Optical recording of epileptiform voltage changes in the neocortical slice

Summary Voltage sensitive probes were used to monitor the development, distribution, and spread of epileptiform potentials with a photodiode array in neocortical slices of guinea pigs. Epileptiform activity was induced by bath application of bicuculline-methiodide or 3,4-diaminopyridine and electrical stimulation of white matter or cortical layer I. Stimulation evoked a primary or early potential which was followed by a delayed epileptiform potential with a larger spatial extent. Shape, duration and amplitude of the delayed epileptiform potential varied strongly among slices and across the recording area and could reach largest amplitudes at a distance from the stimulation point. At a specific recording site, however, with repeated stimulation, potentials were generated in a stereotyped way. Intracellularly recorded delayed epileptiform potentials corresponded very closely at least to the early part of the optical response. Epileptiform activity appeared in layer III as soon as the primary potential reached sufficient amplitude there. Apart from this relationship, the distribution and spread of maximal amplitudes of delayed epileptiform potentials were segregated from those of early potentials. Early potentials reached maximal amplitudes close to the stimulation site. In contrast, the largest amplitudes of delayed epileptiform potentials were always found in layer III. A second maximum occasionally occurred in layer V. The horizontal amplitude distribution of epileptiform potentials was asymmetric, i.e. amplitudes increased to one side and decreased to the other. Early potential maxima spread from deeper to upper layers when initiated by white matter stimulation and from upper to deeper layers when initiated by layer I stimulation. In contrast, delayed epileptiform potentials always spread from layer III to lower layers and to the sides. Velocity of spread of early potentials and delayed epileptiform potentials differed systematically along the vertical and horizontal axis. The distribution of maximal amplitudes, shape, and pattern of spread of epileptiform potentials was the same whether white matter or layer I was stimulated. The independence of delayed epileptiform potential characteristics from the point of stimulation and from early potential characteristics suggests that epileptiform activity is determined by intrinsic properties of the cortex and not by afferent activation.     

Electrophysiological characteristics of neurons in neocortical expiant cultures

Summary We examined the electrophysiological and morphological properties of neocortical neurons maintained in expiant cultures prepared from the parietal cortex of newborn Sprague-Dawley rats. After 3–6 weeks in vitro, cultures showed regional differences in cellular density reminiscent of cortical layering, and an abundance of axonal processes. Pyramidal-shaped neurons with spinous dendrites were the dominant elements revealed by Lucifer yellow injections. Intracellular recordings revealed that many electrophysiological properties of neurons in the explants resembled those of neocortical neurons in vivo and in slice preparations. In response to depolarizing current injection, neurons in the expiants showed the same three patterns of repetitive firing described in neocortical slices, as well as a similar array of responses. Spontaneous synaptic potentials were recorded from all neurons and complex PSPs were evoked in response to focal extracellular stimulation. GABAa receptors mediated a significant component of the evoked responses. Fifteen of sixty neurons generated action potentials that arose spontaneously from resting potentials. Neurons in many slices generated large, prolonged depolarizing potentials that reflected coordinated synaptic activity within the expiants. These results underscore the usefulness of the neocortical explant as a valuable model for studying aspects of the behavior of circuits of cortical neurons.     

Two patterns of firing in human neocortical neurons

Intracellular recordings were made in slices of human neocortex removed for surgical treatment of epilepsy. In response to prolonged suprathreshold current injection, regular spiking neurons (67.5% of sample) responded by repetitive firing throughout the stimulus from all membrane potentials. Bursting neurons (32.5% of sample) responded with a burst of 2–3 spikes which rode upon a voltage-dependent slow depolarization. Bursting behavior was only observed from membrane potentials more negative than −65 mV.     

