Behavior and binding: Correlations between α1-adrenergic stimulation of acoustic startle and α1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in α₁-adrenoceptors and behavioral effects of α₁-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the α₁-adrenergic agonists,d-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of α₁-adrenoceptor occupation measured by [³H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using eitherd-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of α₁-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (K_D) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

1H-MRS in spinal cord injury: acute and chronic metabolite alterations in rat brain and lumbar spinal cord

A variety of tests of sensorimotor function are used to characterize outcome after experimental spinal cord injury (SCI). These tests typically do not provide information about chemical and metabolic processes in the injured CNS. Here, we used (1) H-magnetic resonance spectroscopy (MRS) to monitor long-term and short-term chemical changes in the CNS in vivo following SCI. The investigated areas were cortex, thalamus/striatum and the spinal cord distal to injury. In cortex, glutamate (Glu) decreased 1 day after SCI and slowly returned towards normal levels. The combined glutamine (Gln) and Glu signal was similarly decreased in cortex, but increased in the distal spinal cord, suggesting opposite changes of the Glu/Gln metabolites in cortex and distal spinal cord. In lumbar spinal cord, a marked increase of myo-inositol was found 3 days, 14 days and 4 months after SCI. Changes in metabolite concentrations in the spinal cord were also found for choline and N-acetylaspartate. No significant changes in metabolite concentrations were found in thalamus/striatum. Multivariate data analysis allowed separation between rats with SCI and controls for spectra acquired in cortex and spinal cord, but not in thalamus/striatum. Our findings suggest MRS could become a helpful tool to monitor spatial and temporal alterations of metabolic conditions in vivo in the brain and spinal cord after SCI. We provide evidence for dynamic temporal changes at both ends of the neuraxis, cortex cerebri and distal spinal cord, while deep brain areas appear less affected.     

Exaggerated auditory startle responses in patients with spinal cord injury

OBJECTIVE: Central nervous system reorganization following spinal cord injury (SCI) may cause functional changes in the motor tracts in patients in whom increased auditory startle responses (ASRs) have been previously reported. We hypothesized that if increased ASRs in patients with incomplete SCI were due to compensatory mechanisms, these changes would be related to severity and/or localization of the lesion. METHODS: We examined ASR characteristics in 29 SCI patients and 14 age-matched healthy volunteers. Fourteen patients had incomplete and 15 complete SCI; 10 patients had cervical and 19 thoracolumbar SCI. Five auditory stimuli were applied binaurally to subjects in a sitting position, with a 5-min interstimulus interval. Surface electromyographic recordings were obtained from orbicularis oculi (OOc), sternocleidomastoid (SCM), biceps brachii (BB), and tibialis anterior (TA) muscles. RESULTS: ASR probability was significantly higher and area-under-the-curve was significantly larger in SCM and BB in patients than in controls. ASR latency was significantly shorter in SCM and BB in patients with cervical than in those with thoracolumbar SCI (p < 0.02), but there were no statistically significant differences between complete and incomplete SCI (p > 0.1). Time span since onset correlated significantly with ASR area in OOc, SCM and BB (p < 0.05). CONCLUSION: The capability of the adult central nervous system to reorganize its circuits over time for improved functionality following injury is probably the key to understanding the increased ASRs in patients with SCI. The exaggeration of the startle reflex is potentially important since it may be useful for augmenting voluntary movement in the clinical rehabilitation of patients with SCI.     

Muscarinic binding sites in small homogenates and in autoradiographic sections from rat and human spinal cord

Binding of labelled l-quinuclidinylbenzylate was studied in cryosections and homogenates of human and rat spinal cord. For the cryosections an autoradiographic method was used. With both techniques a higher density of muscarinic binding sites was found in rat than in human spinal cord. In the autoradiograhs very high densities of muscarinic binding sites were observed in the motor neurone area and in the apical part of the dorsal horn. The latter high density region was not always found in homogenates from dissected tissue samples. The autoradiographic technique has a better resolution for detecting discrete regional variations in the receptor content of the spinal cord.     

