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ACITIE' AND C:NIC FEGI'S OF PVIC ~.(7PI1VE IN RATS AND EV'IDIINCF FOR A NONCHOLIMERRGIC SITE OF ACI'ICN . by L. G. Abood, K. Lowy, and H. Rooth Center for Hraj.n Research University of' Rochester Medical Center Rochester, Netw York 14642

Since the discovery that acetylcholine was a neurotra.nsmitter in the autonomic nervous system and that its action~consisted of a muscarinic and nicotinic component, the action of nicotine has been attributed main],y to effects on nicotinic choli.nergic synapses (Domino 1973; Larson and Silvette 1975). Although the action of nicotine in the autonomic nervous system or in certain~ brain areas can be understood in terms of either its agonistic or antagonistic action at nicotinic cholinergic synapses, a close perusal of the ].iterature reveals that its neuropharmacological effects are considerably more cmnpl.ex and cannot be entirely explained by this mechanism. Recently we have observed that when the natural form of nicotine, which is the (-)-isomer, is injec- ted directly into a rat's lateral or third ventricle, there occurs a characteristic prostration-i:irmobilization syndrome lasting for fran 0.5 - 2 mi.n (Abood et al. 1978). The syndrome can be pre- vented or antagonized by the intraventricular administration of a ni,unber of newly synthesized nicotine or piperidine derivatives,, but not by a variety of antinicotinic and nurnerous psychotropic agents. These observations along with those involving electro- physiological and receptor binding measurements led to the conclusion that the prostration syndrome may not be mediated by cho].linergie mechharisms. In the present report are described additional pharmacological and electrophysiological studies on the acute effects of nico- tine along with observations on the chronic effects following continuous intr3vertricular '~influsion of nicotine into the lateral ventricles of freely moving rats. MAl'ERIAhS AND lv=ODS All studies were performed on male Sprague-Dawley rats weighing between 150-200 g. The anesthetized rat was introduced into a Kopf stereotactic instrimient, and a stainless steel cannula (#220 DK 1 rat cannula, obtained from David Kopf) was inserted stereotactically aiming at either the lateral or third ventricles.

<_,YTt was attached to the skull by means of acrylic cement and 4 : small set screws. A bipolar Fornivar coated nich.ropne electrode ,- , of 0.2 nan thickness and 3/4 mm vertical tip separation was in- erted with leads into the region of the dorsal hippocampus. Electrical Recordings From Conscious Rats Electrical recordings were made from freely moving conscious rats by way of a light shielded flexible cable leading into a Grass P-15-B preamplifier set for an amplification of 1000 and a filter band between 3-30 cycles/sec. The amplifier output was connected to an FM tape recorder and a Grass dynagraph. Analysis of elec- trical activity was accomplished by two LINC-8 programs operating on the output from the tape recorder. Spectral analysis was done by means of a fast Fourier transform program modified for use by LZNC-8. Another program was used to plot histogr3ms of amplitude distribution recorded fran representative sections of the tape recordings. . . 3H-Nicotine Binding to Glass and Tissue An extensive investigation was undertaken in an effort to develop a receptor binding assay for nicotine utilizing various neural membrane preparations, synaptornoimes, brain hoinogenates, and brain slices; however, none of these proved to be satisfactory. The ligands used in this exploratory study were 12'I-a-burgaratoXin, 1`'C-d-tubocur~arine, and 3H: nicotine . The techniques for measur- ing binding to~ cell free preparations included equilibrium dialy- sis, centrifugation, gel filtr ation, and filtration through glass fiber filters. Although same stereospecific or specific nitotine binding could be der,umstrated with fresh rat brain slices , the most satisfactory data was obtained by measuring 3H-ndcotine binding directly to Whatrran GB/F glass fiber filters in the absence of any tissue preparations. The procedure for measuring 3H:nicotine binding to glass fiber filters is described elsewhere (Abood et al. 1978'). Briefly, it consisted of filtering a solution (0.05M '`ris, pH 7.5) of 10_8M 3H-nicotine (&.2 curies/ngrzole) in the absence or presence of 10 SM (-) or (+)-nicotine or nicotine analogues and then fil- tering in vacuo through Whatman GF/B glass fiber filters (2.1 cm). After washing the filters with 10 ml 0.05M Tris, pH 7.5, the filter transferred to vials and radioactivity measured by liquid scintillation. Although some success was obtained in measuringg specific nicotine binding to fresh rat brain slices, no specific or stereospecific binding was demonstrable to synaptosones, neural membranes, or brain homogenates, employi.r,g all known techniques for measu_ring binding (see "Results'f ) . Chronic Lntraventr. icular Infl.asion of Nicotine Chronic microinfusion of nicotine into the lateral ventricles of rats was accomplished by mans of Alzet osmotic minipLmps Niodel

