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��

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60

��

1. Friedman A, Kaufer D, Pavlovsky L, Soreq H (1998) Cholinergic excitation

induces activity-dependent electrophysiological and transcriptional responses in

hippocampal slices. J Physiol Paris 92: 329-335.

2. Meshorer E, Erb C, Gazit R, Pavlovsky L, Kaufer D, Friedman A, Glick D, Ben

Arie N, Soreq H (2002) Alternative splicing and neuritic mRNA translocation

under long-term neuronal hypersensitivity. Science 295: 508-512.

3. Pavlovsky L, Browne RO, Friedman A (2003) Pyridostigmine enhances

glutamatergic transmission in hippocampal CA1 neurons. Exp Neurol 179: 181-

187.

4. Browne, R.O., Pavlovsky, L., and Friedman, A. Muscarinic Neuromodulation in

the Hippocampus and Parahippocampal Region. In: Silman, I., Fisher, A.,

Anglister, L. Michaelson, D. and Soreq, H. (eds.) Cholinergic Mechanisms,

Martin Dunitz , London�

61

��

Cholinergic excitation induces activity-dependent electrophysiological and transcriptional

responses in hippocampal slices.

62

��

Alternative splicing and neuritic mRNA translocation under long-term neuronal

hypersensitivity.

Our results identify a clear physiologicfunction for ERb. Estrogen receptors areknown to regulate a number of genes thataffect vascular function (5, 26 ). MultipleERb-regulated gene products may contrib-ute to the abnormal vascular contraction,ion channel dysfunction and hypertensionobserved in ERbKO animals. Our findingsalso support the concept that the transcrip-tion factor ERb controls expression ofgenes critical to normal vascular physiolo-gy in both males and females. Gene targetsof ERb in relevant target tissues may pro-vide insights into the pathophysiology andtreatment of hypertension.

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619 (1994).27. A. Y. Kolyada, N. Savikovsky, N. E. Madias, Biochem.

Biophys. Res. Commun. 220, 600 (1996).28. We are grateful to N. Madias for the iNOS-luciferase

reporter construct, N. Flavahan for guidance in es-tablishing the vascular ring methods, and M. Arono-vitz for superb technical assistance. Supported in partby NIH grants P50 HL63494, R01 HL55309, and R01HL56069 (M.E.M.); NIH grant HL56235 and a Grant-in-Aid from the American Heart Association ( Y.Z.);NIH grant R01 HL61298 (R.H.K.); NIH grantsHD30276 and HL53546 (P.W.S.); NIH grantGM20069 (O.S.); and grants from KaroBio AB and theSwedish Cancer Fund ( J-Å.G.), MFR04764 and Heartand Lung Foundation (P.T.), and from Karolinska In-stitute (1306/2000FFU) ( Z.B.). The reported findingsare solely the responsibility of the authors and do notnecessarily represent the official views of the NIH.

9 August 2001; accepted 27 November 2001

Alternative Splicing andNeuritic mRNA Translocation

Under Long-Term NeuronalHypersensitivity

Eran Meshorer,1 Christina Erb,1 Roi Gazit,2 Lev Pavlovsky,3

Daniela Kaufer,1* Alon Friedman,3 David Glick,1

Nissim Ben-Arie,2 Hermona Soreq1†

To explore neuronal mechanisms underlying long-term consequences of stress,we studied stress-induced changes in the neuritic translocation of acetylcho-linesterase (AChE) splice variants. Under normal conditions, we found thesynaptic AChE-S mRNA and protein in neurites. Corticosterone, anticholines-terases, and forced swim, each facilitated a rapid (minutes), yet long-lasting(weeks), shift from AChE-S to the normally rare AChE-R mRNA, promotedAChE-R mRNA translocation into neurites, and induced enzyme secretion.Weeks after stress, electrophysiological measurements in hippocampus slicesdisplayed apparently normal evoked synaptic responses but extreme hyper-sensitivity to both anticholinesterases and atropine. Our findings suggest thatneuronal hypersensitivity under stress involves neuritic replacement of AChE-Swith AChE-R.

Traumatic stress is often followed by long-term pathological changes (1, 2). In humans,extreme cases of such changes are clinicallyrecognized as posttraumatic stress disorder(PTSD) (3). Although the immediate re-sponse to acute stressful insults has beenextensively studied, the molecular mecha-nisms leading to the long-term neuronal hy-persensitivity that is characteristic of PTSDare yet unknown. Stimulus-induced changesin alternative splicing have recently emergedas a major mechanism of neuronal adaptationto stress, contributing to the versatility andcomplexity of the expression patterns of thehuman genome (4–6). Another stimulus-in-duced post-transcriptional process is dendrit-ic mRNA translocation, which has been de-scribed for several transcripts (7–12). Be-cause psychological, physical, and chemicalstressors all cause neuronal activation andhyperexcitation, dendritic translocation ofspecific target mRNAs may follow.

Acetylcholinesterase (AChE) modulationsprovide an appropriate case study for explor-ing long-term stress effects. Chemical, psy-chological, and physical stresses all shift

splicing from the primary mRNA productthat encodes the synaptic membrane AChE-Smultimeric protein to the normally rare“readthrough” AChE-R transcript, whichyields soluble monomers (13). We thus ex-amined neuronal distributions of the twosplice variants, which have characteristic 39regions (Fig. 1A). A comprehensive search ofthe NCBI GenBank and EST databases re-vealed several AChE-S mRNAs but only asingle AChE-R mRNA of rodent brain origin(GenBank accession number X70141), attest-ing to the scarcity and/or instability of neu-ronal AChE-R mRNA under normal condi-tions. To study changes in gene expression atthe subcellular level, we used double-labelfluorescence in situ hybridization (FISH) ofspecific AChE mRNA splice variants (14)and confocal microscope image analysis.

FISH detection efficiencies likely dependon probe sequences, but subcellular distribu-tions can be reliably compared for singletranscripts in different cells and conditions.Cultured PC12 cells (15), primary cultures ofmouse cerebellar neurons (16), and pyrami-dal neurons in paraffin-embedded sections ofthe prefrontal cortex (17) all displayed alarger fraction of AChE-S mRNA transcriptsin neuronal processes than of AChE-RmRNA (Fig. 1, B through D). Also, both celltypes displayed nuclear localization ofAChE-R but not of AChE-S mRNA (Fig. 1,B and C) (18). To test whether labeling prop-erties prejudiced this conclusion, we reversedthe fluorophores on the two probes (Fig. 1, Band C). In paraffin-embedded brain sectionsfrom naıve mice, cortical pyramidal neuronspresented dispersed AChE-S mRNA through-

Departments of 1Biological Chemistry and 2Cell andAnimal Biology, The Institute of Life Sciences and TheEric Roland Center for Neurodegenerative Diseases,The Hebrew University of Jerusalem, Israel 91904.3Departments of Physiology and Neurosurgery,Zlotowsky Center of Neuroscience, Ben Gurion Uni-versity and Soroka Medical Center, Beersheva, Israel84105.

*Present address: Department of Biological Sciences,Stanford University, Stanford, CA 94305, USA.†To whom correspondence should be addressed. E-mail: [email protected]

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out the processes, while AChE-R mRNA waslocalized to the cell body. In addition, theperikaryal cytoplasm exhibited punctatedconcentrations of AChE-R mRNA alternatingwith double-labeled regions, whereas neu-rites had AChE-S mRNA with foci of bothtranscripts (Fig. 1D). The neurite contents ofcultured PC12 cells and developing cerebel-lar neurons, also known to express ampleAChE (19), and of prefrontal cortex neuronsin vivo were 22 6 3, 28 6 4 and 28 6 7%,respectively, for AChE-S mRNA but only10 6 2, 7 6 2 and 11 6 6% for AChE-RmRNA (P , 0.05, two-tailed Student’s t-test). AChE-S mRNA, thus, preferentially lo-calized in neurites (except under stress, seeFig. 1E, below).

The human ACHE gene includes a glu-cocorticoid response element (GRE) about 17kb upstream from the transcription initiationsite (Fig. 1A). In humans, a deletion adjacentto the GRE causes constitutive overexpres-sion and anti-AChE hypersensitivity (20),which suggests a physiologically significantrole for glucocorticoids in regulating bothneuronal ACHE gene expression and anticho-linesterase hypersensitivity. We thereforecompared PC12 cells treated for 6 hours withcorticosterone (10 mM, 0.1% ethanol) to con-trol cells (0.1% ethanol). Twenty-four hoursafter treatment, catalytic activity againstacetylthiocholine rose by 25 6 14% (P ,0.05) in PC12 cells. Corticosterone, further,increased by 30 to 50% the levels of bothsplice variants within 24 hours (P , 0.05)(Fig. 2, A through H). However, the AChE-SmRNA-labeled area remained essentially un-changed under control conditions (distancefrom nuclear border to limit of mRNA label-ing 5 37 6 13 mm) or corticosterone (34 613 mm). In contrast, AChE-R mRNA labelingextended a smaller distance from the nucleus,25 6 9 mm under control conditions, increas-ing to 33 6 17 mm under corticosterone (P ,0.05).

