Sleep patterning changes in a prenatal stress model of depression.

Clinical depression is accompanied by changes in sleep patterning, which is controlled in a circadian fashion. It is thus desirable that animal models of depression mirror such diurnally-specific state alterations, along with other behavioral and physiological changes. We previously found several changes in behavior indicative of a depression-like phenotype in offspring of rats subjected to repeated, variable prenatal stress (PNS), including increased locomotor activity during specific periods of the circadian cycle. We, therefore, investigated whether PNS rats also exhibit alterations in sleep/wakefulness behavior around the change from light-to-dark phase. Control and PNS Sprague-Dawley rats were implanted with electrodes for continuous monitoring of electroencephalic activity used to determine behavioral state. The distribution of slow-wave sleep (SWS), rapid eye movement sleep (REMS) and wakefulness was compared for periods before and after lights were turned off, between baseline conditions and after exposure to an acute stressor. Both REMS and SWS amounts were increased in PNS rats relative to control animals in the beginning of the dark phase. REMS changes were due to an increase in REMS bout number, rather than in bout duration. During this circadian time period, we did not find any sex differences in the state changes. These results indicate that PNS affects baseline sleep patterning in both male and female rats around active-phase onset.

Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the cellular changes in the LDT induced by exposures to nicotine prenatally, when combined with alterations in other reward-related regions, could contribute to the increased susceptibility to smoking observed in the offspring.

Electrophysiological changes in laterodorsal tegmental neurons associated with prenatal nicotine exposure: implications for heightened susceptibility to addict to drugs of abuse.

Prenatal nicotine exposure (PNE) is a risk factor for developing an addiction to nicotine at a later stage in life. Understanding the neurobiological changes in reward related circuitry induced by exposure to nicotine prenatally is vital if we are to combat the heightened addiction liability in these vulnerable individuals. The laterodorsal tegmental nucleus (LDT), which is comprised of cholinergic, GABAergic and glutamatergic neurons, is importantly involved in reward mediation via demonstrated excitatory projections to dopamine-containing ventral tegmental neurons. PNE could lead to alterations in LDT neurons that would be expected to alter responses to later-life nicotine exposure. To examine this issue, we monitored nicotine-induced responses of LDT neurons in brain slices of PNE and drug naive mice using calcium imaging and whole-cell patch clamping. Nicotine was found to induce rises in calcium in a smaller proportion of LDT cells in PNE mice aged 7-15 days and smaller rises in calcium in PNE animals from postnatal ages 11-21 days when compared with age-matched control animals. While inward currents induced by nicotine were not found to be different, nicotine did induce larger amplitude excitatory postsynaptic currents in PNE animals in the oldest age group when compared with amplitudes induced in similar-aged control animals. Immunohistochemically identified cholinergic LDT cells from PNE animals exhibited slower spike rise and decay slopes, which likely contributed to the wider action potential observed. Further, PNE was associated with a more negative action potential afterhyperpolarization in cholinergic cells. Interestingly, the changes found in these parameters in animals exposed prenatally to nicotine were age related, in that they were not apparent in animals from the oldest age group examined. Taken together, our data suggest that PNE induces changes in cholinergic LDT cells that would be expected to alter cellular excitability. As the changes are age related, these PNE-associated alterations could contribute differentially across ontogeny to nicotine-mediated reward and may contribute to the particular susceptibility of in utero nicotine exposed individuals to addict to nicotine upon nicotine exposure in the juvenile period.

Nicotine during pregnancy: changes induced in neurotransmission, which could heighten proclivity to addict and induce maladaptive control of attention.

Prenatal exposure to nicotine, occurring either via maternal smoking or via use of transdermal nicotine patches to facilitate cigarette abstinence by pregnant women, is associated with ∼ 13% of pregnancies worldwide. Nicotine exposure during gestation has been correlated with several negative physiological and psychosocial outcomes, including heightened risk for aberrant behaviors involving alterations in processing of attention as well as an enhanced liability for development of drug dependency. Nicotine is a terotogen, altering neuronal development of various neurotransmitter systems, and it is likely these alterations participate in postnatal deficits in attention control and facilitate development of drug addiction. This review discusses the alterations in neuronal development within the brain's major neurotransmitter systems, with special emphasis placed on alterations within the laterodorsal tegmental nucleus, in light of the role this cholinergic nucleus plays in attention and addiction. Changes induced within this nucleus by gestational exposure to nicotine, in combination with changes induced in other brain regions, are likely to contribute to the transgenerational burden imposed by nicotine. Although neuroplastic changes induced by nicotine are not likely to act in isolation, and are expected to interact with epigenetic changes induced by preconception exposure to drugs of abuse, unraveling these changes within the developing brain will facilitate eventual development of targeted treatments for the unique vulnerability for arousal disorders and development of addiction within the population of individuals who have been prenatally exposed to nicotine.

