affected by chronic dietary Li (brain levels 0.8 to 1.2 mEq/1 tissue). Our rats receiving 21 daily injections of HAL did show a behavioral supersensitivity to the dopamine agonist, apomorphine, and this effect was attenuated by concurrent treatment with dietary Li (accompanying paper). However, in contrast to previous data, this behavioral attenuation could not be linked to the prevention of increased [3H]spiroperidol binding in the corpus striatum. Furthermore, co-administration of dietary Li to subjects injected with HAL for 3 weeks did not reverse the increased density of [3H]spiroperidol binding sites which developed in the corpus striatum. Neither HAL nor Li treatment altered the affinity of the radioligand for its binding site. In the same animals, neostriatal dopamine-sensitive adenylate cyclase was not affected by either long-term dietary Li or chronic neuroleptic treatment, supporting the view that membrane antagonist and agonist sites differentially adapt to chronic alterations of synaptic input. Taken together, the results are incompatible with the hypothesis that the anti-manic action of Li is related to its ability to prevent dopamine receptor supersensitivity.

Effects of chronic lithium treatment on dopamine receptors in the rat corpus striatum. I. Locomotor activity and behavioral supersensitivity

Spontaneous locomotor activity and dopaminergic responsivity were assessed after long-term dietary treatment with the anti-manic drug lithium. Chronic dietary Li administration was not accompanied by the toxicities often reported with other modes of administration. In addition, the diet reliably yields serum and brain Li levels in the prophylactic range for manic-depressive illness. After 4 weeks exposure to Li, spontaneous locomotor activity was reduced as compared to subjects on the control diet whether or not food intake was restricted. The depression of locomotor activity following an injection of the dopamine agonist, apomorphine, was less severe in animals that ingested Li compared to those with free access to the control diet. Finally, in confirmation of the findings of Pert et al., chronic Li administration led to a partial attenuation of apomorphine-evoked stereotyped behaviors in subjects rendered supersensitive to the drug by daily injections of haloperidol (HAL) for 3 weeks. The findings suggest that the commonly reported suppressant action of Li on spontaneous behavior is not attributable to overt toxicity or to diminished growth rate. Similarly, these health factors do not account for the ability of chronic Li treatment to suppress the behavioral manifestation of dopaminergic supersensitivity associated with long-term HAL exposure.

Effects of chronic lithium on enkephalin systems and pain responsiveness

We have recently undertaken an extensive examination of the effects of chronic dietary lithium treatment on levels of brain leucine-enkephalin immunoreactivity (1-enk-IR), release of endogenous 1-enk-IR from globus pallidus prisms in vitro and behavioral responsiveness to pain. Two LiCl containing diets were used. Rats on the lower strength diet attained brain Li levels of 0.40-0.55 mEq/L, while those on the higher strength diet attained levels of 0.70-1.0 mEq/L. In one series of experiments, we sought to relate alterations of K+-stimulated, Ca++-dependent release of 1-enk-IR to alterations of the content of the peptide. After 1 week on the lower strength diet, neither measure was affected in any of the brain regions examined. Following 2 or 3 weeks of feeding with the lower strength diet, 1-enk-IR levels in the globus pallidus and nucleus accumbens were elevated. However, 3 weeks of the high strength Li diet did not lead to alterations of 1-enk-IR content. In contrast, the release of 1-enk-IR was potentiated but only in subjects in which the brain lithium exceeded a threshold level. In another series of studies, we observed that hot-plate escape latency was significantly elevated in rats fed the high strength Li diet for 3 weeks. Also, the Li-treated animals had a greater morphine-induced elevation of escape latency than controls; this effect was less effectively blocked by naltrexone. These findings suggest that chronic exposure to Li leads to transiently elevated levels of 1-enk, and, when brain Li levels are greater than 0.5 mEq/L, to a potentiation of endogenous enkephalin release. The analgesia in Li-treated subjects may eventually be related to these influences of the anti-manic drug on enkephalinergic neurotransmission.

