Voltage clamp analysis of cholinergic action in the hippocampus

A slow muscarinic EPSP, accompanied by an increase in membrane input resistance, can be elicited in hippocampal CA1 pyramidal cells in vitro by electrical stimulation of cholinergic afferents in the slice preparation. Associated with the slow EPSP is a blockade of calcium-activated potassium afterhyperpolarizations (AHPs) (Cole and Nicoll, 1984a). In this study a single-electrode voltage clamp was used to examine the currents affected by activation of muscarinic receptors, using either bath application of carbachol or electrical stimulation of the cholinergic afferents. The 3 main findings of this study are that (1) of the 2 calcium-activated potassium currents (termed IAHP and IC) in hippocampal pyramidal cells, only IAHP is sensitive to carbachol; (2) IAHP is approximately 10-fold more sensitive to carbachol than is another muscarine-sensitive current, IM; and (3) neither blockade of IAHP nor of IM can account for the production of the slow EPSP. Rather, the slow EPSP appears to be generated by the blockade of a nonvoltage-dependent, resting potassium current. We propose that the muscarinic blockade of IAHP, which largely accounts for spike frequency adaptation, is primarily involved in enhancing action potential discharge to depolarizing stimuli, while the slow EPSP acts directly to cause action potential discharge.     

Evidence that cholinergic axon terminals are equipped with both muscarinic and adenosine receptors

The release of 3H-acetylcholine (ACh) from longitudinal muscle strips of guinea pig ileum, which were previously incubated with 3H-choline, was measured by scintillation spectrometry. The release of ACh evoked by electrical field stimulation was inhibited in the following ways: stimulating muscarinic receptors directly with oxotremorine or indirectly with eserine by increasing ACh concentration in the surrounding axon terminals or stimulating adenosine receptors by increasing the biophase concentration of adenosine with dipyridamole. The muscarinic antagonist atropine and the adenosine receptor antagonist theophylline enhanced ACh release. Atropine prevented the effect of eserine and oxotremorine on ACh release and theophylline counteracted the effect of dipyridamole. When the release of ACh was under the inhibitory effect of muscarinic receptor stimulation theophylline did not increase ACh release. Under these conditions atropine caused an extremely high increase in the release of ACh, which was not further enhanced by theophylline. When the extracellular level of adenosine was increased by dipyridamole, eserine, atropine or eserine and atropine together, they were unable to change the release of ACh, while theophylline increased release of ACh. Therefore, it is concluded that the muscarinic receptor mediated inhibition of ACh release is not due to previously released adenosine. Thus, adenosine and muscarinic feedback systems seem to be independent and each cholinergic nerve ending contains both adenosine and muscarinic receptors.     

Classification of muscarinic responses in hippocampus in terms of receptor subtypes and second-messenger systems: electrophysiological studies in vitro

The hippocampal slice preparation was used to classify cholinergic effects in terms of muscarinic receptor subtypes (M1 or M2) and biochemical effector systems linked to these effects in CA1 pyramidal cells. Based on the action of the M1 antagonist pirenzepine and the M2 antagonist gallamine, the muscarinic-induced membrane depolarization and blockade of the afterhyperpolarization appear to result from activation of an M1 receptor, while the cholinergic depression of the EPSP and the blockade of a potassium current termed the M-current appears to involve the activation of an M2 receptor. All of the muscarinic actions could be observed in pertussis toxin-treated hippocampi, suggesting that a pertussis toxin-sensitive G-protein is not involved in these actions. Cholinergic agents that are weak agonists of phosphoinositide (PI) turnover are fully effective in all of the muscarinic actions except the blockade of the M-current on which they had little agonist activity and actually blocked the action of full agonists. These results strongly suggest that the blockade of the M-current may involve stimulation of PI turnover. In addition, we show that the blockade of the M-current is mimicked by intracellular application of inositol trisphosphate. Our results do not show any obvious relationship between the muscarinic receptor subtypes and the biochemical effector systems.     

