Robert E. Baker

Gabaa receptor maturation in relation to eye opening in the rat visual cortex

Changes in subunit composition of N-methyl-D-aspartate (NMDA) receptors have been reported to be affected by visual experience and may therefore form a major aspect of neuronal plasticity in the CNS during development. In contrast, putative alterations in the expression and functioning of the inhibitory GABAA receptor around eye opening have not been well defined yet. Here we describe the timing of changes in GABAA receptor subunit expression and the related synaptic functioning in the neonatal rat visual cortex and the influence of visual experience on this process. Quantitative analysis of all GABAA receptor subunit transcripts revealed a marked alpha3 to alpha1 subunit switch, in addition to a change in alpha4 and alpha5 expression. The changes were correlated with an acceleration of the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Both changes in receptor expression and synaptic functioning were initiated well before eye opening. Moreover, dark rearing could not prevent the robust upregulation of alpha1 or the change in sIPSC kinetics, indicating that this is not dependent of sensory (visual) input. Upon eye opening a positive correlation was observed between a faster decay of the sIPSCs and an increase in sIPSC frequency, which was absent in dark-reared animals. Thus, lack of extrinsic input to the cortex does not affect overall developmental regulation of synaptic functioning of GABAA receptors. However, we cannot exclude the possibility that visual experience is involved in proper shaping of the inhibitory network of the primary visual cortex.

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones.

Abstract Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 µmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 µmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.

Cocultured, but not isolated, cortical explants display normal dendritic development: a long-term quantitative study.

Dendritic growth has been studied in long-term organotypic neonatal rat occipital neocortex grown either apart as isolated explants or in tandem as cocultures. Quantitative light microscopic measurement of dendritic and axonal branching patterns within the cortical slice was accomplished using rapid Golgi stained materials. In both isolates and cocultures the overall cellular organization of the slice was maintained over 4 weeks in vitro with morphologically distinguishable pyramidal and nonpyramidal neurons located within the same layers and with the same orientations as observed in situ. Long-term increases in the total length of basal dendrites, apical dendrite and axons were observed only in cocultures and were similar to growth patterns reported for in situ materials. Dendritic growth was mainly due to elongation of terminal dendritic segments. Surprisingly, isolated explants showed no long-term increases in total (basal) dendrites, apical dendrites or axons with time in vitro. A transient decrease in the number of basal dendritic segments and increase in terminal segment lengths at the end of the first week in vitro, however, was observed in nonpyramidal neurons. It is hypothesized that (i) afferent inputs and/or efferent targets develop only in cocultures and provide a crucial conditions for the continued growth of dendritic/axonal arborization for neocortical neurons in vitro, (ii) intrinsic interconnectivity within isolated explants is not sufficient to maintain long-term growth of neuritic arbors, and (iii) remodelling of dendritic arbors within isolated explants occurs at the same time as these explants are showing noticeable increases in the level of spontaneous bioelectric activity, which suggests that dendritic growth and network formation may be function dependent.

Chronic blockade of glutamate-mediated bioelectric activity in long-term organotypic neocortical explants differentially effects pyramidal/non-pyramidal dendritic morphology