Three-hour incubation of PC12 cells with1.5 nM EN101, an antisense oligonucleotidethat induces preferential degradation ofAChE-R mRNA (15), affected the selective,yet limited, suppression of AChE-R mRNA(30 6 8%, P , 0.05), but left unchangedAChE-S mRNA levels (Fig. 2, E through H).An inversely oriented sequence, INV101, af-fected neither the AChE-R mRNA level norits distribution in PC12 cells (21), attesting tothe sequence specificity of the antisense ef-fect. The labeled area remained unchangedeither with AChE-S or AChE-R cRNAprobes, following EN101 treatment, suggest-ing full EN101 accessibility to the cell body(Fig. 2, I and J).

To test whether EN101 is effective simi-larly in neurites and perikaryal subcellularsites, we employed cerebellar neurons in pri-mary culture. DIG labeling of AChE-S

mRNA was not affected differently byEN101 and INV101, in either cell body orneurites. In contrast, EN101 reducedAChE-R mRNA labeling in the cell body ofcerebellar neurons in culture, down to almost50% of its level in INV101-treated controls(22). This suppression was completely re-stricted to the perikaryon, with no differenceobserved for neuritic AChE-R mRNA in an-tisense-treated cerebellar neurons.

Cytochemical staining of catalytically ac-tive AChE (17) revealed intensified AChEactivity in the cell bodies of corticosterone-

treated cultured cerebellar neurons (Fig. 3, Aand C). This suggested that the overexpressedAChE mRNA transcripts were translated toyield active enzyme. Immunocytochemicalstaining with an antibody targeted againstrecombinant AChE-S (23) presented appar-ently similar staining patterns in neurites ofcontrol and corticosterone-treated culturedcerebellar neurons (Fig. 3, B and D). Thisindicated that neurites under stress secrete thehormone-induced soluble AChE-R, with noincrease in the synaptic membrane-associatedAChE-S.

Fig. 1. Variant-specific subcellular distribution of AChE mRNAs. (A) Alternative splicing. Shown arethe distal enhancer glucocorticoid response element (GRE) and the mouse ACHE gene (top), as wellas AChE-S mRNA (S) and AChE-R mRNA (R). Linkage of exons 2, 3, and 4 is common to bothvariants (a). The R transcript includes at its 39 terminus pseudointron 4 (green) and exon 5; optionb yields the S transcript by connecting exon 4 to 6 (red). (B through E) Localization of the S andR transcripts in vitro and in vivo. Shown are percentages 6 SD of FISH signals for the S and Rtranscripts (each totaling 100%) in three parts of the perikaryon and two neurite areas of 10 PC12cells (B), cultured primary cerebellar neurons (C), pyramidal neurons from a paraffin-embeddedslice of the prefrontal cortex (D), and cerebellar neurons from an embedded brain slice of a mousethat had been stressed for four consecutive days (23 4 min forced swim), and sacrificed 2 weekslater (E). In control mice (D), the fraction of AChE-R mRNA in segments 415 was 9 6 2% in bothcortical and cerebellar neurons; for poststress mice, it was 24 6 7% (E) (P , 0.05). Note nuclearlabeling of the R transcript in cultured cerebellar neurons (arrowheads) and the punctated patternof transcript accumulation in vivo [arrows in (D) and (E)]. Asterisks indicate columns withsignificant differences between AChE-S and AChE-R mRNAs (P , 0.05).

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We used FISH detection of the intracel-lular AChE-R mRNA transcript to assessthe expression levels of this variant in vivo.Dorsal hippocampus neurons of naıveFVB/N mice express extremely low levelsof AChE-R mRNA under normal condi-tions (Fig. 3E). Two days following thestress of surgical implantation of a micro-dialysis cannula (24 ), the range of neuritelabeling increased from 2.0 6 0.3 to 5.1 61.0 mm from the nucleus (Fig. 3G, P ,0.0005). Injection through the cannula ofthe AChE inhibitor neostigmine (125 nmol)resulted in a more extensive translocationof AChE-R mRNA within 25 min (to 8.5 61.2 mm from the nucleus, Fig. 3I). In neu-rites of untreated hypersensitive AChE-Stransgenic mice, AChE-R mRNA reachedgreater distances from the nucleus (9 6 1mm) than those of their strain-matched con-trols (Fig. 3F, P , 0.0005). Sham injectionfailed to further increase transport (10 6 1mm, Fig. 3H), but AChE-R mRNA reacheddendrite distances of 15 6 2 mm under

neostigmine (Fig. 3J), significantly longerthan either similarly treated nontransgenicanimals, or sham injected transgenics (inboth cases P , 0.0005; Fig. 3, K and L).

Reported rates of mRNA dendrite trans-port range from 10 to 20 mmzhour21 (25) to300 to 360 mmzhour21 (26 ). In our study,assuming a constant rate, AChE-R mRNAtraveled a minimum of 8 6 5 mmzhour21

in anticholinesterase-treated FVB/N mice,which increased to 14 6 7 mmzhour21 insimilarly treated hAChE-S transgenic mice(P , 0.0005). This rate is consistent withthe lower range estimate. The stability ofAChE-R mRNA in the face of an antisenseagent predicted long-lasting poststress neu-ritic presence of this transcript in vivo. Totest this, we subjected FVB/N mice to 4consecutive days of forced swim (two4-min sessions per day). In naıve mice,cerebellar granule neuron processes wereloaded with 28 6 12% of the cellularAChE-S mRNA but only 9 6 2% of theAChE-R mRNA content (Fig. 1C). Twoweeks post-stress, a considerably largerfraction (24 6 7%) of the stress-increasedamount of AChE-R mRNA translocatedinto neurites and displayed patches of con-centrated AChE-R mRNA in both the cellbodies and processes (Fig. 1E). Thus, boththe absolute levels and the neurite fractionof AChE-R mRNA increased considerably.

In nontransgenic mice, hippocampalAChE-R mRNA is generally limited to thegranular layer (17). AChE-R mRNA levelsremained considerably higher than baseline 4weeks after 4 consecutive days of forcedswim or 3-day exposure to very low diiso-propylfluorophosphonate (DFP) levels (0.1mgzkg21zday21, i.p., LD50 5 2.5 mgzkg21).Prestressed or preexposed animals present-ed high-intensity labeling in the hippocam-pus CA1 region, dentate gyrus, and den-drite layer (Fig. 4, A through F), predictingmodified composition of neuritic AChEvariants either after stress or low-level ex-posure to an anticholinesterase.

Released neuronal acetylcholine binds toboth pre- and postsynaptic receptors and isassumed to serve as a modulatory neurotrans-mitter and set the response level of the neu-ronal network to incoming stimuli (27). Thisinvolves electrophysiological mechanismsthat are only partly understood, but dependon synaptic hydrolysis of acetylcholine byAChE-S. To test whether the supplementa-tion of neuritic AChE-S with AChE-R com-promises the capacity to confront intensifiedcholinergic stimuli, we stimulated the CA2/CA3 region of the stratum oriens, a regionrich in cholinergic fibers, and recorded pop-ulation field potentials (pfps), which sum theresponses of a large number of neurons, in thecell body layer of the CA1 region (17). Re-cording in hippocampus slices (28) was per-formed 1 month after exposure to stress or inslices from naıve animals. The anticholines-terase physostigmine induced an increase (426 15%, P , 0.05) in the amplitude of theevoked population spike response of naıvemice (Fig. 4, G and I). This response was70% reversible following the addition of at-ropine to the bathing solution, indicating ma-jor action through muscarinic receptors. In

Fig. 2. Corticosterone induction and antisensesuppression of AChE mRNA in PC12 cells. Shownare confocal micrographs (A through F), averagelabeling intensities (G and H) and distributionsof distances from the nucleus to the cell border(I and J) of the S (red) and R (green) transcriptsin PC12 cells (n 5 40) under control conditions[(A) and (B)], or after 3 hours in 10 mM corti-costerone [(C) and (D)] or 1.5 nM EN101 [(E)and (F)]. Asterisks indicate columns with signif-icant differences from controls (P , 0.05). Pan-els (I) and (J) present the numbers of cells thatcontain AChE-S or AChE-R mRNA versus theirmaximum distance from the nucleus.

Fig. 3. Increased AChE-R expression and den-dritic translocation under hormone, geneticbackground, sham injection, and chemicalstressors. (A through D) Shown are primarycerebellar neurons stained for AChE activityunder vehicle treatment [(A), 0.1% ethanol] orcorticosterone [(C), 10 mM, 6 hours]. (B) and(D) Higher magnification immunocytochemicalAChE-S labeling. (E through J) Confocal fieldimages of hippocampal CA1 neurons from nor-mal FVB/N mice [(E), (G), and (I)] and trans-genic animals overexpressing human AChE [(F),(H), and (J)], under control conditions [(E) and(F)], 2 days following cannula implantation andperfusion with artificial cerebrospinal fluid [(G)and (H)], or following injection through thisprobe of 125 nmol neostigmine [(I) and (J)]. (Kand L) Neuritic translocation of detectableAChE-R mRNA labeling (in mm from nucleus)for 30 neurons from at least two animals ineach group. Significant differences from notedvalues are starred (P , 0.0005).