Chemical and physiological aspects of the actions of lithium and antidepressant drugs

The possible mechanisms underlying the anti-manic actions of lithium have been examined in a variety of interdisciplinary experiments. The possibility that lithium can regulate the sensitivity changes in dopaminergic transmission produced by chronic treatment with haloperidol has been tested. Although a modest modification of behavioral responses to the dopamine agonist apomorphine was found, there was no evidence that this action of lithium reflected alterations of the binding parameters of dopamine-related ligands. In other studies, consistent, dose-dependent increases in brain enkephalin content were found after rats consumed a specially manufactured lithium diet for 2-3 weeks. Not only were brain enkephalin levels increased after this treatment, but some signs of basal analgesic responsiveness also suggested that the elevated levels of enkephalins were functionally significant. To test the possibility that the effects of lithium may not be seen in normal rats, the effects of lithium were compared on spontaneously hypertensive and unaffected, normotensive rats of a related strain. Treatment with lithium altered blood pressure in the hypertensive strain but did not affect blood pressure in the controls. These studies suggest that multiple brain systems may be regulated by treatment with lithium but that the critical pathophysiological process may not be demonstrable in the normal rat.

In vivo release of enkephalin from the globus pallidus

Push-pull cannulae were acutely positioned through previously implanted guides in the globus pallidus of unanesthetized freely moving cats and rats. During slow-flow perfusions, enkephalin release was detected in resting conditions and increased more than 3-fold when both 50 mM K+ and 1.8 mM Ca2+ were present in the perfusing medium. Local perfusion with veratrine also enhanced enkephalin release. Furthermore, in vivo, electrical stimulation of the rat caudo-putamen enhanced enkephalin release in the pallidum. This latter finding is consistent with a functional strio-pallidal enkephalin-containing pathway previously postulated by immunohistochemical or lesion experiments.

Morphological and functional correlates of VIP neurons in cerebral cortex

Vasoactive Intestinal Polypeptide (VIP) promotes the hydrolysis of 3H-glycogen newly synthesized from 3H-glucose by mouse cortical slices. This effect occurs rapidly, approximately 50% of the maximal effect being reached within one minute. The maximal effect is achieved after 5 minutes and maintained for at least 25 minutes. Furthermore the glycogenolytic effect of VIP is reversible, and pharmacologically specific. Thus several neuropeptides present in cerebral cortex such as cholecystokinin-8, somatostatin-28, somatostatin-14, met-enkephalin, leu-enkephalin, do not affect 3H-glycogen levels. VIP fragments 6-28, 16-28 and 21-28 are similarly inactive. Furthermore, among the peptides which share structural homologies with VIP, such as glucagon, secretin, PHI-27 and Gastric Inhibitory Peptide, only secretin and PHI-27 promote 3H-glycogen hydrolysis, with EC50 of 500 and 300 nM respectively, compared to an EC50 of 25 nM for VIP. Immunohistochemical observations indicate that each VIP-containing bipolar cell is identified with a unique radical cortical volume, which is generally between 15-60 micrograms in diameter and overlaps with the contiguous domains of neighbouring VIP-containing bipolar cells. Thus this set of biochemical and morphological observations support the notion that VIP neurons have the capacity to regulate the availability of energy substrates in cerebral cortex locally, within circumscribed, contiguous, radial domains.

Senescent change in tissue weight and immunoreactive β-endorphin, enkephalin, and vasopressin in eight regions of C57BL/6J mouse brain and pituitary

Tissue weights and immunoreactive (IR) content and concentration of beta-endorphin, enkephalin, and vasopressin were assayed for senescent change in anterior pituitary, neurointermediate pituitary, hypothalamus, hippocampus, striatum, dorsolateral cortex, and pons-medulla, as well as residual brain samples remaining after the other dissections. Groups of naive male C57BL/6J mice, 8-12 months old and 28-32 months old, served as subjects. Old mice exhibited significant decline in anterior pituitary and hippocampus weight. Significant increases with age were found in pons-medulla weight. IR beta-endorphin content decreased in hypothalamus and neurointermediate pituitary. IR enkephalin decreased in striatum and dorsolateral cortex. IR vasopressin content increased in hypothalamus and residual brain. Assays were replicated in later experiments using additional subjects, for total Ns of up to 54 mice. Although significant overall results were always consistent in direction from replication to replication, the magnitude of age change exhibited considerable variability. Such results suggest that single experiments on age changes in neuropeptides, particularly those giving negative results, should be carefully scrutinized and replicated before their acceptance as evidence for a transmitters stability or instability throughout the lifespan.