Serotonin1A autoreceptor activation by S 15535 enhances circadian activity rhythms in hamsters: evaluation of potential interactions with serotonin2A and serotonin2C receptors.

Mammalian circadian activity rhythms are generated by pacemaker cells in the suprachiasmatic nucleus (SCN). As revealed by the actions of diverse agonists, serotonergic input from raphe nuclei generally inhibits photic signaling in the suprachiasmatic nucleus. In contrast, the serotonin (5HT)1A partial agonist, 4-(benzodioxan-5-yl)1-(indan2-yl)piperazine (S 15535), was found to enhance the phase-shifting influence of light on hamster circadian rhythms [Gannon, Neuroscience 119 (2003) 567]. Herein, we extend this observation in showing that S 15535 (5.0 mg/kg, i.p.) markedly (275%) enhanced the light-induced phase shift in circadian activity rhythms: further, this action was dose-dependently abolished by the highly-selective 5HT1A receptor antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]N-2-pyridinyl-cyclohexane-carboxamide maleate) (0.1-0.5 mg/kg, i.p.). WAY 100,635, which was inactive alone, shares the antagonist actions of S 15535 at postsynaptic 5HT1A sites, yet blocks its effects at their presynaptic counterparts. Thus, 5HT1A autoreceptor activation must be involved in this effect of S 15535 which contrasts with the opposite, inhibitory influence upon phase shifts of the "full" agonist, 8-OH-DPAT, which acts by stimulation of postsynaptic 5HT1A receptors [Rea et al., J Neurosci 14 (1994) 3635]. Despite the occurrence of 5HT2A and 5HT2C receptors in the (rat) suprachiasmatic nucleus, their influence on circadian rhythms is unknown since actions of selective ligands have never been evaluated. This issue was investigated with the most selective agents currently available. However, the 5HT2A agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.25 and 0.5 mg/kg), and the 5HT2C agonist, alphaS-6-chloro-5-fluoro-a-methyl-1H-indole-1-ethanamine fumarate (Ro-60-0175) (1.0 and 5.0 mg/kg), failed to affect light-induced phase shifts in hamsters. Moreover, even over broad dose-ranges, the 5HT2A antagonist, (+)-(2,3-dimethoxy-phenyl)-[1-[2-(4-fluoro-phenyl)-ethyl]-piperidin-4-yl]methanol (MDL 100,907) (0.1-1.0 mg/kg), and the 5HT2C antagonist, 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl carbamoyl]indoline (SB 242,084) (1.0-10.0 mg/kg), were likewise inactive. In view of evidence that 5HT2A and 5HT2C sites functionally interact with 5HT1A receptors, we also examined the influence of these agents upon the actions of S 15535, but no significant alteration was seen in its enhancement of rhythms. In conclusion, S 15535 elicits a striking enhancement of light-induced phase shifts in circadian rhythms by specifically recruiting 5HT1A autoreceptors, which leads to suppression of serotonergic input to the suprachiasmatic nucleus. Surprisingly, no evidence for a role of 5HT2A or 5HT2C sites was found, though comparable functional studies remain to be undertaken in rats. Indeed, the present work underlines the importance of comparative studies of circadian rhythms in various species, as well as the need for further study of potential interactions among 5HT receptor subtypes in their control.

Serotonergic serotonin (1A) mixed agonists/antagonists elicit large-magnitude phase shifts in hamster circadian wheel-running rhythms.