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nonstimulated slices from animals tested 1month following 4 days of repeated stress,the mean pfp amplitude was similar to that ofnonstimulated controls. However, exposureof slices from stressed mice to physostigmineresulted in a 12-fold larger increase overbaseline in stimulation-evoked populationspike amplitudes than that observed in non-stressed animals. Atropine administration re-versibly blocked this response by more than90%, much more effectively than its capacityto block field potentials in the control brain(Fig. 4H versus 4G). The stronger responsesto both stimulators and antagonists followingstress spanned the entire range of stimulusintensity (Fig. 4, I and J), similar to thehypersensitivity patterns seen in humans asPTSD. As in control mice, the evoked pfpwas blocked by the NMDA and AMPA an-tagonists, aminophosphonopentanoic acid(APV) and dinitroquinoxalinedione (DNQX),respectively, attesting to the glutamatergic,i.e., excitatory, nature of the hypersensitizedsynapses. Neuronal hypersensitivity has longbeen known to follow stressful experiences(29), but the mechanisms leading to it re-mained unknown. Our current findings sug-gest a role for stimulus-induced AChE-Roverexpression and neuritic translocation inthis phenomenon.

Translocation into neuronal processes pre-sumably depends on cis-acting elements and/or secondary structures, primarily within the39 untranslated region (26, 30). AChE-RmRNA includes no known neurite-targetingmotif; its transport into dendrites may thus beassociated with its unique 39 sequence, orwith the stress-induced accumulation ofmany more nascent AChE-R mRNA tran-scripts. The normally short half-life ofAChE-R mRNA, ;4 hours (31), also appearsto be modified following stress. AChE-RmRNA includes a long U-rich element in the39-UTR (positions 13,020 through 13,007 inGenBank accession number AF312033),which may contribute to mRNA destabiliza-tion through the binding of trans-acting pro-teins (32). Although the importance of thiselement to the stability of AChE-R mRNAremains to be tested, the differential resis-tance of neurite AChE-R mRNA to antisenseoligonucleotide-mediated degradation mayreflect low nuclease levels in neurites, espe-cially of ribonuclease H, which targets DNA-RNA hybrids. AChE mRNA had been shownto be stabilized during neuronal differentia-tion (33); our observations suggest that thismay be due, at least partially, to AChE-Rtranscripts being sequestered in neurites.

The weeks-long hypersensitivity of pre-stressed hippocampus to anticholinesterasesattributes a role to the hippocampus in thecholinergic component of post-stress sensiti-zation. The recorded field potentials weresensitive to both cholinergic and glutamater-

gic antagonists, suggesting a long-termchange in the interactions between these twotransmitter systems. Cholinergic-glutamater-gic interactions have been associated withhigher brain functions such as long-term po-tentiation, memory, and behavior (34), all ofwhich are affected by stress. The hypersensi-tization may also reflect an involvement of

neuritic AChE-R in plasticity, due to its ca-pacity to affect cell-cell and cell-matrix inter-actions (13).

In conclusion, we find that the stress-induced increases in cholinergic neurotrans-mission (35) promote a long-term replace-ment of synaptic membrane AChE-S proteinby its soluble AChE-R counterpart. The long-

Fig. 4. Hypersensitivity in hippocampal slices under long-lasting overexpression of AChE-R mRNA.(A through F) Neuronal overexpression. Shown is a comparison of hippocampal slices from controlmice to those stressed 1 month earlier by forced swim [(A) to (D), Fast Red colorimetry] orexposure to DFP [0.1 mg kg21, three consecutive daily injections, (E) and (F), ELF fluorimetry]. (Gthrough J) Cholinergic hypersensitivity. Shown are extracellular recordings (average 6 SD) on theCA1 areas of two to four hippocampus slices from each of three mice, in response to stratum oriensstimulation of slices from controls [(G) and (I)] or 1 month following consecutive daily stresssessions [(H) and (J)]. Above treatment descriptions are shown representative voltage traces.Stimulations (20 s each) were delivered using a bipolar electrode (10 mm insulated tungsten wires,200 mm apart) placed in the Schaffer collaterals. Applied drug concentrations were: physostigmine,10 mM; atropine, 10 mM; APV, 50 mM; DNQX, 20 mM.

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term hypersensitization to repeated stimulishown by cholinergic brain tracts may be dueto this substitution.

References and Notes1. B. S. McEwen, Annu. Rev. Neurosci. 22, 105 (1999).2. R. M. Sapolsky, L. M. Romero, A. U. Munck, Endocr.

Rev. 21, 55 (2000).3. G. Mezey, I. Robbins, Brit. Med. J. 323, 56 (2001).4. J. A. McCormick et al., Mol. Endocrinol. 14, 506

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299 (2001).13. H. Soreq, S. Seidman, Nature Rev. Neurosci. 2, 294

(2001).14. Poly-L-ornithine–coated cover slips [0.5 mg ml21, 10

min at room temperature (RT )] were sterilized byultraviolet irradiation ( TUV/c 8 W, 3 hours at RT ).Cover slip–grown cells were fixed in 3% paraformal-dehyde (20 min), dried (1 hour at 37°C), washed 235 min in phosphate-buffered saline (PBS) and 23 5min in 0.2% Tween-20 PBS (PBT ), incubated with 10mg/ml proteinase K (5 min at RT ), and rewashed(PBT, 23 5 min). Streptavidin (1 mg ml21, 30 min atRT ) served to block nonspecific labeling. Hybridiza-tion was in a humidified chamber with 10 mg ml21

probe in 50% formamide, 53 SSC, 10 mg ml21 tRNA,10 mg ml21 heparin (90 min at 52°C). Washes werein 50% formamide, 53 SSC, and 0.5% sodium dode-cyl sulfate, and in 50% formamide, 23 SSC at 60°C,then at RT in tris-buffered saline (pH 7.5), with 0.1%Tween-20 ( TBST ) including 2 mM levamisole. Fol-lowing blockade in 1% skim milk (30 min), digoxige-nin-labeled probes were detected with fluorescein- orrhodamine-labeled anti-DIG antibodies (1 hour at RT,three washes in TBST ). Biotin-labeled probes weredetected (a) fluorometrically by conjugation withstreptavidin-Cy2 or -Cy3 (Figs. 1 and 2) or (b) the ELFmethod (Molecular Probes, Eugene, OR) (Fig. 4, E andF), or (c) colorimetrically with streptavidin-alkalinephosphatase conjugates using Fast Red (Roche Diag-nostics, Mannheim, Germany) (Fig. 3, E to J, and Fig.4, A to D). Mounting was with Immu-Mount (Shan-don, Pittsburgh, PA).

15. N. Galyam et al., Antisense Nucleic Acid Drug Dev.11, 51 (2001).

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17. D. Kaufer, A. Friedman, S. Seidman, H. Soreq, Nature393, 373 (1998).

18. Following hybridization with an AChE-R cRNA probe,43 of 50 PC12 cells and 45 of 50 cultured primarycerebellar neurons displayed nuclear as well as cyto-plasmic labeling. In contrast, only 9 of 50 PC12 cellsand 5 of 50 primary neurons labeled for AChE-SmRNA showed nuclear labeling, reflecting differentratios of nuclear pre-mRNA to mature varianttranscripts.

19. H. Soreq, D. Zevin-Sonkin, N. Razon, EMBO J. 3, 1371(1984).

20. M. Shapira et al., Hum. Mol. Genet. 9, 1273 (2000).21. When treated with 1.5 nM INV101, an oligonucleo-

tide of sequence inverse to EN101, both PC12 cellsand cultured primary cerebellar neurons were labeledas extensively as untreated cells with both AChE-Sand AChE-R cRNAs (n 5 20 cells; SD , 17 and 12%,respectively, and P . 0.5 for the two probes).

22. Supplementary material is available at www.sciencemag.org/cgi/content/full/295/5554/508/DC1

23. Samples were incubated with monoclonal anti-AChE-S antibodies ( Transduction Laboratories, San

Diego, CA), dilution 1:200, 1 hour at RT overnight4°C, then similar incubations with HRP-tagged goatanti-mouse secondary antibodies (1:500). Detectionwas with Tyramide Signal Amplification system andCy5 fluorophore (NEN Life Science Products, Boston,MA).

24. C. Erb et al., J. Neurochem. 77, 638 (2001).25. L. Davis, B. Burger, G. A. Banker, O. Steward, J. Neu-

rosci. 10, 3056 (1990).26. C. S. Wallace, G. L. Lyford, P. F. Worley, O. Steward,

J. Neurosci. 18, 26 (1998).27. R. Gray, A. S. Rajan, K. A. Radcliffe, M. Yakehiro, J. A.

Dani, Nature 383, 713 (1996).28. A. Friedman, D. Kaufer, L. Pavlovsky, H. Soreq,

J. Physiol. Paris 92, 329 (1998).29. S. M. Antelman, A. J. Eichler, C. A. Black, D. Kocan,

Science 207, 329 (1980).30. A. Blichenberg et al., J. Neurosci. 19, 8818 (1999).