γ-Hydroxybutyric acid induces actions via the GABAB receptor in arousal and motor control-related nuclei: implications for therapeutic actions in behavioral state disorders.

γ-Hydroxybutyric acid (GHB) is used as an effective therapeutic for reducing the hypersomnolence and cataplexy (loss of motor control) of the sleeping disorder, narcolepsy, with an immediate pharmacologic behavioral action of inducing a natural sleep-like state. Despite its clinical use, few studies have examined the cellular actions of this drug on behavioral state-related neurons. Therefore, we monitored GHB-induced responses using calcium imaging within the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), two pontine nuclei important in state and motor control. In addition, we recorded GHB-induced membrane responses using whole cell, patch clamp electrophysiology of immunohistochemically-identified principal neurons within these nuclei. GHB induced GABAB receptor-mediated rises in calcium in neurons of the LDT and the DR. However, the pattern and amplitude of calcium rises differed greatly between these two nuclei. GHB induced GABAB receptor antagonist-sensitive outward currents/hyperpolarizations in immunohistochemically-identified cholinergic LDT and serotonergic DR neurons. However, GHB had this action in a greater proportion of DR cells than LDT neurons. Further, larger inhibitory currents were induced in DR cells when compared to the amplitude of GHB-induced current in LDT-responding cells. Finally, NCS-382 and HOCPCA, a reported antagonist and agonist specific to activity at the putative GHB receptor, respectively, with no demonstrated binding at the GABAB receptor, failed to block GHB-induced effects or elicit any discernible electrophysiological action when applied alone, indicating a lack of involvement of a GHB receptor in mediating GHB actions. Taken together, our data support the conclusion that GHB may be exerting its actions on state and motor control, in part, via an acutely mediated strong inhibition of serotonergic DR neurons and a more modest inhibitory action on a smaller proportion of LDT cholinergic neurons. Given the roles played by these nuclei, these actions are consistent with acute pharmacologic effects of GHB: hypotonia and promotion of sleep, including presence of REM, a sub-state of sleep. Differences in GHB-mediated calcium suggest differential regulation of calcium-dependent processes, which may also contribute to functioning of the LDT and DR in state and motor control and the therapeutic pharmacologic actions of GHB, which develop following chronic administration. These findings add to knowledge of cellular actions of GHB and it is hoped that, combined with findings from other studies examining GHB neurotransmission, these data can contribute to development of highly targeted therapeutics at the GABAB receptor for management of human disorders presenting with alterations in motor and arousal control.

GABAergic actions on cholinergic laterodorsal tegmental neurons: implications for control of behavioral state.

Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl(-) -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABA(A) receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca(2+) solutions. THIP, a selective GABA(A) receptor agonist with a preference for δ-subunit containing GABA(A) receptors, induced inward currents, suggesting the existence of extrasynaptic GABA(A) receptors. LDT cells also possess GABA(B) receptors as baclofen-activated a TTX- and low Ca(2+)-resistant outward current that was attenuated by the GABA(B) antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a G(IRK) mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABA(B) receptors linked to the same G(IRK) activated in these cells by muscarinic receptor stimulation. Activation of GABA(B) receptors inhibited Ca(2+) increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca(2+) were unaltered by prior emptying of intracellular Ca(2+) stores, but were abolished by low extracellular Ca(2+) and pre-application of nifedipine, indicating that activation of GABA(B) receptors inhibits influx of Ca(2+) involving L-type Ca(2+) channels. Presence of GABA(C) receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABA(C) receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABA(A, B and C) receptors, including extrasynaptically located GABA(A) receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT.

Dual orexin actions on dorsal raphe and laterodorsal tegmentum neurons: noisy cation current activation and selective enhancement of Ca2+ transients mediated by L-type calcium channels.

