Inhibition of synaptic transmission and epileptiform activity in central neurones by fluspirilene

1. Recent studies have shown that fluspirilene, a dopamine D₂ receptor antagonist which is a long-acting neuroleptic useful in the maintenance therapy of schizophrenic patients, also displays Ca²⁺ channel blocking activity. In the present study, we have investigated the effect of fluspirilene on synaptic transmission and epileptiform activity induced in slices of hippocampus and amygdala.
2. Fluspirilene reversibly suppressed the field excitatory postsynaptic potential (f-e.p.s.p) in a concentration-dependent manner in the area CAl of the hippocampus without affecting the size and shape of fibre volley. Fluspirilene also inhibited the intracellularly recorded e.p.s.p. in amygdala neurones without affecting the resting membrane potential or neuronal input resistance.
3. Fluspirilene increased the ratio of paired-pulse facilitation suggesting a presynaptic mode of action.
4. Epileptiform activity induced in the disinhibited slices was suppessed by fluspirilene in a concentration-dependent manner. This antiepileptic effect was occluded in slices pretreated with the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA).
5. It is concluded that fluspirilene-induced synaptic inhibition is probably due to a reduction in presynaptic Ca²⁺ currents. In clinical trials, the low incidence of seizures provoked by fluspirilene might be related to its intrinsic ability to inhibit synaptic transmission and epileptiform activity.

     

Presynaptic inhibition of synaptic transmission in the rat hippocampus by activation of muscarinic receptors: involvement of presynaptic calcium influx

1. Modulation of presynaptic voltage-dependent calcium channels (VDCCs) by muscarinic receptors at the CA3–CA1 synapse of rat hippocampal slices was investigated by using the calcium indicator fura-2. Stimulation-evoked presynaptic calcium transients ([Ca_pre]_t) and field excitatory postsynaptic potentials (fe.p.s.ps) were simultaneously recorded. The relationship between presynaptic calcium influx and synaptic transmission was studied.
2. Activation of muscarinic receptors inhibited [Ca_pre]_t, thereby reducing synaptic transmission. Carbachol (CCh, 10 μM) inhibited [Ca_pre]_t by 35% and reduced fe.p.s.p. by 85%. The inhibition was completely antagonized by 1 μM atropine. An approximate 4^th power relationship was found between presynaptic calcium influx and postsynaptic responses.
3. Application of the N-type VDCC-blocking peptide toxin ω-conotoxin GVIA (ω-CTx GVIA, 1 μM) inhibited [Ca_pre]_t and fe.p.s.ps by 21% and 49%, respectively, while the P/Q-type VDCC blocker ω-agatoxin IVA (ω-Aga IVA, 1 μM) reduced [Ca_pre]_t and fe.p.s.ps by 35% and 85%, respectively.
4. Muscarinic receptor activation differentially inhibited distinct presynaptic VDCCs. ω-CTx GVIA-sensitive calcium channels were inhibited by muscarinic receptors, while ω-Aga IVA-sensitive channels were not. The percentage inhibition of ω-CTx GVIA-sensitive [Ca_pre]_t was about 63%.
5. Muscarinic receptors inhibited presynaptic VDCCs in a way similar to adenosine (Ad) receptors. The percentage inhibition of ω-CTx GVIA-sensitive [Ca_pre]_t by Ad (100 μM) was about 59%. There was no significant inhibition of ω-Aga IVA-sensitive channels by Ad. The inhibitions of [Ca_pre]_t by CCh and Ad were mutually occlusive.
6. These results indicate that inhibition of synaptic transmission by muscarinic receptors is mainly the consequence of a reduction of the [Ca_pre]_t due to inhibition of presynaptic VDCCs.

     

Effect of chronic ethanol treatment in vivo on excitability in mouse cortical neurones in vitro

1. The effects of cessation of chronic ethanol ingestion on seizure activity in vivo and on the characteristics of the evoked synaptic potentials in cortical neurones in vitro have been investigated in mice. Withdrawal from chronic ethanol treatment increased handling seizure ratings in mice between 4 and 16 h post-withdrawal. This ethanol-induced increase in seizure rating was unaffected by carbamazepine (30 mg kg⁻¹) but significantly reduced at a higher concentration (130 mg kg⁻¹).
2. Intracellular recordings were made from cortical layer II neurones in vitro from control mice and from mice following chronic ethanol ingestion. Evoked synaptic potentials were generated in these neurones through intralaminar stimulation.
3. Neurones from control mice displayed an evoked potential consisting of a fast excitatory postsynaptic potential (e.p.s.p.) mediated by AMPA-type glutamate receptors and an inhibitory postsynaptic potential (i.p.s.p.) mediated via GABA_A receptors. Application of pentylenetetrazole (PTZ) or bicuculline onto these neurones inhibited the i.p.s.p., caused a large increase in both the amplitude and duration of the e.p.s.p. and initiated spontaneous excitatory activity. The resulting large evoked e.p.s.p. was mediated via both NMDA- and AMPA-type glutamate receptors.
4. Most neurones (77%) from ethanol treated mice displayed an evoked potential which comprised a large e.p.s.p. and no i.p.s.p. The e.p.s.p. consisted of several distinct components and in addition these neurones displayed spontaneous paroxysmal depolarizing shifts. This multi-component e.p.s.p. was mediated through both NMDA- and AMPA-type glutamate receptors. A population (23%) of neurones from ethanol treated mice exhibited evoked potentials which possessed both inhibitory and excitatory components and these neurones were effectively identical to those obtained from control mice.
5. Carbamazepine reduced the duration of the e.p.s.p. in neurones from ethanol treated mice and in PTZ-treated control neurones.
6. Prolonged ethanol ingestion is known to create a neurochemical imbalance in cortical neurones resulting in abnormal neurotransmission. The present study highlights the functional consequences that arise as a result of these neurochemical changes leading to over-excitation of neurones and pronounced epileptiform activity.

