Microinjection Of Cholinergic Agonist Research Articles

Medial Septum Modulates Cellular Response Induced in Hippocampus on Microinjection of Cholinergic Agonists into Hypothalamic Lateral Supramammillary Nucleus.

Cholinergic mechanisms in supramammillary nucleus (SuM), especially the lateral SuM (lSuM) modulates septo-hippocampal neural activity. The lSuM, as compared to the contiguous medial SuM (mSuM) has relatively dense projections to hippocampus and cingulate cortex (Cg). In the present study, we have investigated whether the effects of cholinergic activation of SuM on hippocampal and cortical neural activities involve a cooperative interaction with the medial septum (MS). Microinjection of the broad-spectrum cholinergic agonist, carbachol, or the cholinergic-nicotinic receptor agonist, nicotine, into the lSuM and the mSuM in urethane anesthetized rat evoked a similar pattern of hippocampal theta rhythm. Despite that, only the lSuM microinjections resulted in an increase in expression of c-Fos-like immunoreactivity (c-Fos-ir) in neurons, including interneurons, of the ipsilateral hippocampus with a very dense expression in dentate gyrus. Likewise, a robust induction of c-Fos-ir was also observed in the ipsilateral Cg. Inhibition of the MS with muscimol pre-treatment attenuated both carbachol-evoked c-Fos-ir and theta activation. The findings indicate that cholinergic–nicotinic mechanisms in lSuM evoke not only neural activation via the ascending synchronizing pathway but also an MS-modulated expression of the plasticity-related molecule c-Fos in cortical regions that are strongly innervated by the lSuM.

Inhibition of neuronal nitric oxide synthase (nNOS) reduces cardiovascular responses elicited by microinjection of cholinergic agonists in the Nucleus of the Solitary Tract (NTS) in non‐anesthetized rats

Microinjection of ACh into the NTS of non‐anesthetized rats induces hypotension and bradycardia. We investigated if nNOS inhibitor (TRIM) changes the responses elicited by nicotinic and muscarinic receptors stimulation in the NTS. Five days before the experiments, rats were anesthetized with Ketamin and Xylazin and guide cannulas aiming at the NTS were implanted bilaterally. One day before the experiments, femoral artery was cannulated to record arterial pressure (AP). Unilateral NTS microinjection of nicotine (NIC 0.01 ‐ 10 pmol/100 nl) elicited dose‐dependent hypotension and bradycardia (0.5 pmol δMAP: ‐41±2 mmHg; δHR: ‐141±10 bpm; n=8, p< .05). Responses to 0.5 pmol NIC were reduced to ‐11±2 mmHg and ‐45±12 bpm (n=9, p< .05), 10 min after injection of TRIM (13.3 nmol/100 nl) into the NTS. Unilateral microinjection of pilocarpine (PIL 0.01 ‐ 10 nmol/100 nl) elicited hypotension and bradycardia (0.5 nmol: ‐32±2 mmHg and ‐147±11 bpm; n=9, p< .05). With doses of PIL higher than 0.5 nmol, the hypotension was followed by a significant increase in MAP. Ten min after unilateral injection of TRIM in the NTS, responses to 0.5 nmol PIL were reduced to ‐8±3 mmHg and ‐32±16 bpm; n=4, p< .05. Responses to NIC and PIL returned to control 60 min after TRIM microinjection. We suggest that both nicotinic and muscarinic receptors participate in ACh transmission in the NTS, and NO may modulate their responses. Supported by Fapesp, CNPq.

