Daniel J. Feller

Antisense GAP-43 Inhibits the Evoked Release of Dopamine from PC12 Cells

Abstract: To investigate the role of the neuronal growth‐associated protein GAP‐43 (neuromodulin, B‐50, F1, P‐57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP‐43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP‐43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP‐43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP‐43 RNA in PC12 cells leads to a decrease in GAP‐43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP‐43 in calcium‐dependent neurotransmitter release.

Activation of corticostriatal pathway leads to similar morphological changes observed following haloperidol treatment.

Treatment with haloperidol, a dopamine receptor D‐2 antagonist, for one month resulted in an increase in the mean percentage of asymmetric synapses containing a discontinuous, or perforated, postsynaptic density (PSD) [Meshul et al. (1994) Brain Res., 648:181–195] and a change in the density of striatal glutamate immunoreactivity within those presynaptic terminals [Meshul and Tan (1994) Synapse, 18:205–217]. We speculated that this haloperidol‐induced change in glutamate density might be due to an activation of the corticostriatal pathway. To determine if activation of this pathway leads to similar morphological changes previously described following haloperidol treatment, GABA (10−5 M, 0.5 μl) was injected into the thalamic motor (VL/VM) nuclei daily for 3 weeks. This treatment resulted in an increase in the mean percentage of striatal asymmetric synapses containing a perforated PSD and an increase in the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated or non‐perforated PSD. Subchronic injections of GABA into the thalamic somatosensory nuclei (VPM/VPL) had no effect on the mean percentage of synapses with perforated PSDs but resulted in a small, but significant, increase in density of glutamate immunoreactivity. Using in vivo microdialysis, an acute injection of GABA (10−5 M, 15 μl) into VL/VM resulted in a prolonged rise in the extracellular level of striatal glutamate. The increase in asymmetric synapses with perforated PSDs and in glutamate immunoreactivity within nerve terminals of the striatum following either subchronic haloperidol treatment or GABA injections into VL/VM suggest that an increase in glutamate release may be a common factor in these two experiments. It is possible that the extrapyramidal side effects associated with haloperidol treatment may be due, in part, to an increase in release of glutamate within the corticostriatal pathway.

Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [3H]flunitrazepam and the potency of gamma-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities.

Genetic components of ethanol responses

A powerful technique for determining the role of a particular neurotransmitter in mediating a response to ethanol (EtOH) is the analysis of selectively bred lines of animals. Lines selected for sensitivity and resistance to an EtOH effect differ principally in gene frequencies for genes affecting the selected response. Hence, other differences between the lines are likely due to pleiotropic actions of those genes. We discuss behavioral pharmacological experiments in two sets of selected lines. Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mouse lines were selected for severe and minimal handling-induced convulsions (HIC), respectively, after withdrawal from chronic EtOH inhalation. The HIC is also elevated after acute administration of low doses of convulsant drugs. WSP mice were found to be more sensitive than WSR mice to many such drugs. There was no apparent specificity of such effects to any particular neurotransmitter system. Thus, genetic determination of a behavioral response to EtOH in this case cannot be traced to the influence of a single neurotransmitter system. COLD and HOT mice were selectively bred to show severe and mild hypothermia, respectively, after acute EtOH administration. COLD mice are also more sensitive to a number of other alcohols, barbiturates, and other general central nervous system depressants. When tested for sensitivity to a number of drugs with specific effects on neurotransmitter systems, COLD and HOT mice did not differ in sensitivity to drugs affecting dopaminergic, alpha-adrenergic, or nicotinic acetylcholinergic systems. COLD mice were more sensitive, however, to opioid and serotonergic drugs. Thus, analysis of these selected lines was successful in identifying particular neurotransmitters which may be important in EtOH-induced hypothermia.