     

Plasticity of autonomic nerves: Differential effects of long-term guanethidine sympathectomy on the sensory innervation of the rat uterus during maturation

The sensory nerves, containing substance P and calcitonin gene-related peptide, and noradrenaline-containing sympathetic nerves of the rat uterus were analyzed following long-term sympathectomy with guanethidine in prepubertal (four weeks), young adult (eight weeks) and fully adult animals (18 weeks). Immunohistochemical and histochemical methods were used in association with nerve density measurements and biochemical assays. The main findings were as follows:

1. (1) long-term guanethidine treatment completely abolished the noradrenergic innervation of the uterine horn and parametrial tissue and markedly reduced the tissue levels of noradrenaline in both regions at the three ages analysed;

2. (2) in the uterine hom guanethidine treatment had no effect on the tissue levels of either calcitonin gene-related peptide or substance P or on the density of calcitonin gene-related peptide-containing nerves, at any of the three ages studied;

3. (3) in the parametrial tissue increased levels of calcitonin gene-related peptide were observed at 8 and 18 weeks of age, together with a significant increase in the density of calcitonin gene-related peptide-containing nerves.

Substance P levels showed a transient increase in this tissue at eight weeks. In conclusion, long-term sympathectomy with guanethidine resulted in an increase in calcitonin gene-related peptide and substance P in sensory nerves in the parametrial tissue, but not in the uterine horn. The changes in the parametrial tissue only occurred after puberty. It is suggested that sensory nerves in the uterine horn may be less responsive to sympathetic denervation since loss of sympathetic nerves occurs as part of a normal physiological process during pregnancy in this region.

Velocity invariance of preferred axis of motion for single spot stimuli in simple cells of cat striate cortex

In slices from the visual cortex of kittens maintained in vitro, long-term potentiation of synaptic transmission following high frequency stimuli (10 Hz, 2 min) delivered at low to medium stimulus intensities (80 to 200 A), is accompanied by changes of certain electrophysiological measures recorded intracellularly, such as long-lasting depolarization of membrane potential and decreased threshold to elicitation of an action potential. These parameters have never before been shown to be altered following high frequency stimulation in other systems widely used in studying synaptic plasticity, such as in hippocampal neurons. Another important difference between results from these two systems is that the amplitude of the excitatory post-synaptic potential is enhanced after high frequency stimulation in hippocampal neurons, whereas in striate cortex from young kittens, we observed a decrease. We demonstrate here that this decrease can be reversed to show enhancement from the original amplitude, upon clamp of membrane potential back to the voltage observed prior to stimulation. Thus, what appears to be long-term depression of synaptic transmission, as recorded extracellularly and represented by diminished flow of synaptic current, can be reversed by stepping membrane voltage back to the pre-high frequency stimulation level, to produce responses that then become consistent with long-term potentiation.     

Demonstration of Cerebral Plasticity by Intra-Operative Neurophysiological Monitoring: Report of an Uncommon Case

Summary  It has been postulated long ago that “eloquent” areas shift their location in patients with arteriovenous malformations (AVM). Obviously the “motor region” in not located in the precentral gyrus in a patient with an AVM in the “motor region”.  We report on the case of a 15-year old boy with an AVM in the left sensorimotor cortex, in whom intra-operative mapping showed an inexcitability of the precentral gyrus, while stimulation of the cortex anterior to the primary motor cortex elicited motor responses. This indicates that motor function was translocated from the primary to the supplementary motor cortex. Surgery was performed under general anaesthesia. Neurophysiological monitoring was performed throughout surgery. The central sulcus was identified by phase reversal of the somatosensory evoked potentials. The motor cortex was mapped by direct high-frequency (500 Hz) monopolar anodal stimulation.  In the patient herein reported, stimulation of the “anatomically” defined primary motor cortex induced no motor response, as expected. Motor response was elicited only by stimulation of the cortex anterior to the precentral gyrus. There was no postoperative deterioration of motor function. These observations indicate that the precentral gyrus was functionally “useless”. The motor region was relocated into more rostral areas in the supplementary motor cortex. This translocation of function in the presence of an AVM indicates cerebral plasticity.     