Ultrastructure of fetal spinal cord and cortex implants into adult rat spinal cord

The ultrastructure of cortex and spinal cord from 11-, 12-, and 15-day-old fetuses implanted into the spinal cord of adult rats was studied over 3 months. Under deep Chloropent anesthesia, a 0.5 × 1.0-mm square of fetal cortex or a 1.0-mm segment of fetal spinal cord was implanted subpially between the left dorsal column and the dorsal horn of 70 adult rats. Implants grew toward gray matter, usually interfacing with the host at the isthmus between the horns of the spinal cord. However, implants were observed that occupied the entire left dorsal and ventral horns of the left half of the host spinal cord. Implants had concentric zones: A central zone with basal lamina lined joined channels and subjacent neuroglia; a zone of differentiating implant nervous system; a zone with basal lamina lined implant with overlying pial cells on the dorsal and lateral surfaces of the implant; a zone that interfaced with the host with overlapping neuropil on the lateral and ventral surfaces of the implant. Neuron types were typical for cortical or spinal implants. Implants survived for 3 months and reached stages of neuronal and neuroglial maturation similar to controls. Both fetal spinal cord and brain were successful as implants, had delayed differentiation, and formed complex neuropils. The zone of overlapping interface of the donor and host is an anatomical indication of physiological and functional integration.     

Sexual dimorphism and the influence of neonatal androgen in the dorsolateral motor nucleus of the rat lumbar spinal cord

There is a sexually dimorphic motor nucleus, the spinal nucleus of the bulbocavernosus (SNB) in the fifth and sixth lumbar segments of the rat spinal cord. We now report a second sex difference in the dorsolateral nucleus (DLN) in the ventral horn of the rat lumbar cord, which includes motoneurons innervating the ischiocavernosus muscle, a sexually dimorphic perineal muscle. Adult females possess fewer motoneurons in the DLN, probably because of an absence of neurons innervating the ischiocavernosus muscle, which females lack. The effect of a single dose of testosterone propionate on day 2 of life was confined to a specific rostrocaudal region of the adult DLN in which it partially masculinized the female DLN. Masculinized females have more DLN neurons than control females. The direction of change induced in DLN neuron number by the neonatal hormone treatment is compatible with the hypothesis that androgens are involved with the sexually dimorphic development of the DLN. In another motor nucleus, the retrodorsolateral nucleus, a small sex difference in neuron number was found in one study, but was not replicated in a second experiment.     

Synaptogenesis in the intermediate gray region of the lumbar spinal cord in the postnatal rat

Mid-thoracic spinal cord transection produces dramatically different behavioral results depending upon a rat's age at the time of surgery. The present study was initiated to determine whether the synaptic development in the gray matter of the normal, developing spinal cord differs before and after the period when maximal behavioral recovery occurs. The L6 segments from 10 groups of animals, 0–30 days of age, taken at 3 day intervals (4 animals/group) were studied by light microscopy. Areal measurements of the gray matter were made using an integrating x-y tablet interfaced to a computer. Cell size, cell density and area of neuropil were evaluated in the lateral portions of the intermediate gray matter, laminae VI and VII. Electron microscopic analyses of synaptogenesis were performed on material from the same region in animals 3, 12, 15, 21 and 30 days old using similar morphometric methods while taking note of vesicle, junctional, and mitochondrial morphology. A 60% increase in area of neuropil paralleled a linear increase, of comparable magnitude, in area of the gray matter until 15 days of age when both curves reached a plateau. Neuronal perikaryal size remained constant ( 200 sq. μm in plane of nucleolus) throughout development and so could not have contributed to the increase in area of gray matter. Areal measurements of the size and counts of the number of vesicle containing profiles demonstrated a 50% increase in density of axon terminals between 3 and 12 days of age and a steady decline thereafter. The size of vesicle-containing profiles in laminae VI and VII remained constant at a small value ( 0.35 sq. μm) until 12 days of age, showed rapid growth to 0.54 sq. μm between 12 and 15 days of age, followed by a more moderate increase in sectional area after 15 days. These results suggest that during the period when recovery of function follows spinal injury, synaptogenesis in the intermediate gray region of the lumbar spinal cord proceeds rapidly, while at stages when little recovery of function follows spinal transection, synaptogenesis is essentially complete.     