The antinociception produced by microinjection of a cholinergic agonist in the ventromedial medulla is mediated by noradrenergic neurons in the A7 catecholamine cell group

Activation of neurons in the ventromedial medulla by electrical stimulation or by microinjection of opioid or cholinergic agonists produces antinociception that is mediated in part by spinally-projecting noradrenergic neurons. Several lines of evidence indicate that these noradrenergic neurons are located in the pontine A7 catecholamine cell group. For example, anatomical studies have demonstrated that neurons in the ventromedial medulla project to the noradrenergic neurons in the A7 catecholamine cell group that provide the major noradrenergic innervation of the spinal cord dorsal horn. In addition, electrical and chemical stimulation of A7 neurons produces antinociception that can be reduced by intrathecal injection of α 2-adrenoceptor antagonists. The present studies provide more direct evidence that activation of neurons in the ventromedial medulla produces antinociception by activating noradrenergic neurons in the A7 cell group. Neurons in the ventromedial medulla were stimulated by microinjecting the cholinergic agonist carbachol (5 μg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars α of pentobarbital anesthetized Sprague–Dawley rats. In some experiments, the local anesthetic tetracaine (10 μg) was then microinjected near the A7 cell group to inactivate the spinally-projecting noradrenergic neurons. In other experiments, cobalt chloride (100 mM) was microinjected near the A7 cell group to block synaptic activation of spinally-projecting noradrenergic neurons. Microinjection of carbachol into sites in the ventromedial medulla produced antinociception, assessed using the tail flick test, that lasted more than 60 min. However, the effects of carbachol were attenuated by microinjection of either tetracaine or cobalt into sites near the A7 cell group neurons identified by tyrosine hydroxylase-immunoreactivity. Similar injections of tetracaine or cobalt more than 500 μm from the A7 neurons did not alter the antinociceptive effect of carbachol. These results support the conclusion that the antinociception produced by activating neurons in the ventromedial medulla is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group.

CAMP and protein kinase A modulate cholinergic rapid eye movement sleep generation.

Cholinergic neurotransmission in the medial pontine reticular formation (mPRF) modulates rapid eye movement (REM) sleep generation. Microinjection of cholinergic agonists and acetylcholinesterase inhibitors into the mPRF induces a REM sleep-like state, and microdialysis data reveal increased mPRF levels of acetylcholine during REM sleep. Muscarinic cholinergic receptors (mAChRs) participate in REM sleep generation, and data suggest that mAChRs of a non-M1 subtype modulate REM sleep generation. The signal transduction pathway activated by m2 and m4 mAChRs involves a pertussis toxin-sensitive G protein, adenylate cyclase (AC), adenosine 3',5'-cyclic monophosphate (cAMP), and protein kinase A (PKA). Therefore, the present study tested the hypothesis that cAMP and PKA within the mPRF modulate the carbachol-induced REM sleep-like state. To test this hypothesis, the mPRF was microinjected with compounds known to facilitate the effects of cAMP (dibutyryl cAMP and 8-bromo-cAMP), stimulate PKA (Sp-cAMP[S]), and inhibit PKA (Rp-cAMP[S]). The results showed that compounds that fostered the intracellular effects of cAMP significantly decreased cholinergic REM sleep, while having no effect on spontaneously occurring REM sleep. These data are consistent with the recent finding that within the mPRF, AC and a pertussis toxin-sensitive G protein modulate cholinergic REM sleep generation. These new data suggest a modulatory role for pontine cAMP and PKA in cholinergic REM sleep regulation.

Cholinergic stimulation of rostral and caudal substantia nigra pars compacta produces opposite effects on circling behavior and striatal dopamine release measured by brain microdialysis

