The motivation to obtain nicotine-conditioned reinforcers depends on nicotine dose.

Stimuli associated with nicotine (NIC) can acquire new meaning via Pavlovian conditioning. If a stimulus is associated with the primary reinforcing effects of NIC, the new conditional properties of the stimulus should make it a more valuable reinforcer (i.e., increase the motivation to obtain the stimulus), and this value should be based, in part, on the strength or intensity of the primary reinforcer (i.e., NIC dose). The purpose of the present study was to investigate whether NIC-conditioned reinforcement increased motivation to obtain non-NIC stimuli, as reflected by performance on a progressive ratio (PR) reinforcement schedule, and whether this increased motivation was systematically related to NIC dose. Two Paired groups were allowed to nose-poke for NIC (0.03 or 0.09mg/kg/infusion, IV) accompanied by 15-s illumination of a stimulus light (conditional stimulus or CS). Two Unpaired groups (0.03 or 0.09mg/kg/infusion) could also make a nose-poke response for the CS; however their NIC infusions were controlled by the Paired group (i.e., yoked design). A fifth group (CS-Only) was allowed to nose-poke for CS presentations and saline infusions. After 29 conditioning sessions the nose-poke operant was prevented by obscuring the receptacle and the CS (accompanied by saline infusion for all groups) was made contingent upon a novel operant response (lever press). During the acquisition of this novel response, each CS/saline infusion earned increased the number of responses required to earn the next CS/saline infusion. Pairings with the primary reinforcing effects of NIC resulted the acquisition of a novel response for the CS. Motivation to obtain the CS depended on salience (dose) of the primary reinforcement (NIC).

Nicotine serves as a feature-positive modulator of Pavlovian appetitive conditioning in rats.

The present experiments examined whether a nicotine state could set the occasion for a pairing between visual cues and a rewarding outcome in rats. Following nicotine administration, presentation of a conditional stimulus (CS; light-on) was followed by brief access to a sucrose solution. When saline was administered, the same CS was presented but was not followed by any consequence. In Experiment 1, two groups assessed whether rats could acquire this Pavlovian feature-positive discrimination via different training procedures. An anticipatory food-seeking conditioned response (CR) developed during the CS on nicotine sessions but not on saline sessions in both groups. In Experiment 2, centrally acting antagonists of nicotinic acetylcholine and opiate receptors (mecamylamine and naloxone, respectively) dose-dependently blocked nicotine's control of the CR, whereas the peripherally acting nicotinic antagonist hexamethonium had no effect. Increasing or decreasing the interval between nicotine administration and testing also attenuated the CR. These results are consistent with the hypothesis that nicotine can occasion appetitive Pavlovian relations via its action at central nervous system cholinergic receptors.

COMT haplotypes suggest P2 promoter region relevance for schizophrenia.

A recent study found, in a large sample of Ashkenazi Jews, a highly significant association between schizophrenia and a particular haplotype of three polymorphic sites in the catechol-O-methyl transferase, COMT, gene: an IVS 1 SNP (dbSNP rs737865), the exon 4 functional SNP (Val158Met, dbSNP rs165688), and a downstream SNP (dbSNP rs165599). Subsequently, this haplotype was shown to be associated with lower levels of COMT cDNA derived from normal cortical brain tissue, most likely due to cis-acting element(s). As a first step toward evaluating whether this haplotype may be relevant to schizophrenia in populations other than Ashkenazi Jews, we have studied this haplotype in 38 populations representing all major regions of the world. Adding to our previous data on four polymorphic sites in the COMT gene, including the Val158Met polymorphism, we have typed the IVS 1 rs737865 and 3' rs615599 sites and also included a novel IVS 1 indel polymorphism, yielding seven-site haplotype frequencies for normal individuals in the 38 globally distributed populations, including a sample of Ashkenazi Jews. We report that the schizophrenia-associated haplotype is significantly heterogeneous in populations worldwide. The three-site, schizophrenia-associated haplotype frequencies range from 0% in South America to 37.1% in Southwest Asia, despite the fact that schizophrenia occurs at roughly equal frequency around the world. Assuming that the published associations found between the exon 4 Val158Met SNP and schizophrenia are due to linkage disequilibrium, these new haplotype data support the hypothesis of a relevant cis variant linked to the rs737865 site, possibly just upstream in the P2 promoter driving transcription of the predominant form of COMT in the brain. The previously described HindIII restriction site polymorphism, located within the P2 promoter, varies within all populations and may provide essential information in future studies of schizophrenia.