31. R. Y. Chan, F. A. Adatia, A. M. Krupa, B. J. Jasmin,J. Biol. Chem. 273, 9727 (1998).

32. J. Guhaniyogi, G. Brewer, Gene 265, 11 (2001).33. Z. D. Luo et al., Mol. Pharmacol. 56, 886 (1999).34. T. G. Aigner, Curr. Opin. Neurobiol. 5, 155 (1995).35. A. Imperato, S. Puglisi-Allegra, P. Casolini, L. Ange-

lucci, Brain Res. 538, 111 (1991).36. We thank K. Loffleholz and J. Klein (Mainz) for fruitful

discussions and N. Melamed-Book ( Jerusalem) forassistance. Supported by the U.S. Army Medical Re-search and Materiel Command (DAMD 17-99-1-9547to H.S. and A.F.), U.S.-Israel Binational Science Foun-dation (1999/115 to H.S., 1998/066 to N.B.-A., 1997/00174 to A.F.), European Community (QLG3-CT-2000-00072 to N.B.-A), and Ester Neuroscience, Ltd.(to H.S.). C.E. was a Minerva Foundation postdoctoralfellow.

2 October 2001; accepted 30 November 2001

Dynamics and Constancy inCortical Spatiotemporal Patterns

of Orientation ProcessingDahlia Sharon* and Amiram Grinvald

How does the high selectivity to stimulus orientation emerge in the visualcortex? Thalamic feedforward-dominated models of orientation selectivitypredict constant selectivity during the visual response, whereas intracorticalrecurrent models predict dynamic improvement in selectivity. We imaged thecat visual cortex with voltage-sensitive dyes to measure orientation-tuningdynamics of a large neuronal population. Tuning-curve width did not narrowafter response onset, whereas the difference between preferred and orthogonalresponses (modulation depth) first increased, then declined. We identified asuppression of the evoked responses, referred to as the evoked deceleration-acceleration (DA) notch, which was larger for the orthogonal response. Fur-thermore, peak selectivity of the tuning curves was contemporaneous with theevoked DA notch. These findings suggest that in the cat brain, sustained visualcortical processing does not narrow orientation tuning; rather, intracorticalinteractions may amplify modulation depth and suppress the orthogonal re-sponse relatively more than the preferred. Thus, feedforward models and re-current models of orientation selectivity must be combined.

Visual cortical neurons are highly selectivefor the orientation of stimuli presented withintheir receptive field (1), a property not sharedby their thalamic inputs (2). How orientationselectivity arises in the cortex is still debated.Previous experiments (3–10) have suggestedmechanisms that include feedforward (thal-amically dominated) (1, 11) and recurrent(intracortically dominated) (12–15) models.The input impinging on orientation-selectiveneurons has constant selectivity in feedfor-ward models, whereas recurrent models pre-dict improvement in selectivity during thevisual response. Although single-unit meth-odologies excel at determining the propertiesof individual neurons, these properties are

highly variable, making it extraordinarily dif-ficult to obtain large samples on which tobase estimates of neuronal population behav-ior. In optical imaging with voltage-sensitivedyes (16), the recorded signal accurately rep-resents membrane-potential changes at theneuronal population level (17, 18), emphasiz-ing synaptic potentials in the dendritic tufts ofcortical neurons from superficial and deeplayers. Recently, this method has been im-proved substantially, enabling in vivo imag-ing of cortical neuronal population activitywith millisecond temporal resolution and spa-tial resolution of 50 to 100 mm (19). We there-fore used optical imaging to explore the dynam-ics of orientation selectivity (9, 20–22).

We imaged the responses of area 18 in thecat visual cortex to high-contrast square-wave gratings of six different orientations(23). We examined the recording period start-ing 50 ms before stimulus onset and lasting300 ms (thus avoiding late intrinsic signal

Department of Neurobiology and the Center for Stud-ies of Higher Brain Functions, Weizmann Institute ofScience, Rehovot 76100, Israel.

*To whom correspondence should be addressed. E-mail: [email protected]

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63

�!��

Pyridostigmine enhances glutamatergic transmission in hippocampal CA1 neurons.

Pyridostigmine enhances glutamatergic transmissionin hippocampal CA1 neurons

Lev Pavlovsky, R. Orie Browne, and Alon Friedman*Departments of Physiology and Neurosurgery, Soroka University Medical Center, Ben-Gurion University and

Zlotowski Center of Neuroscience, Beersheva, 84105 Israel

Received 18 March 2002; accepted 27 September 2002

Abstract

Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the autoimmune diseasemyasthenia gravis. Due to its positively charged ammonium group, under normal conditions pyridostigmine cannot cross the blood-brainbarrier (BBB) and penetrate the brain. However, several studies have suggested that under conditions in which the BBB is disrupted,pyridostigmine enters the brain, changes cortical excitability, and leads to long-lasting alterations in gene expression. The aim of this studywas to characterize the mechanisms underlying pyridostigmine-induced changes in the excitability of central neurons. Using whole cellintracellular recordings in hippocampal neurons we show that pyridostigmine decreases repetitive firing adaptation and increases theappearance of excitatory postsynaptic potentials. In voltage clamp recordings, both pyridostigmine and acetylcholine (ACh) increased thefrequency but not the amplitude of excitatory postsynaptic currents. These effects were reversible upon the administration of the muscarinicreceptor antagonist, atropine, and were not blocked by tetrodotoxin. We conclude that pyridostigmine, by increasing free ACh levels, causesmuscarinic-dependent enhancement of excitatory transmission. This mechanism may explain central side effects previously attributed to thisdrug as well as the potency of AChE inhibitors, including nerve-gas agents and organophosphate pesticides, in the initiation of corticalsynchronization, epileptic discharge, and excitotoxic damage.© 2003 Elsevier Science (USA). All rights reserved.

Keywords: Acetylcholine; Acetylcholinesterase inhibitors; Glutamate; Muscarinic receptors; Presynaptic modulation; Synaptic transmission

Introduction

Pyridostigmine, a carbamate acetylcholinesterase inhib-itor (AChE-I), is routinely employed in the treatment of theautoimmune disease myasthenia gravis (Lisak, 1999; Victorand Ropper, 2001) and is also recommended by most armiesfor use as a pretreatment under threat of chemical warfarebecause of its protective effect against organophosphatepoisoning. Under routine treatment, pyridostigmine is ex-cluded from the brain due to a positively charged ammo-nium group that prevents its penetration through the blood-brain barrier (BBB). However, several reports havesuggested that under acute stress pyridostigmine may pen-etrate the mouse brain (Esposito et al., 2001; Friedman et

al., 1996; but see also Grauer et al., 2000), increase neuronalexcitability, and induce long-lasting alterations in gene ex-pression (Friedman et al., 1996; Kaufer et al., 1998).

The mechanisms underlying pyridostigmine-inducedchanges in neuronal excitability are not completely under-stood. While increased free acetylcholine (ACh) levels dueto AChE inhibition may underlie depolarization as well asdecreased adaptation and action potential threshold (Coleand Nicoll, 1984), other mechanisms not related to AChhave been previously reported (Moretto, 1998). Actions ofACh include both altering the conductance of specific ionicchannels and modulating synaptic transmission. For exam-ple, the activation of the nicotinic �7 subunit was demon-strated to increase intracellular Ca2� and to enhance gluta-mate release in single mossy-fiber presynaptic terminals(Gray et al., 1996). The electrophysiological changes sec-ondary to activation of muscarinic receptors were studied

* Corresponding author. Fax: �972-8-6400896.E-mail address: [email protected] (A. Friedman).

R

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more extensively in the CA1 region. It was suggested todecrease conductance of several potassium channels result-ing in a slow depolarization and decreased firing adaptation(Benardo and Prince, 1982a,b; Cole and Nicoll, 1984; Mad-ison et al., 1987; McQuiston and Madison, 1999; Segal,1982), to enhance back propagation of action potentialsalong the dendritic tree (Tsubokawa and Ross, 1997), and toaffect both pre- and postsynaptic processes by decreasingCa2� conductance (Gahwiler and Brown, 1987; Qian andSaggau, 1997).

Anatomical data support the modulatory role of ACh.For example, previous studies showed that cholinergic fi-bers terminating in the stratum radiatum of the CA1 areaexhibit an extremely low (�10%) frequency of junctionalspecialization (Descarries et al., 1997). In the neocortex, theabundance of nonjunctional oppositions of cholinergic pro-files to dendritic spines and axons (Mrzljak et al., 1995) aswell as numerous muscarinic receptors in pre- and postsyn-aptic sites of asymmetric synapses (Mrzljak et al., 1998,1993) supports the notion that paracrine ACh release playsa role in modulating excitatory transmission. However,studies in the human cerebral cortex also show that thedistribution of neurons rich in AChE does not parallel cho-linergic innervation (Mesulam and Geula, 1991). These dataraised the possibility of other, noncatalytic roles for AChE.Sternfeld et al. (1998) gave evidence to support such rolesin neuronal cultures demonstrating that AChE enhancesneuritic growth and synaptic development by a mechanismunrelated to its catalytic function. These studies, togetherwith enhanced AChE transcription following CNS exposureto pyridostigmine (Kaufer et al., 1998), raise the possibilityof noncholinergic responses following such exposure.

In this study, based on intracellular recordings in hip-pocampal CA1 neurons, we propose that increased AChlevels result in alterations in intrinsic and synaptic proper-ties that lead to enhanced neuronal excitability and explainthe central side effects of pyridostigmine and its propensityto excitotoxicity.