Turning in circles is among the behaviors elicited by unilateral cholinergic stimulation of the substantia nigra. Recent studies have shown that microinjection of cholinergic agonists into the substantia nigra pars compacta increases dopamine release and turnover in the striatum of anesthetized rats [Hernández-López et al. (1992) Brain. Res. 598, 114-120; Blaha and Winn (1993) J. Neurosci. 13, 1035–1044]. In this study, the relationship between circling behavior and striatal dopamine release following cholinergic stimulation of the substantia nigra pars compacta neurons was assessed by brain microdialysis in awake rats. The results indicate that cholinergic stimulation of the substantia nigra pars compacta with the mixed nicotinic-muscarinic cholinergic agonist carbachol modulates striatal dopamine release, and this effect is accompanied by circling behavior and stereotypies. Microinjection of carbachol (109nmol) in the caudal portions of the substantia nigra pars compacta induced contralateral circling associated with an increase of dopamine release in neostriatum. On the contrary, ipsilateral circling and reduction of striatal dopamine release was elicited when the same dose of the drug was applied in the rostral portions of the substantia nigra pars compacta. The above findings are in accordance with recent electrophysiological studies suggesting the existence of sub-populations of nigrostriatal dopaminergic neurons, and indicate that the substantia nigra pars compacta is functionally compartmentalized. We conclude that the cholinergic input to the substantia nigra pars compacta could modulate the motor behavior through regulating the firing rate of nigrostriatal dopaminergic neurons and dopamine releasin the neostriatum.

Pontomedullary neurons transsynaptically labeled by laryngeal pseudorabies virus.

The activity of the laryngeal abductor, the posterior cricoarytenoid (PCA) muscles, is diminished with the loss of wakefulness. During sleep, PCA muscles become hypotonic, suggesting that neural mechanisms which control states of consciousness also contribute to state-dependent changes in upper airway patency. The medial pontine reticular formation (mPRF) has long been known to play a key role in sleep control. Microinjection of cholinergic agonists into the mPRF produces a rapid eye movement (REM) sleep-like state and PCA muscle hypotonia, but the neuroanatomical pathways mediating this hypotonia are not clear. Therefore, the present study used retrograde transsynaptic viral labeling to define multisynaptic pathways from the PCA muscles to the mPRF. Pseudorabies virus was injected into the laryngeal PCA muscles of anesthetized rats. The distribution of retrogradely labeled neurons was studied in the brain stem 4 days post-inoculation. The results show that brain stem areas known to be involved in sleep cycle control are part of a multisynaptic pathway providing input to the laryngeal PCA muscles.

Aminergic and cholinergic afferents to REM sleep induction regions of the pontine reticular formation in the rat

Microinjection of cholinergic agonists in a dorsolateral part of the mesopontine tegmentum has been shown to induce a rapid eye movement (REM) sleep-like state. Physiological evidence indicates that not only acetylcholine but also various amine transmitters, including those implicated in behavioral state regulation, affect neuronal activity in this region of the pontine reticular formation. In the present study, sources of select aminergic and cholinergic inputs to this REM sleep induction zone were identified and quantitatively analyzed by using fluorescence retrograde tracing combined with immunofluorescence in the rat. In addition to previously demonstrated cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei, the REM sleep induction zone received various aminergic inputs that originated in widely distributed regions of the brainstem and hypothalamus. Serotoninergic afferents represented a mean of 44% of all aminergic/cholinergic source neurons projecting to the REM sleep induction zone, which was comparable to the mean percentage of 39% represented by cholinergic afferent neurons. The serotoninergic afferents originated from the raphe nuclei at all brainstem levels, with heavier projections from the pontine than from the medullary raphe nuclei. Unexpectedly, an additional major serotoninergic input was provided by serotoninergic neurons in the nucleus prosupralemniscus (B9). Noradrenergic afferent neurons represented a mean of 14% of all aminergic/cholinergic source neurons, which was only about one-third of the mean percentage of either cholinergic or serotoninergic source neurons. These noradrenergic projection neurons were located not only in the locus ceruleus (8%) but also in the lateral tegmentum, including the A5 (4%) and A7 (2%) cell groups. Histaminergic neurons in the tuberomammillary hypothalamic nucleus represented a minor group of afferent neurons (3%), and a still smaller input came from adrenergic C1 neurons. The pattern of these transmitter-specific afferent connections appeared to be similar regardless of the longitudinal level within the REM sleep induction zone. The present results are consistent with previous behavioral and physiological evidence for a role of the pontine REM sleep induction zone in triggering REM sleep. The regulation of REM sleep induction would be best understood in terms of a state-dependent interplay of cholinergic, serotoninergic, and other inputs all acting convergently upon neurons in the REM sleep-inducing region of the pontine reticular formation.

