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Postnatal development of identified medium-sized caudate spiny neurons in the cat.

The morphology of intracellularly recorded neurons in the cat caudate nucleus (Cd) was studied during postnatal development. After intracellular recording of evoked responses in these neurons, horseradish peroxidase (HRP) was injected iontophoretically through the recording micropipette. Fifty-eight Cd neurons in cats ranging from 6 days of age through adulthood were identified morphologically. All of the recovered Cd cells were medium-sized spiny neurons. The basic somatodendritic morphology of these neurons was evident in the youngest kittens. The most striking morphological change was the postnatal formation of an extensive local axonal collateral plexus. The development of these local axonal collaterals was also quantified with computer assistance in medium-sized Cd spiny neurons selected from silver-impregnated material. This analysis showed that the major development of the branches of this local plexus occurred between birth and 3-4 months of postnatal age. Data from both the HRP-filled and silver-stained axons indicated that the postnatal growth of the local axonal collaterals of the medium spiny cells was associated with the elaboration and increasing prevalence of evoked inhibitory postsynaptic potentials in Cd neurons.

Age Factors↗

Late development of intrinsic excitation in the rat neostriatum: an in vitro study.

Functional maturation of intrinsic circuitry in the neostriatum was studied by intracellular recording and intracellular staining with Lucifer yellow in slices obtained from rat pups at postnatal days (P)1-20 and from adult rats. The most striking observation was that intrastriatal stimulation elicited predominantly inhibitory responses in slices obtained from animals of P1-6. In contrast, intrastriatally evoked responses in slices obtained after P10 were predominantly excitatory. The inhibitory postsynaptic potentials (IPSPs) recorded in slices obtained from pups were blocked by bicuculline (50 microM) and exhibited a reversal potential at about -60 mV which shifted in depolarizing direction when intracellular Cl- activity increased. Thus, these IPSPs correspond to IPSPs observed in adult animals. It is concluded that maturation of excitatory synapses is the main change during postnatal development. The changes of postsynaptic potentials were paralleled by the appearance of spines on dendrites around P7 as revealed by intracellular staining. The apparent input resistances and time constants of young neurons were very high and responses to large current injections were often distorted by humps which could not be observed in adult neurons. Only young neurons responded with bursts to synaptic activation in the presence of bicuculline (50 microM). It appears that dendritic conductances have a stronger influence on somatic discharge in the electrotonically compact young neurons than in adult neurons.

Animals↗

Postnatal development of the electrical activity of rat nigrostriatal dopaminergic neurons.

Extra- and intracellular recordings were obtained in vivo from dopaminergic nigrostriatal neurons in rat pups ranging in age from postnatal day (PD) 1 to PD28, and in adult rats. Neurons from PD1-3 rats were active at very low rates in a random pattern, rarely showed bursting activity, and often exhibited long periods of up to several minutes of silence. Spontaneous spikes were of relatively low amplitude and long duration. The mean firing rate increased and became more regular over time, and short bursts consisting of only 2 spikes were observed. By the second postnatal week, the initial segment component of the spontaneous spike resembled that seen in adults, but the somadendritic component was still relatively small, and there was often a very marked temporal delay between the two. Near the end of the second postnatal week, neurons exhibited a transient phase of pacemaker-like activity. Mean firing rates continued to increase with time, as did the incidence and complexity of bursting activity. The spontaneous firing rate, pattern and spike morphology approached adult values by the fourth postnatal week. Antidromic responses from neostriatum were obtained as early as PD1, and consisted of a significantly greater proportion of full initial segment-soma dendritic spikes compared to nigrostriatal neurons from adult rats. There was usually a long delay between the initial segment and somadendritic components of the spike. Mean antidromic latency and mean antidromic threshold did not vary significantly from PD1-3 to adults. Axonal conduction velocity reached maximal adult values by PD16-21. Neostriatal-evoked orthodromic responses consisted principally of a poststimulus inhibition whose duration decreased from PD1 through adulthood. Pure excitatory responses were very rarely observed at any age. Intracellular recordings from PD2, PD3 and PD5 rats revealed striatal-evoked inhibitory postsynaptic potentials in non-dopaminergic nigral neurons with a mean onset latency (9.8 +/- 3.8 ms) which did not differ from that previously reported for adult rats.

Action Potentials↗

Development of GABAergic synaptic connections in vivo and in cultures from the rat superior colliculus.