Dynamical aspects of neocortical histogenesis in the rat

Summary The formation and transformation of embryonic neocortical cell layers has been studied by means of autoradiographic and morphometric methods in the light of the concept of a dual origin of mammalian neocortex (Marin-Padilla, 1978; Raedler and Raedler, 1978).A primordial plexiform layer begins to form on ED 13, when the first postmitotic preneurons enter the area between ventricular layer and pial basement membrane. Before this stage the area was occupied by single fibres and processes of the ventricular cells only. The further development of the primordial plexiform layer is characterized by a linear increase in width, mainly due to the addition of migrating preneurons and ingrowing fibre bundles. The older preneurons of the primordial plexiform layer are situated outermost in the layer, the younger ones at its inner circumference. The differentiation accordingly follows an outside — inside gradient. The preneurons of this layer are designed to become either the Cajal-Retzius cells of Layer I or subcortical neurons (Layer VII) (Rickmann et al., 1977).While the mitotic activity of cells in the ventricular layer decreases only gradually during the course of neurogenesis, the thickness of the ventricular layer (in the lateral part of neocortex) decreases dramatically from ED 16 on. At the same time the cortical plate starts to form. This is brought about by a translocation of ultrastructurally rather undifferentiated ventricular daughter cells from the inner to the outer circumference of the cerebral wall. The width of the cortical plate grows almost exponentially, especially its medial part. Its development is characterized by the addition of preneurons on its outer circumference while maturation takes place at its inner border.The significance of these developmental features with regard to neocortical histogenesis is discussed.     

Comparison of the electrical properties of neocortical neurones in slices in vitro and in the anaesthetized rat

Summary Because neurones in isolated brain slices are abnormal in several respects, we have compared the electrical properties of neocortical neurones from the anaesthetized rat with neurones from slices of the same area of neocortex in vitro. Resting potentials were not significantly different but there was a small increase in mean spike amplitude and threshold in slices. The most striking observation was a doubling of the mean apparent input resistance (R_in) of neurones in slices (36 M cf. 18 M in vivo). To assess the extent to which the loss of surface membrane of neurones in slices might contribute to the raised R_in, we estimated the membrane capacity of neurones in the two samples from calculations of the membrane time constants. The mean membrane capacity was significantly lower in neurones from slices, being 2/3 that of the in vivo sample, showing that recordings were indeed mostly from smaller cells in the slices. Possible causes of the difference in mean input resistance not attributable to neuronal size are also discussed.     

A low-voltage activated,transient calcium current is responsible for the time-dependent depolarizing inward rectification of rat neocortical neurons in vitro

Intracellular recordings were obtained from rat neocortical neurons in vitro. The current-voltage-relationship of the neuronal membrane was investigated using current- and single-electrode-voltage-clamp techniques. Within the potential range up to 25 mV positive to the resting membrane potential (RMP: –75 to –80 mV) the steady state slope resistance increased with depolarization (i.e. steady state inward rectification in depolarizing direction). Replacement of extracellular NaCl with an equimolar amount of choline chloride resulted in the conversion of the steady state inward rectification to an outward rectification, suggesting the presence of a voltage-dependent, persistent sodium current which generated the steady state inward rectification of these neurons. Intracellularly injected outward current pulses with just subthreshold intensities elicited a transient depolarizing potential which invariably triggered the first action potential upon an increase in current strength. Single-electrode-voltage-clamp measurements reveled that this depolarizing potential was produced by a transient calcium current activated at membrane potentials 15–20 mV positive to the RMP and that this current was responsible for the time-dependent increase in the magnitude of the inward rectification in depolarizing direction in rat neocortical neurons. It may be that, together with the persistent sodium current, this calcium current regulates the excitability of these neurons via the adjustment of the action potential threshold.     

Threshold effects of N-methyl D-aspartate (NMDA) and 2-amino 5-phosphono valeric acid (2APV) on the spontaneous activity of neocortical single neurones in the urethane anaesthetised rat

Summary Spontaneous activity of single neurones in neocortex was sampled using pairs of microelectrodes in rats anaesthetised with urethane. In confirmation of previous studies, many cells recorded from middle layers characteristically fired in bursts, the onset times of which were synchronous both unilaterally and bilaterally. Iontophoresis of 2APV onto such cells either caused an abolition of bursts or a reduction in spikes per burst. In the latter case action potentials which occurred later in the burst were preferentially abolished. Iontophoresis of NMDA onto the same cells caused a prolongation of bursts with minimal effect on intraburst interspike interval. In interactive trials with the two drugs the effect of NMDA could be abolished by 2APV, and NMDA counteracted the effect of 2APV. It is concluded that spontaneous burst generation in neocortex during urethane anaesthesia is generated through a cortical NMDA/2APV-sensitive receptor mechanism.     