Muscarinic Modulation of Intrinsic Burst Firing in Rat Hippocampal Neurons

Intracellular recordings in rat hippocampal slices were used to examine how exogenous and endogenous cholinergic agonists modulate the firing pattern of intrinsically burst-firing pyramidal cells. About 24% of CA1 pyramidal cells generated all-or-none, high-frequency bursts of fast action potentials in response to intracellular injection of long positive current pulses. Application of carbachol (5 μM) converted burst firing in these neurons into regular trains of independent spikes. Acetylcholine (5 μM) exerted a similar effect, provided acetylcholine esterase activity was blocked with neostigmine (2 μM). Atropine (1 μM) reversed this cholinergic effect, indicating its mediation by muscarinic receptors. Cholinergic agonists also caused mild neuronal depolarization but the block of intrinsic burst firing was independent of this effect. Repetitive stimulation of cholinergic fibres in the presence of neostigmine (2 μM) evoked a slow cholinergic excitatory postsynaptic potential (EPSP) lasting about a minute. During the slow EPSP, burst firing could not be evoked by depolarizing pulses and the neurons fired in regular mode. These effects were prevented by pretreatment with atropine (1 μM). Exogenously applied cholinergic agonists and endogenously released acetylcholine also reduced spike frequency accommodation and suppressed the long-duration afterhyperpolarization in burst-firing pyramidal cells in an atropine-sensitive manner. A membrane-permeable cAMP analogue (8-bromo-cAMP; 1 mM) also reduced frequency accommodation and blocked the long-duration afterhyperpolarization, but did not affect intrinsic burst firing at all. Taken together, the data show that muscarinic receptor stimulation transforms the stereotyped, phasic response of burst-firing neurons into stimulus-graded, tonic discharge.     

Muscarinic agonist carbachol depresses excitatory synaptic transmission in the rat basolateral amygdala in vitro

Intracellular recordings in slice preparations of the basolateral amygdala were used to test which excitatory amino acid receptors mediate the excitatory postsynaptic potentials due to stimulation of the external capsule. These recordings were also used to examine the action of muscarinic agonists on the evoked excitatory potentials. Intracellular recordings from amygdaloid pyramidal neurons revealed that carbachol (2-20 microM) suppressed, in a dose-dependent manner, excitatory postsynaptic responses evoked by stimulation of the external capsule (EC). This effect was blocked by atropine. The estimated effective concentration to produce half-maximal response (EC(50)) was 6.2 microM. Synaptic suppression was observed with no changes in the input resistance of the recorded cells, suggesting a presynaptic mechanism. In addition, the results obtained using the paired-pulse protocol provided additional support for a presynaptic action of carbachol. To identify which subtype of cholinergic receptors were involved in the suppression of the EPSP, four partially selective muscarinic receptor antagonists were used at different concentrations: pirenzepine, a compound with a similar high affinity for muscarinic M1 and M4 receptors; gallamine, a noncompetitive antagonist for M2; methoctramine, an antagonist for M2 and M4; and 4-diphenylacetoxy-N-methylpiperidine, a compound with similar high affinity for muscarinic receptors M1 and M3. None of them independently antagonized the suppressive effect of carbachol on the evoked EPSP completely, suggesting that more than one muscarinic receptor subtype is involved in the effect. These experiments provide evidence that in the amygdala muscarinic agonists block the excitatory synaptic response, mediated by glutamic acid, by acting on several types of presynaptic receptors.     

Nucleus basalis stimulation facilitates thalamocortical synaptic transmission in the rat auditory cortex

