Glutamate excitation of oxytocin neurones in vitro involves predominantly non-NMDA receptors

Experiments were undertaken to compare effects of the NMDA and non-NMDA receptor antagonists, AP5 (40 μM) and NBQX (10 μM), on glutamate-induced firing in supraoptic oxytocin (OT) and vasopressin (VP) neurones in vitro. In putative OT neurones NBQX caused a significantly greater reduction in firing than AP5, whilst in putative VP neurones both antagonists reduced activity powerfully and to a similar extent. The relatively small effect of AP5 in putative OT neurones was unaffected by the removal of extracellular magnesium. These results suggest that glutamate-induced firing in putative OT neurones is predominantly controlled by non-NMDA receptors.     

Indomethacin prevents the -NAME-induced increase in plasma levels of oxytocin in dehydrated rats

Inhibiting NO synthase (NOS) with N^G-nitro- -arginine methyl ester ( -NAME, 250 μg/5 μl of artificial cerebrospinal fluid (aCSF)) injected intracerebroventricularly (i.c.v.) increased already enhanced levels of oxytocin, but not vasopressin, in conscious adult male Sprague–Dawley rats dehydrated for 24 h. Intracerebroventricular pretreatment with indomethacin (200 μg/5 μl aCSF), an inhibitor of cyclo-oxygenase, but not with losartan (25 μg/5 μl aCSF), an antagonist of angiotensin II (ANG II) AT₁-receptor subtype, nearly prevented the elevation in oxytocin levels after -NAME. Thus, NO inhibits prostaglandin (but not ANG II) mediated the modulatory actions of NO on oxytocin secretion from the hypothalamo-neurohypophysial system (HNS) during water deprivation.     

Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists

Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9–2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3–13 mV and duration 0.5–4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyl-d-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 μM). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-induced depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1–50 μM) induced, in addition to a depolarizing response, the discharge of small (1.5–6.5 mV), brief (7–20 ms) IPSPs which were reversed just below resting membrane potential at −64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs.     

Effects of oxytocin and vasopressin on thoracic sympathetic preganglionic neurones in the cat

When applied by iontophoresis onto single sympathetic preganglionic neurones in the intermediolateral nucleus of segments T₁–T₃ in the cat, oxytocin and vasopressin each had an excitatory effect. This effect consisted of a prolonged (30–300 sec) after-discharge following termination of application. These results indicate that oxytocin and vasopressin each exert excitatory effects on sympathetic preganglionic neurones and support the possibility that they may be chemical mediators of synaptic transmission in the intermediolateral nucleus, perhaps in cardioacceleratory and/or pressor pathways descending from the paraventricular nucleus of the hypothalamus.     

Central interactions between angiotensin II and PGD2 in the regulation of vasopressin and oxytocin secretion in dehydrated rats

Brain-derived angiotensin II (ANG II) and prostaglandins have important roles in the regulation of body fluid and blood pressure homeostasis. In the present studies we investigated the central interactions between these two neurochemical products in regulating the hypothalamo-neurohypophysial system during dehydration. Intracerebroventricular (icv) administration of prostaglandin D₂ (PGD₂; 20 μg/5 μl) to conscious adult male Sprague–Dawley rats deprived of water for 24 h did not alter significantly the already elevated plasma levels of vasopressin or oxytocin. When PGD₂ was administered in combination with losartan, an antagonist of ANG II AT₁-receptor subtype, however, concentrations of both hormones in plasma became further elevated. Icv administration of ANG II (50 ng/5 μl) increased further the enhanced plasma levels of vasopressin and oxytocin, as expected. Pretreatment with indomethacin (200 μg/5 μl; icv), an inhibitor of cyclo-oxygenase, significantly attenuated the ANG II-induced increase in oxytocin secretion only. Independent of the presence of ANG II, however, indomethacin decreased plasma levels of vasopressin, but not oxytocin. These results indicate that a prostaglandin is required for the stimulated release of vasopressin during dehydration and that the elevation of oxytocin secretion in response to ANG II depends largely on activation of cyclo-oxygenase and production of prostaglandins. The oxytocin response to exogenously administered PGD₂, however, can be negatively modulated by a mechanism dependent upon ANG II AT₁ receptors.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 μM) and tyrphostin B42 (100 μM), two PTKs inhibitors, reduced the steady-state barium current (I_Ba). On the other hand, daidzein and genistin (100 μM), two inactive analogues of genistein, had no effect on I_Ba amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant I_Ba, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of ω-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. ω-Conotoxin-MVIIC (3–10 μM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Extracellular potassium, glial and neuronal potentials in the solitary complex of rat brainstem slices

