Samuel B. Kombian

Dendritically Released Peptides Act as Retrograde Modulators of Afferent Excitation in the Supraoptic Nucleus In Vitro

Oxytocin (OXT) and vasopressin (VP) are known to be released from dendrites of magnocellular neurons. Here, we show that these peptides reduced evoked EPSCs by a presynaptic mechanism, an effect blocked by peptide antagonists and mimicked by inhibition of endogenous peptidases. Dendritic release of peptides, elicited with depolarization achieved by high frequency stimulation of afferents or with current injection into an individual neuron, induced short-term synaptic depression similar to that seen following exogenous peptide application and was prevented by peptide antagonists. Thus, dendritically released peptides depress evoked EPSCs in magnocellular neurons by activating presynaptic OXT and/or VP receptors. Such a retrograde modulatory action on afferent excitation may serve as a feedback mechanism to permit peptidergic neurosecretory neurons to autoregulate their own activity.

Oxytocin retrogradely inhibits evoked, but not miniature, EPSCs in the rat supraoptic nucleus: role of N‐ and P/Q‐type calcium channels

1 We previously reported that oxytocin (OXT), released from the dendrites of magnocellular neurons in the supraoptic nucleus (SON), acts retrogradely on presynaptic terminals to inhibit glutamatergic transmission. Here we test the hypothesis that oxytocin reduces calcium influx into the presynaptic terminal. 2 We used nystatin perforated‐patch recording in vitro to first identify the calcium channels involved in glutamatergic transmission in the SON. ω‐Conotoxin GVIA (ω‐CTx) and ω‐Agatoxin TK (ω‐Aga) both reduced evoked EPSC amplitude, while nicardipine and nickel had no effect. A combination of ω‐CTx and ω‐Aga completely abolished the evoked EPSCs. 3 This depressant effect was accompanied by an increase in the paired pulse ratio with no change in the kinetics of the evoked EPSCs, AMPA currents or postsynaptic cell properties. These results suggest that presynaptic N‐ and P/Q‐type calcium channels mediate glutamate release in the SON while L‐, T‐ and R‐type channels make little or no contribution. 4 Oxytocin‐induced reduction of the evoked EPSC was substantially occluded in the presence of ω‐CTx but only partially in the presence of ω‐Aga. 5 Amastatin, an endopeptidase inhibitor that increases the level of endogenous OXT, also reduced the evoked EPSC. This amastatin effect was also occluded by ω‐CTx and ω‐Aga. 6 Miniature EPSCs, which are independent of extracellular calcium, were unaffected by either ω‐CTx or by OXT, thus further substantiating an action of both compounds on calcium channels. 7 Therefore, dendritically released oxytocin acts mainly via a mechanism involving the N‐type channel, and to a lesser extent the P/Q‐type channel, to decrease excitatory transmission.

Chapter 18 Modulation of synaptic transmission by oxytocin and vasopressin in the supraoptic nucleus

It is now generally accepted that magnocellular neurons of the supraoptic and paraventricular nuclei release the neuropeptides oxytocin and vasopressin from their dendrites. Peptide release from their axon terminals in the posterior pituitary and dendrites differ in dynamics suggesting that they may be independently regulated. The dendritic release of peptide within the supraoptic nucleus (SON) is an important part of its physiological function since the local peptides can regulate the electrical activity of magnocellular neurons (MCNs) which possess receptors for these peptides. This direct postsynaptic action would affect the output of peptide in the neurohypophysis. Another way that these peptides can regulate MCN activity would be to modulate afferent inputs unto themselves. Although the influence of afferent inputs (inhibitory and excitatory) on SON magnocellular neuron physiology has been extensively described in the last decade, a role for these locally released peptides on synaptic physiology of this nucleus has been difficult to show until recently, partly because of the difficulty of performing stable synaptic recordings from these cells in suitable preparations that permit extensive examination. We recently showed that under appropriate conditions, oxytocin acts as a retrograde transmitter in the SON. Oxytocin, released from the dendrites of MCNs, decreased evoked excitatory synaptic transmission by inhibiting glutamate release from the presynaptic terminals. It modulated voltage-dependent calcium channels, mainly N-type and to a lesser extent P/Q-type channels, located on glutamatergic terminals. Although evidence is less conclusive, it is possible that vasopressin has similar actions to reduce excitatory transmission. This synaptic depressant effect of oxytocin and/or vasopressin, released from dendrites, would ensure that MCNs regulate afferent input unto themselves using their own firing rate as a gauge. Alternatively, it may only be a subset of afferent terminals that are sensitive to these peptides, thereby providing a means for the MCNs to selectively filter their afferent inputs. Indeed its specificity is partly proven by our observation that oxytocin does not affect spontaneous glutamate release, or GABA release from inhibitory terminals (Brussaard et al., 1996). Thus, the dendrites of MCNs of the supraoptic nucleus serve a dual role as both recipients of afferent input and regulators of the magnitude of afferent input, allowing them to directly participate in the shaping of their output. This adds to a rapidly growing body of evidence in support of the concept of a two-way communication between presynaptic terminals and postsynaptic dendrites, and shows the potential of this nucleus as a model to study such form of synaptic transmission.