Dendritic/axonal growth has been examined in long-term organotypic neocortical explants taken from neonatal rat pups and grown either as isolated slices or as co-cultures. The quantitative light microscopic measurement of dendritic and axonal branching patterns within both types of explants was carried out on Golgi-stained materials. Spontaneous bioelectric activity (SBA) was blocked within both types of explants using a combination of APV and DNQX, NMDA and non-NMDA receptor antagonists, respectively. No extracellularly measurable SBA was observed to occur in the silenced explants in the presence of both antagonists but reappeared following wash-out with control medium. In both control and silenced explants, the overall cellular organization of the slice was maintained throughout the culturing period, with distinguishable pyramidal and non-pyramidal neurons located within the same layers and with the same orientations as observed in situ. The major findings of the present study show the following. (i) Pyramidal neurones chronically exposed to APV/DNQX exhibited no basal dendritic growth in co-cultured explants, while growth of apical dendritic lengths was similar to control values in the absence of SBA. (ii) Pyramidal neurones, nonetheless, exhibited significant terminal segment growth under SBA blockade which was correlated with a concomitant decrease in number of basal dendrites. (iii) Axonal growth in co-cultures was not sustained in silenced pyramidal neurones. (iv) Non-pyramidal neurones showed significant total dendritic and axonal growth in co-cultures following APV/DNQX treatment. (v) Non-pyramidal cells in co-cultures experienced an increase in terminal segment length at 2 weeks which declined in the third week. This increase-decrease was correlated with a decrease-increase in the total number of dendritic segments during the second and third weeks, respectively. (vi) In isolated explants the only departure from control growth curves was a significant increase in terminal segment length which was offset by a similar decrease in number of dendritic segments under APV/DNQX growth conditions. Thus the chronic loss of glutamate-mediated SBA differentially effected pyramidal and non-pyramidal neurones in isolated and co-cultured explants, with pyramidal neurones experiencing the more pronounced effects. We conclude that SBA effects the dynamics of neuritic elongation and branching and that these changes are most dramatically seen in co-cultures which cross-innervate one another, presumably via pyramidal axons. We hypothesize that the activity-dependent changes associated with reduction in pyramidal dendritic and axonal growth may be associated with neurotrophin receptor production/maturation.

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: Differential effects on intrinsically generated bioelectric discharges in a living ‘model’ system for slow-wave sleep activity

Within the context of an updated thorough review of the literature concerning activity-dependent cerebro-cortical development, a survey is made of recent experiments which utilize spontaneous spike-trains as the dependent variable in rodent neocortex cultures when synaptic transmission is interfered with during early ontogeny. Emphasis is placed on the complexity of homeostatic adaptations to reduced as well as intensified firing. Two kinds of adaptation are distinguished: (i) rapid recovery (within several hours) towards baseline levels despite sustained blockade of excitatory synaptic transmission, and (ii) the generation of essentially normal firing patterns in cultures assayed in control medium following development in the presence of excitatory receptor blockers. The former category of homeostatic responses is strongly dependent on the type of preparation, with isolated organotypic explants showing greatly limited plasticity in comparison with co-cultures of matching contralateral pieces of cortical tissue. In such co-cultures, compensatory excitatory drive manifests itself even when all three known types of ionotropic glutamate receptors are chronically blocked, and is then mediated by (muscarinic) cholinergic mechanisms which normally do not contribute measurably to spontaneous activity. The rapid return of high levels of spontaneous firing during sustained selective glutamatergic receptor blockade appears to protect neuronal cultures treated in this way from becoming hyperexcitable. In particular, quasi-epileptiform paroxysmal bursting upon return to control medium, such as appears in preparations where bioelectric activity has been totally suppressed during network formation, fails to appear in chronically receptor blocked cultures. On the contrary, desensitization of blocked glutamate receptors, as a physiological compensation for the up-regulation of non-blocked receptors, could be demonstrated for both the AMPA and the NMDA glutamate receptor sub-types. This wide range of homeostatic responses underscores the importance of spontaneous neuronal discharges for setting and maintaining an optimal balance between excitatory and inhibitory mechanisms in developing neocortical networks.

Homeostatically regulated spontaneous neuronal discharges protect developing cerebral cortex networks from becoming hyperactive following prolonged blockade of excitatory synaptic receptors