1701 (Wei and Loh 1976). -The mirLipunp was inserted subcutaneously and was connected by means of a fine catheter to a 24 gauge stain- ; less steel caruzula, stereotactically implanted into the lateral ventricles. The reservoir of the minipump contained 170 yl of 10 mg/mi solution of nicotine. HC1 and the delivery rate was about 1 u1/hr (i.e. 10 ug nicotine/hr)~. In order to determine the reliability of the minipurrps they were tested for their re- maining contents 1-2 weeks later by either measuring optical absorbance at 260~nM or by the use of 3H-nicotine and measuring residual radioactivity. REsvLTs Psychophanrn.cological Effects of Nicotine Given Intraventricularly Within 1-10 sec following the intraventricular administration of 2-10 ug of (-)-nicotine HC1 the rats became prostrate and imnobile, mariifesting occasional seizures and tremors along with various autonomac changes (tachycardia, hyperpnea, and urination-defe- cation). The dose-response relationship was monotonic in this range. In order to produce a comFarable response with (+)- nicotine, the required dose was at least 100 times greater than for (-)-nicotine. A variety of agents were testedfor their ability to prevent the prostration imimbilization syndrome of (-)-nicotine including a series of synthetic derivatives of nicotine and piperidine (table 1). When 10iug of either the benzyl or 4-azido-2-nitrophenyl derivatives of either piperidine of nicotine was given intraventricularly 2 min prior to 4 ug of (-)-nicotine the prostration syndrome could be prevented. A wide variety of psychotropic neurotransmitters, cholinergic agents (e.g. naloxone, d-tubocurarine anticholinergics, and physostigmir.e) and d-tubocurarine were ineffective. Azapetine, an a-adrenergic blocker having a structural relationship to nicotine, possessed moderate antagonist activity. Neither chlorpromazine or diazepam had any effect in antagonizing nico- tine or on binding. 3H-Nicotine Binding to Glass Fiber Filters In general a good correlation was observed between the behavioral antagonism~ of a given agent andits ability to compete with 3H- nicotine for binding to glass fiber filters (table 1). The most ~ effective antagonists such as N-ANPP, N-benzyl and N-ONPS deri- O vatives of nicotine and piperidine were also the most effective © agents in blocking nicotine binding. The (+)-isomer of nicotine, ~ which possessed about 1 percent of the efficacy of (-)-nicotine ~ in producing the prostration sy^ndrome, was 1/10 as effective as ~ the natural nicotine in blocking binding. Although some stereo- CL~ specific and specific binding of nicotine could be demonstrated with fi°esh~brain slices, the magnitude of the binding was small ~ ar.d somewhat variable; therefore, such data was not included. No specific binding was demonstrable to neural membranes, synaptosomes, or homogenates prepared'from rat brain.

Table 1 "":Antagonism of various agents to prostration syndrome in rats and Fto 3H nicotine binding to glass filters. At least 8 rats were used for behavioral studies. The dose of (-)-nicotine was 4 ti,g, which was given 1 min after the test agent. All drugs were given intraventricularly at doses of 10 pg, except for (+)-nicotine and piperidine, which were given at 100 ug• At least 8 rats were used~for each drug tested behaviorally, while the standard error in the binding studies is within 6 percent of the mean. Binding is expressed as moles x 101`'/glass fiber filter. N-ANPP = 4- azido-2-nitrophenyl; N-CNPS = 2-nitrophenylsulfenyl. Agent % Behavioral H~-nicotine binding Antagonism Moles.x 10. Glass % competition Glass Control - 12.0 (-)-nicotine 0 3.6 70 (+ -nicotine 33 11.0 8 Lbenzyl nicotine 92 7.0 42 L-ANPP-nicotine 83 6.5 45 -benzyl piperidine 75 6.0 50 -ANPP piperidine 72 6.0 50 -ONPS-piperidir.e 35 9.0 25 CNPS-nicoti,ne 40 10.0 17 piperidine 40 11.5 4 butaclamol slight 12.0 0 decam.ethonitun, 0, 0.5 19 -quinuclidiayl benzilate slight 11.8' 2 oxotremorine 0 9.9 17 r.a.loxone 0 12.0 0 a-lobeline 0 11.5 4 azapetine moderate 10.0 17 chlorprana.zi:ne slight 11.5 4 diazepam 0 11:T 2'.