Extracellular K⁺ activities (aK_e) and neuronal and glial membrane potentials were recorded in the nucleus tractus solitarius (NTS) and in the dorsal vagal motor nucleus (DVMN) of rat brainstem slices after orthodromic stimulation of the tractus solitarius (TS). In glial cells, repetitive stimulation of the TS induced depolarizations of up to 30 mV followed by repolarizations which were fitted by exponential curves with a time constant of 1.6–5 s. Similar stimulations induced elevations ofaK_e of up to 8 mM, the recovery of which was fitted by single exponential curves with a time constant ranging between 1.6 and 4 s. These elevations inaK_e were reduced by 75% during blockage of synaptic transmission in low Ca²⁺, high Mg²⁺ solution, and by 24% with application of 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 50 μM). Perfusion with a low Mg²⁺ solution increased theaK_e response to stimulation of the TS, an effect that was reduced by the addition of 2-amino-5-phosphono-valeric acid (AP7, 50 μM) to the bath. No significant change inaK_e and glial potential was seen when superfusing high concentrations of the C-terminal octapeptide of cholecystokinin (CCK₈, 1–5 μM) and C-terminal tetrapeptide (CCK₄, 50–100 μM). The effect of TS stimulations on solitary complex neurons suggests that extracellular K⁺ concentration is increased during synaptic activation of non-NMDA or NMDA ionotropic receptors. Conversely, slow depolarizations elicited by repetitive afferent activity or excitation by CCK agonists develop in neurons in the absence of measurable extracellular K⁺ fluctuations or glial depolarization.     

Action potential-like responses in B 104 cells with low Na channel densities

Action potential generation and Na⁺ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV_1/2 for Na⁺ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na⁺ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowg_K: g_Na ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd²⁺ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na⁺ channels ( < 3/μm² and < 1/μm² in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K⁺ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.     

Vasopressin-induced calcium signaling in cultured hippocampal neurons

We recently demonstrated that the neural peptide vasopressin (AVP) can act as a neurotrophic factor for hippocampal nerve cells in culture. Because the neurotrophic effect of vasopressin is mediated by the V₁ receptor [11], we investigated AVP activation of calcium signaling pathways in cultured hippocampal neurons. Results of this investigation demonstrate that exposure of cultured hippocampal neurons prelabeled with [³H]myo-inositol to vasopressin induced a significant accumulation of [³H]inositol-1-phosphate ([³H]IP₁). The selective V₁ vasopressin receptor agonist, [Phe², Orn²]vasotocin, induced a s significant accumulation of [³H]IP₁ whereas a selective V₂ vasopressin receptor agonist, [deamino¹, d-Arg⁸]-vasopressin, did not. Moreover, V₁ agonist-induced accumulation of [³H]IP₁ was blocked by the selective V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ agonist-induced accumulation of [³H]IP₁ was concentration dependent and exhibited a steep inverted U-shaped curve that included both stimulation and inhibition of [³H]IP₁ accumulation. Time course analysis of V₁ agonist-induced accumulation of [³H]IP₁ revealed significant increase by 20 min which continued to be significantly elevated for 60 min. Investigation of the effect of closely related peptides on [³H]IP₁ accumulation indicated that the vasopressin metabolite peptide AVP_4–9 and oxytocin significantly increased [³H]IP₁ accumulation whereas the vasopressin metabolite peptide AVP_4–8 did not. AVP_4–9 and oxytocin induced [³H]IP₁ accumulation were blocked by the V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ receptor activation was associated with a pronounced rise in intracellular calcium. Results of calcium fluorometry studies indicated that V₁ agonist exposure induced a marked and sustained rise in intracellular calcium that exhibited oscillations. Interestingly, absence of calcium in the extracellular medium abolished both the rise in intracellular calcium and the appearance of oscillations. The loss of the intracellular calcium signal is not due to a failure to induce PIP₂ hydrolysis since activation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V₁ agonist (250 nM) induced a highly significant increase in⁴⁵Ca²⁺ uptake from the extracellular medium within 5 sec of exposure.⁴⁵Ca²⁺ uptake remained significantly greater than basal for 300 sec. The increase in⁴⁵Ca²⁺ uptake was followed by a significant inhibition of uptake by 20 min of exposure. These results indicate that in cultured hippocampal neurons, V₁ vasopressin receptor activation leads to activation of the phosphatidylinositol signaling pathway, uptake of calcium from the extracellular medium and induction of complex intracellular calcium signals. These data provide the first step in delineating the biochemical mechanism that underlies vasopressin-induced neutrophism.     

