Midbrain Dorsal Raphe Research Articles

Chronic benzodiazepine treatment decreases postsynaptic GABA sensitivity.

Benzodiazepines exert most of their pharmacological effects by a selective facilitation of the postsynaptic actions of GABA. Clinical, behavioural and electrophysiological studies have shown reduced drug response following chronic benzodiazepine administration. We present here electrophysiological evidence for decreased postsynaptic sensitivity to GABA following chronic benzodiazepine administration as measured by the direct iontophoretic application of GABA and serotonin onto serotonergic cells in the midbrain dorsal raphe nucleus (DRN), known to receive GABAergic input. The subsensitivity to GABA was found to be dose dependent and was seen when diazepam administration was three weeks or longer. Further, acute injection of the specific benzodiazepine antagonist, Ro15-1788, was found to reverse rapidly the decrease in GABA sensitivity observed in chronically diazepam-treated animals without altering GABA sensitivity in vehicle-treated rats. Decreased response to chronic benzodiazepines does not appear to be consistently related to alterations in the number or affinity of receptors for benzodiazepines. Our studies of radioligand-binding showed a decrease in the ability of GABA to enhance benzodiazepine binding in cerebral cortical membranes from chronic diazepam-treated animals without significant changes in benzodiazepine binding site density or affinity.

Correlative firing patterns of serotonergic neurons in rat dorsal raphe nucleus

In this study, local neuronal interactions in the midbrain dorsal raphe nucleus (DRN) were analyzed by the use of autocorrelation and cross-correlation histograms. The autocorrelograms of serotonin (5-hydroxytryptamine; 5-HT)-containing cells showed an initial trough and subsequent periodicity which corresponded to their regular rhythmic firing pattern. Cross-correlograms revealed that all adjacent 5-HT neurons recorded from single micropipettes displayed complicated patterns of functional interactions. The interactions could be grouped into the following categories: (1) synchronization (interspike intervals less than 10 msec), (2) synchronization and direct inhibition, (3) synchronization and mutual inhibition. In contrast, cross-correlograms generated from adjacent neuronal pairs consisting of one 5-HT cell and one non-5-HT neuron or from non-adjacent 5-HT neuronal pairs usually failed to show functional interactions. The results of the present study are consistent with previous findings of a powerful auto-and mutual regulatory system for 5-HT neurons in the DRN.

The effects of stimulation and lesion of raphe nuclei on luteinizing hormone release in estrogen-progesterone-treated ovariectomized rats

A study was made of the effects of electrochemically stimulating (ECS) and destroying the midbrain dorsal and median raphe nuclei on the estrogen-progesterone- (EP) induced surge of pituitary LH release in ovariectomized rats. ECS and lesions were produced simultaneously with anodal direct current through a chronically implanted stimulation-lesion electrode. Blood for LH assay was collected through a chronic intra-atrial cannula positioned on the day of first treatment with progesterone. ECS of the raphe nuclei exerted a strong inhibition of the LH surge when compared with sham stimulation. On the second EP treatment the onset of the LH surge was advanced both in sham-stimulated animals and in rats with raphe lesions resulting from the anodal direct current of the initial ECS. It was suggested that this apparent facilitation of the LH surge on the second EP treatment was the result of reduced stress or an altered hormonal milieu and not a consequence of destroying the raphe nuclei. The results confirm in a chronic preparation that stimulation of raphe nuclei depresses the EP-induced LH surge and that subsequently in the absence of the raphe nuclei the LH response to EP treatment is apparently normal.

Accelerated recovery from eating behavior deficits after sequential lesions of cortex and hypothalamus: Is dopamine involved?