The role of the anterior intralaminar nuclei and N-methyl D-aspartate receptors in the generation of spontaneous bursts in rat neocortical neurones

Summary The nature of spontaneous unitary activity of rat neocortex was investigated during slow wave sleep and urethane anaesthesia. Neurones in layer IV and V locations fired in a burst-pause pattern at a low burst repetition rate (0.5–4 per second) during both stage 3/4 sleep and urethane anaesthesia. Occasionally an alternative mode of firing (spindle clusters), associated with focal spindle wave activity, was also found to occur in both states. Using dual microelectrode implants it was found that the onset times of bursts (but not spindle clusters), coincided in the same and opposing cortices, whether in functionally similar or disparate areas. The highest probability was that burst onsets occurred simultaneously (resolution =2.56 ms, interquartile range=40 ms). Spontaneous unitary activity was investigated in the thalamus for temporal correlation with spontaneous unitary activity in neocortex under urethane anaesthesia. Neurones of the anterior intralaminar group (aIL) consistently fired in a burst-pause pattern such that each aIL burst showed a strong tendency to precede a cortical burst. Unilateral electrical stimulation of the aIL nuclei evoked widespread bilateral entrainment of cortical bursts. In contrast stimulation of VP1, or cutaneous sites, evoked only short duration spike responses together with burst abolition in the appropriate restricted Sm1 area. Ionophoresis of NMDA (N-Methyl D-Aspartate) onto Sm1 neurones increased the probability of cortical burst responses to aIL stimulation in addition to decreasing the latency by 20–40 ms (n=11). lonophoresis of 2APV (2-amino 5-phosphono valeric acid) caused simultaneous abolition of spontaneous cortical bursts and bursts evoked by aIL stimulation. Short latency responses to cutaneous and VP1 stimulation were unaffected by ionophoresis of 2APV sufficient to cause burst elimination, suggesting that this pathway does not operate via a 2APV sensitive receptor mechanism. Anatomical features of the aIL nuclei and their overall cortical projection pattern are discussed in relationship to these findings. The activation of cortical NMDA/APV sensitive receptors by aIL afferents in the spontaneous generation of bursts in cortical cells is discussed.     

Altered synaptic activities in cultures of neocortical neurons from prenatally X-irradiated rats

Prenatal X-irradiation can induce severe microcephaly in the brains of offspring. The possible alteration of neuronal synapse formation was examined in such X-irradiated rats with microcephaly using the whole-cell current clamp technique. The total number of neocortical cells from prenatally (E16) X-irradiated rats decreased to 16% of the control value, while the ratio of GABA-positive/MAP2-positive neurons increased 2.2-fold. Neocortical neurons from E17 normal rat fetuses cultured on monolayers of astrocytes for 7-10 days exhibited synchronized synaptically-driven rhythmic depolarizing potentials (RDPs). Neocortical neurons from prenatally (E15 or E16) X-irradiated rats also exhibited synchronized RDPs, however, their amplitude and the number of spikes decreased. These results suggest that, although neurons which survive in X-irradiated rats can form synapses, inhibitory inputs are predominant over excitatory inputs. It is possible that not only acute neuronal loss induced by X-irradiation but also increased inhibitory inputs in neocortex give rise to subsequent neurological disorders in X-irradiated rats.     

Sparing of an olfactory discrimination habit following extensive neocortical removals in rats

Adult male albino rats, blinded by enucleation, learned a tap water-acetone discrimination and subsequently sustained bilateral destruction of the occipito-temporal, parietal, lateral fronto-parietal, dorsal fronto-parietal or cingulate areas. The retention test disclosed that the various brain-damaged groups were not significantly inferior to the control group in savings scores. These data, which confirm the findings of Swann [13], suggest that certain olfactory experiences are not stored within the neocortex.     

Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures

Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates a number of critical developmental processes in both vertebrates and invertebrates, including angiogenesis, vasculogenesis and hematopoiesis. We employed an organotypic rat explant model (produced from embryonic day 17 fetuses) to assess the effects of vascular endothelial growth factor on brain microvasculature in general and the ventral midbrain specifically. Immunohistochemistry using antisera to rat endothelial cell antigen and laminin demonstrated a robust, dose-dependent effect of vascular endothelial growth factor, resulting in increased vessel neogenesis, branching and lumen size by three days in vitro. This effect was blocked by addition of an anti-vascular endothelial growth factor antibody. At higher doses of vascular endothelial growth factor, the effect was attenuated, though a statistically significant increase in both astrocyte, and neuronal density was observed using antisera to glial and neuronal markers. Tyrosine hydroxylase-immunoreactive (i.e. dopaminergic) neurons, particularly, exhibited increased survival in response to vascular endothelial growth factor application. Vascular endothelial growth factor had a mitogenic effect on endothelial cells and astrocytes, but not dopaminergic neurons, as demonstrated by the addition of [³H]thymidine to the cultures 2 h after the cultures were established. Similarly, results of a radioreceptor assay indicated that specific vascular endothelial growth factor binding sites were present on blood vessels and astrocytes, and were up-regulated by exposure to vascular endothelial growth factor.

We conclude that, in explants of the ventral mesencephalon, exogenously applied vascular endothelial growth factor is mitogenic for endothelial cells and astrocytes, and promotes growth/survival of neurons in general and dopaminergic neurons in particular.     

Connectivity of fetal neocortical block transplants in the excitotoxically ablated cortex of adult rats

Fetal neocortical block grafts placed into new-born recipients are able to exchange axonal projections with the host central nervous system, as shown in several previous experiments. The present study examined the connectivity of fetal neocortical block transplants placed into the excitotoxically ablated cortex of adult rats. Young adult rats received injections of the excitotoxic amino acid N-methyl-D-aspartate into the sensorimotor cortex area 1 week prior to receiving a fetal (E14–15) neocortical transplant. Afferent and efferent connections of these grafts were examined 3–6 months after transplantation by injecting the transplants with the fluorescent retrograde tracers fast blue and diamidino yellow or with the anterograde tracer Phaseolus vulgaris leucoagglutinin. Retrogradely labeled neurons were observed within several host brain regions including the ipsilateral neocortex, several thalamic nuclei, subcortical areas such as claustrum and lateral hypothalamus, nucleus basalis, dorsal raphe nuclei and locus coeruleus. Fibers labeled with Phaseolus vulgaris leucoagglutinin were found extending throughout the transplants, but with rare exceptions fibers were not observed within the host brain. The experiments showed that neocortical block grafts placed into the excitotoxically ablated neocortex receive afferent input from areas in the host brain that normally innervate the sensorimotor cortex. The extensive Phaseolus vulgaris leucoagglutinin-positive axonal labeling found within the grafts demonstrated the ability of the grafted neurons to establish extensive intrinsic graft connections. Their failure to project out of the grafts suggests that the mature host brain does not provide a permissive environment for neurite extension.     

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infrared-videomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 ± 7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro.     

The involvement of excitatory amino acids in neocortical epileptogenesis: NMDA and non-NMDA receptors

Summary Conventional intracellular recording techniques were used to investigate the N-methyl-D-aspartate (NMDA) and non-NMDA mediated synaptic mechanisms underlying the stimulus-induced paroxysmal depolarization shift (PDS) generated by cells in rat neocortical slices treated with bicuculline methiodide (BMI). The NMDA receptor antagonists CPP or MK-801 were ineffective in abolishing the PDS. However, both drugs were able to attenuate the late phase of the PDS and delay its time of onset. In contrast, the non-NMDA receptor blocker CNQX demonstrated potent anticonvulsant property by reducing the PDS into a depolarizing potential that was graded in nature. This CNQX-resistant depolarizing potential was readily blocked by CPP. Voltage-response analysis of the PDS indicated that the entire response (including its NMDA-mediated phase) displayed conventional voltage characteristics reminiscent of an excitatory postsynaptic potential that is mediated by non-NMDA receptors. We conclude that the activation of non-NMDA receptors is necessary and sufficient to induce epileptiform activity in the neocortex when the GABAergic inhibitory mechanism is compromised. The NMDA receptors contribute to the process of PDS amplification by prolonging the duration and reducing the latency of each epileptiform discharge. However, the participation of NMDA receptors is not essential for BMI-induced epileptogenesis, and their partial involvement in the PDS is dependent upon the integrity of the non-NMDA mediated input. The lack of NMDA-like voltage dependency observed in the PDS's late phase might reflect an uneven distribution of NMDA receptors along the cell and/or an association of this excitatory amino acid receptor subtype in the polysynaptic pathways within the neocortex.     