Cholecystokinin and neurotensin inversely modulate excitatory synaptic transmission in the parabrachial nucleus in vitro

Cholecystokinin and neurotensin are present in fibres innervating the parabrachial nucleus and have previously been shown to modulate the flow of visceral afferent information through the parabrachial nucleus to the cortex in the rat. This study examined the effects of cholecystokinin and neurotensin on synaptic transmission in the parabrachial nucleus using a pontine slice preparation and the nystatin perforated-patch recording technique. Stimulation of the ventral, external lateral portion of the parabrachial nucleus elicited glutamate-mediated, excitatory postsynaptic currents in cells recorded in the parabrachial nucleus. Bath application of neurotensin dose-dependently and reversibly enhanced, while cholecystokinin attenuated, the evoked excitatory postsynaptic current. In addition, the frequency of spontaneous, miniature excitatory postsynaptic currents recorded in parabrachial nucleus cells was significantly increased by neurotensin and decreased by cholecystokinin application. Paired-pulse depression was also enhanced and decreased by neurotensin and cholecystokinin, respectively. These synaptic changes induced by neurotensin and cholecystokinin were not accompanied by changes in input resistance of parabrachial nucleus cells over a wide voltage range (although neurotensin reduced an outwardly rectifying conductance at potentials positive to -20 mV), nor did these peptides alter the inward current induced by a brief bath application of the glutamate agonist, alpha-amino-3-hydroxy-methylisoxazole-4-propionate. The neurotensin antagonist, SR48692 (100 microM), completely and reversibly blocked the neurotensin-induced enhancement of the excitatory postsynaptic current. The non-selective cholecystokinin receptor antagonist, proglumide (100 microM), completely and reversibly blocked the cholecystokinin-induced attenuation of the excitatory postsynaptic current. In addition, the selective cholecystokinin-A receptor antagonist, L-364,718 (10 microM), but not the selective cholecystokinin-B receptor antagonist, L-365,260 (100 microM), blocked the effect of cholecystokinin on synaptic transmission. These results suggest that neurotensin and cholecystokinin act at presynaptic neurotensin and cholecystokinin-A receptors, respectively, to modulate excitatory synaptic transmission in the parabrachial nucleus.

Anticonvulsant enaminones depress excitatory synaptic transmission in the rat brain by enhancing extracellular GABA levels

1 Enaminones are a novel group of compounds that have been shown to possess anticonvulsant activity in in vivo animal models of seizures. The cellular mechanism by which these compounds produce their anticonvulsant effects is not yet known. This study examined the effects of enaminones on excitatory synaptic transmission. 2 We studied the effects of 3‐(4′‐chlorophenyl)aminocyclohex‐2‐enone (E118), methyl 4‐(4′‐bromophenyl)aminocyclohex‐3‐en‐6‐methyl‐2‐oxo‐1‐oate (E139) and ethyl 4‐(4′‐hydroxyphenyl)aminocyclohex‐3‐en‐6‐methyl‐2‐oxo‐1‐oate (E169) on isolated evoked, glutamate‐mediated excitatory synaptic responses by recording whole‐cell currents and potentials in cells of the nucleus accumbens (NAc) contained in forebrain slices. 3 The anticonvulsant enaminones (E118 and E139), but not E169, depressed NMDA and non‐NMDA receptor‐mediated synaptic responses. The inhibition of the non‐NMDA response was concentration‐dependent (1.0–100 μM) with a maximal depression of ∼−30%. E118 and E139 had similar potencies (EC50=3.0 and 3.5 μM, respectively) in depressing this response but E139 was more efficacious (Emax=−31.3±3.8%) than E118 (Emax=−22.6±1.6%). 4 The excitatory postsynaptic current (EPSC) depression caused by 10 μM E139 (−27.7±3.8%) was blocked by 1 μM CGP55845 (6.3±8.1%), a potent GABAB receptor antagonist. 5 Pretreatment of slices with γ‐vinylGABA and 1‐(2‐(((diphenylmethylene)imino)oxy)ethyl)‐1,2,5,6‐tetrahydro‐3‐pyridine‐carboxylic acid (NO‐711), an irreversible GABA transaminase (GABA‐T) inhibitor and a GABA reuptake blocker, respectively, like the anticonvulsant enaminones, also caused a depression of the evoked EPSC (−38.1±14.1 and −24.1±8.9%, respectively). In the presence of these compounds, E139 did not cause a further depression of the EPSC. Our data suggest that anticonvulsant enaminones cause EPSC depression by enhancing extracellular GABA levels possibly through the inhibition of either GABA reuptake or GABA‐T enzyme, or both.