Materials and methods

Slice preparation

Housing and treatment of animals were in accordancewith institutional guidelines. Slices were prepared as previ-ously reported (Fleidervish et al., 1996, 1998). In short,brains of anesthetized mice were quickly removed andplaced in ice-cold artificial cerebrospinal fluid (aCSF) con-taining (in mM): NaCl, 124; KCl, 3; MgSO4, 2; NaH2PO4,1.25; NaHCO3, 26; D-glucose, 10; and CaCl2, 2 (pH 7.4),and saturated with 95% O2 and 5% CO2. In order to pre-serve cholinergic pathways, 400 �M slices were cut in thesaggital plane (Vibroslice, Campden Instruments, Lough-borough, UK) because the major cholinergic input to thehippocampus originates in the anteriorly located septum.

After at least 60 min of equilibration in a storage chamber,slices were transferred to an interface recording chamber,continuously perfused with oxygenated aCSF at 32°C.

Whole cell recordings

This study is based on stable recordings (holding for�15 min, series resistance of �8 M� or Vm � �60 mV)from a total number of 75 neurons, recorded in slices fromyoung mice (12–20 days old, n � 32 animals) of either sex.Whole cell recordings from cells in the CA1 pyramidal celllayer were made using the patch clamp technique (Hamill etal., 1981) as modified for blind recording in slices (Blantonet al., 1989). Patch pipettes, pulled from borosilicate glasscapillaries (Hilgenberg, Germany) on a Narishige PP83puller, had resistances of 3–5 M�. For voltage clamp re-cordings pipettes were filled with a solution consisting of (inmM): CsCl, 110; CsOH, 25; EGTA, 11; CaCl2, 1; MgCl2, 2;and N-2-hydroxyethylpiperazine- N�-2-ethansulfonic acid(Hepes), 10. The pH (7.2) was adjusted with CsOH. Seriesresistance, in the range of 5–8 M�, was compensated by40–85% using the built-in circuitry of an Axopatch-200A(Axon Instruments, Foster City, CA) amplifier.

For current clamp recordings, pipettes were filled with asolution consisting of (in mM): K-gluconate, 135; KCl, 6;MgCl2, 2; Hepes, 10. The pH (7.2) was adjusted with KOH.

Data acquisition and analysis

Signals were filtered at 2 kHz (4-pole Bessel filter),stored on video, and digitized off line at 5–10 kHz usingAxotape (Axon Instruments) or WCP (J. Dempster) soft-ware. Synaptic currents were analyzed using the Mini Anal-ysis program (Jaejin software), demo version. Data areexpressed throughout as means � SEM. Differences be-tween inter-event intervals and amplitude under controlaCSF and following ACh application were determined byStudent’s paired t tests. P � 0.05 was taken as the level ofstatistical significance.

Drug application

Drugs were applied by addition to the bathing solution orby local application as noted in the text. ACh and atropine(both 5 mM dissolved in aCSF) were applied by pressuremicroinjection (20–30 psi) from glass electrodes (tip diam-eter 50–100 �m) using a Picospritzer II (General Valve,Fairfield, NJ). These electrodes were positioned tangentiallyto, and at a distance of 100–200 �m, from the recordingelectrode. In some experiments pyridostigmine, atropine (1�M each), bicuculine-mediodid (BMI, 10 �M), DL-2-ami-no-5-phosphonovaleric acid (AP5), or 6,7-dinitroquinoxa-line-2, 3-dione (DNQX, 20–50 �M each) was added to thebathing solution (all drugs were purchased from Sigma).

182 L. Pavlovsky et al. / Experimental Neurology 179 (2003) 181–187

Results

Current clamp recordings using whole cell patchconfiguration

During current clamp recordings, injection of long de-polarization steps evoked repetitive firing with a character-istic adaptation (Fig. 1A). Firing adaptation was reducedfollowing the addition of pyridostigmine (10 �M) to thebathing solution similar to previous studies with the AChEinhibitor, eserine, using sharp electrodes (Madison et al.,1987). Unlike these previous studies, the high input resis-tance characterizing recordings under the whole cell patchconfiguration technique enables the detection of spontane-ous postsynaptic potentials (PSPs). Pyridostigmine in-creased PSP frequency (Figs. 1B, C), while it did not haveany measurable effect on the passive membrane character-istics (membrane potential, input resistance around restingpotential, and membrane time constant) (Figs. 1A, C).These effects of pyridostigmine were reversible 30 minfollowing the addition of atropine to the extracellular solu-tion (Figs. 1A, B, C). While the effect of AChE-Is on firingadaptation was shown to be due to inhibition of atropine-sensitive slow potassium channels (Benardo and Prince,1982b; Madison et al., 1987), the mechanism underlying theenhanced occurrence of synaptic potentials is not known. Todirectly test AChE-Is effects on excitatory postsynaptic cur-rents (EPSCs) generated along the dendritic tree, the whole

cell patch configuration method was used for voltage clamprecordings.

AChE inhibitors increase EPSC frequency

In these experiments inhibitory transmission wasblocked by extracellular bicuculine monoiodit (GABA-Areceptor antagonist). Intracellular CsCl2 was used to blockGABA-B currents (Ling and Benardo, 1994) as well asmuscarinic-dependent potassium channels (Coggan et al.,1994). Under these conditions, perfusion with the AChE-I,pyridostigmine (1 �M), resulted in increased spontaneousEPSC frequency in 5 out of 6 recorded neurons, suggestingthat the drug enhanced release of neurotransmitter vesicles.In the cell shown in Fig. 2A, under control conditions,EPSC analysis revealed 101 events during a 10-s window(mean interval 100.6 � 87.7 ms) as compared to 155 events30 min following the addition of pyridostigmine (meaninterval 70.9 � 63.7, P � 0.005). The increase in frequencywas not associated with significant changes in EPSC am-plitude (14.4 � 8.4 vs 13.2 � 8.2 pA), rise time (6.4 � 9.9vs 8.4 � 10.7 ms), or decay time (2.7 � 4.9 vs 2.6 � 4.2

Fig. 2. Pyridostigmine increases EPSC frequency. (A) Five consecutivetraces under control conditions (left) and 30 min following the addition ofpyridostigmine to the bathing solution. (B) Frequency histogram of 10-sanalysis of spontaneously occurring EPSCs before (open bars) and afterpyridostigmine (filled bars) showing the increased appearance of events atlow intervals following drug addition. On the right, averaged EPSCscharacteristics following pyridostigmine in percentage change related tocontrol. (C) Frequency histogram of spontaneous EPSCs (left) and aver-aged values of EPSC characteristics (right) under normal aCSF and 30 minafter the addition of atropine. Averaged values are given as percentage ofcontrol. EPSCs were collected and measured at holding potential of�70mV.

Fig. 1. Pyridostigmine enhances neuronal excitability. (A) Representativecurrent clamp recording from a CA1 pyramidal neuron. Note the decreasein firing adaptation following pyridostigmine (pyrido, middle trace) thatreversed following the addition of atropine (right). (B) Three consecutivetraces under control (left), pyridostigmine (middle), and atropine (right),showing increase in the appearance of spontaneous synaptic events. (C)Quantitative analyses of pyridostigmine effects: Left graph: V/I curve.Averaged voltage deflection from 0.5 to 1 s of subthreshold current injec-tion. Middle graph: Firing adaptation vs amplitude of injected current.Firing adaptation was calculated by dividing the first interspike frequency(f1) to the adapted firing frequency (averaged frequency between 0.5 and1 s). Right graph: PSP interval histogram in a 1-min time window under thethree experimental conditions.

183L. Pavlovsky et al. / Experimental Neurology 179 (2003) 181–187

ms), thus indicating a presynaptic site of action (Fig. 2B).Since a pyridostigmine-mediated increase in excitabilitywas shown to depend upon activation of muscarinic recep-tors (Friedman et al., 1998; Kaufer et al., 1998) the non-specific muscarinic antagonist, atropine, was added to thebathing solution. Following atropine perfusion, EPSC fre-quency decreased by �40% with no effect on amplitude orrise or decay time (n � 3, Fig. 2C).

ACh enhances excitatory transmission

Next, we used a drop application of ACh to explorewhether AChE-I effects were mediated through an increasein extracellular ACh concentration (see Materials and meth-ods). In 8 out of 11 cells ACh application caused instanta-neous increase in the frequency of spontaneous events thatreturned to baseline within 10 s (Fig. 3A, inset). While inmost cases response to ACh application was seen within

less than 100 ms, pointing to dendritic action, in two cells,increased frequency was noted only 2–3 s following appli-cation, presumably due to diffusion of the applied ACh. Inthree cells, drop application was associated with a largeinward current (�200 pA amplitude, 50–200 ms duration)followed by frequent EPSCs (Figs. 3A, B). Fig. 3C sum-marizes detailed analysis of three cells showing the meanincrease in cumulative EPSC frequency without an effect onamplitude measured during a 2-s time window followingACh application. When atropine was applied locally, 5–10s prior to ACh, the evoked inward current and frequentappearance of EPSCs were significantly reduced (Fig. 3D).

