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The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express mu- and delta-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of mu and delta, but not kappa, receptor expression in F11 cells. Radioligand binding assays show that F11 cell membranes bind the mu- and delta-opioid receptor agonists, DAGO and DPDPE with Kd = 4.5 and 4.9 nM and Bmax = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K+ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the kappa-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, beta-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K+ current. The opioid-induced increase in voltage-dependent membrane K+ current in F11 cells resembles the inhibitory effect elicited by mu- and delta-opioid agonists in primary cultures of mouse DRG neurons.

Previous studies have shown that chronic morphine increases levels of the G-protein subunits Gia and Goa, adenylate cyclase, cyclic AMP-dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Gia and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Gia seen in the NAc, increased levels of Gia and Goa in amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region.

Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Spinal cord explants with attached dorsal root ganglia (DRGs), from 14-day fetal mice were fixed at 1-3 weeks in vitro and incubated for leucine-enkephalin (LE) immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Results show long processes with labeled varicosities seen more often in dorsal regions of the cord explants. Stained punctate bodies and varicosities were often seen close to large cells in these cultures, whereas no label was detected in neuronal perikarya. A prominent laminar array of stained punctate bodies was noted in one cord explant, concentric with the perimeter of the explant. No LE label was detected in the neuritic outgrowths from the cord-DRG explants, whereas high levels of opiate receptors develop in these outgrowths, primarily on the DRG neurites, by 1-2 weeks in culture. The results indicate the presence of LE in explants of fetal mouse spinal cord with attached DRGs and offer an in vitro model system in which the onset and development of peptidergic neurons can be studied as they form functional cellular interrelationships with neurons bearing opioid and monoaminergic receptors in these organotypic cultures.

Clusters of dorsal root ganglia (DRGs) from 13- to 14-day fetal mice were co-cultured with specific fragments of deafferented spinal cord (0.5-1 mm apart) on collagen-coated coverslips in Maximow slide chambers. Nerve growth factor (NGF) was added to the culture medium (1000 biological units/ml, at explantation) to ensure optimal survival and growth of a large fraction of the fetal DRG neurons. Sequential microscopic observations of the living cultures and cytologic studies after silver impregnation demonstrate that many neurites from isolated DRGs can invade dorsal (DC) regions of co-cultured spinal cord explants, whereas they are deflected from ventral cord (VC) tissue and its neuritic-glial outgrowth. Furthermore, some DRG neurites may become redirected towards distant DC target explants even after long circuitous detours around proximally arrayed VC explants. DRG neurites also show remarkably sharp projections to DC tissue and more complete avoidance of adjacent VC tissue when the DRG neurites approach a suitably arranged DC-VC-DC interface, e.g. forming de novo 'dorsal roots' at the end of a longitudinal strip of whole spinal cord. These experiments suggest that DC-VC boundaries may be particularly effective in guiding DRG neurites to specific regions of the CNS. The present studies of co-cultured fetal mouse DRG and spinal cord explants provide the first demonstration of preferential neuritic growth in vitro in relation to specific CNS target tissues.

Chronic exposure of fetal mouse spinal cord-ganglion explants to the opioid antagonist naloxone (10 microM, 7 days) produced a pronounced upregulation of mu opioid receptors. The antagonist action was stereospecific, as it was produced by (-)-, but not by (+)-naloxone, and was dose-dependent. Half-maximal naloxone-induced receptor upregulation occurred after two days; receptor density was maximal at 5 days. Exposure of the explant cultures to naloxone (10 microM) in the presence of the protein synthesis inhibitor, cycloheximide (1 microM; a concentration which blocks greater than 90% protein synthesis) resulted in receptor density changes that were similar to those observed in cultures exposed to naloxone alone. This finding suggests that antagonist-induced opiate receptor upregulation does not require the synthesis of new receptor molecules.

We have utilized extracellular microiontophoretic injections of horseradish peroxidase into fetal mouse retinal explants to label retinal ganglion cell axons innervating co-cultured tectal explants in a solid Golgi-like manner. Using dorsal root ganglia-tectum and retina-spinal cord co-cultures as controls, our results indicate that retinal neurites show selective growth and arborizations within their appropriate tectal, target tissue. Retinotectal explant co-cultures may be a useful model system for studying aspects of neuronal specificity.

