Regulation of α7 nicotinic acetylcholine receptors in mouse somatosensory cortex following whisker removal at birth

Previous studies in postnatal mouse demonstrating high levels of α7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined α-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [¹²⁵I]α-bungarotoxin binding on postnatal day 6 were significantly higher (23 ± 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 ± 3%) [¹²⁵I]α-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [¹²⁵I]α-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [¹²⁵I]α-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of α7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity. J. Comp. Neurol. 397:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Postnatal development of two nicotinic cholinergic receptors in seven mouse brain regions

The developmental profiles for binding of alpha-[125I]bungarotoxin and L-[3H]nicotine to putative nicotinic cholinergic receptors were determined in seven mouse brain regions. The overall pattern of development of alpha-bungarotoxin binding was similar in all of the regions. Neonatal binding values tended to be greater than those observed in adult brain regions. Maximal binding occurred within 10 days of birth and adult binding values were reached by 20 days of age. The patterns of development of nicotine binding in each of the seven brain regions differed according to region. Gross similarities in developmental profiles for nicotine binding were found among the more caudal and among the more rostral regions. In hindbrain and cerebellum, maximal nicotine binding was found at birth (5 days of age in cerebellum); binding declined approximately 4-fold by 20 days and remained relatively constant thereafter. In midbrain and hypothalamus, a less extensive decrease in nicotine binding occurred from birth to adulthood (midbrain, 25%; hypothalamus, 50%). Nicotine binding in hippocampus and cortex remained unchanged between birth and adulthood. The developmental pattern for nicotine binding in striatum differed from that found in the other brain regions. At 5 days of age, binding was about 65% of adult binding, which was reached at 30 days of age. In most of the brain regions the developmental profile for alpha-bungarotoxin binding was different from that of nicotine. This difference was especially notable in striatum, where adult nicotine binding was higher than neonatal nicotine binding, whereas adult alpha-bungarotoxin binding was lower than neonatal alpha-bungarotoxin binding.     

Facilitation of glutamate release by nicotine involves the activation of a Ca2+/calmodulin signaling pathway in rat prefrontal cortex nerve terminals

The effect of nicotine on evoked glutamate release from isolated nerve terminals (synaptosomes) from rat prefrontal cortex was examined. We found that nicotine significantly potentiated 4-aminopyridine (4AP)-evoked glutamate release, and this potentiatory effect was mimicked by the selective alpha7 nicotinic receptor agonist choline and was blocked by the selective alpha7 nicotinic receptor antagonist methyllycaconitine, indicating its mediation by alpha7 nicotinic receptors. Examination of the effect of nicotine on cytosolic [Ca(2+)] revealed that the potentiation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx through N- and P/Q-type Ca(2+) channels. The potentiatory effect of nicotine on Ca(2+) influx seems to be attributed to its increasing synaptosomal excitability because nicotine significantly increased depolarization-evoked increase in the intrasynaptosomal free Na(+) concentration and 4AP-evoked depolarization of the synaptosomal plasma membrane potential. Also, Ca(2+) ionophore ionomycin-induced glutamate release was enhanced by nicotine, and this action was blocked by methyllycaconitine. These results suggest that nicotine exerts its potentiatory effect presynaptically, likely through the activation of alpha7 nicotinic receptors, resulting in Na(+) influx and local depolarization, which subsequently enhances the Ca(K+) entry through voltage-dependent N-and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat prefrontocortical nerve terminals. Moreover, in this release potentiation may involve an activation of Ca(2+)/calmodulin signaling pathway as nicotine-mediated potentiation of 4AP- and ionomycin-evoked glutamate release were significantly attenuated by KN62, a selective inhibitor of Ca(2+)/calmodulin-dependent kinase II.     