Positive Feedback of Vasopressin on its Own Release in the Central Nervous System: in vitro Studies

In this paper evidence is shown that synthetic arginine vasopressin (AVP) can evoke marked in vitro release of endogenous immunoreactive AVP (I-AVP) from male rat septal and hypothalamic tissue superfused in vitro. The stimulatory action was dosedependent with a maximal amplification factor of 2.3 when using 14 pg of synthetic AVP as the stimulus. It was highly specific since only AVP was effective and not three closely related substances such as lysine vasopressin, oxytocin and a 4–9 C fragment of AVP. This reproducible effect of AVP required, however, effective concentrations of bacitracin (10^?4 to 10^?5 M) in the superfusion medium to inhibit aminopeptidase(s) capable of inactivating AVP. Lastly, the stimulatory action of AVP on its own release was not blocked by a V₁-receptor antagonist of AVP but was blocked by a V₂-antagonist. It is proposed that this novel and robust positive feedback of AVP on its own release may be involved in the mechanism of memory consolidation of certain behavioral tasks known to be affected by AVP.     

[Arg8]Vasotocin Excites Neurones in the Dorsal Vagal Complex in vitro: Evidence for an Action through Novel Class(es) of CNS Receptors

Using extracellular recordings from brainstem slices in vitro, it was demonstrated that a high proportion (38/56) of neurones in the dorsal vagal complex of dioestrus, virgin female rats exhibit an excitatory response to [Arg⁸]-vasotocin (AVT). Pharmacological characterization suggests that these responses cannot be entirely explained by interaction with either of the currently known classes of central receptors for oxytocin (OT) and vasopressin (V_1a). Comparison of the responses with those to the OT receptor-specific agonist [Thr⁴,Gly⁷]-OT (TGOT), showed that not all neurones that responded to TGOT also responded to AVT (3/27). Furthermore, while the effects of 10^?7 M TGOT could be blocked either by the broad-spectrum antagonist d(CH₂)⁵[d-Tyr(OEt)²,Val⁴,Cit⁸]-vasopressin or by the selective OT receptor antagonist d(CH²)₅[Tyr(Me)²,Thr⁴,Orn⁸,Tyr-NH₂⁹]-vasotocin, these peptides did not completely block the responses to AVT, indicating that AVT is unlikely to act through the central OT receptor. The responses to AVT and [Arg⁸]-vasopressin (AVP) indicated the presence of at least 2 classes of receptor with which these agonists could act. Of 42 neurones tested with both AVP and AVT, none responded to AVP in the absence of a response to AVT, while 7/42 responded to AVT without a response to AVP. This might be explained by AVP acting through only the V₁ receptor, while AVT acts through both the V₁ and its own novel class of receptor. This was substantiated by the fact that two OT/V₁ receptor antagonists, d(CH²)₅[d-Tyr(OEt)²,Val⁴,Cit⁸]-VP and d(CH²)₅|Tyr(Me)²,Tyr-NH₂⁹]-AVP, were unable to block completely all the responses to AVT at a dose which suppressed responses to both AVP and TGOT. These data suggest that, in addition to activation by OT and AVP, neurones in the mammalian central nervous system are able to be excited by AVT, possibly involving its own distinct class of receptor.     