The roles of the nigrostriatal dopaminergic system and of the neostriatum in recovery from ingestive behavior deficits and body weight losses after damage to the nigrostriatal bundle in the lateral hypothalamus were investigated. Sequential removals of pairs of neostriatal afferent inputs (from cortex, substantia nigra, and dorsal raphe nucleus) were effected by making an initial lesion of one input followed in 30 days by a test lesion of a second input. The recovery of rats with combination lesions (initial + test) was compared with that of controls with only test lesions. An initial unilateral rostral cortex lesion prior to ipsilateral lateral hypothalamic test damage accelerated recovery of rats with these lesions (initial + test) from the eating behavior deficits and body weight losses suffered by animals with only unilateral hypothalamic test lesions. Because cortical lesions alone had no effect upon neostriatal dopamine content, sparing from subsequent hypothalamic test damage is not dependent on a dopamine increase caused by the initial cortical lesion. Also, this acceleration of recovery from unilateral lateral hypothalamic test damage by a prior lesion is an effect that appears to be specific to the ipsilateral cortex, because initial destruction of the contralateral cortex or of the midbrain dorsal raphe nucleus did not attenuate the deficits caused by hypothalamic test lesions. Furthermore, when an initial unilateral hypothalamic lesion preceded an ipsilateral cortical test lesion, recovery of eating behavior and of body weight was not accelerated. Thus, the order in which our cortical and hypothalamic lesions were made determined whether or not accelerated recovery from eating deficits occurred. Therefore, the ability to make the neuronal alterations underlying this accelerated recovery may be the property of a subcortical system such as substantia nigra, but not of cortex itself.

Serotonin receptors in hippocampus and frontal cortex

The inhibition by various serotonin agonists and antagonists of the binding of 3 nM 3H-d-LSD, 1.7 nM 3H-serotonin and 0.22 nM 3H-spiperone to homogenates of calf hippocampus and frontal cortex was studied. The 50% inhibitory concentrations (IC 50) for these drugs versus 3H-d-LSD binding had similar values to and correlated with corresponding IC 50 values versus 3H-serotonin binding in the hippocampus, suggesting that 3H-LSD and 3H-serotonin label similar sites in this region. In the calf frontal cortex, serotonin revealed a biphasic inhibition against 3H-d-LSD binding and the tryptamines inhibited over a concentration range of 10 000-fold. The IC 25 values of various drugs versus 3H-d-LSD binding correlated with the IC 50 values versus 3H-serotonin, but did not correlate w with the IC 50 values versus 3H-spiperone. These data suggest that 3H-d-LSD bound to more than one serotonin site in the calf frontal cortex and that 3H-spiperone bound to a separate serotonergic site. Scatchard analyses of the binding for these three 3H-ligands indicated that in the calf frontal cortex the density of 3H-d-LSD sites was approximately equal to the sum of the densities for 3H-serotonin (S-1 sites) and 3H-spiperone (S-2 sites). Two weeks after serotonin-depleting radiofrequency heat lesions of the midbrain dorsal and median raphe nuclei in rats, both 3H-serotonin and 3H-LSD showed enhanced binding in the hippocampus. These data support previous suggestions that supersensitivity develops specifically in serotonin receptors following afferent denervation.

Effect of selective midbrain and diencephalic 5,7-dihydroxytryptamine lesions on serotonin content in individual preopticohypothalamic nuclei and on serum luteinizing hormone level.

Injection of the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into the midbrain dorsal (DR) or median (MR) raphe nucleus of castrated and normal male rats was followed by measurement of serum luteinizing hormone (LH) level and the 5-hydroxytryptamine (5-HT) content of several hypothalamic and amygdaloid nuclei. Only the DR lesions lead to a significant decrease (42%) in serum LH level in normal rats. The elevated LH level in castrated animals was not affected by either lesion. The DR lesions were followed by 5-HT reductions only in the medial preoptic area, the arcuate and ventromedial hypothalamic nuclei, and in the basal and central amygdaloid nuclei. In contrast, the 5-HT reductions produced by MR lesions were much more widespread, being found in all nuclei assayed with the exception of the dorso- and ventromedial hypothalamic nuclei. In a second experiment, degeneration of serotonergic terminals in the ventromedial region of the hypothalamus following intradiencephalic injections of 5,7-DHT led to a significant decrease in serum LH level and a 5-HT reduction in the arcuate, ventromedial and dorsomedial hypothalamic nuclei. 5,7-DHT injections into the medial preoptic area and the anterior hypothalamic area did not affect serum LH level. These results suggest that a serotonergic pathway originating in the midbrain dorsal raphe nucleus and innervating the mediobasal hypothalamus has a stimulatory influence on LH secretion.