Corticosterone treatment differentially affects adrenocorticoid receptors expression and binding in the hippocampus and spinal cord of the rat

We have studied the immediate and long-term effects of high doses of corticosterone (CORT) on mRNA expression and binding properties of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and spinal cord of rats. Animals were treated with corticosterone (10 mg/d subcutaneously) for 21 consecutive days, and mineralocorticoid and glucocorticoid receptors were studied either 24 h or 2 wk after the last injection. Major results show that corticosterone treatment reduces mineralocorticoid and glucocorticoid receptor maximum binding capacity (B _max) in both the hippocampus and spinal cord and that this reduction is partially reversed after cessation of treatment. With respect to mRNA expression, in the hippocampus recovery after cessation of treatment is complete. By contrast, in the spinal cord, mineralocorticoid receptor mRNA expression is irreversibly increased after treatment, but the glucocorticoid receptor mRNA level remains unaffected during and after treatment. Thus, these data suggest the presence of distinct regulatory mechanisms for adrenocorticoid receptors in rat brain and spinal cord, in response to long-term exposure to high levels of circulating corticosterone and after recovery from treatment.     

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

Distribution of alpha2-adrenoceptor mRNAs in the rat lumbar spinal cord in normal and axotomized rats.

Using riboprobe in situ hybridization we have studied the distribution of alpha2A-, alpha2B- and alpha2c-adrenoceptor (AR) mRNAs in the lumbar spinal cord in normal rats and after peripheral axotomy. A strong alpha2A- and alpha2C AR mRNA labelling was found in motoneurons and other cells in the ventral horns. In the dorsal horns strong alpha2A-AR mRNA labelling was found in all layers and in the lateral spinal nucleus, whereas alpha2C-AR mRNA was found in lower numbers of cells in various layers. The alpha2B-AR mRNA signal was only detected in some small cells superficially in the dorsal horn. With regard to axotomy only a marginal effect was observed for alpha2C-AR mRNA in the ventral horn. The results suggest that alpha2-ARs are involved both in sensory and motor processing.     

Development of commissural neurons in the embryonic rat spinal cord.

Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.     

Development of interneurons with ipsilateral projections in embryonic rat spinal cord

Considerable progress has been made in recent years in identifying molecules with restricted expression in mammalian spinal cord at early developmental stages. However, the significance of the different expression patterns for most of these molecules is nuclear because so little is known about the development of various classes of spinal interneurons. Recently, we have characterized the development of rat spinal cord interneurons with an axon that crosses in the ventral commissure (Silos-Santiago and Snider, J. Comp. Neurol., 325:514, 1992). In the current study, we describe the morphological development of ipsilaterally projecting spinal interneurons in laminae V–VIII of the thoracic spinal cord. These neurons were labelled by retrograde lateral diffusion of DiI after crystals were placed in various locations in the embryonic thoracic cord. By E14, approximately 48 hours after the first interneurons are generated, eight different groups of ipsilateral interneurons are present in the spinal cord. By E15, these groups of ipsilateral interneurons have reached distinct locations within the gray matter. Even at this early stage, different groups of cells have elaborated characteristic dendritic arborizations. By E19, at least 17 different types of ipsilateral interneurons can be identified on the basis of location and dendritic morphology. In general, ipsilateral interneurons are located more dorsally and laterally than commissural interneurons at all stages of embryonic development. Furthermore, in comparison with commissural neurons, fewer ipsilateral interneurons have dendritic arbors with a mediolateral orientation in the transverse plane. This work demonstrates that rat embryonic spinal cord contains a large number of morphologically distinct classes of interneurons that extend axons into the ipsilateral lateral funiculus. These neurons can be distinguished from commissural neurons on the basis of location and morphology. These results, taken together with those from our previous study, provide a framework for the localization of gene expression to different classes of spinal interneurons at early developmental stages. © 1994 Wiley-Liss, Inc.