Effects of GABA on spontaneous [Ca]c dynamics and electrical properties of rat adrenal chromaffin cells

A large fraction of rat adrenal chromaffin cells (about 60%) shows spontaneous [Ca²⁺]_c oscillations and spontaneous action potentials. In the present study the effects of γ-aminobutyric acid (GABA) on the spontaneous [Ca²⁺]_c oscillations and electrical properties of rat adrenal chromaffin cells were investigated using Fura-2 [Ca²⁺]_c imaging and patch clamp techniques. GABA inhibited the spontaneous [Ca²⁺]_c oscillations in a reversible manner. The effect of GABA was mimicked by the GABA_A and GABA_C receptor agonist, muscimol, but not by the GABA_B receptor agonist, baclofen. Moreover, the effect was antagonized by the selective GABA_A receptor antagonist, bicuculline. The mode of the inhibition was all-or-none, and the threshold concentration at which the inhibition occurred varied widely (50 μM to over 1 μM) from cell to cell. GABA (100 μM) elicited a transient burst of action potentials of diminished amplitude, which was followed by arrest of action potentials. Further analysis showed that GABA (100 μM) induced inward whole-cell currents in voltage-clamp experiments and produced depolarization and membrane conductance increase in current-clamp experiments. The effects appear to be due to an increase in chloride ion conductance since the degree of GABA-induced depolarization depended on the pipette [Cl⁻]. These results suggest that GABA, acting through GABA_A receptor, may play a role in the physiological regulation of rat adrenal chromaffin cells by directly modifying the discharge of spontaneous action potentials and spontaneous [Ca²⁺]_c oscillations.     

Proconvulsive effect of vasopressin; mediation by a putative V2 receptor subtype in the central nervous system

Subcutaneously (s.c.) administered [Arg⁸]vasopressin (AVP) potentiated seizures induced by intracerebroventricular (i.c.v.) injection of 1.95 mg pilocarpine (a muscarinic cholinergic agonist). A bell-shaped relation between dose and effect was found. I.c.v. pretreatment with a V₁, V₂ or oxytocin receptor antagonist was performed to determine whether and what type of receptor is involved in this proconvulsive effect of vasopressin. For these experiments a higher dose of pilocarpine (2.4 mg i.c.v.) was injected. This caused seizures in a slightly but not significantly higher percentage of the rats. A dose-dependent protective action of the V₂ receptor antagonist d(CH₂)₅[d-Ile²,Ile⁴]AVP (effective doses were 25 and 125 ng) on seizures was found. A reduction was observed in the number of animals that developed tonic-clonic convulsions. Neither the V₁ receptor antagonist d(CH₂)₅[Tyr(Me)²]AVP nor the oxytocin receptor antagonist desGly(NH₂)⁹d(CH₂)₅[Tyr(Me)²Thr⁴]OVT possessed anti-convulsive activity. Subsequently the type of receptor was studied in detail with fragments of AVP with either V₁ or V₂ activity. AVP (with V₁ and V₂ affinity) (1 and 3 μg s.c.) potentiated pilocarpine (1.95 mg) induced seizures. Vasotocin and oxytocin were without effect. Interestingly neither s.c. nor i.c.v. administration of the selective kidney type vasopressin receptor (V₂) agonist dDAVP potentiated pilocarpine induced seizures. Several selective antidiuretic agonists (V₂), such as d[Val⁴]AVP, d[Phe²,Val⁴,d-Arg⁸]vasopressin (3 μg), [Val⁴,d-Arg⁸]vasopressin (3 μg) and d[Val⁴,d-Arg⁸]vasopressin (3 μg) were active. Other selective antidiuretic compounds, such as [Val⁴]AVP, dAVP, d[Tyr(Me)²]AVP and HO[d-Arg⁸]vasopressin (3 μg) did not influence seizures. These results demonstrate that a combination of substitution of aminoacid 4 (Gln) by Val and to a lesser extent deamination and the d-arginine form yield an active molecule, which can potentiate pilocarpine induced seizures and suggest the existence of a V₂ receptor subtype in the brain.     