Synaptic activity in the superficial (i.e. visual) layer of the superior colliculus was investigated with intracellular microelectrodes using a preparation of the isolated superfused tectum from neonatal rat. It was found that by postnatal day 9 (i.e. before eye opening) the majority of neurons in the superficial gray layer (SGS, stratum griseum superficiale) were already capable of generating Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) in response to intracollicular stimulation. Properties and development of GABAergic synaptic connections were further characterized in a dissociated cell culture from the SGS. The cultures were prepared from E21 rat embryos and studied between 1 and 38 days in vitro (DIV). gamma-[3H]aminobutyric acid ([3H]GABA) uptake served to identify GABAergic neurons and to estimate their relative density. Axon terminals were labeled by indirect immunostaining for glutamic acid decarboxylase (GAD) and examined with light (LM) and electron microscopy (EM). Responsiveness to exogenous and endogenous GABA was investigated by recording ionic currents with patch clamp techniques. [3H]GABA uptake-positive neurons constituted about 40% of the whole cellular population dissociated from the SGS of E21 rats. After 2 weeks in culture, [3H]GABA uptake was observed in 45-60% of the cells with neuronal features. The relative number of GAD-immunoreactive neuronal perikarya ranged from 28 to 39%, after 2 weeks in vitro. Responsiveness to exogenous GABA was found in all freshly plated neurons. Release of GABA could be demonstrated after 2 DIV by recording spontaneous bicuculline-sensitive Cl- currents. These currents had the characteristics of GABAA receptor-mediated synaptic currents. However, even as late as DIV 6, very few vesicle-containing axonal terminals apposing postsynaptic specializations were revealed with EM. GAD-labeled puncta became clearly visible only after DIV 10-12. Between DIV 14 and 21, the intensity of immunostaining and the density of GAD-labeled synaptic contacts increased, reaching a maximum around DIV 28. GAD-positive puncta covered both neurons and non-neuronal cells. At the level of EM, GAD-positive terminals were shown to establish synaptic contacts with neuronal somata and processes, forming in the majority of cases (22 out of 32 stained terminals) symmetrical contacts. It is concluded that in the SGS of the rat superior colliculus GABAergic neurons and GABAA receptors are present before birth. In dissociated cell cultures ionic currents can be generated in response to endogenous GABA before axonal terminals of GABAergic neurons fully mature. Finally, our experiments show that visual activity is not a prerequisite for the formation of GABAergic synapses between neurons of the SGS.

Action Potentials↗

GABAB receptors and their significance in mammalian pharmacology.

Much progress has been made in GABAB receptor pharmacology since the discovery of this receptor in 1980. Selective agonists and antagonists have been developed and a functional role for the receptor as a mediator of slow inhibitory postsynaptic potentials in many brain regions has emerged. In this article, Norman Bowery discusses the evidence for heterogeneity of GABAB receptors, their possible physiological and pathological roles and the therapeutic potential of GABAB receptor agonists and antagonists.

Animals↗

Dendritic spikes in normal spinal motoneurons of cats.

A spike of about 2 ms in duration and 3-8 mV in amplitude (D-spike) was produced by peripheral nerve (biceps semitendinosus, tibial and sciatic nerves) and dorsal root (L7 and S1) stimulation in normal spinal motoneurons of cats in a decerebrate, non-anesthetized state. The strength of the hyperpolarizing current to abolish the D-spike was always greater than that to abolish the IS-spike. The D-spike triggered the IS-spike at a level more depolarized than that at which the M-spike triggered the IS-spike. An inhibitory postsynaptic potential, which inhibited the IS-spike, could not inhibit the D-spike. Two different D-spikes, as distinguished by a difference in threshold, could occur in the same motoneuron. These results suggested that the D-spike was dendritic in origin.

Action Potentials↗

Age-related alterations in the properties of hippocampal pyramidal neurons among rat strains.

We compared age-related alterations in the electrophysiological and pharmacological properties of CA1 hippocampal pyramidal neurons in three strains of rats (Sprague-Dawley, Fisher 344, and Wistar) at 3-4 and 25-32 months of age, using the in vitro slice preparation. The most consistent age-related alterations in the properties of rat hippocampal neurons were: a decrease in membrane excitability, a decrease in the amplitude and duration of inhibitory postsynaptic potentials and a decreased sensitivity to the effect of the cholinergic agonist carbachol. In contrast, no consistent alterations in calcium-dependent events were observed in these strains of rats. The age-related changes in the duration of the afterhyperpolarizations (AHPs) were different (and even opposite) depending on the strain studied. Our results show that age-related changes observed in a given strain are not necessarily present in all strains of the same species.

Aging↗

Mechanosensory interneurons (MSIs) in the crayfish 6th abdominal ganglion are inhibited by activation of other MSIs.