Nucleus basalis (NB) neurons are a primary source of neocortical acetylcholine (ACh) and likely contribute to mechanisms of neocortical activation. However, the functions of neocortical activation and its cholinergic component remain unclear. To identify functional consequences of NB activity, we have studied the effects of NB stimulation on thalamocortical transmission. Here we report that tetanic NB stimulation facilitated field potentials, single neuron discharges, and monosynaptic excitatory postsynaptic potentials (EPSPs) elicited in middle to deep cortical layers of the rat auditory cortex following stimulation of the auditory thalamus (medial geniculate, MG). NB stimulation produced a twofold increase in the slope and amplitude of the evoked short-latency (onset 3.0 ± 0.13 ms, peak 6.3 ± 0.21 ms), negative-polarity cortical field potential and increased the probability and synchrony of MG-evoked unit discharges, without altering the preceding fiber volley. Intracortical application of atropine blocked the NB-mediated facilitation of field potentials, indicating action of ACh at cortical muscarinic receptors. Intracellular recordings revealed that the short-latency cortical field potential coincided with a short-latency EPSP (onset 3.3 ± 0.20 ms, peak 5.6 ± 0.47 ms). NB stimulation decreased the onset and peak latencies of the EPSP by about 20% and increased its amplitude by 26%. NB stimulation also produced slow membrane depolarization and sometimes reduced a long-lasting IPSP that followed the EPSP. The combined effects of NB stimulation served to increase cortical excitability and facilitate the ability of the EPSP to elicit action potentials. Taken together, these data indicate that NB cholinergic neurons can modify neocortical functions by facilitating thalamocortical synaptic transmission. © 1993 Wiley-Liss, Inc.     

Muscarinic modulation of synaptic transmission in slices of the rat ventral striatum is dependent on the frequency of afferent stimulation

Extracellular, intracellular and tight-seal patch-clamp recordings in ventral striatal slices were used to investigate whether the effectiveness of muscarinic neuromodulation of fast synaptic transmission may be dependent on the frequency of afferent stimulation. In all neurons tested, EPSPs were reversibly attenuated by muscarine or carbachol. This action was completely antagonized by atropine or pirenzepine. Several observations indicated a presynaptic site of action. In extracellular recordings, carbachol reduced the monosynaptic population spike but not the non-synaptic compound action potential. The acetylcholinesterase inhibitors eserine and pyridostigmine also induced an atropin-sensitive reduction of the EPSP. When the rate of afferent stimulation was increased, control EPSPs or EPSCs exhibited a decline in peak amplitude until reaching a steady-state value. Muscarinic modulation of steady-state EPSPs/EPSCs was significantly stronger in the range of lower frequencies (0.25–4 Hz) than at higher frequencies (8 and 12 Hz). The GABA_A and GABA_B-receptor/channel antagonists picrotoxin and 2-hydroxy-saclofen, the opiate receptor antagonist naloxone and atropine failed to alter the shape of the frequency-response curve. These results show that both exogenous and endogenous muscarinic receptor agonists are capable of activating a presynaptic mechanism by which fast excitatory inputs to the ventral striatum are depressed. The depressive effect is clearly stronger at lower rates of afferent stimulation than at high rates. This frequency-dependent attenuation of excitatory synaptic inputs exemplifies a new type of activity-dependent neuromodulation in central neural circuits.     

Phorbol esters mimic some cholinergic actions in hippocampal pyramidal neurons

Muscarinic receptor stimulation in the hippocampus has been associated with inositol phospholipid breakdown. In other systems this leads to the formation of inositol trisphosphate and diacylglycerol, which promotes the activation of protein kinase C. Phorbol esters, which directly activate protein kinase C, exhibit high and specific binding in the hippocampus. This, along with the advantages of the hippocampal slice preparation, including direct pharmacological access to a cell population (CA1 pyramidal cells) having clearly defined muscarinic responses, makes this an ideal preparation to examine whether protein kinase C serves as the intracellular signal for muscarinic receptor occupation. Like muscarinic agonists, phorbol esters abolish the slow calcium-activated potassium afterhyperpolarizing potential (AHP) and its underlying current without reducing calcium action potentials. Those phorbol analogs that do not activate kinase C have no effect, suggesting that activation of this enzyme is required to reduce the AHP. The accommodation of spike discharge normally seen during a long depolarizing stimulus is also markedly reduced by phorbol esters as well as by muscarinic receptor activation. However, unlike muscarinic agonists, phorbol esters have no effect on the muscarine-sensitive, voltage-dependent, potassium current termed IM, nor do they consistently cause an increase in input resistance. Moreover, unlike ACh, they do not appear to have a presynaptic inhibitory action on the fast EPSP elicited by orthodromic stimulation. The slow cholinergic EPSP was blocked by phorbol esters, but this could be accounted for by a postsynaptic action. Thus, if inositol phospholipid turnover is involved in mediating muscarinic responses in the hippocampus, the activation of protein kinase C can account for only part of the electrophysiological response.     