Effects of chronic caffeine pre-exposure on conditioned and unconditioned psychomotor activity induced by nicotine and amphetamine in rats.

Three experiments examined the effects of chronic pre-exposure to caffeine on the subsequent conditioned and unconditioned locomotor activating effects of nicotine or amphetamine in rats. Rats were given daily intraperitoneal injections of caffeine anhydrous (0, 10 or 30 mg/kg base) for 30 days. Conditioning (environment-drug pairings) began after the last day of caffeine pre-exposure. Pre-exposure to 30 mg/kg of caffeine enhanced the acute and chronic locomotor effects of amphetamine (0.5 mg/kg). A similar enhancement of activity was not seen with the high (0.421 mg/kg base) or low dose (0.175 mg/kg) of nicotine. In a drug-free test, the distinct environment paired with amphetamine and the high dose of nicotine evoked increases in activity relative to controls. Caffeine pre-exposure did not affect expression of this conditioned hyperactivity. These effects of caffeine pre-exposure on amphetamine-induced activity could not be attributed to non-specific effects of caffeine.

Chronic caffeine exposure in rats blocks a subsequent nicotine-conditioned taste avoidance in a one-bottle, but not a two-bottle test.

Two experiments were conducted in order to investigate nicotine-conditioned taste avoidance (CTA) following chronic preexposure to caffeine. Rats were given daily intraperitoneal injections of caffeine anhydrous (0, 10, or 30 mg/kg) for 10 or 30 days. Training of the nicotine-CTA began after the last day of caffeine preexposure. On five separate occasions access to a saccharin solution was followed immediately by an injection of 1.2 mg/kg nicotine hydrogen tartrate salt or saline. Nicotine-CTA readily developed in saline-preexposed controls. That is, paired rats drank less saccharin solution than unpaired rats after repeated saccharin-nicotine pairings. A similar pattern of nicotine-CTA was found for rats preexposed to 30 mg/kg caffeine for 10 days. Following 10 days of preexposure to 10 mg/kg caffeine, however, CTA did not develop under standard testing conditions. Thirty days of caffeine preexposure did not affect the development of a nicotine-CTA even though the anorexic effects of caffeine were evident after exposure to 30 mg/kg for this duration. Thus, caffeine exposure appears to weaken acquisition or expression of the conditioned avoidance properties of nicotine. This effect is sensitive to the dose of caffeine and duration of preexposure. Importantly, the pattern of nicotine-CTA does not appear to be due to nonspecific effects of caffeine.

Global variation in the frequencies of functionally different catechol-O-methyltransferase alleles.

BACKGROUND: Catechol-O-methyltransferase (COMT) has been investigated as a candidate gene in many neurologic disorders involving catecholaminergic systems. The NlaIII restriction site polymorphism (RSP) at COMT is a G<-->A (site absent<-->site present) single nucleotide polymorphism (SNP) at nucleotide 322/472 (in the short or long mRNA) that results in a Val<-->Met polymorphism at amino acid 108/158 (in soluble or membrane-bound) COMT protein and different enzyme activity levels, high for Val, low for Met. COMT enzyme activity is known to vary among ethnic groups, presumably as a result of different population frequencies of these COMT alleles. We have undertaken a direct survey of allele frequencies of this polymorphism in a global sample of populations. METHODS: We typed 1314 individuals from 30 different populations using PCR of the relevant region followed by digestion with NlaIII and electrophoresis. RESULTS: The frequencies of the low activity allele (COMT*L, NlaIII site-present) vary significantly from 0.01 to 0.62. Europeans have nearly equal frequencies of the two alleles while the COMT*H allele is much more common in populations in all other parts of the world. Sequencing in nonhuman primates indicates that COMT*H is the ancestral allele in humans. CONCLUSIONS: This is the first global survey of the COMT*L and COMT*H allele frequencies, confirming and extending earlier studies to show significant world-wide variation. This is also the first study establishing the COMT*L allele as the derived allele unique to humans. Henceforth, in any population-based association studies of this polymorphism, the control allele frequencies should be in agreement with these published values for corresponding ethnic groups.

