Effect of osmotic stress on potassium accumulation around glial cells and extracellular space volume in rat spinal cord slices.

In rat brain and spinal cord slices, the local extracellular accumulation of K(+), as indicated by K(+) tail currents (I(tail)) after a depolarization step, is greater in the vicinity of oligodendrocytes than that of astrocytes. It has been suggested that this may reflect a smaller extracellular space (ECS) around oligodendrocytes compared to astrocytes [Chvátal et al. [1997] J. Neurosci. Res. 49:98-106; [1999] J. Neurosci. Res. 56:493-505). We therefore compared the effect of osmotic stress in spinal cord slices from 5-11-day-old rats on the changes in reversal potentials (V(rev)) of I(tail) measured by the whole-cell patch-clamp technique and the changes in ECS volume measured by the real-time iontophoretic method. Cell swelling induced by a 20 min perfusion of hypoosmotic solution (200 mmol/kg) decreased the ECS volume fraction from 0.21 +/- 0.01 to 0.15 +/- 0.02, i.e., by 29%. As calculated from V(rev) of I(tail) using the Nernst equation, a depolarizing prepulse increased [K(+)](e) around astrocytes from 11.0 to 44.7 mM, i.e., by 306%, and around oligodendrocytes from 26.1 to 54.9 mM, i.e., by 110%. The ECS volume fraction decrease had the same time course as the changes in V(rev) of I(tail). Cell shrinkage in hyperosmotic solution (400 mmol/kg) increased ECS volume fraction from 0.24 +/- 0.02 to 0.32 +/- 0.02, i.e., by 33%. It had no effect on [K(+)](e) evoked by a depolarizing prepulse in astrocytes, whereas in oligodendrocytes [K(+)](e) rapidly decreased from 52 to 26 mM, i.e., by 50%. The increase in ECS volume was slower than the changes in [K(+)](e). These data demonstrate that hypoosmotic solution has a larger effect on the ECS volume around astrocytes than around oligodendrocytes and that hyperosmotic solution affects the ECS volume around oligodendrocytes only. This indicates that increased K(+) accumulation in the vicinity of oligodendrocytes could be due to a restricted ECS. Oligodendrocytes in the CNS are therefore most likely surrounded by clusters of "compacted" ECS, which may selectively affect the diffusion of neuroactive substances in specific areas and directions and facilitate spatial K(+) buffering.     

Gliogenesis in rat spinal cord: evidence for origin of astrocytes and oligodendrocytes from radial precursors

We have examined glial cell lineages during rat spinal cord development by using a variety of antibodies that react with immature and mature glia. Radial glia in embryonic cord bound 1) A2B5, an antibody that reacts with a glial precursor cell population in optic nerve; 2) AbR24, which is directed against GD3 ganglioside and binds to immature neuroectodermal cells and to developing oligodendrocytes in forebrain and cerebellum; and 3) an antibody to the intermediate filament, vimentin. With time, two different populations emerged, both of which seemed to be derivatives of radial cells. One cell type expressed the astrocyte intermediate filament, GFAP, in addition to vimentin. GFAP-containing cells eventually took on the forms of astrocytes in gray and white matter. The other type expressed carbonic anhydrase, an enzyme characteristic of oligodendrocytes and enriched in myelin. Carbonic anhydrase-positive cells eventually developed into small cells with oligodendrocyte morphology. Our observations suggest a common lineage for astrocytes and oligodendrocytes from radial cells during spinal cord gliogenesis.     

Serotonin 2A receptor-like immunoreactivity is detected in astrocytes but not in oligodendrocytes of rat spinal cord

The distribution of the serotonin 2A (5-HT2A) receptor in glial cells in the white matter of rat spinal cord was immunohistochemically examined with specific antibodies against the 5-HT2A receptor. 5-HT2A receptor-like immunoreactivity was detected in astrocytes that were identified by an antibody against the glial fibrillary acidic protein. In contrast, 5-HT2A receptor-like immunoreactivity was not observed in oligodendrocytes.     

Internalization of mu-opioid receptors in rat spinal cord slices.