The biological clock that generates circadian rhythms in mammals is located within the suprachiasmatic nuclei at the base of the hypothalamus. The circadian clock is entrained to the daily light/dark cycle by photic information from the retina. The retinal input to the clock is inhibited by exogenously applied serotonin agonists, perhaps mimicking an endogenous inhibitory serotonergic input to the clock arriving from the midbrain raphe. In the present study, a unique class of serotonergic compounds was tested for its ability to modulate retinal input to the circadian clock. The serotonergic ligands 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro(4.5)decane-7,9-dione dihydrochloride (BMY 7378), S 15535, and 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione hydrochloride (MDL 73005 EF) can all be classified as mixed agonists/antagonists at type 1A serotonin receptors. Circadian wheel-running activity rhythms were monitored in Syrian hamsters maintained in constant darkness. Dim white-light pulses administered to the hamsters at circadian time 19 advanced the phase of their running rhythms by 1-2 h. Injection of BMY 7378, S 15535, and to a lesser degree MDL 73005 EF, prior to the light pulses resulted in phase advances from 5 to 6 h, and by as much as 8 h. Neither BMY 7378 nor S 15535 had any effect on light-induced phase delays in hamster activity rhythms at circadian time 14. Further, BMY 7378 is able to phase advance circadian rhythms by approximately 1 h at night even without light exposure. Finally, the effects of BMY 7378 on circadian rhythms is opposite to that observed with the prototypical serotonin 1A agonist (+/-)-8-hydroxy-2-(DI-n-propyl-amino)tetralin hydrobromide (8-OH-DPAT) (8-OH-DPAT elicits non-photic phase advances in the day and inhibits photic-induced phase advances at night). These results suggest that pharmacologically blocking raphe input to the suprachiasmatic circadian clock results in substantially larger photically induced phase advances in wheel-running rhythms. This is further evidence that raphe input to the circadian clock is probably acting to dampen the clock's response to light under certain conditions. The large-magnitude phase shifts, and temporal-activity profile seen with BMY 7378 and S 15535, suggest that compounds with this unique pharmacological profile may be beneficial in the treatment of circadian phase delays recently reported to be a complication resulting from Alzheimer's disease.

5HT7 receptors in the rodent suprachiasmatic nucleus.

Serotonergic modulation of circadian rhythms in rodent model preparations has received considerable attention over the past decade. Investigators have also been trying to determine which of the many serotonin receptor subtypes may be mediating the effects of serotonin in the suprachiasmatic nucleus, the location of the biological clock that generates the circadian rhythms. A single study in 1993 using the in vitro rat hypothalamic slice preparation suggested that serotonergic modulation of circadian rhythms at the level of the suprachiasmatic nucleus was acting via the newly discovered 5HT7 receptor subtype. Since that initial claim, serotonin modulation of circadian rhythms at the level of the suprachiasmatic nucleus has generally been attributed to 5HT7 receptor activation. However, when trying to cite relevant literature in support of 5HT7 involvement, it becomes evident that attributing rhythm-related serotonin activity in the suprachiasmatic nucleus to 5HT7 receptors may be somewhat premature. There are issues related to pharmacological specificity, species-specific results, and significant knowledge gaps that necessitate a careful review of the literature to make a judgment as to whether 5HT7 receptors are responsible for serotonergic activity in the rodent suprachiasmatic nucleus. In addition, there is sufficient data available at present to make an initial determination as to the degree of 5HT7 receptor involvement at any level in the generation or modulation of circadian rhythms in rodent species.

Serotonin transporter localization in the hamster suprachiasmatic nucleus.

Pacemaker cells within the hamster suprachiasmatic nucleus generate circadian rhythms. The suprachiasmatic nucleus is heavily innervated by serotonin axons originating in the median raphe nuclei. Consequently, serotonergic agonists and antagonists or agents that alter levels of serotonin in the synapse following transmission can modulate many aspects of circadian rhythmicity. Examples of the latter are some antidepressants and the stimulant amphetamine that bind to the serotonin transporter and block serotonin reuptake. It has been hypothesized that circadian rhythm dysfunction may be involved in depression, and that the efficacy of certain antidepressants in treating depression may involve an alteration of serotonin levels and certain circadian rhythm parameters. However, although the hamster is the behavioral model of choice for the study of circadian rhythms, the identification of serotonin transporters in this species has not been reported. Therefore, in this report we describe the distribution of the serotonin transporter in the hamster suprachiasmatic nucleus using immunohistochemical techniques. Our results demonstrate a dense labeling of the serotonin transporter throughout the ventral and medial regions of the suprachiasmatic nucleus, a pattern that overlaps the distribution of serotonergic afferents in this nucleus. Amphetamines and certain antidepressants may serve as substrates for this transporter and elicit chronopharmacological activity by elevating serotonin levels in the suprachiasmatic nucleus.

Opioid induced non-photic phase shifts of hamster circadian activity rhythms.

