Ileuron,

Vol. 6, 333-344, March, 1991, Copyright

0 1991 by Cell Press

Control of Postsynaptic Ca*+ Influx in Developing Neocortex by Excitatory and Inhibitory Neurotransmitters Rafael Yuste* and Lawrence C. Katz* I.aboratory of Neurobiology “he Rockefeller University New York, New York 10021

Summary We assessed the pathways by which excitatory and inhibitory neurotransmitters elicit postsynaptic changes in [Ca*+]i in brain slices of developing rat and cat neocortex, using fura 2. Glutamate, NMDA, and quisqualate transiently elevated [Ca*+]i in all neurons. While the quisqualate response relied exclusively on voltage-gated Ca*+ channels, almost all of the NMDA-induced Ca*+ influx was via the NMDA ionophore itself, rather than through voltage-gated Ca*+ channels. Glutamate itself altered ]Ca*+]i almost exclusively via the NMDA receptor. Furthermore, synaptically induced Ca*+ entry relied almost completely on NMDA receptor activation, even with low-frequency stimulation. The inhibitory neurotransrnitter GABA also increased [Ca*+]i, probably via voltage-sensitive Ca*+ channels, whereas the neuromodulag-or acetylcholine caused Ca*+ release from intracellular stores via a muscarinic receptor. low concentrations of these agonists produced nonperiodic [Ca*+]i oscillations, which were temporally correlated in neighboring cells. Optical recording with Ca*+-sensitive indicators may thus permit the visualization of functional networks in developing cortical circuits. &rtroduction luring a restricted period in early postnatal developlnent, neuronal activity shapes and refines connectivty in both visual (Wiesel, 1982) and somatosensory vortices (Van der Loos and Woolsey, 1973). The cellular ,nechanisms responsible for this plasticity are largely .rnknown. By analogy with events required to induce ‘ong-term potentiation (LTP) in the hippocampus(Lynch ?t al., 1983; Malenka et al., 1988,1989; Wigstrom et al., 1986), recent studies have suggested that N-methyl-oaspartate (NMDA) receptor-mediated Ca*+ influx may be a critical step in detecting correlations in pre- and postsynaptic activity that give rise to plastic changes in the developing mammalian cortex (Bear et al., 1990; Fox et al., 1989;Tsumoto et al., 1987; Miller et al., 1989a) and the frog optic tectum (Cline et al., 1987; Udin and Scherer, 1990). Neurotransmitter-induced changes in the intracellular Ca*+ concentration ([Ca”]J are therefore likely to be closely associated with plastic changes in cortical synapses. Currently, however, little is known about the actions of excitatory or inhibitory neurotransmit* Present address: Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, North Carolina 27710.

ters on [Caz+]r in developing neurons. To determine how neurotransmitters regulate [Ca*+]i in developing cortical neurons, we used optical recording with the fluorescent Ca*+ indicator fura 2 (Grynkiewicz et al., 1985) in brain slices of developing somatosensory and visual cortices. In light of the proposed role of NMDA receptormediated neurotransmission in the control of ocular dominance column plasticity in the visual cortex, we were particularly interested in dissecting the actions of glutamate on developing cortical neurons. At first glance, theCa2+-gating propertiesof the NMDA receptor-especially the requirement for prolonged depolarization to remove the voltage-dependent Mg*+ block-seemed difficult to reconcile with the weak excitability of very young developing cortical neurons. In the adult rodent hippocampus, the NMDAmediated component of the Cd*+ influx during lowfrequency synaptic activation is extremely small; most of the postsynaptic voltage change and Ca*+ entry rely on activation of quisqualatelkainate, and not NMDA, receptors (Collingridge et al., 1983; Regehr et al., 1989). A significant NMDA receptor contribution to postsynaptic Ca*+ entry can be detected only at high stimulation frequencies (20-100 Hz), which elicit LTP (Regehr and Tank, 1990). In the developing neocortex, however, major activity-dependent alterations in neural architecture take place even though cells respond sluggishly and fatigue rapidly both in vivo (Hubel and Wiesel, 1963; Purpura et al., 1965; Maffei and GalliRestra, 1990; Armstrong-James, 1975) and in vitro (McCormick and Prince, 1987; Kriegstein et al., 1987), often requiring many seconds or minutes between stimulus presentations to recover. For example, Armstrong-James (1975) found that neurons in l-week-old rat somatosensorycortex could not follow stimuli presented at greater than 0.1 Hz. Thus the conditions necessaryto evoke NMDA-mediated Ca*+ influx in the hippocampus may not occur in the developing cortex. Using electrical and chemical stimulation, we sought to determine the requirements for NMDA-induced Ca2+ influx in developing cortical neurons. To complete our study, we also evaluated the responses to y-aminobutyric acid (GABA), the major inhibitory transmitter in the cortex (Krnjevic, 1987), and acetylcholine, which has been suggested as an important neuromodulator of visual cortical plasticity (Bear and Singer, 1986). Results All experiments used brain slices prepared from somatosensory and visual cortices of Long-Evans rats between postnatal days (P) 1 and 7. These ages correspond to the”critical period”during which the organization of the somatosensory cortex can undergo plastic changes after perturbations of the facial whiskers

.-

40 Figure 1. Staining

of a Living

Brain Slice with

250 [W+]i

>500 (nM)

Fura 2-AM

(A) Layer 2/3 of a living slice (400 pm thick) of the somatosensory cortex of a P3 rat, stained with fura 2-AM. Almost all cells in the layer are clearly labeled with indicator. The pial surface is indicated by the arrow; the area below the pia devoid of labeling corresponds to layer 1. Bar, 100 pm. (B) Higher magnification view of labeled cells. Individual cell bodies and apical dendrites are clearly stained. Bar, 50 urn. (C-F) Pseudocolor ratio images of NMDA-evoked increases in [Ca2’], for a group of cells in the upper layers of a slice prepared from PI rat somatosensory cortex at 0 CC), 110 CD), 170 (E), and 520 (F) s after drug application. NMDA (40 PM) was applied at 0 s and washed out at 384 s. All cells responded to NMDA with an approximately IO-fold increase in [Caz’l,, and all returned to basal levels after removal of the agonist. Bar in (C), 50 urn.