ACh enhances excitatory transmission in the presence ofTTX

Our results point to a presynaptic effect of ACh toenhance transmitter release. One possible explanation is thatACh increased firing in presynaptic axons, perhaps througha reduced potassium conductance or direct action on Na�

channels (Narahashi, 1996). To elucidate such a mecha-nism, ACh was applied in the presence of TTX (10�6 M) inthe bathing solution. TTX efficacy was assured as no actionpotentials were evoked when holding potential was movedto depolarizing levels (��40 mV). Under these conditions,miniature EPSCs (mEPSCs) were recorded with an averageamplitude of 7.5 � 2.8 pA. Although the effects of AChwere less prominent under these conditions, a statisticallysignificant (paired sample t test, P � 0.01) increase in themEPSC frequency was observed in 3 out of 4 cells (meanintervals in ms � SEM for the 4 cells under control condi-tions vs following ACh application: 144.2 � 22.7 vs 82.2 �5.5; 214.2 � 20.6 vs 92.7 � 12.2; 86.7 � 5.1 vs 66.4 � 3.7;217.4 � 33.7 vs 195.2 � 43.2) with no significant change inamplitude (Fig. 4A, B). Similar experiments in the presenceof either the NMDA-receptor (5-APV) or the AMPA-recep-

Fig. 3. ACh increases EPSC frequency. (A) Whole cell patch, voltageclamp recording from a neuron at a holding potential of �70mV. Appli-cation of ACh induced prolonged inward current and transient increase inthe frequency of spontaneous synaptic events. Inset—the bar graph showsthe number of events/s before and after ACh application (drop applied atTime � 0). (B) At a faster time scale, events from the “*” sign showed inA. (C) Cumulative frequency histogram for three cells, showing a de-creased interval between EPSCs with no change in amplitude. (D) Intra-cellular recording from a different neuron at a holding potential of �70mV.ACh application induced inward current and small EPSCs. Prior applica-tion of atropine greatly diminished both ACh-mediated effects.

Fig. 4. ACh enhances glutamatergic transmission in the presence of TTX.(A) A 5-s trace showing an increase in the appearance of miniature EPSCsfollowing application of ACh. (B) Quantitative analysis of three cellsshowing significant increase in the number of mEPSCs during 8-s analysisfollowing ACh application (left). The same analysis showed no significantchange in amplitude of events (right). Cumulated numbers are presented aspercentage of maximum under control conditions.


tor antagonists in the extracellular solution revealed, asexpected, larger non-NMDA-mediated EPSCs at �70mV(9.28 � 0.34, n � 173 and 5.15 � 0.07 pA, n � 285 events,for AMPA- and NMDA-mediated events, respectively). Asignificant interval decrease was measured under both con-ditions following bath application of ACh: from 57 � 2.6 to40.1 � 1.43 ms (n � 187 events) for presumed NMDA-mediated events, and from 74.4 � 4.1 to 38.2 � 1.4 ms (n� 143 events) for presumed non-NMDA-mediated EPSCs(n � 2 cells, P � 0.01).

Discussion

Our results support the following conclusions regardingthe effects of pyridostigmine on CA1 pyramidal neurons:(1) pyridostigmine enhances spontaneous glutamate release;(2) these effects are mediated via increased free ACh levels;and (3) ACh action is mediated through the activation ofmuscarinic receptors presumably located on presynaptic ter-minals.

Previous studies using sharp electrodes for current clamprecordings have suggested that inactivation of several slowpotassium conductances increases excitability of CA1 neu-rons following muscarinic activation (Benardo and Prince,1982,b; Cole and Nicoll, 1984; Madison et al., 1987; Segal,1982). The introduction of the voltage clamp techniqueusing the whole cell patch configuration allowed us toexplore the effects of ACh on fast excitatory synaptic cur-rents that emanate electrotonically distant from the soma.While increasing the occurrence of spontaneous release maynot affect somatic membrane potential because most exci-tatory synapses are located on the electrically isolated spineheads, the concurrent reduction of K� conductance alongthe dendritic tree will cause the cell to be more electricallycompact and thus contribute to the slow EPSPs recorded inthe soma following muscarinic activation (Madison et al.,1987) as well as to the boosting of action potential backpropagation along the dendrites (Tsubokawa and Ross,1997). Interestingly, Seeger and Alzheimer (2001) recentlyshowed m2-dependent activation of an inward-rectifyingpotassium current, suggesting a voltage-dependent inhibi-tion of AMPA-mediated EPSPs. Future studies are awaitedto reveal the ultimate effect of muscarinic activation inrelation to the precise localization of different subtypes ofmuscarinic receptors and dependent channels. In addition,several recent studies have shown that excitatory synapsescan contain NMDA receptor responses in the absence offunctional AMPA receptors and are, therefore, postsynap-tically silent at resting membrane potentials (Liao et al.,1995, 2001; Takumi et al., 1999). ACh-mediated enhancedglutamate release might then activate silent synapses via therapid acquisition of AMPA receptors and may be importantin synaptic plasticity and neuronal development (Liao et al.,2001). Such a mechanism could contribute to the aug-mented NMDA current recorded in the presence of ACh

(Markram and Segal, 1990, 1992), as well as to muscarinic-dependent long-term potentiation (Auerbach and Segal,1994; Sokolov and Kleschevnikov, 1995; Kobayashi et al.,1997) and memory formation (Perry et al., 1999). Underconditions in which ACh levels increase dramatically, suchas following toxic exposure to AChE-Is (Messamore et al.,1993), the increased glutamatergic current may lead to rapidsynchronization of the local network manifested as the char-acteristic seizure often observed after exposure to theseagents. This may explain the similarities reported betweenorganophosphates and excitatory amino acids in inducingneuronal toxicity (Noraberg et al., 1998; Solberg and Bel-kin, 1997).

Interestingly, ACh was reported to activate inhibitoryinterneurons in the CA1 region through the activation ofnicotinic receptors (Alkondon et al., 1997; Jones and Yakel,1997), abundant in interneurons but not in pyramidal cells(Sudweeks and Yakel, 2000). Concurrent activation of bothexcitatory and inhibitory neurotransmission was postulatedto underlie ictal activity due to both GABA-A-dependentdendritic depolarization and increased presynaptic excitabil-ity (Traub et al., 1996).

The mechanism underlying ACh-induced glutamate re-lease is not known. However, a mechanism involving en-hancement of release from presynaptic terminals is sup-ported by our data showing no change in amplitude, risetime, or fall time of EPSCs following administration ofeither ACh, an inhibitor of the hydrolyzing enzyme or amuscarinic receptor antagonist. Our observation that AChaction was still observed following the blockade of presyn-aptic action potentials (Fig. 4), and had a similar effect forboth NMDA- and non-NMDA-mediated EPSCs, furthersupports a direct presynaptic mechanism. Previous reportsshowing a decrease in Ca2� entry to, presumably, presyn-aptic CA3 terminals following carbachol exposure suggestthat voltage-dependent Ca2� channels may not be involved(Qian and Saggau, 1997; Scanziani et al., 1995). In line withthese data, recent experiments in isolated rat brain synap-tosomes showed calcium-independent, voltage-dependentdirect muscarinic control on transmitter release (Ilouz et al.,1999). Future experiments should be directed to test thedetails of muscarinic effects on specific proteins participat-ing in the machinery of transmitter release.

Our data implicate a functional coupling between cho-linergic and glutamatergic transmission in hippocampal py-ramidal cells. As the role of ACh in learning and memory iswell documented, any alteration in coupling properties inthe hippocampal region is expected to result in significantfunctional disturbances. We have recently shown long-last-ing alterations in the expression of key cholinergic proteinsin the neocortex and hippocampus following acute stress orexposure to AChE-Is (Kaufer et al., 1998; Meshorer et al.,2002). These changes in gene expression tend to reduceACh synthesis (by down regulating cholineacetyltrans-ferase) and enhance its hydrolysis (by up-regulation of theread-through form of AChE), potentially altering this func-


tional coupling between the two transmitter systems. In-deed, extreme sensitivity of glutamatergic-mediated synap-tic transmission to either AChE-Is or muscarinic antagonistswas found in mice, 4 weeks following repeated exposure tostress or peripheral injection of low doses of AChE-Is (Me-shorer et al. 2002). Future studies should explore the de-tailed mechanisms underlying these changes in cholinergic-glutamatergic coupling and under what conditions suchdisturbances might occur.

Our data suggest that under conditions in which theblood-brain barrier is disrupted, clinically relevant, low con-centrations of pyridostigmine (Aquilonius and Hartvig,1986) may alter cortical function. Disruption of the BBBhas been reported in a wide range of clinical conditionsincluding primary CNS pathology and systemic disease(Tomkins et al., 2001). Indeed, CNS-related side effectswere occasionally reported following pyridostigmine ad-ministration in patients with myasthenia gravis (Iwasaki etal., 1988), in a soldier with low activity of the serum-scavenging enzyme, butyril-ChE (Loewenstein-Lichten-stein et al., 1995), or under stress (Sharabi et al., 1991).Further research is warranted to search for evidence of BBBdisruption in patients suffering from CNS-related symptomswho are treated with pyridostigmine or other peripherallyacting drugs.

Acknowledgments

Supported by grants from the US-Israeli Binational Sci-ence Foundation Grant 9700174, Grant 4536 from theChief’s Scientist’s Office of the Ministry of Health, Israel,and the U.S. Army Medical Research and Materiel Com-mand (DAMD 17-99-1-9547).