Anoxia-induced depolarization in CA1 hippocampal neurons: role of Na-dependent mechanisms

We have previously shown that (1) removal of extracellular sodium (Na⁺) reduces the anoxia-induced depolarization in neurons in brain-slice preparations and (2) amiloride, which blocks Na⁺-dependent exchangers, prevents anoxic injury in cultured neocortical neurons. Since anoxia-induced depolarization has been linked to neuronal injury, we have examined in this study the role of Na⁺-dependent exchangers and voltage-gated Na⁺ channels in the maintenance of membrane properties of CA1 neurons at rest and during acute hypoxia. We recorded intracellularly from CA1 neurons in hippocampal slices, monitored V_m and measured input resistance (R_m) with periodic injections of negative current. We found that tetrodotoxin (TTX, 1 μM) hyperpolarized CA1 neurons at rest and significantly attenuated both the rate of depolarization (ΔV_m/dt) and the rate of decline of R_m (ΔR_m/dt) by about 60% during the early phase of hypoxia. The effect of TTX was dose-dependent. Amiloride (1 mM) decreased V_m and increased R_m in the resting condition but changed little the effect of hypoxia on neuronal function. Benzamil and 5-(N-ethyl-N-isopropyl)-2′,4′-amiloride (EIPA), two specific inhibitors of Na⁺ dependent exchangers, were similar to amiloride in their effect. We conclude that neuronal membrane properties are better maintained during anoxia by reducing the activity of TTX-sensitive channels and not by the action of Na⁺-dependent exchangers.     

The formation of cerebrospinal fluid in two amphibians,Rana catesbeiana andRana pipiens

The cerebrospinal fluid (CSF) system of two amphibians,Rana catesbeiana andRana pipiens has been perfused from the lateral ventricle to a site in the subarachnoid space close to the roof of the fourth ventricle. The CSF production rate (V_f) was measured by the dilution method using a perfusion fluid containing [¹⁴C]dextran in artificial CSF. ForRana catesbeiana V_f was 1.43 μl min⁻¹ or 0.2 μl min⁻¹ mg choroid plexus⁻¹ and forRana pipiens V_f was 0.2 μl min⁻¹ or 0.1 μl min⁻¹ mg chorotid plexus⁻¹. These control values declined by 35% and 14% respectively in the second half of the experimental period. Ouabain (5 × 10^−5M) in the perfusion fluid for the second half of the experiment resulted in a much greater reduction in V_f, viz. 65% forRana catesbeiana and 71% forRana pipiens. Acetazolamide (1 × 10⁻⁴M) in the perfusion fluid for the second half of the experiment gave no change in V_f values when compared with control experiments. Mean ventricular volume measured inRana catesbeiana was74.5 ± 4.8 μl.     

Differential effects of a benzodiazepine on synaptic transmissions in rat hippocampal neurons in vitro

The effects of midazolam, one of the most popular benzodiazepines, on synaptic transmissions were compared with intracellular recordings between CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. First, we studied the effects of midazolam on orthodromically evoked spikes, membrane properties and synaptic potentials. Secondly, the effects of a GABA_A receptor agonist, muscimol, were examined on membrane properties to determine whether or not the densities of GABA_A receptors are different between CA1-PCs and DG-GCs. Midazolam (75 μM) markedly depressed orthodromically evoked spikes in CA1-PCs, compared with those in DG-GCs. A GABA_A receptor antagonist, bicuculline (10 μM), almost completely antagonized the depressant effects of midazolam on spike generation in CA1-PCs, whereas it had little effect on midazolam in dentate gyrus granule cells. Midazolam produced either depolarizing or hyperpolarizing effects on resting membrane potentials (V_m) with an input resistance decrease in CA1-PCs, whereas it produced depolarized V_m in DG-GCs. Midazolam significantly increased the amplitude of monosynaptic inhibitory postsynaptic potentials in CA1-PCs, whereas midazolam slightly decreased these in DG-GCs. Midazolam significantly decreased the amplitude of excitatory postsynaptic potentials both in CA1-PCs and DG-GCs. Muscimol (100 μM) produced either depolarizing or hyperpolarizing effects on V_m with an input resistance decrease in CA1-PCs, and it depolarized V_m with an input resistance decrease in DG-GCs. These results demonstrate that midazolam has differential effects on excitatory and inhibitory synaptic transmissions in hippocampal neurons. The mechanism of this difference could be partly due to the different types of GABA_A receptors between CA1-PCs and DG-GCs.     