Inhibition of cholinesterase elicits muscarinic receptor-mediated synaptic transmission in the rat adrenal medulla

To determine the role of acetylcholinesterase in cholinergic synaptic transmission in the adrenal medulla in vivo, we applied a dialysis technique to the adrenal medulla of anesthetized rats and examined the effect of acetylcholinesterase inhibitor on the contribution of nicotinic and muscarinic receptors to catecholamine release. Exogenous acetylcholine-induced epinephrine release was inhibited by atropine (a muscarinic receptor antagonist) as well as hexamethonium (a nicotinic receptor antagonist). Endogenous acetylcholine (nerve stimulation)-induced epinephrine release was inhibited by hexamethonium but not atropine. In the presence of neostigmine (an acetylcholinesterase inhibitor), both exogenous and endogenous acetylcholine-induced catecholamine release was enhanced. In either case, epinephrine release was inhibited by atropine as well as hexamethonium. In the presence of eserine (another acetylcholinesterase inhibitor), endogenous acetylcholine-induced epinephrine release was also inhibited by atropine. Exogenous or endogenous acetylcholine-induced norepinephrine release was primarily inhibited by hexamethonium regardless of whether neostigmine was absent or present. In the rat adrenal medulla, the inhibition of acetylcholinesterase not only enhanced cholinergic synaptic transmission but also elicited muscarinic receptor-mediated synaptic transmission for epinephrine release.     

Preliminary observations on the physiology and pharmacology of hippocampal theta-off cells

The discharge patterns of phasic linear and tonic non-linear hippocampal theta-off cells and the simultaneously occurring field activity [theta (theta) and large amplitude irregular activity] were studied in response to sensory input, electrical stimulation of the dorsomedial-posterior hypothalamus and systemic administration of cholinergic agents, in urethane-anesthetized rats. Sensory input, hypothalamic stimulation and the administration of eserine all produced a total reduction of the discharges of phasic linear theta-off cells when the resulting induced theta was of a frequency of 5 Hz and above. The discharges of tonic non-linear theta-off cells were also totally reduced during the same conditions, even at the lowest frequencies of induced theta. Administration of atropine SO4 abolished all theta activity previously elicited by electrical stimulation of the hypothalamus, sensory input and eserine. However, sensory input and hypothalamic stimulation still produced the reduction of discharge rates of both types of theta-off cells in the presence of atropine SO4. The linear relation between the discharge rate of phasic linear theta-off cells and levels of hypothalamic stimulation was also preserved in the presence of atropine. It was suggested that GABA may mediate the discharge properties of hippocampal theta-off cells.     

Interaction of Noradrenergic and Cholinergic Agonists with Ligands Increasing K-conductance of Guinea Pig Hippocampal Neurons,in vitro

Single electrode current clamp and voltage clamp recordings were employed to study the effects of noradrenergic agonists and a cholinergic agonist (carbachol, Cch) on the resting membrane potential of CA3 neurons in guinea pig hippocampal slices. Stimulation of muscarinic and beta-adrenergic receptors depolarized, and stimulation of alpha1-adrenergic receptor hyperpolarized, CA3 neurons but the membrane potential changes were small. Hyperpolarizations or outward currents induced by baclofen, adenosine or serotonin (5-HT) were strongly potentiated by alpha-noradrenergic agonists and suppressed by Cch at concentrations ten times lower than those having any direct effects on membrane potential. Both the enhancement of the baclofen-induced hyperpolarization by phenylephrine and its suppression by Cch were pronounced at low concentrations of baclofen, but diminished at higher concentrations. The modulatory effects persisted after blockade of sodium spikes by tetrodotoxin and after blockade of fast inhibitory and excitatory synaptic transmission by picrotoxin and 6-cyano-7-nitroquinoxaline-2,3-dione. Our data suggest that, through the postsynaptic interaction with ligands activating potassium conductance, noradrenergic and muscarinic receptor stimulation can exert a stronger inhibitory and excitatory effect on CA3 pyramidal neurons at their resting membrane potential than would be expected from the changes in membrane potential induced by these neuromodulators on their own.     