GABA A mediated inhibition and post-inspiratory pattern of laryngeal constrictor motoneurons in rat

Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague-Dawley rats. During extracellular recordings, GABA iontophoresis (0.5M) strongly inhibited LCMN (n=6). Interestingly, BIC iontophoresis (5 mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5+/-1.4 mV before and 1.5+/-0.4 mV after the BIC, P=0.05, n=5), but also the depolarisation during post-inspiration (3.0+/-1.3 mV before and 1.6+/-0.4 mV after the BIC, P=0.02, n=5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABA(A) receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration.     

Unexpected suppression of neuronal G protein-activated, inwardly rectifying K+ current by common phospholipase C inhibitor

Stabilization of the binding of phosphatidylinositol bisphosphate (PIP(2)) to G protein-coupled inward rectifier K+ (GIRK) channels is essential for their activation, whereas hydrolysis of PIP(2) by phospholipase C (PLC) inhibits channel activity. Apparently inconsistent with this mechanism, we found that the commonly used PLC inhibitor, U73122 (1 microM), produced a significant reduction in the amplitude of baclofen (20 microM)-evoked GIRK currents in whole-cell recordings from acutely isolated rat neocortical pyramidal cells. Also, U73122 reduced the percentage of baclofen-responsive neurons from 100% (n=40) to 56% (n=25). Since NCDC (100 microM), a PLC inhibitor of another molecular class, displayed no effect on GIRK current amplitude or responsiveness (100%, n=6), inhibition of PLC is unlikely to account for the effects of U73122 in our preparation. Lending further support to this notion, the structurally closely related compound, U73343, which does not inhibit PLC, proved to be even more efficient in suppressing GIRK current as compared to U73122. In neurons, in which GIRK channels were irreversibly activated by GTPgammaS (n=10), the depressant action of U71322 was fully preserved. These findings hint at a direct interaction of U73122 with the GIRK channel or a closely associated protein. Caution is therefore warranted when employing this compound to examine the role of PLC and PIP(2) in the regulation of GIRK channel activity.     

Are extent and force independent movement parameters? Preparation- and movement-related neuronal activity in the monkey cortex

Movement extent and movement force can be independently controlled in motor performance. Therefore, independent representations of extent and force should exist in the central nervous system (CNS). To test this hypothesis, microelectrode recordings were made in sensorimotor cortex of monkeys trained to perform visually cued wrist flexion movements of two extents, against two levels of frictional resistance. An initial preparatory signal (PS) provided complete, partial or no information about extent and/or force of the movement, which had to be performed in response to a second, response signal (RS). The activity of 511 neurons of the primary motor cortex (MI), the premotor cortex (PM), the postcentral cortex (PC), and the posterior parietal cortex (PA) was recorded in two monkeys. Both reaction time (RT) and neuronal data suggest that there exist independent, neuronal mechanisms responsible for the programming of either parameter. On the one hand, partial information about either movement parameter shortened RT when compared with the condition of no prior information. On the other hand, there were, among others, two discrete populations of neurons, one related only to extent, the other only to force. Preparatory changes in activity related to either movement parameter were mainly located in the frontal cortex, especially in the PM. After occurrence of the RS, the percentage of selective changes in activity increased and tended to extend to the parietal cortex. In particular during the movement, force-related changes in activity have been encountered in PA. Furthermore, we conducted trial-by-trial correlation analyses between RT and preparatory neuronal activity for all conditions of prior information. The mean correlation coefficient was significantly higher in the condition of information about movement extent than of information about movement force and it was significantly higher in MI/ PM than in PC/PA.     

Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology

Fluorescent indicator molecules, such as fura-2, are useful probes for studying the concentrations of ions in single cells. A key feature of these fluorescent dyes is the shift in their excitation spectra upon binding the ion, thus making alternate excitation from two wavelengths desirable. In this report we describe an inexpensive system for making simultancous electrophysiological and dual excitation fluorescence measurements using equipment much of which is available in a typical biophysical laboratory. In order to synchronize the fluorescence signal with the appropriate excitation wavelength we devised a simple computer program which uses the output of photodiodes placed in the excitation beam to determine which wavelength is illuminating the cell. We also describe the use of a liquid light guide to transmit excitation illumination from the light source to the epifluorescence port of the microscope in order to isolate a perfusion chamber from light, electrical noise and vibration. A sensitive light detection system was assembled using a photomultiplier tube and discriminator that took advantage of sampling single unit events obtained with photon counting rather than the analog of annode current. However, rather than employing a sophisticated and expensive photon counting system, a filter was used to integrate the signal so that an analog output could be presented to a multichannel analog to digital converter commonly used for electrophysiological recordings. This apparatus was sensitive enough to allow the determination of the intracellular free Ca²⁺ concentration, [Ca²⁺]_i, using fura-2 in the following situations: (a) in single processes of dorsal root ganglion (DRG) neurons grown in primary culture, (b) the release of Ca²⁺ from intracellular stores in single neurons, (c) the influx of Ca²⁺ through channels activated by excitatory amino acids and (d) it was also possible to measure [Ca²⁺]_i transients while simultaneously recording Ca²⁺ currents at precisely controlled membrane potentials in DRG neurons. The instrumentation described here makes use of a number of innovations which investigators developing similar systems may find useful.Supported by PHS grants DA02121, DA02575 and MH40165 and by grants from Miles Inc, and Marion Labs.Dr. Thayer is supported by National Research Service Award number NS08009.     

Dopaminergic neuronal survival and the effects of bFGF in explant,three dimensional and monolayer cultures of embryonic rat ventral mesencephalon

Embryonic substantia nigra cells when transplanted into the striatum can reverse many of the defects of Parkinson's disease. The efficacy of such grafts is compromised by the poor survival of grafted dopaminergic neurones; typically, 3–10% survive transplantation. We used three tissue culture models to identify stages in the procedure for the preparation and insertion of grafts which might be responsible for this cell death and to identify environments in which survival is optimised. (1) The ventral mesencephalon was dissected from the donor brain, then placed immediately into culture contained in a collagen gel. (2) The dissected tissue fragments were enzymatically dissociated, then the cells placed into monolayer culture. (3) Enzymatically dissociated tissue was packed into 0.5-mm-diameter porous tubes, to simulate the compaction of cells into a graft deposit in the host brain. Dissociation of the tissue by itself caused the death of approximately 30% of dopaminergic neurones, as judged by the difference in cell counts between the intact embryonic day 14 (E14) mesencephalon, and cells dissociated then packed into tubes. Of the dissociated neurones approximately 60% died during the first 24 h and 87% during the first 3 days in monolayer culture, while only 7% of dopaminergic neurones in three-dimensional cultures and 11% of neurones in explant cultures died over the first 3 days. Embryonic dopaminergic neurones are clearly very vulnerable to adverse conditions during the first days after their removal from the donor brain. The excellent survival of neurones in three-dimensional and explant cultures indicates that close association with other cells, which may provide greatly improved access to trophic factors, can enable the cells to survive this period of vulnerability. In contrast to its effects in monolayer cultures, bFGF had no effect on dopaminergic neuronal survival in either explant or three-dimensional cultures.     

Do nigro-striatal neurones possess a discrete dendritic modulatory mechanism? Electrophysiological evidence from the actions of amphetamine in brain slices

Summary Dopamine released from dendrites of nigrostriatal neurones in the substantia nigra exerts an inhibitory action on these cells. However, the spatio-temporal characteristics of the action of dendritic dopamine is still unclear. The aim of the present study was to investigate the responses of these neurones in the guinea-pig to amphetamine, applied locally in the region of the distal dendrites in pars reticulata. During intracellular recording in vitro it was found that amphetamine hyperpolarizes the membrane and causes a decrease in the input resistance, probably by increasing a potassium conductance. This response was resistant to blockade of sodium channels but sensitive to dopamine depletion by reserpine and alpha-methyl-p-tyrosine. The response showed tachyphylaxis and proved to be highly dependent on the site of administration of amphetamine. It is concluded that the release and action of dopamine occurs locally, in a heterogenous pattern, within the dendritic field of nigrostriatal neurones. The possibility is discussed that this phenomenon underlies a modulatory mechanism, localized in dendrites.