Additivity of nicotinic and muscarinic cholinergic stimulation of substantia nigra.

Microinjection of cholinergic agonists into the anterior substantia nigra increases behaviours for which there is a pre-existing tendency and a low current rate. Rats received microinjections of carbachol, nicotine or a combination of both into the anterior substantia nigra. Carbachol and nicotine both stimulated consumption of palatable food in a dose-dependent manner (F(3,24) = 3.99, p < 0.02 and F(3,33) = 7.07, p < 0.01 respectively). Addition of carbachol to each dose of nicotine caused a significant increase in feeding compared with nicotine alone (F(1,23) = 7.01, p < 0.015). This suggests muscarinic or nicotinic cholinergic stimulation of the anterior substantia nigra can have behaviourally similar effects and, at the doses used here, the effects of nicotinic and muscarinic stimulation are additive.

Effect on vasopressin release of microinjection of cholinergic agonists into the rat supraoptic nucleus.

It is likely that central cholinergic pathways to the paraventricular and supraoptic nuclei participate in the control of vasopressin release. We have shown previously that this is due, in part, to activation of muscarinic, but not nicotinic, receptors in the paraventricular nucleus. There is, however, reason to believe that this cholinergic effect in the supraoptic nucleus may be the result of activation of nicotinic receptors. To test this possibility, we have studied in conscious unrestrained rats the effect of microinjection of muscarinic and nicotinic agonists into the supraoptic nucleus on vasopressin release, mean arterial blood pressure, and heart rate. Under ether anesthesia, a stainless steel guide cannula was placed in the supraoptic nucleus 5-7 days before the experiment, and femoral, arterial, and venous catheters were implanted 1 day before the experiment. Microinjection of nicotine into the supraoptic nucleus at doses of 1 and 10 micrograms resulted in transient increases in the plasma vasopressin concentration that were 7-fold and 11-fold greater, respectively, than control values at 3 min. There were also small transient increases in mean arterial blood pressure, but heart rate was unchanged. The microinjection of 2 and 20 ng of oxotremorine, a muscarinic agonist, into the supraoptic nucleus had no effect on the plasma vasopressin concentration, mean arterial blood pressure, or heart rate. These doses of oxotremorine were previously shown to have potent stimulatory effects on vasopressin release when microinjected into the paraventricular nucleus. These findings suggest that the central cholinergic stimulation of vasopressin release is due, in part, to activation of muscarinic receptors in the paraventricular nucleus and nicotinic receptors in the supraoptic nucleus.

Cholinergic stimulation of substantia nigra: abolition of carbachol-induced eating by unilateral 6-hydroxydopamine lesion of nigrostriatal dopamine neurones

Microinjection of cholinergic agonists into the substantia nigra is known to elicit increases in eating, drinking and sexual behaviour under appropriate circumstances. It has been suggested that these effects are dependent on stimulation of nigrostriatal dopamine-containing neurones in the substantia nigra pars compacta, but no direct evidence has confirmed this. The present experiment was therefore undertaken to determine whether unilateral lesions of nigrostriatal dopamine neurones made by 6-hydroxydopamine would attenuate or abolish eating in satiated rats elicited by intranigral microinjection of the muscarinic agonist carbachol. Two groups of rats were tested: a 6-hydroxydopamine- and a sham-lesion group. Before lesions were made intranigral microinjection of 0.5 microgram/0.5 microliter carbachol stimulated significantly more eating than control microinjections in both groups. After 6-hydroxydopamine lesions, microinjection of carbachol elicited no more eating than vehicle alone. Rats given sham lesions (ascorbate-saline vehicle only) showed increased feeding to intra-nigral carbachol before and after sham-lesioning. Post-mortem analysis by HPLC was used to determine the concentration of dopamine, DOPAC, HVA, serotonin and 5-HIAA in the lesioned and non-lesioned hemispheres of both 6-hydroxydopamine- and sham-lesioned rats. In caudate-putamen there were significant reductions in the concentration of DA (to 50.03% of the level in control sides), DOPAC (to 49.34%) and HVA (to 63.98%) in the 6-hydroxydopamine-lesioned but not sham-lesioned rats. The concentration of dopamine, DOPAC and HVA were not affected in the nucleus accumbens. The turnover of dopamine (assessed by calculating the ratio of dopamine to DOPAC) in the caudate-putamen but not nucleus accumbens was also altered by the 6-hydroxydopamine lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Microdialysis of cat pons reveals enhanced acetylcholine release during state-dependent respiratory depression

