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.     

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.     

Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.

It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.     

Metabotropic glutamate receptor agonist ACPD inhibits some,but not all,muscarinic-sensitive K+ conductances in basolateral amygdaloid neurons

Muscarinic agonists produce membrane depolarization and losses of spike frequency accommodation and the slow afterhyperpolarization (AHP) when applied to neurons of the basolateral amygdala (BLA). Underlying these changes are the muscarinic-induced inhibitions of several K⁺ conductances, including the voltage-activated M-current (I_M), a slowly decaying Ca²-activated current (I_AHP), a voltage-insensitive leak current (I_Leak), and the hyperpolarization-activated inward rectifier current (I_IR) Similar depolarizations and losses of the slow AHP have been observed in other neuronal cell types following stimulation of metabotropic glutamate receptors. Therefore, we tested the effects of the metabotropic glutamate receptor agonist, 1-aminocyclopentanels, 3r-dicarboxylic acid (ACPD), on pyramidal neurons impaled with a single microelectrode for current- and voltage-clamp recordings in a brain slice preparation of the rat BLA. Application of ACPD (20 or 100 μM) to BLA neurons inhibited I_M and I_{A HP}, resulting in membrane depolarization and reductions in the amplitude and duration of the slow AHP. However, ACPD did not inhibit the muscarinic-sensitive current I_IR, nor was I_Leak blocked in the majority of neurons examined. These findings suggest the possibility that muscarinic cholinergic and metabotropic glutamatergic receptor agonists may activate separate intracellular transduction pathways which have convergent inhibitory effects onto I_M and I_AHP in BLA pyramidal neurons. © 1994 Wiley-hiss, Inc.     

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.     

Effects of endogenous acetylcholine on spontaneous activity in rat dorsal cochlear nucleus slices

We have examined the contribution of endogenous acetylcholine (ACh) release to the spontaneous firing of both regular (probably fusiform cells) and bursting neurons (probably cartwheel cells) in the dorsal cochlear nucleus (DCN) in rat brainstem slices. The muscarinic antagonists atropine, scopolamine, and tropicamide (1–2 μM) caused substantial decreases of firing rates in a majority of the neurons. Reversible acetylcholinesterase (AChE) inhibitors typically caused large transient increases in firing that decayed more slowly than responses to carbachol. The irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) usually caused a sustained increase, with an initial peak followed by a gradual change to a final level higher than before DFP. Tropicamide caused large decreases in firing after DFP, confirming sustained ACh release. Both neostigmine and DFP applied after AChE inhibition by DFP sometimes elicited a transient response. We conclude that the level of sustained response to DFP is determined by the rate of endogenous ACh release, and that DFP and reversible AChE inhibitors exert an initial transient agonist effect that overlaps the initial effect of acetylcholinesterase inhibition. The slice experiments provide a model for cholinergic mechanisms in vivo, confirm that the release of endogenous ACh increases the firing rates of regular and bursting neurons in superficial DCN, and support the hypothesis that spontaneous firing of DCN neurons is sustained in part by cholinergic inputs.     

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.     

Metrifonate increases neuronal excitability in CA1 pyramidal neurons from both young and aging rabbit hippocampus.

The effects of metrifonate, a second generation cholinesterase inhibitor, were examined on CA1 pyramidal neurons from hippocampal slices of young and aging rabbits using current-clamp, intracellular recording techniques. Bath perfusion of metrifonate (10-200 microM) dose-dependently decreased both postburst afterhyperpolarization (AHP) and spike frequency adaptation (accommodation) in neurons from young and aging rabbits (AHP: p < 0.002, young; p < 0.050, aging; accommodation: p < 0.024, young; p < 0.001, aging). These reductions were mediated by muscarinic cholinergic transmission, because they were blocked by addition of atropine (1 microM) to the perfusate. The effects of chronic metrifonate treatment (12 mg/kg for 3 weeks) on CA1 neurons of aging rabbits were also examined ex vivo. Neurons from aging rabbits chronically treated with metrifonate had significantly reduced spike frequency accommodation, compared with vehicle-treated rabbits. Chronic metrifonate treatment did not result in a desensitization to metrifonate ex vivo, because bath perfusion of metrifonate (50 microM) significantly decreased the AHP and accommodation in neurons from both chronically metrifonate- and vehicle-treated aging rabbits. We propose that the facilitating effect of chronic metrifonate treatment on acquisition of hippocampus-dependent tasks such as trace eyeblink conditioning by aging subjects may be caused by this increased excitability of CA1 pyramidal neurons.     