In order to further examine the role of spontaneous action potential (SAP) discharges in neocortical development, amino-acid-mediated synaptic transmission was selectively blocked in an improved organotypic neocortex culture preparation. Contralateral occipital cortex slices from neonatal rats were co-cultured for several weeks in a ventricle-to-ventricle orientation known to greatly enhance cyto-morphological and electrophysiological maturation. Such preparations are highly resistant to attempts to suppress neuronal firing by blocking ionotropic glutamate receptors: not only can kainate receptors partly substitute for NMDA- and AMPA-mediated neurotransmission when these receptors are pharmacologically blocked, but (muscarinic) cholinergic receptors also begin to drive SAP activity when the kainate receptors, too, are chronically blocked. Only tetrodotoxin proved able to eliminate SAPs altogether in these co-cultures, while GABAergic receptor blockade (using bicucculine) led to persistent epileptiform discharges. Treatment effects were assayed upon transfer to control medium by means of a quantitative analysis of spontaneously occurring polyneuronal spike trains. Total suppression of action potentials for several weeks (by tetrodotoxin treatment) led, as in earlier experiments, to strongly intensified burst firing upon transfer to control medium. Chronic glutamate receptor blocked cultures, on the other hand, showed only minor deviations from control firing levels and patterns when assayed in normal medium. Protection against the development of hyperactivity despite partial blockade of synaptic transmission was roughly proportional to the degree to which spontaneous firing during the treatment period approximated normal SAP levels. This homeostatic response to chronically reduced excitatory drive thus differs from earlier results using isolated organotypic cortex cultures, in which the restoration of SAP activity failed to prevent the development of network hyperactivity. Chronic bicucculine treatment, in contrast, had little or no homeostatic effect on SAP firing patterns; on the contrary, opposite to earlier findings using isolated occipital cortex explants, paroxysmal discharges persisted even after transfer to control medium.

Central neuronal responsiveness to sensory ganglion stimulation is correlated with the incidence of spontaneous bioelectric activity in developing spinal cord cultures.

In spinal cord explants co-cultured with dorsal root ganglion cells for 3–4 weeks in a (horse)serum-containing medium, the spread of ganglion-evoked action potentials from monosynaptic innervation sites (“polysynaptic excitability index”) was not correlated with the incidence of neuronal “background” discharges. Moreover, chronic exposure of serum-grown cultures to tetrodotoxin (TTX) in a dose sufficient to reversibly block bioelectric activity, failed to significantly affect this index. For explants grown in a chemically defined medium (CDM) similar excitability scores were obtained only if a low level of spontaneous activity was measured. The most active preparations scored considerably higher, with intermediate values being found in the moderately active cultures. Chronic TTX-exposure in developing CDM-grown cultures reduced their excitability scores to the level found in weakly active, untreated, explants despite a normal incidence of spontaneous activity. The present study indicates that low levels of spontaneous activity in untreated explants were associated with a similar sluggishness of DRG-evoked responses as previously observed after chronic treatment with TTX. These results give additional grounds for confidence that this reduced responsiveness of spinal cord neurons to sensory input is indeed attributable to prolonged reduction of centrally generated excitation during development in vitro.

SENSORY END ORGAN MODULATION VS NERVE CELL REDUNDANCY AS POSSIBLE MECHANISMS IN THE DEVELOPMENT OF MISDIRECTED REFLEX RESPONSES IN SKIN GRAFTED ANURANS

Publisher Summary This chapter presents a general review of the misdirected reflex phenomenon in anurans, with reasons for rejecting selective peripheral regrowth as a class of mechanism by which cutaneous neurites could effectuate connexions with their appropriate sensory end organs. The refuted mechanisms include (1) peripheral searching, (2) multiple branching, and (3) the degeneration–regeneration of inappropriate neurites. The chapter reviews the data that argues against sensory impulse patterning as the source of cutaneous reflex localization. Behavioral observations, combined with nerve crush experiments, have shown that the peripheral nerve trunks that mediate only dorsal wiping responses in control frogs mediate misdirected, that is, ventrally directed wipes in skin-grafted animals. The cutaneous receptive fields (CRFs) for the nerve trunks are located in the same areas on the body surface in control and in skin-grafted animals, regardless of the type of skin present. Two experimental approaches might be useful to examine the alternative to the mechanism of modulation. One would be the use of early embryonic skin rotations, coupled with behavioral observations immediately after metamorphosis. The second proposed experimental approach would employ H 3 -thymidine autoradiography to establish the birthdates of neurons within the dorsal root ganglion (DRG).