. Electrical Recordings of Dorsal Hippocarmus After (-) and (+)- : ; Nicotine and Benzyl Nicotine ontaneous electrical activity recorded fYrom the dorsal hippo- campus of imanesthetized, freely moving rats before and after the administration of 4 ug of (-)-nicotine bitartrate revealed marked changes in both the frequency and amplitude of the eiectricall pattern (figures 1 a-d; figure 2 a-d). Frequency analysis of the electrical record are presented as oscillographic displays fZ^om fast Fourier transform~(figure 1 a-d). _The control recordindi- cated a frequency range of 6-8 cycles/sec with a minor component in the 12-14 cycles/sec range (figure 1 a). Within 1 min after the.administration of 4 ug of_(-)-nicotine, when the rat was completely prostrate and iL*mobile, the 5-8 cycle/sec activity greatly diminished (figure 1 b). When 10 ug of N-benzyl nicotine was given intraventricularly about 30 sec after the record in figure 1 b, the electrica.Il patternwithin 30 sec reverted to one resembling the control (figure lc ). For _conmarison, a frequency histogram is presented of a record from an animal given 200 ug of (+)-nicotine bitartrate, a dose which produced only minimal prostration, muscle weakmess, and inactivity (figure ld). A slight shift to lower frequencies was noted with a peak value at about 5 cycles/sec. A computer analysis performed on the arrpli- tudes of the electrical recordirgs, and the results are presented the form of histograms (lower tracings, figure 2 a-cl)~. During in the control period the amplitudes varied over a large range between 200-400 uV. After (-)-nicotine when the animal was pros- trate, the higher amplitudes at 400 uV'disappeared, while the major component was at 200 uV. The flattening of the electrical recording after (-)-nicoti,ne is evident in the upper tracing (figure 2b)~. After 10 ug of benzyl nicotine, the electrical record and the amplitude distribution~resembled the control pat- tern; however, the 300-400 uV amplitude components did not reach the control level (figure 2c). After 200 ug of (+)-nicotine, the histograr,i and electrical patternvaried only slightly frorm the control records, the maj or difference being arn increase in the 200 pV component (figure 2d). Electrical Activity of Hippocampus During Nicotine Influsion The electrical activity was recorded from the dorsal hippocarrpus of freely roving rats throughout the period of the intraventri- cular infusion of nicotine at a rate of 10 u&1r (figure 3 a-d). The electrical pattern of the hippocampus is displayed in the upper oscillographic tracings of figure 3a and lb; and ir. the tracing below is displayed a corresponding arrplitude histogram of the upper record. The arrplitude range before the infusion began had a broad spectrum ranging up to 400 uV (figure 3a); but after 48 hours of infusion the amlitude range narrowed consider- ably with a ma.xirrnam of 100 UV (figure 3b). After 6 days of in*- fusion the amplitude histogram and electrical recordresembled the control record (figure 3c) and two days following the removal of the mimip unp the records were unchanged (figure 3d). A spec- $ O O O N ~ N ~'