Neuroendocrine regulation of pulsatile luteinizing hormone release in the rat.

This review considers some of the neuroendocrine factors influencing pulsatile LH secretion. Such release is apparently due to the pulsatile discharge of LHRH from brain peptidergic neurons. This is a physiologically important event since a periodic rather than continuous input signal to the pituitary gland prevents it from becoming refractory to LHRH stimulation. Pulsatile secretion of LH, in the rat at least, does not appear to be regulated sloely by the medial basal hypothalamus. Central noradrenergic, cholinergic, dopaminergic, and serotoninergic systems are involved in influencing episodic LH release, presumably by affecting pulsatile LHRH secretion. Moreover, several hypothalamic as well as extrahypothalamic areas appear to play integral parts in controlling the rhythmic alterations in blood LH levels. These regions include the arcuate and suprachiasmatic nuclei, perisuprachiasmatic area, medial preoptic area, and midbrain dorsal raphe nucleus. Ovarian steroids also exert important influences on pulsatile LH relase, and greatly modify the response of this secretory system to neurotransmitters and stimuli from certain brain regions.

Facilitation of memory consolidation by vasopressin: Mediation by terminals of the dorsal noradrenergic bundle?

Administration of arginine-vasopressin (AVP, 5 μ, s.c.) immediately after the learning trial results in a long-term facilitation of a one-trial learning passive avoidance response. This effect of AVP is absent in animals with prior destruction of the ascending dorsal noradrenergic bundle by bilateral microinjection of 6-hydroxy-dopamine (6-OHDA). Postlearning local microinjection of a minute amount of AVP via chronically implanted cannulae into the locus coeruleus did not influence passive avoidance behavior. Upon injection into the midbrain dorsal raphe nucleus, however AVP facilitated passive avoidance behavior. This effect, however, was absent in rats receiving previous microinjection of 5,6-dihydroxytryptamine (5,6-DHT) or of 6-OHDA into the dorsal raphe nucleus. Bilateral 6-OHDA-induced lesions of the nucleus accumbens or 5,6-DHT-induced destruction of the dorsal raphe nucleus did not prevent the effect of AVP administered subcutaneously. The data suggest that vasopressin facilitates memory consolidation processes by modulating noradrenergic neurotransmission in terminals of the dorsal noradrenergic bundle. The serotoninergic neuronal network originating from the dorsal raphe nucleus has a secondary—norepinephrine-mediated—influence upon these processes.

Serotonergic control of prolactin release in male rats

To further clarify the relationship between the central serotonergic system and the control of prolactin secretion, we studied the effect of dorsal raphe' lesions, electrical stimulation of the midbrain raphe' nucleus and treatment with parachlorophenylalanine (PCPA) on prolactin secretion. Radio frequency destruction of serotonergic cell bodies in the midbrain dorsal raphe' nucleus or PCPA decreased forebrain serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) concentration and prolactin secretion. Electrical stimulation of the raphe' increased forebrain serotonin turnover and prolactin secretion. These observations indicate that serotonergic neurons located in the raphe' nuclei may be involved in regulating prolactin secretion in male rats.

Effects of midbrain raphe lesions or systemic p-chlorophenylalanine on the development of kindled seizures in rats