Differential Effects of Acetylcholine on Neuronal Activity and Interactions in the Auditory Cortex of the Guinea-pig

During normal brain operations, cortical neurons are subjected to continuous cholinergic modulations. In vitro studies have indicated that, in addition to affecting general cellular excitability, acetylcholine also modulates synaptic transmission. Whether these cholinergic mechanisms lead to a modulation of functional connectivity in vivo is not yet known. Herein, the effects were studied of an iontophoretic application of acetylcholine and of the muscarinic agonist, carbachol, on the ongoing activity and co-activity of neurons simultaneously recorded in the auditory cortex of the anaesthetized guinea-pig. Iontophoresis of cholinergic agonists mainly affected the spontaneous firing rates of auditory neurons, affected autocorrelations less (in most cases their central peak areas were reduced), and rarely affected cross-correlations. These findings are consistent with cholinergic agonists primarily affecting the excitability of cortical neurons rather than the strength of cortical connections. However, when changes of cross-correlations occurred, they were usually not correlated with concomitant changes in average firing rates nor with changes in autocorrelations, which suggests a secondary cholinergic effect on specific cortico-cortical or thalamo-cortical connections.     

The vestibulo‐ and preposito‐cerebellar cholinergic neurons of a ChAT‐tdTomato transgenic rat exhibit heterogeneous firing properties and the expression of various neurotransmitter receptors

Cerebellar function is regulated by cholinergic mossy fiber inputs that are primarily derived from the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN). In contrast to the growing evidence surrounding cholinergic transmission and its functional significance in the cerebellum, the intrinsic and synaptic properties of cholinergic projection neurons (ChPNs) have not been clarified. In this study, we generated choline acetyltransferase (ChAT)‐tdTomato transgenic rats, which specifically express the fluorescent protein tdTomato in cholinergic neurons, and used them to investigate the response properties of ChPNs identified via retrograde labeling using whole‐cell recordings in brainstem slices. In response to current pulses, ChPNs exhibited two afterhyperpolarisation (AHP) profiles and three firing patterns; the predominant AHP and firing properties differed between the MVN and PHN. Morphologically, the ChPNs were separated into two types based on their soma size and dendritic extensions. Analyses of the firing responses to time‐varying sinusoidal current stimuli revealed that ChPNs exhibited different firing modes depending on the input frequencies. The maximum frequencies in which each firing mode was observed were different between the neurons that exhibited distinct firing patterns. Analyses of the current responses to the application of neurotransmitter receptor agonists revealed that the ChPNs expressed (i) AMPA‐ and NMDA‐type glutamate receptors, (ii) GABA_A and glycine receptors, and (iii) muscarinic and nicotinic acetylcholine receptors. The current responses mediated by these receptors of MVN ChPNs were not different from those of PHN ChPNs. These findings suggest that ChPNs receive various synaptic inputs and encode those inputs appropriately across different frequencies.     

Cellular bases of neocortical activation: modulation of neural oscillations by the nucleus basalis and endogenous acetylcholine.

In the mammalian neocortex, the EEG reflects the state of behavioral arousal. The EEG undergoes a transformation, known as activation, during the transition from sleep to waking. Abundant evidence indicates the involvement of the neurotransmitter acetylcholine (ACh) in EEG activation; however, the cellular basis of this involvement remains unclear. We have used electrophysiological techniques with in vivo and in vitro preparations to demonstrate actions of endogenous ACh on neurons in auditory neocortex. In vivo stimulation of the nucleus basalis (NB), a primary source of neocortical ACh, (1) elicited EEG activation via cortical muscarinic receptors, (2) depolarized cortical neurons, and (3) produced a change in subthreshold membrane potential fluctuations from large-amplitude, slow (1-5 Hz) oscillations to low-amplitude, fast (20-40 Hz) oscillations. The NB-mediated change in pattern of membrane potential fluctuations resulted in a shift of spike discharge pattern from phasic to tonic. Stimulation of afferents in the in vitro neocortex elicited cholinergic actions on putative layer 5 pyramidal neurons. Acting via muscarinic receptors, endogenous ACh (1) reduced slow, rhythmic burst discharge and facilitated higher-frequency, single-spike discharge in burst-generating neurons, and (2) facilitated the appearance and magnitude of intrinsic membrane potential oscillations. These in vivo and in vitro observations suggest that neocortical activation results from muscarinic modulation of intrinsic neural oscillations and firing modes. Rhythmic-bursting pyramidal neurons in layer 5 may act as cortical pacemakers; if so, then modifying their discharge characteristics could alter local cortical networks. Larger, intercortical networks could also be modified, due to the widespread projections of NB neurons. Thus, NB cholinergic neurons may play a critical role in producing different states of neocortical function.     