17β-Estradiol inhibits outward potassium currents recorded in rat parabrachial nucleus cells in vitro

Evidence is increasingly accumulating in support of a role for the steroid hormone 17beta-estradiol to modify neuronal functions in the mammalian CNS, especially in autonomic centers. In addition to its well known slowly developing and long lasting actions (genomic), estrogen can also rapidly modulate cell signaling events by affecting membrane excitability (non-genomic). Little, however, is known regarding the mechanism(s) by which 17beta-estradiol produces its rapid effects on neuronal membrane excitability. As potassium channels play a crucial role in cell excitability, we hypothesized that 17beta-estradiol caused excitability by modulating potassium flux through the neuronal cell membrane. We tested this hypothesis by examining the effects of 17beta-estradiol on outward potassium currents recorded in cells from the parabrachial nucleus of rats, in vitro. Bath application of 17beta-estradiol (10-100 microM) reversibly reduced voltage-activated outward potassium currents in a concentration-dependent manner. This effect was mimicked by BSA-17beta-estradiol but not mimicked by 17alpha-estradiol and was significantly reduced by ICI 182,780, a selective estrogen receptor antagonist. The inhibitory effect of 17beta-estradiol was dependent on extracellular potassium concentration, with more profound effects observed at lower concentrations. The 17beta-estradiol-induced inhibition of the outward current was blocked by pretreatment with the potassium channel blockers tetraethylammonium and 4-aminopyridine. The time constants of deactivation of tail currents were decreased by 17beta-estradiol over a range of test potentials (-140 to -80 mV). Finally, the inhibitory effect of 17beta-estradiol on the outward potassium currents was blocked following pre-incubation of slices in lavendustin A, a tyrosine kinase inhibitor. Taken together, these results suggest that 17beta-estradiol acts rapidly at an extracellular membrane receptor to reduce tetraethylammonium- and 4-aminopyridine-sensitive outward potassium currents by accelerating the closure of potassium channels. This may be the ionic basis of 17beta-estradiol-induced enhancement of neuronal excitability.

Dopamine depresses glutamatergic synaptic transmission in the rat parabrachial nucleus in vitro.

Nystatin-perforated patch recordings were made from rat parabrachial neurons in an in vitro slice preparation to examine the effect of dopamine on parabrachial cells and on excitatory synaptic transmission in this nucleus. In current clamp mode, dopamine reduced the amplitude of the evoked excitatory postsynaptic potential without significant change in membrane potential. In cells voltage-clamped at -65 mV, dopamine dose dependently and reversibly decreased evoked, pharmacologically isolated, excitatory postsynaptic currents with an EC50 of 31 microM. The reduction in excitatory postsynaptic current was accompanied by an increase in paired pulse ratio (a protocol used to detect presynaptic site of action) with no change in the holding current or in the decay of the evoked excitatory postsynaptic currents. In addition, dopamine altered neither postsynaptic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate-induced currents, nor steady-state current voltage curves. Miniature excitatory postsynaptic current analysis revealed that dopamine caused a rightward shift of the frequency-distribution curve with no change in the amplitude-distribution curve, which is consistent with a presynaptic mechanism. The dopamine-induced attenuation of the excitatory postsynaptic current was almost completely blocked by the D1-like receptor antagonist SCH23390 (10 microM), although the D2-like antagonist sulpiride (10 microM) also partially blocked it. Combined application of both antagonists blocked all dopamine-induced synaptic effects. The synaptic effect of dopamine was mimicked by the D1-like agonist SKF38393 (50 microM), but the D2-1ike agonist quinpirole (50 microM) also had a small effect. Combined application of both agonists did not produce potentiated responses. Dopamines effect on the excitatory postsynaptic current was independent of serotonin, GABA and adenosine receptors, but may have some interactions with adrenergic receptors. These results suggest that dopamine directly modulates excitatory synaptic events in the parabrachial nucleus predominantly via presynaptic D1-like receptors.