Microinjection of cholinergic agonists and acetylcholinesterase inhibitors into the medial pontine reticular formation (mPRF) causes a state that is polygraphically similar to rapid-eye-movement (REM) sleep. Respiratory studies of intact unanesthetized cats during this cholinergically induced REM sleep-like state have shown that the same cholinoceptive pontine reticular regions that mediate REM sleep can also cause state-dependent respiratory depression. The present study investigated the hypothesis that acetylcholine (ACh) release in the mPRF is increased during the respiratory depression that accompanies the cholinergically induced REM sleep-like state. Cats were implanted for polygraphic recording of sleep and wakefulness and with guide tubes aimed for placing a microinjector in one mPRF and a microdialysis probe in the contralateral mPRF. ACh release was measured with high-performance liquid chromatography and electrochemical detection. Compared with waking levels, ACh was significantly increased and respiratory frequency was significantly decreased during the carbachol-induced REM sleep-like state. These results support the hypothesis that endogenous cholinergic neurotransmission in brain regions known to regulate REM sleep can also cause state-dependent changes in respiratory control.

The Effect on Vasopressin Release of Microinjection of Cholinergic Agonists into the Paraventricular Nucleus of Conscious Rats

Abstract We have studied in conscious unrestrained rats under basal conditions the effect of activation of muscarinic and nicotinic receptors in the paraventricular nucleus on vasopressin secretion and mean arterial blood pressure. The microinjection of oxotremorine (0.2, 2 or 20 ng), a specific muscarinic agonist, produced a substantial, dose-dependent, transient increase in the plasma vasopressin concentration. There was also a rise in mean arterial blood pressure and a bradycardia that followed the same time-course as the change in plasma vasopressin levels. The microinjection of nicotine (0.1, 1 or 10 HQ) into the paraventricular nucleus had only questionable effects on vasopressin release and mean arterial blood pressure; heart rate was unaffected. These findings suggest that muscarinic receptors may be of primary importance in the paraventricular nucleus in the Cholinergic stimulation of vasopressin release.

Chapter 12 Cholinergic mechanisms subserving cardiovascular function in the medulla and spinal cord

This investigation was designed to study the role of muscarinic receptor subtypes in the cardiovascular responses elicited by microinjection of cholinergic agonists in the intermediate portion of the NTS, the VLPA and VLDA areas and the IML of the spinal cord. Microinjections of L-glutamate (1.77 nmol in 20-50 nl in 0.9% sodium chloride solution) were used to identify these areas. Bilateral microinjections (0.02-2 nmol/site) of a potent M2 muscarinic receptor agonist, CD, but not those of a relatively selective M1 receptor agonist (McNA343; 3 nmol/site), into the intermediate portion of NTS and the VLDA induced depressor and bradycardic responses. In the VLPA these agonists elicited pressor and tachycardic effects while in the IML at T1-T3 only increase in HR was observed. Previous microinjections of a selective competitive M2 receptor antagonist (AFDX-116; 0.8-1.6 nmol/site), but not those of a potent selective M1 receptor antagonist (PZ; 1.5-2.0 nmol/site), into these areas blocked the effects of CD. These results indicate that the muscarinic receptors of M2 type may play a part in the regulation of cardiovascular function in the above-mentioned cardiovascular areas in a yet unidentified manner.