(Van der Loos and Woolsey, 1973). We confirmed the synaptic stimulation results (see below) in newborn kitten primary visual cortex. After incubation in the acetomethylester form of fura 2 (fura 2-AM), slices

were imaged in a temperature-controlled chamber on the stage of an inverted microscope. Virtually all ceils in layer 2/3 were clearly labeled with fura 2 (Figures IA and IB). We were able to resolve easily individual cell

Yegulation 335

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Induce

Increases

in [Ca2’],

(A) Changes in [Ca2’], in a layer 2/3 cell from the primary visual cortex of a P5 rat, after bath application of agonists to the slice during the time indicated by the bars. Glutamate (100 PM), NMDA (50 PM), and quisqualate (5 uM] induced consecutive elevations in [Ca*+],. The response to quisqualate peaked and declined rapidly in the presence of the agonist. (B) APV blocks responses to glutamate. Changes in [Ca”], in response to successive applications of 100 uM glutamate, 50 uM glutamate, 50 uM glutmate in the presence of 50 uM APV, and 50 uM glutamate after APV was washed out, in a P3 neuron from the somatosensory cortex. APV virtually abolishes the response to glutamate.

bodies in the lower 50-70 pm of the slices. Although some glial cells and radial glial processes were also labeled, neurons could be readily distinguished by their size, pyramidal shape, and presence of an apical dendrite (Figures IA and 18). Resting [Ca2+]i increased somewhat with age, from 16 + 10 nM (n = 70) at PI (mean + SD), to 67k 15 nM (n = 17) at P7. The ages over which we were able to visualize changes in [Ca’+]i, however, were restricted by the behavior of fura 2-AM in older slices: after PIO, fura 2-AM failed to label neocortical cell bodies over a broad range of incubation conditions (see Experimental Procedures). Responses of Cortical Neurons to Glutamate and Its Agonists Bath application of either NMDA, quisqualate, or glutamate caused every labeled neuron to respond with a substantial and completely reversible increase in [Ca”],. A pseudocolor representation of the response

of afield of cells to bath application of NMDA is shown in Figure 1 (C-F). We did a series of dose-response experiments to determine the relative potency of the three agonists in inducing elevations in [Ca”],; in subsequent experiments drug concentrations that resulted in approximately half-maximal responses were used. These agonist concentrations also ensured that we were working well below the limits of saturation of the fura 2 signal. Although at half-maximal concentrations the characteristics of the NMDA and glutamate responses were very similar, both differed substantially from the quisqualate response. The response of a representative cell at P5 to successive application of glutamate, NMDA, and quisqualate is illustrated in Figure 2A. Similar results were observed in 36 slices at all ages examined. In response to bath application of either glutamate (100 PM) or NMDA (50 PM), [Ca2+], increased approximately 6-fold (for example, from 47 + 22 nM to 312 + 118 nM for glutamate, or to 331 + 120 nM for NMDA, n = 32, P5). Using either glutamate or NMDA, [Ca’+]r remained elevated until the agonist was washed out of the slice; [Ca*‘], then returned to baseline values. Bath application of quisqualate (5 PM) increased peak [Ca*+]r to a level similar to that evoked by NMDA and glutamate (from 47 f 22 nM to 380 f 109 nM, n = 32) (Figure 2A). The quisqualate response, however, peaked rapidly and began to declineeven before the solution was changed back to normal artificial cerebrospinal fluid (ACSF). This decline probably reflects the rapid desensitization of the quisqualate receptor (Trussell et al., 1988) or possibly the inactivation of voltage-gated Ca*+ channels (Eckert and Chad, 1984). The quisqualate response was completely blocked by addition of Ni2+ (1 mM) to the perfusate, suggesting that it was due to entry of external Ca2+. We saw no evidenceforquisqualate-induced releaseof Ca2+from iniracellular stores, as has been described in cultured hippocampal cells (Murphy and Miller, 1988), although we did not preload cells by KCI-induced depolarization, which may be necessary to reveal the “metabotropic” glutamate receptor. Nevertheless, we could detect Ca2+ release from intracellular stores. The cholinergic agonist carbachol (50 PM) elicited a Ni2+-insensitive, atropine-sensitive increase in [Ca’+]r, which probably reflects muscarinic receptor-mediated Ca2+ release from intracellular stores (Neher et al., 1988; see below). The shapes of the response curves shown in Figure 2A suggested a strong similarity between the glutamate and NMDA responses. The use of antagonists selective for NMDA and quisqualate receptors demonstrated that the response to bath-applied glutamate was mediated almost exclusively by NMDA, and not quisqualate receptors. When slices were perfused with glutamate (10 slices, PI-P7) in the presence of 50 uM 2-amino-5phosphonovaleric acid (APV), a competitive NMDA antagonist (Davies et al., 1981), the previously measured response to glutamate (50-‘100 PM) was revers-

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Figure 3. Synaptically Induced Ca” Influx Is Triggered by the NMDA Receptor

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Representative single traces of fluorescence changes over background (AF/F) at 380 nm excitation after stimulating the white matter with a single 100 us pulse (arrowhead), while optically recording with a single photodiode from a 200 x 200 urn area in the upper layers of a visual cortical slice (P3). The shutter remained open for IO s. (A) The initial response peaked at a AFIF of about 24% and IastedoverlOs. (B) In the presence of APV (100 PM), AFIF peaked at about 6% and declined to baseline within 2 s. (C) In the presence of APV (100 PM) and DNQX (20 PM), the response was completely abolished. (D). After washing out APV and DNQX, the response was almost identical to that in (A).

(sets)

ibly blocked (Figure 26). For individual cells, the degree of blockade varied between 80% and 100% of the peak response, with a mean of 95% k 4% (n = 70, P3). MK-801 (2 PM), a noncompetitive NMDA antagonist (Wong et al., 1986), had identical actions. Neither APV nor MK-801 had any effect on the response to bathapplied quisqualate. Exogenously applied glutamate thus appears to stimulate primarily NMDA, and not quisqualate, receptors. Since glutamate has been proposed as the principal endogenous excitatory transmitter in the cortex (Mayer and Westbrook, 1987), we next examined the extent to which increases in [Ca’+]i resulting from synaptically released transmitter involved NMDA receptor activation.

Role of the NMDA Receptor in Synaptically Mediated Increases in [Ca*‘]i Afferent fibers were stimulated by a bipolar electrode placed in the white matter while a photodiode recorded changes in fura 2 fluorescence from a 200 x 200 pm region of layer 2/3. Following a single shock, we monitored fura 2 fluorescence continuously for IO s at a single excitation wavelength (380 nm) for better time resolution than our videocameraand ratio imaging could provide. Despite the long illumination time, by using appropriate neutral density filters, the bleachingoffura2fluorescencewas99% (signal within 1% of noise level). When we reversed the experimental procedure and applied DNQX or CNQX (a more potent non-NMDA receptor antagonist [Honore et al., 19881) prior to APV (4 experiments), we observed only a small reduction in the size of the optical signal. An example of such an experiment is shown in Figure 4. CNQX (20 uM) abolished only 24% of the initial signal; the rest was eliminated by APV application. These results were consistently observed when the stimulating electrode was placed in the white matter directly beneath the recording site. If the stimulating electrode was placed >I m m lateral to the recording site, both CNQX and APV abolished most of the response (5 experiments). We interpret these results to indicate that at close distances, where monosynaptic responses should dominate, most of the [Ca*‘li increase is due to entry via the NMDA channel. At greater distances, which should

Regulation

of [W’],

in Developing

Neocortex

337

include polysynaptic responses. Despite these differences, both methods demonstrate that thevast majority of Ca *+ influx after synaptic stimulation results from NMDA receptor activation.