Neurohypophysial peptides as retrograde transmitters in the supraoptic nucleus of the rat

A possible role for vasopressin and oxytocin in the physiology of the supraoptic nucleus was investigated using nystatin‐perforated patch recording in acute brain slices from the rat containing the supraoptic nucleus. We observed that exogenously applied oxytocin reduced glutamate‐mediated synaptic transmission by acting at a presynaptic oxytocin receptor. Endogenous oxytocin, released either by afferent excitation (tetanus) or by postsynaptic depolarization of the recorded magnocellular neurone caused a similar reduction of excitatory input and this could be blocked with an oxytocin antagonist. Thus endogenous oxytocin, released from magnocellular dendrites, acts as a retrograde transmitter to reduce afferent excitation. We discuss the possible significance of these results in terms of the physiological role of oxytocin in the intact animal and suggest possible avenues for further experimentation.

Dopamine and adenosine mediate substance P‐induced depression of evoked IPSCs in the rat nucleus accumbens in vitro

The major projection cells of the nucleus accumbens (NAc) are under a strong inhibitory influence from GABAergic afferents and depend on afferent excitation to produce their output. We have earlier reported that substance P (SP), a peptide which is colocalized with GABA in these neurons, depresses excitatory synaptic transmission in this nucleus (Kombian, S.B., Ananthalakshmi, K.V.V., Parvathy, S.S. & Matowe, W.C. (2003) J. Neurophysiol., 89, 728–738). In order to better understand the role of this peptide in the synaptic physiology of the NAc, it is important to determine its effects on inhibitory synaptic responses. Using whole‐cell recording in rat forebrain slices, we show here that SP also depresses evoked inhibitory postsynaptic currents (IPSCs) in the NAc via intermediate neuromodulators. SP caused a partially reversible, dose‐dependent decrease in evoked IPSC amplitude. This effect was present without measurable changes in the holding current, input resistance of recorded cells or decay rate (τ) of IPSCs. It was mimicked by a neurokinin‐1 (NK1) receptor‐selective agonist, [Sar9, Met (O2)11]‐SP, and blocked by an NK1 receptor‐selective antagonist, L 732 138. The SP‐induced IPSC depression was prevented by SCH23390, a dopamine D1‐like receptor antagonist and by 8‐cyclopentyltheophylline, an adenosine A1 receptor blocker. Furthermore, the SP effect was also markedly attenuated by exogenous adenosine, dipyridamole, rolipram and barium. These data show that SP, acting on NK1 receptors, depresses inhibitory synaptic transmission indirectly by enhancing extracellular dopamine and adenosine levels. SP therefore acts in the NAc to modulate both excitatory and inhibitory afferent inputs using the same mechanism(s).

Cholecystokinin activates CCKB receptors to excite cells and depress EPSCs in the rat rostral nucleus accumbens in vitro

The peptide cholecystokinin (CCK) is abundant in the rat nucleus accumbens (NAc). Although it is colocalized with dopamine (DA) in afferent terminals in this region, neurochemical and behavioural reports are equally divided as to whether CCK enhances or diminishes DAs actions in this nucleus. To better understand the role of this peptide in the physiology of the NAc, we examined the effects of CCK on excitatory synaptic transmission and tested whether these are dependent on DA and/or other neuromodulators. Using whole‐cell recording in rat forebrain slices containing the NAc, we show that sulphated CCK octapeptide (CCK‐8S), the endogenously active neuropeptide, consistently depolarized cells and depressed evoked excitatory postsynaptic currents (EPSCs) in the rostral NAc. It caused a reversible, dose‐dependent decrease in evoked EPSC amplitude that was accompanied by an increase in the decay constant of the EPSC but with no apparent change in paired pulse ratio. It was mimicked by unsulphated CCK‐8 (CCK‐8US), a CCKB receptor‐selective agonist, and blocked by LY225910, a CCKB receptor‐selective antagonist. Both CCK‐8S and CCK‐8US induced an inward current with a reversal potential around −90 mV that was accompanied by an increase in input resistance and action potential firing. The CCK‐8S‐induced EPSC depression was slightly reduced in the presence of SCH23390 but not in the presence of sulpiride or 8‐cyclopentyltheophylline. By contrast, it was completely blocked by CGP55845, a potent GABAB receptor‐selective antagonist. These results indicate that CCK excites NAc cells directly while depressing evoked EPSCs indirectly, mainly through the release of GABA.