RACINE, R. AND D. V. COSCINA. Effects of midbrain raphe lesions or systemic p- chlorophenylalanine on the development of kindled seizures in rats. BRAIN RES. BULL. 4(1) 1–7, 1979.—Previous research has suggested that brain serotonin (5-hydroxytryptamine or 5-HT) neurons inhibit epileptiform seizure activity. To test further this possibility, experiments were performed to determine if brain 5-HT depletion would enhance the occurrence and/or magnitude of seizures “kindled” from the amygdala or neocortex of rats. Two modes of 5-HT depletion were used: (1) radiofrequency heat lesions of the midbrain dorsal and median raphe nuclei, and (2) systemic injection of the 5-HT synthesis inhibitor, p-chlorophenylalanine (pCPA). Both modes of 5-HT depletion reliably enhanced the strength of motor convulsions kindled from the cortex. Systemic pCPA also reduced the duration of after-discharges (ADs) in cortically-stimulated rats. However, pCPA reduced rather than enhanced convulsions kindled from the amygdala. In contrast to this, raphe lesions appeared to sensitize rats to the effects of amygdaloid kindling, i.e., lesions lowered AD thresholds, AD durations and number of ADs to elicit motor convulsions. Viewed together, these data support the hypothesis that 5-HT neurons can serve to inhibit seizures. However, the lack of robustness across parameters of epileptogenesis as well as discrepant findings related to 5-HT depletion mode additionally suggest that kindled seizures affect other neuronal populations in addition to those under serotonergic influence.

Effect of Lesions in the Suprachiasmatic Nucleus-Retrochiasmatic Area on the Inhibition of Pulsatile LH Release Induced by Electrical Stimulation of the Midbrain Dorsal Raphe Nucleus

The present studies were designed to determine if the inhibition of pulsatile luteinizing hormone (LH) secretion reported to occur during midbrain dorsal raphe nucleus (DRN) stimulation involved some portion of the suprachiasmatic nucleus(SCN)-retrochiasmatic area (RCA). In animals having lesions that completely destroyed the SCN but did not extend into the RCA, dorsal raphe-induced inhibition of pulsatile LH release was still present. If, however, a significant portion (more than 40%) or all of the RCA was encompassed by the lesion (with variable or no SCN damage), the inhibition induced by DRN stimulation was prevented. This indicates that the SCN is not necessary for the inhibitory effect of DRN stimulation on pulsatile LH release to occur. However, the pathway responsible for this inhibition does appear to project to or through the RCA. Furthermore, pulsatile LH release was reduced, but present, during the control period prior to stimulation in rats with SCN lesions (and no damage to the RCA), and mean blood LH levels for this group were significantly decreased during the control bleeding period. Therefore, the neuronal pathway activating episodic LH release involves, but is not restricted solely to, the SCN.

Serotonin Involvement in the Inhibition of Episodic Luteinizing Hormone Release during Electrical Stimulation of the Midbrain Dorsal Raphe Nucleus in Ovariectomized Rats*

The effect of electrical stimulation of the dorsal raphe nucleus (DRN) in the midbrain on episodic LH release was studied in ovariectomized, pentobarbital- anesthetized rats. Animals were bled continuously at a rate of 50 μl whole blood/5-6 min through indwelling right atrial cannulae for 90 min before stimulation, 90 min during stimulation, as well as for 30-60 min thereafter. Whole blood was analyzed for LH by RIA. Electrical stimulation near but outside the DRN had no effect on pulsatile LH release. However, stimulation of the DRN completely inhibited episodic LH secretion for the entire period of stimulation. If brain levels of serotonin (5-HT) were decreased by DL-p-chlorophenylalanine (PCPA; 320 mg/kg), given 71 h before stimulation, no change in episodic LH release occurred during DRN stimulation. Repletion of brain 5-HT levels with L-5-hydroxytryptophan (5-HTP; 30 mg/kg), given to PCPA-treated rats 2.0-2.5 h before stimulation, restored the inhibition of pulsatile LH release during DRN stimulation...

Elevations in nociceptive thresholds following locus coeruleus lesions

Serotonin depletion or lesions of the midbrain dorsal raphe nuclei attenuate both morphine-produced and stimulation-produced analgesia. In contrast, norepinephrine depletion enhances both types of analgesia. To extend these findings, the effects of destruction of the noradrenergic locus coeruleus (LC) upon nociceptive flinch-jump thresholds were investigated. After four preoperative baseline sessions, lesions were placed in the LC bilaterally and nociceptive thresholds were determined for up to five weeks postoperatively. The lesions were localized by monoamine histofluorescence procedures together with conventional histological staining techniques. In 9 of 13 animals, the LC or its ascending dorsal noradrenergic bundle sustained either bilateral or unilateral damage, evidenced by green fluorescent back-up caudal to the lesions. Eight of these animals demonstrated significantly increased jump thresholds. In the remaining four animals, both lesions spared the LC and nociceptive thresholds were either unchanged or significantly decreased. In three of the four, raphe damage was noted, evidenced by yellow fluorescent back-up. The results suggest apparently contrasting roles of norepinephrine and serotonin in nociception.

Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique

Afferents to the midbrain dorsal and median raphe nuclei in the rat were studied by means of the horseradish peroxidase (HRP) retrograde transport method. The HRP was given by means of a modified iontophoretic delivery technique. This technique permitted an efficient and localized deposition of a high concentration of HRP into the raphe nuclei. Afferents to the raphe as determined by this method could be categorized into 2 classes: those exclusively to the raphe and those also positive for adjacent reticular formation. The most striking afferent area to the raphe, both in terms of selectivity and density, was the lateral habenula. This result is in accord with previous studies using degeneration methods which indicate an habenular projection to the raphe area. There were afferents exclusively positive for the dorsal raphe nucleus emanating from the nucleus of the solitary tract. Most other raphe afferent areas were also positive for the reticular formation (e.g., prefrontal cortex, medial forebrain bundle, preoptic nuclei, and reticular formation). The existence of a major afferent system from the lateral habenula to the midbrain raphe is consistent with the concept of a “dorsal pathway” which might be responsible for relaying information from forebrain limbic structures to the “midbrain limbic areas”.

Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations.

The axoplasmic retrograde transport of horseradish peroxidase (HRP) from axon terminals to their parent cell bodies and histochemical fluorescence microscopy have been used to study the ipsilateral centrifugal fibers to the olfactory bulbs and anterior olfactory nucleus in the rabbit. Focal injections of peroxidase were placed unilaterally into the main or accessory olfactory bulb or into the anterior olfactory nucleus. In animals with injected HRP confined within the main bulb, perikarya retrogradely labeled with the protein in the ipsilateral forebrain were observed in the anterior prepyriform cortex horizontal limb of the nucleus of the diagonal band, and far lateral preoptic and rostral lateral hypothalamic areas. Brain stem cell groups that contained HRP-positive somata include the locus coeruleus and midbrain dorsal raphe nucleus. Except for the prepyriform cortex, the basal forebrain structures with labeled perikarya correlate well with locations of cell bodies containing acetylcholinesterase and choline acetyltransferase. These somata may represent a cholinergic afferent system to the main olfactory bulb. Peroxidase-labeled cell bodies in the locus coeruleus and midbrain raphe are indicative of noradrenergic and serotonergic innervations respectively of the olfactory bulb. In rabbits in which peroxidase was injected or diffused into the accessory olfactory bulb and anterior alfactory nucleus, HRP-positive somata were identified in the prepyriform cortex bilaterally, the horizontal limb of the diagonal band nucleus, lateral hypothalamic region, nucleus of the lateral olfactory tract, corticomedial complex of the amygdala, mitral and tufted cell layers of the ipsilateral main olfactory bulb, locus coeruleus, and the midbrain raphe. Evidence for centrifugal fibers to the accessory olfactory bulb from the corticomedial complex of the amygdala, locus coeruleus, and possibly the nucleus of the lateral olfactory tract and midbrain raphe is discussed. A similar distribution of labeled perikarya in the forebrain and brain stem was seen in rats in which peroxidase injected into the main olfactory bulb had spread into the accessory bulb and anterior olfactory nucleus. Histochemical fluorescence microscopy of the main and accessory olfactory bulbs in the rabbit and rat revealed fine caliber, green fluorescent fibers and varicosities predominantly in the granule cell layer and less so among cells in the glomerular layer. In sections through the root of the main olfactory bulb, a similar fluorescence was seen in the deep half of the plexiform layer of the pars externa of the anterior alfactory nucleus. These fluorescent fibers likely represent the noradrenergic innervation of the olfactory bulbar and retrobulbar formations. A fluorescent yellow hue was observed in the glomerular layer of the main bulb and may signify a serotonergic innervation of this lamina...