Global variation of a 40-bp VNTR in the 3'-untranslated region of the dopamine transporter gene (SLC6A3).

BACKGROUND: The dopamine transporter (DAT) is the primary mechanism for dopamine clearance from the synapse in midbrain dopaminergic neurons, and the target of psychostimulant and neurotoxic drugs such as cocaine, amphetamine, and MPTP. Consequently, the gene for DAT (SLC6A3) has been the focus of many population-based case-control association studies using a 40-bp VNTR in the 3'-untranslated region. Results have differed depending on the population studied, suggesting allele frequency effects are involved. For this reason, a global survey of allele frequencies for this VNTR polymorphism was performed. METHODS: Individuals (n = 1528) from 30 populations around the world were typed for this VNTR using PCR and agarose gel electrophoresis. RESULTS: As with previous studies, the ten-repeat allele is most common, except for a Middle Eastern population in which the nine-repeat allele is most frequent. Frequencies of the nine- and ten-repeat alleles vary widely even among European populations. CONCLUSIONS: Many previous association studies have used "white" or "black" U.S. populations. However, many different ethnic groups have contributed to these populations. The large variation in allele frequencies observed in this study emphasizes the inadequacy of most past studies using the case-control design and the importance of matching patient and control populations in future association studies.

A latent process regression model for spatially correlated count data.

This paper proposes a regression model for spatially correlated count data that generalizes the work of Zeger (1988, Biometrika 75, 621-629) developed in a time-series setting. In this approach, spatial correlation is introduced through a latent process, and the marginal mean function may contain spatial trends and covariates. Generalized estimating equations are used to estimate and perform marginal inference on the spatial trend and covariate effects. The feasibility of this approach is demonstrated using an example of the distribution of neuronal cell counts in a laboratory culture dish.

Reversal of hemiparkinsonian syndrome in nonhuman primates by amnion implantation into caudate nucleus.

Although recent animal and human experiments suggest that tissue implantation can ameliorate parkinsonism, there is controversy about what mechanism underlies recovery. Secretion of dopamine from the graft seems unlikely to be the sole restorative factor. Regenerative sprouting by the host brain may also underlie behavioral recovery. Fetal amnion and term amnion, which were shown to produce and secrete a factor that supports the outgrowth of neurite processes in vitro, were implanted in hemiparkinsonian monkeys. Fetal amnion implants induced sprouting of dopaminergic fibers from the host brain and behavioral improvement, despite failure of the grafts to survive. Animals implanted with term amnion also had some sprouted dopaminergic fibers and behavioral improvement, but these were limited and were similar to the recovery, in prior experiments using the same primate model of parkinsonism, of animals that received surgical cavitation only. Recovery after central nervous system grafting with fetal amnion, a fetal accessory tissue, does not require secretion of a deficient neurotransmitter(s) from the graft and occurs despite the failure of graft survival. Recovery after cerebral implantation of fetal tissues appears to depend more on the regenerative and recuperative processes of the host brain than on graft replacement of deficient neurotransmitters or development of functional synaptic connections between the graft and the host brain.

Spatial distribution of neurons in tissue culture wells: implications for sampling methods to estimate population size.

Many laboratory procedures require the counting of cells in culture. While many cultured cells may be counted by automated methods, neuronal cultures often require manual cell counting methods that are prohibitively time-consuming. This paper examines methods of sampling from tissue culture wells for estimating total cell counts. Performance of sampling and estimation schemes will depend in part on how the cells distribute themselves within a well. Spatial statistical analysis techniques are applied to the known total number and distribution of neurons in two wells counted in a grid scheme to demonstrate some important features of the neuron distributional patterns. Based on these two wells and simulated realizations from other point processes, a new sampling and estimation technique using open wedge-shaped sampling regions radiating from the centre of the well is proposed. This method is shown to result in more accurate estimates of the total number of neurons in the well than standard methods.

Dopaminergic neuronal sprouting and behavioral recovery in hemi-parkinsonian rats after implantation of amnion cells.