'_tral analysis of the frequency employing fast Fourier transform revealed after 16 hours of nicotine a broad spectrum up to 30 sec with a ma,jor component in 5-7/sec range (figure 4a). After s48 hours of nicotine infhsion the higher frequency components ` aL~st vanished and only the 5-7/sec component remained ( figure -4b). After 6 days of inf;ising nicotine, the frequency spectrum returned to the pattern seen in the control (figure 4c); and two .days after removal _of the min.ipurrp the pattern remained unchanged (fi gure 4d) y , . ... . . _s~ _. . __.. .. . . . . Figure 1 (a-d) Spectral frequency analyses of electrical recordings of rat dorsal hippocampus after (-)- or (+)-nicotine and NLbenzyl nico- tine. a = control, b = 1 min after 4Ug (-)-nicotine, c = same rat given 200 ug (+)-nicotine 2 days later (control record identi- cal), d = a rat given 10 ug N-benzyl nicotine 30 sec after (-)- nicotine. The records are typical of at least 5 individual rats for each drug. See text. Each dot on the abscissa represents a scale of 5 cycles/sec. --------------------------------- Chronic Nicotine Administration and Tolerance In an effort to determine whether tolerance developed with repeated intraventricular administration of nicotine, rats were exposed to various dosage regimes of nicotine (table 2). When rats received 10 j,g of nicotine for 3 consecutive days, they all responded (prostration irrmobilization syndrome) on day 1, while

Figure 2 (a-d) Electrical record and am.plitude histogracrs corresponding to records described in figure 1 a-d_. The bar represents a scale of 10 4 V on the abscissa. Table 2 Effects of various dosage regime on behavioral response of rats to nicotine. From 5-10 rats were represented in each schedule. Nicotine was administered into left lateral ventricles. Dosage Schedule Incidence of Prostration Syndrome 10 ug nicotine daily for 3 All of rats responding day 1, consecutive days 50 percent on day 2, 20 per- cent on day 3, 90 percent response after 3 days recovery. Tolerance 10 ug nicotine every other day st rats fully respo ed ar for 10' da.ys eachda.y drug was given. No tolerance. 2 ug nicotine day 1, ug,day Response on day 'to 10 ug ? 10ug da,Y3 nicotine. Alzet miniptunp (10 ug/rir' Normal response to 10 ug for 10 days. nicotine on day after infu

Figure 3 (a-d) :Amplitude hi;stogram of hippocanmal electrical activity following chronic intraventricular infusion of nicotine. Same rat and legend as infigure 3 a-d. Figure 4 (a-d) F'r equency analysis of hippocar.pal electrical activity following chronic intraventricular infusion of nicotine. a = control, b = 48 hrs of nicotine infusion, c = 6 days of nicotine, d = 2' days after infusion was discontinued.

.`50 percent responded on day 2 and only 20 percent on day 3. If 10 ug of nicotine was given every other day for 10 days, most of ' .;4the rats responded each day the drug was given. After 10 ug of ,Inicotine was given for 5 consecutive days, none of the rats re- sponded to 10 pg of nicotine onday 6; however, most of the ani- mals responded on the second day after discontinuing the nicotine. If rats were given 2~~~ of nicotine on the first day, 4 ug on the second day, and 10 ug of nicotine on the third day, they all respondedito 10 pg of nicotine on the fourth day. ~ Effect of Nicotine on Brain Levels of Met-Enkephalin Regional brain levels of met-enkephalin were measured in rats sacrificed 1-2 minutes following the intraventricular irjection, of 10 ug of nicotine. The anLTals were still prostrate at the time of sacrifice. No significant differences were observed .between the saline and.nicotine - injected rats in any of the three brain regions (table 3). The determinations were by Dr. S. Chanda utilizing the-technique of radioimrnine assay. Table 3 ;Effect of nicotine on enkephalin of rat brain regions. Enkephalin = TYR-GLY-GLY-PHE-r,IET in pmoles/g wet wt. All rats received 10 ug nicotine intraventricula.rly 1-2 minutes before sacrifice. Results are average of 6 rats. - - Nicotine Saline Telenencephalon 128 ± 39 97 ± 29 Diencephalon 149 ± 55 152 ± 36 Mesencephalon 223 ~± 4'3 243 ± 34 D2SOtJSSION! The maj or conclusion fromithi.s study is that the action of nico- tine in producing the prostration itrmobilization syndrome does not appear to be mediated by cholinergic rrechanisms in the brain. Among the reasons for this conclusion are the following: 1) the syndrome cannot be simulated by a number of cholinergic agents in doses up to 100 jig; included among such agents were acetylcholine, oxotremorine, pilocarpine, physostigmine, ar.d' a-lobeline; 2) a variety of agents known to block the nicotinic cholinergic receptor were ineffective in either preventing or reversing the behavioral effect of nicotine; and 3) the nicctine and piperidine analogues which were effective in blocking the behavioral effect of nicotine did not interfere with the binding