Explants of 14-day fetal mouse spinal cord with attached dorsal root ganglia, which had become differentiated over 2-3 weeks in culture, were exposed to 1-2 microM taxol for up to 6 days. The culture medium was supplemented with nerve growth factor (300 units/ml) during exposure to the drug. By 3-6 days in taxol, unusually numerous microtubules were seen in peripheral perikaryal and proximal neuritic regions of ganglion neurons. Microtubules also engirdled massive aggregations of pleomorphic vesicular/cisternal elements in many neurons. These aggregates were visible as unusual 'clear' spheroidal regions in the living cells, and were often as large as the nuclei. Some of the elements comprising these striking vesicular/cisternal accumulations appeared to be portions of disrupted Golgi complexes normally polarized around the cytocentrum, as well as hypertrophied smooth endoplasmic reticulum formations. In other neuronal areas, Golgi complexes and other organelles were altered or disrupted to lesser degrees. Ordered microtubular arrays occurred along endoplasmic reticulum cisternae both in neuron somata and neurites. Over time, a plethora of microtubules assembled throughout the perikarya in various orientations apparently unrelated to microtubule organizing centers. Unlike the effects of other plant alkaloids that interact with tubulin, there was no discernible increase in filaments, although their distribution appeared altered. Concentric ordered microtubular-macromolecular lamellated complexes were seen only in neurites. Neuronal nuclei were misshapen, often displaced, and displayed fine structure reminiscent of chromatolysis. Satellite and Schwann cells contained atypically abundant microtubules, abnormal cisternae, disrupted Golgi complexes, and increased lysosomes. Some nuclei displayed abnormal chromatin, and in rare cases even microtubules. We suggest that taxol alters the distribution, integrity, and/or organization of organelle systems in dorsal root ganglion cells by engendering unusually abundant microtubules in abnormal groupings and aberrant locations in these cells.

The depressant effects of morphine (0.1-1 microM) on sensory-evoked dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia (DRGs) were rapidly restored after addition of 4-aminopyridine (4-AP; 0.1 mM) and major components of these cord responses were stably maintained in the presence of the opiate. Moreover, prior exposure of cord-DRG explants to 0.1 mM 4-AP prevented the depressant effects of 0.1 microM morphine on DRG-evoked dorsal-horn responses, and the effects of 1-10 microM morphine were at least partly antagonized. Increased Ca++ levels (5 microM) attenuated the depression of dorsal horn responses by 1-10 micro M morphine and these effects of Ca++ were greatly enhanced in the presence of 4-AP--in some cultures, concentrations of morphine as high as 100 micro M were strongly antagonized during test periods up to 2 hours. Receptor assays showed that 0.1 mM 4-AP +/- 5 mM Ca++ had no effect on stereospecific opiate binding, indicating that the antagonist actions of these agents in our cultures do not occur at the level of the opiate receptor. The relevance of our in vitro studies of 4-AP antagonism of opiate-depressant effects on sensory-evoked dorsal-horn network responses for analyses of problems in opiate analgesia has been strengthened by a recent report demonstrating that 4-AP does, in fact, reverse morphine analgesia in rats, as determined by tail flick tests.

Extracellular microiontophoretic injections of horseradish peroxidase (HRP) into NGF-enhanced fetal mouse dorsal root ganglia (DRGs) produced an anterograde solid Golgi-like labeling of DRG neurites and their terminal arborizations within co-cultured spinal cord explants. In cultures of spinal cord transverse cross-sections with attached DRGs, the large NGF-enhanced DRGs remained in close proximity to the cord, often adjacent to both dorsal and ventral cord regions. Despite this, nearly all DRG neurites that entered the cord did so via dorsal root fascicles. They branched and ramified extensively within the dorsal region, taking on a wavy or kinky course and showed various types of arborizations. The density of cord innervation was much lower when isolated DRGs and cord explants were co-cultured 0.5-1 mm apart. Although fewer entering DRG fibers were labeled by our HRP injections the same qualitative growth and arborization patterns were seen within dorsal and ventral cord regions as in explants of cord with attached DRGs. When the facing edge contained both dorsal and ventral tissues, HRP-labeled DRG fibers entered dorsal regions selectively. DRG fibers readily entered, ramified and arborized within isolated strips of dorsal cord, whereas they sharply avoided isolated ventral cord explants. The avoidance of ventral cord cannot simply be due to the paucity of specific synaptic targets within the tissue, for larger numbers of DRG fibers entered completely inappropriate CNS target tissues, e.g. superior colliculus explants--though they did not ramify or arborize to any degree comparable to that seen within dorsal cord regions.

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (approximately 10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 microM) for greater than 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 microM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca++ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord in situ. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, i.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca++ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerant cultures may be an expression of opiate dependence.