Cells immunoreactive for the mu-opioid receptor (MOR) in laminae I-II of the spinal cord were identified as small neurons with rostro-caudal dendrites. In spinal cord slices, [D-Ala2,MePhe4-Gly-ol5]enkephalin (DAMGO) or etorphine (1 microM) caused naloxone-sensitive MOR endocytosis in 100% of these neurons, whereas the selective delta- and kappa-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and spiradoline mesylate (U-62,066), respectively, produced negligible internalization at 1 microM. The EC50 for DAMGO was 30 nM, similar to its potency to inhibit cAMP accumulation and to increase [gamma-35S]GTP binding. MOR internalization followed an exponential timecourse with a half-life of 1.7 min. MOR internalization in spinal cord slices was faster and occurred at lower agonist concentrations than in MOR-transfected cells, suggesting that spinal cord neurons have a more effective coupling of MORs to intracellular components mediating endocytosis.     

Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter

Myelencephalon-specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156-8168, 1997b). To determine the cell-specific expression patterns of MSP, we generated MSP-specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP-immunoreactive oligodendroglia, but not by GFAP-immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord.     

Neurochemical identification of afferents onto spinomedullary neurons in the rat spinal cord central gray matter

The synaptic relationship between spinal cord central gray projection neurons and immunocytochemically identified afferents in the rat were examined at the light microscopic level using the combined techniques of retrogradely transported True blue and serotonin (5-HT), enkephalin (ENK), and substance P (SP) immunocytochemistry. At L4-L6, numerous retrogradely labeled neurons could be identified around the central canal after large bulbar injections of True blue. Of these projection neurons, 75% were apposted by 5-HT varicosities, 57% by ENK varicosities and 58% by SP varicosities. Hemisection of the spinal cord produced a marked reduction in the amount of 5-HT immunoreactivity and the number of putative 5-HT contacts observed on neurons of the spinal cord central gray. A small decrease in SP immunoreactivity and putative contacts was seen after dorsal rhizotomy. Neither rhizotomy nor hemisection produced discernable changes in ENK immunofluorescence. Based on the distributions of 5-HT, ENK and SP in the spinal cord, we suggest that a more precise delineation of lamina X in the rat can be made according to immunocytochemical rather than strictly morphological criteria.     

Patch-clamp recordings from white matter glia in thin longitudinal slices of adult rat spinal cord

BACKGROUND: Recent studies have demonstrated spontaneous and prolonged hyperthermia following stroke in both humans and rodents. However, a full characterization of these pyretic changes and the effects of anti-pyretic drugs on outcome is not available. METHODS: The aims of this study were to monitor conscious body temperature (n=10 per group) using programmable microchips for up to 24 h in rats following either permanent (p) or 90 min transient (t) middle cerebral artery occlusion (MCAO) or sham surgery, and to evaluate the relationship to hypothalamic damage. Also, the effects of anti-pyretic drug therapy on body temperature and infarct volume were evaluated in animals treated with vehicle, optimal doses of either aspirin or paracetamol (250 mg/kg i.p.) following pMCAO (n=10 per group). RESULTS: At 1 h, body temperature significantly (P<0.01) increased to 38.6+/-0.2 degrees C following tMCAO and 38.9+/-0.1 degrees C following pMCAO compared with sham-operated animals (37.1+/-0.1 degrees C). Sustained hyperthermia (> or =38.1 degrees C) was observed for up to 24 h following pMCAO but approached baseline within 30 min (37.6+/-0.2 degrees C) following tMCAO with reperfusion. The post-stroke pyrexia was related to the degree of ischemia where hypothalamic damage was observed in (80%) of the animals undergoing pMCAO and (0%) in the tMCAO group (P<0.05). Treatment with paracetamol (250 mg/kg i.p.) significantly attenuated (P<0.05) but did not normalize core body temperature up to 2 h (38.2+/-0.4 degrees C) compared with vehicle treated animals (39.3+/-0.1 degrees C). Aspirin had no effect on temperature under these conditions. Hypothalamic damage and lesion volume were not different between animals treated with paracetamol (253.3+/-8.5 mm(3)), aspirin (264.0+/-11.6 mm(3)) or vehicle (274.4+/-8.2 mm(3)). CONCLUSIONS: This study is the first to demonstrate the utility of programmable microchips to monitor serial changes in post-stroke hyperthermia. The sustained post-stroke pyrexia and negative effects of antipyretic treatment may be attributed to the extensive hypothalamic injury suggesting that better pharmacologic approaches to reduce body temperature should be identified and evaluated for brain protection in severe experimental stroke.     

