Developmental neurotoxicity to methamphetamines.

1. To investigate the long-term changes caused by amphetamines in the developing brain, we used both an in vivo and in vitro model of chronic fetal exposure to methamphetamine and related drugs. 2. Offspring of rats, treated with either saline, 2 mg/kg twice a day (b.i.d.) or 10 mg/kg b.i.d. methamphetamine throughout gestation, were examined at 30 days of age for changes in the monoamine system of their brains. 3. At the lower dose methamphetamine was neurotoxic to specific neuronal populations, mostly serotonergic. At the higher dose, methamphetamine retained its neurotoxic properties, but also stimulated the growth of axonal terminals in specific regions as evidenced by an increase in monoamine uptake sites. The neurochemical changes at the higher dose were correlated with deficits in adult behavioural measures. 4. Corresponding in vitro drug treatments of rat neuroblastomas cells also produced a dose-related effect on cellular growth and differentiation patterns. Neurotoxic as well as stimulatory effects of methamphetamine and some related compounds were seen in culture. 5. Our in vivo and in vitro observations demonstrate neurotoxic effects of amphetamines and the remodelling of synaptic morphology in response.

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns.

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.

An absence of changes in sigma receptor subtypes in the brains of genetically dystonic (dt) rats.

Binding sites for the sigma ligand [3H]di-o-tollylguanidine ([3H]DTG) have been reported to be altered in the brains of genetically dystonic rats. In the present study, selective sigma 1 and sigma 2 assay conditions were utilized in an effort to define which subpopulation of [3H]DTG binding sites is altered in the dystonic strain (dt). Both this approach and a re-examination using conditions similar to the previous report failed to confirm a difference between the brains of dystonic and normal rats in terms of sigma binding. Although not directly negating the possible involvement of sigma receptors in dystonia, these results indicate that sigma 1 and sigma 2 receptors appear unchanged in dystonic rats.

In utero methamphetamine effects: I. Behavior and monoamine uptake sites in adult offspring.

Chronic in utero methamphetamine treatment, throughout gestation in rats, resulted in alterations in both behavior and brain monoamine function in the adult offspring. The higher dose of methamphetamine (10 mg/kg/b.i.d.) caused a significant decrease in square crossing and rearing in an open field, as well as a regional increase of serotonin and dopamine uptake sites. In contrast, the lower dose of in utero methamphetamine (2 mg/kg/b.i.d.) resulted in a significant decrease in regional densities of serotonin and dopamine uptake sites, and only decreased rearing behavior. Across treatment groups, there were significant correlations between open-field square crossing activity and the number of uptake sites in specific brain areas. Other measured behaviors, such as the neonate righting reflex and the adult Morris water maze performance, were unaffected by either in utero drug regimen. These results are discussed in terms of the known neurotoxicity of amphetamines and the ability of the immature nervous system to compensate for fetal exposure to methamphetamine.

Chronic treatment of rats with the specific sigma ligand D-pentazocine fails to modulate dopamine D2 and sigma binding in brain.

Drugs that bind at the sigma site have been proposed to have psychotomimetic and/or antipsychotic activity that is independent from that mediated by the phencyclidine (PCP) receptor. Some evidence suggests that sigma ligands may produce their effects by modulation of dopamine neurotransmission. The present study examined potential interactions between the sigma and dopamine system following chronic treatment with d-pentazocine, a specific sigma ligand that has negligible affinity for either PCP or dopamine receptors. Rats treated for one month with either saline or 10 mg/kg per day d-pentazocine revealed no significant differences in either the KD or Bmax of [3H]haloperidol-labeled sigma or [3H]YM-09151-2-labeled dopamine D2 receptors across multiple brain regions. These results are discussed in relation to the pharmacology of putative sigma-receptor subtypes.

PCP and sigma receptors in brain are not altered after repeated exposure to PCP in humans.

The psychotomimetic effects of phencyclidine (PCP) in humans can persist or reappear months after the drug has been eliminated, suggesting that PCP can induce long-term changes in the brain. The present study examined whether repeated exposure to PCP in a human drug-addicted population was accompanied by alterations in either PCP or sigma binding sites in their postmortem brains as compared to suicide controls. Saturation studies using [3H]TCP and [3H]haloperidol in the presence of spiperone to measure PCP and sigma sites, respectively, revealed no significant differences in the affinity or density of binding sites between these two clinical populations in a variety of brain areas examined. The results suggest that these brain binding sites remain unperturbed in humans despite multiple challenges with PCP. Delayed psychotic episodes following the use of PCP may be attributed to other neurochemical changes that are initiated by interactions of PCP with these two binding sites.