After treatment of mouse spinal cord-ganglion explants with pertussis toxin (PTX), the acute depressant effects of opioids on sensory-evoked dorsal-horn network responses are markedly attenuated, and characteristic cord discharges can then occur even in the presence of greater than 100-fold higher opioid concentrations, as observed after chronic exposure to opioids. The usual acute depressant effects of serotonin, norepinephrine, and oxotremorine on dorsal-horn discharges are similarly attenuated in PTX-treated cultures. These results together with our previous physiologic and biochemical analyses of adenylate cyclase (AC) and cyclic AMP (cAMP) activities in cord-ganglion cultures suggest that the neuromodulatory effects of opioid, monoaminergic and muscarinic agonists on primary afferent networks in the spinal cord may be mediated by binding to neuronal receptor subtypes that are negatively coupled via Gi to a common pool of AC.

Exposure of fetal mouse spinal cord-ganglion explants to morphine (greater than 0.1 microM) results in naloxone-reversible, dose-dependent depression of sensory-evoked dorsal-horn synaptic-network responses within a few minutes. After chronic opiate exposure (1 microM) for 2-3 days, these dorsal cord responses recover and can then occur even in greater than 10 microM morphine. In the present study, when naive explants were treated with forskolin (10-50 microM)--a selective activate activator of cyclase (AC)--for 10-30 min prior to and during exposure to morphine (0.1-0.3 microM) or D-Ala2-D-Leu5-enkephalin (0.03-0.1 microM), the usual opioid depressant effects on dorsal-horn responses generally failed to occur (10-30 min tests). Dibutyryl cyclic AMP (10 microM) or the more lipid-soluble analog, dioctanoyl cyclic AMP (0.1 mM), produced a similar degree of subsensitivity to opiates as 10 microM forskolin. With high levels of forskolin (50 microM), even concentrations of morphine up to 1-10 microM were far less effective in depressing cord responses. These effects of exogenous cAMP analogs and forskolin on cord-ganglion explants are probably both mediated by increases in intracellular cAMP. The marked decrease in opioid sensitivity of cAMP or forskolin-treated cord-ganglion explants provides significant electrophysiologic data compatible with the hypothesis that neurons may develop tolerance and/or dependence during chronic opioid exposure by a compensatory enhancement of their AC/cAMP system following initial opioid depression of AC activity. Previous evidence relied primarily on behavioral tests and biochemical analyses of cell cultures. It will be of interest to determine if dorsal-horn tissues of cord-ganglion explants do, in fact, develop increased AC/cAMP levels as they express physiologic signs of tolerance during chronic exposure to opioids.

Radioimmunoassays of methionine-enkephalin (Met-Enk) in organotypic cultures of 13-day fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) demonstrate a progressive development of immunoreactivity (IR) during 5 weeks in vitro. Met-Enk IR in these cultures increased to levels observed in adult rodent spinal cord. most of the Met-Enk IR assays were made on cord explants excised from cord-DRG cultures. In smaller numbers of assays performed on entire DRG-cord cultures or on cord cultured in the absence of DRGs, similar levels of Met-Enk IR were obtained. Thus most of the Met-Enk IR appeared to be located within the cord tissue. No Met-Enk IR was detected in DRGs cultured in the absence of cord. In contrast, low levels of Met-Enk IR were present in about 50% of the assays of DRGs cultured attached to the cord. Since these assays included the neuritic outgrowths of the cultures, our data do not preclude possible contamination by Met-Enk immunoreactive cord neurites that may have aberrantly projected into the outgrowth zones. Nevertheless, the data raise the possibility of a trophic influence of cord tissue on the development of Met-Enk IR in DRG neurons. The development of Met-Enk IR in cord regions of cord-DRG explants extends previous binding assays demonstrating development of opiate receptors in these cultures and provides further support to electrophysiological analyses suggesting tonic opioid inhibitory networks in these explants.