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64

�"��

Muscarinic Neuromodulation in

the Hippocampus and Parahippocampal Region

1

Muscarinic Neuromodulation in the Hippocampus and Parahippocampal Region

Browne, R.O., Pavlovsky, L., and Friedman, A.*

1Departments of Physiology and Neurosurgery, Soroka University Hospital and

Zlotowski Center of Neuroscience, Ben-Gurion University, Beersheva, Israel.

* To whom correspondence should be addressed

Tel: 972-8-6400893

Fax: 972-8-6400896

Email: [email protected]

2

The cholinergic system, including both muscarinic and nicotinic subsystems, plays an

important role in a variety of cognitive functions including attention, learning and

memory.1 The cholinergic role in the processes underlying these cognitive functions

seems to be mediated by cholinergic modulation of neurons that are simultaneously

being acted upon by other afferents. Here we will focus on muscarinic

neuromodulation in the hippocampus and parahippocampal regions, which are

structures known to be critically involved in both higher cognitive functions, like

learning and memory, as well as in pathological states like epilepsy.2 Studying the

role of muscarinic activation on the overall function of the neuronal circuit can be

achieved by electrophysiological recordings from large regions of the intact brain

(e.g. during electroencephalography) or the isolated brain region in-vitro. However,

the underlying mechanisms of muscarinic effects can be studied only using recordings

from single neurons under specific pharmacological conditions. We will shortly

summarize here results from different approaches, necessary for perceiving a more

complete picture of the role of muscarinic receptors in the healthy and the diseased

brain.

The muscarinic acetylcholine (ACh) receptor is a G protein-coupled metabotropic

receptor. There are five known muscarinic receptor subtypes (labeled M1-M5)

encoded by at least five different genes,3 and there may be as many as nine.4 The

complex diversity in localization of muscarinic receptor subtypes within the central

nervous system (CNS) provides a wide variety of differential responses in distinct

areas. Due to the limited selectivity of ligands, the mechanisms underlying different

responses are not always clear.

3

Anatomy

The hippocampus and parahippocampal region receive a considerable cholinergic

input which originates primarily from the basal forebrain through the fimbria-fornix.5

This projection was shown by anatomical studies using staining for

acetylcholinesterase (AChE) and choline acyltransferase (ChAT) to be derived

primarily from the medial septum and diagonal band of Broca.6-9 Moreover,

stimulation of this pathway leads to increased ACh levels in the hippocampus.

Little is known about the detailed synaptic connectivity of cholinergic inputs to the

hippocampus and parahippocampal region. There is an abundance of cholinergic

fibers in stratum oriens of CA1 and dentate hilus.7-9 However, the detailed anatomical

targets of these synapses are not known. Interestingly, in the neocortex, cholinergic

boutons are frequently observed in close apposition to asymmetrical (presumably

excitatory) synapses on dendritic spines and shafts without forming recognizable

synaptic specializations. Immunohistochemical localization of muscarinic receptors,

the predominant cholinergic receptors in adult brain, confirms the anatomical data on

cholinergic innervation; muscarinic receptors are found on postsynaptic elements

apposed to symmetrical synapses, predominantly on dendritic shafts. In addition,

muscarinic receptors are found in pre- and post-synaptic elements of non-cholinergic,

asymmetrical synapses, including those on dendritic spines. Thus, anatomical data

suggest that ACh affects synaptic integration through activation of both classical

cholinergic synapses and through direct modulation of non-cholinergic synapses.

In cortical cells, there are currently 5 known muscarinic receptor subtypes

expressed.10 Immunocytochemical studies regarding the localization of different

4

muscarinic receptor sub-types show complex patterns and region-specificity. For

example, Rouse and colleagues11 showed that M1-M4 receptors are differentially

expressed in laminar patterns in the molecular layer of the dentate gyrus. Moreover,

while M1 receptors were found in dendrites and spines, M2-M4 were most frequently

found on pre-synaptic terminals. These differences may be functionally important as,

for example, shown in physiological experiments where muscarinic receptor agonists

inhibit synaptic transmission at the medial but not lateral perforant path.12 These

physiological studies, as well as lesion studies, suggest that different muscarinic

receptor subtypes are differentially expressed in different sites of the CNS synapse,

and have different functions.

Physiology

While anatomical details are necessary to identify possible candidates for muscarinic

action in cortical regions, physiological experiments are essential for understanding of

the detailed effects of muscarinic activation. Such experiments suggest a complex and

sometimes conflicting picture regarding muscarinic effects. Here we will briefly

summarize effects of muscarinic receptor activation on brain electrophysiology at

different levels using several experimental approaches.

Rhythmogenesis

The excitation of cholinergic neurons in the basal forebrain is followed by a shift in

the electroencephalogram from low frequency, high-amplitude to high-frequency, low

amplitude fluctuations. Muscarinic activation of the septal nucleus is known to induce

a prominent rhythmic slow activity at the theta range (4-12 Hz)13 in both the

hippocampus and entorhinal cortex.14-17 Theta rhythm oscillations have been shown to

5

facilitate synaptic plasticity in CA1 of the hippocampus18 and, as such, to have an

important role in memory and its cellular representative, long term potentiation

(LTP). Indeed, rhythmic activity is one of the hallmarks of normal brain function and

apparently reflects changes in neuronal properties and their ability to respond to

incoming stimuli, the cellular correlate of alertness and attention. Indeed, frequent

diseases of cortical brain structures, like epilepsy and Alzheimer’s disease are often

characterized by abnormal rhythmogenesis.19-21

Another rhythm frequently studied is the gamma rhythm (20-80 Hz),22,23 which is also

reported to be important for information processing.24,25 Muscarinic receptor

activation (apparently mediated by the M1 receptor subtype) can induce gamma

network oscillations in the hippocampus.26 In mice, knockout of the M1 but not M2-

M5 receptor results in inability to induce gamma oscillations in CA3 region.27

Many studies explored the association of rhythmic activity and pathological

conditions. As shown in Figure 1A, the cholinergic agonist carbamylcholine (CCh,

10-30 µM) causes spontaneous synaptic oscillations and epileptiform discharges,

which are probably driven by cells in layer V and are blocked by the non-specific

muscarinic agonist, atropine.28

Population activity in small neuronal networks

To study the detailed mechanisms underlying changes in activity in groups of

neurons, many investigators use the in-vitro slice preparation, which gives the

advantage of a stable system enabling a wide variety of pharmacological and

6

experimental manipulations. However, it should be remembered that most of these

studies use brain slices which are deafferented from the principal cholinergic nuclei.

Bliss and Lomo originally described LTP of excitatory post-synaptic potentials

(EPSPs) in hippocampal neurons.29 Since that time, LTP has been studied extensively

as a candidate electrophysiological correlate of memory. The literature contains a few

reports of either no cholinergic effect on hippocampal LTP30,31 or inhibition of LTP in

the CA3-mossy fiber system.32 However, most studies suggest cholinergic facilitation

of LTP in the hippocampus.33-41 Facilitation of LTP may be linked to potentiation of

NMDA responses by muscarinic activity42-46 or enhanced release of ACh from

presynaptic terminals.47 It is possible to induce LTP in the deep layers of the

entorhinal cortex in the presence of atropine, but it is significantly suppressed.48

Although induction of LTP in the hippocampus and entorhinal cortex does not appear

to necessitate cholinergic input, it seems that cholinergic modulation does play a

significant role in facilitation of LTP.

A prominent inhibitory activity of muscarinic activation is transient depression of

field EPSPs in both hippocampus and entorhinal cortex by CCh (> 2 µM).48-51 The

amplitude of field EPSPs was depressed as much as 56%.50 However, there is a wide

disperity of results between studies. This short term depression is dose-dependent49

and blocked by atropine,48-50 but not pirenzepine,49 a selective M1 antagonist.

Furthermore, Auerbach and Segal51 showed that the M3 antagonist 4-

diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) blocked depression of

field EPSPs (slope), whereas M1, M2 and M4 antagonists did not block the

depression. Mecamylamine, a nicotinic antagonist had no effect.49 Paired pulse

7

facilitation (PPF) during depression was enhanced in both hippocampus and

entorhinal cortex,48,50,52 suggesting that in some cases muscarinic activation results in

decreased transmitter release. Interestingly, unlike CCh, ACh itself was reported to

enhance release of glutamate from Schaffer collaterals fibers, when inhibition is

blocked.47 Additionally, Massey and colleagues49 reported long lasting depression (24

± 3%, 50 µM CCh) of field EPSPs in layer II/III of the perirhinal cortex following

CCh-induced short lasting depression. This effect is possibly modulated by M1

receptors, as both atropine and pirenzepine blocked the effect.

Interestingly, low-dose CCh (< 1 µM) produced strikingly different results from those

obtained with higher doses. CCh (< 1 µM) caused a transient, reversible depression of

field EPSPs occasionally followed by a long-lasting potentiation,48,50 sometimes

referred to as muscarinic LTP (LTPm).46 LTPm in the rat hippocampus is similar to

LTP produced by tetanic stimulation in that it shows increased activity to afferent

stimulation which starts slowly and persists over time. However, its effects are

mediated by a post-synaptic M2 receptor instead of an NMDA receptor, although it

appears that the same post-receptor mechanism is used for both LTPm and tetanic,

NMDA-dependent LTP. Notably, very low doses of CCh (0.1 µM) facilitated

induction of tetanic LTP without producing an effect on their own. LTPm could be a

pre-synaptic effect,47 a post-synaptic effect50,51 or both. The probability of inducing

LTPm after depression by low-dose CCh was greater in the hippocampus than

entorhinal cortex, possibly due to regional variance in muscarinic receptor subtypes.50

Synaptic transmission

8

Muscarinic receptors modulate inhibition of cholinergic synaptic transmission.