Different uptake of cobalamin (vitamin B12) by astrocytes and oligodendrocytes isolated from rat spinal cord

In the present study, we hypothesized that cobalamin (Cbl) deficiency might affect astrocytes and oligodendrocytes of rat spinal cord (SC) differently. Radiolabeled Cbl ([Cyano-14C]cyano-Cbl) was used to investigate whether the in vitro uptake of Cbl is different in primary cultures of oligodendrocytes and astrocytes. In culture medium supplemented with serum that naturally contains Cbl, the time course of labeled Cbl uptake by neonatal oligodendrocytes had two peaks, at 8 h and 24 h. No uptake was observed when the same cells were cultured in a serum-free medium and consequently in the absence of Cbl. Oligodendrocytes isolated from adult rat SC showed no uptake under any of the tested conditions. Astrocytes isolated from adult Cbl-deficient and newborn rat SC, both cultured in a medium supplemented with serum, showed peak Cbl uptake at 8 h and 12 h, respectively, whereas those isolated from the SC of an adult normal rat cultured under the same conditions showed no uptake throughout the experimental period. Astrocytes isolated from normal, Cbl-deficient adult rats and newborns cultured in a serum-free medium not containing Cbl, showed a similar trend of Cbl uptake with a peak at 24 h. Oligodendrocytes isolated from Cbl-deficient rats showed no uptake when cultured in medium with or without serum. This study provides evidence for a difference in the uptake of labeled Cbl between rat SC astrocytes and oligodendrocytes in relation to (1) age of the donor SC, (2) Cbl status of the donor SC, and (3) Cbl deficiency in the incubation medium that facilitates Cbl uptake in neonatal and adult astrocytes.     

Astrocytes and oligodendrocytes reactions after a total section of the rat spinal cord

Regeneration after an injury in the Central Nervous System is dependent on intrinsic and extrinsic factors. Among the latter are the reactions of glial cells. Using the model of total section of adult rat spinal cord, we have studied the spatial and temporal responses of astrocytes and oligodendrocytes to the lesion of spinal cord axons. We studied at molecular and cellular levels the specific markers GFAP (glial fibrillary acidic protein) for the astrocytes, CNP (2′-3′ cyclic 3′ nucleotide phosphodiesterase) which is principally expressed by immature oligodendrocytes, and MBP (myelin basic protein) implicated later in the myelin compaction, and which is more specific of mature oligodendrocytes. After injury, all astrocytes, but more markedly those of the grey matter, reacted by an increase of GFAP messenger and protein. This increase was very rapid for messenger, and peaked at 3 days. This increase was more protracted for the protein and persisted after 3 weeks. Messenger increase is more marked and more protracted below than above the lesion. Oligodendrocytes also reacted quickly by an increase of CNP and MBP messengers. For CNP, both messenger and protein increased rapidly and returned to control level after 1 week. MBP showed the same time course of changes, with lower and slower decrease above the lesion. Counts of oligodendrocytes showed that the percentage of the less mature form (light oligodendrocytes) increased dramatically above and below the lesion. After 1 week, above the lesion, this percentage was well below that of the control, whereas below the lesion, it reverted to control value. These results indicate that, following a lesion, astrocytes react quickly and intensely, but more so below the lesion; oligodendrocytes resume a sequence of maturation which is eventually completed above the lesion where remyelinisation can occur and which is prematurely interrupted below the lesion. However, intact oligodendrocytes persist below the lesion, where they constitute a potential for remyelinisation of regenerated and/or transplanted axons.     

