Agonist-induced serotonin 2A receptor desensitization in the rat frontal cortex and hypothalamus.

This study examined the time course and possible mechanisms of agonist-induced desensitization of 5-hydroxytryptamine serotonin 2A receptors in the rat frontal cortex and hypothalamic paraventricular nucleus after 1, 4, and 7 days of treatment with (-)-1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCl [(-)-DOI] (1 mg/kg i.p.), a selective 5-HT(2A/2C) receptor agonist. In the frontal cortex, 5-HT-mediated phospholipase C (PLC) enzyme activity decreased by 24 to 30% after 4 to 7 days of (-)-DOI treatment without any significant changes in the guanosine 5'-3-O-(thio)triphosphate-mediated PLC enzyme activity. Additionally, treatment with (-)-DOI did not significantly change the levels of G(alpha11), regulator of G protein signaling (RGS)4, or RGS7 proteins in the frontal cortex, whereas G(alphaq) protein levels in the frontal cortex decreased (47%) only after 7 daily (-)-DOI injections. The functional status of 5-HT(2A) receptors in the hypothalamic paraventricular nucleus was examined using 5-HT(2A) receptor-mediated increases in plasma hormone levels. Plasma adrenocorticotrophic hormone (ACTH) and oxytocin measurements showed that 5-HT(2A) receptor desensitization began after only 1 day of (-)-DOI treatment, and the desensitization continued to increase after 4 and 7 days of treatment (ACTH response decreased 64.2-67.7%; oxytocin response decreased 82.3-90.1%). There were no significant alterations in levels of G(alphaq) or G(alpha11) lamic paraventricular proteins in the hypothanucleus. In conclusion, these results suggest that chronically administered (-)-DOI induces desensitization of 5-HT(2A) receptors in vivo, via a reduction in the ability of 5-HT(2A) receptors to activate G proteins without consistently altering levels of G(alpha) proteins or RGS proteins.

6-hydroxydopamine-induced lesions of dopaminergic neurons alter the function of postsynaptic cholinergic neurons without changing cytoskeletal proteins.

The neuropathological hallmarks of many neurodegenerative diseases are intraneuronal inclusions containing cytoskeletal proteins such as neurofilaments in Lewy bodies in Parkinson's disease and tau in neurofibrillary tangles in Alzheimer's disease. Dysfunction in dopaminergic and cholinergic systems also exist in both Alzheimer's disease and Parkinson's disease. Because the primary pathology in Parkinson's disease is localized to the dopaminergic system, we set out to determine if perturbations in cholinergic systems are a consequence of dopaminergic neuron loss. Therefore, following intracerebral microinjections of 6-hydroxydopamine in rats, the activity of cholinergic neurons was measured by hemicholinium binding in cholinergic terminal fields and perturbations in cytoskeletal proteins were examined in dopaminoceptive neurons using immunocytochemistry. The 6-hydroxydopamine injections robustly reduced the number of monoaminergic cell bodies in the lateral midbrain and dramatically decreased dopamine and its major metabolites in dopaminergic projection sites. This treatment increased hemicholinium binding in the prefrontal cortex (200%) and amygdala (284%); however, despite previous reports to the contrary, there were no increases in immunoreactivity for phosphorylated neurofilaments, microtubule-associated protein (MAP) 2, tau or paired helical filament (PHF) tau. This lack of an increase in cytoskeletal proteins was observed following either injections of moderate doses of the toxin directly into the medial forebrain bundle or after high doses were administered intracerebroventricularly. These results suggest that removal of dopaminergic inputs to the forebrain results in hyperactivity of the cholinergic systems but is not sufficient to induce postsynaptic perturbations in cytoskeletal proteins which occur in neurodegenerative diseases.

Effects of serotonergic 5-HT1A and 5-HT1B ligands on ventral pallidal neuronal activity.