Cells obtained from human, monkey, or rat term amnion membrane produce an activity which, in vitro, increases process outgrowth from rat sympathetic neurons and from dopaminergic neurons of the rat ventral mesencephalon. To determine if these cells could induce sprouting of dopaminergic nerve fibers in vivo, the substantia nigra of rats was lesioned unilaterally with 6-hydroxydopamine and live-rat-term amnion cells, or killed-rat-term amnion cells were implanted into the denervated striata. A control group of rats received saline injections into the denervated striata. Rats implanted with live amnion cells had a significant decrease in turning in response to amphetamine. The lesioned and implanted striata of live-amnion-cell-implanted rats contained significantly greater areas of tyrosine hydroxylase-immunoreactive fibers than the lesioned and implanted striatum of rats in the killed-amnion-cell or saline groups. Differences in the area of tyrosine hydroxylase-immunoreactive fibers in the implanted striata or in amphetamine-induced rotation between killed amnion cell-implanted and saline-injected rats did not reach significance. Implants of live amnion cells into the striatum of a parkinsonian animal model can evoke the de novo appearance of dopaminergic fibers in the denervated striatum and behavioral recovery, most likely through a trophic mechanism.

Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release L-dopa in vitro.

Two PC12 cell-derived lines have been studied following encapsulation into polymer-based hollow fibers with respect to secreted catecholamines and their metabolites. Cellular encapsulation provides a chronic microperfusion environment within which basally secreted PC12 products can be readily measured. Encapsulated PC12 cells grown and held under the conditions specified in this report basally release amounts exceeding their total cellular stores of the dopamine precursor L-DOPA and the electrochemically active dopamine metabolites DOPAC and HVA during 45-min static incubations. Under these same conditions, these cells release less than 0.1% of their total cellular store of dopamine. Depolarizing incubations enhance dopamine secretion eightyfold and enhance secretion of L-DOPA, HVA, and DOPAC about twofold. The relative composition of products basally secreted differs between PC12-derived cell lines, and an inverse relationship exists between basal release of L-DOPA and total cellular store of dopamine. These results further indicate that selected PC12 cell lines are potentially a source of both dopamine and L-DOPA in therapeutic cellular replacement applications.

A novel approach to neural transplantation in Parkinson's disease: use of polymer-encapsulated cell therapy.

Transplantation of dopaminergic neurons derived from fetal or adrenal tissue into the striatum is a potentially useful treatment for Parkinson's disease (PD). Although initially promising, recent clinical studies using adrenal autografts have demonstrated limited efficacy. The use of human fetal cells, despite promising preliminary results, is complicated by tissue availability and ethical concerns. An attractive alternative is based on encapsulating dopamine-producing cells into polymer capsules prior to transplantation. Polymer capsules can be fabricated to surround the cells with a semi-permeable and immunoprotective barrier. The semi-permeable membrane allows nutrients to enter the capsule, so the encapsulated cells will survive and function, and dopamine and other low molecular weight constituents to diffuse out into the host tissue. Thus, the technique allows use of unmatched human tissue (allografts), or even animal tissue (xenografts) without immunosuppression of the recipient. Cell-loaded polymer capsules can also be retrieved if necessary or desired. The demonstration that striatal implants of encapsulated dopamine-producing cells promote behavioral recovery in rodent and primate models of PD further suggests that cellular encapsulation may be a useful strategy for ameliorating the behavioral consequences of PD.

MHC-specific cytotoxic T lymphocyte killing of dissociated sympathetic neuronal cultures.

Experiments were conducted to determine whether neurons in culture can serve as targets for immunologic attack mediated by major histocompatibility complex (MHC)-specific cytotoxic T lymphocytes (CTLs) which recognize Class I antigens. Allogeneic C3H/He primary neuronal cultures were quickly destroyed after CTL addition, while syngeneic C57BL/6J neurons were not lysed. Alterations in the distribution of chromatin were the first ultrastructural changes that occurred, followed by loss of nuclear morphology, cytosolic changes, and eventually fragmentation of both the nucleus and cytosol. With Campenot chambers, it was possible to separate the membrane and nuclear lesions. CTLs exposed to neurites, but separated from the cell body by the chamber barrier, caused degeneration of neurites but did not cause lysis and cell death. Neuronal lysis mediated by antibody and complement appeared to be distinct from CTL-mediated lysis. These experiments demonstrate that neurons in culture are targets for MHC-specific CTLs, and therefore probably express functional levels of Class I antigens. The signal for killing by CTLs is not retrogradely transported from the neurite to the cell body, and morphologic events following CTL-neuron interaction resemble those that occur in dividing tumor target cell populations.