In vivo modulation of 5-hydroxytryptamine release in mouse prefrontal cortex by local 5-HT(2A) receptors: effect of antipsychotic drugs

In the rat, postsynaptic 5-hydroxytryptamine2A receptors medial prefrontal cortex control the activity of the serotonergic system through changes in the activity of pyramidal neurons projecting to the dorsal raphe nucleus. Here we extend these observations to mouse brain. The prefrontal cortex expresses abundant 5- hydroxytryptamine2A receptors, as assessed by immunohistochemistry, Western blots and in situ hybridization procedures. The application of the 5-hydroxytryptamine2A/2C agonist DOI (100 microm) by reverse dialysis in the medial prefrontal cortex doubled the local release of 5-hydroxytryptamine. This effect was reversed by coperfusion of tetrodotoxin, and by the selective 5-hydroxytryptamine2A receptor antagonist M100907, but not by the 5-hydroxytryptamine2C antagonist SB-242084. The effect of DOI was also reversed by prazosin (alpha1-adrenoceptor antagonist), BAY x 3702 (5-hydroxytryptamine1A receptor agonist), NBQX (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate/kainic acid antagonist) and 1S,3S-ACPD (mGluR II/III agonist), but not by dizocilpine (N-methyl-d-aspartate antagonist). alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate mimicked the 5-hydroxytryptamine elevation produced by DOI, an effect also reversed by BAY x 3702. Likewise, the coperfusion of classical (chlorpromazine, haloperidol) and atypical antipsychotic drugs (clozapine, olanzapine) fully reversed the 5-hydroxytryptamine elevation induced by DOI. These observations suggest that DOI increases 5-hydroxytryptamine release in the mouse medial prefrontal cortex through the activation of local 5-hydroxytryptamine2A receptors by an impulse-dependent mechanism that involves/requires the activation of local alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate receptors. This effect is reversed by ligands of receptors present in the medial prefrontal cortex, possibly in pyramidal neurons, which are involved in the action of antipsychotic drugs. In particular, the reversal by classical antipsychotics may involve blockade of alpha1-adrenoceptors, whereas that of atypical antipsychotics may involve 5-hydroxytryptamine2A receptors and alpha1-adrenoceptors.     

Evaluation of (‐)‐[18F]Flubatine‐specific binding: Implications for reference region approaches

We aimed to characterize changes in binding of (‐)‐[¹⁸F]Flubatine to α₄β₂*‐nicotinic acetylcholine receptors (α₄β₂*‐nAChRs) during a tobacco cigarette smoking challenge. Displacement of (‐)‐[¹⁸F]Flubatine throughout the brain was quantified as change in (‐)‐[¹⁸F]Flubatine distribution volume (V_T), with particular emphasis on regions with low V_T. Three tobacco smokers were imaged with positron emission tomography (PET) during a 210 min bolus‐plus‐constant infusion of (‐)‐[¹⁸F]Flubatine. A tobacco cigarette was smoked in the PET scanner ～125 min after the start of (‐)‐[¹⁸F]Flubatine injection. Equilibrium analysis was used to estimate V_T at baseline (90‐120 min) and after cigarette challenge (180‐210 min), at the time of greatest receptor occupancy by nicotine. Smoking reduced V_T by 21 ± 9% (average ±SD) in corpus callosum, 17 ± 9% in frontal cortex, 36 ± 11% in cerebellum, and 22 ± 10% in putamen. The finding of displaceable (‐)‐[¹⁸F]Flubatine binding throughout the brain is an important consideration for reference region‐based quantification approaches with this tracer. We observed displacement of (‐)‐[¹⁸F]Flubatine binding to α₄β₂*‐nicotinic acetylcholine receptors in corpus callosum by a tobacco cigarette challenge. We conclude that reference region approaches utilizing corpus callosum should first perform careful characterization of displaceable (‐)‐[¹⁸F]Flubatine binding and nondisplaceable kinetics in this putative reference region.     

Increases in α-adrenergic receptors in the hypothalamus of the genetically obese mouse (ob/ob)

Despite significantly elevated hypothalamic norepinephrine levels, genetically obese mice (ob/ob) had more hypothalamic α-adrenergic receptors than their lean littermates. This receptor increase appeared to be specific to the α-receptors in the hypothalamus since no change was found in the number of α-receptors in the cortex or in the dopamine and muscarinic receptors in the cortex and striatum.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

α1-adrenergic,D1, and D2 receptors interactions in the prefrontal cortex: Implications for the modality of action of different types of neuroleptics