Glucocorticoid enhancement of memory consolidation in the rat is blocked by muscarinic receptor antagonism in the basolateral amygdala

Glucocorticoid-induced memory enhancement is known to depend on beta-adrenoceptor activation in the basolateral amygdala (BLA). Additionally, inactivation of muscarinic cholinergic receptors in the rat amygdala blocks memory enhancement induced by concurrent beta-adrenergic activation. Together, these findings suggest that glucocorticoid-induced modulation of memory consolidation requires cholinergic as well as adrenergic activation in the BLA. Two experiments investigated this issue. The first experiment examined whether blockade of muscarinic cholinergic receptors in the BLA with atropine alters the memory-enhancing effects of the systemically administered glucocorticoid dexamethasone. Dexamethasone (0.3, 1.0 or 3.0 mg/kg, s.c.) administered to rats immediately after inhibitory avoidance training produced dose-dependent enhancement of 48-h retention. Concurrent bilateral infusions of the muscarinic cholinergic antagonist atropine (0.5 microg in 0.2 microL per side) into the BLA blocked the memory enhancement. The second experiment investigated whether the BLA is a locus of interaction between glucocorticoid and muscarinic activation. The specific glucocorticoid receptor (GR or type II) agonist RU 28362 (1.0, 3.0 or 10 ng) was infused into the BLA either alone or together with atropine immediately after training. The GR agonist produced dose-dependent memory enhancement and atropine blocked the memory enhancement. These findings indicate that muscarinic cholinergic activation within the BLA is critical for enabling glucocorticoid enhancement of memory consolidation and that enhancement of memory induced by GR activation in the BLA requires cholinergic activation within the BLA.     

The pharmacology of cholinergic excitatory responses in hippocampal pyramidal cells

The pharmacology of excitatory cholinergic responses in CA1 pyramidal cells was examined in detail using intracellular recording from the hippocampal slice preparation. Acetylcholine (ACh), carbachol, muscarine and pilocarpine depolarized the membrane potential with an associated increase in input resistance. In addition, these agonists increased cell firing and depressed the afterhyperpolarization (AHP) that is due to a calcium-activated potassium conductance. The weak effects of ACh (20-200 microM) were considerably enhanced by addition of eserine (1-10 microM). All excitatory effects were completely antagonized by atropine (0.1-1 microM) but unaffected by dihydro-beta-erythroidine (DHBE) and gallamine (1-50 microM). In contrast to the muscarinic agonists, the nicotinic agonists nicotine and dimethylphenylpiperazinium (DMPP) had no excitatory effects on CA1 pyramidal cells. Phenyltrimethylammonium (PTMA), at high concentrations did depolarize cells and depress the AHP but these effects were antagonized by atropine and not DHBE or gallamine. The action of the analogue of cyclic GMP, 8-bromo-cyclic GMP, although variable, mimicked the membrane effects of ACh in some cells and depressed the AHP in most cells. Intracellular injection of cyclic GMP routinely depressed the AHP. In summary, we have demonstrated two cholinergic responses of hippocampal pyramidal cells that are mediated purely by muscarinic receptors. We could find no evidence to support a mixed-type receptor or the involvement of nicotinic receptors in the excitation of hippocampal pyramidal cells to cholinergic agents.     