Figure 4. Non-NMDA Receptor Component of the Synaptically

Antagonists Inhibit only a Small Induced Caz+ Influx

Representative traces from an experiment similar to that shown in Figure 3, in which the order of the application of NMDA and non-NMDA antagonists was reversed. A 100 us stimulus was applied to the white matter of a newborn cat primary visual cortex slice, while recording optically with a photodiode from an 200 x 200 urn area in the upper layers. (A) Initial response in standard ACSF. The response peaks at 4.5% AFIF. (B) In the presence of 20 uM CNQX, a specific non-NMDA receptor blocker, the response peaks at 3.4%, which represents a 24% decrease from control. (C) Concomitant application of 100 uM APV and 20 uM CNQX completely abolishes the response. (D) Subtraction of the trace in (B) from the trace in (A) reveals the CNQX-sensitive component, whose magnitude and time course are similar to those of the APV-resistant trace shown in Figure 38.

recruit polysynaptic components, both antagonists are equally effective, presumably because they block long-distance polysynaptic responses. At the low stimulation intensities used, no antidromic component (i.e., glutamic antagonist-insensitive) was observed. Thus, in the developing neocortex, a single pulse of afferent stimulation induces a rise in [Ca’+]r, which we presume to be predominantly monosynaptic, with two components: a fast, but small DNQX-sensitive component and a much larger and slower APV-sensitive component. This APV-sensitive (NMDA-mediated) component contributes approximately 75% of the peak fluorescence change recorded after white matter stimulation, a percentage that agrees quite closely with our findings using bath-applied glutamate stimulation. The differences between the photodiode results and the ratiometric results from bath application of glutamate can be attributed to at least four factors. First, the limited temporal resolution of our ratiometric imaging system may have obscured someof the rapidly desensitizing responses mediated by quisqualate. Second, the photodiode recordings integrate responses from cell bodies and neuropil, whereas the ratiometric measurementswerefrom cell bodies only. Third, the single-wavelength fluorescence changes underestimate the actual changes in [Ca*+], (data not shown). Finally, these responses may

NMDA-Evoked Ca*+ Influx Is Almost Exclusively via the NMDA Receptor lonophore Although we observed that cells in the developing cortex have both functional quisqualate and NMDA receptors, these results suggest that most of the glutamate and synaptically evoked Ca2+ entry in developing rat cortical neurons is either directly or indirectly mediated by NMDA receptor activation. To distinguish between Ca2+ entry via the NMDA receptor channel itself and Ca2+ entry via voltage-sensitive Ca*+ channels that open subsequent to NMDA-induced depolarization, we examined NMDA and non-NMDA responses in the presence of nimodipine, a dihydropyridine blocker of L-type voltage-gated Ca*+ channels (Tsien et al., 1988; Hess et al., 1984) in experiments on 15 slices. The presence of 2 PM nimodipine reduced the response to bath applications of 5 PM quisqualate and 50 PM KCI by 40%-60%, while leaving the response to 50 PM NMDA almost unaltered (Figure 5). Overall, nimodipine blocked 75% of the KCI-induced response was blocked by nimodipine (Nerbonne and Katz, 1990, Sot. Neurosci., abstract). The blockade by nimodipine was dose dependent and reversible, al-

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(A) Application of 50 m M KCI (indicated by KCI on the graph) induces an approximately 6-fold increase in [Caz’], of a single cell in the upper layers of a P4 rat visual cortex. NMDA at 50 uM (indicated by NMDA) resulted in a similar increase in [Ca*‘],. In the presence of 250 nM nimopidine, the response to the same concentration of KCI is diminished approximately 40% (KCII NIMO), while the response to NMDA remains unaltered. (B) In the same cell, after 1 hr of washing with normal ACSF, the response to 50 m M KCI returns to control levels.

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Figure 6. Effects of Nimodipine on Elevation of [Ca*‘], induced by Glutamate Agonists and Depolarization by KCI Pooled data from visual cortex slices from 21 PI-P4 rats using nimodipine concentrations of 62 PM. Responses to NMDA (50 PM) were blocked an average of 5.5% f 2% (n = 74 cells); responses to quisqualate (5 PM) and KCI (50 mM) were reduced 40% f 6% (n = 54) and 56% f 9% 6-r = 26), respectively. The difference between the NMDA group and either the quisqualate or theKCl group is highly significant (t test, pI hr) for recovery. These results suggest that the NMDA-mediated Ca*+ entry we observed occurred through the NMDA receptor itself, rather than through subsequent activation of voltagesensitive Ca2+ channels. Changes in [Ca*‘]i Elicited by GABA GABA is the principal inhibitory neurotransmitter in the neocortex. We were initially interested in determining whether we could observe responses to glutamate and its agonists in the presence of GABA or its agonist muscimol, reasoning that the presence of CABA would keep cells relatively hyperpolarized. We observed, however, that muscimol and GABA themselves consistently induced a significant elevation in [Ca2+],, an effect previously observed in cultured cerebellar granule cells (Connor et al., 1987). Application of 100 PM GABA or 25-50 uM muscimol caused an approximately &fold increase in [Ca”+]i in 14 out of I7 experiments (Figure 7A). These responses occurred in the presence of tetrodotoxin (TTX) and were unaffected by 2 mM kynurenic acid, which blocks both NMDA and non-NMDA receptors. This is consistent with a direct effect of GABA agonist on postsynaptic cells. Responses were transient, however, and even in the continued presence of agonist, [Ca’+]i rapidly returned to close to resting levels. The responses to these inhibitory neurotransmitters were also blocked by 1 mM extracellular Ni2+, suggesting that these responses, like those to glutamate and its analogs, require extracellular Ca2+ entry. The application of muscimol also resulted in a small but consistent elevation of the basal [Ca2+], of about 50%, from 50 + 13 nM to 70 f 13 nM (n = 37), which persisted for at least 8 min. Of all the agonists described in this report, GABA and muscimol were the only ones that consistently produced a persistent elevation of the basal [Ca”]i. The

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a Rise in [Cal*],

(A) Change in [Ca2+],, in an upper layer neuron in a P2 rat visual cortex slice, induced by 50 PM muscimol applied during the period indicated by arrowheads. TTX (1 PM) was present throughout the experiment. Note that after an initial increase, [Ca2+], declines rapidly even in the continued presence of agonist. (B) Small, transient increase in [Ca”], in response to the cholinergic agonist carbachol in a cell from a P3 rat somatosensory cortex slice. Carbachol was applied between the times indicated by the arrowheads; TTX (1 PM) was present throughout the experiment.