A neuroanatomical gradient in the pontine tegmentum for the cholinoceptive induction of desynchronized sleep signs

Microinjection of cholinergic agonists into the tegmentum was used to evoke a state which was polygraphically and behaviorally similar to desynchronized (D) sleep. This study was designed to test the hypothesis that the production of this pharmacologically induced D sleep-like state (D-ACh) was dependent upon the anatomical locus of drug administration within the pottine tegmentum. Four dependent variables of D sleep were measured: D latency, D percentage, D duration and D frequency. Multiple regression analysis and analysis of variance were performed to evaluate the relationship between the three-dimensional coordinates of the injection site (posterior, vertical and lateral) and these 4 dependent measures. The intrapontine site of drug administration accounted for a statistically significant amount of the variance in D latency, D percentage and D duration. There was no significant relationship between the anatomical site of saline injection and the dependent measures of D sleep. A significance increase in D frequency following microinjection of cholinergic agonists was found to be independent of injection site. Pontine injection sites which yielded the shortest D latencies were found to be the same sites from which the highest D percentages were evoked. Rostrodorsal pontine tegmental injection sites were most effective in producing the highest percentages of D-ACh with the shortest latencies to onset. Injections made more caudally and ventrally within the pontine tegmentum produced lower percentages of D-ACh with longer latencies to onset. These data suggest the existence of an anatomical gradient within the pontine tegmentum for the cholinoceptive evocation of a D sleep-like state, and further support the concept that D sleep is generated, in part, by pontine cholinergic mechanisms.

Effects of microinjection of cholinergic agonists into the pontine reticular formation on the gain of vestibulospinal reflexes in decerebrate cats.

1. The question of which pontine neuronal groups and related receptors can mediate the cholinergic induction of the increased gain of vestibulospinal reflexes elicited by sinusoidal stimulation of labyrinth receptors was investigated by injecting in precollicular decerebrate cats either carbachol, which is a mixed muscarinic-nicotinic agonist, or bethanechol, which is a pure muscarinic agonist, via a cannula stereotaxically oriented in different pontine tegmental structures. 2. Injection of 0.1-0.2 microliter of carbachol solution (0.01-0.2 microgram/microliter of sterile saline) into the dorsal aspect of the pontine reticular formation (pRF), which slightly decreased the tonic contraction of limb extensors ipsilateral to the side of the injection, greatly increased the amplitude of the multiunit EMG response of the ipsilateral triceps brachii to roll tilt of the animal at 0.15 Hz, +/- 10 degrees, leading to selective stimulation of labyrinth receptors. Correspondingly, the response gain of the forelimb extensor to labyrinth stimulation increased. Moreover, a slight decrease in phase lead of the responses was observed. These findings were not attributable to decreased postural activity, since they were still observed when postural EMG activity was reflexly maintained by an increased static stretch of the muscle. No changes in the dynamic characteristics of the responses were observed in the contralateral triceps brachii. 3. The changes in posture as well as in response gain produced by the carbachol injection appeared suddenly, but partially declined to reach a plateau level which persisted for several hours before returning to the control level. Moreover, the magnitude of the effects increased in relation to the dose of the cholinergic agonist. 4. Histological controls indicated that the structure responsible for these postural and reflex changes was located in the dorsal aspect of the pontine tegmentum immediately ventral to the principal locus coeruleus (LC); this area corresponds to the peri-LC region and the surrounding pRF including the dorsal aspect of the central tegmental field. The effects were still obtained after chronic kainic acid lesioning of the gigantocellular area of the medulla. 5. An increase in gain of the vestibulospinal reflex which was as potent, dose-dependent, and site-specific as that previously observed with carbachol, appeared after injection of the pure muscarinic agonist bethanechol.(ABSTRACT TRUNCATED AT 400 WORDS)