The hypocretin/orexins (Hcrt/Orxs) are hypothalamic neuropeptides that regulate stress, addiction, feeding, and arousal behaviors. They depolarize many types of central neurons and can increase [Ca2+]i in some, including those of the dorsal raphe (DR) and laterodorsal tegmental (LDT) nuclei-two structures likely to contribute to the behavioral actions of Hcrt/Orx. In this study, we used simultaneous whole cell and Ca2+-imaging methods in mouse brain slices to compare the Hcrt/Orx-activated current in DR and LDT neurons and to determine whether it contributes to the Ca2+ influx evoked by Hcrt/Orx. We found Hcrt/Orx activates a similar noisy cation current that reversed near 0 mV in both cell types. Contrary to our expectation, this current did not contribute to the somatic Ca2+ influx evoked by Hcrt/Orx. In contrast, Hcrt/Orx enhanced the Ca2+ transients produced by voltage steps (-60 to -30 mV) by approximately 30% even in neurons lacking an inward current. This effect was abolished by nifedipine, augmented by Bay-K and abolished by bisindolylmaleimide I. Thus Hcrt/Orx has two independent actions: activation of noisy cation channels that generate depolarization and activation of a protein kinase C (PKC)-dependent enhancement of Ca2+ transients mediated by L-type Ca2+ channels. Immunocytochemistry verified that both these actions occurred in serotonergic and cholinergic neurons, indicating that Hcrt/Orx can function as a neuromodulator in these key neurons of the reticular activating system. Because regulation of Ca2+ transients mediated by L-channels is often linked to the control of transcriptional signaling, our findings imply that Hcrt/Orxs may also function in the regulation of long-term homeostatic or trophic processes.

Calcium dynamics and electrophysiological properties of cerebellar Purkinje cells in SCA1 transgenic mice.

Cerebellar Purkinje cells (PCs) from spinocerebellar ataxia type 1 (SCA1) transgenic mice develop dendritic and somatic atrophy with age. Inositol 1,4,5-trisphosphate receptor type 1 and the sarco/endoplasmic reticulum Ca(2+) ATPase pump, which regulate [Ca(2+)](i), are expressed at lower levels in these cells compared with the levels in cells from wild-type (WT) mice. To examine PCs in SCA1 mice, we used whole-cell patch clamp recording combined with fluorometric [Ca(2+)](i) and [Na(+)](i) measurements in cerebellar slices. PCs in SCA1 mice had Na(+) spikes, Ca(2+) spikes, climbing fiber (CF) electrical responses, parallel fiber (PF) electrical responses, and metabotropic glutamate receptor (mGluR)-mediated, PF-evoked Ca(2+) release from intracellular stores that were qualitatively similar to those recorded from WT mice. Under our experimental conditions, it was easier to evoke the mGluR-mediated secondary [Ca(2+)](i) increase in SCA1 PCs. The membrane resistance of SCA1 PCs was 3.3 times higher than that of WT cells, which correlated with the 1.7 times smaller cell body size. Most SCA1 PCs (but not WT) had a delayed onset (about 50--200 ms) to Na(+) spike firing induced by current injection. This delay was increased by hyperpolarizing prepulses and was eliminated by 4-aminopyridine, which suggests that this delay was due to enhancement of the A-like K(+) conductance in the SCA1 PCs. In response to CF stimulation, most PCs in mutant and WT mice had rapid, widespread [Ca(2+)](i) changes that recovered in <200 ms. Some SCA1 PCs showed a slow, localized, secondary Ca(2+) transient following the initial CF Ca(2+) transient, which may reflect release of Ca(2+) from intracellular stores. Thus, with these exceptions, the basic physiological properties of mutant PCs are similar to those of WT neurons, even with dramatic alteration of their morphology and downregulation of Ca(2+) handling molecules.

Noradrenaline excites non-cholinergic laterodorsal tegmental neurons via two distinct mechanisms.