significance this time.

of this change, if any, is unclear to us at

Carbachol, an Acetylcholine Agonist, Releases Ca2+ from Intracellular Stores The actions of glutamate, its agonists, and GABA could all be blocked by external Ni2+, suggesting that their actions were mediated by extracellular Ca2+ influx. The acetylcholine agonist carbachol also consistently caused a transient rise in [Ca”]i (Figure 7B). The carbachol response was relatively small compared with the responses to excitatory amino acid agonists-usually no more than a 3-fold increase in [Ca2’], (from 48 rt 30 nM to 88 + 31 nM, n = 15, at P3). The response was always transient, and [Ca2+], returned to baseline even before the drug was removed from the perfusate. In contrast to all the other responses we observed, the increased [Ca’+]i evoked by carbachol was unaffected by 1 mM external NP+, but was abolished by 100 f.rM atropine, a muscarinic receptor blocker. Nicotine (1 mM) elicited no observable responses. We therefore conclude that the actions of acetylcholine in these developing cortical cells are linked to activation of a muscarinic recep-

Regulation 339

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Time (set) Figure 8. [Ca*+],Oscillations Induced by Quisqualate

in Developing

Neocortical

Neurons

(A) Successive video frames of a neuron undergoing rapid changes in [Ca*‘],. Between 0 sand 2 s, the fluorescence intensity (with 380 nM excitation) of the pyramidal cell indicated by the large arrow decreased dramatically and then returned to baseline level within 4 s. This cell was recorded from the upper layers of a P5 somatosensorycortex slice; the pial surface is toward the lower right of the figure. The small arrow indicates a cell whose [Ca2+], remains unchanged during the blinking episode of the other cell. Bar, 10 urn. (B) Graph of the fluorescence changes in the pyramidal cells shown in (A). At the arrowhead, 250 nM quisqualate was applied. The star marks the blinking episode illustrated in (A). No TTX was present in this experiment.

tor, which leads to mobilization stores (Neher et al., 1988).

Transmitter-Induced Cortical Neurons

of intracellular

[Ca*+]i Oscillations

Ca2+

in

When we tested the effects of neurotransmitters such as NMDA and quisqualate near or above concentrations that evoked half-maximal responses, [Ca*+], invariably rose and fell smoothly, as seen in Figures 2, 4, and 6. When we tested very low concentrations of drugs, however, [Ca*‘l, of individual neurons frequently exhibited rapid, nonperiodic oscillations of considerable magnitude, which we call “blinking,” based on the rapid changes in fluorescence intensity. A single blink in a cortical pyramidal cell in response to bath application of 500 nM quisqualate is illustrated in Figures 8A and 88. The blinking episodes of 8 cells in the same experiment are illustrated in Figure 9. All our recordings of these events were done using 2 s intervals between video frames; the onset of these events therefore was less than 2 s. Individual neurons exhibited 1-12 blinks, with 4-120 s between events. During the times that cells were actively responding, the blinks had periods of approximately 13 s. Although

Time (sex) Figure 9. Blinking

in 8 Neighboring

Cells

Simultaneous fluorescence changes of 8 cells in the same field shown in Figure 8A. At the arrowheads, 250 nM quisqualate was applied and remained present for the duration of the experiment. The episodes are repetitive and nonperiodic, with considerable heterogeneity in the number of blinks and the intervals between them.

we have not yet analyzed this in detail, we observed that the blinking of nearby cells was often temporally correlated when recordings were done in the absence of TTX. In Figure 10, the peak of each blink of the cells in Figure 9 is indicated by a line segment. Although we did not compute correlation coefficients for each of these cells, even casual inspection reveals that [Ca’+]r peaks in different cellsfrequentlyoccur in near synchrony. Even 4 min after the onset of responses, 5 of 8 cells exhibited a transient [Ca”]i peak within 4 s of one another. Because we recorded these events at a single wavelength, we did not directly quantify the magnitude of the responses; however, the very large AFlF associated with these events suggests that [Ca*+], is changing at least 6-fold during the largest blinks. Although quisqualatewas most effective in eliciting blinking, we observed similar events in response to low concentrations of NMDA (IO pM)and tocarbachol (20 PM). Both NMDA- and quisqualate-evoked blinks were blocked by 1 m M extracellular Ni2+, suggesting thatexternal Ca*+entrywas required (data not shown). In these cells carbachol elicits a rise in [CaZ’], by releasing intracellular Ca2+ stores, but carbachol-induced blinking was blocked by 1 m M NP, suggesting that extracellular Ca2+ entry is necessary. For all three ago-

Neuron 340

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The peak of each blink is represented by a small bar, and each row represents the blinking episodes of a single cell. The stippled vertical lines mark times at which more than one cell blinks in the same 2 s interval (our temporal resolution in this experiment); these are suggestive of correlated changes in [Ca*‘],.

nists, blinks were usually preceded by a slower influx of Ca*+, suggesting that some elevation in [Ca*+]i may be required to initiate these events. Discussion

We found that fura 2-AM can be used to stain neurons in a semi-intact preparation of the postnatal mammalian brain. Since changes in [Ca*+], are closely linked to neuronal activity(Lev-Ram and Grinvald, 1987;Tank et al.,1988; Rossetal.,1990)fura2(andotherCa2’dyes) can serve as optical indicators of neuronal activity. Unlike voltage-sensitive dyes, which undergo only small changes in fluorescence during activity, often less than 0.1% (Grinvald et al., 1988), Ca*+ indicators change their fluorescence by IO%-100%. This greatly reduces the complexity of instrumentation and the signal-to-noise requirements, allowing the use of video cameras rather than photodiode arrays for imaging dynamic patterns of activity (albeit with somewhat poorer time resolution than voltage-sensitive dyes can provide). In principle the techniques we used to image activity in brain slices should be directly applicable to in vivo imaging of neuronal activity, providing a method for optical recording with cellular resolution. We also suspect that visualizing [Ca*+], changes, rather than postsynaptic voltage changes, may provide a more revealing picture of activity-mediated events in developing brains. Relatively small changes in postsynaptic voltage can lead to large changes in [Ca’+]i, especially when the NMDA receptor is involved. Thus, although the number of synapses in the developing cortex is only a tiny percentage of the adult values, and those synapses contain far fewer vesicles than adult synapses (Armstrong-James and Johnson, 1970), activation of these connections appears capable of inducing very large changes in [Ca2+],, even if cells are “silent” by extracellular single unit recordings or field potentials.