The activation of rat mesocortical dopaminergic (DA) neurons evoked by the electrical stimulation of the ventral tegmental area (VTA) induces a marked inhibition of the spontaneous activity of prefrontocortical cells. In the present study, it was first shown that systemic administration of either clozapine (a mixed antagonist of D₁, D₂, and α₁-adrenergic receptors) (3–5 mg/ kg, i.v.), prazosin (an α₁-adrenergic antagonist) (0.2 mg/ kg, i.v.), or sulpiride (a D₂ antagonist) (30 mg/ kg, i.v.), but not SCH 23390 (a D₁ antagonist) (0.2 mg/ kg, i.v.), reversed this cortical inhibition. Second, it was found that following the systemic administration of prazosin, the VTA-induced cortical inhibition reappeared when either SCH 23390 or sulpiride was applied by iontophoresis into the prefrontal cortex. Third, it was seen that, whereas haloperidol (0.2 mg/ kg, i.v.), a D₂ antagonist which also blocks α₁-adrenergic receptors, failed to reverse the VTA-induced inhibition, the systemic administration of haloperidol plus SCH 23390 (0.2 mg/ kg, i.v.) blocked this inhibition. Finally, it was verified that the cortical inhibitions obtained following treatments with either “prazosin plus sulpiride” or “prazosin plus SCH 23390” were blocked by a superimposed administration of either SCH 23390 or sulpiride, respectively. These data indicate that complex interactions between cortical D₂, D₁, and α₁-adrenergic receptors are involved in the regulation of the activity of prefrontocortical cells innervated by the VTA neurons. They confirm that the physiological stimulation of cortical α₁-adrenergic receptors hampers the functional activity of cortical D₁ receptors and suggest that the stimulations of cortical D₁ and D₂ receptors exert mutual inhibition on each other's transmission. Synapse 30:362–370, 1998. © 1998 Wiley-Liss, Inc.     

DMXB (GTS‐21) ameliorates the cognitive deficits in beta amyloid injected mice through preventing the dysfunction of alpha7 nicotinic receptor

Intracerebroventricular injection of beta‐amyloid_25–35 (Aβ_25–35) in mice leads to cognitive deficits with the dysfunction of α7 nicotinic acetylcholine receptor (α7nAChR) within 1–2 weeks in a dose‐dependent manner. The present study focused on the effect of DMXB, a selective α7nAChR agonist, on Aβ_25–35 (3 nmol)‐impaired spatial memory and α7nAChR function. We found that the treatment with DMXB on days 1–10 after Aβ_25–35 injection dose‐dependently prevented Aβ_25–35‐induced impairment of acquisition performance and probe trail test in Morris water maze. Importantly, the treatment with DMXB (1 mg/kg) perfectly prevented Aβ_25–35‐induced depression of α7nAChR response, which was associated with improving the probability of presynaptic glutamate release and the induction of high‐frequency stimulation (HFS)‐dependent long‐term potentiation (LTP) in hippocampal Schaffer collaterale‐CA1 synapse. Furthermore, although either the basal level of extracellular signal‐regulated kinase 2 (ERK2) or its phosphorylation in the hippocampus had no difference between control and Aβ_25–35 mice, the Aβ_25–35 injection significantly attenuated HFS‐triggered increase in ERK2 phosphorylation. The treatment with DMXB also rescued the ERK2 phosphorylation triggered by HFS in Aβ_25–35 mice that is required for LTP induction. This study firstly provides in vivo evidence that the anti‐amnesic effect of DMXB is likely due to preventing the Aβ_25–35‐induced dysfunction of α7nAChR. © 2010 Wiley‐Liss, Inc.     

Dopamine release in the prefrontal cortex during stress is reduced by the local activation of glutamate receptors

Using microdialysis, we investigated the effects of the ionotropic glutamatergic agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on the stress-induced dopamine release in the prefrontal cortex of the freely moving rat. Handling-stress during 40 min increased extracellular dopamine by 195% and dopamine metabolites dihydroxyphenilacetic acid (DOPAC) by 120% and homovallinic acid (HVA) by 155% of baseline, but it did not modify extracellular glutamate, in the prefrontal cortex. Both NMDA (100 microM) and AMPA (20 microM), perfused through the microdialysis probe in the prefrontal cortex simultaneously to stress, significantly reduced the stress-induced dopamine release. These same doses or lower doses of NMDA (20 and 100 microM) and AMPA (1 and 20 microM) did not significantly modify basal dopamine release in the prefrontal cortex, but higher doses of these glutamatergic agonists significantly decreased (NMDA 500 microM) or increased (AMPA 100 microM) basal dopamine release in this area of the brain. These results show that the local activation of prefrontal glutamatergic ionotropic receptors reduces the stress-induced dopamine release in the prefrontal cortex of the rat.     

Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (α4β2) transfected into mouse fibroblasts with ion channel activity

(−)-Nicotine, cytisine and carbachol evoked⁸⁶Rb efflux from mouse fibroblasts stably transfected with α₄β₂ chick brain nicotinic subunits. This response to (−)-nicotine was inhibited by mecamylamine and dihydro-β-erythroidine and was mirrored by a rise in intracellular Ca²⁺ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant⁸⁶Rb from cells and unlike the above agonists displayed significantly different IC₅₀ values for the displacement of [³H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes.     

Effect of acetylcholinesterase inhibitors on the binding of nicotinic alpha4beta2 receptor PET radiotracer, (18)F-nifene: A measure of acetylcholine competition

Acetylcholinesterase inhibitors (AChEI's) are used to treat Alzheimer's disease (AD), and the putative mode of action is to increase acetylcholine (ACh) levels. Our goal is to evaluate competition of ACh with nicotinic alpha4beta2 receptor PET agonist radiotracer, 2-[(18)F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine ((18)F-nifene). This ability to measure ACh-(18)F-nifene competition may have potential to assess efficacy of AChEI's in vivo. In vitro studies in rat brain slices used two AChEI's, physostigmine (PHY) and galanthamine (GAL). Brain slices were incubated with (18)F-nifene and various concentrations of PHY (0.2-20 microM) or GAL (0.4-4 microM) prior to (18)F-nifene treatment. For ACh competition, slices were also incubated with PHY + 100 nM ACh or GAL + 100 nM ACh or 100 nM ACh alone. Nonspecific binding of (18)F-nifene was determined using 300 microM nicotine. In the in vitro rat brain homogenate binding assay, ACh inhibited (3)H-cytisine binding to alpha4beta2 receptors with K(i) values of 19.2 nM (with PHY) and 34.7 microM (no PHY) indicating approximately 1.8 x 10(3) weaker binding of ACh in the absence of AChEI. Binding of (18)F-nifene was not affected by PHY (0.2-20 microM) or ACh 100 nM alone but decreased substantially by PHY + ACh 100 nM in all brain regions (down by >40% of control in thalamus). Similarly, for GAL (4 microM) no effect on (18)F-nifene binding occurred but GAL (0.4-4 microM) + ACh 100 nM showed a reduction of (18)F-nifene binding in all brain regions (down by approximately 15%). The reduction in both cases is a result of ACh competition with (18)F-nifene in the presence of AChEI. These preliminary in vitro results suggest that ACh is able to compete with (18)F-nifene at the alpha4beta2 receptors in the presence of PHY or GAL. The effect is AChEI-concentration dependent and is greater for PHY than GAL. Thus (18)F-nifene has promise for assessing ACh levels and AChEI effects in vivo.     

Modulation of the nicotinic α-bungarotoxin site in chromaffin cells in culture by a factor(s) endogenous to neuronal tissue

An endogenous factor(s) which affects the in vitro binding of α-bungarotoxin (α-BGT) to rat brain membranes has previously been found in brain supernatant. This fraction, as well as a partially purified preparation of this material from bovine brain, is here shown to affect the binding of α-BGT to chromaffin cell membranes. To study possible long term effects, the supernatant extract was added to adrenal medullary chromaffin cells in culture. The cells were incubated for several days and at the end of this time, the medium bathing the cells, which contained the endogenous factor(s), was removed and α-BGT binding to the cells measured. Binding to control cultures had shown that α-BGT bound to the chromaffin cells in a saturable manner, with high affinity (K_d = 1.5 nM) and the specificity of a nicotinic receptor ligand. After incubation of the cells with supernatant factor, a marked decline in the number of a α-BGT binding sites was observed with no change in affinity. This does not appear to be due to a detrimental effect on the cells as cell number did not appear to be decreased in the cultures preincubated with the supernatant extract and the DNA and protein content were similar in the control and treated cultures. The possibility that there was some non-specific detrimental effect to the chromaffin cell membrane was considered; however, the stimulated release of noradrenaline from the cells was not affected by treatment of the cultures in the presence of the supernatant fractions. In addition, tyrosine hydroxylase activity was significantly increased in the treated cultures. d-Tubocurarine, an antagonist at the acetylcholine receptor, caused an increase in α-BGT binding after 7 days of treatment, while the agonist nicotine and choline had no effect. These results suggest that in brain supernatant there may exist an endogenous factor(s), which may function in the regulation of the nicotinic-like α-BGT receptors in neuronal cell.     

Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release

Previously, serotonin (5-HT) was found to induce a marked increase in glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) in apical dendrites of layer V pyramidal cells of prefrontal cortex; this effect was mediated by 5-HT_2A receptors, a proposed site of action of hallucinogenic and atypical antipsychotic drugs. Unexpectedly, although the effect of 5-HT was Ca²⁺-dependent and tetrodotoxin-sensitive, it did not appear to involve the activation of excitatory afferent impulse flow. This paradox prompted us to investigate (in rat brain slices) whether 5-HT was acting through an atypical mode of excitatory transmitter release. We found that the frequency of 5-HT-induced spontaneous EPSCs was fully supported by Sr²⁺ in the absence of added Ca²⁺, implicating the mechanism of asynchronous transmitter release which has been linked to the high-affinity Ca²⁺-sensor synaptotagmin III. Although the early, synchronous component of electrically evoked EPSCs was reduced while 5-HT was being applied, late, nonsynchronous components were enhanced during 5-HT washout and also by the 5-HT₂ partial agonist 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI); the effect of DOI was blocked by a selective 5-HT_2A antagonist (MDL 100,907). This late, nonsynchronous component was distinct from conventional polysynaptic EPSCs evoked in the presence of the GABA_A antagonist bicuculline, but resembled asynchronous glutamatergic excitatory postsynaptic potentials (EPSPs) evoked in the presence of Sr²⁺. An enhancement of asynchronous EPSCs by a specific neurotransmitter receptor has not been reported previously. The possible role of excessive asynchronous transmission in the cerebral cortex in mediating the hallucinogenic effects of 5-HT_2A agonists such as DOI is discussed.     

Functional α7‐containing nicotinic receptors of NG2‐expressing cells in the hippocampus

In the postnatal central nervous system, glial cells expressing the chondroitin sulfate proteoglycan NG2 (NG2‐cells) constitute a cell population exhibiting several properties of oligodendrocyte precursors such as the ability to proliferate. One particular feature of NG2‐cells is that they express several glutamatergic and GABAergic ionotropic receptors activated by synaptic neurotransmitter release. Here, we used patch‐clamp recordings, immunostaining, calcium imaging, and intracellular labeling to test for the presence of ionotropic nicotinic acetylcholine receptors (nAChRs) in NG2‐cells identified in acute hippocampal slices of mice. We demonstrated that these cells express functional nAChRs during the second postnatal week, i.e., the period in which they become the most abundant proliferative cell type of CA1 stratum radiatum. Pharmacological experiments showed that NG2‐cells express α7‐containing nAChRs. In particular, the powerful positive allosteric modulator of these receptors PNU‐120596 induced a 20‐fold increase of agonist‐induced currents and revealed rises in intracellular calcium concentration upon agonist applications. In addition, nanomolar concentrations of nicotine, which did not induce any response in these cells, largely desensitized nAChR‐mediated currents. These data indicate that the functional expression of Ca²⁺‐permeable α7‐containing nAChRs in hippocampal slices is not restricted to neurons and that the receptors of NG2‐cells can be desensitized by low concentrations of nicotine. © 2009 Wiley‐Liss, Inc.     

In vitro regulation of serotonin transporter activity by protein kinase A and nicotinic acetylcholine receptors in the prefrontal cortex of rats

We investigated the effect of in vitro exposure to nicotinic acetylcholine receptors (nAChRs), agonists, antagonists, and protein kinase A (PKA) modulators on the activity of the serotonin transporter (SERT) in prefrontocortical (PFC) synaptosomes. The plasma membrane SERT is an active transport mechanism specific for serotonin. Receptors and second messengers capable of altering transporter activity would be expected to have profound effects on serotonergic neurotransmission and on functions involving serotonergic input, such as cognition, anxiety, and mood. Our data suggest that activation of nAChRs, quite likely via PKA, increase the activity of the SERT in the PFC and, thereby, can alter 5-HT levels in a region important in the behavioral effects of nicotine and 5-HT. Nicotine at 4 microM increased [(3)H]5-HT uptake by 75%. Because the nAChR antagonists mecamylamine and dihydro-beta-erythrodine (DHbetaE) both decreased [(3)H]5-HT uptake into synaptosomes, it appeared that the SERT might be tonically activated by acetylcholine present within our synaptosomal preparations. Blocking PKA significantly decreased [(3)H]5-HT, while stimulation of PKA activity significantly increased the uptake. A 66% decrease compared with control was produced by 100 microM Rp-cAMP, and a 41% increase in 5-HT uptake over control was observed with 30 microM Sp-cAMPs. Furthermore, the enhancement in uptake produced by 4 microM nicotine was inhibited in a time-dependent fashion by preincubation with 10 microM Rp-cAMP. A better understanding of the influence of the cholinergic system and the receptors involved in the trafficking of SERT would help clarify the important relationship between the cholinergic and serotonergic systems and the role these systems play in behavior.     