Muscarinic autoreceptors on pre-synaptic sites in the hippocampus mediate release of

ACh from cholinergic nerve terminals. In a study performed by Zhang and

colleagues53 M2-M4 double knockout mice were used, and it was found that the non-

selective muscarinic agonist, oxotremorine (0.1-10 µM), inhibited K+-stimulated

release of ACh up to 80% in wild type mice, whereas no inhibition was recorded in

double knockout mice. In addition, studies with single knockout mice indicated that

the M2 receptor was principally responsible for inhibition in the hippocampus.53

However, a pharmacological study by Vannucchi and colleagues54 found that the M1

and M4 receptor subtypes were most likely responsible for regulation of ACh release.

As stated above, there are apparently conflicting conclusions in the literature

regarding muscarinic effects on glutamatergic transmission in the hippocampus and

parahippocampal regions. As PPF enhancement during CCh-induced depression of

field potentials may serve as an indirect evidence for decreased excitatory release, a

more direct measure of spontaneous excitatory post-synaptic currents (EPSCs)

showed that ACh increased, and atropine decreased, the frequency of events,

suggesting enhanced release (Figure 1B).47 Increased release was still observed

following addition of the Na+-channel blocker, TTX, to the bath, excluding indirect

effects on pre-synaptic firing. However, it is hard to exclude other effects like post-

synaptic enhancement of NMDA receptors.45 Differences between brain sub-regions

and experimental procedures can also underlie some of these differences.

Intrinsic properties

9

The output from a neuronal circuit is determined in a complex manner by the

properties of individual synapses, synaptic integration and the intrinsic membrane

properties of individual neurons. Membrane properties will determine the passive

response of the cell membrane to synaptic input, electrical length of the neuron,

synaptic integration and action potential characteristics, including spike threshold,

duration and frequency-time characteristics. Thus, a full understanding of muscarinic

activation cannot be achieved without recordings from single neurons, under

conditions in which there is no synaptic interference. Using sharp microelectrodes and

the current-clamp technique Cole and Nicoll55 have shown muscarinic-dependent

slow depolarization and decreased frequency adaptation in CA1 pyramidal neurons.

They, and others,56 concluded that muscarinic receptor activation suppresses a slow

K+ conductance leading to an increase in excitability. At least two different K+

conductances, Im and a slower IAHP are inactivated by muscarinic activation. Recent

studies by Rouse and colleagues57 report that lack of the M1 receptor in knockout

mice doesn’t affect modulation of these K+ currents in mouse hippocampal pyramidal

cells.

Although the distribution of muscarinic-inactivating K+ channels in pyramidal

neurons is not known, the abundance of receptors along the dendritic tree may suggest

significant increase in dendritic membrane resistance and decreased electrotonic

distance following release of ACh. Such changes in sub-threshold membrane

properties may increase the efficacy of distance synapses in input-output relationship

as well as to the boosting of action potential back propagation along the dendrites and

their effect on synaptic properties.58

10

Intracellular recordings from CA1 neurons of rat hippocampus showed that CCh

application induced various reversible changes in action potential characteristics.

While low-dose CCh (2 µM) resulted in an increase in spike duration, higher

concentrations (10-40 µM) decreased spike amplitude and rate of rise, increased spike

duration and raised spike threshold. CCh effects were sensitive to atropine, but also to

Ca2+ channel blockers and Ca2+-free medium. Blockade of Ca2+-dependent K+

currents did not produce effects similar to those of CCh suggesting that these currents

are not involved in the modulation of the action potential. The researchers concluded

that the action potential is modulated by a Ca++ dependent mechanism–possibly

involving protein kinase C.59

The muscarinic-dependent plateau potential is another intrinsic phenomenon mediated

by the cholinergic system (Figure 1C).60 A recent study by Egorov and colleagues61

reported that muscarinic receptor activation inhibits afterhyperpolarization and causes

plateau potentials in entorhinal cortex layer V and II. Interestingly, the plateau

potential did not self-terminate as has been seen in other areas. In addition, the

increase in the plateau potential could facilitate persistent spiking that responded to

stimuli by graded changes in firing frequency. Accumulating evidence suggests that

the excitability of cortical neurons may be modulated by several muscarinic-

modulated conductances besides K+. For example, two mixed cation currents, Ih and

Icat, are potentiated by muscarinic activation.62-64 CCh was also reported to increase

intracellular Ca++ (and hence modulate Ca++ -dependent processes) by activation of

voltage dependent Ca++ channels.23,65

11

To summarize, understanding the overall cholinergic effects on brain function via

muscarinic receptors is still an important challenge for neuroscience. The overall

effect will depend on many factors including specific brain regions, the distribution of

different sub-types of muscarinic receptors, effects of activation on synaptic

transmission, post-synaptic integration and modulation of gene expression, translation

and post-translational processes. This challenge is especially important in light of the

crucial role of the cholinergic system in brain function in health and disease.

Acknowledgements:

Supported by the U.S. Army Medical Research and Materiel Command (DAMD 17-

99-1-9547). Browne, R.O. is a Kreitman Foundation fellow.

12

Figure Legend

Figure 1: Cholinergic Modulation of Neuronal Activity: A. Extracellular recording

in the entorhinal cortex: following perfusion of 50 µM carbamylcholine

(+CCh), spontaneous synchronous rhythmic activity is recorded, which is

abolished by addition of 1 µM pirenzepine (+pirenz), a selective M1

antagonist. B. Intracellular whole cell patch voltage clamp recording. The cell

is held at -70 mV, and spontaneous excitatory post-synaptic currents are

recorded in the presence of intracellular Cs+ (to block K+ conductance) and

extracellular bicuculine (to block GABA-A receptors). Focal drop application

of 5 mM acetylcholine (+ACh) evoked a large inward excitatory synaptic

response. In the presence of 1 µM atropine (+atr) the evoked response was

significantly reduced. C. Intracellular whole cell patch current clamp

recording. In the presence of intracellular Cs+ and blockade of synaptic

transmission, spontaneous Ca++ action potentials were evoked. Following drop

application of ACh, a transient, reversible prolongation of these intrinsic

plateaus is clearly observed.

13

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browne_figure.tif (540x780x32b tiff)

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Abstract

Background: Psychological stress or exposure to acetylcholinesterase inhibitors (AChE-

I) lead to long-lasting brain dysfunction, specifically altered emotional and cognitive

capabilities. In this thesis I studied the neurophysiological mechanisms underlying

hippocampal dysfunction in mice following exposure to stress. Previous works showed

that acute stress is followed by long-lasting changes in the expression of key cholinergic

genes in both the neocortex and hippocampus, thus suggesting altered cholinergic

functions in these regions.

Aims: The present study was performed to reveal the physiologic basis of altered

cholinergic functions in the hippocampus following exposure to stress or

acetylcholinesterase inhibitors.

Methods: All experiments were done in male mice subjected to repeated swim forced

test sessions, as well as single or repeated injections of the potent anticholinesterase DFP.

Non-treated and saline injected age-matched animals served as controls.

Results and Conclusions: In control (naïve) animals we found that cholinergic

activation is associated with enhancement of glutamatergic transmission in CA1

pyramidal cells in the hippocampus. This enhancement was mediated by muscarinic

receptor, and experimental evidence suggest it is pre-synaptic in nature. In animals 4

weeks following repeated (4 days) swim forced stress, extracellular recordings revealed

significant changes in network hippocampal plasticity, observed as enhanced muscarinic-

dependent evoked glutamatergic responses and long-term potentiation (LTP). Intra-

cellular whole cell patch recordings showed that enhanced frequency of muscarinic-

induced glutamatergic post-synaptic currents. Our results indicate that ACh enhances

glutamatergic responses in CA1 pyramidal neurons, probably via pre-synaptic

mechanisms, and that this specific interaction is dramatically altered in stresses animals.

Such changes in hippocampal functions may be important in understanding the

pathophysiology of delayed cognitive and emotional disturbances associated with post-

traumatic stress disorder (PTSD).

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This work was carried out under the supervision of

Dr. Alon Friedman��

In the Department of Physiology

Faculty of Health Sciences.

67

�

Electrophysiological Mechanisms Underlying Delayed Cholinergic Hypersensitivity

Following Psychological Stress. �

Thesis submitted in partial fulfillment

of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

by

Lev Pavlovsky

Submitted to the Senate of Ben-Gurion University

of the Negev

Approved by the advisor

Approved by the Dean of the Kreitman School of Advanced Graduate Studies

��

Beer-Sheva

68

Electrophysiological Mechanisms Underlying Delayed Cholinergic Hypersensitivity

Following Psychological Stress.

Thesis submitted in partial fulfillment

of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

by

Lev Pavlovsky

Submitted to the Senate of Ben-Gurion University

of the Negev

�� !��"

Beer-Sheva�

אסטראז -כולין- מאפיינים אלקטרופיזיולוגיים של תגובת...

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