Opioid agonists selectively decrease the duration of the Ca2+ component of the action potential recorded from embryonic dorsal root ganglion neurons in dissociated cell cultures. In contrast, no significant alterations in the action potentials generated by adult dorsal root ganglion neurons in vivo were detected during opioid exposure. In the present study, the perikaryal opioid sensitivity of fetal mouse dorsal root ganglion neurons was analyzed during maturation in organotypic explant cultures. To determine whether spinal cord might influence this sensitivity, neuron perikarya were tested in ganglia grown: (a) in isolation; (b) attached to spinal cord explants; and (c) attached to spinal cord, but decentralized by a dorsal root transection in mature explants 1-2 weeks before the tests. After 2-8 weeks in culture, the duration of the Ba2+-enhanced Ca2+ component of intracellularly recorded action potentials was measured prior to and during bath exposure to the opioid, [D-Ala2, D-Leu5]enkephalin. Sensitive neurons were characterized by a marked, reversible reduction (averaging about 50%) in the duration of the Ca2+ component (which was antagonized by naloxone). The fraction of opioid-sensitive neuron perikarya in dorsal root ganglia grown attached to cord explants was significantly lower (48%) than in ganglia grown isolated (78%) or decentralized in vitro (79%). The mean duration of the Ca2+ component was significantly shorter in ganglion cells which had been grown attached to cord, or subsequently decentralized, compared to cells grown in isolated ganglia (by 24 and 38%, respectively). This difference was even larger in the opioid-insensitive groups. Although opioid-sensitive perikarya in ganglia grown attached to cord had a significantly longer Ba2+-enhanced Ca2+ component than that of insensitive neurons, some of the insensitive perikarya in all 3 types of explant paradigms displayed Ca2+ components which were as prolonged as those of sensitive cells. The results obtained in this study support the hypothesis that the observed decrease in the fraction of opioid-sensitive perikarya during development of fetal mouse dorsal root ganglia is due to regulation by interactions with their central target tissue, the spinal cord. The developmental decrease in the duration of the Ca2+ component of the action potential of these ganglion cells is also enhanced by the presence of the spinal cord. However, regulation of functional opiate receptors and Ca2+ component duration of the ganglion cell perikarya appear to be independent processes.

After chronic exposure of mouse spinal cord-ganglion explants to morphine, the acute depressant effects of opioids on sensory-evoked dorsal-horn network responses are markedly attenuated, and characteristic cord discharges can then occur even in the presence of greater than 100-fold higher opioid concentrations. The present study demonstrates that a remarkably similar degree of tolerance to opioids develops in these cord-ganglion explants after exposure to pertussis toxin (PTX). The usual acute depressant effects of serotonin, norepinephrine and oxotremorine on dorsal-horn discharges are also similarly attenuated in PTX-treated cultures. PTX is known to interfere with the guanine nucleotide protein Gi that is required for opioid, alpha 2-adrenergic and muscarinic receptor-mediated inhibition of adenylate cyclase in various cells. We have previously found that in cord-dorsal root ganglion explants agents which elevate intracellular cAMP also attenuate opioid depressant effects. Furthermore, these explants contain an opioid-inhibited adenylate cyclase system, and chronic exposure to morphine as well as PTX increases adenylate cyclase activity. These findings together with the present results suggest that the neuromodulatory effects of opioid, monoaminergic and muscarinic agonists on primary afferent networks in the spinal cord may be mediated by binding to neuronal receptor subtypes that are negatively coupled via Gi to a common pool of adenylate cyclase.

Correlative electrophysiologic and cytologic analyses demonstrate that a significant group of neurons in isolated (NGF-enhanced) fetal mouse dorsal root ganglia (DRGs) can grow across a collagen substrate and selectively innervate specific dorsal horn and dorsal column nuclei regions in co-cultured explants of deafferented spinal cord and medulla. Neurites from this group of DRG cells from connections in CNS target zones that generate characteristic primary afferent network responses to sensory stimuli, as observed in cultures of spinal cord with attached DRGs. Systematic microelectrode stimulus-mapping tests revealed that many DRG neurites were preferentially distributed in sensory target zones of co-cultured cord and medulla explants and that few collaterals of these DRG neurons were present in neighboring inappropriate regions, especially in the ventral cord. Another group of DRG neurons appears to be responsible for the less prominent, but clear-cut, innervation that developed in some of the co-cultured ventral cord explants. Fetal DRGs were also able to establish characteristic primary afferent dorsal horn or dorsal column nuclei networks when introduced into cultures of deafferented spinal cord and medulla that had been explanted alone for 1-3 weeks prior to introduction of the DRGs. These experiments demonstrate that CNS target neurons remain receptive to DRG innervation even after 1-3 weeks of maturation in vitro. Our electrophysiologic and cytologic analyses of DRG and CNS explants in organotypic co-cultures provide the first systematic attempt to establish conditions under which preferential neuritic growth to and functional innervation of specific CNS target tissues can occur in vitro. This model system should facilitate analyses of mechanisms underlying development, as well as regeneration, of specific synaptic connections in the CNS.

Exposure of organotypic mouse spinal cord-dorsal root ganglion cultures to 1-2 microM taxol for up to 6 days results in a remarkable increase in cytoplasmic microtubules in differentiated oligodendroglia. In contrast, there is a notably smaller change in the microtubule complement of astroglia. This suggests that the regulatory and/or synthetic systems for tubulin in these glial types may differ, and that oligodendroglia appear more responsive to modulation in the levels of tubulin subunits as a result of the action of taxol.