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Leukodystrophies are often devastating diseases, presented with progressive clinical signs as spasticity, ataxia and cognitive decline, and lack proper treatment options. New therapy strategies for leukodystrophies mostly focus on oligodendrocyte replacement to rescue lack of myelin in the brain, even though disease pathology also often involves other glial cells and the spinal cord. In this study we investigated spinal cord pathology in a mouse model for Vanishing White Matter disease (VWM) and show that astrocytes in the white matter are severely affected. Astrocyte pathology starts postnatally in the sensory tracts, followed by changes in the astrocytic populations in the motor tracts. Studies in post‐mortem tissue of two VWM patients, a 13‐year‐old boy and a 6‐year‐old girl, confirmed astrocyte abnormalities in the spinal cord. For proper development of new treatment options for VWM and, possibly, other leukodystrophies, future studies should investigate spinal cord involvement.     

Astroglial reaction in the gray matter lumbar segments after midthoracic transection of the adult rat spinal cord

Previous studies of cordotomized rats revealed a glial reaction in the gray matter of the spinal cord at sites remote from the lesion, and the present study was done to explore this phenomenon further. Seventy-five young adult female rats were cordotomized and 10 hemicordotomized, both operations at T5. Between 1 and 28 days postoperatively, histologic sections of thoracic and lumbar segments stained by phosphotungstic acid hematoxylin (PTAH, pH 2.37), by periodic acid Schiffdimedon (PAS-D) or by an immunocytochemical method for glial fibrillar acidic protein (GFAP) revealed histological changes as follows: PTAH staining showed that astroglia in thoracic and lumbar regions of the cordotomized rats possessed a swollen, pink-staining cytoplasm and enlarged, thick, dark blue-staining fibrous processes. This response, first noted within 4 days, had intensified by 7 days and was maximal at 14 to 17 days postoperatively. By 28 days, the reaction had diminished but was still readily detectable. The more specific GFAP staining procedure confirmed that the reactive cells were astrocytes and demonstrated that their fibrillar density had increased. The PAS-D reaction revealed glycogen accumulation in glia of the lumbar gray matter within 2 days; this response intensified by 4 days and diminished to normal by 14 days. This reaction was largely concentrated in the perivascular end feet of astroglia, but also appeared in conjunction with perineuronal astroglia. The site of glial reactivity included both dorsal and ventral horns and was particularly noticed in the gray matter surrounding the central canal. In the hemicordotomized rats, the thoracic and lumbar glia response was much more pronounced ipsilaterally than contralaterally. These results support the interpretation that an astroglial response, involving hypertrophy, fibrillogenesis, and glycogen accumulation, occurs in response to degenerating nerve fibers caudal to sites of spinal cord injury.     

Three-dimensional distribution of astrocytes in zebrafish spinal cord.

We prepared a monoclonal antibody (A-22) that recognizes a 60-kDa protein in the zebrafish brain. The antigen is distributed throughout the brain but is not found outside it. The antibody recognizes star-shaped cells with long processes in the spinal cord. All A-22-positive cells are also GFAP-immunopositive, but there are GFAP-positive cells that are A-22-negative. The cells are connected to small veins and to the surface of the spinal cord. Immunopositive cells are generally homogeneous in size and shape and are found not only in the spinal cord but also in several areas of the brain. These results indicate that the stained cell is an astrocyte. Most of these cells (88%) are distributed in the gray matter of the spinal cord; the remainder (12%) are found in the white matter. Most of the cells in the gray matter are found in the ventral and dorsal horns, but some are also present in the central area along the ventricle. Glial cell bodies form an array along the longitudinal axis and are connected to each other by thick projections. The cellular array is not visible in coronal sections. In contrast, thin processes from the cells extend to the surfaces of veins, to neurons, and to the periphery of the spinal cord. We estimate that there are about 13,500 A-22-positive astrocytes in the spinal cord; however, this represents only 26% of the total number of astrocytes in the spinal cord (approximately 52,000).     