Audiogenic Seizure Susceptibility in WSP and WSR Mice

Summary: Mice selectively bred for susceptibility (WSP, withdrawal seizure prone) and resistance (WSR, withdrawal seizure resistant) to ethanol (EtOH) withdrawal seizures were tested for susceptibility to audiogenic sei–zures (AGS). The seizure response of mice was studied at four ages: 17, 22, 28, and 71–78 days. WSR mice exhibited no response at any age, whereas WSP mice were sensitive on days 17, 22, and 28. The maximum number of WSP mice responding to audiogenic stimulation was observed on day 22. However, the frequency and severity of responses by WSP mice was less than that of DBA/2J mice tested under identical conditions (60 vs. 100% showing at least Some response). Overall, these data suggest that susceptibility to AGS and handling‐induced convul‐sions (HIC) during EtOH withdrawal may share some common genetic determinants and presumably some common neurochemical systems. Various treatments have been shown to enhance HIC more in WSP as compared with WSR mice. Acoustic stimulation did not induce AGS in adult mice, but the treatment significantly enhanced HIC in WSP but not WSR mice. These data strongly imply that some common neurochemical pathway may regulate susceptibility to HIC elicited by diverse treatments.

Serotonin and genetic differences in sensitivity and tolerance to ethanol hypothermia

Mice have been selectively bred for genetic sensitivity (COLD) or insensitivity (HOT) to acute ethanol-induced hypothermia. COLD mice readily develop tolerance to the hypothermic effects of ethanol (EtOH) when it is chronically administered, while HOT mice do not. A number of studies have implicated serotonergic systems in both sensitivity and the development of tolerance to the hypothermic and ataxic effects of EtOH. In the experiments reported here, we administered the serotonin (5HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to HOT and COLD mice before the acute and chronic administration of equipotent doses of EtOH. 5,7-DHT lesions significantly reduced (by about 65%) whole brain levels of 5HT in both selected lines. This treatment reduced sensitivity to acute EtOH hypothermia in COLD, but not in HOT mice, and blocked the development of tolerance only in COLD mice. Metabolites of 5HT, norepinephrine, and dopamine were generally increased in hypothalamic and brain stem tissue after acute EtOH injection, but HOT and COLD mice were not differentially susceptible to these effects. These results suggest that genes affecting 5HT systems may mediate some of the differences in response to the hypothermic effects of EtOH characterizing HOT and COLD mice.

μ-Opiate receptor binding and function in HOT and COLD selected lines of mice

mu-Opiate receptor binding and function were examined in mice selectively bred for sensitivity (COLD) and resistance (HOT) to ethanol-induced hypothermia. These mice also have differential hypothermic sensitivity to mu-opiates. mu-Opiate receptor density was higher in the frontal cortex of HOT mice compared with COLD mice, but was the same in other brain areas. In addition, there were no line differences in Kd values. Basal adenylate cyclase (AC) activity was similar in both lines, as was the response to forskolin (FS) stimulation. Morphine was more effective at inhibiting FS-AC activity in the hypothalamus of HOT mice compared with COLD mice but was equally effective in the frontal and parietal cortex. There were no differences between lines in basal Ca2+, Mg2+, or Ca2+/Mg(2+)-ATPase activity. Further, 30 min after treatment ATPase activities were not altered in ethanol- or levorphanol-treated mice. These results suggests that mu-opiate biochemical pathways, but not ATPase enzyme systems, may be involved in mediating differential hypothermic sensitivity observed in HOT and COLD mice.

Olfactory Bulb Kindling in Mice Susceptible and Resistant to Ethanol Withdrawal

Summary: The relation between kindling and susceptibil ity to ethanol withdrawal seizures was investigated using withdrawal seizure‐prone (WSP) and withdrawal seizure resistant (WSR) mice. These lines were developed by se lective breeding to be prone and resistant, respectively, to handling‐induced convulsions after chronic exposure to ethanol. Development of kindled seizures in response to electrical stimulation of the olfactory bulb was inves tigated in mice aged 2 and 8 months with no exposure to ethanol. Older WSP mice kindled more slowly than older WSR mice, requiring significantly more stimulations to reach the first stage 3 and the first stage 5 seizures. In younger mice, there was no significant difference be tween the two lines in the rate of kindling. The lower kindling rate in mature WSP mice is in contrast to their higher sensitivity to handling‐induced convulsions on withdrawal from ethanol and other agents. This finding suggests that separate genetic factors underlie these two models of mouse seizures.

[D-Ala 2 ]Deltorphin

[D-Ala2]Deltorphin I and II, heptapeptide relatives of deltorphin, are isolated from the skin of the Argentinian frog Phyllomedusa bicolor …