Homogeneity

of the Neurotransmitter

Responses

By using video imaging, we could simultaneously monitor the responses to neurotransmitters of many individual cells in different locations in the slice. Anatomically,cortical neuronsareextremelydiverse,with numerous cell classes distinguished by neurotransmitters, synaptic connections, and projection patterns found in each of the six cortical layers. In the developing cortex, however, we detected little difference in the neurotransmitter responses among cells. This result came as a surprise to us, since one of the goals of our experiments was to identify the neurotransmitter responses of different anatomically identified classes of cells. Although we concentrated most of our experiments in layer 2/3, in most slices, all labeled cells in all layers responded similarly to NMDA, quisqualate, glutamate, GABA, and carbachol, even in the presence of 2 mM TTX to attenuate electrical spread of activity. We also did not encounter consistent differences in neurotransmitter responses in different cortical areas: responses were indistinguishable in the rat somatosensory and visual cortices and even in the kitten visual cortex. The few cells that failed to respond to neurotransmitters invariably had high basal [Ca*+],, which indicated dead or dying cells. This commonality at the second messenger level probably reflects the presence of functional NMDA, quisqualate, GABA, and muscarinic receptors on all developing cortical cells and suggests common features in the initial development of neurotransmitter responses in different classes of cortical neurons residing in different layers or cortical areas. Synaptically Induced Ca2+ Entry during Stimulation Is Dominated by Activation NMDA Receptors

Low-Frequency of

Our finding that most synaptically induced Ca2+ entry is blocked by NMDA antagonists is consistent with electrophysiological investigations in vivo demonstrating that application of APV blocks visual responses of single cells in striate cortex of developing cats (Fox et al., 1989; Tsumoto et al., 1987; Miller et al., 1989a). Our results suggest that the decreased visually evoked activitywould result in a significant reduction in postsynaptic Ca*+ influx as well. The only other instances in which pharmacological information about synaptically induced Ca*+ entry in the mammalian CNS is available is the CA1 region of the hippocampus (Regehr et al., 1989; Regehr and Tank, 1990). When compared with the adult hippocampus and the developing cortex, we find at least one major difference: in the developing cortex most of the synaptically induced rise in [Ca*+], is NMDA receptor-mediated, even at low-frequency synaptic stimulation that causes Ca2+ entry only through voltage-gated channels in the hippocampus. This difference suggests that, in the developing cortex, even infrequent or subthreshold postsynaptic activity can elicit dramatic changes in [Ca2+], via the NMDA receptor, which could lead to changes

Regulation 341

of [CP],

in Developing

Neocortex

in synaptic strength or neuronal architecture comparable to those elicited by high-frequency stimulation in the adult hippocampus and adult cortex (Bliss and Ltimo, 1973; Artola and Singer, 1987). In the developing cortex, excitatory postsynaptic potentials are of much longer duration than those in the adult cortex: 100 ms (Purpura et al., 1965) compared with 5-10 ms. Such long duration excitatory postsynaptic potentials could provide the prolonged depolarizations necessary to permit Ca*+ entry via the NMDA ionophore.

Independence Voltage-Cated

of the NMDA lonophore Ca*+ Channels

tial.Thissuggeststhatweaksubthreshold stimulation, which does not raise the postsynaptic cell to threshold, should give rise to Ca*+ influx primarily through the NMDA receptor, whereas very strong depolarizations would activate both NMDA and voltage-sensitive Ca2+ channel gates. In developing tissue, where synapse numbers are low and postsynaptic electrical responses are weak, these differences in the activation range of the two gates could favor Ca*+ entry through the NMDA gate, as we observed in our electrical stimulation expeirments.

and

Because the NMDA receptor is permeable to Ca2+, K+, and Na+, activation of the receptor not only should allow direct entry of Ca2+, but should also result in depolarization of the cell and subsequent activation in voltage-sensitive Ca2+ channels. Thus the elevation in [Ca*‘]i observed after NMDA application could resultfrom somecombinationofentryviathetwogates. Experimentswith nimodipine, however, revealed that L-type voltage-sensitive Ca” channels contributed little to the NMDA response. However, we do not yet know whether low voltage-activated T-type channels might contribute to the NMDA-induced response. In preliminary experiments using dissociated cortical neurons, we detected primarily nimodipine-sensitive responses; ethosuximide, a T-type channel antagonist (Coulter et al., 1989), had only small effects. Nevertheless, it is certainly possible that in a semi-intact tissue like a brain slice, T-type channels might contribute to the early phase of the NMDA response. The observation that [Ca*+], remains elevated as long as NMDA is present argues that most of the Ca2+ influx is probably not through a rapidly inactivating channel. Not surprisingly, the quisqualate response, which, like the responses to KCI, relies on voltage-sensitive Ca2+ channels was significantly blocked by nimodipine. The absence of”cross-talk” between the NMDA ionophore and voltage-sensitive Ca2+ channels may be due at least in part to differences in the voltage sensitivities of the two conductances. The Mg2’ block of the NMDA receptor is mostly removed at about -40 mV, which should permit Ca2+ entry(Mayer and Westbrook, 1985). In contrast, L-type Ca2+ channels, which appearsto bethedominantCa2+channel inourtissue, are activated at considerably more positive potentials (Tsien et al., 1988). Therefore, relatively low doses of NMDAmaydepolarizeacell sufficientlyto releasethe Mg2+ block of the receptor partly, but not sufficiently to activate voltage-sensitive Ca2+ channels. Mayer et al. (1987), studying Ca*+ fluxes in cultured spinal cord neurons usingarsenazo III and voltageclamping,similarly concluded that “Ca2’ influx through NMDA receptor channels.. . may be significant at the resting potential.” Their studies suggest that, even in the presence of near-physiological levels of Mg2+, considerable, albeit submaximal, Ca2+ influx through NMDA receptor channels occurs at or near the resting poten-