To clarify the role of the 5-HT system in limbic outputs, the present study compared the effects of the 5-HT1A agonist 8-OH-DPAT and the 5-HT1B agonist CP-94253 with the non-selective 5-HT agonist TFMPP on the firing rate of ventral pallidal (VP) neurons recorded in chloral hydrate-anesthetized rats. 8-OH-DPAT (0.25-256 microg/kg i.v.) dose-dependently enhanced (9/26 neurons) or suppressed (8/26) activity, and the 5-HT1A antagonist (+)WAY-100135 often attenuated these responses. TFMPP (0.011-1.453 mg/kg i.v.) dose-dependently reduced the firing rate of 7/8 VP neurons tested. In contrast, CP-94253 (0.013-12.8 mg/kg i.v.) had little or no effect. In sum, these data suggest that the 5-HT1A receptor appears to be particularly important in influencing limbic outputs mediated via the VP.

Effects of crus cerebri lesions and repeated amphetamine treatment on the activity of nigral dopaminergic neurons.

Previous research suggests that the firing rate of dopamine (DA) neurons in the substantia nigra pars compacta (SNC) may be altered by repeated DA agonist treatment. Because changes in the frequency of DA activity could reflect the firing patterns (e.g., bursting) of the neurons sampled, this study examined both the firing rate and pattern of SNC DA neurons after long-term amphetamine (AMPH) treatment (5 mg/kg d-AMPH s.c. twice daily for 6 days). To assess the contribution of postsynaptic feedback from the forebrain, unilateral electrolytic lesions were made to the crus cerebri (CC), containing the striatonigral pathway, prior to AMPH treatment. Single-unit activity of presumed SNC DA neurons was recorded in adult male rats under urethane anesthesia. Spontaneous firing rate was reduced by AMPH treatment, relative to saline vehicle, but was unaffected by CC or sham lesions. Neurons categorized as bursting had faster rates of activity than nonbursting cells. AMPH treatment reduced the number of bursts seen in intact rats but increased bursting in lesioned rats. These results suggest that changes in DA firing rate previously found after chronic AMPH may reflect altered patterns of activity. In addition, the effects of long-term AMPH on the firing patterns of DA neurons appear to be mediated by fibers in the CC.

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 alpha2-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 microg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars alpha of pentobarbital anesthetized Sprague-Dawley rats. In some experiments, the local anesthetic tetracaine (10 microg) 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 microm 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.

Localization of motor- and nonmotor-related neurons within the matrix-striosome organization of rat striatum.

Striatal neurons can be classified as movement- and nonmovement-related depending on their ability to change firing rate in close temporal association with spontaneous movement in an open-field arena. The present study assessed the location of these cell types within the compartmental organization of the striatum by combining single-unit recording techniques in freely moving rats with calbindin immunohistochemistry. Movement-related neurons were found predominately either in the matrix or along the matrix-striosome border. Most of these neurons were nonselective in that they increased activity whenever the animals changed from a quiet resting posture to any form of behavioral activation (e.g., grooming, locomotion, rearing). The remaining neurons in this group responded exclusively to movements of the head. Nonselective units discharged at a significantly slower rate than head-movement units during both quiet rest and periods of actual movement. Nonmovement-related neurons, which failed to show a reliable change in activity to overt behavior, comprised a relatively small portion of the neuronal sample but were also located in either the matrix or along the matrix-striosome border. Collectively, these results suggest that even though striatal neurons can be distinguished on the basis of their responsiveness to ongoing behavior in an open-field paradigm, such distinctions are not clearly linked to sites within the matrix or its striosomal borders.

Partial and full dopamine D1 agonists produce comparable increases in ventral pallidal neuronal activity: contribution of endogenous dopamine.

Systemic administration of the partial DA D1 agonist SKF38393 often increases the firing rate of neurons in the VP of rats. This study extended this finding by comparing responses to (+/-)SKF38393 with those produced by two D1 agonists that have greater intrinsic efficacy, (+/-)SKF82958 and (+/-)DHX. The role of endogenous DA in D1 agonist-induced effects also was examined. Extracellular recordings of single VP neurons were obtained in chloral hydrate-anesthetized male rats, to which equimolar doses of SKF38393, SKF82958 or DHX were administered i.v. Each of the agonists increased firing rate in about 45% of the neurons tested. Moreover, each agonist produced the same maximal increase in activity (161% to 178% of spontaneous rate). Acute decreases in synaptic DA, produced by either GBL or combined treatment with reserpine and AMPT, potentiated the maximal increase in activity evoked by SKF38393 or SKF82958. These DA-depleting treatments did not alter the percentage of neurons that displayed this response to D1 agonist challenge. Low doses of the selective D1 antagonists SCH23390 or SCH39166 generally attenuated the agonist-induced changes in firing rate, supporting the conclusion that D1 receptors were activated by SKF38393, SKF82958 and DHX. Thus, these three D1 agonists, which produce different maximal increases in striatal adenylyl cyclase activity, had comparable efficacy to increase VP neuronal activity. A reduction in endogenous DA enhanced the D1 agonist-induced effects, possibly through a reduction in inhibitory influences on VP neurons that are mediated by other DA receptor subtypes.

