Edmund G. Anderson

Reversal of the antinociceptive effects of intrathecally administered serotonin in the rat by a selective 5-HT3 receptor antagonist

The ability of the highly selective 5-HT3 receptor antagonist ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) to block the increase in tail flick (TFL) and hot plate latencies (HPL) produced by intrathecally (i.t.) administered serotonin (5-HT) was examined in pargyline pretreated rats. ICS 205-930 (0.1 microgram, i.t.) blocked the ability of 5-HT (200 micrograms) to increase TFL and HPL. Significant hyperalgesia, as measured by a decrease in TFL and HPL compared to saline controls, also resulted from either the coadministration of ICS 205-930 (10 micrograms) and 5-HT (200 micrograms) or from ICS 205-930 (100 micrograms) alone. These data suggest an important role for 5-HT3 receptors in modulating spinal nociceptive responses.

5-HT3 receptors modulate spinal nociceptive reflexes

The selective 5-HT3 receptor agonist 2-methyl-serotonin (2-Me-5-HT) mimicked the antinociceptive activity of 5-HT when intrathecally administered to rats. Two hundred micrograms (i.t.) doses of these agonists produced similar increases in tail flick latency. However, equal doses of 2-Me-5-HT and 5-HT doubled and tripled, respectively, the mean response latency as measured by the hot plate test. The potent and selective 5-HT3 receptor antagonists ICS 205-930 (3-tropanyl-indole-3-carboxylate) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) antagonized the antinociceptive effects of both 5-HT and 2-Me-5-HT. However, there were differences in the efficacy of these antagonists. Thus, intrathecal pretreatment with ICS 205-930 (0.05 micrograms) or MDL 72222 (0.1 micrograms) blocked the antinociceptive effects of 5-HT (200 micrograms, i.t.) as measured by the tail flick test, however, higher doses (0.1 and 1.0 micrograms, respectively) were required in the hot plate test. Pretreatment with ICS 205-930 (0.1 microgram) or MDL 72222 (0.1 microgram) blocked the effects of 2-Me-5-HT (200 micrograms, i.t.) in both analgesiometric tests. It is concluded that 5-HT3 receptors are intimately involved in the modulation of spinal nociceptive responses.

Long-term potentiation induced by physiologically relevant stimulus patterns

Single pulse stimuli were delivered to the Schaffer collaterals in the in vitro hippocampal slice preparation. Local application of bicuculline to the CA3 region resulted in a series of population cell bursts in CA3, resembling the neuronal patterns which occur in the normal, freely moving rat during hippocampal sharp waves. These bicuculline-induced cell bursts in CA3 resulted in long-term potentiation (LTP) of the CA1 response. These findings suggest that the naturally occurring hippocampal sharp waves may reflect events analogous to artificially induced LTP.

Identification of 5-HT3 binding sites in rat spinal cord synaptosomal membranes

The 5-HT3 receptor antagonists, ICS 205-930 and MDL 72222, displace 47-55% of the specific [3H]serotonin (100 nM) binding to synaptosomal membranes derived from the dorsal, but not ventral, spinal cord of rats with IC50s less than 1.0 nM. Methiothepin (10 microM) increased displacement to 86-94% without shifting these IC50s. Scatchard plots of [3H]5-HT binding in the presence of methiothepin (10 microM) reveal a single population of sites (KD = 11.5 nM, Bmax = 282 fmol mg-1 protein). These results indicate the presence of 5-HT3 binding sites in dorsal spinal cord.

5-HT2 and 5-HT3 receptors mediate two distinct depolarizing responses in rat dorsal root ganglion neurons

The effects of serotonin (5-HT) were studied on transmembrane potentials in 188 rat dorsal root ganglion cells (150 A-type, 16 C-type and 22 unidentified neurons). 5-HT produced a concentration-dependent depolarization in 88% of these neurons. Membrane input resistance (Rin), determined from the slope of current-voltage displacement curves, was increased in 51% and decreased in 41% of the responding neurons. Both responses occurred in 8% of the neurons. No differences in these responses were observed between A- and C-type neurons. Norepinephrine (NE) depolarized 75% (n = 20) of the neurons tested while increasing the Rin. In cells where 5-HT decreased Rin, 2-methyl 5-HT, but not alpha-methyl 5-HT, mimicked the response. The selective 5-HT3 antagonist, ICS 205-930, blocked this response, but ketanserin and methiothepin did not affect it. The 5-HT-induced increase in Rin was blocked by 5-HT2 antagonists (ketanserin, methiothepin and spiperone); mimicked by alpha-methyl 5-HT, but not affected by 2-methyl 5-HT. The selective 5-HT3 antagonist, ICS 205-930, did not antagonize this response. The action of NE but not 5-HT was blocked by the selective alpha 1 antagonist, prazosin. These data indicate that the 5-HT induced depolarization with decreased Rin is mediated by 5-HT3 receptors and the depolarization with increased Rin is mediated by 5-HT2 receptors. Furthermore, these two receptors can occur on the same cell.

Single unit studies of identified bulbospinal serotonergic units.