Compartmental changes in expression of c-Fos and FosB proteins in intact and dopamine-depleted striatum after chronic apomorphine treatment

Chronic administration of dopaminergic agonists to rats with unilateral 6-OH-dopamine (6-OHDA) lesions of nigrostriatal pathway produces behavioral sensitization to subsequent agonist challenges and may serve as a model for DOPA-induced dyskinesias. In order to understand striatal mechanisms behind this long-term behavioral change we examined striatal c-Fos and FosB immunoreactivity induced by apomorphine challenge (5 mg/kg, s.c.) after 3 days of withdrawal following a 2-week administration (5 mg/kg, b.i.d., s.c.) both in intact and 6-OHDA-lesioned animals. In intact rats, c-Fos induction by acute apomorphine exposure showed a striosomal pattern, whereas FosB immunopositivity was diffusely distributed. Following chronic administration, FosB induction turned to a clear striosome dominant pattern similar to c-Fos expression. In denervated striatum, expression of both proteins was profoundly augmented in a homogeneous pattern after a single dose of apomorphine. A distinct striosomal patterning appeared after chronic apomorphine administration in ventromedial part of the denervated striatum with a down-regulation in the matrix and relative enhancement in striosomes. These results suggest that compartmental reorganization of striatal neuronal activity may play a role in long-term behavioral changes induced by chronic dopaminergic treatments both under normal and dopamine-depleted conditions.     

Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses

We describe a method to label gamma-aminobutyric acid (GABA)A receptors on the surface of living hippocampal neurons in primary culture, and we compare the distribution of receptors with that of active synapses. To visualize GABAA receptors, the affinity-purified antibody beta3(1-13), recognizing the extracellular N-termini of the GABAA receptor beta2- and beta3-subunits, was used in combination with fluorescent secondary antibodies. The beta2- and beta3-subunits belong to the predominant GABAA receptor subunits in the hippocampus. As expected for aggregates of GABAA receptors in the somato-dendritic plasma membrane, a patchy staining pattern similar to that seen by labelling neurons after fixation was obtained. An antiserum recognizing an intracellular epitope of GABAA receptor beta3-subunits did not label the receptors in living neurons. Whole-cell recordings of GABA-evoked Cl - currents were not affected after decorating GABAA receptors with antibody beta3(1-13). Combining the staining of GABAA receptors with the labelling of active presynaptic terminals with the fluorescent dyes FM1-43 or FM4-64, consistently resulted in the detection of GABAA receptor clusters that were not located at active synapses. These amounted to approximately 50% of all labelled GABAA receptor clusters. GABAA receptor clusters that were not associated with active presynaptic terminals partially colocalized with the synaptic vesicle marker protein sv2, while another fraction had no presynaptic counterpart at all. These findings suggest the presence of presynaptically silent GABAergic synapses in cultured hippocampal neurons. They also indicate that for the maintenance of GABAA receptor aggregates, the release of GABA from an opposing active terminal is not essential.     

Prior short-term synaptic disinhibition facilitates long-term potentiation and suppresses long-term depression at CA1 hippocampal synapses

Long-term potentiation (LTP) and long-term depression (LTD) are two main forms of activity-dependent synaptic plasticity that have been extensively studied as the putative mechanisms underlying learning and memory. Current studies have demonstrated that prior synaptic activity can influence the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. Here, we show that prior short-term synaptic disinhibition induced by type A gamma-aminobutyric acid (GABA) receptor antagonist picrotoxin exhibited a facilitation of LTP induction and an inhibition of LTD induction. This effect lasted between 10 and 30 min after washout of picrotoxin and was specifically inhibited by the L-type voltage-operated Ca2+ channel (VOCC) blocker nimodipine, but not by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphopentanoic acid (D-APV). Moreover, this picrotoxin-induced priming effect was mimicked by forskolin, an activator of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), and was blocked by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536) and the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). It was also found that following picrotoxin application, CA1 neurons have a higher probability of synchronous discharge in response to a population of excitatory postsynaptic potential (EPSP) of fixed slope (EPSP/spike potentiation). However, picrotoxin treatment did not significantly affect paired-pulse facilitation (PPF). These findings suggest that a brief of GABAergic disinhibition can act as a priming stimulus for the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. The increase in Ca2+ influx through L-type VOCCs in turn triggering a cAMP/PKA signalling pathway is a possible molecular mechanism underlying this priming effect.     

Platelet-activating factor receptor is not required for long-term potentiation in the hippocampal CA1 region

From pharmacological studies, platelet-activating factor (PAF) has been proposed as a retrograde messenger for long-term potentiation (LTP) in the hippocampal CA1 region. We re-examined a possible contribution of PAF to LTP with a more specific approach using mice deficient in the PAF receptor. The PAF receptor-deficient mice exhibited normal LTP and showed no obvious abnormality in excitatory synaptic transmission. We also performed pharmacological experiments on the wild-type mice. Two structurally different antagonists of PAF receptors had no effects on LTP. Furthermore, the application of PAF itself caused no detectable changes in excitatory synaptic transmission. Thus, we conclude that the PAF receptor is not required for LTP in the CA1 region. Introduction