1. Identified mechanosensory interneurons (MSIs) in the 6th abdominal ganglion of the crayfish Procambarus clarkii have been shown to inhibit other projecting MSIs. 2. Interneurons sensitive to water-current stimulation of the tailfan, and which inhibited the tactile response of other MSIs when activated by depolarizing currents, were identified by iontophoresis of fluorescent dye. 3. Ten inhibitory interneurons have been identified, including both non-adapting, directional cells and phasic "touch" cells. 4. Inhibition triggered by activation of the identified cells was not widespread among fibers in the connectives. 5. Inhibition recorded intracellularly was mediated by compound inhibitory postsynaptic potentials of long duration (300-400 msec) and latencies of 13-15 msec, and therefore was apparently polysynaptic. 6. Depolarization and/or activity in MSIs, which modulates the stimulus response characteristics of related cells is a possible mechanism for contrast enhancement among directional or frequency-selective interneurons.

Abdomen↗

A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus.

In response to orthodromic stimulation, granule neurons of the dentate gyrus of the rat hippocampal slice display a late hyperpolarization (LH) with properties which clearly distinguish it from the conventional gamma-aminobutyric ac id (GABA)-mediated chloride (Cl) dependent inhibitory postsynaptic potential (IPSP). The LH is not reduced by antagonists of GABA-mediated Cl conductances such as picrotoxin, and it is probably dependent on increased potassium conductance rather than in increased Cl conductance. The LH can be elicited by orthodromic stimulation which does not elicit a burst of action potentials and, in fact, the LH has been observed following stimulation which did not produce a depolarization detectable at the resting membrane potential. It is suggested that the increased potassium conductance of the LH may be calcium-dependent, or it may be directly elicited by a neurotransmitter.

Animals↗

Pitrazepin, a novel GABAA antagonist.

Pitrazepin is a potent new GABA antagonist which differs from bicuculline in its chemical structure and its interaction with both [3H]muscimol and [3H]flunitrazepam binding sites, whereby the potency of pitrazepin in displacing [3H]muscimol exceeds that of bicuculline by at least a factor of 10. In physiological experiments, blockade of synaptically released GABA by pitrazepin was shown to reduce inhibitory postsynaptic potentials and resulted in the onset of bursting activity which persisted for hours following drug application. The effect of pitrazepin was not tissue specific since it induced a bursting discharge pattern in cultures derived from hippocampus and hypothalamus. Bursting activity was abolished by GABA, baclofen and pentobarbital, but only weakly reduced by midazolam. Pitrazepin also antagonized the action of exogenous GABA, but failed to influence the action of baclofen, an effect which was associated with a decreased membrane input resistance and which was blocked by barium. These results indicate that pitrazepin selectively interacts with the putative GABAA site, thereby antagonizing the chloride-dependent GABA response.

Animals↗

D-baclofen does not antagonize the actions of L-baclofen on rat neocortical neurons in vitro.

The ability of D-baclofen to antagonize the actions of L-baclofen on rat neocortical neurons was investigated. Intracellular recordings were made from neurons in cortical layers 2 and 3 in an in vitro slice preparation. Baclofen stereoisomers were applied at known concentrations in the superfusion medium. At a concentration of 3 microM, L-baclofen produced approximately 70% depressions of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) that were evoked by stimulation of superficial cortical layers. L-baclofen also hyperpolarized neocortical neurons. These hyperpolarizations were accompanied by decreases in neuronal input resistance and in direct excitability. We have shown previously that these latter effects are secondary to the action of baclofen to increase the potassium conductance of neocortical neurons. D-baclofen, at concentrations of 1-100 microM, did not antagonize depressions by L-baclofen of EPSPs and IPSPs nor the action of L-baclofen to increase the potassium conductance of neocortical neurons. At concentrations of 50-100 microM, D-baclofen produced 20-30% effects when applied alone, thus suggesting that these concentrations of D-baclofen produced a significant degree of receptor occupancy. Our results demonstrate that D-baclofen is not an antagonist or high affinity partial agonist at the receptors through which baclofen exerts its effects on single neurons in the rat neocortex.

Animals↗

Novel synaptic responses mediated by dopamine and gamma-aminobutyric acid in neuroendocrine cells of the intermediate pituitary.

Neuroendocrine cells of the pituitary pars intermedia contain pro-opiomelanocortin and secrete mainly alpha-melanocyte-stimulating hormone and, in lesser amounts, corticotropin and endorphin. We have recorded intracellularly from rat pars intermedia cells in vitro and analysed the synaptic inputs to these cells. Electrical stimulation of the pituitary stalk evoked a biphasic postsynaptic response in pars intermedia cells. An initial inhibitory postsynaptic potential was most likely mediated by gamma-aminobutyric acid (GABA) because there was a conductance increase, and it was blocked by bicuculline, a GABA antagonist. A more prolonged hyperpolarization was also observed which could last 10-30 s and was associated with a decreased whole cell conductance. This late hyperpolarization was blocked by the dopamine antagonists, chlorpromazine and domperidone, and was therefore most likely dopamine-mediated.

Animals↗

Picrotoxin induced epileptiform activity in amygdaloid neurons.