The phase of the circadian pacemaker in hamsters can be shifted by the application of certain non-photic stimuli late in the subjective day. A projection from the intergeniculate leaflet of the thalamus to the circadian pacemaker in the suprachiasmatic nucleus is believed to mediate some types of non-photic phase-shifting stimuli. In hamsters, this projection is immunoreactive to both Neuropeptide Y and enkephalin. Previous work in other laboratories has shown that Neuropeptide Y administration is capable of phase shifting circadian rhythms without the application of light. The present study was undertaken to determine if enkephalinergic compounds likewise have the ability to non-photically phase shift hamster activity rhythms. Hamsters were maintained under conditions of constant darkness and circadian wheel running activity was recorded. Agonists and antagonists selective for kappa, mu, and delta opioid receptors were systemically applied without light to hamsters at circadian times 8 and 10 to determine if they were able to elicit phase shifts in wheel running activity rhythms. Of the compounds tested, only the delta opioid agonist BW373U86 significantly affected circadian phase. BW373U86 phase advanced hamster wheel running activity rhythms by approximately 45 min, although total activity levels following drug application were not significantly affected. Changes in the amount of wheel running activity were detected after administration of some mu and kappa opioids, although the circadian phase was not altered. These results indicate that enkephalin-mimetic delta opioid agonists are capable of producing non-photic phase shifts in hamster activity rhythms, and that opioids can independently affect circadian phase and activity levels in hamsters.

Effects of the 5HT1A agonist/antagonist BMY 7378 on light-induced phase advances in hamster circadian activity rhythms during aging.

The entrainment of some circadian rhythms in rodents and humans to the environmental light-dark cycle deteriorates during aging. Recent evidence suggests that the time-keeping ability of the circadian pacemaker maintains its endogenous period in both hamsters and humans. This suggests that any changes in the coupling between environmental cues and the circadian pacemaker are not due to changes in "clock speed," but rather due to a weakened coupling between the afferent systems relaying environmental information and the circadian pacemaker located in the suprachiasmatic nucleus. The suprachiasmatic nucleus receives serotonergic input from the raphe nuclei, and serotonergic 5HT1A,7 agonists have been reported to lose their circadian phase-adjusting efficacy during aging in hamsters. In the present study, the authors report the effects of a novel serotonergic agonist BMY 7378 on light-induced phase advances during aging in the hamster. The present report demonstrates that BMY 7378 is a highly efficacious chronobiotic that more than doubles the magnitude of light-induced phase shifts in hamster wheel-running activity rhythms. Light-induced phase advances in hamster wheel-running activity of at least 6 h following a single systemic dose of BMY 7378 are routinely observed. Furthermore, BMY 7378 potentiation of phase shifts is maintained in old hamsters, suggesting that BMY 7378 has a different site of activity than previously reported 5HT1A,7 agonists that have a diminished effect on circadian phase during aging.

SNC 80, a delta-opioid agonist, elicits phase advances in hamster circadian activity rhythms.

Non-photic stimuli administered to hamsters during the subjective day can cause phase advances in circadian wheel running activity. It is believed that afferent projections from the intergeniculate leaflet of the thalamus to circadian pacemaker cells within the suprachiasmatic nucleus mediate the phase shifting effects of some non-photic stimuli. In hamsters, many of the intergeniculate leaflet afferents contain enkephalin, yet the role of opioids in producing non-photic phase shifts in hamsters has not been reported. In the present study, we show that SNC 80, an agonist for the delta opioid receptor subtype, will phase advance hamster wheel running activity rhythms when administered late in the subjective day. These results indicate that opioids may be involved in modulating the circadian pacemaker in hamsters.

Distribution of delta opioid receptor immunoreactivity in the hamster suprachiasmatic nucleus and intergeniculate leaflet.

The hamster suprachiasmatic nucleus (SCN) is innervated by a dense plexus of enkephalin-containing axons originating from cells in the intergeniculate leaflet (IGL) of the thalamus. However, the distribution of opioid receptors within the hamster SCN has not been reported. Opioid receptors consist of three primary subtypes: mu, delta and kappa opioid receptors. Enkephalins have the highest affinity for delta opioid receptors. Therefore, in the present study, we examined the distribution of delta opioid receptor immunoreactivity in the hamster SCN and the IGL of the thalamus. Coronal sections of the hamster hypothalamus inclusive of the SCN or thalamic regions containing the IGL were prepared at specific times of the day and labeled with anti-delta opioid receptor polyclonal antisera using standard immunohistochemical techniques. delta opioid receptors were heavily distributed within rostral-caudal regions of the SCN, with the densest labeling located in the ventral and medial regions of the mid-SCN. Similar patterns of labeling were observed for tissue prepared during mid-day or mid-night times. In contrast, delta opioid receptor immunoreactivity only sparsely labeled cells in the IGL. Cellular staining in all regions appeared as dark punctate labeling surrounding cells, indicative of terminal boutons. Therefore, it is suggested that delta opioid receptors are located presynaptically on axon terminals within the hamster SCN and IGL. These results suggest that delta opioid receptors may play a role in modulating circadian rhythms generated within the SCN, possibly by regulating transmitter release within the nucleus.