Elevation of [Ca”]; by GABA GABA is widely recognized as the predominant inhibitory neurotransmitter in the mature cerebral cortex (Curtis and Felix, 1971; Scharfman and Sarvey, 1985; Sillito, 1975); in the mature brain GABA usually hyperpolarizes neurons. A growing number of reports suggest, however, that in developing neurons GABA can depolarize cells and lead to increases in neuronal excitability. For example, application of CABA to either the dendrites or cell bodies of immature hippocampal neurons results in a transient depolarization of 5-20 mV (Janigro and Schwartzkroin, 1988; Ben-Ari et al., 1989). In experiments in cultured cerebellar granule cells, Connor et al. (1987) observed (in separate populations of cells) that CABA application caused a small depolarization, a large conductance increase, and a persistent increase in [Ca2+], that lasted for many minutes. In ourcortical slices, GABAor muscimol application caused primarily a transient increase in [Caz+]i. We believe that this increase is consistent with the time course of GABA-induced depolarizations observed by several groups in the hippocampus. Because the GABA-induced increases in [Ca2’], could be blocked by Ni2+, suggesting the involvement of voltage-gated Ca2+ channels, we suspect that the responses are due to the activation of low-threshold, transient Ca2+ channels, or T-type channels (Tsien et al., 1988). In agreement with Connor et al., we did observe a persistent increase in the basal [Ca’+]i after muscimol application, but our increases were only 50% of the basal [Ca2+],, unlike the 2- to 3-fold increases observed in granule cells. This may be attributable to differences in cell type (cortical neurons versus cerebellar granule cells) or to experimental conditions. The experiments of Connor et al. were done on explant cultures, whereas all of our observations were made on acute brain slices. While the functional significance of this GABA-induced Ca2+ influx is currently unknown, we speculate that it may be involved in the development or modification of inhibitory synapses in cortical circuits. Specific inhibitory connections are involved in the generation of many cortical response properties, including end-stop inhibition (Bolz and Gilbert, 1986) and orientation selectivity (Sillito and Versiani, 1977). Most “Hebbian”modelsfortheactivity-dependentdevelopment of cortical circuits deal exclusively with excita-

Neuron 342

tory interactions (e.g., Miller et al., 1989b), postulating that correlated pre- and postsynaptic depolarizations would strengthen synapses, while anti-correlated depolarizations would weaken connections. In the hippocampus, the enhancement of synaptic strength requires influx of Ca2+ through the NMDA receptor. Based on these models, however, it is unclear how inhibitory synapses could be specifically enhanced, added, or removed, since during “normal” inhibitory neurotransmission, depolarization of a GABAergic presynaptic terminal would be associated with hyperpolarization of the postsynaptic cell. If during early development GABA can depolarize cells and, as we have shown, cause a transient increase in postsynaptic [Ca2+],, this would provide a mechanism for the activity-dependent modification of inhibitory synapses. Transmitter-Induced Ca2+ Oscillations Periodic oscillations in [Ca2+], have been observed in a wide variety of neuronal and nonneuronal cells in responseto hormones, mitogens, neuropeptides, and neurotransmitters(Berridgeand lrvine,1989; Rinkand Jacob, 1989). Recently Cornell-Bell et al. (1990) and Jensen and Chiu (1990) demonstrated that glutamate and quisqualateelicit oscillatory behavior in cultured CNS astrocytes. The oscillation periods observed for these glial cells (11-24 s) are similar to the average period of the neuronal oscillations that we observed, although the oscillations in glia appear to be considerably more rhythmic and persistent than those in neurons. We are confident that the cells we observed blinking were in fact neurons and not glia, based on the size of the cells and the presence of apical dendrites. The relationship between these neuronal oscillations and glial oscillations, if any, is presently unclear. If such a relationship exists, however, it could provide a mechanism for locallycorrelatingtheactivityof specific neuronal groups, which could be critical for the emergence of defined cortical circuits. Waves of correlated activity separated by long periods of relative inactivity have been observed in the developing retina using multisite extracellular electrode recording and have been postulated to generate the correlations necessary to segregate the inputs from the two eyes within the lateral geniculate nucleus in the cat (Wong et al., 1990, Sot. Neurosci., abstract). Similar patterns of activity have been observed using optical recordings in the retina with flue3 (Wong and Shatz, personal communication). SinceCa2’fluxesarecloselyassociatedwith neuronal activity (Lev-Ram and Grinvald, 1987; Tank et al., 1988; Ross et al., 1990), our ability to load and image virtually all neurons in a developing cortical slice with Ca2+-sensitive indicators provides a powerful tool for revealing functional interactions between groups of neurons and should reveal patterns of excitability within neuronal networks that would be difficult to detect with conventional techniques.

Experimental

Procedures

Preparation of Brain Slices Coronal brain slices (400 pm thick) were prepared, following previously described procedures (Katz, 1987), from somatosensory and visual cortices of I- to 7-day-old Long-Evans rats and the visual cortex of newborn cats. Rats up to Cdays-old were cryo-anesthetized prior to decapitation; older rats and cats were first anesthetized with Nembutal (50 mg/kg, intraperitoneally). Slices were maintained submerged in wells inside a rotating (60 rpm), heated (33OC) incubation chamber, in standard ACSF containing 2 m M Ca2+ and 1.3 m M Mg2’, bubbled with 95% Od5% COz. Slices were incubated for 1 hr in 10 PM fura- AM, 0.001% pluronic acid and then transferred to a heated (33OC) recording chamber on the stage of aZeiss IM35 inverted microscope. Slices were superfused with oxygenated test solutions at 10 ml/min (chamber volume 0.5 ml). All solutions (except those used in electrical stimulation and oscillation experiments) contained l-2 PM lTX to reduce synaptic release of endogenous neurotransmitter that would otherwise be triggered by bath application of glutamate agonists. L-Glutamate, quisqualate, APV, and TrX were from Sigma. MK-BOI was kindly provided by Dr. P. Anderson (Merck Sharp & Dohme Research Laboratories, West Point, PA). NMDA was obtained from Cambridge Research Biochemicals (Cambridge, MA), and DNQX and CNQX from Tocris Neuramin (Essex,CB),fura2-AM,fura2 K+salt,and pluronicacid from Molecular Probes (Eugene, OR). Nimodipine was kindly provided by Dr. A. Scriabine (Miles Pharmaceuticals, West Haven, CT). Limitations of Fura Z-AM Loading We noted a marked age dependency in the ability of fura 2-AM to load into neocortical neurons. In the rat, virtually every cortical neuron was labeled at birth (PO). With increasing age, labeling became increasingly restricted to the superficial layers, which contain the most recently postmigratory neurons. By PIO, only a few scattered neuronal somata in a slice were loaded with dye, despite considerable staining of other elements of the neuropil. The inability to load cells was not due to an inability of older cells to cleave the ester bond: acutely dissociated cells from all ages loaded beautifully with fura 2-AM. Nor was the inability to accumulate indicator due to the active secretion of fura 2 by mature cells, since intracellular injections of the K+ salt of fura 2 into adult cortical neurons remained in the cytoplasm for hours. Rather, it appears that the structure of the intact slice somehow prevents effectiveloadingof most neurons, by preventing access of the dye to neuronal membranes. We tested different Ca2+ dyes and over 20 detergents, along with osmotic shock, electroporation, lipofection, extracellular esterase inhibitors, secretion inhibitors (e.g., probenicid), and various incubation temperatures and durations; none of these improved the labeling in older animals. Optical Recording Cells in the lower 50 pm of the slices were visualized with Zeiss Plan-Neofluar 25x and 63x oil immersion objectives. Neutral density filters of 1.0-2.0 optical density were used $t all times to preventdye bleaching and phototoxicity. For ratiometric images, the emission at >490 nm was collected for pairs of images at 350 and 380 nm excitation (10 nm bandwidth interference filters; Omega, 75 W xenon lamp) using a computer-controlled filter wheel and either a SIT or ICCD camera (Hamamatsu) coupled to an image processor (Imaging Technology 151). Pairs of images were collected every IO-30 s; each image resulted from the average of 16 video frames. Background-subtracted pairs of images were stored using a high-resolution optical disc recorder (Panasonic TQ 20289. Digital thresholding was used to create a mask that allowed us to visualize cell bodies selectively in the plane of focus and to suppress out-of-focus cells and fluorescence. To estimate [Ca2+li, average pixel values of individual cell bodies