Profile of neuronal excitation following selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro

The excitatory actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar⁹,Met(O₂)¹¹]substance P (SP) were tested on a sample (n=50) of deep dorsal horn neurones in the isolated and hemisected young rat spinal cord. Superfusion of the NK1R agonist (2 μM) elicited a prolonged membrane depolarisation (6.6±0.5 mV) and an increase in action potential firing in 41/50 (82%) neurones. These [Sar⁹,Met(O₂)¹¹]SP-induced depolarisations were attenuated by the selective NK1R antagonist GR82334 (1 μM). An increased neuronal excitability after [Sar⁹,Met(O₂)¹¹]SP application was indicated by an augmented spike frequency generated in response to long duration, step depolarisations. In order to assess whether a direct excitatory action existed, [Sar⁹,Met(O₂)¹¹]SP was re-tested on a sample of TTX-treated neurones (n=14). The majority (9/14) retained agonist sensitivity although the amplitude of the depolarisation was reduced to 48% of the control value. A sample of neurones (n=7) that responded to the NK1R agonist were morphologically characterised after filling with the intracellular dye, biocytin. Dorsal dendrites that clearly penetrated lamina II and that could receive a direct C-afferent input, were identified in only 2/7 neurones. These electrophysiological and neuroanatomical data demonstrate that deep dorsal horn neurones possess functional NK1Rs. The implications of the existence of these NK1Rs in the context of spinal somatosensory systems and SP is considered.     

[H]Prazosin binding in the intermediolateral cell column and the effects of iontophoresed methoxamine on sympathetic preganglionic neuronal activity in the anaesthetized cat and rat

The autoradiographic localization of [³H]prazosin (α₁-adrenoceptor ligand) binding sites was determined in cat spinal cord sections. High levels of [³H]prazosin binding were found in the intermediolateral cell column (IML) at thoracic and lumbar levels. The iontophoresis of theα₁-adrenoceptor agonist methoxamine onto sympathetic preganglionic neurones (SPNs) in anaesthetized cats and rats caused excitation of 8 cat SPNs and 13 rat SPNs. These results suggest an excitatory role for some of the catecholaminergic of the IML.     

Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD67 protein

γ-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD₆₅ and GAD₆₇. The contribution of these isoforms to GABA synthesis flux (V_GAD) is not known quantitatively. In the present study we compared V_GAD in cortex of control and vigabatrin-treated rats under α-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD₆₅ and GAD₆₇) measured by enzymatic assay and quantitative immunoblotting. V_GAD was determined by re-analysis of ¹³C NMR data obtained ex vivo and in vivo during infusions of [1-¹³C]glucose using an extension of a model of glutamate–glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. V_GAD was significantly lower in vigabatrin-treated rats (0.030–0.05 μmol/min per g, P<0.003) compared to the non-treated control group (0.10–0.15 μmol/min per g). The 67–70% decrease in V_GAD was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44±15% decrease in GAD₆₇ protein (from 0.63±0.10 to 0.35±0.08 μg protein/mg tissue, P<0.05); GAD₆₅ protein was unchanged. The reduction in GAD₆₇ protein could account for a maximum of 65% of the decrease in V_GAD in vigabatrin-treated animals suggesting that inhibition of GAD₆₅ must have also occurred in these experiments, although product inhibition of GAD₆₇ by increased GABA could play a role. GAD₆₇ could account for 56–85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.     

Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones

Intracellular free calcium concentration ([Ca²⁺]_i) was measured in cultured explants of myenteric plexus neurones by using the fluorescent calcium indicator Indol in combination with patch-clamp techniques. The basal [Ca²⁺]_i was 94 nM and spontaneous oscillations in the internal free calcium concentration were recorded. These oscillations were associated with bursts of action potentials triggered by spontaneous nicotinic excitatory synaptic potentials. Under voltage clamp conditions, application of the selective nicotinic agonist m-hydroxyphenylpropyltri-methylammonium iodide (10 μM) induced an inward current and increased the intracellular free calcium concentration. We conclude that cholinergic synaptic excitatory activity provide a regular calcium entry in myenteric neurone and suggest that the nicotinic channel might be significantly permeable to calcium.