To determine whether cholinergic spinal cord neurons can develop preferential projections in vitro within sympathetic ganglia (SGs) of appropriate levels of the neuraxis, organotypic explants of fetal mouse spinal cord (E13) from cervical, thoracic (upper and lower) and lumbar segments were co-cultured with either pairs of neonatal SGs: the rostral superior cervical ganglion (SCG) or a caudally located upper lumbar ganglion (LG). After 3.5-4 weeks of co-culture, levels of the enzyme, choline acetyltransferase (ChAT), were measured in individual spinal cord explants and SCGs or double LGs (to match the target mass of a single SCG). Interaction was assumed to occur primarily between an SCG or LG doublet and the adjacent ipsilateral half of the co-cultured cord segment. An index of cholinergic interaction was defined as the ganglion ChAT activity per unit ChAT activity in half co-cultured cord segment. The index of interaction with the SCG was highest with the T1/T2 (1.4) as compared with the T10/T11 (0.79), L1/L2 (0.38) and C2/C3 (0.11) segments. In contrast, the index of cholinergic interaction with double LGs was highest with the more caudally located T10/T11 (0.62) cord segment as compared with the rostral T1/T2 (0.33), cervical C2/C3 (0.2) and lumbar L1/L2 (0.17) segments. Ganglion compound action potentials evoked in LGs by stimulation of the ipsilateral portion of T10/T11 cord were blocked by the ganglionic antagonist, hexamethonium, as previously observed in co-cultures of SCGs with T1/T2 cord. These results indicate that pools of preganglionic neurons in thoracic cord segments can develop in vitro preferential cholinergic projections within SGs of appropriate position. Cervical and lumbar cord segments which contain a preponderance of somatic motoneurons over preganglionic neurons did not interact as effectively with either type of SG. The preferential cholinergic projections from rostral thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within LGs may reflect some degree of positional preference intrinsic to embryonic spinal cord neurons and/or their appropriate target SGs, consistent with the positional specificity expressed by preganglionic neurons and SGs in situ.

Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.

Development and regional localization of patterned spontaneous bioelectric activities, recorded extracellularly, were studied in cultures of frontal sections of spinal cord and medulla, explanted from 13- to 15-day-old fetal mice. Spontaneous single- and multi-unit tonic and phasic spike discharges were recorded after 4-21 days in vitro. Stereotyped slow waves with positive or negative polarity, and durations up to several seconds, often occurred concomitant with spike burst discharges. Microelectrode mapping demonstrated regional localization of spontaneous activity patterns in these cross-sectional central nervous system explants, which were oriented so that dorsal and ventral borders could be regularly identified. Phasic discharges were widely distributed throughout the explants, whereas tonic discharges were located primarily in the dorsal region of the medulla; negative slow waves were generated in central and ventral regions of spinal cord and medulla explants, whereas positivities were located in the peripheral regions of the explants. Phasic discharges were often synchronous when recorded at two symmetrical regions of an oriented explant, as early as 4 days in vitro. The major developmental change in spontaneous activities of these explants was a progressive shortening of both burst and cycle durations of the phasic discharges.

Neurites of nerve growth factor-enhanced fetal mouse dorsal root ganglion cells can not only establish characteristic sensory synaptic network functions in dorsal regions of attached spinal cord explants, but some of the neurites may grow through the cord tissue in these cultures and make similar functional synaptic connections with specific types of "target" neurons in localized zones within nearby medulla explants.

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2-4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrorphan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the delta- and kappa-receptor subtypes appear to be involved since the selective delta-opioid agonist, [D-Pen2, D-Pen5]enkephalin, and the selective kappa-opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-opioid agonist, Tyr-D-Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin; treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxin treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent by opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and serotonin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive Gi protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

The serums from animals with experimental allergic encephalomyelitis and humans with multiple sclerosis produce, in addition to demyelination, rapid, reversible alterations in complex, evoked bioelectric (synaptic) responses of cultured cerebral cortex and spinal cord tissues of the mouse. The active factors are dependent on complement and are not present in serums from normal animals and humans.

Fragments of embryonic spinal cord (human, rat, and chick) can regenerate and differentiate in tissue culture. Complex bioelectric activity evoked by electric stimuli indicates that nerve cells in cultures may maintain, for months in vitro, not only the capacity to propagate impulses along their neurites but also a remarkable degree of functional organization resembling the activity of synaptic networks of the central nervous system.