Cholinergic neurons of the laterodorsal tegmental nucleus have been hypothesized to play a critical role in the-generation and maintenance of rapid eye movement sleep. Less is known about the function of non-cholinergic laterodorsal tegmental nucleus neurons. As part of our ongoing studies of the brainstem circuitry controlling behavioral state, we have begun to investigate the functional properties of these neurons. In the course of these experiments, we have observed a novel response to the neurotransmitter noradrenaline. Whole-cell patch-clamp recordings of laterodorsal tegmental nucleus neurons were carried out in 21- to 35-day-old rat brain slices. A subpopulation of laterodorsal tegmental nucleus cells responded to a 30-s application of 50 microM noradrenaline with depolarization and a decrease in input resistance which lasted several minutes. Following return to resting membrane potential, these cells invariably exhibited barrages of excitatory postsynaptic potentials which lasted at least 12 min. These excitatory postsynaptic potentials were reversibly abolished by bath application of tetrodotoxin, as well as by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, but were insensitive to application of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. To examine whether these neurons were cholinergic, the recorded cells were labeled with biocytin and tested for co-localization with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a marker for laterodorsal tegmental nucleus cholinergic neurons. In every instance, neurons with these properties were non-cholinergic. However, they were always located in close proximity to reduced nicotinamide adenine dinucleotide phosphate-diaphorase-positive laterodorsal tegmental nucleus cells. The present data indicate that noradrenaline, in addition to directly inhibiting cholinergic cells of the laterodorsal tegmental nucleus, also results in the direct and indirect excitation of non-cholinergic cells of the laterodorsal tegmental nucleus. The indirect excitation is long lasting and mediated by glutamatergic mechanisms. Our working hypothesis is that these non-cholinergic cells are local circuit inhibitory interneurons and that prolonged excitation of these neurons by noradrenaline may serve as a mechanism for inhibition of cholinergic laterodorsal tegmental nucleus cells during wakefulness, when noradrenaline tone is high.

Vasoactive intestinal polypeptide excites medial pontine reticular formation neurons in the brainstem rapid eye movement sleep-induction zone.

Although it has long been known that microinjection of the cholinergic agonist carbachol into the medial pontine reticular formation (mPRF) induces a state that resembles rapid eye movement (REM) sleep, it is likely that other transmitters contribute to mPRF regulation of behavioral states. A key candidate is the peptide vasoactive intestinal polypeptide (VIP), which innervates the mPRF and induces REM sleep when injected into this region of the brainstem. To begin understanding the cellular mechanisms underlying this phenomenon, we examined the effects of VIP on mPRF cells using whole-cell patch-clamp recordings in the in vitro rat brainstem slice. VIP directly depolarized cells via activation of an inward current; these effects were attenuated and potentiated in low-sodium and low-calcium medium, respectively. The depolarization induced by VIP was slower in onset and longer-lived than that evoked by carbachol. The VIP-induced depolarization was reduced in a dose-dependent manner by a competitive antagonist of VIP receptors. Effects of VIP were attenuated in the presence of guanosine 5'-O-(2-thiodiphosphate, 2'5'dideoxyadenosine, and PKI15-24 and were nonadditive in the presence of 8-bromo-cAMP. We conclude that VIP excites mPRF neurons by activation of a sodium current. This effect is mediated at least in part by G-protein stimulation of adenylyl cyclase, cAMP, and protein kinase A. These data suggest that VIP may play a physiological role in REM induction by its actions on mPRF neurons.

Effects of excitation of sensory pathways on the membrane potential of cat masseter motoneurons before and during cholinergically induced motor atonia.

Electrical stimulation of the nucleus pontis oralis during wakefulness enhances somatic reflex activity; identical stimuli during the motor atonia of active (rapid eye movement) sleep induces reflex suppression. This phenomenon, which is called reticular response-reversal, is based upon the generation of excitatory postsynaptic potential activity in motoneurons during wakefulness and inhibitory postsynaptic potential activity during the motor atonia of active sleep. In the present study, instead of utilizing artificial electrical stimulation to directly excite brainstem structures, we sought to examine the effects on motoneurons of activation of sensory pathways by exogenously applied stimuli (auditory) and by stimulation of a peripheral (sciatic) nerve. Accordingly, we examined the synaptic response of masseter motoneurons prior to and during cholinergically induced motor atonia in a pharmacological model of active sleep-specific motor atonia, the alpha-chloralose-anesthetized cat, to two different types of afferent input, one of which has been previously demonstrated to elicit excitatory motor responses during wakefulness. Following the pontine injection of carbachol, auditory stimuli (95 dB clicks) elicited a hyperpolarizing potential in masseter motoneurons. Similar responses were obtained upon stimulation of the sciatic nerve. Responses of this nature were never seen prior to the injection of carbachol. Thus, stimulation of two different afferent pathways (auditory and somatosensory) that produce excitatory motor responses during wakefulness instead, during motor atonia, results in the inhibition of masseter motoneurons. The switching of the net result of the synaptic response from one of potential motor excitation to primarily inhibition in response to the activation of sensory pathways was comparable to the phenomenon of reticular response-reversal. This is the first report to examine the synaptic mechanisms whereby exogenously or peripherally applied stimuli that elicit motor excitation during wakefulness instead elicit inhibitory motor responses during the motor atonia of active sleep. Thus, not only are motoneurons tonically inhibited during active sleep, but the selective elicitation of inhibitory motor responses indicates that this inhibition can be phasically increased in response to sensory stimuli, possibly in order to maintain the state of active sleep. The data provided the foundation for the hypothesis that, during naturally occurring active sleep, there is a change in the control of motor systems so that motor suppression occurs in response to stimuli that would otherwise, if present during other behavioral states, result in the facilitation of motor activity.

