Synapse

Recent postmortem studies have suggested that reduced γ‐aminobutyric acid (GABA)ergic activity in limbic cortex may be one component to the pathophysiology of schizophrenia. This hypothesis has underscored the importance of knowing whether midbrain dopamine afferents interact extensively enough with inhibitory interneurons to suggest a direct functional relationship. Toward this end, a double immunofluorescence approach combined with confocal laser scanning microscopy has been used to localize dopamine and GABA simultaneously in rat medial prefrontal cortex. The results confirm studies from other laboratories showing a rich network of dopamine‐immunoreactive fibers forming a gradient across the cortical laminae, with deeper layers having the highest density. When viewed with oil immersion optics, dopamine‐immunoreactive fibers were frequently found to be in close apposition with GABA‐immunoreactive cell bodies. The percentage of GABA‐containing neurons showing such contacts was highest in layer VI (65%) and progressively decreased toward layer I (9%). Varicose regions of the dopamine fibers were typically present at the point of contact with a GABA‐immunoreactive cell body. Using an immunoperoxidase technique to localize dopamine fibers and cresyl violet staining to visualize neurons simultaneously, two separate statistical analyses were performed to assess whether the frequency of contacts between dopamine fibers and cell bodies in general may be due to random effects. In layer VI, a high percentage of both pyramidal and nonpyramidal neurons were found to be in contact with dopamine varicosities (71% and 76%, respectively), but these were not significantly different from that observed for GABA‐containing cells (65%) in double‐immunofluorescence specimens. A Chi‐square statistical test was used to compare the observed and predicted number of varicosities forming cell body contacts. This analysis indicated that the percentage of dopamine varicosities (30%) that form appositions with cell bodies is much greater than would be expected if these appositions were due to random effects (15%). Moreover, using an estimate of intensity for a stationary Poisson process, it was again found that random effects can not account for these interactions (P = 0.01). Taken together with earlier electron microscopic studies from other laboratories, the present findings support the idea that GABAergic interneurons have extensive interactions with dopamine varicosities. While these interactions are not unique to GABAergic cell bodies, they suggest that inhibitory interneurons can play a direct role in mediating the effects of midbrain dopamine afferents in rat medial prefrontal cortex. © 1993 Wiley‐Liss, Inc.

In a recent study in rat medial prefrontal cortex (mPFC), a fluorescently coupled, high‐affinity ligand for the D₁ receptor subtype was localized to nonpyramidal neurons, while a ligand selective for the D₂ subtype was found on neurons with a size distribution overlapping with both small pyramidal and large nonpyramidal cells. These observations raised the possibility that a subpopulation of cortical neurons with an intermediate size range may coexpress both the D1 and D2 receptor subtypes. In the present study, the D₁ and D₂ receptor subtypes have been simultaneously localized in layer VI of rat mPFC using 20 nM SCH 23390‐Bodipy and 20 nM N‐(p‐aminophenethyl) spiperone‐Texas red, respectively, in the presence of 100 nM mianserin (5‐HT₂ receptor antagonist). The localization of receptor binding fluorescence was assessed in paired images using fluoresceiN isothiocyanate (FITC) and rhodamine dichroic filters for the D₁ and D₂ subtypes, respectively. Under the conditions employed here, most cell bodies showed either D_l‐like or D₂‐like receptor binding fluorescence, while a colocalization of both fluoroprobes was observed on only 25% of the labeled cells. When the size of each single‐labeled cell body was measured using the respective FITC (D₁‐probe) and rhodamine (D₂‐probe) epifluorescence filters, the distribution of cells showing only D₁‐like receptor binding fluorescence was similar to nonpyramidal neurons (68.6 ± 1.8 μm²), while that for cells showing only D₂‐like receptor binding fluorescence was similar to that of both large interneurons and small pyramidal cells (106.9 ± 2.4 μm²). Cells showing both D₁‐ and D₂‐like receptor binding fluorescence were found to overlap in size only with nonpyramidal cells when either fluorescent filter was used. These findings are consistent with the hypothesis that the D₁ and D₂ receptor subtypes are most often found on different populations of neurons in mPFC, although approximately 25% of all such cells appear to be nonpyramidal neurons having both D₁ and D₂ receptor binding activity. These findings suggest that the dopamine projections to rat cortex probably engage in a complex interplay with intrinsic cortical neurons and their respective neurotransmitter systems. © 1995 Wiley‐Liss, Inc.

We have compared rapid freezing followed by freeze‐substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 μM without visible ice crystals. Freeze‐substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze‐substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques. © 1993 Wiley‐Liss, Inc.