Selective loss of cerebral cortical sigma, but not PCP binding sites in schizophrenia.

Drugs such as phencyclidine (PCP) that interact with PCP and sigma binding sites can produce psychotomimetic effects that resemble some symptoms of schizophrenia. Therefore, it has been suggested that PCP and sigma receptors may be important in the clinical manifestations of schizophrenia. Assays of these two binding sites in human postmortem brains showed consistent significant reductions in the density of sigma, but not PCP sites, in schizophrenics as compared with age-matched and postmortem interval-matched normal and suicide controls. Reductions in the density of sigma binding sites in schizophrenia were most prominent in temporal cerebral cortex, and were accompanied by a small increase in affinity for the ligand [3H]haloperidol. These data provide the first evidence for alterations in sigma binding sites in schizophrenia, and suggest that selective sigma ligands may be useful in the treatment of the disorder.

In vivo binding of [3H]d-N-allylnormetazocine and [3H]haloperidol to sigma receptors in the mouse brain.

Specific binding to sigma sites has been demonstrated and characterized in vitro using [3H]d-N-allylnormetazocine ([3H]d-NANM) and [3H]haloperidol ([3H]HAL) as ligands. As an extension of these experiments, we examined the regional in vivo specific binding of [3H]d-NANM and [3H]HAL in the mouse brain. Specific in vivo sigma binding was seen with both ligands; average estimates of specific binding across brain regions were 54 per cent and 56 per cent of total brain radioactivity, using [3H]d-NANM and [3H]HAL, respectively. Both ligands showed high levels of specific binding in the cerebellum, medulla-pons and midbrain, and lowest levels in the hippocampus. Estimated average [3H]d-NANM binding to phencyclidine (PCP) receptors across seven brain regions was only 13 per cent of total brain radioactivity, and showed a more uniform regional distribution than sigma binding. While the distributions of in vivo specific binding of [3H]d-NANM and [3H]HAL to sigma sites were comparable to findings obtained in vitro, the present estimates of in vivo [3H]d-NANM binding to PCP sites did not resemble the distribution of PCP receptors found in vitro. The results suggest that radiolabelled d-NANM and HAL may be useful for imaging sigma binding sites in vivo.

Pharmacological characteristics and distributions of sigma- and phencyclidine receptors in the animal kingdom.

The phylogenetic distributions of sigma- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [3H]haloperidol and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to sigma- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [3H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that sigma- and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.

Effects of self-administered phencyclidine on regional uptake of 2-deoxy-D-[1-14C]glucose in brain.

Phencyclidine profoundly alters cerebral metabolism in the rat. This study explored whether cerebral metabolic effects of phencyclidine differed when the drug was self-administered by trained rats, compared with when it was given acutely to naive rats. The regional cerebral uptake of 2-deoxy-D-[1-(D14C] glucose (DG) was examined following two injections of phencyclidine (0.5 mg/kg/injection, i.v.) or saline in freely-moving, drug-experienced rats. Naive controls received phencyclidine or saline according to an identical dose regimen. In self-administering and naive rats, phencyclidine produced many of the same effects on uptake of DG, including the following: decreases in the habenula, inferior colliculus, sensory cortical areas and corresponding thalamic relay nuclei; and increases in limbic areas (entorhinal and retrosplenial cortices, subicular areas). Some regions (auditory and motor cortices, medial geniculate body, globus pallidus) showed different effects in self-administering and naive rats. Another study, in which rats were not self-administering phencyclidine, but had histories of treatment with drugs similar to those of the self-administering rats, indicated that chronic exposure to drug accounted for some of the differences. Furthermore, differences between the effects of phencyclidine in self-administering, versus non-self-administering rats with similar histories suggested that activity in some regions of the brain may relate to training in drug self-administration and/or behavior.

Sigma receptors in post-mortem human brains.