Regulation 343

of [Ca”], in Developing

Neocortex

were determined at each wavelength and at each time point, yielding values for R that were used in the formula [Ca2+], = & St& KR - RmMRma. - R)]. This system was calibrated with high and low [Ca2+], solutions in a 170 urn thick calibration chamber and with slices kept under low and high [Ca*+], conditions (low [Ca2+],condition: 6 hr incubation in Ca2+-free ACSFcontaining 10 m M EGTA; high [Ca*+], condition: 30 min in ACSF containing 10 m M Ca*+ and 1% Triton-X). The values of the constants used in the formula were Kd = 220, R,,. = 0.13, R,, = 3.5, and St/S, = 10. All [CaZ+], values are expressed as the mean + SD. For single-wavelength recordings, excitation at 380 nm was used and averages of 4-16 video frames were collected every 2 s. Our estimates of the actual [Ca2+], are subject to several errors, the most serious of which is contributions from out-of-focus labeled cells. Because almost all cells throughout the thickness of the slice were labeled with fura 2, the signal recorded at each cell body in focus contains contributions from out-of-focus and neuropil. However, the changes of the out-of-focus signals were in all cases of identical time course and direction to the behavior of individual in-focus cells, but of lower magnitude.

Berridge, M. J., and Irvine, R. F. (1989). lnositol cell signalling. Nature 347, 197-205.

Electrical Stimulation and Photodiode Recordings For recording responses to electrical stimuli, fluorescence at a single wavelength (380 mM) was measured by a single photodiode (EC & G) in place of the video camera. The time constant of the photodiodewas approximately2 ms. Singleelectrical shocks (100 us duration) were delivered from a stimulus isolator via a bipolar tungsten electrode placed in the cortical white matter directly underneath the diode recording site. The photodiode signals were recorded on tape and subsequently digitized and analyzed using pClamp (Axon Instruments).

Cornell-Bell, A. H., Finkbeiner, S. M., Cooper, M. S., and Smith, S. J. (1990). Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247, 470-473. Coulter, D.A., Huguenard, J. R., and Prince, D.A. (1989). Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons. Ann. Neural 25, 582-593.

Acknowledgments

Davies, J., Francis, A. A., Jones, A. W., and Watkins, J. C. (1981). 2-Amino-5phosphonovalerate (P-APV), a potent and selective antagonist of amino acid-induced and synaptic excitation. Neurosci. Lett. 27, 77-81.

We thank Dr. S. Smith for providing our initial image processor programs, M. Johnson, P. Huang, and K. Christian for extensive computer programming, and P. Peirce for photography. Drs. T. Bonhoeffer, A. Grinvald, R. Lewis, J. Nerbonne, and T. Wiesel provided critical comments and technical assistance. L. C. K. is a Lucille P. Markey scholar, and this work was supported by a grant from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

20, 1990; revised

January

Armstrong-James, M. (1975). The functional status and columnar organization of singlecells responding to cutaneous stimulation in neonatal rat somatosensory cortex Sl. J. Physiol. 246,501-538. Armstrong-James, M., and Johnson, R. (1970). Quantitative studies of postnatal changes in synapses in rat superficial motor cortex. Z. Zellforsch. Mikrosk. Anat. 710, 559-568. A., and Singer. W. (1987). Long-term receptors in rat visual cortex. Nature

potentiation 330, 649-652.

and

Bliss, T. V. P., and LBmo, T. (1973). Long-lasting potentiation of synaptic transmission in the dentate area of the unanesthetized rabbit following stimulation of the perforant path. J. Physiol.232, 331-356. Bolz, J., and Gilbert, in the visual cortex 362-365.

C. D. (1986). Generation of end-inhibition via interlaminar connections. Nature 320,

Cline, H. T., Debski, E. A., and Constantine-Paton, M. (1987). N-methyl-o-aspartate receptor antagonist desegregates eyespecific stripes. Proc. Natl. Acad. Sci. USA 84, 4342-4345. Collingridge,C. L., Kehl, S. J.,and McLennan, H. (1983). Excitatory amino acids in synaptic transmission in the Schaffer collateralcommissural pathway of the rat hippocampus. J. Physiol. 334, 33-46. Connor, J. A., Tseng, H.-Y., and Hockberger, P. E. (1987). Depolarization-and transmitter-induced changes in intracellular Ca*+ of rat cerebellar granule cells in explant cultures. J. Neurosci. 7, 1384-1400.

Curtis, D. R., and Felix, D. (1971). The effect of bicuculline synaptic inhibition in the cerebral and cerebellar cortices cat. Brain Res. 34, 301-321.

Eckert, R., and Chad, J. E. (1984). Inactivation Prog. Biophys. Mol. Biol. 44, 215-267.

upon of the

of Ca2+ channels.

Fox, K., Sato, H., and Daw, N. (1989). The location and function of NMDA receptors in cat and kitten visual cortex. J. Neurosci. 9, 2443-2454. Grinvald, A., Frostig, R. D., Lieke, E., and Hildesheim, R. (1988). Optical imaging of neuronal activity. Physiol. Rev. 68,1285-1366. Grynkiewicz, C., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca*+ indicatorswith greatly improved fluorescence prop erties. J. Biol. Chem. 260, 3440-3450. Hess, P., Lansman, J. B., and Tsien, R. W. l(1984). Different modes of Caz+ channel gating behavior favoured by dihydropyridine Ca2+ agonists and antagonists. Nature .?‘17, 538-544.

8,1991

References

Artola, NMDA

phosphates

and

Honore, T., Davies, S. N., Drejer, J.. Fletcher, E. J., Jacobsen, P., Lodge, D., and Nielsen, F. E. (1988). Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science 247, 701-703. Hubel, D. H., and Wiesel, T. N. (1963). Receptor fields of cells in striate cortex of very young, visually inexperienced kittens. J. Neurophysiol. 26, 994-1002. Janigro, D., and Schwartzkroin, P. A. (1988). Effects of GABA and baclofen on pyramidal cells in the developing rabbit hippocampus: an in vitro study. Dev. Brain Res. 47, 171-183.

of visual cortical Nature 320, 172-

Jensen, A. M., and Chiu, S. Y. (1990). Fluorescence measurement of changes in intracellular calcium induced by excitatory amino acids in cultured cortical astrocytes. J. Neurosci. 70, 1165-1175.