A methodology for determining the patch-matrix compartmental location of extracellular single-unit recordings in the striatum of freely moving rats.

A methodology was developed to combine extracellular electrophysiological recording techniques in awake, behaving rats with immunohistochemical protocols to determine the placement of recording sites in the patch (striosome) or matrix (extrastriosome) regions of the striatum. The recording system includes a 3-barrel glass micropipette, which can be used to deposit Pontamine Sky Blue to mark a small number of neurons at the recording site. Subsequent immunostaining for calbindin allows the site to be localized within the patch-matrix organization. Other dyes or neuroanatomical probes can be ejected from other barrels of the recording pipette to label afferent and efferent structures. The methodology can be applied to many brain regions, providing for integrative studies of behavior and nervous system structure and function.

A simple micromanipulator for multiple uses in freely moving rats: electrophysiology, voltammetry, and simultaneous intracerebral infusions.

An inexpensive, easily fabricated micromanipulator is described that can be used for single-unit recording or voltammetry in freely moving rats. The basic design is configured around the standard coupling system between a plastic syringe and corresponding needle hub. The device can be used with glass or metal microelectrodes for electrophysiology or carbon-fiber or carbon-disk microelectrodes for voltammetry. With either recording technique, the micromanipulator also can accommodate a 33-ga infusion cannula, which allows drugs to be administered directly to the recording site. The entire assembly is lightweight and can be used with a head-mounted amplifier system for relatively noise-free recording.

Serotonergic dorsal raphe neurons: changes in spontaneous neuronal activity and responsiveness to 5-MeODMT following long-term amphetamine administration.

Single-unit activity, characteristic of serotonergic neurons, was recorded in the dorsal raphe nucleus of urethane-anesthetized rats pretreated twice daily with saline or with 10.0 mg/kg D-amphetamine for 6 days. Compared to controls, amphetamine-pretreated animals showed a trend toward increased spontaneous firing rate and decreased responsiveness to 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a serotonergic autoreceptor agonist. The most pronounced effect of amphetamine pretreatment, however, was a highly significant correlation between spontaneous neuronal activity, measured as either firing rate or interspike interval, and the 5-MeODMT response. Faster firing cells required predictably higher doses of 5-MeODMT to produce an inhibition. No such relationship was observed in control animals. Taken together, these results suggest that repeated administration of relatively high doses of amphetamine produces complex changes in the dorsal raphe including a shift in the sensitivity of serotonergic autoreceptors.

Serotonergic dorsal raphe neurons: subsensitivity to amphetamine with long-term treatment.

Rats were pretreated twice daily for six consecutive days with either saline or 1.0, 5.0, or 10.0 mg/kg (+)-amphetamine. On the following day, single-unit recording techniques were used to identify serotonin-containing neurons in the dorsal raphe nucleus (DRN). Pretreatment with amphetamine did not alter the mean spontaneous firing rate of these cells, but in some instances it appeared to produce periods of irregular bursting. Moreover, the response to challenge injections of amphetamine was reduced significantly by pretreatment with the large dose. Thus, whereas an intravenous challenge of approximately 3.0 mg/kg produced a greater than 50% inhibition of activity in the dorsal raphe nucleus in rats pretreated with saline, 1.0 or 5.0 mg/kg (+)-amphetamine, more than twice the challenge dose was required to suppress the activity of serotonergic neurons in rats pretreated with 10.0 mg/kg (+)-amphetamine. These results parallel those previously obtained with dopaminergic neurons, suggesting that both types of cells lose their sensitivity to amphetamine with repeated injections.