Relationship between sensory stimuli-elicited IPSPs in motoneurons and PGO waves during cholinergically induced muscle atonia.

Inhibitory postsynaptic potentials (IPSPs) can be produced in masseter motoneurons by sensory stimuli after the injection of carbachol into the nucleus pontis oralis (NPO) of alpha-chloralose-anesthetized cats. We have postulated previously that these IPSPs, which are induced in masseter motoneurons by sensory stimuli, arise as the result of phasic activation of the motor inhibitory system that mediates atonia occurring spontaneously during active sleep. In the present study, we determined that sensory stimuli, which excite different sensory pathways, somatosensory and auditory, also elicit ponto-geniculo-occipital (PGO) waves during the carbachol-induced state. Because the elicitation of PGO waves has been hypothesized to be a central sign of activation of alerting mechanisms, we suggest that these stimuli also excite those CNS structures that are involved in the alerting network. The temporal association of the sensory stimuli-elicited IPSPs and PGO waves also was examined by correlating the intracellular response of masseter motoneurons and the extracellular response of lateral geniculate nuclei neurons to somatosensory and auditory stimuli. Sensory stimuli produced an IPSP that had a similar latency from the foot of the elicited PGO wave as that of spontaneously occurring motoneuron IPSPs and PGO waves that occur during both carbachol-induced muscle atonia and naturally occurring active sleep. In addition, the intensity of the stimulus necessary for elicitation of PGO waves was found to be lower than that required for the elicitation of IPSPs in motoneurons. Additionally, evoked responses in masseter motoneurons during the carbachol-induced state were graded in response to increases in stimulus intensity. The preceding data suggest that some type of processing of sensory input occurs such that only those stimuli that are capable of activating alerting mechanisms involved in the generation of PGO waves result in an increase in activity in the motor inhibitory system. We conclude that there may be a functional link between alerting mechanisms involved in the generation of PGO waves and the motor inhibitory system that generates IPSPs in motoneurons. This functional link may serve to preserve atonia, and thus the state of active sleep, from potentially disruptive PGO-related influences that, during other behavioral states, result in motor activation.

Strychnine blocks inhibitory postsynaptic potentials elicited in masseter motoneurons by sensory stimuli during carbachol-induced motor atonia.

In previous studies we reported that large-amplitude inhibitory potentials were elicited in masseter motoneurons by auditory stimuli (95-dB clicks) and stimulation of the sciatic nerve in alpha-chloralose-anesthetized cats [Kohlmeier K. A. et al. (1994) Soc. Neurosci. Abstr. 20, 1218; Kohlmeier K. A. et al. (1995) Sleep Res. 24, 9]. These potentials were always elicited during motor atonia induced by the pontine injection of carbachol into the nucleus pontis oralis and were never elicited prior to atonia. In the present report, the hyperpolarizing potentials that arose in response to clicks and stimulation of the sciatic nerve were blocked following the juxtacellular application of strychnine, a glycinergic antagonist. In contrast, bicuculline, a GABA(A) receptor antagonist, did not suppress the carbachol-dependent hyperpolarizing potentials elicited by these stimuli. In some motoneurons, blockade of the inhibitory potential by strychnine revealed a depolarizing potential. These data suggest that clicks and stimulation of the sciatic nerve not only elicit inhibition of motoneurons but also activate an excitatory drive which is masked by elicited inhibitory postsynaptic potentials. These findings suggest that glycine is likely to be the neurotransmitter that is responsible for the inhibitory postsynaptic potentials elicited in masseter motoneurons following the presentation of auditory and somatosensory stimuli during carbachol-induced motor atonia. We suggest that the same system that mediates glycinergically-dependent motor atonia during naturally occurring active sleep [Chase M. H. et al. (1989) J. Neurosci. 9, 743-751] also mediates the carbachol-dependent response of motoneurons to sensory stimuli.