Non-serotonergic bulbospinal neurons were identified by conduction velocities greater than 6 m/s. These units were found to fire at rates from 0 to 22 Hz, to respond to sensory stimuli with either excitation or inhibition, and to have unremarkable spike shapes. In iontophoretic experiments, both excitation and inhibition were observed in response to acetylcholine, norepinephrine and serotonin. Serotonergic bulbospinal neurons were identified by their conduction velocities below 6 m/s. These neurons, (which have been shown to be destroyed by 5,7-dihydroxytryptamine), exist as two groups: a a slower-conducting group with conduction velocities below 1.2 m/s, and a faster-conducting group with conduction velocities between 2 and 6 m/s. The neurons of the faster-conducting group were found to be similar to the non-serotonergic group in their firing, spike shapes and responses to sensory stimuli; while the units of the slower-conducting group were consistently found to fire between 0.03 and 6 Hz, to respond to sensory stimuli only with excitation, and to have distinctive spike shapes. Despite these differences, both groups of serotonergic units were found to be consistently inhibited by ACh, NE and 5-HT. In contrast to reports of serotonergic neurons in the midbrain, these units were not generally found to be inhibited by i.v. LSD. It is concluded that the serotonergic neurons of the medullary raphe are distinct both from the non-serotonergic neurons, and from serotonergic neurons in other parts of the brain.

The effect of the GABA antagonists bicuculline and picrotoxin on primary afferent terminal excitability.

Summary (1) The effects of the convulsants bicuculline, picrotoxin, and strychnine on the polarization of gastrocnemius Ia primary afferent terminals have been ascertained with the excitability testing technique. Phasic increases in the excitability of these terminals were induced by conditioning stimuli applied to the hamstring nerve. (2) Bicuculline blocked the phasic excitability increases without appreciably altering the tonic state of terminal excitability. (3) Picrotoxin also blocked phasic increases and in addition increased the tonic excitability of the terminal. (4) Convulsant doses of strychnine failed to block phasic excitability increases but in larger doses increased tonic excitability. (5) The capacity of the GABA antagonists bicuculline and picrotoxin to block phasic increases in terminal excitability supports the hypothesis that the presynaptic inhibitory pathway contains a GABA synapse. (6) Although its position remains uncertain, these data can best be explained by locating this synapse at a site removed from the afferent terminal, where it functions to disinhibit tonically active interneurons which depolarize the terminal.

Serotonin preferentially hyperpolarizes capsaicin-sensitive C type sensory neurons by activating 5-HT1A receptors

The effects of serotonin (5-HT) were investigated by intracellular recording from 179 dorsal root ganglion (DRG) cells classified by conduction velocity. Bath applied 5-HT depolarized 82% and hyperpolarized 4% of the A-type cells. In C-type cells, 5-HT depolarized only 41%, but hyperpolarized 39% of the cells. The depolarizing responses were of two types; an increase or decrease in R(in), mediated by 5-HT2or3 receptors, respectively. These receptors were observed in both A- and C-type cells. Hyperpolarizing responses were largely confined to A(delta)- and C-type cells. Carboxamidotryptamine and 8-OH-dipropylamino-tetralin were full agonists in eliciting hyperpolarization, and metitepin, spiperone and spiroxitrine behaved as competitive antagonists. This indicated that hyperpolarization was mediated by a 5-HT1A receptor. A 5-HT1A&3 receptor were found co-localized on some C-type cells. A strong depolarizing response to capsaicin was observed in the subgroup of C-type neurons that were also hyperpolarized by 5-HT. Thus a co-localization of capsaicin and 5-HT1A receptors was also observed.

The influence of semicarbazide-induced depletion of γ-aminobutyric acid on presynaptic inhibition

Summary Administration of semicarbazide to acute spinal cats resulted in a gradual and complete suppression of the dorsal root reflex and dorsal root potential. Semicarbazide also produced marked reduction in the long-latency inhibition as measured by monosynaptic testing. The time course of these effects correlated with the time course of the semicarbazide-induced depletion of GABA in the spinal cord. Semicarbazide had little or no effect on mono- and polysynaptic reflexes or postsynaptic inhibition. These results are consistent with the hypothesis that GABA is involved in the mediation of presynaptic inhibition.

Influence of serotonin antagonists on bulbospinal systems

Abstract We attempted to elucidate the function of the serotonin-containing bulbospinal neurons by directly stimulating the raphe nucleus of unanesthetized decerebrates cats. Such stimuli evoked both a dorsal and a ventral root potential, and produced a complex time-dependent facilitation and inhibition of segmentally evoked monosynaptic reflexes (MSR). The entire time course of the curve representing the brain stem-evoked facilitation and inhibition of the MSR was displaced in a facilitatory direction following administration of the serotonin (5-HT) antagonists cinanserin or methysergide. The apparent block of descending inhibition probably resulted from increased descending facilitation. These results occurred without an increase in motoneuronal excitability, since the segmental MSR remained unchanged. The 5-HT antagonists increased the brain stem-evoked dorsal root potential (DRP), but did not alter the ventral root potential. In contrast, these agents depressed the segmentally evoked DRP. Measurements of the DRP length constants in afferent fibers revealed that brain stem stimulation evoked DRPs in fibers of smaller diameter than dorsal root stimulation. Apparently none of the potentials evoked by raphe stimulation were mediated by serotonergic fibers since these potentials were not reduced by 5-HT antagonists. It is suggested that the serotonergic system is tonically active in the decerebrate cat, and that the increased facilitatory action on the conditioned MSR and brain stem DRP following a 5-HT antagonist results from release of tonic serotonergic inhibition of descending facilitatory fibers.