Bear, M. F., Kleinschmidt, A., Cu, Q., and Singer, W. (1990). Disruption of experience-dependent synaptic modifications in striate cortex by infusion of an NMDA receptor antagonist. J. Neurosci. 70, 909-925.

Katz, L. C. (1987). Local circuitry of identified projection neurons in cat visual cortex brain slices. J. Neurosci. 7, 1223-1249.

Bear, M. F., and Singer, W. (1986). Modulation plasticity by acetylcholine and noradrenaline. 176.

Ben-Ari, Y., Cherubini, E., Corradetti, Giant synaptic potentials in immature rones. J. Physiol. 476, 303-325.

R., and Caiarsa, J.-L. (1989). rat CA3 hippocampal neu-

Kriegstein, A. R., Suppes, T., and Prince, D. A. (1987). Cellular and synaptic physiology and epileptogenesis of developing rat neocortical neurons in vitro. Dev. Brain Res. 34, 161-171. Krnjevic, K. (1987). GABAergic Mind Behav. 8, 537-547.

inhibition

in the

neocortex.

J.

NeWOIl 344

Lev-Ram, V., and Grinvald, A. (1987). Activity-dependent calcium transients in central nervous system myelinated axons revealed by the calcium indicator fura-2. Biophys. J. 52, 571-576.

Trussell, L. O., Thio, L. L., Zorumski, C. F., and Fischbach, G. D. (1988). Rapid desensitization of glutamate receptors in vertebrate central neurons. Proc. Natl. Acad. Sci. USA 85, 2834-2838.

Lynch, C., Larson, J., Kelso, S., Barrionuevo, C., and Schottler, F. (1983). Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305, 719-721.

Tsien, R. W., Lipscombe, D., Madison, D. V., Bley, K. R., and Fox, A. P. (1988). Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci. 77, 431-438.

Maffei, L., and Calli-Resta, L. (1990). Correlation in the discharges of neighboring rat retinal ganglion cells during prenatal life. Proc. Natl. Acad. Sci. USA 87, 2861-2864.

Tsumoto, T., Hagikara, K., Sato, H., and Hata, Y. (198;3. NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats. Nature 327, 513-514.

Malenka, R. C., Kauer, J. A., Zucker, R. S., and Nicoll, R. A. (1988). Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science 242, 81-84.

Udin, S. B., and Scherer, W. J. (1990). Restoration of the plasticity of binocular maps by NMDA after the critical period in Xenopus. Science 249, 669-672.

Malenka, R. C., Kauer, J. A., Perkel, D. J., and Nicoll, R. A. (1989). The impact of postsynaptic calcium on synaptic transmissionits role in long-term potentiation. Trends Neurosci. 72, 444-450.

Van der Loos, H., and Woolsey, T. A. (1973). Somatosensory cortex: structural alterations following early injury to sense organs. Science 779, 395-398.

Mayer, M. L., and Westbrook, C. L. (1985). Theaction of N-methyl-oaspartic acid on mouse spinal neurones in culture. J. Physiol. 367, 65-90.

Wiesel, T. N. (1982). Postnatal development and the influence of environment. Nature

of the visual cortex 299, 583-592.

Mayer, M. L., and Westbrook, G. L. (1987). The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28, 197-276.

Wigstrom, H., Gustafsson, B., Huang, Y.-Y., and Abraham, W. C. (1986). Hippocampal long-term potentiation is induced by pairing single afferent volleys with intracellularly injected depolarizing current pulses. Acta Physiol. Stand. 726, 317-319.

Mayer, M. L., MacDermott, A. B., Westbrook, G. L., Smith, S. J., and Barker, J. L. (1987). Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo Ill. J. Neurosci. 7, 3230-3244.

Wong, E. H. F., Kemp, J. A., Priestley, T., Knight, A. R., Woodruff, G. N., and Iversen, L. L. (1986). The anticonvulsant MK-801 is a potent N-methyl-o-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83, 7104-7108.

McCormick, D. A., and Prince, D. A. (1987). Post-natal development of electrophysiological properties of rat cerebral cortical pyramidal neurones. J. Physiol. 393, 743-762. Miller, K. D., Chapman, B., and Stryker, M. P. (1989a). Visual responses in adult cat visual cortex depend on N-methyl-easpartate receptors. Proc. Natl. Acad. Sci. USA 86, 5183-5187. Miller, K. II., Keller,J. B., and Stryker, M. P. (1989b). Ocular dominance column development: analysis and simulation. Science 245, 605-615. Murphy, S. N., and Miller, R. J. (1988). Aglutamate receptor regulates Caz+ mobilization in hippocampal neurons. Proc. Natl. Acad. Sci. USA 85, 8737-8741. Neher, E., Marty, A., Fukuda, K., Kubo, T., and Numa, S. (1988). Intracellular calcium release mediated by two muscarinic receptor subtypes. FEBS Lett. 240, 88-94. Purpura, D. P., Shofer, R. J., and Scarff, T. (1965). Properties of synaptic activities and spike potentials in immature neocortex. J. Neurophysiol. 28, 925-942. Regehr, W. C., and Tank, D. W. (1990). Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites. Nature 345, 807-810. Regehr, W. C., Connor, J. A., and Tank, D. W. (1989). Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation. Nature 347, 533-536. Rink, T. J., and Jacob, R. (1989). Calcium excitable eels. Trends Neurosci. 72, 43-46.

oscillations

in non-

Ross, W. N., Lasser-Ross, N., and Werman, R. (1990). Spatial and temporal analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites. Proc. R. Sot. (Lond.) B 240, 173-185. Scharfman, H. E., and Sarvey, 1. M. (1985). Responses to gammaaminobutyric acid applied to cell bodies and dendrites of rat visual cortical neurons. Brain Res. 358, 385-389. Sillito, A. M. (1975). The effectiveness of bicucullineas an antagonist of GABA and visually evoked inhibition in the cat’s striate cortex. J. Physiol. 250, 287-304. Sillito, A. M., and Versiani, V. (1977). The contribution atory and inhibitory inputs to the length preference complex cells in layers II and III of the cat’s striate Physiol. 273, 775-790.

of excitof hypercortex. J.

Tank, D. W., Sugimori, M., Connor, J. A., and Llinas, R. R. (1988). Spatially resolved calcium dynamicsof mammalian Purkinjecells in cerebellar slices. Science 242, 773-777.

Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters.

We assessed the pathways by which excitatory and inhibitory neurotransmitters elicit postsynaptic changes in [Ca2+]i in brain slices of developing rat...
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