The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia

We have previously shown that certain commercially available lots of α-bungarotoxin block transmission in ciliary and choroid neurons of both pigeon and chicken ciliary ganglia at a concentration of 10 μg/ml (1.2 μM). The blockade is antagonized by pre-incubation with 100 μM tubocurarine.Further evidence that this blockade is produced by a postsynaptic action, as one would expect of an α-neurotoxin, are our findings that: (a) exposure to the toxin prevents the depolarization of ganglion cells normally seen in response to the cholinergic agonist, carbachol; and (b) the blocking activity of the toxin is removed by treatment with membranes purified from Torpedo electric organ containing an excess of α-neurotoxin binding sites.A high affinity binding site for [¹²⁵I]α-bungarotoxin was characterized in the chicken ciliary ganglion. However, since it is labelled equally well by lots of α-bungarotoxin which block transmission and those that do not, this site does not appear to be involved in the blockade of transmission.α-Cobratoxin (fromNaja naja siamensis), the α-neurotoxin L.s. III (fromLaticauda semifasciata) and certain lots of α-bungarotoxin produce a partial blockade of transmission in ciliary neurons of the pigeon ciliary ganglion at a concentration of 10 μg/ml (1.2 μM), but have no effect on transmission in choroid neurons. Two other α-neurotoxins fromLaticauda semifasciata, erabutoxin a and erabutoxin b, have no effect on transmission in either cell population at this concentration. None of the α-neurotoxins tested had any effect on transmission in either the rat superior cervical ganglion or the rat pelvic ganglion at concentrations up to 100 μg/ml (12 μM). Collagenase treatment of these ganglia, in an attempt to increase access of the toxins to ganglion cells, did not alter these negative results.β-Bungarotoxin (0.5 μg/ml, 0.02 μM) produces a complex blockade of transmission in both avian ciliary ganglia and rat superior cervical ganglia. Unlike the action of α-bungarotoxin, the blockade of ciliary ganglion transmission by β-bungarotoxin is irreversible and is not prevented by pretreatment with tubocurarine.     

Endogenously released ACh and exogenous nicotine differentially facilitate long-term potentiation induction in the hippocampal CA1 region of mice

We examined the role of α7- and β2-containing nicotinic acetylcholine receptors (nAChRs) in the induction of long-term potentiation (LTP). Theta-burst stimulation (TBS), mimicking the brain's naturally occurring theta rhythm, induced robust LTP in hippocampal slices from α7 and β2 knockout mice. This suggests TBS is capable of inducing LTP without activation of α7- or β2-containing nAChRs. However, when weak TBS was applied, the modulatory effects of nicotinic receptors on LTP induction became visible. We showed that during weak TBS, activation of α7 nAChRs occurs by the release of ACh, contributing to LTP induction. Additionally, bath-application of nicotine activated β2-containing nAChRs to promote LTP induction. Despite predicted nicotine-induced desensitization, synaptically mediated activation of α7 nAChRs still occurs in the presence of nicotine and contributed to LTP induction. Optical recording of single-stimulation-evoked excitatory activity with a voltage-sensitive dye revealed enhanced excitatory activity in the presence of nicotine. This effect of nicotine was robust during high-frequency stimulation, and was accompanied by enhanced burst excitatory postsynaptic potentials. Nicotine-induced enhancement of excitatory activity was observed in slices from α7 knockout mice, but was absent in β2 knockout mice. These results suggest that the nicotine-induced enhancement of excitatory activity is mediated by β2-containing nAChRs, and is related to the nicotine-induced facilitation of LTP induction. Thus, our study demonstrates that the activation of α7- and β2-containing nAChRs differentially facilitates LTP induction via endogenously released ACh and exogenous nicotine, respectively, in the hippocampal CA1 region of mice.