Glial fibrillary acidic protein immunohistochemistry of spinal cord astrocytes after induction of ischemia or anoxia in culture

The effects of ischemia (removal of oxygen and glucose for 4 h) and anoxia (removal of oxygen alone) on astrocytes were studied in dissociated cultures of E14 spinal cord containing both neurons and astrocytes. In addition, a group of cultures was treated with a low Na+, low Ca2+, and high K+ medium during the 4-h ischemic period (ischemia-protected group), a process that protects neurons from ischemic damage under identical conditions. Astrocytes were examined immunohistochemically using glial fibrillary acidic protein (GFAI) antiserum 24 h after insult. Densitometry and statistical analysis (1-way analysis of variance [ANOVA], a priori; 2-tailed Tukey-t, a posteriori) of the digitized images of the somata and processes of astrocytes in the anti-GFAP reacted cultures showed significant differences between the groups; a significant increase (P less than 0.01) in the GFAP-positive reaction in the somata of ischemic astrocytes and a significant decrease (P less than 0.01) in the GFAP-positive reaction in the processes of ischemic, ischemia-protected, and anoxic astrocytes. There were no significant differences in the GFAP immunoreactivity of somata between control, ischemia-protected, and anoxic astrocytes or of processes from ischemic, ischemia-protected, and anoxic astrocytes. These data show that following ischemia cultured astrocytes increase somatic GFAP immunoreactivity compared to all other groups tested whereas the staining intensity for GFAP was decreased in the processes of all three experimental groups compared to controls. Ischemia protection resulted in the absence of the enhancement of somatic GFAP immunoreactivity. The relationship of the astrocytic response and the type of cellular stress is discussed.     

Selective necrosis of spinal cord gray matter. A complication of dissecting aneurysm of the aorta

Summary A 56 year old man with known hypertension developed a dissecting aneurysm of the aorta accompanied by transient paralysis of the lower extremities. The aneurysm was operated (fenestration procedure) but the next day the dissection extended further distally and the patient became oliguric and completely paraplegic. Sensation of the legs remained relatively intact. He died 14 days after surgery, from rupture of the aorta. Autopsy revealed a dissecting aneurysm extending from the left subclavian artery to the bifurcation of the aorta, with dissection around the intercostal and lumbar arteries. The spinal cord showed selective necrosis of the gray matter with preservation of the white matter in the lower thoracic and upper lumbar segments. The review of the literature suggests that a whole spectrum exists from no cord lesions at all (despite severance of intercostal and lumbar arteries) to massive necrosis of the cord, in extreme cases even with associated necrosis of the vertebral bodies. Selective necrosis such as in the case presented is most likely the result of a borderline collateral circulation sufficient to meet the needs of the white matter but unable to cope with the greater oxygen need of the gray matter.

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Zusammenfassung Bei einem 56 jährigen Hochdruck-Patienten entwickelte sich ein Aneurysma dissecans der Aorta, begleitet von vorübergehender Lähmung der unteren Extremitäten. Das Aneurysma wurde operiert (Fenestration), am nächsten Tage aber dehnte sich die Dissektion weiter nach distal aus: der Patient wurde oligurisch und komplett gelähmt. Die Sensibilität der Beine blieb relativ intakt. 14 Tage nach dem chirurgischen Eingriff verstarb er an Aortenruptur. Die Autopsie ergab ein von der linken A. subclavia bis zur Bifurkation der Aorta ausgedehntes dissezierendes Aneurysma mit Dissektion um die intercostalen und lumbalen Arterien. Das Rückenmark zeigte eine selektive Nekrose der grauen Substanz in den unteren Brust- und oberen Lumbalsegmenten mit Verschonung der weißen Substanz. Die Literaturübersicht ergibt, daß ein breites Spektrum von Läsionen von völliger Verschonung (trotz Schädigung der intercostalen und lumbalen Arterien) bis zur massiven Rückenmarksnekrose, in den extremen Fällen auch verbunden mit Nekrose der Wirbelkörper, möglich ist. Eine selektive Nekrose wie in dem mitgeteilten Fall ist vermutlich das Ergebnis eines insuffizienten Grenzzonenkollateralkreislaufes, der zur Ernährung der weißen Substanz ausreicht, aber den größeren Sauerstoffbedarf der grauen Substanz nicht decken kann.