Recording of dorsal raphe unit activity in vitro

The spontaneous electrical activity of single neurons of the midbrain dorsal raphe nucleus in the rat was recorded in vitro in 400 μm thick slices. The majority of raphe neurons exhibited the slow and regular discharge pattern that characterizes the activity of raphe neurons when recorded in vivo. The viability of these neurons in vitro thus provides a preparation for analyzing endogenous and exogenous factors that regulate the activity of these serotonin-containing neurons.

Inhibition of firing of raphe neurones by tryptophan and 5-hydroxytryptophan: Blockade by inhibiting serotonin synthesis with Ro-4-4602

In these studies, serotonergic ncuronal activity was monitored by extracellular recording from identified cells in the midbrain dorsal raphe nucleus. The systemic administration of l-tryptophan. the initial precursor in the biosynthesis of brain serotonin (5-HT). was found to depress the firing of serotonergic neurones. The systemic administration of l-5-hydroxytryptophan, the immediate precursor of 5-HT. in combination with a low dose of Ro4-4602 (50 mg/kg, which inhibits mainly peripheral decarboxylase) was also found to depress raphe firing. The tryptophan-induced depression of raphe activity was prevented by pretreatment of the animal with an l-aromatic amino acid decarboxylase inhibitor, Ro4-4602, in a dose (800 mg/kg) sufficient to block the formation of 5-HT in raphe neurones. p-Chlorophenylalanine. another 5-HT synthesis inhibitor, was found to be ineffective in preventing the depression of raphe firing by tryptophan. However, previous studies have shown that p-chlorophenylalanine does not prevent the accumulation of 5-HT within the raphe perikarya following loading doses of tryptophan (Aghajanian, Kuhar and Roth. 1973). Raphe neurones were also inhibited by the local (microiontophoretic) administration of l-tryptophan or l-5-hydroxytryptophan. We conclude that (1) both 5-HT precursors (tryptophan and 5-hydroxytryptophan) inhibit raphe firing through their enzymatic conversion to 5-HT: (2) a local increase in 5-HT within the raphe perikarya is sufficient to lead to a decrease in raphe cellular activity.

174) - The effect of morphine on single unit activity of midbrain dorsal raphe in cats

174) - The effect of morphine on single unit activity of midbrain dorsal raphe in cats

Inhibition of both noradrenergic and serotonergic neurons in brain by the α-adrenergic agonist clonidine

By means of single unit recording techniques it was found that a small systemically administered (intravenous) dose of the α-adrenergic agonist clonidine inhibited the spontaneous firing of brain norepinephrine (NE)-containing neurons in the locus coeruleus. In addition, the NE neurons were consistently inhibited by the direct (microiontophoretic) application of minute amounts of NE or clonidine. Intravenous clonidine also inhibited the firing of the great majority of 5-hydroxytryptamine (5-HT) neurons in the midbrain dorsal raphe nucleus. However, this action does not appear to be a direct one since clonidine (and NE) had relatively weak or variable effects when applied microiontophoretically to raphe neurons. The clonidine-induced depression of raphe firing may be secondary to an impairment in adrenergic transmission since (1) the depression could be reversed by the NE-releasing agents d- and l-amphetamine, (2) high doses of clonidine itself (which have been reported to have postsynaptic α-agonistic activity) reversed the depression produced by a low dose of clonidine and (3) prior destruction of NE neurons by 6-hydroxydopamine (7–12 days) rendered raphe neurons insensitive to the depressant effect of i.v. clonidine. Dopaminergic (substantia nigra, zona compacta) neurons did not respond to either low or high doses of clonidine. These results are consistent with previous data showing that clonidine decreases NE and 5-HT but not dopamine turnover. We conclude that systemically administered clonidine inhibits the firing of brain NE neurons by acting directly upon adrenergic receptors located on or near the soma of these neurons but that the concomitant inhibition of 5-HT neurons is an indirect effect (possibly secondary to an impairment in noradrenergic transmission).