Local administration of serotonin agonists blocks light-induced phase advances of the circadian activity rhythm in the hamster.

Circadian rhythms in mammals are synchronized to environmental light-dark cycles through a direct retinal projection to the suprachiasmatic nucleus (SCN), a circadian clock. This process is thought to be modulated by other afferents to the SCN, including a dense serotonergic projection from the midbrain raphe. Previous work from this laboratory demonstrated that a systemically administered 5-hydroxytryptamine1A/7 (5-HT1A/7) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) dose dependently attenuates light-induced phase shifts of the circadian activity rhythm of the Syrian hamster. In this study, we demonstrate that local injections (1-100 microM) of the 5-HT1A/7 agonists 8-OH-DPAT or 5-carboxamidotryptamine into the region of the SCN inhibit light-induced phase advances of the circadian wheel-running rhythm. In addition, the inhibitory effects of systemically administered 8-OH-DPAT were unaffected by either radiofrequency-induced lesions of the intergeniculate leaflet or 5,7-dihydroxytryptamine-induced lesions of serotonergic projections to the SCN. These findings support a modulatory role of serotonin in photic regulation of circadian phase through an action at the level of the SCN.

GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus.

The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABAA agonist muscimol (10 microM) potentiated SCN field potentials by 23%, while application of the GABAA antagonist bicuculline (10 microM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA, agonist baclofen (1.0 microM) inhibited SCN field potentials by 48%, while the GABAB antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABAA activity tends to increase, and GABAB activity tends to decrease the response of SCN neurons to optic nerve stimulation.

Twelve-hour phase shifts of hamster circadian rhythms elicited by voluntary wheel running.

Running in a novel wheel can serve as a nonphotic zeitgeber to entrain or phase shift circadian rhythms in hamsters. In this study, hamsters were entrained to a light:dark schedule of 14:10 h but had no access to running wheels. At four different phase points of the light cycle, hamsters were transferred to constant darkness and provided with running wheels. All hamsters began running shortly after transfer and were allowed to continue running at their own volition. Approximately 20% of the hamsters transferred at zeitgeber time (ZT) 23 (ZT 12 = lights out) ran more than 4 h after transfer and showed phase advances of the circadian activity rhythm by as much as 15 h, while hamsters that ran less than 4 h on average did not phase shift. A similar result was observed for hamsters transferred at ZT 2. Hamsters transferred at ZT 5 and 8 also did not phase shift if they ran less than 4 h, although the relation between longer runs and phase shifts became less evident. A sustained run in excess of 4 h appeared to be associated with large phase advances. These results show that under certain conditions, a single sustained bout of wheel-running activity is capable of phase shifting the circadian pacemaker by more than 12 h.

cGMP-dependent protein kinase inhibitor blocks light-induced phase advances of circadian rhythms in vivo.

The suprachiasmatic nucleus (SCN) contains the primary mammalian circadian clock. Light synchronizes these circadian rhythms through a mechanism involving the release of excitatory amino acids (EAA) and synthesis of nitric oxide (NO) in the SCN. In the current study, we investigated whether cGMP-mediated activation of cGMP-dependent protein kinase (PKG) is associated with light-induced phase shifts of the circadian oscillator. Local administration of the specific PKG inhibitor, KT-5823, significantly attenuated light-induced advances in the phase of activity rhythms when administered during late subjective night (CT 19). Similar treatment at CT 14 had no significant effect on light-induced phase delays. These results are the first to implicate PKG in the biochemical pathway(s) responsible for photic phase advances, and suggest a divergence in biochemical pathways involved in photic phase shifts.