The pharmacological profile and regional distribution of sigma receptors in human autopsy brains were determined using [3H]-haloperidol as the ligand, in the presence of 50 nM spiperone to block binding to D2 dopamine sites. Specific binding in the cerebellum was to a single and saturable class of receptors with Kd = 0.95 +/- 0.12 nM and maximum binding = 358 +/- 13 fmol/mg of protein. Inhibition studies of the nondopamine [3H]haloperidol binding site in human cerebellar membranes revealed stereospecific binding and a pharmacological profile similar to that of specific sigma binding sites characterized in rodent brains using [3H]haloperidol, N-[3H]allylnormetazocine and di-o-[3H]-tolylguanidine as radioligands. The densities of sigma sites in the brain were highest in the cerebellum, nucleus accumbens and cerebral cortex. A post-mortem simulation study was performed with guinea pig brains due to the concern that sigma receptors might deteriorate in the human brain before assay. The results showed that this site was remarkably stable and insensitive to long periods of cooling or freezing.

Cocaine produces fine structural nuclear alterations in cultured neuroglioblastoma cells.

NG 108-15 neuroglioblastoma cells were grown in culture medium containing either 10(-3), 10(-6) or 10(-9) M cocaine for 1-3 days. Some cultures were assessed for viability while others were processed for electron microscopy. Following 1-3 days of cocaine, no cytoplasmic alterations were observed compared to control; however, numerous dense bodies were present in some cells cultured with the highest doses. After 2 days of treatment with 10(-3) and 10(-6) M cocaine, nuclear invaginations were found filled with vesicles, and the nuclear membrane surrounding the vesicles was disrupted. Following 3 days of treatment with 10(-3) and 10(-6) M cocaine, patches of vesicles and tubules were also seen in the nucleus, but without surrounding membranes. The vesicles ranged in size from 0.05 to 0.8 micron. Cell counts revealed a significant slowing in the rate of cell division after two days of exposure to 10(-3) M cocaine. All concentrations of cocaine caused a significant decrease in cell viability by the third day of treatment. These results suggest that cocaine may interfere with cell replication and also may have a neurotoxic effect.

Alterations in local cerebral glucose utilization induced by phencyclidine.

The effects of phencyclidine (PCP; 0.5, 1,5, 10 mg/kg, i.v.) on local cerebral glucose utilization (LCGU) in the rat were studied with the 2-deoxy-D-[1-14C]glucose method. Significant findings were obtained in 41 of 87 brain regions of PCP-treated rats (25-270% of control). Rates of LCGU increased throughout the limbic system, except the habenula. Although LCGU increased in most sensory structures, it decreased in specific layers of the somatosensory and auditory cortices and the inferior colliculus. Evidence was seen for dissociation between LCGU responses of specific thalamic relay areas and their terminal fields in the cortex. Increases in LCGU occurred throughout the motor system, manifesting a striking pattern of columnar activity in the motor cortex. However, LCGU was reduced in the frontal cortical pole. Elevated LCGU was observed in the pontine nuclei and the nuclei and the nucleus solitarius. Effects of 5 mg/kg PCP diminished with time although 8 regions maintained a metabolic alteration at 180 min. PCP induced several behaviors, including stereotypies, which varied with the dose and time after drug administration. The results demonstrate a PCP-induced activation of various functional circuits in the brain, especially the limbic system, and may provide a physiological basis for PCP's psychotomimetic properties.

Extraordinary postmortem stability of kappa opioid receptors in guinea-pig brain.

Postmortem delay can be an important variable in biochemical studies on autopsy tissue. We subjected guinea-pig brains to gradual cooling, simulating temperature conditions of human postmortem brains, in order to assess the sensitivity of kappa receptors to postmortem degradation. Kappa receptor specific binding was defined as the (-)-[3H]ethylketocyclazocine bound in the presence of 100 nM D-ala2-D-leu5-enkephalin and 30 nM morphine. Postmortem delays of up to 16 h did not alter the affinity or density of kappa binding sites. The remarkable stability of kappa receptors may greatly facilitate the study of this opioid receptor subtype in human brain.

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays.

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.

A flexible high density multi-channel electrode array for long-term chronic implantation.

The construction of a high density multi-channel fluid potential electrode array is described. The resulting electrode array which is flat, flexible and non-irritating, is designed for long-term chronic implantation over cortical surfaces of compound curvature, as well as over restricted regions, such as the visual areas located along the medial bank of the occipital cortex. Electrophysiological data and gross anatomy obtained from animals with implantation times of up to one year are displayed.