Effects of cholinergic agonists and antagonists on interleukin-2-induced corticotropin-releasing hormone release from the mediobasal hypothalamus

In previous research we found that interleukin-2 (IL-2)-induced corticotropin-releasing hormone (CRH) release in vitro is mediated by cholinergic activation of nitric oxidergic (NOergic) neurons. The NOergic neurons release nitric oxide that stimulates CRH release. To further characterize the mechanism of IL-2-induced CRH release, the possible role of nicotinic as well as muscarinic receptors in IL-2-stimulated CRH release was evaluated. Medial hypothalamic (MH) explants from adult male rats were preincubated in Krebs-Ringer (KRB) buffer for 45 min followed by incubation for an additional 30 min in fresh KRB or KRB containing various compounds. As previously reported, acetylcholine (ACH) stimulated CRH release in a dose-related fashion. IL-2 (10(-13) M) stimulation of CRH release was unaffected by the lower concentration of ACH (10(-9) M), but surprisingly was inhibited by a 100-fold higher concentration. Atropine (ATR) (10(-7) M) blocked CRH release induced by ACH (10(-7) M) and the release of CRH induced by IL-2. The cholinergic agonist carbachol (CAR) (10(-7) M) also released CRH and this action was blocked by ATR (10(-7) M). CRH release in the presence of CAR was lowered below basal when the concentration of ATR was increased to 10(-6) M. In contrast to ACH, CAR had an additive effect to release CRH when combined with IL-2 (10(-13) M). Nicotine (10(-7) M) also stimulated CRH release and this stimulation was completely blocked by 10(-6) M but not by 10(-7) M of the nicotinic receptor blocker, hexamethonium (HEX). The lower concentration of HEX blocked the stimulatory effect of ACH (10(-7) M) and IL-2 on CRH release. Combined blockade with ATR plus HEX completely blocked the action of ACH and even reduced the CRH concentration to below basal values. Furthermore, combined blockade completely blocked the release of CRH induced by IL-2. We conclude that nicotinic as well as muscarinic receptors play an important role in CRH release, and that they both act to mediate IL-2-stimulated CRH release.     

Septal modulation of the population spike in the fascia dentata produced by perforant path stimulation in the rat

Electrical stimulation of the perforant path, which originates in the entorhinal cortex, produces a characteristic excitatory postsynaptic field potential (extracellular EPSP) which can be recorded in the fascia dentata. This evoked response may include a population spike, if stimulation is sufficient. In the anaesthetized rat, stimulation of the medial septum, when paired with perforant path stimulation, was found to augment the population spike component of the evoked field potential. Stimulation of the septum alone produced no apparent field potential. The augmentation effect was found to have a rapid onset (4 ms), which is sufficient for the participation of interneurons, and a relatively long time course (150 ms). Presynaptic mechanisms of facilitation were ruled out as there was no concurrent alteration of the extracellular EPSP. A change in population spike threshold, compatible with a postsynaptic mechanism, was observed and some possible models of action discussed. Augmentation survived depletion of hippocampal norepinephrine caused by injections of 6-hydroxydopamine into the dorsal noradrenergic bundle, indicating that the facilitation was not due to an activation of the ascending noradrenergic fibres of passage originating from the locus coeruleus. The cholinergic septo-hippocampal pathway was ruled out as a likely candidate for the modulation as the augmentation survived injections of the muscarinic antagonists atropine and scopolamine and the nicotinic antagonists tubocurarine and dihydro-beta-erythroidine. A relationship between the septal modulation and hippocampal theta was suggested.     

Effects of atropine on hippocampal theta cells and complex-spike cells

The medial septal nuclei are essential for the naturally occurring hippocampal theta rhythm. Evidence that the rhythmic activity of the septum is carried via cholinergic afferents to the hippocampus has been: (a) the existence of a cholinergic septo-hippocampal projection, and (b) the sensitivity of one type of theta rhythm to antimuscarinic agents or cholinergic depletion. The muscarinic action of acetylcholine on pyramidal cells, however, is too slow to carry even a 4 Hz signal. Recent in vitro studies have confirmed a fast excitatory response by some hippocampal interneurons to muscarinic agonists. In urethane anesthetized rats, iontophoretic application of atropine to 17 hippocampal theta cells (presumed interneurons) during the theta rhythm, reduced their firing rates to an average of 24% of control rates. The effect of iontophoretic atropine application to 4 CA1 complex-spike cells (presumed pyramidal cells) was a selective elimination of their bursting activity with no significant effect on overall firing rate. The data suggest that: (1) interneuronal firing, during the hippocampal theta rhythm, is dominated by an excitatory cholinergic input and not by excitatory collaterals of pyramidal cells; and (2) somatic burst firing by CA1 pyramidal cells requires the presence of acetylcholine.     