Nitric oxide synthase inhibitor blocks light-induced phase shifts of the circadian activity rhythm, but not c-fos expression in the suprachiasmatic nucleus of the Syrian hamster.

Circadian rhythms in mammals are entrained to the environmental light cycle by daily adjustments in the phase of the circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Brief exposure of hamsters maintained under constant darkness to ambient light during subjective nighttime produces both phase shifts of the circadian activity rhythm and characteristic patterns of c-fos protein (Fos) immunoreactivity in the SCN. In this study, we demonstrate that light-induced phase shifts of the circadian activity rhythm are blocked by intracerebroventricular (i.c.v.) injection of the competitive nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), but not by the inactive isomer, D-NAME. The effects of L-NAME are reversible and dose-related, and are countered by co-injection of arginine, the natural substrate for NOS. While effects on behavioral rhythms are pronounced, similar treatment does not alter the pattern of light-induced Fos immunoreactivity in the SCN. These results suggest that nitric oxide is a component of the signal transduction pathway that communicates photic information to the SCN circadian pacemaker, and that nitric oxide production is either independent of, or downstream from, pathways involved in induction of c-fos expression.

Serotonergic potentiation of photic phase shifts of the circadian activity rhythm.

Recent evidence suggests that serotonin may function to regulate the sensitivity of the circadian clock to the resetting effect of environmental light. Here we report that systemic administration of NAN-190, a drug that acts at both postsynaptic and somatodendritic serotonin receptors, potentiates light-induced phase shifts by as much as 250%. The effects of the drug are dose-related and are significant at light intensities between 0.2 and 200 lux. It is proposed that drugs with pharmacological properties similar to NAN-190 may prove useful as chronobiologics to adjust the sensitivity of the circadian system to natural 'zeitgebers'.

In situ hybridization of antisense mRNA oligonucleotides for AMPA, NMDA and metabotropic glutamate receptor subtypes in the rat suprachiasmatic nucleus at different phases of the circadian cycle.

Eleven oligonucleotides directed against mRNA for AMPA, NMDA and metabotropic glutamate receptor subtypes were hybridized to rat coronal brain sections containing the suprachiasmatic nucleus (SCN). These oligonucleotides were hybridized to tissue samples collected at midday and midnight phases of the circadian cycle. Glutamate receptor mRNA for the AMPA subunits GluR1, GluR2 and GluR4, and the NMDA receptor subtype NMDAR1, were heavily expressed in the SCN and surrounding areas. The mRNA for the metabotropic glutamate subunit mGluR1 was only lightly expressed in the SCN. In contrast, mRNA for NMDAR2A, NMDAR2B, NMDAR2C and GluR3 was not detected in the SCN. The mRNA found to be expressed in the rat SCN was similar in samples collected at midday and midnight, suggesting no circadian variation in endogenous SCN glutamate receptors at these two times of the light-dark cycle.

Glutamate receptor immunoreactivity in the rat suprachiasmatic nucleus.

Antibodies selective for the glutamate receptor subunits GluR1 and GluR2/3 were used to localize glutamate receptor immunoreactivity in the rat suprachiasmatic nuclei (SCN). These antisera identified two distinct cell populations within the SCN. Cells immunoreactive to GluR2/3 antiserum were located predominantly in the ventral SCN while antiserum selective for GluR1 stained a population located along the dorsal and lateral borders of the nucleus. There were no apparent day-night differences in GluR immunoreactivity observed in the SCN.

Transduction of a protein kinase C-generated signal into the long-lasting facilitation of glutamate release.

The present study investigated the delayed and persistent effects of 4 beta-phorbol 12,13-dibutyrate (PDBu) on the K(+)-evoked release of endogenous glutamate and dynorphin B-like immunoreactivity from a subcellular fraction (P3) that is enriched in hippocampal mossy fiber synaptosomes. It is demonstrated that the alpha, beta, gamma, epsilon, and zeta isoforms of protein kinase C (PKC) are present in the P3 fraction obtained using the guinea pig hippocampus as starting tissue. The K(+)-evoked release of glutamate was found to be selectively enhanced when mossy fiber-enriched synaptosomes were preincubated with PDBu for 15 minutes and extensively washed with a PDBu-free medium. The persistent enhancement of glutamate release observed under this condition was not reversed by the protein kinase inhibitor staurosporine and was desensitized to the potentiating effects of an acute reexposure to PDBu. The overall content and activity of PKC was not substantially altered during the initial 15 minutes of treatment with PDBu (10 microM). More prolonged pretreatments with PDBu altered the substrate specificity of PKC and decreased the content of all PKC isoforms, but did not reverse the facilitation of glutamate release that followed preincubation in the presence of PDBu. It is concluded that the persistent activation of PKC enhances K(+)-evoked glutamate release from hippocampal mossy fiber-enriched synaptosomes and that, once established, this presynaptic facilitation is sustained by a process that is no longer directly dependent on continued PKC phosphotransferase activity.