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With 3 Figures in the Text     

The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K+ contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K+-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K+-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K+-rich solutions adequately models the localized K+ accumulation which occurs physiologically during afferent activity. K+-induced depolarizations were decreased in the presence of 20 mM Mg2+; this effect was due to a direct decrease in the membrane response to K+ and not to blockade of K+-induced transmitter release onto primary afferents. The depolarization caused by a K+ concentration comparable to a maximum estimate of the K+ accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K+ levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K+-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.     

Reactive astrocytes in lesioned rat spinal cord: effect of neural transplants

The astroglial reaction following a laceration-type surgical lesion of rat spinal cord is recognized by hypertrophy of astrocytes. This phenomenon can be readily demonstrated by enhanced immunoreactivity for glial fibrillary acidic protein (GFAP) and a recently discovered 30 kD protein (J1-31 antigen). The results reported in this article lead to the conclusion that the astroglial reaction is not influenced significantly by the transplantation of embryonic neocortical tissue to the cavity of a laceration-type lesion. These observations could be relevant to the assessment of strategies for treatment of spinal cord injury.     

A comparison of the regeneration potential of dorsal root fibers into gray or white matter of the adult rat spinal cord.

To assess the role of white matter inhibition as a barrier to neurite outgrowth in vivo, we unilaterally transected three consecutive lumbar dorsal roots (L4-L6), incised the spinal cord, and transplanted the peripheral stump of L4 either medially onto the white matter of the dorsal columns or laterally, just superficial to the gray matter of the dorsal horn at the level of L5. Three weeks to seven months later, the translocated root was retransected, and its central stump was anterogradely labeled with HRP. The staining pattern demonstrated that regenerating sensory axons had entered the spinal cord from both medially and laterally placed roots. Axonal staining from medially placed dorsal roots (onto the white matter of the dorsal columns) was sparse and limited to the white matter. Staining of laterally placed roots revealed a small subpopulation of regenerating axons which had entered the gray matter and formed terminal arbors. Successful axonal regeneration into the gray matter, albeit minimal, was associated with a localized and limited inflammatory response near the sites of axonal ingrowth.     

The effect of morphine on the potassium evoked release of tritiated 5-hydroxytryptamine from spinal cord slices in the rat

The effect of morphine on the potassium (40 mM) evoked release of exogenous [3H]5-HT from slices of the dorsal spinal cord of the rat was studied. The effects of in vitro applied morphine on the slices were compared to those produced by systemic morphine applied to the animals before preparation of the slices. The in vitro application of morphine (10(-6) to 10(-5) M) did not affect the release of [3H]5-HT. By contrast, it was observed that the potassium evoked release of [3H]5-HT from the slices of the spinal cord of rats which had received 10 mg/kg s.c. of morphine 30 min beforehand was significantly increased. The effect of systemic morphine was dose-dependent (in the range of 1.5-10 mg/kg s.c.) and could be blocked by prior administration of naloxone (1 mg/kg i.m.) 2 min before the morphine. The acute administration of 10 mg/kg s.c. of morphine, which did not induce analgesia in rats rendered tolerant to morphine, did not modify the [3H]5-HT release. Higher doses of morphine, which have been shown to restore analgesia in these rats, induced an increase in the release which was significant for a dose of 100 mg/kg s.c. These results demonstrating a specific and dose-dependent increase in the potassium evoked release of [3H]5-HT from spinal dorsal cord slices after systemic administration of morphine, emphasize the role of serotonergic systems in such analgesia. The lack of effect of the drug directly applied in vitro favours a supraspinal site of action of the drug and is in good agreement with recent results in the literature.     

The effect of injection technique on the passive spread of astrocytes following transplantation into rat spinal cord white matter

Bisbenzimide-labelled astrocytes were transplanted into the spinal white matter of the rat using three different injection techniques and the variability of the longitudinal distance over which they were found was compared 30 min later. Cells spread up to 5.02 mm and the greatest variability was seen when they were injected as a bolus (54%), compared with 26% when injected over 2 min. These results show the importance of establishing the extent of passive spread of cells and its variability when performing studies in cell migration.