State-dependent phenomena in cat masseter motoneurons.

In the present study we explored the mechanisms of carbachol-induced muscle atonia in the alpha-chloralose-anesthetized animal. We compared our findings to those that have been previously obtained in unanesthetized cats during muscle atonia occurring during natural active sleep. Accordingly, in cats anesthetized with alpha-chloralose, intracellular records were obtained from masseter motoneurons before and after carbachol-induced motor atonia. Following the induction of atonia, the membrane potential activity was dominated by high-frequency, discrete, hyperpolarizing potentials. These hyperpolarizing potentials were reversed in polarity by the intracellular injection of chloride ions and abolished by the application of strychnine. These findings indicate that they were inhibitory postsynaptic potentials (IPSPs) mediated by glycine. These IPSPs appeared exclusively during muscle atonia. In addition, masseter motoneurons were significantly hyperpolarized and their rheobase increased. There was a decrease in input resistance and membrane time constant. In the alpha-chloralose-anesthetized preparation, stimulation of the nucleus pontis oralis (NPO) induced IPSPs in masseter motoneurons following, but never prior to, the pontine injection of carbachol. Thus, this is the first demonstration that "reticular response-reversal' may be elicited in an anesthetized preparation. Another state-dependent phenomenon of active sleep, the occurrence of IPSPs in motoneurons that are temporally correlated with ponto-geniculo-occipital (PGO) waves, was also observed in this preparation only after carbachol administration. Based on the data in this report, we conclude that the inhibitory system that mediates atonia during the state of active sleep can be activated in an animal that is anesthetized with alpha-chloralose. Specifically, the neuronal groups that generate spontaneous IPSPs, those that mediate the phenomenon of reticular response-reversal, and those involved in the generation of PGO waves are capable of being activated and remain functional during alpha-chloralose-anesthesia.

Muscle atonia can be induced by carbachol injections into the nucleus pontis oralis in cats anesthetized with alpha-chloralose.

Cholinergic excitation of structures in the pontine reticular formation appears to be a key step in the generation of active sleep. For example, muscle atonia which occurs as a result of the postsynaptic inhibition of motoneurons during active sleep is also present after carbachol, a cholinergic agonist, is injected into the nucleus pontis oralis. In the present study, in order to obtain information regarding the mechanisms that generate atonia during active sleep and to provide a paradigm for studying atonia in anesthetized cats, we determined whether cholinergically induced atonia could be generated in an animal that was anesthetized with alpha-chloralose. Cats which were initially anesthetized with alpha-chloralose (40 mg/kg, I.V.) exhibited spikes in the EEG, hippocampus and lateral geniculate nuclei. Muscle atonia occurred after carbachol (200 mM) was injected by microiontophoresis (300-500 nA) into the nucleus pontis oralis; the spikes in the EEG, hippocampus and lateral geniculate nuclei were still present. We believe that the atonia induced by carbachol in alpha-chloralose-anesthetized cats is mediated by the same mechanisms that operate during active sleep in the unanesthetized animal for the following reasons. First, in the same cats when they were not anesthetized with alpha-chloralose, carbachol injections in the identical brainstem sites induced active sleep with its accompanying pattern of muscle atonia. Second, after carbachol was injected into the same sites in alpha-chloralose-anesthetized cats, intracellular recordings from lumbar motoneurons revealed that inhibitory postsynaptic potentials were bombarding motoneurons; these inhibitory potentials were similar to those which are present during naturally occurring active sleep. In addition, stimulation of the nucleus reticularis gigantocellularis (NRGc) was found to induce large amplitude depolarizing potentials in lumbar motoneurons in alpha-chloralose-anesthetized cats prior to the administration of carbachol, whereas after its administration, accompanying muscle atonia there were large amplitude hyperpolarizing potentials and a reduction in the amplitude of depolarizing potentials. We therefore conclude that the cholinergically induced processes that initiate and maintain muscle atonia are not blocked by the actions of alpha-chloralose.