An analysis of cholinoceptive neurons in the hippocampal formation by direct microinfusion

Microinfusions of cholinergic agents were made in various sites in the dorsal hippocampal formation of urethane anaesthetized rats. Infusions of eserine or carbachol elicited hippocampal theta activity when made in areas containing high levels of cholinergic markers: the stratum oriens and radiatum of the CA1 and CA3, the stratum moleculare and stratum granulosum of the dentate gyrus and the infragranular region of the hilus. Subsequent infusions of atropine sulfate antagonized the theta activity. Control infusions of equal volumes of saline in active sites were without effect. Infusions of eserine or carbachol in the vicinity of the hippocampal fissure, the stratum lacunosum/moleculare of the CA1 or CA3, in the deep regions of the hilus, and in the lateral ventricle and overlying neocortex, were also without effect. Furthermore, in active sites, the latency to onset of theta and subsequent theta frequency, were both directly related to the total amount of carbachol infused. Thus, areas in which theta could be elicited with a cholinergic agonist (carbachol), or an anticholinesterase (eserine) and antagonized with atropine, were found to correspond well to areas previously found to contain a high density of cholinoceptive neurons, using autoradiographic and immunohistochemical techniques. These results provide further support for the involvement of acetylcholine as a neurotransmitter in the generation of type 2 theta in the hippocampal formation.     

Atropine enhances nicotinic cholinergic EPSPs in rat neostriatal slices

Atropine enhances and muscarinic agonists reduce the amplitudes of EPSPs evoked by intrastriatal stimulation in rat neostriatal slices. The mechanism of action suggested is presynaptic muscarinic modulation of an EPSP which is caused by cholinergic activation of nicotinic receptors on the cholinoceptive neuron.     

Theta in hippocampal slices: relation to synaptic responses of dentate neurons

The effects of cholinergic agents on isolated dentate neurons were studied to characterize cellular mechanisms underlying carbachol-induced 'theta' EEG activity. Carbachol, eserine, and acetylcholine produced a synchronization of slow wave activity (theta) accompanied by depression of perforant path to dentate field potentials. These effects were antagonized by atropine but not d-tubocurarine. The results suggest that muscarinic receptors mediate theta activity resulting from a depolarization of dentate neurons.     

Differential effects of atropine, procaine and dopamine in the rat ventral tegmentum on lateral hypothalamic rewarding brain stimulation

Microinjections of the muscarinic antagonist, atropine, of dopamine, or of the local anesthetic, procaine, in the ventral tegmentum elevated frequency thresholds for lateral hypothalamic self-stimulation. The largest and most robust effects were observed following atropine (30 or 60 micrograms) microinjections. The most sensitive sites for the atropine effect were near dopamine cells. In order to determine if the effects of atropine can be reversed by pretreatment with a cholinergic agonist, carbachol (1-3 micrograms) was microinjected 15 min prior to atropine. Carbachol pretreatment attenuated the frequency threshold elevation of atropine by 47-95%. Since atropine is a local anesthetic, the effects of procaine on self-stimulation thresholds were tested as well. Procaine (100 or 250 micrograms) in ventral tegmentum elevated frequency thresholds by much less than atropine. Therefore, while atropine attenuates reward primarily through blockade of muscarinic receptors, the local anesthetic effect of atropine may enhance the threshold elevation. Dopamine (1-10 micrograms) also elevated frequency thresholds, but when dopamine injections were repeated daily, the threshold elevations were attenuated. This attenuation contrasted with the robust effects of atropine, and may reflect the development of autoreceptor subsensitivity. Hence, both dopaminergic and muscarinic receptors in ventral tegmentum are involved in lateral hypothalamic brain stimulation reward.