Desensitization of muscarinic acetylcholine receptors: possible relation to receptor heterogeneity and phosphoinositides

The increases in firing rates of hippocampal cells were examined following microiontophoretic application of several muscarinic cholinergic receptor agonists. The agonists studied had been pharmacologically characterized previously and divided into two classes: class A agonists (e.g. acetylcholine, carbamylcholine, and oxotremorine-M) which maximally stimulate PI turnover and reveal mAChR heterogeneity, and class B agonists (e.g. bethanecol and oxotremorine-1) which poorly stimulate PI turnover and do not alter mAChR conformation/orientation in the hippocampus. While comparable stimulatory effects on hippocampal pyramidal cell firing rates were seen with both classes of agonists during short (20 s) ejection periods, longer applications (greater than 25 s) produced class-dependent differential firing patterns. Prolonged ejection of class A agonists selectively desensitized cells to further, continued application in the same ejection period, and the firing rates declined. Class B agonists produced stimulatory responses in hippocampal cells during the entire ejection period, and DE was not observed. This desensitization effect (DE) was observed only for bursts and not for simple spikes.     

Spontaneous activity in rat vestibular nuclei in brain slices and effects of acetylcholine agonists and antagonists

Extracellular recording was used to investigate spontaneously active neurons in all four major nuclei of the rat vestibular nuclear complex (VNC) in brainstem slices. The density of spontaneously active neurons was highest in the medial vestibular nucleus (MVN), slightly lower in the superior (SuVN) and spinal (SpVN) nuclei, and lowest in the lateral vestibular nucleus (LVN). We compared the effects of acetylcholine agonists and antagonists on spontaneously discharging neurons in MVN, SuVN, and SpVN with those in the nearby dorsal cochlear nucleus (DCN). The proportion of neurons responding to carbachol was greatest in DCN and smallest in SpVN. Unlike in DCN, some neurons in MVN, SuVN, and SpVN showed decreased firing during carbachol or muscarine. Magnitudes of responses to carbachol and muscarine were closely correlated (P<0.01). MVN neurons possessed nicotinic as well as muscarinic receptors. Activation of either type was unaffected by blocking synaptic transmission. The IC(50) values for the muscarinic subtype-preferential antagonists were compared, and tropicamide, preferential for M(4), was the most potent. Our results suggest that: (1) the relative numbers of spontaneously active neurons in rat VNC differ among nuclei; (2) acetylcholine agonists elicit changes in mean firing rates of neurons in MVN, SuVN and SpVN, but fewer neurons respond, and responses are smaller than in DCN; (3) both muscarinic and nicotinic acetylcholine receptors are present on MVN neurons, but muscarinic receptors may be more prominent.     

Effects of dithiothreitol, a sulfhydryl reducing agent, on CA1 pyramidal cells of the guinea pig hippocampus in vitro

The radioprotectant, dithiothreitol (DTT) has been shown to increase excitability in the hippocampal slice preparation. In the present study, intracellular recording techniques were used to further examine the actions of DTT. Electrophysiological recordings from CA1 pyramidal cells were obtained prior to, during and after DTT exposure. DTT caused a small depolarization without altering membrane resistance. DTT induced spontaneous firing and occasional burst firing in normally silent neurons. These effects were accompanied by a reduction in spike frequency adaptation but no change in the afterhyperpolarization following a train of action potentials. Following DTT exposure, orthodromic stimulation produced multiple firing. Subthreshold excitatory postsynaptic potentials (EPSPs) were significantly prolonged. Isolating the CA1 subfield, attenuated the prolongation of the EPSP by DTT. Recurrent inhibitory postsynaptic potentials were unaffected by DTT. The actions of DTT are likely to result from DTT-induced reduction of disulfide bonds since the reduced form of DTT does not cause a similar hyperexcitability.     

Induction of activity-dependent LTD requires muscarinic receptor activation in medial prefrontal cortex

The medial prefrontal cortex (mPFC) forms part of a neural circuit involved in the formation of lasting associations between objects and places. Cholinergic inputs from the basal forebrain innervate the mPFC and may modulate synaptic processes required for the formation of object-in-place memories. To investigate whether acetylcholine regulates synaptic function in the rat mPFC, whole-cell voltage-clamp recordings were made from pyramidal neurons in layer V. Bath application of the cholinergic agonist carbachol caused a potent and long-term depression (LTD) of synaptic responses that was blocked by the muscarinic receptor antagonist scopolamine and was mimicked, in part, by the M(1) receptor agonists McN-A-343 or AF102B. Furthermore, inhibition of PKC blocked carbachol-mediated LTD. We next determined the requirements for activity-dependent LTD in the prefrontal cortex. Synaptic stimulation that was subthreshold for producing LTD did, however, result in LTD when acetylcholine levels were enhanced by inhibition of acetylcholinesterase or when delivered in the presence of the M(1)-selective positive allosteric modulator BQCA. Increasing the levels of synaptic stimulation resulted in M(1) receptor-dependent LTD without the need for pharmacological manipulation of acetylcholine levels. These results show that synaptic stimulation of muscarinic receptors alone can be critical for plastic changes in excitatory synaptic transmission in the mPFC. In turn, these muscarinic mediated events may be important in the formation of object-in-place memories. A loss of basal forebrain cholinergic neurons is a classic hallmark of Alzheimer's dementia and our results provide a potential explanation for the loss of memory associated with the disease.     