The cloned 5‐HT₃ receptor from NCB‐20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5‐HT₃ receptors were voltage‐clamped at −70 mV. Anandamide, at the concentration range of 0.1–100 μM, reversibly inhibited 1 μM 5‐HT induced currents. The inhibition of 5‐HT induced currents by anandamide was concentration‐dependent with an EC₅₀ of 3.7 μM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5‐HT‐induced currents. In the presence of 10 μM anandamide, the maximum 5‐HT‐induced response was also inhibited and the respective EC₅₀ values were 3.4 μM and 3.1 μM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5‐HT₃ receptors. CB₁ receptor antagonist SR‐141716A (1 μM) and pertussis toxin (5 μg/ml) did not cause a significant change on the inhibition of 5‐HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8‐Br‐cAMP, a membrane‐permeable analog of cAMP, or Sp‐cAMPS (0.1 mM), a membrane‐permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G‐proteins. In conclusion, we demonstrated that the endogenous cannabinioid anandamide inhibits the function of 5‐HT₃ receptors expressed in Xenopus oocytes in a cannabinoid‐receptor independent and noncompetitive manner. Synapse 46:150–156, 2002. Published 2002 Wiley‐Liss, Inc.

The transcription factor ΔFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of ΔFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Δ⁹‐THC, robustly induces ΔFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of ΔFosB induction in dorsal striatum. In addition, all four drugs induced ΔFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced ΔFosB to a small extent in amygdala. Furthermore, all drugs induced ΔFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of ΔFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of ΔFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces ΔFosB in a region‐specific manner in brain. Synapse 358–369, 2008. © 2008 Wiley‐Liss, Inc.

The effects of cocaine on dopamine (DA) neurotransmission were evaluated by in vivo microdialysis in the striatum of halothane‐anesthetized rats. Intravenous cocaine produced a dose‐dependent, transient increase of the extracellular concentration of DA, with a peak response within 10 min and a return to control level by 30 min. The sharp DA response pattern was abolished in a calcium‐free environment, indicating that DA release enhanced by cocaine originates from a vesicular storage pool. Continuous administration of cocaine (via the perfusion medium) directly into the nigrostriatal terminal region also produced a dose‐dependent increase in DA release. Low concentrations (10⁻⁵ M and 10⁻⁶ M) of cocaine maintained DA at a constant stable level, consistent with the effects observed after potent DA uptake inhibitory agents (e.g., nomifensine and Lu19005). However, continuous exposure to high concentrations (< 10⁻⁴ M) induced a transient elevation of DA within 20 min, following which DA decreased to a stable but high level; this decrease might reflect tolerance to the effect of cocaine. Administration of cocaine (10⁻³ M) into the substantia nigra did not change striatal DA release. The local striatal action of cocaine was less potent than amphetamine in elevating DA overflow and in its effect on DA metabolism. These findings suggest that the fast transient enhancement of DA by intravenous cocaine is most likely a consequence of the transient presence of cocaine in the terminal region, correlating with the well‐known rapid pharmacokinetic and behavioral aspects of the drug.

Most forms of neuronal plasticity are associated with induction of the transcription factor zif268 (egr1). Down‐regulation of cdc20 (p55^cdc)—a regulatory protein for the anaphase‐promoting complex, which controls access of specific substrates to the proteasome—was observed after transfection of a neuronal cell line with zif268. Treatment of cultured hippocampal neurones with NMDA, which elevates endogenous zif268 levels, also decreased cdc20 levels. Conversely, the levels of cdc20 were found to be increased in the cerebral cortex of mice with targeted deletion of the zif268 gene, when compared with wild‐type controls. Our findings indicate that expression of the cdc20 gene is down‐regulated by zif268 in neuronal cells, and provide new evidence that altered expression of proteasome‐regulatory genes following zif268 induction may be a key component of long‐lasting CNS plasticity. Synapse 61:463‐468, 2007. © 2007 Wiley‐Liss, Inc.

Agmatine, a metabolite of L‐arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training‐induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold‐labeling and electron‐microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague–Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning‐induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter. Synapse, 2011. © 2010 Wiley‐Liss, Inc.

Phencyclidine exerts psychotomimetic effects in humans and is used as a pharmacological animal model for schizophrenia. We, and others, have demonstrated that phencyclidine induces cognitive deficits in rats that are associated with schizophrenia. These cognitive deficits can be normalized by inhibition of nitric oxide synthase. The development of selective microelectrochemical nitric oxide sensors may provide direct evidence for the involvement of nitric oxide in these effects. The aim of the present study was to use LIVE (long term in vivo electrochemistry) to investigate the effect of phencyclidine, alone or in combination with the nitric oxide synthase inhibitor L‐NAME, on nitric oxide levels in the medial prefrontal cortex of freely moving rats. Phencyclidine (2 mg kg⁻¹) produced an increase in cortical nitric oxide levels and this increase was ameliorated by L‐NAME (10 mg kg⁻¹). Tentatively, the results from the present study provide a biochemical rationale for the involvement of nitric oxide in the phencyclidine model of schizophrenia. Synapse 63:1083–1088, 2009. © 2009 Wiley‐Liss, Inc.