Lester M. Partlow

Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates

A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85-90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiencies greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.

Neuronal stimulation of (3H)thymidine incorporation by primary cultures of highly purified non-neuronal cells.

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-3H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [3H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [3H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [3H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [3H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [3H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.

Calcium channel blockers and behavioral sensitization

Behavioral sensitization to amphetamine-induced stereotypy was previously shown to consist of two separable phenomena, induction and expression, both of which involve the excitatory amino acids (EAA). In the present experiments, the calcium channel blockers (CCB), nifedipine, diltiazem and verapamil, were shown to block both phenomena; these results are similar to those reported earlier for DNQX, an antagonist of the non-N-methyl-D-aspartate receptors for the EAA. The CCB, like DNQX, affect only that percentage of the stereotypic response which results from the sensitization reaction, without affecting the quantitative portion of the response attributable to the acute effect of amphetamine. The results support previous conclusions that the sensitization response consists of two quantitative components, only one of which involves the EAA. The antagonism exhibited by the CCB suggests that behavioral sensitization involves Ca++ and L-type calcium channels.

Passive avoidance impairment in rats following cycloheximide injection into the amygdala

Cycloheximide (CHX:1, 10 or 20 microg) was injected via indwelling cannulas into various regions of the rat brain and its effects on passive avoidance training were studied. Rats with 10 or 20 microg of CHX injected into the amygdala immediately after the training footshock exhibited amnesia for the learning experience when tested after 24 h. In contrast, animals injected with 20 microg of CHX at a site either in the internal capsule only 2 mm above the amygdaloid injection site or in the frontal cortex showed no retention deficit when tested after 24 h. A quantitative examination of protein synthesis in brain halves 30 min after unilateral injection of 20 microg of CHX into the amygdala demonstrated that total protein synthesis was inhibited by less than 10%. Autoradiographic studies revealed that this inhibition resulted from a profound, highly localized inhibition of protein synthesis in areas immediately adjacent to the cannula. A comparison of the regional patterns of protein synthesis inhibition caused by injection of CHX into either the amygdala or internal capsule suggested that CHX might produce amnesia by virtue of its localized effect on the amygdala. Control experiments revealed that injection of 20 microg CHX into the amygdala had no effect on short-term retention, or short-term performance. Injection of 20 microg of CHX into the amygdala 12 h after the footshock had no effect on long-term retention. The observed impairment of retention was shown to be dose-dependent as injection of 1 microg of CHX into the amygdala was without effect. In addition, it was demonstrated that the CHX-induced amnesia did not result from induction of local seizure activity. These data show that localized injections of small amounts of CHX into the amygdala can produce deficient memory of a training experience even though total brain protein synthesis is only slightly inhibited.

Stimulation of non-neuronal cell proliferation in vitro by mitogenic factors present in highly purified sympathetic neurons

Sympathetic neurons have been demonstrated to contain one or more mitogens which are active on highly purified non-neuronal cells cultured in medium containing an optimal concentration of fetal calf serum. Neurons and homologous non-neuronal cells were separated by a method recently developed in this laboratory. The highly purified neurons were either sonicated or homogenized prior to addition to nonneuronal cultures. The presence of neuronal sonicate (1) greatly stimulated [3H]thymidine incorporation into acid-precipitable macromolecules without altering the soluble [3H]thymidine pool, (2) increased both the fraction of non-neuronal cells which took up [3H]thymidine and the density of labeling as observed by autoradiography, and (3) increased the number of cells present in treated cultures after 40 h. The enhancement of [3H]thymidine incorporation was dose-dependent and did not involve cyclic AMP. Addition of neuronal sonicate also caused marked non-neuronal cell elongation which resulted in the elaboration of very long cell processes. The active factor(s) in the neuronal sonicate were partially heat-labile. Norepinephrine was ruled out as a possible mitogenic factor.

An Investigation of the Thermal and Athermal Effects of Microwave Irradiation on Erythrocytes

Erythrocytes heated by either microwave irradiation or conventional techniques were examined for loss of hemoglobin (Hb) and potassium (K+). In all experiments, microwave heated red blood cells (RBCs) were directly compared with RBCs warmed to the same temperature by use of an adjustable incubator and with RBCs maintained at room temperature. Microwave irradiation was carried out at either 2.45 GHz or in selected 0.5 GHz swept frequency regions in the 12.5-18 GHz range. Sample temperature was continuously monitored in both microwave heated and conventionally heated samples by use of a relatively nonperturbing liquid crystal optic fiber temperature probe. When rabbit RBCs were warmed by 3.7°C for 45 min by either irradiation (2.45 GHz, 10 mW/cm2) or conventional heating, no additional Hb or K+ was released into the supernatant. In contrast, when rabbit RBCs were rapidly warmed from room temperature to 37°C by either technique, the heated erythrocytes lost significantly more of both Hb and K+ than equivalent RBCs maintained at room temperature. In addition, RBCs warmed to 41.5°C by either technique lost far more Hb and K+ than those warmed to 37°C. In all experiments, Hb and K+ were lost in equal amounts by microwave heated and conventionally heated erythrocytes warmed at the same rate to the same final temperature. Thus, at all frequencies and power levels tested, any increased loss of either Hb or K+ from microwave irradiated rabbit RBCs should be ascribed to thermal effects on the stability and/or permeability of the erythrocyte membrane.