The effects of the epileptogenic agent, picrotoxin, on inhibitory postsynaptic potentials (IPSPs) were studied in the amygdala in vitro slice preparation. Picrotoxin was superfused onto the tissue and intracellular recordings were obtained from basolateral amygdaloid neurons (BLANs). Stimulation of the stria terminalis pathway evoked an excitatory postsynaptic potential (EPSP)--IPSP sequence. Depolarization of the membrane potential increased the amplitude of the IPSP, whereas hyperpolarization of the membrane potential decreased the amplitude of the IPSP. The IPSP reversed polarity at -74 mV. Application of picrotoxin reduced the IPSP and resulted in the development of paroxysmal depolarizing shifts. Picrotoxin itself had no apparent effect on the resting membrane potential or input resistance. These results indicate that epileptiform activity induced by picrotoxin in the basolateral amygdaloid neuron is of synaptic origin.

Action Potentials↗

Effects of chloralose-urethane anesthesia on single-axon reciprocal Ia IPSPs in the cat.

Reciprocal Ia inhibitory postsynaptic potentials (IPSPs) generated by single afferents have been recorded with signal averaging in unanesthetized ischemic-decapitate cats for comparison with measurements previously obtained from preparations anesthetized with a mixture of chloralose and urethane. The results are similar to those which we obtained recently for single-axon recurrent IPSPs. Together, the studies show that chloralose-urethane anesthesia has a depressant effect on two widely studied circuits in the mammalian spinal cord.

Anesthesia↗

Outward chloride/cation co-transport in mammalian cortical neurons.

The mechanism underlying outward chloride transport in guinea pig cingulate cortical neurons of in vitro slices was characterized with respect to its pharmacological antagonists and anion selectivity, and the nature of other ion movements coupled to Cl- transport. Changes in intracellular Cl- concentration, following iontophoresis of Cl- from KCl-filled intracellular recording electrodes, were estimated from changes in the amplitude of GABAergic, Cl(-)-mediated inhibitory postsynaptic potentials (IPSPs). The rate of outward Cl- transport was found to be reduced by bumetanide but not by SITS. SCN-, but not NO3-, was found to be actively transported. Increasing the extracellular K+ concentration ([K+]o) from 2.5 to 10 mM was found to inhibit Cl- extrusion. These data suggest that active Cl- extrusion from mammalian cortical neurons is mediated by an outwardly directed chloride/cation cotransport mechanism. Inhibition of this process by elevated [K+]o may be important in epilepsy.

Animals↗

Microiontophoresis of flurazepam on inspiratory and postinspiratory neurons in the ventrolateral medulla of cats: an intracellular study in vivo.

Effects of flurazepam on the periodic inhibitory postsynaptic potentials (IPSPs) and on the action of locally applied gamma-aminobutyric acid (GABA) were studied in bulbar respiratory neurons of decerebrate cats using concentric multibarrelled electrodes for intracellular recording and extracellular iontophoresis. Iontophoresis of flurazepam augmented spontaneous IPSPs and increased the hyperpolarization induced by GABA. Iontophoretic application of bicuculline suppressed the action of flurazepam. The reversal potential for spontaneous IPSPs was similar to that for the GABA-response. Intracellular Cl- injection shifted both the IPSP wave and the GABA response in a depolarizing direction. Flurazepam enhanced these depolarizing responses. These results suggest that GABA mediates the postsynaptic inhibition in bulbar respiratory neurons.

Animals↗

Converse motor output of inspiratory bulbospinal premotoneurones during vomiting.

Intracellular recordings of the activities of 10 inspiratory bulbospinal neurones of the medulla were performed in decerebrate cats. Fictive vomiting was induced by repetitive stimulation of the supra-diaphragmatic vagus nerves and was defined by series of synchronous large bursts in both the phrenic (inspiratory) and abdominal (expiratory) nerves. During these synchronous bursts the inspiratory bulbospinal neurones of both the dorsal and ventral respiratory groups were strongly hyperpolarized by chloride-dependent inhibitory postsynaptic potentials (IPSPs). We concluded that during vomiting the central pattern generator is inhibited, and that another pattern generator drives the spinal respiratory motoneurones.

Action Potentials↗

Excitation and inhibition of medullary inspiratory neurons by two types of burst inspiratory neurons in the cat.

In Nembutal-anesthetized and artificially ventilated cats, we studied the connectivity of burst inspiratory (I) neurons in the Bötzinger complex and the ventral respiratory group (VRG) with spike-triggered averaging methods. Burst I neurons exhibited tonic (I-TON) or decrementing (I-DEC) firing patterns. Spikes of I-TON neurons induced monosynaptic EPSPs in intracellularly recorded I neurons of both the VRG and the dorsal respiratory group (DRG). Spikes of I-DEC neurons induced monosynaptic inhibitory postsynaptic potentials (IPSPs) in both VRG and DRG I neurons.

Action Potentials↗