Cholinergic modulation of nucleus accumbens medium spiny neurons

The rat nucleus accumbens contains acetylcholine-releasing interneurons, presumed to play a regulatory role in the electrical activity of medium spiny output neurons. In order to examine this issue in detail, we made electrophysiological recordings in rat nucleus accumbens slices. These experiments showed that gamma-aminobutyric acid-mediated inhibition of the output neurons might be facilitated by activation of nicotinic acetylcholine receptors, in addition to being suppressed via activation of muscarinic acetylcholine receptors. In contrast, glutamatergic excitation of output neurons appeared to be inhibited by activation of muscarinic acetylcholine receptors and to be insensitive to activation of nicotinic acetylcholine receptors. The spontaneous firing frequency of cholinergic neurons appeared to be under control of both a muscarinic and a nicotinic pathway in a bi-directional manner. Finally, we made paired recordings in which the functional connection between cholinergic neurons and output neurons was monitored. Driving the cholinergic neurons at physiological firing frequencies stimulated gamma-aminobutyric acid-mediated inhibition of the output neurons, via activation of nicotinic acetylcholine receptors. The onset of this effect was slow and lacked a fixed delay. These data indicate that activation of nicotinic acetylcholine receptors in rat nucleus accumbens may mediate the facilitation of gamma-aminobutyric acid-mediated inhibition of medium spiny output neurons. Possible mechanisms of neurotransmission, mediating this cholinergic modulation are discussed.     

Cholinergic Modulation of the Action Potential in Rat Hippocampal Neurons

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 μM) caused an increase in spike duration; high doses (10–40 μM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca²⁺-free medium and by Ca²⁺-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca²⁺-dependent K⁺ currents I_c or I_AHP, suggesting that these K⁺ currents are not involved in the modulation.     

Distinct classes of pyramidal cells exhibit mutually exclusive firing patterns in hippocampal area CA3b

It is thought that CA3 pyramidal neurons communicate mainly through bursts of spikes rather than so-called trains of regular firing action potentials. Reports of both burst firing and nonburst firing CA3 cells suggest that they may fire with more than one output pattern. With the use of whole-cell recording methods we studied the firing properties of rat hippocampal pyramidal neurons in vitro within the CA3b subregion and found three distinct types of firing patterns. Approximately 37% of cells were regular firing where spikes generated by minimal current injection (rheobase) were elicited with a short latency and with stronger current intensities trains of spikes exhibited spike frequency adaptation (SFA). Another 46% of neurons exhibited a delayed onset at rheobase with a weakly-adapting firing pattern upon stronger stimulation. The remaining 17% of cells showed a burst-firing pattern, though only elicited in response to strong current injection and spontaneous bursts were never observed. Control experiments indicated that the distinct firing patterns were not due to our particular slicing methods or recording techniques. Finally, computer modeling was used to identify how relative differences in K+ conductances, specifically K(C), K(M), and K(D), between cells contribute to the different characteristics of the three types of firing patterns observed experimentally.     

Presynaptic inhibition of Schaffer collateral synapses by stimulation of hippocampal cholinergic afferent fibres

It has been known for decades that muscarinic agonists presynaptically inhibit Schaffer collateral synapses contacting hippocampal CA1 pyramidal neurons. However, a demonstration of the inhibition of Schaffer collateral synapses induced by acetylcholine released by cholinergic hippocampal afferents is lacking. We present original results showing that electrical stimulation at the stratum oriens/alveus with brief stimulus trains inhibited excitatory postsynaptic currents evoked by stimulation of Schaffer collaterals in CA1 pyramidal neurons of rat hippocampal slices. The increased paired-pulse facilitation and the changes in the variance of excitatory postsynaptic current amplitude that paralleled the inhibition suggest that it was mediated presynaptically. The effects of oriens/alveus stimulation were inhibited by atropine, and blocking nicotinic receptors with methyllycaconitine was ineffective, suggesting that the inhibition was mediated via the activation of presynaptic muscarinic receptors. The results provide a novel demonstration of the presynaptic inhibition of glutamatergic neurotransmission by cholinergic fibres in the hippocampus, implying that afferent cholinergic fibres regulate the strength of excitatory synaptic transmission.     

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.