A Quantitative regional analysis of protein synthesis inhibition in the rat brain following localized injection of cycloheximide

Previous work has shown that bilateral injection of as little as 10 microgram of cycloheximide (CHX) into the amygdala, but not into the internal capsule, caused a time-dependent disruption of long-term retention of passive avoidance training. Under these conditions, protein synthesis in the entire brain was inhibited by less than 10%. The present study was undertaken to quantify the resulting inhibition of protein synthesis in various brain regions (amygdala, internal capsule, caudate, cortex, hippocampus, thalamus, hypothalamus and the entire half brain). Rats were subcutaneously injected with L-[14C-methyl]methionine following unilateral administration of CHX via a cannula implanted in either the amygdala or the internal capsule. Regional inhibition of protein synthesis was determined by analysis of autoradiograms from different brain levels using an image analyzing computer to measure the optical densities of microscopic areas corresponding to discrete neuroanatomical structures. Regional patterns of inhibition were assessed: (a) after injection of different doses of CHX (10 or 20 microgram) into the amygdala; (b) after injection of 20 microgram of CHX into the amygdala or internal capsule; and (c) at different times (0.5, 3, 6 and 24 h) after injection of 20 microgram of CHX into the amygdala. Quantitative results are presented for the temporal and spatial patterns of protein synthesis inhibition caused by CHX injection. Since injection of CHX into the amygdala resulted in a profound inhibition of protein synthesis in both the amygdala and internal capsule while injection into the thermal capsule only caused a marked inhibition in the capsule itself, these results provide a possible explanation for our earlier observation that injection of CHX into the amygdala produced a retention deficit while injection into the adjacent internal capsule had no effect on memory function. These observations on protein synthesis inhibition support our earlier hypothesis that CHX injected into the amygdala might impair memory by virtue of its action on amygdaloid function rather than as a result of its effect on the brain as a whole.

Preparation and partial characterization of highly purified primary cultures of neurons and non-neuronal (glial) cells from embryonic chick cerebral hemispheres and several other regions of the nervous system

Purified cultures of neurons and non-neuronal (glial) cells were prepared from the cerebral hemispheres of 10-day chick embryos by a method previously used for embryonic chick sympathetic ganglia 16. This technique separates these cell types on the basis of both: (1) differences in the adhesiveness of neurons and non-neuronal cells to a collagen substrate; and (2) the capacity of neurons to form homotypic aggregates. Purity of the cerebral non-neuronal cultures was determined to be greater than or equal to 99.5% by microscopic examination, while that of the cerebral neuronal cultures was only 92%. Modification of the technique by periodic redissociation of the neuronal aggregates during cell separation increased the purity of the neuronal cultures to greater than or equal to 97% as determined both by microscopic examination and by measurement of levels of butyrylcholinesterase, an enzyme present in the non-neuronal cells. Highly purified cultures of neurons were also prepared from the optic lobes of 10-day chick embryos (greater than or equal to 98%), but attempts to obtain non-neuronal cultures of reasonable density from this tissue were unsuccessful. In addition, highly purified non-neuronal cultures (greater than or equal to 99.5%) were prepared from the dorsal root ganglia of 12-day chick embryos, but cultures enriched with dorsal root neurons could only be partially purified (82%). Specific activity of butyrylcholinesterase in cerebral non-neuronal cells was found to vary inversely with the density of non-neuronal cells.

Delta-9-tetrahydrocannabinol depresses inward sodium current in mouse neuroblastoma cells

Whole-cell voltage-clamp techniques were used in order to define the effects of delta-9-tetrahydrocannabinol (THC) on the voltage-gated sodium current in neuroblastoma cells. With regard to the inward sodium current, THC decreased the peak amplitude and increased both the time to peak and tau for recovery. The reversal potential was unchanged, suggesting that channel selectivity for sodium was not altered by the drug. With regard to the outward sodium current, THC had no effect on the peak amplitude, time to peak or tau for recovery. This functional alteration of the voltage-gated sodium channel may contribute to the depressant effects of the cannabinoid.

Characterization of a variety of standard collagen substrates: ultrastructure, uniformity, and capacity to bind and promote growth of neurons

SummaryCollagen substrates were characterized after preparation by the four methods most commonly used for tissue culture (saline precipitation, exposure to ammonium hydroxide vapor, exposure to ultraviolet light, and air drying). Although roughly equivalent percentages of collagen were precipitated by each technique (87 to 97%), marked differences were found in surface uniformity and ultrastructure. Substrates were quite uniform if precipitated by exposure to ammonium hydroxide or ultraviolet light, of intermediate uniformity if saline precipitated, and not at all uniform if air dried. Scanning electron microscopy revealed that (a) ammonium hydroxide and saline precipitation primarily resulted in formation of collagen fibrils, (b) air drying produced a small number of fibrils plus a large amount of amorphous material, and (c) exposure to ultraviolet light only resulted in the formation of globular, nonfibrillar collagen aggregates.The capacity of collagen substrates to bind and grow neurons differed markedly with the method of preparation and the amount of collagen plated per unit area. Quantification of binding and growth of both cerebral and sympathetic neurons revealed that these are separate measures of the biocompatibility of a surface and that growth was uniformly inferior on globular collagen that had been precipitated by ultraviolet light. Long-term (≥2 wk) growth of sympathetic neurons was optimal on thick beds of saline-precipitated collagen, whereas short-term growth was best on thin layers of either saline or ammonium hydroxide-precipitated collagen. Cerebral neurons bound and grew optimally on thick collagen beds after both short- and long-term culture. In addition, cerebral neurons were found to be more dependent on the method of precipitation of the thin collagen substrates than were sympathetic neurons.