Sympathetic neuronal destruction in macaque monkeys by guanethidine and guanacline.

To determine whether the peripheral sympathetic neurons of subhuman primates are destroyed by guanacline treatment, we treated Macaca fasicularis with 2 or 20 mg/kg of guanethidine, guanacline, or the saturated analog of guanacline (SAG) 5 times per week for 4 or 12 weeks. All monkeys given 20 mg/kg of guanethidine, guanacline, or SAG showed a marked loss of neurons in the ganglia of the peripheral sympathetic nervous system. Treatment of macaques with 2 mg/kg of the guanidinium compounds resulted in patches of small-cell infiltrate, slight neuronal loss, and degenerative alterations in the sympathetic ganglia. Neuronal alterations in sympathetic ganglia of all treated monkeys were accompanied by a prominent heterogeneous infiltrate of mononuclear cells arranged primarily in a perivascular distribution and extending into the ganglionic neuropil. Peripheral sensory ganglia were unaffected. These histological findings are similar to those described in the guanethidine-induced immune-mediated sympathectomy, which has been extensively studied in the rat.

Species and structural specificity of the lipopigment accumulation and neuronal destruction induced by N-(2-guanidinoethyl)-4-methyl-1,2,5,6-tetrahydropyridine (guanacline).

Guanacline, a guanidinium adrenergic neuron blocking agent similar to guanethidine, was studied clinically and experimentally during the late 1960s. Like guanethidine, it has been reported to produce sympathetic neuronal destruction in rats. Unlike guanethidine, it has been reported to produce irreversible sympathetic deficits in man and to produce fluorescent lipopigment in rat sympathetic neurons. Guanacline and its derivative in which the double bond of the tetrahydropyridine ring is reduced (saturated analog of guanacline, SAG) were prepared. Several species were treated chronically with varying doses of guanethidine, guanacline or SAG; the superior cervical ganglia were examined light microscopically for neuronal destruction and for osmiophilic fluorescent lipopigment accumulation. All 3 drugs produced rapid neuronal destruction in rats accompanied by massive small-cell infiltration. In striking contrast, treatment for many weeks with doses up to 100 mg/kg/day produced no small-cell infiltration or apparent neuronal destruction in mice or guinea pigs. The neuronal destruction produced by guanacline and SAG in the rat, like that caused by guanethidine, was prevented by immunosuppression or gamma-irradiation, indicating that all 3 agents produce neuronal destruction in rats by an immune-mediated mechanism. Thus, the ability of the drug to produce sympathectomy is species specific but not drug specific. The opposite was found with respect to fluorescent lipopigment accumulation. Guanacline, but not guanethidine or SAG, produced fluorescent lipopigment in all species examined. Therefore, the double bond of the tetrahydropyridine ring plays a critical role in the production of the fluorescent lipopigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of retrogradely transported endogenous nerve growth factor in axons of sympathetic neurons.

The presence of retrogradely transported endogenous nerve growth factor (NGF) in sympathetic nerves of the guinea pig was demonstrated directly by fluorescent and peroxidase immunohistochemistry in the ligated superior postganglionic nerve of the superior cervical ganglion. Fixed, frozen sections of previously ligated nerve were incubated with either rabbit antiserum against guinea pig NGF (gpNGF), rabbit antibodies against gpNGF purified on a mouse NGF (mNGF) affinity column, the portion of rabbit antiserum against gpNGF that did not bind to the mNGF affinity column, or nonimmune rabbit serum. Positive staining on the peripheral side of the ligation was obtained only with unfractionated antiserum against gpNGF and with purified antibodies against gpNGF. The staining properties of the various antiserum preparations correlated with their ability to block gpNGF- and mNGF-induced neurite outgrowth in the embryonic chick dorsal root ganglion bioassay and in the PC12 bioassay. Homogenates of superior postganglionic nerve supported growth of embryonic chick dorsal root ganglia and differentiation of PC12 cells. This support was blocked by the specific antisera against NGF used in the immunohistochemistry experiments. These experiments demonstrate that endogenous NGF, presumably released by peripheral target tissues, is retrogradely transported in vivo.