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes.

Opioid agonists specific for the mu, delta, and kappa opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The kappa opioid agonists U-62,066E and (-) ethylketocyclazocine, but not the mu agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAGO) nor the delta agonist [D-Pen2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca2+ levels, indicating a possible mechanism for kappa agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca2+ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca2+ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the delta-opioid specific antagonist ICI 174,864 and the mu opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that kappa opioid agonists inhibit transmitter release from mossy fiber terminals through both kappa opioid and non-kappa opioid receptor mediated mechanisms.

Kainic acid depresses the ex vivo release of dynorphin B and glutamate from rat hippocampal mossy fiber synaptosomes.

This experiment examined the effects of intracerebroventricularly (i.c.v.) administered kainic acid (KA) on the subsequent ex vivo release of L-glutamate (Glu) and dynorphin B-like immunoreactivity (Dyn B-LI) from isolated rat hippocampal mossy fiber (MF) synaptosomes at 4.5 h, 20 h or 48 h after administration of 0.5 microgram/microliter KA. The Dyn B-LI content in the synaptosomal fraction initially decreased at 4.5 h and then rebounded and remained elevated above control levels at 20 h and 48 h. The K(+)-evoked release of Dyn B-LI from the synaptosomes was markedly depressed at 4.5 h after KA and remained significantly below control levels at 20 h and 48 h. In contrast, KA caused no change in the K(+)-evoked release of Glu at 4.5 h as compared to control levels, but did result in a significant decrease in Glu release at 20 h and 48 h. These data indicate a persistent effect of i.c.v. KA on neurotransmission at MF-CA3 synapses in rat hippocampus, resulting in a suppression of the release of Glu as well as the opioid peptide, Dyn B.

A presynaptic role for protein kinase C in hippocampal mossy fiber synaptic transmission.

It has been suggested that the maintenance of long-term potentiation (LTP) in the hippocampal mossy fiber (MF) synapse involves a presynaptic mechanism that does not require the activation of protein kinase C (PKC), since this enzyme appears to be absent in the MF presynaptic terminals. In the present study the authors evaluated this proposal by directly comparing the metabolic properties of hippocampal MF synaptosomes and a conventional P2B synaptosomal preparation prepared from the same hippocampal tissue. Protein kinase C-dependent histone phosphotranferase activity was found to be comparable in MF and P2B synaptosomes. Western blot analysis was performed using antisera prepared against four of the PKC isoforms, and the results demonstrate that the alpha, beta, and gamma PKC isoforms are present in relatively equivalent amounts in these two subcellular fractions. However, the cytosolic fraction derived from the hippocampal MF synaptosomes appeared to contain a greater amount of the PKC-epsilon isoform when compared to the P2B synaptosomal preparation. Four distinct endogenous substrates present in the MF synaptosomes are shown to be phosphorylated in response to PKC activation. A functional role for PKC in the hippocampal MF nerve endings seems to be indicated by the finding that 4 beta-phorbol 12,13-dibutyrate (PDBu) and 4 beta-phorbol 12,13-diacetate produce a dose-dependent potentiation of the K(+)-evoked release of endogenous glutamate and dynorphin B, while the inactive 4-alpha-phorbol was without effect. The PDBu-induced enhancement of transmitter release was blocked by the PKC inhibitor, staurosporine. In addition, PDBu significantly facilitated the rise in cytosolic free calcium that immediately followed depolarization of the MF synaptosomal membrane. It is concluded that hippocampal MF presynaptic terminals possess a variety of PKC isoforms and that their activation may have an important facilitory influence on MF synaptic transmission and plasticity.